CN109749981B - Hepatocyte-like cells derived from human adipose-derived stem cells, and preparation method and application thereof - Google Patents
Hepatocyte-like cells derived from human adipose-derived stem cells, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a method for preparing hepatocyte-like cells derived from human adipose-derived stem cells and a hepatocyte-like cell population obtained by the method. The method comprises the use of specific combinations of cytokines in specific induced differentiation steps, thereby enabling highly mature hepatocyte-like cells to be derived from human adipose stem cells for the treatment of acute liver failure.
Description
Technical Field
The invention relates to the field of stem cells and biological medicines. In particular, the invention relates to hepatocyte-like cells (Hepatocyte like cell, HLC) derived from Human adipose stem cells (Human-Adipose Derived Stem Cell, hADSC), a preparation method thereof and application thereof in treating diseases such as acute liver failure.
Background
Liver failure (Liver failure) is a serious threat to human health. In situ liver transplantation is the best treatment means at present, but the treatment cost is high and limited by liver sources; stem cells have strong self-renewal capacity and multi-directional differentiation potential across germ layers, and have become hot spots for treating liver diseases such as acute liver failure.
Human mesenchymal stem cells (Mesenchymal stem cell, hMSC) have been demonstrated to be induced to differentiate into hepatocyte-like cells (Hepatocyte like cell, HLC) by the addition of a combination of several cytokines in a culture system, and hMSC are widely available and promising for the treatment of severe liver diseases such as acute liver failure (Acuteliver failure, ALF). However, the hepatic differentiation ability of stem cells derived from different tissues is not consistent, and it is not known which stem cells derived from tissues are more likely to differentiate into hepatocytes, and the treatment of liver failure is more favorable after differentiation. In addition, it is not known how stem cells derived from a specific tissue are liver-differentiated into hepatocyte-like cells having a high degree of maturation that can be used for the treatment of acute liver failure.
Therefore, there is an urgent need in the art for a method capable of obtaining hepatocyte-like cells having a high degree of maturation from hepatic differentiation of stem cells derived from a specific tissue, which can be used for the treatment of acute liver failure, and such hepatocyte-like cells.
Disclosure of Invention
The invention aims to provide a hepatocyte-like cell which has the advantages of high maturity and the like, thereby being capable of being used for treating acute liver failure.
It is a further object of the present invention to provide a method for hepatic differentiation of stem cells from specific tissue sources to obtain hepatocyte-like cells having a high degree of maturation that can be used in the treatment of acute liver failure.
In a first aspect, the present invention provides a method of preparing highly mature human adipose stem cell-derived hepatocyte-like cells, said method comprising the steps of:
1) Culturing human adipose-derived stem cells by using a culture medium containing Activin A;
2) Inducing the cultured human adipose-derived stem cells obtained in the step (1) to differentiate into hepatocyte-like cells by using a hepatic differentiation medium; and
3) Maturing the hepatocyte-like cells obtained in step (2) by using a liver maturation medium, thereby obtaining highly matured hepatocyte-like cells derived from human adipose-derived stem cells;
wherein the liver differentiation medium comprises one or more of the following components:
HGF, EGF, IGF, FGF4 insulin transferrin sodium selenite additive, dexamethasone, fetal bovine serum, penicillin-streptomycin solution, optionally DMEM-F12 medium;
the liver maturation medium comprises one or more of the following:
HGF, EGF, IGF, FGF4, ITS, dexamethasone, niacinamide, oncoinhibin M (OSM), BSA, penicillin-streptomycin solution, optionally DMEM-F12 medium.
In a specific embodiment, said step 1) is carried out for 18-30 hours, preferably 22-26 hours, most preferably 24 hours; said step 2) is carried out for 5-9 days, preferably 6-8 days, most preferably 7 days; said step 3) is carried out for 5-9 days, preferably 6-8 days, most preferably 7 days.
In a preferred embodiment, the human adipose stem cells are pre-treated with a DNA methylase inhibitor prior to step 1).
In a preferred embodiment, the DNA methylase inhibitor includes, but is not limited to, 5-azacytidine.
In a preferred embodiment, the DNA methylase inhibitor is pre-treated at a dose of 10ug/L for a period of 24 hours.
In particular embodiments, the resulting highly mature human adipose stem cell-derived hepatocyte-like cells possess one or more of the following characteristics:
1) More than 40%, preferably more than 50%, most preferably more than 60% of said hepatocyte-like cells express ALB, AFP, HNF alpha, HNF4 alpha, CYP3A4mRNA; alternatively, the expression of ALB, AFP, HNF α, hnf4α, CYP3A4mRNA in the hepatocyte-like cells is increased by 30% or more, preferably by 50% or more, most preferably by 70% or more compared to before induction;
2) More than 20%, preferably more than 50%, most preferably more than 75% of said hepatocyte-like cells express Alb and AFP proteins; alternatively, the expression of Alb and AFP proteins in the hepatocyte-like cells is increased by more than 10%, preferably more than 20%, most preferably more than 30% compared to before induction;
3) More than 10%, preferably more than 30%, most preferably more than 60% of the hepatocyte-like cells are cell glycogen staining positive cells;
4) More than 5%, preferably more than 20%, most preferably more than 30% of said hepatocyte-like cells ingest ICG; and
5) The expression of urea in the hepatocyte-like cells is increased by more than 10%, preferably more than 20%, most preferably more than 30% compared to before induction.
In a preferred embodiment, the hepatocyte-like cells present more human Alb antibody positive cells around the central vein on day 1 after transplantation.
In a preferred embodiment, the hepatocyte-like cells of the invention have an increase of more than 20%, preferably more than 100%, most preferably more than 300% in anti-inflammatory factors such as IL-1ra, IL-13 etc. on day 1 after transplantation.
In a specific embodiment, about 40 or more, preferably about 50 or more, and most preferably about 50-60, highly mature human adipose stem cell-derived hepatocyte-like cells are obtained per 100 initial human adipose stem cells.
