WO2019021990A1 - Cellules de type entérocyte - Google Patents

Cellules de type entérocyte Download PDF

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WO2019021990A1
WO2019021990A1 PCT/JP2018/027454 JP2018027454W WO2019021990A1 WO 2019021990 A1 WO2019021990 A1 WO 2019021990A1 JP 2018027454 W JP2018027454 W JP 2018027454W WO 2019021990 A1 WO2019021990 A1 WO 2019021990A1
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cells
differentiation
small intestine
culture
drug
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PCT/JP2018/027454
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English (en)
Japanese (ja)
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水口　裕之
和雄 高山
亮介 根来
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国立大学法人大阪大学
国立研究開発法人医薬基盤・健康・栄養研究所
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Priority to JP2019532586A priority Critical patent/JP7181556B2/ja
Publication of WO2019021990A1 publication Critical patent/WO2019021990A1/fr

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    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the present invention relates to a method for inducing differentiation of pluripotent stem cells into small intestinal epithelial-like cells (ELC), and further relates to small intestinal epithelial-like cells obtained by the method for inducing differentiation. Furthermore, the present invention relates to a method for evaluating drug toxicity and / or a method for evaluating pharmacokinetics using the above-mentioned small intestine epithelial-like cells. Furthermore, the present invention relates to a medium composition for inducing differentiation into the small intestine epithelial-like cells and a kit for preparation of the medium.
  • Pluripotent stem cells are undifferentiated cells having pluripotency and self-replication ability, and it has been suggested that cells induced to differentiate from pluripotent stem cells have tissue repair ability after tissue damage. For this reason, pluripotent stem cells and their differentiated cells are being studied extensively as being useful in screening of substances for treating various diseases and in the field of regenerative medicine.
  • pluripotent stem cells iPS cells are released from somatic cells by introducing specific transcription factors such as OCT3 / 4, SOX2, KLF4 and C-MYC into somatic cells such as fibroblasts. It is an induced pluripotent stem cell produced by differentiation.
  • the pluripotent cells can theoretically be induced to differentiate into all tissues and organs including small intestinal epithelial cells and the like.
  • Non-Patent Document 1 (Nature, 2011 Feb 3; 470 (7332): 105-9) is the first paper in the world to produce small intestine tissue from human pluripotent stem cells. In this paper, we show that it is possible to produce organoids that contain small intestinal epithelial cells, Panate cells, Goblet cells and intestinal epithelial endocrine cells present in the small intestine.
  • Non-Patent Document 2 (Stem Cell Reports, 2014 Jun 3; 2 (6): 838-52) is a paper that reports that long-term self-replicatable small intestinal stem cells can be produced from human pluripotent stem cells.
  • the small intestinal stem cells prepared in this paper can be differentiated into organoids including all of the small intestinal epithelial cells, Panate cells, Goblet cells, and intestinal epithelial endocrine cells present in the small intestine, as in Non-Patent Document 1.
  • Non-patent document 3 (Stem Cells, 2013 Jun; 31 (6): 1086-96) induces differentiation of mouse and human pluripotent stem cells into cells of the small intestine lineage, BIO (6-GSK-3 Inhibitor IX) Using Bromoindirubin-3'-oxime), DAPT (N-[(3,5-Difluorophenyl) acetyl] -L-alanyl-2-phenyl] glycine-1,1-dimethylethyl ester) as a ⁇ -secretase inhibitor, etc.
  • BIO 6-GSK-3 Inhibitor IX
  • DAPT N-[(3,5-Difluorophenyl) acetyl] -L-alanyl-2-phenyl] glycine-1,1-dimethylethyl ester
  • CDX2 cartilage type homeobox 2
  • BIO and DAPT it becomes possible to efficiently induce differentiation of CDX2 (caudal type homeobox 2) positive cells from pluripotent stem cells.
  • CDX2 is a master transcription factor that controls intestinal differentiation which is expressed in any of hindgut, small intestine stem cells, intestinal progenitor cells and small intestinal epithelial cells.
  • Non-Patent Document 4 (Drug Metab Pharmacokinet, 2014; 29 (1): 44-51) is an article that attempts to differentiate human pluripotent stem cells into small intestinal epithelial-like cells.
  • small intestinal epithelium-like cells expressing small intestine markers such as SI (Sucrase Isomaltase), SLC 15 A1 (solute carrier family 15 member 1) / PEPT1 (Peptide transporter 1), LGR 5 (leucine-rich repeat containing G protein-coupled receptor 5) Cells can be generated.
  • SI Sudcrase Isomaltase
  • SLC 15 A1 solute carrier family 15 member 1
  • PEPT1 Peptide transporter 1
  • LGR 5 leucine-rich repeat containing G protein-coupled receptor 5
  • the prepared small intestinal epithelial-like cells can take in the dipeptide ⁇ -Ala-Lys-AMCA ( ⁇ -Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid).
  • drug metabolizing enzyme CYP3A4 Cytochrome P450 3A4
  • Non-Patent Document 5 is also a paper in which differentiation of human pluripotent stem cells into small intestinal epithelial-like cells is attempted.
  • small intestinal epithelial-like cells expressing small intestinal markers such as SI, Villin, SLC15A1 / PEPT1, BCRP (Breast cancer resistance protein) can be prepared. Furthermore, it is shown in non-patent document 6 that this small intestinal epithelial-like cell has PEPT1 activity and BCRP activity (Drug Metab Dispo, 2016 Oct; 44 (10): 0. doi: 10.1124 / dmd. 116.069336. Epub 2016 Jul 14.) It is indicated by. However, the problem is that the expression of the drug transporter ABCB1 (ATP-binding cassette sub-family B member 1) / MDR1 (multidrug resistance protein 1) is extremely low (about 1/100) compared to human small intestine. is there.
  • ABCB1 ATP-binding cassette sub-family B member 1
  • MDR1 multidrug resistance protein 1
  • Non-Patent Document 1-6 research is being conducted on the development of techniques for inducing differentiation into small intestinal epithelial cells, but these methods can simultaneously evaluate drug metabolism and drug absorption from human pluripotent stem cells.
