WO2014188994A1 - Procédé d'amplification de cellules utilisant une préparation d'acides aminés - Google Patents

Procédé d'amplification de cellules utilisant une préparation d'acides aminés Download PDF

Info

Publication number
WO2014188994A1
WO2014188994A1 PCT/JP2014/063181 JP2014063181W WO2014188994A1 WO 2014188994 A1 WO2014188994 A1 WO 2014188994A1 JP 2014063181 W JP2014063181 W JP 2014063181W WO 2014188994 A1 WO2014188994 A1 WO 2014188994A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
hepatocytes
amino acid
liver
undifferentiated
Prior art date
Application number
PCT/JP2014/063181
Other languages
English (en)
Japanese (ja)
Inventor
英樹 谷口
貴則 武部
康晴 上野
博之 小池
Original Assignee
公立大学法人横浜市立大学
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 公立大学法人横浜市立大学 filed Critical 公立大学法人横浜市立大学
Priority to JP2015518230A priority Critical patent/JP6265385B2/ja
Publication of WO2014188994A1 publication Critical patent/WO2014188994A1/fr

Links

Images

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
    • C12N5/067—Hepatocytes
    • 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
    • C12N2500/32—Amino acids

Definitions

  • the present invention relates to a cell amplification method using an amino acid preparation, and more particularly to a method for amplifying a cell by culturing the cell using the amino acid preparation.
  • Non-patent Document 1 In general, attempts have been made to maintain and amplify stem cells and induce differentiation into various functional cells by adding protein preparations such as cytokines to a pluripotent stem cell culture system (Patent Documents 1 to 6). 3. Non-patent document 1).
  • An object of the present invention is to provide a method for efficiently and inexpensively creating functional cells from undifferentiated organ cells.
  • the inventors of the present invention focused on the difference in metabolic characteristics between undifferentiated cells existing in the intermediate differentiation stage and cells existing in other differentiation stages, which are the origins of organ cells that attempt mass creation.
  • metabolome analysis and transcriptome analysis were performed on the liver from the early embryonic period to adulthood for the purpose of detecting differences in metabolic characteristics in cells at different stages of differentiation.
  • the branched-chain amino acid degrading enzyme branched-chain aminotransferase: BCAT1
  • BCAT1 branched-chain aminotransferase
  • the branched chain amino acid has an action of inducing differentiation into a functional cell through induction of transient initial amplification of undifferentiated organ cells.
  • the gist of the present invention is as follows.
  • (2) The culture of undifferentiated hepatocytes in the presence of a branched-chain amino acid at a concentration of 1 mM or more amplifies undifferentiated hepatocytes and induces differentiation into hepatocytes (1) Method.
  • a method for inducing differentiation into hepatocytes comprising culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more.
  • Hepatocytes comprising amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more.
  • Preparation method Preparation method.
  • (6) A method for selecting cells capable of differentiating into hepatocytes comprising culturing undifferentiated hepatocytes in the presence of a branched-chain amino acid at a concentration of 1 mM or more.
  • a medium containing a branched chain amino acid at a concentration of 1 mM or more A medium containing a branched chain amino acid at a concentration of 1 mM or more.
  • the medium according to (7) which is used for amplifying undifferentiated hepatocytes.
  • the medium according to (7) which is used for inducing differentiation of undifferentiated hepatocytes into hepatocytes.
  • the culture method and medium provided by the present invention enable functional cells to be created efficiently and inexpensively from undifferentiated organ cells such as tissue stem cells derived from ES / iPS cells. This is expected to become a cell manipulation technique useful for creating a large amount of human hepatocytes necessary for drug discovery screening.
  • pluripotent stem cells in addition to obtaining a large amount of cells, pluripotent stem cells before tissue stem cells that cause tumor formation and ectopic tissue formation and cells that have differentiated into other organs It is essential to remove it.
