WO2022073211A1 - 诱导分化细胞成多潜能内胚层干细胞的方法及其应用 - Google Patents

诱导分化细胞成多潜能内胚层干细胞的方法及其应用 Download PDF

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WO2022073211A1
WO2022073211A1 PCT/CN2020/120038 CN2020120038W WO2022073211A1 WO 2022073211 A1 WO2022073211 A1 WO 2022073211A1 CN 2020120038 W CN2020120038 W CN 2020120038W WO 2022073211 A1 WO2022073211 A1 WO 2022073211A1
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cells
induced pluripotent
endoderm
stem cells
smensc
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PCT/CN2020/120038
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French (fr)
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程新
邓小刚
吴佳颖
付天龙
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中国科学院分子细胞科学卓越创新中心
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Priority to AU2020471426A priority Critical patent/AU2020471426A1/en
Priority to PCT/CN2020/120038 priority patent/WO2022073211A1/zh
Priority to US18/248,443 priority patent/US20230365925A1/en
Priority to EP20956526.6A priority patent/EP4227402A4/en
Publication of WO2022073211A1 publication Critical patent/WO2022073211A1/zh

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Definitions

  • the present invention relates to the fields of biotechnology and cell therapy.
  • the present invention relates to methods of inducing differentiated cells into multipotent endodermal stem cells and uses thereof.
  • De-differentiation refers specifically to differentiated cells that lose their specific functions and return to the earlier developmental stages under the stimulation of certain external conditions, which is a common phenomenon in nature. Precursor/stem cells obtained by degeneration are of great significance for tissue damage repair.
  • hEnSCs can expand indefinitely and differentiate into functional endoderm-derived cells in vitro, but their growth depends on mouse fibroblasts as trophoblasts and high concentrations of matrigel, limiting their clinical application.
  • the invention discloses a method for inducing differentiated cells into pluripotent endoderm stem cells (small molecule induced Endoderm Stem Cells, smEnSCs).
  • the invention discloses a method for inducing differentiated cells into multipotent endoderm stem cells (small molecule induced Endoderm Stem Cells, smEnSCs) by using a combination of small molecular compounds and cytokines
  • the invention also discloses the use of small molecules to induce the degeneration of differentiated cells in vitro for the first time, and to establish an induced pluripotent endoderm stem cell line (smEnSCs)
  • the invention also discloses that, by optimizing the culture method, smEnSCs can be infinitely expanded in vitro independent of trophoblast cells.
  • the invention also discloses that a differentiation system is established in which smEnSCs are differentiated into hepatic parenchyma cells, bile duct epithelial cells and small intestinal epithelial cells and other endoderm-derived cells in vitro.
  • a method for inducing human differentiated cells to differentiate into induced pluripotent endoderm stem cells characterized in that it comprises the steps of:
  • the differentiated cells are cultured in a culture system to obtain induced pluripotent endodermal stem cells (smEnSC); wherein the culture system includes EGF, A83-01 and CHIR99021.
  • smEnSC induced pluripotent endodermal stem cells
  • the differentiation is degeneration.
  • the first culture condition contains a first culture medium.
  • the first medium is selected from the group consisting of SFD, DMEM, DMEM/F12, or a combination thereof.
  • the culture system further comprises one or more additives selected from the group consisting of: Ascorbic Acid Phosphate Magnesium, L-glutamine, MTG, bFGF .
  • the differentiated cells include adult cells.
  • the adult cells are selected from the group consisting of hepatocytes, gastric epithelial cells, pancreatic islet cells, small intestinal epithelial cells, or a combination thereof.
  • the hepatocytes include primary hepatocytes and hepatocytes derived from stem cell differentiation.
  • the adult cells are derived from adult tissue isolation, in vitro directed differentiation of stem cells (eg, endoderm stem cells, induced pluripotent stem cells).
  • stem cells eg, endoderm stem cells, induced pluripotent stem cells.
  • the endoderm stem cells are derived from human embryonic stem cells or human induced pluripotent stem cells.
  • the pluripotent stem cells are selected from the group consisting of human embryonic stem cell lines (such as H9, Hes2), human induced pluripotent stem cell lines (such as ZRZ-iPSCs, WT6 iPSCs), or a combination thereof.
  • the endoderm stem cells are selected from the group consisting of H9, Hes2, ZRZ-iPSC EnSC, WT6 iPSC EnSC, or a combination thereof.
  • the method further comprises step (b): differentiating the induced pluripotent endoderm stem cells (smEnSC) into endoderm-derived cells.
  • smEnSC induced pluripotent endoderm stem cells
  • the endoderm-derived cells include hepatocytes, bile duct epithelial cells, small intestinal epithelial cells, pancreatic islet beta cells, and gastric epithelial cells.
  • the step (b) does not contain trophoblast cells.
  • step (b) comprising:
  • the induced pluripotent endoderm stem cells are cultured in the culture system to obtain small intestinal cells.
  • the second culture condition contains a second medium.
  • the second medium includes a liver-oriented specialized medium, a liver-oriented maturation medium I and a liver-oriented maturation medium II.
  • the liver-specific medium is selected from the group consisting of SFD, DMEM/F12, DMEM, or a combination thereof.
  • the hepatic maturation medium I is selected from the group consisting of SFD, DMEM/F12, DMEM, or a combination thereof.
  • the hepatic maturation medium II is selected from the group consisting of SFD, HCM, DMEM/F12, DMEM, or a combination thereof.
  • the liver-specific culture medium further contains one or more additives selected from the group consisting of:
  • BMP4 FGF, A83-01, SB431542, IWP2, Dexamethasone, DMSO.
  • the liver maturation medium I further comprises one or more additives selected from the group consisting of:
  • HGF HGF
  • Dexamethasone OSM
  • C-E C-E
  • A83-01 EGFi
  • VK1 VK1
  • the liver maturation medium II further comprises one or more additives selected from the group consisting of HGF, Dexamethasone, OSM, C-E, A83-01, EGFi, VK1.
  • the third culture condition contains a third medium.
  • the third medium includes a bile duct-specific medium and a bile duct maturation medium.
  • the third medium is selected from the group consisting of SFD, DMEM/F12, DMEM, William's E, or a combination thereof.
  • the third culture condition also contains one or more additives selected from the group consisting of:
  • FGF10 HGF
  • TGF ⁇ TGF ⁇
  • EGF Activin A
  • Dexamethasone Dexamethasone
  • the fourth culture condition contains a fourth culture medium.
  • the fourth medium includes expansion medium, hindgut specialization medium, small intestine maturation medium I and small intestine maturation medium II.
  • the fourth medium is selected from the group consisting of SFD, DMEM/F12, DMEM, or a combination thereof.
  • the fourth culture condition also contains one or more additives selected from the group consisting of:
  • FGF4 EGF, A83-01, CHIR99321, Wnt3a, R-spondin, EGF, Noggin.
  • the expansion medium is selected from the group consisting of SFD, DMEM/F12, DMEM, or a combination thereof.
  • the hindgut-specific medium is selected from the group consisting of SFD, DMEM/F12, DMEM, or a combination thereof.
  • the small intestine maturation medium I is selected from the group consisting of SFD, DMEM/F12, DMEM, or a combination thereof.
  • the small intestine maturation medium II is selected from the group consisting of SFD, DMEM/F12, DMEM, or a combination thereof.
  • the expansion medium further comprises one or more additives selected from the group consisting of:
  • FGF4 EGF, A83-01.
  • the hindgut specialization medium further comprises one or more additives selected from the group consisting of:
  • the small intestine maturation medium I further comprises one or more additives selected from the group consisting of:
  • FGF4 CHIR99321, EGF, Noggin.
  • the small intestine maturation medium II further comprises one or more additives selected from the group consisting of CHIR99321, EGF, and Noggin.
  • step (a) the adult cells are cultured under the first culture condition for 3-18 days, preferably, 4-16 days, more preferably, 6-12 days.
  • step (b1) the induced pluripotent endoderm stem cells are cultured under the second culture condition for 15-40 days, preferably, 18-36 days, more preferably, 20-25 days .
  • step (b2) the induced pluripotent endoderm stem cells are cultured under the third culture condition for 10-30 days, preferably, 15-28 days, more preferably, 15-21 days .
  • step (b3) the induced pluripotent endoderm stem cells are cultured under the fourth culture condition for 20-50 days, preferably, 22-45 days, more preferably, 25-40 days .
  • the method has one or more features selected from the group consisting of:
  • a high differentiation rate of induced pluripotent endoderm stem cells is 80-98.5%, preferably 90-95%;
  • the culture system does not contain or contains very little extracellular matrix.
  • the extracellular matrix is selected from the group consisting of: Matrigel, Laminin, Basement Membrane Extracts, or a combination thereof
  • the content (v/v) of the extracellular matrix is 0.5%-5%, preferably 1%-3%, more preferably 1%-2% .
  • the method includes therapeutic and non-therapeutic.
  • the density of adult cells is 0.5 ⁇ 10 6 -4 ⁇ 10 6 cells/plate, preferably, 1 ⁇ 10 6 -2 ⁇ 10 6 cells/plate.
  • the volume of the culture system is 10-25ml, preferably 10-20ml, and most preferably 10-15ml.
  • the ratio M2/M1 of the number M2 of the obtained induced pluripotent endoderm stem cells to the number M1 of the adult cells is 5-15, preferably 8-14, more preferably 8 -10.
  • the induced pluripotent endoderm stem cells are functional induced pluripotent endoderm stem cells.
  • the endoderm-derived cells are functional endoderm-derived cells.
  • the second aspect of the present invention provides an induced pluripotent endoderm stem cell, the induced pluripotent endoderm stem cell is prepared by the method of claim 1 .
