CN115558632A - Method for inducing gallbladder-derived stem cells to differentiate into functional liver parenchymal cells and application - Google Patents

Method for inducing gallbladder-derived stem cells to differentiate into functional liver parenchymal cells and application Download PDF

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CN115558632A
CN115558632A CN202211258248.9A CN202211258248A CN115558632A CN 115558632 A CN115558632 A CN 115558632A CN 202211258248 A CN202211258248 A CN 202211258248A CN 115558632 A CN115558632 A CN 115558632A
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gallbladder
cells
derived stem
stem cells
additive
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陈费
王敏君
金宜强
徐守佳
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Shanghai Beixian Biotechnology Co ltd
Second Military Medical University SMMU
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Shanghai Beixian Biotechnology Co ltd
Second Military Medical University SMMU
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Abstract

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

Description

Method for inducing gallbladder-derived stem cells to differentiate into functional liver parenchymal cells and application
Technical Field
The invention relates to the field of functional liver parenchymal cells, in particular to a method for inducing a gallbladder-derived stem cell to be differentiated into the functional liver parenchymal cell and application thereof.
Background
China is a country with high incidence of liver diseases, acute and chronic liver diseases caused by viruses, medicines, foods, heredity and the like can cause the liver to have functional failure, wherein chronic injuries are more important causes of hepatitis, cirrhosis and liver cancer, and timely intervention and treatment can effectively avoid death of patients (Changing global epidemiology of liver cancer from 2010to 2019 NASH is the fast growing cause of liver cancer PMID. The cell replacement therapy provides a candidate scheme for treating liver diseases, and the transplantation of functional liver cells is expected to solve some problems of clinical treatment of liver diseases, such as liver organ donor shortage, drug-induced liver dysfunction, side effect injury and the like. More importantly, research reports that the hepatocyte transplantation has obvious treatment effect on rare diseases such as hepatolenticular degeneration, tyrosinemia type 1 and other chromosome genetic metabolic diseases, and can help patients to spend dangerous periods for further treatment.
Obtaining functional hepatocytes may provide a source of cells for cell therapy of liver diseases. The current common methods include: liver in-situ separation, induction of embryonic stem cell directed differentiation, somatic cell reprogramming and induction of differentiation of other source cells, and the like. Different ways of obtaining liver-like cells are different in different seasons: 1) The method for obtaining functional liver cells in vitro by digesting, separating and enriching liver tissues has the advantages that the liver cells have complete functions, and generally have obvious treatment effect after being transplanted; however, it is difficult to use it widely because its source is limited and it cannot be amplified in a large amount. 2) The embryonic stem cells have sufficient sources, can be directionally induced and differentiated to obtain hepatocyte-like cells, and have partial hepatocyte functions; the disadvantages are the limited differentiation efficiency, the tumorigenicity of the residual stem cells and the constant ethical debate. 3) The reprogrammed hepatocyte-like cells have wide sources, are the hot spots of the current research, and the improvement of the induction efficiency and the enhancement of the function are the technical bottlenecks to be overcome. 3) Other cells such as mesenchymal stem cells, hematopoietic stem cells, etc. have also been reported to have the ability to differentiate into liver-like cells.
Gallbladder tissues are important components of a liver bile duct system, the gallbladder and the liver are homologously developed, the tissue sources are wide, theoretically, if the gallbladder-derived cells are differentiated into hepatocyte-like cells, the gallbladder-derived cells have certain feasibility, the cell source problem can be well solved, and more importantly, adult tissue cells generally do not have tumorigenic characteristics. Precursor research shows that the gallbladder can separate dry cells with certain differentiation capacity. Therefore, a gallbladder-derived stem cell P production system is established, and directional induction and differentiation are performed, so that a new functional hepatocyte source can be provided, and a candidate cell source with application value and competitiveness is hopefully provided for cell therapy of clinical liver diseases.
Disclosure of Invention
The present invention aims to provide a method and an application for inducing gallbladder-derived stem cells to differentiate into functional parenchymal liver cells, so as to solve the problems in the background art.
In order to solve the technical problems, the invention provides the following technical scheme:
a method of inducing the differentiation of gallbladder-derived stem cells into functional liver parenchymal cells, comprising the steps of:
s1: pretreating gallbladder tissue, inoculating in digestion solution, filtering, centrifuging, and collecting cell precipitate; culturing cell sediment in a culture system under a first culture system to obtain gallbladder-derived stem cells;
s2: amplifying, digesting and directionally differentiating the 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;
s3: and culturing the hepatocyte precursor cells in a culture system under a third culture system to obtain functional liver parenchymal cells.