  • tissue stem cells that cause tumor formation and ectopic tissue formation and cells that have differentiated into other organs It is essential to remove it.
  • target undifferentiated organ cells can be purified in large quantities at low cost. It is expected. Since there is no need to use complicated techniques such as genetic modification and FACS as in the prior art, it is an inexpensive and simple method with extremely high advantages.
  • the present invention provides a culture method capable of efficiently amplifying and sorting cells using an amino acid preparation that is less expensive than conventionally used protein preparations.
  • B Confirmation of mouse branching difference amino acid metabolizing enzyme (Bcat1) expression using quantitative RT-PCR. Similar to the results of (A), it is confirmed that the expression of Bcat1 is increased only in the early stage of liver development.
  • C Confirmation of Bcat1 expression in early embryonic mouse fetal liver (embryonic day 11.5) by immunohistochemical staining. Bcat1 expression is confirmed in fetal liver stained with Ck8 / 18. Red: Bcat1, green: Ck8 / 18 (epithelial cell marker), blue: DAPI. He: Sakai Heart, Sakai Li: Samurai Liver. Comparison of amino acid content in livers at different stages of differentiation by metabolomic analysis.
  • A Branching difference amino acid metabolic pathway diagram.
  • Bcat is responsible for the initial stage of metabolism of branched-chain amino acids (BCAA), and branched-chain amino acid metabolites are finally taken into the citric acid cycle and used for energy production.
  • B Comparison of amino acid concentrations in liver tissues of embryonic day 11.5 and 19.5 day mouse fetuses and 8-week-old adult mice, calculated from metabolomic analysis.
  • Leu leucine, al Val: valine, Ile: isoleucine, Arg: arginine, n Asn: asparagine, Asp, Cys: cysteine, Glu: glutamate, Phe: phenylalanine, Pro: proline, Ser: serine, Thr: ⁇ ⁇
  • the unit of the vertical axis is nmol / g. It is confirmed that the content of divergent amino acids (Leu, Val, Ile) is low in the liver on embryonic day 11.5. Evaluation of the effect on fetal liver formation when a diet containing no branched-chain amino acids is given to pregnant mice.
  • (A) Mouse fetuses fed a valine-free diet also have a reduced proportion of hepatic progenitor cells in the liver. Proliferation of mouse liver cells in the early stages of development is enhanced under culture conditions rich in L-valine among branched chain amino acids.
  • (A) A branched chain amino acid (L-valine, L-isoleucine, L-leucine) is added to a medium having a basic composition at a concentration in the range of 0.4 mM to 4 mM, and embryonic day 11 mouse fetal liver-derived cells are added for 6 days. Change in the number of colonies composed of 90 or more after culturing.
  • Cont indicates a group not added with amino acid, and the rate of increase relative to the value of Cont is indicated in%.
  • B Branched-chain amino acids (L-valine, L-isoleucine, L-leucine) and non-essential amino acids were added to a basic composition medium at a concentration of 4 mM, and embryonic day 11.5, 13.5, 15.5 mouse embryonic liver-derived cells Change in the number of colonies composed of 90 or more after 6 days of culture. The left is a photograph of a colony on the 6th day of culture when the control group and L-valine were added. The scale bar is 200 ⁇ m.
  • BCAA is L-valine, L-isoleucine and L-leucine added at 4 mM each, NEAA is a non-essential amino acid addition group, and the rate of increase relative to the value of Cont is indicated in%.
  • BCAA is L-valine, L-isoleucine and L-leucine added at 4 mM each, NEAA is a non-essential amino acid addition group, and the rate of increase relative to the value of Cont is indicated in%.
  • From the top row data for embryonic day 11.5, embryonic day 13.5, and embryonic day 15.5. Error bars are standard errors. Significant difference test is Mann-Whitney ’s U test.
  • N is 3-12. Under culture conditions rich in L-valine, differentiation of mouse hepatic stem / progenitor cells into hepatocytes is promoted.
  • A Immunostained image of colonies after adding 6 mM of branched chain amino acids and culturing for 6 days.
  • Red Amber albumin (ALB)
  • Green Amber cytokeratin (Ck) 7