  • the induced pluripotent endoderm stem cells have one or more characteristics selected from the following:
  • the induced pluripotent endoderm stem cells have one or more characteristics selected from the following:
  • the liver-directed marker gene is selected from the group consisting of ALB, AAT, CYP3A7, CYP3A4, or a combination thereof.
  • the precursor cell marker gene is selected from the group consisting of CDX2, SOX17, or a combination thereof.
  • the endoderm-specific gene is selected from the group consisting of SOX17, CDX2, FOXA2, or a combination thereof.
  • the liver precursor cell-specific gene is selected from the group consisting of SOX9, TBX3, or a combination thereof.
  • the induced pluripotent endoderm stem cells are obtained by induction from adult cells.
  • a third aspect of the present invention provides an endoderm-derived cell, wherein the endoderm-derived cell has a three-dimensional spherical structure, wherein the diameter of the balloon of the endoderm-derived cell is 100-500 ⁇ m.
  • the endoderm-derived cells have one or more characteristics selected from the following:
  • Hepatocyte spheroids are hollow spheres with a diameter of 150-500 ⁇ m;
  • the bile duct cell structure is a spheroid structure or a tubular structure, the diameter of the spheroid is 150-500 ⁇ m, the inner diameter of the tubular structure is 20-80 ⁇ m, and the length is 500 ⁇ m-3 mm;
  • the cell structure of the small intestine includes solid cell spheroids, cell spheroids with lumen, and tubular structures, wherein the diameter of the solid cell spheroids and the cell spheroids with lumen is 100-500 ⁇ m, the inner diameter of the tubular structure is 20-80 ⁇ m, and the length is 500 ⁇ m- 5mm.
  • the endoderm-derived cells include hepatocytes, bile duct epithelial cells, small intestinal epithelial cells, pancreatic islet beta cells, and gastric epithelial cells.
  • the fourth aspect of the present invention provides the use of the induced pluripotent endoderm stem cell described in the second aspect of the present invention, or the endoderm-derived cell described in the third aspect of the present invention, for preparing a medicament or a preparation, the medicament or preparations for (a) treatment of liver, bile duct-related diseases; and/or (b) intestinal diseases; and/or (c) diabetes.
  • the liver and bile duct-related diseases are selected from the group consisting of genetic metabolic liver disease, chronic/acute liver failure, Alagille syndrome, Crigler-Najjar syndrome type 1 (Crigler-Najjar syndrome type 1), or a combination thereof.
  • the hereditary liver disease is selected from the group consisting of: hepatolenticular degeneration (Wilson's disease, WD), glycogen storage disease type Ia, ⁇ -antitrypsin deficiency, hemochromatosis, congenital biliary tract Atresia, sitrin deficiency, familial hypercholesterolemia, or a combination thereof.
  • the intestinal disease is selected from the group consisting of intestinal injury, short bowel syndrome, or a combination thereof.
  • the diabetes is selected from the group consisting of type I diabetes, type II diabetes, or a combination thereof.
  • a fifth aspect of the present invention provides an induction medium, the induction medium contains a basal medium and supplements; wherein the basal medium is selected from the group consisting of SFD, DMEM, DMEM/F12, BDM, or a combination thereof ; and, the additives include EGF, A83-01 and CHIR99021.
  • the additive further includes bFGF, VEGF, EGF, HGF, OSM, and Dexamethasone.
  • the additive is selected from one or more additives of the following group: Ascorbic Acid Phosphate Magnesium, L-glutamine, MTG, bFGF.
  • the sixth aspect of the present invention provides a use of the induction medium according to the fifth aspect of the present invention, for inducing differentiated cells to differentiate into induced pluripotent endodermal stem cells (smEnSC).
  • smEnSC induced pluripotent endodermal stem cells
  • a seventh aspect of the present invention provides a method for screening or determining a potential therapeutic agent that promotes differentiation of differentiated cells into induced pluripotent endodermal stem cells (smEnSC), comprising the steps of:
  • test compound is a potential therapeutic agent for promoting the differentiation of differentiated cells into induced pluripotent endoderm stem cells
  • test compound is a potential therapeutic agent for promoting the differentiation of differentiated cells into induced pluripotent endodermal stem cells.
  • the "significantly higher” refers to M1/M2 ⁇ 2, preferably ⁇ 3, more preferably ⁇ 4.
  • the method is non-diagnostic and non-therapeutic.
  • Figure 1 shows in vitro induction of differentiated hepatocyte degeneration and establishment of smEnSC cell line
  • C Gene expression changes after 7 days of Eheps degeneration. Hepatic genes (ALB, AAT, CYP3A7 and CYP3A4) were turned off and precursor cell marker genes (CDX2 and SOX17) were turned on.
  • (H) smEnSCs can be established from single cells and thus have stemness at the clonal level.
  • Figure 2 shows in vitro induction of smEnSCs to differentiate into endoderm-derived cells
  • A smEnSC-Hep gene expression profile.
  • the differentiated smEnSC-Hep liver-oriented genes such as ALB and CYPs were significantly up-regulated.
  • AL stands for adult liver tissue.
  • smEnSCs were induced into bile duct epithelial cells (smEnSC-Cho) in vitro, and qPCR experiments showed that they expressed bile duct epithelial cell marker genes.
  • FIG. 3 shows that smEnSC-Hep can rescue liver failure model FRG mice; among them,
  • smEnSC-Hep transplantation can significantly improve the survival of FRG mice.
  • Sham represents the untransplanted smEnSC-Hep group
  • smEnSC-Hep represents the transplanted smEnSC-Hep group.
  • induced pluripotent endodermal stem cells smEnSC
  • smEnSC induced pluripotent endodermal stem cells
  • the differentiation rate of pluripotent endoderm stem cells is >90%, and the induced pluripotent endoderm stem cells have >90% purity.
  • the induced pluripotent endoderm stem cells of the present invention can be further differentiated into endoderm-derived cells (such as liver parenchyma cells, bile duct cells, small intestinal cells, pancreatic islet beta cells, etc.), in addition, the induced pluripotent endoderm stem cells of the present invention also It can be used to treat liver, bile duct related diseases and diabetes. On this basis, the present inventors have completed the present invention.
  • endoderm-derived cells such as liver parenchyma cells, bile duct cells, small intestinal cells, pancreatic islet beta cells, etc.
  • the present inventors invented a method for inducing differentiated cells into pluripotent endoderm stem cells (small molecule induced Endoderm Stem Cells, smEnSCs) using a combination of small molecule compounds and cytokines.
  • stem cell-derived hepatocytes and other differentiated cells are cultured with a combination of specific small molecule compounds and cytokines to induce their degeneration, and to establish a novel endoderm that does not depend on trophoblast cells and can proliferate indefinitely.
  • stem cell lines which were then further induced to differentiate into hepatocytes, bile duct epithelial cells, intestinal epithelial cells, and pancreatic islet beta cells in vitro.
  • A83-01 has the chemical formula C25H19N5S and has a CAS number of 909910-43-6;
  • panel refers to panels of various sizes, including 58 mm 2 panels.
  • induced pluripotent endoderm stem cells As used herein, the terms “induced pluripotent endoderm stem cells”, “smEnSC” refer to induced pluripotent endoderm stem cells produced by the methods of the present invention, said induced pluripotent endoderm stem cells having one or more of the following characteristics :
  • differentiated cells refer to differentiated cells, which refer to cell types that are further differentiated from the fertilized egg from the perspective of developmental biology. They are cells of a specific lineage and cannot proliferate.
  • stem cells obtained by directed differentiation are included. of mature cells as well as primary isolated adult cells.
  • differentiated cells include adult cells.
  • Adult cells refer to primary cells directly isolated from adult tissues. Adult cells are terminally differentiated cell types and do not have the ability to proliferate.
  • the starting cells of the induced pluripotent endoderm stem cells of the present invention are human differentiated cells (including adult cells), which are cultured under first culture conditions to obtain induced pluripotent endoderm stem cells (smEnSCs), wherein the first Culture systems under culture conditions included EGF, A83-01 and CHIR99021.
  • smEnSCs induced pluripotent endoderm stem cells
  • Hepatic Maturation Medium I includes HGF, Dexamethasone, OSM, C-E, A83-01, EGFi
  • Hepatic Maturation Medium II includes Dexamethasone, C-E, A83-01, EGFi
  • the third medium such as SFD
  • supplements including FGF10, HGF, TGF ⁇ , dexamethasone
  • fourth medium including expansion medium, hindgut specialization
  • the culture system of the present invention does not contain or contains little Matrigel.
  • SFD Human free differentiation medium
  • HCM medium is commercially available (Commercial medium: Lonza: CC-4182) (Ogawa, S., Surapisitchat, J., Virtanen, C., Ogawa, M., Niapour, M., Sugamori, K.S., ... & Zhao, B. (2013). Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development, 140(15), 3285-3296.).
  • smEnSCs induced pluripotent endodermal stem cells
  • smEnSC induced pluripotent endodermal stem cells
  • test compound is a potential therapeutic agent for promoting the differentiation of differentiated cells into induced pluripotent endoderm stem cells
  • test compound is a potential therapeutic agent for promoting the differentiation of differentiated cells into induced pluripotent endodermal stem cells.
  • the "significantly higher” refers to M1/M2 ⁇ 2, preferably ⁇ 3, more preferably ⁇ 4.
  • the method is non-diagnostic and non-therapeutic.
  • the present invention finds for the first time that, by culturing differentiated cells (such as adult cells) in a culture system, functional induced pluripotent endoderm stem cells with a very high differentiation rate (>90%) can be obtained, and the induced multipotent endoderm stem cells of the present invention The purity of the potential endoderm stem cells is also very high, up to 99%.
