WO2008066199A1 - Method of inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method and use of the same - Google Patents

Method of inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method and use of the same Download PDF

Info

Publication number
WO2008066199A1
WO2008066199A1 PCT/JP2007/073352 JP2007073352W WO2008066199A1 WO 2008066199 A1 WO2008066199 A1 WO 2008066199A1 JP 2007073352 W JP2007073352 W JP 2007073352W WO 2008066199 A1 WO2008066199 A1 WO 2008066199A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
insulin
medium
cell
secreting
Prior art date
Application number
PCT/JP2007/073352
Other languages
French (fr)
Japanese (ja)
Inventor
Naoya Kobayashi
Jorge David Rivas-Carrillo
Noriaki Tanaka
Original Assignee
Naoya Kobayashi
Jorge David Rivas-Carrillo
Noriaki Tanaka
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Naoya Kobayashi, Jorge David Rivas-Carrillo, Noriaki Tanaka filed Critical Naoya Kobayashi
Priority to JP2008547073A priority Critical patent/JPWO2008066199A1/en
Publication of WO2008066199A1 publication Critical patent/WO2008066199A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to a method for inducing differentiation of embryonic stem cells (hereinafter also referred to as ES cells) into insulin-secreting cells, insulin-secreting cells induced by the method, a therapeutic agent for diabetes, a bioartificial organ, research
  • ES cells embryonic stem cells
  • the present invention relates to reagents and drug discovery model animals.
  • Yeouido is a group of endocrine cells, consisting of ⁇ cells, / 3 cells, ⁇ cells, and ⁇ cells. Insulin, the only hormone in the body that has the effect of lowering blood sugar, is secreted from the ⁇ cells of Yeouido. Isojima transplantation aims to replace and regenerate a hypoglycemic system that has been abolished by isolating this island from prey and transplanting it to a patient with insulin-dependent diabetes.
  • ⁇ cells account for 80-85% of all cells that make up the islands. ⁇ -cells don't just secrete insulin; more importantly, they can sense sugar in the blood.
  • the main body of regulation of insulin secretion in glucose metabolism depends on a complex system involving multiple organs! /! In other words, it is possible to adjust blood glucose to a very narrow range by reacting quickly to blood glucose that changes rapidly during exercise or eating alone and secreting the appropriate amount of insulin. Is possible. Sensing the effect target and adjusting the action The fact that a single type of cell is used alone and that the cell occupies most of the island is the rationale for the island transplant.
  • Diabetes mellitus due to insulin deficiency or deficiency is indicated for Kashiwajima transplantation.
  • Diabetes is largely divided into two categories based on the cause of the decrease in activity. One is the destruction of ⁇ / 3 cells due to some cause, damage and loss of insulin secretion, the other is the presence or absence of insulin in the blood normal or normal S insulin resistance is peripheral Since it exists in the tissue, the action of insulin is reduced.
  • the former is called type 1 diabetes or juvenile diabetes, and the latter is called type 2 diabetes.
  • the indication for Kashiwajima transplantation is type 1 diabetes.
  • type 2 diabetes Even in type 2 diabetes, as the stage progresses, insulin deficiency is caused by glucose toxicity and / 3 cell exhaustion, but type 2 diabetes is not currently indicated for Kashiwajima transplantation. The reason is that insulin secretion is maintained to some extent.Insulin is hardly secreted against type 2 diabetes.In type 1 diabetes, blood glucose control by insulin therapy is very difficult. This is because of the insulin resistance in the base, and there is concern about the effects of the island transplantation. Although strict regulation of blood glucose works well in prognosis even in type 2 diabetes, there is a severe fact that there is a shortage of transplanted islands, and only type 1 diabetes is the subject of transplantation. In the situation where the supply of transplanted islands will be abundant in the future, non-insulin-dependent diabetes mellitus triggered by insulin resistance is likely to be indicated.
  • Intense insulin therapy reduced the incidence or progression of long-term complications from 39% to 76% compared to conventional insulin therapy, and at the same time increased the incidence of hypoglycemia three-fold.
  • This clinical trial has shown that strict regulation of blood glucose is very useful in avoiding long-term complications of diabetes, and that some of the goal can be achieved with insulin-enhanced therapy. This also means that insulin therapy does not allow perfect blood glucose control and limits the prevention of long-term complications.
  • Type 1 diabetes is caused by abnormal destruction of ⁇ ⁇ ⁇ / 3 cells that produce insulin due to abnormal autoimmunity (Atkinson MA et al., ⁇ Engl. J. Med., (1994) 331). , P.1428-1436.
  • transplantation which is one of the regenerative replacement therapy for ⁇ / 3 cells.
  • This method includes spleen transplantation and Yeouido transplantation.
  • the purpose of these two transplants is to allow very strict glycemic control and avoid the development of hypoglycemia and even long-term complications.
  • the purpose of insulin therapy is not just to improve the quality of life (QOU) by freeing patients from the daily annoyance of insulin therapy.
  • Transplant therapy is an insulin therapy as a means to move insulin-dependent diabetes toward the ultimate goal of healing. It is a treatment method with much more potential, but it has a big problem with the current Kashiwajima transplantation, but there is a shortage of transplanted Kashiwajima.
  • ⁇ ⁇ 2603_2609 ⁇ are effective in inducing differentiation into insulin-secreting cells. Is low (less than 3%), and the amount of insulin secreted is less than 1 / 1,000 per mg protein compared to normal Kashiwajima. This is because both methods perform embryoid body formation in the first step for initial differentiation to induce insulin-secreting cells from undifferentiated ES cells. Differentiation is induced by embryoid body formation, but the cells are differentiated in all directions, such as ectoderm, endoderm, and mesoderm. Therefore, the target insulin-secreting cells belong to the endoderm. Therefore, since cells that differentiate into other ectoderm or mesoderm cannot be trapped, there is a disadvantage that the final differentiation induction rate is low.
  • the insulin secretion amount of the insulin-secreting cells obtained by the conventional technique is as low as 1/100 to 1/500 of the normal island. This is because during the treatment process in the Damour method! /, And during the development of the viscera, the differentiation into ⁇ -cells is suppressed (Miralles F et al., I nt J Dev Biol. 2006; 50 (1): 17_26.) and fibers reported to induce exocrine cells (see Bhushan A et al., Development. 2001; 128 (24): 5109_17) One factor is the use of blast growth factor 10 (FGF-10)!
  • An object of the present invention is to provide a sufficiently functional, safe insulin-secreting cell that can be supplied in large quantities, a therapeutic agent for diabetes, a bioartificial viscera, a research reagent, and a drug model animal using the cell. It is to be.
  • the present invention relates to a method for inducing differentiation of embryonic stem cells into insulin-secreting cells, wherein (a) embryonic stem cells are cultured in the presence of fountain fibroblast growth factor and activin.
  • a step of differentiating into an endoderm (b) a step of differentiating the resulting definitive endoderm into a primitive gland by culturing in a conditioned medium in the presence of a fibroblast growth factor, (c) obtained Culturing primordial buds in a conditioned medium to increase the number of neurogenin 3 (Ngn 3) -expressing cells to obtain neurogenin 3 expressing cells; and (d) the resulting neurogenin 3 expressing cells.
  • a method comprising the step of differentiating into insulin-secreting cells by stimulation in a cell culture medium having a high glucose concentration.
  • the fountain fibroblast growth factor is a fountain fibroblast growth factor other than fountain fibroblast growth factor 10.
  • the activin is preferably activin A! /.
  • the concentration of activin is 2 to 200 ng / mL! /.
  • Conditioned medium strength used in step (b) It is preferable that the human glioblastoma cell line T98G is cultured in a serum-free cell culture medium.
  • Conditioned medium strength S used in step (c), human liver endothelial cell line TMNK-1, or vascular endothelial cells are preferably cultured in a low glucose cell culture medium.
  • the culture in each step is collagen type IV, matrigel, fibronectin, respectively.
  • Preferred is a three-dimensional culture using a biocompatible material selected from the group consisting of: dielatin, polyornithine and laminin.
  • the biocompatible material used in step (a) is preferably collagen type IV.
  • the biocompatible material used in step (a) is preferably laminin.
  • the concentration of the fibroblast growth factor is preferably 2 to 20 ng / mL.
  • the medium used in the step (a) preferably contains 0.2 to 3% ushi fetal serum and 2 to 3% ushi serum albumin in a serum-free cell culture medium. .
  • the medium used in step (b) contains retinoic acid in a conditioned medium in an amount of 1-50 11101 /
  • the medium used in step (1) contains 0.25-2 11101 / ⁇ -secretase inhibitor XVIII and 5-5013 ⁇ 4 / 111 fibroblast growth factor in the conditioned medium, and epithelial growth It is preferable to contain 5 to 50 ng / mL of the factor.
  • step (c) it is preferable to select using a neurogenin 3 expressing cell force cell sorter.
  • the medium used in the step (d) contains 2 to 50 ng / mL of hepatocyte growth factor, contains !! to 1 Ommol / L of nicotinamide, and contains 5 to 1 OOnmol / L of exedine 4. It is preferable to contain 1 to 10 mol / L of troglitazone and 1 to 20 mol / L of zinc sulfate.
  • Step (d) comprises a step of repeatedly stimulating and culturing neurogenin 3 expressing cells in a cell culture medium having a high glucose concentration and then in a cell culture medium having a low glucose concentration. It is preferable to repeat 1 to 3 times of stimulation culture for 2 hours per day in the medium for 1 to 10 days.
  • the medium used in step (d) is a lipid in cell culture medium having a high glucose concentration. It preferably contains amino acids, vitamins and / or minerals.
  • step (d) includes enhancing Maf A gene expression in insulin-secreting cells by introducing and expressing the Maf A gene in a neurogenin 3 expressing cell.
  • the embryonic stem cell is derived from a mammal.
  • the mammal is most preferably a mouse. .
  • the present invention also relates to insulin secreting cells induced by the differentiation induction method.
  • Insulin-secreting cell strength S and insulin-secreting cells forming spheroids are preferably contained.
  • the present invention further relates to a therapeutic agent for diabetes containing the insulin-secreting cells.
  • the present invention further relates to a bioartificial viscera containing the insulin-secreting cells.
  • the present invention further relates to a research reagent containing the insulin-secreting cells.
  • the present invention further relates to a drug discovery model animal containing the insulin-secreting cells.
  • FIG. 1 Photograph showing the determination of gene expression of definitive endoderm-specific genes Foxa2 and Soxl 7 in mouse definitive endoderm differentiated from mouse ES cells by the method of the present invention by RT-PCR. It is. Lanes 1 to 4 show the gene expression of mouse definitive endoderm cultured in collagen type IV, matrigel, laminin type 1, and dielatin, respectively.
  • FIG. 2 Expression of definitive endoderm-specific genes Foxa2 and Soxl 7 in mouse definitive endoderm differentiated from mouse ES cells using culture dishes processed with collagen type IV by the method of the present invention. It is a photograph determined by PCR method. Lanes ! to 4 show gene expression of mouse definitive endoderm cultivated by adding and cultivating activin A at a concentration of 3 ⁇ 4 ng / ml, 10 ng / ml, lOOng / ml, and 200 ng / ml, respectively.
  • FIG. 3 Gene expression of the primordial acupuncture-specific gene Pdx-1 in the mouse primitive gland, in which mouse definitive endoderm differentiation was induced by the method of the present invention, Shh gene expression as a negative control, and endogenous standard This is a photograph in which GAPDH gene expression is determined by RT-PCR. Lanes 1 to 4 have retinoic acid concentrations of 1 ⁇ , ⁇ , ⁇ , 100 The gene expression of the primitive mouse mouse added and cultured at 11 M and 1000 M is shown.
  • FIG. 4 (a)] is a microscopic image showing the expression of Maf A gene in insulin-secreting cells induced from mouse ES cells by the differentiation induction method of the present invention. 1 indicates the nucleus, and 2 indicates the entire insulin-secreting cell that is spherical.
  • 4 (b)] is a microscopic image showing the morphology of insulin-secreting cells derived from mouse ES cells by the differentiation induction method of the present invention. 1 indicates the nucleus and 2 indicates the insulin-secreting cells that are spherical.
  • Insulin-secreting cells derived from mouse ES cells using low glucose RCNK-1 conditioned medium in step (d) by the differentiation induction method of the present invention and mouse ES cells by the conventional differentiation induction method 2 is a graph showing the insulin secretory capacity of insulin secreting cells derived from the serotype. 1-10 on the horizontal axis in the medium after the insulin-secreting cells of the present invention were cultured in step (d)! /, After being cultured in low gnorecose RCNK-1 conditioned medium for 2 days after being stimulated with “gnorecose stimulation: low ⁇ high” Insulin secretion amount (1 on the horizontal axis), the insulin-secreting cells of the present invention in step (d)!
  • the amount of insulin secretion in the medium after “low ⁇ high ⁇ low” (4 on the horizontal axis), insulin secretion of the present invention Insulin secretion amount (5 on the horizontal axis) in the medium after culturing the vesicles in step (d) for 7 days in low glucose RCNK-1 conditioned medium after “glucose stimulation: low ⁇ high”
  • the amount of insulin secretion in the medium after “Glucose Stimulation: Low ⁇ High ⁇ Low” after culturing for 7 days in a low glucose RCNK-1 conditioned medium (6 on the horizontal axis), the conventional Schroder method The amount of insulin secreted in the medium after ⁇ glucose stimulation: low ⁇ high '' (7 on the horizontal axis), and the insulin-secreting cells of the conventional shredder method ⁇ glucose stimulation: low ⁇ high '' ⁇ Insulin secretion in the medium after low (8 on the horizontal axis), Insulin secretion in the medium after ⁇ Darcose
  • FIG. 7 A graph showing blood glucose levels after transplantation of insulin secreting cells, undifferentiated mouse ES cells, or mouse islets derived from mouse ES cells by the differentiation induction method of the present invention and transplanted to diabetic mice.
  • healthy mice and non-transplanted diabetic mice are used.
  • healthy mice and transplantation should be carried out! /, NA! / And diabetic mice were injected with 2 units (IU) of intermediate human insulin NPH subcutaneously, and the subsequent 5 days were treated with intermediate humans.
  • One unit of insulin NPH is injected subcutaneously.
  • FIG. 8 is a diagram showing an embodiment of the bioartificial viscera of the present invention.
  • the bioartificial spleen is a cell inlet 3 for injecting spherical insulin-secreting cells 7, a lid 4, a polyethylene butyl alcohol membrane (pore size 30 nm) for blocking the permeation of immunocompetent cells 5, It consists of PTAU nonwoven fabric 6 with PAU processing.
  • the bioartificial spleen of the present invention filled with insulin-secreting cells, undifferentiated mouse ES cells, or mouse islets derived from mouse ES cells by the differentiation-inducing method of the present invention is embedded in a dialysis mouse and implanted. It is the graph which measured the blood glucose level after.
  • As a control healthy mice and non-transplanted diabetic mice are used. After transplantation During the first 10 days, healthy mice and non-transplanted diabetic mice are injected with 2 units (IU) of human intermediate insulin NPH subcutaneously!
  • the present invention provides a method in which definitive endoderm differentiated from undifferentiated ES cells using activin is differentiated into neurogenin 3 expressing cells using a conditioned medium, and then the cells are enriched in a high-darcose medium.
  • ES cells that are preferably derived from mammals include mice, humans, monkeys, and the like.
  • induced pluripotent stem cells also called induced pluripotent stem cells
  • iPS cells differentiate pluripotent stem cells having differentiation pluripotency like ES cells instead of ES cells are also used in the present invention.
  • iPS cells are expected to lead to cell transplantation therapy for many diseases such as spinal cord injury and juvenile diabetes. When iPS cells are induced from the patient's own somatic cells, immune rejection after transplantation can be overcome. (Home page http://www.jst.go.jp/pr/info/info320/index.html).
  • ES cells can be produced from embryos, which are the early stages in which a fertilized egg continues to grow.
  • a fertilized egg with a single ovum and sperm is divided into two, four, eight, etc. in the middle of growing into a fetus, and is called a blastocyst on days 5 and 6 It becomes a state.
  • the blastocyst is composed of a blastocoel that holds an inner cell mass that is a spherical mass about 0.1 mm in diameter.
  • the inner cell mass eventually grows into endoderm, mesoderm, and ectoderm and forms all the cells of the body, and the trophectoderm forms their placenta and isolates the embryo from the outside world Shape bag To do.
  • ES cells can be produced by removing the cells and culturing these cells in an environment where they can proliferate and do not differentiate. Therefore, ES cells can be produced as described above.
  • Such ES cells were used in mice in 1981 in Evans et al. (1981, Nature 292: 154-6 ⁇ ) and Martin et al. (Martin GR. Et al., 1981, Proc Natl Acad Sci 78: 7634- 8.) and can be purchased from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan).
  • Nestin-positive stem cells isolated from islets are cultured in the presence of activin A and HGF! / Activin A and betacellulin or nicotinamide and ethasendin-4 to the same extent in rats and humans.
  • ES cells derived from any species, particularly human-derived ES cells are induced to differentiate into insulin-secreting cells according to the present invention. It is obvious to those skilled in the art (Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas M, Habener JF. Multipotential nes ti. n-positive stem cells isolated from adult pancreatic islets differentiated ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001; 50 (3): 521_3 3).
  • iPS cells can be prepared by introducing specific factors into somatic cells. Mau Introducing four factors (Oct3 / 4, Sox2, c-Myc, Klf4) into the somatic cells using retroviral vectors, iPS cells with similar morphology and proliferative potential to ES cells and pluripotency (Takahashi, Yamanaka S., Cell. 25; 126 (4): 663_76, 2006 ⁇ ).
  • Human HPS cells can also be produced by introducing the same 4 factors as mouse into somatic cells derived from human adult skin using retroviral vectors and then culturing under the conditions of human ES cells (Ta kahashi., Anabe K, Ohnuki, Nanta M, IchisaKa ⁇ , omoda, YamanaKa Ce 11. 2007 131 (5): 861-72 ⁇ ).
  • iPS cells can be preferably used because they have few ethical problems that do not require the use of fertilized eggs. In addition, these cells are highly likely to contribute greatly to elucidating the cause of diseases and developing new therapeutic drugs.
  • the medium used in the cell culturing method of the present invention may be of any composition, as long as it can grow cells, minerals, sugars, amino acids, peptides, vitamins, organic acids, nucleic acids. Any component that contains components necessary for cell culture, such as a pH adjuster and an enzyme, may be used.
  • the vessel used for ES cell differentiation induction culture was coated with a skiafold using a biocompatible material in terms of differentiation induction ability, function expression ability, viability, and the like. It is preferred to use a culture dish.
  • Such skifolds include laminin, fibronectin, collagen (collagen type IV and collagen type I, etc.), dielatin, enteractin, polyornithine, self-assembling peptide-hide mouth gel, Poly (p-N-vinylbenzyl- D-lactonamide) (PVLA) (polystyrene derivative having latatose in the side chain).
  • Matrigel Matrigel, 56% laminin, collagen type IV31%, Entactin 8%, Betaton Dickinson Fund Company
  • Growth Factor Reduced Matrigel GFR Matrigel, Laminin 61%, Collagen IV 30%, Entactin 7%, Betaton Dickinson & Company
  • Pura Matrix Pura Matrix (PuraM a tri x , amino acid 16 residues (Ac— (RA DA) CONH)
  • a peptide-hide mouth gel a strain of about 5 nm long oligopeptide
  • ES cell to embryo In the step of inducing differentiation culture into the endoderm (step (a)), a culture dish processed with collagen type IV or matrigel is most preferred, followed by fibronectin, dielatin, laminin, and non-processed order. In the step of differentiating definitive endoderm into primitive gills (step (b)), a culture dish processed with collagen type IV or matrigel is most preferred, followed by fibronectin, dielatin, laminin, and non-processed order.
  • the culture dish processed with laminin is the most preferred, followed by matrigel, fibronectin, collagen type IV, dielatin, and non-processed order. preferable.
  • matrigel processed culture dishes are most preferred, polyornitine, fibronectin, laminin, collagen type IV, dielatin, unprocessed Order is preferred.
  • a medium used in the step of inducing differentiation culture from ES cells of the present invention into definitive endoderm for example, (1) a medium for serum-free cell culture supplemented with hormone growth factor, (2 ) ES differentiation induction medium (DMEM F12) [DMEM (manufactured by Invitrogen) and nutrient mixture Ham F-12 (manufactured by Invitrogen) are mixed at a ratio of 1: 1 volume ratio, 4.5 mg / mL glucose, 20 % FBS, 2 mmol / L glutamine, 25 mmol / L HEPES (manufactured by Invitrogen), 100 mg / mL penicillin, 100 mg / mL stref.
  • DMEM F12 ES differentiation induction medium
  • serum-free cell culture media supplemented with hormone growth factors are preferred as serum-free cell culture media: RPMI 1640 medium (Sigma), DMEM medium (Invitrogen), Ham F-12 medium. (Invitrogen) is preferred, and a mixed medium composed of mixed components of RPMI1640 medium, DMEM medium and Ham F-12 medium is preferred! /.
  • hormone growth factors include insulin, transferrin, sodium selenite, ethanolamine and the like.
  • a preferred commercially available medium is S-Clone medium (manufactured by Sanko Junyaku Co., Ltd.), a serum-free basic medium for hematopoietic stem cell research. It is preferable to use Cron SF-03 medium (Sanko Junyaku Co., Ltd.)!
  • the cell concentration during culture is preferably 1 ⁇ 10 5 to 4 ⁇ 10 5 cells / mL, and most preferably 1.75 ⁇ 10 5 cells / mL.
  • the culture temperature is preferably in the range of 22 to 37 ° C.
  • the pH is preferably in the range of 7.2 to 7.4.
  • the culture period in step (a) is about 1 to 7 days.
  • a differentiation-inducing factor a cell growth factor, or the like to the medium used in the step (a).
  • a differentiation-inducing factor a cell growth factor, or the like
  • the activin include activin A, activin B, and activin AB.
  • Preferred examples include activin A, and then activin AB and activin B.
  • Concentrations added to the medium are 2 to 200 ng / mL for activin, 5 to 20 ng / mL for fibroblast growth factor, preferably 5 to for activin; 10 to 150 ng / mL for fibroblast growth factor More preferably 10 to 20 ng / mL for activin, most preferably 20 ng / mL for fibroblast growth factor.
  • concentration of the fibroblast growth factor is less than the above range, the cell tends to die, whereas when it exceeds the above range, it tends to differentiate into a mesoderm.
  • serum such as rabbit fetal serum and human serum and serum albumin to the medium.
  • the cells are cultured for 48 to 96 hours in a medium supplemented with 2.5% ushi serum albumin.
  • Examples of the medium used in the step of differentiating definitive endoderm of the present invention into primitive pods include, for example, a conditioned medium (conditioning medium) obtained by culturing a human cell line in a serum-free cell culture medium.
  • a conditioned medium conditioning medium
  • Human cell lines include human glioblastoma Cell tumor-derived cell line T98G (ATCC number, CRL— 1690), neuroectodermal tumor cell line (SK—— PN—— DW (ATCC ⁇ , CRL—— 2 ⁇ 39), etc.
  • T98G ATCC number, CRL— 1690
  • SK—— PN——— DW ATCC ⁇ , CRL—— 2 ⁇ 39
  • the following cell lines derived from human neuroblastoma can be used (all are available from ATCC (USA); see www.at cc.org for the homepage).
  • VIT1 VIT1 Primary Pancreatic Mesenchymal Cell Line
  • a conditioned medium obtained by culturing human glioblastoma cell line T98G (ATCC number, CRL-1690) in S'cron SF-03 medium is most preferable to use.
  • the cell culture concentration at the time of preparation is preferably 5 x 10 5 to 1 x 10 6 cells / mL, most preferably 8 x 10 5 cells / mL! /.
  • the culture temperature during preparation of the conditioned medium is preferably in the range of 35 to 37 ° C.
  • the pH is preferably in the range of 7.2 to 7.5.
  • the culture period is about 3-5 days. It is preferable to remove the cells with a filter, etc., before using the conditioned medium.
  • the cell concentration in the germ layer culture is preferably 1 10 5 to 8 10 5 cells / 111, most preferably 1.75 ⁇ 10 5 cells / mL.
  • the culture temperature is preferably in the range of 22 to 37 ° C, and the pH is preferably in the range of 7.2 to 7.5.
  • the culture period is about 4-10 days.
  • retinoic acid and fountain fibroblast growth factor it is preferable to add fibroblast growth factor and / or retinoic acid.
  • the concentration to be added to the medium is 1-50 ⁇ mol / L for retinoic acid and 5-20 ng / mL for fibroblast growth factor, 10-20 ⁇ mol / L for retinoic acid, and fibroblasts.
  • cell growth factor it is 10-20 ng / mL.
  • retinoic acid 10 11101 / re fibroblast growth factor is 20 ng / mL for force S.
  • Examples of the medium used in the step (step (c)) of obtaining neurogenin 3-expressing cells from the primitive silkworm of the present invention include a conditioned medium obtained by culturing a human cell line in a low glucose medium.
  • Examples of human cell lines include human liver endothelial cell lines and human vascular endothelial cell lines.
  • the following cell lines can be used (PJ "> b from ScienCell Research Laboratories (http: / / www.sciencellonline.com/ products / endothelial. htm ⁇ ) Bruno 0
  • HBMEC Human Brain Microvascular En dothelial Cells
  • HCMEC Human Cardiac Microvascula Endothelial Cells
  • HUVEC Human Umbilical Vein Endothelial Cells
  • a human hepatic endothelial cell line or a human vascular endothelial cell line it is preferable to use a human hepatic endothelial cell line or a human vascular endothelial cell line.
  • a human immortalized hepatic sinusoidal endothelial cell line TMNT-1 cell (deposited organization: National Institute of Advanced Industrial Science and Technology) Patent Biological Depositary Center, Address: Tsukuba Sakai Higashi 1-chome, 1-chome, 1-chome, Ibaraki, Japan 1st post (305-8566), date of deposit: April 16, 2002, deposit number: FER M BP—8017 ) Is most preferred.
  • This TMNT-1 cell line uses the Simian virus 40 tumor antigen (SV40T) gene-expressing retroviral vector SSR # 69 and the telomerase gene (hTERT) -expressing retroviral vector SSR # 197, and is used in normal human adult hepatic sinusoidal endothelium. It is a hepatic sinusoidal endothelial cell line that has established differentiation ability by immortalizing cells. Cell concentration at the time of the conditioned medium produced is 5 X 10 5 ⁇ ; and most preferably from 10 X 10 5 cells / mL is preferred signaling 8 X 10 5 cells / mL.
  • the culture temperature during preparation of the conditioned medium is preferably in the range of 35 to 37 ° C.
  • the pH is preferably in the range of 7.2 to 7.5.
  • the culture period is about 3-5 days. It is preferable to remove the cells with a filter before using the conditioned medium.
  • a plasmid vector that transiently expresses an enhanced yellow fluorescent protein ngn3—Spel—e YFP (the plasmid vector is available from the following paper: Mellitzer G, Martin M, Sidhoum-Jenny M, Orvain, Barths J, Seymour PA, Sander M, radwohi G. Pancreatic islet progenitor cells in neurogenin 3-yellow fluorescent protein knock-add -on mice.Mole Endocrinol. 18 (11): 2765_76 100 X 10 5 ⁇ 500 X 10 5 cells / that it is mL is preferred instrument 100 X 10 5 ⁇ 200 X 10 5 cells / mL force S most preferred les.
  • the culture temperature during gene introduction is preferably in the range of 35 to 37 ° C.
  • the pH is preferably in the range of 7.2 to 7.5.
  • the introduction period is about 1 to 5 seconds.
  • Cell concentration in the culture after gene transfer and selection should be between 1 X 10 4 and 1 X 10 5 cells / mL 3 10 4 to 8 10 4 cells / 111 is more preferred 5 X 10 4 cells / mL is the most preferred force S.
  • the culture temperature is preferably in the range of 35 to 37 ° C.
  • the pH is preferably in the range of 7.2 to 7.5.
  • the culture period is about 3-10 days.
  • a cell growth factor or the like it is preferable to add to the conditioned medium used in the step (c) in consideration of cell differentiation induction efficiency. For example, it is particularly preferred to add both epidermal growth factor and fibroblast growth factor, for which it is preferred to add epidermal growth factor and / or fibroblast growth factor.
  • Concentrations added to the medium are 5-50 ng / mL for epidermal growth factor and 5-50 ng / mL for fibroblast growth factor, 10-50 ng / mL for epidermal growth factor, fibroblasts
  • 10-50 ng / mL is even more preferred, with epidermal growth factor being 20 ng / mL, and for fibroblast growth factor, 20 ng / mL is most preferred!
  • concentration of fibroblast growth factor is less than the above range, cell proliferation tends to be attenuated.
  • ⁇ -secretase inhibitor XVIII it is preferable to add to the conditioned medium in the range of 0 ⁇ 25 to 2 11101 /, more preferably in the range of 0.5 to 5 mol / L. 1 mol / L Most preferably, it is added.
  • ⁇ -secretase inhibitor XVIII When cultured without the addition of ⁇ -secretase inhibitor XVIII, cell viability tends to decrease.
  • a medium used in the step of differentiating neurogenin 3 expressing cells of the present invention into insulin-secreting cells a conditioned medium obtained by culturing human Kashiwajima in a low glucose medium, RPMI1640 medium, William s E medium, CMRL1066 medium (manufactured by Invitrogen), low glucose DMEM medium (manufactured by Invitrogen), and the like. From the viewpoint of preventing exhaustion of insulin-producing cells, it is preferable to use a low glucose medium. From the point of increasing glucose responsiveness of insulin-secreting cells, during culture using low glucose medium, daily in high glucose culture medium without serum;!
  • Stimulated culture is preferred.
  • the culture temperature is preferably in the range of 22 to 37 ° C.
  • the pH is preferably in the range of 7.2 to 7.5.
  • the culture period is preferably 1 to 10 days, and most preferably 5 to 7 days.
  • hepatocyte growth factor it is preferable to add one or more of hepatocyte growth factor, nicotinamide, zinc sulfate, troglitazone, and etasdin-4 (exendin-4) to the medium used in the step (d). It is particularly preferable to add all types. Concentrations added to the medium include 2-20 ng / mL for hepatocyte growth factor, !-10 mmol / L for nicotinamide,!
  • Exodine 4 is preferred to be 5 to 100 nmol / L for hepatocyte growth factor 2 to 10 ng / mL, nicotinamide 5 to 10 mmol / L, zinc sulfate 10 to 20 mol / L, troglitazone 5 to 10 H mol / L, 10 to 100 nmol / L is more preferable for Exdin-4, 10 ng / mL for hepatocyte growth factor, 10 mmol / L for nicotinamide, and 16.7 nmol / L for zinc sulfate
  • 10 ⁇ 11101 / Lexdin-1 is most preferably 100 nmol / L.
  • serum such as urchin fetal serum and human serum
  • serum such as urchin fetal serum and human serum
  • 3 to 5% urine fetal serum It is more preferable to culture for 3 to 5 days in a medium supplemented with 5% ushi fetal serum after culturing for 2 to 4 days in a medium supplemented with 3% urchin fetal serum. It is most preferable to culture for 4 days in a medium supplemented with 5% urchin fetal serum after culturing for 3 days in a medium supplemented with.
  • the high glucose medium used for stimulating the cells can be a high-dulose DMEM medium or the like.
  • the high glucose DMEM medium and the like can further contain lipids, amino acids, vitamins and the like. It is most preferable to use high glucose DMEM medium with addition of lipids, amino acids, vitamins and minerals.
  • the human islet used for preparation of the conditioned medium is obtained by immortalizing and proliferating healthy isolated human islet and then removing the immortalized gene.
  • Healthy isolated human islets can be isolated from human spleen according to known methods (Staudacher C, Ricordi C, Stela M, Socci, Cammelli L, Ferrari, Oicarlo V. Minerva Chir. 31: 1665-1668 1985 or Ricordi C, Finke EH, Lacy IV, Diabetes 35: 649-653, 1986).
  • Immortalized human Kashiwajima can be prepared by introducing an immortalized gene sandwiched between a pair of site-specific recombination sequences into Kashiwajima using a vector.
  • an immortalized gene examples include the SV40T gene, which is a tumor antigen (T antigen) gene of a DNA type tumor virus, and the human telomerase reverse transcriptase (hTERT) gene.
  • T antigen tumor antigen
  • hTERT human telomerase reverse transcriptase
  • Examples of the immortalized gene transfer vector include pYK-1 vectors containing the SV40T gene, and those skilled in the art will know Kobayashi who is one of the present inventors if the vector, vector transfer method, and SV40T gene excision method are known to those skilled in the art.
  • the high glucose medium refers to a medium having a glucose concentration of 3600 mg / L to 4500 mg / L in a serum-free cell culture medium such as RPMI1640 medium, DMEM medium, Ham F-12 medium, etc.
  • the medium means a medium having a glucose concentration of 600 mg / L to 1000 mg / L contained in the serum-free cell culture medium.
  • Preferred high-Dalcos medium and low-glucose medium are high-glucose DMEM medium (manufactured by Invitrogen) and conditioned medium obtained by cultivating human islets in low-glucose medium and low-glucose DMEM medium (manufactured by Invitrogen), respectively. Can be mentioned.
  • fibroblast growth factor (FGF) used in each differentiation induction step of the present invention include FGF-2, FGF-5, and the like. 10 is mentioned.
  • insulin-secreting cell means a cell having the ability to synthesize insulin and secrete it outside the cell.
  • the term “definitive endoderm” is definitive endoderm in English, and the mouse embryonic day 3.5 is called a blastocyst and is distinguished by the outer vegetative ectoderm and the inner cell mass that forms the future body. The Although the inner cell mass is still undifferentiated, it undergoes initial differentiation into endoderm, mesoderm and ectoderm by gastrulation. Differentiation of the endoderm is a bit complicated, and the definitive endoderm (Definitive Endoderm), which is incorporated into the embryo in the future, and the visceral endoderm (Visceral), which creates the extraembryonic endoderm. Endoderm).
  • mesendoderm mesendoderm
  • primordial mushroom is called primitive pancreas in English.
  • both planar culture and three-dimensional culture can be used.
  • cells have been tertiaryized on a skifold (scaffold). It has been known that conditions that are closer to the in vivo environment can be created by culturing in the original, and the cell function is improved.
  • Three-dimensional culture is preferable from the viewpoint of improving insulin secretion ability.
  • the skiafold used for three-dimensional culture includes collagen such as laminin, fibronectin, collagen IV, collagen I, dielatin, entactin, peptide-hide mouth gel, poly (pN-vinylbenzyto D-lactonamide) (PVLA ) And the like.
  • collagen such as laminin, fibronectin, collagen IV, collagen I, dielatin, entactin, peptide-hide mouth gel, poly (pN-vinylbenzyto D-lactonamide) (PVLA )
  • PVLA poly (pN-vinylbenzyto D-lactonamide)
  • Examples of commercially available products include Matrigel, Growth Factor Reduced Matrigel, and PuraMatrix. Furthermore, the nonwoven fabric etc. which are mentioned later can be used.
  • the definitive endoderm, primitive gland, neurogenin 3 expressing cells and insulin secreting cells induced to differentiate from ES cells by the method of the present invention are observed for morphological characteristics and / or reverse transcription polymerase chain reaction. (RT—PCR).
  • Morphological confirmation methods include transmission electron microscopy, insulin staining, etc., and confirmation of morphological characteristics specific to cells.
  • RT-PCR the characteristics of cells induced to differentiate can be evaluated from gene expression.
  • genes for definitive endoderm markers Foxa2 hepatocyte nuclear factor 3, beta
  • Sox 17 sex determining region Y, sex determining region Y
  • Foxa2 hepatocyte nuclear factor 3, beta
  • Sox 17 sex determining region Y
  • Pdx—1 ⁇ transcription factors, pancreat ic transcription factor
  • definitive endoderm differentiated from undifferentiated ES cells using activin is treated with neurogenin 3 using a conditioned medium.
  • the neurogenin 3 expressing cells can be stimulated with a high-gnosoleic medium to induce insulin secreting cells with high differentiation / induction efficiency, and moreover secrete insulin efficiently.
  • the differentiation-inducing efficiency and the insulin secretion ability of the cells obtained are 3 to 5 times or more, respectively, compared to the differentiation-inducing method that forms embryoid bodies, which is a conventional technique. It is preferably about 3 to 5 times or more, more preferably about 5 to 10 times or more and about 5 to 10 times or more.
  • Insulin secretion capacity can be calculated as the amount of insulin secretion (ng) relative to the total amount of protein secreted into the medium (mg). Measurement is performed after glucose stimulation, and the insulin secretion capacity is calculated by calculating the insulin secretion capacity. Cell function can be evaluated.
  • the efficiency of inducing differentiation into insulin-secreting cells is determined by the number of cells that do not secrete insulin used in the final step of the differentiation-inducing method (in the present invention, obtained in step (c) of the differentiation-inducing method, and in step (d) It can be evaluated by the number of insulin-secreting cells relative to the number of neurogenin 3 expressing cells used).
  • the differentiation induction method of the present invention it is possible to obtain insulin secreting cells with a differentiation induction efficiency of 40 to 60%. This efficiency is considerably higher than the differentiation induction efficiency (3 to 12%, average 7.3%) described in Damour et al., Nat. Biotech., Published online, 19 October 2006, doi: 10.1038 / nbtl259. high.
