WO2017206837A1 - 将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、重编程方法及应用 - Google Patents

将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、重编程方法及应用 Download PDF

Info

Publication number
WO2017206837A1
WO2017206837A1 PCT/CN2017/086336 CN2017086336W WO2017206837A1 WO 2017206837 A1 WO2017206837 A1 WO 2017206837A1 CN 2017086336 W CN2017086336 W CN 2017086336W WO 2017206837 A1 WO2017206837 A1 WO 2017206837A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
reprogramming
combination
hiendopcs
bay
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/086336
Other languages
English (en)
French (fr)
Inventor
王韫芳
王术勇
张文成
覃金华
王璇
常铭洋
闫舫
裴雪涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute OF TRANSFUSION MEDICINE ACADEMY OF MILITARY MEDICAL SCIENCES PEOPLE'S LIBRATION ARMY OF CHINA
Original Assignee
Institute OF TRANSFUSION MEDICINE ACADEMY OF MILITARY MEDICAL SCIENCES PEOPLE'S LIBRATION ARMY OF CHINA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute OF TRANSFUSION MEDICINE ACADEMY OF MILITARY MEDICAL SCIENCES PEOPLE'S LIBRATION ARMY OF CHINA filed Critical Institute OF TRANSFUSION MEDICINE ACADEMY OF MILITARY MEDICAL SCIENCES PEOPLE'S LIBRATION ARMY OF CHINA
Priority to JP2018563588A priority Critical patent/JP7005528B2/ja
Priority to EP17805782.4A priority patent/EP3483262B1/en
Priority to US16/306,646 priority patent/US11149253B2/en
Publication of WO2017206837A1 publication Critical patent/WO2017206837A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/065—Modulators of histone acetylation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/23—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the gastro-intestinal tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the invention belongs to the field of cell engineering technology and relates to a technology for reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells, including specific small molecule compound combinations, reprogramming kits, and heavy weights used in reprogramming.
  • Stem cell technology has been rated as the most promising technology in the life sciences field in the 21st century by "Nature” and “Science” magazines. It is a brand-new medical treatment that overcomes the major diseases that traditional medical treatment methods cannot reach. technology. Stem cells have the potential of self-renewal and multi-directional differentiation, and have broad application prospects in the fields of organ tissue regeneration, disease model establishment, developmental biology and drug research and development. In the past 10 years, stem cell theory and technology research has achieved rapid development, and it is expected to become a new disease treatment method, promote the paradigm shift of medical treatment, and lead a new medical revolution. Stem cells have a variety of different classification methods. From a developmental perspective, stem cells are roughly classified into two categories: Embryonic stem cells (ESCs) and adult stem cells.
  • ESCs Embryonic stem cells
  • adult stem cells are roughly classified into two categories: Embryonic stem cells (ESCs) and adult stem cells.
  • ESCs are derived from the inner cell mass (ICM) of the blastocyst stage and are a class of pluripotent stem cells with infinite self-renewal and differentiation into all cell types of the human body.
  • ICM inner cell mass
  • hESCs Thomson established human embryonic stem cell lines
  • ESCs have produced a variety of functional cells, including cardiomyocytes, nerve cells, blood cells, and liver cells [Murry CE, Keller G. Differentiation of Embryonic Stem Cells to  Clinically Relevant Populations: Lessons from Embryonic Development. Cell. 132: 661-80.].
  • ESCs faces international ethical controversy, and ESCs-derived cells will produce immune rejection after transplantation, and undifferentiated ESCs will form teratoma and many other problems, thus greatly limiting their clinical application. .
  • iPSCs induced Pluripotent Stem Cells
  • OCT4, SOX2, KLF4, and c-MYC induced Pluripotent Stem Cells
  • iPSCs have a milestone significance: First, it breaks through the classical epigenetic panoramic mode, greatly enriching the theory of cell fate conversion, leading the paradigm shift of cell fate research and stem cell research, and the basis for regenerative medicine. Research and clinical applications have opened up broad prospects. Second, iPSCs have the potential for unlimited self-renewal of ESCs and differentiation into all cell types in the body, while avoiding ethical controversy in the study and application of ESCs. Thirdly, since fibroblasts can be taken from the patient's own body, the immune rejection problem caused by the transplantation of terminal cells derived from the differentiation of ESCs into the body is also avoided. Finally, patient-derived iPSCs can establish individual-specific disease cell models and individualized drug screening platforms.
  • iPSCs pluripotent stem cells
  • iPSCs have many advantages and are very promising cell types, it is still difficult to avoid the risk of teratoma due to the similar pluripotency of ESCs.
  • iPSCs are differentiated into functional cells. The problem of random differentiation leads to a variety of unintended cell types that pose a safety risk for large-scale clinical applications.
  • lineage reprogramming technology based on the development of iPSCs technology and principle has received increasing attention.
  • Lineage reprogramming mainly uses lineage specific transcriptional factors rather than pluripotent transcription factors (OSKM) to overexpress the starting cells, mainly skin fibroblasts or blood cells, and then The starting cells are directly converted into cells of interest.
  • OSKM pluripotent transcription factors
  • pedigree reprogramming avoids the teratogenic risk of iPSCs because it does not pass through the pluripotency stage of iPSCs.
  • lineage reprogramming can directly obtain terminal mature functional cells depending on the transcription factors used and culture conditions.
  • Adult stem cells with expansion ability, and the obtained target cell types are relatively uniform; again, lineage reprogramming also has the advantages of iPSCs without immune rejection and individualization, thus showing more in stem cell and regenerative medicine research and disease treatment.
  • pancreatic exocrine cells The lineage reprogramming of pancreatic exocrine cells to endocrine beta cells in the same germ layer was first realized in 2008 [Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.
  • fibroblasts have been transformed into various types of neurons, neural stem cells, etc.; in the mesoderm, cardiomyocytes, cardiac progenitor cells, hematopoietic stem/progenitor cells, etc. have been obtained; islet cells and hepatocytes have been obtained in the endoderm. , liver stem cells, etc. Chinese scientists have made a series of breakthroughs in this field, especially in the field of lineage reprogramming of liver cells.
  • the domestic ⁇ successfully converted mouse fibroblasts into hepatocytes using a combination of hepatocyte-specific transcription factors for the first time [Huang P, He Z, Ji S, Sun H, Xiang D, Liu C , et al.Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.Nature.2011;475:386-9.].
  • Hu Yiping's laboratory used transcription factor combinations to obtain mouse liver stem cells with self-renewal and differentiation into hepatocytes and biliary cells [Yu B, He ZY, You P, Han QW, Xiang D, Chen F, et al .Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.
  • small molecule compounds Compared with transcription factors, small molecule compounds have obvious advantages in many aspects. Small molecule compounds can be freely permeable to cells, easy to synthesize, cost-effective, non-immunogenic, acting on protein levels without integration with genomes, and can be standardized and scaled. Production. It is easier to achieve protein regulation intensity and spatiotemporal control by changing the concentration and duration of action of small molecules. In recent years, many studies have been devoted to screening small molecules for improving the efficiency of iPSCs production, shortening the generation time of iPSCs, and reducing the use of transcription factors. The ultimate goal is to completely replace the classical Yamanaka four factors with small molecules. Avoid integration of the virus in host cells [Li W, Li K, Wei W, Ding S.
  • Endoderm stem/progenitor cells are the source of development of internal organs such as liver, pancreas, stomach, and intestine, and endodermal stem/progenitor cells are obtained, and a large number of amplifications are performed, and then induced to differentiate, and a large number of functional livers are obtained.
  • Cells, pancreatic cells or intestinal epithelial cells will have great significance for the treatment of various types of endoderm organ diseases. As we all know, China is a big country with liver diseases. There are currently 97 million hepatitis B virus carriers and 10 million hepatitis C virus carriers. In recent years, the development trend of alcoholic fatty liver disease and nonalcoholic fatty liver disease in China has also increased significantly.
  • liver diseases Including drug-induced liver damage, immune liver disease, etc., all of which seriously affect the health of our nationals [Wang FS, Fan JG, Zhang Z, Gao B, Wang HY. The global burden of liver disease: the major impact of China. Hepatology.2014 ;60:2099-108.].
  • Various liver diseases if lack of timely and effective treatment, will gradually progress to the stage of end-stage liver disease (ESLD) represented by liver cirrhosis, liver failure and liver cancer.
  • ESLD end-stage liver disease
  • the treatment of artificial liver is not mature enough to be carried out on a large scale.
  • the medical treatment can only adopt a conservative and palliative approach.
  • ESLD orthotopic liver transplantation
  • T1DM type 1 diabetes
  • T1DM type 1 diabetes
  • islet ⁇ cells and current research has made remarkable progress
  • the lack of exogenous islets or beta cells severely hampers the application of this treatment.
  • endodermal stem/progenitor cells can be obtained and extensively expanded and then induced to differentiate, a large number of functional hepatocytes, pancreatic beta cells or intestinal epithelial cells will be treated for cells of liver disease, cells of diabetes Treatment and even the treatment of intestinal diseases bring great hope.
  • endodermal stem/progenitor cells are obtained from pluripotent stem cell ESCs or iPSCs and induced to differentiate into the liver and pancreas [Sneddon JB, Borowiak M, Melton DA. Self-renewal of embryonic-stem-cell -derived progenitors by organ-matched mesenchyme.Nature.2012;491:765-8.Cheng X,Ying L,Lu L,Galvao AM,Mills JA,Lin HC,et al.Self-renewing endodermal progenitor lines generated from human pluripotent Stem cells. Cell Stem Cell.
  • fibroblasts are relatively easy to acquire than other adult cells.
  • Skin biopsy is available and therefore a major advantage for clinical applications, however, for the desired cells of interest such as endodermal progenitor cells, hepatocytes or pancreatic cells, there is an epigenetic barrier starting from fibroblasts.
  • Fibroblasts are derived from the mesoderm, while the endodermal progenitor cells of the target belong to the endoderm. The transition from mesoderm to endoderm is a great obstacle, especially for small molecules with milder effects. .
  • fibroblasts belong to mesenchymal cells in histological classification, while endodermal progenitor cells belong to epithelial cells.
  • the transition from fibroblasts to endoderm progenitors requires a process of interstitial epithelial transition (MET).
  • MET has been proven to be the first important obstacle to break through in the production of iPSCs [Li R, Liang J, Ni S, Zhou T, Qing X, Li H, et al.
  • iPSCs Li R, Liang J, Ni S, Zhou T, Qing X, Li H, et al.
  • a mesenchymal-to-epithelial transition initiates and is required For the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell. 2010; 7: 51-63.]
  • the same MET is also an important obstacle to pedigree reprogramming.
  • trophoblast cells can secrete a variety of cytokines, soluble extracellular matrix or microRNA, and in close contact with the cultured cells to provide a suitable extracellular microenvironment for cell growth and survival.
  • Trophoblast cells are often necessary for stem/progenitor cell culture and reprogramming, and the most commonly used trophoblast cells, whether reprogramming or lineage reprogramming, are mouse embryonic fibroblasts (MEFs), human foreskin. Fibroblasts (HFFs) or mesenchymal stem cells (MSCs), etc., which provide only a basic stem cell growth environment and do not have lineage-specific support and induction.
  • MEFs mouse embryonic fibroblasts
  • HFFs human foreskin.
  • MSCs mesenchymal stem cells
  • dry/progenitor cells are “stem cells” or “progenitor cells”, which are referred to as the same type of cells, and are collectively referred to as “dry/progenitor cells” because of their inconsistent naming in the industry.
  • the invention provides a combination of small molecule compounds capable of reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells selected from the group consisting of a TGF- ⁇ signaling pathway, an epigenetic modifier, and a calcium channel agonist And functional groups such as metabolic pathway regulators; representative combinations of 8 small molecule compounds (8M), including FBP, All or a plurality of compounds including Bay SB or A83 in Bay K 8644 (Bay), Bix01294 ((Bix), SB431542 (SB) or A83-01 (A83), VPA, RG108 (RG), PD0325901, and PS48 combination.
  • 8M small molecule compounds capable of reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells selected from the group consisting of a TGF- ⁇ signaling pathway, an epigenetic modifier, and a calcium channel agonist And functional groups such as metabolic pathway regulators
  • 8M 8 small molecule compounds
  • the combination of 8 small molecule compounds is: SB43154: VPA: PD0325901: RG108: Bix01294: Bay K 8644: PS48: FBP molar ratio is 50:12500:12.5:1:12.5:50:125 : a combination of 87500; or A83: VPA: PD0325901: RG108: Bix01294: Bay K 8644: PS48: FBP molar ratio of 12.5: 12500: 12.5: 1:12.5: 50: 125: 87500;
  • the 8M combination needs to satisfy the RG use concentration of 0.01 to 1 ⁇ M, preferably 0.04 ⁇ M.
  • the small molecule compound that can reprogram the digestive tract-derived epithelial cells into endoderm stem/progenitor cells is a combination comprising four small molecule compounds, such as Bix01294 (Bix), Bay K 8644 (Bay), RG108 (RG). And SB431542 (SB), referred to as BBRS combination, or A83-01 (A83), Bay K 8644 (Bay), RG108 (RG) and SB431542 (SB), referred to as BBRA combination.
  • the concentration of each compound in the small molecule compound combination is SB43152 (1 ⁇ 10 ⁇ M), A83 is (0.4 ⁇ 1 ⁇ M), RG108 is (0.01 ⁇ 1 ⁇ M), Bix01294 is (0.1 ⁇ 2 ⁇ M), Bay K 8644 is (1 to 4 ⁇ M).
  • the BBRS combination is a combination of each compound in a SB:RG:Bix:Bay molar ratio of 50:1:12.5:50
  • the BBRA combination is a combination of each compound according to A83:RG:Bix:Bay molar ratio of 12.5:1:12.5:50.
  • each compound is used at a concentration of SB43152 of 2 ⁇ M, A83 of 0.5 ⁇ M, RG108 of 0.04 M, Bix01294 of 0.5 ⁇ M, and Bay K 8644 of 2 ⁇ M.
  • the small molecule compound combination can reprogram the digestive tract-derived epithelial cells into endoderm stem/progenitor cells with the support of gastric myofibroblasts or small intestinal muscle fibroblasts as trophoblast cells.
  • the invention provides a reprogramming kit capable of reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells, comprising the above-mentioned small molecule compound combination capable of reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells , and instructions for use of the compound.
  • Each compound is packaged separately, or each compound is mixed and packaged in an 8M combination or a BBRS combination or a BBRA combination, and the specification describes the use concentration range or specific concentration of each compound.
  • the reprogramming kit further includes trophoblast cells which are stromal cells derived from the digestive tract, such as gastric myofibroblasts or intestinal muscle fibroblasts, and instructions for use thereof.
  • trophoblast cells which are stromal cells derived from the digestive tract, such as gastric myofibroblasts or intestinal muscle fibroblasts, and instructions for use thereof.
  • the reprogramming kit further comprises a basal medium Advanced DMEM/F12, a cell culture base addition component glutamine (Glutamax) and an antibiotic (SP), and instructions for use thereof, wherein the relative medium DMEM/F12 basal medium, glutamine
  • the amide is used at a concentration of 2 mM (1 ⁇ )
  • the antibiotic is cyan-streptomycin
  • the concentration of the Advanced DMEM/F12 basal medium is 100 U/mL penicillin + 0.1 mg/mL streptomycin, and each substance is packaged or pressed separately.
  • the concentrations used are mixed with the basal medium and packaged.
  • the invention also provides a reprogramming of digestive tract-derived epithelial cells into endoderm stem/progenitor cells Medium.
  • the reprogramming medium is formed by blending the small molecule compound combination with a basal medium and a cell culture base addition component.
  • the optimized reprogramming medium was formulated with 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL penicillin and 0.1 mg/mL streptomycin), 2 ⁇ M SB43154, 0.5 mM in Advanced DMEM/F12. VPA, 0.5 ⁇ M PD0325901, 0.04 ⁇ M RG108, 0.5 ⁇ M Bix01294, 2 ⁇ M Bay K8644, 5 ⁇ M PS48, 3.5 mM FBP.
  • concentrations listed are the concentrations used, ie the concentrations of the components in Advanced DMEM/F12 (solvent).
  • a more preferred reprogramming medium formulation is: Advanced DMEM/F12 containing 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL penicillin and 0.1 mg/mL streptomycin), 1 to 10 ⁇ M of SB43152, 0.01 to 1 ⁇ M of RG108, 0.1 to 2 ⁇ M of Bix01294, and 1 to 4 ⁇ M of Bay K 8644; here most preferably Advanced DMEM/F12 contains 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL penicillin and 0.1 mg) /mL streptomycin), 2 ⁇ M SB43152, 0.04 ⁇ M RG108, 0.5 M Bix01294, 2 ⁇ M Bay K 8644.
  • Advanced DMEM/F12 containing 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL penicillin and 0.1 mg/mL streptomycin), 0.4-1 ⁇ M A83-01, 0.01 to 1 ⁇ M RG108, 0.1 to 2 ⁇ M Bix01294, 1 to 4 ⁇ M Bay K 8644; here most preferably Advanced DMEM/F12 contains 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL) Penicillin and 0.1 mg/mL streptomycin), 0.5 ⁇ M A83-01, 0.04 ⁇ M RG108, 0.5 M Bix01294, 2 ⁇ M Bay K 8644.
  • the present invention also provides the use of the above small molecule test compound combination or kit or reprogramming medium, which is the basis of trophoblasts in gastrointestinal stromal cells using the above small molecule compound combination or kit or reprogramming medium.
  • the digestive tract-derived epithelial cells are reprogrammed into endoderm stem/progenitor cells.
  • the reprogramming method for reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells comprises the following steps:
  • a primary isolated digestive tract-derived epithelial cell is used as a starting cell to expand and culture the digestive tract-derived epithelial cells;
  • step 3 adding the trophoblast cells prepared in the step 2) to the step 1) expanding the cultured digestive tract-derived epithelial cells, and continuing co-cultivation overnight;
  • the starting cells in the step 1) are gastrointestinal-derived epithelial cells, including gastric and duodenal epithelial cells.
  • gastric epithelial cells are more easily obtained, and therefore are preferably gastric epithelial cells (hGECs), and particularly preferably gastric epithelial cells (hGECs) positive for NCAM (neural cell adhesion molecule).
  • hGECs gastric epithelial cells
  • NCAM-positive gastric epithelial cells are used as starting cells, and cultured in Kubota medium at 37 ° C in a 5% CO 2 incubator for 5 days.
  • the trophoblast cells in the step 2) are stromal cells derived from the digestive tract, including gastric myofibroblasts or small intestinal muscle fibroblasts, preferably human gastric myofibroblasts (aGSEMFs).
  • gastric myofibroblasts or small intestinal muscle fibroblasts preferably human gastric myofibroblasts (aGSEMFs).
  • aGSEMFs Human gastric myofibroblasts
  • mitomycin-C preferably treated with mitomycin-C for 2-3 hours, the cells are washed with PBS, and the cells are digested with TrypLE enzyme.
  • Step 3) Preferably, the trophoblast cells prepared in step 2) are added to the step 1) cultured in 5 days of digestive tract-derived epithelial cells at a density of 1 to 3 ⁇ 10 5 per square centimeter at 37 ° C, 5% CO 2 In the incubator overnight (12-16 hours).
  • the reprogramming medium in the step 4) is a mixture of a small molecule compound combination and a basal medium and a cell culture base addition component, the specific composition of which is as described above.
  • a method of reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells comprises the following steps:
  • the primary isolated digestive tract-derived epithelial cells were used as the starting cells, and cultured for 5 days in a serum-free Kubota's medium at 37 ° C in a 5% CO 2 incubator;
  • the trophoblast cells were treated with mitomycin-C for 2-3 hours, the cells were washed with PBS, and the cells were digested with TrypLE enzyme;
  • step 3 Add the trophoblast cells prepared in step 2) at a density of 1 to 3 ⁇ 10 5 per square centimeter in the step 1) to culture the starting epithelial cells for 4-5 days at 37 ° C, 5% CO 2 conditions. Cultivate overnight (12-16 hours);
  • the medium was changed to reprogramming medium, and the medium was changed every 2-3 days. After 7-15 days of culture, clones of induced human endodermal progenitor cells were obtained.
  • Endoderm stem/progenitor cells obtained by the above reprogramming method are also within the scope of the invention.
  • the invention also provides a method for passage of endoderm stem/progenitor cells (hiEndoPCs), comprising the steps of:
  • hiEndoPCs endoderm stem/progenitor cells clones and divide them into small pieces at a ratio of about 1:3-, placing them in FN+AWF, CT+AWF or Feeder [trophoblast cells, Mitomycin-C (10 ⁇ g/mL) treated adult gastric myofibroblasts (aGSEMFs) + A medium was subcultured in a 37 ° C, 5% CO 2 incubator to obtain passaged endoderm stem/progenitor cells. .
  • endodermal stem/progenitor cells hiEndoPCs
  • passaged endoderm stem/progenitor cells obtained above for inducing differentiation into liver cells, pancreatic beta cells and intestinal cells is also within the scope of the invention.
  • the present invention provides a combination of small molecule compounds, reprogramming kits, reprogramming medium, and reprogramming of digestive tract-derived epithelial cells into endoderm stem/ancestors, which can reprogram gut-derived epithelial cells into endoderm stem/progenitor cells.
  • Cell method human gastric epithelial cells (hGECs) are used as starting cells, and adult gastric muscle fibroblasts (aGSEMFs) are obtained as trophoblasts from human gastric tissue muscle layer, and combined with small molecule compounds under the support of trophoblast cells.
  • the human gastric epithelial cell lineage is reprogrammed into endoderm stem/progenitor cells and can be further subcultured and expanded.
  • endoderm stem/progenitor cells obtained by the present invention or their passage cells in combination with the corresponding induced differentiation system, mature liver cells, pancreatic ⁇ cells and intestinal cells can be obtained, and cell therapy for liver diseases, diabetes and intestinal diseases is expected. Providing an ideal source of seeds with broad application prospects.
  • Figure 1 is a technical roadmap of the present invention after determining the starting cells, the initial small molecules, and the trophoblast cells;
  • Figure 2 is a morphological view of gastric epithelial cells (hGECs) and duodenal epithelial cells (hDECs) cultured for 4-5 days under a phase contrast microscope (scale: 100 ⁇ m);
  • Figure 3 is a diagram showing the morphology of gastric myofibroblasts subcultured under a phase contrast microscope (scale: 100 ⁇ m);
  • Figure 4 is a graph showing the expression of gastric-specific and endodermal progenitor-specific proteins in gastric epithelial cells
  • Figure 5 is a dynamic process cell morphology map of human gastric epithelial cells (hGECs) reprogrammed to endodermal progenitor cells (hiEndoPCs) (scale: 100 ⁇ m);
  • hGECs human gastric epithelial cells
  • hiEndoPCs endodermal progenitor cells
  • Figure 6 is a diagram showing the morphology of human duodenal epithelial cells (hDECs) and their derived endoderm progenitor cells (hiEndoPCs) (scale: 100 ⁇ m);
  • Figure 7 is a cell morphology diagram of a reprogramming process starting from human gastric epithelial cells (hGECs) (A) or gastrointestinal myofibroblasts (GSEMFs) (B), respectively (scale: 100 ⁇ m);
  • hGECs human gastric epithelial cells
  • GEMFs gastrointestinal myofibroblasts
  • Figure 8 is a panoramic scan of the clonal morphology, showing different clones on day 7 (A) and day 15 (B) (outline outline with dashed lines), C, D, E, F are specific forms of representative clones;
  • Figure 9 is a schematic diagram of a grid of clones of different sizes in the calculation of cloned area (scale: 250 ⁇ m);
  • Figure 10 is a histogram of the efficiency of cloning formation of small molecule compounds (8M) with various trophoblast cells, extracellular matrix or conditioned medium (reprogramming efficiency under different support conditions);
  • Figure 11 is a cell morphology diagram supported by MSC, MEFs and aGSEMF-CM (scale: 100 ⁇ m);
  • Figure 12 is a histogram of the formation efficiency of clones of 8 small molecule compounds (8M) subtracted one by one;
  • Figure 13 is a bar graph showing the cloning formation efficiency of four small molecule compounds of Bix01294 (Bix), Bay K 8644 (Bay), RG108 (RG) and SB431542 (SB);
  • Figure 14 is a cloning pattern of four small molecules of Bix01294 (Bix), Bay K 8644 (Bay), RG108 (RG) and SB431542 (SB) subtracted one by one (scale: 100 ⁇ m);
  • Figure 15A is a histogram of clone formation efficiency based on SB431542 and in combination with other three small molecule compounds;
  • Figure 15B is a histogram of clone formation efficiency based on A83-01 and in combination with other three small molecule compounds;
  • Figure 16 is a cloning pattern diagram based on SB431542 and combined with other three small molecule compounds
  • Figure 17 is a flow chart of the optimization process of the small molecule compound
  • Figure 18 is a preliminary mapping staining of the starting cells by fluorescence in situ hybridization (FISH) (A: FISH staining after reprogramming of male-derived hGECs; B: FISH staining after reprogramming of female-derived hGECs);
  • FISH fluorescence in situ hybridization
  • Figure 19 is a diagram showing immunofluorescence staining of gastric antrum tissue CD56 (NCAM) (scale: 50 ⁇ m);
  • Figure 20 is a schematic diagram showing the NCAM flow sorting of the starting human human gastric epithelial cells (hGECs) and the reprogramming of the corresponding subpopulations (the lines outline the outlines of different clones);
  • Figure 21 is a flow level detection map of endodermal progenitor-specific proteins in induced endodermal progenitor cells (hiEndoPCs);
  • Figure 22 is a staining diagram showing the expression of gastric-specific and endodermal progenitor-specific protein expression in induced endodermal progenitor cells (hiEndoPCs);
  • Figure 23 is a bar graph showing the results of endodermal progenitor cell marker gene expression analysis in induced endodermal progenitor cells (hiEndoPCs), (A) and gastric-specific gene expression analysis results (B);