  • ALK5 inhibitor SB 431 542
  • Wnt 3a Wnt 3a
  • EGF epidermal growth factor
  • Patent Document 1 discloses that the efficiency of differentiation into small intestinal epithelial cells is improved by introducing the CDX2 gene and / or the FOXA2 gene into cells using an adenoviral vector (hereinafter referred to as "Ad vector"). ing.
  • Ad vector an adenoviral vector
  • Caco-2 cells (a cell line derived from human colon cancer) capable of forming strong tight junctions and predicting drug permeation in the small intestine can be put to practical use as a model cell of the small intestine in vitro absorption evaluation system. ing.
  • the problem is that the above cells are derived from cancer cells and, unlike human small intestinal epithelial cells, hardly express the drug metabolizing enzyme CYP3A4, etc., it is a stable test regarding drug metabolism and permeability in the small intestine. There is a great need for possible superior cells.
  • the present inventors conducted further studies on culture solution and culture time based on the conventional method for producing small intestine epithelial-like cells in order to achieve the above-mentioned problems, and as a result, pluripotent stem cells (definitive endoderm) were obtained.
  • pluripotent stem cells definitive endoderm
  • LY2090314 is added to a culture system and cultured, and it is found that differentiation of pluripotent stem cells to small intestine epithelial-like cells can be effectively induced by preparing intestinal progenitor cells, the present invention Completed.
  • the present invention consists of the following.
  • a method for inducing differentiation of pluripotent stem cells into intestinal epithelial-like cells comprising the following steps: 1) inducing differentiation of pluripotent stem cells into endoderm cells; 2) A step of culturing the endoderm cells obtained by the differentiation induction in a system containing LY 2090314 to induce differentiation into intestinal progenitor cells.
  • the small intestinal epithelial-like cells obtained by the method for inducing differentiation according to any one of the above 1 to 4. 6.
  • a medium for inducing differentiation to small intestine epithelial-like cells which comprises LY2090314.
  • a medium for inducing differentiation into small intestine epithelial-like cells which comprises Wnt3a and EGF.
  • 10. 10 The culture medium for differentiation induction according to the above 9, which comprises any two or more humoral factor compounds selected from Wnt3a, EGF, p38 MAPK inhibitor, IGF-1, R-spondin, Noggin and DEX. 11. 11.
  • the differentiation-inducing medium according to item 10 which comprises Wnt3a, EGF, p38 MAPK inhibitor, IGF-1, R-spondin, Noggin and DEX. 12. At least two or more reagents including at least two or more types of liquid factor compounds selected from LY2090314, Wnt3a, EGF, p38 MAPK inhibitor, IGF-1, R-spondin, Noggin and DEX , A kit for preparing a medium for inducing differentiation into small intestine epithelial-like cells.
  • the liquid factor compound is added to the basic medium for induction of differentiation and the cells are cultured, without performing the gene transfer operation with the Ad vector or the like.
  • pluripotent stem cells can be easily and efficiently induced to differentiate into small intestinal epithelial-like cells.
  • pluripotent stem cells are induced to differentiate into endoderm cells, and then LY2090314 is added to the culture system and cultured to produce intestinal progenitor cells.
  • the differentiation induction method of the present invention effectively produces pluripotent stem cells. Can induce differentiation into small intestinal epithelial-like cells.
  • CDX2 gene in pluripotent stem cell-derived small intestinal epithelial cells is human by culturing in a medium containing Wnt3a, EGF, p38 MAPK inhibitor, IGF-1, R-spondin, Noggin and DEX in intestinal progenitor cells Because it is higher than the adult small intestine (Adult Intestine: AI), it is considered that highly efficient differentiation induction could be achieved without gene transfer using an Ad vector.
  • the differentiation induction method of the present invention provides stable small intestinal epithelial-like cells stably. It has become possible.
  • the obtained cells have the properties of various small intestinal epithelial-like cells, and are excellent in terms of the expression amounts of drug metabolizing enzymes and drug transporters. According to the above, with regard to drug metabolism and permeability in the small intestine, it is possible to simultaneously evaluate drug metabolism and drug absorption under a homogeneous evaluation system using stably excellent small intestinal epithelial-like cells.
  • FIG. 1 It is a figure which shows the experimental protocol for the screening of the GSK3 (beta) inhibitor effective in the differentiation induction to an intestinal progenitor cell (intestinal progenitor cells) from a definitive endoderm cell.
  • Example 2 It is the figure which confirmed expression of various gene markers when endodermal cell culture is culture
  • Example 2 It is a photograph figure which shows the result of having analyzed the protein expression level of CDX2 when culturing endodermal cell culture in the culture system containing various GSK3 beta inhibitors by the western blot method.
  • Example 2 It is the figure which computed the CDX2 positive cell rate by the effect
  • Example 2 It is the photograph figure which confirmed the expression of CDX2 by the effect
  • Example 2 It is the figure which confirmed expression of various gene markers when endodermal cells are cultured by the culture system which changed the use density
  • Example 3) It is a photograph which shows the result of having analyzed the protein expression level of CDX2 when the use density
  • Example 3 It is the figure which computed the CDX2 positive cell ratio when the use density
  • Example 3 It is a figure which shows the experimental protocol for the screening of the humoral factor compound effective in differentiation-induction from intestinal progenitor cells (intestinal progenitor cells) to small intestinal epithelial-like cells (enterocyte-like cells).
  • Example 4 It is the figure which confirmed expression of a Villin or ISX gene marker when a gut precursor cell is culture
  • Example 4 It is the figure which confirmed expression of a Villin or ISX gene marker when an intestinal progenitor cell is culture
  • Example 4 It is the figure which confirmed expression of Villin or CDX2 gene marker after culture
  • Example 4 It is a figure which shows the experimental protocol for confirming the culture
  • Example 5 It is a figure which confirmed expression of Villin, ISX, or a CDX2 gene marker after culture for each period.
  • Example 5 It is the figure which confirmed the expression of Villin, ISX, CDX2 or ANP EP gene marker of the small intestine epithelial-like cell (ELC) obtained by culture
  • Example 5 It is a figure which shows the experimental protocol for confirming the combination of the humoral factor compound effective in the differentiation induction from a intestinal progenitor cell (intestinal progenitor cells) to a small intestinal epithelial-like cell (enterocyte-like cells).
