WO2005095589A1 - Souches de precurseurs pancreatiques transdifferentiees des cellules pancreatiques acinaires - Google Patents

Souches de precurseurs pancreatiques transdifferentiees des cellules pancreatiques acinaires Download PDF

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WO2005095589A1
WO2005095589A1 PCT/KR2004/002669 KR2004002669W WO2005095589A1 WO 2005095589 A1 WO2005095589 A1 WO 2005095589A1 KR 2004002669 W KR2004002669 W KR 2004002669W WO 2005095589 A1 WO2005095589 A1 WO 2005095589A1
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pancreatic
cell
cells
transdifferentiated
acinar
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Si-Young Song
Ji-Eun Lee
Han-Soo Kim
Jing Wen
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Yonsei University
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    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention relates to a pancreatic precursor cell line transdifferentiated from pancreatic acinar cells. More particularly, the present invention relates to a pancreatic precursor cell line transdifferentiated from pancreatic acinar cells, which expresses both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development, and a preparation method thereof. Also, the present invention is concerned with pancreatic islet cells transdifferentiated from the pancreatic precursor cell line and expressing a pancreatic islet cell marker gene, and a preparation method thereof.
  • pancreas The mammalian pancreas consists of (1) the ductal tree, (2) the exocrine acini that produce digestive enzymes and (3) the endocrine tissue that produces hormones.
  • Pancreatic ductal epithelial cells, acinar cells and islet cells all are derived from the primitive ductal structure formed during the protodifferentiated state of the early pancreatic development (J. Slack, Development, 1995, vol.121, pp.1569-1580).
  • the more advanced stage of pancreatic morphogenesis is achieved by the expression of insulin promoter factor (IPF) as well as the interaction among ductal cells, islet cells and acinar cells. After this embryonic development, ductal cells become remarkably reduced in their capacity to differentiate into acinar cells (M.
  • IPF insulin promoter factor
  • pancreatic ductal cells of the adult pancreas can differentiate into islet cells under certain conditions suitable for intercellular interaction (S. Bonner- Weir et al. Diabetes, 1993, vol.42, pp.1715-1720; L. Rosenberg, Cell Transplant, 1995, vol.4, pp.371-383).
  • the differentiation of pancreatic ductal cells into endocrine cells producing hormones is similar to the ontogeny of the pancreatic development.
  • Various pancreatic islet cells have been prepared using the differentiation capacity of pancreatic ductal cells. For example, U.S. Patent No.
  • 5,888,705 discloses a method of inducing the differentiation of isolated human mature pancreatic cells into islet cells using hepatocyte growth factor/scatter factor (HGF/SF).
  • HGF/SF hepatocyte growth factor/scatter factor
  • PCT Publication No. WO97/15310 discloses a method of inducing mature pancreatic ductal cells to differentiate into islet cells including culturing the mature pancreatic ductal cells in a serum-containing, low-glucose medium and then culturing the cultured cells in a medium with higher glucose content.
  • U.S. Patent No. 5,834,308 discloses a method of producing islet cells, including isolating precursor cells from prediabetic adults and culturing the precursor cells in a medium that stimulates the growth of functional islet cells.
  • pancreatic precursor cells are differentiated only into islet cells.
  • Pancreatic precursor cells of the aforementioned studies do not have the capacity to differentiate into both endocrine and exocrine cell types.
  • the ideal population of pancreatic precursor cells should be able to differentiate into endocrine cells (i.e., islet- ⁇ , islet- ⁇ , islet- ⁇ and islet-PP cells) and exocrine cells (i.e., acinar cells), as well as ductal cells. This is because these precursor cells are clinically useful.
  • WO 01/77300 discloses a population of pancreatic epithelial precursor cells that are capable of differentiating into functional endocrine and exocrine cells, methods of isolating the pancreatic epithelial precursor cells, characterization of pancreatic epithelial precursor cells, and application of the pancreatic epithelial precursor cells.
  • the pancreatic epithelial precursor cells are isolated from the human fetal pancreatic tissue, this patent is problematic in that the raw material is difficult to obtain.