  • Figure 24 shows the DNA methylation chip showing changes in epigenetic changes in whole genome DNA before and after reprogramming (A: cluster analysis of hGECs and hiEndoPCs; B: GO analysis of hiEndoPCs compared to hGECs hypomethylated genes);
  • Figure 25A is a volcano plot showing the number of genes whose promoter methylation levels change in hiEndoPCs compared to hGECs; B is a comparison of methylation levels of hiEndoPCs and hGECs in the promoter regions of FOXA2 and GATA4 genes;
  • Figure 26 is a correlation analysis of RNA deep sequencing, showing the relationship between hiEdoPs and ESC-derived DE, PGT, and PFG;
  • Figure 27 is a PCA analysis chart showing hGECs, hDECs, hiEndoPCs derived from hGECs, and hDECs. The positional relationship of the source hiEndoPCs, ESCs-derived DE, and GSEMFs on the whole gene expression profile;
  • Figure 28 is a microscopic morphology diagram of hGECs and hiEndoPCs, (scale: 2 ⁇ m (A-D), 100 nm (E));
  • Figure 29 shows the dynamics of hiEndoPCs during reprogramming.
  • A is the axial map of the period in the reprogramming process of hiEndoPCs
  • B is the graph of the proliferation rate in different periods
  • C is the dynamics of the cell morphology in Phase II and Phase III. Change map
  • Figure 30 is a cell morphology diagram of the hiEndoPCs after passage in different support environments (scale: 100 ⁇ m);
  • Figure 31 is a cell morphology diagram of hiEndoPCs during passage (scale: 200 ⁇ m);
  • Figure 32 is a histogram of the number of passages of hiEndoPCs and the number of cells per generation
  • Figure 33 shows the results of differentiation of hiEndoPCs into the pancreas
  • A is a three-dimensional morphological map of hiEndoPC-Pans
  • B is a histioPC-Pans DTZ staining map
  • C and D are hiEndoPC-Pans pancreas-specific protein expression maps (scale: 50 ⁇ m); Pancreatic specific gene expression map of hiEndoPC-Pans;
  • F is the insulin release response map of hiEndoPC-Pans;
  • Figure 34 shows the results of differentiation of hiEndoPCs into the intestine, A is the morphological map of hiEndoPC-Ints; B is the intestinal-specific gene expression map of hiEndoPC-Ints; C is the intestinal specific protein staining map of hiEndoPC-Ints (scale: 50 ⁇ m) );
  • Figure 35 shows the results of induced differentiation of hiEndoPCs into the liver,
  • A is the liver-specific gene expression map of hiEndoPC-Heps;
  • B is the AFP and CK18 staining map of hiEndoPC-Heps (scale: 50 ⁇ M);
  • C is the ALB and AFP of hiEndoPC-Heps Flow level detection map;
  • Figure 36 is a bar graph showing the gene expression level of hiEndoPCs induced differentiation into thyroid (A) and lung (B).
  • the technical route of the present invention (shown in FIG. 1) is: obtaining human gastric epithelial cells (hGECs) as starting cells, separating from the muscle layer of human gastric tissue. Cultured the digestive tract myofibroblasts (aGSEMFs) as a trophoblast, screened appropriate small molecule combinations and reprogrammed the human gastric epithelial cell lineage to endoderm stem/progenitor cells with the support of trophoblast cells.
  • hGECs human gastric epithelial cells
  • aGSEMFs digestive tract myofibroblasts
  • hGECs human gastric-derived epithelial cells
  • Eight preliminary combinations of small molecules were determined by preliminary screening: SB431542+VPA+PD0325901+RG108+Bix01294+BayK8644+PS48+FBP
  • Human gastric epithelial cells (hGECs) can be reprogrammed into endodermal progenitor cells (hiEndoPCs)-like clones under conditions of human gastric-derived myofibroblasts (GSEMFs) as a trophoblast.
  • GSEMFs human gastric-derived myofibroblasts
  • trophoblast cells human gastric myofibroblasts (GSEMFs)
  • GEMFs human gastric myofibroblasts
  • the optimal reprogramming system is BBRS+GSEMFs, in which the efficiency of reprogramming is 4%-6%;
  • endodermal progenitor cells hiEndoPCs
  • hiEndoPCs endodermal progenitor cells
  • hiEndoPCs induced endoderm progenitor cells
  • Endodermal progenitor cells hiEndoPCs
  • Other dry/progenitor cell characteristic proteins include CXCR4, EPCAM, LGR5, CK19 and gastric epithelial-specific markers M ⁇ C6 and GASTRIN.
  • hiEndoPCs In the expression of hiEndoPCs, it was found that the endoderm-characterized genes were significantly up-regulated in hiEndoPCs compared with hGECs, while the gastric-specific genes were not expressed, which confirmed the successful reprogramming of hGECs to endoderm progenitor cells. Epigenetic analysis also showed significant changes in cells after reprogramming, with molecular features of endodermal progenitor cells. The precise development of the developmental stage of hiEndoPCs revealed that hiEndoPCs were between the two endoderm developmental stages of the original digestive tract (PGT) and the posterior intestine (PFG) of hESCs, and closer to PFG.
  • PTT endoderm developmental stages of the original digestive tract
  • PFG posterior intestine
  • hiEndoPCs have the characteristics of microscopic levels of stem cells and can be passaged 4-6 times. Although compared to ESCs or iPSCs, hiEndoPCs the limited expansion potential, however, starting at 106 cells as a starting point and reprogramming through 4-6 generations amplification about 109 cells can be obtained, so that a sufficient number of cell therapy And within a limited number of passages, the genome of the cell is relatively stable, safer, and more conducive to future cell therapy.
  • endodermal progenitor cells have the potential to differentiate into endoderm organs such as pancreas, liver, intestine, lung, and thyroid gland during development
  • hiEndoPCs are also found to form functional pancreatic beta cells, hepatocytes, and intestines using corresponding induced differentiation conditions. The ability of cells, lung cells and thyroid cells.
  • the percentage concentration is mass/mass (W/W, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, unless otherwise specified). Percent concentration in units of mL/100 mL).
  • Example 1 Establishment of a system for inducing conversion of human gastric epithelial cells to endoderm progenitor cells by small molecule compounds
  • the target cells for reprogramming of the present invention are endodermal progenitor cells, and the liver, pancreas, biliary tract, stomach, intestine, etc. are derived from endoderm from a developmental point of view, and are homologous to endodermal progenitor cells, and epigenetic similarity
  • the reprogramming disorder is relatively small, making it a good starting cell type.
  • gastrointestinal surgery such as major gastrectomy or gastric cancer resection, some normal gastric or duodenal tissues are inevitably abandoned, and gastric ulcer or duodenal ulcer is cured by gastroscopy.
  • the normal stomach or duodenal tissue is also available for follow-up, so the source of the starting cells is also feasible.
  • the present invention establishes a culture system of gastric epithelial cells and trophoblast cells, and then performs reprogramming of endodermal progenitor cells using a total of eight small molecules selected from the four major classes and using the digestive tract myofibroblasts as a trophoblast. Under these conditions, endodermal stem/progenitor-like clones were successfully produced in gastric epithelial or duodenal epithelial cells. After the reprogramming was successful, the system was further optimized to find the optimal combination of reprogramming small molecules and the smallest combination of small molecules. Finally, the starting cells were grouped to identify a subset of the starting cells that were successfully reprogrammed.
  • the liver, pancreas, gastrointestinal and other internal organs are derived from endodermal progenitor cells, which are all homologous in development, and thus are closer to each other than other germ layer-derived cells.
  • the kinship more epigenetic similarities.
  • they are all epithelial tissue type cells, and do not need to undergo MET barriers during the conversion process, so in theory, cell type conversion between them is relatively easy.
  • gastrointestinal tissue cells are rapidly renewed, a large number of cells are actively proliferating, and proliferating cells or progenitor cells are more easily reprogrammed.
  • the GI-tract is a channel that connects various organs of the internal organs.
  • GI-tract-derived human gastric epithelial cells are used as the starting cells to reprogram endodermal progenitor cells, which has the advantage of apparent homology compared with fibroblasts.
  • Epithelial sources sexual advantage, the advantages of proliferating progenitor cells and also the feasibility of clinical operation.
  • TGF- ⁇ signaling pathway inhibitor SB431542 (SB) or A83-01 (A83)
  • TGF- ⁇ signaling pathway inhibitor SB or A83
  • SB or A83 can replace Sox2, and combine with Oct4, Klf4, c-Myc to produce iPSCs, another Is the MAPK/ERK signaling pathway inhibitor PD0325901 (PD), and the combination of PD and SB (or A83) can increase the reprogramming efficiency by 100. Multiple times, and greatly shorten the reprogramming time;
  • VPA histone deacetylase inhibitor
  • VPA can even replace Klf4
  • Sox2 can promote the formation of iPSCs, histone methyltransferase inhibition
  • the agent Bix01294 (BIX) and the DNA methyltransferase inhibitor RG108 (RG) can significantly improve the reprogramming efficiency;
  • Metabolic pathway regulator phosphoinositide-dependent protein kinase 1 agonist PS48, phosphofructokinase agonist FBP, which facilitates the energy metabolism of mature cell oxidative phosphorylation to the stem cell anaerobic glycolysis pathway, thereby improving Reprogramming efficiency.
  • trophoblast cells in order to promote the production and maintenance of endodermal progenitor cells, digestive tract myofibroblasts closely related to endoderm organs were selected as trophoblast cells, which will pass through the early stage of development. The role of secretion affects the development of endoderm organs and supports the expansion of endoderm progenitor cells.
  • Stomach and duodenal tissues from surgical or biopsy are provided by the General Hospital of the Chinese People's Liberation Army and General Surgery of 307 Hospital.
  • the aborted fetal tissue is provided by the General Hospital of the People's Liberation Army. All patients providing the organization are informed and signed informed consent. Use the General Hospital of the People's Liberation Army and the 307 Hospital Ethics Committee.
  • Laser confocal microscopy (Zeiss), constant temperature water bath (long wind), cryogenic centrifuge (Eppendorf), inverted phase contrast microscope (Leica), surgical instruments (surgical handle, blade, ophthalmic straight, curved, scissors), laser Confocal small dish (NEST).
  • KM medium a bag of RPMI 1640 (Gibco) powder dissolved in 1 L of deionized water, adding 1 ⁇ cyanine-streptomycin, 10 -9 M Zinc SuLfate heptahydrate (zinc sulfate heptahydrate, Sigma) ), 0.54g Nicotinamide (nicotinamide, Sigma), 5mg InsuLin (insulin, Sigma), 10 -6 M hydrocortisone (hydrocortisone, Sigma), 2g NaHCO 3 (Sigma), 5 ⁇ 10 -5 M beta-mercaptoethanol ( ⁇ -mercaptoethanol, Sigma), 30 nM Selenium (selenium, Sigma), free fatty acid (Sigma), 10 ⁇ g/mL High density lipoprotein (Sigma), 1 g Bovine Serum Albumin (Born Serum Albumin, available from Gibco) ), 5 mg Transferrin (transferrin, Sigma), 2 mM Glutamax
  • Mg 2+ PBS preparation use 1L of deionized water to dissolve 0.24g KH 2 PO 4 , 8.0g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 , can be added according to the volume of the preparation Reagents. After the preparation is completed, the 0.45 ⁇ M filter is filtered and then autoclaved or directly filtered through a 0.22 filter.
  • Antibody the company Item number Primary species Dilution ratio CXCR4 (C-X-C motif chemokine receptor 4)
  • Gastric epithelial cells can be purchased directly; the gastric antrum, pylorus or duodenal mucosal epithelial cells can also be obtained by isolation and culture as follows:
  • step 5 Repeat step 5 until no significant chunks of tissue are terminated.
  • pre-cooled PBS was added to the collected supernatant, centrifuged at 4 ° C, 1200 rpm (revolution / minute) for 5 minutes, washed 3-4 times, fully remove the residual digestive enzymes in the cells.
  • the mucosal layer and the muscular layer were obtained by separating the clinically obtained gastric or duodenal tissue.
  • the mucosal layer was used to separate the gastric or duodenal epithelial cells
  • the muscular layer was used to separate the trophoblast cells.
  • Fibroblasts According to the method of tissue separation of the stomach or duodenal epithelium in the experimental procedure, cells which are close to the crypt are usually obtained.
  • Human serum epithelial cells (hGECs) and duodenal epithelial cells (hDECs) were cultured in a typical dance-like epithelial morphology using serum-free epithelial cell culture medium Kubota's medium. 2)).
  • Myofibroblasts can be purchased directly; gastrointestinal myofibroblasts can also be isolated and cultured by the following adherence methods:
  • the stomach or duodenum muscle layer obtained as described above is fully mashed into small tissue blocks using a surgical blade.
  • the tissue fragments were wetted in a sugar medium or KM medium, and the tissue fragments were evenly spread in a 10 cm culture dish using a 1 ml small dropper, and placed in a 37 ° C incubator for 30 minutes. The remaining stromal cells were isolated and cultured as described above.
  • the stromal cell growth medium was added along the edge of the culture dish, and the cells were cultured in an incubator at 37 ° C, 5% CO 2 .
  • fetal fibroblasts fetal fibroblasts
  • aGSEMFs adult gastric myofibroblasts
  • fISEMFs fetal intestinal myofibroblasts
  • fDCs fetal transverse stromal cells
  • the obtained gastric or duodenal muscular layer was mechanically mashed into small pieces and the myofibroblasts (GSEMFs) were isolated and cultured using tissue adherence method.
  • the tissue is attached for about 4-7 days, and it can be seen that the fusiform cells climb out of the tissue block and can be passaged for 10-14 days.
  • the subcultured myofibroblasts are typically long spindle-like fibroblasts. (As shown in Figure 3).
  • hGECs human gastric epithelial cells
  • the corresponding secondary antibody was incubated for 1 hour at room temperature in the dark, and washed 2-3 times with PBS.
  • DAPI was incubated for 15 minutes at room temperature in the dark.
  • RESULTS As shown in Figure 4, immunofluorescence staining revealed that human gastric epithelial cells (hGECs) underexpressed endodermal progenitor cells markers CK19, FOXA2, CXCR4, SOX9, LGR5, EPCAM, etc., while highly expressing the gastric characteristic markers MUC6 and GAST.
  • hGECs human gastric epithelial cells
  • the first part, "Isolation, culture and phenotypic identification of gastric epithelial cells” successfully established an isolation culture system for gastric epithelial cells and myofibroblasts in gastric antrum or pylorus.
  • Human gastric epithelial cells (hGECs) cultured in vitro showed typical epithelial-like appearance. form. Since the cells in the crypt are usually primitive (compared to the type of mature gastric cells), a certain degree of proliferation can be achieved.
  • hGECs Human gastric epithelial cells express high expression of gastric-specific markers and low expression of early progenitor early progenitor markers, consistent with our previous speculation, due to their partial expression of some markers of endoderm progenitor cells, inward Reprogramming disorders during reprogramming of germ layer progenitors are relatively small and are therefore a good source of starting cells for transformation to endoderm progenitor cells.
  • the characteristic markers of the gastric epithelium of human gastric epithelial cells (hGECs) must be lost during reprogramming. The source and culture problems of the starting cells were solved, and the characteristics were also preliminarily determined. The next step was to reprogram the endodermal progenitor cells with the starting cells.
  • small molecule compounds and trophoblast cells induce the conversion of gastric epithelial cells to endoderm progenitor cells
  • hGECs Human gastric epithelial cells
  • aGSEMFs adult gastric myofibroblasts
  • Advanced DMEM/F-12 medium Advanced RPMI 1640 medium, NEAA (non-essential amino acids), TrypLE digestive enzymes, glutamine (Glutamax), Dispase (discrete enzyme) were purchased from Gibco, cyan-streptomycin and Mitomycin-C (Sigma).
  • Ultra concentration means the concentration of each compound in a solvent of Advanced DMEM/F-12 medium.
  • the concentration of each component in the formulation is its concentration in Advanced DMEM/F-12 medium.
  • hGECs human gastric epithelial cells
  • hDECs duodenal epithelial cells
  • hiEndoPCs induced endoderm progenitor cells
  • the specific method includes the following steps:
  • hGECs Primary isolated human gastric epithelial cells
  • hDECs duodenal epithelial cells
  • Kubta medium was used (prepared by reference to literature, Kubota, H) ., and Reid, LM (2000).
  • Clonogenic hepatoblasts common precursors for hepatocytic and biliary lineages, and lack lacking classical major histocompatibility complex class I antigen. Proc. Natl. Acad. Sci. USA 97, 12132–12137), at 37 ° C, The starting cells were expanded by culturing for 4-5 days under a 5% CO 2 incubator condition.
  • trophoblast cells Preparation of trophoblast cells: treatment of trophoblast cells of adult gastric myofibroblasts (aGSEMFs) with mitomycin-C (10 ⁇ g/mL for the purpose of mitochondrial cells losing mitosis) Tissue matrix layer is obtained, cultured and expanded, frozen in large amount, and resuscitated in advance for 2-3 hours, washed 3-4 times with PBS, TrypLE enzyme digests cells, and then washes cells;
  • aGSEMFs adult gastric myofibroblasts
  • mitomycin-C 10 ⁇ g/mL for the purpose of mitochondrial cells losing mitosis
  • hGECs human gastric epithelial cells
  • hDECs duodenal epithelial cells
  • the treated gastric myofibroblasts are at a suitable density (density is generally 1 to 3 ⁇ 10 5 Added to human gastric epithelial cells (hGECs) or duodenal epithelial cells (hDECs) in culture overnight at 37 ° C in a 5% CO 2 incubator (12-16 hours);
  • Reprogramming culture Change to 8M reprogramming medium on the second day after adding gastric myofibroblasts, change the medium every 2-3 days, continuously observe, generally culture for 1 week or more (7- 15 days), induced endodermal progenitor cells (hiEndoPCs) were obtained.
  • hGECs human gastric epithelial cells
  • hiEndoPCs induced endodermal progenitor cells
  • hGECs gastric epithelial cells
  • G-hiEndoPCs typical endodermal stem/progenitor cell-like clone
  • hDECs human duodenal epithelial cells
  • GEMFs isolated cultured gastrointestinal myofibroblasts
  • Typical endoderm stem/progenitor cell clones can also be formed (as shown in Figure 6, the left margin is duodenal epithelial cells (hDECs), and the right panel is produced by duodenal epithelial cell reprogramming.
  • Endodermal stem/progenitor cells D-hiEndoPCs
  • morphology is almost similar to G-hiEndoPCs ( Figure 5). Due to limited sources of duodenal tissue, human gastric epithelial cells (hGECs) are often used as reprogramming initiation cells.
  • hGECs human gastric epithelial cells
  • GSEMFs gastrointestinal myofibroblasts
  • hiEndoPCs endodermal progenitor cells
  • Endodermal stem/progenitor cell-like clones could not be formed without the use of small molecule compounds, but only human gastric epithelial cells (hGECs) or gastrointestinal myofibroblasts (GSEMFs) were used as starting cells, respectively, in 8M reprogramming medium. No endodermal stem/progenitor cell-like clones were formed in culture for 15 days (as shown in Figure 7), indicating small molecule compounds (8M), human gastric epithelial cells (hGECs), and gastrointestinal myofibroblasts (GSEMFs). The combination is an indispensable factor in the formation of endodermal progenitor cells (hiEndoPCs).
  • the specific method includes the following steps:
  • the time nodes fuse together to form large clones (B of Figure 8), so the number of clones is less in the calculation, but the total area is increasing. For more objective reaction reprogramming effects, the total number of clones in this experiment is comprehensive.
  • the number of clones and the cloned area under different areas were used as a method for calculating the reprogramming efficiency.
  • the area of the different clones was calculated using the microscope grid line: the area of each microscope microgrid was 0.0625 mm 2 , and the number of grids occupied by the clone was multiplied by 0.0625 mm 2 , which is the area of each clone, and the calculation method of the clone area of different sizes. As shown in Figure 9, the calculation results are shown in Table 4.
  • the second part successfully reprogrammed human gastric epithelial cells (hGECs) into endodermal stem/progenitor cells under the condition of eight small molecules (8M) and adult gastric myofibroblasts (aGSEMFs) as the trophoblasts.
  • 8M small molecules
  • aGSEMFs adult gastric myofibroblasts
  • the stage of hiEndoPCs since the initial significant change that occurs during reprogramming is cell morphology, the appearance of stem/progenitor cell clones is used as an initial judgement for successful reprogramming.
  • Human duodenal epithelial cells (hDECs) can also be reprogrammed into endoderm stem/progenitor cells (hiEndoPCs) using the same method. Small molecule compounds and trophoblast cells are essential elements during reprogramming.
  • hGECs Human gastric epithelial cells
  • fGSEMFs fetal gastric myofibroblasts
  • aGSEMFs adult gastric myofibroblasts
  • fISEMFs fetal intestinal myofibroblasts
  • fDCs fetal transverse stromal cells
  • MSCs Mesenchymal stem cells
  • MEFs mouse embryonic fibroblasts
  • HFFs adult skin fibroblasts
  • Basic reagents Advanced DMEM/F-12 medium, Advanced RPMI 1640 medium, NEAA (non-essential amino acid), and glutamine (Glutamax) were purchased from Gibco; cyan-streptomycin, mitomycin- C was purchased from Sigma; TrypLE, Dispase digestase was purchased from Invitrogen; Matrigel gum was purchased from BD; Gelatin was purchased from Sigma.
  • fISEMFs fetal intestinal myofibroblasts
  • aGSEMFs adult gastric myofibroblasts
  • hGECs human gastric epithelial cells
  • hiEndoPCs endodermal progenitor cells
  • the specific method includes the following steps:
  • hGECs human gastric epithelial cells
  • the reprogramming method is the same as the method in the second part.
  • hGECs human gastric epithelial cells
  • hiEndoPCs endodermal progenitor cells
  • 8M 8 small molecule compounds
  • aGSEMFs adult gastric myofibroblasts
  • stromal cells derived from the digestive tract include fetal gastric myofibroblasts (fGSEMFs), fetal intestinal myofibroblasts (fISEMFs), fetal transverse stromal cells (fDCs), and other commonly used trophoblast cells such as mesenchyme Stem cells (MSCs), mouse embryonic fibroblasts (MEFs), adult dermal fibroblasts (HFFs), etc., also tested a variety of extracellular matrices such as Matrigel gelatin, gelatin, etc., as well as fetal intestinal myofibroblasts (fISEMFs) Sources and conditioned media from adult gastric myofibroblasts (aGSEMFs) were used to compare reprogramming.
  • fGSEMFs fetal gastric myofibroblasts
  • fISEMFs fetal intestinal myofibroblasts
  • fDCs fetal transverse stromal cells
  • trophoblast cells such as mesenchyme Stem cells (MSCs),
  • fISEMFs fetal intestinal myofibroblasts
  • aGSEMFs-CM conditioned medium derived from adult gastric myofibroblasts
  • the cells were continuously apoptotic as the culture time prolonged (as shown in Figure 11, the picture in the upper right or upper left corner of the figure is small). Enlarged view in the box). Due to the limited source of fetal tissue, clinically adult gastric specimens are the most accessible, and a large number of adult gastric myofibroblasts (aGSEMFs) can be obtained, and aGSEMFs can maintain high reprogramming efficiency, so aGSEMFs are a class. Good reprogramming of trophoblast cells, which will support reprogramming in subsequent experiments with trophoblast cells.
  • the trophoblast cells selected by the second part are used as supporting cells, and the molecules are reduced one by one based on the above-mentioned culture conditions of 8 small molecule compounds (8M), and the indispensable small molecules are first screened.
  • step 2) Based on the combination of small molecule compounds screened in step 1), successively reduce the combination of small molecule compounds that ultimately find the best (highest reprogramming efficiency) and the smallest combination of small molecules that ensure the formation of clones.
  • step (2) the most reasonable trophoblast cells, adult gastric myofibroblasts (aGSEMFs), were identified as support cells for optimization of small molecule combinations. Based on the combination of 8 small molecule compounds (8M), the molecules were reduced one by one. When the PD0325901, PS48, and FBP were subtracted, the formation efficiency of the clones increased, indicating that these small molecule compounds inhibited reprogramming.
  • aGSEMFs adult gastric myofibroblasts
  • BBRS combination induced endodermal progenitor cells
  • SB431542 is the most critical, it is based on SB431542 and combined with any of the other three small molecules (Bix01294 (Bix), Bay K 8644 (Bay), RG108 (RG)), and found that SB431542 is able to guarantee cloning.
  • the indispensable role of SB431542 has important implications for the mechanism of action of small molecule compounds during reprogramming.
  • SB431542 (SB) is the smallest small molecule compound capable of reprogramming successfully in the reprogramming system
  • SB431542 is a TGF- ⁇ signaling pathway inhibitor
  • A83-01 (A83) replaces the optimization experiment of SB repeat small molecule compound combination.
  • the result A83 (using concentration 0.5 ⁇ M) can produce similar or better SB either alone or in combination with other small molecules (referred to as BBRA).
  • BBRA represents the other three small molecules Bix01294 (B), Bay K 8644 (B), RG108 (R), respectively.
  • hGECs human gastric epithelial cells
  • hiEndoPCs endodermal progenitor cells
  • aGSEMFs adult gastric myofibroblasts
  • hGECs Human gastric epithelial cells
  • aGSEMFs adult gastric myofibroblasts
  • aGSEMFs adult gastric antrum tissue
  • BD Flow cytometry
  • Zeiss laser confocal microscopy
  • Leica inverted phase contrast microscope
  • Advanced DMEM/F-12 medium, NEAA (non-essential amino acid), glutamine (Glutamax) were purchased from Gibco; cyanine-streptomycin, Accutase, mitomycin-C were purchased from Sigma TrypLE digestive enzyme was purchased from Invitrogen.
  • Small molecule compounds SB431542, Bix01294, RG108, Bay K 8644 were purchased from Stemgent Corporation.
  • CD56-PE eBioscience
  • rabbit anti-human CD56 NCAM
  • murine anti-human IgG1 MUC5AC Abeam
  • Alexa 647Goat Anti-Mouse IgG2b ⁇ 2b
  • Alexa 568 Goat Anti-Mouse IgG1 ⁇ 1 was purchased from Invitrogen.
  • FISH Fluorescence in situ hybridization
  • the specific method includes the following steps:
  • RESULTS Since two cell types are involved in the reprogramming system, the starting cells - human gastric epithelial cells (hGECs) and trophoblast cells - adult gastric myofibroblasts (aGSEMFs), the foregoing have confirmed that they cannot be individually Programming to induce endodermal progenitor cells (hiEndoPCs), only the combination of the two and reprogramming in BBRS reprogramming medium can be successful.
  • hGECs human gastric epithelial cells
  • aGSEMFs trophoblast cells - adult gastric myofibroblasts
  • hiEndoPCs are indeed derived from gastric epithelial cells rather than trophoblast cells.
  • FISH fluorescence in situ hybridization
  • the specific method includes the following steps:
  • hGECs Human gastric epithelial cells