  • the present invention utilizes small molecules to induce the degeneration of differentiated cells in vitro for the first time, and establishes an induced pluripotent endoderm stem cell line (smEnSCs).
  • smEnSCs induced pluripotent endoderm stem cell line
  • the smEnSCs of the present invention can be infinitely expanded in vitro independent of trophoblast cells, and can be re-differentiated into endoderm-derived cells such as hepatocytes, bile duct epithelial cells and small intestinal epithelial cells under specific induced differentiation conditions.
  • the smEnSCs of the present invention are expected to replace hEnSCs in many fields such as regenerative medicine.
  • this degeneration system can also serve as an ideal model for the study of degeneration mechanisms.
  • the stem cell-derived hepatocytes of the present invention are easy to obtain, can be cultured in vitro for a long time, and can be genetically manipulated, so they have the potential to replace human primary hepatocytes for research on degeneration mechanisms.
  • the present invention utilizes a combination of small molecular compounds to induce the degeneration of Eheps to establish smEnSCs, with low cost and high stability.
  • the culture of smEnSCs of the present invention does not depend on trophoblast cells such as MEFs, and meets the requirements of clinical treatment.
  • the smEnSCs of the present invention can expand indefinitely in vitro, do not form tumors in vivo, and re-differentiate into endoderm-derived cell types such as hepatocytes, bile duct epithelial cells, and small intestinal epithelial cells under specific in vitro induction conditions. Therefore, it can be used for the large-scale production of functional cells that meet clinical needs in vitro.
  • Induced pluripotent endoderm stem cell (smEnSC) medium SFD medium with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ml, Wako, cat#013-19641), L-glutamine (1X, Cellgro), MTG (4.5 ⁇ 10 -4 M), bFGF (10ng/ml), EGF (10ng/ml), A83-01 (0.5 ⁇ M); CHIR99021 (3 ⁇ M);
  • smEnSC Hepatocyte Differentiation Medium Liver Specialized Medium, SFD Medium Supplemented with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ml, Wako, cat#013-19641), L-glutamine (1X, Cellgro), MTG ( 4.5 ⁇ 10 -4 M), hBMP4 (50ng/ml); bFGF (20ng/ml), A83-01 (0.5 ⁇ M), IWP2 (5 ⁇ M), Dexamethasone (40ng/ml), DMSO (1% v/v) Liver to maturation medium I, SFD medium supplemented with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ml, Wako, cat#013-19641), L-glutamine (1X, Cellgro), MTG (4.5 ⁇ 10 -4 M) , HGF (25ng/ml), Dexamethasone (40ng/ml), OSM (20ng/ml), CE (0.1uM
  • Liver maturation medium II HCM medium supplemented with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ml, Wako, cat#013-19641), L-glutamine (1X, Cellgro), MTG (4.5 ⁇ 10 -4 M) , HGF (25ng/ml), Dexamethasone (40ng/ml), CE (0.1uM), A83-01 (0.5uM), EGFi (2uM).
  • smEnSC bile duct cell differentiation medium bile duct-specific medium, SFD medium with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ml, Wako, cat#013-19641), L-glutamine (1X, Cellgro), MTG (4.5 ⁇ 10 -4 M), ActivinA (50ng/ml), FGF10 (50ng/ml); bile duct maturation medium, SFD medium with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ml, Wako, cat#013-19641) , L-glutamine (1X, Cellgro), MTG (4.5 ⁇ 10 -4 M), HGF (25ng/ml), TGF ⁇ (5ng/ml), dexamethasone (40ng/ml).
  • smEnSC small intestinal cell differentiation medium expansion medium, SFD medium with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ml, Wako, cat#013-19641), L-glutamine (1X, Cellgro), MTG (4.5 ⁇ 10 -4 M), FGF4 (10ng/ml), EGF (10ng/ml), A83-01 (0.5 ⁇ M); Hindgut specialization medium, SFD medium was added with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ ml, Wako, cat#013-19641), L-glutamine (1X, Cellgro), MTG (4.5 ⁇ 10 -4 M), FGF4 (500ng/ml), CHIR99321 (3 ⁇ M); Small Intestinal Maturation Medium I, SFD culture The base species were added with L-Ascorbic Acid Phosphate Magnesium (50 ⁇ g/ml, Wako, cat#013-19641), L-glutamine (1X, Cellgro
  • the differentiated and mature Eheps were digested with collagenase B (1 mg/ml, Roche) at 37°C for 2 hours, then digested with 0.25% trypsin for 5 minutes, and then mechanically blown into single cells, which were seeded in matrigel-coated six-well plates. 1-2 x 105 cells per well were cultured with smEnSC medium (Fig. 1A). Cell proliferation was clearly observed under light microscopy (Fig. 1B). qPCR results showed that after culturing Eheps in our invented medium for 7 days, the liver-directed marker genes AAT, ALB, CYP3A7, and CYP3A4 were significantly down-regulated (Fig. 1C).
  • the expressions of early endoderm-related genes SOX17 and CDX2 were significantly up-regulated, which indicated that the The function of the liver parenchyma cells was greatly degraded or even disappeared.
  • the first 2 passages were passaged every 6 days, and the latter passages were passaged every 3-4 days.
  • the number of cells in the starting Eheps is 0.5-2 ⁇ 10 5
  • the number of cells that can obtain smEnSC after culture is 1-2 ⁇ 10 6 .
  • Stable smEnSC cell lines exhibited uniform epithelioid cell morphology (Fig. 1D) and in addition were able to expand indefinitely in vitro (Fig. 1E).
  • Gene expression analysis showed that smEnSCs expressed the liver-enriched transcription factor HNF4A, as well as the definitive endoderm (DE) and hEnSC key transcription factor SOX17, ( Figure 1F).
  • smEnSCs did not express the transcription factor EOMES, while FOXA1, TBX3, SOX9, CEBPA, PROX1 and other key transcription factors in early liver development were significantly up-regulated (Fig. 1F).
  • smEnSCs expressed CDX2, a key transcription factor for hindgut fate determination, but CDX2 was not expressed in DE (Fig. 1F).
  • Flow cytometry showed that smEnSC co-expressed SOX17 and FOXA1, EpCAM and SOX9 (Fig. 1G).
  • smEnSCs were transplanted into SCID-Beige mice (20 mice, 5 ⁇ 10 6 cells/mouse), and no tumor formation was found in vivo after 4 months of follow-up (Fig. 1I). This result indicates that smEnSCs do not form tumors in vivo, which is the future A safe seed cell for cell therapy applications.
  • 1 ⁇ 10 5 smEnSCs were first cultured in smEnSC medium for two days, and then replaced with liver-specific medium for four days. Digest with 0.05% trypsin to form clumps of 20-50 cells, combine 2 wells of cells into 1 well, add 80 ⁇ l matrigel, transfer to low adsorption culture plate, and continue to culture for 2 days with liver-specific medium. Afterwards, the cells were cultured for 12 days in Hepatic Maturation Medium I and 6 days in Hepatic Maturity Medium II.
  • Hepatocytes differentiated from smEnSCs express a series of adult hepatocyte-specific proteins, including secreted proteins Albumin, AAT, a key enzyme involved in glycogen accumulation, G6PC, and surface markers of some adult hepatocytes ASGPR1.
  • smEnSC-Hep expressed a series of liver transcription factors including HNF4A, CEBPA, HNF6, etc., and the expression levels were similar to those of adult liver (Fig. 2A).
  • smEnSC-Hep expresses CYP3A4, CYP3A7, CYP2C9, CYP2C19, and CYP2B6 ( FIG. 2A ).
  • smEnSC-Hep expressed biphasic enzymes such as EPHX1 and a series of transporter proteins, such as NTCP, UGT1A1, MRP2, BCRP and BSEP (Fig. 2A).
  • Flow cytometric analysis found that more than 90% of cells in smEnSC-Hep were AFP+ and 70% were ALB+ (Fig. 2B).
  • the expression of CYP3A4 was significantly up-regulated after induction (Fig. 2E).
  • Hepatic parenchyma cells are spherical structures, hollow spheres, with a diameter of about 300 ⁇ m.
  • smEnSCs 1 ⁇ 10 5 smEnSCs were first cultured in smEnSC medium for two days, and then replaced with liver-specific medium for four days.
  • 20-50 cells were digested with 0.05% trypsin, 2 wells of cells were synthesized into 1 well, 80 ⁇ l of matrigel was added, and the cells were transferred to a low-adsorption culture plate and cultured in liver-specific medium for 2 days. Subsequently, the bile duct-specific medium was replaced and cultured for 4 days. Then transfer to bile duct maturation medium and continue to culture for 7 to 10 days.
  • smEnSC-Cho The bile duct epithelial cells smEnSC-Cho obtained from smEnSC differentiation showed a three-dimensional organoid shape (Fig. 2I), and after re-attaching on matrigel, they could form a specific structure with its own lumen. From the results of gene expression analysis, smEnSC-Cho expressed the key transcription factors HNF1B and SOX9 in bile duct epithelial cells, while the liver transcription factor HNF4A was significantly down-regulated (Fig. 2F). smEnSC-Cho, expresses a variety of receptor proteins and transporters related to bile duct function, including CTFR, AQP1, SCR, GGT, etc. (Fig. 2F).
  • Notch signaling pathway is important for bile duct development.
  • Notch signaling was maintained and activated in smEnSC and smEnSC-Cho, and its downstream target gene HES1 was highly expressed (Fig. 2F).