  • Insulin-secreting cells obtained by the method for inducing differentiation of the present invention have an insulin secretion ability that is 1/20 to 1/50 of that of normal Kashiwajima. This secretory capacity is considerably higher than the insulin secretory capacity of the insulin-secreting cells obtained by the conventional technique (from 1/100 to 1/500 of the normal island).
  • the insulin-secreting cells express the Maf A gene.
  • MafA is a transcription factor that is essential for insulin secretion in the vicinal ⁇ -cell that binds to the insulin promoter.
  • MafAKO knockout mice MafA is clearly an important factor in glucose metabolism due to abnormal Ngernoens construction and abnormal insulin secretion (http: //www.md.tsukuba. ac.jp/basic—med/anatomy/embryology/Maf.html ⁇ : Insulin-secreting cells obtained by the separation induction method of the present invention have the ability to express the MafA gene.
  • a cell having the ability to express the MafA gene is an indicator that the cell has glucose responsiveness and is mature as an insulin-secreting cell.
  • MafA is naturally expressed in insulin-secreting cells
  • a vector containing the MafA gene is introduced into the cell in step (d) in order to increase the maturity of the cell and increase glucose responsiveness. It is possible to enhance the expression of the MafA gene by expressing it.
  • spheroid formation (spherical, with a diameter force of 00 to 300, 1 m) of insulin-secreting cells is possible.
  • spheroid means a three-dimensional spherical cell mass. Forming and / or maintaining spheroids indicates that the spheroid's physiology is closer to living tissue compared to solitary cells or distorted spheroids.
  • the spheroids composed of insulin-secreting cells obtained by the differentiation induction method of the present invention are morphologically 3 points or more in shape, 3 or more in edge shape, 3 or more in integration, and 3 or more in diameter. 3 points or more, dyeing uniformity of 3 points or more, and a total evaluation value of 15 points or more is preferable 4 points or more, edge shape is 4 points or more, integration is 4 points or more.
  • the diameter is 4 points or more, the uniformity of dyeing is 4 points or more, the total evaluation value is 20 points or more, 5 points are more preferable, the shape of the edge is 5 points, the integration is 5 points Most preferably, the diameter is 5 points, the uniformity of dyeing is 5 points, and the total evaluation value is 25 points.
  • the function of insulin-secreting cells obtained by the culture method of the present invention is 1.5 times or more at high glucose compared to that at low glucose compared to glucose stimulation. More preferably, it is more preferably 5 times or more.
  • the shape "flat” means that the long / short axis ratio is 10 or more when the cell is regarded as an elliptical sphere, and “substantially flat” means that the long Indicates that the shaft / short axis ratio is 5 or more and less than 10, and the “loose spherical shape” means that the long / short axis ratio is 2 or more and less than 5 ⁇ Almost spherical '' means that the major / minor axis ratio is 1.2 or more and less than 2, and ⁇ spherical '' means that the major / minor axis ratio is less than 1.2. Indicates that there is.
  • Edge shape force S “Unevenness” means that 90% or more of the edges of the spheroids are bumpy and slippery, lacking! “Sora! /,” Means that more than 50% to less than 90% of the edges of the spheroids are irregular! /, And “Slightly balanced” means 20% of the edges of the spheroids. More than 50% is uneven, ⁇ almost balanced '' means that more than 10% to less than 20% of the spheroid edges are uneven, and ⁇ balanced '' means Indicates that less than 10% of the spheroid edges are uneven.
  • Integration is "fragmented” means that more than 80% of the spheroids are constricted, and “almost fragmented” means that spheroids are constricted 60 to 80% of the total, “Slightly solid / compact” means that 40% to 60% of the spheroids are constricted, and “Almost solid / compact” The number of constrictions in the id is 20 to 40% of the total, and “solid / compact” indicates that the constriction in the spheroids is less than 20% of the total.
  • “individual spheroids of less than 100” means that the diameter of each spheroid is less than 100 mm
  • “spheroid strength of 00 ⁇ ; 150 ⁇ m” means spheroid.
  • the individual diameter is in the range of 100 to 150 ⁇ 111
  • “Spheroid individual is 125 to 175 ⁇ 111” means that the individual diameter of the spheroid is in the range of 125 to 175 ⁇ 111.
  • Spheroid's individual force 50-200 ⁇ m means that the spheroid's individual diametric force is in the range of 50-200 ⁇ m, and “10% or more of each spheroid is greater than 200 ⁇ m” Indicates that more than 10% of each spheroid is larger than 200 mm.
  • Insulin secretion in response to glucose stimulation is the insulin-secreting cell force, for example, insulin secretion by sensing a change from low glucose concentration to high darucose concentration (glucose stimulation) in the medium.
  • Insulin is the only hormone that acts to lower blood sugar, and is secreted by / 3 cells of the presumptive Langernos island (Iwajima) in response to blood levels of darcose. If the function of insulin-secreting cells is good, the ability to properly secrete insulin in response to fluctuations in glucose concentration. The responsiveness is poor.
  • Insulin-secreting cells the ratio of insulin secreted at low glucose concentration to insulin secreted at high glucose concentration (insulin secretion at high glucose concentration / low Darcos) (Stimulation index; SI), which has been compared (Bergert H, Knoch P, Meisterreld R, Jager M, OuwendijKj, Kersting Saeger HD, Solimena M, Effect of oxygenated) Cell Transplant. 2005; 14 (7): 441_8), it is preferred that the SI straight is 1 ⁇ 5 or more, more preferably 2 or more, and more 5 or more. perfluorocarbons on isolated rat pancreatic islets inculture. Most preferably.
  • High glucose culture means that the glucose concentration in the medium is 3600 mg / L to 4500 mg / L
  • low glucose culture means that the glucose concentration in the medium is 600 mg / L to 1000 mg / L. .
  • the embryoid endoderm differentiated from undifferentiated ES cells using activin is differentiated into neurogenin 3 expressing cells using a conditioned medium, and then the cells are differentiated.
  • the cells are differentiated.
  • By inducing differentiation efficiently into insulin-secreting cells by stimulating them with a high glucose medium a large amount of insulin-secreting cells having high insulin-secreting ability and high differentiation-inducing efficiency can be supplied.
  • Diabetes therapeutics, bioartificial viscera, research reagents, or drug discovery model animals that can benefit everyone by using this as the cell source for diabetes therapeutics, bioartificial victories, research reagents, or drug discovery model animals The development of can be greatly expected.
  • another embodiment of the present invention is a therapeutic agent for diabetes, a bioartificial viscera, a research reagent, and a drug discovery model animal containing insulin secreting cells differentiated from ES cells.
  • the therapeutic agent for diabetes includes a cell mass such as a pellet obtained by concentrating insulin-secreting cells as it is or by filtering. Furthermore, the therapeutic agent for diabetes can be cryopreserved by adding a protective agent such as DMSO. In order to use it more safely as a therapeutic agent for diabetes, it can be treated under conditions that change the protein of the pathogen, such as heat treatment, radiation treatment, or mitomycin C treatment, while retaining its functions as a therapeutic agent for diabetes. can do.
  • the method of performing cell transplantation using Eco 1 has a method of transplanting into the spleen and liver by directly puncturing the spleen under the preferred abdominal echo guide because it is less invasive, and the liver is most preferred.
  • the dosage (transplantation amount) of the cell preparation is preferably 1 X 10 8 to 1 X io 1Q cells / individual, more preferably 5 X 10 8 to; more preferably 1 X io 1Q cells / individual, 1 X 10 8 to 1 X io 1Q cells / individuals are most preferred.
  • the dose (transplant amount) can be appropriately changed depending on the age, weight, symptoms, etc. of the patient to be administered.
  • Examples of the administration form (transplantation method) of the therapeutic agent for diabetes using the insulin-secreting cells to the mouse include a method of incising the mouse and transplanting it under the renal capsule.
  • the dose (transplantation amount) of the cell preparation is preferably 3 X 10 6 to 1 X 10 7 cells / individual 4 X 10 6 to 8 X 10 6 cells / individual is more preferable 5 X 10 6 cells / Individual is most preferred.
  • the bioartificial viscera of the present invention it is preferable to culture cells three-dimensionally by using a skifold such as a nonwoven fabric.
  • a skifold such as a nonwoven fabric.
  • three-dimensional culture of cells on skifolds can create conditions that are closer to the in vivo environment and improve cell functions.
  • Cells are cultured on skifolds such as nonwoven fabrics. This is effective for improving the functions.
  • Insulin-secreting cells can be encapsulated in a device made of a polymer material and engrafted in a state isolated from the immune system, reducing the physical burden on patients who use biotechnology. Is done. Therefore, the biomedical organ of the present invention is expected to play a central role in the treatment of diabetes in the 21st century.
  • Examples of the artificial artificial viscera include a hybrid artificial viscera combining a hollow fiber type reactor (device) and a separated / cultured cell.
  • a bioartificial viscera is placed outside the body and connected to the blood vessel, placed in the body and connected to the blood vessel, or placed in the abdominal cavity without connecting to the blood vessel, but placed subcutaneously without connecting to the blood vessel. 4 forms There is.
  • the insulin-secreting cells of the present invention can be used for any form of bioartificial viscera, ES cells lose their ability to proliferate when induced to differentiate, but ES cells that have been poorly differentiated remain.
  • a subcutaneous implantable body type is preferable from the viewpoint that a risk associated with cell transfer and the like can be avoided and a reactor can be arbitrarily taken out.
  • Bioreactors can be used as indwelling artificial bags (for example, Ale jandro soto- Gutierrez, Naoya obayashi, Jorge David Rivas-arrillo, Nalu Navarro- Alvarez, Debaio Zhao, Teru Okitsu, Hirofumi Noguchi, Hesham Basma, Yashuhiko T abata, Yong Chen, imiaki Tanaka, Michiki Narushima, Atsushi Mki, Tadayoshi Ue da, Hee- Sook Jun, J To Won Yoon, Jane Lebkowski, Noriaki i, anaka & Ira J Fox, ⁇ Rev ersal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytesj nature biotechnology Volume 24, No.
  • MELS Modular Extracorporeal Liver
  • Various types such as System
  • These reactors can be used in the present invention because there is no scaffold for the attachment of insulin secreting cells, so the cells are simply filled into the space force inside the hollow fiber, the space outside the hollow fiber. There is a tendency to float only by itself. Insulin-secreting cells tend to have insufficient differentiation function in a floating state, and collide with surrounding cells and are susceptible to stress stimulation.
  • a hollow space is provided so that a scaffold can be provided to insulin-secreting cells.
  • a reactor comprising a skid fold such as yarn and nonwoven fabric is preferred.
  • any membrane can be used as long as cells do not adhere to the membrane surface and hinder the substance exchange. Specifically, it has been conventionally used for medical purposes.
  • Commercially available products such as polysulfone membranes and ethylene acetate butyl random copolymer saponified membranes (for example, trade names: EVAL, manufactured by Kuraray Medical Co., Ltd.) are preferred.
  • the pore sizes of commercially available hollow fiber membranes include dialysis membranes ( ⁇ 5 nm), plasma component separation membranes (20-30 nm), plasma separation membranes (30-200 nm), and so on. From the viewpoint of the permeability of the substance, a plasma separation membrane (30 to 200 nm) is preferable.
  • the immunocompetent cells and immunoglobulins in the blood flowing in the hollow fiber should not come into direct contact with the cells packed on the skifold such as the nonwoven fabric outside the hollow fiber.
  • a pore size of 100 nm is most preferred.
  • the nonwoven fabric is preferably processed and modified so that the cells can adhere.
  • non-woven fibers include polytetrafluoroethylene (PTFE). Of these, polytetrafluoroethylene having been subjected to polyamino acid urethane (P AU) processing is preferred from the viewpoint of processing.
  • PTFE polytetrafluoroethylene
  • P AU polyamino acid urethane
  • Bioartificial spleen is a polyethylene bulal alcohol membrane (pore size is 3 Onm) (eg, Eval membrane) to block permeation of immune-bearing cells 5, PAU-treated PTFE nonwoven fabric 6 for cell adhesion, and insulin secretion It consists of a cell inlet 3 for injecting cells 7 and a lid 4.
  • the PTFE nonwoven fabric 6 has a two-layer structure covered with a polyethylene-but alcohol film 5.
  • Insulin-secreting cells 7 that are spherical (spheroids) are attached to the PTFE nonwoven fabric and can remain inside the bioartificial viscera.
  • a reactor consisting of a three-layer structure force covered with a PE mesh (pore size of about 1 OOnm) on the outside of a polyethylene bural alcohol membrane without a PE mesh is also possible.
  • a PE mesh pore size of about 1 OOnm
  • blood vessels extend into the PE mesh, and the nutrient supply of the cells in the bioartificial viscera becomes possible.
  • a preferred embodiment is a bioreactor having a two-layer structure shown in FIG.
  • the artificial viscera can be produced by those skilled in the art by referring to the “SG literature”.
  • Examples of methods for filling a cell or cell culture into a nanoreactor include, for example, a method in which a bioreactor is pre-implanted in the body and then filled with insulin-secreting cells, and cell differentiation is induced in a test tube. After making ES cells into insulin-secreting cells, the bioreactor is filled with the necessary number of cells, and the bioreactor is pre-processed with a skifold such as matrigel, and the insulin-secreting cells are There is a method of inducing differentiation.
  • a method is preferred in which a back-type bioreactor is embedded in the body in advance, and a blood vessel extends around the bioreactor, and then insulin-secreting cells are filled into the bioreactor.
  • the cell suspension is preferably attached to the reactor using a 10-50 mL syringe! /, Preferably injected from the cell inlet! / ,.
  • the bioreactor of the present invention is also useful for the production of useful substances, the investigation and exploration of the functions of tissues and organs, screening for new drugs, and alternative methods for animal experiments to evaluate the effects of endocrine disrupting substances. It can be suitably applied.
  • the bioartificial viscera of the present invention can be used, for example, for the production of insulin, which is a production component inherent to Yeouido.
  • Insulin can be produced by purifying the obtained medium by a method usually used for protein purification, such as an affinity column.
  • the bioartificial viscera of the present invention can be transplanted, for example, to diabetic patients that are increasing year by year not only in Japan but also internationally. Prevention and control of human diabetes 21st century It is no exaggeration to say that this is a major issue for centuries in the era. Therefore, many pharmacists are working on the development of diabetes drugs.
  • the research reagent containing insulin-secreting cells obtained by the differentiation induction method of the present invention means, for example, a research reagent used for screening of a new drug.
  • the research reagent may be used in any form, such as a method in which a new drug is added as it is to a culture vessel in which insulin-secreting cells are cultured, a method in which the cells are contained in a bioreactor, and the like Is mentioned.
  • insulin-secreting cells obtained by the differentiation induction method of the present invention by evaluating medicinal effects using tolptamide marketed as a hypoglycemic agent instead of a new drug are also useful as research reagents. It shows that.
  • Tolptamide is a sulfonylurea hypoglycemic agent. Tolptamide acts on the viscera and increases insulin secretion, thereby reducing blood glucose levels. Mainly used for type 2 diabetes (non-insulin dependent diabetes). Pharmacological action stimulates insulin secretion by stimulating 0 cells of Langeron's islet in the viscera (acupuncture). However, since it is not a substitute for insulin, it is ineffective for type 1 diabetes (insulin-dependent diabetes) where no insulin is secreted (http: ⁇ www.interq.or.jp/ox/dwm/se/se39). / s e3961006.html).
  • Tg trussianic
  • diabetic Tg animals with various unique characteristics have been born.
  • the establishment, maintenance, breeding and supply of strains is a daunting task. Establishing a model animal takes many years and a lot of effort, patience, and as the flow of time has become faster as in recent years, it is no longer an issue that can be tackled carefully.
  • breeding and maintenance of the strain requires manpower and equipment, and in the case of a model animal that has finished its role, there is a risk that it will disappear.
  • due to the differences between human diabetes and diabetes model animals there is a problem that there are many dropouts in the clinical trial stage of the developed drug.
  • the bioartificial viscera of the present invention is preferable because these problems can be solved.
  • the drug discovery model animal refers to an animal containing insulin-secreting cells obtained by the differentiation induction method of the present invention.
  • a chimeric mouse having human-derived insulin-secreting cells can be mentioned, and it can be used for drug discovery and pharmacological testing for human Kashiwajima. it can.
  • mice-derived ES cells purchased from Sumitomo Dainippon Pharma Co., Ltd.
  • feeder cells that have been introduced with a neomycin resistance gene
  • mouse-derived embryonic fibroblasts purchased from Sumitomo Dainippon Pharma Co., Ltd.
  • Culture flask T-75 Fecon, Becton Decktonson and Cannony (Becton), coated with 0.1% gelatin water (catalog number: R—ES — 006B, purchased from Dainippon Sumitomo Pharma Co., Ltd.) , Dickinson and Company)).
  • ES medium (R—ES—101: purchased from Dainippon Sumitomo Pharma Co., Ltd.): Dulbecco's Modified Eagle (DMEM) medium (1: 1 volume ratio) 15% Fetal serum (FBS), 1% non-essential amino acid, 1% nucleoside, l lO ⁇ mol / L 2-mercaptoethanol (purchased from Dainippon Sumitomo Pharma Co., Ltd.), 1% penicillin and streptomycin, 1% gnoretamic acid, In addition, 500 U / mL mouse-derived leukemia inhibitory factor (LIF) (purchased from Dainippon Sumitomo Pharma Co., Ltd.) was used.
  • LIF mouse-derived leukemia inhibitory factor
  • the culture medium was changed every day, and ES cells were passaged every 3 days.
  • the proportion of ES cells in the culture plate reached 80-90%, in a two-step procedure, first add 0.25% trypsin EDTA (Invitrogen). 45 5 seconds later, remove the trypsin EDTA solution to remove the feeder cells together (the ES cells still remain in the culture plate at this stage), and then 2 minutes later, add the ES medium and detach As a result, ES cells remaining in the culture plate were recovered.
  • trypsin EDTA Invitrogen
  • the undifferentiated ES cells obtained in Production Example 1 were cultured at a reduced cell culture concentration (cell culture concentration: 17,000,000 cells / mU, collagen type IV, matrigenore, laminin type 1, or dielatin)
  • Activin A catalog number: 338 -AC, R & D
  • FGF-2 catalog number: 233—FB / CF, R & D
  • Manufactured by Company D (20 ng / mU, sullon fetal serum (FBS), and sullon serum albumin (BSA) with S-cron SF-03 medium (Product No.
  • RNA reverse transcriptase 22 ° C for 10 minutes, 42 ° C for 20 minutes, 99 ° C for 5 minutes, and then allowed to stand at 4 ° C for more than 5 minutes
  • PCR was performed according to the protocol using the obtained 2 ag reverse transcription product at 20 pmol / mL of each primer. Used for amplification. The PCR reaction was repeated at 95 ° C for 10 minutes and then at each cycle for 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds for the number of cycles. Then, it left still at 4 degreeC for 5 minutes or more.
  • the primers and PCR conditions for each gene are described below.
  • 5 'Primer 5' — TGGTCACTGGGGACAAGGGAA (Self column number 1)
  • 3 'Primer 5'-GCAACAACAGCAATAGAGAAC (SEQ ID NO: 2) Soxl 7it1 ⁇ 4-(210bp, 60. C, 35 cycles)
  • 5 'Primer 5' — GCCAAAGACGAACGCAAGCGGT (Self Row Number 3)
  • 3 'Primer 5' — TCATGCGCTTCACCTGCTTG (SEQ ID NO: 4)
  • the concentrations of activin A are 2 ng / ml, 10 ng / ml, lOOng / ml, and 200 ng / ml, respectively.
  • the concentration of activin A is most preferably lOng / ml, followed by lOOng / ml, followed by 200 ng / ml.
  • Figures 1 and 2 show that undifferentiated mouse ES cells were collagen type IV, fibroblast growth factor (FGF-2) (20ng / mL), urchin fetal serum (FBS), and urine serum albumin (BSA). ) Cron SF-03 medium (Product No.
  • step (a) requires 100 ng / mL of activin ⁇ , and low concentration (30 ng / mU of activin A has low differentiation induction efficiency.
  • step (a) of the differentiation induction method of the present invention the process is lower than that of the conventional Damour method. It became clear that ES cells could be induced to differentiate even with a concentration of activin A.
  • Example 2 After the differentiation induction described in Example 1 was performed for 2 days, primitive embryos were formed using the embryoid body obtained in the step (a).
  • Human glioblastoma cell line T98G (ATCC number: C RL— 1690, available from ATCC (USA)) cultured in SF-03 medium for 1 day (cell culture concentration: 8 X 10 5 cells / mL, culture) Temperature: 37 ° C, pH: 7.4)
  • Use conditioned medium obtained obtained
  • the definitive endoderm obtained in the step (a) was cultured for 5 days (culture temperature: 37 ° C., pH: 7.4).
  • the above conditioned medium is supplemented with retinoic acid (1 11101 / relo ⁇ mol / L, lOO ⁇ mol / L or 1000 a mol / L) and FGF-2 (20 ng / mU), and collagen type IV caloculture.
  • Dish T 25 flask (cell culture concentration 175,000 cells / mL, 700,000 cells in total) was used.
  • the cells obtained in step (b) include Foxa2 (+), Soxl 7 (+), Pdx— The gene expression of 1 (+) and Shh (—) was shown, confirming that ES cells had started to form primitive pods.
  • RNA reverse transcriptase 22 ° C for 10 minutes, 42 ° C for 20 minutes, 99 ° C for 5 minutes, and then left at 4 ° C for more than 5 minutes.
  • PCR was performed according to the protocol using the obtained 2 ag reverse transcription product at 20 pmol / mL of each primer. Used for amplification. The PCR reaction was repeated at 95 ° C for 10 minutes and then at each cycle for 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds for the number of cycles. Then, it left still at 4 degreeC for 5 minutes or more.
  • the primers and PCR conditions for each gene are described below.
  • 5 'primer 5' — ACCATGAACAGTGAGGAGCA (SEQ ID NO: 5)
  • GAPDH gene (171bp, 60.C, 35 cycles)
  • Lanes 1 to 4 show retinoic acid concentrations of 1 M, 10 M, 100 ⁇ M, and 1000 ⁇ M, respectively.
  • 10M retinoic acid the expression of Pdx-1 gene, a marker gene specific to primordial acupuncture, is strongly confirmed, but Shh is not expressed. This result strongly supports that this method can efficiently induce the differentiation of primitive moths!
  • Step (c) Selection of neurogenin 3 expressing cells
  • Neurogenin 3 expressing cells were increased by culturing the primitive koji obtained in step (b) using a conditioned medium.
  • Human immortalized hepatic sinusoidal endothelial cell line TMNT— 1 cell (Depositor: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, Address: 1-chome, Tsukuba, Ibaraki, Japan 1-Chuo 6 ( Postal code 305—8566), deposit date: April 16, 2002, deposit number: FERM BP—8017, available to everyone, low glucose D MEM medium (glucose concentration: 1000 mg / L, catalog number 31600— 083, manufactured by Invitrogen) for 1 day (cell culture concentration: 8 X 10 5 cells / mL, culture temperature: 37 ° C, pH: 7.4), ⁇ -secretase inhibitor XVIII (Calbiochem (catalog number 565779), or Alexis Biochemicals (catalog number: ALX—270—415—C250, or ALX—270—415—
  • the eYFP-expressing cells were selected using a cell sorter (MoFlo) as an indicator.
  • ngn3—Spel—eYFP was introduced temporarily using Nucelofector (Amaxa, which can be used by everyone according to the handling rules).
  • For the operation method please refer to the following website: http: ⁇ www.wako-chem. Co.jp/siyaku/info/gene/article/amaxa/df/Manual.pdf)
  • V Cell culture concentration: 5 ⁇ 10 5 cells / mL, culture temperature: 37 ° C., pH: 7.4
  • Spherical cuts from healthy isolated human islets provided by the University of Alberta, Canada (available to everyone from Canada, University of Alberta, Human Islet Transplant Program, Dr. Jonathan RT. Lakey) ! /, Which was in the form of! /, Was selected using a hand pickup method under a stereomicroscope (ST EMI, Carl Zeiss, Germany) and seeded in a T25 culture flask.
  • ST EMI Carl Zeiss, Germany
  • CMRL1066, 10% ⁇ shea womb ⁇ Qing Invitrogen) FCS, Sigma
  • 10- 7 mol / l insulin Sigma
  • 10- 6 mol / l dexamethasone Sigma
  • 25 g / ml epidermal growth factor EGF, Sigma
  • 10 mM nicotinamide Sigma
  • antibiotic penicillin G / streptomycin Sigma
  • Neurogenin 3 expressing cells obtained in step (c) are cultured in 3% or 5% urine fetal serum (3 days in 3% urine fetal serum and then 4 days in 5% urinary fetal serum) , Using low glucose DME M medium supplemented with 10 ng / mL hepatocyte growth factor, 10 mmol / L nicotinamide, 4 100 nmol / L dexdin, 10 mol / L troglitazone, 16.7 ⁇ mol / L zinc sulfate The cells were cultured for a day (culture temperature: 37 ° C, pH: 7.4). As a culture dish, a 6-well-plate processed with Matrigel was used.
  • step (d) culture is performed daily for 2 hours in a high glucose DMEM medium (glucose concentration: 4500 mg / L, catalog number: 128 00-082, manufactured by Invitrogen) without serum (culture temperature: 37 ° C). , PH: 7.4), and then cultured for 22 hours in the low glucose DMEM medium for 7 days.
  • Hepatocyte growth factor 10 ng / mL, nicotinamide 10 mmol / L, dexdin 4 100 nmol / L, troglitazone; 10 ⁇ mol / L, zinc sulfate 16 ⁇ 7 ⁇ mol / L were also added to the high glucose medium. .
  • Neurogenin 3 expressing cells obtained in step (c) were induced to differentiate into insulin secreting cells using a low glucose RCNK-1 conditioned medium.
  • RCNK a non-replicating recombinant adenovector that produces Cre recombinase labeled with a nuclear localization signal (NLS) when a single cell is cultured in low-Darcose DMEM + 10% FBS and filled to a flask.
  • Ax CANCre (3 X 10 8 pfu / ml) (available from RIKEN GENE BANK, RDB No.
  • MOI multipleplicity of infection
  • 10-50 15 is preferred
  • RCNK-1 cells were infected and the immortalized gene SV40T was removed with the Cre / loxP system (culture temperature: 35-37 ° C).
  • RCNK-1 cells were cultured in low glucose DMEM + 5% FBS for 2 days (48 hours), and then cultured in low glucose DMEM alone.
  • the low glucose RCNK-1 conditioned medium was collected. This operation can be performed 4 to 5 times until cells weaken because it is serum-free.
  • KRBB medium Kerbs Ringer Balanced Buffered (KRBB) (14 3 mM Na, 5.8 mM K, 2.5 mM Ca, 1.2 mM Mg2, 124. ImM CI, 1.2 mM PO-4, 1.2 mM SO—4, 25 mM HCO—3, lOmM HEPE S, 0.2% BSA, including details in the following paper: Srivastava S, Goren HJ .Insulin constitutively secreted by beta-cells is necessary for glucose-stimulated ins ulin secretion. Diabetes.
  • KRBB medium Kerrebs Ringer Balanced Buffered (KRBB) (14 3 mM Na, 5.8 mM K, 2.5 mM Ca, 1.2 mM Mg2, 124. ImM CI, 1.2 mM PO-4, 1.2 mM SO—4, 25 mM HCO—3, lOmM HEPE S, 0.2% BSA, including details in the following paper: Sri
  • Insulin-secreting cells obtained by the differentiation-inducing method of the present invention were observed under a phase contrast microscope (see FIG. 4 (a)).
  • Nucleus 1 is stained red in a dot shape, and the entire insulin-secreting cell group 2 having a spherical shape with a diameter of about 200 m is stained green, confirming the expression of the MafA gene.
  • MafA gene expression (MafA antibody (BL1069, catalog number: A300-611A, manufactured by BETHYL L ABORATORIES, INC. For the method of use, refer to the instruction manual attached to the product).
  • the stained color is not expressed because it is converted into a gradation, whereas the insulin-secreting cells obtained by the differentiation induction method of the present invention have the ability to express the Maf A gene, whereas It has been reported that insulin-secreting cells induced to differentiate by the damour method, which expresses the MafA gene! /, Is! // (Dam I Nore et al., Nat. Biotech., Published online , 19 October 2006, doi: 10.1038 / nbtl25 9.) This result also revealed that the differentiation induction method of the present invention is superior to the prior art.
  • FGF-10 suppresses differentiation to / 3 cells during the development of viscera (Mirares et al., Int J Dev Biol. 2006). ; 50 (1): 17_26)) and induced exocrine cells (see Bushshan et al., Development. 2001; 128 (24): 5109_17), whereas EGF and FGF-2 Promotes differentiation into / 3 cells during development of the spleen (Cras-Meneur C, El ghazi L, zermcnow P, ⁇ charfmann R. Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differe ntiation. Diabetes. 2001 Jul; 50 (7): 1571-9).
  • the morphology of insulin-secreting cells obtained by the differentiation induction method of the present invention was observed under a phase contrast microscope (see FIG. 4 (b)).
  • nucleus 1 is dyed red
  • the entire insulin-secreting cell group 2 having a spherical shape with a diameter of about 200 m 2 is dyed green indicating insulin secretion.
  • Insulin was detected using an anti-insulin antibody (Sta. Cruz sc-9168, refer to the instruction manual attached to the product for usage). Insulin-secreting cells were confirmed to increase in the number of culture days! The attached photo is converted to black and white with two gradations, so the dyed color is not represented. Both scale bars in the lower right of the photo in Fig. 4 indicate 200 m. Insulin-secreting cells have formed a spherical shape with the number of days of culture. [0133] Morphological study of cell differentiation on day 2
  • Shape is “loose spherical: 3 points”, 2. Edge shape is “slightly balanced: 3 points”, 3. Consistency is “fragmented: 1 point”, 4 The diameter was 125 to 175 111: 3 points for each of the cultured islands. 5. The uniformity of staining was “slightly uniform: 3 points”. The total of the evaluation values was 13 points (see FIG. 4).
  • Shape is “spherical: 5 points” 2. Edge shape is “slightly balanced: 3 points” 3. Integration is “slightly solid / compact: 3 points” 4. Diameter is “culture” 10% or more of the islands were larger than 200 in: 5 points, and 5. Dyeing uniformity was “slightly uniform: 3 points”. The total of the evaluation values was 19 points (see Figure 4).
  • Shape is “spherical: 5 points” 2. Edge shape is “balanced: 5 points” 3. Integration is “solid / compact: 5 points” 4. Diameter is "culture pot” More than 10% of the islands are larger than 200 m! / ⁇ : 5 points ”, 5. Dyeing uniformity is“ completely uniform: 5 points ”The total evaluation value was 25 points (see Figure 4) . On the other hand, insulin-secreting cells obtained by the Damour method were not spherical.
  • the shape is “flat: 1 point” 2.
  • the edge shape is “uneven: 1 point” 3.
  • the integrity is “fragmented: 1 point” 4.
  • the diameter is “the whole culture island” Is less than 100 m: 1 point ”, 5. Uniformity of staining is“ Slightly uniform: 3 points ”.
  • the total of the evaluation values was 7 points (Dam Ill et al., Nat. Biotech., Published online, 19 October 2006 , Doi: 10.1038 / nbtl259.)
  • Insulin-secreting cells obtained by culturing using the DMEM medium of Example 4 or insulin-secreting cells obtained by culturing using the RCNK-1 conditioned medium of Example 4 were used.
  • Each of DMEM (see Fig. 5 (a) and Table 1 (a)) or KRBB (see Fig. 5 (b) and Table 1 (b)) has a concentration of 3.3 mM (low nocturne) or 25 mM (high).
  • glucose stimulation low ⁇ high
  • Dalcos stimulation low ⁇ high ⁇ low
  • the insulin-secreting cells obtained by the differentiation-inducing method of the present invention were ⁇ glucose-stimulated: low to high '' every time the culture days increased to 2, 4, and 7 with low glucose DMEM.
  • the amount of insulin secretion when “glucose stimulation: low ⁇ high ⁇ low” was observed. It decreased (see 2, 4, and 6 on the horizontal axis in Fig. 5), indicating that insulin-secreting cells have glucose responsiveness.
  • the amount of insulin secreted in the medium after “insulin-secreting cells: low to high” was compared with the conventional Schroder method. Insulin secretion in the medium after “cell-stimulated: low ⁇ high ⁇ low” (see 8 on the horizontal axis in FIG.
  • insulin secretion in the medium after the glucose-stimulated cells by the Damour method were "glucose stimulated: low ⁇ high ⁇ low" (horizontal in Fig. 5) Axis 10) was measured.
  • the insulin-secreting cells obtained by the differentiation-inducing method of the present invention have superior insulin secretion ability and excellent glucose responsiveness as compared with insulin-secreting cells according to the prior art.
  • Insulin-secreting cells obtained in Example 4 were measured using a low glucose concentration KRBB as the medium, and the amount of insulin secretion was measured (see Fig. 6 (a) and Table 2).
  • the amount of insulin secreted in the medium after stimulation with KRBB with 10 mol / L tolptamide (Sigma) for 1 hour was measured (see Fig. 6 (b) and Table 2).
  • the insulin-secreting cells obtained by the differentiation induction method of the present invention showed a higher insulin-secreting ability than the conventional technique.
  • Streptozotocin (220 mg / kg) was intraperitoneally administered to severe immunodeficiency (SCID (severe combined immunodeficiency)) mice (body weight 20 g, Japan Clare Co., Ltd.), and blood glucose level increased to 360 mg / dL or more after 1 week.
  • SCID severe immunodeficiency
  • I was diagnosed with diabetes.
  • the diabetic mice were used for transplantation experiments. Insulin secreting cells (5 ⁇ 10 6 cells) obtained by the differentiation induction method of the present invention, undifferentiated mouse ES cells (5 ⁇ 10 6 cells), and 450 normal mouse islets were used as transplanted cells. .
  • Cell transplantation was performed on 5 diabetic mice each. Normal healthy mice (3 mice) and non-transplanted diabetic mice were used as controls.
  • Figure 7 and Table 3 show the average blood glucose levels after transplantation under the left renal capsule of mice.
  • 2 units of intermediate human insulin NPH Novo Nordisk
  • Figure 7 shows the average blood glucose level after transplantation. Thereafter, blood glucose level was measured every 3 days, and after 3 weeks after transplantation, blood glucose level was measured once a week using a Portable glucose meter FreeStlyle TM (TheraSence).
  • mice transplanted with mouse ES cells the blood glucose level again increased to 360 mg / dL after NPH administration, indicating that there was no therapeutic effect.
  • a mouse transplanted with insulin-secreting cells obtained by the differentiation induction method of the present invention can maintain a glucose concentration of about 100 mg / dL, similar to a mouse transplanted with healthy mice Kashiwajima. It was. This indicates that the function of the insulin-secreting cells is similar to that of normal mouse Kashiwajima and has a therapeutic effect. All diabetic mice not treated (non-treated group) and all mice transplanted with ES cells died of hyperglycemia within 10 weeks.
  • a diabetic mouse (produced by the method described in Example 9) to be transplanted with a bioartificial spleen is opened, and a bag-type bioreactor (see Fig. 8; the production method is described in the SG document) is subcutaneously embedded in the mouse.
  • Vascular extension around the bioreactor was performed over a period of days.
  • Insulin secretory cells (5 ⁇ 10 6 cells), undifferentiated mouse ES cells (5 ⁇ 10 6 cells) obtained by the differentiation induction method of the present invention 3 days and 10 days after implantation of the bioreactor, and Cell filling was performed on bioreactors in which 450 normal mice, Kashiwajima, were embedded in 5 diabetic mice each.
  • mice Normal healthy mice (3 mice) and non-transplanted diabetic mice were used as controls.
  • the average mouse blood glucose level after cell loading is shown in Figure 9 and Table 4.
  • 2 units of intermediate human insulin NPH Novo Nordisk
  • blood glucose level was measured every 3 days, and after 3 weeks after transplantation, blood glucose level was measured once a week using Portable glucose meter FreeStlyle TM (TheraSence) .
  • mice embedded with bioartificial viscera filled with mouse ES cells showed a blood glucose level of 360 mg / dL or more again after NPH administration, indicating that there was no therapeutic effect.
  • a mouse equipped with a bioartificial organ embedded with insulin-secreting cells obtained by the differentiation-inducing method of the present invention is the same as a mouse equipped with a bioartificial viscera filled with healthy mice.
  • the glucose concentration could be maintained at about 100 mg / dL. This indicates that the function of the insulin-secreting cells is similar to that of normal mouse Kashiwajima and has a therapeutic effect. All untreated diabetic mice (non-treated group) and all mice embedded with bioartificial embryos filled with ES cells died of hyperglycemia within 10 weeks.
  • the differentiation inducing method of the present invention it is possible to induce insulin secreting cells that are sufficiently functional to replace healthy human spleen cells and that can be supplied in large quantities and are safe.
  • the therapeutic agent for diabetes, the bioartificial viscera, the research reagent and the drug discovery model animal of the present invention can realize high safety / treatment for diabetes.
  • SEQ ID NO: 1 5 for polymerase chain reaction to detect Foxa2 gene
  • SEQ ID NO: 2 3' primer for polymerase chain reaction to detect Foxa2 gene
  • SEQ ID NO: 3 to detect Soxl 7 gene
  • Primer for Polymerase Chain Reaction ⁇ Primer SEQ ID NO: 4 For Polymerase Chain Reaction to detect Soxl 7 gene
  • T Primer SEQ ID NO: 5 Pdx—Primer for Polymerase Chain Reaction to detect ⁇ ⁇ ⁇ gene
  • SEQ ID NO: 6 Pdx —For polymerase chain reaction to detect sputum gene
  • T primer SEQ ID NO: 7 For polymerase chain reaction to detect Shh gene
  • Primer SEQ ID NO: 8 For polymerase chain reaction to detect Shh gene
  • T primer SEQ ID NO: 9 Polymerase chain reaction ⁇ primer for detecting GAPDH gene
  • SEQ ID NO: 10 GAPDH gene For polymerase chain reaction to detect T; primer

