WO2017054783A1 - 诱导人干细胞向肝细胞定向分化的新方法 - Google Patents

诱导人干细胞向肝细胞定向分化的新方法 Download PDF

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WO2017054783A1
WO2017054783A1 PCT/CN2016/107647 CN2016107647W WO2017054783A1 WO 2017054783 A1 WO2017054783 A1 WO 2017054783A1 CN 2016107647 W CN2016107647 W CN 2016107647W WO 2017054783 A1 WO2017054783 A1 WO 2017054783A1
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cells
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differentiation
stem cells
inhibitor
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张培霖
陈立新
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海门雨霖细胞科技有限责任公司
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Definitions

  • the present invention belongs to the fields of biology and medicine; more specifically, the present invention relates to a novel method for directly inducing direct differentiation of human stem cells, such as human embryonic stem cells or induced pluripotent stem cells, into liver cells using only small molecule combination multi-target orientation, and application.
  • a dedicated direct differentiation medium for this new method is a dedicated direct differentiation medium for this new method.
  • liver disease According to the World Health Organization, millions of people worldwide die from liver disease every year. China is a big country with liver disease. There are 140 million people with hepatitis B and hepatitis C virus alone, accounting for 28% of the world. Acute and chronic liver failure caused by various reasons, critical illness, prognosis is dangerous, and the mortality rate is high ( 70 to 80%). Hepatocyte transplantation and bioartificial liver replacement therapy can not only treat liver failure, but also treat liver genetic metabolic diseases and neuropsychiatric disorders caused by elevated liver ammonia due to liver dysfunction; hepatocyte transplantation can also promote acute liver failure. Endogenous liver regeneration in patients.
  • liver source deficiency is a global problem, therefore, the use of human embryonic stem cells (embryonic stem cells, ES cells) and the multipotential differentiation potential of induced pluripotent stem cells (iPS cells), and the induction of differentiation into hepatocytes became one of the global hotspots for obtaining hepatocyte sources; while iPS cells broke through ethics. The restrictions are therefore more valued by researchers.
  • iPS cells Human iPS cells are induced to form pluripotent stem cells by introducing four genes (Oct4, Sox2, Klf4, c-Myc or Oct4, Sox2, Nanog, Lin28) into somatic cells (Takahashi K. Cell 2007; 131:861- 872, Yu J, et al. Science 2007; 318: 1917-1920).
  • iPS cells have similar properties to ES cells and can differentiate into the inner, middle and outer germ layers under specific induction conditions.
  • the iPS cells can be used for infinite expansion and multi-directional differentiation potential in vitro, and by direct induction of differentiation of iPS cells into hepatocytes, sufficient hepatocyte sources can be obtained.
  • the personalized hepatocytes obtained by the method can be maximized. To limit or reduce the immune rejection caused by allogeneic cell transplantation.
  • ES/iPS cells have been shown to differentiate into functional hepatocytes in vitro as early as 2007 (Cai J, et al, Hepatology 2007, 45(5): 1229-1239). With the deepening and extensive research, more and more effective methods of induced differentiation have emerged. At present, the most common differentiation method is to induce pluripotent stem cell differentiation by growth factor: firstly, the pluripotent stem cells differentiate into anterior definitive endoderm cells through Nodal signal and FGF signal, and then in BMP4 and FGF growth factors.
  • HGF hepatocyte growth factor
  • OSM oncostatin M
  • the ES/iPS cell culture conditions of most differentiation methods still need to be
  • the mouse feed cell layer is cultured or the culture medium contains animal-derived components.
  • the liver cells obtained by such differentiation are difficult to be clinically applied due to the possibility of unknown animal pathogens; 2.
  • Most differentiation methods are not efficient, and differentiated hepatocytes require additional purification steps because of low purity. The production cost also reduces the viability and biological activity of the cells, which is difficult to meet the requirements of clinical application; 4.
  • the surface markers of mature hepatocytes are lacking or low in ASGPR (Takayama K, et al, J Hepatol 2012, 57(3): 628-36); 5. Differentiated hepatocyte insufficiency, especially P450 metabolic enzymes are often missing or low (Schwartz RE, et al, Biotechnol Adv 2014, pii:S0734-9750(14)00005-6); 6. Introduction of foreign genes can greatly improve the above defects, however, the introduced foreign genes may change the genetic structure and lead to an increased risk of cancer, which cannot be practically applied. Clinical; 7. The proliferation, passage, cryopreservation and resuscitation of differentiated hepatocytes are closely related to clinical application, and almost all reports do not mention this problem.
  • hepatocytes obtained by differentiating ES/iPS cells by the existing differentiation method cannot be practically applied to clinical liver cell transplantation and bioartificial liver replacement therapy because they cannot meet clinical requirements.
  • Japanese scientist Takebe obtained "liver bud”-micro-hepatic tissue by co-cultivating iPSC and MSC with vascular endothelial cells Takebe T, et al, Nature 2013, 499 (7459): 481-4. Although it has a significant improvement in metabolism and survival rate in mice with liver injury.
  • this "liver bud” tissue has the urea synthesis function of normal liver tissue or hepatocytes; and whether the method can be applied to human liver cell differentiation is still unknown, and there is still a certain distance from the actual application;
  • this research provides new ideas for the clinical application research and development of ES/iPS cell differentiation hepatocytes.
  • Chemically induced cell direct reprogramming is the process of altering cell fate by chemical factors that induce regulation of cellular signaling pathways, epigenetic and chemical biological changes without altering the cellular gene sequence.
  • the invention is based on the direct reprogramming mechanism of cells, does not require stepwise induction, and does not require the use of cell growth factors, and uses only chemical small molecule induction regulation to directly differentiate human stem cells into high-purity, high-quality hepatocytes, without any Reported in the literature.
  • the basic mechanism of the present invention is mainly through the combination of two small molecules of GSK3 ⁇ inhibitor and TGF ⁇ inhibitor, multi-target induction of GSK3 ⁇ , TGF ⁇ signaling pathway change, and synergistic action of retinoids, and then regulation of stem cell signaling pathway and Epigenetic changes that directly differentiate stem cells into hepatocytes.
  • GSK3 ⁇ inhibitors and TGF ⁇ inhibitors comprise two classes, the same function, or a series of small molecules that induce the same target, and different combinations formed can induce stem cells to differentiate into hepatocytes to different degrees. Therefore, it is necessary to protect similar small molecule compounds that have the same function or induce the same target, have the same effect on the same signaling pathway, and constitute a patent for a small molecule combination capable of inducing direct differentiation of stem cells into hepatocytes.
  • a medium for inducing direct differentiation of human stem cells into hepatocytes comprising: a cell differentiation basic medium;
  • GSK3 ⁇ inhibitor final concentration of 0.5-8 uM
  • TGF ⁇ inhibitor final concentration of 0.1-10 uM
  • Retinoic acid compound final concentration is 0.001-10 uM
  • the medium is capable of inducing direct differentiation of human stem cells into hepatocytes, and obtaining human liver precursor cells or liver mature cells.
  • GSK3 ⁇ inhibitor final concentration of 0.5-5 uM
  • TGF ⁇ inhibitor final concentration of 0.5-8 uM
  • Retinoic acid compound final concentration is 0.01-5 uM.
  • the GSK3 ⁇ inhibitor is CHIR-99021 or / and CHIR-98014, the final concentration of which is 0.5-8 uM, preferably 0.5-5 uM;
  • the TGF ⁇ inhibitor is SB431542 or / and A83-01 or / and RepSox, the final concentration is 0.1-10 uM, preferably the amount is: 0.5-8 uM;
  • the retinoic acid compound is retinoic acid and has a final concentration of 0.001 to 10 uM; a preferred amount is 0.01 to 5 uM.
  • the medium may also be added to include one or more components selected from the group consisting of:
  • Rock inhibitor has a final concentration of 0.5-50 uM; preferably 1-20 uM; and/or
  • HGF Liver growth factor
  • Oncostatin M a final concentration of 1-100 ng/ml; preferably 5-50 ng/ml; and/or
  • Dexamethasone final concentration is 0.5-20 uM; preferably 2.5-10 uM;
  • the medium to which the above ingredients are added can increase cell survival rate, or promote differentiation and maturation of ES/iPS cells into hepatocytes, and maintain growth of liver mature cells.
  • the GSK3 ⁇ inhibitor comprises: CHIR-99021, BIO, AZD2858, TWS119, CHIR-98014, etc., having the same function, or inducing the same target.
  • the TGF ⁇ inhibitor comprises: SB431542, A83-01, SB525334, LY2109761, RepSox, etc., the same type of TGF ⁇ signaling pathway inhibitor or compound, or a combination thereof, which has the same function or induces the same target;
  • the ground is TGF ⁇ inhibitor SB431542 or / and A83-01;
  • the retinoids are natural or synthetic, including: Retinoic acid (RA), also known as: all trans retinoic acid (ATRA); 13-cis retinoic acid (13) -cis retinoic acid, 13-CRA), 9-cis-retinoic acid (9-CRA), etc., which have the same function or induce the same target. Or a compound, or a combination thereof; preferably retinoic acid (RA);
  • the Rock inhibitor includes: the same type of Rock signaling pathway inhibitor or compound represented by Y-27632 (alias: Y-27632 2HCI), GSK429286A, RKI-1447, etc., having the same function, or inducing the same target, Or a combination thereof; preferably a Rock inhibitor Y-27632.
  • the cell differentiation basic medium in another preferred embodiment, is 0.5% N2, 1% B27, 1% Non-AA, 1% Sodium pyruvate added to the basal cell culture medium, preferably Also added 1% streptomycin; wherein, the percentage of each component of the cell differentiation basic medium can also be up and down by 50%; preferably up and down 30%; more preferably up and down 20%, such as 10 %, 5%;
  • the basal cell culture medium includes, but is not limited to, DMEM/F12, MEM, DMEM, RPMI1640, Neuronal basal or Fischers, and the like.
  • composition comprising a GSK3 ⁇ inhibitor, a TGF ⁇ inhibitor, and a retinoid compound, and the use of the medium for inducing direct differentiation of human stem cells into hepatocytes to obtain human liver Precursor cells or liver mature cells; preferably, the composition further comprises a component selected from the group consisting of Rock inhibitor, liver growth factor, Oncostatin M or dexamethasone.
  • the GSK3 ⁇ inhibitor, the TGF ⁇ inhibitor, and the retinoid compound are in a molar ratio (which can also be converted into a weight ratio): (0.5-8): (0.1-10): ( 0.001-10); Preferably, (0.5-5): (0.5-8): (0.01-5) is present.
  • a kit for inducing direct differentiation of human stem cells into hepatocytes comprising a GSK3 ⁇ inhibitor, a TGF ⁇ inhibitor, and a retinoid; is used for inducing human stem cells to be directed to liver cells Direct differentiation; preferably, further comprising a component selected from the group consisting of a Rock inhibitor, a liver growth factor, Oncostatin M or dexamethasone; for increasing the direct differentiation of human stem cells into hepatocytes Cell viability, or promoting differentiation of ES/iPS cells into functional liver mature cells, and maintaining growth of liver mature cells; or the kit includes any of the media described above.
  • a method for inducing direct differentiation of human stem cells into hepatocytes comprising: directing human stem cells to direct differentiation into hepatocytes using a medium as described above; preferably The method steps include:
  • Human liver precursor cell differentiation initiation the culture plate is priming with matrigel, rat tail glue, gelatin, fibronectin, and glass adhesion protein for 30 minutes to 24 hours; then human stem cells are applied at the application site.
  • the hepatocyte differentiation medium or the hepatocyte differentiation-enhancing medium is preferably suspended in a hepatocyte differentiation medium, plated; 37 ⁇ 1° C., 5% CO 2 culture, and changed every 72 hours;
  • Subculture step Digest the differentiated human ES/iPS cells into single cells with digestive juice, including trypsin, EDTA, Acutase, Tryple E, etc., and resuspend and pass through 1:2-1:5; according to method steps (1) The method subcultures differentiated cells;
  • Human liver precursor cell differentiation obtained human liver precursor cells obtained by differentiation and culture for 10-15 days according to the method steps (1) and (2); obtained human liver precursor cells can be used for freezing
  • the cells can be further differentiated into functional human liver mature cells by storage, resuscitation, and passage; preferably, the subculture method is: subculture the differentiated human liver precursor cells according to the method described in the step (2);
  • Human liver precursor cell maturation culture human liver precursor cells obtained by the method step (3) differentiation culture are applied to the hepatocyte differentiation medium or hepatocyte differentiation-enhancing medium; preferably, in hepatocytes
  • the differentiation culture medium is further differentiated and cultured, 37 ⁇ 1°C, 5% CO 2 differentiation culture for 7-15 days, and functional human liver mature cells can be obtained; the obtained functional human liver mature cells can be used for cryopreservation, resuscitation, Limited passage.
  • the human stem cells include, but are not limited to, human embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, cord blood stem cells, and the like, and have humanized stem cells having multipotential differentiation potential;
  • the human stem cells are human embryonic stem cells or induced pluripotent stem cells.