  • Example 6 In the small intestine epithelial-like cells (ELC) prepared by the method of the present invention, the expression levels of various genes highly expressed in the small intestine are relatively compared with the expression levels in human adult small intestine (AI) and human adult colon (colon) FIG.
  • Experimental Example 6-1 In the small intestine epithelial-like cells (ELC) produced by the method of the present invention, the expression levels of various genes highly expressed in the large intestine are relatively compared with the expression levels in human adult small intestine (AI) and human adult colon (colon) FIG.
  • Experimental Example 6-1 Regarding small intestine epithelial-like cells (ELC) produced by the method of the present invention, the results of comparing the gene expression levels of various drug-metabolizing enzymes relative to the expression levels in human adult small intestine (AI) and human adult colon (colon) FIG.
  • Example 6-2 It is a figure which shows the result of having analyzed the drug metabolising enzyme CYP3A4 inducibility about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • Example 6-2 It is a figure which shows the result of having confirmed the activity of carboxylesterase 2 (CES2) about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • CES2 carboxylesterase 2
  • Experimental Example 6-3 The expression of various drug transporter-related genes expressed on the apical membrane side of the small intestine epithelial-like cells (ELCs) prepared by the method of the present invention is expressed relative to the amount expressed in human adult small intestine (AI) and human adult colon (colon) It is a figure showing the result of comparing.
  • ELCs small intestine epithelial-like cells
  • the expression of various drug transporter-related genes expressed on the basement membrane side is expressed relative to the amount expressed in human adult small intestine (AI) and human adult colon (colon) It is a figure showing the result of comparing.
  • Example 6-4 It is the figure which confirmed the cell membrane barrier ability by the membrane permeability coefficient of cell membrane resistance (TEER) and lucifer yellow (Lucifer yellow: LY) about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • Example 6-5 It is a figure which shows the result of having evaluated the drug transport function by MDR1 transport capacity about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • Example 6-6 It is a figure which shows the result of having evaluated the drug transport function by PEPT1 transport capacity about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • E-cadherin E-cadherin
  • B.I It is a figure which shows the expression result of ZO-1.
  • E-cadherin E-cadherin
  • B.I It is a figure which shows the expression result of ZO-1.
  • E-cadherin E-cadherin
  • B.I It is a figure which shows the expression result of ZO-1.
  • E-cadherin E-cadherin
  • B.I It is a figure which shows the expression result of ZO-1.
  • CDX2 CDX2
  • E-cadherin E-cadherin about the intestinal-progenitor cell produced by the method of this invention.
  • Villin and PEPT1 Villin and PEPT1 for evaluating whether it has polarity about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • Experimental Example 7-3 It is a figure which shows the ALP staining result for evaluating ALP activity about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • Experimental Example 7-4 It is a figure which shows the transmission microscope observation result for carrying out a morphological evaluation about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • (Experimental example 7-5) It is a figure which shows the expression result of Chromogranin A, lysozyme, and Mucin2 about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • Example 7-6 It is a figure which shows the result of having performed the comprehensive gene analysis by the microarray in order to confirm a small intestine type or a large intestine type characteristic about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • Example 7-7 It is the figure which confirmed the cell membrane barrier ability by the membrane permeability coefficient of FD4 about the small intestine epithelial-like cell (ELC) produced by the method of this invention.
  • the present invention relates to a method for selectively inducing differentiation of pluripotent stem cells (PSCs) to small intestinal epithelial-like cells (ELCs), and further to excellent expression of drug metabolizing enzymes and drug transporters.
  • PSCs pluripotent stem cells
  • ELCs small intestinal epithelial-like cells
  • Small intestinal epithelial-like cells Small intestinal epithelial-like cells.
  • Caco-2 cells are human colon cancer-derived cell lines and can form strong tight junctions, they are widely used as an in vitro absorption evaluation system as a model for predicting drug permeation in the small intestine.
  • the main drug-metabolizing enzyme in small intestinal epithelial cells is CYP3A4
  • Caco-2 cells unlike human small intestine, hardly express drug-metabolizing enzymes. Therefore, drug metabolism can not be evaluated in Caco-2 cells. At present, no experimental system has been established that can simultaneously evaluate drug metabolism and drug absorption in the small intestine.
  • CYP1A2, CYP2B6, CYP3A4 drug metabolizing enzymes
  • CYP induction The induction of CYP leads to a large change in drug metabolism rate from that in non-induction.
  • CYP3A4 which is the main CYP molecular species in the small intestine, is induced by drugs such as VD3 (active vitamin D3) and RIF (rifampicin). Therefore, the present inventors aimed to produce small intestinal epithelial-like cells applicable to evaluation of drug metabolism and drug absorption from human pluripotent stem cells.
  • pluripotent stem cells may be undifferentiated cells having pluripotency and / or self-replication ability, and are not particularly limited, but iPS cells (induced pluripotent stem cells) or ES cells (embryonic stem cells) And other pluripotent stem cells. Particularly preferred are iPS cells.
  • iPS cells induce the reprogramming of differentiated cells without using fertilized eggs, surplus embryos or ES cells by introducing several kinds of genes into somatic cells, and pluripotency and proliferation ability similar to ES cells And induced pluripotent stem cells, which were first made in the world from mouse fibroblasts in 2006 (Cell 126: 663-676, 2006). Furthermore, it has been successfully established human iPS cells by introducing into human derived fibroblasts OCT3 / 4, SOX2, KLF4 and C-MYC, which are human homologous genes of 4 genes used for establishing mouse iPS cells. It has been reported (Cell 131: 861-872, 2007).
  • the iPS cells used in the present invention may be iPS cells produced by a method known per se as described above, or iPS cells produced by a new method to be developed in the future.
  • Tic JCRB1331
  • YO2 and iHC1 can be used.
  • ES cells are pluripotent stem cells isolated as an undifferentiated stem cell population by transferring a cell mass generally called the inner cell mass inside the blastocyst stage embryo to in vitro culture. It is.
  • ES cell lines pluripotent in mice by GRMartin (Natl. Acad. Sci. USA, 78, 7634, 1981) followed by MJEvans & MHKaufman (Nature, 292, 154, 1981) It was established. Many strains of human-derived ES cells have already been established, and they can be obtained from ES Cell International, Wisconsin Alumni Research Foundation, National Stem Cell Bank (NSCB), and the like.