  • CD56 NCAM, a neuronal cell adhesion molecule, a surface protein expressed on the cell membrane
  • CD56 is immunofluorescent staining, and the cell immunofluorescence staining method is the same as the first part.
  • the starting cells were derived from gastric epithelial cells rather than trophoblast cells. Since the gastric epithelial cells belong to the primary cells and the cell types are mixed, in order to further clarify which subgroup of the starting cells has undergone the conversion to hiEndoPCs, the starting cells need to be subdivided. Firstly, it is necessary to clarify the surface markers of hGECs. Since the isolated hGECs are mainly derived from the crypts of the gastric antrum, it has been reported that NCAM is a surface marker expressed in a variety of endodermal-derived tissues, especially on primitive cells.
  • NCAM in situ staining of gastric antrum tissue revealed that NCAM (CD56) is mainly expressed in different degrees of deep gastric tissue (ie, crypt part) (as shown by the red fluorescence in the left part of Figure 19), shallow Gastric tissue is hardly expressed.
  • the superficial tissue mainly expresses MUC5AC (as shown in the middle green fluorescence of Figure 19, and the right panel shows the synthetic map of NCAM staining and MUC5AC staining). Therefore, NCAM is considered to be used for initial cell division. Reliable sign.
  • the cultured hGECs were flow-classified using NCAM as a sorting marker, and divided into two groups of NCAM-positive and NCAM-negative cells. Reprogramming was then performed with NCAM positive and negative cells as starting cells, respectively. The results showed that NCAM-positive starting cells were able to reprogram successfully, while negative cells were almost impossible (as shown in Figure 20).
  • the starting cells were derived from gastric epithelial cells instead of trophoblast cells.
  • NCAM-positive, but not NCAM-negative, gastric epithelial cells undergo cell type switching during reprogramming, thereby achieving precise localization of the starting cells.
  • Endoderm progenitor cells are thought to be the origin of liver, pancreas, intestine, stomach, lung, thyroid and other internal organs, and have the ability to proliferate, thus acquiring functional hepatocytes, pancreatic cells, intestinal cells, lungs and thyroid cells. Good seed cells. Previous studies have been conducted to obtain endodermal progenitor cells starting from Embryonic stem cells (ESCs) or induced Pluripotent Stem Cells (iPSCs), which were identified by reprogramming in this example. The endoderm progenitor cells lay a good foundation.
  • ESCs Embryonic stem cells
  • iPSCs induced Pluripotent Stem Cells
  • Example 1 we successfully generated endodermal stem/progenitor-like clones (hiEndoPCs) in human gastric epithelial cells (hGECs) using a combination of BBRS and adult gastric myofibroblasts (aGSEMFs). However, this is only a preliminary morphological change, and the endodermal progenitor cell properties are confirmed. PCR and immunoprotein staining are used to identify the characteristic gene expression of induced endodermal stem/progenitor cells (hiEndoPCs), and the whole genome expression is detected by gene chip. The methylation chip was subjected to epigenetic analysis, and its proliferative properties were also tested. The microscopic characteristics were observed by electron microscopy.
  • hiEndoPCs endodermal progenitor-like cells
  • aGSEMFs adult gastric myofibroblasts
  • hGECs human gastric epithelial cells
  • hiEndoPCs induced endoderm progenitor cells
  • ESCs H9 human embryonic stem cells
  • the antibodies were as in the first part of Example 1, the immunohistochemistry kit (Vector Lab), RA and LDN 193189 were purchased from Sigma; A83-01 was purchased from Stemgent; b-FGF, Wnt3a, Activin A, FGF10 were purchased from R&D.
  • the protein immunofluorescence staining method was the same as in Example 1.
  • Extracting reprogrammed cloned RNA, reverse transcription and real-time PCR the specific method includes the following steps:
  • RNA extraction kit purchased from QIAGEN
  • Buffer RW1 provided by RNA extraction kit, available from QIAGEN
  • 700 ⁇ L of Buffer RW1 was added to spincolumn, centrifuged at 12,000 rpm for 30 s, and the filtrate was discarded.
  • Buffer RPE provided by RNA extraction kit, purchased from QIAGEN
  • 500 ⁇ L of Buffer RPE was added to spincolumn, centrifuged at 12,000 rpm for 30 s, and the filtrate was discarded.
  • Human embryonic stem cell H9 which is in good growth state, was inoculated in a suitable density in a Matrigel gel-coated well plate in advance, and the medium was changed to DE induction medium the following day: Advanced RPMI 1640+1% (W/V) B27 (none Serum nerve cell additive) + ActivinA (activin A, 100 ng / mL) + CHIR99021 (3 ⁇ M, purchased The medium was changed from Stemgent for 1 day, and the medium was changed to: Advanced RPMI 1640 + 1% B27 + Activin A (100 ng / mL) on day 2 and day 3.
  • PGT induction medium Advanced (Chinese name: Advanced) RPMI 1640 + 2% (V / V) FBS + 50 ng / mL FGF10 (fibril growth factor 10) + Cyclopamine (cyclopamine, 0.25 ⁇ M), PGT can be obtained in 3 days.
  • the stem/progenitor cell-specific transcription factor FOXA2 (red fluorescence, expression 96 ⁇ 2.6%), SOX9 (red fluorescence, expression 97 ⁇ 1.18%) and other endodermal stem/progenitor cell-associated markers such as CXCR4 (green fluorescence) , expression amount 98 ⁇ 0.56%), EPCAM (red fluorescence, expression amount 97 ⁇ 2.7%), LGR5 (green fluorescence, expression amount 92 ⁇ 4.3%), CK19 (green fluorescence, expression amount 91 ⁇ 4.2%).
  • Flow-level detection is consistent with immunoprotein staining, which further confirms the characteristics of endodermal progenitor cells of hiEndoPCs.
  • FIG. 23A A, Fig. 23A).
  • the middle column group is hECs, DE, PGT, PFG, hiEndoPCs, P1, hiEndoPCs, P4 and hGECs from left to right.
  • hiEndoPCs no longer express the gastric-specific markers MUC6, PGC, GIF, and GAST (B of Figure 23, and the column population in Figure 23B is hGECs, hiEndoPCs, and human gastric tissue from left to right).
  • hiEndoPCs Inducible endoderm progenitor cells obtained after reprogramming of human gastric epithelial cells (hGECs) lose their characteristic markers such as MUC6, GIF, PGC, GAST, etc., and begin to express high endoderm early ancestors. Symbols of cell marker genes such as transcription factors FOXA2, SOX9, HNF1B, GATA4, PDX1, HOXA3 and other endodermal progenitor cells such as CXCR4, CK19, LGR5, EPCAM. Both transcript levels and protein expression levels indicate endodermal progenitor characteristics of hiEndoPCs. However, in order to fully clarify the molecular characteristics, developmental stage characteristics and spatiotemporal localization of hiEndoPCs, it is necessary to analyze the whole gene expression or epigenetic expression profile.
  • aGSEMFs adult gastric myofibroblasts
  • hGECs human gastric epithelial cells
  • hDECs duodenal epithelial cells
  • hiEndoPCs induced endoderm progenitor cells
  • DE qualitative endoderm
  • PTT primitive digestive tract
  • PGT the posterior intestine
  • RNA extraction and reverse transcription methods of hGECs, hiEndoPCs, PGT, and PFG are the same as those in the first step of the present embodiment.
  • the extracted hGECs, hiEndoPCs, PGT, and PFG RNA were first amplified, and the bioacylated cRNA was generated from the total RNA according to the procedure provided by the Illumina TotalPrep kit, and then the cRNA was hybridized using a Sentrix Chip Array, and the hybridization was performed according to Illumina.
  • Illumina TotalPrep kit Provides methods for processing data using Illumina BeadStudio software, uploading raw data to Gene Expression Omnibus Database(accession number GSE69706).
  • hiEndoPCs The identification of a pair of hiEndoPCs is limited to certain characteristic markers of endoderm stem/progenitor cells.
  • hiEndoPCs and hGECs are indeed two different types of cells.
  • the results of the cluster analysis showed that hiEndoPCs and hGECs had different apparent patterns (A panel of Fig. 24), which clearly confirmed that the cells had undergone major changes after reprogramming.
  • RNA deep sequencing revealed that hiEndoPCs were between PGT and PFG and closer to PFG (Figure 26).
  • PCA principal component analysis showed that the epithelial-derived hiEndoPCs were very similar to the duodenal epithelial-derived hiEndoPCs in the whole gene expression profile (PCA principal component analysis refers to the linear transformation of multiple variables to select fewer a multivariate statistical analysis method for the number of important variables), which is very similar to the cell morphology observed in the previous Consistent and different from their starting cells, they are also significantly different from ESCs-derived qualitative endoderm (DE) and trophoblast cells (GSEMFs) (Figure 27).
  • DE ESCs-derived qualitative endoderm
  • GEMFs trophoblast cells
  • hGECs are significantly different from hiEndoPCs.
  • hiEndoPCs acquire the molecular characteristics of endoderm stem/progenitor cells, so the conversion of hGECs to hiEndoPCs is indeed a reprogramming process.
  • RNA deep sequencing confirmed that hiEndoPCs are closer to PFG between PGT and PFG at the developmental stage, thus realizing the temporal and spatial localization of hiEndoPCs.
  • hGECs Human gastric epithelial cells
  • hiEndoPCs induced endodermal progenitor cells
  • H7650 transmission electron microscope H7650 Electron Microscopy (electron microscope), AMT XR16M CCD Digital Camera (AMT), AMT Capture Engine Software Version 600.259 (capture engineering software), detachable 96-well plate ( Corning).
  • Polybed 812epoxy resin (Multiple Bed Epoxy Resin) was purchased from Polysciences, Inc., Warrington, PA, Reynolds'lead citrate (provided by the National Instruments Analytical Testing Center of the Academy of Military Medical Sciences), aqueous uranyl acetate Uranyl acetate, provided by the National Instrumental Analysis and Testing Center of the Academy of Military Medical Sciences).
  • the specific method includes the following steps:
  • hiEndoPCs and hGECs are planted in detachable 96-well plates.
  • Uranium was provided by the National Instrumental Analysis and Testing Center of the Academy of Military Medical Sciences. It was stained for 15 minutes and then stained with Reynolds'lead citrate for 7 minutes using Reynolds' lead citrate (provided by the National Instruments Analytical Testing Center of the Academy of Military Medical Sciences).
  • the cytoplasm is rich in content and contains a large amount of mitochondria, microvilli and vacuoles (Fig. 28, B).
  • the cells of hiEndoPCs are smaller, more closely connected, and the nucleoli are more obvious.
  • the nucleoplasmic ratio is larger (Fig. 28, C), the cytoplasm is less, contains less mitochondria, and a small amount of mitochondria and endoplasmic reticulum are distributed in the nuclear membrane. Around (D of Figure 28), and the cell junction has a finger-like connection of immature cells (Fig. 28, E). Therefore, microscopic analysis shows that hiEndoPCs are significantly different from hGECs, hiEndoPCs exhibit microscopic characteristics of stem cells, and hGECs are characterized by terminally differentiated mature cells.
  • aGSEMFs induced endodermal progenitor cells
  • hiEndoPCs induced endodermal progenitor cells
  • Fibronectin (FN, fibronectin), Cell-TAK gel (CT) were purchased from BD; serum-free cell rapid freezing solution (Bio-Tool); A83-01 (Stemgent); bFGF (basic fibroblast growth factor b) Wnt3a was purchased from R&D; mitomycin-C (Sigma); Advanced DMEM/F12, Dispase (discrete) were purchased from Gibco.
  • Cryopreservation The manually picked clones were digested with 5 mg/mL Dispase enzyme at 37 ° C for 5 minutes, and blown into small pieces. The cells were washed twice with medium, and then frozen rapidly with an appropriate amount of serum-free cells. The cells were resuspended in a stock solution, placed in a cryotube, and stored directly at -80 °C.
  • hiEndoPCs are endodermal progenitor cells, they must have certain potential for passage amplification.
  • the inventors have repeatedly screened the passage conditions to determine whether Fibronectin (FN) gel or BD Cell-TAK (CT) gel is used as the cell.
  • the external matrix is combined with Advanced DMEM/DF12+AWF (A83-01 0.5 ⁇ M+Wnt3a50ng/mL+bFGF 10ng/mL), ie, FN+AWF (Fig. 30A) or CT+AWF (Fig. 30B). Under the conditions of the cells, the clones can be well grown and maintained in the state of the original clones after passage.
  • the original stem/progenitor cell clone morphology was also maintained under the conditions of aGSEMFs as a trophoblast and combined with Advanced DMEM/DF12+A (A83-01 0.5 ⁇ M), ie, Feeder+A (Fig. 30C).
  • hiEndoPCs can also be cryopreserved, and the condition of the original clone can be maintained by using the condition of Feeder+A after cloning and resuscitation (Fig. 30D, Post-Thawed cells, after cryopreservation).
  • hiEndoPCs can be expanded for about 4-6 generations under the aforementioned subculture conditions, and the cell morphology remains basically unchanged during the passage (Fig. 31, the lower row of pictures is an enlargement of the upper row of the picture box). After the passage of 4 generations, the cell proliferation rate slowed down, and the number of cells in the 6th generation reached a peak, indicating that the proliferation potential was limited.
  • hGECs human gastric epithelial cells
  • hiEndoPCs approximately 109 numbers of stem/progenitor cells (hiEndoPCs) can be obtained (Figure 32). ).
  • hiEndoPCs can be expanded for 4-6 passages using the appropriate passage conditions.
  • hGECs Human gastric epithelial cells
  • hiEndoPCs induced endoderm progenitor cells
  • ESCs H9 human embryonic stem cells
  • Antibody the company Item number Primary resistance source Dilution ratio AFP Sigma A8452 Mouse immunoglobulin 2a 200 ALB Abcam Ab10241 Mouse immunoglobulin 2b 400 CK18 Santa Cruz Sc-6259 Mouse immunoglobulin 1 100 Lgr5 Sigma HPA012530 rabbit 350 CDX2 R&D AF3665 Goat immunoglobulin 100 Muc2 Abcam Ab118964 Mouse immunoglobulin 1 100 Somatostatin Millipore AB5494 rabbit 100 InsuLin Abcam Ab7842 Guinea pig 100 Glucagon Sigma G2654 Mouse immunoglobulin 1 200 ProinsuLin R&D MAB13361 Mouse immunoglobulin 2a 200 C-peptide Millipore 05-1109 Mouse immunoglobulin 1 100 --amylase Sigma A8273 rabbit 200 PDX1 Abcam Ab47308 Guinea pig 200
  • Advanced DMEM/DF12, MCDB131 (low protein, serum-free medium 131), CMRL 1066, Advanced RPMI 1640, GlutaMax (glutamine), NEAA (non-essential amino acids), N2 (serum-free nerve cell additive N2) and B27 (serum-free nerve cell additive B27) were purchased from Gibco; ITS-X (insulin-transferrin-selenium-ethanolamine complex solution, Life Technologies); T3, Ascorbic acid (vitamin) C, Sigma); HM (hepatocyte culture medium, Sciencell); Lanminin (laminin), Fibronectin (fibronectin, FN), Collagen IV (type 4 collagen) were purchased from BD.
  • Cytokines b-FGF (basic fibroblast growth factor b), Wnt3a, ActivinA (activin A), FGF4 (fibroblast growth factor 4), HGF (hepatocyte growth factor), OSM (ostomalin) M), FGF10 (fibroblast growth factor 10), FGF7 (fibroblast growth factor 7), EGF (epidermal growth factor), Noggin (head protein), BMP4 (bone morphogenetic protein 4, IGF (insulin-like growth factor), TSH (thyroid stimulating hormone), InsuLin (insulin) were purchased from R&D; NaI (sodium iodide, Sigma).
  • Kit human albumin ELISA kit (human albumin assay kit, purchased from Bethyl).
  • the specific method includes the following steps:
  • Collegan IV/Matrigel/Laminin/KM is mixed at a ratio of 1:3:1:5, then uniformly applied to the well plate with an appropriate volume of the glue mixture, and dried at room temperature for 3-5 hours.
  • the hiEndoPCs were induced to differentiate by a staged method.
  • the specific method includes the following steps:
  • the differentiated cells are induced to differentiate into the pancreas according to four stages.
  • 2.1MCDB 131medium (purchased from Gibco) + 1.5 g / L sodium bicarbonate (sodium bicarbonate) + 2 mM (concentration) 2 mM Glutamax (glutamine) + 10 mM final glucose concentration (glucose) + 2% BSA (bovine serum albumin) +0.25mM ascorbic acid + 50ng/mL of FGF7 (fibril growth factor 7) + 0.25 ⁇ M SANT-1+1 ⁇ M retinoic acid (retinoic acid) (RA) +100nM LDN193189+1: 200ITS-X (insulin - Transferrin-selenium-ethanolamine complex solution) + 200 nM TPB, cultured for 2 days.
  • the second stage cells were digested with 5 mg/mL dispase (discrete enzyme) at 37 ° C for 5 minutes, then mechanically blown into small pieces and transferred to a low adsorption well plate to change the medium: MCDB 131medi ⁇ m + 1.5g / L sodium Bicarbonate+2mM Gl ⁇ tamax+20mM final glucose concentration+2%BSA+0.25 ⁇ M SANT-1+0.05 ⁇ M retinoic acid+100nM LDN193189+1:200ITS-X+1 ⁇ M T3 (insulin-transferrin-selenium-ethanolamine complex solution)+ 10 ⁇ M ALK5 inhibitor II + 10 ⁇ M zinc suLfate (zinc sulfate) + 10 ⁇ g/mL of heparin (heparin), cultured for 3 days.
  • dispase discrete enzyme
  • the specific method includes the following steps:
  • RPMI 1640medium 2 mM Glutama + 100 U / mL penicillin + 0.1 mg / mL streptomycin + 500 ng / mL FGF4 (fibroblast growth factor 4) + 500 ng / mL Wnt3a (WNT signaling pathway protein ligand 3A) + 100 ng / mL EGF (epidermal growth factor) + 3 ⁇ M CHIR99021 (Stemgent), cultured for 3 days.
  • the specific method includes the following steps:
  • the specific method includes the following steps:
  • the protocol is performed according to the requirements on the c-peptide (C-peptide) ELISA kit.
  • the specific method includes the following steps:
  • the standard and the supernatant sample to be detected are added to a 96-well plate coated with a c-peptide antibody, each of which is 25 ⁇ L, and each sample is set to three replicates.
  • wash buffer washing solution
  • the well plate was placed on a microporous shaker and incubated at 800 rpm for 1 hour at room temperature.
  • the OD value was measured at a wavelength of 450 nm on the microplate reader within 13 and 30 minutes, and the results were calculated.
  • the specific method includes the following steps:
  • the DTZ stock solution (purchased from Sigma) was diluted at a ratio of 1:20, and then filtered through a 0.45 ⁇ m filter to prepare a working solution.
  • RESULTS Endodermal progenitor cells eventually developed into organs such as the pancreas, liver, intestines, lungs, and thyroid gland. To confirm that hiEndoPCs are endodermal progenitor cells, the present invention tested their potential to induce differentiation in multiple directions.
  • pancreatic induction differentiation protocol Pancreatic Felicia W, Millman Jeffrey R, Gürtler M, Segel M, Van Dervort A, Ryu Jennifer H, et al. Generation of Functional Human Pancreatic ⁇ Cells In Vitro. Cell. 2014; 159: 428-39 .Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, Asadi A, et al. Resal of diabetes with insuLin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol. 2014; 32: 1121-33. HiIntoPCs were induced to differentiate into the pancreas.
  • hiEndoPCs-derived pancreatic cells exhibited three-dimensional sprouting morphology of islets in vivo (Fig. 33A).
  • DTZ dithizone staining showed that redEndoPC-Pans appeared red, indicating that the cytoplasm of the cells contained zinc ions, which is characteristic of islet beta cells (Fig. 33B).
  • Pancreatic specific protein staining of hiEndoPC-Pans showed significant expression of pancreatic specific transcription factors PDX1 and NKX6.1, islet ⁇ cell marker GCG, islet ⁇ cell marker C-PEP and PRO-INS, and islet ⁇ cell marker SST. (Fig. 33C and D).
  • hiEndoPC-Ints The hiEndoPC-Ints midgut-specific transcription factor CDX2, small intestinal cell marker VIL1, goblet cell marker MUC2, small intestinal endocrine cell marker CHGA, and small intestinal pancreatic cell marker LYSO were significantly up-regulated after induction (Fig. 34B). Moreover, at the protein level, the markers characteristic of the intestines VIL1, MUC2, CDX2, LGR5, and ECAD were also significantly expressed (Fig. 35C), confirming that hiEndoPC-Ints had initially possessed characteristics of the intestine.
  • hiEndoPC-Heps hiEndoPCs-derived hepatocytes
  • hiEndoPC-Heps hiEndoPCs-derived hepatocytes
  • AFP alpha-fetoprotein
  • CK18 cytokeratin 18
  • hiEndoPCs induce differentiation into the thyroid [Longmire TA, Ikonomou L, Hawkins F, ChristodouLou C, Cao Y, Jean JC, et al. Efficient derivation of p ⁇ rified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell. 2012; 10:398 -411.], the specific transcription factor NKX2.1, thyroid-specific transcription factor Pax8, thyroglobulin (Tg), thyroid stimulating hormone receptor shared by thyroid and lung in hiEndoPCs-derived thyroid cells (hiEndoPCs-Thyroid) The expression level of TSHR) was significantly higher than before induction (Fig. 36A).
  • hiEndoPCs The endocytic differentiation potential of hiEndoPCs was identified by a strategy that promoted differentiation into the pancreas, liver, intestine, thyroid and lung. The results showed that after the differentiation of hiEndoPCs into five directions, the expression of related genes and protein expression were significantly improved. The function has also appeared, more clearly confirmed that hiEndoPCs are endoderm progenitors The cells also suggest that hiEndoPCs can provide ideal seed cells for cell therapy such as diabetes, liver disease and intestinal diseases.
  • Example 3 Small molecule compound combination capable of reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells
  • the four small molecule compounds are subjected to the same method in different concentration ranges SB43152 (1 to 10 ⁇ M), RG108 (0.01 to 1 ⁇ M), Bix01294 (0.1 to 2 ⁇ M), and Bay K 8644 (1 to 4 ⁇ M).
  • the production of hiEndoPCs was verified (see Table 9), and SB108 (0.01 ⁇ 1 ⁇ M) was combined with other three small molecules at different concentrations with A83-01 (A83, 0.4 ⁇ 1 ⁇ M), Bix01294 (0.1 ⁇ 2 ⁇ M), Bay K8644 (1 to 4 ⁇ M) was used to verify the production of hiEndoPCs (Table 10).
  • the endoderm-related gene, protein, epigenetic modification and differentiation function were identified by referring to the method of Example 2 for the endoderm progenitor cells obtained in the combination of Table 9 and Table 10. The results showed that the induced endoderm obtained by the above combination was obtained.
  • Example 4 Preparation of a reprogramming kit for reprogramming gut-derived epithelial cells into endoderm stem/progenitor cells
  • the invention can reprogram the digestive tract-derived epithelial cells into reprogramming kits of endoderm stem/progenitor cells, including a small molecule compound combination capable of reprogramming the digestive tract-derived epithelial cells into endoderm stem/progenitor cells, specifically
  • the small molecule compound combination consists of 8 (8M) small molecule compounds: FBP (fructose diphosphate), Bay K 8644, Bix01294, SB431542 or A83-01, Valproic Acid (VPA) (valproic acid), RG108 , PD0325901 and PS48.
  • a preferred combination of small molecule compounds that can reprogram gut-derived epithelial cells to endoderm stem/progenitor cells includes the following four small molecule compounds, wherein the BBRS combination is Bix01294 (Bix), Bay K 8644 (Bay), RG108 (RG And SB431542 (SB), BBRA is combined into Bix01294 (Bix), Bay K 8644 (Bay), RG108 (RG) and A83-01 (A83).
  • each compound may be packaged separately, or the compounds may be mixed and packaged according to the 8M combination or the BBRS combination or the BBRA combination; when the compounds are separately packaged, the use concentration of each compound is described in the specification of the kit, and the specific concentration value may be referred to Example 1 and Example 3.
  • the kit also includes a basic medium Advanced DMEM/F12 and a cell culture base addition component Glutamine (Glutamax) and an antibiotic SP, and instructions for use thereof, wherein the concentration of glutamine is relatively high relative to the Advanced DMEM/F12 basal medium.
  • Glutamine Glutamax
  • antibiotic SP an antibiotic SP
  • concentration of glutamine is relatively high relative to the Advanced DMEM/F12 basal medium.
  • 2 mM (1 ⁇ ) antibiotics (such as cyanine-streptomycin) are used at a concentration of 100 U/mL penicillin + 0.1 mg/mL streptomycin, and the components are packaged separately or mixed at the concentrations indicated and packaged.
  • the kit may also combine the small molecule compound combination with the basal medium and the cell culture base addition component to form a reprogramming medium, and the reprogramming medium is formulated as: Advanced DMEM/F12+2 mM glutamine (Glutamax) + ⁇ -streptomycin (100 U/mL penicillin + 0.1 mg/mL streptomycin) + SB43154 (2 ⁇ M) or A83-01 (0.5 ⁇ M) + VPA (0.5 mM) + PD0325901 (0.5 ⁇ M) + RG108 (0.04 ⁇ M) + Bix01294 (0.5 ⁇ M) + Bay K 8644 (2 ⁇ M) + PS48 (5 ⁇ M) + FBP (3.5 mM).
  • the reprogramming medium is formulated as: Advanced DMEM/F12+2 mM glutamine (Glutamax) + ⁇ -streptomycin (100 U/mL penicillin + 0.1 mg/mL streptomycin) + SB43154 (2 ⁇ M
  • the preferred reprogramming medium formulation is: Advanced DMEM/F12 containing 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL penicillin and 0.1 mg/mL streptomycin), 1 to 10 ⁇ M of SB43152, 0.01 ⁇ 1 ⁇ M RG108, 0.1-2 ⁇ M Bix01294, 1-4 ⁇ M Bay K 8644; more preferably Advanced DMEM/F12 contains 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL penicillin and 0.1 mg/mL) Streptomycin), 2 ⁇ M SB43152, 0.04 ⁇ M RG108, 0.5 M Bix01294, 2 ⁇ M Bay K 8644.
  • Advanced DMEM/F12 containing 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL penicillin and 0.1 mg/mL streptomycin), 0.4 to 1 ⁇ M of A83- 01, 0.01 to 1 ⁇ M of RG108, 0.1 to 2 ⁇ M of Bix01294, 1 to 4 ⁇ M of Bay K 8644; more preferably Advanced DMEM/F12 contains 2 mM glutamine (Glutamax), cyan-streptomycin (100 U/mL penicillin and 0.1) Mg/mL streptomycin), 0.5 ⁇ M A83-01, 0.04 ⁇ M RG108, 0.5 M Bix01294, 2 ⁇ M Bay K8644.
  • the kit may further include trophoblast cells which are stromal cells derived from the digestive tract, such as gastric myofibroblasts or intestinal muscle fibroblasts, and instructions for use thereof.
  • trophoblast cells which are stromal cells derived from the digestive tract, such as gastric myofibroblasts or intestinal muscle fibroblasts, and instructions for use thereof.
  • the kit further includes instructions for use, which describe the actual composition and method of use of the kit, including reprogramming of digestive tract-derived epithelial cells into endodermal stem/progenitor cells using related reagents.
  • instructions for use which describe the actual composition and method of use of the kit, including reprogramming of digestive tract-derived epithelial cells into endodermal stem/progenitor cells using related reagents. The description in the example.
  • the invention provides a small molecule compound combination, reprogramming method and application for reprogramming digestive tract-derived epithelial cells into endoderm stem/progenitor cells, which are human gastric epithelial cells (hGECs) as starting cells and human gastric muscles.
  • hGECs human gastric epithelial cells
  • Fibroblasts as a trophoblast, with a combination of compounds including SB or A83 selected from FBP, Bay K 8644, Bix01294, SB431542 or A83-01, VPA, RG108, PD0325901 and PS48
  • the digestive tract-derived epithelial cells are reprogrammed into endoderm stem/progenitor cells, which can be used to induce differentiation into liver cells, pancreatic beta cells, and intestinal cells, and the present invention can be applied industrially.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Physiology (AREA)
  • Nutrition Science (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、重编程方法及应用，以人胃上皮细胞(hGECs)作为起始细胞，以人胃肌成纤维细胞(aGSEMFs)来作为滋养层，用选自FBP、Bay K 8644、Bix01294、SB431542或A83-01、VPA、RG108、PD0325901和PS48中的全部或多个包括SB或A83在内的化合物组合将消化道来源上皮细胞重编程为内胚层干/祖细胞，该内胚层干/祖细胞可用于向肝脏细胞、胰腺β细胞和肠细胞方向诱导分化。