  • Blue Amber DAPI. From the top, no addition group, L-valine, L-isoleucine, L-leucine addition group.
  • B Comparison of the abundance of each lineage cell in the colony when branched-chain amino acids were added, calculated from immunostained images.
  • the black part in the pie chart is the ratio of ALB positive and Ck7 negative cells, the gray part is ALB negative and Ck7 positive cells, and the white part is the ratio of ALB / Ck7 co-positive cells.
  • Stages 1, 2, 3, and 4 were defined for each time point at which the factors added to the culture system were changed.
  • B Comparison of human BCAT1 gene expression levels in human iPS-derived liver cells and human liver cells in each differentiation stage. In the differentiation process of iPS cells, it was confirmed that the expression of BCAT1 in Stage2 was the highest.
  • C Proliferative change of human iPS-derived hepatic endoderm cells upon addition of branched chain amino acids.
  • D Change in differentiation of human iPS-derived hepatic endoderm cells upon addition of branched chain amino acids. Error bars are standard errors. Significant difference test is Mann-Whitney ’s U test. N is 4-8.
  • a branched-chain amino acid (L-valine, L-isoleucine, L-leucine) is added to a medium having a basic composition at a concentration in the range of 0.4 mM to 20 mM, and embryonic day 11 mouse fetal liver-derived cells are added for 6 days. Change in the number of colonies composed of 90 or more after culturing.
  • the horizontal axis indicates the concentration of each branched chain amino acid added (mM), and the rate of increase relative to the value of the non-added group (0 ⁇ m) is indicated in%. Error bars are standard errors.
  • N is 3-12.
  • the present invention provides a method for amplifying undifferentiated hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched chain amino acid having a concentration of 1 mM or more.
  • the concentration of the branched chain amino acid is 1 ⁇ m or more, preferably 1 to 20 mM, and more preferably 2 to 10 mM.
  • branched chain amino acids examples include amino acids (both D and / or L-amino acids) such as valine, leucine, isoleucine and derivatives thereof, and combinations thereof, and valine is preferred.
  • Undifferentiated of undifferentiated hepatocytes refers to a state in which differentiation is not completely completed, and undifferentiated hepatocytes is a concept including all cells that can differentiate into hepatocytes.
  • undifferentiated hepatocytes include pluripotent stem cells such as iPS cells and ES cells, and undifferentiated organ (eg, liver) cells derived from living tissue.
  • the undifferentiated hepatocytes are preferably in a stage where the expression of BCAT1 is enhanced.
  • Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel. Examples of the coating agent for the cell culture container include matrigel, laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO 2 concentration is preferably 2% to 10%, and most preferably 5%.
  • the present invention also provides a method for inducing differentiation into hepatocytes, including culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more.
  • Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel. Examples of the coating agent for the cell culture container include matrigel, laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO 2 concentration is preferably 2% to 10%, and most preferably 5%.
  • Amplification of undifferentiated hepatocytes and induction of differentiation into hepatocytes by the method of the present invention can be used for the preparation of hepatocytes. Accordingly, the present invention includes amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 ⁇ mM or higher. A method for preparing hepatocytes is also provided.
  • mice when branched chain amino acids are added to liver-derived undifferentiated cells in the early embryonic period (E11.5), they are composed of cells that strongly express albumin, a hepatocyte-specific marker. Appeared frequently. Therefore, in the method of the present invention, undifferentiated hepatocytes cultured in the presence of a branched chain amino acid at a concentration of 1 ⁇ mM or higher are cells at a differentiation stage level corresponding to the period of E11.5 in mice. It is preferable. This differentiation stage is a stage in which a large amount of liver stem / progenitor cells are present.