  • these embryonic cells are highly apt to differentiate into unwanted cells so that they may be transformed to cancer cells.
  • acinar cells have been reported to participate in the histogenesis of ductal adenocarcinoma. When pancreatic acinar cells are chemically induced to cause carcinogenesis, they dedifferentiate and thus produce ductal-like cells (A. Krapp et al. Genes Dev. 1998, vol.12, pp.3752-3763).
  • the present invention provides pancreatic precursor cells transdifferentiated from pancreatic acinar cells, which express both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development.
  • the present invention provides a method of preparing pancreatic precursor cells transdifferentiated from pancreatic acinar cells, comprising (1) isolating the pancreatic acinar cells from an adult, (2) in vitro culturing of the pancreatic acinar cells in a medium for mammalian cell culture and (3) isolating the pancreatic precursor cells expressing both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development during the in vitro culturing.
  • the present invention provides pancreatic islet cells that are transdifferentiated from the pancreatic precursor cells and express a pancreatic islet cell marker gene.
  • the present invention provides a method of preparing pancreatic islet cells transdifferentiated from the pancreatic precursor cells, comprising (1) contacting the pancreatic precursor cells, prepared as described above, with a growth factor, (2) culturing the pancreatic precursor cells in a medium for mammalian cell culture and (3) isolating the pancreatic islet cells expressing a pancreatic islet cell marker gene during the culturing.
  • Fig. 1 photographically shows the morphological changes of pancreatic acinar cells according to culture time ⁇ A-F: ⁇ 200; G-H: lOO; after culturing, day 0 (A), day 1 (B), day 3 (C), day 5 (D), day 10 (E), day 15 (F); YGIC4 (G), YGIC5 (H) ⁇ ; Fig.
  • FIG. 3 is a graph showing amylase activity of pancreatic acinar cells according to culture time; Fig.
  • FIG. 4 photographically shows the morphological changes of pancreatic acinar cells upon three-dimensional culture according to culture time ⁇ after culturing in collagen, day 0 (A), day 3 (B), day 5 (C); after culturing in matrigel, day 0 (D), day 3 (E), day 5 (F) ⁇ ;
  • Fig. 5 shows the results of BrdU incorporation assay for measuring the proliferative ability of pancreatic precursor cells obtained in the present invention;
  • Fig. 6 shows the results of Western blotting for expression markers of YGIC4 and YGIC5 cell lines according to the present invention; and
  • Fig. 7 shows the results of Western blotting for expression markers of pancreatic islet cells transdifferentiated from YGIC4 and YGIC5 cell lines according to the present invention.
  • pancreatic precursor cells refer to pluripotential cells obtained by the transdifferentiation of pancreatic acinar cells, which lose characteristics of pancreatic acinar cells and come to have characteristics of pancreatic ductal cells and characteristics of pancreatic precursor cells capable of differentiating into all types of pancreatic cells (that is, acinar cells, ductal cells and islet cells).
  • pancreatic precursor cells it is necessary to plainly define the terms used in the present specification and the characteristics of differentiated cells constituting the pancreas.
  • pancreatic acinar cells are cells that secrete various digestive enzymes participating in the degradation of proteins, lipids and polysaccharides.
  • Pancreatic acinar cell marker genes include, but are not limited to, amylase and p48.
  • Pancreatic islet cells may be classified into four cell types according to their functions: (1) ⁇ cells that produce glucagon; (2) ⁇ cells that produce insulin; (3) ⁇ cells that produce somatostatin; and (4) pp cells that produce pancreatic polypeptides.
  • pancreatic islet cells ovoid in shape and about 75 ⁇ m to 175 ⁇ m in size (long axis), produce the pancreas-derived hormones and secrete the hormones into the blood stream.
  • Pancreatic islet cell marker genes include the aforementioned various hormones.
  • Pancreatic ductal cells are cuboid epithelial cells which are about 10 ⁇ m in diameter, tightly dense in appearance and relatively small and rounded compared to acinar cells.