  • smEnSC-Cho expresses the important keratins CK19 and CK7 for bile duct epithelial cells. 1 ⁇ 10 5 smEnSCs can obtain 2-3 ⁇ 10 6 bile duct epithelial cells after a complete differentiation process.
  • the bile duct cell structure is a spheroid or tubular structure, the diameter of the spheroid is 280 ⁇ m, the inner diameter of the tubular structure is 50 ⁇ m, and the length is 1 mm;
  • 1 x 105 smEnSCs were first cultured in expansion medium for 2-3 days. It was then replaced with hindgut-specific medium and cultured for 4 days. After that, trypLE or 0.05% trypsin was used to digest into 20-50 cell clumps, 2 wells of cells were combined into 1 well, 80 ⁇ l of matrigel was added, and transferred to low-adsorption culture plates with small intestine maturation medium I for further culturing for 3 days. Afterwards, the cells were cultured in small intestine maturation medium for 18-24 days.
  • HIOs obtained from in vitro differentiation of smEnSCs expressed the transcription factors CDX2 and SOX9, which determine and maintain intestinal fate, as in adult intestinal tissues (Fig. 2G).
  • HIOs obtained by in vitro differentiation of smEnSCs also expressed the Enterocyte-specific marker VILLIN, enterocyte-specific marker Chromogranin A (CHGA), and Paneth cell-specific marker Lysozyme (LYZ); however, Goblet cell marker Mucin2 ( MUC2) (Fig. 2G).
  • HIOs formed by 3D suspension differentiation had a highly ordered tissue structure with internal organ cavities, similar to previously reported organoid structures established from primary intestinal cells (Fig. 2I).
  • HIO expresses transporter proteins such as MRP2, etc., which is beneficial to simulate the absorption of small intestinal substances in vitro (Fig. 2G).
  • 1 ⁇ 10 5 smEnSC starting cells can obtain 2-3 ⁇ 10 6 intestinal cells after a complete differentiation process.
  • the small intestinal cell structure includes solid cell spheroids, lumen cell spheroids and tubular structures, wherein the solid cell spheroids and lumen cell spheroids have a diameter of 300 ⁇ m, and the tubular structure has an inner diameter of 60 ⁇ m and a length of 2 mm.
  • 1 ⁇ 10 5 smEnSCs were first cultured in expansion medium for 2-3 days, and then induced into Pancreatic Foregut, Pancreatic Endoderm and Pancreatic Progenitor under corresponding culture conditions. After digestion with Dispase or 0.05% trypsin, 20-50 cells were formed into clumps, 2 wells of cells were combined into 1 well, 80 ⁇ l of matrigel was added, and transferred to a low-adsorption culture plate to continue to differentiate into mature islet ⁇ cells smEnSC- ⁇ Cell .
  • qPCR experiments showed that smEnSC- ⁇ Cell expressed pancreatic ⁇ -cell markers INS, MAFB, PDX1 and NKX6.1, while smEnSC-specific genes CDX2, SOX17, FOXA1 and SOX9 were significantly down-regulated (Fig. 2H).
  • Islet ⁇ -cells are three-dimensional solid spherical structures with structural features such as diameters ranging from 200 ⁇ m to 500 ⁇ m (Fig. 2I).
  • FRG mice were withdrawn from drinking water starting 6-7 days before transplantation.
  • smEnSC-heps were digested with collagenase B (1 mg/ml) for 1 h and then treated with 0.25% Trypsin-EDTA for 5 min.
  • the digested cells were filtered through a 70 ⁇ m filter, and finally 1 ⁇ 10 6 Eheps were resuspended in 100-150 ⁇ l PBS, placed on ice, and the cells were transplanted from the spleen.
  • the mice were weighed every 3 days after transplantation and the death of the mice was recorded. Mice that were not transplanted with cells and lost 30% of their body weight were used as negative controls. Mice that were still alive 8 weeks after cell transplantation were processed, and blood and liver samples were collected for later analysis.
  • Cell degeneration is a ubiquitous biological phenomenon in nature and plays an active role in the repair of individual damage.
  • the use of terminally differentiated cells to degenerate into proliferative cells is a new idea for establishing new stem cell lines. Due to the lack of ideal in vitro cell models, the molecular mechanisms of degeneration are currently unclear.
  • hEnSCs-derived hepatic parenchymal cells Eheps are used to conduct degeneration research, and the results show that the inhibition of TGF ⁇ signaling pathway and the activation of MAPK signaling pathway play an important role in the degeneration of differentiated cells.
  • the present invention utilizes differentiated cells to establish a novel induced pluripotent endodermal stem cell line (smEnSCs) that can be stably passaged in vitro.
  • smEnSCs novel induced pluripotent endodermal stem cell line
  • This cell line can efficiently differentiate into endoderm-derived cells such as liver parenchyma, bile duct epithelium, and small intestine, providing a novel seed cell for regenerative medicine.
