Method and application of inducing gallbladder-derived stem cells to differentiate into functional hepatocytes

Abstract

The present invention relates to the field of biotechnology and cell therapy, and discloses a method and application of inducing gallbladder-derived stem cells to differentiate into functional hepatocytes; the method comprises the steps of: culturing gallbladder-derived stem cells in a culture system in the presence of a first culture medium, a second culture medium, and a third culture medium, thereby obtaining functional hepatocytes. The functional hepatocytes obtained by the present invention have a high differentiation efficiency and have the effect of treating liver-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of functional hepatocytes, and in particular to a method and application of inducing gallbladder-derived stem cells to differentiate into functional hepatocytes. Background Art

[0002] my country is a country with a high incidence of liver diseases. Acute and chronic liver diseases caused by viruses, drugs, food, and genetics can lead to functional liver failure. Chronic damage is an important cause of hepatitis, cirrhosis, and liver cancer. Timely intervention and treatment will effectively prevent the death of patients (Changing global epidemiology of liver cancer from 2010 to 2019: NASH is the fastest growing cause of liver cancer; PMID: 33734139). Cell replacement therapy provides a candidate for the treatment of liver diseases. Transplanting functional liver cells is expected to solve some problems in the clinical treatment of liver diseases, such as the shortage of liver organ donors, drug-induced liver dysfunction, and side effects. More importantly, studies have reported that liver cell transplantation has a significant therapeutic effect on rare diseases such as hepatolenticular degeneration, type 1 tyrosinemia, and other chromosomal genetic metabolic diseases, which can help patients survive the dangerous period and wait for further treatment.

[0003] Obtaining functional hepatocytes can provide a cell source for cell therapy of liver diseases. Commonly used methods include: in situ separation of liver, induced differentiation of embryonic stem cells, somatic cell reprogramming, and induced differentiation of cells from other sources. Hepatocytes obtained by different methods have their own advantages: 1) Digestion, separation, and enrichment of liver tissue is a classic method for obtaining functional hepatocytes in vitro. The advantage is that hepatocytes are fully functional and generally have significant therapeutic effects after transplantation; however, their sources are limited and cannot be amplified in large quantities, so they are difficult to use widely. 2) There is a sufficient source of embryonic stem cells, which can be induced to differentiate into hepatocyte-like cells with some hepatocyte functions; the disadvantage is that the differentiation efficiency is limited, the residual stem cells are tumorigenic, and have been subject to ethical controversy. 3) Reprogrammed hepatocyte-like cells have a wide range of sources and are currently a hot topic of research. Improving induction efficiency and enhancing function are technical bottlenecks that need to be overcome. 3) Others, such as mesenchymal stem cells and hematopoietic stem cells, have also been reported to have the ability to differentiate into hepatocytes.

[0004] Gallbladder tissue is an important part of the liver bile duct system. The gallbladder and liver are homologous in development and have a wide range of tissue sources. Theoretically, if cells derived from the gallbladder differentiate into hepatocyte-like cells, it is feasible to do so and can solve the problem of cell sources. More importantly, adult tissue cells generally do not have tumorigenic characteristics. Precursor studies have shown that cells with stem characteristics can be isolated from the gallbladder and have a certain differentiation ability. Therefore, establishing a gallbladder-derived stem cell P production system and directed induction of differentiation can provide a new source of functional hepatocytes, which is expected to provide a candidate cell source with application value and competitiveness for cell therapy of clinical liver diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of inducing gallbladder-derived stem cells to differentiate into functional hepatic parenchymal cells, so as to solve the problems raised in the above-mentioned background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for inducing gallbladder-derived stem cells to differentiate into functional hepatocytes, comprising the following steps:

[0008] S1: pre-treating the gallbladder tissue and adding it to the digestion solution for incubation, filtering, and centrifuging to collect the cell precipitate; culturing the cell precipitate in the culture system under the first culture system to obtain gallbladder-derived stem cells;

[0009] S2: amplifying, digesting, and directing differentiation of gallbladder-derived stem cells to obtain differentiated cells; culturing the differentiated cells in a culture system under a second culture system to obtain hepatocyte precursor cells;

[0010] S3: Under the third culture system, hepatocyte precursor cells are cultured in the culture system to obtain functional hepatocytes.