Further, in the step S1, the culture system is a first culture medium; the first medium comprises the following components: liquid basal medium, 1 XB 27 additive, 1 XN 2 additive, 1 Xqing streptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng/mL R-spondin, 1-1000mM nicotinamide, 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, 0.1-100 μ M ALK5 inhibitor, 0.1-100ng/mL recombinant human Noggin protein, 0.1-100 μ M PKC inhibitor, 0.1-100 μ M MATPase inhibitor, 0.1-100 μ M Rock inhibitor;
wherein the liquid basal medium: b27 additive: n2 additive: the volume ratio of the streptomycin is 1:1:1:1.
further, in the step S2, the culture system is a second culture medium; the second culture medium comprises the following components: liquid basal medium, 1 XB 27 additive, 1 XN 2 additive, 1 Xpenicillin, 0.1-50mM N-acetylcysteine, 0.1-100ng/mL R-spondin, 1-1000mM nicotinamide, 0.1-100ng/mL recombinant human hepatocyte growth factor, 0.1-100 μ M TGF beta inhibitor, 0.1-100 μ M gamma-Secretase inhibitor, 0.1-100 μ M EGFR inhibitor, 0.1-100 μ M Rock inhibitor;
wherein the liquid basal medium: b27 additive: n2 additive: the volume ratio of the streptomycin is 1:1:1:1.
further, in the step S3, the culture system is a third culture medium; the third medium comprises the following components: liquid basal medium, 1 XB 27 additive, 1 XN 2 additive, 1 Xstreptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng/mL R-spondin, 1-1000mM nicotinamide, 0.1-100ng/mL recombinant human hepatocyte growth factor, 0.1-100ng/mL recombinant oncostatin M, 0.1-100 μ M gamma-Secretase inhibitor, 0.1-100 μ M MALK5 inhibitor, 0.1-100 μ M dexamethasone;
wherein the liquid basal medium: b27 additive: n2 additive: the volume ratio of the streptomycin is 1:1:1:1.
further, the gallbladder-derived stem cell is any one of gallbladder tissue removed in cholecystitis and gallbladder tissue discarded in liver transplantation operation.
Further, in the step S1, the gallbladder-derived stem cells are cultured for 1 to 15 days under the first culture strip system; preferably, the culture time is 10-12 days;
in the step S2, the gallbladder-derived stem cells are cultured for 10to 30 days in a second culture system; preferably, the culture time is 15-20 days;
in the step S3, under a third culture system, the culture time for culturing the hepatocyte precursor cells is 1 to 10 days; preferably, the cultivation time is 2 to 4 days.
Further, the method has one or more of the following features:
high differentiation efficiency, wherein the differentiation rate is 75-90%;
during the culture process, every 1ml of culture solution is inoculated with 1x10 5 The individual gallbladder-derived stem cell can produce 0.5-0.8x10 6 And (4) hepatic parenchymal cells.
Further, the methods include therapeutic and non-therapeutic.
Further, the differentiation promoting substances are matrigel, type 1 collagen and a mixture of matrigel and type 1 collagen in a volume ratio of 1:1, any one of the mixtures.
Further, the inducing composition comprises a first inducing factor, a second inducing factor and a differentiation promoting substance; wherein.
Further, the first inducing factor composition comprises a TGF beta inhibitor, a gamma-Secretase inhibitor and an EGFR inhibitor; the second inducing factor comprises recombinant oncostatin M, gamma-Secretase inhibitor, ALK5 inhibitor and 0.1-100 mu M dexamethasone; the differentiation promoting substances are matrigel, type 1 collagen and matrigel and type 1 collagen 1:1, any one of the mixtures.
Further, the inducing composition is used for inducing the gall bladder-derived stem cells to differentiate into liver parenchymal cells.
A functional liver parenchymal cell comprising the following components: mature hepatocyte markers albumin, HNF4A, CYP3A4, AAT.
Further, the mature hepatocyte marker albumin is any one of EPCAM, CK19, TROP2 and SOX 9.
Further, the functional liver parenchymal cells are flat paving stone-like, have a diameter of 15-20 μm, and have a function of storing glycogen and synthesizing fat.
Further, the parenchymal hepatic cells have one or more of the following characteristics:
5-90% of the cells are positive for HNF4A expression;
40-70% are AAT positive;
30-60% are CYP positive.