  • Figure 1 Comparison of morphology of human ES cells and differentiated liver precursor cells; left: human ES cells; right: human ES cells differentiated liver precursor cells.
  • Figure 3 Comparison of morphology of human ES cells with differentiated liver mature cells and human primary hepatocytes; left panel: human ES cells; middle panel: human ES cells differentiated liver mature cells; right panel: human primary hepatocytes.
  • FIG. 4 Flow cytometric analysis of liver-specific marker staining of human ES cell-differentiated liver mature cells. The results showed that the proportion of cells in which the obtained hepatocyte-specific markers of the differentiated liver mature cells were positive was very high.
  • FIG. 1 Comparison of human iPS cell differentiation liver mature cells and primary hepatocyte albumin production, wherein PHH is: primary hepatocytes; iPSC-Hep is: human iPS cells differentiate liver mature cells; C1-C4 is: 4 Culture conditions.
  • FIG. 6 Comparison of urea production between human iPS cells differentiated liver mature cells and primary hepatocytes.
  • PHH is: primary hepatocytes
  • iPSC-Hep is: human iPS cells differentiate liver mature cells
  • C1-C4 are: 4 culture conditions.
  • the source of urea is blood ammonia in the blood, and its production amount reflects the detoxification ability of liver cells.
  • FIG. 1 Glycogen staining of human ES cells differentiated liver mature cells. The depth of staining shows the ability of hepatocytes to store glycogen.
  • FIG. 8 Induction of P450 enzyme (CYP3A4 and CYP1A2) activity by human ES cells differentiated liver mature cells. Left panel: CYP3A4 increased activity under different concentrations of rifampicin; right panel: CYP1A2 increased activity under different concentrations of omeprazole.
  • Figure 9 Comparison of morphology of human ES cells and liver cells and liver cells obtained by direct differentiation; left: human ES cells; in the figure: differentiation of liver precursor cells for 6 days; right: differentiation of liver cells for 15 days.
  • Figure 9 shows that the GSK3 ⁇ inhibitor BIO combined with a small molecule composed of a TGF ⁇ inhibitor and retinoic acid can also induce direct differentiation of stem cells into liver precursor cells and liver mature cells.
  • Figure 10 Comparison of morphology of human iPS cells and liver cells and liver cells obtained by direct differentiation; left: human iPS cells; in the figure: differentiation of liver precursor cells for 6 days; right: differentiation of liver cells for 20 days.
  • Figure 10 shows that the TGF ⁇ inhibitor RepSox combined with a small molecule composed of a GSK3 ⁇ inhibitor and retinoic acid can also induce direct differentiation of stem cells into liver precursor cells and liver mature cells.
  • Figure 11 Comparison of morphology of human ES cells with hepatocytes derived from direct differentiation; left: human ES cells; in the figure: differentiation of 6-day liver precursor cells; right: differentiation of 16-day liver cells.
  • Figure 11 shows that the small molecule combination of GSK3 ⁇ inhibitor CHIR-98014 and TGF ⁇ inhibitor SB525334 and retinoic acid can also induce stem cells to directly differentiate into liver precursor cells and liver mature cells.
  • the inventors have intensively studied to induce direct differentiation of human stem cells, preferably human embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) into hepatocytes.
  • the new method and its dedicated direct differentiation medium does not need to introduce a foreign gene into a stem cell, does not require a growth factor, and does not require stepwise induction, and can realize liver differentiation of stem cells by conventional culture, and the obtained differentiated human liver cell has typical characteristics of human liver cells.
  • the obtained human liver precursor cells can be maintained for a long time, can also be frozen, resuscitated, proliferated and passaged; the mature differentiation culture can be continued to obtain functional human liver mature cells; the obtained functional human liver mature cells can be frozen. Deposit, recovery, limited passage, and maintenance.
  • the method has simple culture conditions, low cost, and is safe and stable.
  • the term “comprising” or “including” includes “comprising”, “consisting essentially of”, “consisting essentially of” and “consisting of.”
  • the induced direct differentiation target is human stem cells, including but not limited to: human embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, cord blood stem cells, and other human stem cells having multipotential differentiation potential; The best is: human embryonic stem cells or induced pluripotent stem cells;
  • the present inventors provide a novel method for inducing human stem cells, preferably to induce direct differentiation of human embryonic stem cells or induced pluripotent stem cells into hepatocytes, and a dedicated direct differentiation medium thereof.
  • the dedicated direct differentiation medium includes "hepatocyte differentiation medium” and "hepatocyte differentiation-enhancing medium”.
  • the hepatocyte differentiation medium includes: a GSK3 ⁇ inhibitor, a TGF ⁇ inhibitor, a retinoid compound.
  • the above components are added to the cell differentiation basic medium in a suitable ratio to induce human stem cells to be directed to the liver cells. Differentiation.
  • the "GSK3 ⁇ inhibitor” refers to a general term for inhibitors capable of inhibiting the GSK3 ⁇ signaling pathway in cells, including but not limited to: CHIR-99021, BIO, AZD2858, TWS119, CHIR-98014, and the like.
  • CHIR-99021 (CT99021), which is a GSK-3 ⁇ and ⁇ inhibitor with IC50 of 10 nM and 6.7 nM, respectively, which is 500 times more potent than CDC2, ERK2 and other kinases;
  • CHIR-99021 (CT99021) HCl a hydrochloride salt of CHIR-99021, is a GSK-3 ⁇ / ⁇ inhibitor with an IC50 of 10 nM/6.7 nM in a cell-free assay, which can be used to distinguish between GSK-3 and its most Close homologs Cdc2 and ERK2;
  • BIO which is a specific GSK-3 inhibitor
  • the IC50 of GSK-3 ⁇ / ⁇ in a cell-free assay is 5 nM
  • AZD2858 a selective GSK-3 inhibitor with an IC50 of 68 nM, enhances the Wnt signaling pathway
  • TWS119 which is a GSK-3 ⁇ inhibitor with an IC50 of 30 nM in a cell-free assay
  • CHIR-98014 which is a potent GSK-3 ⁇ / ⁇ inhibitor with an IC50 of 0.65 nM/0.58 nM in a cell-free assay
  • AR-A014418 an ATP competitive and selective GSK3 ⁇ inhibitor with IC50 and Ki of 104 nM and 38 nM in a cell-free assay
  • LY2090314 which is a potent GSK-3 inhibitor, acting on GSK-3 ⁇ / ⁇ with an IC50 of 1.5 nM/0.9 nM;
  • SB216763 which is a potent, selective GSK-3 ⁇ / ⁇ inhibitor with an IC50 of 34.3 nM;
  • AZD1080 an orally bioavailable, selective, brain-permeable GSK3 inhibitor that inhibits human GSK3 ⁇ and GSK3 ⁇ , Ki of 6.9 nM and 31 nM, respectively, is more selective than CDK2, CDK5, CDK1 and Erk2 14 times higher.
  • the GSK3 ⁇ inhibitor is CHIR-99021, which is nicknamed CT99021; and its molecular structural formula is as shown in the following formula (I):
  • TGF ⁇ inhibitor refers to a general term for inhibitors capable of inhibiting the TGF ⁇ signaling pathway in cells, including but not limited to: SB431542, A83-01, SB525334, LY2109761, RepSox, and others having the same function.
  • SB-431542 which is a potent, selective ALK5 inhibitor with an IC50 of 94 nM, comparable to p38, MAPK It is 100 times more potent than other kinases;
  • a 83-01 which is an inhibitor of ALK5, ALK4 and ALK7 with IC50 of 12, 45 and 7.5 nM, respectively;
  • SB525334 a potent, selective TGF ⁇ receptor I (ALK5) inhibitor with an IC50 of 14.3 nM in a cell-free assay, 4 times lower than ALK5 and no activity against ALK2, 3, and 6;
  • LY2109761 which is a novel, selective TGF- ⁇ receptor type I/II (T ⁇ RI/II) dual inhibitor, with a Ki of 38 nM and 300 nM in a cell-free assay, respectively;
  • RepSox a potent, selective TGF ⁇ R-1/ALK5 inhibitor, binds ATP to ALK5 and ALK5 autophosphorylate, with IC50 of 23 nM and 4 nM in cell-free assays, respectively.
  • SD-208 a selective TGF- ⁇ RI (ALK5) inhibitor with an IC50 of 48 nM and a selectivity more than 100-fold higher than TGF- ⁇ RII;
  • GW788388 a potent, selective ALK5 inhibitor with an IC50 of 18 nM in a cell-free assay, also inhibits TGF-beta type II receptor and activin type II receptor activity, but does not inhibit BMP type II receptor;
  • SB505124 a selective TGF ⁇ R inhibitor that acts on ALK4 and ALK5, with IC50 of 129 nM and 47 nM in cell-free assays, also inhibits ALK7, but does not inhibit ALK1, 2, 3 or 6;
  • EW-7197 a highly potent, selective, orally bioavailable TGF- ⁇ receptor ALK4/ALK5 inhibitor with IC50 of 13 nM and 11 nM, respectively.
  • the TGF ⁇ inhibitor is SB 431542 (or SB-431542); and its molecular structural formula is as shown in the following formula (II):
  • the TGF ⁇ inhibitor is A83-01 (or referred to as A8301); and its molecular structural formula is as shown in the following formula (III):
  • the retinoids include: Retinoic acid (RA), alias: all trans retinoic acid (ATRA); 13-cis retinoic acid (13-cis) Retinoic acid, 13-CRA), 9-cis-retinoic acid (9-CRA), etc., the same type of retinoid differentiation agent or compound that has the same function or induces the same target. , or a combination thereof:
  • Retinoic acid compounds are a group of oxidative metabolites or derivatives of vitamin A (retinol) and synthetic structures with similar structures to vitamin A, both natural and synthetic. Mainly include: Retinoic acid (RA), alias: retinoic acid, all trans retinoic acid (ATRA); 13-cis retinoic acid (13-CRA), 9-cis-retinoic acid (9-CRA), isotretinoin, dimension Fenretinide, Acitretin, Etretinate, Tazarotene, Adapalene and TTNPB, CD437, Targretin and the like.
  • RA Retinoic acid
  • ATRA all trans retinoic acid
  • 13-CRA 13-cis retinoic acid
  • 9-CRA 9-cis-retinoic acid
  • isotretinoin dimension Fenretinide, Acitretin, Etretinate, Tazarotene, Adapalene and TTNPB, CD437, Targretin and the
  • Retinoic acid compounds are characterized by the regulation of differentiation, proliferation, and apoptosis of many cell types in vitro and in vivo. Many of the retinoids and their isomer derivatives have the same or similar functions. Therefore, it has become the most important type of differentiation-inducing agent and has been used for clinical treatment of skin diseases.
  • the retinoic acid (RA), or all trans retinoic acid (ATRA), retinoic acid, vitamin A acid, vitamin formic acid, retinoic acid, All-trans retinoic acid and retinoic acid have a molecular structural formula as shown in the following formula (IV):
  • Rock inhibitor refers to a general term for inhibitors capable of inhibiting the Rock signaling pathway in cells, including but not limited to: Y-27632, GSK429286A, RKI-1447, etc., having the same function. , or the same class of inhibitors that induce the same target:
  • Y-27632 (Y-27632 2HCl), a selective ROCK1 (p160ROCK) inhibitor with a K i of 140 nM in a cell-free assay, compared to other kinases including PKC, cAMP-dependent protein kinase, MLCK and PAK More than 200 times stronger;
  • GSK429286A is a selective ROCK1 and ROCK2 inhibitor with IC50 of 14 nM and 63 nM, respectively;
  • RKI-1447 is a potent ROCK1 and ROCK2 inhibitor with IC50 of 14.5nM and 6.2nM, respectively, with anti-invasive and anti-tumor activity;
  • Thiazovivin is a novel ROCK inhibitor with an IC50 of 0.5 ⁇ M in a cell-free assay that promotes the survival of human embryonic stem cells (hESC) after single cell isolation;
  • RO-3306 is an ATP-competitive selective CDK1 inhibitor with a K i of 20 nM and a selectivity more than 15 times that of other human kinases.
  • the Rock inhibitor is Y-27632 (Y-27632 2HCI), which is synonymous with Y-27632dihydrochloride; Y-27632 2HCI; and its molecular structural formula is represented by the following formula (V).
  • the present inventors first proposed the down-regulation of GSK3 ⁇ signaling pathway and TGF ⁇ signaling pathway in the field, and combined with retinoic acid (RA) can promote direct differentiation of human pluripotent stem cells into hepatocytes. It should be understood that the same technical effects can be achieved by inhibiting the GSK3 ⁇ signaling pathway and the TGF ⁇ signaling pathway other than the specific GSK3 ⁇ inhibitor and TGF ⁇ inhibitor enumerated in the examples of the present invention, and should also be included in the present invention. in.
  • RA retinoic acid
  • Rock inhibitor in addition to the specific retinoic acid (RA)-inducing differentiation agent and the Rock inhibitor as exemplified in the examples of the present invention, other inhibitors of the retinoids and the Rock signaling pathway having the same function or inducing the same target may be used. Real The same technical effects are now also included in the present invention.