  • Pancreatic ductal cell marker genes include cytokeratins, such as CK 7, CK 8, CK 18 and CK 19, CFTR, mucin MUC1, carbonic anhydrase II and carbohydrate antibody 19.9 (syalyl-Lewis-a).
  • transdifferentiation typically means that cells in a differentiated state are converted to ones in another differentiated state. Since transdifferentiation employs completely differentiated cells as a starting material, it differs from stem cell-derived neogenesis. During transdifferentiation, numerous genes switch on and off, and thus a dramatic change may be obtained in morphologies and phenotypes of cells.
  • the present invention the
  • pancreatic precursor cells are characterized in that expression of an acinar cell marker gene (e.g., amylase, p48) decreases, the endoplasmic reticulum and Golgi apparatus in the cytoplasm degenerate and zymogen granules are reduced, whereas a pancreatic ductal cell marker gene (e.g., CK 19, CFTR) is expressed, and the pancreatic precursor cells have the morphology of pancreatic ductal cells.
  • an acinar cell marker gene e.g., amylase, p48
  • a pancreatic ductal cell marker gene e.g., CK 19, CFTR
  • pancreatic precursor cells express a gene participating in the pancreatic development, and thus possess a multipotency to differentiate into all types of cells constituting the pancreas.
  • Non-limiting examples of the gene include Notch- 1, Jagged- 1, Wnt-4, ⁇ -catenin, neurogenin 3, Neuro D, Pax 4, Pax 6, Pdx-1 , PTFl/p48 and Tcf-4.
  • the pancreatic precursor cell of the present invention may express a marker gene specifically expressed only in stem cells with limited Differentiation, such as hematopoietic stem cells, nerve stem cells and hepatic stem cells. Examples of such a marker gene include SCF/c-kit and vimentin.
  • Pancreatic acinar cells as a starting material for obtaining the pancreatic precursor cells of the present invention, are preferably isolated from the adult mammalian pancreas. In particular, since the pancreatic precursor cells possess the multipotency to differentiate into several types of cells constituting the pancreas, it may be used for the treatment of various pancreatic diseases, which requires the transplantation of the cells, tissue or organ of the pancreas.
  • pancreatic precursor cells of the present invention are more preferably prepared using pancreatic acinar cells isolated from the individual into who it will be transplanted, in order to prevent the graft from being rejected.
  • pancreatic acinar cells were isolated from rats and transdifferentiated to produce two pancreatic precursor cell lines, which were designated as "YGIC4" and "YGIC5", respectively.
  • the "YGIC5" was deposited at an international depositary authority, Korean Cell Line Research Foundation (KCLRF; Cancer Research Institute, Seoul National University College of Medicine, Yungon- dong, Chongno-gu, Seoul, Republic of Korea) on January 29, 2004, and was assigned accession number KCLRF-BP-00093.
  • the transdifferentiation for preparing the pancreatic precursor cells according to the present invention may be achieved by a method known in the art.
  • transdifferentiation of pancreatic acinar cells into precursor cells may be induced by in vitro culturing of pancreatic tissue. Compared to other tissues, for acinar cells derived from the pancreas, intercellular interaction is more important for the maintenance and growth of the cells.
  • the pancreatic precursor cells according to the present invention may be prepared by a method comprising (1) isolating the pancreatic acinar cells from an adult, (2) in vitro culturing of the pancreatic acinar cells in a medium for mammalian cell culture and (3) isolating the pancreatic precursor cells expressing both a pancreatic ductal cell marker gene and a gene participating in the pancreatic development during the in vitro culture.
  • Isolation of pancreatic acinar cells First, pancreas tissue is isolated from the adult pancreas and microdissected.
  • pancreatic tissue may be microdissected by a physical means using, for example, a homogenizer, mortar and pestle, a blender, scalpels, syringes, forceps or an ultrasonic device.
  • the pancreatic tissue may be microdissected by enzymatic digestion.
  • available enzymes include neutral proteases, serine proteases, including trypsin, chymotrypsin, elastase and collagenase.
  • the physical means may be used in combination with enzyme treatment.
  • the pancreas tissue microdissected as described above is preferably preserved in a buffer solution, for example, containing bovine serum albumin (BSA), pyruvate or a trypsin inhibitor.