In a second aspect, the present invention provides a highly mature human adipose stem cell-derived hepatocyte-like cell population, said hepatocyte-like cell population having one or more of the following characteristics:
1) More than 40%, preferably more than 50%, most preferably more than 60% of said hepatocyte-like cells express ALB, AFP, HNF alpha, HNF4 alpha, CYP3A4mRNA; alternatively, the expression of ALB, AFP, HNF α, hnf4α, CYP3A4mRNA in the hepatocyte-like cells is increased by 30% or more, preferably by 50% or more, most preferably by 70% or more compared to before induction;
2) More than 20%, preferably more than 50%, most preferably more than 75% of said hepatocyte-like cells express Alb and AFP proteins; alternatively, the expression of Alb and AFP proteins in the hepatocyte-like cells is increased by more than 10%, preferably more than 20%, most preferably more than 30% compared to before induction;
3) More than 10%, preferably more than 30%, most preferably more than 60% of the hepatocyte-like cells are cell glycogen staining positive cells;
4) More than 5%, preferably more than 20%, most preferably more than 30% of said hepatocyte-like cells ingest ICG; and;
5) The expression of urea in the hepatocyte-like cells is increased by more than 10%, preferably more than 20%, most preferably more than 30% compared to before induction.
In a preferred embodiment, the hepatocyte-like cell population showed more human Alb antibody positive cells around the central vein on day 1 post-transplantation.
In a preferred embodiment, the hepatocyte-like cells of the invention have an increase of more than 20%, preferably more than 100%, most preferably more than 300% in anti-inflammatory factors such as IL-1ra, IL-13 etc. on day 1 after transplantation.
In a specific embodiment, the hepatocyte-like cells are prepared by the method of the first aspect.
In a third aspect, the present invention provides a pharmaceutical composition comprising a population of highly mature human adipose-derived stem cell-derived hepatocyte-like cells as described in the second aspect or a highly mature human adipose-derived stem cell-like cell prepared by the method of the first aspect, and optionally a pharmaceutically acceptable carrier.
In a fourth aspect, the present invention provides a kit comprising a population of highly mature human adipose-derived stem cell-derived hepatocyte-like cells as defined in the second aspect, or a highly mature human adipose-derived stem cell-like cell prepared by the method of the first aspect, or the pharmaceutical composition of claim 7, and optionally instructions for use of the kit.
In a fifth aspect, the present invention provides the use of a population of highly mature human adipose stem-derived hepatocyte-like cells as described in the second aspect or prepared by the method of the first aspect for the preparation of a medicament for the treatment of a disease, wherein said disease comprises but is not limited to acute liver failure, liver fibrosis, liver cirrhosis and the like.
In a specific embodiment, the disease is acute liver failure.
In a sixth aspect, the present invention provides a method of treating a disease comprising the step of treating a subject in need thereof with the population of highly mature human adipose stem-derived hepatocyte-like cells of the second aspect, or the highly mature human adipose stem-derived hepatocyte-like cells prepared by the method of the first aspect, or the kit of the fourth aspect, wherein the disease comprises but is not limited to acute liver failure, liver fibrosis, cirrhosis, etc.
In a preferred embodiment, the disease is acute liver failure.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the morphology and identification of hASC and hUCMSC;
FIG. 2 shows hepatocyte-specific mRNA detection before and after hUCMSC differentiation;
FIG. 3 shows the expression of Alb and AFP during the differentiation of hASC into HLC by cellular immunofluorescence;
FIG. 4 shows the expression of Alb and AFP during the differentiation of hUCMSC into HLC by cellular immunofluorescence;
FIG. 5 shows the results of functional identification of hASC-induced and hUCMSC-induced cells;
FIG. 6 shows liver function index detection results of hASC and hUCMSC derived HLC treated ALF rats;
FIG. 7 shows that immunohistochemical staining suggests greater migration of hASC and hASC-HLC to liver tissue;
FIG. 8 shows Ki67 staining and Tunnel test suggest that hASC, hASC-HLC has stronger regeneration promoting effect and apoptosis inhibiting effect on injured liver tissue;
FIG. 9 shows protein chip detection of inflammatory and chemokine levels in rat serum at day 1 post-implantation;
FIG. 10 shows protein chip detection of inflammatory and chemokine levels in rat serum at day 7 post-implantation.
Detailed Description
The inventors have conducted extensive and intensive studies to isolate hASC and hUCMSC in vitro, induce differentiation of these two stem cells into hepatocytes in vitro using a variety of cytokines such as Activin A (Activin A), HGF, EGF, FGF4, etc., the difference of hepatic differentiation capacity of stem cells from two tissues is discussed through fluorescence quantitative PCR, cellular immunofluorescence, glycogen PAS staining, ICG uptake experiments and the like; and the two stem cells before and after differentiation were used to treat D-galactosamine (D-gal) induced acute liver failure rats, respectively, and their effects on rat survival and liver function reconstruction were observed.
The present inventors have unexpectedly found that the use of specific combinations of cytokines in specific induced differentiation steps can result in highly mature hepatocyte-like cells from human adipose stem cells, and thus can be used in the treatment of acute liver failure. The present invention has been completed on the basis of this finding.
Human adipose-derived stem cells
Adipose stem cells (ADSCs) are a type of stem cells isolated from adipose tissue that have multipotent differentiation potential. It is a mesenchymal stem cell derived from adipose tissue, and can be differentiated into mesenchymal cells such as bone cells, cartilage cells or fat cells. ADSC cells can be stably proliferated in vitro and have low decay rate, meanwhile, the ADSC cells have the advantages of easily obtained materials, capability of obtaining a large number of stem cells by a small amount of tissues, suitability for large-scale culture, small damage to organisms and the like, and have wide sources and large in-vivo reserve quantity, are suitable for autologous transplantation and gradually become one of new research hotspots in recent years.
In recent years, multipotent human adipose-derived stem cells (hADSCs) have become a good source of cells for the treatment of serious diseases in humans, such as spinal cord injury, cerebral stroke, acute liver failure, and the like.
For purposes of illustration and convenience, embodiments of the present invention isolate human adipose-derived stem cells (hADSCS) from adipose tissue obtained by liposuction in humans. It is well known in the art that human adipose-derived stem cells (hADSC) can be obtained from a variety of sources, including but not limited to commercial sources.