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Diabetes (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Obesity (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • External Artificial Organs (AREA)
  • Prostheses (AREA)
  • Materials For Medical Uses (AREA)

Abstract

A method comprising differentiating embryonic endoderm, which has been differentiated from undifferentiated embryonic stem cells with the use of activin, into neurogenin 3-expressing cells using a conditioned medium, and then stimulating these cells in a glucose-enriched medium to thereby efficiently induce the differentiation thereof into insulin-secreting cells. In addition to the method of inducing the differentiation of embryonic stem cells into insulin-secreting cells, the insulin-secreting cells induced by this method, a remedy for diabetes, a biological artificial pancreas, a research reagent and a model animal for drug design, each containing the above-described cells, are also provided.

Description

明 細 書  Specification
胚性幹細胞のインスリン分泌細胞への分化誘導方法、該方法により誘導 されるインスリン分泌細胞およびその用途  Method for inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method, and use thereof
技術分野  Technical field
[0001] 本発明は、胚性幹細胞（以下、 ES細胞とも言う）のインスリン分泌細胞への分化誘 導方法および該方法により誘導されるインスリン分泌細胞、ならびに糖尿病治療薬、 バイオ人工勝臓、研究試薬および創薬モデル動物に関する。  [0001] The present invention relates to a method for inducing differentiation of embryonic stem cells (hereinafter also referred to as ES cells) into insulin-secreting cells, insulin-secreting cells induced by the method, a therapeutic agent for diabetes, a bioartificial organ, research The present invention relates to reagents and drug discovery model animals.
背景技術  Background art
[0002] 2000年カナダ.エドモントンのアルバータ大学から臨床瞵島移植 7症例について の報告がなされた（シャピロ（Shapiro AM)ら、 Ν· Engl. J. Med., (2000)343， p.230- 238.参照）。後に'エドモントン 'プロトコール'と呼ばれる免疫抑制剤の斬新な使用法 によって瞵島移植を受けた全ての 1型糖尿病症例でインスリン療法から離脱できたと 言うものである。瞵島移植はインスリン依存型糖尿病患者にとって現在もっとも理想に 近い治療法である。勝臓は外分泌腺と内分泌腺から構成されている。勝臓全容積の 98%以上を外分泌腺が占め、内分泌腺は 2%以下である。 1869年ランゲルノ、ンス によって見!/、だされた内分泌腺組織は、光学顕微鏡像にお!/、て外分泌腺組織の海 の中に浮かぶように存在する島のように見えることから瞵島（islet)と呼ばれる。瞵島 は内分泌細胞の集団であり、 α細胞、 /3細胞、 ΡΡ細胞、 δ細胞などからなっている。 体内のホルモンで唯一血糖を下げる作用をもつインスリンは瞵島の β細胞から分泌 される。この瞵島を勝臓から単離し、インスリン依存型糖尿病の患者に移植して一旦 廃絶した血糖降下システムの置換再生を目指すのが瞵島移植である。 β細胞は瞵 島を構成する全細胞の 80〜85%を占めている。 β細胞は単にインスリンを分泌する のみではない.さらに重要なのは、血中の糖分を感知することができると言うことであ る。糖代謝におけるインスリン分泌調節の主体は複数の器官が関与した複雑な系に 依存して!/、な!/、。すなわち瞵島単独で運動時や摂食時などの急激に変化する血糖 に対して迅速に反応し、それにみあった適量のインスリンを分泌することによって血 糖を非常に狭い範囲に調節することが可能である。効果対象の感知と作用の調節が 一種類の細胞単独でおこなわれ、その細胞が瞵島の大部分を占めていると言う事実 が瞵島移植の理論的根拠となってレ、る。 [0002] 2000 Canada. University of Alberta, Edmonton reported on seven cases of clinical Kashiwajima transplantation (Shapiro AM et al., S. Engl. J. Med., (2000) 343, p.230- 238.). The novel use of an immunosuppressant, later called the 'Edmonton' Protocol ', was able to withdraw from insulin therapy in all patients with type 1 diabetes who had undergone Kashiwajima transplantation. Kashiwajima transplantation is currently the most ideal treatment for insulin-dependent diabetes patients. The viscera is composed of exocrine and endocrine glands. Exocrine glands occupy over 98% of the total visceral volume, and less than 2% of endocrine glands. In 1869, Langerno, sees the endocrine gland tissue that appears in the optical microscopic image! / And appears to be an island that floats in the sea of the exocrine gland tissue. islet). Yeouido is a group of endocrine cells, consisting of α cells, / 3 cells, ΡΡ cells, and δ cells. Insulin, the only hormone in the body that has the effect of lowering blood sugar, is secreted from the β cells of Yeouido. Isojima transplantation aims to replace and regenerate a hypoglycemic system that has been abolished by isolating this island from prey and transplanting it to a patient with insulin-dependent diabetes. β cells account for 80-85% of all cells that make up the islands. β-cells don't just secrete insulin; more importantly, they can sense sugar in the blood. The main body of regulation of insulin secretion in glucose metabolism depends on a complex system involving multiple organs! /! In other words, it is possible to adjust blood glucose to a very narrow range by reacting quickly to blood glucose that changes rapidly during exercise or eating alone and secreting the appropriate amount of insulin. Is possible. Sensing the effect target and adjusting the action The fact that a single type of cell is used alone and that the cell occupies most of the island is the rationale for the island transplant.
[0003] 瞵島単離成功の是非は、単離に用いる勝臓自体の善し悪しにかかっている。良好 な瞵島単離のためには外分泌腺組織の状態も良い必要がある。瞵島単離を目的とし た勝臓摘出には想像以上の繊細さが要求される。例えば、勝臓にさわって圧力をか けるだけでも、瞵外分泌細胞はその含有酵素である蛋白分解酵素が放出されるため 自己融解がはじまる。また、遠隔地で勝臓が摘出された場合、瞵島単離施設までの 輸送中の保存についても充分な配慮が必要となる。  [0003] The pros and cons of the success of Isojima Island depend on the quality of the viscera used for isolation. The condition of the exocrine gland tissue also needs to be good for good isolation of Kashiwajima. For the purpose of isolating the islands for the purpose of isolating the islands of the islands, it is necessary to be more delicate than expected. For example, even by touching the viscera and applying pressure, the exocrine cells start autolysis because the proteolytic enzyme that is the enzyme contained in them is released. In addition, when a viscera is extracted at a remote site, sufficient consideration is required for preservation during transportation to the Isojima Isolation Facility.
[0004] インスリンの不足あるいは欠乏がその原因となっている糖尿病が瞵島移植の適応と なる。活性の低下の原因に基づいて糖尿病は大きく二つに分類される。一つは何ら かの原因によって瞵 /3細胞が破壊、障害されてインスリンの分泌がなくなるもの、もう 一つは血中にインスリンが正常あるいは正常以上に存在するのだ力 Sインスリン抵抗性 が末梢組織に存在するためインスリンの作用が低下しているものである。前者が 1型 糖尿病あるいは若年型糖尿病、後者が 2型糖尿病と呼ばれる。瞵島移植の適応は 1 型糖尿病である。 2型糖尿病においても病期が進むと糖毒性や /3細胞の疲弊によつ てインスリンの不足を来すが、 2型糖尿病は現在のところ瞵島移植の適応とはなって いない。理由は、インスリンの分泌がある程度保たれている 2型糖尿病に対してインス リンがほとんど分泌されていない 1型糖尿病ではインスリン療法による血糖調節が非 常に困難であること、 2型糖尿病はその病態の基盤にインスリン抵抗性をもっため瞵 島移植の効果が懸念されるためである。 2型糖尿病でも血糖の厳格な調節は予後に 良好に働くが、移植瞵島の不足と言う厳然たる事実があり 1型糖尿病のみが瞵島移 植の対象となっている。将来移植瞵島の供給が豊富となるような状況下では、インス リン抵抗性を契機としたインスリン非依存型糖尿病もその適応となる可能性は充分に ある。  [0004] Diabetes mellitus due to insulin deficiency or deficiency is indicated for Kashiwajima transplantation. Diabetes is largely divided into two categories based on the cause of the decrease in activity. One is the destruction of 瞵 / 3 cells due to some cause, damage and loss of insulin secretion, the other is the presence or absence of insulin in the blood normal or normal S insulin resistance is peripheral Since it exists in the tissue, the action of insulin is reduced. The former is called type 1 diabetes or juvenile diabetes, and the latter is called type 2 diabetes. The indication for Kashiwajima transplantation is type 1 diabetes. Even in type 2 diabetes, as the stage progresses, insulin deficiency is caused by glucose toxicity and / 3 cell exhaustion, but type 2 diabetes is not currently indicated for Kashiwajima transplantation. The reason is that insulin secretion is maintained to some extent.Insulin is hardly secreted against type 2 diabetes.In type 1 diabetes, blood glucose control by insulin therapy is very difficult. This is because of the insulin resistance in the base, and there is concern about the effects of the island transplantation. Although strict regulation of blood glucose works well in prognosis even in type 2 diabetes, there is a severe fact that there is a shortage of transplanted islands, and only type 1 diabetes is the subject of transplantation. In the situation where the supply of transplanted islands will be abundant in the future, non-insulin-dependent diabetes mellitus triggered by insulin resistance is likely to be indicated.
[0005] 統計によると欧米では年間人口比（10万人中） 12人から 35人（日本では 1. 5人） 力 ^型糖尿病を発症している。欧米社会において糖尿病は解決すべき大きな社会問 題である。糖尿病の一次予防と、合併症の二次予防のための対策が急務であると言 X·る。 [0006] 1993年に糖尿病コントロールと合併症に関する臨床試験（Diabetes Control and C omplications Trial, DCCT)によるインスリン強化療法の合併症に対する効果が発表 されている (The Diabetes Control and Complications Trial Research Group. N. Engl. J. Med., (1993)329， p.977-986.参照）。これは、可能な限り厳格なインスリン療法で あるインスリン強化療法によって長期合併症の予防が可能かどうかとの疑問に対する 臨床試験である。インスリン強化療法は従来のインスリン療法に比べて長期合併症の 発症あるいは進行を 39%から 76%減少させ、同時に低血糖発症率を 3倍に増やし たと言う結果であった。この臨床試験によって、糖尿病の長期合併症を回避するため には血糖の厳格な調節が非常に有用であり、インスリン強化療法によってある程度そ の目標は達成可能であることが示された。だ力 このことは同時にインスリン療法では 完璧な血糖調節は不可能であり、長期合併症予防にも限界があることを意味した。 1 型糖尿病は自己免疫の異常によって、インスリンを産生する瞵 /3細胞が特異的に破 壊されることで発症する（アトキンソン（Atkinson MA)ら、 Ν· Engl. J. Med. , (1994)331， p.1428-1436.参照）。その根本的治療には、瞵 /3細胞の再生置換療法のひとつであ る移植が考えられる。この方法として瞵臓移植と瞵島移植がある。これら二つの移植 の目的は、非常に厳格な血糖調節を可能とし、低血糖さらには長期合併症の発症を 回避することである。単にインスリン療法による日常の煩わしさから患者を開放し生活 の質（QOUを向上させる事だけが目的ではない。移植療法はインスリン依存性糖尿 病を治癒と言う最終目標に向かわせる手段として、インスリン療法よりは遙かに潜在 能力を持った治療法である。し力もながら、現行の瞵島移植には大きな問題点がある 。移植瞵島の不足である。現在の瞵島単離技術がいかに向上しようがこれを解決す ることは不可能である。需要と供給のバランスが違いすぎる。瞵島移植が一般的移植 医療とはなり得てもそのためのウェイティング 'リストは膨大なものとなり、 1型糖尿病 患者が実際に瞵島移植の恩恵を得るには数年力 十数年を要すると言う事態になる 。すなわち 1型糖尿病患者が日常臨床で提示される治療法の選択肢として瞵島移植 が挙げられることはありえない。 According to statistics, in Europe and America, annual population ratio (out of 100,000 people) 12 to 35 people (1.5 people in Japan) has developed type ^ diabetes. Diabetes is a major social problem to be solved in Western society. X. It is said that there is an urgent need for measures to prevent primary diabetes and prevent secondary complications. [0006] In 1993, the clinical trial on diabetes control and complications (Diabetes Control and Complications Trial, DCCT) published the effect of insulin enhancement therapy on complications (The Diabetes Control and Complications Trial Research Group. N. Engl. J. Med., (1993) 329, p.977-986.). This is a clinical trial on the question of whether long-term complications can be prevented with insulin-enhanced therapy, the strictest possible insulin therapy. Intense insulin therapy reduced the incidence or progression of long-term complications from 39% to 76% compared to conventional insulin therapy, and at the same time increased the incidence of hypoglycemia three-fold. This clinical trial has shown that strict regulation of blood glucose is very useful in avoiding long-term complications of diabetes, and that some of the goal can be achieved with insulin-enhanced therapy. This also means that insulin therapy does not allow perfect blood glucose control and limits the prevention of long-term complications. Type 1 diabetes is caused by abnormal destruction of さ れ る / 3 cells that produce insulin due to abnormal autoimmunity (Atkinson MA et al., Ν Engl. J. Med., (1994) 331). , P.1428-1436. For the fundamental treatment, transplantation, which is one of the regenerative replacement therapy for 瞵 / 3 cells, is considered. This method includes spleen transplantation and Yeouido transplantation. The purpose of these two transplants is to allow very strict glycemic control and avoid the development of hypoglycemia and even long-term complications. The purpose of insulin therapy is not just to improve the quality of life (QOU) by freeing patients from the daily annoyance of insulin therapy. Transplant therapy is an insulin therapy as a means to move insulin-dependent diabetes toward the ultimate goal of healing. It is a treatment method with much more potential, but it has a big problem with the current Kashiwajima transplantation, but there is a shortage of transplanted Kashiwajima. However, it is impossible to solve this problem.The balance between supply and demand is too different.While the island transplantation can be a general transplantation medical treatment, the waiting list for it is huge. Diabetes patients will actually take several decades to get the benefits of Kashiwajima transplantation, ie Kashiwajima transplantation is an option for treatment presented to patients with type 1 diabetes in daily clinical practice. It can't be lost.
[0007] すでに臨床瞵島移植で実証された瞵島細胞の有効性と移植瞵島の絶対的不足と 言う 2つの現実を前にして、瞵島あるいは瞵 /3細胞に匹敵する機能をもつ細胞を作 製することは高い社会的貢献と大きな医療経済的影響を有するものである。また、近 年急速に進歩し注目を集めて!/、る幹細胞研究でも瞵内分泌細胞への分化誘導の可 能性が示されており（ルメルスキー（Lumelsky N)ら、 Science, (2001)， Mayl8;292(552 0)， ρ· 1389-1394·およびアツサディ（Assady S)ら、 Diabetes, (2001)， 50 ρ· 1691-1697. 参照）、人為的瞵 /3細胞産生に対する関心をさらに助長する要因となっている。 [0007] Cells that have functions comparable to those of Kashiwajima or Kashiwa / 3 cells in front of the two realities of the effectiveness of Kashiwajima cells already demonstrated in clinical Kashiwajima transplantation and the absolute lack of transplanted Kashiwajima Make Manufacturing has a high social contribution and great medical economic impact. In recent years, stem cell research that has made rapid progress and attracted attention has shown the possibility of inducing differentiation into 瞵 endocrine cells (Lumelsky N et al., Science, (2001), Mayl8). ; 292 (552 0), ρ · 1389-1394 · and Assady S et al., Diabetes, (2001), 50 ρ · 1691-1697.), Further fostering interest in artificial sputum / 3 cell production It is a factor.
[0008] 糖尿病の患者数は 1型および 2型糖尿病ともに増加しており、糖尿病に伴う失明、 腎不全および心血管系病変などの重篤な合併症が多発していることからも、急増す るであろう当該医療費に対する対策は、高齢化社会をむかえるにあたって、医療上 および社会的な緊急課題である。現在臨床で用いられて!/、るインスリン投与療法で は、たとえ厳格な監視下でも 1型糖尿病の血糖コントロール、 2型糖尿病の合併症の 発症防止は極めて困難である。血糖を感知してそれに対する過不足ない量のインス リンを分泌する細胞を移入することで 1型糖尿病を完治できると言う考えが今や世界 中で一般的となった。し力もながら、勝臓移植および瞵島単離に要する脳死体からの 摘出瞵はそれを必要とする患者数に対して圧倒的に少なぐ今後解消される見込み はない。 [0008] The number of patients with diabetes has increased in both type 1 and type 2 diabetes, and the number has increased rapidly due to the frequent occurrence of serious complications such as blindness, renal failure and cardiovascular lesions. Measures against such medical expenses will be a medical and social urgent issue when facing an aging society. Insulin therapy currently in clinical use! /, It is extremely difficult to control glycemic control of type 1 diabetes and prevent complications of type 2 diabetes even under strict monitoring. The idea that type 1 diabetes can be cured completely by transfecting cells that sense blood glucose and secrete a sufficient amount of insulin for it is now common throughout the world. However, there is no expectation that the number of patients who need it will be resolved in the future because the number of patients who need it will be overwhelmingly small.
[0009] そこで、ブタ組織や細胞を利用した研究が進む一方、人畜共通感染症、生体組織 適合性や倫理的問題も浮上してきた。とくにウィルスの潜在的危険性が大きな問題と なってきた。ブタの臓器や細胞が保有するブタ由来のウィルス（とりわけ、内因性ブタ 特異的レトロウイノレス（porcine endogenous retrovirus； PERV)は、染色体に糸且み込ま れているために排除は不可能である）がレシピエントに感染して病気を起こす危険性 、それが家族や医療スタッフに感染を広げ、さらに社会に新しいウィルス感染を広げ る可能性である（ペイシヤンス（Patience C)ら、 Nat. Med., (1997)， 3(3)， p.282-286.参 昭）  [0009] Therefore, while research using pig tissues and cells has progressed, zoonotic diseases, biotissue compatibility and ethical issues have also emerged. In particular, the potential danger of viruses has become a major problem. Swine-derived viruses possessed by porcine organs and cells (especially endogenous porcine specific retrovirus (PERV) cannot be eliminated because it is inserted into the chromosome). The risk of infecting recipients and causing illness, which can spread infection to families and medical staff and further spread new viral infections to society (Patience C et al., Nat. Med., ( 1997), 3 (3), p.282-286.
[0010] よって、ヒト成熟瞵島 0細胞にかわる細胞の供給源として、胚性幹細胞（ES細胞と も言う）や組織幹細胞からのインスリン分泌細胞の分化誘導は魅力的である。よって、 これまで多くの研究者によって、当該研究に関する報告がなされている（ルメルスキ 一ら、 Science, (2001)， Mayl8;292(5520), ρ· 1389- 1394·、ホリ (Hori Y)ら、 Proc. Natl. Acad. Sci. USA., (2002) Decl0;99(25)， ρ· 16105- 16110·、ラジャゴパル（Rajagopal J) ら、 Science, (2003) Janl7;299(5605)， ρ·363·、シビオネ（Sipione S)ら、 Diabetologia, ( 2004) 47(3)， P.499- 508·、ミヤザキ（Miyazaki S)ら、 Diabetes. (2004) 53(4)， p.1030-10 37·、ハンソン（Hansson M)ら， Diabetes, (2004) 53(10)， ρ·2603_2609·およびダムール (D ' Amour A)ら、 Nat. Biotech. , published online, 19 October 2006， doi: 10.1038/ nbtl259.参照）。 [0010] Therefore, induction of differentiation of insulin-secreting cells from embryonic stem cells (also referred to as ES cells) and tissue stem cells is attractive as a source of cells that replace the mature human islet 0 cells. Thus, many researchers have reported on the research (Lumerski et al., Science, (2001), Mayl8; 292 (5520), ρ · 1389-1394 ·, Hori Y et al., Proc. Natl. Acad. Sci. USA., (2002) Decl0; 99 (25), ρ · 16105-16110 ·, Rajagopal J Science, (2003) Janl7; 299 (5605), ρ · 363 ·, Sipione S et al., Diabetologia, (2004) 47 (3), P.499-508 ·, Miyazaki S et al., Diabetes. (2004) 53 (4), p.1030-10 37 ·, Hansson M et al., Diabetes, (2004) 53 (10), ρ · 2603_2609 ·, and D'Amour A) et al., Nat. Biotech., Published online, 19 October 2006, doi: 10.1038 / nbtl259.).
[0011] しかしながら、ルメルスキーら、 Science, (2001)， Mayl8;292(5520), ρ· 1389_1394·、 ホリら、 Proc. Natl. Acad. Sci. USA., (2002) Decl0;99(25)， ρ· 16105- 16110·、ラジャゴ パルら、 Science, (2003) Janl7;299(5605)， ρ·363·、シビオネら、 Diabetologia, (2004) 4 7(3)， ρ·499- 508·、ミヤザキら、 Diabetes. (2004) 53(4)， ρ· 1030-1037.およびハンソンら 、 Diabetes, (2004) 53(10)， ρ·2603_2609·に記載された手法はインスリン分泌細胞へ の分化誘導効率が低く（3%以下である）、かつ分泌されるインスリン量も正常瞵島と 比較すると mg蛋白あたり 1 , 000分の一と少ない。これは、いずれの手法も未分化な ES細胞からインスリン分泌細胞を誘導するための初期分化のために、胚様体形成を 最初の工程で行っているためである。胚様体形成を行うことで、分化が誘導されるが 、細胞が外胚葉、内胚葉、中胚葉と全ての方向への分化してしまうため、 目的とする インスリン分泌細胞は、内胚葉に属するために、他の外胚葉ゃ中胚葉へと分化してく る細胞を淘汰することができないため、最終的な分化誘導率が低く成ってしまうと言う 欠点、を有する。  [0011] However, Lumelsky et al., Science, (2001), Mayl8; 292 (5520), ρ · 1389_1394 ·, Hori et al., Proc. Natl. Acad. Sci. USA., (2002) Decl0; 99 (25), ρ · 16105-16110 ·, Rajago Pal et al., Science, (2003) Janl7; 299 (5605), ρ · 363 ·, Sibione et al., Diabetologia, (2004) 4 7 (3), ρ · 499-508 ·, Miyazaki Diabetes. (2004) 53 (4), ρ · 1030-1037. And Hanson et al., Diabetes, (2004) 53 (10), ρ · 2603_2609 · are effective in inducing differentiation into insulin-secreting cells. Is low (less than 3%), and the amount of insulin secreted is less than 1 / 1,000 per mg protein compared to normal Kashiwajima. This is because both methods perform embryoid body formation in the first step for initial differentiation to induce insulin-secreting cells from undifferentiated ES cells. Differentiation is induced by embryoid body formation, but the cells are differentiated in all directions, such as ectoderm, endoderm, and mesoderm. Therefore, the target insulin-secreting cells belong to the endoderm. Therefore, since cells that differentiate into other ectoderm or mesoderm cannot be trapped, there is a disadvantage that the final differentiation induction rate is low.
[0012] 一方で、未分化な ES細胞を胚様体を形成させることなく胚体内胚葉を形成させ、ィ ンスリン分泌細胞へ分化誘導させる手法が報告されてレ、る（「ダムール法」と言うことも ある） (ダム一ルら、 Nat. Biotech., published online, 19 October 2006， doi: 10.1038/ nbtl259.参照）。胚様体形成を行わないこの手法は、胚様体形成を行う手法と比較し て効率よくインスリン分泌細胞を得ることが可能である。し力もながら、ダムールら、 Na t. Biotech., published online, 19 October 2006， doi: 10.1038/nbtl259.に記載された 従来技術によるインスリン分泌細胞への分化誘導効率は 3〜； 12%とかなり低い効率 である。また、該従来技術により得られたインスリン分泌細胞のインスリン分泌量が正 常瞵島の 100分の 1から 500分の 1と低い効率である。これは、ダムール法では処置 過程にお!/、て、勝臓の発生過程で β細胞への分化を抑制（ミラレス（Miralles F)ら、 I nt J Dev Biol. 2006; 50(1): 17_26.参照）し、瞵外分泌細胞を誘導する（ブシュシャン（ Bhushan A)ら、 Development. 2001;128(24):5109_17参照）と報告されている線維芽 細胞増殖因子 10 (FGF- 10)を使用して!/、ることが要因のひとつである。 [0012] On the other hand, a technique has been reported in which undifferentiated ES cells are formed into definitive endoderm without forming embryoid bodies and induced to differentiate into insulin-secreting cells (referred to as the “Damur method”). (See Dam Iru et al., Nat. Biotech., Published online, 19 October 2006, doi: 10.1038 / nbtl259.). This technique that does not perform embryoid body formation can yield insulin-secreting cells more efficiently than the technique that performs embryoid body formation. However, the efficiency of inducing differentiation into insulin-secreting cells according to the conventional technique described in Damour et al., Nat. Biotech., Published online, 19 October 2006, doi: 10.1038 / nbtl259. Is considerably low at 3 to 12%. Efficiency. In addition, the insulin secretion amount of the insulin-secreting cells obtained by the conventional technique is as low as 1/100 to 1/500 of the normal island. This is because during the treatment process in the Damour method! /, And during the development of the viscera, the differentiation into β-cells is suppressed (Miralles F et al., I nt J Dev Biol. 2006; 50 (1): 17_26.) and fibers reported to induce exocrine cells (see Bhushan A et al., Development. 2001; 128 (24): 5109_17) One factor is the use of blast growth factor 10 (FGF-10)!
発明の開示  Disclosure of the invention
[0013] 本発明の目的は、充分に機能的であり、大量供給が可能で安全なインスリン分泌 細胞ならびに該細胞を用いた糖尿病治療薬、バイオ人工勝臓、研究試薬および創 薬モデル動物を提供することである。  [0013] An object of the present invention is to provide a sufficiently functional, safe insulin-secreting cell that can be supplied in large quantities, a therapeutic agent for diabetes, a bioartificial viscera, a research reagent, and a drug model animal using the cell. It is to be.
[0014] 前記問題点に鑑み鋭意検討した結果、ァクチビンを用いて未分化な ES細胞から 分化させた胚体内胚葉を、馴化培地を用いてニューロジェニン 3発現細胞へ分化さ せたのちに該ニューロジェニン 3発現細胞を高グルコース培地で刺激することにより、 インスリン分泌細胞へ効率的に分化誘導できることを見出し、本発明を完成した。  [0014] As a result of intensive studies in view of the above problems, definitive endoderm differentiated from undifferentiated ES cells using activin was differentiated into neurogenin 3 expressing cells using conditioned medium, and then The inventors have found that by stimulating Jenin 3 expressing cells with a high glucose medium, differentiation can be efficiently induced into insulin-secreting cells, and the present invention has been completed.
[0015] すなわち、本発明は、胚性幹細胞のインスリン分泌細胞への分化誘導方法であつ て、（a)泉維芽細胞増殖因子およびァクチビンの存在下に胚性幹細胞を培養するこ とにより胚体内胚葉へ分化させる工程、（b)得られた胚体内胚葉を、線維芽細胞増 殖因子の存在下、馴化培地を用いて培養することにより原始瞵へ分化させる工程、（ c)得られた原始瞵を、馴化培地を用いて培養することによりニューロジェニン 3 (Ngn 3)発現細胞数を増加させ、ニューロジェニン 3発現細胞を得る工程、および (d)得ら れたニューロジェニン 3発現細胞を、高グルコース濃度の細胞培養用培地中で刺激 してインスリン分泌細胞へ分化させる工程を含む方法に関する。  [0015] That is, the present invention relates to a method for inducing differentiation of embryonic stem cells into insulin-secreting cells, wherein (a) embryonic stem cells are cultured in the presence of fountain fibroblast growth factor and activin. A step of differentiating into an endoderm, (b) a step of differentiating the resulting definitive endoderm into a primitive gland by culturing in a conditioned medium in the presence of a fibroblast growth factor, (c) obtained Culturing primordial buds in a conditioned medium to increase the number of neurogenin 3 (Ngn 3) -expressing cells to obtain neurogenin 3 expressing cells; and (d) the resulting neurogenin 3 expressing cells. And a method comprising the step of differentiating into insulin-secreting cells by stimulation in a cell culture medium having a high glucose concentration.
[0016] 泉維芽細胞増殖因子は泉維芽細胞増殖因子 10以外の泉維芽細胞増殖因子で あることが好ましい。  [0016] Preferably, the fountain fibroblast growth factor is a fountain fibroblast growth factor other than fountain fibroblast growth factor 10.
[0017] ァクチビンはァクチビン Aであることが好まし!/、。  [0017] The activin is preferably activin A! /.
[0018] ァクチビンの濃度が 2〜200ng/mLであることが好まし!/、。  [0018] Preferably, the concentration of activin is 2 to 200 ng / mL! /.
[0019] 工程 (b)において用いられる馴化培地力 ヒト神経膠芽細胞腫由来細胞株 T98G を無血清細胞培養培地中で培養したものであることが好ましい。  Conditioned medium strength used in step (b) It is preferable that the human glioblastoma cell line T98G is cultured in a serum-free cell culture medium.
[0020] 工程（c)において用いられる馴化培地力 S、ヒト肝内皮細胞株 TMNK— 1または血 管内皮細胞を低グルコース細胞培養培地中で培養したものであることが好ましい。  [0020] Conditioned medium strength S used in step (c), human liver endothelial cell line TMNK-1, or vascular endothelial cells are preferably cultured in a low glucose cell culture medium.
[0021] 各工程における培養がそれぞれ、コラーゲンタイプ IV、マトリゲル、フイブロネクチン 、ジエラチン、ポリオル二チンおよびラミニンよりなる群から選ばれる生体適合性材料 を用いる三次元培養であることが好ましレ、。 [0021] The culture in each step is collagen type IV, matrigel, fibronectin, respectively. Preferred is a three-dimensional culture using a biocompatible material selected from the group consisting of: dielatin, polyornithine and laminin.
[0022] 工程（a)において用いられる生体適合性材料がコラーゲンタイプ IVであることが好 ましぐ工程 ）において用いられる生体適合性材料がラミニンであることが好ましい[0022] The biocompatible material used in step (a) is preferably collagen type IV. The biocompatible material used in step (a) is preferably laminin.
〇 Yes
[0023] 工程（a)における細胞培養濃度が 1 X 105〜4 X 105細胞/ mLであることが好ましく 、工程 (b)における細胞培養濃度が 1 X 105〜8 X 105細胞/ mLであることが好まし い。 [0023] Step preferably cell culture concentration of 1 X 10 5 ~4 X 10 5 cells / mL in (a), the cell culture density at step (b) is 1 X 10 5 ~8 X 10 5 cells / Preferably it is mL.
[0024] 工程（a)および/または (b)において、線維芽細胞増殖因子の濃度が 2〜20ng/ mLであることが好ましい。  [0024] In the step (a) and / or (b), the concentration of the fibroblast growth factor is preferably 2 to 20 ng / mL.
[0025] 工程 (a)において用いられる培地が、無血清細胞培養用培地中に、ゥシ胎仔血清 を 0. 2〜3%含有し、ゥシ血清アルブミンを 2〜3%含有することが好ましい。 [0025] The medium used in the step (a) preferably contains 0.2 to 3% ushi fetal serum and 2 to 3% ushi serum albumin in a serum-free cell culture medium. .
[0026] 工程 (b)において用いられる培地が、馴化培地中にレチノイン酸を 1〜50 11101/[0026] The medium used in step (b) contains retinoic acid in a conditioned medium in an amount of 1-50 11101 /
L含有することが好ましい。 It is preferable to contain L.
[0027] 工程 ）において用いられる培地が、馴化培地中に γ—セクレターゼ阻害剤 XVIII を 0. 25〜2 11101/し含有し、線維芽細胞増殖因子を5〜501¾/111し含有し、上皮 増殖因子を 5〜50ng/mL含有することが好ましい。 [0027] The medium used in step (1) contains 0.25-2 11101 / γ-secretase inhibitor XVIII and 5-501¾ / 111 fibroblast growth factor in the conditioned medium, and epithelial growth It is preferable to contain 5 to 50 ng / mL of the factor.
[0028] 工程（c)において、ニューロジェニン 3発現細胞力 セルソーターを用いて選別され ることが好ましい。 [0028] In the step (c), it is preferable to select using a neurogenin 3 expressing cell force cell sorter.
[0029] 工程（d)において用いられる培地が、肝細胞増殖因子を 2〜50ng/mL含有し、二 コチンアミドを;!〜 1 Ommol/L含有し、ェクセディン 4を 5〜 1 OOnmol/L含有し、 トログリタゾン 1〜10 mol/Lおよび硫酸亜鉛を 1〜20 mol/L含有することが好 ましい。  [0029] The medium used in the step (d) contains 2 to 50 ng / mL of hepatocyte growth factor, contains !! to 1 Ommol / L of nicotinamide, and contains 5 to 1 OOnmol / L of exedine 4. It is preferable to contain 1 to 10 mol / L of troglitazone and 1 to 20 mol / L of zinc sulfate.
[0030] 工程（d)が、ニューロジェニン 3発現細胞を高グルコース濃度の細胞培養用培地、 次いで低グルコース濃度の細胞培養用培地中で繰り返し刺激培養する工程からなり 、高グルコース濃度の細胞培養用培地中で 1回あたり 2時間の刺激培養を、 1日あた り 1〜3回行うことを 1〜10日間繰り返すことが好ましい。  [0030] Step (d) comprises a step of repeatedly stimulating and culturing neurogenin 3 expressing cells in a cell culture medium having a high glucose concentration and then in a cell culture medium having a low glucose concentration. It is preferable to repeat 1 to 3 times of stimulation culture for 2 hours per day in the medium for 1 to 10 days.
[0031] 工程 (d)において用いられる培地が、高グルコース濃度の細胞培養培地中に脂質 、アミノ酸、ビタミンおよび/またはミネラルを含有することが好ましい。 [0031] The medium used in step (d) is a lipid in cell culture medium having a high glucose concentration. It preferably contains amino acids, vitamins and / or minerals.
[0032] 工程（d)が、ニューロジェニン 3発現細胞に Maf A遺伝子を導入し発現させることに より、インスリン分泌細胞の Maf A遺伝子発現を増強させることを含むことが好ましい[0032] Preferably, step (d) includes enhancing Maf A gene expression in insulin-secreting cells by introducing and expressing the Maf A gene in a neurogenin 3 expressing cell.
〇 Yes
[0033] 胚性幹細胞が哺乳類由来のものであることが好ましぐ哺乳類がマウス、ヒト、サル よりなる群力、ら選ばれること力 Sさらに好ましく、哺乳類がマウスであることが最も好まし い。  [0033] It is preferable that the embryonic stem cell is derived from a mammal. The ability to be selected from a group consisting of mice, humans, and monkeys. S is more preferable. The mammal is most preferably a mouse. .
[0034] 本発明はまた、前記分化誘導方法により誘導されるインスリン分泌細胞に関する。  [0034] The present invention also relates to insulin secreting cells induced by the differentiation induction method.
[0035] インスリン分泌細胞力 S、スフエロイドを形成しているインスリン分泌細胞を含有するこ とが好ましい。 [0035] Insulin-secreting cell strength S and insulin-secreting cells forming spheroids are preferably contained.
[0036] 本発明はさらに、前記インスリン分泌細胞を含有する糖尿病治療剤に関する。  [0036] The present invention further relates to a therapeutic agent for diabetes containing the insulin-secreting cells.
[0037] 本発明はさらに、前記インスリン分泌細胞を含有するバイオ人工勝臓に関する。 [0037] The present invention further relates to a bioartificial viscera containing the insulin-secreting cells.
[0038] 本発明はさらに、前記インスリン分泌細胞を含有する研究試薬に関する。 [0038] The present invention further relates to a research reagent containing the insulin-secreting cells.
[0039] 本発明はさらに、前記インスリン分泌細胞を含有する創薬モデル動物に関する。 [0039] The present invention further relates to a drug discovery model animal containing the insulin-secreting cells.
図面の簡単な説明  Brief Description of Drawings
[0040] [図 1]本発明の方法によりマウス ES細胞から分化誘導されたマウス胚体内胚葉にお ける胚体内胚葉特異的遺伝子 Foxa2および Soxl 7の遺伝子発現を RT— PCR法に て判定した写真である。レーン 1〜4は、それぞれコラーゲンタイプ IV、マトリゲル、ラ ミニンタイプ 1、ジエラチンで培養したマウス胚体内胚葉の遺伝子発現を示す。  [0040] [Fig. 1] Photograph showing the determination of gene expression of definitive endoderm-specific genes Foxa2 and Soxl 7 in mouse definitive endoderm differentiated from mouse ES cells by the method of the present invention by RT-PCR. It is. Lanes 1 to 4 show the gene expression of mouse definitive endoderm cultured in collagen type IV, matrigel, laminin type 1, and dielatin, respectively.
[図 2]本発明の方法によりコラーゲンタイプ IVで加工した培養皿を用いてマウス ES細 胞から分化誘導されたマウス胚体内胚葉における胚体内胚葉特異的遺伝子 Foxa2 および Soxl 7の遺伝子発現を RT— PCR法にて判定した写真である。レーン;!〜 4 は、それぞれァクチビン Aの濃度力 ¾ng/ml、 10ng/ml, lOOng/ml, 200ng/ mlで添加し培養したマウス胚体内胚葉の遺伝子発現を示す。  [Fig. 2] Expression of definitive endoderm-specific genes Foxa2 and Soxl 7 in mouse definitive endoderm differentiated from mouse ES cells using culture dishes processed with collagen type IV by the method of the present invention. It is a photograph determined by PCR method. Lanes !! to 4 show gene expression of mouse definitive endoderm cultivated by adding and cultivating activin A at a concentration of ¾ ng / ml, 10 ng / ml, lOOng / ml, and 200 ng / ml, respectively.
[図 3]本発明の方法によりマウス胚体内胚葉力 分化誘導されたマウス原始瞵におけ る原始瞵特異的遺伝子 Pdx— 1の遺伝子発現、ネガティブコントロールである Shh遺 伝子発現、および内因性標準である GAPDH遺伝子発現を RT— PCR法にて判定 した写真である。レーン 1〜4は、それぞれレチノイン酸濃度が 1 Μ、 ΙΟ , Μ, 100 11 M、 1000 Mで添加し培養したマウス原始瞵の遺伝子発現を示す。 [Fig. 3] Gene expression of the primordial acupuncture-specific gene Pdx-1 in the mouse primitive gland, in which mouse definitive endoderm differentiation was induced by the method of the present invention, Shh gene expression as a negative control, and endogenous standard This is a photograph in which GAPDH gene expression is determined by RT-PCR. Lanes 1 to 4 have retinoic acid concentrations of 1 Μ, ΙΟ, Μ, 100 The gene expression of the primitive mouse mouse added and cultured at 11 M and 1000 M is shown.
園 4(a)]本発明の分化誘導方法によりマウス ES細胞から誘導されたインスリン分泌細 胞の Maf A遺伝子発現を示す顕微鏡像である。 1は核を示し、 2は球状を呈すインス リン分泌細胞全体を示す。 4 (a)] is a microscopic image showing the expression of Maf A gene in insulin-secreting cells induced from mouse ES cells by the differentiation induction method of the present invention. 1 indicates the nucleus, and 2 indicates the entire insulin-secreting cell that is spherical.
園 4(b)]本発明の分化誘導方法によりマウス ES細胞から誘導されたインスリン分泌細 胞の形態を示す顕微鏡像である。 1は核を示し、 2は球状を示すインスリン分泌細胞 を示す。 4 (b)] is a microscopic image showing the morphology of insulin-secreting cells derived from mouse ES cells by the differentiation induction method of the present invention. 1 indicates the nucleus and 2 indicates the insulin-secreting cells that are spherical.
園 5(a)]本発明の分化誘導方法により工程 (d)において低グルコース DMEMを用い てマウス ES細胞から誘導されたインスリン分泌細胞および従来技術の分化誘導方法 によりマウス ES細胞から誘導されたインスリン分泌細胞のインスリン分泌能を示すグ ラフである。横軸の 1〜10は、本発明のインスリン分泌細胞を工程（d)において低グ ルコース DMEMで 2日間培養後「グルコース刺激：低→高」した後の培地中のインス リン分泌量 (横軸の 1)、本発明のインスリン分泌細胞を工程（d)において低ダルコ一 ス DMEMで 2日間培養後「グルコース刺激：低→高→低」した後の培地中のインスリ ン分泌量 (横軸の 2)、本発明のインスリン分泌細胞を工程（d)において低グルコース DMEMで 4日間培養後「グルコース刺激：低→高」した後の培地中のインスリン分泌 量 (横軸の 3)、本発明のインスリン分泌細胞を工程（d)において低グルコース DME Mで 4日間培養後「グルコース刺激：低→高→低」した後の培地中のインスリン分泌 量 (横軸の 4)、本発明のインスリン分泌細胞を工程（d)において低グルコース DME Mで 7日間培養後「グルコース刺激：低→高」した後の培地中のインスリン分泌量 (横 軸の 5)、本発明のインスリン分泌細胞を工程（d)において低グルコース DMEMで 7 日間培養後「グルコース刺激：低→高→低」した後の培地中のインスリン分泌量 (横 軸の 6)、従来技術であるシュローダー法のインスリン分泌細胞を「グルコース刺激:低 →高」した後の培地中のインスリン分泌量 (横軸の 7)、従来技術であるシュローダー 法のインスリン分泌細胞を「グルコース刺激:低→高→低」した後の培地中のインスリ ン分泌量 (横軸の 8)、従来技術であるダムール法のインスリン分泌細胞を「ダルコ一 ス刺激:低→高」した後の培地中のインスリン分泌量 (横軸の 9)、従来技術であるダム ール法のインスリン分泌細胞を「グルコース刺激:低→高→低」した後の培地中のイン スリン分泌量 (横軸の 10)をそれぞれ示す。 5 (a)] Insulin-secreting cells derived from mouse ES cells using low glucose DMEM in step (d) by the differentiation induction method of the present invention and insulin induced from mouse ES cells by the differentiation induction method of the prior art It is a graph showing the insulin secretory ability of secretory cells. 1 to 10 on the horizontal axis indicate the amount of insulin secreted in the medium after the insulin-secreting cells of the present invention were cultured in low glucose DMEM for 2 days in step (d) and then “glucose stimulation: low → high” (horizontal axis). 1) Insulin-secreting cells of the present invention in the step (d) for 2 days after culturing with low darcosose DMEM and after “glucose stimulation: low → high → low”, the amount of insulin secreted in the medium (on the horizontal axis) 2) Insulin-secreting cells of the present invention are cultured in low glucose DMEM for 4 days in step (d) and then the amount of insulin secreted in the medium after “glucose stimulation: low → high” (3 on the horizontal axis), Insulin secreting cells cultured in low glucose DME M in step (d) for 4 days and then “glucose stimulation: low → high → low” insulin secretion amount in the medium (4 on the horizontal axis), insulin secreting cells of the present invention After culturing for 7 days with low glucose DME M in step (d) Insulin secretion amount in the medium after glucose stimulation: low → high (5 on the horizontal axis), and after culturing the insulin secreting cells of the present invention in low glucose DMEM in step (d) for 7 days, “glucose stimulation: low → high → Insulin secretion in the medium after `` low '' (6 on the horizontal axis), insulin secretion in the medium after `` glucose stimulation: low → high '' for the Schroder method insulin-secreting cells (horizontal axis) 7) Insulin secretion in the medium after `` glucose stimulation: low → high → low '' of the Schroder method insulin-secreting cells of the conventional technology (8 on the horizontal axis), the conventional technology of the Damour method insulin The amount of insulin secreted in the medium after `` Darcose stimulation: low → high '' of the secretory cells (9 on the horizontal axis), the insulin secretion cells of the conventional Damole method were `` glucose stimulated: low → high → In the medium after `` low '' Each shows the amount of secretion of slin (10 on the horizontal axis).
