Method for inducing pluripotent stem cells to differentiate into hepatic stellate cells and application of method

Abstract

The invention discloses a method for inducing pluripotent stem cells to differentiate into hepatic stellate cells and application of the method. The method comprises the following steps: S1, performing induced differentiation on the induced pluripotent stem cells to obtain parenchymal hepatic cells; s2, carrying out passage on the parenchymal hepatic cells in the step S1, continuously culturing the parenchymal hepatic cells for 3-7 days by using a selective enrichment culture medium for hepatic stromal cells, carrying out passage again, and continuously culturing the parenchymal hepatic cells for 3-7 days by using the selective enrichment culture medium for hepatic stromal cells to obtain hepatic stromal cells; and S3, carrying out passage on the hepatic stromal cells in the step S2, and continuously culturing the hepatic stromal cells for 3-7 days by using a hepatic stellate cell induction culture medium to obtain the hepatic stellate cells. According to the method, the efficiency and functional maturity of differentiation from the human induced pluripotent stem cells to the hepatic stellate cells are remarkably improved, and a brand-new research platform is provided for deep understanding of a development regulation mechanism, homeostasis maintenance and pathological activation of the hepatic stellate cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a method for inducing pluripotent stem cells to differentiate into hepatic stellate cells and an application thereof. Background Art

[0002] The liver is a vital organ in the human body, and liver fibrosis is a major liver disease. It is a chronic liver injury caused by multiple factors, including viral infection, alcoholism, and autoimmune diseases. Liver fibrosis has the risk of irreversibly developing into liver failure and liver cancer. Activation of hepatic stellate cells is the main driver of liver fibrosis. When the liver is continuously damaged, activated hepatic stellate cells produce large amounts of extracellular matrix (ECM), becoming the main fibrotic cell type in the liver. Hepatic stellate cells (HSCs) are specialized pericytes located in the sinusoids of the liver. They play an important role in vitamin A storage, ECM maintenance, and liver damage repair.

[0003] Therefore, in-depth research on hepatic stellate cells is urgent. However, primary human hepatic stellate cells (pHSCs) have disadvantages such as difficulty in obtaining, heterogeneity, limited proliferation capacity, and inability to remain quiescent in vitro; therefore, the understanding of the important functional characteristics and activation phenotypes of pHSCs is limited. Currently, there have been reports showing methods for generating induced hepatic stellate cells (iHSCs) from human induced pluripotent stem cells (hiPS Cs). However, the differentiation protocols are mostly based on the combination of a single cytokine chemical small molecule, making it difficult to obtain functionally complete cells that are highly similar to HSCs. During embryonic development, the generation of HSCs is closely dependent on a complex and delicate microenvironment. In addition, the presence of liver sinusoidal endothelial cells and surrounding matrix during embryonic development also provides a supportive microenvironment for the development of HSCs.

[0004] The liver microenvironment is crucial for the differentiation of hepatic stellate cells. The liver microenvironment comprises multiple components, including the extracellular matrix, cytokines, and growth factors. The extracellular matrix provides physical support and biochemical signals for hepatic stellate cells, and changes in its composition and structure can affect cell morphology and function. Various cytokines, such as transforming growth factor-β, can activate hepatic stellate cells, prompting their differentiation into myofibroblast-like cells, which in turn secrete extracellular matrix and participate in the process of liver fibrosis. Growth factors, such as platelet-derived growth factor, promote the proliferation and migration of hepatic stellate cells and play an important regulatory role in their differentiation during injury repair and pathological conditions. Furthermore, other cells in the liver, such as Kupffer cells and endothelial cells, also participate in regulating the differentiation of hepatic stellate cells through paracrine signaling. In summary, the liver microenvironment, through the synergistic action of multiple factors, finely regulates the differentiation of hepatic stellate cells and plays a key role in maintaining liver physiological function and pathological processes. Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a method for inducing the differentiation of pluripotent stem cells into hepatic stellate cells. This method not only significantly improves the efficiency and functional maturity of human induced pluripotent stem cells (hiPSCs) differentiation into hepatic stellate cells (HSCs), but also provides a new research platform for in-depth understanding of the developmental regulation mechanism, homeostasis maintenance, and pathological activation of HSCs.

[0006] The present invention also provides a kit.

[0007] The present invention also provides an application.

[0008] According to a first aspect of the present invention, a method for inducing pluripotent stem cells to differentiate into hepatic stellate cells is provided, the method comprising the following steps:

[0009] S1: Induced pluripotent stem cells are differentiated into hepatocytes;

[0010] S2: subculturing the hepatocytes described in step S1 and continuing to culture them in a hepatic stromal cell selective enrichment medium for 3 to 7 days, and then repeating the subculturing 1 to 5 times, each time for 3 to 7 days, to obtain hepatic stromal cells;

[0011] S3: The hepatic stromal cells described in step S2 are passaged and cultured in a hepatic stellate cell induction medium for 3 to 7 days to obtain hepatic stellate cells;

[0012] Wherein, the hepatic stromal cell selective enrichment medium comprises dexamethasone, IL-6, EGF, Wnt3a, A83-01, FGF2, fetal bovine serum and HGF;

[0013] The hepatic stellate cell induction medium comprises SB-431542, FGF2, VEGF, Dorsomorphin, Y-27632 and fetal bovine serum.

[0014] In some embodiments of the present invention, the hepatic stromal cell selective enrichment medium in step S2 includes 1-3 mmol / L dexamethasone, 5-20 μg / mL IL-6, 10-30 μg / mL EGF, 5-20 μg / mL Wnt3a, 1-5 μmol / LA83-01, 5-20 μg / mL FGF2, 1%-3% fetal bovine serum and 10-30 μg / mL HGF.

[0015] In some embodiments of the present invention, the hepatic stromal cell selective enrichment medium in step S2 includes 2-3 mmol / L dexamethasone, 5-15 μg / mL IL-6, 15-25 μg / mL EGF, 5-15 μg / mL Wnt3a, 1-3 μmol / L LA83-01, 5-15 μg / mL FGF2, 1.5%-2.5% fetal bovine serum and 15-25 μg / mL HGF.

[0016] In some embodiments of the present invention, the basal medium of the hepatic stromal cell selective enrichment medium in step S2 is SFD medium.

[0017] In some embodiments of the present invention, the SFD medium is based on IMDM medium (GIBCO, 12440053) and Ham's F-12K medium (GIBCO, 21127022), with the mixing ratio of IMDM to Ham's F-12K medium being 3:1, and 1% N2 (GIBCO, 17502048), 1% B27 (GIBCO, 17504044), 450 mmol / L 1-thioglycerol (Sigma, M6145), 1% Glutamax (ThermoFisher, 35050061), 1% PS (GIBCO, 15140122), 0.05% BSA (Sigma-Aldrich, V900933-100G) and 0.5 mmol / L L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate (Sigma, A8960) are added.