Liver failure and adipose stem cell-derived hepatocyte-like cells
Liver failure is a serious liver injury caused by various factors, and causes serious dysfunction or decompensation of functions such as synthesis, detoxification, excretion, bioconversion and the like. Clinically, the patients can have a series of symptoms such as coagulation mechanism disorder, jaundice, hepatic encephalopathy, ascites and the like. The treatment of liver failure is an international problem, none of the current approaches is effective in curing, and the advent of stem cell technology provides another solution for the treatment of liver failure.
The inventor separates adult stem cells from human fat, cord blood, umbilical cord and placenta in sequence, and carries out hASC transplantation in an acute liver failure rat model through various ways such as femoral vein and spleen injection, and the like, and finds that hASC contributes to the improvement of the survival rate of the rat and the recovery of liver function [1]; however, liver cell-associated proteins such as Alb were not expressed 7 days after hASC transplantation, and it was presumed that they did not exert "cell replacement effect" in a short period of time. Through further studies, it was found that its main mechanism of action may be hASC paracrine mediated hepatocyte regeneration [2]. However, the treatment of liver failure strives for seconds, and this "cell replacement" is more important. Therefore, in vitro direct manipulation of hASC transdifferentiation into hepatocytes and use in liver failure rat therapy, the time for in vivo transdifferentiation of hASC was shortened artificially, so that it directly exerted "hepatocyte replacement effect", and it was highly likely to further improve the efficacy of hASC in treating liver failure.
Embryonic stem cells, induced pluripotent stem cells, and adult stem cells can all be induced to differentiate into HLCs in vitro. Among them, adipose stem cells and umbilical cord mesenchymal stem cells are more available and have fewer ethical problems. However, in vitro culture conditions are difficult to simulate the complex microenvironment in which in vivo cells are located, and it is difficult to obtain fully functional mature hepatocytes through the internationally existing hepatic differentiation system.
The present inventors creatively used a combination of three-stage induction (initiation, differentiation, maturation) with specific cytokines (including aFGF, bFGF, FGF-4, HGF, EGF, IGF, OSM, etc.) to induce hASC differentiation towards HLC. A series of cytokines were explored for their role in the directed differentiation of hASC into HLC, and it was found that FGF-4 and HGF were used to induce hASC differentiation into HLC during the differentiation period (but not efficiently). In addition to HGF and FGF-4, the inventors have combined EGF, ITS, OSM and other factors, and on the 3 rd day of induction, part of cells are changed in morphology, and on the 11 th day of induction, the round-like cells are further increased, and have been identified to have the functions of liver cells such as protein secretion, glycogen synthesis, urea synthesis, ICG uptake and excretion and the like. With the method of the present invention, more than 40, preferably more than 50, most preferably 50-60, highly mature human adipose stem cell-derived hepatocyte-like cells can be obtained per 100 initial human adipose stem cells.
Considering the tissue heterogeneity of stem cells, the present inventors examined the hepatic differentiation ability of stem cells of various tissue sources. The inventor simulates the development process in the liver, and simultaneously induces hASC and hUCMSC to differentiate into HLC by using a stepwise induction strategy, and detects the specific mRNA of the liver cells to find that the hASC is easier to induce and differentiate into HLC than the hUCMSC, under the same in vitro differentiation condition, the hASC expresses higher liver cell related genes and proteins such as Alb, HNF1α, HNF4α, CYP3A4 and the like, and the expression of CYP450 enzyme is currently internationally recognized HLC functional maturation standard; the detection of cellular immunofluorescence also suggests that hASC synthesizes more Alb. In subsequent animal experiments, hASC-derived HLC increased the survival rate and promoted recovery of liver function of liver-depleted rats more significantly; through cell proliferation antigen Ki67 staining and Tunnel test, we find that hASCs and hASCs-HLC have stronger regeneration promoting effect on damaged liver tissue than hUCMSC and hUCMSCs-HLC, and can obviously inhibit hepatocyte apoptosis. Notably, protein chip testing of rat serum suggests that the hASC treatment group appears to exert a more potent anti-inflammatory effect than the hASC-HLC treatment group.
Further, the present inventors found that when hASCs are pretreated with a DNA methylase inhibitor, if the dose and duration of action of 5-azacytidine are strictly controlled, the time for hASCs to differentiate toward HLCs is significantly shortened, and that 5-azacytidine might promote DNA demethylation by inhibiting DNA methylase, up-regulating the expression of hepatocyte-related transcription factors such as HNF1 and HNF4, thereby promoting hepatic differentiation of hASCs. Lateral differentiation may be another mechanism by which mesenchymal transition to epithelial (MET) occurs during hepatic differentiation of MSCs, the reverse of epithelial to mesenchymal transition (EMT).
In the research, we find that the hASC from adipose tissue has stronger hepatic differentiation capacity and better curative effect than the hUCMSC from umbilical cord huatong gum, and is more suitable for treating liver diseases such as acute liver failure. In view of the fact that a molecular regulatory network in the liver differentiation process of the hASC is not clear at present, how to search a key factor of the regulatory network, and by intervening the key factor, the differentiation efficiency of the hASC is further improved, and the functional maturation of HLC is promoted, which is a main direction of stem cell differentiation research in the future. However, at the present stage, the search for a more suitable source of stem cells may be of higher clinical value for the current situation where an effective hepatic differentiation strategy is currently lacking.
The use of the human adipose-derived stem cell-derived hepatocyte-like cell population of the present invention
It will be apparent to those skilled in the art in view of the teachings of the present invention that the hepatocyte-like cells or hepatocyte-like cell populations obtained according to the invention may be used for the treatment of acute liver failure, liver fibrosis, cirrhosis, etc.
Pharmaceutical composition and application thereof
The invention also provides a pharmaceutical composition based on the hepatocyte-like cells or the hepatocyte-like cell populations derived from the human adipose-derived stem cells, which contains an effective amount of the hepatocyte-like cells or the hepatocyte-like cell populations derived from the human adipose-derived stem cells and a pharmaceutically acceptable carrier.
Typically, the hepatocyte-like cells or population of hepatocyte-like cells derived from human adipose stem cells of the invention may be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, wherein the pH is typically about 5-8; preferably, the pH is about 7-8.
As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to a human and/or animal.