  • endoderm-derived cells such as liver parenchyma, bile duct epithelium, and small intestine.
  • Eheps can establish a novel endodermal stem cell line, smEnSCs, through the induction of degeneration by small molecule compounds, which provides an ideal in vitro model for studying the mechanism of primary liver parenchymal cell degeneration and provides a new type of seed cells for regenerative medicine.
  • the present invention utilizes single-cell level transcriptome analysis combined with epigenetic analysis to study the dynamic molecular regulation mechanism in the above-mentioned degeneration process.
  • single-cell sequencing technology to study the dynamic changes of the Ehep degeneration process, on the one hand, it can track the changes of each cell group in the process, and infer which cell subsets can respond to exogenous inducing signals; on the other hand, it can comprehensively reveal the degeneration process. key signaling pathways.
  • hepatic parenchymal cell culture methods have many defects in cell proliferation, serum dependence, etc., and are difficult to meet the application requirements.
  • hepatic parenchymal cell degeneration under this induction condition could not obtain endodermal stem cells equivalent to hEnSC or smEnSC stage, suggesting that the epigenetic and transcriptional status of differentiated cells may affect the process and outcome of degeneration.
  • the present invention uses hEnSC-derived Eheps to simulate the degeneration process of primary liver parenchyma cells in vitro, and establishes a novel endodermal stem cell line smEnSCs with infinite expansion ability and multi-differentiation potential.
  • the present invention expects to use a similar method to achieve in vitro degeneration of human adult hepatocytes and other terminally differentiated cells and to establish stably proliferating tissue-specific stem cells, providing a new cell source for regenerative medicine applications.

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Abstract

提供一种诱导分化细胞成多潜能内胚层干细胞的方法及其应用，具体地，提供了一种诱导人类分化细胞(Differentiated Cell)分化为诱导多潜能内胚层干细胞(smEnSC)的方法，包括步骤：(a)在第一培养条件下，在培养体系中培养分化细胞，从而获得诱导多潜能内胚层干细胞(smEnSC)；其中，所述培养体系包括EGF、A83-01和CHIR99021。功能性的诱导多潜能内胚层干细胞具有非常高的分化率和纯度。

Description

诱导分化细胞成多潜能内胚层干细胞的方法及其应用 技术领域
本发明涉及生物技术和细胞治疗领域。具体地，本发明涉及诱导分化细胞成多潜能内胚层干细胞的方法及其应用。
背景技术
细胞退分化(De-differentiation)特指分化细胞(differentiated cells)在某些外界条件刺激下失去其特有功能并返回上级发育阶段(earlier developmental stages)，是自然界中普遍存在的现象。退分化获得的前体/干细胞对组织损伤修复等有着重要意义。
hEnSCs在体外能够无限扩增并分化成功能性内胚层衍生细胞，但其生长依赖于小鼠成纤维细胞作为滋养层及高浓度的matrigel，限制了其临床应用。
原代肝实质细胞的退分化现象在其分离培养过程特别明显，许多功能性蛋白基因尤其是参与异物质代谢的一相酶P450家族，在培养的早期迅速丢失。关于人原代肝实质细胞的退分化机制目前尚未明了，然而由于人原代肝实质细胞不易获得、在体外不能长期培养、难以进行遗传操作，因此不适合用于退分化机制的研究。
因此，本领域迫切需要开发一种诱导分化细胞成多潜能内胚层干细胞(small molecule induced Endoderm Stem Cells，smEnSCs)的方法。
发明内容
本发明公开了一种诱导分化细胞成多潜能内胚层干细胞(small molecule induced Endoderm Stem Cells，smEnSCs)的方法。
本发明公开了一种利用小分子化合物及细胞因子组合诱导分化细胞成多潜能内胚层干细胞(small molecule induced Endoderm Stem Cells，smEnSCs)的方法
本发明还公开了，利用小分子首次在体外诱导分化细胞的退分化，并建立诱导多潜能内胚层干细胞系(smEnSCs)
本发明还公开了，通过优化培养方法，smEnSCs能够不依赖于滋养层细胞在体外无限扩增。
本发明还公开了，建立了smEnSCs在体外分化成肝实质细胞、胆管上皮细胞以及小肠上皮细胞等内胚层衍生细胞的分化体系。
在本发明第一方面，提供了一种诱导人类分化细胞分化为诱导多潜能内胚层干细胞(smEnSC)的方法，其特征在于，包括步骤：
(a)在第一培养条件下，在培养体系中培养分化细胞，从而获得诱导多潜能内胚层干细胞(smEnSC)；其中，所述培养体系包括EGF、A83-01和CHIR99021。
在另一优选例中，所述分化为退分化。
在另一优选例中，所述第一培养条件含有第一培养基。
在另一优选例中，所述第一培养基选自下组：SFD、DMEM、DMEM/F12、或其组合。
在另一优选例中，所述培养体系还包括选自下组的一种或多种添加物：抗坏血酸磷酸镁(Ascorbic Acid Phosphate Magnesium)、L-谷氨酰胺(L-glutamine)、MTG、bFGF。
在另一优选例中，所述分化细胞包括成体细胞。
在另一优选例中，所述成体细胞选自下组：肝实质细胞、胃上皮细胞、胰岛细胞、小肠上皮细胞、或其组合。
在另一优选例中，所述肝实质细胞包括原代肝实质细胞、干细胞分化来源的肝实质细胞。
在另一优选例中，所述成体细胞来源于成体组织分离、干细胞(如内胚层干细胞、诱导多能干细胞)的体外定向分化。
在另一优选例中，所述的内胚层干细胞来源于人胚胎干细胞或人诱导多能干细胞。
在另一优选例中，所述多能干细胞选自下组：人胚胎干细胞系(如H9、Hes2)、人诱导多能干细胞系(如ZRZ-iPSCs、WT6iPSCs)、或其组合。
在另一优选例中，所述的内胚层干细胞选自下组：H9、Hes2、ZRZ-iPSC EnSC、WT6iPSC EnSC、或其组合。
在另一优选例中，所述方法还包括步骤(b)：将诱导多潜能内胚层干细胞(smEnSC)分化为内胚层衍生细胞。
在另一优选例中，所述内胚层衍生细胞包括肝实质细胞、胆管上皮细胞、小肠上皮细胞、胰岛β细胞、胃上皮细胞。
在另一优选例中，所述步骤(b)中不含有滋养层细胞。
在另一优选例中，所述步骤(b)中，包括：
(b1)在第二培养条件下，在培养体系中培养诱导多潜能内胚层干细胞，从而获得肝实质细胞；或
(b2)在第三培养条件下，在培养体系中培养诱导多潜能内胚层干细胞，从而获得胆管细胞；或
(b3)在第四培养条件下，在培养体系中培养诱导多潜能内胚层干细胞，从而获得小肠细胞。
在另一优选例中，所述第二培养条件含有第二培养基。
在另一优选例中，所述第二培养基包括肝向特化培养基、肝向成熟培养基I和肝向成熟培养基II。
在另一优选例中，所述肝向特化培养基选自下组：SFD、DMEM/F12、DMEM、或其组合。
在另一优选例中，所述肝向成熟培养基I选自下组：SFD、DMEM/F12、DMEM、或其组合。
在另一优选例中，所述肝向成熟培养基II选自下组：SFD、HCM、DMEM/F12、DMEM、或其组合。
在另一优选例中，所述肝向特化培养基还含有选自下组的一种或多种添加物：
BMP4、FGF、A83-01、SB431542、IWP2、Dexamethasone、DMSO。
在另一优选例中，所述肝向成熟培养基I还包括选自下组的一种或多种添加物：
HGF、地塞米松(Dexamethasone)、OSM、C-E、A83-01、EGFi、VK1。
在另一优选例中，所述肝向成熟培养基II还包括选自下组的一种或多种添加物：HGF、Dexamethasone、OSM、C-E、A83-01、EGFi、VK1。
在另一优选例中，所述第三培养条件含有第三培养基。
在另一优选例中，所述第三培养基包括胆管特化培养基和胆管成熟培养基。
在另一优选例中，所述第三培养基选自下组：SFD、DMEM/F12、DMEM、William’s E、或其组合。
在另一优选例中，所述第三培养条件还含有选自下组的一种或多种添加物：
FGF10、HGF、TGFβ、EGF、Activin A、地塞米松(Dexamethasone)。
在另一优选例中，所述第四培养条件含有第四培养基。
在另一优选例中，所述第四培养基包括扩增培养基、后肠特化培养基、小肠成熟培养基I和小肠成熟培养基II。
在另一优选例中，所述第四培养基选自下组：SFD、DMEM/F12、DMEM、或其组合。
在另一优选例中，所述第四培养条件还含有选自下组的一种或多种添加物：