  • BSA bovine serum albumin
  • pyruvate pyruvate
  • trypsin inhibitor a trypsin inhibitor
  • Substances that can be used to create a density gradient include, but are not limited to, serum (BSA), ovalbumin, nonionic synthetic polymers of sucrose (FicollTM), colloidal polyvinylpyrrolidone-coated silica (PercollTM), polyvinylpyrrolidone or PVP, and methylcellulose.
  • BSA serum
  • FicollTM nonionic synthetic polymers of sucrose
  • PercollTM colloidal polyvinylpyrrolidone-coated silica
  • PVP polyvinylpyrrolidone or PVP
  • methylcellulose methylcellulose.
  • the microdissected pancreas tissue cells are is centrifuged, and the resulting cell suspension passes through a filter to give desired pancreatic acinar cells.
  • the isolated pancreatic acinar cells may be cultured in vitro in a typical medium for mammalian cell culture.
  • pancreatic precursor cells may be isolated by a single cell isolation method.
  • Media useful for the in vitro culture according to the present invention are commercially available, or may be prepared, for example, according to components and their ratio described in the catalogue from the American Type Culture Collection (ATCC). Examples of available media include Ham medium, IMDM Iscove's medium, Leibovitz L15 medium, Mac Coy 5A medium, M199 medium, Melnick's medium, MEM Eagle's Minimum Essential medium, NCTN medium, Puck's medium, RPMI medium, Swim S77 medium, Trowell T8 medium, Waymouth medium and Williams E medium. In particular, Waymouth medium is preferred in the present invention. In addition, the
  • Waymouth medium is preferably supplemented with an about 0.1-1% antibiotic and about 1-10% FBS.
  • a substance capable of suppressing acute differentiation of cells is introduced at the early stage of the in vitro culture.
  • the differentiation suppressor useful in the present invention includes steroid substances known in the art, whereas dexamethasone and its derivatives are preferred. Dexamethasone is used in a suitable amount of 5 to 15 ⁇ g/ml. Also, after the pancreatic acinar cells clearly display the characteristics of pancreatic ductal cells during in vitro culture (12 to 16 days after the culture starts), the cells are preferably cultured in a medium not containing the differentiation suppressor.
  • the pancreatic acinar cells may be induced to transdifferentiate into pancreatic precursor cells by secondary culture (for example, in plates, flasks, roller bottles and petri dishes) as well as tertiary culture.
  • the tertiary culture is not limited to, but may use matrigel or collagen.
  • Matrigel a mixture of extracellular basement membrane components, is preferably used for cells that are unable to survive as single cells or for identifying the three-dimensional structure of cells in vitro (example, formation of cystic structure).
  • Collagen serves as an intercellular connecting agent, and is suitable for identifying intercellular interaction.
  • (3) Evaluation of the differentiated degree of cells The differentiated degree of cells may be determined by morphology or expression of marker genes or a combination thereof. The morphology and marker genes of various cells constituting the pancreas are the same as described above. Changes in morphology during transdifferentiation may be easily observed using an inverted light microscope or electron microscope. Change in phenotype, is not limited to, but may be detected by RT-PCR .
  • the pancreatic precursor cell line obtained as described above is capable of differentiating into all types of cells constituting the pancreas.
  • the present invention provides a pancreatic islet cell that is transdifferentiated from the pancreatic precursor cell according to the present invention and expresses a pancreatic islet cell marker gene. Examples of the pancreatic islet cell marker gene are the same as described above.
  • the pancreatic islet cells transdifferentiated from the pancreatic precursor cells may be prepared by a method comprising (1) contacting the pancreatic precursor cells, prepared as described above, with a growth factor, (2) culturing the pancreatic precursor cells in a medium for mammalian cell culture and (3) isolating the pancreatic islet cells expressing a pancreatic islet cell marker gene during the culturing.
  • the growth factor that can be used in step (1) of the above method includes, but is not limited to, activin A, Betacellulin and GLP-1.