As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
The pharmaceutical compositions of the present invention comprise carriers including, but not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In general, the pharmaceutical formulations should be compatible with the mode of administration, and the pharmaceutical compositions of the present invention may be formulated as injectable formulations, for example, using physiological saline or aqueous solutions containing glucose and other adjuvants, by conventional methods. The pharmaceutical compositions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into sustained release preparation.
The effective amount of the hepatocyte-like cells or hepatocyte-like cell populations derived from human adipose stem cells of the invention may be specifically selected depending on the mode of administration and the severity of the disease to be treated. For example, it may be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). Such factors include, but are not limited to: such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc.
The pharmaceutical composition of the present invention is preferably used with subcutaneous injection of an agent and intravenous injection of an agent. In another preferred embodiment, the concentration of the hepatocyte-like cells or hepatocyte-like cell populations derived from human adipose stem cells injected subcutaneously with the agent is 10 7 Each ml, preferably 10 8 Each ml, more preferably 10 9 And each ml.
In the present invention, the administration site of hepatocyte-like cells or hepatocyte-like cell populations derived from human adipose stem cells to a subject in need thereof is an in situ injection or intravenous injection at the damaged area of the subject.
The main advantages of the invention include:
1. the invention develops a method for preparing a highly mature hepatocyte-like cell or hepatocyte-like cell population derived from human adipose-derived stem cells, thereby obtaining the hepatocyte-like cell or hepatocyte-like cell population derived from human adipose-derived stem cells with higher maturity than that obtained by the prior art;
2. The method is simple and easy to operate; and
3. the human adipose-derived stem cell-derived hepatocyte-like cells or hepatocyte-like cell populations of the present invention have a higher degree of maturation and are thus more readily useful in clinical therapies, such as acute liver failure and the like.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Examples
Materials and methods
Isolation and culture of hASC
The fat tissue of a young healthy human body after liposuction operation (supplied by a tenth people hospital of the university of the same university) is taken, washed 3 times by PBS liquid and sheared into small pieces of about 1mm3, and 0.1% |collagenase (Gibco, 17100-017) is added for digestion for 30min. Preparing a complete culture medium: 500ml DMEM-F12 medium (WISENT INC., 319-075-CL), 10% FBS (Gibco, 10099-141), 10ng/ml bFGF (Sino Biological Inc., 10014-HNAE), and an equal volume of complete medium are added to terminate digestion and centrifuged at 1000rpm for 10min. The cells were resuspended, filtered through a 100 μm sieve, inoculated into T25 cell culture flasks at 1X106/ml, incubated in a 5% CO2 saturated humidity incubator at 37℃for 48 hours, and then replaced. Cells were observed to 80% confluency, digested with 0.25% pancreatin (Gibco, 25200-056) at 1: and 3, subculturing. The cell morphology of the generation P0, P5, P10 and P15 was observed under a microscope.
Isolation and culture of hUCMSC
The umbilical cord (supplied by the tenth people's hospital at the university of the same aid) after the caesarean section of the young healthy puerpera is taken, the umbilical cord is peeled off by forceps to obtain Huatong gum, the gum is soaked in 75% alcohol for 2min, the gum is washed 3 times by PBS liquid and then sheared into small pieces of about 1mm3, and 0.2% |collagenase, 0.2% type IV collagenase (Gibco), 0.1% pancreatin (Gibco, 27250-018) and 0.1% hyaluronidase (Beijing Biodee Biotechnology, 9004-61-9) and 15IU/ml DNase (Beijing Biodee Biotechnology, R0110) are added for digestion for 4h. The digestion was stopped by adding an equal volume of complete medium and centrifuged at 1000rpm for 10min. The cells were resuspended in complete medium, filtered through a 100 μm sieve, inoculated into T25 cell flasks at 1X 106/ml, incubated in a 5% CO2 saturated humidity incubator at 37℃for 48 hours, and the medium was changed. Cells were observed to 80% confluency, digested with 0.25% pancreatin, at 1: and 3, subculturing. The cell morphology of the generation P0, P5, P10 and P15 was observed under a microscope.
3. Detection of hASCs and hUCMSCs surface molecular markers by flow cytometry
The P4 generation hASC and hUCMSC are respectively added with 0.25 percent of pancreatin to be digested to prepare 500ul single cell suspension. CD90-FITC, CD44-PE, CD73-APC, CD105-PerCP-CP5.5 antibody were added to each of the human mesenchymal stem cell assay kit (BD, 562245) and 5. Mu.l each, incubated on ice for 30min, centrifuged at 1000rpm for 10min, and three times washed with PBS followed by detection by flow cytometry (BD FACSVerse).
4. Adipogenic osteogenic differentiation
1) Adipogenic induced differentiation: the cells were mixed at 1X 10 4 A density of/ml was inoculated into 24-well plates and cells were observed to 80% confluency using a lipid-forming basal medium (R&D, CCM 007) and adipogenic culture additives (R&D, 390415), induced differentiation for 2 weeks, oil red O solution (Beijing Solarbio Science&Technology co., ltd, G1260) staining observations.
2) Osteogenic induced differentiation: the cells were mixed at 1X 10 4 A density of/ml was inoculated into 24-well plates, and cells were observed to 50% confluency using an osteogenic basal medium (R&D, CCM 007) and osteogenic culture additives (R&D, 390416), induced differentiation for 2 weeks, alizarin (sigma) staining observations.
5. Hepatic differentiation
hASC and hUCMSC were respectively used in 1X 10 4 Inoculating at 10cm dish/ml density, observing cell until 50% fusion, adding complete medium containing 100ng/ml Activin A (Shanghai ExCell Biology, inc., CB 031-0033), and inducing with liver differentiation medium the next dayGuiding for 1 week: 100ng/ml HGF (Sino Biological Inc., 10463-HNAS), 20ng/ml EGF (R)&D，236-EG-200)、20ng/ml IGF(R&D, 291-G1-200), 20ng/ml FGF4 (Shanghai ExCell Biology, inc., CB 055-0343), 10 μg/ml Insullin-transferring in-Sodium Selenite Supplement (Insulin Transferrin sodium selenite supplement, roche, 11074547001), 1 μM dexamethasone (sigma, D4902-25 MG), 2MG/ml fetal bovine serum (BSA, EMB Millipore Corp.,126575-10 GM), 1%Penicillin Streptomycin (Gibco, 15070-063), DMEM-F12 medium; liver maturation medium was then changed for 1 week: 40ng/ml HGF, 20ng/ml EGF, 20ng/ml IGF, 20ng/ml FGF4, 10. Mu.g/ml ITS, 1. Mu.M dexamethasone, 1mM nicotinamide (Sigma, N0636-100G), 20ng/ml OSM (Sino Biological Inc., 10452-HNAH), 2mg/ml BSA, 1% diabody, DMEM-F12 medium.