  • the present invention also includes equivalent compounds, analogs, derivatives and/or salts, hydrates or precursors thereof having the same function as the above respective compounds or inducing the same target.
  • Biochemical reagents and pharmaceutical preparations prepared from the above respective compounds are also included in the present invention.
  • Analogs of the compounds include, but are not limited to, isomers, racemates of the compounds.
  • the compound has one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis or trans isomers.
  • the “salt” includes, but is not limited to, (1) a salt formed with an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.; (2) a salt formed with an organic acid such as acetic acid, oxalic acid, or dibutyl Acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine.
  • Other salts include salts formed with alkali metals or alkaline earth metals such as sodium, potassium, calcium or magnesium.
  • the "precursor of a compound” means a compound which can be converted into any one of the above compounds in a medium, or a compound of any of the above compounds, after being administered or treated by an appropriate method.
  • the "cell differentiation basic medium” is a basic nutrient maintenance medium used in the art for performing human stem cells, such as human embryonic stem cells or induced pluripotent stem cell differentiation culture.
  • human stem cells such as human embryonic stem cells or induced pluripotent stem cell differentiation culture.
  • the "cell differentiation basic medium” cannot be used to cause human embryonic stem cells or induction.
  • the pluripotent stem cells are directed to differentiate directly into the liver cells.
  • the "cell differentiation basic medium” is such that 0.5% N2, 1% B27, 1% Non-AA, 1% Sodium pyruvate is added to the basal cell culture medium, preferably 1 is also added. % streptomycin (percentage is in v/v, wherein each percentage can also float up and down 50%; preferably up and down 30%; better up and down 20%, such as 10%, 5%) .
  • the "cell differentiation basic medium” may also be commercially available.
  • the basal cell culture medium may be, but not limited to, DMEM/F12, MEM, DMEM, RPMI1640, Neuronal basal or Fischers, and the like. It will be understood that those skilled in the art are familiar with the formulation or purchase route of the basal cell culture medium described, and therefore, the basal cell culture medium is not limited to those exemplified in the present invention.
  • the "hepatocyte differentiation medium" of the present invention is prepared as follows:
  • GSK3 ⁇ inhibitor CHIR-99021 final concentration is 0.5-8 uM; preferred amount is: 0.5-5 uM;
  • TGF ⁇ inhibitor SB431542 or / and A83-01 final concentration is 0.1-10 uM; preferred amount is: 0.5-8 uM;
  • Retinoic acid (RA) The final concentration is 0.001-10 uM; the preferred amount is 0.01-5 uM.
  • the "hepatocyte differentiation medium” of the "new method for inducing differentiation of human stem cells into hepatocytes" of the present invention can be obtained by the above formulation.
  • hepatocyte differentiation-enhancing medium can be obtained by adding any one or more of the following components:
  • Rock inhibitor see the foregoing for details of the Rock inhibitor or preferably the Rock inhibitor Y-27632: the final concentration is 0.5-50 uM; preferred amount: 1-20 uM; and/or
  • HGF Liver growth factor
  • Oncostatin M final concentration is 1-100 ng/ml; preferred amount: 5-50 ng/ml; and/or:
  • Dexamethasone The final concentration is 0.5-20 ⁇ M; the preferred amount is 2.5-10 ⁇ M.
  • hepatocyte differentiation-enhancing medium can be obtained which increases the cell viability during the direct differentiation of human stem cells into hepatocytes; or promotes the differentiation of ES/iPS cells, or liver precursor cells into Functional liver mature cells; or enhance certain biological behaviors and functions of differentiated hepatocytes; and maintain the growth of mature liver cells for a long time
  • the present invention also discloses a novel method for inducing human stem cells using only small molecule combinations, preferably to induce direct differentiation of human embryonic stem cells or induced pluripotent stem cells into hepatocytes.
  • human ES/iPS cells are induced to differentiate and culture in the "hepatocyte differentiation medium” and/or "hepatocyte differentiation-enhancing medium" of the present invention, and high-purity liver precursor cells or liver mature cells can be obtained.
  • the method for culturing the liver precursor cells is: the culture plate is primed for 1-24 hours, and then the human ES/iPS cells are added to the hepatocyte differentiation medium or the hepatocyte differentiation-enhancing medium. It is preferably suspended in hepatocyte differentiation medium, plated; cultured at 37 ⁇ 1°C, 5% CO2, once every 72 ⁇ 1 hours; cultured for 10-15 days, liver precursor cells can be obtained.
  • the method for priming the culture plate is well known in the art, and the materials that can be used for the culture plate priming include, but are not limited to, matrigel, rat tail glue, gelatin, fibronectin, villogen, etc., and optionally One kind of making a base.
  • the cells can be subcultured at a ratio of 1:2-1:5.
  • the subculture step is: digesting human ES/iPS cells in the differentiation culture into single cells with a digestive juice, and resuspending and subculturing the differentiated cells at a ratio of 1:2-1:5.
  • the digestive juice may be a solution comprising one or more digestive enzymes such as trypsin, EDTA, Acutase, Tryple E and the like.
  • the obtained liver precursor cells can be used for cryopreservation, resuscitation, passage, and can be further induced into functional human liver mature cells.
  • the method of inducing liver precursor cells to become functional human liver mature cells comprises: adding hepatocyte precursor cells to said hepatocyte differentiation medium or hepatocyte differentiation-enhancing medium, preferably hepatocyte differentiation.
  • the culture medium was suspended, 37 ⁇ 1° C., 5% CO 2 , and further matured and cultured for 7-15 days to obtain functional liver mature cells.
  • the liver mature cells obtained by the method of the invention can be cryopreserved, resuscitated, limited passage, and maintained for a long time; can also be applied to cell transplantation for liver disease, bioartificial liver construction, new drug liver toxicity test, drug efficacy evaluation, drug target identification It can provide sufficient hepatocyte source or hepatocyte model for basic research and clinical application of biology, medicine and pharmacy; its differentiation process can also provide the best research platform for human liver cell development and differentiation process, and its application prospect is very broad.
  • human embryonic stem cells involved in the present invention are commercially available commercially. Commercially available embryonic stem cell lines, such as those listed in Table 1, do not involve the use of human embryos.
  • WiCell an unofficial, non-profit affiliate that distributes human embryonic stem cells to qualified scientists at low cost.
  • the mechanism for providing such ready-made human embryonic stem cells includes NSCB (National Stem Cell Bank), ES CELL INTERNATIONAL, nov cell, TECHNION-HOME TO ISRAEL'S NOBEL SCIENTISTS, and UCSF (University of California San Fransisco). Therefore, human embryonic stem cells can be obtained by other means of "taking from human embryos".
  • the direct differentiation purity is high without additional purification means.
  • the results of flow cytometry show that the obtained differentiated liver cells have a purity of more than 90%; the mature liver cell surface markers of hepatocytes obtained after differentiation and culture Asgp-positive cells are >90% (20-60% in other reports); other hepatocyte-specific immune markers such as ALB, CYP3A, and HNF4a-positive cells are above 80%; this indicates the high quality of differentiated liver cells. And effectively reduce costs;
  • hepatocyte The universality and reproducibility of directly differentiated hepatocytes are good.
  • the inventors have verified that 9 human ES/iPS (including 2 human ES cells and 7 human iPS cell lines) cell lines are all differentiated into morphological functions. Hepatocyte;
  • hepatocytes have a number of specific functions of hepatocytes, such as albumin production, urea synthesis, and P450 enzyme (CYP enzyme: CYP3A4, CYP1A2) activity induction, glycogen storage.
  • CYP enzyme CYP3A4, CYP1A2
  • the hepatocytes obtained by the method of the invention are functional hepatocytes;
  • the method of the present invention is an animal-free culture method, which can culture ES/iPS cells without using animal feeder cells, and can be directly applied to mass production and clinical application of GMP standards. This feature has not been reported; There is contamination of animal-derived substances;
  • the differentiation stage is clear and the quality is easy to control
  • liver precursor cells obtained by direct differentiation can be proliferated, frozen and resuscitated; and can continue to differentiate into mature functional hepatocytes; the obtained liver mature cells can be frozen, resuscitated, limited passage, and maintained.
  • Example 1 Preparation of a medium for inducing human embryonic stem cells or inducing direct differentiation of pluripotent stem cells into hepatocytes
  • the cell differentiation basic medium was prepared in accordance with a conventional method. Namely: in DMEM/F12 (basal cell culture medium): 0.5% N2, 1% B27, 1% Non-AA, 1% Sodium pyruvate, 1% streptomycin mixture (100x) was added. Among them, the percentages are all in v/v.
  • TGF ⁇ inhibitor SB431542 5 uM
  • TGF ⁇ inhibitor A83-01 3uM
  • TGF ⁇ inhibitor SB431542 2uM
  • TGF ⁇ inhibitor A83-01 3uM
  • TGF ⁇ inhibitor SB431542 5uM
  • TGF ⁇ inhibitor SB431542 5uM
  • TGF ⁇ inhibitor SB525334 6uM
  • Dexamethasone The final concentration is 5 uM.
  • Oncostatin M final concentration 20 ng/ml.
  • HGF Human liver growth factor
  • hepatocyte differentiation medium 2 or 8 Based on the above “hepatocyte differentiation medium 2 or 8" formula, add:
  • Example 2 Human hepatocyte precursor cell differentiation culture and liver mature cell culture using hepatocyte differentiation medium 1 and hepatocyte differentiation-enhancing medium 2
  • the culture plate was primed with Matrigel for 12 hours, hepatocyte differentiation medium 1 was added to the culture plate, and then human embryonic stem cells (ES) (see the left panel of Fig. 1) were suspended in Hepatocyte Differentiation Medium 1, Laying; culture at 37 ° C, 5% CO 2 , changing every 72 hours.
  • ES human embryonic stem cells
  • Human embryonic stem cells in hepatocyte differentiation medium 1 Human embryonic stem cells in hepatocyte differentiation medium 1, human liver precursor cells obtained by differentiation and culture at 37 ° C, 5% CO 2 for 10-15 days, human ES cells and liver precursor cells obtained by differentiation are shown in the figure.
  • the right picture shows liver precursor cells obtained by differentiation culture for 15 days, which can be used for cryopreservation, resuscitation, passage, and further induction into functional human liver mature cells.
  • the differentiated cultured cells When the differentiated cultured cells reach 90% confluence during the differentiation culture, they can be passaged 1:2 to 1:5.
  • Subculture step human ES cells in the differentiation process are digested into single cells by digestive juice (containing trypsin), resuspended and passaged 1:2 to 1:5; subcultured and differentiated according to the method described in the above "1" Cells; subculture results are shown in Figure 2.
  • human liver precursor cells obtained by culturing or subculture were cultured or subcultured according to the methods described in the above 1, 2, for differentiation induction culture: human liver precursor cells were cultured in "hepatocyte differentiation-enhancing medium 2", 37 ° C, 5% CO 2 Mature culture for 7-15 days to obtain functional human liver mature cells.
  • the functional human liver mature cells obtained by mature culture for 7-15 days can be frozen, resuscitated, limited passage, maintained for a long time, and used for various functional tests.
  • Example 3 Flow cytometric analysis of hepatic-specific marker staining for differentiation of human ES cells into hepatic mature cells using hepatocyte differentiation medium 4
  • the method of inducing differentiation of human ES cells into hepatocytes is the same as in Example 2. The difference is that hepatocyte differentiation medium 4 has been used for culture.
  • liver-specific marker staining of human ES cells differentiated into liver mature cells The liver mature cells obtained by inducing differentiation of human ES cells by the above experimental steps were subjected to immunostaining using human hepatocyte-specific markers (AAT, ALB, Asgpr, CYP3A, HNF4a) by a conventional immunostaining method.
  • the immunostaining method is:
  • the proportion of cells in which the obtained hepatocyte-specific markers of differentiated liver mature cells were positive was very high. Therefore, it was confirmed that the culture medium and the culture method of the present invention obtained functional human liver mature cells.
  • Example 4 using hepatocyte differentiation medium 1, 2 and hepatocyte differentiation-enhancing medium 1, 4 (corresponding to C1 to C4, respectively) and hepatocyte differentiation-enhancing medium 2, respectively, were induced to differentiate human iPS cells into liver. Comparison of mature cells and albumin production in primary hepatocytes
  • the method of inducing differentiation of human iPS cells into hepatic mature cells was the same as in Example 2 except that the differentiation culture was simultaneously carried out by grouping 4 different differentiation medium groups and induction of culture of human iPS cells.
  • the obtained four groups of induced liver immature cells differentiated from human iPS cells were compared with human primary hepatocytes for albumin production.
  • the specific method is:
  • the hepatic maturation cells and the commercially available human primary hepatocytes obtained by the differentiation of the four groups of induced human iPS cells obtained by the above experimental steps were subjected to albumin secretion function detection by an ELISA kit; for the detection procedure, refer to the kit manual (Bioassay System, USA) /DIAG-250, BCG Albumin assay kit).