園 5(b)]本発明の分化誘導方法により工程 (d)において低グルコース RCNK—1馴 化培地を用いてマウス ES細胞から誘導されたインスリン分泌細胞および従来技術の 分化誘導方法によりマウス ES細胞から誘導されたインスリン分泌細胞のインスリン分 泌能を示すグラフである。横軸の 1〜10は、本発明のインスリン分泌細胞を工程（d) にお!/、て低グノレコース RCNK— 1馴化培地で 2日間培養後「グノレコース刺激：低→ 高」した後の培地中のインスリン分泌量 (横軸の 1)、本発明のインスリン分泌細胞を 工程（d)にお!/、て低グルコース RCNK— 1馴化培地で 2日間培養後「グルコース刺 激:低→高→低」した後の培地中のインスリン分泌量 (横軸の 2)、本発明のインスリン 分泌細胞を工程（d)にお!/、て低グルコース RCNK— 1馴化培地で 4日間培養後「グ ルコース刺激:低→高」した後の培地中のインスリン分泌量 (横軸の 3)、本発明のイン スリン分泌細胞を工程（d)において低グルコース RCNK— 1馴化培地で 4日間培養 後「グルコース刺激:低→高→低」した後の培地中のインスリン分泌量 (横軸の 4)、本 発明のインスリン分泌細胞を工程（d)において低グルコース RCNK— 1馴化培地で 7 日間培養後「グルコース刺激：低→高」した後の培地中のインスリン分泌量 (横軸の 5 )、本発明のインスリン分泌細胞を工程（d)において低グルコース RCNK— 1馴化培 地で 7日間培養後「グルコース刺激：低→高→低」した後の培地中のインスリン分泌 量 (横軸の 6)、従来技術であるシュローダー法のインスリン分泌細胞を「グルコース刺 激:低→高」した後の培地中のインスリン分泌量 (横軸の 7)、従来技術であるシュロー ダ一法のインスリン分泌細胞を「グルコース刺激:低→高→低」した後の培地中のイン スリン分泌量 (横軸の 8)、従来技術であるダムール法のインスリン分泌細胞を「ダルコ ース刺激:低→高」した後の培地中のインスリン分泌量 (横軸の 9)、従来技術である ダムール法のインスリン分泌細胞を「グルコース刺激:低→高→低」した後の培地中 のインスリン分泌量 (横軸の 10)をそれぞれ示す。 5 (b)] Insulin-secreting cells derived from mouse ES cells using low glucose RCNK-1 conditioned medium in step (d) by the differentiation induction method of the present invention and mouse ES cells by the conventional differentiation induction method 2 is a graph showing the insulin secretory capacity of insulin secreting cells derived from the serotype. 1-10 on the horizontal axis in the medium after the insulin-secreting cells of the present invention were cultured in step (d)! /, After being cultured in low gnorecose RCNK-1 conditioned medium for 2 days after being stimulated with “gnorecose stimulation: low → high” Insulin secretion amount (1 on the horizontal axis), the insulin-secreting cells of the present invention in step (d)! /, Low glucose RCNK-1 after culturing for 2 days in conditioned medium, “glucose stimulation: low → high → low Insulin secretion amount in the culture medium (2 on the horizontal axis), the insulin-secreting cells of the present invention were used in step (d)! /, And cultured for 4 days in low glucose RCNK-1 conditioned medium. : Low → high ”insulin secretion in the medium (3 on the horizontal axis), and insulin-secreting cells of the present invention were cultured in low glucose RCNK-1 conditioned medium for 4 days in step (d). The amount of insulin secretion in the medium after “low → high → low” (4 on the horizontal axis), insulin secretion of the present invention Insulin secretion amount (5 on the horizontal axis) in the medium after culturing the vesicles in step (d) for 7 days in low glucose RCNK-1 conditioned medium after “glucose stimulation: low → high” In the step (d), the amount of insulin secretion in the medium after “Glucose Stimulation: Low → High → Low” after culturing for 7 days in a low glucose RCNK-1 conditioned medium (6 on the horizontal axis), the conventional Schroder method The amount of insulin secreted in the medium after `` glucose stimulation: low → high '' (7 on the horizontal axis), and the insulin-secreting cells of the conventional shredder method `` glucose stimulation: low → high '' → Insulin secretion in the medium after low (8 on the horizontal axis), Insulin secretion in the medium after `` Darcose stimulation: low → high '' for insulin secreting cells of the conventional Damour method (9 on the horizontal axis), the conventional technology Damoo Law of insulin secretion cells: show "glucose-stimulated low → high → low" and amount of insulin secreted in the medium after the (10 on the horizontal axis), respectively.
園 6(a)]本発明の分化誘導方法によりマウス ES細胞から誘導されたインスリン分泌細 胞および従来技術の分化誘導方法によりマウス ES細胞から誘導されたインスリン分 泌細胞のトルプタミド刺激前のインスリン分泌能を示すグラフである。横軸の 1〜5は、 本発明のインスリン分泌細胞を工程（d)において低グルコース DMEMで 2日間培養 (横軸の 1)、本発明のインスリン分泌細胞を工程（d)において低グルコース DMEM で 4日間培養 (横軸の 2)、本発明のインスリン分泌細胞を工程 (d)において低ダルコ ース DMEMで 7日間培養（横軸の 3)、従来技術であるシュローダー法によるインスリ ン分泌細胞 (横軸の 4)、および従来技術であるダムール法によるインスリン分泌細胞 (横軸の 5)を示す。 6 (a)] insulin secretion cells induced from mouse ES cells by the differentiation induction method of the present invention and insulin secretion before tolptamide stimulation of insulin secretory cells induced from mouse ES cells by the differentiation induction method of the prior art. It is a graph which shows performance. 1 to 5 on the horizontal axis indicate that the insulin-secreting cells of the present invention are cultured in low glucose DMEM for 2 days in step (d) (1 on the horizontal axis), the insulin-secreting cells of the present invention were cultured in low glucose DMEM for 4 days in step (d) (2 on the horizontal axis), and the insulin-secreting cells of the present invention were cultured in step (d) for low dalcose DMEM. 7 shows the culture for 7 days (3 on the horizontal axis), insulin-secreting cells by the Schroder method (horizontal axis 4), and the insulin-secreting cells by the conventional Damour method (5 on the horizontal axis).
園 6(b)]本発明の分化誘導方法によりマウス ES細胞から誘導されたインスリン分泌細 胞および従来技術の分化誘導方法によりマウス ES細胞から誘導されたインスリン分 泌細胞のトルプタミド刺激後のインスリン分泌能を示すグラフである。横軸の 1〜5は、 本発明のインスリン分泌細胞を工程（d)において低グルコース DMEMで 2日間培養 (横軸の 1)、本発明のインスリン分泌細胞を工程（d)において低グルコース DMEM で 4日間培養 (横軸の 2)、本発明のインスリン分泌細胞を工程 (d)において低ダルコ ース DMEMで 7日間培養（横軸の 3)、従来技術であるシュローダー法によるインスリ ン分泌細胞 (横軸の 4)、および従来技術であるダムール法によるインスリン分泌細胞 (横軸の 5)を示す。 6 (b)] Insulin secretion after tolptamide stimulation of insulin-secreting cells induced from mouse ES cells by the differentiation induction method of the present invention and insulin-secreting cells induced from mouse ES cells by the differentiation induction method of the prior art It is a graph which shows performance. 1 to 5 on the horizontal axis show that the insulin-secreting cells of the present invention were cultured in low glucose DMEM for 2 days in step (d) (1 on the horizontal axis), and the insulin-secreting cells of the present invention were cultured in low glucose DMEM in step (d). Culture for 4 days (horizontal axis 2), insulin-secreting cells of the present invention in low-darcose DMEM in step (d) for 7 days (horizontal axis 3), insulin-secreting cells by the Schroder method (prior art) ( 4) on the horizontal axis, and insulin-secreting cells (5 on the horizontal axis) by the conventional Damour method.
園 7]本発明の分化誘導方法によりマウス ES細胞から誘導されたインスリン分泌細胞 、未分化マウス ES細胞、またはマウス瞵島を糖尿病マウスへ移植し、移植後の血糖 値を測定したグラフである。コントロールとして、健常マウスおよび移植を行っていな い糖尿病マウスを用いている。移植後の最初の 5日間は健常マウスおよび移植を行 つて!/、な!/、糖尿病マウス共に中間型ヒトインスリン NPHを 2単位（IU)皮下注射し、そ の後の 5日間は中間型ヒトインスリン NPHを 1単位皮下注射している。 FIG. 7] A graph showing blood glucose levels after transplantation of insulin secreting cells, undifferentiated mouse ES cells, or mouse islets derived from mouse ES cells by the differentiation induction method of the present invention and transplanted to diabetic mice. As controls, healthy mice and non-transplanted diabetic mice are used. During the first 5 days after transplantation, healthy mice and transplantation should be carried out! /, NA! / And diabetic mice were injected with 2 units (IU) of intermediate human insulin NPH subcutaneously, and the subsequent 5 days were treated with intermediate humans. One unit of insulin NPH is injected subcutaneously.
園 8]本発明のバイオ人工勝臓の一実施態様を示す図である。バイオ人工勝臓は球 状を呈しているインスリン分泌細胞 7を注入する細胞注入口 3、蓋 4、免疫担当細胞 の透過を阻止するためのポリエチレン ビュルアルコール膜（ポアサイズは 30nm) 5 、細胞接着のための PAU加工した PTFE不織布 6からなる。 FIG. 8 is a diagram showing an embodiment of the bioartificial viscera of the present invention. The bioartificial spleen is a cell inlet 3 for injecting spherical insulin-secreting cells 7, a lid 4, a polyethylene butyl alcohol membrane (pore size 30 nm) for blocking the permeation of immunocompetent cells 5, It consists of PTAU nonwoven fabric 6 with PAU processing.
園 9]本発明の分化誘導方法によりマウス ES細胞から誘導されたインスリン分泌細胞 、未分化マウス ES細胞、またはマウス瞵島を充填した本発明のバイオ人工勝臓を糖 尿病マウスへ埋め込み、埋め込み後の血糖値を測定したグラフである。コントロール として、健常マウスおよび移植を行っていない糖尿病マウスを用いている。移植後の 最初の 10日間は健常マウスおよび移植を行っていない糖尿病マウス共に中間型ヒト インスリン NPHを 2単位（IU)皮下注射して!/、る。 9] The bioartificial spleen of the present invention filled with insulin-secreting cells, undifferentiated mouse ES cells, or mouse islets derived from mouse ES cells by the differentiation-inducing method of the present invention is embedded in a dialysis mouse and implanted. It is the graph which measured the blood glucose level after. As a control, healthy mice and non-transplanted diabetic mice are used. After transplantation During the first 10 days, healthy mice and non-transplanted diabetic mice are injected with 2 units (IU) of human intermediate insulin NPH subcutaneously!
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0041] 本発明は、ァクチビンを用いて未分化な ES細胞から分化させた胚体内胚葉を、馴 化培地を用いてニューロジェニン 3発現細胞へ分化させたのちに該細胞を高ダルコ ース培地で刺激することにより、インスリン分泌細胞へ効率的に分化誘導する方法に 関する。 [0041] The present invention provides a method in which definitive endoderm differentiated from undifferentiated ES cells using activin is differentiated into neurogenin 3 expressing cells using a conditioned medium, and then the cells are enriched in a high-darcose medium. The method of efficiently inducing differentiation into insulin secreting cells by stimulation with.
[0042] 本発明の ES細胞からインスリン分泌細胞を分化誘導する方法には、（a)線維芽細 胞増殖因子およびァクチビンの存在下に胚性幹細胞を培養することにより胚体内胚 葉へ分化させる工程、（b)得られた胚体内胚葉を、線維芽細胞増殖因子の存在下、 馴化培地を用いて培養することにより原始瞵へ分化させる工程、（c)得られた原始瞵 を、馴化培地を用いて培養することによりニューロジェニン 3発現細胞数を増加させ、 ニューロジェニン 3発現細胞を得る工程、および（d)得られたニューロジェニン 3発現 細胞を、高グルコース濃度の細胞培養用培地中で刺激してインスリン分泌細胞へ分 化させる工程を含む方法が含まれる。  [0042] In the method of inducing differentiation of insulin secreting cells from ES cells of the present invention, (a) embryonic stem cells are cultured in the presence of fibroblast growth factor and activin to differentiate into definitive endoderm. (B) differentiating the resulting definitive endoderm into a primitive pod by culturing it in a conditioned medium in the presence of fibroblast growth factor, (c) Increasing the number of cells expressing neurogenin 3 by culturing with, and (d) obtaining the cells expressing neurogenin 3 in the medium for cell culture of high glucose concentration. A method comprising the step of stimulation and differentiation into insulin secreting cells is included.
[0043] ES細胞としては、哺乳類由来のものが好ましぐ哺乳類としては、マウス、ヒト、サル などが挙げられる。また、 ES細胞に代えて ES細胞のように分化多能性を有する誘導 多能性幹細胞（または人工多能性幹細胞ともいう） (induced pluripotent stem cell, iP S細胞)も、本発明において同様に使用することができる。 iPS細胞は脊髄損傷、若 年型糖尿病など多くの疾患に対する細胞移植療法につながることが期待されており 、患者自身の体細胞から iPS細胞を誘導した場合、移植後の免疫拒絶反応も克服で きる (ホームへージ http://www.jst.go.jp/pr/info/ info320/index.html)。  [0043] Examples of ES cells that are preferably derived from mammals include mice, humans, monkeys, and the like. In addition, induced pluripotent stem cells (also called induced pluripotent stem cells) (differentiated pluripotent stem cells, iPS cells) having differentiation pluripotency like ES cells instead of ES cells are also used in the present invention. Can be used. iPS cells are expected to lead to cell transplantation therapy for many diseases such as spinal cord injury and juvenile diabetes. When iPS cells are induced from the patient's own somatic cells, immune rejection after transplantation can be overcome. (Home page http://www.jst.go.jp/pr/info/info320/index.html).
[0044] ES細胞は、受精卵が成長を続ける初期の段階である胚から製造することができる。  [0044] ES cells can be produced from embryos, which are the early stages in which a fertilized egg continues to grow.
卵子と***が 1つになった受精卵は、胎児へと成長していく途中で 2つ、 4つ、 8つ · · •と***を繰り返し、 5、 6日目には胚盤胞と呼ばれる状態になる。胚盤胞は直径 0. 1 mmほどの球状のかたまりである内部細胞塊を抱く胞胚腔から構成される。内側の細 胞塊は、いずれ内胚葉、中胚葉、外胚葉へと成長し、体のあらゆる細胞を形作ってい く部分で、栄養外胚葉はそれらの胎盤を形成し、また胚を外界から隔離する袋を形 成する。この内部細胞塊をほぐしたのち、細胞を取り出し、これらの細胞を増殖可能 でかつ分化しない環境で培養することにより ES細胞を製造することができる。したが つて、 ES細胞は、前記のとおり胚力 製造することができる。 A fertilized egg with a single ovum and sperm is divided into two, four, eight, etc. in the middle of growing into a fetus, and is called a blastocyst on days 5 and 6 It becomes a state. The blastocyst is composed of a blastocoel that holds an inner cell mass that is a spherical mass about 0.1 mm in diameter. The inner cell mass eventually grows into endoderm, mesoderm, and ectoderm and forms all the cells of the body, and the trophectoderm forms their placenta and isolates the embryo from the outside world Shape bag To do. After loosening the inner cell mass, ES cells can be produced by removing the cells and culturing these cells in an environment where they can proliferate and do not differentiate. Therefore, ES cells can be produced as described above.
[0045] このような ES細胞は、マウスでは 1981年にエバンスら（Evans et al.， 1981， Nature 292: 154-6·)や、マーチンら（Martin GR. et al.， 1981， Proc Natl Acad Sci 78: 7634- 8. )によって樹立され、商品としては大日本住友製薬株式会社 (大阪、 日本）などから 購入可能である。 [0045] Such ES cells were used in mice in 1981 in Evans et al. (1981, Nature 292: 154-6 ·) and Martin et al. (Martin GR. Et al., 1981, Proc Natl Acad Sci 78: 7634- 8.) and can be purchased from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan).
[0046] このような ES細胞は、ヒトでは 1998年にトムソンら（Thomson et al. , Science, 1998,28 2: 1145-7.)によって樹立され、 WiCell研究施設（WiCell Research Institute,ウェブサ イト: http：〃 www.wicell.org八マジソン、ウィスコンシン州、米国）から入手可能である。  [0046] Such ES cells were established in 1998 by Thomson et al., Science, 1998, 28 2: 1145-7. In the WiCell Research Institute (Website: http: 〃 www.wicell.org Eight Madison, Wisconsin, USA).
[0047] 我国においては、ヒト ES細胞の研究はナショナルバイオリソースプロジェクトに指定 されており、京都大学再生医科学研究所附属幹細胞医学研究センターで樹立され たヒト ES細胞が、「再生医科学研究所ヒト ES細胞分配規定」（平成 15年 1 1月 26日 施行）にしたがって、文部科学大臣の確認 (承認）を得るなどの一定の条件下に入手 が可能であるため、万人が実験を自由に行うことはできない状況がある。し力もながら 、ダム一ノレら、 Nat. Biotech. , published online, 19 October 2006， doi: 10.1038/nbtl2 59.においてダムールらがヒト ES細胞からインスリン分泌細胞が誘導できることを、ま た、成体の瞵島から分離されたネスチン陽性の幹細胞をァクチビン Aと HGFな!/、し はァクチビン Aとベータセルリンないしは、ニコチン酸アミドとエタセンディンー4の存 在下で培養することでラットおよびヒトで同程度に種を問わずインスリン分泌細胞へ誘 導できることを報告している事実を鑑みると、いずれの種由来の ES細胞、特にヒト由 来の ES細胞を使用しても、本発明にしたがってインスリン分泌細胞へ分化誘導する ことができることは当業者に明らかである（Zulewski H， Abraham EJ， Gerlach MJ, Dan iel PB， Moritz W， Muller B， Vallejo M， Thomas M , Habener JF. Multipotential nes tin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001 ;50(3):521_3 3参照)。  [0047] In Japan, human ES cell research has been designated as a National BioResource Project, and human ES cells established at the Center for Stem Cell Medicine, Kyoto University Research Institute for Regenerative Medicine are In accordance with the ES Cell Distribution Regulations (enforced January 26, 2003), it can be obtained under certain conditions, such as obtaining confirmation (approval) by the Minister of Education, Culture, Sports, Science and Technology. There are situations that cannot be done. However, in Dam Inomre et al., Nat. Biotech., Published online, 19 October 2006, doi: 10.1038 / nbtl2 59., it was confirmed that Damul et al. Can induce insulin-secreting cells from human ES cells. Nestin-positive stem cells isolated from islets are cultured in the presence of activin A and HGF! / Activin A and betacellulin or nicotinamide and ethasendin-4 to the same extent in rats and humans. In light of the fact that it has been reported that it can be induced to insulin-secreting cells regardless of the type, ES cells derived from any species, particularly human-derived ES cells, are induced to differentiate into insulin-secreting cells according to the present invention. It is obvious to those skilled in the art (Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas M, Habener JF. Multipotential nes ti. n-positive stem cells isolated from adult pancreatic islets differentiated ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 2001; 50 (3): 521_3 3).
[0048] iPS細胞は、体細胞に特定の因子を導入することにより作製することができる。マウ ス体細胞に 4つの因子（Oct3/4、 Sox2、 c-Myc, Klf4)をレトロウイルスベクターで導入 することにより、形態や増殖能が ES細胞と類似し、分化多能性も有する iPS細胞を作 製することができる（Takahashi , Yamanaka S.， Cell. 25; 126(4):663_76， 2006·)。ヒト 成人皮膚に由来する体細胞に、マウスと同じ 4因子をレトロウイルスベクターで導入し 、その後、ヒト ES細胞の条件で培養することでヒ HPS細胞も作製することができる（Ta kahashi ., anabe K, Ohnuki , Nanta M, IchisaKa Γ, omoda , YamanaKa Ce 11. 2007 131(5):861-72·)。 [0048] iPS cells can be prepared by introducing specific factors into somatic cells. Mau Introducing four factors (Oct3 / 4, Sox2, c-Myc, Klf4) into the somatic cells using retroviral vectors, iPS cells with similar morphology and proliferative potential to ES cells and pluripotency (Takahashi, Yamanaka S., Cell. 25; 126 (4): 663_76, 2006 ·). Human HPS cells can also be produced by introducing the same 4 factors as mouse into somatic cells derived from human adult skin using retroviral vectors and then culturing under the conditions of human ES cells (Ta kahashi., Anabe K, Ohnuki, Nanta M, IchisaKa Γ, omoda, YamanaKa Ce 11. 2007 131 (5): 861-72 ·).
[0049] このように、 iPS細胞は、受精卵を使用する必要がなぐ倫理面の問題が少ないた め、好ましく用いること力 Sできる。またこれらの細胞は、疾患の原因の解明や新治療薬 の開発に大きく寄与する可能性が高い。  [0049] As described above, iPS cells can be preferably used because they have few ethical problems that do not require the use of fertilized eggs. In addition, these cells are highly likely to contribute greatly to elucidating the cause of diseases and developing new therapeutic drugs.
[0050] 本発明の細胞の培養方法において用いられる培地は、細胞を増殖させることがで きるものであればいかなる組成のものでもよぐ無機質、糖、アミノ酸、ペプチド、ビタミ ン、有機酸、核酸、 pH調整剤、酵素等の細胞の培養に必要な成分を含有するもの であればよい。  [0050] The medium used in the cell culturing method of the present invention may be of any composition, as long as it can grow cells, minerals, sugars, amino acids, peptides, vitamins, organic acids, nucleic acids. Any component that contains components necessary for cell culture, such as a pH adjuster and an enzyme, may be used.
[0051] 本発明において、 ES細胞の分化誘導培養に用いる容器としては、分化誘導能、機 能発現能、および生存能等の点から生体適合性材料を用いたスキヤフォールドでコ ートされた培養皿を使用するのが好ましい。該スキヤフォールドとしては、ラミニン、フ イブロネクチン、コラーゲン（コラーゲンタイプ IVやコラーゲンタイプ Iなど）、ジエラチン 、ェンタクチン、ポリオル二チン、 自己組織化能を有するペプチドハイド口ゲル、 Poly (p - N - vinylbenzyl - D - lactonamide) (PVLA) (ラタトースを側鎖にもつポリス チレン誘導体）などが挙げられる。市販品としてはマトリゲル (Matrigel、ラミニン 56 %、コラーゲンタイプ IV31 %、ェンタクチン 8%からなる、ベタトン ディッキンソン ァ ンド カンパニー製）、グロースファクターリデューストマトリゲル（GFR Matrigel、ラミ ニン 61 %、コラーゲン IV30%、ェンタクチン 7%からなる、ベタトン ディッキンソン アンド カンパニー製）、ピュラマトリックス（PuraMatrix、アミノ酸 16残基 (Ac— (RA DA) CONH )で長さが約 5nmのオリゴペプチドであるペプチドハイド口ゲル、株[0051] In the present invention, the vessel used for ES cell differentiation induction culture was coated with a skiafold using a biocompatible material in terms of differentiation induction ability, function expression ability, viability, and the like. It is preferred to use a culture dish. Such skifolds include laminin, fibronectin, collagen (collagen type IV and collagen type I, etc.), dielatin, enteractin, polyornithine, self-assembling peptide-hide mouth gel, Poly (p-N-vinylbenzyl- D-lactonamide) (PVLA) (polystyrene derivative having latatose in the side chain). Commercially available products include Matrigel (Matrigel, 56% laminin, collagen type IV31%, Entactin 8%, Betaton Dickinson Fund Company), Growth Factor Reduced Matrigel (GFR Matrigel, Laminin 61%, Collagen IV 30%, Entactin 7%, Betaton Dickinson & Company, Pura Matrix (PuraM a tri x , amino acid 16 residues (Ac— (RA DA) CONH), a peptide-hide mouth gel, a strain of about 5 nm long oligopeptide, strain
4 2 4 2
式会社スリ一 'ディー'マトリックス ·ジャパン製）、 Poly (p - N -vinylbenzyl— D— la ctonamide) (PVLA) (有限会社セラジッタス製）などが挙げられる。 ES細胞から胚 体内胚葉への分化誘導培養する工程（工程 (a) )においては、コラーゲンタイプ IVも しくはマトリゲルで加工した培養皿が最も好ましぐ次いでフイブロネクチン、ジエラチ ン、ラミニン、非加工の順で好ましい。胚体内胚葉を原始瞵へ分化させる工程（工程（ b) )においては、コラーゲンタイプ IVもしくはマトリゲルで加工した培養皿が最も好ま しぐ次いでフイブロネクチン、ジエラチン、ラミニン、非加工の順が好ましい。原始瞵 力、らニューロジェニン 3発現細胞を得る工程（工程 (c) )においては、ラミニンで加工し た培養皿が最も好ましぐ次いでマトリゲル、フイブロネクチン、コラーゲンタイプ IV、 ジエラチン、非加工の順が好ましい。ニューロジェニン 3発現細胞からインスリン分泌 細胞へ分化させる工程（工程 (d) )においては、マトリゲル加工培養皿が最も好ましく 、ポリオル二チン（polyornitine)、フイブロネクチン、ラミニン、コラーゲンタイプ IV、 ジエラチン、非加工の順が好ましい。 Examples include the company Suriichi 'D' Matrix Japan), Poly (p-N-vinylbenzyl—D-lactonamide) (PVLA) (manufactured by Sera Jitters). ES cell to embryo In the step of inducing differentiation culture into the endoderm (step (a)), a culture dish processed with collagen type IV or matrigel is most preferred, followed by fibronectin, dielatin, laminin, and non-processed order. In the step of differentiating definitive endoderm into primitive gills (step (b)), a culture dish processed with collagen type IV or matrigel is most preferred, followed by fibronectin, dielatin, laminin, and non-processed order. In the process of obtaining neurogenin 3 expressing cells (step (c)), the culture dish processed with laminin is the most preferred, followed by matrigel, fibronectin, collagen type IV, dielatin, and non-processed order. preferable. In the step of differentiation from neurogenin 3 expressing cells into insulin-secreting cells (step (d)), matrigel processed culture dishes are most preferred, polyornitine, fibronectin, laminin, collagen type IV, dielatin, unprocessed Order is preferred.
本発明の ES細胞から胚体内胚葉への分化誘導培養する工程（工程 (a) )において 用いられる培地としては、たとえば、（1)ホルモン成長因子を添加した無血清細胞培 養用培地、（2) ES分化誘導培地（DMEM F12)〔DMEM (インビトロジェン社製） と栄養素混合物ハム F— 12 (インビトロジヱン社製）が 1： 1容量比の割合で混合され、 4. 5mg/mL濃度のグルコース、 20%FBS、 2mmol/L グルタミン、 25mmol/ L HEPES (インビトロジェン社製）、 100mg/mLペニシリン、 100mg/mLストレフ。 トマイシン（シグマ アルドリッチ コーポレーション製）を加え、 LIFを加えないもの〕、 (3)霊長類 ES細胞用培地（ (株)リブロセル製など）、 (4)マウス胚性線維芽細胞用馴 化培地 (R&Dシステム製など）などが挙げられる。なかでもホルモン成長因子を添カロ した無血清細胞培養用培地が好ましぐ無血清細胞培養用培地としては、 RPMI16 40培地（シグマ社製）、 DMEM培地（インビトロジェン社製）、ハム F— 12培地（イン ビトロジェン社製）などが挙げられ、 RPMI1640培地、 DMEM培地およびハム F—1 2培地の混合成分から構成される混合培地が好まし!/、。ホルモン成長因子としては、 インスリン、トランスフェリン、亜セレン酸ナトリウム、エタノールァミンなどが挙げられ、 インスリン、トランスフェリン、亜セレン酸ナトリウムおよびエタノールアミンを全て添加 すること力 S好ましい。市販されている好ましい培地としては、エス'クロン（S— Clone) 培地（三光純薬株式会社製）が挙げられ、造血幹細胞研究用無血清基本培地エス- クロン SF— 03培地（三光純薬株式会社製）を用いることが好まし!/、。培養時の細胞 濃度は 1 X 105〜4 X 105細胞/ mLであることが好ましぐ 1. 75 X 105細胞/ mLで あることが最も好ましい。培養温度は 22〜37°Cの範囲が好ましぐ pHは 7. 2〜7. 4 の範囲が好ましい。工程 (a)の培養期間は 1〜7日程度である。 As a medium used in the step of inducing differentiation culture from ES cells of the present invention into definitive endoderm (step (a)), for example, (1) a medium for serum-free cell culture supplemented with hormone growth factor, (2 ) ES differentiation induction medium (DMEM F12) [DMEM (manufactured by Invitrogen) and nutrient mixture Ham F-12 (manufactured by Invitrogen) are mixed at a ratio of 1: 1 volume ratio, 4.5 mg / mL glucose, 20 % FBS, 2 mmol / L glutamine, 25 mmol / L HEPES (manufactured by Invitrogen), 100 mg / mL penicillin, 100 mg / mL stref. Tomycin (with Sigma-Aldrich Corporation) but without LIF), (3) Primate ES cell medium (such as Librocell), (4) Conditioned medium for mouse embryonic fibroblasts (R & D) System-made). Among these, serum-free cell culture media supplemented with hormone growth factors are preferred as serum-free cell culture media: RPMI 1640 medium (Sigma), DMEM medium (Invitrogen), Ham F-12 medium. (Invitrogen) is preferred, and a mixed medium composed of mixed components of RPMI1640 medium, DMEM medium and Ham F-12 medium is preferred! /. Examples of hormone growth factors include insulin, transferrin, sodium selenite, ethanolamine and the like. It is preferable to add all of insulin, transferrin, sodium selenite and ethanolamine. A preferred commercially available medium is S-Clone medium (manufactured by Sanko Junyaku Co., Ltd.), a serum-free basic medium for hematopoietic stem cell research. It is preferable to use Cron SF-03 medium (Sanko Junyaku Co., Ltd.)! The cell concentration during culture is preferably 1 × 10 5 to 4 × 10 5 cells / mL, and most preferably 1.75 × 10 5 cells / mL. The culture temperature is preferably in the range of 22 to 37 ° C. The pH is preferably in the range of 7.2 to 7.4. The culture period in step (a) is about 1 to 7 days.
[0053] 前記工程 (a)に用いる培地には、分化誘導因子、細胞増殖因子などを加えることが 好ましい。たとえば、ァクチビンおよび/または泉維芽細胞成長因子を加えることが 好ましぐァクチビンおよび泉維芽細胞成長因子の両方を加えることがとくに好ましい 。ァクチビンとしては、ァクチビン A、ァクチビン B、ァクチビン AB等が挙げられ、好ま しいものとしてァクチビン Aが挙げられる、次いでァクチビン AB、ァクチビン Bである。 培地に加える濃度としては、ァクチビンでは 2〜200ng/mL、線維芽細胞成長因子 では 5〜20ng/mLであることが好ましぐァクチビンでは 5〜； 150ng/mL、線維芽 細胞成長因子では 10〜20ng/mLであることがさらに好ましぐァクチビンでは 10 〜； 100ng/mL、線維芽細胞成長因子では 20ng/mLであることが最も好ましい。 線維芽細胞増殖因子の濃度が前記範囲未満の場合、細胞が死滅する傾向があり、 一方前記範囲を超える場合、中胚葉に分化する傾向がある。さらに前記培地には、 ゥシ胎仔血清、ヒト血清などの血清および血清アルブミンを添加することが好ましい。 たとえば、 0. 2〜3%のゥシ胎仔血清および 2〜3%ゥシ血清アルブミンを添加するこ と力 S好ましく、 0. 2%のゥシ胎仔血清および 2. 5%ゥシ血清アルブミンを添加した培 地で 6〜24時間培養後、 1 %のゥシ胎仔血清および 2. 5%ゥシ血清アルブミンを添 加した培地で 6〜48時間培養し、次!/、で 3%のゥシ胎仔血清および 2. 5%ゥシ血清 アルブミンを添加した培地で 12〜96時間培養後することがより好ましぐ 0. 2%のゥ シ胎仔血清および 2. 5 %ゥシ血清アルブミンを添加した培地で 12〜 24時間培養後 、 1 %のゥシ胎仔血清および 2. 5%ゥシ血清アルブミンを添加した培地で 24〜48時 間培養し、次!、で 3%のゥシ胎仔血清および 2. 5%ゥシ血清アルブミンを添加した培 地で 48〜96時間培養することが最も好ましい。  [0053] It is preferable to add a differentiation-inducing factor, a cell growth factor, or the like to the medium used in the step (a). For example, it is particularly preferred to add both activin and / or fountain fibroblast growth factor, where it is preferred to add activin and / or fountain fibroblast growth factor. Examples of the activin include activin A, activin B, and activin AB. Preferred examples include activin A, and then activin AB and activin B. Concentrations added to the medium are 2 to 200 ng / mL for activin, 5 to 20 ng / mL for fibroblast growth factor, preferably 5 to for activin; 10 to 150 ng / mL for fibroblast growth factor More preferably 10 to 20 ng / mL for activin, most preferably 20 ng / mL for fibroblast growth factor. When the concentration of the fibroblast growth factor is less than the above range, the cell tends to die, whereas when it exceeds the above range, it tends to differentiate into a mesoderm. Furthermore, it is preferable to add serum such as rabbit fetal serum and human serum and serum albumin to the medium. For example, it is preferable to add 0.2 to 3% urine fetal serum and 2 to 3% urinary serum albumin. After culturing for 6 to 24 hours in the added medium, incubate for 6 to 48 hours in a medium supplemented with 1% urine fetal serum and 2.5% urine serum albumin. It is more preferable to culture for 12 to 96 hours in medium supplemented with fetal serum and 2.5% urine serum albumin 0.2% urinary fetal serum and 2.5% urine serum albumin added After culturing for 12 to 24 hours in the same medium, incubate for 24 to 48 hours in medium supplemented with 1% ushi fetal serum and 2.5% ushi serum albumin. Most preferably, the cells are cultured for 48 to 96 hours in a medium supplemented with 2.5% ushi serum albumin.
[0054] 本発明の胚体内胚葉を原始瞵へ分化させる工程（工程 (b) )において用いる培地と しては、たとえば、ヒト細胞株を無血清細胞培養用培地で培養した馴化培地（コンデ イシヨンド メディウム（CM)とも言う）が挙げられる。ヒト細胞株としては、ヒト神経膠芽 細胞腫由来細胞株 T98G (ATCC番号、 CRL— 1690)、神経外胚葉性腫瘍細胞株 (human neuroectodermal tumor cell lineノ SK—— PN—— DW (ATCC^号、 CRL—— 2丄 39)などが挙げられ、ヒト神経芽種由来細胞株としては、たとえば、以下のものが利用 可能である（いずれも、 ATCC (米国）から入手可能である。ホームページは、 www.at cc.org参照)。 [0054] Examples of the medium used in the step of differentiating definitive endoderm of the present invention into primitive pods (step (b)) include, for example, a conditioned medium (conditioning medium) obtained by culturing a human cell line in a serum-free cell culture medium. Medium (CM)). Human cell lines include human glioblastoma Cell tumor-derived cell line T98G (ATCC number, CRL— 1690), neuroectodermal tumor cell line (SK—— PN—— DW (ATCC ^, CRL—— 2 丄 39), etc. For example, the following cell lines derived from human neuroblastoma can be used (all are available from ATCC (USA); see www.at cc.org for the homepage).
[0055] ヒト神経芽細胞株（embryonal neuroblastoma) SK— N— DZ、 ATCC番号： CRL— 2 149  [0055] Human neuroblastoma SK— N—DZ, ATCC number: CRL— 2 149
ヒト神経芽細胞株 SK— N— AS、 ATCC番号： CRL - 2137  Human neuroblastoma cell line SK— N— AS, ATCC No .: CRL-2137
ヒト神経芽細胞株 SK— N— FI、 ATCC番号： CRL— 2142  Human neuroblastoma cell line SK— N—FI, ATCC number: CRL— 2142
ヒト脳由来；線維芽細胞種 IMR— 32、 ATCC番号： CCL 127  Human brain derived; fibroblast type IMR-32, ATCC number: CCL 127
ヒト神経芽細胞株 CHP - 212, ATCC番号： CRL— 2273  Human neuroblastoma cell line CHP-212, ATCC number: CRL-2273
ヒト神経芽細胞株 SH— SY5Y、 ATCC番号： CRL— 2266  Human neuroblastoma cell line SH—SY5Y, ATCC number: CRL— 2266
ヒト神経芽細胞株 BE (2)—Ml 7、 ATCC番号： CRL— 2267  Human neuroblastoma cell line BE (2) —Ml 7, ATCC number: CRL— 2267
ヒト神経芽細胞株 BE (2)— C、 ATCC番号： CRL— 2268  Human neuroblastoma cell line BE (2) — C, ATCC number: CRL— 2268
ヒト神経芽細胞株 SK— N— BE (2)、 ATCC番号： CRL— 2271  Human neuroblastoma cell line SK— N— BE (2), ATCC number: CRL— 2271
ヒト神経芽細胞株 SK— N— SH、 ATCC番号： HTB - 11  Human neuroblastoma cell line SK—N—SH, ATCC No .: HTB-11
[0056] また、以下の細胞株も利用可能である（Chemiconから入手可能である。ホームぺ ーン (ュ、 www.chemicon.com参照)。 [0056] The following cell lines are also available (available from Chemicon. Home pane (see www.chemicon.com)).
不死化勝間葉細胞株 LT2 (LT2 Immortalized Pancreatic Mesenchymal Cell Line) 、カタログ番号： SCR013  Immortalized mesenchymal cell line LT2 (LT2 Immortalized Pancreatic Mesenchymal Cell Line), catalog number: SCR013
原始瞵間葉細胞株 VIT1 (VIT1 Primary Pancreatic Mesenchymal Cell Line)、カタ ログ番号： SCR014  Primitive mesenchymal cell line VIT1 (VIT1 Primary Pancreatic Mesenchymal Cell Line), catalog number: SCR014
[0057] なかでも、ヒト神経膠芽細胞腫由来細胞株 T98G (ATCC番号、 CRL— 1690)を エス'クロン SF— 03培地で培養した馴化培地を用いることが最も好ましい。馴化培地 作製時の細胞培養濃度は 5 X 105〜1 X 106細胞/ mLであることが好ましぐ 8 X 105 細胞/ mLであることが最も好まし!/、。馴化培地作製時の培養温度は 35〜37°Cの範 囲が好ましぐ pHは 7. 2〜7. 5の範囲が好ましい。培養期間は 3〜5日程度である。 馴化培地を使用する前にはフィルターなどで細胞を除去することが好ましレ、。胚体内 胚葉の培養の細胞濃度は1 105〜8 105細胞/111しでぁることが好ましぐ 1. 75 X 105細胞/ mLであることが最も好ましい。培養温度は 22〜37°Cの範囲が好ましく 、 pHは 7. 2〜7. 5の範囲が好ましい。培養期間は 4〜10日程度である。 [0057] Among them, it is most preferable to use a conditioned medium obtained by culturing human glioblastoma cell line T98G (ATCC number, CRL-1690) in S'cron SF-03 medium. Conditioned medium The cell culture concentration at the time of preparation is preferably 5 x 10 5 to 1 x 10 6 cells / mL, most preferably 8 x 10 5 cells / mL! /. The culture temperature during preparation of the conditioned medium is preferably in the range of 35 to 37 ° C. The pH is preferably in the range of 7.2 to 7.5. The culture period is about 3-5 days. It is preferable to remove the cells with a filter, etc., before using the conditioned medium. Embryonic body The cell concentration in the germ layer culture is preferably 1 10 5 to 8 10 5 cells / 111, most preferably 1.75 × 10 5 cells / mL. The culture temperature is preferably in the range of 22 to 37 ° C, and the pH is preferably in the range of 7.2 to 7.5. The culture period is about 4-10 days.
[0058] 前記工程 (b)に用いる培地には、たとえば、線維芽細胞成長因子および/または レチノイン酸を加えることが好ましぐレチノイン酸および泉維芽細胞成長因子の両方 を加えることがとくに好ましい。培地に加える濃度としては、レチノイン酸では 1〜50 μ mol/L,線維芽細胞成長因子では 5〜20ng/mLであることが好ましぐレチノィ ン酸では 10〜20 μ mol/L、線維芽細胞成長因子では 10〜20ng/mLであること 力さらに好ましぐレチノイン酸では 10 11101/レ線維芽細胞成長因子では 20ng /mLであること力 S最も好ましレ、。線維芽細胞増殖因子の濃度が前記範囲未満の場 合、細胞が死滅する傾向があり、一方前記範囲を超える場合、中胚葉に分化する傾 向がある。 [0058] It is particularly preferable to add both retinoic acid and fountain fibroblast growth factor to the medium used in step (b), for example, it is preferable to add fibroblast growth factor and / or retinoic acid. . The concentration to be added to the medium is 1-50 μmol / L for retinoic acid and 5-20 ng / mL for fibroblast growth factor, 10-20 μmol / L for retinoic acid, and fibroblasts. For cell growth factor it is 10-20 ng / mL. For retinoic acid 10 11101 / re fibroblast growth factor is 20 ng / mL for force S. When the concentration of the fibroblast growth factor is less than the above range, the cell tends to die, whereas when it exceeds the above range, the cell tends to differentiate into mesoderm.
[0059] 本発明の原始瞵からニューロジェニン 3発現細胞を得る工程（工程 (c) )において用 いる培地としては、ヒト細胞株を低グルコース培地で培養した馴化培地が挙げられる 。ヒト細胞株としては、ヒト肝内皮細胞株、ヒト血管内皮細胞株、が挙げられ、たとえば 、以下の細胞株が利用可能である（ScienCell Research Laboratoriesから入手 PJ"目 bであ (http:/ /www.sciencellonline.com/ products/ endothelial. htm^ )ノ 0 [0059] Examples of the medium used in the step (step (c)) of obtaining neurogenin 3-expressing cells from the primitive silkworm of the present invention include a conditioned medium obtained by culturing a human cell line in a low glucose medium. Examples of human cell lines include human liver endothelial cell lines and human vascular endothelial cell lines. For example, the following cell lines can be used (PJ "> b from ScienCell Research Laboratories (http: / / www.sciencellonline.com/ products / endothelial. htm ^) Bruno 0
[0060] カタログ番号 1000 HBMEC (ヒト脳血管内皮細胞（Human Brain Microvascular En dothelial Cells; )  [0060] Catalog No. 1000 HBMEC (Human Brain Microvascular En dothelial Cells;)
カタログ番号 1300 HCPEC (ヒト脳脈絡叢血管内皮細胞（Human Choroid Plexus E ndothelial Cells; )  Catalog Number 1300 HCPEC (Human Choroid Plexus Endothelial Cells;)
カタログ番号 2900 HIMEC (ヒト小腸血管内皮細胞（Human Intestinal Microvascul ar Maothelial Cells^ )  Catalog No. 2900 HIMEC (Human Intestinal Microvascular Maothelial Cells ^)
カタログ番号 6000 HCMEC (ヒト心臓血管内皮細胞（Human Cardiac Microvascula r Endothelial Cells)  Catalog No. 6000 HCMEC (Human Cardiac Microvascula Endothelial Cells)
カタログ番号 6100 HAEC (ヒト大動脈血管内皮細胞（Human Aortic Endothelial C ells) )  Catalog No. 6100 HAEC (Human Aortic Endothelial Cells)
カタログ番号 6530 HREC (ヒト網膜血管内皮細胞（Human Retinal Endothelial Cell s) ) Catalog Number 6530 HREC (Human Retinal Endothelial Cell s))
カタログ番号 8000 HUVEC (ヒト臍帯静脈血管内皮細胞（Human Umbilical Vein E ndothelial Cells; )  Catalog No. 8000 HUVEC (Human Umbilical Vein Endothelial Cells;)
カタログ番号 8010 HUAEC (ヒト臍帯動脈血管内皮細胞（Human Umbilical Artery Endotnelial Cells J )  Catalog No. 8010 HUAEC (Human Umbilical Artery Endotnelial Cells J)
[0061] なかでもヒト肝内皮細胞株またはヒト血管内皮細胞株を用いることが好ましぐ特にヒ ト不死化肝類洞内皮細胞株 TMNT— 1細胞（寄託機関：独立行政法人産業技術総 合研究所 特許生物寄託センター、あて名：日本国茨城県つくば巿東 1丁目 1番地 1 中央第 6 (郵便番号 305— 8566)、寄託日：平成 14年 4月 16日、受託番号： FER M BP— 8017)を用いることが最も好ましい。この TMNT— 1細胞株は、シミアンゥ ィルス 40腫瘍抗原（SV40T)遺伝子発現レトロウイルスベクター SSR # 69およびテ ロメラーゼ遺伝子（hTERT)発現レトロウイルスベクター SSR # 197を使用し、正常ヒ ト成人肝類洞内皮細胞を不死化して樹立した分化能を有する肝類洞内皮細胞株で ある。馴化培地作製時の細胞濃度は 5 X 105〜； 10 X 105細胞/ mLであることが好ま しぐ 8 X 105細胞/ mLであることが最も好ましい。馴化培地作製時の培養温度は 35 〜37°Cの範囲が好ましぐ pHは 7· 2〜7. 5の範囲が好ましい。培養期間は 3〜5日 程度である。馴化培地を使用する前にはフィルターなどで細胞を除去することが好ま しい。 [0061] In particular, it is preferable to use a human hepatic endothelial cell line or a human vascular endothelial cell line. Particularly, a human immortalized hepatic sinusoidal endothelial cell line TMNT-1 cell (deposited organization: National Institute of Advanced Industrial Science and Technology) Patent Biological Depositary Center, Address: Tsukuba Sakai Higashi 1-chome, 1-chome, 1-chome, Ibaraki, Japan 1st post (305-8566), date of deposit: April 16, 2002, deposit number: FER M BP—8017 ) Is most preferred. This TMNT-1 cell line uses the Simian virus 40 tumor antigen (SV40T) gene-expressing retroviral vector SSR # 69 and the telomerase gene (hTERT) -expressing retroviral vector SSR # 197, and is used in normal human adult hepatic sinusoidal endothelium. It is a hepatic sinusoidal endothelial cell line that has established differentiation ability by immortalizing cells. Cell concentration at the time of the conditioned medium produced is 5 X 10 5 ~; and most preferably from 10 X 10 5 cells / mL is preferred signaling 8 X 10 5 cells / mL. The culture temperature during preparation of the conditioned medium is preferably in the range of 35 to 37 ° C. The pH is preferably in the range of 7.2 to 7.5. The culture period is about 3-5 days. It is preferable to remove the cells with a filter before using the conditioned medium.
[0062] ニューロジェニン 3 (Ngn3)プロモーター下に eYFP (enhanced yellow fluorescence  [0062] eYFP (enhanced yellow fluorescence) under the neurogenin 3 (Ngn3) promoter
；増強黄色蛍光タンパク質）を一過性に発現するプラスミドベクター ngn3— Spel— e YFP (以下の論文から当該プラスミドベクターは入手可能である。 Mellitzer G， Martin M, Sidhoum-Jenny M, Orvainし， Barths J, Seymour PA, Sander M, radwohi G. Pa ncreatic islet progenitor cells in neurogenin 3-yellow fluorescent protein knock-add -on mice. Mol Endocrinol. 18(11):2765_76· 2004)を導入する時の細胞濃度は 100 X 105〜500 X 105細胞/ mLであることが好ましぐ 100 X 105〜200 X 105細胞/ mLであること力 S最も好ましレ、。遺伝子導入時の培養温度は 35〜37°Cの範囲が好ま しぐ pHは 7. 2〜7. 5の範囲が好ましい。導入期間は 1〜5秒程度である。遺伝子導 入および選別後の培養における細胞濃度は 1 X 104〜1 X 105細胞/ mLであること が好ましぐ 3 104〜8 104細胞/111しでぁることがさらに好ましぐ 5 X 104細胞/ mLであること力 S最も好ましい。培養温度は 35〜37°Cの範囲が好ましぐ pHは 7. 2 〜7. 5の範囲が好ましい。培養期間は 3〜10日程度である。 A plasmid vector that transiently expresses an enhanced yellow fluorescent protein ngn3—Spel—e YFP (the plasmid vector is available from the following paper: Mellitzer G, Martin M, Sidhoum-Jenny M, Orvain, Barths J, Seymour PA, Sander M, radwohi G. Pancreatic islet progenitor cells in neurogenin 3-yellow fluorescent protein knock-add -on mice.Mole Endocrinol. 18 (11): 2765_76 100 X 10 5 ~500 X 10 5 cells / that it is mL is preferred instrument 100 X 10 5 ~200 X 10 5 cells / mL force S most preferred les. The culture temperature during gene introduction is preferably in the range of 35 to 37 ° C. The pH is preferably in the range of 7.2 to 7.5. The introduction period is about 1 to 5 seconds. Cell concentration in the culture after gene transfer and selection should be between 1 X 10 4 and 1 X 10 5 cells / mL 3 10 4 to 8 10 4 cells / 111 is more preferred 5 X 10 4 cells / mL is the most preferred force S. The culture temperature is preferably in the range of 35 to 37 ° C. The pH is preferably in the range of 7.2 to 7.5. The culture period is about 3-10 days.