[0018] In some embodiments of the present invention, the number of repeated subcultures in step S2 is 1 to 3 times.

[0019] In some embodiments of the present invention, the hepatic stellate cell induction medium in step S3 comprises 3-8 μmol / L SB-431542, 5-20 μg / mL FGF2, 5-20 μg / mL VEGF, 0.1-1 μmol / L Dorsomorphin, 5-20 μmol / L Y-27632 and 1%-3% fetal bovine serum.

[0020] In some embodiments of the present invention, the hepatic stellate cell induction medium in step S3 comprises 4-6 μmol / L SB-431542, 5-15 μg / mL FGF2, 5-15 μg / mL VEGF, 0.4-0.6 μmol / L Dorsomorphin, 5-15 μmol / L Y-27632 and 1.5%-2.5% fetal bovine serum.

[0021] In some embodiments of the present invention, the basal medium of the hepatic stellate cell induction medium in step S3 is StemPro TM -34SFM medium.

[0022] In some embodiments of the present invention, step S1 includes culturing induced pluripotent stem cells using culture medium A for 2 to 5 days, subculturing and continuing to culture using culture medium B for 3 to 7 days, and changing the medium to continue to culture using culture medium C for 7 to 12 days to obtain hepatocytes.

[0023] After the hepatocytes are induced in step S1, a liver biomimetic microenvironment is constructed, thereby better inducing hepatic stellate cells in the subsequent steps. In some embodiments of the present invention, the culture medium A comprises B27, Activin A, and CHIR99021.

[0024] In some embodiments of the present invention, the culture medium A comprises 0.5% to 2% B27, 50 to 200 μg / mL Activin A and 1 to 5 μmol / L CHIR99021.

[0025] In some embodiments of the present invention, the culture medium A comprises 0.5% to 1.5% B27, 80 to 120 μg / mL Activin A and 2 to 4 μmol / L CHIR99021.

[0026] In some embodiments of the present invention, the basal medium of the culture medium A is RPMI1640 medium.

[0027] In some embodiments of the present invention, the culture medium B comprises KSR, NEAA, Glutamax, dimethyl sulfoxide and dimethyl ethanol.

[0028] In some embodiments of the present invention, the culture medium B comprises 10% to 30% KSR, 0.5% to 2% NEAA, 0.5% to 2% Glutamax, 0.5% to 2% dimethyl sulfoxide and 30 to 70 nmol / L dimethyl ethanol.

[0029] In some embodiments of the present invention, the culture medium B comprises 15% to 25% KSR, 0.5% to 1.5% NEAA, 0.5% to 1.5% Glutamax, 0.5% to 1.5% dimethyl sulfoxide and 40 to 60 nmol / L dimethyl ethanol.

[0030] In some embodiments of the present invention, the basal medium of the culture medium B is KO-DMEM medium.

[0031] In some embodiments of the present invention, the culture medium C comprises dexamethasone, OSM, FGF2, nicotinamide and HGF.

[0032] In some embodiments of the present invention, the culture medium C comprises 1-6 mmol / L dexamethasone, 5-20 μg / mL OSM, 5-20 μg / mL FGF2, 0.1-1 g / mL nicotinamide and 10-30 μg / mL HGF.

[0033] In some embodiments of the present invention, the culture medium C comprises 2-3 mmol / L dexamethasone, 5-15 μg / mL OSM, 5-15 μg / mL FGF2, 0.3-0.6 g / mL nicotinamide and 15-25 μg / mL HGF.

[0034] In some embodiments of the present invention, the basal medium of the culture medium C is SFD medium.

[0035] In some embodiments of the present invention, the cell seeding density of the passage and the re-passage in step S2 is 1-5×10 3 pieces / cm 2 .

[0036] In some embodiments of the present invention, the cell seeding density in step S3 is 1 to 5×10 3 pieces / cm 2 .

[0037] The core innovation of the method provided in the first aspect of the present invention is that a biomimetic liver microenvironment is systematically introduced into the differentiation process of hiPSCs to HSCs for the first time. In order to construct a biomimetic microenvironment, multi-stage induction factors are added during the culture process to simulate the physiological process of liver development. In the first stage (0-2 days), Activin A and CHIR99021 are used to activate the TGF-β / Nodal and Wnt / β-catenin signaling pathways to simulate the directional differentiation of the endoderm during the gastrulation period of the embryo. Activin A drives the expression of the definitive endoderm characteristic genes SOX17 / FOXA2 through SMAD2 / 3 phosphorylation, while CHIR99021 stabilizes β-catenin by inhibiting GSK-3β, synergistically enhancing endoderm specification. In the second stage (3-8 days), DMSO is added to induce polarization of hepatic progenitor cells, which promotes epithelial morphogenesis by regulating cell membrane fluidity; KSR provides the growth factors required for liver bud development, and dithiothreitol maintains intracellular redox homeostasis, simulating the metabolic characteristics in the fetal liver microenvironment. During the maturation phase (9-18 days), dexamethasone is used to activate the glucocorticoid receptor and induce the expression of the CYP450 enzyme system. OSM promotes terminal differentiation of hepatocytes through the gp130-JAK / STAT3 pathway. HGF and FGF2 synergistically activate MET / ERK signaling, driving hepatocyte functional maturation. Nicotinamide, as an NAD+ precursor, regulates the sirtuin pathway, supporting energy metabolism homeostasis. Simultaneously, thioglycerol and ascorbic acid in the SFD culture medium recreate the microenvironmental characteristics of the hepatic lobule structure by scavenging ROS and promoting collagen deposition. This combination of temporal signals and metabolic factors systematically reconstructs the molecular spatiotemporal program of liver development. As a key regulator of HSC development and functional maintenance, the liver microenvironment is composed of biochemical and mechanical signals from the extracellular matrix (ECM), intercellular interactions, and dynamic gradients of local signaling molecules (such as growth factors and cytokines). Studies have shown that ECM components (such as laminin and type IV collagen) regulate the adhesion, migration and phenotypic stability of HSCs through integrin-mediated mechanical signaling (such as the FAK-YAP / TAZ pathway). At the same time, paracrine signals between hepatocytes and endothelial cells (such as HGF and Wnt2) coordinate the differentiation timing and functional maturation of HSCs by activating the Notch pathway. In addition, the spatiotemporal dynamic distribution of local signaling molecules (such as TGF-β1 and PDGF-BB) further simulates the microenvironmental response mechanism of HSCs during liver development and fibrosis. The construction of this biomimetic microenvironment not only significantly improves the efficiency and functional maturity of hiPSCs differentiation into HSCs, but also provides a new research platform for in-depth understanding of the developmental regulation mechanism, homeostasis maintenance and pathological activation of HSCs.