[0011] Further, in step S1, the culture system is a first culture medium; the first culture medium includes the following components: liquid basal culture medium, 1×B27 additive, 1×N2 additive, 1×penicillin-streptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng / mL R-spondin, 1-1000mM niacinamide, 0.1-100ng / mL recombinant human epidermal growth factor, 0.1-100ng / mL recombinant human fibroblast growth factor, 0.1-100ng / mL recombinant human hepatocyte growth factor, 0.1-100μM ALK5 inhibitor, 0.1-100ng / mL recombinant human Noggin protein, 0.1-100μM PKC inhibitor, 0.1-100μM ATPase inhibitor, 0.1-100μM Rock inhibitor;

[0012] Among them, the volume ratio of liquid basic culture medium: B27 additive: N2 additive: penicillin and streptomycin is 1:1:1:1.

[0013] Further, in step S2, the culture system is a second culture medium; the second culture medium includes the following components: liquid basal culture medium, 1×B27 additive, 1×N2 additive, 1×penicillin-streptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng / mL R-spondin, 1-1000mM niacinamide, 0.1-100ng / mL recombinant human hepatocyte growth factor, 0.1-100μM TGFβ inhibitor, 0.1-100μM γ-Secretase inhibitor, 0.1-100μM EGFR inhibitor, 0.1-100μM Rock inhibitor;

[0014] Among them, the volume ratio of liquid basic culture medium: B27 additive: N2 additive: penicillin and streptomycin is 1:1:1:1.

[0015] Further, in step S3, the culture system is a third culture medium; the third culture medium includes the following components: liquid basal culture medium, 1×B27 additive, 1×N2 additive, 1×penicillin-streptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng / mL R-spondin, 1-1000mM niacinamide, 0.1-100ng / mL recombinant human hepatocyte growth factor, 0.1-100ng / mL recombinant oncostatin M, 0.1-100μM γ-Secretase inhibitor, 0.1-100μM ALK5 inhibitor, 0.1-100μM dexamethasone;

[0016] Among them, the volume ratio of liquid basic culture medium: B27 additive: N2 additive: penicillin and streptomycin is 1:1:1:1.

[0017] Furthermore, the gallbladder-derived stem cells are any one of gallbladder tissue removed during cholecystitis and gallbladder tissue discarded during liver transplantation.

[0018] Furthermore, in step S1, the gallbladder-derived stem cells are cultured for 1-15 days under the first culture strip system; preferably, the culture time is 10-12 days;

[0019] In step S2, the gallbladder-derived stem cells are cultured in the second culture system for 10-30 days; preferably, the culture time is 15-20 days;

[0020] In the step S3, under the third culture system, the hepatocyte precursor cells are cultured for 1-10 days; preferably, the culture time is 2-4 days.

[0021] Furthermore, the method has one or more of the following features:

[0022] High differentiation efficiency, the differentiation rate is 75-90%;

[0023] During the culture process, 1x10 5 Gallbladder-derived stem cells can produce 0.5-0.8x10 6 hepatocytes.

[0024] Furthermore, the methods include therapeutic and non-therapeutic methods.

[0025] Furthermore, the differentiation-promoting substance is any one of matrix gel, type 1 collagen, and a mixture of matrix gel and type 1 collagen in a volume ratio of 1:1.

[0026] Furthermore, the induction composition comprises a first induction factor, a second induction factor and a differentiation-promoting substance; wherein.

[0027] Furthermore, the first induction factor composition includes TGFβ inhibitor, γ-Secretase inhibitor, and EGFR inhibitor; the second induction factor composition includes recombinant oncostatin M, γ-Secretase inhibitor, ALK5 inhibitor, and 0.1-100μM dexamethasone; the differentiation-promoting substance is any one of matrix gel, type 1 collagen, and a 1:1 mixture of matrix gel and type 1 collagen.

[0028] Furthermore, the induction composition is used to induce gallbladder-derived stem cells to differentiate into hepatic parenchymal cells.

[0029] A functional hepatocyte comprises the following components: albumin, HNF4A, CYP3A4 and AAT, which are markers of mature hepatocytes.

[0030] Furthermore, the mature hepatocyte marker albumin is any one of EPCAM, CK19, TROP2, and SOX9.

[0031] Furthermore, the functional hepatocytes are flat paving stone-like, with a diameter of 15-20 μm, and have the functions of storing glycogen and synthesizing fat.

