Preparation method and application of hepatocyte-like cell sheet derived from human embryonic stem cells

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a preparation method and application of a hepatocyte-like cell sheet derived from human embryonic stem cells. The preparation method of the hepatocyte-like cell sheet derived from the human embryonic stem cells comprises the following steps: providing the human embryonic stem cells, carrying out induced differentiation on the human embryonic stem cells to a definitive endoderm, and carrying out directional differentiation on the induced cells to hepatocyte cells, and culturing the directionally differentiated cells until the cells are mature, and preparing the hepatocyte-like cell sheet derived from the human embryonic stem cells. According to the method provided by the invention, the hESC-HPLCs tablet is prepared from hepatocyte-like cells induced and differentiated by human embryonic stem cells, and is used for treating a DILF mouse subjected to APAP modeling. The hESC-HPLCs tablet prepared by the method disclosed by the invention can obviously improve the survival rate of mice, reduce the liver injury degree of the mice and accelerate the recovery of injured tissues, so that the recovery process is accelerated.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to a method for preparing a hepatocyte-like cell sheet derived from human embryonic stem cells and its application. Background Art

[0002] Drug-induced liver failure (DILF) is acute liver failure (ALF) caused by liver damage induced by various prescription or over-the-counter chemical drugs, biological agents, traditional Chinese medicine (TCM), natural medicine (NM), health products (HP), dietary supplements (DS) and their metabolites and even excipients. It is the most serious clinical type of drug-induced liver injury (DILI), with a poor prognosis and a mortality rate of 24-52%. At present, the treatment of DILF mainly includes conservative medical treatment, artificial liver support system, granulocyte colony stimulating factor (G-CSF) treatment, in vivo hepatocyte transplantation and liver transplantation. Liver transplantation is currently the most effective way to treat DILF, but objective factors such as shortage of donor livers, immune response and high cost make it unable to meet the clinical treatment needs, limiting its clinical application and promotion.

[0003] As an emerging treatment option, cell therapy has obvious advantages in reducing inflammation, improving scarring, replacing tissue and organ functions, and promoting tissue and organ regeneration and repair. It is a very promising treatment for various intractable diseases. Human embryonic stem cells (hESCs) are considered to be one of the best candidate cells in the field of regenerative medicine because of their multidirectional differentiation potential and other characteristics. Functional cells derived from hESCs are expected to become a new treatment strategy for end-stage liver diseases such as liver failure. In cell therapy, cell delivery is one of the key links. Currently, the cell delivery methods used in cell therapy include cell suspension, biodegradable scaffolds, organoids, and cell sheet engineering technology. Among them, the most widely used is suspension injection, but suspension therapy has no cell-to-cell interaction, which is not conducive to cell survival, long-term maintenance of related functions and characteristics; at the same time, abnormal colonization may occur. The cell sheet engineering technology is basically mature, eliminating the limitations of traditional tissue engineering. Cell sheet-related research based on thermo-responsive polyisopropylacrylamide (PIPAAm) culture dishes has involved multiple types of cells and can deliver denser cell populations to the desired site in some cases.

[0004] However, at present, there are no reports on the combination of cell sheet technology and embryonic stem cell directed differentiation technology and its application in liver diseases. Summary of the invention

[0005] Based on this, an embodiment of the present application provides a method for preparing a hepatoid cell sheet derived from human embryonic stem cells and its application.

[0006] On one hand, the present application provides a method for preparing a hepatoid cell sheet derived from human embryonic stem cells, comprising: providing human embryonic stem cells, inducing the human embryonic stem cells to differentiate into defined endoderm, and collecting the induced cells;

[0007] Then, directing differentiation of the induced cells into hepatic cells; and,

[0008] The cells after directed differentiation are cultured until maturity, and a hepatocyte-like cell sheet derived from human embryonic stem cells is prepared using a temperature-responsive culture container.

[0009] In one embodiment, inducing differentiation of the human embryonic stem cells into defined endoderm comprises:

[0010] The human embryonic stem cells are first cultured in a first culture medium for 1 to 2 days, and then cultured in a second culture medium for 2 to 4 days;

[0011] The first culture medium comprises RPMI-1640 culture medium, human activin A and Wnt3a;

[0012] The second culture medium comprises RPMI-1640 culture medium, human activin A and fetal bovine serum.

[0013] In one embodiment, the first culture medium comprises RPMI-1640 culture medium, human activin A with a final concentration of 5 ng / mL to 300 ng / mL, and Wnt3a with a final concentration of 20 ng / mL to 100 ng / mL; and or,

[0014] The second culture medium comprises RPMI-1640 culture medium, human activin A with a final concentration of 10 ng / mL to 200 ng / mL, and fetal bovine serum with a final concentration of 0.2% to 2%.

[0015] In one embodiment, directing the differentiation of the induced cells into hepatic cells comprises:

[0016] The induced cells are first cultured in the third culture medium for 6 to 12 days, and then cultured in the fourth culture medium for 5 to 12 days;

[0017] The third culture medium comprises KO / DMEM culture medium, keratinocyte growth factor and fetal bovine serum;

[0018] The fourth culture medium comprises KO / DMEM medium, as well as serum replacement (SR), L-glutamine, non-essential amino acids, β-mercaptoethanol, and dimethyl sulfoxide.