[0038] According to a second aspect of the present invention, a kit is provided, comprising a hepatocyte induction medium, a hepatic stromal cell selective enrichment medium, and a hepatic stellate cell induction medium;

[0039] The hepatic stromal cell selective enrichment medium comprises dexamethasone, IL-6, EGF, Wnt3a, A83-01, FGF2, fetal bovine serum and HGF; the hepatic stellate cell induction medium comprises SB-431542, FGF2, VEGF, Dorsomorphin, Y-27632 and fetal bovine serum.

[0040] In some embodiments of the present invention, the hepatocyte induction medium includes medium A, medium B and medium C.

[0041] In some embodiments of the present invention, the culture medium A comprises B27, ActivinA and CHIR99021.

[0042] In some embodiments of the present invention, the culture medium A comprises 0.5% to 2% B27, 50 to 200 μg / mL Activin A and 1 to 5 μmol / L CHIR99021.

[0043] In some embodiments of the present invention, the culture medium A comprises 0.5% to 1.5% B27, 80 to 120 μg / mL Activin A and 2 to 4 μmol / L CHIR99021.

[0044] In some embodiments of the present invention, the basal medium of the culture medium A is RPMI1640 medium.

[0045] In some embodiments of the present invention, the culture medium B comprises KSR, NEAA, Glutamax, dimethyl sulfoxide and dimethyl ethanol.

[0046] In some embodiments of the present invention, the culture medium B comprises 10% to 30% KSR, 0.5% to 2% NEAA, 0.5% to 2% Glutamax, 0.5% to 2% dimethyl sulfoxide and 30 to 70 nmol / L dimethyl ethanol.

[0047] In some embodiments of the present invention, the culture medium B comprises 15% to 25% KSR, 0.5% to 1.5% NEAA, 0.5% to 1.5% Glutamax, 0.5% to 1.5% dimethyl sulfoxide and 40 to 60 nmol / L dimethyl ethanol.

[0048] In some embodiments of the present invention, the basal medium of the culture medium B is KO-DMEM medium.

[0049] In some embodiments of the present invention, the culture medium C comprises dexamethasone, OSM, FGF2, nicotinamide and HGF.

[0050] In some embodiments of the present invention, the culture medium C comprises 1-6 mmol / L dexamethasone, 5-20 μg / mL OSM, 5-20 μg / mL FGF2, 0.1-1 g / mL nicotinamide and 10-30 μg / mL HGF.

[0051] In some embodiments of the present invention, the culture medium C comprises 2-3 mmol / L dexamethasone, 5-15 μg / mL OSM, 5-15 μg / mL FGF2, 0.3-0.6 g / mL nicotinamide and 15-25 μg / mL HGF.

[0052] In some embodiments of the present invention, the basal medium of the culture medium C is SFD medium.

[0053] In some embodiments of the present invention, the hepatic stellate cell induction medium comprises 3-8 μmol / L SB-431542, 5-20 μg / mL FGF2, 5-20 μg / mL VEGF, 0.1-1 μmol / L Dorsomorphin, 5-20 μmol / L LY-27632 and 1%-3% fetal bovine serum.

[0054] In some embodiments of the present invention, the hepatic stellate cell induction medium comprises 4-6 μmol / L SB-431542, 5-15 μg / mL FGF2, 5-15 μg / mL VEGF, 0.4-0.6 μmol / L Dorsomorphin, 5-15 μmol / L Y-27632 and 1.5%-2.5% fetal bovine serum.

[0055] In some embodiments of the present invention, the basal medium of the hepatic stellate cell induction medium is StemPro TM -34SFM medium.

[0056] In some embodiments of the present invention, the hepatic stromal cell selective enrichment medium includes 1-3 mmol / L dexamethasone, 5-20 μg / mL IL-6, 10-30 μg / mL EGF, 5-20 μg / mL Wnt3a, 1-5 μmol / LA83-01, 5-20 μg / mL FGF2, 1%-3% fetal bovine serum and 10-30 μg / mL HGF.

[0057] In some embodiments of the present invention, the hepatic stromal cell selective enrichment medium comprises 2-3 mmol / L dexamethasone, 5-15 μg / mL IL-6, 15-25 μg / mL EGF, 5-15 μg / mL Wnt3a, 1-3 μmol / LA83-01, 5-15 μg / mL FGF2, 1.5%-2.5% fetal bovine serum and 15-25 μg / mL HGF.

[0058] In some embodiments of the present invention, the basal medium of the hepatic stromal cell selective enrichment medium is SFD medium.

[0059] According to the third aspect of the present invention, a use of the kit according to the second aspect of the present invention in inducing pluripotent stem cells to differentiate into hepatic stellate cells is provided.

[0060] The present invention has at least the following beneficial effects:

[0061] The method of inducing pluripotent stem cells to differentiate into hepatic stellate cells provided by the present invention can induce high-purity, high-functional hepatic stellate cells, and the phenotype of these hepatic stellate cells is very close to that of primary isolated hepatic stellate cells. This innovative strategy has opened up a new path for liver disease modeling and regenerative medicine research, and has important scientific significance and application value. This new method has the potential to stably and scalably produce functional HSCs, opening up a new path for studying the progression of liver diseases and related cellular microenvironments. In addition, the above-mentioned homologous multicellular integration strategy also provides a more accurate model for studying liver development, disease mechanisms and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0063] Figure 1 Schematic diagram of the method for inducing pluripotent stem cells to differentiate into hepatic stellate cells in Example 1 of the present invention;

[0064] Figure 2 Figures show the cell morphology results at different stages of the induction process in the experimental examples of the present invention; hiPSC stands for induced pluripotent stem cells, DE stands for definitive endoderm, HE stands for hepatic stem cells, and MH stands for hepatocytes. The scale bar is 100 μm.