Description

将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、重编程方法及应用 技术领域
本发明属于细胞工程技术领域，涉及一种可将消化道来源上皮细胞重编程为内胚层干/祖细胞技术，包括在重编程过程中所用到的特定小分子化合物组合、重编程试剂盒、重编程方法及该内胚层干/祖细胞进一步向实质细胞诱导分化的应用。
背景技术
干细胞技术多次被《自然》、《科学》等杂志评为二十一世纪生命科学领域最具发展前景的技术，是攻克各种传统医学治疗手段力所不及的重大疾病的一种全新的医疗技术。干细胞具有自我更新和多向分化潜能，在器官组织再生、疾病模型建立、发育生物学及药物研发等领域具有广阔的应用前景。近10年来，干细胞理论与技术研究取得了飞速的发展，有望成为全新的疾病治疗方式，推动医学治疗的范式转换，引领新的医学革命。干细胞有多种不同的分类方法，从发育的角度来看，干细胞大致分为胚胎干细胞(Embryonic stem cells)(ESCs)和成体干细胞(Adult stem cells)两大类。ESCs来源于囊胚期的内细胞团(Inner cell mass)(ICM)，是一类具有无限自我更新和向人体所有细胞类型进行分化的多能性干细胞。1998年，Thomson在世界上首次建立了人胚胎干细胞系(hESCs)[Thomson JA,Itskovitz-Eldor J,Shapiro SS,Waknitz MA,Swiergiel JJ,Marshall VS,et al.Embryonic Stem Cell Lines Derived from Human Blastocysts.Science.1998；282:1145-7.]，由于其能够产生成体所有细胞类型，理论上能为基础研究、疾病治疗和药物研发提供理想的细胞来源。多年来利用ESCs已经产生了多种功能性细胞，包括心肌细胞、神经细胞、血细胞和肝脏细胞等[Murry CE,Keller G.Differentiation of Embryonic Stem Cells to&#xa0；Clinically Relevant Populations:Lessons from Embryonic Development.Cell.132:661-80.]。然而，ESCs的使用在国际上面临着伦理学的争议，而且ESCs来源的细胞移植后会产生免疫排斥反应，未分化的ESCs会形成畸胎瘤等诸多问题，因此大大限制了其走向临床的应用。而成体干细胞存在于出生后各个阶段的多种组织中，具有一定的自我更新能力并且能够向其来源组织中的成熟细胞进行分化，如造血干细胞、间充质干细胞、肝脏干细胞和神经干细胞等。由于其来源于成体组织且只能向固定的方向分化，因此不具有成畸胎瘤的风险，也没有伦理学的问题，所以是临床疾病治疗的良好种子细胞。但其来源毕竟有限，只能从人体组织中分离获得，而且分化潜能也很局限只能生成其所在谱系内的一种或几种成熟细胞类型，体外 也很难大规模的扩增，所以其临床应用也有很大的局限性。多年来，众多的研究都在致力于寻求更为理想的细胞类型来进行疾病的治疗、药物研发和机制研究。
传统的表观遗传学观点认为谱系的特化、细胞的分化是单向的、不可逆的过程，多能的细胞状态只能向终末成熟的细胞状态进行命运特化(Waddington’epigenetic landscape)。而诱导多能性干细胞(induced Pluripotent Stem Cells)简称iPSCs的出现深刻的改变了这一观念。2006年日本科学家Yamanaka利用ESCs特异性的4个多能性相关的转录因子OCT4、SOX2、KLF4、c-MYC(OSKM)成功的将成纤维细胞转换为类似ESCs的多能性干细胞iPSCs，首次证实了终末分化的细胞在多能性相关转录因子的作用下可以重新获得分化多能性，这就是经典的重编程技术[Takahashi K,Yamanaka S.Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors.Cell.126:663-76.Takahashi K,Tanabe K,Ohnuki M,Narita M,Ichisaka T,Tomoda K,et al.Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors.Cell.131:861-72.]。iPSCs的出现具有里程碑式的意义：首先，它突破了经典的表观遗传全景模式，极大的丰富了细胞命运转换的理论，引领了细胞命运研究和干细胞研究的范式转换，给再生医学的基础研究和临床应用开辟了广阔前景。其次，iPSCs具有ESCs的无限自我更新和向体内所有细胞类型进行分化的潜能，同时避免了ESCs研究和应用的伦理学争议。再次，由于成纤维细胞可取自病人自体因此也避免了ESCs分化产生的终末细胞移植入体内带来的免疫排斥问题。最后，病人自体来源的iPSCs可以建立个体特异性的疾病细胞模型和个体化的药物筛选平台。iPSCs的出现不仅给疾病的基础研究，药物筛选带来新的研究模式，而且使多能性干细胞(PSCs)走向临床应用跨进一大步。利用iPSCs已经成功的产生了多种功能性细胞类型，而且iPSCs来源的视网膜色素上皮治疗致盲性眼病的临床试验正在开展。Yamanaka也因此项技术而获得2012年的诺贝尔生理学或医学奖。
尽管iPSCs具备多方面的优势，是一种非常具有治疗前景的细胞类型，然而由于同样具有ESCs类似的多能性依然难以避免致畸胎瘤的风险，此外iPSCs向功能性细胞进行分化的过程中存在随机性分化的问题，会产生多种非预期细胞类型，这些问题都会对大规模的临床应用带来安全性的风险。为了弥补这一缺陷，近年来基于iPSCs技术和原理发展而来的谱系重编程(lineage reprogramming)技术日益受到广泛的重视。谱系重编程主要使用目的细胞谱系特异性的转录因子(lineage specific transcriptional factors)而非多能性的转录因子(OSKM)在起始细胞主要是皮肤成纤维细胞或血细胞中进行过表达，进而将起始细胞直接转换为目的细胞。谱系重编 程的出现同样是干细胞领域具有里程碑意义的重要事件。首先，由于不经过iPSCs的多能性阶段，谱系重编程避免了iPSCs的致畸胎瘤风险；其次，谱系重编程可以根据使用的转录因子和培养条件的不同直接获得终末成熟的功能细胞或者具有扩增能力的成体干细胞，而且得到的目的细胞类型相对均一；再次，谱系重编程也具有iPSCs的无免疫排斥及个体化的优势，因此在干细胞与再生医学研究和疾病治疗上显示出了更为广阔的应用前景[Sancho-Martinez I,Baek SH,Izpisua Belmonte JC.Lineage conversion methodologies meet the reprogramming toolbox.Nat Cell Biol.2012；14:892-9.]。最后，谱系重编程的出现极大的丰富了细胞命运转换和重编程理论的内涵，使人们对于细胞命运特化和调控的理解更为全面和深刻，并将这一概念应用到更为广阔的领域。
从2008年首次实现在同一胚层内的胰腺外分泌细胞向内分泌β细胞的谱系重编程[Zhou Q,Brown J,Kanarek A,Rajagopal J,Melton DA.In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.Nature.2008；455:627-32.]到2010年开始实现跨胚层的成纤维细胞向神经细胞的转换[Vierbuchen T,Ostermeier A,Pang ZP,Kokubu Y,Sudhof TC,Wernig M.Direct conversion of fibroblasts to functional neurons by defined factors.Nature.2010；463:1035-41.]，谱系重编程领域取得了一个又一个的巨大突破。目前使用病毒介导转录因子在体细胞中过表达的方式，已在三个胚层的的多种细胞类型中成功的实现了命运转换。在外胚层中已经将成纤维细胞转换为多种类型的神经元、神经干细胞等；在中胚层已经获得了心肌细胞、心肌祖细胞、造血干/祖细胞等；在内胚层获取了胰岛细胞、肝细胞、肝干细胞等。在这一领域尤其是肝系细胞的谱系重编程领域，中国科学家取得了一系列突破。2011年国内恵利建实验室在国际上首次使用肝细胞特异性的转录因子组合成功的将小鼠成纤维细胞转换为肝细胞[Huang P,He Z,Ji S,Sun H,Xiang D,Liu C,et al.Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.Nature.2011；475:386-9.]。2013年胡以平实验室使用转录因子组合又获得了具有自我更新和向肝细胞、胆管细胞进行分化的小鼠肝干细胞[Yu B,He ZY,You P,Han QW,Xiang D,Chen F,et al.Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.Cell Stem Cell.2013；13:328-40.]。2015年惠利建和邓宏魁实验室分别独立的使用不同的转录因子组合成功的将人的成纤维细胞转变成功能性肝细胞，为肝病治疗、药物筛选提供了良好的种子来源[Du Y,Wang J,Jia J,Song N,Xiang C,Xu J,et al.Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming.Cell Stem Cell. 2014；14:394-403.Huang P,Zhang L,Gao Y,He Z,Yao D,Wu Z,et al.Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.Cell Stem Cell.2014；14:370-84.]。尽管这些年来利用谱系重编程已获取了人或鼠的多种细胞类型，而且已经弥补了iPSCs应用上的多个缺陷，然而目前绝大多数谱系重编程的研究都是依赖于慢病毒或逆转录病毒介导的转录因子过表达的方式，这些病毒载体进入目的细胞后存在与宿主基因组整合，由此可能导致宿主基因组不稳定，为将来的临床应用带来安全性隐患，此外转录因子的操作过程复杂、效率较低下，不同实验室操作规程不一，难以大规模的扩展应用。因此诸多的研究目前致力于寻找更为安全的、高效的方式来替代转录因子。
与转录因子相比，小分子化合物具有明显的多方面的优势，小分子化合物可以自由通透细胞、易于合成、经济高效、无免疫源性、作用于蛋白水平不与基因组整合、可以标准化和规模化生产。通过改变小分子的浓度和作用时间较容易实现蛋白调控的强度和时空控制。近年来众多的研究都在致力于筛选小分子用于提高iPSCs的生成效率，缩短iPSCs的生成时间，减少转录因子的使用，最终目的都是为了完全使用小分子来替代经典的Yamanaka四因子，从而避免病毒在宿主细胞中整合[Li W,Li K,Wei W,Ding S.Chemical approaches to stem cell biology and therapeutics.Cell Stem Cell.2013；13:270-83.]。2013年国内邓宏魁实验室，在这方面取得了突破性的进展，其团队通过多次的筛选，找到了合适的小分子化合物组合能够在不依赖任何外源转录因子的情况下实现小鼠成纤维细胞向iPSCs的重编程，尽管效率只有0.2％并且过程复杂，但这项突破为基于小分子的重编程和谱系重编程带来了理论及现实的依据[Hou P,Li Y,Zhang X,Liu C,Guan J,Li H,et al.Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds.Science.2013；341:651-4.]。在研究小分子化合物介导iPSCs产生的同时，筛选合适的小分子用于调控谱系重编程甚至完全替代谱系特异性转录因子，从而产生安全高效可控的目的细胞的研究也在国内外如火如荼的开展[Li K,Zhu S,Russ HA,Xu S,Xu T,Zhang Y,et al.Small molecules facilitate the reprogramming of mouse fibroblasts into pancreatic lineages.Cell Stem Cell.2014；14:228-36.Zhu S,Rezvani M,Harbell J,Mattis AN,Wolfe AR,Benet LZ,et al.Mouse liver repopulation with hepatocytes generated from human fibroblasts.Nature.2014；508:93-7.Zhu S,Russ HA,Wang X,Zhang M,Ma T,Xu T,et al.Human pancreatic beta-like cells converted from fibroblasts.Nat Commun.2016；7:10080.]。在这一领域，中国科学家同样取得了令人瞩目的成就，裴刚实验室近期筛选到合适的小分子组合将阿尔茨 海默病病人来源的成纤维细胞重编程为成熟的功能性神经元[Hu W,Qiu B,Guan W,Wang Q,Wang M,Li W,et al.Direct Conversion of Normal and Alzheimer's Disease Human Fibroblasts into Neuronal Cells by Small Molecules.Cell Stem Cell.2015；17:204-12.]，而邓宏魁实验室则成功的使用小分子组合将小鼠的成纤维细胞转换为神经元[Li X,Zuo X,Jing J,Ma Y,Wang J,Liu D,et al.Small-Molecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons.Cell Stem Cell.2015；17:195-203.]，LeiZhang等完全使用小分子将人星形胶质细胞谱系重编程为功能性神经元[Zhang L,Yin JC,Yeh H,Ma NX,Lee G,Chen XA,et al.Small Molecules Efficiently Reprogram Human Astroglial Cells into Functional Neurons.Cell Stem Cell.2015；17:735-47.]。这些突破性的进展为完全使用小分子将成体细胞转换为其他类型的功能性细胞或干细胞奠定了坚实的理论基础，也带来巨大希望。《体细胞重编程技术在肝脏再生中的应用基础研究》[张文成，2012年博士论文]讨论了利用小分子化合物谱系重编程消化道细胞获得诱导内胚层多能干细胞(iEMSC)，实验中使用SB431542、VPA、Y27632小分子化合物以及成分未公开的1#-6#小分子组合进行了诱导重编程效率的比较。
内胚层干/祖细胞是肝脏、胰腺、胃、肠等内脏器官发育的源泉，获得内胚层干/祖细胞，并进行大量的扩增，然后再对其进行诱导分化，获得大量的功能性肝细胞、胰腺细胞或肠上皮细胞将对多种类型的内胚层器官疾病的治疗具有巨大的意义。众所周知，我国是肝病大国，目前有9700万的乙肝病毒携带者，1000万的丙肝病毒携带者，近年来酒精性脂肪性肝病和非酒精性脂肪性肝病在我国的发展趋势也明显升高，其它包括药物性肝损伤、免疫性肝病等都严重影响我国的国民身体健康[Wang FS,Fan JG,Zhang Z,Gao B,Wang HY.The global burden of liver disease:the major impact of China.Hepatology.2014；60:2099-108.]。各种肝病如果缺乏及时有效的治疗都将逐渐进展到以肝硬化，肝衰竭和肝癌为代表的终末期肝病(ESLD)的阶段。我国庞大的肝病基础人群造成了每年数十万人罹患ESLD。而对于ESLD而言，目前的治疗手段非常有限，人工肝的治疗还未成熟难以大规模开展，内科治疗只能采取保守姑息的方式，对于ESLD目前公认的最有效的治疗方式是原位肝移植，但又严重受限于肝源的匮乏。据估计我国每年死于ESLD的人群超过30万例，给万千家庭带来巨大痛苦，给社会带来沉重的医疗负担。而功能性肝细胞移植被认为是肝移植的良好替代治疗方式，但同样也受限于来源的匮乏。此外，无论是我国还是世界上其他国家，都拥有庞大的糖尿病人群，而且还呈现逐年增加的趋势。国际糖尿病联盟(International Diabetes Federation)的分析显示在不远的将来糖尿病将影响世 界上超过30亿的人口。然而目前糖尿病的治疗方法同样有限而且效果不一，尤其是Ⅰ型糖尿病(T1DM)只能通过终生注射外源性的胰岛素进行治疗，长期注射胰岛素费用昂贵，还会产生胰岛素抵抗，降低治疗效果以及带来感染、低血糖、过敏反应等一系列慢性并发症的风险。而T1DM被认为可通过外源性移植胰岛β细胞来治愈并且目前的研究已取得令人瞩目的进展[Bellin MD,Barton FB,Heitman A,Alejandro R,Hering BJ.Potent induction immunotherapy promotes long-term insulinindependence after islet transplantation in type 1diabetes.American Journal of Transplantation.2012；12:1576-83.]，但外源性胰岛或β细胞的缺乏严重阻碍了这种治疗方式的应用。如果能够获得内胚层干/祖细胞，并进行大量的扩增然后再对其进行诱导分化进而获得大量的功能性肝细胞、胰腺β细胞或肠上皮细胞将会对肝病的细胞治疗，糖尿病的细胞治疗甚至肠道疾病的治疗带来巨大的希望。
多项研究已经报道从多能性干细胞ESCs或iPSCs来获得内胚层干/祖细胞并将其向肝脏和胰腺进行诱导分化[Sneddon JB,Borowiak M,Melton DA.Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme.Nature.2012；491:765-8.Cheng X,Ying L,Lu L,Galvao AM,Mills JA,Lin HC,et al.Self-renewing endodermal progenitor lines generated from human pluripotent stem cells.Cell Stem Cell.2012；10:371-84.]，然而由于其来源于多能性干细胞，同样难以回避前述的多能性干细胞应用面临的问题。目前国际上多项研究都在致力于使用小分子将人的成体细胞重编程为内胚层祖细胞或者其来源的肝脏细胞和胰腺细胞。2014年丁盛实验室使用Yamanaka四因子OSKM在人成纤维细胞中短暂过表达并且结合小分子使细胞首先进入一个不同于ESCs或iPSCs多能性状态的一个可塑中间态，然后再给予内胚层的诱导条件将成纤维细胞重编程到内胚层祖细胞阶段(EPs)，接着使用肝细胞的序贯诱导方法最终获得了类肝细胞样的细胞[Zhu S,Rezvani M,Harbell J,Mattis AN,Wolfe AR,Benet LZ,et al.Mouse liver repopulation with hepatocytes generated from human fibroblasts.Nature.2014；508:93-7.]。近期使用类似的方案，他们将人成纤维细胞重编程到内胚层祖细胞阶段后，进而向胰腺进行诱导分化，从而获取了功能性的β细胞[Zhu S,Russ HA,Wang X,Zhang M,Ma T,Xu T,et al.Human pancreatic beta-like cells converted from fibroblasts.Nat Commun.2016；7:10080.]。尽管这一方式不经过多能性状态，因此避免了多能性干细胞的成畸胎瘤性，但其依然需要OSKM四因子的始发驱动，因此还是一个部分依赖于转录因子的策略，还未做到完全依赖小分子的作用。分析目前整个重编程领域还未实现仅使用小分子来介导谱系重编程获得上皮源性的内胚层谱系细胞类型包括内 胚层祖细胞、肝细胞或胰腺细胞的原因，我们认为存在以下几个关键的科学问题：
1、起始细胞的问题：首先，无论是经典重编程iPSCs还是谱系重编程，绝大多数都是以成纤维细胞为起始细胞，尽管成纤维细胞在获取上比其它成体细胞相对容易，通过皮肤活检即可获得，因此也是临床应用的一大优势，然而对于期望获得的目的细胞如内胚层祖细胞、肝细胞或胰腺细胞而言，以成纤维细胞为起始存在表观遗传的障碍。成纤维细胞来源于中胚层，而目的细胞内胚层祖细胞则属于内胚层，由中胚层向内胚层的跨胚层转换是一个巨大障碍，尤其是对于作用较为温和的小分子而言显得更为困难。其次，成纤维细胞在组织学分类上属于间质类细胞，而内胚层祖细胞则属于上皮类细胞，由成纤维细胞向内胚层祖细胞的转换需要经历间质上皮转换(MET)的过程，而MET已经被证实是iPSCs产生过程中需要突破的首个重要障碍[Li R,Liang J,Ni S,Zhou T,Qing X,Li H,et al.A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts.Cell Stem Cell.2010；7:51-63.]，同样MET也是谱系重编程的重要障碍。
2、小分子的选择问题：小分子的选择与起始细胞的特性密切相关，不同的起始细胞决定了在向内胚层祖细胞的转换过程所需要的小分子组合也不尽相同。由于目前还没有完全使用小分子获取内胚层的任何一种细胞类型的报道来作为参考，因此在小分子的选择上要经过详尽的调研和筛选。
3、滋养层细胞的使用问题：滋养层细胞可以分泌多种细胞因子，可溶性细胞外基质或MicroRNA等，并且和培养的细胞密切接触，给细胞的生长和存活提供合适的细胞外微环境。滋养层细胞对于干/祖细胞的培养和重编程来说通常是必须的，而目前无论是重编程还是谱系重编程最常用的滋养层细胞都是小鼠胚胎成纤维细胞(MEFs)、人包皮成纤维细胞(HFFs)或间充质干细胞(MSCs)等，这些细胞仅能提供基础的干细胞生长环境并不具有谱系特异的支持和诱导作用。
发明内容
本发明的目的是提供可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、及能将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程试剂盒和重编程方法。
本发明中，“干/祖细胞”的概念为“干细胞”或“祖细胞”，所指为同一种细胞，因业内命名不统一，统称为“干/祖细胞”。
本发明所提供的可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合，所述小分子化合物选自TGF-β信号通路、表观修饰剂、钙离子通道激动剂和代谢通路调节剂等功能群；代表性的含有8个小分子化合物的组合(8M)，包括FBP、 Bay K 8644(Bay)、Bix01294((Bix)、SB431542(SB)或A83-01(A83)、VPA、RG108(RG)、PD0325901和PS48中的全部或多个包括SB或A83在内的化合物的组合。
其中，包括8个小分子化合物的组合(简称8M)为：SB43154：VPA：PD0325901：RG108：Bix01294：Bay K 8644：PS48：FBP按摩尔比为50：12500：12.5：1:12.5：50:125：87500的组合；或A83：VPA：PD0325901：RG108：Bix01294：Bay K 8644：PS48：FBP按摩尔比为12.5：12500：12.5：1:12.5：50:125：87500的组合；按以上配比的8M组合在使用中需满足RG使用浓度为0.01～1μM，优选0.04μM。
优选的可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合为包括4个小分子化合物的组合，为Bix01294(Bix)、Bay K 8644(Bay)、RG108(RG)和SB431542(SB)，简称BBRS组合，或A83-01(A83)、Bay K 8644(Bay)、RG108(RG)和SB431542(SB)，简称BBRA组合。
所述小分子化合物组合中各化合物使用浓度分别为：SB43152为(1～10μM)，A83为(0.4～1μM)，RG108为(0.01～1μM)，Bix01294为(0.1～2μM)，Bay K 8644为(1～4μM)。
其中BBRS组合为各化合物按SB：RG：Bix：Bay摩尔比50:1:12.5:50的组合，BBRA组合为各化合物按A83：RG：Bix：Bay摩尔比12.5:1:12.5:50的组合；优选各化合物使用浓度SB43152为2μM，A83为0.5μM，RG108为0.04M，Bix01294为0.5μM，Bay K 8644为2μM。
本发明中，所述小分子化合物组合在以胃肌成纤维细胞或小肠肌成纤维细胞为滋养层细胞的支持下能将消化道来源的上皮细胞重编程为内胚层干/祖细胞。
本发明所提供的可将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程试剂盒，包括上述可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合，以及化合物的使用说明书。各化合物分别包装，或按8M组合或BBRS组合或BBRA组合将各化合物混合后包装，说明书记载各化合物的使用浓度范围或具体浓度。
所述重编程试剂盒还包括滋养层细胞及其使用说明，所述滋养层细胞为消化道来源的基质细胞，如胃肌成纤维细胞或小肠肌成纤维细胞。
所述重编程试剂盒还包括基础培养基Advanced DMEM/F12，细胞培养基础添加成分谷氨酰胺(Glutamax)和抗生素(SP)，及其使用说明，其中相对Advanced DMEM/F12基础培养基，谷氨酰胺的使用浓度为2mM(1×)，抗生素为青-链霉素,相对Advanced DMEM/F12基础培养基的使用浓度为100U/mL青霉素+0.1mg/mL链霉素，各物质分别包装或按所列使用浓度与基础培养基混合后包装。
本发明还提供一种可将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程 培养基。该重编程培养基由所述小分子化合物组合与基础培养基和细胞培养基础添加成分共混而成。
优化的重编程培养基的配方为：Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，2μM的SB43154，0.5mM的VPA，0.5μM的PD0325901，0.04μM的RG108，0.5μM的Bix01294，2μM的Bay K8644，5μM的PS48，3.5mM的FBP。所列浓度均为使用浓度，即各组分在Advanced DMEM/F12(溶剂)中的浓度。
更优选的重编程培养基配方为：Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，1～10μM的SB43152，0.01～1μM的RG108，0.1～2μM的Bix01294，1～4μM的Bay K 8644；这里最优选Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，2μM的SB43152，0.04μM的RG108，0.5M的Bix01294，2μM的Bay K 8644。
另一更优选的重编程培养基配方为：Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，0.4～1μM的A83-01，0.01～1μM的RG108，0.1～2μM的Bix01294，1～4μM的Bay K 8644；这里最优选Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，0.5μM的A83-01，0.04μM的RG108，0.5M的Bix01294，2μM的Bay K 8644。
本发明还提供上述小分子试化合物组合或剂盒或重编程培养基的应用，该应用是使用上述小分子化合物组合或试剂盒或重编程培养基在消化道来源的基质细胞做滋养层的基础下将消化道来源上皮细胞重编程为内胚层干/祖细胞。
本发明所提供的将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程方法，包括以下步骤：
1)将原代分离的消化道来源上皮细胞作为起始细胞，扩增培养所述消化道来源上皮细胞；
2)用丝裂霉素-C处理滋养层细胞后清洗，并用酶消化细胞后备用；
3)将步骤2)准备的滋养层细胞添加于步骤1)扩增培养的消化道来源上皮细胞中，继续共培养过夜；
4)第2天更换为重编程培养基，每2-3天换一次培养基，培养7-15天，得到诱导内胚层干/祖细胞(hiEndoPCs)克隆。
在上述方法中，所述步骤1)中的起始细胞为消化道来源上皮细胞，包括胃、十 二指肠上皮细胞。从临床获取的难易程度考虑，胃上皮细胞更易获取，因此优选为胃上皮细胞(hGECs)，尤其优选为NCAM(神经细胞黏附分子)阳性的胃上皮细胞(hGECs)。步骤1)优选以NCAM阳性的胃上皮细胞为起始细胞，用Kubota(库部塔)培养基在37摄氏度，5％CO2培养箱条件下培养5天。
所述步骤2)中的滋养层细胞为消化道来源的基质细胞，包括胃肌成纤维细胞或小肠肌成纤维细胞，优选人胃肌成纤维细胞(aGSEMFs)。步骤2)优选用丝裂霉素-C处理人胃肌成纤维细胞(aGSEMFs)2-3小时，用PBS清洗细胞，用TrypLE酶消化细胞。
步骤3)优选按1～3×105每平方厘米的密度将步骤2)准备的滋养层细胞添加于步骤1)培养到5天的消化道来源上皮细胞中，在37℃、5％CO2培养箱中过夜(12-16小时)。
步骤4)中所述重编程培养基为小分子化合物组合与基础培养基和细胞培养基础添加成分的混合物，其具体组成如前所述。
具体的，将消化道来源上皮细胞重编程为内胚层干/祖细胞的方法，包括以下步骤：
1)将原代分离的消化道来源上皮细胞作为起始细胞，用无血清Kubota’s medium(库部塔培养基)在37摄氏度，5％CO2培养箱条件下培养5天；
2)用丝裂霉素-C处理滋养层细胞2-3小时，用PBS清洗细胞，用TrypLE酶消化细胞；
3)按1～3×105每平方厘米的密度将步骤2)准备的滋养层细胞添加于步骤1)培养了4-5天的起始上皮细胞中，在37℃、5％CO2条件下共培养过夜(12-16小时)；
4)第2天起，更换为重编程培养基，每2-3天换液一次，培养7-15天后，获得诱导内胚层干/祖细胞(hiEndoPCs,human induced endodermal progenitor cells)克隆。
用上述重编程方法获得的内胚层干/祖细胞(hiEndoPCs)也属于本发明。
本发明还提供了一种对内胚层干/祖细胞(hiEndoPCs)进行传代的方法，包括以下步骤：
1)传代前准备：在孔板中接种经丝裂霉素-C(10μg/mL)处理的成人胃肌成纤维细胞(aGSEMFs)或提前约3个小时将Fibronectin(FN，纤连蛋白)、Cell-TAK(CT，细胞组织粘附剂)胶铺于孔板中，室温晾干；
2)配制传代培养基：Advanced DMEM/DF12+AWF(A83-01 0.5μM+Wnt3a50ng/mL+bFGF 10ng/mL)或Advanced DMEM/DF12+A(A83-01，0.5μM)；
3)手动挑取内胚层干/祖细胞(hiEndoPCs)克隆，并将其划成小块，比例约1:3-，将其置于FN+AWF、CT+AWF或Feeder[滋养层细胞，经丝裂霉素-C(10μg/mL)处理的成人胃肌成纤维细胞(aGSEMFs)]+A培养基中在37℃、5％CO2培养箱中进行传代培养得到传代内胚层干/祖细胞。
以上获得的内胚层干/祖细胞(hiEndoPCs)或传代内胚层干/祖细胞在向肝脏细胞、胰腺β细胞和肠细胞诱导分化中的应用也属于本发明。