  • ES cells are generally established by culturing early embryos, but it is also possible to produce ES cells from early embryos obtained by nuclear transfer of somatic cell nuclei. Furthermore, a blastocyst stage embryo-like cell structure is prepared by transferring a cell nucleus of a desired animal to cell vesicles (cytoplasts, ooploidoids) obtained by dividing egg cells of heterologous animals or enucleated egg cells into multiple cells, There is also a method of producing ES cells based on it.
  • cell vesicles cytoplasts, ooploidoids
  • an ES cell with genetic information of somatic cell nucleus by generating parthenogenetic embryos to a stage equivalent to the blastocyst stage and preparing ES cells therefrom, or by fusing ES cells with somatic cells How to make a has also been reported.
  • the ES cells used in the present invention may be ES cells produced by a method known per se as described above, or ES cells produced by a new method to be developed in the future.
  • ES cells for example, human ES cell lines (KhES3) can be used.
  • the term "enterocyte-like cells” refers to cells produced by the method for inducing differentiation of the present invention or the treatment for inducing differentiation.
  • the small intestine epithelial-like cells produced by the method of the present invention may be simply abbreviated as “ELC”.
  • the intestinal progenitor cells shown in the above step 2) refer to cells before being induced to differentiate into the small intestinal epithelial cells of the present invention, and are synonymous with, for example, small intestinal progenitor cells.
  • a method known per se can be applied to the method of differentiating pluripotent stem cells into endoderm cells in the step 1) in the preparation of small intestinal epithelial-like cells.
  • activin A it is necessary to use activin A in a culture system of pluripotent stem cells.
  • Activin A can be added to the culture system at 3 to 200 ng / ml, preferably about 100 ng / ml. When the concentration of activin A is lower than 3 ng / ml, it is considered that differentiation to endodermal cells can not be efficiently promoted.
  • the time to add activin A to the culture system is not particularly limited as long as differentiation of pluripotent stem cells into endoderm cells is possible, for example, 0 to 6 days after the start of pluripotent stem cell culture, for example Can be from 1 to 7 days.
  • step 2) it is necessary to culture endoderm cells obtained by the differentiation induction in a system containing a GSK3 ⁇ inhibitor.
  • a GSK3 ⁇ inhibitor LY2090314 is suitable, and 3 to 100 nM, preferably about 20 nM can be added to the culture system. When the concentration of LY2090314 is lower than 3 nM, it is considered that differentiation can not be efficiently promoted.
  • the time when LY 2090314 is added to the culture system is not particularly limited as long as it is after differentiation induction into endoderm cells, but for example, it is added 0 to 11 days after endodermal cell culture start, preferably 0 to 4 days And the preferred addition period may be 4 days.
  • endoderm cells can be induced to differentiate into intestinal progenitor cells. The induction of differentiation into intestinal progenitor cells can be confirmed by CDX2 expression.
  • the cells can be further cultured in a system containing Wnt 3a and EGF (epidermal growth factor) in the step 3) to induce differentiation into small intestine epithelial-like cells.
  • Wnt 3a and EGF epidermal growth factor
  • the step 3 it is more preferable to culture in a system comprising Wnt3a and EGF, and any two or more selected from p38 MAPK inhibitors, IGF-1, R-spondin, Noggin and DEX. Can be induced to differentiate into small intestinal epithelial-like cells.
  • step 3 culturing in a system containing Wnt3a, EGF, p38 MAPK inhibitor, IGF-1, R-spondin, Noggin and DEX is most preferable for inducing differentiation into small intestinal epithelial-like cells.
  • 0.1 nM to 10 mM can be contained in the culture system.
  • EGF 0.5 to 5000 ng / ml, preferably about 50 ng / ml can be added to the culture system.
  • p38 MAPK inhibitors include SB202190, SB203580, SB706504 and the like, and most preferred is SB202190.
  • 0.1 to 1000 ⁇ M preferably about 10 ⁇ M can be added to the culture system.
  • IGF-1 0.2 to 2000 ng / ml, preferably about 20 ng / ml can be added to the culture system.
  • 0.1 nM to 10 mM can be contained in the culture system.
  • 0.1 nM to 10 mM can be contained in the culture system.
  • DEX 0.01 to 100 ⁇ M, preferably about 1 ⁇ M can be added to the culture system.
  • the humoral factor compound to be added is Wnt3a (0.1 nM to 10 mM), EGF (50 ng / ml), p38 MAPK inhibitor (10 ⁇ M), IGF-1 (20 ng / ml),
  • Wnt3a 0.1 nM to 10 mM
  • EGF 50 ng / ml
  • p38 MAPK inhibitor 10 ⁇ M
  • IGF-1 20 ng / ml
  • R-spondin 0.1 nM to 10 mM
  • Noggin 0.1 nM to 10 mM
  • DEX 1 ⁇ M
  • a liquid factor or compound such as BIO or DAPT may be added to the endodermal cell culture system.
  • BIO 0.01 to 10 ⁇ M, preferably about 5 ⁇ M can be added to the culture system. If the concentration of BIO is lower than 0.01 ⁇ M, the effect of promoting differentiation to small intestinal epithelial-like cells may not be confirmed, and if it is higher than 10 ⁇ M, cytotoxicity may occur.
  • DAPT 0.02 to 20 ⁇ M, preferably about 10 ⁇ M can be added to the culture system.
  • BIO and DAPT are added to the culture system is not particularly limited as long as differentiation of endoderm cells into small intestinal epithelial cells is possible, for example, 0 to 30 days after endoderm cell culture is started, preferably It can be added for 30 days. Either BIO or DAPT may be added first, or may be added simultaneously.
  • the culture solution exemplified below can be used as a basic medium that can be used in the differentiation induction method of the present invention.
  • Substances to be added to each culture solution can be appropriately increased or decreased depending on the purpose.
  • the manufacturers and distributors are not limited to the following as long as the reagents used can exert the same function.
  • ReproStem trade name
  • iPSellon trade name
  • Essential 8 trade name
  • TeSR-E8 trade name
  • StemFit R
  • AK03N trade name
  • stemFit R
  • AK02N trade name
  • a medium for differentiation induction for example, a medium containing 1 ⁇ Glutamax (Thermo fisher scientific), B27 Supplement (Thermo fisher scientific), and penicillin / streptomycin in RPMI 1640 medium (Sigma) can also be used.