  • liver differentiation markers eg, albumin (ALB), ⁇ -fetoprotein (AFP), transthyretin (TTR), forkhead box protein A2 (FOXA2), Hepatocyte nuclear factor 4 alpha (HNF4A), retinol binding protein 4 (RBP4), cytochrome P450 (CYP) 3A4, CYP3A7, glucose-6-phosphatase (G6PC), GATA binding protein 4/6 (GATA4 / 6), asialoglycoprotein receptor 1 (ASGR1) , Angiotensinogen (AGT), transferrin (TRF), apolipoprotein A-1 (APOA1), fibrinogen ⁇ chain (FGA), homogentisic acid (HGD), Carbamoyl-Phosphate Synthase 1 (CPS1) Can be guessed.
  • albumin e.g, albumin (ALB), ⁇ -fetoprotein (AFP), transthyretin (TTR), forkhead box protein A2 (FOXA2)
  • the level of differentiation stage corresponding to the time of mouse E11.5 It can be assumed that it is a cell. Also, if the expression level of markers such as ALB, RBP4, ASGR1, AGT, TRF, FGA, APOA1, HGD, CPS1 is reduced compared to cells in other differentiation stages, it corresponds to the time of mouse E11.5 It can be assumed that the cells are at the level of differentiation stage. On the other hand, it is also possible to estimate using the expression of BCAT1 as an index. Specifically, if the expression level of BCAT1 is increased as compared with ES cells or iPS cells, it can be assumed that the cells are at the level of differentiation corresponding to the time of mouse E11.5.
  • the present invention also provides a method for selecting cells capable of differentiating into hepatocytes, including culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more.
  • Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel.
  • coating agents for cell culture containers include laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO 2 concentration is preferably 2% to 10%, and most preferably 5%.
  • the present invention provides a medium containing a branched chain amino acid having a concentration of 1 mM or more.
  • the medium of the present invention can be used for amplifying undifferentiated hepatocytes.
  • the medium of the present invention can be used for inducing differentiation of undifferentiated hepatocytes into hepatocytes.
  • the culture medium of the present invention can also be used to select cells that can differentiate into hepatocytes.
  • the medium of the present invention contains amino acids other than branched chain amino acids (glutamine, alanine, arginine, asparagine, cysteine, glutamic acid, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine, and these Amino acids such as derivatives (both D and / or L-amino acids)), phenol red, pyruvate, HEPES, trace metals, phosphates, acetates, vitamins, ascorbic acid, nicotinamide, 2-mercaptoethanol, Dexamethasone, insulin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), activin A, basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP) 4, oncostatin M, hydrocortisone, heparin, blood vessel Endothelial cell growth factor (VEGF), insulin
  • the medium of the medium of the present invention water, serum, pH buffer solution or the like can be used.
  • commercially available media eg Dulbecco's Modified Eagle Medium (DMEM), E-MEM, IMDM, lactose-containing glucose-free DMEM, Ham F12, RPMI-1640, Williams E, etc., and mixtures thereof
  • DMEM Dulbecco's Modified Eagle Medium
  • E-MEM E-MEM
  • IMDM lactose-containing glucose-free DMEM
  • Ham F12 RPMI-1640
  • Williams E etc.
  • a branched chain amino acid may be added to) so as to contain a branched chain amino acid having a concentration of 1 mM or more.
  • the undifferentiated hepatocytes are described above.
  • branched-chain amino acids can promote amplification of undifferentiated hepatocytes. Therefore, the present invention provides an undifferentiated hepatocyte amplification promoter comprising a branched chain amino acid.
  • branched chain amino acids can induce differentiation from undifferentiated hepatocytes to hepatocytes. Therefore, the present invention provides an agent for inducing differentiation into hepatocytes containing a branched chain amino acid.
  • the present invention is a basic culture technique capable of drastically reducing costs for industrial production of human organ cells by using an inexpensive and simple method of adding an amino acid preparation (branched chain amino acid).
  • tissue / Organ Production Method WO2013 / 047639
  • the amino acid is an L-amino acid.