[0032] Furthermore, the hepatocytes have one or more of the following characteristics:

[0033] 5-90% of cells were positive for HNF4A expression;

[0034] 40-70% are AAT positive;

[0035] 30-60% are CYP positive.

[0036] An application of inducing human gallbladder-derived stem cells to differentiate into functional liver parenchymal cells for preparing a composition for treating liver-related diseases.

[0037] Furthermore, the liver-related disease is any one or more of acute hepatitis, liver failure, cirrhosis, hepatolenticular degeneration and tyrosinemia.

[0038] Furthermore, the composition is any one of a pharmaceutical composition, a food composition and a health product composition.

[0039] Furthermore, the dosage form of the composition is any one of an injection, a lyophilized preparation and a solution preparation.

[0040] Furthermore, an induction medium is provided, which contains a basal medium core additive; wherein the basal medium is selected from the following group: DMEM, F12 combination.

[0041] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0042] The present invention uses human gallbladder stem cells as starting cells for the first time and efficiently prepares functional liver parenchymal cells under appropriate in vitro conditions, providing a candidate cell source for the treatment of liver diseases.

[0043] The present invention firstly finds that gallbladder-derived stem cells cultured in a culture system in the presence of a first culture medium, a second culture medium, and a third culture medium can obtain functional hepatic parenchymal cells with a high differentiation rate.

[0044] The present invention prepares hepatic parenchymal cells derived from gallbladder stem cells for the first time by optimizing conditions, and proves that the hepatic parenchymal cells can cure liver failure after being transplanted into animals.

[0045] The gallbladder-derived stem cells obtained by the present invention and the functional hepatic parenchymal cells differentiated therefrom have broad application prospects: 1) providing candidate cell therapy options for patients with liver failure; 2) providing potentially available functional cells for rare diseases and genetic diseases related to liver metabolism; 3) providing seed cells for in vitro artificial liver support devices; 4) providing a cell screening platform for liver drug screening.

[0046] The invention provides a cell source for constructing a tissue engineering liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0048] Figure 1This is a typical growth morphology diagram of human gallbladder-derived stem cells of the present invention;

[0049] Figure 2 This is a typical growth curve of human gallbladder-derived stem cells of the present invention;

[0050] Figure 3 The human gallbladder-derived stem cells of the present invention express liver stem cell markers;

[0051] Figure 4 The human gallbladder-derived stem cells of the present invention have the molecular phenotype of mature hepatocytes after being induced to differentiate;

[0052] Figure 5 The human gallbladder-derived stem cells of the present invention express mature hepatocyte markers after being induced to differentiate;

[0053] Figure 6 The human gallbladder-derived stem cells of the present invention have hepatocyte function after being induced to differentiate;

[0054] Figure 7 The transplanted gallbladder-derived stem cells of the present invention have the ability to cure mice with liver failure after being induced to differentiate. DETAILED DESCRIPTION

[0055] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] In the following examples, B27 additive was provided by Gibco, model number 12587010; N2 additive was provided by Gibco, model number 17502048; penicillin-streptomycin was provided by Gibco, model number 15140122; N-acetylcysteine ​​was provided by Sigma, model number A9165; R-spondin was provided by Peprotech, model number 120-38; niacinamide was provided by Sigma, model number N0636; recombinant human epidermal growth factor was provided by Peprotech, model number AF10015; recombinant human fibroblast growth factor was provided by Peprotech, model number AF10026; recombinant human hepatocyte growth factor was provided by Peprotech, model number 10039H; ALK5 inhibitor was provided by Selleck, model number S7692; recombinant human Noggin protein was provided by Peprotech, model number 12010C; PKC inhibitor was provided by Selleck k was provided by Selleck, model number S6577; ATPase inhibitor was provided by Selleck, model number S7099; Rock inhibitor was provided by Selleck, model number S6390; TGFβ inhibitor was provided by Selleck, model number S1067; γ-Secretase inhibitor was provided by Selleck, model number S2215; EGFR inhibitor was provided by Selleck, model number S1143; recombinant oncostatin M was provided by R&D, model number 295-OM; dexamethasone was provided by Selleck, model number S1322; sterile PBS was provided by Solaribio, model number P1020; Accutase digestion solution was provided by Gibco, model number A1110501; Matrigel was provided by Corning, model number 354234; type 1 collagen was provided by Corning, model number 356236; TrypLE was provided by Gibco, model number 12563011.