  • Example 5 using hepatocyte differentiation medium 1, 2 and hepatocyte differentiation-enhancing medium 1, 4 (corresponding to C1 to C4, respectively) and hepatocyte differentiation medium 3 were respectively induced to induce human iPS cells to differentiate into liver maturation. Comparison of urea production between cells and human primary hepatocytes
  • the method of inducing differentiation of human iPS cells into hepatic mature cells is the same as in Example 2, except that the differentiation of the four groups of different differentiation media is simultaneously carried out and differentiation of human iPS cells is induced.
  • the urea synthesis function and the commercially available human primary hepatocytes obtained from the differentiation of the four groups of human iPS cells obtained in the above experiment were tested for urea synthesis function by the urea nitrogen detection kit; for the detection procedure, refer to the kit manual (Bioassay System, USA/ DIUR-500,Urea assay kit);
  • the source of urea is blood ammonia in the blood, and its production amount reflects the detoxification ability of liver cells.
  • the results of comparison of urea production between 4 groups of human iPS cell-differentiated hepatocytes and human primary hepatocytes are shown in Fig. 6.
  • the obtained human iPS cell-differentiated liver mature cells have a function of generating urea specific to hepatocytes.
  • Example 6 Using glycogen differentiation medium 5 and hepatocyte differentiation-enhancing medium 3 to induce glycogen staining of human ES cells to differentiate liver mature cells
  • the method of inducing differentiation of human ES cells into liver mature cells is the same as in Example 2 except that the hepatocyte differentiation medium 5 is used for differentiation culture and hepatocyte differentiation enhancement medium 3 for differentiation and maturation culture.
  • Liver mature cells obtained by differentiation of human ES cells are subjected to glycogen staining.
  • the depth of staining shows the ability of hepatocytes to store glycogen.
  • Hepatic glycogen staining was performed using the Schiff method. The specific method is:
  • liver mature cells obtained by the method of the present invention have the same glycogen storage activity as human liver cells.
  • Example 7 Induction of P450 enzymes (CYP3A4 and CYP1A2) activity in hepatic mature cells obtained by inducing differentiation of human ES cells by using hepatocyte differentiation-enhancing medium 4 and hepatocyte differentiation-enhancing medium 2
  • the method for inducing differentiation of human ES cells into liver mature cells is the same as in Example 2, except that the hepatocyte differentiation-enhancing medium 4 is used for differentiation culture; and the hepatocyte differentiation-enhancing medium 2 is used for further differentiation and maturation.
  • a liver mature cell obtained by inducing differentiation of human ES cells by the above experimental procedure was subjected to an increase in P450 enzyme (CYP3A4) induced by Rifampicine.
  • the method for inducing P450 enzyme activity is as follows:
  • liver mature cells obtained by inducing human ES cell differentiation in the above experimental procedure were subjected to an increase in P450 enzyme (CYP1A2) induced by omeprazole.
  • P450 enzyme CYP1A2
  • the treatments were treated with different concentrations of omeprazole (1 uM, 10 uM, 25 uM), and the treatment group without the addition of omeprazole and other conditions was used as a control.
  • the CYP1A2 gene expression was quantitatively detected by qRT-PCR on the mature liver cells obtained by the differentiation of the induced ES cells.
  • the method for inducing differentiation of human ES cells into liver precursor cells and liver (mature) cells is the same as in Example 3; the results are shown in FIG. 9;
  • Figure 9 shows that the GSK3 ⁇ inhibitor BIO combined with a small molecule composed of a TGF ⁇ inhibitor and retinoic acid can also induce direct differentiation of stem cells into liver precursor cells and liver mature cells.
  • the method for inducing differentiation of human iPS cells into liver precursor cells and liver (mature) cells is the same as in Example 3; the results are shown in FIG. 10;
  • FIG. 10 shows that the TGF ⁇ inhibitor RepSox combined with a small molecule composed of a GSK3 ⁇ inhibitor and retinoic acid can also induce direct differentiation of stem cells into liver precursor cells and liver mature cells.
  • Example 10 Comparison of Morphological Differentiation of Human ES Cells Directly Differentiated into Liver Precursor Cells and Hepatocytes by Hepatocyte Differentiation Medium 8
  • the method for inducing differentiation of human ES cells into liver precursor cells and liver (mature) cells is the same as in Example 3; the results are shown in FIG. 11;
  • Figure 11 shows that the small molecule combination of GSK3 ⁇ inhibitor CHIR-98014 and TGF ⁇ inhibitor SB525334 and retinoic acid can also induce stem cells to directly differentiate into liver precursor cells and liver mature cells.
  • the novel method for inducing direct differentiation of human stem cells (such as embryonic stem cells or induced pluripotent stem cells) into hepatocytes of the present invention has the following characteristics:
  • the differentiation stage is clear and the quality is easy to control
  • the direct differentiation efficiency is high, and 1 ⁇ 10 6 human ES/iPS cells can differentiate into functional liver cells of 5-10 ⁇ 10 6 or more;
  • hepatocytes are of high purity without additional purification means (Asgp ⁇ 85%); therefore, the cell activity is not damaged, the operation is simple, and the cost is reduced;
  • Directly differentiated hepatocytes have complete functions, such as albumin production, urea synthesis, glycogen storage and P450 enzyme activity induction;
  • the direct differentiation method has good universality and reproducibility. Two human ES cells and seven human iPS cell lines are differentiated into functional liver mature cells;