An application of inducing human gallbladder-derived stem cells to differentiate into functional liver parenchymal cells is used for preparing a composition for treating liver-related diseases.
Further, the liver-related disease is any one or more of acute hepatitis, liver failure, liver cirrhosis, hepatolenticular degeneration and tyrosinemia.
Further, the composition is any one of a pharmaceutical composition, a food composition and a health product composition.
Further, the composition is in the form of any one of injection, freeze-dried preparation and solution preparation.
Further, an induction medium is provided, the induction medium containing a basal medium nuclear supplement; wherein the basal medium is selected from the group consisting of: DMEM, F12 combination.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes human gallbladder stem cells as initial cells for the first time, efficiently prepares functional parenchymal hepatocytes under proper conditions in vitro, and provides a candidate cell source for treating liver diseases.
The invention firstly discovers that functional liver parenchymal cells with high differentiation rate can be obtained from 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.
The invention prepares the liver parenchymal cells derived from the gall bladder stem cells by optimizing conditions for the first time, and proves that the liver parenchymal cells have the effect of treating liver failure after being transplanted into an animal body.
The gallbladder-derived stem cells and the differentiated functional liver parenchymal cells thereof have wide application prospects: 1) Providing a candidate cell therapy regimen for a patient with liver failure; 2) (ii) a Provides potential usable functional cells for liver metabolism-related rare diseases and genetic diseases; 3) Providing seed cells for an in vitro artificial liver support device; 4) Provides a cell screening platform for screening liver drugs.
The invention provides a cell source for constructing the tissue engineering liver.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a typical growth morphology of human gallbladder-derived stem cells of the present invention;
FIG. 2 is a typical growth curve of human gallbladder-derived stem cells of the present invention;
FIG. 3 shows that human gallbladder-derived stem cells of the present invention express hepatic stem cell markers;
FIG. 4 shows that the human gallbladder-derived stem cells of the present invention have mature hepatocyte molecular phenotype after induced differentiation;
FIG. 5 shows the expression of mature hepatocyte markers after induced differentiation of human gallbladder-derived stem cells of the present invention;
FIG. 6 shows that the human gallbladder-derived stem cells of the present invention have hepatocyte functions after induced differentiation;
FIG. 7 shows that the transplanted human gallbladder-derived stem cell of the present invention has the ability to rescue mice with liver failure after induced differentiation.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the B27 additive was supplied by Gibco under the model number 12587010; n2 additive was supplied by Gibco, model number 17502048; penicillin streptomycin is supplied by Gibco, and has the model of 15140122; n-acetylcysteine supplied by Sigma, model A9165; r-spondin supplied by Peprotech, model number 120-38; nicotinamide, supplied by Sigma, model number N0636; the recombinant human epidermal growth factor is provided by Peprotech, and the model is AF10015; the recombinant human fibroblast growth factor is provided by Peprotech and has the model of AF10026; the recombinant human hepatocyte growth factor is provided by Peprotech, and the model is 10039H; ALK5 inhibitors are supplied by seleck, model S7692; the recombinant human Noggin protein is provided by Peprotech and has the model number of 12010C; PKC inhibitors are supplied by seleck, model S6577; ATPase inhibitors are supplied by seleck, model S7099; rock inhibitors are supplied by seleck, model S6390; TGF β inhibitor is supplied by seleck, model S1067; the gamma-Secretase inhibitor is provided by belleck, model number S2215; EGFR inhibitors are supplied by seleck, model S1143; the recombinant oncostatin M is provided by R & D and has the model of 295-OM; dexamethasone was supplied by belleck, type S1322; sterile PBS supplied by solarbio, model number P1020; accutase digestive juice is provided by Gibco, and the model is A1110501; matrigel was supplied by Corning, model number 354234; type 1 collagen is supplied by Corning, model number 356236; trypLE is supplied by Gibco, model number 12563011.