[0057] Example

[0058] The first culture medium includes the following components: 1 mL of liquid basal culture medium DMEM / F12, 1 mL of B27 additive, 1 mL of N2 additive, 1 mL of penicillin-streptomycin, 1 mM N-acetylcysteine, 25 ng / mL of R-spondin, 10 mM of niacinamide, 50 ng / mL of recombinant human epidermal growth factor, 50 ng / mL of recombinant human fibroblast growth factor, 25 ng / mL of recombinant human hepatocyte growth factor, 0.1 μM of ALK5 inhibitor, 0.1 ng / mL of recombinant human Noggin protein, 0.1 μM of PKC inhibitor, 10 μM of ATPase inhibitor, and 1 μM of Rock inhibitor;

[0059] The second culture medium includes the following components: 1 mL of liquid basal culture medium DMEM / F12, 1 mL of B27 additive, 1 mL of N2 additive, 1 mL of penicillin-streptomycin, 1 mM N-acetylcysteine, 25 ng / mL of R-spondin, 10 mM of niacinamide, 25 ng / mL of recombinant human hepatocyte growth factor, 10 μM of TGFβ inhibitor, 1 μM of γ-Secretase inhibitor, 10 μM of EGFR inhibitor, and 1 μM of Rock inhibitor;

[0060] The third culture medium includes the following components: 1 mL of liquid basal culture medium DMEM / F12, 1 mL of B27 additive, 1 mL of N2 additive, 1 mL of penicillin-streptomycin, 1 mM N-acetylcysteine, 25 ng / mL of R-spondin, 10 mM of niacinamide, 25 ng / mL of recombinant human hepatocyte growth factor, 25 ng / mL of recombinant oncostatin M, 1 μM of γ-secretase inhibitor, 10 μM of ALK5 inhibitor, and 2 μM of dexamethasone;

[0061] S1: Obtain human gallbladder tissue under sterile conditions and transfer it to the GMP workshop for processing. First, cut the tissue and wash it several times with sterile PBS until the solution is clear. Then cut the tissue into small pieces, add Accutase digestion solution and incubate at 37°C for half an hour. The digestion solution containing cells is filtered through a 70μm filter mesh, and the cell precipitate is collected by centrifugation. Prepare a 1:1 matrix gel / type 1 collagen mixture with an overall concentration of 2%, evenly spread it in the cell culture bottle, cover the bottom surface, and aspirate and discard it before use. Maintain the growth of gallbladder-derived stem cells under the first culture medium conditions for 15 days to obtain gallbladder-derived stem cells;

[0062] S2: After the gallbladder-derived stem cells were prepared by the above steps, they were cultured at a rate of 1.4×10 6 The cells were inoculated into single-well density of 1×10 cells in a six-well plate for expansion. 5 The cells were seeded into culture flasks at a cell density of 100 mL per ml for directed differentiation. After 16 days under the second culture medium conditions, hepatocyte precursor cells were obtained;

[0063] S3: Culture continuously for 4 days under the third culture medium conditions to obtain hepatocytes.

[0064] In the above differentiation process, the medium was changed every two days.

[0065] like Figure 1 As shown, the growth morphology of gallbladder-derived stem cells was photographed under a microscope from day 1 to day 15.

[0066] Comparative Example

[0067] The following comparative examples, Kubota culture medium was prepared according to J Hepatol, 2014, 60(6): 1194-1202;

[0068] S1: Gallbladder tissue was processed in the same manner as in Example S1. Cells were grown in Kubota medium for 10 days.

[0069] S2: The gallbladder epithelial cells obtained by the above steps cannot proliferate in KM culture medium, and as the culture time increases, the epithelial cells gradually die, and the fibroblasts show a growth advantage, and the expanded culture operation cannot be performed.

[0070] test:

[0071] 1. Cell growth curve drawing

[0072] The cells were routinely digested and counted. 5 Cells were inoculated into 24-well plates at a density of 10 cells / ml. During the continuous culture days, the cells were digested and counted at a fixed time every day. The cell growth curve was drawn with time as the horizontal axis and the number of cells as the vertical axis.

[0073] like Figure 2 As shown in the figure, after the cells are inoculated, the cells are counted in consecutive growth days and a growth curve is drawn. From left to right, the third, sixth, and ninth generations are cultured (marked as P3, P6, and P9 in the figure). The results show that the cells present a typical S-shaped growth curve in the early generations (P3, P6). Starting from the ninth generation, the cell proliferation ability decreases. The horizontal axis represents the number of cell growth days (in days), and the vertical axis represents the number of cells (in ×10 4 cells).