  • the differentiated liver precursor cells can be proliferated, frozen and resuscitated; they can continue to differentiate into functional liver mature cells; the obtained liver mature cells can be frozen, resuscitated, limited passage, and maintained.
  • the morphological function of the hepatocytes obtained by differentiation is highly consistent with human primary hepatocytes.
  • ES/iPS cells are not cultured using animal feeder cells, so there is no contamination of animal-derived substances;
  • GSK3 ⁇ inhibitors, TGF ⁇ inhibitors contain two classes, the same function, or a series of small molecules that induce the same target, the different combinations formed can induce stem cells to differentiate into hepatocytes to different degrees.
  • the method is simple, easy to operate; does not need to form an embryo body (EB);

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Abstract

一种使用小分子组合多靶点定向诱导人干细胞，如人胚胎干细胞（EmbryonicStemcells，ES细胞）或诱导多能干细胞（InducedPluripotentStemcells，iPS细胞）向肝细胞直接分化的方法。一种人干细胞向肝细胞定向直接分化培养的培养基及培养方法。上述方法不需要在干细胞内导入外源基因，不需要分步诱导，也不需要各种细胞生长因子，只使用化学小分子，即可实现人干细胞向肝细胞定向直接分化，所获得的分化人肝细胞具有人肝细胞的典型特征，分化的肝前体细胞可以长期传代；分化的肝成熟细胞可有限传代。并且，所述方法为常规培养，操作简单，成本低廉且安全稳定。

Description

诱导人干细胞向肝细胞定向分化的新方法 技术领域
本发明属于生物学和医学领域；更具体地，本发明涉及仅使用小分子组合多靶点定向诱导人干细胞，如人胚胎干细胞或诱导多能干细胞向肝细胞直接分化的新方法，以及应用于该新方法的专用直接分化培养基。
背景技术
根据世界卫生组织统计，全球每年有上百万的人死于肝病。中国是一个肝病大国，仅乙型和丙型肝炎病毒带原者就有1.4亿，约占全球的28％；由各种原因导致的急慢性肝衰竭，病情危重，预后凶险，病死率高(70～80％)。而肝细胞移植和生物人工肝替代治疗不仅可以治疗肝衰竭，还可以治疗肝遗传代谢性疾病以及因为肝脏功能障碍，血氨升高而导致的神经精神疾患；肝细胞移植还可以促进急性肝衰竭病人的内源性肝再生。然而除肝细胞移植和生物人工肝构建外，新药肝毒检测、肝病研究等方面，都需要大量合格的人肝细胞；但肝源缺乏是全球问题，因此，利用人胚胎干细胞(embryonic stem cells，ES细胞)和诱导多能干细胞(induced pluripotent stem cells，iPS细胞)的多向分化潜能，定向诱导其分化为肝细胞就成为获得肝细胞来源的全球热点研究之一；而iPS细胞突破了伦理上的限制，因此受到研究者更多的重视。
人iPS细胞是通过将4个基因(Oct4，Sox2，Klf4，c-Myc或者Oct4，Sox2，Nanog，Lin28)导入体细胞，而诱导形成的多潜能干细胞(Takahashi K.Cell 2007；131：861-872,Yu J，et al.Science 2007；318：1917-1920)。iPS细胞具有和ES细胞类似的性质，在特定的诱导条件下可向内、中、外三个胚层分化。利用iPS细胞可以在体外无限扩增和多向分化潜能，通过定向诱导iPS细胞分化为肝细胞，就可获得足够的肝细胞来源。如果能够通过获得病人的体细胞并建立与患者具有相同遗传背景的iPS细胞系，诱导其分化为病人所需的肝细胞用于患者的肝细胞移植治疗，该方法获得的个性化肝细胞可以最大限度地避免或者降低由异体细胞移植导致的免疫排斥反应。
ES/iPS细胞早在2007年就被证明可在体外分化为功能性的肝细胞(Cai J，et al，Hepatology 2007，45(5):1229-1239)。随着研究的深入和广泛开展，更多，更有效的诱导分化方法涌现出来。目前最常见的分化方法是用生长因子分步诱导多潜能干细胞分化：首先通过Nodal信号和FGF信号，使多潜能干细胞分化发育为前层确定性内胚层细胞，之后再在BMP4和FGF生长因子的作用下向肝细胞方向分化，最后用肝细胞生长因子(HGF)和制瘤素M(OSM)促进分化肝细胞进一步成熟(Agarwal S，et al，Stem Cells 2008，26(5):1117-1127，Hay DC，et al，PNAS 2008，105(34):12301-12306，Song Z，et al，Cell Res 2009，19(11):1233-1242，Touboul T，et al，Hepatology 2010，51(5):1754-1765，Duan Y，et al，Stem Cells 2010，28(4):674-686，Si-Tayeb K，et al，Hepatology 2010，51(1):297-305.Sullivan GJ，et al，Hepatology 2010，51(1):329-335，Liu H，et al，Sci Trans Med 2011， 3(82):82ra39，Kajiwara M，et al，PNAS 2012，109(31):12538-43，2012)。其他方法包括用人的MSC(Mesenchymal Stem Cells)作为饲细胞诱导(Mallanna SK，et al，Curr Protoc Stem Cell Biol 2013.26:Unit 1G.4)，三维培养(Mobarra N，et al，Int J Hematol Oncol Stem Cell Res 2014，8(4):20-9)或导入外源基因(Takayama K，et al，PLoS One 2011，6(7):e21780，Takayama K，et al，Mol Ther 2012，20(1):127-37)，Takayama K，et al，Biomaterials 2013.34(7):1781-9)等方式。尽管定向诱导人ES/iPS细胞分化为肝细胞的研究取得了较大进展，目前的研究结果仍有这样那样的缺陷和问题，如：1.大多分化方法的ES/iPS细胞培养条件仍然需要在小鼠饲细胞层上培养或者培养液含有动物源成分，用这样方法分化获得的肝细胞由于可能存在未知的动物病原而很难临床应用；2.诱导分化步骤多，所用的细胞生长因子多，因而很难控制分化的阶段以及最终的质量控制，这也直接导致分化成本高昂，难以实际应用；3.大多分化方法分化效率不高，分化的肝细胞因为纯度低而需要额外的纯化步骤，增加了生产成本，也减少了细胞的活力和生物活性，很难达到临床应用的要求；4.成熟肝细胞表面标志ASGPR缺乏或较低(Takayama K，et al，J Hepatol 2012，57(3):628-36)；5.分化的肝细胞功能不全，尤其是P450代谢酶常常缺失或低下(Schwartz RE，et al，Biotechnol Adv 2014，pii:S0734-9750(14)00005-6)；6.导入外源基因可极大改善以上缺陷，然而导入的外源基因可能使基因结构发生改变从而导致致癌风险增加而无法实际应用于临床；7.分化的肝细胞的增殖，传代，冻存和复苏等情况均和临床应用密切相关，而几乎所有的报道都未提及这一问题。因而用现有的分化方法分化ES/iPS细胞所获得的肝细胞，因为不能满足临床的要求而无法实际应用于临床肝细胞移植和生物人工肝替代治疗。2013年，日本科学家Takebe通过将iPSC和MSC与血管内皮细胞共培养而获得“肝芽”-微小肝组织(Takebe T，et al，Nature 2013，499(7459):481-4)。虽然其对肝损伤小鼠的代谢和生存率均显著改善。但并没有报道这种“肝芽”组织具有正常肝组织或肝细胞所具有的尿素合成功能；且该方法能否应用于人体肝细胞分化，还是一个未知数，离实际应用还有一定的距离；不过，这一研究成果为ES/iPS细胞分化肝细胞的临床应用研究和发展提供了新的思路。
综上，在诱导人源干细胞定向分化肝细胞领域，还需要进行深入的研究，以期获得能够提供医药、临床用的合格人肝细胞的真正适用方法和产品。
化学诱导细胞直接重编程是在不改变细胞基因序列的情况下，通过化学因子诱导调控细胞信号通路、表观遗传及化学生物学变化来改变细胞命运的过程。本发明是借鉴细胞直接重编程机理，不需要分步诱导，亦无需使用细胞生长因子，仅使用化学小分子诱导调控，将人干细胞直接分化为高纯度、高质量的肝细胞方法，未见任何文献报道。
发明内容
本发明的目的在于提供无需分步诱导，亦无需使用各种细胞生长因子，且只通过常规培养方式，仅使用小分子诱导人干细胞，优选地诱导人胚胎干细胞或诱导多能干细胞向肝 细胞直接分化的新方法及其专用直接分化培养基。
本发明已基本明确的机理，主要是通过GSK3β抑制剂、TGFβ抑制剂两类小分子组合，多靶点诱导GSK3β、TGFβ信号通路改变，以及维甲酸类化合物的协同作用，继而调控干细胞信号通路和表观遗传的改变，从而直接分化干细胞为肝细胞。需要说明的是，GSK3β抑制剂、TGFβ抑制剂包含了两种类别，功能相同，或诱导相同靶点的系列小分子，所形成的不同组合，都能够不同程度地诱导干细胞直接分化为肝细胞。因此需要保护其功能相同或诱导相同靶点，对同一条信号通路起相同效应的同类小分子化合物，以及所构成的能够诱导调控干细胞直接分化为肝细胞的小分子组合的专利权。
在本发明的第一方面，提供一种用于诱导人干细胞定向直接分化为肝细胞的培养基，所述的培养基包括：细胞分化基本培养基；以及
GSK3β抑制剂：终浓度为0.5-8uM；
TGFβ抑制剂：终浓度为0.1-10uM；和
维甲酸类化合物：终浓度为0.001-10uM；
所述培养基能够诱导人干细胞向肝细胞定向直接分化，获得人肝前体细胞或肝成熟细胞。
在一个优选例中，所述的培养基中：
GSK3β抑制剂：终浓度为0.5-5uM；
TGFβ抑制剂：终浓度为0.5-8uM；和
维甲酸类化合物：终浓度为0.01-5uM。
在另一优选例中，所述的培养基中：
GSK3β抑制剂是CHIR-99021或/和CHIR-98014，其终浓度为0.5-8uM，优选量为0.5-5uM；
TGFβ抑制剂是SB431542或/和A83-01或/和RepSox，其终浓度为0.1-10uM，优选量为：0.5-8uM；
维甲酸类化合物是维甲酸，其终浓度为0.001-10uM；优选量为：0.01-5uM。
在另一优选例中，所述培养基还可添加包括选自下组的一种或多种成分：
Rock抑制剂       终浓度为0.5-50uM；较佳地为1-20uM；和/或
肝生长因子(HGF)：终浓度为5-100ng/ml；较佳地为5-40ng/ml；和/或
制瘤素M：           终浓度为1-100ng/ml；较佳地为5-50ng/ml；和/或
地塞米松：         终浓度为0.5-20uM；较佳地为2.5-10uM；
所添加上述成分后的培养基能够增加细胞存活率，或促进ES/iPS细胞分化成熟为肝细胞，并维持肝成熟细胞的生长。
在另一优选例中，所述的培养基中，所述的GSK3β抑制剂包括：CHIR-99021、BIO、AZD2858、TWS119、CHIR-98014等为代表的，具相同功能，或诱导相同靶点的同一类GSK3β信号通路抑制剂或化合物，或其组合；较佳地为GSK3β抑制剂CHIR-99021；
所述的TGFβ抑制剂包括：SB431542、A83-01、SB525334、LY2109761，RepSox等为代表的，具相同功能，或诱导相同靶点的同一类TGFβ信号通路抑制剂或化合物，或其组合；较佳地为TGFβ抑制剂SB431542或/和A83-01；
所述的维甲酸类化合物是天然或人工合成的，包括：维甲酸(Retinoic acid，RA)，又称：全反式维甲酸(all trans retinoic acid，ATRA)；13-顺式维甲酸(13-cis retinoic acid，13-CRA)、9-順式维甲酸(9-cis-retinoic acid，9-CRA)等为代表的，具相同功能，或诱导相同靶点的同一类维甲酸类分化剂或化合物，或其组合；较佳地为维甲酸(Retinoic acid，RA)；
所述的Rock抑制剂包括：Y-27632(别名：Y-27632 2HCI)、GSK429286A、RKI-1447等为代表的，具相同功能，或诱导相同靶点的同一类Rock信号通路抑制剂或化合物，或其组合；较佳地为Rock抑制剂Y-27632。
在另一优选例中，所述的培养基中，所述细胞分化基本培养基为在基础细胞培养基中加入0.5％N2、1％B27、1％Non-AA、1％Sodium pyruvate，较佳地还加入1％青链霉素；其中，该细胞分化基本培养基各组分的百分含量还可上下浮动50％；较佳地上下浮动30％；更佳地上下浮动20％，如10％，5％；较佳地，所述的基础细胞培养基包括但不限于：DMEM/F12、MEM、DMEM、RPMI1640、Neuronal basal或Fischers等。
在本发明的另一方面，提供GSK3β抑制剂、TGFβ抑制剂和维甲酸类化合物所构成的组合物和所述的培养基的用途，用于诱导人干细胞向肝细胞定向直接分化，获得人肝前体细胞或肝成熟细胞；较佳地，所述的组合物中还包括选自下组的成分：Rock抑制剂、肝生长因子、制瘤素M或地塞米松。