  • the pancreatic precursor cells transdifferentiated from pancreatic acinar cells are directly treated with a growth factor, or are transfected with a vector expressing a growth factor.
  • the vector may be introduced into the pancreatic precursor cells by various methods including calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • the medium for mammalian cell culture, used in steps (2) and (3) of the above method, and the detection of the differentiation of the pancreatic precursor cells into pancreatic islet cells and the degree of the differentiation may be obtained and achieved according to the aforementioned method of preparing pancreatic precursor cells transdifferentiated from pancreatic acinar cells.
  • a better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.
  • pancreas was isolated from male Sprague-Dawley rats with an initial weight of about 100-150 g (Jackson Laboratory, Bar Harbor, Maine, USA), homogenized in a spinner flask, and washed twice with a pancreas solution A (4 g glucose, 16.36 g NaCl, 1 M KC1, 1 M MgCl , 1 M CaCl ) containing 500 mg bovine serum albumin, 500 mg pyruvate and 600 mg trypsin inhibitor. Subsequently, the tissue was treated with collagenase type IN (Sigma, St. Louis, MO, USA) at a 37°C water bath for 6-8 min, and centrifuged at 1000 rpm for 10 sec.
  • collagenase type IN Sigma, St. Louis, MO, USA
  • the resulting cell suspension passed through a 200- ⁇ m nylon mesh filter.
  • the isolated acinar cells were washed and then cultured in Waymouth medium supplemented with 10% FBS (HyClone, Logan, Utah, USA) and 10 ⁇ g/ml dexamethasone (Sigma) at 37°C in a 5% CO2/95% air humidified incubator.
  • the cells were observed under an inverted light microscopy to monitor their morphological changes. Identical cells were captured as images with the passage of time (Fig. 1), and collected on days 0, 1, 3, 5, 10, 15 and 20 after starting the primary culture.
  • morphologically identical cells were selected from single cells and cloned using cloning cylinders. As shown in Fig. 1 , on day 2 and day 3 of the primary culture, aggregates of the pancreatic acinar cells, attached to the bottom of plates, were detected, and from day 4 to and day 5, they were expanded. For the above five days of the primary culture, the pancreatic cells displayed the morphology of acinar cells, but, from day 6 to day 8, the cells morphology were gradually changed to that of cuboid like ductal cells.
  • EXAMPLE 2 Structural analysis of cultured cells using electron microscope
  • the cultured cells prepared in Example 1 were analyzed for their structure.
  • the cells were washed with PBS (pH 7.4) twice and fixed in PBS containing 1% glutaraldehyde. Then, the cells were postfixed in osmium tetroxide (OsO 4 ), and samples were observed under a transmission electron microscope. As a result, on day 0, the cells isolated from the pancreas were at a state of cytodifferentiation of acinar cells.
  • acinar cells As a secretory organelle prominent in acinar cells, granules filled with high-density zymogens were observed, which were located in the apical region of cells and connected to the juxtanuclear Golgi and rough endoplasmic reticulum, positioned at the basal region of cells (Fig. 2). On day 2, acinar cells displayed major morphological changes, and in particular, high-density granules were observed at the edge of cells. On day 2 to day 4, the morphology of exocrine acinar cells observed on day 0 was not observed any more.
  • transitional cells with a decrease in the secretory organelle and zymogen granules indicate that the cells are derived from the acinar cell lineage.
  • This transdifferentiation of acinar cells occurred simultaneously with the increase of non-differentiated cells in a culture, and this change was continued until day 3 to day 5.
  • acinar cells continuously underwent degranulation (the D panel of Fig. 2).
  • primitive microvilli were developed around cell aggregates (the E panel of Fig. 2).
  • the overall morphological appearance of the transdifferentiated ductal cells was fairly similar to the profile of non-differentiated ductal cells.
  • Amylase activity was determined by measuring the amount of degraded starch when a culture supernatant was reacted in a substrate buffer solution containing starch for a predetermined period of time, wherein the amount of degraded starch was calculated from the difference between initial starch and residual starch (Fig. 3).