  • the medium used to induce differentiation of endodermal cells is not limited to the above as long as it can exert the same function.
  • any of compounds selected from LY2090314, p38 MAPK inhibitor, IGF-1, R-spondin, Noggin and DEX in the process of differentiation from pluripotent stem cells to small intestinal epithelial-like cells can be used by optionally adding them to the above-mentioned basic medium for differentiation induction as needed.
  • a medium containing LY2090314 (20 nM) in the basic medium shown in (C) above is used as a differentiation-inducing medium be able to.
  • the present invention also extends to a culture medium for inducing differentiation which is used in a method for inducing differentiation of pluripotent stem cells into intestinal epithelial-like cells. Furthermore, the present invention extends to the above-mentioned reagent kit for preparing a culture medium for differentiation induction.
  • the kit comprises a reagent comprising LY2090314 or one or more liquid factor compounds selected from p38 MAPK inhibitor, DEX, EGF, IGF-1, Wnt3a, R-spondin, Noggin etc. Can be configured. For example, for Wnt3a, R-spondin and Noggin, cell culture supernatants expressing these factors can be used.
  • a solution containing a basement membrane matrix can be overlaid on cells in culture and further cultured.
  • the basement membrane matrix is a supramolecular structure existing outside cells in an organism, also called extracellular matrix (ECM), abbreviated as ECM.
  • ECM extracellular matrix
  • a basement membrane matrix that can be used in the method of the present invention, for example, Matrigel (trade name) marketed for "soluble basement membrane extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma" can be mentioned.
  • the overlaying of the basement membrane matrix or the like on the culture substrate can be performed by a method known per se or a method to be developed in the future.
  • a culture substrate such as a culture vessel used for culturing the cells of the present invention can be cultured using a basement membrane matrix or the like coated.
  • the small intestinal epithelial-like cells obtained by the differentiation induction method of the present invention are small intestinal epithelial-like cells obtained by artificially inducing differentiation from pluripotent stem cells.
  • the small intestine epithelial-like cells are characterized by expressing a drug metabolizing enzyme and / or a drug transporter.
  • the small intestine epithelial-like cells of the present invention are positive for small intestinal epithelial cell markers Villin and SI.
  • the small intestine epithelial-like cells of the present invention show significantly higher expression levels of CYP3A4 which is a drug-metabolizing enzyme and PEPT1 which is a drug transporter, compared to human large intestine, and values close to human small intestine Show.
  • small intestinal epithelial cells are tightly linked with each other to form tight junctions, but small intestinal epithelial-like cells obtained by the differentiation induction method of the present invention are also one of cell membrane resistance (TEER) and tight junction lining proteins. It has excellent tight junction function according to the measurement value of certain ZO-1 (Zonula (Zona) occludens 1 protein, aka: Tight-junction protein-1: TJP-1).
  • the present invention extends to intestinal epithelial-like cells obtained by the method for inducing differentiation.
  • the present invention extends to cultures cultured from the above pluripotent stem cells artificially subjected to the differentiation induction method.
  • the present invention also extends to methods of using small intestine epithelial-like cells obtained by the above-described differentiation induction method for drug toxicity evaluation or pharmacokinetic evaluation. Furthermore, the present invention extends to a drug toxicity evaluation method and / or a pharmacokinetic evaluation method characterized by using the small intestine epithelial-like cells obtained by the above-mentioned differentiation induction method. Furthermore, the present invention also extends to a method for testing drug-drug interactions and a method for inducing drug metabolizing enzyme induction characterized by using the small intestine epithelial-like cells. Drug metabolism / drug absorption, drug toxicity and / or pharmacokinetics, drug-drug interaction, drug metabolizing enzyme induction etc.
  • the method for inducing differentiation of the present invention allows stable and superior small intestinal epithelial-like cells to be obtained. It can be provided.
  • Medium 1 Various stem cell maintenance media such as ReproStem, iPSellon, E8, mTeSR, StemFit (R) AK03N, StemFit (R) AK02N can be used as human ES / iPS cell undifferentiated maintenance media.
  • the medium is referred to as "medium 1".
  • Medium 2 A medium containing 1 ⁇ GlutaMAX (Thermo fisher scientific), B27 Supplement (Thermo fisher scientific), penicillin / streptomycin in RPMI 1640 medium (Sigma) can be used. Hereinafter, the medium is referred to as "medium 2".
  • DMEM-high Glucose medium 10% Knock Serum Replacement (Thermo fisher scientific)), 1% Non Essential Amino Acid Solution (Thermo fisher scientific), penicillin / streptomycin, 1 for differentiation induction after endodermal cells It is possible to use DMEM-high Glucose medium (Wako) containing x GlutaMAX (Thermo fisher scientific).
  • DMEM-high Glucose medium Wako
  • GlutaMAX Thermo fisher scientific
  • Example 1 Preparation of endoderm cells
  • Tic JCRB13311
  • GFR Growth Factor Reduced
  • R Matrigel
  • Matrix Matrix
  • Activin A 100 ng / ml of Activin A was added to the culture system of Tic (JCRB1331), and culture was carried out for 4 days for differentiation induction treatment to prepare endoderm cells for preparation of ELC according to the following examples and comparative examples.
  • Example 2 Preparation of Intestinal Progenitor Cells
  • the step of inducing differentiation from endoderm cells to intestinal progenitor cells in the step of inducing differentiation to ELC will be described.
  • Activation of Wnt / ⁇ catenin signal is important for differentiation of endoderm cells into intestinal progenitor cells.
  • FIG. As a positive control, previously reported BIO ⁇ DAPT (Stem Cells. 2013 Jun; 31 (6): 1086-96.) Was used.
  • pancreatic progenitor cell marker PDX1 Pancreatic and duodenal homeobox 1
  • hepatic progenitor cell marker AFP alpha fetoprotein
  • intestinal progenitor cell marker CDX2 intestinal progenitor cell marker
  • the protein expression level of CDX2 was analyzed by Western blotting. Also in the protein expression level of CDX2, the expression level equivalent to that of the previously reported method was shown by the action of LY2090314 (FIG. 3).