  • Example 1 result Comparison of metabolic characteristics in livers of different differentiation stages by metabolomic and transcriptome analysis To detect differences in the expression of metabolism-related genes in cells of different differentiation stages, we used mouse liver cells from early embryonic stages to adulthood. Transcriptome analysis was performed. As a result of comparing the expression of representative molecules in each metabolic pathway, the liver in the early developmental stage (embryonic day 9.5 to 11.5) in which undifferentiated hepatic stem / progenitor cells are present at a high frequency is observed in the subsequent developmental stage.
  • Bcat1 is an enzyme responsible for the initial metabolic reaction of branched chain amino acids (L-valine, L-leucine, L-isoleucine).
  • Metabolome analysis revealed that among amino acids, the concentration of branched-chain amino acids (L-valine, L-leucine, L-isoleucine) was low in the early stage of liver development (embryonic day 11) (Fig. 2).
  • Fig. 2 the concentration of branched-chain amino acids
  • L -leucine, L-isoleucine, L-valine which is responsible for the metabolic reaction of BCAT1, which enhances the proliferation of mouse hepatic stem / progenitor cells in culture conditions rich in L-valine, and frequently used in conventional culture systems
  • BCAT1 which enhances the proliferation of mouse hepatic stem / progenitor cells in culture conditions rich in L-valine, and frequently used in conventional culture systems
  • E13.5 and E15.5 liver-derived cells which were more advanced, were similarly evaluated for the effect of addition of amino acids on cell proliferation.
  • the appearance frequency of highly proliferative colonies was the same or decreased compared to the control when any branched-chain amino acid solution was added.
  • the growth-promoting effect of L-valine was not confirmed by analysis using liver cells in the middle of the embryonic period (E13.5 ⁇ ), so only in undifferentiated liver cells present in the early embryonic period. It was shown that the growth promoting effect by adding L-valine specifically appears.
  • L-valine-rich culture conditions enhance the differentiation of mouse hepatic stem / progenitor cells into hepatocytes.
  • L-leucine, L-isoleucine, and L-valine were examined the effects of L-leucine, L-isoleucine, and L-valine on cell differentiation. .
  • the effect of each branched-chain amino acid addition was compared with the non-amino acid addition group using liver-derived undifferentiated cells in the early embryonic period (E11.5). After culturing for 6 days in a medium supplemented with 4 mM branched-chain amino acid, the differentiation state of hepatic stem / progenitor cells was evaluated by immunostaining.
  • Undifferentiated liver cells can be repeatedly used for the creation of functional cells by induction of differentiation according to a previous patent (WO2009 / 139419) by repeating subculture while maintaining the properties.
  • the fact that 1.5-fold increase in proliferation can be induced in the undifferentiated cells can be expanded thereafter exponentially (power of 1.5), and a large amount of functional cells It becomes a very useful technique to obtain.
  • mice C57BL6 / J purchased from Japan SLC were used. All animal experiments were conducted with the approval of the committee based on the guidelines for animal experiments at Yokohama City University Fukuura Campus.
  • fetal liver cells After anesthetizing a wild-type pregnant mouse, the fetus was taken out from the abdominal cavity and the fetal liver was collected under a binocular stereomicroscope. The collected fetal liver was immersed in DMEM / F12 (Invitrogen) containing 0.2% trypsin and 5% fetal bovine serum (FBS: ICN), incubated on ice for 30 minutes, and then shaken at 37 ° C. for 15 minutes. Thereafter, the cells were dispersed by gentle pipetting. After centrifugation, the plate was washed twice with DMEM / F12 containing 5% FBS. The obtained fetal cells were passed through a nylon mesh (diameter 40 ⁇ m) to collect only single cells and used for the subsequent experiments.
  • DMEM / F12 Invitrogen
  • FBS fetal bovine serum
  • Culture of liver cells Isolated fetal liver cells were seeded on a 6-well plate (BD) coated with Laminin (BD) at 1000 cells / cm 2 and cultured. A uniquely prepared medium was used for cell culture.