[0065] Figure 3 Graphs showing the results of gene expression testing of the stemness of hiPSCs during induction and the endoderm at the definitive endoderm stage in the experimental examples of the present invention;

[0066] Figure 4 Graph showing the results of gene expression detection of hepatocytes in the experimental examples of the present invention;

[0067] Figure 5 Graph showing the results of gene expression detection in liver non-parenchymal cells in the experimental example of the present invention;

[0068] Figure 6 Graphs showing cell morphology during the selective enrichment and expansion of hepatic stromal cells in the experimental examples of the present invention; the scale bar for the first row of results is 200 μm, and the scale bar for the second row of results is 50 μm;

[0069] Figure 7 The figure shows the morphological results of iHSCs induced in the experimental example of the present invention; the scale bar of the result on the left is 200 μm, and the scale bar of the result on the right is 50 μm;

[0070] Figure 8 Graph showing the gene expression detection results during the dynamic expression of HSCs-related specific genes in the experimental examples of the present invention;

[0071] Figure 9 Graph showing the results of gene expression testing of iHSCs and positive reference HSCs in the experimental examples of the present invention;

[0072] Figure 10 Figure 2 is a flow cytometry result diagram of PDGFRB in a test example of the present invention;

[0073] Figure 11 The figure shows the results of immunofluorescence staining to detect iHSCs markers in the experimental example of the present invention; wherein, the scale bar is 50 μm;

[0074] Figure 12 This is a graph showing the results of UV detection of vitamin A storage in iHSCs in an experimental example of the present invention, where the scale bar is 50 μm;

[0075] Figure 13 This is a flow cytometry result diagram of detecting the vitamin A content of iHSCs in a test example of the present invention;

[0076] Figure 14 This is a graph showing the co-expression results of vitamin A and PDGFRB in iHSCs detected by flow cytometry in a test example of the present invention;

[0077] Figure 15 This is a PCA analysis result diagram in the experimental example of the present invention; wherein the HSC group marked in blue represents pHSCs;

[0078] Figure 16 This is a heat map analysis of all genes in the experimental examples of the present invention; the blue-marked HSC group represents pHSCs;

[0079] Figure 17 This is a heat map analysis of HSCs-related genes in the experimental examples of the present invention;

[0080] Figure 18 Graph showing the similarity analysis results between iHSCs and pHSCs in the experimental examples of the present invention;

[0081] Figure 19 This is a UMAP diagram showing the relative distribution of HSCs in the liver in the experimental example of the present invention;

[0082] Figure 20 This is the gene distribution map with high specificity in HSCs displayed by UMAP in the experimental example of the present invention;

[0083] Figure 21 Graph showing the detection results of mRNA expression levels of HSCs-related marker genes in the experimental examples of the present invention. DETAILED DESCRIPTION

[0084] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0085] Example 1

[0086] This embodiment provides a method for inducing pluripotent stem cells to differentiate into hepatic stellate cells. The method includes three stages: induction of hepatocytes, selective enrichment of hepatic stromal cells, and generation of hepatic stellate cells. The schematic diagram of the process is shown in FIG. Figure 1 As shown, the entire cell differentiation process was cultured at 37°C and 5% CO2, specifically including the following steps:

[0087] 1. Induction of hiPSCs into hepatocytes

[0088] (1) Induction to definitive endoderm (DE):

[0089] The day before, plate 3-4×10 hiPSCs. 4 pieces / cm 2hiPSCs were cultured in RPMI 1640 medium (GIBCO, 11875093) supplemented with 1% (v / v) B27 (GIBCO, A1895601), 100 μg / mL Activin A (Nacalai Tesque, 18585-81), and 3 μmol / L CHIR99021 (Tocris, 4423) on day 0. From day 1 to day 2, the medium was replaced with RPMI 1640 medium containing 1% (v / v) B27 and 100 μg / mL Activin A and culture continued.

[0090] (2) Induction into hepatic stem cells (HE):

[0091] From day 3 to day 8, KO-DMEM (GIBCO, 10829018) was used as the basal culture medium. An induction culture medium containing 20% (v / v) KSR (GIBCO, 10828028), 1% (v / v) NEAA (GIBCO, 11140050), 1% (v / v) Glutamax (ThermoFisher, 35050061), 1% (v / v) DMSO (Solarbio, D8371-50 mL), 1% (v / v) PS (GIBCO, 15140122) and 50 nmol / L dimercaptoethanol (GIBCO, 2392436) was prepared. On day 3, the endoderm cells induced in step (1) were digested and cultured at a rate of 3 to 4 × 10 4 pieces / cm 2 The cells were re-inoculated at a density of 100 μg / mL in the above induction medium and cultured until the 8th day.

[0092] (3) Induction into mature hepatocytes (MH) / hepatocytes:

[0093] From day 9 to day 18, cells were cultured in a mature hepatocyte medium using SFD medium as the basal medium. The medium was supplemented with 2.5 mmol / L dexamethasone (Sigma, D2915), 10 μg / mL OSM (Brand, 295-OM-010), 10 μg / mL FGF2 (R&D Systems, 233-FB), 0.407 g / mL Nicotinamide (Sigma, NO636), and 20 μg / mL HGF (R&D Systems, 100-39H). The medium was changed on days 8, 9, 10, 12, 14, and 16, and MH was obtained on day 18.

[0094] The SFD medium used in this example was based on IMDM medium (GIBCO, 12440053) and Ham's F-12K medium (GIBCO, 21127022). The mixing ratio of IMDM to Ham's F-12K medium was 3:1. 1% N2 (GIBCO, 17502048), 1% B27 (GIBCO, 17504044), 450 mmol / L 1-thioglycerol (Sigma, M6145), 1% Glutamax (ThermoFisher, 35050061), 1% PS (GIBCO, 15140122), 0.05% BSA (Sigma-Aldrich, V900933-100G), and 0.5 mmol / L L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate (Sigma, A8960) were added.

[0095] 2. Selective Enrichment and Expansion of Hepatic Stromal Cells

[0096] (1) Selective enrichment of hepatic stromal cells:

[0097] SFD medium was used as the basal medium to prepare the hepatic stromal cell selective enrichment medium, which contained 2.5 mmol / L dexamethasone (Sigma, D2915), 10 μg / mL IL6 (R&D Systems, 206-IL), 20 μg / mL EGF (R&D Systems, 236-EG-200), 10 μg / mL Wnt3a (R&D Systems, 5036-WN-010), 2 μmol / L A83-01 (Tocris Bioscience, 2939), 2% FBS (GIBCO, 10091148), 10 μg / mL FGF2 (R&D Systems, 233-FB) and 20 μg / mL HGF (R&D Systems, 100-39H).

[0098] After the first stage of hepatocyte culture for 18 days, the hepatocytes were digested into single cells using Accutasse cell digestion solution, and about 3×10 4 cells (corresponding to 3×10 3 pieces / cm 2 ) Culture the cells in a Matrix511-coated six-well tissue culture plate using the aforementioned liver stromal cell selective enrichment medium, replacing the medium every two days. During the culture process, the hepatic parenchymal-like cells are gradually eliminated, resulting in the target liver stromal cells.