  • the percentage of CDX2 positive cells was calculated using flow cytometry. As a result, the percentage of CDX2 positive cells became about 50% by the action of LY2090314 (FIG. 4).
  • the expression of CDX2 was analyzed by immunostaining. Also in immunostaining, the expression of CDX2 could be confirmed by the action of LY2090314 (FIG. 5).
  • Example 3 Use concentration of LY2090314
  • differentiation to endodermal cells to intestinal progenitor cells is most effectively differentiated to intestinal progenitor cells when LY2090314 is allowed to act From the fact that it was confirmed, the most effective LY2090314 used concentration was confirmed.
  • Tic JCRB13311
  • LY2090314 is allowed to act on endodermal cells at various concentrations for 4 days to be a pancreatic progenitor cell marker PDX1, a hepatic progenitor cell
  • the gene expression levels of the marker AFP and the intestinal progenitor cell marker CDX2 were analyzed by a quantitative RT-PCR method.
  • the gene expression level of pancreatic progenitor cell marker PDX1 and liver progenitor cell marker AFP is reduced compared to other concentrations, and gene expression level of intestinal progenitor cell marker CDX2 was the highest ( Figure 6).
  • the protein expression level of CDX2 was analyzed by Western blotting. On the protein expression level of CDX2, the action of 10 nM and 20 nM LY2090314 showed high expression level of CDX2 (FIG. 7). In order to evaluate the induction of differentiation into intestinal progenitor cells, the percentage of CDX2 positive cells was calculated using flow cytometry. As a result, due to the action of LY2090314 at 10 nM and 20 nM, the percentage of CDX2 positive cells became approximately 41% and approximately 50%, respectively (FIG. 8). From these results, it was suggested that a concentration of 10 nM or more is suitable for differentiation of endoderm cells into intestinal progenitor cells using LY2090314.
  • Example 4 Preparation of ELC (Screening of Liquid Compound) 1
  • a step of inducing differentiation from an endoderm cell to an ELC via an intestinal progenitor cell in the step of inducing differentiation to ELC will be described.
  • An experimental protocol for screening of humoral factor compounds effective for inducing differentiation of intestinal progenitor cells to ELC is shown in FIG.
  • LY2090314 (20 nM) is allowed to act on endodermal cells for 4 days to induce differentiation into intestinal progenitor cells, and then to ELC We examined the differentiation induction method.
  • LY2090314 (10 nM), SB202190 (10 ⁇ M), Nicotinamide (10 mM), DEX (1 ⁇ M), Triiodothyronine (T3, 1 ⁇ M), SB431542 (10 ⁇ M), HGF (20 ng / ml), EGF (50 ng) / ml), IGF-1 (20 ng / ml), Wnt3a (25% Wnt3a expressing cell culture supernatant), Wnt3a + R-spondin + Noggin (WRN, 25% Wnt 3a + R-spondin + Noggin expressing cell culture supernatant ), [Leul5] -Gastrin 1 (Gastrin, 10 nM) and Trichostatin A (100 nM) were cultured for 12 days. The previously reported BIO • DAPT was used as a positive control, and DMSO was used as a negative control. In addition, the medium exchange in between was performed once every two days.
  • Villin gene and ISX gene as markers of small intestinal epithelial cells was analyzed by quantitative RT-PCR on the 20th day of culture starting from human iPS cells (12th day of culture in medium containing various humoral factor compounds) .
  • the gene expression levels of Villin and ISX which are small intestinal epithelial cell markers, by the action of SB202190, DEX, T3, SB431542, EGF, IGF-1, Wnt3a, Wnt3a + R-spondin + Noggin among various humoral factor compounds. Rose ( Figure 10).
  • Example 5 Preparation of ELC (Optimization of Culture Period)
  • optimization of the culture period in induction of differentiation into ELC was examined.
  • the experimental protocol is shown in FIG.
  • Example 2 In the same manner as in Example 1, after Tic (JCRB1331) was induced to differentiate into endodermal cells, LY2090314 (20 nM) was allowed to act on endodermal cells for 4 days to induce differentiation into intestinal progenitor cells, as shown in FIG. Each culture period was changed for 12 days, 16 days or 20 days in a culture medium containing humoral factor compounds at various concentrations, and optimization of the period for induction of differentiation to ELC was confirmed. After each period of culture, the expression of Villin gene, ISX gene and CDX2 gene as small intestinal epithelial cell markers was analyzed by quantitative RT-PCR. As a result, each gene expression level showed the highest value on the 28th day (20th day of culture in the medium containing each humoral factor compound) from the start of culture from human iPS cells (FIG. 14).
  • the value of human adult small intestine (AI) is 1, and gene expression level of small intestine epithelial cell marker is determined by quantitative RT-PCR method It analyzed.
  • the gene expression levels of Villin, ISX, CDX2, and ANPEP (alanyl aminopeptidase, membrane) in ELC showed values close to those of human adult small intestine (AI).
  • the gene expression level of SI was about 62 times smaller than that of human adult small intestine (AI) (FIG. 15).
  • Villin positive cell rate was about 95%
  • SI positive cell rate was about 88% (FIG. 16).
  • the expression of Villin was analyzed by immunostaining. The expression of Villin could also be confirmed in the immunostaining (FIG. 17).
  • Example 6 Preparation of ELC
  • an ELC for confirming the properties was prepared by the following Experimental Examples 6-1 to 6-6.
  • the experimental protocol is shown in FIG.
  • Tic JCRB13311
  • LY2090314 (20 nM) is allowed to act on endodermal cells for 4 days to induce differentiation into intestinal progenitor cells, Wnt3a, R- 20 days in a medium containing EGF (50 ng / ml), IGF-1 (20 ng / ml), SB202190 (10 ⁇ M), DEX (1 ⁇ M) in medium 3 containing spondin and Noggin (from culture initiation from human iPS cells) 28 days) cultured.
  • the ELCs prepared in this example were used in the following Experimental Examples 6-1 to 6-6.
  • the ELCs prepared in the following Experimental Example 6-5 were obtained by differentiation induction on Cell Culture insert.
  • Example 6-1 Expressed Genes of ELC With respect to the ELCs prepared in Example 6, various expressed genes associated with intestine were confirmed.