Examples
The first culture medium comprises the following components: 1mL of liquid basal medium DMEM/F12, 1mLB27 additive, 1mLN2 additive, 1mL of streptomycin, 1mM of N-acetylcysteine, 25ng/mL of R-spondin, 10mM of nicotinamide, 50ng/mL of recombinant human epidermal growth factor, 50ng/mL of recombinant human fibroblast growth factor, 25ng/mL of recombinant human hepatocyte growth factor, 0.1 μ M of ALK5 inhibitor, 0.1ng/mL of recombinant human Noggin protein, 0.1 μ M of PKC inhibitor, 10 μ M of ATPase inhibitor, 1 μ M of Rock inhibitor;
the second culture medium comprises the following components: 1mL of liquid basal medium DMEM/F12, 1mLB27 additive, 1mLN2 additive, 1mL of streptomycin, 1mM of N-acetylcysteine, 25ng/mL of R-spondin, 10mM of nicotinamide, 25ng/mL of recombinant human hepatocyte growth factor, 10 mu M of TGF beta inhibitor, 1 mu M of gamma-Secretase inhibitor, 10 mu M of EGFR inhibitor and 1 mu M of Rock inhibitor;
the third culture medium comprises the following components: 1mL of liquid basal medium DMEM/F12, 1mLB27 additive, 1mLN2 additive, 1mL of streptomycin, 1mM of N-acetylcysteine, 25ng/mL of R-spondin, 10mM of nicotinamide, 25ng/mL of recombinant human hepatocyte growth factor, 25ng/mL of recombinant oncostatin M, 1 μ M of gamma-Secretase inhibitor, 10 μ M of ALK5 inhibitor and 2 μ M of dexamethasone;
s1: human gallbladder tissues are obtained under aseptic conditions and transferred to a GMP workshop for processing. The tissue was first cut open and washed several times with sterile PBS until the solution was clear. The tissue was then minced into small pieces and incubated with Accutase digest for half an hour at 37 ℃. The cell-containing digest was filtered through a 70 μm filter screen and centrifuged to collect cell pellets. Preparing 1:1 matrigel/type 1 collagen mixture, evenly spread on a cell culture flask, cover the bottom surface, suck off and discard before use. Maintaining the growth of the gallbladder-derived stem cells for 15 days under the condition of a first culture medium to obtain the gallbladder-derived stem cells;
s2: after the gallbladder-derived stem cells are prepared and obtained by the steps, the gallbladder-derived stem cells are prepared according to the proportion of 1.4 multiplied by 10 6 Single well density of individual cells was seeded in six well plates for expansion. The cells were TrypLE-digested into single cells at 1X10 5 The cells are inoculated in culture bottles at the cell density of each milliliter for directional differentiation. Obtaining hepatocyte precursor cells after 16 days under the condition of a second culture medium;
s3: culturing for 4 days under the third culture medium condition to obtain liver parenchymal cells.
Wherein, the liquid is changed every two days in the differentiation process.
As shown in FIG. 1, the growth morphology of gallbladder-derived stem cells was microscopically observed for 1 to 15 days.
Comparative example
Comparative examples, preparation of Kubota Medium reference J Hepatol,2014,60 (6): 1194-1202;
s1: gallbladder tissue was treated in the same manner as in example S1. Cells were maintained under Kubota medium conditions for 10 days of growth.
S2: the gallbladder epithelial cells obtained by the steps can not proliferate in a KM culture medium, and with the prolonging of the culture time, the epithelial cells die gradually, and fibroblasts show growth advantages and can not be subjected to expanded culture operation.
And (3) testing:
1. cell growth curve mapping
Cells were counted after routine digestion. According to 1x10 5 Cells were seeded at a density of one cell/ml in 24-well plates, and cells were digested and counted at fixed times daily for a continuous culture period. Cell growth curves were plotted with time as the horizontal axis and cell number as the vertical axis.
As shown in FIG. 2, after the inoculation of cells, the cells were counted for each of the consecutive days of growth, and a growth curve was plotted. From left to right, the third, sixth and ninth generations (marked as P3, P6 and P9 in the figure) were cultured. The results showed that the cells exhibited a typical sigmoidal growth curve in the early passages (P3, P6), and the cell proliferation capacity decreased at the beginning of the ninth passage. The abscissa represents the number of days in which the cells were grown (in days), and the ordinate represents the number of cells (in. Times.10) 4 Individual cells).
2. Cellular immunofluorescence staining
After the cells were grown to a suitable density, the culture supernatant was discarded, the liquid was discarded after PBST washing three times, 4% PFA solution was added and incubated at room temperature for one hour, after washing, treated with 0.1% Trition X-100 for five minutes, and after PBST washing, blocked at room temperature for one hour using a calf serum blocking solution. Adding various primary antibodies overnight at four degrees, cleaning, and incubating corresponding secondary antibodies respectively at 37 ℃ for half an hour; after washing, DAPI counterstaining is carried out. The fluorescence microscope was photographed and analyzed.