6. Fluorescent quantitative PCR
hASCs and hUCMSCs were isolated at 1X 10 4 The cells were inoculated at a density of/ml into 10cm dish, and RNA was extracted from cells before induction, at 7 th and 15 th days after induction using Trizol (Invitrogen, 15596-026), reverse transcribed into cDNA using RT-PCR kit (Fermentas, K1622), and the expression of ALB, AFP, CK, GATA4, HNF1α, HNF4α, CYP2B6, CYP3A4mRNA was detected by real-time fluorescent quantitative PCR. The PCR reaction conditions were: 2min at 95 ℃, 10min at 95 ℃, 30sec at 60 ℃ and 40cycles. The software automatically analyzes and plots the PCR product dissolution curve. The final data were analyzed with 2- ΔΔct. The primers are shown in Table 1.
TABLE 1 liver cell related gene primer
7. Cellular immunofluorescence
hASC and hUCMSC cells were grown at 1X 10, respectively 4 The cells were fixed with 4% paraformaldehyde, and the cells were fixed with Triton X100-blocked cells before and after induction, respectively, at 3, 7, 11, and 15 days after induction, and human albumin antibodies (R were added to the cells at a density of/ml&D, MAB 1455) and human alpha fetoprotein antibody (R&D, MAB 13691) 1. Mu.l/well, 4℃overnight, mouse secondary antibody (R)&D, NL 007) 1 μl/well, anti-fluorescence decay caplets after incubation in the dark (Beijing Solarbio Science)&Technology Co., ltd, S2100) and fluorescent microscopy (Nikon, ti-S, eclipse) to observe the expression of ALB and AFP proteins in cells.
PAS staining for detecting glycogen synthesis function of liver-like cells
hASC and hUCMSC cells were grown at 1X 10, respectively 4 The cells were fixed with 4% paraformaldehyde at a density of/ml in 24-well plates before, at 7 th and 15 th days after induction, respectively, and glycogen PAS staining solution (Beijing Solarbio Science)&Technology co., ltd, G1281) was stained with glycogen and observed under a microscope (Nikon, ti-S, eclipse).
9. ICG uptake assay for liver-like cells
hASC and hUCMSC cells were grown at 1X 10, respectively 4 The density per ml was inoculated into 24-well plates, and 200ul of 1mg/ml indocyanine green was added before induction, at 7 th and 15 th days after induction, and observed under a microscope (Nikon, ti-S, eclipse) after 30 min.
ELISA detection
hASC and hUCMSC cells were grown at 1X 10, respectively 4 The culture supernatants of each group were collected before and after induction at days 7 and 15, respectively, and inoculated into 24-well plates at a density of/ml, and the culture supernatants were subjected to a procedure according to the instructions of human albumin ELISA Kit (Abcam, ab 108788) and human alpha fetoprotein ELISA Kit (Abcam, ab 108838), absorbance was measured at a wavelength of 450nm, and the concentrations of ALB and AFP proteins in the culture supernatants were calculated from a standard curve. NH4Cl was added before induction, at days 7 and 15 after induction, and after 24 hours, each group of culture supernatants was taken and operated as described by the human Urea ELISA Kit (Shanghai Enzyme-linked Biotechnology co., ltd., ML 07367), absorbance was measured at a wavelength of 450nm, and the concentration of Urea in the culture supernatant was calculated from a standard curve.
11. Acute liver failure model
Healthy male SPF-grade SD rats (supplied by Shanghai Laike laboratory animal Co., ltd., [ license number: SCXK (Shanghai) 2007-0005) ], 6-8 weeks old, and about 150-180 g in weight. D-galactosamine was intraperitoneally injected into rats at a dose of 1g/kg body weight D-gal for 2 times at 12h intervals [3].
12. Femoral vein grafting
The 75 acute liver failure model rats were randomly divided into 5 groups of 15 rats each. The next day after the establishment of the rat acute liver failure model, a transverse incision is made in the right inguinal line of the rat on an ultra clean bench, and 500ul PBS, 500ul hASC, 500ul liver-differentiated hASC (hASC-HLC) on day 15, 500ul hUCMSC, and 500ul liver-differentiated hUCMSC-HLC on day 15 are respectively injected into the femoral vein 5 group by using a disposable 1ml syringe, wherein the cell number is 2-5×10 6 And each. After injection, 75% alcohol cotton ball is used for hemostasis by compression, wound is sutured, and the operation process strictly complies with the aseptic operation principle. Survival rates were counted for both groups of rats. The levels of ALT, AST, ALB, DB, LDH, ALP and gamma-GT in serum were detected by a full-automatic biochemical analyzer by tail-breaking and blood-taking 1 day after molding, 1 day after transplanting and 7 days respectively.
13. Immunohistochemistry
The expression of ALB in rat liver tissue was examined by immunohistochemical method 1 day and 7 days after transplantation, respectively, using human albumin antibody (Abcam, ab 137885) and human alpha fetoprotein antibody (Abcam, ab 133617).
Ki67 staining
Taking liver tissue after cell treatment, preparing paraffin section, dewaxing, placing in a repair box filled with EDTA antigen repair buffer solution (pH9.0), and performing antigen repair in a microwave oven. The addition of hydrogen peroxide blocks endogenous peroxidases. The tissue was covered with 5% BSA, diluted 1:300, of Ki-67 antibody (Novusbio, NB 500-170) dropwise. Sections were placed flat in wet boxes and incubated overnight at 4 ℃. The next daily sections were washed 3 times with shaking on a decolorizing shaker in PBS for 5min each, and then with addition of rabbit secondary antibodies (R & D, NL 004) and observed under a fluorescent microscope (Nikon, 80i, eclipse).