  • Culture medium is 10% FBS, 1 ⁇ g / mL Insulin (Wako), 1 x 10 -7 M Dexamethasone (Sigma), 10 mmol / L nicotinamide (Sigma), 2 mmol / L L-glutamine (Invitrogen), 50 mmol / L 2-mercaptoethanol (Invitrogen), 5 mmol / L HEPES (Dojindo) 2.6 x 10 -4 mol / L L-Ascorbic acid 2-Phosphate (Sigma), DMEM / F12 medium containing 1 x penicillin / streptomycin (Invitrogen) Is added to the branched chain amino acid solution (isoleucine: Ile, leucine: Leu, valine: Val),
  • the influence on culture was evaluated. An equal amount of solvent (PBS) was added to the control. After cell seeding, when the cell adhesion is confirmed, the medium is changed, and the medium before replacement is further added with 20 ng / ml Epidermal growth factor (Sigma) and 25 ng / mL Hepatocyte growth factor (Kringle Pharma). The culture was continued. Incubation was performed at 37 ° C. in a 5% CO 2 gas phase.
  • the plate was washed three times with PBS containing 0.05% Tween 20, sealed with a trend protector (Vector), and observed under an upright fluorescence microscope (Zeiss Axio system).
  • the primary antibodies used were mouse anti-Ck7 antibody (Dako) (1: 100), rabbit anti-Albumin antibody (Biogenesis) (1: 500), and rabbit anti-AFP antibody (MP bio) (1: 200).
  • Secondary antibodies are Alexa488-labeled goat anti-mouse IgG1 antibody (Molecular Probe) (1: 500) and Aelxa555 (Molecular Probe) -labeled goat anti-rabbit IgG antibody (Molecular Probe) (1: 500).
  • Quantitative PCR Quantification was performed using the cDNA solution as a template.
  • the quantitative analysis used the comparative CT method.
  • relative quantification was performed by correcting the amount of cDNA used in the reaction based on the measured value of the GAPDH gene in each sample.
  • correction was performed based on the expression level of 18S.
  • HMT Human Metabolome Technologies
  • a methanol solution containing a mouse liver sample and an internal standard substance of 50 ⁇ M was placed in a crushing tube, and crushed using a desktop crusher (bms) under cooling. Chloroform and Milli-Q water were added to this and stirred, followed by centrifugation. After centrifugation, the aqueous layer was transferred to an ultrafiltration tube. This was centrifuged and subjected to ultrafiltration treatment. The filtrate was dried and dissolved in Milli-Q water again for measurement.
  • Detected peaks were automatically extracted using automatic integration software (MasterHandsver.2.9.0.9), and mass charge cost, migration time and peak area values were obtained as peak information. The obtained peak area value was converted into a relative area value. These data included adduct ions such as Na + and K + and fragment ions such as dehydration and deammonium, so these molecular weight related ions were deleted. For the examined peaks, the peaks between each sample were collated and aligned.
  • liver cells differentiated in this laboratory from iPS cells donated by Professor Nakauchi of the University of Tokyo were created by co-culturing three types of umbilical vein endothelial cells (Lonza) and mesenchymal stem cells (Lonza). Liver buds created by mixing cells at a ratio of 10: 7: 2 and culturing them in a microwell plate are then a medium rich in specific amino acids or a small amount of amino acids (RPMI). And EGM mixed 1: 1) for 2 weeks, and the size was measured. The size of the liver bud was measured using IN Cell Analyzer 2000 based on the fluorescence exhibited by the liver bud.
  • Example 2 In order to clarify the effective concentration of L-valine for promoting cell growth, L-valine was added at various concentrations, and the effect of promoting cell growth on embryonic day 11.5 mouse fetal liver cells was examined. As a result, the cell growth promoting effect was the highest when 4 mM of L-valine was added, and the tendency to promote the growth was observed when the concentration range was 0.8-20 mM (FIG. 9). From these results, it was suggested that the addition of L-valine is effective in enhancing cell proliferation in the range of 0.8-20 mM.
  • the present invention can be used as a cell culture technique or a cell manipulation technique for regenerative medicine or drug screening.
  • the present invention leads to the development of a medium preparation for amplifying organ cells, a medium preparation for sorting hepatocytes induced to differentiate from organ cells, and the like.