[0019] In one embodiment, the third culture medium comprises KO / DMEM culture medium, keratinocyte growth factor at a final concentration of 15 ng / mL to 100 ng / mL, and fetal bovine serum at a final concentration of 2% to 3%;

[0020] The fourth culture medium comprises KO / DMEM culture medium, and serum replacement (SR) with a final concentration of 18% to 22%, L-glutamine with a final concentration of 1mM to 3mM, non-essential amino acids with a final concentration of 0.1% to 15%, β-mercaptoethanol with a final concentration of 0.1mM to 0.12mM, and dimethyl sulfoxide with a final concentration of 0.2% to 2%.

[0021] In one embodiment, culturing the directed differentiated cells to maturity comprises culturing in a fifth culture medium for 6 to 10 days;

[0022] The fifth culture medium comprises L-15 culture medium, bovine serum, hepatocyte growth factor, oncostatin, dexamethasone and L-glutamine;

[0023] Optionally, the fifth culture medium comprises L-15 culture medium and fetal bovine serum with a final concentration of 8% to 12%, hepatocyte growth factor with a final concentration of 10 ng / mL to 300 ng / mL, oncostatin with a final concentration of 5 ng / mL to 150 ng / mL, dexamethasone with a final concentration of 0.05 μM to 1.2 μM and L-glutamine with a final concentration of 2 mM to 3 mM.

[0024] In one embodiment, preparing a hepatocyte-like cell sheet derived from human embryonic stem cells using a temperature-responsive culture container comprises:

[0025] The mature human embryonic stem cell-derived hepatocytes are digested into single cells, inoculated into a temperature-responsive culture container, and cultured for 3 to 4 days. After the human embryonic stem cell-derived hepatocytes in the temperature-responsive culture container establish a connection, the cells automatically separate from the bottom of the container in the form of cell sheets, thereby preparing human embryonic stem cell-derived hepatocytes sheets.

[0026] In one embodiment, the culture medium is replaced every 22 to 26 hours;

[0027] In one embodiment, the culture medium is replaced with RPIM-1640 for 1 to 2 washes.

[0028] On the other hand, the present application provides a human embryonic stem cell-derived liver-like cell sheet prepared by the method for preparing the human embryonic stem cell-derived liver-like cell sheet.

[0029] On the other hand, the present application also provides the method for preparing the hepatic cell sheet derived from human embryonic stem cells, and the use of the hepatic cell sheet derived from human embryonic stem cells in preparing drugs for preventing and treating liver diseases;

[0030] In one embodiment, the drug for preventing or treating liver disease prevents or treats acute drug-induced liver failure.

[0031] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0033] Figure 1 The morphological images of hESCs under the feeder layer system and the cells at various stages during the differentiation of hESCs into hepatocytes (gray is the light microscopy image, green is the ZsGreen fluorescence image);

[0034] Figure 2 The expression of related genes after inducing hESCs into hepatocytes and preparing hepatocyte sheets in the examples of this application;

[0035] Figure 3 The expression of hepatocyte-related marker proteins ALB, AFP, and HNF4α in hESC-HPLCs of the present application example;

[0036] Figure 4 This is the identification of liver cell related functions in hESC-HPLCs in the present application example;

[0037] Figure 5 This is the preparation and harvesting process of hESC-HPLCs slices in the present application example, scale bar 1mm;

[0038] Figure 6 This is the immunofluorescence identification of ALB, AAT, and HNF4α of the hESC-HPLCs slice in the present application example, scale bar 100 μm;

[0039] Figure 7 Comparison of the survival rates of modeling mice in different APAP dosage groups in the present application example;

[0040] Figure 8The observation scores of the survival status of mice in different dosage groups in the examples of this application;

[0041] Fig. 9 This is the pathological analysis of mouse liver tissue 72 hours after modeling in the embodiment of the present application;

[0042] Fig.10 Analysis of the survival rate in the tolerance assessment of mouse surgery in the examples of this application;

[0043] Fig.11 Scoring the mouse status in the surgical tolerance assessment of the present application example;

[0044] Fig.12 This is the survival rate analysis in the effectiveness evaluation of hESC-HPLCs tablets in treating APAP-induced DILF mice in the present application example;

[0045] Fig.13 The gross changes in the liver of mice in different treatment groups in the examples of this application;

[0046] Fig.14 The pathological results of the liver of mice in different treatment groups in the examples of this application;

[0047] Fig.15 This is the TUNEL staining analysis of the liver tissues of mice in the different treatment groups in the examples of this application;

[0048] Fig.16 Analysis of serum biochemical changes in mice in different treatment groups in the examples of this application;

[0049] Fig.17 qPCR detection of PI3K-AKT-mTOR signaling pathway in liver tissue of mice in different treatment groups in the examples of this application;

[0050] Fig.18 Western blot was used to detect the PI3K-AKT-mTOR signaling pathway in the liver tissues of mice in different treatment groups in the examples of this application;