[0074] 2. Cell Immunofluorescence Staining

[0075] After the cells grow to a suitable density, discard the culture supernatant, wash three times with PBST and discard the liquid, add 4% PFA solution and incubate at room temperature for one hour, wash with 0.1% Trition X-100 for five minutes, wash with PBST and block with calf serum blocking solution at room temperature for one hour. Add various primary antibodies and incubate overnight for four times, wash and incubate with corresponding secondary antibodies at 37℃ for half an hour; wash and counterstain with DAPI. Take pictures and analyze with a fluorescence microscope.

[0076] like Figure 3 As shown, immunofluorescence staining shows that the cells express EPCAM, CK19 (red) and TROP2, SOX9 (green), and the cell nucleus is stained with DAPI (blue). The picture was taken at a magnification of 200 times under a microscope;

[0077] like Figure 5As shown, immunofluorescence staining shows that the cells express AAT, CYP3A4, and CYP2E1 (green), and the cell nucleus is stained with DAPI (blue). The picture was taken at a magnification of 200 times under a microscope.

[0078] 3. RNA Extraction and Real-time Quantitative PCR

[0079] After washing the cells, TRIzol RNA isolation solution (Thermo, 15596018) was added to extract RNA according to the instructions. After obtaining RNA, cDNA synthesis was completed using a reverse transcription kit (Thermo, 12183555). After the reaction solution was prepared using SYBRGreen real-time PCR premix (1176202K), the real-time quantitative PCR operation was completed on a Roche Light Cycler 480 instrument and the data were analyzed.

[0080] like Figure 4 As shown, quantitative PCR detection showed that after the cells were induced to differentiate, the expression levels of mature hepatocyte markers AAT, FAH, ALB, CYP1A2, CYP3A4, and G6P gradually increased. hGBSCs: human gallbladder-derived stem cells grown under the first culture condition; Phase 1: human gallbladder-derived stem cells cultured under the second culture condition for 16 days; Phase 2: human gallbladder-derived stem cells cultured under the second culture condition for 4 days; Liver: human liver tissue.

[0081] 4. Liver function tests (glycogen staining, LDL uptake, BIODIPY, rhodamine uptake)

[0082] LDL (Thermo, L34355), BODIPY (Thermo, D3922), and rhodamine (MERCK, 62669709) were all used for live cell staining. They were added to the cell culture medium according to the concentration in the reagent instructions. After incubation for the corresponding time, they were observed under an inverted microscope and photographed for analysis. Glycogen PAS staining was performed according to the instructions of the kit (Solaribio, G1360). After staining, the staining was completed and the cells were examined under a microscope and photographed.

[0083] like Figure 6 As shown, A: Glycogen staining shows that most cells are positive (purple-red colored part) after being treated with differentiation conditions, indicating that cells can store glycogen. The picture was taken with a microscope at 100 times magnification.

[0084] B: Live cell staining with the fluorescent dye Bodipy 493 / 503, taken under a fluorescent microscope, shows that differentiated cells have the ability to synthesize fat (green fluorescent part). The cell nucleus is stained with DAPI (blue). The picture was taken under a microscope with a magnification of 200 times.

[0085] C: Fluorescence microscopy after rhodamine uptake shows that differentiated cells have the ability to take up the dye (the cell cluster takes up the green dye and gathers in the middle). After adding the cell transport receptor inhibitor Verapamil to the system, the cell cluster cannot take up the dye (the middle of the cell cluster is dark). The picture was taken with a microscope at 200 times magnification.

[0086] 5. Experiment on treatment of acute liver failure in rats

[0087] Rats weighing 220-270 g were selected and intraperitoneally injected with 1.2 g / kg body weight of acetaminophen (MACKLIN, A800441) solution to prepare an acute liver failure rat model. 7 The cells were injected into the animals through the portal vein at a dose of 10 cells / rat. The survival of the animals was observed, and serum and liver samples were collected at different times for analysis.