[0099] From the perspective of mechanism of action, during the enrichment of hepatic stromal cells, the cytokines supplemented by the present invention promote the apoptosis of hepatic parenchymal cells and support the proliferation and survival of hepatic stromal cells by selectively regulating signaling pathways and microenvironmental pressure:

[0100] ① IL-6 induces inflammatory stress in hepatocytes by activating the JAK / STAT3 pathway, while enhancing the anti-apoptotic ability of stromal cells (such as upregulating BCL-2), thereby accelerating the metabolic exhaustion of parenchymal cells.

[0101] ②EGF selectively stimulates the EGFR receptor on the surface of stromal cells such as hepatic stellate cells (HSCs), driving their proliferation through the PI3K / AKT pathway, while mature hepatic parenchymal cells cannot respond to this signal due to low EGFR expression.

[0102] ③Wnt3a activates the Wnt / β-catenin pathway, inhibits the expression of terminal differentiation markers of hepatocytes (such as ALB), and promotes the maintenance of stromal cell stemness and mesenchymal phenotype transformation.

[0103] ④A83-01, as a TGF-β receptor inhibitor, blocks the TGF-β / Smad pathway's maintenance of hepatocyte survival (such as inhibiting the expression of hepatocyte-specific metabolic genes) and alleviates the inhibitory effect of TGF-β on stromal cell proliferation.

[0104] ⑤2% FBS introduces exogenous growth factors (such as PDGF) and adhesion proteins, preferentially supporting the adhesion and expansion of stromal cells; while components such as lipid peroxides in the serum can aggravate the oxidative damage of mature hepatocytes, causing them to detach and die.

[0105] ⑥Matrix511 coating (containing laminin-511) enhances the adhesion advantage of stromal cells through integrin α6β1 signaling, while hepatic parenchymal cells are gradually eliminated in a low-density environment without E-cadherin support.

[0106] The synergistic effect of the above factors reconstructs the selective pressure microenvironment, ultimately achieving the advantageous enrichment of liver stromal cells.

[0107] (2) Hepatic stromal cell expansion:

[0108] After about 5 days (about the 23rd day) of enrichment culture in step (1), the cell confluence reaches 90%. At this time, there are still a small number of aging hepatocytes in the culture system, which need to be passaged again. After digestion, about 3×10 4 cells (corresponding to 3×10 3 pieces / cm 2) were inoculated into Matrix511-coated six-well tissue culture plates and cultured in a liver stromal cell selective enrichment medium for 5 days (about the 28th day). At this time, the liver stromal cells proliferated significantly and no liver parenchymal cells were observed under a microscope, preparing for the differentiation of hepatic stellate cells.

[0109] 3. Induction of Hepatic Stellate Cells from Hepatic Stromal Cells

[0110] The liver stromal cells are further differentiated into HSC cells in HSC induction medium. The composition of HSC induction medium includes: the basic medium is StemPro TM -34SFM medium (GIBCO, 10639011) was added with 5.4 μmol / L SB-431542 (Selleck, S1067-10 mg), 10 μg / mL FGF2 (R&D Systems, 233-FB), 10 μg / mL VEGF (R&D Systems, 293-VE), 0.5 μmol / L Dorsomorphin-2HCl (Selleck, S7306-10 mg), 10 μmol / L Y-27632-2HCl (Selleck, S1049) and 2% FBS (GIBCO, 10091148). The hepatic stromal cells obtained by the second stage of expansion were digested into single cells, and about 3×10 4 cells (corresponding to 3×10 3 pieces / cm 2 ) were inoculated into a Matrix511-coated six-well tissue culture plate and cultured using the above-mentioned HSC induction medium. The medium was not changed the next day after inoculation (approximately day 29), and the HSC medium was changed every 2 days thereafter. iHSCs were successfully induced after 5 days of culture.

[0111] From the perspective of mechanism of action, during the induction of hepatic stellate cells, the optimization of HSC induction medium and the combination of cytokines drive the directional differentiation of hepatic stromal cells into HSCs by precisely regulating key signaling pathways:

[0112] ①SB-431542 (5.4 μmol / L) acts as a TGF-β receptor inhibitor, blocking the TGF-β / Smad signaling pathway and inhibiting the transformation of hepatic stromal cells into myofibroblasts or other fibrotic phenotypes. At the same time, it may promote the expression of HSC-specific phenotypes by relieving TGF-β's inhibition of HSC marker genes (such as GFAP and Desmin).

[0113] ②FGF2 (10 μg / mL) activates the FGFR-MAPK / ERK pathway, enhances cell proliferation ability, and synergistically maintains the inactivated state of HSC, simulating the maintenance effect of paracrine signals on HSC in the liver microenvironment.

[0114] ③ VEGF (10 μg / mL) activates the PI3K / AKT pathway by binding to VEGFR2, inducing perivascular characteristics of HSCs (such as α-SMA expression) and promoting their interaction with hepatic sinusoidal endothelial cells, simulating the localization and function of HSCs under physiological conditions.

[0115] ④Dorsomorphin (0.5 μmol / L) prevents liver stromal cells from shifting toward the osteoblast or adipocyte lineage by inhibiting the BMP signaling pathway (blocking ALK2 / 3 / 6), thereby ensuring the specificity of differentiation direction.

[0116] ⑤Y-27632 (ROCK inhibitor) reduces cell apoptosis and enhances cell adhesion ability by inhibiting the Rho / ROCK pathway, especially maintaining the survival and morphological expansion of HSCs in high-density culture.

[0117] ⑥2% FBS provides basic growth factors (such as PDGF) and adhesion proteins to support the adherent proliferation of HSCs. At the same time, the lipid components it contains may simulate the physiological microenvironment for HSCs to store retinol.

[0118] ⑦StemPro TM The serum-free 34SFM matrix utilizes a precise nutrient profile to avoid interference from exogenous factors and ensure the specificity of differentiation signals. The synergistic effect of these factors reconstructs the microenvironment for HSC development, ultimately achieving the efficient transformation of liver stromal cells into functional HSCs.

[0119] Test example

[0120] This experimental example examined the induced differentiation of cells obtained at various stages of the induction process for differentiating induced pluripotent stem cells into hepatic stellate cells provided in Example 1, including observations of cell morphology, expression of specific genes, expression of surface markers, and storage of vitamin A. The specific experimental methods and results are as follows:

[0121] 1. Differentiation of hiPSCs into hepatocytes at different stages

[0122] 1) During the first stage of induction in Example 1, the dynamic changes of cell morphology were tracked, and the results were as follows: Figure 2 shown.