在一个优选例中，所述的组合物中，GSK3β抑制剂、TGFβ抑制剂和维甲酸类化合物以摩尔比(也可换算成重量比)：(0.5-8)：(0.1-10)：(0.001-10)；较佳地，(0.5-5)：(0.5-8)：(0.01-5)存在。
在本发明的另一方面，提供一种用于诱导人干细胞向肝细胞定向直接分化的试剂盒，其中包括GSK3β抑制剂、TGFβ抑制剂和维甲酸类化合物；用于诱导人干细胞向肝细胞定向直接分化；较佳地，其中还包括选自下组的成分：Rock抑制剂、肝生长因子、制瘤素M或地塞米松；用于在诱导人干细胞向肝细胞定向直接分化过程中，增加细胞存活率，或促进ES/iPS细胞向功能性肝成熟细胞分化，并维持肝成熟细胞的生长；或所述的试剂盒中包括前面任一所述的培养基。
在本发明的另一方面，提供一种诱导人干细胞向肝细胞定向直接分化的方法，所述方法包括：应用前面任一所述的培养基诱导人干细胞向肝细胞定向直接分化；较佳地，所述方法步骤包括：
(1)人肝前体细胞分化起始：将培养板用基质胶、鼠尾胶、明胶、纤维连接蛋白、玻璃粘连蛋白其中一种打底30分钟-24小时；然后将人干细胞在应用所述肝细胞分化培养基或肝细胞分化强化培养基，较佳地,在肝细胞分化培养基中混悬，铺板；37±1℃，5％CO2 培养，每72小时换液一次；
(2)传代培养：如细胞达到90％融合，可按1:2-1:5传代；
传代培养步骤：用消化液，包括胰酶，EDTA，Acutase，Tryple E等将分化的人ES/iPS细胞消化为单细胞，重悬后按1:2-1:5传代；按方法步骤(1)所述方法传代培养分化细胞；
(3)人肝前体细胞分化获得：按方法步骤(1)、(2)所述方法分化培养10-15天，可获得的人肝前体细胞；获得的人肝前体细胞可用于冻存、复苏、传代，也可进一步诱导分化为功能性人肝成熟细胞；较佳地，传代培养方法为：按步骤(2)所述方法传代培养分化所得人肝前体细胞；
(4)人肝前体细胞成熟培养：将方法步骤(3)分化培养所得的人肝前体细胞在应用所述肝细胞分化培养基或肝细胞分化强化培养基；较佳地,在肝细胞分化强化培养基中继续分化培养，37±1℃，5％CO2分化培养7-15天，可获得功能性人肝成熟细胞；获得的功能性人肝成熟细胞，可用于冻存、复苏、有限传代。
在另一优选例中，所述的人干细胞包括但不限于：人胚胎干细胞、诱导多能干细胞、间充质干细胞、脂肪干细胞、脐血干细胞等具有多向分化潜能的人源干细胞；较佳地，所述的人干细胞为人胚胎干细胞或诱导多能干细胞。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、人ES细胞与分化所得肝前体细胞形态比较；图左：人ES细胞；图右：人ES细胞分化肝前体细胞。
图2、人ES细胞分化肝细胞的高密度和低密度传代培养。
图3、人ES细胞与分化所得肝成熟细胞及人原代肝细胞的形态比较；左图：人ES细胞；中图：人ES细胞分化肝成熟细胞；右图：人原代肝细胞。
图4、人ES细胞分化肝成熟细胞的肝特异标志物染色的流式细胞分析结果。结果显示所获得的分化肝成熟细胞的肝细胞特异性标志物呈现阳性的细胞比例非常高。
图5、人iPS细胞分化肝成熟细胞与原代肝细胞白蛋白生成的比较，其中，PHH为：原代肝细胞；iPSC-Hep为：人iPS细胞分化肝成熟细胞；C1-C4为：4种培养条件。
图6、人iPS细胞分化肝成熟细胞与原代肝细胞尿素生成的比较。其中，PHH为：原代肝细胞；iPSC-Hep为：人iPS细胞分化肝成熟细胞；C1-C4为：4种培养条件。尿素来源为血液中的血氨，其生成量体现肝细胞的解毒能力。
图7、人ES细胞分化肝成熟细胞的糖原染色。染色的深浅展现肝细胞储存糖原的能力。
图8、人ES细胞分化肝成熟细胞的P450酶(CYP3A4和CYP1A2)活性诱导。左图：CYP3A4在不同浓度利福平诱导下的活性升高；右图：CYP1A2在不同浓度奥美拉措诱导下的活性升高。
图9、人ES细胞与直接分化所得肝前体细胞、肝细胞的形态比较；图左：人ES细胞；图中：分化6天肝前体细胞；图右：分化15天肝细胞。图9显示，GSK3β抑制剂BIO与TGFβ抑制剂及维甲酸构成的小分子组合，也能够诱导干细胞直接分化为肝前体细胞及肝成熟细胞。
图10、人iPS细胞与直接分化所得肝前体细胞、肝细胞的形态比较；图左：人iPS细胞；图中：分化6天肝前体细胞；图右：分化20天肝细胞。图10显示，TGFβ抑制剂RepSox与GSK3β抑制剂及维甲酸构成的小分子组合，也能够诱导干细胞直接分化为肝前体细胞及肝成熟细胞。
图11、人ES细胞与直接分化所得肝前体细胞、肝细胞的形态比较；图左：人ES细胞；图中：分化6天肝前体细胞；图右：分化16天肝细胞。图11显示，GSK3β抑制剂CHIR-98014与TGFβ抑制剂SB525334及维甲酸构成的小分子组合，也能够诱导干细胞直接分化为肝前体细胞及肝成熟细胞。
具体实施方式
本发明人经过深入的研究，开发了一种诱导人干细胞、较佳地人胚胎干细胞(embryonic stem cells，ES细胞)或诱导多能干细胞(induced pluripotent stem cells，iPS细胞)向肝细胞定向直接分化的新方法及其专用直接分化培养基。所述方法不需要在干细胞内导入外源基因，不需要生长因子，也不需要分步诱导，常规培养即可实现干细胞的肝向分化，所获得的分化人肝细胞具有人肝细胞的典型特征；所获得的人肝前体细胞可长期维持，也可冻存、复苏，增殖传代；还可继续成熟分化培养，以获得功能性人肝成熟细胞；获得的功能性人肝成熟细胞，可冻存、复苏、有限传代、维持培养。并且，所述方法培养条件简单，成本低廉且安全稳定。
如本发明所用，术语“含有”或“包括”包括了“包含”、“主要由……构成(制成)”、“基本上由……构成”和“由……构成”。
本发明中，定向诱导直接分化对象为人干细胞，包括但不限于：人胚胎干细胞、诱导多能干细胞、间充质干细胞、脂肪干细胞、脐血干细胞及其他具有多向分化潜能的人源干细胞；较佳地为：人胚胎干细胞或诱导多能干细胞；
培养基
本发明人提供了用于诱导人干细胞，优选地诱导人胚胎干细胞或诱导多能干细胞向肝细胞定向直接分化的新方法及其专用直接分化培养基。所述的专用直接分化培养基包括“肝细胞分化培养基”和“肝细胞分化强化培养基”。
所述的肝细胞分化培养基包括：GSK3β抑制剂，TGFβ抑制剂，维甲酸类化合物。上述组分以适合的比例添加到细胞分化基本培养基中，可以诱导人干细胞向肝细胞定向直 接分化。
如本发明所用，所述的“GSK3β抑制剂”是指能够抑制细胞中GSK3β信号通路的抑制剂的总称，包括但不限于：CHIR-99021，BIO、AZD2858、TWS119、CHIR-98014等为代表的，具相同功能，或诱导相同靶点的同一类抑制剂：
CHIR-99021(CT99021)，其是GSK-3α和β抑制剂，IC50分别为10nM和6.7nM，比对CDC2，ERK2及其他激酶的抑制性强500倍；
CHIR-99021(CT99021)HCl，其是CHIR-99021的盐酸盐，是一种GSK-3α/β抑制剂，在无细胞试验中IC50为10nM/6.7nM，可用于区分GSK-3和其最接近的同源物Cdc2与ERK2；
BIO，其是一种特异性的GSK-3抑制剂，无细胞试验中作用于GSK-3α/β的IC50为5nM；
AZD2858，其是一种选择性的GSK-3抑制剂，其IC50为68nM，增强了Wnt信号通路；
TWS119，其是一种GSK-3β抑制剂，在无细胞试验中IC50为30nM；
CHIR-98014，其是一种有效的GSK-3α/β抑制剂，无细胞试验中IC50为0.65nM/0.58nM；
Tideglusib，其是一种不可逆的，非ATP竞争性的GSK-3β抑制剂，无细胞试验中IC50为60nM；
AR-A014418，其是一种ATP竞争性和选择性的GSK3β抑制剂，无细胞试验中IC50和Ki为104nM和38nM；
LY2090314，其是一种有效的GSK-3抑制剂，作用于GSK-3α/β，IC50为1.5nM/0.9nM；
SB216763，其是一种有效的，选择性GSK-3α/β抑制剂，IC50为34.3nM；
AZD1080，其是一种口服生物有效的，选择性的，可透过大脑的GSK3抑制剂，抑制人类GSK3α和GSK3β，Ki分别为6.9nM和31nM，比作用于CDK2，CDK5，CDK1和Erk2选择性高14倍以上。
作为本发明的优选方式，所述的GSK3β抑制剂是CHIR-99021，其别名为CT99021；其分子结构式如以下式(I)所示：
Figure PCTCN2016107647-appb-000001
如本发明所用，所述的“TGFβ抑制剂”是指能够抑制细胞中TGFβ信号通路的抑制剂的总称，包括但不限于：SB431542、A83-01、SB525334、LY2109761、RepSox及其他具相同功能的同一类抑制剂：
SB-431542，其是有效的，选择性的ALK5抑制剂，IC50为94nM，比对p38、MAPK 和其他激酶的抑制性强100倍；
A 83-01，其是ALK5，ALK4和ALK7的抑制剂，IC50分别为12，45和7.5nM；
SB525334，其是一种有效的，选择性TGFβreceptor I(ALK5)抑制剂，无细胞试验中IC50为14.3nM，作用于ALK4比作用于ALK5效果低4倍，对ALK2，3，和6没有活性；
LY2109761，其是一种新型的，选择性TGF-βreceptor type I/II(TβRI/II)双重抑制剂，无细胞试验中Ki分别为38nM和300nM；
RepSox，其是一种有效的，选择性TGFβR-1/ALK5抑制剂，作用于ATP与ALK5结合以及ALK5自磷酸化，无细胞试验中IC50分别为23nM和4nM。
SD-208，其是一种选择性TGF-βRI(ALK5)抑制剂，with IC50为48nM，选择性比TGF-βRII高100多倍；
GW788388，其是一种有效的，选择性ALK5抑制剂，无细胞试验中IC50为18nM，也抑制TGF-βII型受体和activin II型受体活性，但不抑制BMP II型受体；
SB505124，其是一种选择性TGFβR抑制剂，作用于ALK4和ALK5，无细胞试验中IC50分别为129nM和47nM，也抑制ALK7，但不抑制ALK1，2，3或6；
EW-7197，其是一种高效的，选择性的，口服生物有效的TGF-βreceptor ALK4/ALK5抑制剂，IC50分别为13nM和11nM。
作为本发明的优选方式，所述的TGFβ抑制剂是SB 431542(或称为SB-431542)；其分子结构式如以下式(II)所示：
Figure PCTCN2016107647-appb-000002
作为本发明的优选方式，所述的TGFβ抑制剂是A83-01(或称为A8301)；其分子结构式如以下式(III)所示：
Figure PCTCN2016107647-appb-000003
如本发明所用，所述的维甲酸类化合物，包括：维甲酸(Retinoic acid，RA)，别名：全反式维甲酸(all trans retinoic acid，ATRA)；13-順式维甲酸(13-cis retinoic acid，13-CRA)、9-順式维甲酸(9-cis-retinoic acid，9-CRA)等为代表的，具相同功能，或诱导相同靶点的同一类维甲酸类分化剂或化合物，或其组合：
维甲酸类化合物是一组维生素A(视黄醇)的氧化代谢产物或衍生物以及与维生素A具有类似结构的人工合成物，包括天然和合成两大类。主要包括：维甲酸(Retinoic acid，RA)，别名：维A酸、全反式维甲酸(all trans retinoic acid，ATRA)；13-順式维甲酸(13-cis retinoic acid，13-CRA)、9-順式维甲酸(9-cis-retinoic acid，9-CRA)，异维甲酸(Isotretinoin)、维 胺酯(Fenretinide)、依曲替酸(Acitretin)、依曲替酯(Etretinate)、他扎罗汀(Tazarotene)、阿达帕林(Adapalene)及TTNPB、CD437、Targretin等。维甲酸类化合物的特点是在体内外调节许多细胞类型的分化、增生、凋亡。维甲酸类化合物及其异构体衍生物中许多都具有相同或相类似的功能。因此成为诱导分化剂中最为重要并且已经被用于临床治疗皮肤病的一类药物。
作为本发明的优选方式，所述的维甲酸(Retinoic acid，RA)，或称全反式维甲酸(all trans retinoic acid，ATRA)、维A酸、维生素A酸、维生素甲酸、视黄酸、全反式维A酸、维A甲酸，其分子结构式如以下式(IV)所示：
Figure PCTCN2016107647-appb-000004
如本发明所用，所述的“Rock抑制剂”是指能够抑制细胞中Rock信号通路的抑制剂的总称，包括但不限于：Y-27632、GSK429286A、RKI-1447等为代表的，具相同功能，或诱导相同靶点的同一类抑制剂：
Y-27632(Y-27632 2HCl)，其是一种选择性ROCK1(p160ROCK)抑制剂，无细胞试验中Ki为140nM，比对其他激酶包括PKC，cAMP依赖性蛋白激酶，MLCK和PAK的作用强200多倍；
GSK429286A是一种选择性的ROCK1和ROCK2抑制剂，IC50分别为14nM和63nM；
RKI-1447是一种有效的ROCK1和ROCK2抑制剂，IC50分别为14.5nM和6.2nM，有抗侵入和抗肿瘤活性；
Thiazovivin是一种新型ROCK抑制剂，无细胞试验中IC50为0.5μM，在单细胞分离后，促进人胚胎干细胞(hESC)的存活；
RO-3306是一种ATP竞争性的选择性CDK1抑制剂，Ki为20nM，选择性是其他各种人类激酶的15倍多。
作为本发明的优选方式，所述的Rock抑制剂为Y-27632(Y-27632 2HCI)，其别名为Y-27632dihydrochloride；Y-27632 2HCI；其分子结构式如以下式(V)所示。
Figure PCTCN2016107647-appb-000005
本发明人在前期研究中，在本领域中首次提出GSK3β信号通路、TGFβ信号通路的下调，联合维甲酸(RA)能够促进人多能干细胞向肝细胞定向直接分化。应理解，除了本发明实施例中所列举的具体GSK3β抑制剂、TGFβ抑制剂以外的其它可抑制GSK3β信号通路、TGFβ信号通路的抑制剂也可实现同样的技术效果，也应被包含在本发明中。
同样，除了本发明实施例中所列举的具体维甲酸(RA)诱导分化剂和Rock抑制剂以外的其它具相同功能，或诱导相同靶点的维甲酸类化合物和Rock信号通路的抑制剂也可实 现同样的技术效果，也应被包含在本发明中。