As shown in fig. 3, immunofluorescence staining showed cells expressing EPCAM, CK19 (red) and TROP2, SOX9 (green) and cell nuclei were DAPI stained (blue). The figure is a microscope image shot at 200 times magnification;
as shown in fig. 5, immunofluorescence staining showed that cells expressed AAT, CYP3A4, CYP2E1 (green) and nuclei were DAPI stained (blue). The figure is a microscope image taken at 200 times magnification.
RNA extraction and real-time quantitative PCR
After washing the cells, TRIzol RNA isolation solution (Thermo, 15596018) was added to the cells, and RNA extraction was performed according to the instruction. After obtaining RNA, cDNA synthesis was completed using a reverse transcription kit (Thermo, 12183555). After reaction solution was prepared using SYBR Green real-time PCR premix (1176202K), real-time quantitative PCR was performed and data was analyzed in a Roche Light Cycler 480 instrument.
As shown in FIG. 4, quantitative PCR detection showed that the expression levels of the mature hepatocyte markers AAT, FAH, ALB, CYP1A2, CYP3A4 and G6P were gradually increased after the cells were induced to differentiate. hGBSCs: growing the human gallbladder-derived stem cells under a first culture condition; phase 1: culturing the human gallbladder-derived stem cells under a second culture condition for 16 days; phase 2: culturing the human gallbladder-derived stem cells under a second culture condition for 4 days; liver: human liver tissue.
4. Liver function test (glycogen staining, LDL uptake, BIODIPY, rhodamine uptake)
LDL (Thermo, L34355), BODIPY (Thermo, D3922) and rhodamine (MERCK, 62669709) intakes were stained for viable cells, added to the cell culture medium at reagent instruction concentrations, incubated for the corresponding time, observed under an inverted microscope and analyzed by photography. Glycogen PAS staining was performed according to the kit (solarbio, G1360) instructions, and after staining, microscopic examination and photography were performed
As shown in fig. 6, a: glycogen staining showed that most cells were positive after differentiation conditions (purple-red colored fraction), indicating that the cells could store glycogen. The figure is a 100 times magnified field of view image of a microscope.
B: the living cells were stained with the fluorescent dye Bodipy 493/503, and the cells were shown to have the ability to synthesize fat after differentiation as photographed by a fluorescence microscope (green fluorescent moiety). Nuclei were stained with DAPI (blue). The figure is a microscope image taken at 200 times magnification.
C: fluorescence microscopy images after rhodamine uptake show that the differentiated cells have uptake capacity (the cell mass takes in and aggregates the green dye in the middle). After adding Verapamul, a cell transfer receptor inhibitor, the cell mass can not take in the dye (the middle of the cell mass is in a dark field). The figure is a microscope image taken at 200 times magnification.
5. Acute hepatic failure rat rescue experiment
Selecting rats with body weight of 220-270g, and performing intraperitoneal injection of 1.2g/kg body weight acetaminophen (MACKLIN, A800441) solution to prepare acute liverFailure rat model. According to 1x10 7 Cell/rat dose, cells were injected into animals via portal vein. And (4) observing the survival condition of the animal, and collecting serum samples and liver samples according to time for corresponding analysis.
As shown in fig. 7, the example was the treatment group, and the comparative example was the control group;
a: mouse liver appearance photographs. A control reagent (culture medium) and a target cell are transplanted into an acute liver failure mouse respectively. In the left control group, the liver surface showed large areas of necrosis (dark red portions) and the liver as a whole showed an ischemic state (yellowish brown liver phenotype). The right panel is the treatment group (transplanted differentiated cells), and the liver appeared more normal (ruddy and shiny) with fewer surface necrotic areas.
B: and (5) counting the damaged area of the liver. Tissue sections were prepared by collecting mouse livers, stained with H & E and the area of injury calculated with software. The statistical method is that at least ten liver tissues at different positions are randomly collected, ten random visual fields are selected for each tissue after section staining, and the damage area and the total area are calculated. The results showed that the tissue damage area of the cell-transplanted mice (treated group) was significantly smaller than that of the control group. * Indicating that p is less than 0.05.