[0123] Depend on Figure 2 It can be seen that the cell morphology starts from the initial induced pluripotent stem cell morphology with clear edges and flat and smooth shape. After 18 days of induction, the cell morphology becomes polygonal with multiple faces, and the nucleus is round and located in the center or off-center of the cell, which is the typical morphology of hepatocytes.

[0124] 2) At the initial stage of induction (day 0 and day 3), the mRNA expression levels of the stemness gene markers OCT4 and NANOG, the endoderm markers CXCR4 and SOX17, and the mesoderm markers T and MI XL1 in hiPSCs were detected by real-time quantitative PCR. Figure 3 shown.

[0125] Depend on Figure 3 It can be seen that hiPSCs express a high degree of stemness, and in the later stages of development, both mesoderm and endoderm genes are expressed to varying degrees.

[0126] 3) At the hepatocyte stage, the mRNA expression levels of genes related to hepatocytes and non-hepatic cells were detected by real-time quantitative PCR. The results were as follows: Figure 4 ALB, AAT, and AFP are classic hepatocyte markers, and primary human hepatocytes (PHH) were used as a positive reference for hepatocytes.

[0127] Depend on Figure 4 It can be seen that with the development of hiPSCs to MH, the expression of ALB, AAT and AFP gradually increased, but there was still a certain gap from primary hepatocytes.

[0128] CD34 and CD31 are specific markers of vascular endothelium and are also expressed in a certain amount in the hepatocyte stage. CK19 and EPCAM are specific markers of bile duct, LYVE1 and STAB2 are specific markers of liver sinusoidal endothelium, and both specific markers are expressed in the late stage of hepatocytes ( Figure 5 ).

[0129] The above results indicate that the first stage of the induction method provided by the present invention successfully induced hepatic parenchymal cells, providing a large bionic microenvironment for the subsequent induction of hepatic stellate cells and an important basis for subsequent experiments.

[0130] 2. Morphological changes of hepatic stromal cells during enrichment and expansion

[0131] The cell morphology changes during the second stage of hepatic stromal cell enrichment and expansion in Example 1 were observed under a microscope. Figure 6 shown.

[0132] Depend on Figure 6 It can be seen that the liver stromal cells proliferated in large quantities, and no liver parenchymal cells were observed under a microscope, indicating that the method provided by the present invention can successfully induce differentiation to obtain pure liver stromal cells.

[0133] 3. Cell Morphology of Hepatic Stellate Cells

[0134] After the induction of hepatic stellate cells in the third stage of Example 1 was completed, the cell morphology was observed under a microscope and photographed. The results were as follows: Figure 7 shown.

[0135] Depend on Figure 7 It can be seen that in Example 1, after 5 days of culture in the culture dish in the third stage, hepatic stellate cells were successfully induced. The cell morphology was obviously spindle-shaped, and the cell nucleus became elongated or irregular.

[0136] 4. Dynamic changes of HSC-related specific genes during differentiation

[0137] The cells in the differentiation process of Example 1 were sampled and RNA was extracted. The dynamic changes of mRNA expression levels of HSCs-related specific genes during the differentiation process were detected by real-time quantitative PCR. The results were as follows: Figure 8 As shown; MCstage in the figure refers to cells in the selective enrichment stage of hepatic stromal cells, and MC expanding refers to cells in the expansion stage of hepatic stromal cells.

[0138] Depend on Figure 8 As can be seen, HSC-specific genes such as VIM, ALCAM, PDGFRB, DES, ACTA2, and COL1A1 gradually increase during differentiation; compared to iPSCs, these genes are significantly elevated in the final induced HSC group. In particular, PDGFRB, ACTA2, DES, and COL1A1 show a qualitative leap compared to cells at other stages. These results demonstrate that iHSCs induced in Example 1 of the present invention exhibit elevated expression levels of HSC-specific genes.

[0139] Furthermore, the induced iHSCs were compared with the positive reference LX2 (human hepatic stellate cells) and NPC (hepatic non-parenchymal cells) in HSCs-related specific genes, and the results were as follows Figure 9 shown.

[0140] Depend on Figure 9 It can be seen that, except for ALCAM and DES, the other HSCs-related specific genes all showed certain significant differences compared with the positive reference, further verifying that the iHSCs induced in Example 1 of the present invention have the characteristics of HSCs.

[0141] 5. Flow cytometry detection of PDGFRB expression in cells at different induction stages

[0142] The cell populations during HSC induction were further analyzed by flow cytometry. At each induction stage, cells were collected, processed, and analyzed by flow cytometry. Figure 10 shown.

[0143] Depend on Figure 10 It can be seen that PDGFRB is one of the best membrane markers for detecting hepatic stellate cells. During the differentiation process, the positive expression rate of PDGFRB becomes higher and higher with the advancement of differentiation, which also proves that the differentiation is getting closer to HSCs. This result is consistent with the above qPCR analysis results.

[0144] 6. Immunofluorescence staining to assess cell induction

[0145] The iHSCs were verified at the protein level by immunofluorescence staining, and the expression of vitamin A, collagen, and α-SMA were detected. The results are as follows Figure 11 shown.

[0146] Depend on Figure 11 It can be seen that the hepatic stellate cell markers α-smooth muscle actin (α-SMA) and collagen were expressed, further confirming the successful induction of iHSCs.

[0147] 7. Detection of intracellular vitamin A content

[0148] HSCs have an important function of storing vitamin A in the cytoplasmic lipid droplets of hepatic stellate cells. Vitamin A is mainly stored in the form of retinol. Therefore, this experimental example verifies whether the iHSCs induced by the present invention contain vitamin A.

[0149] First, place the cells under UV light and observe whether there is blue-purple light to determine the vitamin A content in the cells. The results are as follows: Figure 12 shown.

[0150] Depend on Figure 12 It can be seen that using mesenchymal cells (MSCs) as a negative control, under the same conditions, iHSCs showed significant storage of vitamin A.

[0151] Next, flow cytometry was used to further verify the vitamin A expression and cell ratio of iHSCs. The PB450 channel was used to detect the vitamin A storage and cell ratio of iHSCs, and iPSCs were used as a positive reference. The results are as follows: Figure 13 shown.

[0152] Depend on Figure 13 It can be seen that the iHSCs induced by the present invention showed obvious vitamin A positivity after flow cytometry analysis, and more than 80% of the cells expressed vitamin A, further proving that the iHSCs induced by the present invention are functional HSCs.

[0153] In addition, after verifying that iHSCs expressed both PDGFRB and vitamin A, flow cytometry was used to detect their co-positive expression, and the results were as follows: Figure 14 shown.