FGF4、EGF、A83-01、CHIR99321、Wnt3a、R-spondin、EGF、Noggin。
在另一优选例中，所述扩增培养基选自下组：SFD、DMEM/F12、DMEM、或其组合。
在另一优选例中，所述后肠特化培养基选自下组：SFD、DMEM/F12、DMEM、或其组合。
在另一优选例中，所述小肠成熟培养基I选自下组：SFD、DMEM/F12、DMEM、或其组合。
在另一优选例中，所述小肠成熟培养基II选自下组：SFD、DMEM/F12、DMEM、或其组合。
在另一优选例中，所述扩增培养基还包括选自下组的一种或多种添加物：
FGF4、EGF、A83-01。
在另一优选例中，所述后肠特化培养基还包括选自下组的一种或多种添加物：
FGF4、CHIR99321。
在另一优选例中，所述小肠成熟培养基I还包括选自下组的一种或多种添加物：
FGF4、CHIR99321、EGF、Noggin。
在另一优选例中，所述小肠成熟培养基II还包括选自下组的一种或多种添加物：CHIR99321、EGF、Noggin。
在另一优选例中，在步骤(a)中，将成体细胞在第一培养条件下培养3-18天，较佳地，4-16天，更佳地，6-12天。
在另一优选例中，在步骤(b1)中，将诱导多潜能内胚层干细胞在第二培养条件下培养15-40天，较佳地，18-36天，更佳地，20-25天。
在另一优选例中，在步骤(b2)中，将诱导多潜能内胚层干细胞在第三培养条件 下培养10-30天，较佳地，15-28天，更佳地，15-21天。
在另一优选例中，在步骤(b3)中，将诱导多潜能内胚层干细胞在第四培养条件下培养20-50天，较佳地，22-45天，更佳地，25-40天。
在另一优选例中，所述方法具有选自下组的一个或多个特征：
(i)高诱导多潜能内胚层干细胞分化率，所述分化率为80-98.5％，较佳地，90-95％；
(ii)在培养过程中，每1ml的培养液中接种0.5-2×10 5个成体细胞，可产生0.5-3×10 6个诱导多潜能内胚层干细胞。
在另一优选例中，所述培养体系中不含有或含有很少的细胞外基质。
在另一优选例中，所述细胞外基质选自下组：基质胶(Matrigel)、层粘连蛋白(Laminin)、基底膜提取物(Basement Membrane Extracts)、或其组合
在另一优选例中，在培养体系中，所述细胞外基质的含量(v/v)为0.5％－5％，较佳地，1％—3％，更佳地，1％－2％。
在另一优选例中，所述方法包括治疗性和非治疗性的。
在另一优选例中，所述的培养体系中，成体细胞的密度为0.5×10 6-4×10 6细胞/平板，较佳地，1×10 6-2×10 6细胞/平板。
在另一优选例中，所述的培养体系为体积10-25ml，较佳地10-20ml，最佳地为10-15ml。
在另一优选例中，所述的获得的诱导多潜能内胚层干细胞的数量M2与所述成体细胞的数量M1之比M2/M1为5-15，较佳地8-14，更佳地8-10。
在另一优选例中，所述诱导多潜能内胚层干细胞为功能性的诱导多潜能内胚层干细胞。
在另一优选例中，所述内胚层衍生细胞为功能性的内胚层衍生细胞。
本发明第二方面提供了一种诱导多潜能内胚层干细胞，所述的诱导多潜能内胚层干细胞由权利要求1所述的方法制备获得。
在另一优选例中，所述的诱导多潜能内胚层干细胞具有选自以下一种或多种特征：
(a)80-90％的细胞为肝向标志基因表达阴性；
(b)85-98％的细胞为前体细胞标志物基因表达阳性；
(c)85-98％的细胞为内胚层特异基因表达阳性；
(d)80-90％的细胞为肝脏前体细胞特异基因表达阳性；
(e)85-90％的细胞为FOXA1、EpCAM和SOX9表达阳性；
(f)85-95％的细胞为CD31表达阳性。
在另一优选例中，所述的诱导多潜能内胚层干细胞具有选自以下一种或多种特征：
(a)肝向标志基因低表达或不表达；
(b)前体细胞标志物基因高表达；
(c)内胚层特异基因高表达；
(d)肝脏前体细胞特异基因高表达；
(e)FOXA1、EpCAM和SOX9高表达；
(f)CD31高表达。
在另一优选例中，所述肝向标志基因选自下组：ALB、AAT、CYP3A7、CYP3A4、或其组合。
在另一优选例中，所述前体细胞标志物基因选自下组：CDX2、SOX17、或其组合。
在另一优选例中，所述内胚层特异基因选自下组：SOX17、CDX2、FOXA2、或其组合。
在另一优选例中，所述肝脏前体细胞特异基因选自下组：SOX9、TBX3、或其组合。
在另一优选例中，所述的诱导多潜能内胚层干细胞由成体细胞诱导获得。
本发明第三方面提供了一种内胚层衍生细胞，所述内胚层衍生细胞为三维球状结构，其中，所述内胚层衍生细胞的球囊直径大小为100－500μm。
在另一优选例中，所述的内胚层衍生细胞具有选自以下的一种或多种特征：
(a)肝实质细胞球为空心球，直径在150－500μm；
(b)胆管细胞结构为细胞球结构或者管状结构，细胞球直径为150-500μm，管状结构内径为20-80μm，长500μm-3mm；
(c)小肠细胞结构包括实心细胞球、带内腔的细胞球以及管状结构，其中实心细胞球、带内腔的细胞球的直径在100－500μm，管状结构内径在20-80μm，长500μm-5mm。
在另一优选例中，所述内胚层衍生细胞包括肝实质细胞、胆管上皮细胞、小肠上皮细胞、胰岛β细胞、胃上皮细胞。
本发明第四方面提供了一种本发明第二方面所述的诱导多潜能内胚层干细 胞、或本发明第三方面所述的内胚层衍生细胞的用途，用于制备药物或制剂，所述药物或制剂用于(a)治疗肝脏、胆管相关疾病；和/或(b)肠道疾病；和/或(c)糖尿病。
在另一优选例中，所述肝脏、胆管相关疾病选自下组：遗传代谢性肝病、慢/急性肝衰竭、Alagille综合症、Crigler-Najjar综合征1型(Crigler–Najjar syndrome type 1)、或其组合。
在另一优选例中，所述遗传性肝病选自下组：肝豆状核变性(Wilson’s disease，WD)、糖原累积病Ia型、α-抗胰蛋白酶缺乏症、血色病、先天性胆道闭锁、希特林蛋白缺乏症、家族型高胆固醇症、或其组合。
在另一优选例中，所述肠道疾病选自下组：肠道损伤、短肠综合征、或其组合。
在另一优选例中，所述糖尿病选自下组：I型糖尿病、II型糖尿病、或其组合。
本发明第五方面提供了一种诱导培养基，所述诱导培养基含有基础培养基和添加物；其中所述基础培养基选自下组：SFD、DMEM、DMEM/F12、BDM、或其组合；并且，所述添加物包括EGF、A83-01和CHIR99021。
在另一优选例中，所述添加物还包括bFGF、VEGF、EGF、HGF、OSM、和地塞米松(Dexamethasone)。
在另一优选例中，所述添加物选自下组的一种或多种添加物：抗坏血酸磷酸镁(Ascorbic Acid Phosphate Magnesium)、L-谷氨酰胺(L-glutamine)、MTG、bFGF。
本发明第六方面提供了一种本发明第五方面所述的诱导培养基的用途，用于诱导分化细胞分化为诱导多潜能内胚层干细胞(smEnSC)。
本发明第七方面提供了一种筛选或确定促进分化细胞分化为诱导多潜能内胚层干细胞(smEnSC)的潜在治疗剂的方法，包括步骤：
(a)在测试组中，在培养体系中，在测试化合物的存在下，培养分化细胞一段时间T1，检测测试组所述培养体系中的诱导多潜能内胚层干细胞的数量M1；
并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中的诱导多潜能内胚层干细胞的数量M2；和
(b)将上一步骤所检测的M1、M2进行比较，从而确定所述测试化合物是否是促进分化细胞分化为诱导多潜能内胚层干细胞的潜在治疗剂；
其中，如果M1显著高于M2，则表示所述测试化合物是促进分化细胞分化为诱导多潜能内胚层干细胞的潜在治疗剂。
在另一优选例中，所述“显著高于”指M1/M2≥2,较佳地，≥3，更佳地，≥4。
在另一优选例中，所述的方法是非诊断和非治疗性的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1表示体外诱导分化肝实质细胞退分化并建立smEnSC细胞系；其中，
(A)诱导成熟分化的Eheps成smEnSCs流程图。
(B)Eheps退分化过程中细胞形态图。D3，D7分别表示诱导退分化的时间。比例尺：200μm。
(C)Eheps退分化7天后基因表达变化。肝向基因(ALB、AAT、CYP3A7和CYP3A4)关闭，前体细胞标志物基因(CDX2和SOX17)开启。
(D)smEnSCs形态图。呈均一的上皮样形态。比例尺：200μm。
(E)smEnSCs能够在体外无限传代。
(F)smEnSCs基因表达情况。P3，P4代表smEnSCs的代数，DE-T5表示定型内胚层。smEnSC表达内胚层特异基因SOX17、CDX2及FOXA2，肝脏前体细胞特异基因SOX9与TBX3。
(G)流式细胞分析表明，smEnSCs表达SOX17和FOXA1，EpCAM和SOX9。
(H)smEnSC可从单细胞建立，因此具有克隆水平上的干性。
(I)smEnSCs在体内不成畸胎瘤。
图2表示体外诱导smEnSCs分化成内胚层衍生细胞；其中，
(A)smEnSC-Hep基因表达概况。分化获得的smEnSC-Hep肝向基因如ALB、CYPs等明显上调。AL代表成体肝组织。
(B)共染AFP和ALB揭示smEnSCs体外分化成肝实质细胞的效率。AFP阳性率高达90％以上，ALB阳性率也达到近70％。
(C)ELISA检测smEnSC-Heps能够分泌白蛋白。
(D)PAS染色表明smEnSC-Heps具有糖原积累的能力。比例尺：500μm。
(E)smEnSC-Heps能够响应利福平诱导CYP3A4的表达。
(F)smEnSCs体外诱导成胆管上皮细胞(smEnSC-Cho)，qPCR实验表明其表达胆管上皮细胞标志基因。
(G)smEnSCs体外诱导成小肠上皮细胞(smEnSC-HIO)。
(H)smEnSCs体外诱导成胰岛β细胞(smEnSC-βcell)。
(I)smEnSCs体外分化的肝实质细胞、胆管上皮细胞、小肠上皮细胞及胰岛β细胞形态图。
图3表示smEnSC-Hep能够拯救肝衰模型FRG小鼠；其中，
(A)smEnSC-Hep移植能够显著改善FRG小鼠生存。Sham表示未移植smEnSC-Hep组，smEnSC-Hep表示移植smEnSC-Hep组。
(B)移植smEnSC-Hep存活的FRG小鼠血清中能够检测到人源白蛋白。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，在本发明的第一培养条件下，在培养体系中培养成体细胞，能够获得诱导多潜能内胚层干细胞(smEnSC)，并且本发明的诱导多潜能内胚层干细胞的分化率＞90％，诱导多潜能内胚层干细胞具有＞90％的纯度。并且，本发明的诱导多潜能内胚层干细胞还可以进一步分化为内胚层衍生细胞(如肝实质细胞、胆管细胞、小肠细胞、胰岛β细胞等)，此外，本发明的诱导多潜能内胚层干细胞还可用于治疗肝脏、胆管相关疾病和糖尿病。在此基础上，本发明人完成了本发明。
具体地，本发明人发明了一种利用小分子化合物及细胞因子组合诱导分化细胞成多潜能内胚层干细胞(small molecule induced Endoderm Stem Cells，smEnSCs)的方法。通过将干细胞来源的肝实质细胞等分化细胞用含特定的小分子化合物及细胞因子组合培养，诱导其退分化，并通过传代的方式建立不依赖于滋养层细胞的、可无限增殖的新型内胚层干细胞系，随后进一步在体外对其进行诱导分化成肝实质细胞、胆管上皮细胞、小肠上皮细胞及胰岛β细胞。