  • 1 unit of Caraway (Asan Pharm. Co., Hwa-Sung, Korea), used in this assay, refers to the amount of an enzyme capable of digesting 100 mg starch at 37°C for 30 min.
  • Fig. 3 when acinar cells underwent transdifferentiation during in vitro culturing, amylase expression levels decreased, and amylase activity was thus reduced.
  • Matrigel (Becton Dickinson, Franklin lakes, NJ, USA) was thawed according to the manufacturer's protocol and sprayed into each well of culture plates placed on ice. The plates were warmed to room temperature for several minutes, and a suitable density of cells was mixed with matrigel and cultured. The cells were cultured in a new medium, Waymouth medium supplemented with 10% FBS, and this medium was replaced with a fresh one twice per week. Separately, another cell substrate, collagen I gel, was prepared and suitably mixed with cells, and the cells were cultured. The cells were observed under an inverted light microscope to monitor mo ⁇ hological changes of the cells. Day 1, day 5 and day 10 after the overlay of matrigel or collagen gel, images for identical cells were captured.
  • acinar cells were also found to transdifferentiate into ductal cells (Fig. 4).
  • the pancreatic acinar cells started to form the ductal structure on day 5 after the primary culture (the A and B panels of Fig. 4), and on day 10, most cells formed a cystic structure
  • EXAMPLE 6 Evaluation of proliferation and tumor-forming ability and Western blotting for pancreatic cell markers of YGIC4 and YGIC5 cell lines
  • YGIC4 and YGIC5 were individually plated at a density of 5x10 4 cells/ml and reached confluence after 4.5 days.
  • YGIC4 and YGIC5 cells were found to have doubling times of 35 hrs and 32 hrs, respectively.
  • YGIC4 and YGIC5 cells were grown without a feeder after twelve passages.
  • YGIC cells were grown in monolayers, they exhibited the typical irregular and cobblestone-like morphology of fusiform epithelial cells. In addition, these cells did not contain any zymogen granules, and primitive microvilli emerged around cell aggregates.
  • YGIC cells were stored in liquid nitrogen upon rounds of subculturing, and were regenerated by thawing for use in experiments. These cells were found to maintain the initial morphology even after fifty passages.
  • YGIC4 and YGIC5 cells were subcutaneously injected into six-week-old ICR/nude mice. YGIC cells were found not to proliferate when subcutaneously injected into ICR/nude mice. This result indicates that the cell lines are normal cells not forming tumors. In addition, the cell lines were found not to possess a K-ras codon 12 mutation (found in 90% of pancreatic cancer). The YGIC cells obtained according to the present invention were found to have immortal morphological features and growth properties and not to form tumors. C.
  • Western blotting Western blotting was carried out to determine cell types with the following primary antibodies: mouse monoclonal anti-cytokeratine 19 (1 :1000, DAKO), rabbit polyclonal anti-amylase (1 :2000, Sigma), rabbit polyclonal anti-insulin, rabbit polyclonal anti-48, rabbit polyclonal anti-Pdxl (1 :1000), mouse monoclonal anti-CFTR, mouse monoclonal anti-vimentin, rabbit polyclonal anti-Notch- 1, rabbit polyclonal anti-Tcf-4, rabbit polyclonal anti-Wnt, rabbit polyclonal anti- ⁇ -catenin (1 :1000, Santa Cruz Biotechnology Inc.
  • the YGIC cells were found to express a pancreatic ductal cell marker CFTR and cytokeratine 19, while not expressing exocrine cell markers, amylase and p48, and an endocrine beta cell marker, insulin. Also, the YGIC cells were found to express genes participating in the pancreatic development, Notch- 1, Jagged- 1, Wnt-4, ⁇ -catenin, Pdx-1 and Tcf-4. Further, the YGIC cells expressed SCF/c-kit and vimentin.
  • EXAMPLE 7 Transdifferentiation of YGIC4 YGIC5 cells into pancreatic islet cells
  • the YGIC cell lines prepared in Example 6 were cultured in Waymouth medium supplemented with an antibiotic (penicillin, streptomycin or amphotericin B) and 10% FBS in a 5% CO2/95% air humidified incubator.