本发明提供了可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、重编程试剂盒、重编程培养基以及将消化道来源上皮细胞重编程为内胚层干/祖细胞的方法。本发明将人胃上皮细胞(hGECs)作为起始细胞，从人胃组织肌层分离培养获取成人胃肌成纤维细胞(aGSEMFs)来作为滋养层，用小分子化合物组合在滋养层细胞的支持下将人胃上皮细胞谱系重编程为内胚层干/祖细胞，并可进一步进行传代培养和扩增。利用本发明获得的内胚层干/祖细胞或其传代细胞并结合使用相应的诱导分化体系，可获取成熟的肝脏细胞、胰腺β细胞和肠细胞，期望给肝病、糖尿病以及肠道疾病的细胞治疗提供理想的种子来源，应用前景广阔。
下面结合具体实施例对本发明做进一步详细说明。
附图说明
图1为确定了起始细胞、初始小分子以及滋养层细胞后的本发明的技术路线图；
图2为相差显微镜下培养4-5天的胃上皮细胞(hGECs)和十二指肠上皮细胞(hDECs)形态图(标尺：100μm)；
图3为相差显微镜下传代培养的胃肌成纤维细胞形态图(标尺：100μm)；
图4为胃上皮细胞中胃特异性及内胚层祖细胞特异性蛋白表达图；
图5为人胃上皮细胞(hGECs)向内胚层祖细胞(hiEndoPCs)重编程的动态过程细胞形态图(标尺：100μm)；
图6为人十二指肠上皮细胞(hDECs)及其来源的内胚层祖细胞(hiEndoPCs)形态图(标尺：100μm)；
图7为分别以人胃上皮细胞(hGECs)(A)或胃肠道肌成纤维细胞(GSEMFs)(B)为起始细胞的重编程过程细胞形态图(标尺：100μm)；
图8为克隆形态全景扫描图，显示第7天(A)和第15天(B)的不同克隆(用虚线勾画大致轮廓)，C、D、E、F为代表性克隆的具体形态；
图9为克隆面积计算中不同大小克隆所占格子示意图(标尺：250μm)；
图10为小分子化合物(8M)与各种滋养层细胞、细胞外基质或条件培养基支持下的克隆形成效率柱状图(不同的支持条件下重编程的效率)；
图11为MSC、MEFs和aGSEMF-CM支持作用下的细胞形态图(标尺：100μm)；
图12为8个小分子化合物(8M)逐一减去的克隆形成效率柱状图；
图13为Bix01294(Bix)、Bay K 8644(Bay)、RG108(RG)和SB431542(SB)4个小分子化合物逐一减去的克隆形成效率柱状图；
图14为Bix01294(Bix)、Bay K 8644(Bay)、RG108(RG)和SB431542(SB)4个小分子逐一减去的克隆形态图(标尺：100μm)；
图15A为以SB431542为基础并和其它三种小分子化合物组合下的克隆形成效率柱状图；
图15B为以A83-01为基础并和其它三种小分子化合物组合下的克隆形成效率柱状图；
图16为以SB431542为基础并和其它三种小分子化合物组合下的克隆形态图；
图17为小分子化合物优化过程的流程图；
图18为用荧光原位杂交(FISH)进行起始细胞的初步定位染色图(A:男性来源的hGECs重编程后的FISH染色情况；B：女性来源的hGECs重编程后的FISH染色情况)；
图19为胃窦部组织CD56(NCAM)免疫荧光染色图(标尺：50μm)；
图20为起始细胞人胃上皮细胞(hGECs)的NCAM流式分选及相应亚群的重编程情况示意图(线条勾画的是不同克隆的大致轮廓)；
图21为诱导内胚层祖细胞(hiEndoPCs)中内胚层祖细胞特异性蛋白的流式水平检测图；
图22为诱导内胚层祖细胞(hiEndoPCs)中胃特异性及内胚层祖细胞特异性蛋白表达检测染色图；
图23为诱导内胚层祖细胞(hiEndoPCs)中内胚层祖细胞标志基因表达分析结果柱状图，(A)和胃特异性基因的表达分析结果(B)；
图24为DNA甲基化芯片显示重编程前后全基因组DNA表观遗传的改变图(A:hGECs与hiEndoPCs的聚类分析；B：hiEndoPCs比hGECs低甲基化基因的GO分析)；
图25A为火山图，显示hiEndoPCs与hGECs相比，启动子区域甲基化水平发生改变的基因数量；B为hiEndoPCs与hGECs在FOXA2和GATA4基因启动子区域的甲基化水平比较图；
图26为RNA深度测序相关性分析图，显示hiEdoPs与ESC来源的DE、PGT、PFG的关系；
图27为PCA分析图，显示hGECs、hDECs、hGECs来源的hiEndoPCs、hDECs来 源的hiEndoPCs、ESCs来源的DE、GSEMFs在全基因表达谱上的位置关系；
图28为hGECs与hiEndoPCs微观形态图，(标尺:2μm(A-D)，100nm(E))；
图29为hiEndoPCs在重编程过程中的动力学特征图，A为hiEndoPCs重编程过程中的时期划分轴向图，B为不同时期的增殖速率曲线图，C为PhaseⅡ和PhaseⅢ期的细胞形态的动态变化图；
图30为传代后的hiEndoPCs在不同支持环境下的细胞形态图(标尺：100μm)；
图31为hiEndoPCs在传代过程中的细胞形态图(标尺：200μm)；
图32为hiEndoPCs传代次数及每代的细胞数量柱状图；
图33为hiEndoPCs向胰腺诱导分化结果，A为hiEndoPC-Pans的三维形态学图；B为hiEndoPC-Pans DTZ染色图；C和D为hiEndoPC-Pans胰腺特异性蛋白表达图(标尺：50μm)；E为hiEndoPC-Pans的胰腺特异性基因表达图；F为hiEndoPC-Pans的胰岛素释放反应图；
图34为hiEndoPCs向肠道诱导分化结果，A为hiEndoPC-Ints的形态学图；B为hiEndoPC-Ints肠特异性的基因表达图；C为hiEndoPC-Ints的肠特异性蛋白染色图(标尺：50μm)；
图35为hiEndoPCs向肝脏的诱导分化结果，A为hiEndoPC-Heps的肝特异性基因表达图；B为hiEndoPC-Heps的AFP和CK18染色图(标尺：50μM)；C为hiEndoPC-Heps的ALB和AFP流式水平检测图；
图36为hiEndoPCs向甲状腺(A)和肺(B)诱导分化的基因表达水平检测柱状图。
具体实施方式
在确定了起始细胞、初始小分子以及滋养层细胞后，本发明的技术路线(如图1所示)为：获取人胃上皮细胞(hGECs)作为起始细胞，从人胃组织肌层分离培养获取消化道肌成纤维细胞(aGSEMFs)来作为滋养层，筛选合适的小分子组合并在滋养层细胞的支持下将人胃上皮细胞谱系重编程为内胚层干/祖细胞。
首先，分离培养人胃来源的上皮细胞(hGECs)作为谱系重编程的起始细胞，通过初步筛选，确定八个小分子化合物组合：SB431542+VPA+PD0325901+RG108+Bix01294+BayK8644+PS48+FBP，并在人胃来源的肌成纤维细胞(GSEMFs)作为滋养层的条件下可以将人胃上皮细胞(hGECs)重编程为内胚层祖细胞(hiEndoPCs)样的克隆。以能够出现内胚层祖细胞样克隆为标准，然后对重编程的体系—小分子化合物和滋养层细胞进行了优化，最终确定Bix01294+BayK8644+RG108+SB431542(BBRS)为最优的小分子化合物组合，而且确定 了能够实现重编程的必需小分子化合物是SB431542。由于滋养层细胞—人胃肌成纤维细胞(GSEMFs)的获取最为便利，因此确立了重编程最优的体系是BBRS+GSEMFs，在这样的体系中重编程的效率为4％-6％；此外，本发明还证实了内胚层祖细胞(hiEndoPCs)来源于NCAM阳性的hGECs，实现了起始细胞的精确定位。
其次，还对诱导的内胚层祖细胞(hiEndoPCs)从内胚层特征性的基因、蛋白表达水平、表观遗传水平、全基因组表达水平、细胞微观形态水平及细胞增殖能力、内胚层分化潜能对诱导内胚层祖细胞(hiEndoPCs)做了全方位鉴定。首先检测了常见的内胚层祖细胞特征性的转录因子FOXA2、SOX9、HNF1B、PDX1、GATA4，其它干/祖细胞特征性蛋白包括CXCR4、EPCAM、LGR5、CK19以及胃上皮特异性的标志MΜC6、GASTRIN等在hiEndoPCs中的表达情况，发现与hGECs相比内胚层标志性的基因在hiEndoPCs中明显上调，而胃特异性的基因则不表达，初步证实了hGECs向内胚层祖细胞重编程成功。表观遗传分析也显示重编程后细胞发生了显著改变，具备了内胚层祖细胞的分子特征。对hiEndoPCs的发育阶段进行精确定位，深度测序的结果显示hiEndoPCs处于hESCs来源的原始消化管(PGT)和前肠后段(PFG)这两个内胚层发育阶段之间，且更接近PFG。此外，hiEndoPCs具有干细胞微观水平的特征并能进行4-6次的传代扩增。尽管与ESCs或iPSCs相比，hiEndoPCs的扩增潜能有限，然而以106起始细胞为起点经过重编程和4-6代的扩增可以获得约109的细胞，这样的数量足以进行细胞治疗且在有限的传代次数内细胞的基因组相对稳定，更加安全，也更加有利于未来的细胞治疗。由于发育过程中内胚层祖细胞具有向胰腺、肝脏、肠、肺和甲状腺等内胚层器官进行分化的潜能，因此使用相应的诱导分化条件发现hiEndoPCs同样具有形成功能性胰腺β细胞、肝细胞、肠道细胞、肺细胞和甲状腺细胞的能力。
下述实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《MolecμLar Cloning:A Laboratory Manμal》(Sambrook，J.，Rμssell，David W.，MolecμLar Cloning:A Laboratory Manμal，3rd edition，2001，NY，Cold Spring Harbor)。
所述百分比浓度如无特别说明均为质量/质量(W/W，单位g/100g)百分比浓度、质量/体积(W/V，单位g/100mL)百分比浓度或体积/体积(V/V，单位mL/100mL)百分比浓度。
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。
实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。
实施例1、小分子化合物诱导人胃上皮细胞向内胚层祖细胞转换的体系建立
本发明重编程的目的细胞是内胚层祖细胞，考虑到肝脏、胰腺、胆道、胃、肠等从发育的角度来看都来源于内胚层，与内胚层祖细胞发育同源，表观遗传相似，重编程障碍相对较小，确定其是良好的起始细胞类型。其次，临床上胃大部切除术或胃癌切除术等消化道手术后，不可避免的会有部分正常的胃或十二指肠组织被遗弃，而且胃溃疡或十二指肠溃疡愈后胃镜活检随诊，也可获得的正常的胃或十二指肠的组织，因此起始细胞的来源也是可行的。本发明建立胃上皮细胞和滋养层细胞的培养体系，然后使用选择的四大类共八个小分子并且以消化道肌成纤维细胞做滋养层来进行向内胚层祖细胞的重编程。在这样的条件下成功的在胃上皮或十二指肠上皮细胞中产生了内胚层干/祖细胞样的克隆。重编程成功后，进一步对这个体系进行了优化，找到了最优的重编程小分子组合和最少的小分子组合。最后对起始细胞进行了分群，明确了能够重编程成功的起始细胞亚群。
本发明将消化道来源上皮细胞重编程为内胚层干/祖细胞的总体思路和策略如下：
1、在起始细胞的选择上，肝脏、胰腺、胃肠等内脏器官都来源于内胚层祖细胞，它们在发育上同源，因此与其它胚层来源的细胞相比，它们之间具有更近的亲缘关系，更多的表观遗传相似性。此外，它们都是上皮组织类型细胞，在转换的过程中不需要经历MET的障碍，因此在理论上它们之间的细胞类型转换也就相对容易。此外，胃肠道组织细胞更新速度较快，大量细胞处于活跃增殖状态，增殖快速的细胞或祖细胞更易被重编程。胃肠道(GI-tract)是连接内脏各个器官的通道，各种消化道手术都会有大量切除的正常组织被遗弃，同时胃十二指肠组织通过活检也可获得。因此以GI-tract来源的人胃上皮细胞(hGECs)为起始细胞向内胚层祖细胞进行重编程，在与以成纤维细胞为起始相比，具有表观同源性的优势，上皮源性的优势，增殖祖细胞的优势和也具有临床操作的可行性。
2、在小分子的选择上，确定了四大类功能明确的，能够正向调控重编程的小分子群，分别是：
(1)信号通路抑制剂：TGF-β信号通路抑制剂SB431542(SB)或A83-01(A83)，SB或A83可以替代Sox2，与Oct4、Klf4、c-Myc组合就可产生iPSCs，另一个是MAPK/ERK信号通路抑制剂PD0325901(PD)，PD与SB(或A83)组合可将重编程效率提高100 多倍，而且大大缩短重编程时间；
(2)表观修饰剂：组蛋白去乙酰化酶抑制剂VPA，VPA甚至可以取代Klf4、c-Myc两个转录因子，与Oct4、Sox2组合可以促进iPSCs的生成，组蛋白甲基转移酶抑制剂Bix01294(BIX)、DNA甲基转移酶抑制剂RG108(RG)都可明显提高重编程效率；
(3)钙离子通道激动剂Bay K 8644(Bay)，Bay与Bix的组合则可取代Sox2与c-Myc；
(4)代谢通路调节剂：磷酸肌醇依赖性蛋白激酶1激动剂PS48、磷酸果糖激酶激动剂FBP，有利于成熟细胞氧化磷酸化的能量代谢向干细胞无氧糖酵解的途径转换，从而提高重编程效率。
3、在滋养层细胞的选择上，为了促进内胚层祖细胞的产生和维持，选择了与内胚层器官紧密相关的消化道肌成纤维细胞作为滋养层细胞，这些细胞在发育的早期会通过旁分泌的作用影响内胚层器官的发生和支持内胚层祖细胞的扩增。
一、胃上皮细胞的分离培养及表型鉴定
材料与方法
以下所列材料与方法源自实验的原始记录。在本发明实际应用中，可以使用商业来源的材料和符合工业应用的方法实施，不受所列实验的限制。
(一)实验材料
(1)实验组织
手术或者活检来源的胃、十二指肠组织由中国人民解放军总医院和307医院普外科提供，流产胎儿组织由解放军总医院提供，所有提供组织的患者均已知情并签署知情同意书，组织标本使用经解放军总医院和307医院伦理委员会同意。
(2)实验器材
激光共聚焦显微镜(Zeiss)、恒温水浴锅(长风)、低温离心机(Eppendorf)、倒置相差显微镜(Leica)、手术器械(手术刀柄、刀片、眼科直镊、弯镊、剪刀)、激光共聚焦小皿(NEST)。
(3)主要试剂及配制
1、Kubota Medium(KM)培养基的配制：一袋RPMI 1640(Gibco)粉末溶解于1L去离子水中，加入1×青-链霉素，10-9M Zinc SuLfate heptahydrate(七水硫酸锌，Sigma)，0.54g Nicotinamide(烟酰胺，Sigma)，5mg InsuLin(胰岛素，Sigma)，10-6M hydrocortisone(氢化可的松，Sigma)，2g NaHCO3(Sigma)，5×10-5M beta-mercaptoethanol(β巯基乙醇，Sigma)，30nM Selenium(硒，Sigma)，游离脂肪酸(Sigma)，10μg/mL High density lipoprotein(高密度脂蛋白，Sigma)， 1g Bovine Serum Albumin(牛血清白蛋白，购自Gibco)，5mg Transferrin(转铁蛋白，Sigma)，2mM Glutamax(谷氨酰胺，购自Gibco)。
2、无Ca2+、Mg2+PBS的配制：使用1L去离子水溶解0.24g KH2PO4，8.0g NaCl，0.2g KCl，1.44g Na2HPO4，可根据配制的体积等比例添加试剂。配制完成后0.45μM滤膜过滤后高压蒸汽灭菌或直接0.22滤膜过滤后使用。
3、主要抗体：
表1 用于人胃上皮细胞(hGECs)免疫荧光检测的第一抗体
一抗 公司 货号 一抗种属 稀释比
CXCR4(C-X-C基序趋化因子受体4) Abcam ab77909 兔 200
EPCAM(上皮细胞粘附分子) Neomarker MS-181 小鼠免疫球蛋白1 200
FOXA2(叉头编码框蛋白A2) R&D AF2400 山羊免疫球蛋白 100
SOX9(性别决定区域Y基因9) Abcam ab76997 小鼠免疫球蛋白2a 50
CK19(细胞角蛋白19) Abcam ab7754 小鼠免疫球蛋白2a 200
LGR5(富含重复亮氨酸G蛋白偶联受体5) Sigma HPA012530 兔 350
GASTRIN(胃泌素,GAST) Santa Cruz sc-7783 山羊 50
MUC6(黏膜蛋白-6) Abcam ab49462 小鼠免疫球蛋白1 50
表2 用于人胃上皮细胞(hGECs)免疫荧光检测的第二抗体
Figure PCTCN2017086336-appb-000001
4、0.075mg/mLⅣ型胶原酶的配制：200mL Advanced RPMI 1640培养基(购自Gibco)中加入20mgⅣ型胶原酶(Sigma)和6mg DNase A。
5、其它试剂和材料：TrypLE消化酶(购自Invitrogen)、4％(V/V)多聚甲醛(购自Sigma)、DNase A(Invitrogen)、0.2％(V/V)Triton X-100(聚乙二醇辛基苯基醚，购自Sigma)、胎牛血清(FBS，Gibco)、枸橼酸钠缓冲液(购自Sigma)、DAPI(4',6-二脒基-2-苯基吲哚，Sigma)、免疫组织化学试剂盒(Vector lab)。
(二)实验方法及结果
(1)获取胃上皮细胞
胃上皮细胞可直接购买获得；也可用以下操作分离培养获得胃窦、幽门部或十二指肠粘膜上皮细胞：
1)、提前将配制好的Ⅳ型胶原酶放置于室温或37℃平衡，将PBS置于冰上预冷，准备1000×青-链霉素，75％(V/V)酒精消毒手术器械。
2)、从医院获取新鲜的胃手术后胃窦、幽门或十二指肠组织标本，尽量半个小时内用冰盒运输到实验室。
3)、在预冷的PBS中加入5×的青-链霉素并用其清洗获取的组织标本4-5次，充分洗去残余血污。
4)、钝性分离清洗后的胃组织粘膜层与肌层，同样用含5×青-链霉素的冷PBS分别清洗粘膜层和肌层，而后进行不同的处理。
5)、使用剪刀和手术刀结合将粘膜层剪碎或剁碎，加入适量的Ⅳ型胶原酶(0.075mg/mL)37℃水浴消化，每2-3分钟振荡一次，8-10分钟沉降一次分离物，每次冰上沉降2-3分钟，然后滴管小心收集上清，余下部分继续加适量的酶消化。
6)、重复步骤5，直至无明显的大块组织即终止。
7)、在收集的上清中加入预冷PBS，4℃、1200rpm(转/每分钟)离心5分钟，洗涤3-4遍，充分去除细胞中残留的消化酶。
8)、用含有8％(V/V)胎牛血清(FBS)和1×青-链霉素的KM培养基以合适密度接种细胞，贴壁过夜，次日换为无血清的KM培养基进行培养。
9)、在培养的过程中每2-3天换液，大概培养4-5天即可开始重编程。
结果：将从临床获取的胃或十二指肠组织进行分离可得到粘膜层和肌层，粘膜层用于分离胃或十二指肠上皮细胞，肌层则用于分离滋养层细胞—肌成纤维细胞。按照实验步骤中胃或十二指肠上皮的组织分离方法，通常得到的是接近隐窝部的细胞。使用无血清的上皮细胞筛选培养基库部塔(Kubota’s medium)培养，人胃上皮细胞(hGECs)和十二指肠上皮细胞(hDECs)在培养中都呈现典型的舞蹈样的上皮形态(如图2所示)。经过观察，大约第4-5天细胞的数量达到最多，此后细胞进入逐渐凋亡的状态。因此，通常以培养到4-5天后的细胞，来作为向内胚层祖细胞重编程的起始细胞。而且经过对几十例胃和十二指肠不同部位细胞分离培养的观察和分析来看，发现胃窦、幽门或十二指肠来源的组织无论是在细胞贴附还是生长方面都最好，胃体部或胃底、贲门部组织则几乎不能贴附或很难培养。又由于十二指肠组织来源相对较少，因此在后续的实验中除非特别提及，都以胃窦部或胃幽门部组织作为来源，分离获取胃上皮细胞来作为重编程的起始细胞。
(2)肌成纤维细胞的获得
肌成纤维细胞可直接购买获得；也可用以下贴壁法分离和培养胃肠道肌成纤维细胞：
1)、对前述获得的胃或十二指肠肌层使用手术刀片充分剁碎为小组织块，越小越好，加入少量的含15％(V/V)胎牛血清(FBS)的高糖培养基或KM培养基润湿组织碎块，使用1毫升小滴管将组织碎块均匀涂抹于10cm培养皿中，倒置于37℃培养箱30分钟。其余基质细胞分离培养方法如上述。
2)、贴附30分钟后，沿培养皿边缘加入基质细胞生长培养基，将细胞置于37℃、5％CO2的培养箱中培养。
3)、经过5-7天培养可见有梭形细胞从组织块周围爬出，2周后大量细胞爬出并生长，此时进行细胞传代、冻存。这样即可获得胎儿胃的肌成纤维细胞(fGSEMFs)、成人胃的肌成纤维细胞(aGSEMFs)、胎儿肠肌成纤维细胞(fISEMFs)、胎儿横隔基质细胞(fDCs)。冻存后的细胞可为以后重编程提供滋养层细胞。
结果：将得到的胃或十二指肠肌层机械法剁碎成小块使用组织贴壁法分离培养肌成纤维细胞(GSEMFs)。组织贴附约4-7天，可以看到有梭状的细胞从组织块中爬出，10-14天可以进行传代，传代培养的肌成纤维细胞呈典型的长梭样的成纤维细胞形态(如图3所示)。
(3)胃上皮细胞的免疫荧光检测
对人胃上皮细胞(hGECs)特性进行免疫荧光鉴定，包括以下操作(一抗、二抗分别如表1和表2所示)：
1)、4％(V/V)多聚甲醛固定细胞10-15min，PBS清洗2遍。
2)、0.2％(V/V)Triton X-100破膜10分钟，PBS清洗一遍。
3)、二抗种属来源的血清封闭1小时，PBS清洗一遍。
4)、相应一抗孵育4℃过夜，PBS清洗2-3遍。
5)、相应二抗室温避光孵育1小时，PBS清洗2-3遍。
6)、DAPI室温避光孵育15分钟。
7)、激光共聚焦荧光显微镜下观察并摄像。
结果：如图4所示，免疫荧光染色发现人胃上皮细胞(hGECs)低表达内胚层祖细胞标志CK19、FOXA2、CXCR4、SOX9、LGR5、EPCAM等，而高表达胃特征性标志MUC6和GAST。
第一部分“胃上皮细胞的分离培养及表型鉴定”成功建立了胃窦或幽门部胃上皮细胞和肌成纤维细胞的分离培养体系，体外培养的人胃上皮细胞(hGECs)呈典型的上皮样形态。由于隐窝部细胞通常较为原始(与胃成熟细胞类型相比)，可以进行一定程度的增殖。人胃上皮细胞(hGECs)高表达胃特异性标志，低表达内胚层早期祖细胞标志，与我们先前的推测一致，因其部分表达内胚层祖细胞的某些标志，在向内 胚层祖细胞的重编程过程中重编程障碍会相对较小，因此是向内胚层祖细胞进行转换的良好起始细胞来源。但是人胃上皮细胞(hGECs)的胃上皮特征性的标记在重编程的过程中则必须要丢失。解决了起始细胞的来源和培养问题，而且也初步明确了其特性，下一步将以其为起始细胞进行向内胚层祖细胞的重编程。
二、小分子化合物和滋养层细胞诱导胃上皮细胞向内胚层祖细胞转换
材料与方法
(一)实验材料
(1)实验细胞
人胃上皮细胞(hGECs)和成人胃肌成纤维细胞(aGSEMFs)在步骤一中制备并保存。
(2)实验器材
倒置相差显微镜(Leica)、显微镜格子线、低温离心机(Eppendorf)、12孔板。
(3)主要试剂及配制
1、基础试剂
Advanced DMEM/F-12培养基、Advanced RPMI 1640培养基、NEAA(非必需氨基酸)、TrypLE消化酶、谷氨酰胺(Glutamax)、Dispase(离散酶)均购自Gibco公司，青-链霉素和丝裂霉素-C(Sigma)。
2、小分子化合物
表3 8个小分子化合物(8M)
小分子名称 使用浓度 厂家
FBP(二磷酸果糖) 3.5mM Sigma
Bay K 8644(氟硝尼啶) 2μM Stemgent
Bix01294 0.5μM Stemgent
SB431542 2μM Stemgent
Valproic Acid(VPA，丙戊酸) 0.5mM Stemgent
RG108 0.04μM Stemgent
PD0325901 0.5μM Stemgent
PS48 5μM Stemgent
“使用浓度”是指各化合物以Advanced DMEM/F-12培养基为溶剂的浓度。
3、8M重编程培养基配制
配方：Advanced DMEM/F12+2mM谷氨酰胺(Glutamax)+青-链霉素(100U/mL青霉素+0.1mg/mL链霉素)+SB43154(2μM)+VPA(0.5mM)+PD0325901(0.5μM)+RG108(0.04μM)+Bix01294(0.5μM)+Bay K 8644(2μM)+PS48(5μM)+FBP(3.5mM)。
该配方中各组分浓度均为其以Advanced DMEM/F-12培养基为溶剂的浓度。
(二)实验方法及结果
(1)小分子化合物介导人胃上皮细胞(hGECs)或十二指肠上皮细胞(hDECs)向诱导内胚层祖细胞(hiEndoPCs)转换
具体方法包括以下步骤：
1、起始细胞的扩增：原代分离的人胃上皮细胞(hGECs)或十二指肠上皮细胞(hDECs)作为起始细胞，用Kubta培养基(参照文献进行配制，文献来源Kubota,H.,and Reid,L.M.(2000).Clonogenic hepatoblasts,common precursors for hepatocytic and biliary lineages,are lacking classical major histocompatibility complex class I antigen.Proc.Natl.Acad.Sci.USA 97,12132–12137)在37℃、5％CO2培养箱条件下培养4-5天，将起始细胞进行扩增。
2、滋养层细胞的准备：用丝裂霉素-C(10μg/mL，目的是使滋养层细胞失去有丝分裂的能力))处理滋养层细胞的成人胃肌成纤维细胞(aGSEMFs，该细胞由胃组织基质层分离获得，培养扩增后大量冻存，需要时提前复苏备用)2-3小时，PBS洗3-4遍，TrypLE酶消化细胞，而后洗涤细胞；
3、当待人胃上皮细胞(hGECs)或十二指肠上皮细胞(hDECs)培养至第5-6天时，将已处理的胃肌成纤维细胞以适宜密度(密度一般为1～3×105每平方厘米)添加于培养中的人胃上皮细胞(hGECs)或十二指肠上皮细胞(hDECs)中，在37℃、5％CO2培养箱中过夜(12-16小时)；
4、重编程培养：在添加了胃肌成纤维细胞的第二天更换为8M重编程培养基，每2-3天换一次培养基，连续观察，一般培养1周或2周以上(7-15天)，得到诱导内胚层祖细胞(hiEndoPCs)。
结果：
(1.1)人胃上皮细胞(hGECs)向诱导内胚层祖细胞(hiEndoPCs)的转换
利用8个小分子化合物(8M，如表3所示)，在8M重编程培养基和分离培养的胃肠道肌成纤维细胞(GSEMFs)作为滋养层细胞的培养条件下，人胃上皮细胞(hGECs)的形态不断发生明显变化，由开始的形态较大的舞蹈样或多角样上皮，在第7天开始出现边界较清晰的小的细胞克隆，在第15天左右，人胃上皮细胞(hGECs)已经被重编程为边界清晰，细胞小而紧密，形态较为均一，核质比较大的典型内胚层干/祖细胞样克隆(G-hiEndoPCs)，如图5所示(图5中下排图片为上排图片方框中的放大部分)。
(1.2)人十二指肠上皮细胞(hDECs)向内胚层祖细胞(hiEndoPCs)的转换
同样，以人十二指肠上皮细胞(hDECs)为起始，在8M重编程培养基和分离培养的胃肠道肌成纤维细胞(GSEMFs)作为滋养层细胞的作用下，经过15天的诱导也可形成典型的内胚层干/祖细胞克隆(D-hiEndoPCs)(如图6所示，左幅为十二指肠上皮细胞(hDECs)，右幅为十二指肠上皮细胞重编程产生的内胚层干/祖细胞(D-hiEndoPCs)，形态与G-hiEndoPCs(图5)几乎类似。由于十二指肠组织来源有限，通常以人胃上皮细胞(hGECs)作为重编程的起始细胞。
(1.3)仅以人胃上皮细胞(hGECs)或胃肠道肌成纤维细胞(GSEMFs)为起始细胞向内胚层祖细胞(hiEndoPCs)的转换
不使用小分子化合物，不能形成内胚层干/祖细胞样克隆，但是分别仅以人胃上皮细胞(hGECs)或胃肠道肌成纤维细胞(GSEMFs)为起始细胞，在8M重编程培养基中培养15天也均不能形成内胚层干/祖细胞样克隆(如图7所示)，说明小分子化合物(8M)、人胃上皮细胞(hGECs)和胃肠道肌成纤维细胞(GSEMFs)的组合是内胚层祖细胞(hiEndoPCs)形成不可或缺的因素。
(2)克隆形成效率的计算
具体方法包括以下步骤：
1、计算克隆的总数。
2、使用显微镜格子线计算不同克隆的面积大小：每个显微镜微格的面积为0.0625mm2，用克隆所占的格子数乘以0.0625mm2则是每个克隆的面积。
3、综合克隆的数目和克隆的面积大小来评价重编程的效率。
结果：在前述的观察中已经发现在重编程第7天时已经有小的细胞克隆开始出现，孔板全景扫描显示这样的小克隆界限清晰，数量非常多(图8之A)，在形态上已经接近经典的内胚层干/祖细胞克隆形态(图8之C和D)。由于在重编程第15天产生的克隆形态最典型(图8之E和F)，通常在这个时间点计算克隆形成效率，但第7天至15天克隆的生长速度较快，许多克隆在计算的时间节点融合在一起形成大的克隆(图8之B)，因此克隆的数目在计算时较少，但是总的面积在增大，为了更加客观的反应重编程效果，本实验综合克隆的总数目以及不同面积下的克隆数目和克隆面积来作为重编程效率计算方法。使用显微镜格子线计算不同克隆的面积大小：每个显微镜微格的面积为0.0625mm2，用克隆所占的格子数乘以0.0625mm2则是每个克隆的面积，不同大小的克隆面积计算方法如图9所示，计算结果参见表4。
表4:不同大小的克隆面积计算结果
  方格数 面积
a 12 0.750
b 0.1 0.006
c 6 0.375
第二部分在初步选择的八个小分子(8M)和成人胃肌成纤维细胞(aGSEMFs)作为滋养层的条件下成功的将人胃上皮细胞(hGECs)重编程到内胚层干/祖细胞(hiEndoPCs)的阶段，由于在重编程的过程中发生的最初的明显改变是细胞形态，因此将干/祖细胞克隆的出现作为重编程成功的初始判断。使用相同的方法同样可以将人十二指肠上皮细胞(hDECs)重编程为内胚层干/祖细胞(hiEndoPCs)。在重编程过程中，小分子化合物和滋养层细胞是必需的要素。尽管初步实现了我们的设想，但仍然需要找到最佳的重编程方案，尽可能的提高重编程效率，减少小分子化合物数目，而且找到关键的小分子将会对解析重编程的机制有着十分重要的提示作用。
三、小分子化合物和滋养层细胞重编程体系的优化
材料与方法
(一)实验材料
(1)实验细胞
人胃上皮细胞(hGECs)、胎儿胃肌成纤维细胞(fGSEMFs)、成人胃的肌成纤维细胞(aGSEMFs)、胎儿肠肌成纤维细胞(fISEMFs)、胎儿横隔基质细胞(fDCs)在步骤一中制备；间充质干细胞(MSCs)、小鼠胚胎成纤维细胞(MEFs)、成人皮肤成纤维细胞(HFFs)来源于包皮组织，由本实验室分离培养并保存。
(2)实验器材
倒置相差显微镜(Leica)、显微镜格子线、12孔板。
(3)主要试剂
1、基础试剂：Advanced DMEM/F-12培养基、Advanced RPMI 1640培养基、NEAA(非必须氨基酸)、谷氨酰胺(Glutamax)均购自Gibco公司；青-链霉素、丝裂霉素-C购自Sigma公司；TrypLE、Dispase消化酶购自Invitrogen公司；Matrigel胶购自BD公司；Gelatin购自Sigma公司。
2、小分子化合物组合(8M)：种类和使用浓度与第二部分相同(见表3)。
(二)实验方法及结果
(1)胎儿肠肌成纤维细胞(fISEMFs)或成人胃的肌成纤维细胞(aGSEMFs)条件培养基制备：用浓度10μg/mL的丝裂霉素C处理fISEMFs细胞或aGSEMFs细胞2-3小时，PBS洗3-4遍，然后加上Advanced(先进)DMEM/F-12培养基培养，每天收集培养基，0.22μM滤器过滤，保存于-80℃。
(2)不同的滋养层细胞支持人胃上皮细胞(hGECs)重编程为内胚层祖细胞(hiEndoPCs)
具体方法包括以下步骤：
1)、起始细胞人胃上皮细胞(hGECs)的处理与步骤二中的方法相同。
2)、各种滋养层细胞的准备与第二部分中的方法相同。
3)、重编程方法与第二部分中的方法相同。
4)、在重编程第15天计算小分子化合物(8M)与各种滋养层细胞、细胞外基质或条件培养基下支持下的克隆形成效率，进而找到合适的滋养层细胞。