[0154] Depend on Figure 14 It can be seen that the positive rate of iHSCs induced by the present invention that simultaneously express vitamin A and PDGFRB can reach more than 80%.

[0155] The above results indicate that the iHSCs induced by the present invention not only express PDGFRB, but also express vitamin A, proving that this group of iHSCs has good characteristics of HSCs.

[0156] 8. Gene sequencing analysis of the similarities and differences in expression profiles between iHSCs and pHSCs

[0157] 1) The expression profiles of iHSCs induced by the present invention were compared with those of primary pHSCs and aHSCs / qHSCs induced by traditional methods (for traditional induction methods, see “KOUI Y, HIMENO M, MORI Y, et al. Development of human iPSC-derived quiescent hepatic stellate cell-like cells for drug discovery and invitro disease modeling[J]. Stem Cell Reports, 2021, 16(12): 3050-3063.”) by PCA analysis. The results are as follows: Figure 15 shown.

[0158] Depend on Figure 15 It can be seen that in a large number of RNA sequencing analyses, compared with pHSCs and aHSCs differentiated by traditional induction methods, PCA analysis showed that the iHSCs induced by the present invention showed a high similarity with pHSCs in terms of gene expression profiles or cell characteristics.

[0159] 2) Hierarchical clustering display based on global gene expression heat map ( Figure 16 ), compared with aHSCs and qHSCs induced by traditional methods, the expression of iHSCs and pHSCs was more similar.

[0160] The above results verified from the overall gene level that the iHSCs and pHSCs induced by the present invention showed a consistent trend in results, whether based on PCA analysis results or overall gene heat map analysis results - iHSCs and pHSCs were similar.

[0161] 3) In order to further verify the similarity between iHSCs and pHSCs, narrow the gene range and verify the similarity between iHSCs and pHSCs in HSCs-related gene expression. Therefore, this experimental example identified and compiled a list of 72 highly hepatic stellate cell-specific genes based on the liver-related dataset (E-MTAB-7407) of the EMBL European Bioinformatics Institute and previously reported genes associated with HSCs. These genes are closely related to the characteristics and functions of hepatic stellate cells and are key markers for evaluating the similarity between iHSCs and pHSCs. The heat map of the expression of these hepatic stellate cell-related genes is arranged from high to low, and the results are shown as follows Figure 17 shown.

[0162] Depend on Figure 17 It can be seen that iHSCs and pHSCs show high similarity at the gene expression level, such as high expression of IGF BP5, COL1A1, MMP2 and LAMB1.

[0163] Correlation analysis further showed that iHSCs were more similar to pHSCs, highlighting the effectiveness and superiority of this in vitro differentiation system ( Figure 18 ).

[0164] 4) To further verify the similarity between iHSCs and pHSCs, this study also identified other hepatic stellate cell-specific marker genes.

[0165] First, a hepatic stellate cell population dataset was collected. Single-cell RNA sequencing data from the European Bioinformatics Institute Human Fetal Liver, Skin, and Kidney Dataset (E-MTAB-7407) were used to analyze gene expression profiles. The Uniform Manifold Approximation and Projection (UMAP) method was used to compare hepatic stellate cells with other major liver cell types (including hepatocytes, T cells, B cells, bile duct cells, macrophages, monocytes, and endothelial cells), thereby revealing the relative spatial distribution of hepatic stellate cells in the entire cell lineage. The results are shown in Figure 2. Figure 19 shown.

[0166] Furthermore, bioinformatics analysis of the European Bioinformatics Institute data, numbered E-MTAB-7407, revealed 41 nonspecific marker genes expressed not only in HSCs but also in fibroblasts and smooth muscle cells, including ADCY5, DBH, ELN, HGF, MYL9, GGT5, GEM, and NEXN. Furthermore, six previously reported HSC-associated marker genes were confirmed: ACTA2, COL1A1, DES, PD-GFRB, ALCAM, and NGFR. Notably, the present invention discovered a series of new candidate HSC marker genes, including ASPN, BGN, COL6A3, COL5A1, COL25A1, COLEC11, DCN, FBLN5, HHIP, IGFBP5, LAMB1, NID2, and SVEP1 (see Table 1 for details).

[0167] Table 1 HSCs-related marker genes

[0168]

[0169]

[0170] These newly discovered markers may help to more fully understand the molecular characteristics of HSCs and provide valuable references for future research in this field. Among them, BGN, COL6A3, COLEC10, SVEP1, DCN, HHIP, IGFBP3 and LAMB1 have extremely high specificity in HSCs, providing more sensitive markers for further understanding of HSCs (such as Figure 20 shown).

[0171] 5) This study further conducted qRT-PCR to evaluate the expression of the above-mentioned HSCs-related marker genes in different cell populations, including iPSCs, iHSCs, NPCs, LX2, and aHSCs and qHSCs induced by Miyajima laboratory. The results are shown in Figure 5. Figure 21 shown.

[0172] Depend on Figure 21As shown, compared with the induction protocol developed by the Miyajima laboratory, the expression levels of FBLN5, NID2, LAMB1, IGFBP5, and SVEP1 in iHSCs induced by the present invention were significantly higher, indicating that the induction protocol provided by the present invention improves induction efficiency. Recent reports indicate that FBLN5, NID2, and IGFBP5 are significantly expressed during the differentiation and development of hepatic stellate cells, which is consistent with the current findings of the present invention. However, two other genes, LAMB1 and SVEP1, while expressed at high levels in iHSCs, have not been reported to date. They could serve as novel biomarkers for hepatic stellate cells in future studies and warrant further investigation.

[0173] These results demonstrate that the present invention successfully induced hepatic stellate cells. Transcriptome analysis revealed that iHSCs exhibited high similarity to pHSCs in gene expression profiles (e.g., LGALS1 and IGFBP5). Both at the global gene level and at the level of genes associated with HSCs, iHSCs exhibited high similarity to pHSCs.

[0174] In summary, the present invention provides an efficient and reproducible induction strategy for generating iPSCs-derived HSCs. By simulating the multicellular interactions and signal transduction in the liver microenvironment, the biological behavior of HSCs in vivo can be more accurately reproduced, thereby providing a reliable platform for liver disease modeling and regenerative medicine research. The complexity of the liver microenvironment plays a key role in the occurrence and development of liver diseases (such as liver fibrosis, cirrhosis and liver cancer). In the field of regenerative medicine, the construction of a bionic liver microenvironment provides a new approach for generating functional HSCs. These HSCs can not only be used to construct liver organoids, but also as a tool for cell therapy to repair damaged liver tissue. In short, the liver microenvironment plays a vital role in the development and functional maturation of HSCs. By systematically integrating the bionic liver microenvironment into the process of iPSCs differentiation into HSCs, not only the differentiation efficiency and functional maturity of iHSCs are significantly improved, but also a new perspective is provided for understanding the biological behavior of HSCs. More interestingly, through single-cell analysis, some markers commonly found in fetal HSCs (such as NID2, FBLN5, LAMB1, IGFBP5 and SVEP1) were also expressed in iHSCs, which provides potential markers for tracking the early stages of HSC development and paves the way for further research on the biological functions of HSCs.