C: and (5) serum liver function index detection. Peripheral blood of the mouse after 24 hours of transplantation is respectively collected, serum is centrifugally collected, and liver function related indexes ALT (alanine aminotransferase) and AST (aspartate aminotransferase) are detected by a biochemical analyzer. The results showed that the treated group was significantly lower than the control group. * Indicating that p is less than 0.05.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method of inducing the differentiation of gallbladder-derived stem cells into functional liver parenchymal cells, comprising: the method comprises the following steps:
s1: pretreating gallbladder tissues, adding the pretreated gallbladder tissues into a digestion solution for inoculation, filtering, and centrifugally collecting cell precipitates; culturing the cell sediment in a first culture system to obtain the gallbladder-derived stem cells;
s2: amplifying, digesting and directionally differentiating the gallbladder-derived stem cells to obtain differentiated cells; culturing the differentiated cells in a second culture system to obtain hepatocyte precursor cells;
s3: and culturing the hepatocyte precursor cells in a third culture system to obtain functional liver parenchymal cells.
2. The method of inducing differentiation of gallbladder-derived stem cells into functional hepatocytes as claimed in claim 1, wherein: in step S1, the first culture system is a first culture medium; the first medium comprises the following components: liquid basal medium, B27 additive, N2 additive, streptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng/mL R-spondin, 1-1000mM nicotinamide, 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, 0.1-100 muM ALK5 inhibitor, 0.1-100ng/mL recombinant human Noggin protein, 0.1-100 muM PKC inhibitor, 0.1-100 muM ATPase inhibitor, 0.1-100 muM Rock inhibitor;
wherein the liquid basal medium: b27 additive: n2 additive: the volume ratio of the streptomycin is 1:1:1:1.
3. the method of inducing the differentiation of gallbladder-derived stem cells into functional liver parenchymal cells according to claim 1, wherein: in step S2, the second culture system is a second culture medium; the second culture medium comprises the following components: liquid basal medium, B27 additive, N2 additive, streptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng/mL R-spondin, 1-1000mM nicotinamide, 0.1-100ng/mL recombinant human hepatocyte growth factor, 0.1-100 MuM TGF beta inhibitor, 0.1-100 MuM gamma-Secretase inhibitor, 0.1-100 MuM EGFR inhibitor and 0.1-100 MuM Rock inhibitor;
wherein the liquid basal medium: b27 additive: n2 additive: the volume ratio of the streptomycin is 1:1:1:1.
4. the method of inducing the differentiation of gallbladder-derived stem cells into functional liver parenchymal cells according to claim 1, wherein: in step S3, the third culture system is a third culture medium; the third medium comprises the following components: liquid basal medium, B27 additive, N2 additive, streptomycin, 0.1-50mM N-acetylcysteine, 0.1-100ng/mL R-spondin, 1-1000mM nicotinamide, 0.1-100ng/mL recombinant human hepatocyte growth factor, 0.1-100ng/mL recombinant oncostatin M, 0.1-100 μ M gamma-Secretase inhibitor, 0.1-100 μ M ALK5 inhibitor, 0.1-100 μ M dexamethasone;
wherein the liquid basal medium: b27 additive: n2 additive: the volume ratio of the streptomycin is 1:1:1:1.
5. the method of inducing the differentiation of gallbladder-derived stem cells into functional liver parenchymal cells according to claim 1, wherein: step S1-S3 can be added with differentiation promoting substances; the differentiation promoting substance is any one of matrigel and type 1 collagen or the compound of the matrigel and the type 1 collagen; wherein, the volume ratio is 1:1.
6. the method of inducing differentiation of gallbladder-derived stem cells into functional hepatocytes as claimed in claim 1, wherein: in step S1, the culture time is 2-14 days under the first culture system.
7. The method of inducing the differentiation of gallbladder-derived stem cells into functional liver parenchymal cells according to claim 1, wherein: in step S2, the culture time in the culture system is 10-30 days under the second culture system.
8. The method of inducing differentiation of gallbladder-derived stem cells into functional hepatocytes as claimed in claim 1, wherein: in step S3, under the third culture system, the culture time in the culture system is 1-10d.
9. The functional liver parenchymal cells prepared by the method for inducing the gallbladder-derived stem cells to differentiate into the functional liver parenchymal cells according to any one of claims 1 to 8.
10. The application of inducing the gallbladder-derived stem cells to be differentiated into functional liver parenchymal cells is characterized in that: the use of functional liver parenchymal cells according to claim 9 for the preparation of a composition for the treatment of a liver-related disease.
CN202211258248.9A 2022-10-14 2022-10-14 Method for inducing gallbladder-derived stem cells to differentiate into functional liver parenchymal cells and application Pending CN115558632A (en)

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