Tunnel detection
An in situ apoptosis kit (Roche, 11684817910) was used. Taking liver tissue after cell treatment, preparing paraffin section, dewaxing, hydrating, adding TUNEL reaction liquid after cell permeation, adding converter-POD, reacting with substrate DAB for color development, and observing under a fluorescence microscope (Nikon, 80i, eclipse).
15. High throughput qualitative analysis of protein chips
High-throughput qualitative analysis of relative expression levels of cytokines, chemokines and the like in rat tissues by using a protein chip: the expression of IL-1ra, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IL-10, IL-13, IL-17, IFN-gamma, TNF-alpha, GM-CSF, CCL3, CCL5, CCL20, CINC-1, CINC-2 alpha/beta, CINC-3, CNTF, CX3CL1, CXCL7, CXCL9, CXCL10, LECAM-1, LIX, sICAM-1, TIMP-1, VEGF in rat serum was qualitatively analyzed at high throughput using a rat cytokine protein chip (R & D, ARY 008) protein chip, respectively, 1 day and 7 day after the transplantation. Gel imager (GE, LAS4000 mini) was photographed and analyzed Mean Pixel Density using HLImage software.
16. Statistical analysis
Results are expressed as mean ± standard deviation (X ± SD). Survival analysis was performed using Kaplan-Meier analysis and SPSS22.0 statistical software.
EXAMPLE 1 morphology and identification of hASC and hUCMSC
Morphological identification of hascs and hucmscs was performed as described in materials and methods.
The results were: three days after the primary inoculation of hASC and hUCMSC, cell colonies are formed, cells in the colonies are in a long spindle shape, have uniform size and are closely arranged to radially grow; no obvious changes were made in the morphology and proliferation rate of the P5, P10 and P15 generation cells (see FIG. 1A). Flow cytometry detection of P3 generation hascs and humsc: CD90, CD44, CD73, CD105 were all expressed positively, while CD34, CD45, CD11B, CD19, HLA-DR were expressed negatively (see fig. 1B). After the hASC and the hUCMSC are subjected to adipogenesis and induction for two weeks, oil red O staining can be used for observing that a large number of transparent red lipid drops with good refractive index exist in hASC cytoplasm, and only a small number of tiny red stained particles exist in hUCMSCs cytoplasm; two weeks after osteoinduction of hASCs and hUCMSCs, alizarin red staining was seen for most of the cells (see FIG. 1C).
EXAMPLE 2 differentiation of hASC and hUCMSC into hepatocytes
Hepatic differentiation of hascs and hucmscs was performed as described in materials and methods.
The results were: the results of fluorescent quantitative PCR showed that ALB, AFP, HNF. Alpha., HNF 4. Alpha., CYP3A4mRNA expression was significantly enhanced in the hASC-induced group as compared to the hUCMSC-induced group, and the expression level was increased with the extension of the induction time (see FIG. 2).
EXAMPLE 3 Alb and AFP cellular immunofluorescence observations
Alb and AFP expression levels of hASC-induced cells and hUCMSC-induced cells were measured according to the materials and methods described herein.
The results were: in the hASCs, the expression levels of Alb and AFP increased with the prolongation of the induction time, and the expression level of Alb increased significantly on day 11 (as shown in FIG. 3). In the hucMSC-induced group, however, the ALB and AFP expression levels were not significantly increased (as shown in FIG. 4).
Example 4 functional identification
The glycogen storage, ICG uptake and protein synthesis of hASC-induced and hUCMSC-induced cells were tested as described in materials and methods.
The results were:
1) Glycogen storage level: on day 11 of induction, glycogen staining positive was detected in some cells of the hASC-induced group and the hUCMSC-induced group; on day 15 of induction, glycogen staining positive cells increased, with the hASC induction group being more pronounced (see figure 5A, B).
2) ICG uptake experiments: on day 11 of induction, few cells of hASC-induced and hUCMSC-induced groups can ingest ICG, green particles are visible in the cytoplasm; on day 15 of induction, ICG-ingestible cells were significantly increased, and the hASCs-induced group was more pronounced (see fig. 5C).
3) Protein synthesis function: as the induction time was prolonged, both the expression levels of Alb and urea in the hASC-induced group were increased (see FIGS. 5E-G).
EXAMPLE 5 femoral vein grafting
Femoral vein grafting studies were performed as described in materials and methods, and the results are as follows:
five groups of rats died 8 hours after treatment, all of the PBS control group rats died within 48 hours, and none of the rats died 72 hours later. The survival rates of the hASC treated group, hASC-HLC treated group, hUCMSC treated group and hUCMSC-HLC treated group rats were 26.7% (4/15), 33.3% (5/15), 13.3% (2/15), 6.7% (1/15), respectively (FIG. 6A).
The ALB levels of the four rats in the treatment group recovered to normal on the day after treatment, AST levels were significantly reduced on the day after treatment, and the differences compared with the PBS control group were all statistically significant (P < 0.05) (fig. 6D, E); the rat DB levels in both the hASC-treated group and the hASC-HLC-treated group were decreased and the rat DB levels in both the hUCMSC-treated group and the hUCMSC-HLC-treated group were increased as compared to the control group, wherein the differences between the hASC-treated group and the hUCMSC-HLC-treated group were statistically significant (P < 0.05) (FIG. 6F).
EXAMPLE 6 immunohistochemical staining
Immunohistochemical staining was performed according to the materials and methods described below, with the following results:
normal rats and PBS control group rats liver tissues were not stained with human Alb antibodies (see fig. 7A, B); human albumin antibody staining was negative on day 1 after hASC transplantation (see fig. 7C), with more human Alb positive cells around the central vein on day 7 (see fig. 7D); whereas more human Alb antibody positive cells appeared around the central vein on day 1 after the hASC-HLC transplantation (see fig. 7E), there was a slight decrease in Alb positive cells on day 7 (see fig. 7F). The number of Alb positive cells was smaller than that of the hUCMSC and hUCMSC-HLC treated groups on days 1 and 7 after the hUCMSC-HLC transplantation (see FIGS. 7G-J).