[0063] 前記工程 (c)に用いる馴化培地には、細胞増殖因子などを加えることが細胞の分 化誘導効率を考慮すると好ましい。たとえば、上皮細胞増殖因子および/または線 維芽細胞成長因子を加えることが好ましぐ上皮細胞増殖因子および線維芽細胞成 長因子の両方を加えることがとくに好ましい。培地に加える濃度としては、上皮細胞 増殖因子では 5〜50ng/mL、線維芽細胞成長因子では 5〜50ng/mLであること が好ましぐ上皮細胞増殖因子では 10〜50ng/mL、線維芽細胞成長因子では 10 〜50ng/mLであることがさらに好ましぐ上皮細胞増殖因子では 20ng/mL、線維 芽細胞成長因子では 20ng/mLであることが最も好まし!/、。線維芽細胞増殖因子の 濃度が前記範囲未満の場合、細胞の増殖が減弱する傾向がある。さらに前記馴化 培地には、 γ—セクレターゼインヒビター XVIIIを 0· 25〜2 11101/し添加することが 好ましく、 0. 5〜； ! · 5 mol/L添加することがより好ましぐ 1 mol/L添加すること が最も好ましい。 Ίーセクレターゼインヒビター XVIIIを添加せずに培養を行うと、細 胞の生存率が低下する傾向がある。  [0063] It is preferable to add a cell growth factor or the like to the conditioned medium used in the step (c) in consideration of cell differentiation induction efficiency. For example, it is particularly preferred to add both epidermal growth factor and fibroblast growth factor, for which it is preferred to add epidermal growth factor and / or fibroblast growth factor. Concentrations added to the medium are 5-50 ng / mL for epidermal growth factor and 5-50 ng / mL for fibroblast growth factor, 10-50 ng / mL for epidermal growth factor, fibroblasts For growth factors, 10-50 ng / mL is even more preferred, with epidermal growth factor being 20 ng / mL, and for fibroblast growth factor, 20 ng / mL is most preferred! When the concentration of fibroblast growth factor is less than the above range, cell proliferation tends to be attenuated. Furthermore, it is preferable to add γ-secretase inhibitor XVIII to the conditioned medium in the range of 0 · 25 to 2 11101 /, more preferably in the range of 0.5 to 5 mol / L. 1 mol / L Most preferably, it is added. When cultured without the addition of 培養 -secretase inhibitor XVIII, cell viability tends to decrease.
[0064] 本発明のニューロジェニン 3発現細胞からインスリン分泌細胞へ分化させる工程（ェ 程（d) )において用いる培地としては、ヒト瞵島を低グルコース培地で培養した馴化培 地、 RPMI1640培地、 William' s E培地、 CMRL1066培地（インビトロジェン社製 )、低グルコース DMEM培地（インビトロジェン社製）、などが挙げられ、インスリン分 泌細胞の疲弊を防ぐ点から、低グルコース培地を用いることが好ましい。インスリン分 泌細胞のグルコース応答性を高める点から、低グルコース培地を用いた培養中、血 清無添加の高グルコース培養培地に毎日;!〜 3回、それぞれ約 2時間（限定されない 、 1時間〜 3時間が好ましぐ 1時間半力 2時間半が更に好ましい。）刺激培養す ること力 り好ましい。細胞培養濃度は 1 X 105〜； 10 X 105細胞/ mLであることが好 ましぐ 5 105〜7 105細胞/111しでぁることが最も好ましぃ。培養温度は 22〜37 °Cの範囲が好ましぐ pHは 7· 2〜7. 5の範囲が好ましい。培養期間は 1〜10日であ る事が好ましぐ 5〜7日であることが最も好ましい。 [0065] 前記工程 (d)に用いる培地には、肝細胞増殖因子、ニコチンアミド、硫酸亜鉛、トロ グリタゾン（troglitazone)、ェタスディン 4 (exendin-4)の 1種または 2種以上加えるこ とが好ましぐ全種類加えることがとくに好ましい。培地に加える濃度としては、肝細胞 増殖因子では 2〜20ng/mL、ニコチンアミドでは;!〜 10mmol/L、硫酸亜鉛では ；!〜 20 n mol/L、トログリタゾンでは 1〜10 μ mol/L、ェクスディン 4では 5〜100n mol/Lであることが好ましぐ肝細胞増殖因子では 2〜； 10ng/mL、ニコチンアミド では 5〜； 10mmol/L、硫酸亜鉛では 10〜20 mol/L、トログリタゾンでは 5〜10 H mol/L,ェクスディンー4では 10〜100nmol/Lであることがさらに好ましぐ肝 細胞増殖因子では 10ng/mL、ニコチンアミドでは 10mmol/L、硫酸亜鉛では 16 . 7 n mol/L,トログリタゾンでは 10〃11101/レェクスディン一 4では 100nmol/L であることが最も好ましい。さらに低グルコース培地には、ゥシ胎仔血清、ヒト血清など の血清を添加することが好ましぐ 3〜5%のゥシ胎仔血清を添加することが好ましい 。 3%のゥシ胎仔血清を添加した培地で 2〜4日間培養後 5%のゥシ胎仔血清を添カロ した培地で 3〜5日間培養することがより好ましぐ 3%のゥシ胎仔血清を添加した培 地で 3日間培養後 5%のゥシ胎仔血清を添加した培地で 4日間培養することが最も好 ましい。 [0064] As a medium used in the step of differentiating neurogenin 3 expressing cells of the present invention into insulin-secreting cells (step (d)), a conditioned medium obtained by culturing human Kashiwajima in a low glucose medium, RPMI1640 medium, William s E medium, CMRL1066 medium (manufactured by Invitrogen), low glucose DMEM medium (manufactured by Invitrogen), and the like. From the viewpoint of preventing exhaustion of insulin-producing cells, it is preferable to use a low glucose medium. From the point of increasing glucose responsiveness of insulin-secreting cells, during culture using low glucose medium, daily in high glucose culture medium without serum;! ~ 3 times, each about 2 hours (not limited, 1 hour ~ 3 hours is preferred 1 hour and a half force 2 hours and a half is more preferred.) Stimulated culture is preferred. Cell culture concentration 1 X 10 5 ~; 10 X 10 5 cells / be mL virtuous Mashigu 5 10 5-7 10 5 cells / 111 Shidearu most Konomashii. The culture temperature is preferably in the range of 22 to 37 ° C. The pH is preferably in the range of 7.2 to 7.5. The culture period is preferably 1 to 10 days, and most preferably 5 to 7 days. [0065] It is preferable to add one or more of hepatocyte growth factor, nicotinamide, zinc sulfate, troglitazone, and etasdin-4 (exendin-4) to the medium used in the step (d). It is particularly preferable to add all types. Concentrations added to the medium include 2-20 ng / mL for hepatocyte growth factor, !!-10 mmol / L for nicotinamide,! -20 nmol / L for zinc sulfate, 1-10 μmol / L for troglitazone, Exodine 4 is preferred to be 5 to 100 nmol / L for hepatocyte growth factor 2 to 10 ng / mL, nicotinamide 5 to 10 mmol / L, zinc sulfate 10 to 20 mol / L, troglitazone 5 to 10 H mol / L, 10 to 100 nmol / L is more preferable for Exdin-4, 10 ng / mL for hepatocyte growth factor, 10 mmol / L for nicotinamide, and 16.7 nmol / L for zinc sulfate For troglitazone, 10 ~ 11101 / Lexdin-1 is most preferably 100 nmol / L. Furthermore, it is preferable to add serum such as urchin fetal serum and human serum to the low glucose medium. It is preferable to add 3 to 5% urine fetal serum. It is more preferable to culture for 3 to 5 days in a medium supplemented with 5% ushi fetal serum after culturing for 2 to 4 days in a medium supplemented with 3% urchin fetal serum. It is most preferable to culture for 4 days in a medium supplemented with 5% urchin fetal serum after culturing for 3 days in a medium supplemented with.
[0066] 工程 (d)において、細胞を刺激するときに用いる高グルコース培地には、高ダルコ ース DMEM培地などをそのまま用いることもできる力 高グルコース DMEM培地な どにさらに脂質、アミノ酸、ビタミンおよび/またはミネラルを添加して用いることが好 ましぐ高グルコース DMEM培地に脂質、アミノ酸、ならびにビタミンおよびミネラル を添加して用いることが最も好まし!/、。  [0066] In the step (d), the high glucose medium used for stimulating the cells can be a high-dulose DMEM medium or the like. The high glucose DMEM medium and the like can further contain lipids, amino acids, vitamins and the like. It is most preferable to use high glucose DMEM medium with addition of lipids, amino acids, vitamins and minerals.
[0067] 工程 (d)において、馴化培地作製に使用するヒト瞵島は、健常分離ヒト瞵島を不死 化し増殖した後に不死化遺伝子を除去して得ることが好ましい。健常分離ヒト瞵島は 、公知の方法に従ってヒト瞵臓から分離することができる（Staudacher C， Ricordi C， S tella M, Socciし， Cammelli L, Ferrari , Oicarlo V. Minerva Chir. 31 : 1665-1668， 1 985または Ricordi C， Finke EH, Lacy ΡΕ·， Diabetes 35: 649-653， 1986参照）。不 死化ヒト瞵島は、一対の部位特異的組換え配列に挟まれた不死化遺伝子をベクター により瞵島に導入することにより作製することができる。部位特異的組換え配列として は、 LoxP配列や FRT配列などが挙げられ、不死化遺伝子としては、 DNA型腫瘍ゥ ィルスの腫瘍抗原 (T抗原）遺伝子である SV40T遺伝子およびヒトテロメラーゼ逆転 写酵素 (hTERT)遺伝子などがあげられる。不死化遺伝子導入ベクターとしては、 S V40T遺伝子を含有する pYK—lベクターが挙げられ、当該ベクター、ベクター導入 方法、 SV40T遺伝子の切り出し方法は当業者であれば、本発明者の一人である小 林らの手法：国際公開第 2005/100546号パンフレットおよび/または Miki A, Nar ushima M, Okitsu , Ί akeno Y, soto— Gutierrez A, Rivas—し arrillo JD, Navarro-Alva rez N, Chen Y, Tanaka , Noguchi H, Matsumoto S, ohara M,Lakey JR, obayash i E, Tanaka N, obayashi N. Maintenance of mouse, rat, and pig pancreatic islet fun ctions by coculture with human islet-derived fibroblasts. Cell Transplant. 2006; 15(4) :325-34.を参照し作製することができる。 [0067] In step (d), it is preferable that the human islet used for preparation of the conditioned medium is obtained by immortalizing and proliferating healthy isolated human islet and then removing the immortalized gene. Healthy isolated human islets can be isolated from human spleen according to known methods (Staudacher C, Ricordi C, Stela M, Socci, Cammelli L, Ferrari, Oicarlo V. Minerva Chir. 31: 1665-1668 1985 or Ricordi C, Finke EH, Lacy IV, Diabetes 35: 649-653, 1986). Immortalized human Kashiwajima can be prepared by introducing an immortalized gene sandwiched between a pair of site-specific recombination sequences into Kashiwajima using a vector. As a site-specific recombination sequence Examples of such an immortalized gene include the SV40T gene, which is a tumor antigen (T antigen) gene of a DNA type tumor virus, and the human telomerase reverse transcriptase (hTERT) gene. Examples of the immortalized gene transfer vector include pYK-1 vectors containing the SV40T gene, and those skilled in the art will know Kobayashi who is one of the present inventors if the vector, vector transfer method, and SV40T gene excision method are known to those skilled in the art. Et al .: WO 2005/100546 pamphlet and / or Miki A, Narushima M, Okitsu, Ί akeno Y, soto—Gutierrez A, Rivas— and arrillo JD, Navarro-Alva rez N, Chen Y, Tanaka, Noguchi H, Matsumoto S, ohara M, Lakey JR, obayash i E, Tanaka N, obayashi N. Maintenance of mouse, rat, and pig pancreatic islet fun ctions by coculture with human islet-derived fibroblasts.Cell Transplant. 2006; 15 ( 4) Can be prepared with reference to: 325-34.
[0068] 高グルコース培地とは、 RPMI1640培地、 DMEM培地、ハム F— 12培地などの 無血清細胞培養用培地中に含まれるグルコース濃度が 3600mg/L〜4500mg/ Lである培地を示し、低グルコース培地とは、無血清細胞培養用培地中に含まれるグ ルコース濃度が 600mg/L〜1000mg/Lである培地を示す。好ましい高ダルコ一 ス培地および低グルコース培地としては、それぞれ高グルコース DMEM培地（インビ トロジェン社製）ならびにヒト瞵島を低グルコース培地で培養した馴化培地および低グ ルコース DMEM培地（インビトロジェン社製）がそれぞれ挙げられる。  [0068] The high glucose medium refers to a medium having a glucose concentration of 3600 mg / L to 4500 mg / L in a serum-free cell culture medium such as RPMI1640 medium, DMEM medium, Ham F-12 medium, etc. The medium means a medium having a glucose concentration of 600 mg / L to 1000 mg / L contained in the serum-free cell culture medium. Preferred high-Dalcos medium and low-glucose medium are high-glucose DMEM medium (manufactured by Invitrogen) and conditioned medium obtained by cultivating human islets in low-glucose medium and low-glucose DMEM medium (manufactured by Invitrogen), respectively. Can be mentioned.
[0069] 本発明の分化誘導工程それぞれで用いる線維芽細胞増殖因子（FGF)の好まし!/ヽ 例としては、 FGF— 2、 FGF— 5などが挙げられ、好ましくない例としては、 FGF— 10 が挙げられる。  [0069] Preferable examples of fibroblast growth factor (FGF) used in each differentiation induction step of the present invention include FGF-2, FGF-5, and the like. 10 is mentioned.
[0070] 用語「インスリン分泌細胞」とは、インスリンを合成し、細胞外へ分泌する能力を有す る細胞を意味する。  [0070] The term "insulin-secreting cell" means a cell having the ability to synthesize insulin and secrete it outside the cell.
[0071] 用語「胚体内胚葉」とは英語では definitive endodermと言い、マウスの 3. 5日胚 は胚盤胞と呼ばれ、外側を包む栄養外胚葉と将来体をつくる内部細胞塊に区別され る。内部細胞塊はまだ未分化な状態だが、原腸形成によって内胚葉、中胚葉および 外胚葉へと最初の分化を遂げる。内胚葉の分化は少々複雑で、将来胚に取り込まれ る胚体内胚葉（Definitive Endoderm)と、胚体外内胚葉をつくる臓側内胚葉（Visceral Endoderm)に分けられる。興味深いことに、胚体内胚葉の一部は、中内胚葉（Mesen doderm)と呼ばれる内胚葉と中胚葉の共通の前駆細胞に由来している（http：〃 www. cdb.riKen.jp/jp/04_news/ articles/pdf/05120 l.nishikawa.mesendoderm.pdf^ )。 を意味する。 [0071] The term “definitive endoderm” is definitive endoderm in English, and the mouse embryonic day 3.5 is called a blastocyst and is distinguished by the outer vegetative ectoderm and the inner cell mass that forms the future body. The Although the inner cell mass is still undifferentiated, it undergoes initial differentiation into endoderm, mesoderm and ectoderm by gastrulation. Differentiation of the endoderm is a bit complicated, and the definitive endoderm (Definitive Endoderm), which is incorporated into the embryo in the future, and the visceral endoderm (Visceral), which creates the extraembryonic endoderm. Endoderm). Interestingly, part of the definitive endoderm is derived from a common progenitor cell, called the mesendoderm (Mesen doderm) (http: 〃 www.cdb.riKen.jp/jp/) 04_news / articles / pdf / 05120 l.nishikawa.mesendoderm.pdf ^). Means.
[0072] 用語「原始瞵」とは英語では primitive pancreasと言い、肝芽の少し尾方の腹側 力、ら生じる前腸から発生する腹側勝臓芽 (ventral pancreatic bud)と、中腸の背側か ら発生する背側瞵臓芽（dorsal pancreatic bud)を併せたもの（http：〃 www.ams-jusei. jp/09/amsnews_pdf/2005-10_21.pdf参照）を意味する。  [0072] The term “primordial mushroom” is called primitive pancreas in English. The ventral force of the caudal tail of the liver bud, the ventral pancreatic bud generated from the foregut, and the back of the midgut. It means a combination of dorsal pancreatic buds (http: 〃 www.ams-jusei.jp/09/amsnews_pdf/2005-10_21.pdf).
[0073] 本発明にお!/、て、分化誘導を行なう培養工程には、平面培養と三次元培養の!/、ず れも使用できるが、近年、スキヤフォールド（足場）上で細胞を三次元培養することで より生体内環境に近い条件を作ることができ、細胞機能が向上することが知られてき ており、インスリン分泌能の向上などの点から三次元培養が好ましい。  [0073] In the present invention, in the culture process for inducing differentiation, both planar culture and three-dimensional culture can be used. In recent years, cells have been tertiaryized on a skifold (scaffold). It has been known that conditions that are closer to the in vivo environment can be created by culturing in the original, and the cell function is improved. Three-dimensional culture is preferable from the viewpoint of improving insulin secretion ability.
[0074] 三次元培養に使用するスキヤフォールドとしては、ラミニン、フイブロネクチン、コラ 一ゲン IV、コラーゲン Iなどのコラーゲン、ジエラチン、ェンタクチン、ペプチドハイド口 ゲル、 poly (p-N-vinylbenzyト D-lactonamide) (PVLA)などが挙げられる。市販品と しては、前記マトリゲル（Matrigel)、グロースファクターリデューストマトリゲル（GFR Ma trigel)、ピユラマトリックス（PuraMatrix)などが挙げられる。さらに、後述する不織布な どが使用できる。  [0074] The skiafold used for three-dimensional culture includes collagen such as laminin, fibronectin, collagen IV, collagen I, dielatin, entactin, peptide-hide mouth gel, poly (pN-vinylbenzyto D-lactonamide) (PVLA ) And the like. Examples of commercially available products include Matrigel, Growth Factor Reduced Matrigel, and PuraMatrix. Furthermore, the nonwoven fabric etc. which are mentioned later can be used.
[0075] 本発明の方法により ES細胞より分化誘導された胚体内胚葉、原始瞵、ニューロジ ェニン 3発現細胞およびインスリン分泌細胞は、形態学的特徴の観察および/また は逆転写ポリメラ—ゼ連鎖反応 (RT— PCR)で確認できる。  [0075] The definitive endoderm, primitive gland, neurogenin 3 expressing cells and insulin secreting cells induced to differentiate from ES cells by the method of the present invention are observed for morphological characteristics and / or reverse transcription polymerase chain reaction. (RT—PCR).
[0076] 形態学的な確認方法としては、透過電子顕微鏡検査、インスリン染色などが挙げら れ、細胞に特異的な形態学的特徴を確認することが挙げられる。  [0076] Morphological confirmation methods include transmission electron microscopy, insulin staining, etc., and confirmation of morphological characteristics specific to cells.
[0077] また、 RT— PCRにより、分化誘導された細胞の形質を遺伝子の発現から評価する こともできる。分化誘導の進行を表わす指標として、工程 ωにおいて胚体内胚葉の マーカーである Foxa2 (肝細胞核因子 3 β、 hepatocyte nuclear factor 3， beta)、 Sox 17 (性決定領域丫、 Sex determining region Y)の遺伝子発現が認められる。工程（b) において原始瞵のマーカーである Foxa2、 Soxl 7、 Pdx—1 (瞵転写因子、 pancreat ic transcription factor)の遺伝子発現が認められる。 Shh (ソニックヘッジホッグ）の遺 伝子発現が認められない。 [0077] In addition, by RT-PCR, the characteristics of cells induced to differentiate can be evaluated from gene expression. As an indicator of the progression of differentiation induction, genes for definitive endoderm markers Foxa2 (hepatocyte nuclear factor 3, beta), Sox 17 (sex determining region Y, sex determining region Y) in process ω Expression is observed. Foxa2, Soxl 7, and Pdx—1 (瞵 transcription factors, pancreat ic transcription factor) gene expression. Shh (Sonic hedgehog) gene expression is not observed.
[0078] 本発明に係る ES細胞のインスリン分泌細胞への分化誘導方法によれば、ァクチビ ンを用いて未分化な ES細胞から分化させた胚体内胚葉を、馴化培地を用いてニュ 一ロジェニン 3発現細胞へ分化させたのちに該ニューロジェニン 3発現細胞を高グノレ コース培地で刺激することにより、高!/、分化誘導効率でインスリン分泌細胞を誘導で き、さらにはインスリンを効率よく分泌する細胞の製造が可能となる。本発明の分化誘 導方法においては、分化誘導効率および得られる細胞のインスリン分泌能は、従来 の手法である胚様体を形成する分化誘導方法と比較してそれぞれ 3〜5倍以上およ び 3〜5倍以上となることが好ましぐそれぞれ約 5〜； 10倍以上および約 5〜； 10倍以 上となることがさらに好ましい。  [0078] According to the method for inducing differentiation of ES cells into insulin-secreting cells according to the present invention, definitive endoderm differentiated from undifferentiated ES cells using activin is treated with neurogenin 3 using a conditioned medium. After differentiation into expressing cells, the neurogenin 3 expressing cells can be stimulated with a high-gnosoleic medium to induce insulin secreting cells with high differentiation / induction efficiency, and moreover secrete insulin efficiently. Can be manufactured. In the differentiation-inducing method of the present invention, the differentiation-inducing efficiency and the insulin secretion ability of the cells obtained are 3 to 5 times or more, respectively, compared to the differentiation-inducing method that forms embryoid bodies, which is a conventional technique. It is preferably about 3 to 5 times or more, more preferably about 5 to 10 times or more and about 5 to 10 times or more.
[0079] インスリン分泌能は、培地中に分泌された総タンパク質量 (mg)に対するインスリン 分泌量 (ng)で算出することができ、グルコース刺激後に測定を行いインスリン分泌能 を算出することによりインスリン分泌細胞の機能を評価することができる。  [0079] Insulin secretion capacity can be calculated as the amount of insulin secretion (ng) relative to the total amount of protein secreted into the medium (mg). Measurement is performed after glucose stimulation, and the insulin secretion capacity is calculated by calculating the insulin secretion capacity. Cell function can be evaluated.
[0080] インスリン分泌細胞への分化誘導効率は、分化誘導方法における最終工程に用い るインスリンを分泌しない細胞数 (本発明では、分化誘導方法の工程 (c)で得られ、 工程 (d)に用いるニューロジェニン 3発現細胞数が該当する）に対するインスリン分泌 細胞数で評価することができる。  [0080] The efficiency of inducing differentiation into insulin-secreting cells is determined by the number of cells that do not secrete insulin used in the final step of the differentiation-inducing method (in the present invention, obtained in step (c) of the differentiation-inducing method, and in step (d) It can be evaluated by the number of insulin-secreting cells relative to the number of neurogenin 3 expressing cells used).
[0081] 本発明の分化誘導方法によれば、 40〜60%の分化誘導効率でインスリン分泌細 胞を得ることが可能である。この効率は、ダムールら、 Nat. Biotech., published online, 19 October 2006， doi: 10.1038/nbtl259.に記載された分化誘導効率（3〜； 12%、平 均 7. 3%)と比較するとかなり高い。また、本発明の分化誘導方法により得られたイン スリン分泌細胞は、正常瞵島の 20分の 1から 50分の 1のインスリン分泌能を有する。 この分泌能は、該従来技術により得られたインスリン分泌細胞のインスリン分泌能（正 常瞵島の 100分の 1から 500分の 1)と比較するとかなり高い。  [0081] According to the differentiation induction method of the present invention, it is possible to obtain insulin secreting cells with a differentiation induction efficiency of 40 to 60%. This efficiency is considerably higher than the differentiation induction efficiency (3 to 12%, average 7.3%) described in Damour et al., Nat. Biotech., Published online, 19 October 2006, doi: 10.1038 / nbtl259. high. Insulin-secreting cells obtained by the method for inducing differentiation of the present invention have an insulin secretion ability that is 1/20 to 1/50 of that of normal Kashiwajima. This secretory capacity is considerably higher than the insulin secretory capacity of the insulin-secreting cells obtained by the conventional technique (from 1/100 to 1/500 of the normal island).
[0082] また、インスリン分泌細胞は、 Maf A遺伝子を発現することが好ましい。 MafAはイン スリンプロモーター上に結合する勝臓の β細胞におけるインスリンの分泌に必須であ る転写因子である。 MafAKOノックアウトマウスを作製した研究では、このマウスはラ ンゲルノヽンス島の構築異常や、インスリン分泌異常を示すことから、 MafAは糖代謝 に非常に重要な因子であることが明ら力、となった（ホームページ http：〃 www.md.tsuku ba.ac.jp/basic—med/anatomy/embryology/Maf.html^:照)。本発明の分ィ匕誘導万法 により得られたインスリン分泌細胞は、 MafA遺伝子を発現する能力を有する。これに 対し従来技術であるダムール法で分化誘導されたインスリン分泌細胞は MafA遺伝 子を発現していないことが報告されている (ダムールら、 Nat. Biotech., published onlin e， 19 October 2006， doi: 10.1038/nbtl259.参照）。 MafA遺伝子を発現する能力を 有する細胞であることは、その細胞がグルコース応答性を有していること、インスリン 分泌細胞として成熟していることの指標となる。 [0082] Moreover, it is preferable that the insulin-secreting cells express the Maf A gene. MafA is a transcription factor that is essential for insulin secretion in the vicinal β-cell that binds to the insulin promoter. In studies that created MafAKO knockout mice, MafA is clearly an important factor in glucose metabolism due to abnormal Ngernoens construction and abnormal insulin secretion (http: //www.md.tsukuba. ac.jp/basic—med/anatomy/embryology/Maf.html^: Insulin-secreting cells obtained by the separation induction method of the present invention have the ability to express the MafA gene. On the other hand, it has been reported that insulin-secreting cells induced to differentiate by the conventional Damour method do not express the MafA gene (Damur et al., Nat. Biotech., Published online, 19 October 2006, doi : 10.1038 / nbtl259.) A cell having the ability to express the MafA gene is an indicator that the cell has glucose responsiveness and is mature as an insulin-secreting cell.
[0083] MafAはインスリン分泌細胞がみずから発現していることが好ましいが、当該細胞の 成熟度を高め、グルコース応答性を高めるために、 MafA遺伝子を含有するベクター を工程 (d)において細胞に導入し発現させることで、 MafA遺伝子発現を増強させる こと力 S好まし!/、態様として挙げられる。  [0083] Although it is preferable that MafA is naturally expressed in insulin-secreting cells, a vector containing the MafA gene is introduced into the cell in step (d) in order to increase the maturity of the cell and increase glucose responsiveness. It is possible to enhance the expression of the MafA gene by expressing it.
[0084] また、本発明の分化誘導方法によれば、インスリン分泌細胞のスフエロイド形成（球 形で、直径力 00〜300 ,1 mである）が可能となる。  [0084] Further, according to the differentiation inducing method of the present invention, spheroid formation (spherical, with a diameter force of 00 to 300, 1 m) of insulin-secreting cells is possible.
[0085] 用語「スフエロイド」とは、三次元的球状細胞塊を意味する。スフエロイドを形成およ び/または維持することは、孤立細胞またはいびつなスフエロイドと比較して、前記ス フエロイドの生理機能が生体組織により近いことを示す。  [0085] The term "spheroid" means a three-dimensional spherical cell mass. Forming and / or maintaining spheroids indicates that the spheroid's physiology is closer to living tissue compared to solitary cells or distorted spheroids.
[0086] 培養インスリン分泌細胞の生理機能を検査する方法としては、形態学的検査項目と して下記 4項目を 5段階（1〜5点）評価する方法が挙げられる（文献: Matsumoto Sら 、 Transplantation, 2002;74: 1414·参照）  [0086] As a method for examining physiological functions of cultured insulin-secreting cells, there are methods for evaluating the following four items as morphological examination items in five stages (1 to 5 points) (reference: Matsumoto S et al., (See Transplantation, 2002; 74: 1414)
1.形状 (shape)：  1. Shape:
「扁平 (flat) : 1点」、「ほぼ扁平： 2点」、「いびつな球状： 3点」、「ほぼ球状: 4点」、お よび「球状（spherical) : 5点」の 5段階評価する。この中では、「球状： 5点」が最も好ま しい。  Five-point evaluation: “flat: 1 point”, “almost flat: 2 points”, “loose spherical: 3 points”, “almost spherical: 4 points”, and “spherical: 5 points” To do. Among these, “spherical: 5 points” is most preferable.
2.辺縁の形状（border)：  2.Border shape:
「不ぞろいの（irregular) : 1点」、「ほぼ不ぞろいの： 2点」、「やや均整のとれた： 3点」、 「ほぼ均整のとれた： 4点」、および「均整のとれた（well-rounded)： 5点」の 5段階評価 する。この中では、「均整のとれた： 5点」が最も好ましい。 “Irregular: 1 point”, “Almost uneven: 2 points”, “Slightly balanced: 3 points”, “Almost well balanced: 4 points”, and “Well-balanced (well) -rounded): 5-point rating To do. Among these, “balanced: 5 points” is most preferable.
3.統合性（integrity)：  3. Integrity:
「断片化した (fragmented) : 1点」、「ほぼ断片化した： 2点」、「ややソリッド/コンパク ト： 3 、」、「ほほノリット /コンノヽクト： 4点ヽ」、および「ソリッド/コンノヽクト (solid/ compact ) : 5点」の 5段階評価する。この中では、「ソリッド/コンパクト： 5点」が最も好ましい。  “Fragmented: 1 point”, “substantially fragmented: 2 points”, “slightly solid / compact: 3,”, “hohonorit / connect: 4 points”, and “solid / 5 grades of “solid / compact: 5 points”. Among these, “solid / compact: 5 points” is most preferable.
4.直径 (diameter)：  4. Diameter:
「スフエロイドの個々力 00〃 m未満（allく 100〃 m)： 1点」、「スフエロイドの個々が 100〜； 150〃 m : 2点」、「スフエロイドの個々力 25〜； 175〃 m : 3点」、「スフエロイド の個々力 50〜200 ^ m : 4点」、および「スフエロイドの個々中 10%以上が 200 μ m より大きい（〉10%〉200 m) : 5点」の 5段階評価する。その中では、「スフエロイド の個々中 10%以上が 200 mより大きい： 5点」が最も好ましい。  “Individual force of spheroids less than 00 〃 m (all 〃 100 〃 m): 1 point”, “Individual spheroids from 100 to 150 〃 m: 2 points”, “Individual force of spheroids 25 to 175 〃 m: 3 "Score", "Spheroid individual force 50-200 ^ m: 4 points", and "Spheroids more than 10% of each individual is larger than 200 μm (> 10%> 200 m): 5 points" . Among them, “10% or more of each spheroid is larger than 200 m: 5 points” is most preferable.
5.染色の均一性 (uniformity staining)：  5. Uniformity staining:
「均一ではない（not uniform) : 1点」、「ほぼ均一でない： 2点」、「やや均一： 3点」、「 ほぼ均一： 4点」および「完全に均一（perfectly uniform)： 5点」の 5段階評価する。そ の中では、「完全に均一： 5点」が最も好ましい。  “Not uniform: 1 point”, “not nearly uniform: 2 points”, “slightly uniform: 3 points”, “almost uniform: 4 points” and “perfectly uniform: 5 points” The 5 grades are evaluated. Among them, “completely uniform: 5 points” is most preferable.
[0087] 本発明の分化誘導方法で得られるインスリン分泌細胞からなるスフエロイドは、形態 学的には、形状が 3点以上、辺縁の形状が 3点以上、統合性が 3点以上、直径が 3点 以上、染色の均一性が 3点以上、評価値の合計が 15点以上であることが好ましぐ形 状が 4点以上、辺縁の形状が 4点以上、統合性が 4点以上、直径が 4点以上、染色の 均一性が 4点以上、評価値の合計が 20点以上であることがより好ましぐ形状が 5点 、辺縁の形状が 5点、統合性が 5点、直径が 5点、染色の均一性が 5点、評価値の合 計が 25点であることが最も好ましい。また、本発明の培養方法で得られるインスリン分 泌細胞の機能は、グルコース刺激に対してインスリン分泌が低グルコース時と比べ、 高グルコース時に 1. 5倍以上になることが好ましぐ 2倍以上になることがより好ましく 、 5倍以上になることが最も好ましい。  [0087] The spheroids composed of insulin-secreting cells obtained by the differentiation induction method of the present invention are morphologically 3 points or more in shape, 3 or more in edge shape, 3 or more in integration, and 3 or more in diameter. 3 points or more, dyeing uniformity of 3 points or more, and a total evaluation value of 15 points or more is preferable 4 points or more, edge shape is 4 points or more, integration is 4 points or more The diameter is 4 points or more, the uniformity of dyeing is 4 points or more, the total evaluation value is 20 points or more, 5 points are more preferable, the shape of the edge is 5 points, the integration is 5 points Most preferably, the diameter is 5 points, the uniformity of dyeing is 5 points, and the total evaluation value is 25 points. In addition, it is preferable that the function of insulin-secreting cells obtained by the culture method of the present invention is 1.5 times or more at high glucose compared to that at low glucose compared to glucose stimulation. More preferably, it is more preferably 5 times or more.
[0088] 上記検査項目において、形状が「扁平」とは細胞を楕円状の球体とみなした際に、 長軸/短軸比が 10以上であることを示し、「ほぼ扁平」とは前記長軸/短軸比が 5以 上 10未満であることを示し、「いびつな球状」とは前記長軸/短軸比が 2以上 5未満 であることを示し、「ほぼ球状」とは前記長軸/短軸比が 1. 2以上 2未満であることを 示し、「球状」とは前記長軸/短軸比が 1. 2未満であることを示す。 [0088] In the above inspection items, the shape "flat" means that the long / short axis ratio is 10 or more when the cell is regarded as an elliptical sphere, and "substantially flat" means that the long Indicates that the shaft / short axis ratio is 5 or more and less than 10, and the “loose spherical shape” means that the long / short axis ratio is 2 or more and less than 5 `` Almost spherical '' means that the major / minor axis ratio is 1.2 or more and less than 2, and `` spherical '' means that the major / minor axis ratio is less than 1.2. Indicates that there is.
[0089] 辺縁の形状力 S「不ぞろいの」とはスフエロイドの辺縁の 9割以上がでこぼこしており滑 らカ、さを欠!/、た辺縁であることを示し、「ほぼ不ぞろ!/、の」とはスフエロイドの辺縁の 5 割以上〜 9割未満が不ぞろ!/、であることを示し、「やや均整のとれた」とはスフエロイド の辺縁の 2割以上〜 5割未満が不ぞろいであることを示し、「ほぼ均整のとれた」とは スフエロイドの辺縁の 1割以上〜 2割未満が不ぞろいであることを示し、「均整がとれ た」とはスフエロイドの辺縁の 1割未満が不ぞろいであることを示す。  [0089] Edge shape force S “Unevenness” means that 90% or more of the edges of the spheroids are bumpy and slippery, lacking! "Sora! /," Means that more than 50% to less than 90% of the edges of the spheroids are irregular! /, And "Slightly balanced" means 20% of the edges of the spheroids. More than 50% is uneven, `` almost balanced '' means that more than 10% to less than 20% of the spheroid edges are uneven, and `` balanced '' means Indicates that less than 10% of the spheroid edges are uneven.
[0090] 統合性が「断片化した」とはスフエロイドの中にくびれがあるものが全体の 8割以上 であることを示し、「ほぼ断片化した」とはスフエロイドの中にくびれがあるものが全体 の 6〜8割であることを示し、「ややソリッド/コンパクト」とはスフエロイドの中にくびれ があるものが全体の 4〜6割であることを示し、「ほぼソリッド/コンパクト」とはスフエロ イドの中にくびれがあるものが全体の 2〜4割であることを示し、「ソリッド/コンパクト」 とはスフエロイドの中にくびれがあるものが全体の 2割以下であることを示す。  [0090] Integration is "fragmented" means that more than 80% of the spheroids are constricted, and "almost fragmented" means that spheroids are constricted 60 to 80% of the total, "Slightly solid / compact" means that 40% to 60% of the spheroids are constricted, and "Almost solid / compact" The number of constrictions in the id is 20 to 40% of the total, and “solid / compact” indicates that the constriction in the spheroids is less than 20% of the total.
[0091] 直径について、「スフエロイドの個々が 100 未満」とはスフエロイド個々の直径が全 て 100〃 m未満であることを示し、「スフエロイドの固々力 00〜； 150 μ m」とはスフエ ロイド個々の直径が 100〜150〃111の範囲であることを示し、「スフエロイドの個々が 1 25〜175〃111」とはスフェロィド個々の直径が125〜175〃111の範囲でぁることを示 し、 「スフエロイドの固々力 50〜200 μ m」とはスフエロイド固々の直径力 50〜200 〃mの範囲であることを示し、「スフエロイドの個々中 10%以上が 200〃 mより大きい 」とはスフエロイド個々中 10%以上が 200〃 mより大きいことを示す。  [0091] Regarding the diameter, “individual spheroids of less than 100” means that the diameter of each spheroid is less than 100 mm, and “spheroid strength of 00 ～; 150 μm” means spheroid. The individual diameter is in the range of 100 to 150〃111, and “Spheroid individual is 125 to 175〃111” means that the individual diameter of the spheroid is in the range of 125 to 175〃111. , “Spheroid's individual force 50-200 μm” means that the spheroid's individual diametric force is in the range of 50-200 μm, and “10% or more of each spheroid is greater than 200 μm” Indicates that more than 10% of each spheroid is larger than 200 mm.
[0092] グルコース刺激に対するインスリン分泌（グルコース応答性インスリン分泌とも言う） とは、インスリン分泌細胞力 例えば培地中において低グルコース濃度から高ダルコ ース濃度への変化（グルコース刺激)を感知してインスリン分泌を促進させることを言 う。インスリンは、血糖を下げる方向に作用する唯一のホルモンであり、血中のダルコ ース濃度に応答して勝臓のランゲルノヽンス島（瞵島）の /3細胞から分泌される。インス リン分泌細胞の機能が良好であれば、グルコース濃度の変動に応じて、適切にイン スリンが分泌される力 インスリン分泌細胞の機能が不良であれば、こうしたダルコ一 ス応答性が不良となる。インスリン分泌細胞では、細胞の機能を評価するために、高 グルコース濃度時に分泌されるインスリン分泌量に対する低グルコース濃度時に分 泌されるインスリン分泌量の比率（高グルコース濃度時のインスリン分泌量/低ダルコ ース濃度時のインスリン分泌量） (stimulation index； SI)として表し比較検討されてお り (Bergert H, Knoch P, Meisterreld R， Jager M, OuwendijK j, Kersting Saeger H D， Solimena M,. Effect of oxygenated perfluorocarbons on isolated rat pancreatic isl ets inculture. Cell Transplant. 2005; 14(7):441_8参照）、 SIィ直が 1 · 5以上であること が好ましぐ 2以上であることがより好ましぐ 5以上であることが最も好ましい。 [0092] Insulin secretion in response to glucose stimulation (also referred to as glucose-responsive insulin secretion) is the insulin-secreting cell force, for example, insulin secretion by sensing a change from low glucose concentration to high darucose concentration (glucose stimulation) in the medium. Say to promote. Insulin is the only hormone that acts to lower blood sugar, and is secreted by / 3 cells of the presumptive Langernos island (Iwajima) in response to blood levels of darcose. If the function of insulin-secreting cells is good, the ability to properly secrete insulin in response to fluctuations in glucose concentration. The responsiveness is poor. Insulin-secreting cells, the ratio of insulin secreted at low glucose concentration to insulin secreted at high glucose concentration (insulin secretion at high glucose concentration / low Darcos) (Stimulation index; SI), which has been compared (Bergert H, Knoch P, Meisterreld R, Jager M, OuwendijKj, Kersting Saeger HD, Solimena M, Effect of oxygenated) Cell Transplant. 2005; 14 (7): 441_8), it is preferred that the SI straight is 1 · 5 or more, more preferably 2 or more, and more 5 or more. perfluorocarbons on isolated rat pancreatic islets inculture. Most preferably.
[0093] 高グルコース培養とは、培地中のグルコース濃度が 3600mg/L〜4500mg/L であり、低グルコース培養とは、培地中のグルコース濃度が 600mg/L〜1000mg /Lである培養条件を示す。  [0093] High glucose culture means that the glucose concentration in the medium is 3600 mg / L to 4500 mg / L, and low glucose culture means that the glucose concentration in the medium is 600 mg / L to 1000 mg / L. .
[0094] 本発明にお!/、ては、ァクチビンを用いて未分化な ES細胞から分化させた胚体内胚 葉を、馴化培地を用いてニューロジェニン 3発現細胞へ分化させたのちに該細胞を 高グルコース培地で刺激することにより、インスリン分泌細胞へ効率的に分化誘導を 行なうことで、分化誘導効率が高ぐ高いインスリン分泌能を有するインスリン分泌細 胞が大量に供給できる。これを糖尿病治療剤、バイオ人工勝臓、研究試薬または創 薬モデル動物の細胞源とすることで万人が恩恵を受けることができる糖尿病治療剤、 バイオ人工勝臓、研究試薬または創薬モデル動物の開発が大いに期待できる。  [0094] According to the present invention, the embryoid endoderm differentiated from undifferentiated ES cells using activin is differentiated into neurogenin 3 expressing cells using a conditioned medium, and then the cells are differentiated. By inducing differentiation efficiently into insulin-secreting cells by stimulating them with a high glucose medium, a large amount of insulin-secreting cells having high insulin-secreting ability and high differentiation-inducing efficiency can be supplied. Diabetes therapeutics, bioartificial viscera, research reagents, or drug discovery model animals that can benefit everyone by using this as the cell source for diabetes therapeutics, bioartificial victories, research reagents, or drug discovery model animals The development of can be greatly expected.
[0095] よって、本発明のもう 1つの態様は、 ES細胞から分化誘導させたインスリン分泌細 胞を含有する糖尿病治療剤、バイオ人工勝臓、研究試薬および創薬モデル動物で ある。 [0095] Therefore, another embodiment of the present invention is a therapeutic agent for diabetes, a bioartificial viscera, a research reagent, and a drug discovery model animal containing insulin secreting cells differentiated from ES cells.
[0096] 本明細書において糖尿病治療剤とは、インスリン分泌細胞をそのまま、もしくはフィ ルター濾過などにより濃縮したペレットなどの細胞塊などがあげられる。さらに、前記 糖尿病治療剤は、 DMSOなどの保護剤を加え、凍結保存することもできる。糖尿病 治療剤として、より安全に利用するために、加熱処理、放射線処理、あるいはマイトマ イシン C処理など、糖尿病治療剤としての機能を残しつつ、病原体のタンパク質が変 性する程度の条件下で処理をすることができる。  [0096] In the present specification, the therapeutic agent for diabetes includes a cell mass such as a pellet obtained by concentrating insulin-secreting cells as it is or by filtering. Furthermore, the therapeutic agent for diabetes can be cryopreserved by adding a protective agent such as DMSO. In order to use it more safely as a therapeutic agent for diabetes, it can be treated under conditions that change the protein of the pathogen, such as heat treatment, radiation treatment, or mitomycin C treatment, while retaining its functions as a therapeutic agent for diabetes. can do.
[0097] 前記インスリン分泌細胞を用いた糖尿病治療剤のヒトへの投与形態 (移植方法）とし ては、例えば、ヒト患者の右下腹部に小切開を置き、腸間膜の細い血管を露出して 直視下にカテーテルを揷入して細胞を移植する方法、エコーにて肝臓の門脈を同定 して、カテーテルを穿刺して細胞を移植する方法、または腹部エコーガイド下に脾臓 を直接穿刺することにより脾臓に移植する方法（Nagata H， Ito M， Shirota C， Edge A, McCowan TC， Fox IJ: Route of hepatocyte delivery affects hepatocyte engraftment in the spleen. Transplantation, 76(4):732- 4， 2003.参照）が挙げられる。なかでも、ェ コ一にて細胞移植を行なう方法の方力 侵襲が少ないため好ましぐ腹部エコーガイ ド下に脾臓を直接穿刺することにより脾臓や肝臓に移植する方法があり、肝臓が最も 好ましい。細胞製剤の投与量 (移植量）は、 1 X 108〜1 X io1Q細胞/個体が好ましく 、 5 X 108〜； 1 X io1Q細胞/個体がさらに好ましく、 1 X 108〜； 1 X io1Q細胞/個体が 最も好ましい。また、投与量 (移植量）は、投与される患者の年齢、体重、症状などに よって適宜変更することができる。 [0097] As an administration form (transplantation method) to humans for a therapeutic agent for diabetes using the insulin-secreting cells For example, a small incision is made in the right lower abdomen of a human patient, a thin blood vessel in the mesentery is exposed, a catheter is inserted under direct viewing, and cells are transplanted. Identify and puncture the catheter and transplant the cells, or transplant the spleen directly by puncturing the spleen under abdominal echo guide (Nagata H, Ito M, Shirota C, Edge A, McCowan TC, Fox IJ: Route of hepatocyte delivery affects hepatocyte engraftment in the spleen. See Transplantation, 76 (4): 732-4, 2003.). Among them, the method of performing cell transplantation using Eco 1 has a method of transplanting into the spleen and liver by directly puncturing the spleen under the preferred abdominal echo guide because it is less invasive, and the liver is most preferred. The dosage (transplantation amount) of the cell preparation is preferably 1 X 10 8 to 1 X io 1Q cells / individual, more preferably 5 X 10 8 to; more preferably 1 X io 1Q cells / individual, 1 X 10 8 to 1 X io 1Q cells / individuals are most preferred. In addition, the dose (transplant amount) can be appropriately changed depending on the age, weight, symptoms, etc. of the patient to be administered.
[0098] 前記インスリン分泌細胞を用いた糖尿病治療剤のマウスへの投与形態 (移植方法） としては、例えば、マウスを切開して腎皮膜下に移植する方法が挙げられる。細胞製 剤の投与量 (移植量）は、 3 X 106〜1 X 107細胞/個体が好ましぐ 4 X 106〜8 X 10 6細胞/個体がさらに好ましぐ 5 X 106細胞/個体が最も好ましい。 [0098] Examples of the administration form (transplantation method) of the therapeutic agent for diabetes using the insulin-secreting cells to the mouse include a method of incising the mouse and transplanting it under the renal capsule. The dose (transplantation amount) of the cell preparation is preferably 3 X 10 6 to 1 X 10 7 cells / individual 4 X 10 6 to 8 X 10 6 cells / individual is more preferable 5 X 10 6 cells / Individual is most preferred.
[0099] 本発明のバイオ人工勝臓を製造する場合、不織布などのスキヤフォールドを使用 することで細胞を三次元培養することが好ましい。近年、スキヤフォールド上で細胞を 三次元培養することでより生体内環境に近い条件を作ることができ細胞機能が向上 することが知られてきており、不織布などのスキヤフォールド上で細胞を培養すること が機能の向上に有効である。また、インスリン分泌細胞を高分子素材で作製したデバ イス中に封入して免疫系から隔離した状態で生着させることが可能なため、バイオ人 ェ勝臓を使用する患者の身体的負担が軽減される。したがって、本発明のバイオ人 ェ勝臓は 21世紀の糖尿病治療における中心的役割を果たすものとして期待される。  [0099] When producing the bioartificial viscera of the present invention, it is preferable to culture cells three-dimensionally by using a skifold such as a nonwoven fabric. In recent years, it has been known that three-dimensional culture of cells on skifolds can create conditions that are closer to the in vivo environment and improve cell functions. Cells are cultured on skifolds such as nonwoven fabrics. This is effective for improving the functions. Insulin-secreting cells can be encapsulated in a device made of a polymer material and engrafted in a state isolated from the immune system, reducing the physical burden on patients who use biotechnology. Is done. Therefore, the biomedical organ of the present invention is expected to play a central role in the treatment of diabetes in the 21st century.