  • the cultured YGIC cell lines were transfected with pBX322/GLP-l (10 nmol/L) using Lipofectamine.
  • the transfected YGIC cell lines were evaluated with regards to the expression of stem cell factor (SCF), c-kit, PDX-1, Pax-4, Pax-6, Ngn-3, NeuroD, insulin, glucagons and CFTR by Western blotting, RT-PCR and ELISA (Fig. 7).
  • SCF stem cell factor
  • c-kit PDX-1
  • Pax-4 Pax-6
  • Ngn-3 NeuroD
  • insulin insulin
  • glucagons and CFTR by Western blotting, RT-PCR and ELISA
  • Fig. 7 As shown in Fig. 7, by treatment of the GLP-1, the cells displayed no change in expression levels of factors participating in the embryonic pancreatic development, Hes-1, Notch- 1, neuroD and sonic hedgehog (SHH), but exhibited reduced expression levels of neurogenin-3 (ngn-3).
  • the GLP-1 resulted in increased expression of PDX-1, a regulator of the pancreatic ⁇ -islet cell development, whereas resulting in decreased expression of Pax-6. Further, the GLP-1 resulted in increased expression of stem cell markers SCF and c-kit, whereas resulting in decreased expression of a pancreatic ductal cell marker CFTR. GLP-1 promoted the expression of insulin and glucagon, but did not affect the expression of Glut-2 and glucokinase.
  • pancreatic precursor cells derived from pancreatic acinar cells according to the present invention are capable of differentiating into all types of cells constituting the pancreas, and are thus useful for treating pancreatic diseases requiring organ or tissue transplantation.

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Abstract

L'invention concerne des précurseurs pancréatiques différentiés des cellules pancréatiques acinaires, qui expriment un gène marqueur et un gène participant au développement pancréatique, ainsi qu'un procédé de préparation de ces cellules pancréatiques, qui consiste en ce qui suit: (1) isoler les cellules pancréatiques acinaires chez un adulte, (2) cultiver in vitro les cellules pancréatiques acinaires dans un milieu destiné à la culture cellulaire mammalienne et (3) isoler les précurseurs pancréatiques isolant le précurseur pancréatique exprimant à la fois un gène marqueur de cellules ductales pancréatiques et un gène participant au développement pancréatique durant la culture in vitro. En outre, la présente invention concerne des cellules pancréatiques in vitro qui sont transdifférentiées à partir des cellules précurseurs pancréatiques et expriment un gène marqueur de cellules d'îlots pancréatiques, de même qu'un procédé pour préparer ces cellules d'îlots pancréatiques, qui consiste (1) à mettre en contact les précurseurs pancréatiques, préparés selon le procédé ci-décrit, avec un facteur de croissance, (2) cultiver les précurseurs pancréatiques dans un milieu destiné à la culture de cellules mammaliennes et (3) isoler les cellules d'îlots pancréatiques exprimant un gène marqueur de cellules d'îlots pancréatiques pendant la culture.
PCT/KR2004/002669 2004-04-02 2004-10-18 Souches de precurseurs pancreatiques transdifferentiees des cellules pancreatiques acinaires WO2005095589A1 (fr)

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JP2020182498A (ja) * 2012-11-30 2020-11-12 アクセラレイテッド・バイオサイエンシズ・コーポレーション ｍｉＲ−１２４を調節することにより幹細胞を分化させる方法

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KR101259074B1 (ko) * 2010-11-23 2013-04-29 서울대학교산학협력단 분비신호펩타이드가 연결된 엑센딘-4를 도입한 췌장소도세포 및 이를 이용한 당뇨병 치료방법

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JP2020182498A (ja) * 2012-11-30 2020-11-12 アクセラレイテッド・バイオサイエンシズ・コーポレーション ｍｉＲ−１２４を調節することにより幹細胞を分化させる方法
JP7082646B2 (ja) 2012-11-30 2022-06-08 アクセラレイテッド・バイオサイエンシズ・コーポレーション ｍｉＲ－１２４を調節することにより幹細胞を分化させる方法

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