  • ELC of the present invention has properties of intestinal epithelial cells of small intestine type
  • gene expression of Apoa4 apolipoprotein A4
  • Apoc2, Apoc3 and Fgf19 fibroblast growth factor 19
  • the amount was analyzed by quantitative RT-PCR.
  • the gene expression level of Apoa4, Apoc2, Apoc3 and Fgf19 in the ELC was higher than that of human adult colon (colon), and showed a value close to that of human adult small intestine (AI) ( Figure 19).
  • Car1 carbonic anhydrase 1
  • Car2, Slc2a2 solute carrier family 2 member 2
  • Slc9a3 genes highly expressed in the large intestine
  • the amount was analyzed by quantitative RT-PCR.
  • the gene expression levels of Car1, Car2, Slc2a2 and Slc9a3 in the ELC were lower than the gene expression levels of human adult colon (colon), and showed values close to the gene expression levels of human adult small intestine (AI) (FIG. 20). From the above results, it was suggested that the ELC may be a small intestine type intestinal epithelial cell.
  • Example 6-2 Drug-Metabolizing Enzyme-Drug Conjugating Enzyme Function of ELC
  • the drug-metabolizing enzyme-drug-conjugated enzyme in the ELC prepared in Example 6 was CYP2C9 (cytochrome P450 family 2 subfamily C member 9), CYP2J2, CYP3A4, The gene expression levels of UGT1A1 (UDP glucuronosyltransferase family 1 member A1), UGT1A3, and CES2 (carboxylesterase 2) were analyzed by a quantitative RT-PCR method.
  • CYP2J2, CYP3A4, UGT1A1, UGT1A3 and CES2 in the ELC showed lower values than human adult small intestine (AI).
  • the gene expression level of CYP3A4 was higher than that of human adult colon (FIG. 21).
  • Example 6-3 Activity of Carboxylesterase 2 (CES2) in ELC CES 2 is localized in the small intestine and is an enzyme involved in membrane permeability.
  • the activity of CES2 in the ELC prepared in Example 6 was evaluated using FD (Fluorescein diacetate) which is a substrate of CES2.
  • loperamide (1 mM) was used as a CES2 inhibitor.
  • the activity of CES2 in the ELC was significantly inhibited by the CES2 inhibitor loperamide. From the above results, it was suggested that the ELC could evaluate the activity of CES2 (FIG. 23).
  • Example 6-4 Expression of Drug Transporter Gene in ELC
  • the drug transporter MDR1 multidrug resistance protein 1
  • BCRP breast cancer resistance protein
  • the gene expression levels of PEPT1 peptide transporter 1
  • MRP2 multidrug resistance-associated protein 2
  • MRP4 and MRP6 were analyzed by quantitative RT-PCR.
  • the gene expression levels of BCRP, PEPT1, MRP2, MRP4 and MRP6 in the ELC showed values close to human adult small intestine (AI)
  • the gene expression level of MDR1 was about 1 compared to human adult small intestine (AI) It was / 66.
  • the gene expression level of PEPT1 was over 100 times higher than that of human adult colon (FIG. 24).
  • MRP1, MRP3, MRP5, OST ⁇ (organic soluble transporter alpha) and OST ⁇ which are drug transporters expressed on the basement membrane side in the ELC.
  • the gene expression levels of MRP1, MRP5, OST ⁇ and OST ⁇ in the ELC showed values close to human adult small intestine (AI).
  • the gene expression level of MRP3 was about 1/16 that of human adult small intestine (AI) (FIG. 25).
  • Example 6-5 Barrier Function of ELC The barrier ability of ELC was evaluated by the membrane permeability coefficient of cell membrane resistance (TEER) and lucifer yellow (LY).
  • TEER membrane permeability coefficient of cell membrane resistance
  • LY lucifer yellow
  • ELCs induced to differentiate on Cell Culture insert by the method of Example 6 were used. Differentiation-induced ELCs on Cell Culture inserts are suitable for cell transport assays across membranes.
  • the TEER at ELC was measured using a Millicell ERS-2 resistance measurement system (Merck Millipore).
  • the TEER value of the ELC was about 656 ⁇ ⁇ cm 2 .
  • the TEER value was significantly reduced by the action of capric acid (C10, 10 mM) (FIG. 26A).
  • the membrane permeability coefficient of LY was increased by C10, a tight junction opener (FIG. 26B). It is thought that the ELC may be applied to the development of absorption enhancers because it has barrier ability and LY permeation amount is increased by C10.
  • Example 6-6 Drug Transport Function in ELC
  • MDR1 transportability was measured using Rhodamine 123 (Rhodamine 123), with the amount of permeation from the apical membrane side to the basement membrane side as an index.
  • Rhodamine 123 Rhodamine 123
  • the permeation amount of rhodamine 123 was increased by the action of cyclosporin A (CysA, 10 ⁇ M), which is an MDR1 inhibitor, in the ELC. From the above results, it is suggested that the ELC may be able to evaluate the transport ability of MDR1 from the apical membrane side to the basement membrane side (FIG. 27).
  • the drug transport function in the ELC was evaluated by PEPT1 transport ability.
  • PEPT1 transportability was measured by incorporation of the fluorescent peptide using the fluorescent peptide ⁇ -Ala-Lys-AMCA.
  • the uptake of the fluorescent peptide ⁇ -Ala-Lys-AMCA was reduced by the action of the PEPT1 inhibitor captopril (100 ⁇ M) in the ELC. From the above results, it was suggested that the ELC could evaluate the transport ability of PEPT1 (FIG. 28).
  • Example 7 Preparation of Intestinal Epithelial-Like Cells 2 Intestinal epithelial-like cells were produced from human iPS cell line Tic (JCRB1331) according to the protocol described in FIG. 18 in the same manner as the method described in Example 6.
  • intestinal epithelial-like cells obtained also in the present example are simply referred to as "ELC”.
  • Anti-E Cadherin antibody and Anti-ZO-1 antibody were used as a primary antibody, and Donkey anti-rabbit IgG Secondary Antibody and Alexa Fluor 594 conjugate were used as a secondary antibody. Cell immunostaining confirmed the expression of E-cadherin and ZO-1 (FIG. 29 AB).