结果：在前述的实验中使用8个小分子化合物(8M)和成人胃肌成纤维细胞(aGSEMFs)实现了人胃上皮细胞(hGECs)向内胚层祖细胞(hiEndoPCs)的转换。接下来确定更佳的滋养层细胞或实现无滋养层细胞的重编程。使用了多种消化道来源的基质细胞包括胎儿胃肌成纤维细胞(fGSEMFs)、胎儿肠肌成纤维细胞(fISEMFs)、胎儿横隔基质细胞(fDCs)以及其它常用的滋养层细胞如间充质干细胞(MSCs)、小鼠胚胎成纤维细胞(MEFs)、成人皮肤成纤维细胞(HFFs)等，还测试了多种细胞外基质如Matrigel胶、明胶等，以及胎儿肠肌成纤维细胞(fISEMFs)来源的和成人胃肌成纤维细胞(aGSEMFs)来源的条件培养基，分别来进行重编程的比较。在8M重编程培养基条件下，发现fISEMFs、fGSEMFs、aGSEMFs等消化道基质细胞和横隔基质细胞都可以成功的支持hGECs产生hiEndoPCs，其中又以胎儿肠肌成纤维细胞(fISEMFs)的重编程效率最高，其它的支持介质如MEFs、HFFs、MSCs、Matrigel、明胶以及条件培养基等则很少或几乎不能支持重编程(如图10所示)。以MSCs或MEFs为支持细胞会产生少量的不典型的上皮样细胞克隆。在成人胃肌成纤维细胞(aGSEMFs)来源的条件培养基(aGSEMFs-CM)中随着培养时间的延长，细胞在不断凋亡(如图11所示，图中右上角或左上角图片为小方框中的放大图)。由于胎儿组织来源有限，而临床上成人的胃手术标本最容易获取，可以得到足量的成人胃肌成纤维细胞(aGSEMFs)，而且aGSEMFs也可以维持较高的重编程效率，因此aGSEMFs是一类良好的重编程滋养层细胞，后续的实验中将以其为滋养层细胞来支持重编程。
(3)小分子组合的优化—重编程必要小分子的筛选
1)、以第二部分筛选的滋养层细胞为支持细胞，在前述的8个小分子化合物(8M)培养条件的基础上逐个递减小分子，首先筛选到不可或缺的小分子。
2)、再以第1)步筛选的小分子化合物组合为基础，依次逐个递减最终找到最佳(重编程效率最高)的小分子化合物组合和最少(保证克隆能够形成)的小分子化合物组合。
(4)统计学分析
所有数据均来自于3次及以上独立重复试验，除特殊说明外，数据均描述为平均值±标准差。两组数据间统计学分析用SPSS软件进行学生双尾t检验，P<0.05时两组之间的差异被认为具有统计学意义。
结果：在步骤(2)中确定了最合理的滋养层细胞—成人胃肌成纤维细胞(aGSEMFs)，以其作为支持细胞，来进行小分子组合的优化。以8个小分子化合物的组合(8M)为基础然后逐个递减小分子，当减去PD0325901、PS48、FBP后克隆的形成效率有所提高，说明这些小分子化合物在重编程中起到一定的抑制作用，应该舍去；当减去VPA后，克隆形成的效率没有明显变化，表明VPA可有可无，基于简单化原则，应该去掉；但当减去Bix01294、Bay K 8644、RG108时克隆的形成效率明显减少，尤其是当SB431542减去后克隆不能产成(如图12所示)，表明这四个小分子是必要的。
在前述必须的4个小分子化合物Bix01294、Bay K 8644、RG108、SB431542(简称BBRS组合)的组合上，诱导内胚层祖细胞(hiEndoPCs)同样可以产生而且效率比8个小分子化合物(8M)条件下更高。然后再以Bix01294、Bay K 8644、RG108、SB431542(BBRS组合)为基础再进行优化，当去除Bix01294(Bix)、Bay K 8644(Bay)、RG108(RG)任何一个小分子，克隆的形成效率都会明显下降(如图13所示)，但克隆的形态与BBRS组没有明显差异，当减去SB431542(SB)后则没有克隆形成(如图14所示)。因此确定了重编程最佳的组合是Bix01294、Bay K 8644、RG108、SB431542(BBRS)，在BBRS组合条件下重编程的效率约为4％-6％(如图13所示)。
由于SB431542的作用最为关键，因此以SB431542为基础，再与另外三个小分子(Bix01294(Bix)、Bay K 8644(Bay)、RG108(RG))的任意一个进行组合，发现SB431542是能够保证克隆形成的最少小分子组合，尽管效率不及最优组合BBRS(如图15A所示)，但形态都无明显差异(如图16所示)。SB431542不可或缺的作用对重编程过程中小分子化合物的作用机制有重要的提示作用。
(5)重编程必要小分子化合物的优化
在证实了重编程体系中SB431542(SB)是最少的能够保证重编程成功的小分子化合物后，由于SB431542是TGF-β信号通路抑制剂，发明人尝试使用另一种TGF-β信号通路抑制剂A83-01(A83)替代SB重复小分子化合物组合的优化实验，结果A83(使用浓度0.5μM)无论是单独作用还是与其他小分子组合(简称BBRA组合)都可以产生和SB类似甚至更好的重编程作用。结果见图15B，其中BBR分别代表另外三个小分子Bix01294(B)、Bay K 8644(B)、RG108(R)。
第三部分通过对滋养层细胞的筛选，确定了多种消化道肌成纤维细胞都可以支持人胃上皮细胞(hGECs)向内胚层祖细胞(hiEndoPCs)的转换，尤其是胎儿来源的基质细胞支持作用更强，提示与消化道相关的滋养层细胞对于内胚层祖细胞重编程的独特支持作用。考虑到细胞来源的充足情况，确定了成人胃肌成纤维细胞(aGSEMFs)为滋养层细胞。在aGSEMFs为支持细胞的基础上，通过3轮小分子的优化过程筛选到了最佳的重编程小分子组合Bix01294、Bay K 8644、RG108、SB431542(BBRS)或Bix01294、Bay K 8644、RG108、A83-01(BBRA)以及必要小分子SB431542或A83-01(A83)，其筛选过程如图17所示。后面实施例3中，发明人将以BBRS+aGSEMFs为例作为重编程条件，对人胃上皮细胞(hGECs)向内胚层祖细胞(hiEndoPCs)重编程做详细描述。
四、重编程起始细胞的精确定位
材料与方法
(一)实验材料
(1)实验细胞、组织
人胃上皮细胞(hGECs)、成人胃的肌成纤维细胞(aGSEMFs)、成人胃窦部组织(解放军总医院提供)。
(2)实验器材
流式细胞分析仪(BD)，激光共聚焦显微镜(Zeiss)，倒置相差显微镜(Leica)。
(3)主要试剂
基础试剂：Advanced DMEM/F-12培养基、NEAA(非必须氨基酸)、谷氨酰胺(Glutamax)均购自Gibco公司；青-链霉素、Accutase、丝裂霉素-C均购自Sigma公司；TrypLE消化酶购自Invitrogen公司。
小分子化合物：SB431542、Bix01294、RG108、Bay K 8644均购自Stemgent公司。
抗体：CD56-PE(eBioscience)、兔抗人CD56(NCAM)(Abcam)、鼠抗人IgG1MUC5AC(Abcam)；Alexa
Figure PCTCN2017086336-appb-000002
647Goat Anti-Mouse IgG2b(γ2b)、Alexa
Figure PCTCN2017086336-appb-000003
568Goat Anti-Mouse IgG1(γ1)均购自Invitrogen公司。
(二)实验方法及结果
(1)荧光原位杂交(FISH)—起始细胞的初步定位
具体方法包括以下步骤：
1)、将男性来源的胃上皮细胞接种到女性来源的滋养层细胞上进行重编程或者反之。
2)、重编程成功后对原孔的细胞用甲醇和冰醋酸的混合物(3:1)固定20分钟，送公司进行X和Y染色体的原位杂交。
3)、处理完后的细胞在共聚焦显微镜下观察性别染色体分别在hiEndoPCs和滋养层细胞上的染色情况。
结果：由于在重编程体系中涉及到两种细胞类型，起始细胞—人胃上皮细胞(hGECs)和滋养层细胞—成人胃肌成纤维细胞(aGSEMFs)，前述已经证实它们单独都不能被重编程为诱导内胚层祖细胞(hiEndoPCs)，只有两者组合并在BBRS重编程培养基的培养下经重编程才能成功。为了进一步证实hiEndoPCs确实来源于胃上皮细胞而不是滋养层细胞，进行了性别错配结合荧光原位杂交(FISH)的实验：使用男性来源的hGECs作为起始细胞，以女性来源的胃肠道肌成纤维细胞(aGSEMFs)为滋养层细胞进行重编程，荧光原位杂交(FISH)的结果显示，重编程得到的hiEndoPCsX和Y染色体都是阳性，表明是确实是男性来源，而滋养层细胞则仅是X染色体阳性表明是女性来源(图18A)；而当以女性来源的hGECs作为起始细胞，以男性来源的胃肠道肌成纤维细胞(aGSEMFs)为滋养层细胞进行重编程，荧光原位杂交(FISH)的结果则显示hiEndoPCs仅X染色阳性，表明是女性来源，而滋养层细胞X、Y染色体均阳性表明是男性来源(图18B)。本实验充分证明了hiEndoPCs起源于胃上皮细胞而非滋养层细胞。
(2)起始细胞人胃上皮细胞(hGECs)表面标志的确定—CD56标记的流式分选、接种和重编程
具体方法包括以下步骤：
1)、用Accutase(细胞消化液)将人胃上皮细胞(hGECs)消化成单细胞，1000rpm离心5min，弃上清。
2)、用PBS洗1遍。
3)、用PBS重悬并加入适量CD56-PE，对照组加PE(PE，一种荧光标记染料)同型对照抗体。
4)、置于4℃摇床45min。
5)、用PBS洗3遍。
6)、用适量PBS重悬。
7)、用流式细胞仪进行分析并筛选，分别保留CD56阳性细胞和阴性细胞(全程注意无菌)。
8)、将CD56阳性和阴性的细胞以同样的数量接种于提前用丝裂霉素C处理好的成人胃肌成纤维细胞(aGSEMFs)上，细胞贴附过夜(12-16小时)后更换为BBRS重 编程培养基，进行重编程。
对胃窦部组织CD56(NCAM，神经细胞粘附分子，是表达于细胞膜上的一种表面蛋白)免疫荧光染色，细胞免疫荧光染色方法同第一部分。
结果：通过FISH实验明确证实了起始细胞来源于胃上皮细胞而非滋养层细胞。由于胃上皮细胞属于原代细胞，细胞类型较为混杂，为了进一步明确起始细胞中哪一类亚群发生了向hiEndoPCs的转换，需要对起始细胞进行细分。首先需要明确hGECs的表面标记，由于分离的hGECs主要来源于胃窦部的隐窝，有研究报道NCAM是表达于多种内胚层来源组织的一种表面标志尤其是表达于较原始的细胞上，因此对胃窦部组织进行NCAM原位染色，发现NCAM(CD56)主要在深部胃组织(即隐窝部分)中有强弱不同程度的表达(如图19左幅红色荧光所示),浅部胃组织几乎不表达，浅部组织主要表达MUC5AC(如图19中间幅绿色荧光所示，图19右幅为显示NCAM染色和MUC5AC染色的合成图)，因此认为NCAM是可以用于起始细胞分群的可靠标志。
以NCAM作为分选标记对培养的hGECs进行流式分类，分为NCAM阳性和NCAM阴性的两群细胞。然后以NCAM阳性和阴性的细胞为起始细胞分别进行重编程。结果显示NCAM阳性的起始细胞能够重编程成功，而阴性的细胞几乎不能(如图20所示)。
第四部分通过性别错配实验证实起始细胞来源于胃上皮细胞而非滋养层细胞。通过对起始细胞进行细分类，进一步证实了是NCAM阳性而非NCAM阴性的胃上皮细胞在重编程的过程中发生了细胞类型转换，从而实现了对起始细胞的精确定位。
实施例2、诱导内胚层祖细胞的表型鉴定及分化潜能
内胚层祖细胞被认为是肝、胰、肠，胃、肺、甲状腺等内脏器官发育的起始，而且具有增殖能力，因此是获取功能性肝细胞、胰腺细胞、肠细胞、肺和甲状腺细胞的良好种子细胞。之前有多项以胚胎干细胞(Embryonic stem cells)(ESCs)或诱导多能性干细胞(induced Pluripotent Stem Cells)(iPSCs)为起始获取内胚层祖细胞的研究，为本实施例中鉴定重编程获得的内胚层祖细胞奠定了良好的基础。在实施例1中我们使用BBRS和成人胃肌成纤维细胞(aGSEMFs)的组合成功的在人胃上皮细胞(hGECs)中产生了内胚层干/祖细胞样的克隆(hiEndoPCs)。然而这只是初步的形态改变，还要证实其内胚层祖细胞属性，使用PCR和免疫蛋白染色的方法鉴定诱导内胚层干/祖细胞(hiEndoPCs)特征性基因表达，基因芯片检测其全基因组表达，甲基化芯片进行表观遗传分析，还测试了其增殖特性，使用电镜观察其微观特性，采用不同的诱导分化体系对其向肝脏、胰腺、肠道、肺和甲状腺分化的潜能进行了鉴定，来证实通过谱系重编程确实获得了内胚层祖细胞样的细胞(hiEndoPCs)并且挖掘 hiEndoPCs细胞的特有属性。
一、诱导内胚层祖细胞标志性基因的表达
材料与方法
(一)实验材料
(1)实验细胞
成人胃肌成纤维细胞(aGSEMFs)、人胃上皮细胞(hGECs)、诱导内胚层祖细胞(hiEndoPCs)、H9人胚胎干细胞(ESCs)购自Wicell公司。
(2)实验器材
实时荧光定量PCR仪(Bio-Rad)、普通PCR仪(Eppendorf)、倒置相差显微镜(Leica)、实时定量96孔板及封闭膜(Bio-Rad)、12孔板、激光共聚焦荧光显微镜(Zeiss)、细胞免疫共聚焦小皿(NEST)。
(3)主要试剂
抗体同实施例1第一部分、免疫组化试剂盒(Vector Lab)、RA和LDN193189均购自Sigma公司；A83-01购自Stemgent公司；b-FGF、Wnt3a、ActivinA、FGF10购自R&D公司。
(4)引物序列
表5 引物序列
基因名称 引物序列(F:5'->3') 引物序列(R:5'->3')
GAPDH GAGTCAACGGATTTGGTCGT TTGATTTTGGAGGGATCTCG
FOXA2 GCGACCCCAAGACCTACAG GGTTCTGCCGGTAGAAGGG
GATA4 CCCAGACGTTCTCAGTCAGTG GCTGTTCCAAGAGTCCTGCT
ONECUT2 CGATCTTTGCGCAGAGGGTGCTGT TTTGCACGCTGCCAGGCGTAAG
HNF1B TGTACGCACACAAGCAGGAA GTTGGTGAGTGTACTGATGCTG
HOXA3 AGCAGCTCCAGCTCAGGCGAAA TGGCGCTCAGTGAGGTTCAG
NANOG ACAACTGGCCGAAGAATAGCA GGAGGAAGCTGACAACAATGAAA
SOX9 AGCGAACGCACATCAAGAC GCTGTAGTGTGGGAGGTTGAA
OCT4 CTTGAATCCCGAATGGAAAGGG GTGTATATCCCAGGGTGATCCTC
SOX2 TACAGCATGTCCTACTCGCAG GAGGAAGAGGTAACCACAGGG
C-MYC TCGGAAGGACTATCCTGCTG GTGTGTTCGCCTCTTGACATT
PDX1 TTAGGATGTGGACGTAATT GGTCAAGTTCAACATGACAG
GAST ATGCAGCGACTATGTGTGTATG GCCCCTGTACCTAAGGGTG
GIF ACTCATGGAGAACTCGGTGAC GGGCCTTCAAGTTGTAGGCTC
PGC AGTCTATCCGTGAGACCATGAA GCGGTACTTCCAAGCAGGA
(二)实验方法及结果
(1)诱导内胚层祖细胞(hiEndoPCs)的特征性蛋白表达分析
蛋白免疫荧光染色方法同实施例1。
(2)诱导内胚层祖细胞(hiEndoPCs)中特异性基因的表达分析
提取重编程克隆的RNA、反转录及实时荧光定量PCR，具体方法包括以下步骤：
1)、手动挑取重编程产生的细胞克隆，尽量避免滋养层细胞的混入，然后用RNA提取试剂盒(购自QIAGEN)将挑取的细胞按说明书的要求加入适量裂解液Buffer RLT,充分裂解细胞，并继续按下述步骤来提取RNA：
2)、加入等体积的70％(V/V)乙醇到裂解液里，用移液枪吹打混匀。
3)、将混匀液包括沉淀加入到spincolumn(离心柱，购自QIAGEN)并置于2mL收集管中，12000rpm离心30s，弃掉滤液。
4)、加入700μL的Buffer RW1(RNA提取试剂盒提供，购自QIAGEN)到spincolumn中，12000rpm离心30s，弃掉滤液。
5)、加入500μL Buffer RPE(RNA提取试剂盒提供，购自QIAGEN)到spincolumn中，12000rpm离心30s，弃掉滤液。
6)、加入500μL Buffer RPE到spincolumn中，12000rpm离心2min，弃掉滤液，然后再空离1min。
7)、将spincolumn移至新的1.5mL收集管中，加入30μL RNeasy-free water(无核糖核酸酶水)到spincolumn膜的中央，12000rpm离心1min，弃去spin column，收集管内即为提取的RNA溶液。
8)、使用分光光度计测定洗脱的RNA样品浓度。
9)、按1μg的RNA总量吸取相应体积的RNA，并加入RNA-free water(无核糖核酸酶水)至16μL，65℃加热5min，然后快速将离心管转移到冰上冰浴2分钟。
10)、冰浴结束后加入4μL RNA反转录试剂盒中的5×RT反转录试剂，使用普通PCR仪反转录程序为：37℃15分钟，50℃5分钟，98℃5分钟，4℃终止。
11)、按需求吸取反转录获取的cDNA、三蒸水、SYBR，提前于96孔板中加入检测引物，体系(具体为：cDNA加水总共9μL，SYBR 10μL，引物1μL)配制完成后，封闭膜封闭孔板，放入实时荧光定量PCR仪中，程序：95℃3分钟，95℃10秒，60℃35秒，65℃5秒，95℃5秒，总共循环45次。
(3)胚胎干细胞(ESCs)向定性内胚层(DE)定向诱导分化
提前将生长状态良好的人胚胎干细胞H9以适宜密度接种接种于Matrigel胶包被的孔板里，次日更换培养基为DE诱导培养基：Advanced RPMI 1640+1％(W/V)B27(无血清神经细胞添加剂)+ActivinA(激活素A，100ng/mL)+CHIR99021(3μM，购 自Stemgent)作用1天，第2天和第3天更换培养基为：Advanced RPMI 1640+1％B27+ActivinA(100ng/mL)。
(4)胚胎干细胞(ESCs)向原始消化管(PGT)定向诱导分化
向定性内胚层(DE)诱导后将培养基更换为PGT诱导培养基：Advanced(中文名称：先进)RPMI 1640+2％(V/V)FBS+50ng/mL FGF10(成纤维生长因子10)+cyclopamine(环巴胺，0.25μM)，作用3天即可得到PGT。
(5)胚胎干细胞(ESCs)向前肠后段(PFG)定向诱导分化
向定性内胚层(DE)诱导后将培养基更换为PFG诱导培养基：Advanced RPMI 1640+RA(维甲酸，2μM)+LDN193189(0.25μM，购自Sigma)，作用3天即可得到PFG。
(三)统计学分析
所有数据均来自于三次及以上独立重复试验，除特殊说明外，数据均描述为平均值±标准差。两组数据间统计学分析用SPSS软件进行学生双尾t检验，P<0.05时两组数据之间的差异被认为具有统计学意义。
结果：
(1)诱导内胚层祖细胞(hiEndoPCs)的特征性蛋白表达分析
对重编程后的克隆进行内胚层特异性蛋白的流式分析发现，hiEndoPCs均一性高表达FOXA2(叉头编码框蛋白A2)、SOX9(性别决定区域Y基因9)、SOX17(性别决定区域Y基因17)、LGR5(富含重复亮氨酸G蛋白偶联受体5)、EPCAM(上皮细胞粘附分子)，如图21所示(Isotype为同种型)。对hiEndoPCs进行免疫荧光染色发现，如图22所示，hiEndoPCs不再表达胃特异性的标志MUC6(黏膜蛋白-6)和GAST(胃泌激素)，失去了胃的属性，而开始明显表达内胚层干/祖细胞特异性的转录因子FOXA2(红色荧光，表达量96±2.6％)、SOX9(红色荧光，表达量97±1.18％)和其它内胚层干/祖细胞相关的标志如CXCR4(绿色荧光，表达量98±0.56％)、EPCAM(红色荧光，表达量97±2.7％)、LGR5(绿色荧光，表达量92±4.3％)、CK19(绿色荧光，表达量91±4.2％)。流式水平的检测与免疫蛋白染色相一致，更加印证了hiEndoPCs的内胚层祖细胞特征。
(2)诱导内胚层祖细胞(hiEndoPCs)中特异性基因的表达分析
转录水平的结果也显示与起始细胞人胃上皮细胞(hGECs)相比，内胚层祖细胞(hiEndoPCs)中内胚层早期发育相关的基因FOXA2(叉头编码框蛋白A2)、SOX9(性别决定区域Y基因9)、GATA4(GATA结合蛋白4)、HNF1B(肝细胞核因子同源框蛋白B)、PDX1(胰十二指肠同源异型盒基因1)、ONECUT2(一切除域蛋白家族2)、HOXA3(同源框蛋白A3)明显上调，甚至与胚胎干细胞(ESCs)来源的内胚层早期发 育阶段-定性内胚层(DE)、原始消化管(PGT)、前肠后段(PFG)中早期内胚层基因的表达可比，尤其与PFG的表达模式最为相似(图23的A幅，图23A中柱群从左至右依序为hECs、DE、PGT、PFG、hiEndoPCs,P1、hiEndoPCs,P4和hGECs)。而且hiEndoPCs不再表达胃特异性标志MUC6、PGC、GIF和GAST(图23的B幅，图23B中柱群从左至右依序为hGECs、hiEndoPCs和人胃组织)。
小结：起始细胞人胃上皮细胞(hGECs)重编程后得到的诱导内胚层祖细胞(hiEndoPCs)失去了胃特征性的标志如MUC6、GIF、PGC、GAST等，而开始高表达内胚层早期祖细胞标志性基因如转录因子FOXA2、SOX9、HNF1B、GATA4、PDX1、HOXA3和其它内胚层祖细胞的标志如CXCR4、CK19、LGR5、EPCAM。转录水平和蛋白表达水平都显示了hiEndoPCs的内胚层祖细胞特征。然而，想要全面明确hiEndoPCs分子特性、发育阶段特征和时空定位，还需对其进行全基因表达或表观遗传表达谱的分析。
二、诱导内胚层祖细胞表观遗传分析和发育阶段定位
材料与方法
(一)实验材料
(1)实验细胞
成人胃肌成纤维细胞(aGSEMFs)、人胃上皮细胞(hGECs)、十二指肠上皮细胞(hDECs)、诱导内胚层祖细胞(hiEndoPCs)、定性内胚层(DE)、原始消化管(PGT)、前肠后段(PFG)。
(2)实验器材
普通PCR仪(Eppendorf)。
(3)主要试剂
Illumina TotalPrep kit(伊鲁米娜完全预备试剂盒，Ambion)、Sentrix Chip Array(圣翠科斯芯片阵列，Human HT-12)、QIAamp DNA Micro Kit(微量DNA提取试剂盒，Qiagen)、EZ DNA Methylation-Gold kit(脱氧核糖核酸甲基化金试剂盒，Zymo Research)。
(二)实验方法及结果
(1)hGECs、hiEndoPCs、PGT、PFG的RNA提取、反转录方法同本实施例步骤一。
(2)深度测序处理及分析(中科院北京基因组所协作完成)
提取的hGECs、hiEndoPCs、PGT、PFG RNA先扩增，按照Illumina TotalPrep试剂盒提供的步骤从总RNA中产生生物酰化的cRNA，然后使用Sentrix Chip Array对cRNA进行杂交，杂交后的处理按照Illumina公司提供的方法，使用Illumina BeadStudio软件对数据进行处理，原始数据上传至Gene Expression Omnibus  database(accession number GSE69706)。
(3)DNA甲基化处理及分析(中科院北京基因组所协作完成)
首先进行DNA文库构建、上机测序和数据分析，收集来自两个不同标本的hGECs和相应的两个hiEndoPCs单克隆，用QIAamp DNA Micro Kit提取基因组DNA，用EZ DNA Methylation-Gold kit(Zymo Research，货号D5005)进行亚硫酸转化，然后用高通量测序平台HiSeq 2500(Illumina)进行测序。原始数据提交至Gene Expression Omnibus database(accession number：GSE69706)。
结果：
(1)诱导内胚层祖细胞(hiEndoPCs)的全基因组DNA表观遗传分析
步骤一对hiEndoPCs的鉴定局限在内胚层干/祖细胞的某些特征性的标记上，为了证实hiEndoPCs与hGECs确实是两类不同的细胞，我们又使用全基因组DNA甲基化芯片检测来对hiEndoPCs与hGECs从表观遗传修饰角度进行分析。聚类分析结果显示hiEndoPCs与hGECs拥有不同的表观模式(图24的A幅)，明确证实了重编程后细胞发生了重大改变。Gene Ontology(GO)分析显示，与hGECs相比，hiEndoPCs中低甲基化状态的基因，也就是处于活跃状态的基因分类主要与胚胎发育或干细胞发育相关(图24的B幅)。这正与hiEndoPCs的内胚层早期属性和干/祖细胞属性相一致。
(2)诱导内胚层祖细胞(hiEndoPCs)中的特征性基因的启动子区表观修饰
甲基化分析显示，与hGECs相比，在hiEndoPCs中启动子区域甲基化发生上调和下调的基因分别有519和857个(图25之A)，其中内胚层特异性的转录因子FOXA2(叉头编码框蛋白A2)和GATA4(GATA结合蛋白4)的启动子区域甲基化水平在hiEndoPCs中明显降低(图25之B)，说明FOXA2和GATA4的基因表达处于转录激活状态，这与hiEndoPCs中FOXA2和GATA4基因高表达相一致。
(3)诱导内胚层祖细胞(hiEndoPCs)的发育阶段定位
在从多个水平证实了hiEndoPCs的内胚层干/祖细胞的特征后，还需要对hiEndoPCs所处的发育阶段进行定位以进一步明确其特征。由于hiEndoPCs自然状态下的阳性对照难以得到，因此以较为公认的胚胎干细胞(ESCs)来源的按照发育过程诱导的定性内胚层(DE)、原始消化管(PGT)、前肠后段(PFG)等早期内胚层阶段作为阳性对照来与重编程获得的hiEndoPCs进行全基因组表达谱的比较，RNA深度测序显示hiEndoPCs处于PGT与PFG之间且更接近于PFG(图26)。此外，PCA主成分分析显示胃上皮细胞来源的hiEndoPCs与十二指肠上皮细胞来源的hiEndoPCs在全基因表达谱上非常相似(PCA主成分分析是指将多个变量通过线性变换以选出较少个数重要变量的一种多元统计分析方法)，这与前面发现的它们在细胞形态上极其类似相 一致，并且都与它们的起始细胞不同，也与ESCs来源的定性内胚层(DE)、滋养层细胞(GSEMFs)明显不同(图27)。
小结：全基因组DNA甲基化水平分析进一步证实了hGECs与hiEndoPCs明显的不同，重编程后hiEndoPCs获得了内胚层干/祖细胞的分子特征，因此hGECs向hiEndoPCs的转换确实是重编程的过程。而且RNA深度测序明确了hiEndoPCs在发育阶段上处于PGT和PFG之间更接近于PFG，因此实现了hiEndoPCs的时空定位。
三、诱导内胚层祖细胞的微观特征
材料与方法
(一)实验材料
(1)实验细胞
人胃上皮细胞(hGECs)、诱导内胚层祖细胞(hiEndoPCs)。
(2)实验器材
H7650transmission electron microscope(透射电镜，HITACHI)、H7650Electron Microscopy(电子显微镜)、AMT XR16M CCD Digital Camera(数码相机电耦合器件，AMT)、AMT Capture Engine Software Version 600.259(捕获工程软件)、可拆卸96孔板(Corning)。
(3)主要试剂
Polybed 812epoxy resin(多床环氧树脂)购自Polysciences,Inc.,Warrington,PA，Reynolds’lead citrate(雷若兹柠檬酸铅，由军事医学科学院国家仪器分析测试中心提供)、aqueous uranyl acetate(含水乙酸双氧铀，由军事医学科学院国家仪器分析测试中心提供)。
(二)实验方法及结果
具体方法包括以下步骤：
1、hiEndoPCs和hGECs种植于可拆卸的96孔板中。
2、PBS清洗，使用3％glutaraldehyde(戊二醛)与0.1M sodium cacodylate(二甲胂酸钠，购自Sigma)混合、pH 7.4的固定液过夜(12-16小时)固定细胞。
3、用sodium cacodylate buffer(二甲胂酸钠，购自Sigma)清洗3次后，再用1％osmium tetroxide(四氧化锇，购自Sigma)与0.1sodium cacodylate buffer(二甲胂酸钠缓冲液，购自Sigma)混合再固定1小时。
4、去离子水清洗后，脱水再埋入Polybed 812epoxy resin(多床环氧树脂)中。
5、将组织切成70nm的切片，用4％aqueous uranyl acetate(含水乙酸双氧 铀)由军事医学科学院国家仪器分析测试中心提供，染色15分钟再用Reynolds’lead citrate(雷若兹柠檬酸铅，由军事医学科学院国家仪器分析测试中心提供)染7分钟。
6、染色后的切片用H7650透射电镜观察。
7、使用AMT XR16M CCD和AMT 600.259(捕获工程软件)进行摄像。
结果：如图28所示(图中，Microvilli(微绒毛，Mv)，Terminal web of actin microfilaments(肌动蛋白微丝，AM)，Mitochondria(线粒体，Mi)，Vacuoles(液泡，V)，Endoplasmic reticuLum(内质网，ER)，Nucleus(核，N)，nucleolus(核仁，Nu))，在对hGECs和hiEndoPCs进行电镜水平的观察发现，hGECs呈现成熟细胞的特征包括细胞相对较大，细胞间紧密连接，核质比较小，有许多的肌动蛋白纤维网(图28之A幅)，胞浆内容物丰富，含有大量的线粒体，微绒毛和空泡(图28之B幅)。而hiEndoPCs细胞更小，连接更为紧密，核仁明显，核浆比更大(图28之C幅)，胞浆内容物较少，含线粒体较少，少量线粒体和内质网分布于核膜的周围(图28之D幅)，并且细胞连接处具有不成熟细胞的指状突起连接(图28之E幅)。因此微观水平的分析显示，hiEndoPCs与hGECs明显不同，hiEndoPCs呈现干细胞的微观特征，而hGECs呈现终末分化成熟细胞的特征。
小结：电镜水平的分析进一步证实了hiEndoPCs属于干/祖细胞特征，而hGECs则具有成熟细胞的特征，hGECs向hiEndoPCs转换是成熟细胞向干/祖细胞的转换。
四、诱导内胚层祖细胞的增殖和传代扩增特性
材料与方法
(一)实验材料
(1)实验细胞
成人胃肌成纤维细胞(aGSEMFs)、诱导内胚层祖细胞(hiEndoPCs)。
(2)实验器材
倒置相差显微镜(Leica)、MltraVIEW(超视，PerkinElmer)。
(3)主要试剂
Fibronectin(FN，纤连蛋白)、Cell-TAK胶(CT)均购自BD；无血清细胞快速冻存液(Bio-Tool)；A83-01(Stemgent)；bFGF(碱性成纤维生长因子b)、Wnt3a均购自R&D；丝裂霉素-C(Sigma)；Advanced DMEM/F12、Dispase(离散酶)均购自Gibco。
(二)实验方法及结果
(1)hiEndoPCs的传代
1、传代前准备：提前以合适密度在孔板中接种丝裂霉素C处理的aGSEMFs或提前约3个小时将Fibronectin(FN)、Cell-TAK(CT)胶铺于孔板中，室温晾干。
2、配制传代培养基：Advanced DMEM/DF12+AWF(配方：A83-01 0.5μM+Wnt3a50ng/mL+bFGF 10ng/mL)或Advanced DMEM/DF12+A(A83-01，0.5μM)。
3、传代时手动挑取克隆，并将其划成小块，比例约1:3-4。将其置于FN+AWF、CT+AWF或Feeder(滋养细胞)+A培养基中在37℃、5％CO2培养箱中进行传代培养。
(2)hiEndoPCCs的冻存与复苏
1、冻存：用5mg/mL的Dispase酶将手动挑取的克隆在37℃消化5分钟，并吹打成小块，使用培养基将细胞洗涤2次，然后用适量的无血清细胞快速冻存液重悬细胞，置于冻存管中，直接放于-80℃保存。
2、复苏：将冻存的细胞42℃快速解冻，使用10倍体积的培养基洗涤，离心，然后用Advanced DMEM/DF12+A(A83-01 0.5μM)的培养基重悬并接种于前述准备好的滋养层细胞上进行培养。
结果：
(1)hiEndoPCs在重编程过程中的增殖特征