[0175] This innovative strategy has opened up a new path for liver disease modeling and regenerative medicine research, and has important scientific significance and application value. This new method has the potential to stably and scalably produce functional HSCs, opening up a new path for studying the progression of liver diseases and related cellular microenvironments. In addition, the above-mentioned homologous multicellular integration strategy also provides a more accurate model for studying liver development, disease mechanisms and drug screening. For example, in the study of liver fibrosis and liver cancer, homologous organoids can better simulate cell-to-cell interactions and signal transduction, thereby revealing the molecular mechanisms of disease occurrence and development. In the future, this technology is expected to promote the development of personalized medicine by generating autologous organoids from patient-specific hiPSCs to achieve personalized disease modeling and optimization of treatment strategies. Finally, the method provided by the present invention not only fills the technical gap in liver organoid research, but also opens up a new path for regenerative medicine and precision medicine.

[0176] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A method for inducing pluripotent stem cells to differentiate into hepatic stellate cells, characterized in that: The method comprises the following steps: S1: Induced pluripotent stem cells are differentiated into hepatocytes; S2: subculturing the hepatocytes described in step S1 and continuing to culture them in a hepatic stromal cell selective enrichment medium for 3 to 7 days, and then repeating the subculturing 1 to 5 times, each time for 3 to 7 days, to obtain hepatic stromal cells; S3: The hepatic stromal cells described in step S2 are passaged and cultured in a hepatic stellate cell induction medium for 3 to 7 days to obtain hepatic stellate cells; Wherein, the hepatic stromal cell selective enrichment medium comprises dexamethasone, IL-6, EGF, Wnt3a, A83-01, FGF2, fetal bovine serum and HGF; The hepatic stellate cell induction medium comprises SB-431542, FGF2, VEGF, Dorsomorphin, Y-27632 and fetal bovine serum.

2. The method according to claim 1, characterized in that The hepatic stromal cell selective enrichment medium in step S2 includes 1-3 mmol / L dexamethasone, 5-20 μg / mL IL-6, 10-30 μg / mL EGF, 5-20 μg / mL Wnt3a, 1-5 μmol / L A83-01, 5-20 μg / mL FGF2, 1%-3% fetal bovine serum and 10-30 μg / mL HGF.

3. The method according to claim 1, characterized in that The hepatic stellate cell induction medium in step S3 comprises 3-8 μmol / L SB-431542, 5-20 μg / mL FGF2, 5-20 μg / mL VEGF, 0.1-1 μmol / L Dorsomorphin, 5-20 μmol / L Y-27632 and 1%-3% fetal bovine serum.

4. The method according to claim 1, wherein Step S1 includes culturing induced pluripotent stem cells using medium A for 2 to 5 days, subculturing and continuing to culture using medium B for 3 to 7 days, and then changing the medium to culture using medium C for 7 to 12 days to obtain hepatocytes; Preferably, the culture medium A comprises B27, ActivinA and CHIR99021; Preferably, the culture medium B comprises KSR, NEAA, Glutamax, dimethyl sulfoxide and dimethyl ethanol; Preferably, the culture medium C comprises dexamethasone, OSM, FGF2, nicotinamide and HGF.

5. The method according to claim 4, characterized in that The culture medium A includes 0.5% to 2% B27, 50 to 200 μg / mL Activin A and 1 to 5 μmol / L CHIR99021; Preferably, the culture medium B comprises 10% to 30% KSR, 0.5% to 2% NEAA, 0.5% to 2% Glutam ax, 0.5% to 2% dimethyl sulfoxide and 30 to 70 nmol / L dimethyl ethanol; Preferably, the culture medium C comprises 1-6 mmol / L dexamethasone, 5-20 μg / mL OSM, 5-20 μg / mL FGF2, 0.1-1 g / mL nicotinamide and 10-30 μg / mL HGF.

6. A kit, characterized in that The kit includes a hepatocyte induction medium, a hepatic stromal cell selective enrichment medium, and a hepatic stellate cell induction medium; The hepatic stromal cell selective enrichment medium comprises dexamethasone, IL-6, EGF, Wnt3a, A83-01, FGF2, fetal bovine serum and HGF; the hepatic stellate cell induction medium comprises SB-431542, FGF2, VEGF, Dorsomorphin, Y-27632 and fetal bovine serum.

7. The kit according to claim 6, characterized in that The hepatocyte induction medium includes medium A, medium B and medium C; Preferably, the culture medium A comprises B27, ActivinA and CHIR99021; Preferably, the culture medium B comprises KSR, NEAA, Glutamax, dimethyl sulfoxide and dimethyl ethanol; Preferably, the culture medium C comprises dexamethasone, OSM, FGF2, nicotinamide and HGF.

8. The kit according to claim 6, characterized in that The hepatic stromal cell selective enrichment medium comprises 1-3 mmol / L dexamethasone, 5-20 μg / mL IL-6, 10-30 μg / mL EGF, 5-20 μg / mL Wnt3a, 1-5 μmol / L A83-01, 5-20 μg / mL FGF2, 1%-3% fetal bovine serum and 10-30 μg / mL HGF; And / or, the hepatic stellate cell induction medium comprises 3-8 μmol / L SB-431542, 5-20 μg / mL FGF 2, 5-20 μg / mL VEGF, 0.1-1 μmol / L Dorsomorphin, 5-20 μmol / L Y-27632 and 1%-3% fetal bovine serum.

9. The kit according to claim 7, characterized in that The culture medium A includes 0.5% to 2% B27, 50 to 200 μg / mL Activin A and 1 to 5 μmol / L CHIR99021; Preferably, the culture medium B comprises 10% to 30% KSR, 0.5% to 2% NEAA, 0.5% to 2% Glutam ax, 0.5% to 2% dimethyl sulfoxide and 30 to 70 nmol / L dimethyl ethanol; Preferably, the culture medium C comprises 1-6 mmol / L dexamethasone, 5-20 μg / mL OSM, 5-20 μg / mL FGF2, 0.1-1 g / mL nicotinamide and 10-30 μg / mL HGF.

10. Use of the kit according to any one of claims 6 to 9 for inducing differentiation of pluripotent stem cells into hepatic stellate cells.

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