[0100] ノィォ人工勝臓としては、中空糸型のリアクター（デバイス）と分離 ·培養細胞を組 み合わせたハイブリッド型の人工勝臓などが挙げられる。バイオ人工勝臓は、体外に 装着して血管に接続するもの、体内に留置して血管に接続するもの、または血管に 接続せずに腹腔内に留置するものの、血管に接続せず皮下に留置する 4つの形態 がある。本発明のインスリン分泌細胞は、いずれの形態のバイオ人工勝臓にも使用 可能であるが、 ES細胞は、分化誘導すると増殖能力を失うが、分化が不充分であつ た ES細胞が遺残した場合、細胞を生体内に移植すると奇形種を発症する可能性が あるため、細胞移入などに伴う危険性を回避し、任意にリアクターを取り出せると言う 点から皮下埋め込み体内型であることが好ましい。 [0100] Examples of the artificial artificial viscera include a hybrid artificial viscera combining a hollow fiber type reactor (device) and a separated / cultured cell. A bioartificial viscera is placed outside the body and connected to the blood vessel, placed in the body and connected to the blood vessel, or placed in the abdominal cavity without connecting to the blood vessel, but placed subcutaneously without connecting to the blood vessel. 4 forms There is. Although the insulin-secreting cells of the present invention can be used for any form of bioartificial viscera, ES cells lose their ability to proliferate when induced to differentiate, but ES cells that have been poorly differentiated remain. In this case, since cells may be transplanted into a living body, a deformed species may develop, and therefore, a subcutaneous implantable body type is preferable from the viewpoint that a risk associated with cell transfer and the like can be avoided and a reactor can be arbitrarily taken out.
[0101] ノィォ人工勝臓の開発においては、リアクターの設計 ·開発も重要な要素である。  [0101] The design and development of reactors is also an important factor in the development of Neo artificial vices.
バイオリアクターとしては、体内に留置して使用するバッグ型人工勝臓 (たとえば、 Ale jandro soto- Gutierrez, Naoya obayashi, Jorge David Rivas-し arrillo, Nalu Navarro- Alvarez, Debaio Zhao, Teru Okitsu, Hirofumi Noguchi, Hesham Basma, Yashuhiko T abata, Yong Chen, imiaki Tanaka, Michiki Narushima, Atsushi Mki, Tadayoshi Ue da, Hee- Sook Jun, Jト Won Yoon, Jane Lebkowski, Noriaki i，anaka & Ira J Fox、「Rev ersal of mouse hepatic failure using an implanted liver-assist device containing ES c ell-derived hepatocytesj nature biotechnology Volume24、 No.11、 pl412 - 1419(Pu blished online: 05 November 2006 | doi: 10.1038/nbtl257)参照。以下「SG文献」と言 うこともある。当業者であれば、 SG文献を参照することで、バッグ型人工勝臓を製造 することが可能である。）、サーシ バイオメディカル社（Circe Biomedical Inc.) (レキ シントン、マサチューセッツ州、米国）の支援下でシダーズサイナイ医療センター（Ce dars-Sinai Medical Center) (ロサンジェルス、カリフォルニア州、米国）のディメトリュー (Demetriou)らを中心としたブタ肝細胞を用いたバイオ人工肝臓治療用のへパトァシ スト (HepatAssist) (Hui T， Rozga J, Demetriou AA. J Hepatobiliary Pancreat Surg 20 01; 8: 1-15·)や、ブタ肝細胞を使用したドイツのゲルラック（Gerlach)らの MELS (モ ジュラー体外肝臓システム（Modular Extracorporeal Liver System) )など、様々なタイ プが知られている。これらのリアクターは、もちろん本発明において使用することがで きる力 インスリン分泌細胞が付着するための足場が無いため、細胞はただ単に、中 空糸内スペース力、、中空糸外スペースに充填されるのみで浮遊した状態となる傾向 がある。インスリン分泌細胞は、浮遊状態では、分化機能が充分に発現されない傾向 があり、さらに周りの細胞と衝突し、ストレス刺激を受けやすい。  Bioreactors can be used as indwelling artificial bags (for example, Ale jandro soto- Gutierrez, Naoya obayashi, Jorge David Rivas-arrillo, Nalu Navarro- Alvarez, Debaio Zhao, Teru Okitsu, Hirofumi Noguchi, Hesham Basma, Yashuhiko T abata, Yong Chen, imiaki Tanaka, Michiki Narushima, Atsushi Mki, Tadayoshi Ue da, Hee- Sook Jun, J To Won Yoon, Jane Lebkowski, Noriaki i, anaka & Ira J Fox, `` Rev ersal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytesj nature biotechnology Volume 24, No. 11, pl412-1419 (Published online: 05 November 2006 | doi: 10.1038 / nbtl257). A person skilled in the art can produce a bag-type artificial viscera by referring to the SG literature.), Circe Biomedical Inc. (Lexington, Massachusetts, USA) HepatAssist for bioartificial liver treatment using porcine hepatocytes centered on Demetriou et al. Of Cedars-Sinai Medical Center (Los Angeles, California, USA) Hui T, Rozga J, Demetriou AA. J Hepatobiliary Pancreat Surg 20 01; 8: 1-15 ·), German Gellac et al. Using MELS (Modular Extracorporeal Liver) Various types such as System)) are known. These reactors, of course, can be used in the present invention because there is no scaffold for the attachment of insulin secreting cells, so the cells are simply filled into the space force inside the hollow fiber, the space outside the hollow fiber. There is a tendency to float only by itself. Insulin-secreting cells tend to have insufficient differentiation function in a floating state, and collide with surrounding cells and are susceptible to stress stimulation.
[0102] したがって、本発明においては、インスリン分泌細胞に足場が提供できるよう、中空 糸と不織布などのスキヤフォールドからなるリアクターが好ましい。 [0102] Therefore, in the present invention, a hollow space is provided so that a scaffold can be provided to insulin-secreting cells. A reactor comprising a skid fold such as yarn and nonwoven fabric is preferred.
[0103] 中空糸膜としては、膜表面に細胞が付着して物質交換が妨げられることがなければ どのようなものでも使用することができ、具体的には、従来医療用に用いられている 市販の物、たとえば、ポリスルフォン膜、エチレン 酢酸ビュルランダム共重合体けん 化物膜 (たとえば、商品名：ェバール、クラレメディカル株式会社製など）などが好まし い。市販の中空糸膜のポアサイズは、その用途から透析膜（〜5nm)、血漿成分分離 膜（20〜30nm)、血漿分離膜（30〜200nm)などがある。物質の透過性の点からは 、血漿分離膜（30〜200nm)が好ましい。拒絶反応の危険性を回避するため、中空 糸内を流れる血液中の免疫担当細胞や免疫グロブリンが中空糸外の不織布などの スキヤフォールド上に充填した細胞と直接接触することがないよう、 30〜100nmのポ ァサイズが最も好ましい。 [0103] As the hollow fiber membrane, any membrane can be used as long as cells do not adhere to the membrane surface and hinder the substance exchange. Specifically, it has been conventionally used for medical purposes. Commercially available products such as polysulfone membranes and ethylene acetate butyl random copolymer saponified membranes (for example, trade names: EVAL, manufactured by Kuraray Medical Co., Ltd.) are preferred. The pore sizes of commercially available hollow fiber membranes include dialysis membranes (~ 5 nm), plasma component separation membranes (20-30 nm), plasma separation membranes (30-200 nm), and so on. From the viewpoint of the permeability of the substance, a plasma separation membrane (30 to 200 nm) is preferable. In order to avoid the risk of rejection, the immunocompetent cells and immunoglobulins in the blood flowing in the hollow fiber should not come into direct contact with the cells packed on the skifold such as the nonwoven fabric outside the hollow fiber. A pore size of 100 nm is most preferred.
[0104] 不織布としては、細胞が接着することが出来るように加工 '修飾されているものが好 ましい。不織布の線維としては、ポリ四フッ化工チレン (PTFE)などが挙げられる。な かでも、その加工のしゃすさの点から、ポリ四フッ化工チレンにポリアミノ酸ウレタン（P AU)加工を施したものが好ましい。 [0104] The nonwoven fabric is preferably processed and modified so that the cells can adhere. Examples of non-woven fibers include polytetrafluoroethylene (PTFE). Of these, polytetrafluoroethylene having been subjected to polyamino acid urethane (P AU) processing is preferred from the viewpoint of processing.
[0105] 本発明のバイオ人工勝臓の一実施態様を図 8に示す。バイオ人工勝臓は、免疫担 当細胞の透過を阻止するためのポリエチレン ビュルアルコール膜（ポアサイズは 3 Onm) (例えば、ェバール膜） 5、細胞接着のための PAU加工した PTFE不織布 6、 およびインスリン分泌細胞 7を注入する細胞注入口 3および蓋 4からなる。 PTFE不織 布 6はポリエチレン—ビュルアルコール膜 5に覆われた 2層構造となっている。球状（ スフエロイド）を呈しているインスリン分泌細胞 7は PTFE不織布に付着して存在し、バ ィォ人工勝臓内部に留まることが可能である。このバイオ人工勝臓を体内に埋め込 むと、ポリエチレン—ビュルアルコール膜の周囲に血管が伸びてきて、バイオ人工瞵 臓内の細胞の栄養供給が可能となる。 PEメッシュを有しないポリエチレン ビュルァ ルコール膜の外側にさらに PEメッシュ（ポアサイズは約 1 OOnm)で覆われた 3層構造 力、ら構成されるリアクターも可能である。 3層構造の場合、 PEメッシュ内に血管が伸び てきて、バイオ人工勝臓内の細胞の栄養供給が可能となる。好ましい態様としては、 図 8に示す 2層構造のバイオリアクターが挙げられる。 2層のバイオリアクターは、 PE メッシュがない分、注入されたインスリン分泌細胞とポリエチレン ビュルアルコール 膜の周囲に誘導された血管がより近くに位置するため、血糖値のコントロールが容易 となるため好ましい。なお、当該人工勝臓は、「SG文献」を参照することにより当業者 であれば作製することが可能である。 One embodiment of the bioartificial viscera of the present invention is shown in FIG. Bioartificial spleen is a polyethylene bulal alcohol membrane (pore size is 3 Onm) (eg, Eval membrane) to block permeation of immune-bearing cells 5, PAU-treated PTFE nonwoven fabric 6 for cell adhesion, and insulin secretion It consists of a cell inlet 3 for injecting cells 7 and a lid 4. The PTFE nonwoven fabric 6 has a two-layer structure covered with a polyethylene-but alcohol film 5. Insulin-secreting cells 7 that are spherical (spheroids) are attached to the PTFE nonwoven fabric and can remain inside the bioartificial viscera. When this bioartificial viscera is embedded in the body, blood vessels extend around the polyethylene-butanol alcohol film, and nutrition of cells in the bioartificial organ can be supplied. A reactor consisting of a three-layer structure force covered with a PE mesh (pore size of about 1 OOnm) on the outside of a polyethylene bural alcohol membrane without a PE mesh is also possible. In the case of the three-layer structure, blood vessels extend into the PE mesh, and the nutrient supply of the cells in the bioartificial viscera becomes possible. A preferred embodiment is a bioreactor having a two-layer structure shown in FIG. 2 layer bioreactor, PE Since there are no meshes, the injected insulin-secreting cells and the blood vessels induced around the polyethylene butyl alcohol membrane are located closer to each other, which makes it easier to control the blood glucose level. The artificial viscera can be produced by those skilled in the art by referring to the “SG literature”.
[0106] ノ^オリアクターに細胞または細胞培養物を充填させる方法としては、例えば、バイ オリアクターをあらかじめ体内に埋め込んでおき、その後インスリン分泌細胞を充填さ せる方法、試験管内で細胞を分化誘導し ES細胞をインスリン分泌細胞としたのちに 、必要な細胞数をバイオリアクターに充填させる方法と、バイオリアクター内部をマトリ ゲルなどのスキヤフォールドであらかじめ加工しておき、そのなかでインスリン分泌細 胞を分化誘導する方法とがある。ノ^オリアクター力 バック型である場合、バック型 バイオリアクターをあらかじめ体内に埋め込んでおき、バイオリアクターの周辺に血管 が伸長してから、インスリン分泌細胞をバイオリアクターに充填させる方法が好ましい 。いずれの場合も、細胞の充填には、細胞懸濁液を 10〜50mLの注射器を使用して 、リアクターに装着されて!/、る細胞注入口より注入することが好まし!/、。  [0106] Examples of methods for filling a cell or cell culture into a nanoreactor include, for example, a method in which a bioreactor is pre-implanted in the body and then filled with insulin-secreting cells, and cell differentiation is induced in a test tube. After making ES cells into insulin-secreting cells, the bioreactor is filled with the necessary number of cells, and the bioreactor is pre-processed with a skifold such as matrigel, and the insulin-secreting cells are There is a method of inducing differentiation. In the case of the back-reactor type, a method is preferred in which a back-type bioreactor is embedded in the body in advance, and a blood vessel extends around the bioreactor, and then insulin-secreting cells are filled into the bioreactor. In any case, for cell filling, the cell suspension is preferably attached to the reactor using a 10-50 mL syringe! /, Preferably injected from the cell inlet! / ,.
[0107] また、このようなバイオリアクターを使用するバイオ人工臓器による治療は、体外で 使用する場合は、安全かつ科学的に施行するために、 1)人工臓器リアクターの流入 圧と流出圧のリアルタイムでのモニタリング、 2)気泡が発生した際のアラームの作動、 3)リアクターの温暖化（37°C)などができる機能を一体化した装置で実施されること が好ましい。  [0107] In addition, when using bioartificial organs using such a bioreactor, in order to safely and scientifically perform the treatment when used outside the body, 1) Real-time inflow pressure and outflow pressure of the artificial organ reactor are used. It is preferable that the system is integrated with 2) monitoring, 2) alarm operation when bubbles are generated, and 3) reactor warming (37 ° C).
[0108] 本発明のバイオリアクターはまた、有用物質生産、組織や器官'臓器の機能の調査 •探索、新薬のスクリーニングや内分泌撹乱物質等の影響を評価するための動物実 験代替法等にも好適に適用できる。  [0108] The bioreactor of the present invention is also useful for the production of useful substances, the investigation and exploration of the functions of tissues and organs, screening for new drugs, and alternative methods for animal experiments to evaluate the effects of endocrine disrupting substances. It can be suitably applied.
[0109] 本発明のバイオ人工勝臓は、例えば、瞵島に本来備わっている産生成分であるィ ンスリンの製造に使用することができる。インスリンの製造は、得られた培地をァフィ二 ティーカラムなど、通常タンパク質の精製に使用される方法によって精製することによ り fiなうことができる。  [0109] The bioartificial viscera of the present invention can be used, for example, for the production of insulin, which is a production component inherent to Yeouido. Insulin can be produced by purifying the obtained medium by a method usually used for protein purification, such as an affinity column.
[0110] 本発明のバイオ人工勝臓は、例えば、国内のみならず国際的にも年々増加の一途 をたどっている糖尿病患者に移植が可能である。ヒト糖尿病の予防と制圧力 21世 紀における人類の大きな課題であるといっても過言ではない。そこで、多くの製薬メ 一力一が糖尿病薬の開発に取り組んでいる。 [0110] The bioartificial viscera of the present invention can be transplanted, for example, to diabetic patients that are increasing year by year not only in Japan but also internationally. Prevention and control of human diabetes 21st century It is no exaggeration to say that this is a major issue for mankind in the era. Therefore, many pharmacists are working on the development of diabetes drugs.
[0111] 本発明の分化誘導方法により得られたインスリン分泌細胞を含有する研究試薬は、 例えば、新薬のスクリーニングに用いる研究試薬を意味する。当該研究試薬は、どの ような態様で用いてもよいが、インスリン分泌細胞を培養している培養容器にそのまま 新薬などを添加し評価する方法、バイオリアクター内に当該細胞を封じ込めて評価 する方法などが挙げられる。本明細書では、新薬のかわりに血糖降下薬として市販さ れているトルプタミドを用いて薬効評価を行うことで本発明の分化誘導方法により得ら れたインスリン分泌細胞が研究試薬としても有用であることを示す。 [0111] The research reagent containing insulin-secreting cells obtained by the differentiation induction method of the present invention means, for example, a research reagent used for screening of a new drug. The research reagent may be used in any form, such as a method in which a new drug is added as it is to a culture vessel in which insulin-secreting cells are cultured, a method in which the cells are contained in a bioreactor, and the like Is mentioned. In the present specification, insulin-secreting cells obtained by the differentiation induction method of the present invention by evaluating medicinal effects using tolptamide marketed as a hypoglycemic agent instead of a new drug are also useful as research reagents. It shows that.
[0112] トルプタミドとは、スルフォニル尿素系の血糖降下薬である。トルプタミドは、勝臓に 働きかけインスリンの分泌を増やすことで、血糖値を低下させる作用を有する。おもに 、 2型糖尿病 (インスリン非依存型糖尿病）に用いられる。薬理作用は、勝臓にあるラ ンゲルノヽンス島の 0細胞を刺激してインスリン分泌を促進する（瞵作用）。ただし、ィ ンスリンの代わりにはならないので、インスリンがまったく分泌されない 1型糖尿病（ィ ンスリン依存型糖尿病）には無効である（http：〃 www.interq.or.jp/ox/dwm/se/se39/s e3961006.html参照）。  [0112] Tolptamide is a sulfonylurea hypoglycemic agent. Tolptamide acts on the viscera and increases insulin secretion, thereby reducing blood glucose levels. Mainly used for type 2 diabetes (non-insulin dependent diabetes). Pharmacological action stimulates insulin secretion by stimulating 0 cells of Langeron's islet in the viscera (acupuncture). However, since it is not a substitute for insulin, it is ineffective for type 1 diabetes (insulin-dependent diabetes) where no insulin is secreted (http: 〃 www.interq.or.jp/ox/dwm/se/se39). / s e3961006.html).
[0113] 近年、トラスジエニック (Tg)動物の作成が容易になり、種々のユニークな特性を持 つ糖尿病 Tg動物が誕生している。これらの Tg動物の場合、系統の確立、維持、繁殖 、供給は大変な作業である。モデル動物の確立には多年月と多大な労力'忍耐がか かり、近年のように時の流れが速くなる中、なかな力、じっくりと取り組める課題ではなく なってきている。さらに、系統の繁殖 ·維持にはマンパワーと設備が必要であり、役割 が終了したモデル動物の場合、消滅してしまう危険もある。また、ヒト糖尿病と糖尿病 モデル動物との相違点から、開発医薬品の臨床試験段階でのドロップアウトが多レ、 のが問題となっている。本発明のバイオ人工勝臓は、こうした問題点を解決できるた め好ましい。  [0113] In recent years, it has become easier to create trussianic (Tg) animals, and diabetic Tg animals with various unique characteristics have been born. For these Tg animals, the establishment, maintenance, breeding and supply of strains is a daunting task. Establishing a model animal takes many years and a lot of effort, patience, and as the flow of time has become faster as in recent years, it is no longer an issue that can be tackled carefully. In addition, breeding and maintenance of the strain requires manpower and equipment, and in the case of a model animal that has finished its role, there is a risk that it will disappear. In addition, due to the differences between human diabetes and diabetes model animals, there is a problem that there are many dropouts in the clinical trial stage of the developed drug. The bioartificial viscera of the present invention is preferable because these problems can be solved.
[0114] 本明細書において創薬モデル動物とは、本発明の分化誘導方法により得られたィ ンスリン分泌細胞を含有する動物を言う。例えば、ヒト由来のインスリン分泌細胞を有 するキメラマウスが挙げられ、ヒト瞵島に対する創薬および薬理試験等に用いることが できる。 [0114] In this specification, the drug discovery model animal refers to an animal containing insulin-secreting cells obtained by the differentiation induction method of the present invention. For example, a chimeric mouse having human-derived insulin-secreting cells can be mentioned, and it can be used for drug discovery and pharmacological testing for human Kashiwajima. it can.
[0115] 以下、本発明をヒト由来の ES細胞を用いた実施例をあげて説明する力 本発明は これらに限定されるものではない。  [0115] Hereinafter, the present invention will be described with reference to examples using human-derived ES cells. The present invention is not limited to these examples.
実施例  Example
[0116] 製造例 1 [0116] Production Example 1
ES細胞の調製  ES cell preparation
129vマウス由来の ES細胞（大日本住友製薬株式会社より購入）を、ネオマイシン 耐性遺伝子が導入されているフィーダ一細胞（マウス由来の胚性線維芽細胞、大日 本住友製薬株式会社より購入）を用いて、 0. 1 %ゼラチン水（カタログ番号: R— ES — 006B、大日本住友製薬株式会社より購入）でコートした培養フラスコ T— 75 (ファ ノレコン社製、べクトン デッキンソン アンド カンノ ニー (Becton, Dickinson and Com pany)販売）を使用して培養した。細胞培養用培地には、 ES培養液 (R— ES— 101： 大日本住友製薬株式会社より購入）：ダルベッコ改変イーグル (DMEM)培養液の混 合液（1： 1容量比）に 15%ゥシ胎仔血清 (FBS)、 1 %非必須アミノ酸、 1 %ヌクレオシ ド、 l lO ^ mol/L 2—メルカプトエタノール (大日本住友製薬株式会社より購入）、 1 %ペニシリンおよびストレプトマイシン、 1 %グノレタミン酸、ならびに 500U/mLマウ ス由来白血病抑制因子 (LIF) (大日本住友製薬株式会社より購入)を添加したもの を使用した。培養液は毎日交換し、 ES細胞は、 3日毎に継代した。培養プレート中に ES細胞が占める割合が 80〜90%の状態になったところで、 2段階式処置にて、す なわち、まず、 0· 25%濃度のトリプシン一 EDTA (インビトロジェン社製）を添加し、 4 5秒間経過後、トリプシン EDTA液を取り除くことでフィーダ一細胞を一緒に除去し (ES細胞はまだこの段階では培養プレートに残っている）、ついでその 2分後に ES 培養液を加え剥離することによって、培養プレートに残っている ES細胞を回収した。  129v mouse-derived ES cells (purchased from Sumitomo Dainippon Pharma Co., Ltd.) and feeder cells that have been introduced with a neomycin resistance gene (mouse-derived embryonic fibroblasts, purchased from Sumitomo Dainippon Pharma Co., Ltd.) Culture flask T-75 (Fanorecon, Becton Decktonson and Cannony (Becton), coated with 0.1% gelatin water (catalog number: R—ES — 006B, purchased from Dainippon Sumitomo Pharma Co., Ltd.) , Dickinson and Company)). For cell culture media, ES medium (R—ES—101: purchased from Dainippon Sumitomo Pharma Co., Ltd.): Dulbecco's Modified Eagle (DMEM) medium (1: 1 volume ratio) 15% Fetal serum (FBS), 1% non-essential amino acid, 1% nucleoside, l lO ^ mol / L 2-mercaptoethanol (purchased from Dainippon Sumitomo Pharma Co., Ltd.), 1% penicillin and streptomycin, 1% gnoretamic acid, In addition, 500 U / mL mouse-derived leukemia inhibitory factor (LIF) (purchased from Dainippon Sumitomo Pharma Co., Ltd.) was used. The culture medium was changed every day, and ES cells were passaged every 3 days. When the proportion of ES cells in the culture plate reached 80-90%, in a two-step procedure, first add 0.25% trypsin EDTA (Invitrogen). 45 5 seconds later, remove the trypsin EDTA solution to remove the feeder cells together (the ES cells still remain in the culture plate at this stage), and then 2 minutes later, add the ES medium and detach As a result, ES cells remaining in the culture plate were recovered.
[0117] 実施例 1 [0117] Example 1
工程 (a)：未分化 ES細胞の胚体内胚葉への分化  Step (a): Differentiation of undifferentiated ES cells into definitive endoderm
製造例 1で得られた未分化 ES細胞は、細胞培養濃度を少なくし (細胞培養濃度： 1 75, 000細胞/ mU、コラーゲンタイプ IV、マトリゲノレ、ラミニンタイプ 1、ジエラチン のいずれかで加工した培養皿を用いてァクチビン A (Activin A、カタログ番号： 338 -AC, R&D社製） (Ong/mL, 2ng/mL, lOng/mL, lOOng/mL,または 20 Ong/mU、線維芽細胞成長因子（FGF— 2、カタログ番号： 233— FB/CF、 R& D社製）（20ng/mU、ゥシ胎仔血清（FBS)、およびゥシ血清アルブミン（BSA)を カロえたエス ·クロン SF— 03培地 (製品番号 SS— 1303、三光純薬株式会社製）で計 2日間培養（培養温度： 37°C、 pH： 7. 4)を行った。 2日間の培養におけるゥシ胎仔 血清およびゥシ血清アルブミン (BSA)の濃度は次のように変化させた。すなわち 0. 2%FBSおよび 2. 5%BSAで 12時間培養、 1 %FBSおよび 2. 5%BSAで 12時間 培養後 3%FBSおよび 2. 5%BSAで 24時間 ES細胞を培養した。当該胚体内胚葉 への分化判定には、 Foxa2と Soxl 7の遺伝子発現を RT— PCR法にて判定した。 The undifferentiated ES cells obtained in Production Example 1 were cultured at a reduced cell culture concentration (cell culture concentration: 17,000,000 cells / mU, collagen type IV, matrigenore, laminin type 1, or dielatin) Activin A, catalog number: 338 -AC, R & D) (Ong / mL, 2ng / mL, lOng / mL, lOOng / mL, or 20 Ong / mU, fibroblast growth factor (FGF-2, catalog number: 233—FB / CF, R & D) Manufactured by Company D) (20 ng / mU, sullon fetal serum (FBS), and sullon serum albumin (BSA) with S-cron SF-03 medium (Product No. SS-1303, manufactured by Sanko Junyaku Co., Ltd.) Culture was performed for a total of 2 days (culture temperature: 37 ° C, pH: 7.4) The concentration of urchin fetus serum and urine serum albumin (BSA) in the 2-day culture was changed as follows. That is, the cells were cultured with 0.2% FBS and 2.5% BSA for 12 hours, cultured with 1% FBS and 2.5% BSA for 12 hours, and then ES cells were cultured with 3% FBS and 2.5% BSA for 24 hours. To determine differentiation into definitive endoderm, Foxa2 and Soxl 7 gene expression was determined by RT-PCR.
[0118] 試験例 1  [0118] Test Example 1
誘導された胚体内胚葉における遺伝子発現  Gene expression in induced definitive endoderm
実施例 1に記載の分化誘導された胚体内胚葉において、 RT— PCR法により、胚体 内胚葉特異的な遺伝子である Foxa2および Soxl 7遺伝子の発現を調べた。内因性 コントロールとして GAPDH遺伝子の発現も調べた。 RT— PCR法では、細胞を 0. 2 5%トリプシン一 EDTA (インビトロジェン社製）で処理したのちに回収し、 RNAトリゾ ール (インビトロジェン社製）を用いて製品の取扱説明書にしたがって RNAを抽出し た。抽出した RNA2 gを用いて RNA逆転写酵素による逆転写反応（22°Cで 10分 、さらに 42°C20分、 99°C5分、その後 4°Cで 5分以上静置）を行った。  In the differentiation-induced definitive endoderm described in Example 1, expression of Foxa2 and Soxl 7 genes, which are definitive endoderm-specific genes, was examined by RT-PCR. GAPDH gene expression was also examined as an endogenous control. In the RT-PCR method, cells are treated with 0.25% trypsin EDTA (Invitrogen) and then recovered, and RNA is extracted using RNA Trizol (Invitrogen) according to the product instruction manual. Extracted. Using 2 g of the extracted RNA, a reverse transcription reaction with RNA reverse transcriptase (22 ° C for 10 minutes, 42 ° C for 20 minutes, 99 ° C for 5 minutes, and then allowed to stand at 4 ° C for more than 5 minutes) was performed.
[0119] 得られた 2 a gの逆転写産物を、各プライマー 20pmol/mLで、 AmpliTag Gold キット（パーキン一エルマ一/シータス社、ノーウォーク、コネチカット州、米国）を用 い、そのプロトコールにしたがって PCR増幅に用いた。 PCR反応は、 95°C10分、そ れから各サイクルで 95°C30秒、 60°C30秒、 72°C30秒をサイクル数だけ繰り返した 。その後、 4°Cで 5分以上静置した。各遺伝子に対するプライマーおよび PCR条件を 以下に記載する。  [0119] Using the AmpliTag Gold kit (Perkin-Elmer / Cetus, Norwalk, CT, USA), PCR was performed according to the protocol using the obtained 2 ag reverse transcription product at 20 pmol / mL of each primer. Used for amplification. The PCR reaction was repeated at 95 ° C for 10 minutes and then at each cycle for 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds for the number of cycles. Then, it left still at 4 degreeC for 5 minutes or more. The primers and PCR conditions for each gene are described below.
[0120] Foxa2遺伝子（288bp、 60。C、 30サイクル）  [0120] Foxa2 gene (288bp, 60.C, 30 cycles)
5' プライマー： 5' — TGGTCACTGGGGACAAGGGAA (酉己列番号 1) 3' プライマー： 5' - GCAACAACAGCAATAGAGAAC (配列番号 2) Soxl 7it¼ - (210bp, 60。C、 35サイクル） 5' プライマー： 5' — GCCAAAGACGAACGCAAGCGGT (酉己列番号 3) 3' プライマー： 5' — TCATGCGCTTCACCTGCTTG (配列番号 4) 5 'Primer: 5' — TGGTCACTGGGGACAAGGGAA (Self column number 1) 3 'Primer: 5'-GCAACAACAGCAATAGAGAAC (SEQ ID NO: 2) Soxl 7it¼-(210bp, 60. C, 35 cycles) 5 'Primer: 5' — GCCAAAGACGAACGCAAGCGGT (Self Row Number 3) 3 'Primer: 5' — TCATGCGCTTCACCTGCTTG (SEQ ID NO: 4)
[0121] 当該胚体内胚葉への分化判定には、 Foxa2と Soxl 7の遺伝子発現を RT— PCR 法にて判定した。レーン 1から 4は、それぞれ、コラーゲンタイプ IV、マトリゲル、ラミニ ンタイプ 1、ジエラチンを示す。コラーゲンタイプ IVにて、胚体内胚葉特異的なマーカ 一遺伝子である Foxa2および Soxl 7遺伝子の発現が最も強く確認される。この結果 は、本手法により胚体内胚葉の分化誘導が効率よく誘導できることを強く支持してレヽ る。図 2にコラーゲンタイプ IVにて同様に未分化マウス ES細胞を培養した際の Foxa 2と Soxl 7の遺伝子発現を示す。この場合は、ァクチビン Aを各種濃度で添加した。 レーン 1から 4は、それぞれ、ァクチビン Aの濃度が 2ng/ml、 10ng/ml, lOOng/ ml、 200ng/mlである。ァクチビン Aの濃度が lOng/mlが最も好ましぐ lOOng/ mlが次いで好ましぐ 200ng/mlが次いで好ましい。図 1、 2の結果は未分化マウス ES細胞をコラーゲンタイプ IVにて、線維芽細胞成長因子（FGF— 2) (20ng/mL) 、ゥシ胎仔血清（FBS)、およびゥシ血清アルブミン（BSA)を加えたエス'クロン SF— 03培地 (製品番号 SS— 1303、三光純薬株式会社製）で計 2日間、ァクチビン Aの 濃度が 10ng/mlで培養することで、胚体内胚葉の分化誘導が効率よく誘導できるこ とを強く支持している。従来技術であるダムール法では、工程 (a)の分化誘導は 100 ng/mLのァクチビン Αが必要であること、低濃度（30ng/mUのァクチビン Aでは 分化誘導効率が低いことが記載されている（ダムールら、 Nat. Biotech., published onl ine, 19 October 2006， doi: 10.1038/nbtl259.参照）。本発明の分化誘導方法の工程 (a)では、従来技術であるダムール法と比較して低濃度のァクチビン Aでも ES細胞を 分化誘導しうることが明らかとなった。 [0121] To determine differentiation into the definitive endoderm, the gene expression of Foxa2 and Soxl 7 was determined by RT-PCR. Lanes 1 to 4 show collagen type IV, matrigel, laminin type 1 and dielatin, respectively. Collagen type IV shows the strongest expression of Foxa2 and Soxl 7 genes, which are definitive endoderm-specific markers. This result strongly supports that this method can efficiently induce definitive endoderm differentiation. Figure 2 shows the expression of Foxa 2 and Soxl 7 genes when similarly cultured undifferentiated mouse ES cells in collagen type IV. In this case, activin A was added at various concentrations. In lanes 1 to 4, the concentrations of activin A are 2 ng / ml, 10 ng / ml, lOOng / ml, and 200 ng / ml, respectively. The concentration of activin A is most preferably lOng / ml, followed by lOOng / ml, followed by 200 ng / ml. Figures 1 and 2 show that undifferentiated mouse ES cells were collagen type IV, fibroblast growth factor (FGF-2) (20ng / mL), urchin fetal serum (FBS), and urine serum albumin (BSA). ) Cron SF-03 medium (Product No. SS 1303, Sanko Junyaku Co., Ltd.) added for a total of 2 days, culturing at a concentration of 10 ng / ml of activin A, induces definitive endoderm differentiation. Strongly supports the efficient guidance. The prior art Damour method describes that differentiation induction in step (a) requires 100 ng / mL of activin Α, and low concentration (30 ng / mU of activin A has low differentiation induction efficiency. (See Damour et al., Nat. Biotech., Published online, 19 October 2006, doi: 10.1038 / nbtl259.) In the step (a) of the differentiation induction method of the present invention, the process is lower than that of the conventional Damour method. It became clear that ES cells could be induced to differentiate even with a concentration of activin A.
[0122] 実施例 2 [0122] Example 2
工程 (b) :原始瞵の形成  Process (b): Formation of primitive ridge
実施例 1に記載の分化誘導を 2日間行った後に、工程 (a)で得た胚体胚様体を用 いて原始瞵の形成を行った。ヒト神経膠芽細胞腫由来細胞株 T98G (ATCC番号: C RL— 1690、 ATCC (米国）から入手可能）を SF— 03培地で 1日間培養（細胞培養 濃度： 8 X 105細胞/ mL、培養温度：37°C、 pH : 7. 4)して得られた馴化培地を使用 して、工程 (a)で得られた胚体内胚葉を 5日間培養（培養温度： 37°C、 pH： 7. 4)した 。上記馴化培地には、レチノイン酸（1 11101/レ lO ^ mol/L, lOO ^ mol/Lまた は 1000 a mol/L)および FGF— 2 (20ng/mUを添加し、コラーゲンタイプ IVカロ ェ培養皿 T— 25フラスコ（細胞培養濃度 175, 000細胞/ mL、計 700, 000細胞） を使用した。工程 (b)で得た細胞には、 Foxa2 ( + )、 Soxl 7 ( + )、Pdx— 1 ( + )、 S hh (—）の遺伝子発現を示しており、 ES細胞が原始瞵への形成を開始していること が確認された。 After the differentiation induction described in Example 1 was performed for 2 days, primitive embryos were formed using the embryoid body obtained in the step (a). Human glioblastoma cell line T98G (ATCC number: C RL— 1690, available from ATCC (USA)) cultured in SF-03 medium for 1 day (cell culture concentration: 8 X 10 5 cells / mL, culture) Temperature: 37 ° C, pH: 7.4) Use conditioned medium obtained Then, the definitive endoderm obtained in the step (a) was cultured for 5 days (culture temperature: 37 ° C., pH: 7.4). The above conditioned medium is supplemented with retinoic acid (1 11101 / relo ^ mol / L, lOO ^ mol / L or 1000 a mol / L) and FGF-2 (20 ng / mU), and collagen type IV caloculture. Dish T—25 flask (cell culture concentration 175,000 cells / mL, 700,000 cells in total) was used.The cells obtained in step (b) include Foxa2 (+), Soxl 7 (+), Pdx— The gene expression of 1 (+) and Shh (—) was shown, confirming that ES cells had started to form primitive pods.
[0123] 試験例 2 [0123] Test example 2
誘導された原始瞵における遺伝子発現  Gene expression in induced primitive cocoons
実施例 2に記載の誘導された原始瞵において、 RT— PCR法により、原始瞵特異的 な遺伝子である Pdx—l遺伝子と Shh遺伝子の発現を調べた。内因性コントロールと して GAPDH遺伝子の発現も調べた。また、胚発生の時期に特異的に発現する遺伝 子である Shh遺伝子の発現も調べた。 RT— PCR法では、細胞を 0. 25%トリプシン — EDTA (インビトロジェン社製）で処理したのちに回収し、 RNAトリゾール（インビト ロジェン社製）を用レ、て製品の取扱説明書にしたがって RNAを抽出した。抽出した R ΝΑ2 ;^を用いて RNA逆転写酵素による逆転写反応（22°Cで 10分、さらに 42°C20 分、 99°C5分、その後 4°Cで 5分以上静置）を行った。  In the induced primitive pods described in Example 2, expression of the Pdx-l gene and the Shh gene, which are specific to the primitive pods, was examined by RT-PCR. GAPDH gene expression was also examined as an endogenous control. We also examined the expression of the Shh gene, a gene that is specifically expressed during embryonic development. In the RT-PCR method, cells are treated with 0.25% trypsin-EDTA (Invitrogen) and collected, and then RNA trizol (Invitrogen) is used. Extracted. Using the extracted R ΝΑ2; ^, a reverse transcription reaction with RNA reverse transcriptase (22 ° C for 10 minutes, 42 ° C for 20 minutes, 99 ° C for 5 minutes, and then left at 4 ° C for more than 5 minutes) was performed. .
[0124] 得られた 2 a gの逆転写産物を、各プライマー 20pmol/mLで、 AmpliTag Gold キット（パーキン一エルマ一/シータス社、ノーウォーク、コネチカット州、米国）を用 い、そのプロトコールにしたがって PCR増幅に用いた。 PCR反応は、 95°C10分、そ れから各サイクルで 95°C30秒、 60°C30秒、 72°C30秒をサイクル数だけ繰り返した 。その後、 4°Cで 5分以上静置した。各遺伝子に対するプライマーおよび PCR条件を 以下に記載する。 [0124] Using the AmpliTag Gold kit (Perkin Elmer / Cetus, Norwalk, Conn., USA), PCR was performed according to the protocol using the obtained 2 ag reverse transcription product at 20 pmol / mL of each primer. Used for amplification. The PCR reaction was repeated at 95 ° C for 10 minutes and then at each cycle for 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds for the number of cycles. Then, it left still at 4 degreeC for 5 minutes or more. The primers and PCR conditions for each gene are described below.
[0125] Pdx- 1遺伝子（451bp、 60。C、 40サイクル） [0125] Pdx-1 gene (451 bp, 60. C, 40 cycles)
5' プライマー： 5' — ACCATGAACAGTGAGGAGCA (配列番号 5) 5 'primer: 5' — ACCATGAACAGTGAGGAGCA (SEQ ID NO: 5)
3' プライマー： 5' — TCCTCTTGTTTTCCTCGGGT (配列番号 6) Shh遺伝子（248bp、 60。C、 35サイクル） 3 'primer: 5' — TCCTCTTGTTTTCCTCGGGT (SEQ ID NO: 6) Shh gene (248 bp, 60. C, 35 cycles)
5' プライマー： 5' - CCTCTCCTGCTATGCTCCTG (配列番号 7) 3' プライマー： 5' — GTGGCGGTTACAAAGCAAAT (配列番号 8) 5 'primer: 5'-CCTCTCCTGCTATGCTCCTG (SEQ ID NO: 7) 3 'primer: 5' — GTGGCGGTTACAAAGCAAAT (SEQ ID NO: 8)
GAPDH遺伝子（171bp、 60。C、 35サイクル）  GAPDH gene (171bp, 60.C, 35 cycles)
5' プライマー： 5' — ACCCAGAAGACTGTGGATGG (酉己列番号 9) 5 'Primer: 5' — ACCCAGAAGACTGTGGATGG (Self column number 9)
3' プライマー： 5' — CACATTGGGGGTAGGAACAC (配列番号 10) 3 'primer: 5' — CACATTGGGGGTAGGAACAC (SEQ ID NO: 10)
[0126] 結果を図 3に示す。レーン 1〜4は、それぞれレチノイン酸濃度が 1 M 、 10 M、 100〃M、 1000〃Mを示す。 10〃Mのレチノイン酸で (ま、原台膝 ίこおレヽて (ま、 原始瞵特異的なマーカー遺伝子である Pdx— 1遺伝子の発現が強く確認されるが、 Shhは発現していな力、つた。この結果は、本手法により原始瞵の分化誘導が効率よく 誘導できることを強く支持して!/、る。 The results are shown in FIG. Lanes 1 to 4 show retinoic acid concentrations of 1 M, 10 M, 100 〃M, and 1000 〃M, respectively. With 10M retinoic acid, the expression of Pdx-1 gene, a marker gene specific to primordial acupuncture, is strongly confirmed, but Shh is not expressed. This result strongly supports that this method can efficiently induce the differentiation of primitive moths!
[0127] 実施例 3 [0127] Example 3
工程 (c)ニューロジェニン 3発現細胞の選別  Step (c) Selection of neurogenin 3 expressing cells
工程 (b)で得られた原始瞵を馴化培地を用いて培養することによりニューロジェ二 ン 3発現細胞を増加させた。ヒト不死化肝類洞内皮細胞株 TMNT— 1細胞（寄託機 関:独立行政法人産業技術総合研究所 特許生物寄託センター、あて名：日本国茨 城県つくば巿東 1丁目 1番地 1 中央第 6 (郵便番号 305— 8566)、寄託日：平成 14 年 4月 16日、受託番号： FERM BP— 8017、万人が使用できる）を低グルコース D MEM培地（グルコース濃度： 1000mg/L、カタログ番号 31600— 083、インビトロ ジェン社製)で 1日間培養（細胞培養濃度： 8 X 105細胞/ mL、培養温度： 37°C、 pH : 7. 4)して得られた馴化培地に γ—セクレターゼインヒビター XVIII (Calbiochem 社（カタログ番号 565779)、または Alexis Biochemicals社（カタログ番号： ALX— 270— 415— C250、または ALX— 270— 415— M001)力、ら購人可倉）、 FGF— 2 (20ng/mU、 EGF (20ng/mUを添加したものを使用して、ラミニンー 1 (濃度は 5 a g/cm2) (BD Biosciences社、カタログ番号： 354232)で加工した培養皿 T— 25フラスコ（細胞培養濃度 50, 000細胞/ mL、計 200, 000細胞）でニューロジェ二 ン 3発現細胞を 4日間培養（培養温度： 37°C、 pH : 7. 4)して誘導した。ニューロジェ ニン 3発現細胞の選別には、ニューロジェニン 3遺伝子のプロモーターの下流に増強 黄色蛍光タンパク質（eYFP)をコードして!/、る遺伝子を導入し eYFP発現細胞を指 標としてセルソーター（MoFlo)を用いて選別した。 eYFP遺伝子の導入には、 Ngn3 プロモーター下に eYFP (enhanced yellow fluorescence；増強黄色蛍光タンパク質）を 発現するプラスミドベクター： ngn3 - Spel - eYFP (以下の論文から当該プラスミドべ クタ一は入手可能である。 Mellitzer G, Martin M, Sidhoum— Jenny M, Orvain C， Bart hs J, Seymour PA, Sander M, Gradwohl . Pancreatic islet progenitor cells in neuro genin 3-yellow fluorescent protein knock-add-on mice.Mol Endocrinol.18(11):2765~ 76.2004)を用いた。その後、一過性に ngn3— Spel— eYFPを Nucelofector (Ama xa社、取り扱い規約に則れば万人が使用可能である）を使用して導入した。ベクター ： ngn3 - Spel- eYFP (Mellitzer Gら、 Mol. Endocrinol. (2004) Nov; 18(l l):2765- 76 参照。 Mellitzerからベクターを万人が提供を受けることができる。）をヌクレオフエク ター（Amaxa社製、操作方法は以下のホームページを参照 http：〃 www.wako-chem. co.jp/ siyaku/info/ gene/ article/ amaxa/ df/Manual.pdf)を用 Vヽ飞退 子専人 (細胞 培養濃度： 5 X 105細胞/ mL、培養温度： 37°C、 pH : 7. 4)した。 48時間後に、当該 細胞を、セルソーター MoFloを使用して eYFPの発現を検討したところ、原始瞵の 3 分の 1力 SeYFPを発現して!/、た。 eYFP発現細胞（ニューロジェニン発現細胞）を Mo Floを使用して回収した。そして、 Ngn3発現細胞を MoFlo (Dako— Cytomation社 、取り扱い規約に則れば万人が使用可能である）を使用して回収した。 Neurogenin 3 expressing cells were increased by culturing the primitive koji obtained in step (b) using a conditioned medium. Human immortalized hepatic sinusoidal endothelial cell line TMNT— 1 cell (Depositor: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, Address: 1-chome, Tsukuba, Ibaraki, Japan 1-Chuo 6 ( Postal code 305—8566), deposit date: April 16, 2002, deposit number: FERM BP—8017, available to everyone, low glucose D MEM medium (glucose concentration: 1000 mg / L, catalog number 31600— 083, manufactured by Invitrogen) for 1 day (cell culture concentration: 8 X 10 5 cells / mL, culture temperature: 37 ° C, pH: 7.4), γ-secretase inhibitor XVIII (Calbiochem (catalog number 565779), or Alexis Biochemicals (catalog number: ALX—270—415—C250, or ALX—270—415—M001), available for purchase), FGF—2 (20ng / mU , using a material obtained by adding EGF (20 ng / mU, Raminin 1 (concentration 5 ag / cm 2) (BD Biosciences Cultivated in a T-25 flask (cell culture concentration 50, 000 cells / mL, total 200,000 cells) processed for 4 days (culture temperature: 37 °) C, pH: 7.4) In order to select for neurogenin 3 expressing cells, a gene encoding the enhanced yellow fluorescent protein (eYFP) downstream of the neurogenin 3 gene promoter! The eYFP-expressing cells were selected using a cell sorter (MoFlo) as an indicator. Plasmid vector that expresses eYFP (enhanced yellow fluorescence protein) under the promoter: ngn3-Spel-eYFP (This plasmid vector is available from the following paper. Mellitzer G, Martin M, Sidhoum— Jenny M, Orvain C, Bart hs J, Seymour PA, Sander M, Gradwohl. Pancreatic islet progenitor cells in neuro genin 3-yellow fluorescent protein knock-add-on mice.Mol Endocrinol. 18 (11): 2765 ~ 76.2004) It was. After that, ngn3—Spel—eYFP was introduced temporarily using Nucelofector (Amaxa, which can be used by everyone according to the handling rules). Vector: ngn3-Spel-eYFP (see Mellitzer G et al., Mol. Endocrinol. (2004) Nov; 18 (ll): 2765-76. Vectors are available to everyone from Mellitzer). For the operation method, please refer to the following website: http: 〃 www.wako-chem. Co.jp/siyaku/info/gene/article/amaxa/df/Manual.pdf) V (Cell culture concentration: 5 × 10 5 cells / mL, culture temperature: 37 ° C., pH: 7.4). After 48 hours, when the cells were examined for eYFP expression using the cell sorter MoFlo, they expressed SeYFP! eYFP-expressing cells (neurogenin-expressing cells) were collected using Mo Flo. Then, Ngn3-expressing cells were collected using MoFlo (Dako-Cytomation, which can be used by everyone according to the handling rules).
製造例 2 Production example 2
RCNK— 1細胞株の樹立  RCNK—Establishment of one cell line
カナダのアルバータ大学から提供された健常分離ヒト瞵島（カナダ、アルバータ大 学、ヒト瞵島移植プログラム、ジョナサン レイキー（Jonathan RT. Lakey)博士より、万 人が入手可能である）から、球形のきれ!/、な形態を呈して!/、るものを実体顕微鏡（ST EMI, Carl Zeiss社、ドイツ）下に hand pickup法にて 10個選別し、これを T25培 養フラスコに播いた。培養液は、 CMRL1066,インビトロジェン社製）に 10%ゥシ胎 仔血清（FCS、シグマ社）、 10— 7mol/lインスリン（シグマ社）、 10— 6mol/lデキサメタ ゾン（シグマ社）、 25 g/ml上皮成長因子（EGF、シグマ社）、 10mMニコチンアミ ド（シグマ社）、抗生物質のペニシリン G/ストレプトマイシン（シグマ社）の,袓成のもの を基本培養液として用いた。当該勝島を、ヌクレオフエクタ一システム用緩衝液 (Nucl eofector™ Solution,和光純薬工業株式会社製） 1mlで希釈し、そこに pYK— 1のプ ラスミド DNA (国際公開第 2005/100546号パンフレットを参照）の TE緩衝液（TE buffer,シグマ社製）の懸濁液（1 n ^/ ιΛ) 2 1を添加して、ヌクレオフエクターシステ ム（Nucleofector™ system,和光純薬工業株式会社製）により、該システムのプロトコ ールにしたがって遺伝子導入した。得られた細胞を T— 25フラスコに播種し、 CS - C 無血清培地（CS— SF— 4Z0— 500、大日本製薬株式会社販売）にて培養を継続し た。遺伝導入して 48時間後に、 100
Figure imgf000041_0001