[0088] like Figure 7 As shown, the embodiment is the treatment group, and the comparative example is the control group;

[0089] A: Photograph of the appearance of mouse liver. Control reagent (culture medium) and target cells were transplanted into mice with acute liver failure. The left side is the control group, where a large area of ​​necrosis (black-red part) appeared on the surface of the liver, and the liver as a whole showed an ischemic state (khaki liver phenotype). The right side is the treatment group (transplanted with differentiated cells), where the liver has a relatively normal appearance (ruddy and shiny), with less necrotic part on the surface.

[0090] B: Statistics of liver injury area. Mouse livers were collected, tissue sections were prepared, and the injury area was calculated using software after H&E staining. The statistical method was to randomly collect at least ten liver tissues located in different positions, select ten random fields of view for each tissue after section staining, and calculate the injury area and total area. The results showed that the tissue injury area of ​​mice transplanted with cells (treatment group) was significantly smaller than that of the control group. * indicates p is less than 0.05.

[0091] C: Serum liver function index detection. Peripheral blood of mice was collected 24 hours after transplantation, and serum was collected by centrifugation and tested by biochemical analyzer for liver function related indicators ALT (alanine aminotransferase) and AST (aspartate aminotransferase). The results showed that the treatment group was significantly lower than the control group. * indicates p less than 0.05.

[0092] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for inducing gallbladder-derived stem cells to differentiate into functional hepatocytes, characterized in that: The following steps are involved: S1: pre-treating the gallbladder tissue and adding it to the digestion solution for incubation, filtering, and centrifuging to collect the cell precipitate; culturing the cell precipitate in the first culture system to obtain gallbladder-derived stem cells; S2: amplifying, digesting, and directing differentiation of gallbladder-derived stem cells to obtain differentiated cells; culturing the differentiated cells in a second culture system to obtain hepatocyte precursor cells; S3: culturing hepatocyte precursor cells in the third culture system to obtain functional hepatocytes; In step S1, the first culture system is a first culture medium; the first culture medium includes the following components: liquid basal culture medium, B27 additive, N2 additive, penicillin-streptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng / mL R-spondin, 1-1000mM niacinamide, 0.1-100ng / mL recombinant human epidermal growth factor, 0.1-100ng / mL recombinant human fibroblast growth factor, 0.1-100ng / mL recombinant human hepatocyte growth factor, 0.1-100μM ALK5 inhibitor, 0.1-100ng / mL recombinant human Noggin protein, 0.1-100μM PKC inhibitor, 0.1-100μM ATPase inhibitor, 0.1-100μM Rock inhibitor; Among them, the volume ratio of liquid basal medium: B27 additive: N2 additive: penicillin-streptomycin is 1:1:1:1; In step S2, the second culture system is a second culture medium; the second culture medium comprises the following components: liquid basal culture medium, B27 additive, N2 additive, penicillin-streptomycin, 0.1-50 mM N-acetylcysteine, 0.1-100 ng / mL R-spondin, 1-1000 mM nicotinamide, 0.1-100 ng / mL recombinant human hepatocyte growth factor, 0.1-100 μM TGFβ inhibitor, 0.1-100 μM γ-Secretase inhibitor, 0.1-100 μM EGFR inhibitor, 0.1-100 μM Rock inhibitor; Among them, the volume ratio of liquid basal medium: B27 additive: N2 additive: penicillin-streptomycin is 1:1:1:1; In step S3, the third culture system is a third culture medium; the third culture medium includes the following components: liquid basal culture medium, B27 additive, N2 additive, penicillin-streptomycin, 0.1-50 mM N-acetylcysteine, 0.1-100 ng / mL R-spondin, 1-1000 mM niacinamide, 0.1-100 ng / mL recombinant human hepatocyte growth factor, 0.1-100 ng / mL recombinant oncostatin M, 0.1-100 μM γ-Secretase inhibitor, 0.1-100 μM ALK5 inhibitor, 0.1-100 μM dexamethasone; Among them, the volume ratio of liquid basal medium: B27 additive: N2 additive: penicillin-streptomycin is 1:1:1:1; In step S1, the culture time is 2-14 days under the first culture system; In step S2, under the second culture system, the culture time in the culture system is 10-30 days; In step S3, under the third culture system, the culture time in the culture system is 1-10 days.

2. The method of inducing gallbladder-derived stem cells to differentiate into functional hepatocytes according to claim 1, characterized in that: In steps S1-S3, a differentiation-promoting substance can be added; the differentiation-promoting substance is any one of matrix gel and type 1 collagen or a combination of the two; wherein the volume ratio during the combination is 1:1.

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