本发明还包括上述各化合物的具相同功能，或诱导相同靶点的等效化合物、类似物、衍生物和/或它们的盐、水合物或前体。由上述各化合物制备而成的生化试剂、药剂制品也被包含在本发明中。
所述化合物的类似物包括但不限于：所述化合物的异构体、外消旋体。化合物具有一个或多个不对称中心。所以，这些化合物可以作为外消旋的混合物、单独的对映异构体、单独的非对映异构体、非对映异构体混合物、顺式或反式异构体存在。
所述的“盐”包括但不限于：(1)与如下无机酸形成的盐：如盐酸、硫酸、硝酸、磷酸等；(2)与如下有机酸形成的盐，如乙酸、草酸、丁二酸、酒石酸、甲磺酸、马来酸、或精氨酸等。其它的盐包括与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐等。
所述的“化合物的前体”指当用适当的方法施用或处理后，该化合物的前体在培养基中可转变成上述任一化合物的一种化合物，或上述任一化合物的一种化合物所组成的盐或溶液。
如本发明所用，所述的“细胞分化基本培养基”是本领域用于进行人干细胞，如常用于人胚胎干细胞或诱导多能干细胞分化培养的基本营养维持培养基。在不添加本发明所述的肝细胞分化培养基及肝细胞分化强化培养基，或其中的组合物有效成分的情况下，采用所述的“细胞分化基本培养基”无法使得人胚胎干细胞或诱导多能干细胞向肝细胞定向直接分化。
作为本发明的优选方式，所述的“细胞分化基本培养基”为在基础细胞培养基中加入0.5％N2、1％B27、1％Non-AA、1％Sodium pyruvate，较佳地还加入1％青链霉素(百分数均以v/v计，其中，各百分含量还可上下浮动50％；较佳地上下浮动30％；更佳地上下浮动20％，如10％，5％)。或者，所述的“细胞分化基本培养基”也可以是商业购买的。所述的基础细胞培养基可以是但不限于：DMEM/F12、MEM、DMEM、RPMI1640、Neuronal basal或Fischers等。应理解，本领域技术人员熟悉所述的基础细胞培养基的配制或购买途径，因此，基础细胞培养基并不限于本发明中所举例的这些。
作为本发明的优选方式，本发明所述的“肝细胞分化培养基”如下配制：
在细胞分化基本培养基中添加下列成分：
(1)GSK3β抑制剂CHIR-99021：终浓度为0.5-8uM；优选量为：0.5-5uM；
(2)TGFβ抑制剂SB431542或/和A83-01：终浓度为0.1-10uM；优选量为：0.5-8uM；
(3)维甲酸(RA)：终浓度为0.001-10uM；优选量：0.01-5uM。
如上配方，即可获得本发明的“诱导人干细胞向肝细胞定向分化的新方法”的专用“肝细胞分化培养基”。
在上述“肝细胞分化培养基”配方基础上，还可添加以下任何一种或多种成分，便可获得“肝细胞分化强化培养基”：
(1)Rock抑制剂(Rock抑制剂详细说明见前述)或优选Rock抑制剂Y-27632：终浓度为 0.5-50uM；优选量：1-20uM；和/或
(2)肝生长因子(HGF)：终浓度为5-100ng/ml；优选量：5-40ng/ml；和/或：
(3)制瘤素M：终浓度为1-100ng/ml；优选量：5-50ng/ml；和/或：
(4)地塞米松：终浓度为0.5-20μM；优选量：2.5-10μM。
如上配方，即可获得“肝细胞分化强化培养基”，该培养基可在诱导人干细胞向肝细胞直接分化过程中增加细胞存活率；或促进ES/iPS细胞，或肝前体细胞继续分化为功能性肝成熟细胞；或增强分化肝细胞的某些生物学行为和功能；并可长时间维持肝成熟细胞的生长
培养方法
本发明还公开了一种仅使用小分子组合诱导人干细胞，优选地诱导人胚胎干细胞或诱导多能干细胞向肝细胞直接分化的新方法。该方法是将人ES/iPS细胞在本发明的“肝细胞分化培养基”和/或“肝细胞分化强化培养基”中诱导分化培养，可获得高纯度的肝前体细胞或肝成熟细胞。
作为本发明的优选方式，培养肝前体细胞的方法是：将培养板打底1-24小时，然后将人ES/iPS细胞加入所述肝细胞分化培养基或肝细胞分化强化培养基，较佳的为肝细胞分化培养基中混悬，铺板；37±1℃、5％CO2培养，每72±1小时换液一次；培养10-15天，可获得肝前体细胞。
所述的培养板打底的方法是本领域熟知的，可以用于培养板打底的材料包括但不限于：基质胶、鼠尾胶、明胶、纤维连接蛋白、玻璃粘连蛋白等，可选择其中的一种进行打底。
在人ES/iPS分化培养过程中，如细胞达到85-90％融合，可按1:2-1:5的比例进行细胞传代培养。作为本发明的优选方式，传代培养步骤为：用消化液将分化培养中的人ES/iPS细胞消化为单细胞，重悬后按1:2-1:5的比例传代培养分化细胞。所述的消化液可以是包括胰酶，EDTA，Acutase，Tryple E等一种或多种消化酶的溶液。
所获得的肝前体细胞可用于冻存、复苏、传代，也可进一步诱导为功能性人肝成熟细胞。
作为本发明的优选方式，诱导肝前体细胞成为功能性人肝成熟细胞的方法包括：肝前体细胞加入所述肝细胞分化培养基或肝细胞分化强化培养基，较佳的为肝细胞分化强化培养基中混悬，37±1℃、5％CO2，继续分化成熟培养7-15天，获得功能性的肝成熟细胞。
本发明的方法所获得的肝成熟细胞可冻存、复苏、有限传代、长时间维持培养；也可应用于细胞移植治疗肝病、生物人工肝脏构建、新药肝毒检测、药效评估、药靶鉴定；可为生物、医学、药学的基础研究和临床应用提供充足的肝细胞来源或肝细胞模型；其诱导分化过程也可为人类肝脏细胞发育分化过程提供最佳研究平台，应用前景十分广阔。
此外，还应理解，本发明中涉及应用的人胚胎干细胞，均为市场上可从商业途径购 买到的胚胎干细胞系，例如表1中所列举的，并不涉及人胚胎的应用。
表1、商品化的人胚胎干细胞系
Figure PCTCN2016107647-appb-000006
早在1998年人胚胎干细胞和胚胎生殖细胞就已经建系成功，例如1998年Thomson领导的小组从14个囊胚中最终建立起5个人类ES细胞系：H1、H13、H14、H7和H19；Gearhart领导的小组从5-9周龄的流产胎儿的性腺嵴及肠系膜中分离原始的干细胞，以期避免因直接利用胚胎所造成的伦理学上的麻烦。见晁岚等，“人胚胎干细胞的研究进展”，《现代妇产科进展》，2003年7月第12卷第4期。基于上述工作，在2000年的2月份，Wisconsin Alumni Research Foundation(WARF)建立了WiCell，WiCell是一家非官方的、非盈利性的附属机构，它以低费用向合格的科学家分配人胚胎干细胞。此外，提供这种现成的人胚胎干细胞的机构还包括NSCB(National Stem Cell Bank)、ES CELL INTERNATIONAL、nov cell、TECHNION-HOME TO ISRAEL’S NOBEL SCIENTISTS、UCSF(University of California San Fransisco)等机构。因此，人胚胎干细胞完全可以通过“取自人胚胎”的其它途径获得。
本发明的方法的有益技术效果体现在：
1、仅使用小分子组合多靶点定向诱导人ES细胞或iPS细胞直接分化为肝细胞，不需要分步诱导，也不使用价格昂贵的各种细胞生长因子，且只需常规培养；由于小分子性 质稳定，因此分化结果稳定安全，且成本大幅降低。
2、不导入外源基因，也不改变基因结构，仅使用小分子组合定向诱导人ES细胞或iPS细胞直接分化为肝细胞；无外源基因导入和因基因结构改变导致的实验干扰和致癌风险(区别于肝肿瘤细胞、永生性肝细胞和外源基因导入iPS细胞转化肝细胞)；
3、直接分化效率高，一百万(1×106)的人ES或iPS细胞可转化为5-10×106以上的功能性肝细胞(所有报道均未提及这一点)；且分化肝细胞形态功能与人原代肝细胞高度吻合；
4、直接分化纯度高而不需要额外的纯化手段，流式细胞分析(FACS)结果表明，所获得的分化肝细胞纯度高达90％以上；在分化培养后获得的肝细胞的成熟肝细胞表面标志Asgp阳性细胞>90％(其他报道中一般为20-60％)；其它各项肝细胞特有免疫标志物如ALB、CYP3A、HNF4a阳性细胞均在80％以上；这显示分化所得肝细胞的高质量，并有效降低了成本；
5、直接分化的肝细胞的普适性和可重复性好，本发明人验证了9个人ES/iPS(包括2个人ES细胞和7个人iPS细胞系)细胞系均被分化为形态功能一致的肝细胞；
6、直接分化的肝细胞具有多项肝细胞特有的功能，如生成白蛋白，合成尿素，和P450酶(CYP酶:CYP3A4、CYP1A2)活性诱导，糖原储存等。因而本发明的方法所获得的肝细胞是功能性肝细胞；
7、本发明的方法为无动物源性的培养方法，可不使用动物饲养层细胞培养ES/iPS细胞，可直接应用于GMP标准的批量生产和临床应用，此特点未见任何报道；该方法不存在动物源性物质污染；
8、采用直接分化的技术，不需要形成拟胚体(embryo body，EB)，方法简单，易于操作，成本低廉，适合批量生产。
9、分化阶段明晰，质量容易控制；
10、直接分化所得的肝前体细胞，可以增殖传代，冻存复苏；还可继续分化为成熟的功能性肝细胞；所获得的肝成熟细胞可冻存、复苏、有限传代、维持培养。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、诱导人胚胎干细胞或诱导多能干细胞向肝细胞直接分化的培养基的配制
1、细胞分化基本培养基的配制
细胞分化基本培养基按照常规方法制备。即：在DMEM/F12(基础细胞培养基)中加入：0.5％N2、1％B27、1％Non-AA、1％Sodium pyruvate、1％青链霉素混合液(100x)。其中，百分数均以v/v计。
2、肝细胞分化培养基配制
(1)肝细胞分化培养基1
在上述“细胞分化基本培养基”中添加下列终浓度的成分：
GSK3β抑制剂CHIR-99021：2uM；
TGFβ抑制剂SB431542：5uM；
维甲酸(RA)：2uM。
(2)肝细胞分化培养基2
在上述“细胞分化基本培养基”中添加下列终浓度的成分：
GSK3β抑制剂CHIR-99021：2uM；
TGFβ抑制剂A83-01：3uM；
维甲酸(RA)：5uM。
(3)肝细胞分化培养基3
在上述“细胞分化基本培养基”中添加下列终浓度的成分：
GSK3β抑制剂CHIR-99021：1uM；
TGFβ抑制剂SB431542：2uM；
维甲酸(RA)：0.1uM。
(4)肝细胞分化培养基4
在上述“细胞分化基本培养基”中添加下列终浓度的成分：
GSK3β抑制剂CHIR-99021：2uM；
TGFβ抑制剂A83-01：3uM；
维甲酸(RA)：0.5uM。
(5)肝细胞分化培养基5
GSK3β抑制剂CHIR-99021：3uM；
TGFβ抑制剂SB431542：5uM；和
维甲酸(RA)：3uM；
(6)肝细胞分化培养基6
GSK3β抑制剂BIO：3uM；
TGFβ抑制剂SB431542：5uM；和
维甲酸(RA)：2uM；
(7)肝细胞分化培养基7
GSK3β抑制剂CHIR-99021：3uM；
TGFβ抑制剂RepSox：5uM；和
维甲酸(RA)：1uM；
(8)肝细胞分化培养基8
GSK3β抑制剂CHIR-98014：2uM；
TGFβ抑制剂SB525334：6uM；和
维甲酸(RA)：5uM；
3、肝细胞分化强化培养基配制
(1)肝细胞分化强化培养基1
在上述“肝细胞分化培养基1”配方基础上，添加：
Rock抑制剂(Y-27632)：终浓度10uM；
地塞米松：终浓度为5uM。
(2)肝细胞分化强化培养基2
在上述“肝细胞分化培养基3”配方基础上，添加：
Rock抑制剂(Y-27632)：终浓度5uM；
制瘤素M：终浓度20ng/ml。
(3)肝细胞分化强化培养基3
在上述“肝细胞分化培养基4”配方基础上，添加：
Rock抑制剂(Y-27632)：终浓度2.5uM；
人肝生长因子(HGF)：终浓度20ng/ml。
(4)肝细胞分化强化培养基4
在上述“肝细胞分化培养基2或8”配方基础上，添加：
Rock抑制剂(Y-27632)：终浓度15uM。
实施例2、利用肝细胞分化培养基1和肝细胞分化强化培养基2进行人肝前体细胞分化培养和肝成熟细胞培养
1、人肝前体细胞分化起始
将培养板用基质胶打底12小时，将肝细胞分化培养基1加入培养板中，然后将人胚胎干细胞(ES)(见图1左图)在“肝细胞分化培养基1中混悬，铺板；37℃，5％CO2培养，每72小时换液一次。
人胚胎干细胞在肝细胞分化培养基1中，37℃，5％CO2分化培养10-15天可获得的人肝前体细胞，人ES细胞与分化获得的肝前体细胞形态对比图见图，右图为分化培养15天获得的肝前体细胞，可用于冻存、复苏、传代，也可进一步诱导为功能性人肝成熟细胞。
2、传代培养
当在分化培养过程中，分化培养的细胞达到90％融合，可按1:2～1:5传代。
传代培养步骤：用消化液(含有胰酶)将分化过程中的人ES细胞消化为单细胞，重悬后按1:2～1:5传代；按前述“1”所述的方法传代培养分化细胞；传代培养结果见图2。
3、人肝前体细胞成熟培养
按前述1、2所述的方法培养或传代培养所得的人肝前体细胞进行成熟诱导分化培养：将人肝前体细胞在“肝细胞分化强化培养基2”中，37℃，5％CO2成熟培养7-15天，获得功能性人肝成熟细胞。
人ES细胞与分化所得肝成熟细胞及人原代肝细胞的形态比较见图3。图左：ES细胞，图中：分化的肝成熟细胞，图右：原代肝细胞。
成熟培养7-15天获得的功能性人肝成熟细胞，可冻存、复苏、有限传代、较长时间维持培养；并用于各项功能测试。
实施例3、利用肝细胞分化培养基4进行诱导人ES细胞分化为肝成熟细胞的肝特异标志物染色的流式细胞分析
诱导人ES细胞分化为肝细胞的方法同实施例2。不同之处是一直使用肝细胞分化培养基4培养。
对人ES细胞分化为肝成熟细胞的肝特异标志物染色的流式细胞分析。采用常规的免疫染色方法，将上述实验步骤诱导人ES细胞分化后获得的肝成熟细胞进行人肝细胞特异性标志物(AAT，ALB，Asgpr，CYP3A，HNF4a)免疫染色。免疫染色方法为：
(1)弃细胞培养液，PBS漂洗1次，
(2)0.05％胰酶，37℃，5分钟消化，用胰酶终止剂，或者含血清/白蛋白的细胞培养液终止胰酶作用，800-1000rpm离心3-5分钟,弃上清；
(3)2％多聚甲醛固定10分钟后，PBS漂洗5分钟×3次，
(4)10％羊血清封闭：室温，60分钟，
(5)0.1％Triton：5-10min，