じ無血清 培地にて、耐性クローンの選択を行なった。選択開始から 2週間後に耐性クローンの 出現を認め、 4週間後にクローニングリングを使用し、 RCNK— 1細胞株を樹立した。 Spherical cuts from healthy isolated human islets provided by the University of Alberta, Canada (available to everyone from Canada, University of Alberta, Human Islet Transplant Program, Dr. Jonathan RT. Lakey) ! /, Which was in the form of! /, Was selected using a hand pickup method under a stereomicroscope (ST EMI, Carl Zeiss, Germany) and seeded in a T25 culture flask. Culture solution, CMRL1066, 10% © shea womb仔血Qing Invitrogen) (FCS, Sigma), 10- 7 mol / l insulin (Sigma), 10- 6 mol / l dexamethasone (Sigma), 25 g / ml epidermal growth factor (EGF, Sigma), 10 mM nicotinamide (Sigma), and antibiotic penicillin G / streptomycin (Sigma) were used as the basic culture solution. Dilute Katsushima with 1 ml of Nucleofecta System Buffer (Nucle eofector ™ Solution, Wako Pure Chemical Industries, Ltd.) and add pYK-1 Add a suspension (1 n ^ / ιΛ) 2 1 of TE buffer (TE buffer, manufactured by Sigma) of rasmid DNA (see WO 2005/100546 pamphlet) Nucleofector ™ system (manufactured by Wako Pure Chemical Industries, Ltd.) was used for gene transfer according to the protocol of the system. The obtained cells were seeded in a T-25 flask and continued to be cultured in a CS-C serum-free medium (CS-SF-4Z0-500, sold by Dainippon Pharmaceutical Co., Ltd.). 48 hours after gene transfer, 100
Figure imgf000041_0001
Resistant clones were selected in the same serum-free medium. The emergence of resistant clones was observed 2 weeks after the start of selection, and the RCNK-1 cell line was established using cloning rings 4 weeks later.
[0129] 実施例 4 [0129] Example 4
工程 (d)インスリン分泌細胞への分化  Step (d) Differentiation into insulin secreting cells
(DMEM培地を用 1/、る方法）  (How to use DMEM medium 1 /)
工程 (c)で得られたニューロジェニン 3発現細胞を、 3%または 5%のゥシ胎仔血清 (3%ゥシ胎仔血清にて 3日間、ついで 5%ゥシ胎仔血清にて 4日間培養）、肝細胞増 殖因子 10ng/mL、ニコチンアミド 10mmol/L、ェクスディン一 4 100nmol/L, トログリタゾン 10 mol/L、硫酸亜鉛 16· 7 μ mol/Lを加えた低グルコース DME M培地を用いて 7日間培養（培養温度： 37°C、 pH : 7. 4)した。培養皿としては、マト リゲルで加工した 6—well— plateを使用した。工程（d)においては、毎日、血清非添 加の高グルコース DMEM培地（グルコース濃度： 4500mg/L、カタログ番号： 128 00— 082、インビトロジェン社製）にて 2時間培養（培養温度： 37°C、 pH : 7. 4)した 後、前記低グルコース DMEM培地で 22時間の培養を 7日間継続した。なお、高グ ルコース培地にも肝細胞増殖因子 10ng/mL、ニコチンアミド 10mmol/L、ェクス ディン一 4 100nmol/L,トログリタゾン; 10〃 mol/L、硫酸亜鉛 16 · 7〃 mol/Lを 加えた。  Neurogenin 3 expressing cells obtained in step (c) are cultured in 3% or 5% urine fetal serum (3 days in 3% urine fetal serum and then 4 days in 5% urinary fetal serum) , Using low glucose DME M medium supplemented with 10 ng / mL hepatocyte growth factor, 10 mmol / L nicotinamide, 4 100 nmol / L dexdin, 10 mol / L troglitazone, 16.7 μmol / L zinc sulfate The cells were cultured for a day (culture temperature: 37 ° C, pH: 7.4). As a culture dish, a 6-well-plate processed with Matrigel was used. In step (d), culture is performed daily for 2 hours in a high glucose DMEM medium (glucose concentration: 4500 mg / L, catalog number: 128 00-082, manufactured by Invitrogen) without serum (culture temperature: 37 ° C). , PH: 7.4), and then cultured for 22 hours in the low glucose DMEM medium for 7 days. Hepatocyte growth factor 10 ng / mL, nicotinamide 10 mmol / L, dexdin 4 100 nmol / L, troglitazone; 10 〃 mol / L, zinc sulfate 16 · 7 〃 mol / L were also added to the high glucose medium. .
[0130] (RCNK 1馴化培地を用いる方法）  [0130] (Method using RCNK 1 conditioned medium)
工程 (c)で得られたニューロジェニン 3発現細胞を、低グルコース RCNK— 1馴化 培地を用いてインスリン分泌細胞へ分化誘導を行った。 RCNK— 1細胞を低ダルコ ース DMEM+ 10%FBSで培養し、フラスコに一杯になった時点で核内限局信号（ NLS)標識された Cre組換え酵素を産生する複製不可能な組換えアデノベクター Ax CANCre (3 X 108pfu/ml) (理研ジーンバンクより入手可能、 RDB No. 1748)を MOI (感染多重度） = 10〜50 (15が好ましい）で 2〜4時間（3時間が好ましい）感染 させ、 RCNK—1細胞を Cre/loxPシステムで不死化遺伝子の SV40Tを取り除い た（培養温度： 35〜37°C)。感染後は、 RCNK— 1細胞を低グルコース DMEM+ 5 %FBSで 2日間（48時間）培養して、その後、低グルコース DMEMのみで培養した 。 24時間培養した後に、当該低グルコース RCNK— 1馴化培地を回収した。なお当 該操作は、無血清であるため細胞が弱るまで 4回から 5回可能である。当該低ダルコ ース RCNK— 1馴化培地に 3 %または 5 %のゥシ胎仔血清（3 %ゥシ胎仔血清にて 3 日間、ついで 5%ゥシ胎仔血清にて 4日間培養）、肝細胞増殖因子 10ng/mL、ニコ チンアミド 10mmol/L、ェクスディン一 4 100nmol/L,トログリタゾン 10 μ ol/ L、硫酸亜鉛 16. 7 ,1 mol/Lを加えた培地を用いて 7日間培養（培養温度： 37°C、 p H : 7. 4)した。低グルコース RCNK— 1馴化培地は、培養皿としては、マトリゲルで加 ェした 6—well— plateを使用した。工程（d)においては、毎日、血清非添加の高グ ルコース濃度に調製した KRBB培地（Krebs Ringer Balanced Buffered (KRBB) (14 3mM Na, 5. 8mM K, 2. 5mM Ca, 1. 2mM Mg2, 124. ImM CI, 1. 2mM PO-4, 1. 2mM SO— 4, 25mM HCO— 3, lOmM HEPE S , 0. 2% BSAを含む、以下の論文に詳細に書いてある。 Srivastava S， Goren HJ .Insulin constitutively secreted by beta-cells is necessary for glucose-stimulated ins ulin secretion. Diabetes. 2003 ;52(8):2049_56.参照））に肝細胞増殖因子 lOng/m L、ニコチンアミド 10mmol/L、ェクスディン一 4 100nmol/L,トログリタゾン 10 μ mol/L,硫酸亜鉛 16. 7 μ mol/Lを加えた培地で 2時間培養（培養温度： 37°C、 p H： 7. 4)した後、前記 RCNK— 1馴化培地で 22時間の培養を 7日間継続した。 実施例 5 Neurogenin 3 expressing cells obtained in step (c) were induced to differentiate into insulin secreting cells using a low glucose RCNK-1 conditioned medium. RCNK— a non-replicating recombinant adenovector that produces Cre recombinase labeled with a nuclear localization signal (NLS) when a single cell is cultured in low-Darcose DMEM + 10% FBS and filled to a flask. Ax CANCre (3 X 10 8 pfu / ml) (available from RIKEN GENE BANK, RDB No. 1748) MOI (multiplicity of infection) = 10-50 (15 is preferred) 2-4 hours (3 hours preferred) RCNK-1 cells were infected and the immortalized gene SV40T was removed with the Cre / loxP system (culture temperature: 35-37 ° C). After infection, RCNK-1 cells were cultured in low glucose DMEM + 5% FBS for 2 days (48 hours), and then cultured in low glucose DMEM alone. After culturing for 24 hours, the low glucose RCNK-1 conditioned medium was collected. This operation can be performed 4 to 5 times until cells weaken because it is serum-free. 3% or 5% urine fetal serum (3 days in 3% urine fetal serum, then 4 days in 5% urinary fetal serum) in the low-darcose RCNK-1 conditioned medium, hepatocyte proliferation Culture for 7 days in a medium containing 10ng / mL of factor, 10mmol / L of nicotinamide, 100nmol / L of Exdin, 10μol / L of troglitazone, 16.7,1mol / L of zinc sulfate (culture temperature: 37 ° C, pH: 7.4). As the low-glucose RCNK-1 conditioned medium, a 6-well-plate added with Matrigel was used as a culture dish. In step (d), KRBB medium (Krebs Ringer Balanced Buffered (KRBB) (14 3 mM Na, 5.8 mM K, 2.5 mM Ca, 1.2 mM Mg2, 124. ImM CI, 1.2 mM PO-4, 1.2 mM SO—4, 25 mM HCO—3, lOmM HEPE S, 0.2% BSA, including details in the following paper: Srivastava S, Goren HJ .Insulin constitutively secreted by beta-cells is necessary for glucose-stimulated ins ulin secretion. Diabetes. 2003; 52 (8): 2049_56))) and hepatocyte growth factor lOng / ml, nicotinamide 10 mmol / l, 4 After culturing for 2 hours in a medium supplemented with 100 nmol / L, troglitazone 10 μmol / L, and zinc sulfate 16.7 μmol / L (culture temperature: 37 ° C, pH: 7.4), the above RCNK — 22 hours of culture in 1 conditioned medium continued for 7 days. Example 5
インスリン分泌細胞の Maf A遺伝子発現 Maf A gene expression in insulin-secreting cells
本発明の分化誘導方法により得られたインスリン分泌細胞（工程 (d)の培養 7日目 ) を位相差顕微鏡下にて観察した（図 4 (a)参照）。核 1はドット状に赤く染まっており、 直径が約 200 mの球状を呈しているインスリン分泌細胞群全体 2は緑に染まってい ることから MafA遺伝子の発現が確認できた。 MafA遺伝子の発現は、 MafA抗体を 用いて行った（MafA抗体（BL1069、カタログ番号： A300— 611A、 BETHYL L ABORATORIES, INC.社製。使用方法に関しては、製品に添付の取扱説明書を 参照）。なお、添付の写真は白黒 2階調に変換したものであるので、染色された色は 表現されていない。本発明の分化誘導方法により得られたインスリン分泌細胞は、 M af A遺伝子を発現する能力を有するのに対し、従来技術であるダムール法で分化誘 導されたインスリン分泌細胞は MafA遺伝子を発現して!/、な!/、ことが報告されて!/、る ( ダム一ノレら、 Nat. Biotech., published online, 19 October 2006， doi: 10.1038/nbtl25 9.参照）。この結果からも、本発明の分化誘導方法は従来技術と比較して優れている ことが明らかとなった。本発明が優れている要因としては、ダムール法では FGF— 10 を使用しているのに対し、本発明では工程 (c)において EGFおよび FGF— 2を使用 した点が考えられる。 FGF— 10は、勝臓の発生過程で /3細胞への分化を抑制（ミラ レスら、 Int J Dev Biol. 2006;50(1): 17_26·参照）し、瞵外分泌細胞を誘導する（ブシュ シャンら、 Development. 2001; 128(24):5109_17参照）ことが報告されている。一方、 E GFと FGF— 2が瞵臓の発生過程で /3細胞への分化を促進する（Cras-Meneur C， El ghazi L,し zermcnow P, ^charfmann R. Epidermal growth factor increases undifferent iated pancreatic embryonic cells in vitro: a balance between proliferation and differe ntiation. Diabetes. 2001 Jul;50(7): 1571-9)ことが報告されている。 Insulin-secreting cells obtained by the differentiation-inducing method of the present invention (the 7th day of culture in step (d)) were observed under a phase contrast microscope (see FIG. 4 (a)). Nucleus 1 is stained red in a dot shape, and the entire insulin-secreting cell group 2 having a spherical shape with a diameter of about 200 m is stained green, confirming the expression of the MafA gene. MafA gene expression (MafA antibody (BL1069, catalog number: A300-611A, manufactured by BETHYL L ABORATORIES, INC. For the method of use, refer to the instruction manual attached to the product). The stained color is not expressed because it is converted into a gradation, whereas the insulin-secreting cells obtained by the differentiation induction method of the present invention have the ability to express the Maf A gene, whereas It has been reported that insulin-secreting cells induced to differentiate by the damour method, which expresses the MafA gene! /, Is! // (Dam I Nore et al., Nat. Biotech., Published online , 19 October 2006, doi: 10.1038 / nbtl25 9.) This result also revealed that the differentiation induction method of the present invention is superior to the prior art. In contrast to the damul method, which uses FGF-10, In the invention, EGF and FGF-2 are considered to be used in step (c) FGF-10 suppresses differentiation to / 3 cells during the development of viscera (Mirares et al., Int J Dev Biol. 2006). ; 50 (1): 17_26)) and induced exocrine cells (see Bushshan et al., Development. 2001; 128 (24): 5109_17), whereas EGF and FGF-2 Promotes differentiation into / 3 cells during development of the spleen (Cras-Meneur C, El ghazi L, zermcnow P, ^ charfmann R. Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differe ntiation. Diabetes. 2001 Jul; 50 (7): 1571-9).
実施例 6 Example 6
インスリン分泌細胞のインスリン分泌能の確認および形態学的検討 Confirmation and morphological examination of insulin secretory capacity of insulin secreting cells
本発明の分化誘導方法により得られたインスリン分泌細胞の形態を位相差顕微鏡 下にて観察した（図 4 (b)参照）。なお、核 1は赤く染まっており、分化誘導 4、 7日目に おいては直径が約 200 mの球状を呈しているインスリン分泌細胞群全体 2はインス リン分泌を示す緑に染まつている。インスリンの検出は抗インスリン抗体を用レ、て行つ た（Sta. Cruz sc— 9168,使用方法は製品に添付させている取り扱い説明書を 参照）。インスリン分泌細胞は培養日数の増加に伴!/、緑色を呈して!/、ること力 S確認さ れた。なお、添付の写真は白黒 2階調に変換したものであるので、染色された色は表 現されていない。図 4の写真右下のスケールバーは共に 200 mを示す。インスリン 分泌細胞は培養日数と共に球状の形態を形成してきている。 [0133] 分化誘導 2日目の細胞の形態学検討 The morphology of insulin-secreting cells obtained by the differentiation induction method of the present invention was observed under a phase contrast microscope (see FIG. 4 (b)). In addition, nucleus 1 is dyed red, and on the 4th and 7th days of differentiation induction, the entire insulin-secreting cell group 2 having a spherical shape with a diameter of about 200 m 2 is dyed green indicating insulin secretion. . Insulin was detected using an anti-insulin antibody (Sta. Cruz sc-9168, refer to the instruction manual attached to the product for usage). Insulin-secreting cells were confirmed to increase in the number of culture days! The attached photo is converted to black and white with two gradations, so the dyed color is not represented. Both scale bars in the lower right of the photo in Fig. 4 indicate 200 m. Insulin-secreting cells have formed a spherical shape with the number of days of culture. [0133] Morphological study of cell differentiation on day 2
1.形状が「いびつな球状： 3点」、 2.辺縁の形状が「やや均整のとれた： 3点」、 3.統 合性が「断片化した (fragmented) : 1点」、 4.直径が「培養瞵島の個々が 125〜； 175 111 : 3点」、 5.染色の均一性が「やや均一： 3点」評価値の合計が 13点であった（図 4参照)。  1. Shape is “loose spherical: 3 points”, 2. Edge shape is “slightly balanced: 3 points”, 3. Consistency is “fragmented: 1 point”, 4 The diameter was 125 to 175 111: 3 points for each of the cultured islands. 5. The uniformity of staining was “slightly uniform: 3 points”. The total of the evaluation values was 13 points (see FIG. 4).
[0134] 分化誘導 4日目の細胞の形態学検討  [0134] Morphological study of cell differentiation on day 4
1.形状が「球状： 5点」、 2.辺縁の形状が「やや均整のとれた： 3点」、 3.統合性が「 ややソリッド/コンパクト： 3点」、 4.直径が「培養瞵島の個々中 10%以上が 200 in より大きい： 5点」、 5.染色の均一性が「やや均一： 3点」評価値の合計が 19点であつ た（図 4参照)。  1. Shape is “spherical: 5 points” 2. Edge shape is “slightly balanced: 3 points” 3. Integration is “slightly solid / compact: 3 points” 4. Diameter is “culture” 10% or more of the islands were larger than 200 in: 5 points, and 5. Dyeing uniformity was “slightly uniform: 3 points”. The total of the evaluation values was 19 points (see Figure 4).
[0135] 分化誘導 7日目の細胞の形態学検討  [0135] Morphological study of cell differentiation on day 7
1.形状が「球状： 5点」、 2.辺縁の形状が「均整のとれた： 5点」、 3.統合性が「ソリツ ド/コンパクト： 5点」、 4.直径が「培養瞵島の個々中 10%以上が 200 mより大き!/ヽ : 5点」、 5.染色の均一性が「完全に均一： 5点」評価値の合計が 25点であった（図 4 参照）。一方、ダムール法により得られたインスリン分泌細胞は球状にはなっていなか つた。  1. Shape is "spherical: 5 points" 2. Edge shape is "balanced: 5 points" 3. Integration is "solid / compact: 5 points" 4. Diameter is "culture pot" More than 10% of the islands are larger than 200 m! / ヽ: 5 points ”, 5. Dyeing uniformity is“ completely uniform: 5 points ”The total evaluation value was 25 points (see Figure 4) . On the other hand, insulin-secreting cells obtained by the Damour method were not spherical.
[0136] ダムール法による細胞の形態学的検討  [0136] Morphological study of cells by Damour method
1.形状が「扁平： 1点」、 2.辺縁の形状が「不ぞろいの：1点」、 3.統合性が「断片化 した： 1点」、 4.直径が「培養瞵島の全体が 100 m未満： 1点」、 5.染色の均一性が 「やや均一： 3点」評価値の合計が 7点であった（ダム一ルら、 Nat. Biotech., published online, 19 October 2006， doi: 10.1038/nbtl259.参照）。  1. The shape is “flat: 1 point” 2. The edge shape is “uneven: 1 point” 3. The integrity is “fragmented: 1 point” 4. The diameter is “the whole culture island” Is less than 100 m: 1 point ”, 5. Uniformity of staining is“ Slightly uniform: 3 points ”. The total of the evaluation values was 7 points (Dam Ill et al., Nat. Biotech., Published online, 19 October 2006 , Doi: 10.1038 / nbtl259.)
形態学的検討結果より、本発明の分化誘導方法により得られたインスリン分泌細胞 は従来技術 (ダムール法）より優れていることが明ら力、となった。  From the result of morphological examination, it became clear that insulin-secreting cells obtained by the differentiation induction method of the present invention are superior to the prior art (Damur method).
[0137] 実施例 7 [0137] Example 7
本発明の手法により得られたインスリン分泌細胞の機能評価  Functional evaluation of insulin-secreting cells obtained by the method of the present invention
1)インスリン分泌  1) Insulin secretion
実施例 4の DMEM培地を用いた培養により得られたインスリン分泌細胞または実 施例 4の RCNK— 1馴化培地を用いた培養により得られたインスリン分泌細胞を、そ れぞれ DMEM (図 5 (a)および表 1 (a)参照）または KRBB (図 5 (b)および表 1 (b)参 照）に、 3. 3mM濃度（低グノレコース）ないしは 25mM濃度（高グノレコース）のグノレコ ースを添加して、各 2時間培養（培養温度： 37°C、 pH : 7. 4)して、培地中のインスリ ン分泌、直を測 し (Ultrasensitive insulin kit、 Mercodia Ultrasensitive M ouse Insulin ELISA、製品番号： 10— 1150— 01、 Mercodia社製）従来技術 ( ダム一ノレ法、およびシュローダー法 (シュローダーら、 nature protocols vol.1, no.2, p. 495-507， 2006) )との比較を行った（図 5、表 1 (a)および表 1 (b)参照）。すなわち、低 グルコースにて 2時間刺激後高グルコースにて 2時間刺激（「グルコース刺激：低→高 」と略す）した後の培地中のインスリン分泌量を測定、または低グルコースにて 2時間 刺激後高グルコースにて 2時間刺激後再度低グルコースにて 2時間刺激（「ダルコ一 ス刺激：低→高→低」と略す）した後の培地中のインスリン分泌量を測定した。その結 果、本発明の分化誘導方法により得られたインスリン分泌細胞は、低グルコース DM EMで 2日、 4日、 7日、と培養日数が増加するごとに、「グルコース刺激:低→高」した 際のインスリン分泌量が増加しており（それぞれ、図 5の横軸の 1、 3、 5を参照。）、一 方で「グルコース刺激：低→高→低」した際のインスリン分泌量は減少しており（それ ぞれ、図 5の横軸の 2、 4、 6を参照）、インスリン分泌細胞がグルコース応答性を有す ることが明らかとなった。なお比較として、従来技術であるシュローダー法によるインス リン分泌細胞を「ダルコース刺激：低→高」した後の培地中のインスリン分泌量（図 5の 横軸の 7を参照）、ダムール法によるインスリン分泌細胞を「グルコース刺激:低→高 →低」した後の培地中のインスリン分泌量（図 5の横軸の 8を参照）、ダムール法による インスリン分泌細胞を「グルコース刺激:低→高」した後の培地中のインスリン分泌量（ 図 5の横軸の 9を参照）、ダムール法によるインスリン分泌細胞を「グルコース刺激：低 →高→低」した後の培地中のインスリン分泌量 (図 5の横軸の 10を参照）を測定した。 その結果、本発明の分化誘導方法により得られたインスリン分泌細胞は、従来技術 によるインスリン分泌細胞と比較して優れたインスリン分泌能を有し、グルコース応答 性も優れていることが明らかとなった。 Insulin-secreting cells obtained by culturing using the DMEM medium of Example 4 or insulin-secreting cells obtained by culturing using the RCNK-1 conditioned medium of Example 4 were used. Each of DMEM (see Fig. 5 (a) and Table 1 (a)) or KRBB (see Fig. 5 (b) and Table 1 (b)) has a concentration of 3.3 mM (low nocturne) or 25 mM (high). Add gnoleose) and incubate for 2 hours each (culture temperature: 37 ° C, pH: 7.4) to measure insulin secretion and straightness in the medium (Ultrasensitive insulin kit, Mercodia Ultrasensitive) Mouse Insulin ELISA, product no .: 10— 1150— 01, Mercodia) Conventional technology (Dam I Nore method and Schroder method (Schroder et al., Nature protocols vol.1, no.2, p. 495-507, 2006) )) (See Fig. 5, Table 1 (a) and Table 1 (b)). That is, measure insulin secretion in the medium after stimulation with low glucose for 2 hours and stimulation with high glucose for 2 hours (abbreviated as “glucose stimulation: low → high”), or after stimulation with low glucose for 2 hours The amount of insulin secretion in the medium after stimulation with high glucose for 2 hours and stimulation with low glucose again for 2 hours (abbreviated as “Dalcos stimulation: low → high → low”) was measured. As a result, the insulin-secreting cells obtained by the differentiation-inducing method of the present invention were `` glucose-stimulated: low to high '' every time the culture days increased to 2, 4, and 7 with low glucose DMEM. (See 1, 3, and 5 on the horizontal axis in Fig. 5, respectively.) On the other hand, the amount of insulin secretion when “glucose stimulation: low → high → low” was observed. It decreased (see 2, 4, and 6 on the horizontal axis in Fig. 5), indicating that insulin-secreting cells have glucose responsiveness. For comparison, the amount of insulin secreted in the medium after “insulin-secreting cells: low to high” (see 7 on the horizontal axis in FIG. 5) and insulin secretion by the Damour method were compared with the conventional Schroder method. Insulin secretion in the medium after “cell-stimulated: low → high → low” (see 8 on the horizontal axis in FIG. 5), after “Glucose-stimulated: low → high” by the Damour method Of insulin secretion in the medium (see 9 on the horizontal axis in Fig. 5), insulin secretion in the medium after the glucose-stimulated cells by the Damour method were "glucose stimulated: low → high → low" (horizontal in Fig. 5) Axis 10) was measured. As a result, it was clarified that the insulin-secreting cells obtained by the differentiation-inducing method of the present invention have superior insulin secretion ability and excellent glucose responsiveness as compared with insulin-secreting cells according to the prior art. .
[表 1(a)]
Figure imgf000046_0001
Figure imgf000046_0002
[Table 1 (a)]
Figure imgf000046_0001
Figure imgf000046_0002
表 1 (b) Table 1 (b)
Figure imgf000047_0001
Figure imgf000047_0001
本発明の手法により得られたインスリン分泌細胞の機能評価 Functional evaluation of insulin-secreting cells obtained by the method of the present invention
2)トルプタミドによる刺激 2) Stimulation with tolptamide
実施例 4により得られたインスリン分泌細胞を、低グルコース濃度の KRBBを培地と して用レ、てインスリン分泌量を測定（図 6 (a)および表 2参照）後、培地を低ダルコース 濃度の KRBBに 10 mol/Lトルプタミド（シグマ社製）で 1時間刺激した際の培地 中のインスリン分泌量を測定（図 6 (b)および表 2参照）した。その結果、本発明の分 化誘導方法により得られたインスリン分泌細胞は従来技術と比較して高いインスリン 分泌能を示した。  Insulin-secreting cells obtained in Example 4 were measured using a low glucose concentration KRBB as the medium, and the amount of insulin secretion was measured (see Fig. 6 (a) and Table 2). The amount of insulin secreted in the medium after stimulation with KRBB with 10 mol / L tolptamide (Sigma) for 1 hour was measured (see Fig. 6 (b) and Table 2). As a result, the insulin-secreting cells obtained by the differentiation induction method of the present invention showed a higher insulin-secreting ability than the conventional technique.
[表 2] [Table 2]
Figure imgf000049_0001
Figure imgf000049_0002
Figure imgf000049_0001
Figure imgf000049_0002
Figure imgf000049_0003
Figure imgf000049_0003
3)糖尿病マウスへの移植実験 3) Transplant experiment to diabetic mice
重症免疫不全 (SCID (severe combined immunodeficiency) )マウス（体重 20g、 日 本クレア株式会社）にストレブトゾトシン（220mg/kg)を腹腔内に投与し、一週後に 血糖値が 360mg/dL以上に 2日続けてなった際に、糖尿病と判断した。当該糖尿 病マウスを移植実験に使用した。本発明の分化誘導方法にて得られたインスリン分 泌細胞（5 X 106細胞）、未分化なマウス ES細胞（5 X 106細胞）、および正常マウス瞵 島 450個を移植細胞として用いた。それぞれ 5匹の糖尿病マウスに細胞移植を行つ た。なお、正常な健常マウス（3匹）および移植しない糖尿病マウスをコントロールとし て使用した。マウスの左腎皮膜下に移植した後の血糖値の平均を図 7および表 3に 示す。移植後の最初の 5日間は中間型ヒトインスリン NPH (Novo Nordisk社製）を 2単位皮下注射し、その後の 5日間は中間型ヒトインスリン NPH (Novo Nordisk社 製）を 1単位皮下注射した場合の移植後の血糖値の平均を図 7に示す。その後、 3日 おきに血糖値を測定し、移植後 3週目以降は一週間に一回の割合で血糖値を Porta ble glucose meter FreeStlyle TM (TheraSence社製）を使用して測定した。 マウス ES細胞を移植したマウスでは、 NPH投与を終えた後に血糖値が再度 360mg /dL以上になり、治療効果がないことが明らかとなった。これとは対照的に、本発明 の分化誘導方法により得られたインスリン分泌細胞を移植したマウスは、健常マウス 瞵島を移植したマウスと同様にグルコース濃度が 100mg/dL程度を維持することが できた。これは、当該インスリン分泌細胞の機能が健常マウス瞵島と同程度であり、治 療効果があることを示す。なお、治療を行わない糖尿病マウス全て（非治療群）およ び ES細胞を移植したマウス全ては 10週間以内に高血糖により死亡した。 Streptozotocin (220 mg / kg) was intraperitoneally administered to severe immunodeficiency (SCID (severe combined immunodeficiency)) mice (body weight 20 g, Japan Clare Co., Ltd.), and blood glucose level increased to 360 mg / dL or more after 1 week. When I continued the day, I was diagnosed with diabetes. The diabetic mice were used for transplantation experiments. Insulin secreting cells (5 × 10 6 cells) obtained by the differentiation induction method of the present invention, undifferentiated mouse ES cells (5 × 10 6 cells), and 450 normal mouse islets were used as transplanted cells. . Cell transplantation was performed on 5 diabetic mice each. Normal healthy mice (3 mice) and non-transplanted diabetic mice were used as controls. Figure 7 and Table 3 show the average blood glucose levels after transplantation under the left renal capsule of mice. In the first 5 days after transplantation, 2 units of intermediate human insulin NPH (Novo Nordisk) were injected subcutaneously, and the next 5 days were injected with 1 unit of intermediate human insulin NPH (Novo Nordisk). Figure 7 shows the average blood glucose level after transplantation. Thereafter, blood glucose level was measured every 3 days, and after 3 weeks after transplantation, blood glucose level was measured once a week using a Portable glucose meter FreeStlyle ™ (TheraSence). In mice transplanted with mouse ES cells, the blood glucose level again increased to 360 mg / dL after NPH administration, indicating that there was no therapeutic effect. In contrast, a mouse transplanted with insulin-secreting cells obtained by the differentiation induction method of the present invention can maintain a glucose concentration of about 100 mg / dL, similar to a mouse transplanted with healthy mice Kashiwajima. It was. This indicates that the function of the insulin-secreting cells is similar to that of normal mouse Kashiwajima and has a therapeutic effect. All diabetic mice not treated (non-treated group) and all mice transplanted with ES cells died of hyperglycemia within 10 weeks.
[表 3] 移植後の日数 （日） 0 3 6 9 12 15 18 21 28 35 42 49 56 本発明のインスリン分泌細胞 (5百万個）を移植した糖尿病マウスの血糖値 (mg/dL)[Table 3] Days after transplantation (days) 0 3 6 9 12 15 18 21 28 35 42 49 56 Blood glucose level of diabetic mice transplanted with insulin-secreting cells of the present invention (5 million cells) (mg / dL)
Figure imgf000051_0001
動物 no.l 392 189 146 117 106 99 100 104 103 105 107 103 104 動物 no.2 379 168 167 131 126 111 103 95 106 95 104 110 108 動物 no.3 383 143 179 166 157 124 109 94 98 96 95 97 96 動物 no.4 389 154 156 155 140 110 107 103 102 106 109 115 102 動物 no.5 367 163 123 157 123 112 106 98 97 98 96 103 100 平均 382.0 163.4 154.2 145,2 130.4 111.2 105.0 98.8 101.2 100.0 102.2 105.6 102.0 標準偏差 8.76 15.37 19.09 18.25 17.14 7.93 3.16 4.07 3.31 4.60 5.71 6.25 4.00 マウス ES細胞（5百万個）を移植した糖尿病マウスの血糖値 （mg/dL)
Figure imgf000051_0001
Animal no.l 392 189 146 117 106 99 100 104 103 105 107 103 104 Animal no.2 379 168 167 131 126 111 103 95 106 95 104 110 108 Animal no.3 383 143 179 166 157 124 109 94 98 96 95 97 96 animals no.4 389 154 156 155 140 110 107 103 102 106 109 115 102 animals no.5 367 163 123 157 123 112 106 98 97 98 96 103 100 Average 382.0 163.4 154.2 145,2 130.4 111.2 105.0 98.8 101.2 100.0 102.2 105.6 102.0 Standard deviation 8.76 15.37 19.09 18.25 17.14 7.93 3.16 4.07 3.31 4.60 5.71 6.25 4.00 Mouse Blood glucose level (mg / dL) in diabetic mice transplanted with ES cells (5 million)
面物 no.l 387 211 256 266 374 393 395 389 398 401 412 421 動物 no.2 369 164 213 247 372 378 388 404 409 414  No.l 387 211 256 266 374 393 395 389 398 401 412 421 Animal no.2 369 164 213 247 372 378 388 404 409 414
動物 no.3 371 172 241 253 358 386 377 388 389 395 389 392 389 動物 no.4 382 188 231 289 367 394 386 393 399 387 379 386 397 動物 no.5 388 181 254 303 383 396 405 400 404 411 418  Animal no.3 371 172 241 253 358 386 377 388 389 395 389 392 389 Animal no.4 382 188 231 289 367 394 386 393 399 387 379 386 397 Animal no.5 388 181 254 303 383 396 405 400 404 411 418
平均 379.4 183.2 239.0 271.6 370.8 389.4 390.2 394.8 399.8 401.6 399.5 399.7 393.0 標準傷差 7.96 16.09 15.86 21.31 8.23 6.62 9.37 6.24 6.68 9.99 16.04 15.28 4.00 正常マウス陴島 (450辉島）を移植した糖尿病-マウスの血糖値 （r tig/dL)  Average 379.4 183.2 239.0 271.6 370.8 389.4 390.2 394.8 399.8 401.6 399.5 399.7 393.0 Standard wound difference 7.96 16.09 15.86 21.31 8.23 6.62 9.37 6.24 6.68 9.99 16.04 15.28 4.00 Diabetes transplanted with normal mice Yeouido (450 Yeouido) (tig / dL)
動物 no.l 388 159 96 105 119 108 104 103 108 110 105 107 111 動物 no.2 394 141 98 94 123 103 114 115 108 103 98 103 106 動物 πο.3 373 134 101 93 115 117 127 133 124 112 104 110 108 動物 no.4 369 139 117 98 110 119 124 123 125 114 98 98 100 動物 no.5 375 129 114 112 129 120 124 141 131 127 123 131 127 平均 379.8 140.4 105.2 100.4 119,2 113,4 118.6 123 119.2 113.2 105.6 109.8 110.4 標準偏差 9.54 10.19 8.61 7.17 6.52 6,71 8.52 13.33 9.45 7.83 9.18 11.34 9.05 健常マウス (非洽療群)の血糖値 （ mg/ dL)  Animal no.l 388 159 96 105 119 108 104 103 108 110 105 107 111 Animal no.2 394 141 98 94 123 103 114 115 108 103 98 103 106 Animal πο.3 373 134 101 93 115 117 127 133 124 112 104 110 108 animals no.4 369 139 117 98 110 119 124 123 125 114 98 98 100 animals no.5 375 129 114 112 129 120 124 141 131 127 123 131 127 Average 379.8 140.4 105.2 100.4 119,2 113,4 118.6 123 119.2 113.2 105.6 109.8 110.4 Standard deviation 9.54 10.19 8.61 7.17 6.52 6,71 8.52 13.33 9.45 7.83 9.18 11.34 9.05 Blood glucose level (mg / dL) in healthy mice (non-treatment group)
動物 no.l 83 88 80 87 89 94 88 85 87 90 82 89 87 動物 no.2 64 63 78 84 86 79 63 79 86 78 80 77 80 動物 no.3 73 84 88 79 78 86 73 74 80 83 96 91 94 平均 73.3 78.3 82.0 83.3 84.3 86.3 74.7 79.3 84.3 83.7 86.0 85.7 87.0 標準偏差 7.76 10.96 4.32 3.30 4.64 6.13 10.27 4.50 3.09 4.92 7.12 6.18 5.72 Animal no.l 83 88 80 87 89 94 88 85 87 90 82 89 87 Animal no.2 64 63 78 84 86 79 63 79 86 78 80 77 80 Animal no.3 73 84 88 79 78 86 73 74 80 83 96 91 94 Average 73.3 78.3 82.0 83.3 84.3 86.3 74.7 79.3 84.3 83.7 86.0 85.7 87.0 Standard deviation 7.76 10.96 4.32 3.30 4.64 6.13 10.27 4.50 3.09 4.92 7.12 6.18 5.72
本発明の手法により得られたインスリン分泌細胞の機能評価 Functional evaluation of insulin-secreting cells obtained by the method of the present invention
4)バイオ人工勝臓を用いた糖尿病マウスの治療実験 4) Treatment experiment of diabetic mice using bioartificial viscera
バイオ人工瞵臓を移植する糖尿病マウス（実施例 9記載の方法で作製)を切開し、 マウス皮下にバッグ型のバイオリアクター（図 8参照。 SG文献に作製方法が記載され ている）を埋め込み 3日間かけてバイオリアクター周囲への血管伸長を行った。バイ オリアクターを埋め込んでから 3日後および 10日後に本発明の分化誘導方法にて得 られたインスリン分泌細胞（5 X 106細胞）、未分化なマウス ES細胞（5 X 106細胞）、 および正常マウス瞵島 450個をそれぞれ 5匹の糖尿病マウスに埋め込んだバイオリ アクターに細胞充填を行った。なお、正常な健常マウス（3匹）および移植しない糖尿 病マウスをコントロールとして使用した。細胞を充填した後のマウス血糖値の平均を 図 9および表 4に示す。充填後の最初の 10日間（細胞を充填してから、細胞のインス リン分泌能が回復するまでの間に相当する）は、中間型ヒトインスリン NPH (Novo N ordisk社製）を 2単位皮下注射した。 NPH注射終了後、 3日おきに血糖値を測定し、 移植後 3週目以降は 1週間に 1回の割合で血糖値を Portable glucose meter F reeStlyle TM (TheraSence社製）を使用して測定した。マウス ES細胞を充填した バイオ人工勝臓を埋め込んだマウスでは、 NPH投与を終えた後に血糖値が再度 36 0mg/dL以上になり、治療効果がないことが明ら力、となった。これとは対照的に、本 発明の分化誘導方法により得られたインスリン分泌細胞を埋め込んだバイオ人工瞵 臓を装着したマウスは、健常マウス瞵島を充填したバイオ人工勝臓を装着したマウス と同様にグルコース濃度が 100mg/dL程度を維持することができた。これは、当該 インスリン分泌細胞の機能が健常マウス瞵島と同程度であり、治療効果があることを 示す。なお、治療を行わない糖尿病マウス全て（非治療群）および ES細胞を充填し たバイオ人工勝臓を埋め込んだマウス全ては 10週間以内に高血糖により死亡した。 A diabetic mouse (produced by the method described in Example 9) to be transplanted with a bioartificial spleen is opened, and a bag-type bioreactor (see Fig. 8; the production method is described in the SG document) is subcutaneously embedded in the mouse. Vascular extension around the bioreactor was performed over a period of days. Insulin secretory cells (5 × 10 6 cells), undifferentiated mouse ES cells (5 × 10 6 cells) obtained by the differentiation induction method of the present invention 3 days and 10 days after implantation of the bioreactor, and Cell filling was performed on bioreactors in which 450 normal mice, Kashiwajima, were embedded in 5 diabetic mice each. Normal healthy mice (3 mice) and non-transplanted diabetic mice were used as controls. The average mouse blood glucose level after cell loading is shown in Figure 9 and Table 4. During the first 10 days after filling (corresponding to the period between filling the cells and restoring the insulin secretion capacity of the cells), 2 units of intermediate human insulin NPH (Novo Nordisk) were injected subcutaneously did. After completion of NPH injection, blood glucose level was measured every 3 days, and after 3 weeks after transplantation, blood glucose level was measured once a week using Portable glucose meter FreeStlyle ™ (TheraSence) . Mice embedded with bioartificial viscera filled with mouse ES cells showed a blood glucose level of 360 mg / dL or more again after NPH administration, indicating that there was no therapeutic effect. In contrast, a mouse equipped with a bioartificial organ embedded with insulin-secreting cells obtained by the differentiation-inducing method of the present invention is the same as a mouse equipped with a bioartificial viscera filled with healthy mice. In addition, the glucose concentration could be maintained at about 100 mg / dL. This indicates that the function of the insulin-secreting cells is similar to that of normal mouse Kashiwajima and has a therapeutic effect. All untreated diabetic mice (non-treated group) and all mice embedded with bioartificial embryos filled with ES cells died of hyperglycemia within 10 weeks.
[表 4] 表 4 [Table 4] Table 4
細胞充填後の曰数 0 3 6 9 12 15 18 21 28 35 42 49 56 本発明のインスリン分泌細胞（5百万個）を充填したバイオ人工應臓を埋め込んだ糖尿病マウスの血糖値 (mg /dL) Number of cells after cell filling 0 3 6 9 12 15 18 21 28 35 42 49 56 Blood glucose level of diabetic mice embedded with bioartificial embryo filled with insulin-secreting cells ( 5 million) of the present invention ( mg / dL )
動物 no.l 391 211 216 208 166 189 158 149 151 128 135 118 115 動物 no.2 395 170 175 186 177 193 173 178 149 158 137 144 151 動物 no.3 363 158 189 174 143 143 153 157 127 108 103 105 98 動物 no.4 381 188 197 179 161 154 155 140 123 117 130 115 112 動物 no.5 375 181 206 202 156 163 179 173 151 137 121 124 111 平均 381.0 181.6 196.6 189.8 160.6 168.4 163.6 159.4 140.2 129.6 125.2 121.2 117.4
Figure imgf000053_0001
Animal no.l 391 211 216 208 166 189 158 149 151 128 135 118 115 Animal no.2 395 170 175 186 177 193 173 178 149 158 137 144 151 Animal no.3 363 158 189 174 143 143 153 157 127 108 103 105 98 Animals no.4 381 188 197 179 161 154 155 140 123 117 130 115 112 Animals no.5 375 181 206 202 156 163 179 173 151 137 121 124 111 Average 381.0 181.6 196.6 189.8 160.6 168.4 163.6 159.4 140.2 129.6 125.2 121.2 117.4
Figure imgf000053_0001
標準偏差 11.45 17.87 14.07 13.12 11.22 19.55 10.42 14.29 12.50 17.26 12.40 12.95 17.78 マウス ES細胞（5百万個）を充填したバイオ人工 i 臓を埋め込んだ糖尿病マウスの血糖値 （mg/dL)  Standard deviation 11.45 17.87 14.07 13.12 11.22 19.55 10.42 14.29 12.50 17.26 12.40 12.95 17.78 Mouse Blood glucose level (mg / dL) of diabetic mice implanted with bioartificial i organs filled with ES cells (5 million)
動物 no.l 377 221 217 214 208 371 389 394 391 397 395 392 391 動物 no.2 364 189 201 195 193 362 372 368 382 374 377 389 383 動物 no.3 377 231 216 211 219 390 388 388 398 404 408 412 416 動物 no.4 388 218 231 219 227 394 386 393 399 387 379 386 397 動物 rio.5 J68 211 204 203 213 396 405 400 404 411 418 423 431 平均 374.8 214.0 213.8 208.4 212.0 382.6 388.0 388.6 394.8 394.6 395.4 400.4 403,6 標準偏 8.33 14.06 10.68 8.48 11.42 13.59 10.49 10.98 7.63 13.00 15.98 14.51 17.50 正常マウス膝島（450鸱島）を充填したバイオ人工膝臓を埋め込んだ糖尿病マウスの血糖値 (mg/dL)  Animal no.l 377 221 217 214 208 371 389 394 391 397 395 392 391 Animal no.2 364 189 201 195 193 362 372 368 382 374 377 389 383 Animal no.3 377 231 216 211 219 390 388 388 398 404 408 412 416 animals no.4 388 218 231 219 227 394 386 393 399 387 379 386 397 animals rio.5 J68 211 204 203 213 396 405 400 404 411 418 423 431 average 374.8 214.0 213.8 208.4 212.0 382.6 388.0 388.6 394.8 394.6 395.4 400.4 403, 6 Standard deviation 8.33 14.06 10.68 8.48 11.42 13.59 10.49 10.98 7.63 13.00 15.98 14.51 17.50 Blood glucose level (mg / dL) of diabetic mice embedded with bioartificial knees filled with normal mouse knee islands (450 Yeouido)
動物 no.l 378 177 149 137 110 100 104 103 108 110 105 10? 111 動物 no.2 393 157 170 151 123 111 114 115 113 106 118 114 121 動物 πο.3 366 147 187 110 115 98 100 96 103 102 104 110 108 動物 πο.4 373 147 146 119 100 97 95 97 93 87 88 93 94 動物 no.5 384 166 146 120 96 95 90 91 93 84 8? 84 91 平均 378.8 158.8 159.6 127.4 108.8 100.2 100.6 100.4 102.0 97.8 100.4 101.6 105.0 標準偏差 9.24 11.53 16.38 14.68 9.83 5.64 8.19 8.24 8.00 10.40 11.64 11.29 11.12 健常マウス (非治療群)の血糖値 (mg/dL)  Animal no.l 378 177 149 137 110 100 104 103 108 110 105 10? 111 Animal no.2 393 157 170 151 123 111 114 115 113 106 118 114 121 Animal πο.3 366 147 187 110 115 98 100 96 103 102 104 110 108 Animal πο.4 373 147 146 119 100 97 95 97 93 87 88 93 94 Animal no.5 384 166 146 120 96 95 90 91 93 84 8? 84 91 Average 378.8 158.8 159.6 127.4 108.8 100.2 100.6 100.4 102.0 97.8 100.4 101.6 105.0 Standard deviation 9.24 11.53 16.38 14.68 9.83 5.64 8.19 8.24 8.00 10.40 11.64 11.29 11.12 Blood glucose level (mg / dL) in healthy mice (non-treatment group)
動物 no.l 88 94 88 86 95 97 92 90 84 92 88 93 97 動物 no.2 64 63 78 84 86 79 63 79 86 78 80 77 80 動物 no.3 73 84 88 79 78 86 73 74 80 83 96 91 94 平均 75.0 80.3 84.7 83.0 86.3 87.3 76.0 81.0 83.3 84.3 88.0 87.0 90.3 標準儷差 9.90 12.92 4.71 2.94 6.94 7.41 12.03 6.68 2.49 5.79 6.53 7.12 7.41 Animal no.l 88 94 88 86 95 97 92 90 84 92 88 93 97 Animal no.2 64 63 78 84 86 79 63 79 86 78 80 77 80 Animal no.3 73 84 88 79 78 86 73 74 80 83 96 91 94 Average 75.0 80.3 84.7 83.0 86.3 87.3 76.0 81.0 83.3 84.3 88.0 87.0 90.3 Standard deviation 9.90 12.92 4.71 2.94 6.94 7.41 12.03 6.68 2.49 5.79 6.53 7.12 7.41
[0146] 本発明の分化誘導方法により、健常ヒト勝臓細胞を代替するのに充分機能的であり 、大量供給が可能で安全なインスリン分泌細胞を誘導することができる。また、本発 明の糖尿病治療剤、バイオ人工勝臓、研究試薬および創薬モデル動物は、糖尿病 に対し安全性の高!/、治療が実現できる。 [0146] According to the differentiation inducing method of the present invention, it is possible to induce insulin secreting cells that are sufficiently functional to replace healthy human spleen cells and that can be supplied in large quantities and are safe. In addition, the therapeutic agent for diabetes, the bioartificial viscera, the research reagent and the drug discovery model animal of the present invention can realize high safety / treatment for diabetes.
配列表フリーテキスト  Sequence listing free text
[0147] 配列番号 l : Foxa2遺伝子を検出するためのポリメラーゼ連鎖反応用 5' プライマー 配列番号 2 : Foxa2遺伝子を検出するためのポリメラーゼ連鎖反応用 3' プライマー 配列番号 3 : Soxl 7遺伝子を検出するためのポリメラーゼ連鎖反応用^ プライマー 配列番号 4 : Soxl 7遺伝子を検出するためのポリメラーゼ連鎖反応用; T プライマー 配列番号 5 : Pdx—Ι遺伝子を検出するためのポリメラーゼ連鎖反応用^ プライマ 配列番号 6 : Pdx—Ι遺伝子を検出するためのポリメラーゼ連鎖反応用; T プライマ 配列番号 7： Shh遺伝子を検出するためのポリメラーゼ連鎖反応用^ プライマー 配列番号 8： Shh遺伝子を検出するためのポリメラーゼ連鎖反応用; T プライマー 配列番号 9 : GAPDH遺伝子を検出するためのポリメラーゼ連鎖反応用^ プライマ 配列番号 10 : GAPDH遺伝子を検出するためのポリメラーゼ連鎖反応用; T プライ マー [0147] SEQ ID NO: 1 5 'primer for polymerase chain reaction to detect Foxa2 gene SEQ ID NO: 2 3' primer for polymerase chain reaction to detect Foxa2 gene SEQ ID NO: 3 to detect Soxl 7 gene Primer for Polymerase Chain Reaction ^ Primer SEQ ID NO: 4: For Polymerase Chain Reaction to detect Soxl 7 gene; T Primer SEQ ID NO: 5: Pdx—Primer for Polymerase Chain Reaction to detect プ ラ イ gene SEQ ID NO: 6: Pdx —For polymerase chain reaction to detect sputum gene; T primer SEQ ID NO: 7: For polymerase chain reaction to detect Shh gene ^ Primer SEQ ID NO: 8: For polymerase chain reaction to detect Shh gene; T primer SEQ ID NO: 9: Polymerase chain reaction ^ primer for detecting GAPDH gene SEQ ID NO: 10: GAPDH gene For polymerase chain reaction to detect T; primer