在证实了hiEndoPCs的内胚层祖细胞的分子特征后，对其增殖特性进行分析。仔细观察hiEndoPCs在重编程过程中的动力学特征，将其分为三个阶段：第0-7天有些紧密的、边缘锐利的是细胞克隆逐渐出现(PhaseⅠ)；在7-10天克隆有一个缓慢增殖的过程(PhaseⅡ)，克隆由小变大；在第10-15天是克隆迅速增殖的阶段(PhaseⅢ)，许多小的克隆迅速汇合成面积较大的克隆，在此阶段细胞的倍增时间大概为36.1±4.7小时(参见图29)。
(2)hiEndoPCs的传代培养条件筛选
由于hiEndoPCs是内胚层祖细胞，因此其必须具备一定的传代扩增潜能，发明人经过对传代条件的多次筛选，最终确定在以Fibronectin(FN)胶或BD Cell-TAK(CT)胶作为细胞外基质结合使用Advanced DMEM/DF12+AWF(A83-01 0.5μM+Wnt3a50ng/mL+bFGF 10ng/mL)的条件下也即FN+AWF(图30A)或CT+AWF(图30B)等无滋养层细胞的条件下，克隆传代后可以良好生长并维持原始克隆的状态。在以aGSEMFs为滋养层，结合Advanced DMEM/DF12+A(A83-01 0.5μM)也即Feeder+A(图30C)的条件下，也能维持原始的干/祖细胞克隆形态。hiEndoPCs也可进行冻存，克隆复苏后使用Feeder+A的条件，同样可维持原始克隆的形态(图30D，Post-Thawed cells，冻存复苏后)。
(3)hiEndoPCs的传代扩增特性
hiEndoPCs在前述的传代培养条件下，大约可扩增4-6代，传代过程中细胞形态基本维持不变(图31，下排图片为上排图片方框的放大)。传到4代之后细胞增殖速度减慢，第6代细胞数量达到顶峰，说明其增殖潜力有限。以每次大约可获取的106个人胃上皮细胞(hGECs)为起始，经过重编程和大约4-6次的扩增，可以获得约109数量的干/祖细胞(hiEndoPCs)(图32)。
小结：使用相应的传代条件可以将hiEndoPCs进行4-6代的扩增。
五、诱导内胚层祖细胞分化潜能鉴定
材料与方法
(一)实验材料
(1)实验细胞
人胃上皮细胞(hGECs)、诱导内胚层祖细胞(hiEndoPCs)、H9人胚胎干细胞(ESCs)购自Wicell公司。
(2)实验器材
实时荧光定量PCR仪(Bio-Rad)、普通PCR仪(Eppendorf)、正置荧光显微镜(Leica)、实时定量96孔板及封闭膜(Bio-Rad)、12孔板、激光共聚焦荧光显微镜(Zeiss)、倒置相差显微镜(Leica)、细胞免疫共聚焦小皿(NEST)。
(3)主要试剂
1、主要抗体：
表6 用于内胚层祖细胞(hiEndoPCs)免疫荧光检测的第一抗体
一抗 公司 货号 一抗种属来源 稀释比
AFP Sigma A8452 小鼠免疫球蛋白2a 200
ALB Abcam ab10241 小鼠免疫球蛋白2b 400
CK18 Santa Cruz sc-6259 小鼠免疫球蛋白1 100
Lgr5 Sigma HPA012530 兔 350
CDX2 R&D AF3665 山羊免疫球蛋白 100
Muc2 Abcam ab118964 小鼠免疫球蛋白1 100
Somatostatin Millipore AB5494 兔 100
InsuLin Abcam ab7842 豚鼠 100
Glucagon Sigma G2654 小鼠免疫球蛋白1 200
ProinsuLin R&D MAB13361 小鼠免疫球蛋白2a 200
c-peptide Millipore 05-1109 小鼠免疫球蛋白1 100
α-amylase Sigma A8273 兔 200
PDX1 Abcam ab47308 豚鼠 200
NKX6.1 R&D AF5857 山羊免疫球蛋白 200
Villin Abcam ab201989 小鼠免疫球蛋白1 100
E-Cadherin BD 610181 小鼠免疫球蛋白2a 100
表7 用于内胚层祖细胞(hiEndoPCs)免疫荧光检测的第一抗体
Figure PCTCN2017086336-appb-000004
2、主要培养基、添加物及基质胶：Advanced DMEM/DF12、MCDB131(低蛋白、无血清培养基131)、CMRL 1066、Advanced RPMI 1640、GlutaMax(谷氨酰胺)、NEAA(非必须氨基酸)、N2(无血清神经细胞添加剂N2)、B27(无血清神经细胞添加剂B27)均购自Gibco；ITS-X(胰岛素-转铁蛋白-硒-乙醇胺复合溶液，Life Technologies)；T3、Ascorbic acid(维生素C，Sigma)；HM(肝细胞培养基，Sciencell)；Lanminin(层黏连蛋白)、Fibronectin(纤连蛋白，FN)、CollagenⅣ(4型胶原)购自BD。
3、细胞因子：b-FGF(碱性成纤维细胞生长因子b)、Wnt3a、ActivinA(激活素A)、FGF4(成纤维生长因子4)、HGF(肝细胞生长因子)、OSM(抑瘤素M)、FGF10(成纤维生长因子10)、FGF7(成纤维生长因子7)、EGF(表皮生长因子)、Noggin(头蛋白)、BMP4(骨形态发生蛋白4、IGF(胰岛素样生长因子)、TSH(促甲状腺激素)、InsuLin(胰岛素)购自R&D公司；NaI(碘化钠，Sigma)。
4、小分子化合物：DEX(地塞米松)、RA(维甲酸)、LDN193189、SANT-1均购自Sigma；TPB购自EMD MilliPore；ALK5inhibitor II购自Enzo Life Sciences；gamma secretase inhibitor XX(γ-分泌酶抑制剂XX)购自EMD MilliPore；R428 购自SelleckChem；Chir99021购自Stemgent。
5、试剂盒：human albumin ELISA试剂盒(人白蛋白检测试剂盒，购自Bethyl)。
6、其它试剂：1mg/mL的ICG(靛氰绿)溶液、苏木素、Periodic Acid(过碘酸)、Triton X-100、Schiff’s(聚乙二醇辛基苯基醚，Sigma)、枸橼酸钠缓冲液、4％多聚甲醛。
(4)引物序列
表8 引物序列
基因名称 引物序列(F:5'->3') 引物序列(R:5'->3')
HNF4A ACGGACAGATGTGTGAGTGG CAGGAGCTTATAGGGCTCAGA
AFP CTTGCACACAAAAAGCCCACT GGGATGCCTTCTTGCTATCTCAT
ALB TTTATGCCCCGGAACTCCTTT ACAGGCAGGCAGCTTTATCAG
TF CCTCCTACCTTGATTGCATCAG TTTTGACCCATAGAACTCTGCC
AAT ATGCTGCCCAGAAGACAGATA TTGTTGAAGGTTGGGTGATCC
GGT GGGGAGATCGAGGGCTATGAG GATGACGGTCCGCTTGTTTTC
G6PC TCAGGGAAAGATAAAGCCGACC AGGTAGATTCGTGACAGACAGAC
CYP1A2 ATGGCATTGTCCCAGTCTGTT TGGCTCTGGTGGACTTTTCAG
CYP3A4 AAGTCGCCTCGAAGATACACA AAGGAGAGAACACTGCTCGTG
CYP3A7 AAGGTCGCCTCAAAGAGACA TGCACTTTCTGCTGGACATC
CEBPA GCGGGAACGCAACAACATC GTCACTGGTCAACTCCAGCAC
CEBPB CTTCAGCCCGTACCTGGAG GGAGAGGAAGTCGTGGTGC
ΜGT1A1 TAAGTGGCTACCCCAAAACG GCTTTGCATTGTCCATCTGA
ΜGT1A3 TCAGATGGACAATGCAAAGCGC GGCGCATGATGTTCTCCTTGTA
PDX1 TTAGGATGTGGACGTAATT GGTCAAGTTCAACATGACAG
NKX6.1 AGGACGACGACTACAATAAGCCTCT GCGCTGCTGGACTTGTGCTTCT
NEUROG 3 GGAGTCGGCGAAAGAAGGC TACAAGCTGTGGTCCGCTATG
INSULIN GCAGCCTTTGTGAACCAACAC CCCCGCACACTAGGTAGAGA
SST GCTGCTGTCTGAACCCAAC CGTTCTCGGGGTGCCATAG
AMY2A TTCAGACCTTGGTGGGAAAGA ACGAACCCCAACATTGTTACAT
GLUCAGON GACAAGCGCCATTCACAGG TGACGTTTGGCAATGTTATTCCT
CDX2 GGCAGCCAAGTGAAAACCAG GGTGATGTAGCGACTGTAGTAA
MUC2 TGCAGTGTGATGTCTCTGTTGGGT ATCCATGGGCCAGCAACAATTGAC
VIL1 AGCTCCTCTACAGGCTTGTTCACT GGACGTGTTCAATGCTAACAGCAACC
CHGA TGACCTCAACGATGCATTTC CTGTCCTGGCTCTTCTGCTC
LYSO CTTGTCCTCCTTTCTGTTACGG CCCCTGTAGCCATCCATTCC
AQP5 GCCATCCTTTACTTCTACCTGCTC GCTCATACGTGCCTTTGATGATGG
CC-10 TCATGGACACACCCTCCAGTTATGAG TGAGCTTAATGATGCTTTCTCTGGGC
NKX2.1 CGGCATGAACATGAGCGGCAT GCCGACAGGTACTTCTGTTGCTTG
SPA GTGCGAAGTGAAGGACGTTTGTGT TTTGAGACCATCTCTCCCGTCCC
SPB TCTGAGTGCCACCTCTGCATGT TGGAGCATTGCCTGTGGTATGG
SPC CCTTCTTATCGTGGTGGTGGTGGT TCTCCGTGTGTTTCTGGCTCATGT
PAX8 ACTACAAACGCCAGAACCCTACCA TGTCATTGTCACAGACGCCCTCA
TG ACGGTTCCTCGCAGTTCAAT GCAGCTTGGAACATAGGGGT
TSHR AGCCACTGCTGTGCTTTTAAG CCAAAACCAATGATCTCATCC
(二)实验方法及结果
(1)免疫荧光染色方法与前述相同。
(2)Q-PCR方法与前述相同。
(3)向肝细胞的诱导分化
具体方法包括以下步骤：
1、将ColleganⅣ/Matrigel/Laminin/KM按照1:3:1:5的比例进行混合，然后使用适量体积的胶混合液均匀涂抹于孔板中，置于室温干燥3-5个小时。
2、手动挑取hiEndoPCs并划成合适大小的块接种于上述处理的培养板中，使用加入了8％(V/V)FBS及10uM Y27632的人胃上皮细胞(hGECs)重编程培养基(配方：Advanced(先进)DMEM/F12+2mM谷氨酰胺+青-链霉素+SB43154(2μM)+RG108(0.04μM)+BIX01294(0.5μM)+Bay K 8644(2μM))培养过夜。
3、待细胞团块充分贴壁后，用分阶段法对hiEndoPCs进行诱导分化。第一阶段：KM(配方见实施例第一部分)+25ng/mL BMP4+25ng/mL FGF4+50ng/mL Wnt3a培养3天；第二阶段：HM(商品化培养基，购自ScienCell)+20ng/mL HGF+10ng/mL OSM+1μM Dex培养10-15天。
(4)向胰岛β细胞的诱导分化
具体方法包括以下步骤：
1、将Matrigel/KM按照1:1的比例进行混合，然后使用适量体积的胶混合液均匀涂抹于孔板中，置于室温干燥3-5个小时。细胞贴附方法与前述相同。
2、将贴附的细胞按照4个阶段向胰腺进行诱导分化
2.1MCDB 131medium(购自Gibco)+1.5g/L sodium bicarbonate(碳酸氢钠)+2mM(浓度)2mM Glutamax(谷氨酰胺)+10mM final glucose concentration(葡萄糖)+2％BSA(牛血清白蛋白)+0.25mM ascorbic acid(维生素C)+50ng/mL of FGF7(成纤维生长因子7)+0.25μM SANT-1+1μM retinoic acid(维甲酸)(RA)+100nM LDN193189+1:200ITS-X(胰岛素-转铁蛋白-硒-乙醇胺复合溶液)+200nM TPB，培养2天。
2.2MCDB 131medium+1.5g/L sodium bicarbonate+2mM Glutamax+10mM final  glucose concentration+2％BSA+0.25mM ascorbic acid+2ng/mL of FGF7+0.25μM SANT-1+0.1μM retinoic acid+200nM LDN193189+1:200ITS-X+100nM TPB，培养2天。
2.3 2天后第二阶段细胞用5mg/mL dispase(离散酶)在37℃消化5分钟，然后机械性吹成小块并转移到低吸附孔板中更换培养基：MCDB 131mediμm+1.5g/L sodium bicarbonate+2mM Glμtamax+20mM final glucose concentration+2％BSA+0.25μM SANT-1+0.05μM retinoic acid+100nM LDN193189+1:200ITS-X+1μM T3(胰岛素-转铁蛋白-硒-乙醇胺复合溶液)+10μM ALK5inhibitor II+10μM zinc suLfate(硫酸锌)+10μg/mL of heparin(肝素)，培养3天。
2.4CMRL 1066+2mM Glutamax+2％BSA+100nM LDN193189+1:200ITS-X+1μM T3+10μM ALK5inhibitor II+10μM zinc sμLfate+100nM gamma secretase inhibitor XX(γ-分泌酶抑制剂XX)+2μM R428培养7天或更长。
(5)向肠细胞的诱导分化
具体方法包括以下步骤：
1、RPMI 1640medium+2mM Glutama+100U/mL青霉素+0.1mg/mL链霉素+500ng/mL FGF4(成纤维生长因子4)+500ng/mL Wnt3a(WNT信号通路蛋白配体3A)+100ng/mL EGF(表皮生长因子)+3μM CHIR99021(Stemgent),培养3天。
2、手动挑取第一阶段细胞并机械性吹成小块然后埋入matrigel(人工基底膜)中，37℃、7-10分钟，待胶凝固后加培养基:Advanced DMED/F12+2mM Glutamax+100U/mL青霉素+0.1mg/mL链霉素+1％N2(无血清神经细胞添加剂N2)+1％B27(无血清神经细胞添加剂B27)+100ng/mL EGF(表皮生长因子)+100ng/mL Noggin(头蛋白)，培养1周以上。
(6)向甲状腺的诱导分化
具体方法包括以下步骤：
1、Advanced DMEM/F12+L-Glutamax+B27(1％)+N2(1％)+Noggin(200ng/mL)+SB431542(10μM)作用3天。
2、Advanced DMEM/F12+L-Glutamax+B27(1％)+N2(1％)+Wnt3a(100ng/mL)+EGF(20ng/mL)+BMP4(10ng/mL)+FGF10(10ng/mL)+FGF7(10ng/mL)+TSH(1μg/mL)作用3天。
3、Advanced DMEM/F12+L-Glutamax+B27(1％)+N2(1％)+TSH(1μg/mL)+IGF(50ng/mL)+insuLin(5mg/mL)+NaI(100μM)作用4天。
(7)向肺的诱导分化
具体方法包括以下步骤：
1、Advanced DMEM/F12+L-Glutamax+B27(1％)+N2(1％)+Noggin(200ng/mL)+SB431542(10μM)作用4天。
2、Advanced DMEM/F12+L-Glutamax+B27(1％)+N2(1％)+Wnt3a(100ng/mL)，EGF(20ng/mL)+BMP4(10ng/mL)+FGF10(10ng/mL)+FGF7(10ng/mL)培养3天。
3、Advanced DMEM/F12+L-Glutamax+B27(1％)+N2(1％)+Wnt3a(100ng/mL)+FGF10(10ng/mL)+FGF7(10ng/mL)+Dexamethasone(地塞米松)(50nM)作用3天。
(8)C肽含量的ELISA检测
操作流程按照c-peptide(C肽)ELISA试剂盒上的要求进行，具体方法包括以下步骤：
1、制备工作液：提前把试剂盒中的试剂按照说明分别稀释成工作液，室温平衡20分钟。
2、制备标准品：在5个标准品中每个都加入1mL的去离子水，充分混匀后分装成小样，-20℃冰箱保存。
3、将标准品和待检测的细胞上清样品加入已包被c-peptide抗体的96孔板中，每孔各25μL，每组样品设三个重复。
4、每孔加入缓冲液50μL。
5、将96孔板置于微孔摇床上，800rpm室温孵育1小时。
6、弃去96孔板中液体，每孔再加入350μL wash buffer(洗涤液)，继而倒掉洗液，滤纸吸干，如此重复4-5次。
7、每孔加入Enzyme conjugate solution(酶结合溶液)200μL。
8、孔板置于微孔摇床上，800rpm室温孵育1小时。
9、弃掉液体，每孔加入350μL wash buffer(洗涤液)，重复步骤6。
10、每孔加入底物TMB(四甲基联苯胺)200μL。
11、孔板置于摇床，室温避光孵育30分钟。
12、每孔加入50μL stop solution(终止液)，避光，置于摇床震动5秒。
13、30分钟内在酶标仪上450nm波长处测定OD值，计算结果。
(9)双硫腙(DTZ)染色
具体方法包括以下步骤：
1.将DTZ贮存液(购自Sigma)按1:20的比例进行稀释，然后用0.45μm滤膜过滤后配成工作液。
2.用PBS洗涤待染色的细胞。
3.将配制好的DTZ工作液加入待染色细胞中，37℃孵育10min后显微镜下观察并摄像。
(10)统计学分析
所有数据均来自于三次及以上独立重复试验，除特殊说明外，数据均描述为平均值±标准差。两组数据间统计学分析用SPSS软件进行学生双尾t检验，P<0.05时两组之间的差异被认为具有统计学意义。
结果：体内内胚层祖细胞最终发育形成胰腺、肝脏、肠、肺和甲状腺等器官。为了证实hiEndoPCs是内胚层祖细胞，本发明对其测试了向多个方向诱导分化的潜能。
(1)内胚层祖细胞(hiEndoPCs)向胰腺的诱导分化
根据经典的胰腺诱导分化方案[Pagliuca Felicia W,Millman Jeffrey R,Gürtler M,Segel M,Van Dervort A,Ryu Jennifer H,et al.Generation of Functional Human PancreaticβCells In Vitro.Cell.2014；159:428-39.Rezania A,Bruin JE,Arora P,Rubin A,Batushansky I,Asadi A,et al.Reversal of diabetes with insuLin-producing cells derived in vitro from human pluripotent stem cells.Nat Biotechnol.2014；32:1121-33.]将hiEndoPCs向胰腺进行诱导分化。在诱导2周后，hiEndoPCs来源的胰腺细胞(hiEndoPC-Pans)呈现体内胰岛三维的出芽生长形态(图33A)。DTZ(双硫腙)染色显示hiEndoPC-Pans呈现红色，表明细胞胞浆中含有锌离子，这是胰岛β细胞的特征(图33B)。对hiEndoPC-Pans进行胰腺特异性蛋白染色显示胰腺特异性转录因子PDX1和NKX6.1、胰岛α细胞标志GCG、胰岛β细胞标志C-PEP和PRO-INS以及胰岛δ细胞的标志SST都有明显表达(图33C和D)。转录水平的分析显示多种胰腺特征性基因在诱导后表达上调(图33E)，这与免疫染色结果相一致。为了证实hiEndoPCs-Pans是否具有胰岛β细胞最重要的合成和释放胰岛素的能力，检测了hiEndoPCs-Pans在多种刺激条件下的C肽释放能力。当使用高糖刺激时，与低糖组相比，C肽的释放明显增多。当使用其他的胰岛素分泌促进剂KCL、Tolbμtamiden作用时，C肽的释放也明显增多。而且hiEndoPCs-Pans对刺激的胰岛素释放反应可与ESCs来源的胰岛细胞相当(图33F)。hiEndoPCs-Pans的糖刺激反应性对于以后的糖尿病高血糖的治疗具有十分重要的临床应用意义。
(2)hiEndoPCs向肠道细胞的诱导分化
根据经典的肠细胞诱导方案[Spence JR,Mayhew CN,Rankin SA,Kuhar MF,Vallance JE,Tolle K,et al.Directed differentiation of human pluripotent  stem cells into intestinal tissue in vitro.Nature.2010；470:105-9.Watson CL,Mahe MM,Múnera J,Howell JC,Sundaram N,Poling HM,et al.An in vivo model of human small intestine using pluripotent stem cells.Nature Medicine.2014；20:1310-4.]对hiEndoPCs向肠道分化进行检测。诱导2周后发现hiEndoPCs来源的肠道类器官(hiEndoPC-Ints)呈现体外肠道原代培养的形态特征(图34A)。hiEndoPC-Ints中肠特异性转录因子CDX2、小肠细胞标志VIL1、杯状细胞标志MUC2、小肠内分泌细胞标志CHGA、小肠潘氏细胞标志LYSO在诱导之后表达明显上调(图34B)。而且在蛋白水平，肠特征性的标志VILl、MUC2、CDX2、LGR5、ECAD也都有明显的表达(图35C)，证实了hiEndoPC-Ints已经初步具备了肠的特征。
(3)hiEndoPCs向肝细胞的诱导分化
采用经典的肝向诱导方案[Gouon-Evans V,Boussemart L,Gadμe P,Nierhoff D,Koehler CI,Kubo A,et al.BMP-4is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm.Nat Biotechnol.2006；24:1402-11.]将hiEndoPCs向肝细胞进行诱导分化，发现hiEndoPCs来源的肝细胞(hiEndoPC-Heps)开始表达原代肝细胞特征性的多种功能基因包括HNF4A、CEBPA、CEBPB、TF、AATGGT、G6PC、CYP1A1、CYP3A4、CYP3A7、ΜGT1A1、ΜGT1A3等(图35A)。同时hiEndoPC-Heps中AFP(甲胎蛋白)、CK18(细胞角质蛋白18)的染色阳性(图35B)；在流式水平ALB(白蛋白)阳性的细胞比例高达90％以上(图35C)，表明hiEndoPC-Heps初步具备了肝细胞的特征。
(4)hiEndoPCs向甲状腺和肺的诱导分化
hiEndoPCs向甲状腺进行诱导分化[Longmire TA,Ikonomou L,Hawkins F,ChristodouLou C,Cao Y,Jean JC,et al.Efficient derivation of pμrified lung and thyroid progenitors from embryonic stem cells.Cell Stem Cell.2012；10:398-411.]后，hiEndoPCs来源的甲状腺细胞(hiEndoPCs-Thyroid)中甲状腺和肺共有的特异性转录因子NKX2.1、甲状腺特异性转录因子Pax8、甲状腺球蛋白(Tg)、促甲状腺激素受体(TSHR)表达水平比诱导前有非常明显的提升(图36A)。而hiEndoPCs来源的肺细胞(hiEndoPCs-Lμng)中肺特异性转录因子NKX2.1、肺Clare细胞标志CC-10、一型肺泡细胞的标志水通道蛋白AQP5、二型肺泡细胞的标志表面活性蛋白SPA、SPB、SPC等表达明显上调(图36B)。
采用能够促进向胰腺、肝脏、肠道、甲状腺和肺的诱导分化策略，对hiEndoPCs的内胚层分化潜能做鉴定，结果表明hiEndoPCs向5个方向分化后，相关的基因表达，蛋白表达明显提高，部分功能也已显现，更加明确的证实了hiEndoPCs是内胚层祖细 胞，同时提示了hiEndoPCs对于糖尿病、肝病和肠道疾病等的细胞治疗可以提供理想的种子细胞。
实施例3、可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合
小分子组合4M中单个小分子浓度改变对诱导内胚层祖细胞产生的影响：前述已说明在四个小分子化合物BBRS小分子组合中，在SB43152(SB，2μM)，RG108(RG，0.04μM)，Bix01294(Bix，0.5μM)，Bay K 8644(Bay，2μM)4个小分子使用浓度下，可以重编程获得内胚层祖细胞(hiEndoPCs)，效率4-6％。本实施例用相同的方法对所述4个小分子化合物在不同浓度范围SB43152(1～10μM)，RG108(0.01～1μM)，Bix01294(0.1～2μM)，Bay K 8644(1～4μM)下对hiEndoPCs的产生情况进行了验证(参见表9)，同时用A83-01(A83,0.4～1μM)替换SB后再与其他三个小分子在不同浓度组合RG108(0.01～1μM)，Bix01294(0.1～2μM)，Bay K8644(1～4μM)下对hiEndoPCs的产生情况进行了验证(表10)。
表9：不同使用浓度BBRS小分子组合及其重编程获得hiEndoPCs效率
Figure PCTCN2017086336-appb-000005
Figure PCTCN2017086336-appb-000006
表10：不同使用浓度BBRA小分子组合及其重编程获得hiEndoPCs效率
Figure PCTCN2017086336-appb-000007
参照实施例2的方法对表9和表10组合中所获得的内胚层祖细胞做了内胚层相关的基因、蛋白、表观修饰检测及分化功能鉴定，结果显示：以上组合获得的诱导内胚层均能高表达内胚层祖细胞特征的标志FOXA2、SOX9、GATA4、HNF1B、HOXA3、PDX1、CXCR4、EPCAM、CK19和LGR5，不表达胃细胞特征性的标志MUC6、GAST。其具有内胚层祖细胞特征的表观修饰特点，在向肝细胞进行诱导分化后能够表达肝细胞特异性的标志AFP、ALB、HNF4A、CK18、CYP3A4等；在向胰腺β细胞分化后开始表 达NKX6.1、PDX1、GCG、SST、INS等胰腺特异性标记，并且在刺激的条件下能够释放胰岛素；在向肠细胞进行诱导分化后表达CDX2、MUC2、VIL1、CHGA和LYSO等肠特异性标志；在向肺和甲状腺细胞进行分化后分别表达各自细胞特异性的标志。证实了表9-表10小分子在所列浓度范围内同样可以获得内胚层祖细胞。
实施例4、制备可将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程试剂盒
本发明可将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程试剂盒，包括可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合，具体来讲，小分子化合物组合由8个(8M)小分子化合物组成，分别为：FBP(二磷酸果糖)、Bay K 8644、Bix01294、SB431542或A83-01、Valproic Acid(VPA)(丙戊酸)、RG108、PD0325901和PS48。
优选的可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合包括以下4个小分子化合物，其中BBRS组合为Bix01294(Bix)、Bay K 8644(Bay)、RG108(RG)和SB431542(SB)，BBRA组合为Bix01294(Bix)、Bay K 8644(Bay)、RG108(RG)和A83-01(A83)。
试剂盒中，各化合物可分别包装，或按8M组合或BBRS组合或BBRA组合将各化合物混合后包装；化合物分别包装时，试剂盒中的说明书中记载各化合物的使用浓度，具体浓度数值可参见实施例1和实施例3。
试剂盒中还包括基础培养基Advanced DMEM/F12和细胞培养基础添加成分谷氨酰胺(Glutamax)和抗生素SP，及其使用说明，其中相对Advanced DMEM/F12基础培养基，谷氨酰胺的使用浓度为2mM(1×)，抗生素(如青-链霉素)使用浓度为100U/mL青霉素+0.1mg/mL链霉素，各成分分别包装或按所列使用浓度混合后包装。
试剂盒还可将所述小分子化合物组合与所述的基础培养基和细胞培养基础添加成分组合形成重编程培养基，重编程培养基的配方为：Advanced DMEM/F12+2mM谷氨酰胺(Glutamax)+青-链霉素(100U/mL青霉素+0.1mg/mL链霉素)+SB43154(2μM)或A83-01(0.5μM)+VPA(0.5mM)+PD0325901(0.5μM)+RG108(0.04μM)+Bix01294(0.5μM)+Bay K 8644(2μM)+PS48(5μM)+FBP(3.5mM)。
优选的重编程培养基配方为：Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，1～10μM的SB43152，0.01～1μM的RG108，0.1～2μM的Bix01294，1～4μM的Bay K 8644；更优选Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)， 2μM的SB43152，0.04μM的RG108，0.5M的Bix01294，2μM的Bay K 8644。
另一优选重编程培养基配方为：Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，0.4～1μM的A83-01，0.01～1μM的RG108，0.1～2μM的Bix01294，1～4μM的Bay K 8644；更优选Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，0.5μM的A83-01，0.04μM的RG108，0.5M的Bix01294，2μM的Bay K8644。
试剂盒中，还可包括滋养层细胞及其使用说明，所述滋养层细胞为消化道来源的基质细胞，如胃肌成纤维细胞或小肠肌成纤维细胞。
试剂盒中所有细胞、化合物和试剂均可按前述实施例提示的来源商购获得。
试剂盒中还包括使用说明书，说明书中记载试剂盒的实际组成及使用方法，其中包括使用相关试剂将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程方法，该方法内容参考实施例中的描述。
工业应用性
本发明提供了将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、重编程方法及应用，是以人胃上皮细胞(hGECs)作为起始细胞，以人胃肌成纤维细胞(aGSEMFs)来作为滋养层，用选自FBP、Bay K 8644、Bix01294、SB431542或A83-01、VPA、RG108、PD0325901和PS48中的全部或多个包括SB或A83在内的化合物组合将消化道来源上皮细胞重编程为内胚层干/祖细胞，该内胚层干/祖细胞可用于向肝脏细胞、胰腺β细胞和肠细胞方向诱导分化，本发明能够在产业上应用。