Abstract

L'invention concerne un procédé de production d'une cellule fonctionnelle à partir d'une cellule non différenciée d'un organe avec efficacité élevée et faible coût. Un procédé d'amplification d'une cellule hépatique non différenciée, qui comprend la culture de la cellule hépatique non différenciée en la présence d'un acide aminé ramifié à une concentration de 1 mM ou plus. L'invention concerne également : un procédé d'induction de la différenciation en une cellule hépatique ; un procédé de préparation d'une cellule hépatique ; un procédé de sélection d'une cellule qui peut être différenciée en une cellule hépatique ; un milieu de culture ; un promoteur pour la prolifération d'une cellule hépatique non différenciée ; et un inducteur de la différenciation en une cellule hépatique.
PCT/JP2014/063181 2013-05-20 2014-05-19 Procédé d'amplification de cellules utilisant une préparation d'acides aminés WO2014188994A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2015518230A JP6265385B2 (ja) 2013-05-20 2014-05-19 アミノ酸製剤による細胞増幅法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2013106289 2013-05-20
JP2013-106289 2013-05-20

Publications (1)

Publication Number Publication Date
WO2014188994A1 true WO2014188994A1 (fr) 2014-11-27

Family

ID=51933547

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2014/063181 WO2014188994A1 (fr) 2013-05-20 2014-05-19 Procédé d'amplification de cellules utilisant une préparation d'acides aminés

Country Status (2)

Country Link
JP (1) JP6265385B2 (fr)
WO (1) WO2014188994A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017068801A1 (fr) * 2015-10-23 2017-04-27 株式会社島津製作所 Méthode d'évaluation de l'état de différenciation de cellules
WO2020080270A1 (fr) * 2018-10-15 2020-04-23 公立大学法人横浜市立大学 Composition nutritionnelle

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005047454A1 (fr) * 2003-11-13 2005-05-26 Ajinomoto Co., Inc. Plaque contenant un milieu a base d'acides amines et procede de criblage au moyen de cette plaque
JP3696297B2 (ja) * 1994-06-23 2005-09-14 中外製薬株式会社 肝再生用治療剤
WO2012111790A1 (fr) * 2011-02-17 2012-08-23 味の素株式会社 Potentialisateur de l'activité antitumorale d'un agent chimiothérapique
WO2013018851A1 (fr) * 2011-08-02 2013-02-07 独立行政法人国立がん研究センター Procédé d'induction de la différentiation hépatique à partir d'une cellule souche hépatique induite et d'une cellule progénitrice hépatique induite

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3696297B2 (ja) * 1994-06-23 2005-09-14 中外製薬株式会社 肝再生用治療剤
WO2005047454A1 (fr) * 2003-11-13 2005-05-26 Ajinomoto Co., Inc. Plaque contenant un milieu a base d'acides amines et procede de criblage au moyen de cette plaque
WO2012111790A1 (fr) * 2011-02-17 2012-08-23 味の素株式会社 Potentialisateur de l'activité antitumorale d'un agent chimiothérapique
WO2013018851A1 (fr) * 2011-08-02 2013-02-07 独立行政法人国立がん研究センター Procédé d'induction de la différentiation hépatique à partir d'une cellule souche hépatique induite et d'une cellule progénitrice hépatique induite