Claims

請求の範囲 The scope of the claims
[1] 胚性幹細胞のインスリン分泌細胞への分化誘導方法であって、  [1] A method for inducing differentiation of embryonic stem cells into insulin-secreting cells,
(a)泉維芽細胞増殖因子およびァクチビンの存在下に胚性幹細胞を培養することに より胚体内胚葉へ分化させる工程、  (a) a step of differentiating into definitive endoderm by culturing embryonic stem cells in the presence of fountain fibroblast growth factor and activin,
(b)得られた胚体内胚葉を、線維芽細胞増殖因子の存在下、馴化培地を用いて培 養することにより原始瞵へ分化させる工程、  (b) the step of differentiating the resulting definitive endoderm into a primitive cocoon by culturing using a conditioned medium in the presence of fibroblast growth factor,
(c)得られた原始瞵を、馴化培地を用いて培養することによりニューロジェニン 3発現 細胞数を増加させ、ニューロジェニン 3発現細胞を得る工程、および  (c) increasing the number of neurogenin 3 expressing cells by culturing the obtained primitive koji using a conditioned medium to obtain neurogenin 3 expressing cells; and
(d)得られたニューロジェニン 3発現細胞を、高グルコース濃度の細胞培養用培地中 で刺激してインスリン分泌細胞へ分化させる工程  (d) A step of stimulating the obtained neurogenin 3 expressing cells in a cell culture medium having a high glucose concentration to differentiate into insulin-secreting cells.
を含む方法。  Including methods.
[2] 泉維芽細胞増殖因子が泉維芽細胞増殖因子 10以外の泉維芽細胞増殖因子で ある請求の範囲第 1項記載の方法。  [2] The method according to claim 1, wherein the fountain fibroblast growth factor is a fountain fibroblast growth factor other than the fountain fibroblast growth factor 10.
[3] ァクチビンがァクチビン Aである請求の範囲第 1項または第 2項記載の方法。 [3] The method according to claim 1 or 2, wherein the activin is activin A.
[4] ァクチビンの濃度が 2〜200ng/mLである請求の範囲第 1項〜第 3項のいずれか に記載の方法。 [4] The method according to any one of claims 1 to 3, wherein the concentration of activin is 2 to 200 ng / mL.
[5] 工程 (b)において用いられる馴化培地力 ヒト神経膠芽細胞腫由来細胞株 T98G を細胞培養培地中で培養したものである請求の範囲第 1項〜第 4項のいずれかに記 載の方法。  [5] Conditioned medium force used in step (b) The human glioblastoma cell line T98G is cultured in cell culture medium, as described in any one of claims 1 to 4. the method of.
[6] 工程 ）において用いられる馴化培地力 S、ヒト不死化肝類洞内皮細胞株 TMNT— 1細胞（寄託機関:独立行政法人産業技術総合研究所 特許生物寄託センター、あ て名：日本国茨城県つくば巿東 1丁目 1番地 1 中央第 6 (郵便番号 305— 8566)、 寄託日：平成 14年 4月 16日、受託番号： FERM BP— 8017)または血管内皮細胞 を低グルコース細胞培養培地中で培養したものである請求の範囲第 1項〜第 5項の いずれかに記載の方法。  [6] Process conditioned medium power used in S), human immortalized hepatic sinusoidal endothelial cell line TMNT-1 cell (Depositor: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Address: Ibaraki, Japan) Prefectural Tsukuba Sakai Higashi 1-chome 1 Chuo 6th (Postal Code 305-8566), Deposit Date: April 16, 2002, Deposit Number: FERM BP-8017) or Vascular Endothelial Cells in Low Glucose Cell Culture Medium The method according to any one of claims 1 to 5, wherein the method is cultured in a cell.
[7] 各工程における培養がそれぞれ、コラーゲンタイプ IV、マトリゲル、フイブロネクチン 、ジエラチン、ポリオル二チンおよびラミニンよりなる群から選ばれる生体適合性材料 を用いる三次元培養である請求の範囲第 1項〜第 6項のいずれかに記載の方法。 [7] The culture in each step is a three-dimensional culture using a biocompatible material selected from the group consisting of collagen type IV, matrigel, fibronectin, dielatin, polyornithine and laminin, respectively. 7. The method according to any one of items 6.
[8] 工程（a)において用いられる生体適合性材料がコラーゲンタイプ IVである請求の 範囲第 7項記載の方法。 8. The method according to claim 7, wherein the biocompatible material used in step (a) is collagen type IV.
[9] 工程 ）において用いられる生体適合性材料がラミニンである請求の範囲第 7項ま たは第 8項記載の方法。 [9] The method according to claim 7 or 8, wherein the biocompatible material used in step (5) is laminin.
[10] 工程（a)における細胞培養濃度が 1 X 105〜4 X 105細胞/ mLである請求の範囲 第 1項〜第 9項のいずれかに記載の方法。 [10] The method according to any one of [1] to [9] above, wherein the cell culture concentration in the step (a) is 1 × 10 5 to 4 × 10 5 cells / mL.
[11] 工程 (b)における細胞培養濃度が 1 X 105〜8 X 105細胞/ mLである請求の範囲 第 1項〜第 10項のいずれかに記載の方法。 [11] The method according to any one of [1] to [10], wherein the cell culture concentration in the step (b) is 1 × 10 5 to 8 × 10 5 cells / mL.
[12] 工程（a)および/または (b)における線維芽細胞増殖因子の濃度が 2〜20ng/m[12] The concentration of fibroblast growth factor in step (a) and / or (b) is 2-20 ng / m
Lである請求の範囲第 1項〜第 11項の!/、ずれかに記載の方法。 12. The method according to any one of claims 1 to 11, which is L.
[13] 工程 (a)において用いられる培地が、細胞培養用培地中に、ゥシ胎仔血清を 0. 2[13] The medium used in step (a) is a cell culture medium containing urchin fetal serum 0.2
〜3%含有し、ゥシ血清アルブミンを 2〜3%含有する請求の範囲第 1項〜第 12項の いずれかに記載の方法。 13. The method according to any one of claims 1 to 12, which comprises ˜3% and ushi serum albumin 2˜3%.
[14] 工程 (b)において用いられる培地が、馴化培地中にレチノイン酸を 1〜50 11101/[14] The medium used in step (b) contains 1-50 11101 / retinoic acid in the conditioned medium.
L含有する請求の範囲第 1項〜第 13項のいずれかに記載の方法。 14. The method according to any one of claims 1 to 13, wherein L is contained.
[15] 工程 ）において用いられる培地が、馴化培地中に γ—セクレターゼ阻害剤 XVIII を 0. 25〜2 11101/し含有し、線維芽細胞増殖因子を5〜501¾/111し含有し、上皮 増殖因子を 5〜50ng/mL含有する請求の範囲第 1項〜第 14項のいずれかに記載 の方法。 [15] The medium used in step) contains 0.25 to 2 11101 / γ-secretase inhibitor XVIII and 5 to 501¾ / 111 fibroblast growth factor in the conditioned medium, and epithelial growth The method according to any one of claims 1 to 14, which comprises 5 to 50 ng / mL of a factor.
[16] 工程 ）において、ニューロジェニン 3発現細胞がセルソーターを用いて選別される 請求の範囲第 1項〜第 15項のいずれかに記載の方法。  [16] The method according to any one of claims 1 to 15, wherein in step (1), the neurogenin 3 expressing cells are selected using a cell sorter.
[17] 工程（d)において用いられる培地が、肝細胞増殖因子を 2〜50ng/mL含有し、二 コチンアミドを;!〜 1 Ommol/L含有し、ェクセディン 4を 5〜 1 OOnmol/L含有し、 トログリタゾン 1〜10 mol/Lおよび硫酸亜鉛を;!〜 20 μ mol/L含有する請求の 範囲第 1項〜第 16項のいずれかに記載の方法。  [17] The medium used in step (d) contains 2 to 50 ng / mL of hepatocyte growth factor, contains! ~ 1 Ommol / L of nicotinamide, and contains 5 to 1 OOnmol / L of exedine 4 The method according to any one of claims 1 to 16, wherein troglitazone is contained in an amount of 1 to 10 mol / L and zinc sulfate;! To 20 µmol / L.
[18] 工程 (d)が、ニューロジェニン 3発現細胞を高グルコース濃度の細胞培養用培地、 次いで低グルコース濃度の細胞培養用培地中で繰り返し刺激培養する工程からなり 、高グルコース濃度の細胞培養用培地中で 1回あたり 2時間の刺激培養を、 1日あた り 1 3回行うことを 1〜； 10日間繰り返す請求の範囲第 1項〜第 17項のいずれかに記 載の方法。 [18] Step (d) comprises a step of repeatedly stimulating and culturing neurogenin 3 expressing cells in a cell culture medium having a high glucose concentration and then in a cell culture medium having a low glucose concentration. Stimulated culture for 2 hours per time in medium, 1 day The method according to any one of claims 1 to 17, wherein 1 to 3 times are repeated for 10 days.
[19] 工程 (d)において用いられる培地が、高グルコース濃度の細胞培養培地中に脂質 [19] The medium used in step (d) is a lipid in cell culture medium having a high glucose concentration.
、アミノ酸、ビタミンおよび/またはミネラルを含有する請求の範囲第 1項〜第 18項の いずれかに記載の方法。 The method according to any one of claims 1 to 18, which comprises an amino acid, a vitamin and / or a mineral.
[20] 工程（d)が、さらにニューロジェニン 3発現細胞に Maf A遺伝子を導入し発現させる ことにより、インスリン分泌細胞の MafA遺伝子発現を増強させることを含む請求の範 囲第 1項〜第 19項のいずれかに記載の方法。 [20] The range of claims 1 to 19, wherein step (d) further comprises enhancing MafA gene expression in insulin-secreting cells by introducing and expressing the MafA gene in neurogenin 3 expressing cells. A method according to any of the paragraphs.
[21] 胚性幹細胞が哺乳類由来のものである請求の範囲第 1項〜第 20項のいずれかに 記載の方法。 21. The method according to any one of claims 1 to 20, wherein the embryonic stem cell is derived from a mammal.
[22] 哺乳類がマウス、ヒト、サルよりなる群から選ばれる請求の範囲第 21項記載の方法  [22] The method according to claim 21, wherein the mammal is selected from the group consisting of a mouse, a human and a monkey.
[23] 哺乳類がマウスである請求の範囲第 22項記載の方法。 23. The method according to claim 22, wherein the mammal is a mouse.
[24] 請求の範囲第 1項〜第 23項のいずれかに記載の方法により誘導されるインスリン 分泌細胞。  [24] An insulin-secreting cell induced by the method according to any one of claims 1 to 23.
[25] インスリン分泌細胞力 スフエロイドを形成しているインスリン分泌細胞を含有する請 求の範囲第 24項記載のインスリン分泌細胞。  [25] Insulin-secreting cell force The insulin-secreting cell according to claim 24, comprising an insulin-secreting cell forming a spheroid.
[26] 請求の範囲第 24項または第 25項記載のインスリン分泌細胞を含有する糖尿病治 療剤。 [26] A therapeutic agent for diabetes containing the insulin-secreting cells according to claim 24 or 25.
[27] 請求の範囲第 24項または第 25項記載のインスリン分泌細胞を含有するバイオ人 ェ瞵臓。  [27] A bio-human organ containing the insulin-secreting cells according to claim 24 or 25.
[28] 請求の範囲第 24項または第 25項記載のインスリン分泌細胞を含有する研究試薬  [28] A research reagent comprising insulin-secreting cells according to claim 24 or 25
[29] 請求の範囲第 24項または第 25項記載のインスリン分泌細胞を含有する創薬モデ ノレ動物。 [29] A drug discovery mode animal comprising the insulin secreting cells according to claim 24 or 25.
PCT/JP2007/073352 2006-12-01 2007-12-03 Method of inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method and use of the same WO2008066199A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2008547073A JPWO2008066199A1 (en) 2006-12-01 2007-12-03 Method for inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method, and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006326261A JP5135577B2 (en) 2006-12-01 2006-12-01 Method for inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method, and use thereof
JP2006-326261 2006-12-01

Publications (1)

Publication Number Publication Date
WO2008066199A1 true WO2008066199A1 (en) 2008-06-05

Family

ID=39467980

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2007/073352 WO2008066199A1 (en) 2006-12-01 2007-12-03 Method of inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method and use of the same

Country Status (2)

Country Link
JP (2) JP5135577B2 (en)
WO (1) WO2008066199A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011016485A1 (en) * 2009-08-04 2011-02-10 国立大学法人岡山大学 METHOD FOR INDUCING DIFFERENTIATION OF iPS CELLS INTO HEPATIC PARENCHYMAL CELLS
JP2013512658A (en) * 2008-12-03 2013-04-18 インターナショナル ステム セル コーポレイション Method for obtaining differentiated cells from stem cells
WO2013176249A1 (en) 2012-05-25 2013-11-28 学校法人埼玉医科大学 Method for producing pancreatic hormone-producing cell, pancreatic hormone-producing cell, and differentiation/induction promoter
WO2014189127A1 (en) 2013-05-24 2014-11-27 学校法人埼玉医科大学 New peptide and application thereof
WO2014199622A1 (en) 2013-06-10 2014-12-18 株式会社クラレ Tissue structure and manufacturing method therefor
US8932853B2 (en) 2009-12-29 2015-01-13 Takeda Pharmaceutical Company Limited Method for manufacturing pancreatic-hormone-producing cells
US9157069B2 (en) 2010-08-09 2015-10-13 Takeda Pharmaceutical Company Limited Method of producing pancreatic hormone-producing cells
CN111269833A (en) * 2018-12-05 2020-06-12 中国科学院大连化学物理研究所 Human pancreatic island organoid model construction method based on organ chip

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6378183B2 (en) 2013-08-07 2018-08-22 国立大学法人京都大学 Method for producing pancreatic hormone-producing cells
JP6122817B2 (en) 2014-03-26 2017-04-26 株式会社Ｓｃｒｅｅｎホールディングス Spheroid evaluation method and spheroid evaluation apparatus
JP6596708B2 (en) * 2014-05-20 2019-10-30 国立大学法人東京工業大学 Method for inducing differentiation of insulin producing cells
CN106536718B (en) 2014-05-21 2021-04-27 国立大学法人京都大学 Method for producing pancreatic islet cells and therapeutic agent for pancreatic disease containing pancreatic islet cells
US11338068B2 (en) 2016-11-08 2022-05-24 W. L. Gore & Associates, Inc. Two-part implantable therapy delivery device
JP2019097442A (en) * 2017-11-30 2019-06-24 株式会社日立製作所 Immunoisolation device

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050827A2 (en) * 2002-12-05 2004-06-17 Technion Research & Development Foundation Ltd. Cultured human pancreatic islets, and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050827A2 (en) * 2002-12-05 2004-06-17 Technion Research & Development Foundation Ltd. Cultured human pancreatic islets, and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
D'AMOUR K.A. ET AL.: "Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells", NAT. BIOTECHNOL., vol. 24, no. 11, November 2006 (2006-11-01), pages 1392 - 1401 *
NISHIMURA W. ET AL.: "A switch from MafB to MafA expression accompanies differentiation to pancreatic beta-cells", DEV. BIOL., vol. 293, no. 2, 15 May 2006 (2006-05-15), pages 526 - 539, XP024943980, DOI: doi:10.1016/j.ydbio.2006.02.028 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013512658A (en) * 2008-12-03 2013-04-18 インターナショナル ステム セル コーポレイション Method for obtaining differentiated cells from stem cells
WO2011016485A1 (en) * 2009-08-04 2011-02-10 国立大学法人岡山大学 METHOD FOR INDUCING DIFFERENTIATION OF iPS CELLS INTO HEPATIC PARENCHYMAL CELLS
US8932853B2 (en) 2009-12-29 2015-01-13 Takeda Pharmaceutical Company Limited Method for manufacturing pancreatic-hormone-producing cells
US9157069B2 (en) 2010-08-09 2015-10-13 Takeda Pharmaceutical Company Limited Method of producing pancreatic hormone-producing cells
WO2013176249A1 (en) 2012-05-25 2013-11-28 学校法人埼玉医科大学 Method for producing pancreatic hormone-producing cell, pancreatic hormone-producing cell, and differentiation/induction promoter
WO2014189127A1 (en) 2013-05-24 2014-11-27 学校法人埼玉医科大学 New peptide and application thereof
US9856304B2 (en) 2013-05-24 2018-01-02 Saitama Medical University Peptide and application thereof
WO2014199622A1 (en) 2013-06-10 2014-12-18 株式会社クラレ Tissue structure and manufacturing method therefor
KR20160018460A (en) 2013-06-10 2016-02-17 가부시키가이샤 구라레 Tissue structure and manufacturing method therefor
EP3633030A1 (en) 2013-06-10 2020-04-08 Corning Incorporated Tissue structure and manufacturing method therefor
US11060065B2 (en) 2013-06-10 2021-07-13 Corning Incorporated Tissue structure and preparation method thereof
CN111269833A (en) * 2018-12-05 2020-06-12 中国科学院大连化学物理研究所 Human pancreatic island organoid model construction method based on organ chip

Also Published As

Publication number Publication date
JP2009225661A (en) 2009-10-08
JPWO2008066199A1 (en) 2010-03-11
JP5135577B2 (en) 2013-02-06

Similar Documents

Publication Publication Date Title
JP5135577B2 (en) Method for inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method, and use thereof
US8415153B2 (en) Differentiation and enrichment of islet-like cells from human pluripotent stem cells
CN103981147B (en) A kind of new method for preparing hepatic parenchymal cells
US20070020242A1 (en) Method for transdifferentiation of non-pancreatic stem cells to the pancreatic pathway
JP2005523328A (en) Placenta-derived stem cells and uses thereof
US9428733B2 (en) Method for obtaining pancreatic endocrine cells from adipose tissue-origin cells
US20060084167A1 (en) Formation of Hybrid Cells by Fusion of Lineage Committed Cells with Stem Cells
Mishra et al. Stem cells as a therapeutic target for diabetes
Shaer et al. miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells
JP2021054869A (en) Improved methods for pancreatic islet transplantation
KR102058259B1 (en) Stem cells and pancreatic cells useful for the treatment of insulin-dependent diabetes mellitus
CA2497954A1 (en) Pancreatic small cells and uses thereof
Bahrebar et al. Generation of islet-like cell aggregates from human adipose tissue-derived stem cells by lentiviral overexpression of PDX-1
WO2011016485A1 (en) METHOD FOR INDUCING DIFFERENTIATION OF iPS CELLS INTO HEPATIC PARENCHYMAL CELLS
JPWO2016093362A1 (en) Artificial tissue and manufacturing method thereof
Hefei et al. Morphological characteristics and identification of islet‐like cells derived from rat adipose‐derived stem cells cocultured with pancreas adult stem cells
WO2004074465A1 (en) Human salivary gland-origin stem cell
Moshrefi et al. Transplantation of differentiated umbilical cord mesenchymal cells under kidney capsule for control of type I diabetes in rat
Xiao et al. Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats
CN106367380A (en) Cell co-culture method capable of realizing in-vitro preparation of liver buds and liver buds
WO2017159088A1 (en) Method for preparing cultured cells or cultured tissue for transplantation
Khan et al. In vitro insulin production and analysis of pancreatic transcription factors in induced human hepatic progenitor cells
Lim et al. Generating pancreatic beta-like cells from human pluripotent stem cells
Li Generation of Human Pancreatic Beta Like Cells from Pluripotent Stem Cells
Ham et al. Generation of insulin producing cells from the mouse primary hepatocytes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07850002

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2008547073

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07850002

Country of ref document: EP

Kind code of ref document: A1