Example 7 Ki67 staining and Tunnel test
Ki67 staining and Tunnel tests were performed as described in materials and methods, with the following results:
after immunohistochemical staining of normal liver tissue and PBS control, a small number of cells were stained with Ki67 antibody. Part of Ki67 positive cells were visible on day 1 after the transplantation of the four treatment groups, and Ki67 positive cells were further increased on day 7; of these, both the hASCs and hASCs-HLC treated groups had a greater number of Ki67 positive cells (see FIG. 8A).
Positive cells were not seen after the normal liver tissue sections were stained with Tunnel, and a small number of positive cells were seen on day 1 after hASC and hASC-HLC transplantation; whereas hUCMSC, hUCMSC-HLC treated and PBS control groups both seen a large number of positive cells; each group was found to be only very few cell positives on day 7 (see fig. 8B).
Example 8 protein chip detection
After high throughput qualitative analysis of the protein chips according to the materials and methods described in "materials and methods" for hASC and hASC-HLC transplantation, the relative expression levels of cytokines, chemokines, etc. in rat tissues resulted in the following:
the inflammatory factors such as IL-1 alpha, IL-6 and the like in the serum of the rats are obviously increased and the anti-inflammatory factors such as IL-1ra, IL-13 and the like are obviously increased on the 1 st day after the hASC and hASC-HLC transplantation, wherein the IL-1ra is highest in the hASC treatment group, and the IL-13 is highest in the hASC-HLC treatment group; both groups of chemokines were elevated (see figure 9).
The inflammatory factors such as IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, etc. in the serum of the rats at day 7 after the hASC and hASC-HLC transplantation were reduced earlier, and the hASC-treated group was almost recovered to normal level; while the hASC-HLC treated group was still at higher levels of the anti-inflammatory factors IL-13 and chemokines (see FIG. 10).
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
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Claims (27)
1. A method of preparing highly mature human adipose stem cell-derived hepatocyte-like cells, the method comprising the steps of:
1) Culturing human adipose-derived stem cells by using a culture medium containing Activin A;
2) Inducing the cultured human adipose-derived stem cells obtained in the step (1) to differentiate into hepatocyte-like cells by using a hepatic differentiation medium; and
3) Maturing the hepatocyte-like cells obtained in step (2) by using a liver maturation medium, thereby obtaining highly matured hepatocyte-like cells derived from human adipose-derived stem cells;
wherein, the liver differentiation medium consists of the following components:
HGF, EGF, IGF, FGF4 insulin transferrin sodium selenite additive, dexamethasone, fetal bovine serum, penicillin-streptomycin solution, DMEM-F12 medium;
The liver maturation medium consists of the following components:
HGF, EGF, IGF, FGF4, ITS, dexamethasone, niacinamide, oncostatin M (OSM), BSA, penicillin-streptomycin solution, DMEM-F12 medium;
the hepatocyte-like cells derived from highly mature human adipose-derived stem cells possess one or more of the following characteristics:
1) More than 40% of the hepatocyte-like cells express ALB, AFP, HNF1 alpha, HNF4 alpha, CYP3A4 mRNA; alternatively, the expression of ALB, AFP, HNF α, hnf4α, CYP3A4 mRNA in the hepatocyte-like cells is increased by 30% or more compared to before induction;
2) More than 20% of the hepatocyte-like cells express Alb and AFP proteins; alternatively, the expression of Alb and AFP proteins in the hepatocyte-like cells is increased by more than 10% compared to before induction;
3) More than 10% of the hepatocyte-like cells are cell glycogen staining positive cells;
4) More than 5% of the hepatocyte-like cells ingest ICG; and
5) The expression of urea in the hepatocyte-like cells is increased by more than 10% compared to before induction.
2. The method of claim 1, wherein obtaining highly mature human adipose stem cell-derived hepatocyte-like cells is characterized by one or more of:
1) More than 50% of the hepatocyte-like cells express ALB, AFP, HNF1 alpha, HNF4 alpha, CYP3A4 mRNA; alternatively, the expression of ALB, AFP, HNF α, hnf4α, CYP3A4 mRNA in the hepatocyte-like cells is increased by 50% or more compared to before induction;
2) More than 50% of the hepatocyte-like cells express Alb and AFP proteins; alternatively, the expression of Alb and AFP proteins in the hepatocyte-like cells is increased by more than 20% compared to before induction;
3) More than 30% of the hepatocyte-like cells are cell glycogen staining positive cells;
4) More than 20% of the hepatocyte-like cells ingest ICG; and
5) The expression of urea in the hepatocyte-like cells is increased by more than 20% compared to before induction.
3. The method of claim 2, wherein obtaining highly mature human adipose stem cell-derived hepatocyte-like cells is characterized by one or more of:
1) More than 60% of the hepatocyte-like cells express ALB, AFP, HNF1 alpha, HNF4 alpha, CYP3A4 mRNA; alternatively, the expression of ALB, AFP, HNF a, HNF4 a, CYP3A4 mRNA in the hepatocyte-like cells is increased by more than 70% compared to before induction;
2) More than 75% of the hepatocyte-like cells express Alb and AFP proteins; alternatively, the expression of Alb and AFP proteins in the hepatocyte-like cells is increased by more than 30% compared to before induction;
3) More than 60% of the hepatocyte-like cells are cell glycogen staining positive cells;
4) More than 30% of the hepatocyte-like cells ingest ICG; and
5) The expression of urea in the hepatocyte-like cells is increased by more than 30% compared to before induction.
4. The method of claim 1, wherein step 1) is performed for 18 to 30 hours; the step 2) is carried out for 5 to 9 days; the step 3) is carried out for 5-9 days.
5. The method of claim 4, wherein step 1) is performed for 22 to 26 hours; the step 2) is carried out for 6 to 8 days; the step 3) is carried out for 6-8 days.
6. The method of claim 5, wherein step 1) is performed for 24 hours; the step 2) is carried out for 7 days; the step 3) is carried out for 7 days.
7. The method of claim 1, wherein the human adipose stem cells are pre-treated with a DNA methylase inhibitor prior to step 1).
8. The method of claim 7, wherein the DNA methylase inhibitor comprises 5-azacytidine.
9. The method of claim 7, wherein the DNA methylase inhibitor is pre-treated at a dose of 10ug/L for a period of 24 hours.