术语
如本文所用，术语“A83-01”化学式为C 25H 19N 5S，CAS编号为909910-43-6；
“C-E”的化学式C 27H 24F 2N 4O 3，CAS编号为209986-17-4；
“EGFi”化学式为C 22H 23N 3O 4.HCl，CAS编号为183319-69-9；
SB431542的化学式为C 22H 16N 4O 3，CAS编号为301836-41-9。
如本文所用，术语“平板”指各个规格的平板，包括58mm 2的平板。
如本文所用，术语“诱导多潜能内胚层干细胞”、“smEnSC”指的是经本发明方法制备获得的诱导多潜能内胚层干细胞，所述诱导多潜能内胚层干细胞具有以下一种或多种特征：
(a)肝向基因低表达或不表达；
(b)前体细胞标志物基因高表达；
(c)内胚层特异基因高表达；
(d)肝脏前体细胞特异基因高表达；
(e)FOXA1、EpCAM和SOX9高表达；
(f)CD31高表达；
分化细胞
在本发明中，分化细胞指分化细胞指的是从发育生物学角度讲从受精卵往下进一步分化的细胞类型，其为某一特定谱系的细胞且无法增殖，本专利中包括干细胞定向分化获得的成熟细胞以及原代分离的成体细胞。
在本发明中，分化细胞包括成体细胞。
成体细胞指从成体组织直接分离获取的原代细胞，成体细胞属于终末分化细胞类型，不具备增殖能力。
诱导多潜能内胚层干细胞的诱导培养方法
本发明的诱导多潜能内胚层干细胞的起始细胞为人类分化细胞(包括成体细胞)，在第一培养条件下进行培养，从而获得诱导多潜能内胚层干细胞(smEnSC)，其中，所述第一培养条件下的培养体系包括EGF、A83-01和CHIR99021。
接着，在适合培养的条件下(如在含有第二培养基，包括肝向特化培养基、肝向成熟培养基I和肝向成熟培养基II)和添加物(肝向特化培养基包括BMP4、FGF、A83-01、IWP2、Dexamethasone，肝向成熟培养基I包括HGF、Dexamethasone、 OSM、C-E、A83-01、EGFi，肝向成熟培养基II包括Dexamethasone、C-E、A83-01、EGFi)的条件下或在含有第三培养基(如SFD)和添加物(包括FGF10、HGF、TGFβ、地塞米松)的条件下或在含有第四培养基(包括扩增培养基、后肠特化培养基、小肠成熟培养基I和小肠成熟培养基II)和添加物(扩增培养基包括FGF4、EGF、A83-01，后肠特化培养基包括FGF4、CHIR99321，小肠成熟培养基I包括FGF4、CHIR99321、EGF、Noggin，小肠成熟培养基II包括CHIR99321、EGF、Noggin)的条件下)，对诱导多潜能内胚层干细胞进一步培养，获得内胚层衍生细胞(如肝实质细胞、胆管细胞、小肠细胞、胰岛β细胞等)。
在本发明中，本发明的培养体系中不含或含有很少的基质胶(Matrigel)。
在一优选实施方式中，SFD(Serum free differentiation medium)包括以下组分：
75％Iscove的改良Dulbecco's培养基(IMDM)(Cellgro)，添加有0.5倍N2和B27补充剂(Gibco-BRL)，1％青霉素/链霉素，0.05％牛血清白蛋白的25％Ham's F12培养基(Cellgro)。
HCM培养基可市售获得(Commercial medium:Lonza:CC-4182)(Ogawa,S.,Surapisitchat,J.,Virtanen,C.,Ogawa,M.,Niapour,M.,Sugamori,K.S.,...&Zhao,B.(2013).Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes.Development,140(15),3285-3296.)。
筛选或确定促进分化细胞分化为诱导多潜能内胚层干细胞(smEnSC)的潜在治疗剂的方法
在本发明中，提供了筛选或确定促进分化细胞分化为诱导多潜能内胚层干细胞(smEnSC)的潜在治疗剂的方法，包括步骤：
(a)在测试组中，在培养体系中，在测试化合物的存在下，培养分化细胞一段时间T1，检测测试组所述培养体系中的诱导多潜能内胚层干细胞的数量M1；
并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中的诱导多潜能内胚层干细胞的数量M2；和
(b)将上一步骤所检测的M1、M2进行比较，从而确定所述测试化合物是否是促进分化细胞分化为诱导多潜能内胚层干细胞的潜在治疗剂；
其中，如果M1显著高于M2，则表示所述测试化合物是促进分化细胞分化为诱导多潜能内胚层干细胞的潜在治疗剂。
在另一优选例中，所述“显著高于”指M1/M2≥2,较佳地，≥3，更佳地，≥4。
在另一优选例中，所述的方法是非诊断和非治疗性的。
本发明的主要优点包括：
(1)本发明首次发现，本发明在培养体系中培养分化细胞(如成体细胞)，可获得极高分化率(＞90％)的功能性诱导多潜能内胚层干细胞，并且本发明的诱导多潜能内胚层干细胞的纯度也非常高，高达99％。
(2)本发明利用小分子首次在体外诱导分化细胞的退分化，并建立诱导多潜能内胚层干细胞系(smEnSCs)。
(3)本发明的smEnSCs能够不依赖于滋养层细胞在体外无限扩增，且在特定的诱导分化条件下可以重新分化成肝实质细胞、胆管上皮细胞以及小肠上皮细胞等内胚层衍生细胞，因此本发明的smEnSC有望替代hEnSCs应用于再生医学等许多领域。此外，该退分化体系也可作为退分化机制研究的理想模型。
(4)本发明的干细胞来源的肝实质细胞易于获得、能够在体外长期培养、可进行遗传操作，因此具有替代人原代肝实质细胞进行退分化机制研究的潜力。
(5)本发明利用小分子化合物组合诱导Eheps退分化建立smEnSCs，成本低、稳定性高。
(6)本发明的smEnSCs的培养不依赖于如MEF等滋养层细胞，符合临床治疗的要求。
(7).本发明的smEnSCs能够在体外无限扩增，体内不成瘤，且在特定体外诱导条件下重新分化成肝实质细胞、胆管上皮细胞以及小肠上皮细胞等内胚层衍生细胞类型。因此，可用于在体外规模化制备符合临床需求的功能细胞。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非另有说明，否则本发明实施例中所用的试剂和材料均为市售产品。
通用材料和方法
细胞培养基
诱导多潜能内胚层干细胞(smEnSC)培养基：SFD培养基种加入L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，bFGF(10ng/ml),EGF(10ng/ml),A83-01(0.5μM)；CHIR99021(3μM)；
smEnSC肝实质细胞分化培养基：肝向特化培养基，SFD培养基添加L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，hBMP4(50ng/ml)；bFGF(20ng/ml)，A83-01(0.5μM)，IWP2(5μM)，Dexamethasone(40ng/ml)，DMSO(1％v/v)；肝向成熟培养基Ⅰ，SFD培养基添加L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，HGF(25ng/ml)，Dexamethasone(40ng/ml)，OSM(20ng/ml),C-E(0.1uM),A83-01(0.5uM)，EGFi(2uM)。；肝向成熟培养基Ⅱ，HCM培养基添加L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，HGF(25ng/ml)，Dexamethasone(40ng/ml),C-E(0.1uM),A83-01(0.5uM)，EGFi(2uM)。
smEnSC胆管细胞分化培养基：胆管特化培养基，SFD培养基种加入L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，ActivinA(50ng/ml),FGF10(50ng/ml)；胆管成熟培养基，SFD培养基种加入L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，HGF(25ng/ml)，TGFβ(5ng/ml),地塞米松(40ng/ml)。
smEnSC小肠细胞分化培养基：扩增培养基，SFD培养基种加入L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，FGF4(10ng/ml),EGF(10ng/ml),A83-01(0.5μM)；；后肠特化培养基，SFD培养基种加入L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，FGF4(500ng/ml)，CHIR99321(3μM)；小肠成熟培养基Ⅰ，SFD培养基种加入L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，FGF4(100 ng/ml)，CHIR99321(3μM)，EGF(100ng/ml)，Noggin(100ng/ml)；小肠成熟培养基Ⅱ，SFD培养基种加入L-Ascorbic Acid Phosphate Magnesium(50μg/ml，Wako,cat#013-19641)，L-glutamine(1X，Cellgro)，MTG(4.5×10 -4M)，CHIR99321(3μM)，EGF(100ng/ml)，Noggin(100ng/ml)。
实施例1诱导Eheps成smEnSC
分化成熟的Eheps经胶原酶B(1mg/ml，Roche)37℃消化2个小时，再用0.25％胰酶消化5分钟，后机械力吹达成单细胞，种植在matrigel包被的六孔板，每孔1-2×10 5细胞，用smEnSC培养基培养(图1A)。光学显微镜下能明显观察到细胞增殖(图1B)。qPCR结果显示Eheps在我们发明的培养基中培养7天后，肝向标志基因AAT、ALB、CYP3A7、CYP3A4显著下调(图1C)早期内胚层相关的基因SOX17和CDX2的表达显著上调，这都表明原先的肝实质细胞功能发生大幅度退化甚至消失。前面2代每6天进行一次传代，后期3-4天传代一次。起始Eheps的细胞数量为0.5-2×10 5，培养后可获得smEnSC的细胞数量为1-2×10 6个。
稳定的smEnSC细胞系呈均一的上皮样细胞形态(图1D)，此外能在体外无限扩增(图1E)。基因表达分析表明，smEnSCs表达肝脏富集转录因子HNF4A，以及定型内胚层(Definitive Endoderm，DE)和hEnSC关键转录因子SOX17，(图1F)。但与DE和hEnSC不同的是，smEnSCs不表达转录因子EOMES，而FOXA1、TBX3、SOX9、CEBPA、PROX1等早期肝脏发育关键转录因子有明显上调(图1F)。此外，smEnSC表达后肠命运决定的关键转录因子CDX2，但CDX2在DE中并不表达(图1F)。流式结果显示，smEnSC共表达SOX17和FOXA1，EpCAM和SOX9(图1G)。这些结果表明，分化成熟的肝实质细胞在“F”培养基中发生了退分化，由此建立的新型内胚层干细胞系smEnSC，在发育阶段上晚于DE或hEnSCs。