  • Example 7-2 Expression of CDX2 and E-cadherin in Human iPS Cell-Derived Intestinal Progenitor Cells
  • the expression of CDX2 and E-cadherin in the intestinal progenitor cells obtained in the ELC preparation step of Example 7 was the same as that described above It was evaluated by cell immunostaining using Anti-CDX2 antibody and Anti-E cadherin antibody were used as a primary antibody, Donkey anti-mouse IgG Secondary Antibody, Alexa Fluor 488 conjugate and Donkey anti-rabbit IgG Secondary Antibody and Alexa Fluor 594 conjugate were used as a secondary antibody.
  • Example 7-3 Expression of villin and Peptide transporter 1 (PEPT1) in ELC It was evaluated whether the ELC produced in Example 7 had polarity. When the expression of villin and PEPT1 expressed on the apical membrane side was confirmed by immunostaining, ELC showed a columnar epithelial-like morphology, the expression of villin and PEPT1 could be observed on the apical membrane side, and it was confirmed to have polarity. ( Figure 31).
  • Example 7 Calyphosphatase (ALP) Activity in ELC It was evaluated whether the ELC prepared in Example 7 had ALP activity. For ALP activity, cells were stained with ALP (Blue-Color Staining Kit, System Bioscience) using Blue-Color Staining Kit (System Bioscience). ELC was shown to have ALP activity (FIG. 32).
  • Example 7-5 Morphological evaluation 1 in ELC
  • the ELC produced in Example 7 was observed using a transmission electron microscope (TEM), and morphological evaluation was performed.
  • the ELC exhibited columnar epithelial-like morphology, and the microvilli structure in the apical membrane could be observed (black arrow) (FIG. 33).
  • ELC Morphological evaluation 2 in ELC
  • enteroendocrine cells panate cells and goblet cells, which are cells constituting the small intestine, were present.
  • positive cells of the enteroendocrine cell marker Chromogranin A, positive cells of lysozyme, which is a panate cell marker, and positive cells of a mucin cell marker, which is a goblet cell marker can be observed (FIG. 34).
  • ELC showed morphological characteristics similar to those of the small intestine epithelial cells of the living body, and it was suggested that there are four types of cells constituting the small intestine like the living body in the cell population.
  • Example 7-7-7 Characteristics of ELC
  • the ELC produced in Example 7 was confirmed to be intestinal epithelial cells of small intestine type or large intestine type. A comprehensive analysis of gene expression levels was performed using a microarray for evaluation.
  • the ELC prepared in Example 7 showed a gene expression pattern more similar to human adult small intestine (AI) than human adult colon (FIG. 35). From the above results, it was suggested that the ELC of Example 7 may be a small intestine type intestinal epithelial-like cell.
  • Experimental Example 7-8 Membrane Permeability of ELC 1
  • the barrier ability in ELC induced to differentiate on Cell Culture insert as in Experimental Example 6-5 is evaluated by the membrane permeability coefficient of FITC-dextran (Fluorescein isothiocyanate-dextran: FD4) having an average molecular weight of 3000-5000. did.
  • the HBSS solution containing 1 mg / ml FD4 (Sigma-Aldrich) was added to the cells, and the FD4 permeation amount was measured when reacted at 37 ° C. for 90 minutes.
  • the amount of FD4 transmission was measured using a fluorescence plate reader (TroStar LB941, Berthold) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
  • the tight junction opener capric acid C10, 10 mM
  • Example 7-9 Membrane Permeability of ELC 2
  • the permeation amounts of FD4, lucifer yellow (LY) and rhodamine 123 were measured over time for ELCs induced to differentiate on Cell Culture insert by the method of Example 7.
  • the membrane permeability by LY or rhodamine 123 was measured using a fluorescence plate reader (TroStar LB941, Berthold) in the same manner as the measurement of FD4 described above. As a result, it was confirmed that the transmission of FD4, LY and rhodamine 123 increased with time.
  • the cells obtained by the differentiation induction method of the present invention express each marker expressed by small intestinal epithelial cells, express a drug metabolizing enzyme and a drug transporter, and have a tight junction function.
  • Intestinal epithelial-like cells of the small intestine type In particular, differentiation induction processing can be performed more simply in that differentiation can be induced without introducing a gene using gene recombination technology.
  • the differentiation induction method of the present invention provides stable small intestinal epithelial-like cells stably. It has become possible.
  • the obtained cells have the properties of various small intestinal epithelial-like cells, and are excellent in terms of the expression amounts of drug metabolizing enzymes and drug transporters.
  • drug metabolism and drug absorption can be simultaneously evaluated under a homogeneous evaluation system using stable and excellent small intestinal epithelial-like cells, It is expected to be able to greatly contribute to the analysis, development, etc. of food etc. and is useful.

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Abstract

La présente invention concerne un procédé d'induction sélective de la différenciation de cellules souches pluripotentes en cellules de type entérocyte. L'invention concerne également des cellules de type entérocyte excellentes pour exprimer des enzymes métabolisant des médicaments et des transporteurs de médicaments. Plus particulièrement, l'invention concerne des cellules de type entérocyte de type intestin grêle qui présentent des propriétés plus proches des entérocytes de culture primaire difficiles à acquérir. Ceci est obtenu par un procédé qui contient une étape dans laquelle des cellules endodermiques, obtenues par induction de la différenciation de cellules souches pluripotentes, sont induites pour se différencier en cellules progénitrices intestinales par culture dans un système contenant LY2090314. Les cellules progénitrices intestinales résultantes sont cultivées dans un système qui contient au moins deux composés de facteurs humoraux choisis parmi Wnt3a, EGF, inhibiteur de MAPK p38, IGF-1, R-spondine, noggine et DEX. Les cellules résultantes présentent diverses propriétés de cellules de type entérocyte et sont excellentes par rapport à la quantité d'expression d'enzymes de métabolisation de médicament et de transporteurs de médicament et le métabolisme de médicament et l'absorption de médicament peuvent être évalués simultanément dans un système d'évaluation homogène à l'aide des cellules de type entérocyte très stables.
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WO2024053406A1 (fr) * 2022-09-09 2024-03-14 国立大学法人大阪大学 Cellules de type épithélium de l'intestin de petite taille et leur procédé de production

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