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHIORI IJICHI ET AL.: "Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 303, no. 1, 2003, pages 59 - 64, XP008162560, DOI: doi:10.1016/S0006-291X(03)00295-X *
GIULIO MARCHESINI ET AL.: "Branched-Chain Amino Acid Supplementation in Patients with Liver Diseases", J. NUTRITION, vol. 135, no. 6, 2005, pages 1596S - 1601S *
NANHO HO ET AL., SAIBO BAIYO NARUHODO Q&A IGAI TO SHIRANAI KISO CHISHIKI + TOSSA NI YAKUDATSU TECHNIQUE, 10 February 2005 (2005-02-10), pages 82 - 83 *
TOMOAKI TOMIYA ET AL.: "Branched-chain amino acids, hepatocyte growth factor and protein production in the liver", HEPATOLOGY RESEARCH, vol. 30, 2004, pages 14 - 18 *
TOMOAKI TOMIYA ET AL.: "Leucine no HGF Sansei Sokushin Sayo to sono Rinshoteki Igi", JAPANESE JOURNAL OF NUTRITIONAL ASSESSMENT, vol. 20, no. 5, 2003, pages 495 - 498 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017068801A1 (fr) * 2015-10-23 2017-04-27 株式会社島津製作所 Méthode d'évaluation de l'état de différenciation de cellules
WO2020080270A1 (fr) * 2018-10-15 2020-04-23 公立大学法人横浜市立大学 Composition nutritionnelle
EP3868869A4 (fr) * 2018-10-15 2022-08-03 Public University Corporation Yokohama City University Composition nutritionnelle

Also Published As

Publication number Publication date
JP6265385B2 (ja) 2018-01-24
JPWO2014188994A1 (ja) 2017-02-23

Similar Documents

Publication Publication Date Title
Asai et al. Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells
JP6884908B2 (ja) 成体肝前駆細胞を作製する方法
Katsuda et al. Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes
KR102101060B1 (ko) 성체 간 전구 세포의 제조 방법
KR102598772B1 (ko) 상피 세포의 생체외 증식
Doddapaneni et al. Overexpression of microRNA‐122 enhances in vitro hepatic differentiation of fetal liver‐derived stem/progenitor cells
JP6812003B2 (ja) 肝細胞及び肝非実質細胞、並びにそれらの調製方法
EP3401392B1 (fr) Procédé de production de cellules souches/précurseurs hépatiques à partir de cellules hépatiques matures à l'aide d'un composé de faible poids moléculaire
CN104781393A (zh) 用于生产诱导肝细胞的方法和组合物
AU2012211793A1 (en) Highly functional liver cells derived from pluripotent stem cells, method for producing same, and method for testing metabolism/toxicity of drug
Qu et al. Apoptotic vesicles inherit SOX2 from pluripotent stem cells to accelerate wound healing by energizing mesenchymal stem cells
Merkely et al. Signaling via PI3K/FOXO1A pathway modulates formation and survival of human embryonic stem cell-derived endothelial cells
Winkler et al. Mouse white adipose tissue‐derived mesenchymal stem cells gain pericentral and periportal hepatocyte features after differentiation in vitro, which are preserved in vivo after hepatic transplantation
US10093903B2 (en) Production of virus-receptive pluripotent stem cell (PSC)-derived hepatocytes
US20220016178A1 (en) Generation Of Uniform Hepatocytes From Human Embryonic Stem Cells By Inhibiting TGF-BETA and Methods Of Maintaining Hepatic Cultures
CN115109740A (zh) 肝细胞调控制剂及其制备方法和应用
Gomes-Alves et al. In vitro expansion of human cardiac progenitor cells: exploring'omics tools for characterization of cell-based allogeneic products
JP6265385B2 (ja) アミノ酸製剤による細胞増幅法
US9243228B2 (en) Process for obtaining myofibroblasts
Kotasová et al. Expandable lung epithelium differentiated from human embryonic stem cells
Wu et al. Hepatic differentiation of mouse bone marrow‑derived mesenchymal stem cells using a novel 3D culture system
Pettinato et al. Development of a scalable three-dimensional culture of human induced pluripotent stem cells-derived liver organoids
He et al. Low serum concentration facilitates the differentiation of hepatic progenitor cells
JP6486619B2 (ja) 薬物評価用細胞及び薬物評価方法
Zhang et al. Characterization of human induced pluripotent stem cell (iPSC) line from a 72 year old male patient with later onset Alzheimer's disease

Legal Events

Date Code Title Description
DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14800230

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2015518230

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14800230

Country of ref document: EP

Kind code of ref document: A1