(6)一抗(兔抗ALB抗体，鼠抗CYP3A抗体或兔抗Asgpr抗体)室温1小时或4℃过夜，
(7)PBS漂洗5分钟×3次，
(8)二抗(Cy3标记的羊抗兔抗体，FITC标记的羊抗鼠或羊抗兔抗体)室温45-60min，
(9)PBS洗净，5min×3次。
然后进行流式细胞分析，结果见图4。
由图4可知，所获得的分化肝成熟细胞的肝细胞特异性标志物呈现阳性的细胞比例非常高。因此，可以证明本发明的培养基及培养方法获得了功能性的人肝成熟细胞。
实施例4、利用肝细胞分化培养基1、2和肝细胞分化强化培养基1、4(依次分别对应C1～C4)分别与肝细胞分化强化培养基2进行4组诱导人iPS细胞分化为肝成熟细胞与原代肝细胞白蛋白生成的比较
诱导人iPS细胞分化为肝成熟细胞的方法同实施例2，不同处仅在于利用4组不同分化培养基分组同时进行分化培养以及诱导培养人iPS细胞。
将所获得的4组诱导人iPS细胞分化所得肝成熟细胞与人原代肝细胞进行白蛋白生成的比较。具体方法为：
通过ELISA试剂盒对上述实验步骤所获得的4组诱导人iPS细胞分化所得肝成熟细胞和商购的人原代肝细胞进行白蛋白分泌功能检测；检测实施步骤参见试剂盒说明书(美国Bioassay System公司/DIAG-250,BCG Albumin assay kit)。
4组人iPS细胞分化所得肝成熟细胞与人原代肝细胞进行白蛋白生成的比较结果见图5。由结果可见，所获得的人iPS细胞分化肝成熟细胞具有人肝细胞特有的生成白蛋白的功能。
实施例5、利用肝细胞分化培养基1、2和肝细胞分化强化培养基1、4(依次分别对应C1～C4)分别与肝细胞分化培养基3进行4组诱导人iPS细胞分化为肝成熟细胞与人原代肝细胞尿素生成的比较
诱导人iPS细胞分化为肝成熟细胞的方法同实施例2，不同处仅在于利用4组不同分化培养基分组同时进行分化培养以及诱导分化人iPS细胞。
将所获得的4组诱导人iPS细胞分化所得的肝成熟细胞与原代肝细胞进行尿素生成的比较；具体方法为：
通过尿素氮检测试剂盒对上述实验所获得的4组人iPS细胞分化所得肝成熟细胞和商购的人原代肝细胞进行尿素合成功能检测；检测实施步骤参见试剂盒说明书(美国Bioassay System公司/DIUR-500，Urea assay kit)；
尿素来源为血液中的血氨，其生成量体现肝细胞的解毒能力。4组人iPS细胞分化肝细胞与人原代肝细胞尿素生成的比较结果见图6。
由图6见，所获得的人iPS细胞分化肝成熟细胞具有肝细胞特有的生成尿素的功能。
实施例6、利用肝细胞分化培养基5和肝细胞分化强化培养基3进行诱导人ES细胞分化肝成熟细胞的糖原染色
诱导人ES细胞分化为肝成熟细胞的方法同实施例2，不同处仅在于利用肝细胞分化培养基5进行分化培养和肝细胞分化强化培养基3进行分化成熟培养。
将人ES细胞分化所得的肝成熟细胞进行糖原染色。染色的深浅展现肝细胞储存糖原的能力。用Schiff方法作肝糖原染色。具体方法为：
(1)弃细胞培养液，PBS漂洗1次；
(2)4％多聚甲醛固定10分钟后，PBS漂洗5分钟×3次；
(3)加入PAS-I液10min，流水冲洗；
(4)加入PAS-II液1-2min，流水冲洗；
(5)显微镜取照片。
人ES细胞分化肝成熟细胞的糖原染色结果如图7。培养后的细胞肝糖原染色阳性， 由结果可见，本发明的方法获得的肝成熟细胞具有人肝细胞相同的糖原储存活性。
实施例7、利用肝细胞分化强化培养基4和肝细胞分化强化培养基2进行诱导人ES细胞分化所得肝成熟细胞的P450酶(CYP3A4和CYP1A2)活性诱导
诱导人ES细胞分化为肝成熟细胞的方法同实施例2，不同处仅在于利用肝细胞分化强化培养基4进行分化培养；用肝细胞分化强化培养基2进行继续分化成熟培养。
将人ES细胞分化所得的肝成熟细胞的P450酶(CYP3A4和CYP1A2)活性诱导：
(1)将上述实验步骤诱导人ES细胞分化所得肝成熟细胞进行P450酶(CYP3A4)在利福平(Rifampicine)诱导下升高的情况。P450酶活性诱导的方法如下：
用利福平不同浓度(1uM、10uM、25uM)处理，以不加入利福平且其它条件相同的处理组作为对照。通过PromegaP450-GloTM Assays试剂盒对所处理各组诱导人ES细胞分化所得肝成熟细胞进行P450酶(CYP3A4)检测；实施步骤参见试剂盒说明书(美国Promega公司)；
(2)将上述实验步骤诱导人ES细胞分化所得肝成熟细胞进行P450酶(CYP1A2)在奥美拉措诱导下升高的情况。P450酶活性诱导的方法如下：
用奥美拉措不同浓度(1uM、10uM、25uM)处理，以不加入奥美拉措且其它条件相同的处理组作为对照。通过qRT-PCR对所处理各组诱导人ES细胞分化所得肝成熟细胞进行CYP1A2基因表达定量检测。
P450酶(CYP3A4和CYP1A2)活性诱导结果见图8(图左：CYP3A4在不同浓度利福平诱导下的活性升高；图右：CYP1A2在不同浓度奥美拉措诱导下的活性升高)。由图8可见，本发明的方法获得的成熟肝细胞的P450代谢酶活性很高。
实施例8、利用肝细胞分化培养基6诱导分化人ES细胞直接分化为肝前体细胞以及肝细胞的形态比较
诱导人ES细胞分化为肝前体细胞及肝(成熟)细胞的方法同实施例3；结果见图9；
图左：人ES细胞；图中：分化肝前体细胞；图右：分化肝细胞。
图9显示，GSK3β抑制剂BIO与TGFβ抑制剂及维甲酸构成的小分子组合，也能够诱导干细胞直接分化为肝前体细胞及肝成熟细胞。
实施例9、利用肝细胞分化培养基7诱导分化人iPS细胞直接分化为肝前体细胞以及肝细胞的形态比较
诱导人iPS细胞分化为肝前体细胞及肝(成熟)细胞的方法同实施例3；结果见图10；
图左：人iPS细胞；图中：分化肝前体细胞；图右：分化肝细胞。
图10显示，TGFβ抑制剂RepSox与GSK3β抑制剂及维甲酸构成的小分子组合，也能够诱导干细胞直接分化为肝前体细胞及肝成熟细胞。
实施例10、利用肝细胞分化培养基8诱导分化人ES细胞直接分化为肝前体细胞以及肝细胞的形态比较
诱导人ES细胞分化为肝前体细胞及肝(成熟)细胞的方法同实施例3；结果见图11；
图左：人ES细胞；图中：分化肝前体细胞；图右：分化肝细胞。
图11显示，GSK3β抑制剂CHIR-98014与TGFβ抑制剂SB525334及维甲酸构成的小分子组合，也能够诱导干细胞直接分化为肝前体细胞及肝成熟细胞。
综上，本发明诱导人干细胞(如胚胎干细胞或诱导多能干细胞)向肝细胞直接分化的新方法具有以下的特点：
1、仅使用小分子组合多靶点定向诱导人ES细胞或iPS细胞直接分化为肝细胞。不需要分步诱导，不使用各种细胞生长因子；由于小分子性质稳定，因此分化结果稳定安全，且成本大幅降低；
2、分化阶段明晰，质量容易控制；
3、直接分化效率高，1×106的人ES/iPS细胞可分化为5-10×106以上的功能性肝细胞；
4、直接分化的肝细胞纯度高而不需要额外的纯化手段(Asgp≥85％)；因此不损伤细胞活性，操作简单，成本降低；
5、直接分化的肝细胞功能齐全，具有白蛋白生成，尿素合成，糖原储存及P450酶活性诱导等能力；
6、该直接分化方法普适性和可重复性好，2个人ES细胞和7个人iPS细胞系均被分化为功能性肝成熟细胞；
7、分化所得的肝前体细胞，可以增殖传代，冻存复苏；还可以继续分化为功能性肝成熟细胞；所获得的肝成熟细胞可冻存、复苏、有限传代、维持培养。
8、所分化获得的肝细胞形态功能与人原代肝细胞高度吻合。
9、不使用动物饲养层细胞培养ES/iPS细胞，故不存在动物源性物质污染的现象；
10、GSK3β抑制剂、TGFβ抑制剂包含了两种类别，功能相同，或诱导相同靶点的系列小分子，所形成的不同组合，都能够不同程度地诱导干细胞直接分化为肝细胞。
11、方法简单，易于操作；不需要形成拟胚体(embryo body，EB)；
12、常规培养、周期短、适于批量生产和易于产业化等特点。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种用于诱导人干细胞向肝细胞定向直接分化的培养基，其特征在于，所述的培养基包括：细胞分化基本培养基；以及
    GSK3β抑制剂：终浓度为0.5-8uM；
    TGFβ抑制剂：终浓度为0.1-10uM；和
    维甲酸类化合物：终浓度为0.001-10uM；
    所述培养基能够诱导人干细胞向肝细胞定向直接分化，获得人肝前体细胞或肝成熟细胞。
  2. 如权利要求1所述的培养基，其特征在于，其中：
    GSK3β抑制剂：终浓度为0.5-5uM；
    TGFβ抑制剂：终浓度为0.5-8uM；和
    维甲酸类化合物：终浓度为0.01-5uM。
  3. 如权利要求1所述的培养基，其特征在于，所述的GSK3β抑制剂选自：CHIR-99021、BIO、AZD2858、TWS119、CHIR-98014等为代表的，具相同功能，或诱导相同靶点的同一类GSK3β信号通路抑制剂或化合物，或其组合；
    所述的TGFβ抑制剂选自：SB431542、A83-01、SB525334、LY2109761，RepSox等为代表的，具相同功能，或诱导相同靶点的同一类TGFβ信号通路抑制剂或化合物，或其组合；
    所述的维甲酸类化合物是天然或人工合成的，包括：维甲酸(别名：全反式维甲酸)、13-顺式维甲酸、9-順式维甲酸等为代表的，具相同功能，或诱导相同靶点的同一类维甲酸类分化剂或化合物，或其组合；
    所述的Rock抑制剂选自：Y-27632(别名：Y-27632 2HCI)、GSK429286A、RKI-1447等为代表的，具相同功能，或诱导相同靶点的同一类Rock信号通路抑制剂或化合物，或其组合。
  4. 如权利要求3所述的培养基，其特征在于，所述的GSK3β抑制剂选自：GSK3β抑制剂CHIR-99021；或
    所述的TGFβ抑制剂选自：TGFβ抑制剂SB431542或/和A83-01；或
    所述的维甲酸类化合物是维甲酸；或
    所述的Rock抑制剂为Rock抑制剂Y-27632。
  5. 如权利要求1所述的培养基，其特征在于，其中：
    GSK3β抑制剂是CHIR-99021或/和CHIR-98014，其终浓度为0.5-8uM；
    TGFβ抑制剂是SB431542或/和A83-01或/和RepSox，其终浓度为0.1-10uM；
    维甲酸类化合物是维甲酸，其终浓度为0.001-10uM。
  6. 如权利要求5所述的培养基，其特征在于，其中，
    GSK3β抑制剂是CHIR-99021或/和CHIR-98014，其终浓度为0.5-5uM；
    TGFβ抑制剂是SB431542或/和A83-01或/和RepSox，其终浓度为0.5-8uM；
    维甲酸类化合物是维甲酸，其终浓度为0.01-5uM。
  7. 如权利要求1所述的培养基，其特征在于，所述培养基还可添加包括选自下组的一种或多种成分：
    Rock抑制剂：终浓度为0.5-50uM；和/或
    肝细胞生长因子：：终浓度为5-100ng/ml；和/或
    制瘤素M：终浓度为1-100ng/ml；和/或
    地塞米松：终浓度为0.5-20uM；
    所添加上述成分后的培养基能够增加细胞存活率，或促进ES/iPS细胞分化成熟为肝细胞，并维持肝成熟细胞的生长。
  8. 如权利要求7所述的培养基，其特征在于，所述培养基还可添加包括选自下组的一种或多种成分：
    Rock抑制剂：终浓度为1-20uM；和/或
    肝细胞生长因子：：终浓度为5-40ng/ml；和/或
    制瘤素M：终浓度为5-50ng/ml；和/或
    地塞米松：终浓度为2.5-10uM。
  9. 如权利要求1所述的培养基的用途，其特征在于，用于诱导人干细胞向肝细胞定向直接分化，获得人肝前体细胞或肝成熟细胞。
  10. 如权利要求9所述的用途，其特征在于，所述的组合物中还包括选自下组的成分：Rock抑制剂、肝生长因子、制瘤素M或地塞米松。
  11. 一种用于诱导人干细胞向肝细胞定向直接分化的试剂盒，其特征在于，其中包括GSK3β抑制剂、TGFβ抑制剂和维甲酸类化合物；用于诱导人干细胞向肝细胞定向直接分化。
  12. 如权利要求11所述的试剂盒，其特征在于，其中还包括选自下组的成分：Rock抑制剂、肝生长因子、制瘤素M或地塞米松；用于在诱导人干细胞向肝细胞定向直接分化过程中增加细胞存活率，或促进ES/iPS细胞向功能性肝成熟细胞分化，并维持肝成熟细胞的生长；或
    所述的试剂盒中包括权利要求1-8任一所述的培养基。
  13. 一种诱导人干细胞向肝细胞定向直接分化的方法，其特征在于，所述方法包括：应用权利要求1-8任一所述的培养基诱导人干细胞向肝细胞定向直接分化。
  14. 如权利要求13所述的方法，其特征在于，所述方法步骤包括：
    (1)人肝前体细胞分化起始：将培养板用基质胶、鼠尾胶、明胶、纤维连接蛋白、玻璃粘连蛋白其中一种打底30分钟-24小时；然后将人干细胞在应用权利要求1或2或3或4所述的培养基中混悬，铺板；37±1℃，5％CO2培养，每72小时换液一次；
    (2)传代培养：如细胞达到90％融合，可按1:2-1:5传代；
    传代培养步骤：用消化液，包括胰酶，EDTA，Acutase，Tryple E等将分化的人ES/iPS细胞消化为单细胞，重悬后按1:2-1:5传代；按方法步骤(1)所述方法传代培养分化细胞；
    (3)人肝前体细胞分化获得：按方法步骤(1)、(2)所述方法分化培养10-15天，可获得的人肝前体细胞；获得的人肝前体细胞可用于冻存、复苏、传代，也可进一步诱导分化为功能性人肝成熟细胞；较佳地，传代培养方法为：按步骤(2)所述方法传代培养分化所得人肝前体细胞；
    (4)人肝前体细胞成熟培养：将方法步骤(3)分化培养所得的人肝前体细胞在应用权利要求1或2或3或4所述的培养基中继续分化培养，37±1℃，5％CO2分化培养7-15天，可获得功能性人肝成熟细胞；获得的功能性人肝成熟细胞，可用于冻存、复苏、有限传代。
  15. 如权利要求1-8任一所述的培养基、权利要求9或10所述的用途、权利要求11或12所述的试剂盒、权利要求13或14所述的方法，其特征在于，所述的人干细胞包括但不限于：人胚胎干细胞、诱导多能干细胞、间充质干细胞、脂肪干细胞、脐血干细胞等具有多向分化潜能的人源干细胞；较佳地为人胚胎干细胞或诱导多能干细胞。
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