10. The method of any one of claims 1-9, wherein the hepatocyte-like cells present human Alb antibody positive cells around the central vein on day 1 after transplantation.
11. The method of any one of claims 1-9, wherein the hepatocyte-like cells are elevated by more than 20% with respect to antiinflammatory factors IL-1ra, IL-13 on day 1 post-implantation.
12. The method of claim 11, wherein the hepatocyte-like cells have an increase in anti-inflammatory factors IL-1ra, IL-13 of greater than 100% on day 1 post-transplantation.
13. The method of claim 12, wherein the hepatocyte-like cells have an increase in anti-inflammatory factors IL-1ra, IL-13 of more than 300% at day 1 post-transplantation.
14. The method of claim 7, wherein greater than 40 highly mature human adipose-derived stem cell-derived hepatocyte-like cells are obtained per 100 primary human adipose stem cells.
15. The method of claim 14, wherein greater than 50 highly mature human adipose-derived stem cell-derived hepatocyte-like cells are obtained per 100 primary human adipose stem cells.
16. The method of claim 15, wherein 50-60 highly mature human adipose-derived stem cell-derived hepatocyte-like cells are obtained per 100 primary human adipose stem cells.
17. A population of highly mature human adipose stem cell-derived hepatocyte-like cells, said population of hepatocyte-like cells being characterized by one or more of the following:
1) More than 40% of the hepatocyte-like cells express ALB, AFP, HNF1 alpha, HNF4 alpha, CYP3A4 mRNA; alternatively, the expression of ALB, AFP, HNF α, hnf4α, CYP3A4 mRNA in the hepatocyte-like cells is increased by 30% or more compared to before induction;
2) More than 20% of the hepatocyte-like cells express Alb and AFP proteins; alternatively, the expression of Alb and AFP proteins in the hepatocyte-like cells is increased by more than 10% compared to before induction;
3) More than 10% of the hepatocyte-like cells are cell glycogen staining positive cells;
4) More than 5% of the hepatocyte-like cells ingest ICG; and;
5) The expression of urea in the hepatocyte-like cells is increased by more than 10% compared to before induction;
the hepatocyte-like cell population prepared by the method of any one of claims 1-16.
18. A population of highly mature human adipose stem cell-derived hepatocyte-like cells according to claim 17, said population of hepatocyte-like cells being characterized by one or more of the following:
1) More than 50% of the hepatocyte-like cells express ALB, AFP, HNF1 alpha, HNF4 alpha, CYP3A4 mRNA; alternatively, the expression of ALB, AFP, HNF α, hnf4α, CYP3A4 mRNA in the hepatocyte-like cells is increased by 50% or more compared to before induction;
2) More than 50% of the hepatocyte-like cells express Alb and AFP proteins; alternatively, the expression of Alb and AFP proteins in the hepatocyte-like cells is increased by more than 20% compared to before induction;
3) More than 30% of the hepatocyte-like cells are cell glycogen staining positive cells;
4) More than 20% of the hepatocyte-like cells ingest ICG; and;
5) The expression of urea in the hepatocyte-like cells is increased by more than 20% compared to before induction.
19. A population of highly mature human adipose stem cell-derived hepatocyte-like cells according to claim 18, said population of hepatocyte-like cells being characterized by one or more of the following:
1) More than 60% of the hepatocyte-like cells express ALB, AFP, HNF1 alpha, HNF4 alpha, CYP3A4 mRNA; alternatively, the expression of ALB, AFP, HNF a, HNF4 a, CYP3A4 mRNA in the hepatocyte-like cells is increased by more than 70% compared to before induction;
2) More than 75% of the hepatocyte-like cells express Alb and AFP proteins; alternatively, the expression of Alb and AFP proteins in the hepatocyte-like cells is increased by more than 30% compared to before induction;
3) More than 60% of the hepatocyte-like cells are cell glycogen staining positive cells;
4) More than 30% of the hepatocyte-like cells ingest ICG; and;
5) The expression of urea in the hepatocyte-like cells is increased by more than 30% compared to before induction.
20. A population of highly mature human adipose stem cell-derived hepatocyte-like cells of claim 17, wherein the population of hepatocyte-like cells present human Alb antibody positive cells around the central vein on day 1 post-transplantation.
21. A population of highly mature human adipose stem cell-derived hepatocyte-like cells of claim 17, wherein the hepatocyte-like cells have an increase in anti-inflammatory factors IL-1ra, IL-13 of more than 20% on day 1 post-transplantation.
22. A population of highly mature human adipose stem cell-derived hepatocyte-like cells of claim 21, wherein the hepatocyte-like cells have an increase of more than 100% in anti-inflammatory factors IL-1ra, IL-13 at day 1 post-transplantation.
23. A population of highly mature human adipose stem cell-derived hepatocyte-like cells of claim 22, wherein the hepatocyte-like cells have an increase of more than 300% in anti-inflammatory factors IL-1ra, IL-13 at day 1 post-transplantation.
24. A pharmaceutical composition comprising the population of highly mature human adipose stem cell-derived hepatocyte-like cells of any one of claims 17-23 or the highly mature human adipose stem cell-derived hepatocyte-like cells prepared by the method of any one of claims 1-16, and optionally a pharmaceutically acceptable carrier.
25. A kit comprising a population of highly mature human adipose stem cell-derived hepatocyte-like cells of any one of claims 17-23, or a highly mature human adipose stem cell-derived hepatocyte-like cell prepared by the method of any one of claims 1-16, or the pharmaceutical composition of claim 24, and optionally instructions for use of the kit.
26. Use of a population of highly mature human adipose stem cell-derived hepatocyte-like cells of any one of claims 17-23 or prepared by the method of any one of claims 1-16 in the manufacture of a medicament for treating a disease, wherein the disease comprises acute liver failure, liver fibrosis or liver cirrhosis.
27. The use according to claim 26, wherein the disease is acute liver failure.
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| 体内外人脂肪间充质干细胞向肝样细胞分化的实验研究;殷莉波，等;《中华肝胆外科杂志》;20110430;第17卷(第4期);第322-327页 * |
| 殷莉波，等.体内外人脂肪间充质干细胞向肝样细胞分化的实验研究.《中华肝胆外科杂志》.2011,第17卷(第4期), * |
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