此外，单克隆生长实验表明一个smEnSC能够长成smEnSC克隆并建系(图1H)，这在克隆水平上验证了smEnSCs的干性。
将smEnSCs移植到SCID-Beige小鼠(20只，5×10 6细胞/小鼠)，追踪4个月后未发现任何体内成瘤情况(图1I),这个结果表明smEnSCs体内不成瘤，是未来细胞治疗应用的一个安全的种子细胞。
实施例2诱导smEnSCs成肝实质细胞
1×10 5的smEnSC首先用smEnSC培养基培养两天，后更换肝向特化培养基4天。用0.05％胰酶消化成20-50个细胞的团块，2孔的细胞合为1孔，加入80μl matrigel，转入低吸附培养板中用肝向特化培养基继续培养2天。后在肝向成熟培养基Ⅰ中培养12天，肝向培养基Ⅱ中培养6天。
smEnSCs分化获得的肝实质细胞(smEnSC-Hep)表达一系列成体肝实质细胞特异的蛋白，包括分泌型蛋白Albumin、AAT，参与糖原积累的关键酶G6PC，以及部分成体肝实质细胞的表面标志物ASGPR1。而在转录因子方面，smEnSC-Hep表达一系列肝脏转录因子包括HNF4A、CEBPA、HNF6等，且表达水平与成体肝相近(图2A)。在肝实质细胞的成熟度的重要标志P450家族酶类中，smEnSC-Hep表达CYP3A4、CYP3A7、CYP2C9、CYP2C19以及CYP2B6等(图2A)。此外，smEnSC-Hep表达EPHX1等二相酶以及一系列转运体蛋白，如NTCP、UGT1A1、MRP2、BCRP及BSEP等(图2A)。流式细胞分析发现，smEnSC-Hep中超过90％的细胞为AFP+，70％的细胞为ALB+(图2B)。smEnSC-Heps形成的细胞团——“肝球”(Liver balls)(图2I)具有特殊的极性结构，能够分泌白蛋白(图2C)，并积累糖原(图2D)，经利福平诱导后能显著上调CYP3A4的表达(图2E)。这些结果表明，smEnSC能够在现有的三维系统里高效分化成功能性肝实质细胞。1×10 5的smEnSC起始细胞量经过完整的分化流程后可获得2-3×10 6的肝实质细胞。
肝实质细胞为球状结构，为空心球，直径为300μm左右。
实施例3诱导smEnSCs成胆管上皮细胞
1×10 5的smEnSC首先用smEnSC培养基培养两天，后更换肝向特化培养基4天。用0.05％胰酶消化成20-50个细胞的团块，2孔的细胞合成成1孔，加入80μlmatrigel，转入低吸附培养板中用肝向特化培养基继续培养2天。随后更换胆管特化培养基培养4天。后转入胆管成熟培养基继续培养7至10天。
smEnSC分化获得的胆管上皮细胞smEnSC-Cho呈三维类器官形态(图2I)，且重新在matrigel上贴壁后能够形成自带管腔的特定结构。而从基因表达分析的结果看，smEnSC-Cho表达胆管上皮细胞的关键转录因子HNF1B和SOX9，而肝脏转录因子HNF4A显著下调(图2F)。smEnSC-Cho，表达多种与胆管功能相关的受体蛋白和转运体，包括CTFR，AQP1，SCR，GGT等(图2F)。 Notch信号通路对胆管发育具有重要意义，我们发现在smEnSC及smEnSC-Cho中Notch信号维持激活，其下游靶基因HES1高表达(图2F)。是，smEnSC-Cho表达胆管上皮细胞重要的角蛋白CK19和CK7。1×10 5的smEnSC经过完整的分化流程后可获得2-3×10 6的胆管上皮细胞。这些结果表明，smEnSCs在体外可以高效分化成胆管上皮细胞。
胆管细胞结构为细胞球结构或者管状结构，细胞球直径为280μm，管状结构内径为50μm，长1mm；
实施例4诱导smEnSCs成小肠细胞
1×10 5的smEnSC首先在扩增培养基中培养2-3天。后更换为后肠特化培养基培养4天。后用TrypLE或者0.05％胰酶消化成20-50个细胞的团块,2孔的细胞合为1孔，加入80μlmatrigel，转入低吸附培养板中用小肠成熟培养基Ⅰ继续培养3天。后在小肠成熟培养基中继续培养18-24天。
从基因表达来看，与成体小肠组织一样，smEnSCs体外分化获得的HIO表达小肠命运决定与维持的转录因子CDX2和SOX9(图2G)。此外，smEnSCs体外分化获得的HIO还表达Enterocyte特有的标志物VILLIN，肠内分泌细胞特有的标志物Chromogranin A(CHGA),Paneth cell特有的标志物Lysozyme(LYZ)；然而，Goblet cell的标志物Mucin2(MUC2)(图2G)。此外，经三维悬浮分化形成的HIO具有高度有序的组织结构，含有内部官腔，与之前报到的从原代小肠细胞建立的类器官结构相似(图2I)。并且，在我们的分化系统里还出现了较长的管状结构，这在小肠分化的相关研究中未见报道。HIO表达转运体蛋白如MRP2等，有利于体外模拟小肠物质吸收等(图2G)。这些结果表明，smEnSC能够在体外分化成具有一定结构及多种小肠细胞类型的小肠组织。
1×10 5的smEnSC起始细胞量经过完整的分化流程后可获得2-3×10 6的小肠细胞。
小肠细胞结构包括实心细胞球、带内腔的细胞球以及管状结构，其中实心细胞球、带内腔的细胞球的直径在300μm，管状结构内径在60μm，长2mm。
实施例5诱导smEnSCs成胰岛β细胞
1×10 5的smEnSC首先在扩增培养基中培养2-3天，随后在相应的培养条件下分别诱导成Pancreatic Foregut，Pancreatic Endoderm，Pancreatic Progenitor。 后用Dispase或者0.05％胰酶消化成20-50个细胞的团块，2孔的细胞合为1孔，加入80μlmatrigel，转入低吸附培养板中用继续分化为成熟的胰岛β细胞smEnSC-βCell。qPCR实验表明，smEnSC-βCell表达胰岛β细胞标志物INS，MAFB，PDX1及NKX6.1，而smEnSC特有的基因CDX2,SOX17,FOXA1及SOX9明显下调(图2H)。
胰岛β细胞为三维实心球结构，直径为200μm-500μm等结构特征(图2I)。
实施例6 smEnSC-Hep移植FRG小鼠实验
FRG小鼠移植前6-7天开始撤除饮水中的NTBC。smEnSC-heps经胶原酶B(1mg/ml)消化1h后，在用0.25％Trypsin-EDTA处理5分钟。消化好的细胞经70μm滤膜过滤，最后用100-150μl PBS重悬1×10 6Eheps，置于冰上，从脾脏移植细胞。移植后每3天称重一次并记录小鼠死亡情况。未移植细胞且体重下降30％的小鼠作为阴性对照。细胞移植后8周仍然存活的小鼠进行处理，并采集血液及肝脏样本进行后面的分析。
结果显示，经smEnSC-Hep移植后FRG小鼠血清中可检测到人源白蛋白(图3B)，小鼠生存明显改善(图3A)。这些结果表明，smEnSC-Hep对肝衰竭等疾病具有较好的治疗效果。
讨论
细胞退分化，是自然界中普遍存在的生物学现象，在个体损伤修复等发挥着积极的作用。利用终末分化的细胞退分化为可增殖的细胞是建立新型干细胞系的新思路。由于缺乏理想的体外细胞模型，退分化的分子机制目前尚不清楚。
本发明中利用hEnSCs来源的肝实质细胞Eheps进行退分化的研究，结果显示TGFβ信号通路的抑制及MAPK信号通路的激活对分化细胞的退分化具有重要作用。
通过操纵这些信号通路，本发明利用分化细胞建立了新型的可在体外稳定传代的诱导多潜能内胚层干细胞系(smEnSCs)。该细胞系可以高效分化为肝实质、胆管上皮和小肠等内胚层衍生细胞，为再生医学提供了新型种子细胞。与hEnSCs相比，smEnSCs的培养不依赖于滋养层细胞，符合临床应用的要求。
综上所述，Eheps经小分子化合物诱导退分化可建立新型内胚层干细胞系 smEnSCs，为研究原代肝实质细胞退分化机制提供了理想的体外模型，为再生医学提供了新型种子细胞。
本发明利用单细胞水平的转录组分析，结合表观遗传组学分析，研究上述退分化过程中的动态分子调控机制。利用单细胞测序技术研究Ehep退分化过程的动态变化，一方面能够跟踪该过程中各细胞类群的变化，并推断具体哪些细胞亚群能够响应外源诱导信号；另一方面可全面揭示退分化过程中的关键信号通路。
现有的其它肝实质细胞培养方法，在细胞增殖、血清依赖等方面存在多种缺陷，难以满足应用需求。然而，该诱导条件下的肝实质细胞退分化并不能获得等同hEnSC或smEnSC阶段的内胚层干细胞，提示分化细胞的表观遗传及转录状态可能影响退分化的进程及结果。
综上所述，本发明利用hEnSC来源的Eheps在体外模拟原代肝实质细胞退分化过程，并建立具有无限扩增能力及多分化潜能的新型内胚层干细胞系smEnSCs。本发明期待利用类似方式可实现人成体肝实质细胞以及其它终末分化细胞的体外退分化并建立稳定增殖的组织特异性干细胞，为再生医学应用提供新细胞来源。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种诱导人类分化细胞分化为诱导多潜能内胚层干细胞(smEnSC)的方法，其特征在于，包括步骤：
    (a)在第一培养条件下，在培养体系中培养分化细胞，从而获得诱导多潜能内胚层干细胞(smEnSC)；其中，所述培养体系包括EGF、A83-01和CHIR99021。
  2. 如权利要求1所述的方法，其特征在于，所述分化为退分化。
  3. 如权利要求1所述的方法，其特征在于，所述分化细胞包括成体细胞。
  4. 如权利要求3所述的方法，其特征在于，所述成体细胞选自下组：肝实质细胞、胃上皮细胞、胰岛细胞、小肠上皮细胞、或其组合。
  5. 一种诱导多潜能内胚层干细胞，其特征在于，所述的诱导多潜能内胚层干细胞由权利要求1所述的方法制备获得。
  6. 一种内胚层衍生细胞，其特征在于，所述内胚层衍生细胞为三维球状结构，其中，所述内胚层衍生细胞的球囊直径大小为100－500μm。
  7. 一种权利要求5所述的诱导多潜能内胚层干细胞、或权利要求6所述的内胚层衍生细胞的用途，其特征在于，用于制备药物或制剂，所述药物或制剂用于(a)治疗肝脏、胆管相关疾病；和/或(b)肠道疾病；和/或(c)糖尿病。
  8. 一种诱导培养基，其特征在于，所述诱导培养基含有基础培养基和添加物；其中所述基础培养基选自下组：SFD、DMEM、DMEM/F12、BDM、或其组合；并且，所述添加物包括EGF、A83-01和CHIR99021。
  9. 一种权利要求8所述的诱导培养基的用途，其特征在于，用于诱导分化细胞分化为诱导多潜能内胚层干细胞(smEnSC)。
  10. 一种筛选或确定促进分化细胞分化为诱导多潜能内胚层干细胞(smEnSC)的潜在治疗剂的方法，其特征在于，包括步骤：
    (a)在测试组中，在培养体系中，在测试化合物的存在下，培养分化细胞一段时间T1，检测测试组所述培养体系中的诱导多潜能内胚层干细胞的数量M1；
    并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中的诱导多潜能内胚层干细胞的数量M2；和
    (b)将上一步骤所检测的M1、M2进行比较，从而确定所述测试化合物是否是促进分化细胞分化为诱导多潜能内胚层干细胞的潜在治疗剂；
    其中，如果M1显著高于M2，则表示所述测试化合物是促进分化细胞分化为 诱导多潜能内胚层干细胞的潜在治疗剂。
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