Claims (20)

  1. 可将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合，所述小分子化合物选自TGF-β信号通路、表观修饰剂、钙离子通道激动剂和代谢通路调节剂等功能群；代表性的含有8个小分子化合物的组合(8M)，包括FBP、Bay K 8644(Bay)、Bix01294((Bix)、SB431542(SB)或A83-01(A83)、VPA、RG108(RG)、PD0325901和PS48中的全部或多个包括SB或A83在内的化合物的组合。
  2. 根据权利要求1所述的小分子化合物组合，其特征在于：包括8个小分子化合物的组合为：SB43154：VPA：PD0325901：RG108：Bix01294：Bay K 8644：PS48：FBP按摩尔比为50：12500：12.5：1:12.5：50:125：87500的组合；或A83：VPA：PD0325901：RG108：Bix01294：Bay K 8644：PS48：FBP按摩尔比为12.5：12500：12.5：1:12.5：50:125：87500的组合。
  3. 根据权利要求1所述的小分子化合物组合，其特征在于：包括4个小分子化合物的组合为：Bix01294(Bix)、Bay K 8644(Bay)、RG108(RG)和SB431542(SB)，简称BBRS组合，或A83-01(A83)、Bay K 8644(Bay)、RG108(RG)和SB431542(SB)，简称BBRA组合。
  4. 根据权利要求3所述的小分子化合物组合，其特征在于：组合中各化合物使用浓度为：SB43152为(1～10μM)，A83为(0.4～1μM)，RG108为(0.01～1μM)，Bix01294为(0.1～2μM)，Bay K 8644为(1～4μM)。
  5. 根据权利要求3或4所述的小分子化合物组合，其特征在于：BBRS组合为各化合物按SB：RG：Bix：Bay摩尔比50:1:12.5:50的组合，BBRA组合为各化合物按A83：RG：Bix：Bay摩尔比12.5:1:12.5:50的组合；优选各化合物使用浓度SB43152为2μM，A83为0.5μM，RG108为0.04M，Bix01294为0.5μM，Bay K 8644为2μM。
  6. 一种可将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程试剂盒，包括权利要求1至5任一所述小分子化合物组合以及化合物的使用说明书；各化合物分别包装，或按8M组合或BBRS组合或BBRA组合将各化合物混合后包装，说明书记载各化合物的使用浓度。
  7. 根据权利要求6所述重编程试剂盒，其特征在于，还包括滋养层细胞及其使用说明，所述滋养层细胞为消化道来源的基质细胞，如胃肌成纤维细胞或小肠肌成纤维细胞。
  8. 根据权利要求6或7所述重编程试剂盒，其特征在于，还包括基础培养基Advanced DMEM/F12，细胞培养基础添加成分谷氨酰胺(Glutamax)和抗生素(SP)， 及其使用说明，其中相对Advanced DMEM/F12基础培养基，谷氨酰胺的使用浓度为2mM(1×)，抗生素为青-链霉素,相对Advanced DMEM/F12基础培养基的使用浓度为100U/mL青霉素+0.1mg/mL链霉素，各物质分别包装或按所列使用浓度混合后包装。
  9. 一种可将消化道来源上皮细胞重编程为内胚层干/祖细胞的重编程培养基，由权利要求1至5任一所述小分子化合物组合与权利要求8中提到的基础培养基、细胞培养基础添加成分共混而成。
  10. 根据权利要求9所述重编程培养基，其配方为：Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，2μM的SB43154或0.5μM的A83-01，0.5mM的VPA，0.5μM的PD0325901，0.04μM的RG108，0.5μM的Bix01294，2μM的Bay K8644，5μM的PS48，3.5mM的FBP。
  11. 根据权利要求9所述重编程培养基，其配方为：Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，1～10μM的SB43152，0.01～1μM的RG108，0.1～2μM的Bix01294，1～4μM的Bay K 8644；优选Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，2μM的SB43152，0.04μM的RG108，0.5M的Bix01294，2μM的Bay K 8644。
  12. 据权利要求9所述重编程培养基，其配方为：Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，0.4～1μM的A83-01，0.01～1μM的RG108，0.1～2μM的Bix01294，1～4μM的Bay K 8644；优选Advanced DMEM/F12中含2mM谷氨酰胺(Glutamax)，青-链霉素(100U/mL青霉素和0.1mg/mL链霉素)，0.5μM的A83-01，0.04μM的RG108，0.5M的Bix01294，2μM的Bay K 8644。
  13. 权利要求1至5任一所述小分子化合物或权利要求6或7或8所述重编程试剂盒或权利要求9至12任一所述重编程培养基在将消化道来源上皮细胞重编程为内胚层干/祖细胞中的应用；所述消化道来源上皮细胞包括胃上皮细胞和十二指肠上皮细胞，优选胃上皮细胞(hGECs)，最优为NCAM阳性的人胃上皮细胞(hGECs)。
  14. 将消化道来源上皮细胞重编程为内胚层干/祖细胞的方法，作为权利要求13所述应用中的一种，包括以下步骤：
    1)将原代分离的消化道来源上皮细胞作为起始细胞，扩增培养所述消化道来源上皮细胞；
    2)用丝裂霉素-C处理滋养层细胞后清洗，并用酶消化细胞后备用；
    3)将步骤2)准备的滋养层细胞添加于步骤1)扩增培养的消化道来源上皮细胞 中，继续共培养过夜；
    4)第2天更换为重编程培养基，每2-3天换一次培养基，培养7-15天，得到诱导内胚层干/祖细胞(hiEndoPCs)克隆。
  15. 根据权利要求14所述方法，其特征在于：所述步骤1)中的起始细胞为消化道来源上皮细胞，包括胃上皮细胞和十二指肠上皮细胞，优选为胃上皮细胞(hGECs)，尤其优选为NCAM(神经细胞黏附分子)阳性的胃上皮细胞；
    步骤1)优选以NCAM阳性的胃上皮细胞为起始细胞，用Kubota(库部塔)培养基在37摄氏度，5％CO2培养箱条件下培养5天。
  16. 根据权利要求14或15所述方法，其特征在于：所述步骤2)中的滋养层细胞为消化道来源的基质细胞，包括胃肌成纤维细胞或小肠肌成纤维细胞，优选人胃肌成纤维细胞(aGSEMFs)；
    步骤2)优选用丝裂霉素-C处理人胃肌成纤维细胞(aGSEMFs)2-3小时，用PBS清洗细胞，用TrypLE酶消化细胞。
  17. 根据权利要求14或15或16所述方法，其特征在于：步骤3)优选按1～3×105每平方厘米的密度将步骤2)准备的滋养层细胞添加于步骤1)培养到5天的消化道来源上皮细胞中，在37℃、5％CO2培养箱中过夜(12-16小时)。
  18. 用权利要求14至17任一所述方法获得的内胚层干/祖细胞(hiEndoPCs)。
  19. 一种对内胚层干/祖细胞(hiEndoPCs)进行传代的方法，包括以下步骤：
    1)传代前准备：在孔板中接种经丝裂霉素-C(10μg/mL)处理的成人胃肌成纤维细胞(aGSEMFs)或提前约3个小时将Fibronectin(FN，纤连蛋白)、Cell-TAK(CT，细胞组织粘附剂)胶铺于孔板中，室温晾干；
    2)配制传代培养基：Advanced DMEM/DF12+AWF(A83-01 0.5μM+Wnt3a50ng/mL+bFGF 10ng/mL)或Advanced DMEM/DF12+A(A83-01 0.5μM)；
    3)挑取内胚层干/祖细胞(hiEndoPCs)克隆，并将其划成小块，比例约1:3-4，将其置于FN+AWF、CT+AWF或Feeder(滋养层细胞，经丝裂霉素-C(10μg/mL)处理的成人胃肌成纤维细胞(aGSEMFs))+A培养基中在37℃、5％CO2培养箱中进行传代培养，得到传代内胚层干/祖细胞。
  20. 权利要求18所述内胚层干/祖细胞(hiEndoPCs)或权利要求19得到的传代内胚层干/祖细胞在向肝脏细胞、胰腺β细胞和肠细胞诱导分化中的应用。
PCT/CN2017/086336 2016-06-03 2017-05-27 将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、重编程方法及应用 Ceased WO2017206837A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2018563588A JP7005528B2 (ja) 2016-06-03 2017-05-27 消化管由来上皮細胞を内胚葉幹細胞／前駆細胞に初期化するための低分子化合物の組み合わせ、初期化方法および適用
EP17805782.4A EP3483262B1 (en) 2016-06-03 2017-05-27 Small molecule compound combination for reprogramming digestive tract derived epithelial cells to endodermal stem/progenitor cells, reprogramming method and application
US16/306,646 US11149253B2 (en) 2016-06-03 2017-05-27 Small molecule compound combination for reprogramming digestive tract derived epithelial cells to endodermal stem/progenitor cells, reprogramming method and application

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610391309.7 2016-06-03
CN201610391309 2016-06-03

Publications (1)

Publication Number Publication Date
WO2017206837A1 true WO2017206837A1 (zh) 2017-12-07

Family

ID=59660019

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/086336 Ceased WO2017206837A1 (zh) 2016-06-03 2017-05-27 将消化道来源上皮细胞重编程为内胚层干/祖细胞的小分子化合物组合、重编程方法及应用

Country Status (5)

Country Link
US (1) US11149253B2 (zh)
EP (1) EP3483262B1 (zh)
JP (1) JP7005528B2 (zh)
CN (3) CN107142240B (zh)
WO (1) WO2017206837A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110551688A (zh) * 2019-09-26 2019-12-10 上海交通大学医学院附属瑞金医院 一种诱导体细胞重编程为造血干/祖细胞且促进造血干/祖细胞体外扩增的组合物及其应用
CN111394302A (zh) * 2020-02-24 2020-07-10 中山大学 人睾丸间质干细胞的分离与培养方法
US20220177852A1 (en) * 2019-04-11 2022-06-09 Genex Health CO., LTD. Method for culturing primary cells of gastric cancer and gallbladder and bile duct cancer, and supporting reagents
CN120118841A (zh) * 2025-03-24 2025-06-10 瓯江实验室 一种化学重编程诱导功能性 ciRPE 细胞的组合物、诱导方法及应用

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109486744A (zh) * 2017-09-11 2019-03-19 中国科学院广州生物医药与健康研究院 一种用于制备内胚层细胞系的培养体系以及制备内胚层细胞系的方法
ES3062949T3 (en) * 2018-10-15 2026-04-14 Evia Life Sciences Inc Method for producing stem/precursor cells, by using low molecular weight compound, from cells derived from endodermal tissue or organ
CN110055217B (zh) * 2019-01-29 2021-03-30 中国人民解放军军事科学院军事医学研究院 获得巨核细胞及血小板的方法
AU2022223410A1 (en) * 2021-02-19 2023-09-07 Deepcell, Inc. Systems and methods for cell analysis
CN114958716A (zh) * 2021-02-23 2022-08-30 中国人民解放军海军军医大学第三附属医院 一种化学诱导成纤维细胞重编程为肺干细胞的方法
CN114736870B (zh) * 2022-04-20 2023-11-17 上海交通大学医学院附属第九人民医院 一种唾液腺腺样囊性癌类器官及其培养方法、培养基和应用
WO2023217123A1 (zh) * 2022-05-10 2023-11-16 上海赛立维生物科技有限公司 肺前体样细胞的制备方法和应用
CN115354026B (zh) * 2022-08-16 2023-08-18 中国人民解放军军事科学院军事医学研究院 获得造血干/祖细胞的培养基及培养方法和用途
CN116474120A (zh) * 2023-04-03 2023-07-25 臻赫医药(杭州)有限公司 重编程因子抗衰老mRNA组合物、制备方法及应用
CN120555324B (zh) * 2024-02-28 2026-03-24 广州国家实验室 一种构建肠类器官的试剂组合或试剂盒及其应用
CN120330129B (zh) * 2025-06-19 2025-09-09 淇嘉科技(苏州)有限公司 原始消化管分区通用型诱导方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101445791A (zh) * 2008-12-04 2009-06-03 北京大学 一种筛选多潜能细胞的方法及其专用培养基
WO2012006577A2 (en) * 2010-07-08 2012-01-12 Duke University Direct reprogramming of cells to cardiac myocyte fate
CN102959076A (zh) * 2010-03-31 2013-03-06 斯克里普斯研究所 重编程细胞
CN104120107A (zh) * 2013-04-28 2014-10-29 北京大学 一种新的诱导体细胞重编程的方法，试剂盒及用途
CN104673741A (zh) * 2015-02-04 2015-06-03 广东省中医院 高效非整合性人类iPSC诱导平台
CN104694570A (zh) * 2008-03-17 2015-06-10 斯克里普斯研究所 用于产生诱导的多能干细胞的组合化学遗传方法
US20150322405A1 (en) * 2011-12-13 2015-11-12 Flinders University Of South Australia Method of producing multipotent stem cells
WO2016022992A1 (en) * 2014-08-07 2016-02-11 Duke University Compositions and methods for the reprogramming of cells into cardiomyocytes

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060281136A1 (en) * 2003-05-20 2006-12-14 Shinichi Nishikawa Preparation of endodermal stem cells
WO2009096049A1 (ja) 2008-02-01 2009-08-06 Kyoto University 人工多能性幹細胞由来分化細胞
KR101874463B1 (ko) 2009-10-31 2018-08-02 뉴 월드 레보러토리즈 인코포레이티드. 세포의 재프로그램화 방법 및 이의 용도
US9453205B2 (en) * 2009-10-31 2016-09-27 Genesis Technologies Limited Methods for reprogramming cells and uses thereof
WO2011056971A2 (en) 2009-11-04 2011-05-12 Cellular Dynamics International, Inc. Episomal reprogramming with chemicals
CA2840192A1 (en) * 2011-06-23 2012-12-27 The Children's Hospital Of Philadelphia Self-renewing endodermal progenitor lines generated from human pluripotent stem cells and methods of use thereof
AU2014248167B2 (en) * 2013-04-03 2019-10-10 FUJIFILM Cellular Dynamics, Inc. Methods and compositions for culturing endoderm progenitor cells in suspension
CN104278008B (zh) * 2013-07-12 2020-08-21 北京宏冠再生医学科技有限公司 一种通过小分子化合物处理来制备多潜能干细胞的方法、试剂盒和用途
KR101782488B1 (ko) 2015-05-19 2017-09-28 주식회사 스템랩 Oct4가 도입된 인간체세포로부터 직접적 리프로그래밍을 통한 희소돌기아교 전구세포를 유도하는 방법

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104694570A (zh) * 2008-03-17 2015-06-10 斯克里普斯研究所 用于产生诱导的多能干细胞的组合化学遗传方法
CN101445791A (zh) * 2008-12-04 2009-06-03 北京大学 一种筛选多潜能细胞的方法及其专用培养基
CN102959076A (zh) * 2010-03-31 2013-03-06 斯克里普斯研究所 重编程细胞
WO2012006577A2 (en) * 2010-07-08 2012-01-12 Duke University Direct reprogramming of cells to cardiac myocyte fate
US20150322405A1 (en) * 2011-12-13 2015-11-12 Flinders University Of South Australia Method of producing multipotent stem cells
CN104120107A (zh) * 2013-04-28 2014-10-29 北京大学 一种新的诱导体细胞重编程的方法，试剂盒及用途
WO2016022992A1 (en) * 2014-08-07 2016-02-11 Duke University Compositions and methods for the reprogramming of cells into cardiomyocytes
CN104673741A (zh) * 2015-02-04 2015-06-03 广东省中医院 高效非整合性人类iPSC诱导平台

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP3483262A4 *
WANG, Y.F. ET AL.: "Conversion of Human Gastric Epithelial Cells to Multipotent Endodermal Progenitors Using Defined Small Molecules", CELL STEM CELL, vol. 19, no. 4, 21 July 2016 (2016-07-21), pages 449 - 461, XP029761238 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220177852A1 (en) * 2019-04-11 2022-06-09 Genex Health CO., LTD. Method for culturing primary cells of gastric cancer and gallbladder and bile duct cancer, and supporting reagents
CN110551688A (zh) * 2019-09-26 2019-12-10 上海交通大学医学院附属瑞金医院 一种诱导体细胞重编程为造血干/祖细胞且促进造血干/祖细胞体外扩增的组合物及其应用
CN110551688B (zh) * 2019-09-26 2023-03-21 上海交通大学医学院附属瑞金医院 一种诱导体细胞重编程为造血干/祖细胞且促进造血干/祖细胞体外扩增的组合物及其应用
CN111394302A (zh) * 2020-02-24 2020-07-10 中山大学 人睾丸间质干细胞的分离与培养方法
CN120118841A (zh) * 2025-03-24 2025-06-10 瓯江实验室 一种化学重编程诱导功能性 ciRPE 细胞的组合物、诱导方法及应用

Also Published As

Publication number Publication date
CN113151157A (zh) 2021-07-23
CN107099499A (zh) 2017-08-29
EP3483262A1 (en) 2019-05-15
JP2019521669A (ja) 2019-08-08
CN113151157B (zh) 2022-06-28
US20200385682A1 (en) 2020-12-10
US11149253B2 (en) 2021-10-19
CN107142240B (zh) 2021-01-29
CN107099499B (zh) 2021-02-02
JP7005528B2 (ja) 2022-01-21
EP3483262B1 (en) 2021-11-17
CN107142240A (zh) 2017-09-08
EP3483262A4 (en) 2019-12-25

Similar Documents

Publication Publication Date Title
US11149253B2 (en) Small molecule compound combination for reprogramming digestive tract derived epithelial cells to endodermal stem/progenitor cells, reprogramming method and application
CN111004770B (zh) 功能性肝细胞诱导方法及其专用三维诱导培养基和应用
JP7752651B2 (ja) オルガノイドを入手するための組成物および方法
US8278105B2 (en) Induction, propagation and isolation of liver progenitor cells
CN111004771B (zh) 建立体外模拟肝病模型的方法及其专用三维培养基
CN102985556B (zh) 分离高纯度细胞群体的生理学方法
JP2017012159A (ja) 多能性幹細胞から誘導した細胞を精製するための方法
CN107208056A (zh) 原始肠内胚层细胞及其制作方法
KR20170087520A (ko) 만능성 줄기 세포의 현탁 배양
KR102403165B1 (ko) 만능성 줄기 세포의 장의 중장 내배엽 세포로의 분화
CN107787363B (zh) 真正的胰腺祖细胞的分离
JP2008514214A (ja) ヒト胚盤胞由来幹細胞（ｈＢＳ）から肝細胞様細胞を発生させる方法
US20250164464A1 (en) Liver organoid-derived cultured hepatocytes
CN114395523A (zh) 一种诱导干细胞分化为肝细胞的试剂盒及其应用
US20200140826A1 (en) Maintenance and Expansion of Pancreatic Progenitor Cells
Petrakova et al. Comparative analysis reveals similarities between cultured submandibular salivary gland cells and liver progenitor cells
KR102879106B1 (ko) 3차원 장관 오가노이드 유래 장 줄기세포 집합체 배양을 위한 화학적 조성이 명확한 2차원 배양법
CN116731961A (zh) 一种第三咽囊内胚层的培养方法及应用
CN111718890B (zh) 一种将成纤维细胞转分化为腺上皮细胞的方法及其培养体系和应用
KR20260053025A (ko) 인공 고환 세포 및 이의 제조 방법
KR100861632B1 (ko) 줄기세포의 분화유도 방법
Azar et al. International Journal of Molecular Sciences Review The Use of Stem Cell-Derived Organoids in Disease Modeling: An Update
Parashurama Stem cell engineering of the endoderm: Approaches to controlling endoderm induction and differentiation from embryonic stem cells
HK1243458B (zh) 原始肠内胚层细胞及其制作方法

Legal Events

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

Ref document number: 17805782

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2018563588

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017805782

Country of ref document: EP

Effective date: 20190103