[0051] Fig.19 H&E staining of mouse liver + hESC-HPLCs slice 30 days after transplantation of hepatoid cell slices in the example of this application;

[0052] Fig. 20 This is the hALB immunohistochemical staining of mouse liver + hESC-HPLCs slice 14 days after the transplantation of hepatoid cell sheets in the example of this application. DETAILED DESCRIPTION

[0053] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0055] the term

[0056] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0057] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0058] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0059] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0060] The term "human embryonic stem cells (hESCs)" Human embryonic stem cells (hESCs) are highly pluripotent cells that are derived from the inner cell mass of the early embryo. These cells have the ability to self-renew and can differentiate into almost all types of cells in the human body under appropriate conditions. hESCs have great potential in the field of medical research and treatment.

[0061] The term "HPLCs" refers to hepatocyte-like cells (HLCs or HPLCs) obtained by inducing differentiation of hESCs (human embryonic stem cells) or iPSCs (induced pluripotent stem cells), which are similar to mature hepatocytes in morphology, function and gene expression.

[0062] This application prepares hESC-HPLCs sheets from hepatic-like cells derived from human embryonic stem cells based on the directed induction and differentiation of human embryonic stem cells into hepatic-like cells. The method provided in this application combines the induced differentiation system with the cell sheet engineering technology, making up for the shortcomings of conventional cell delivery methods, opening up new research areas for tissue engineering research, and also providing new ideas and methods for the field of stem cells and regenerative medicine.

[0063] On the one hand, the present application provides a method for preparing a hepatoid cell sheet derived from human embryonic stem cells, comprising:

[0064] Providing human embryonic stem cells, inducing the human embryonic stem cells to differentiate into defined endoderm, and collecting the induced cells;

[0065] Then, directing differentiation of the induced cells into hepatic cells; and,

[0066] The cells after directed differentiation are cultured until maturity, and a hepatocyte-like cell sheet derived from human embryonic stem cells is prepared using a temperature-responsive culture container.

[0067] Definitive endoderm (DE) is an important cell type in the embryonic development process, which plays a key role in the subsequent differentiation of multiple cell types.

[0068] Providing human embryonic stem cells includes establishing hESCs lines that stably express luciferase (Luciferase, Luc) and ZsGreen green fluorescent protein and performing pluripotency identification.

[0069] In a specific example, inducing differentiation of the human embryonic stem cells into defined endoderm comprises:

[0070] The human embryonic stem cells are first cultured in a first culture medium for 1 to 2 days, and then cultured in a second culture medium for 2 to 4 days;

[0071] The first culture medium comprises RPMI-1640 culture medium, human activin A and Wnt3a;

[0072] The second culture medium comprises RPMI-1640 culture medium, human activin A and fetal bovine serum.

[0073] In a specific example, the first culture medium comprises RPMI-1640 culture medium, and human activin A with a final concentration of 5 ng / mL to 300 ng / mL and Wnt3a with a final concentration of 20 ng / mL to 100 ng / mL; and or,

[0074] The second culture medium comprises RPMI-1640 culture medium, human activin A with a final concentration of 10 ng / mL to 200 ng / mL, and fetal bovine serum with a final concentration of 0.2% to 2%.

[0075] Optionally, directing the differentiation of the induced cells into hepatic cells comprises:

[0076] The induced cells are first cultured in the third culture medium for 6 to 12 days, and then cultured in the fourth culture medium for 5 to 12 days;

[0077] The third culture medium comprises KO / DMEM culture medium, keratinocyte growth factor and fetal bovine serum.

[0078] The fourth culture medium comprises KO / DMEM medium, serum replacement (SR), L-glutamine, non-essential amino acids, β-mercaptoethanol and dimethyl sulfoxide.

[0079] Further optionally, the third culture medium comprises KO / DMEM culture medium, keratinocyte growth factor with a final concentration of 15 ng / mL to 100 ng / mL, and fetal bovine serum with a final concentration of 2% to 3%.

[0080] The fourth culture medium comprises KO / DMEM culture medium, and serum replacement (SR) with a final concentration of 18% to 22%, L-glutamine with a final concentration of 1mM to 3mM, non-essential amino acids with a final concentration of 0.1% to 15%, β-mercaptoethanol with a final concentration of 0.1mM to 0.12mM, and dimethyl sulfoxide with a final concentration of 0.2% to 2%.

[0081] In a specific example, culturing the directed differentiated cells to maturity includes culturing in a fifth culture medium for 6 to 10 days; inducing differentiation of an embryonic stem cell line stably expressing Luc and ZsGreen green fluorescent proteins (chHES-90-Luc-ZsGreen) into hESC-HPLCs.

[0082] The fifth culture medium comprises L-15 culture medium, bovine serum, hepatocyte growth factor, oncostatin, dexamethasone and L-glutamine.

[0083] Optionally, the fifth culture medium comprises L-15 culture medium and fetal bovine serum with a final concentration of 8% to 12%, hepatocyte growth factor with a final concentration of 10 ng / mL to 300 ng / mL, oncostatin with a final concentration of 5 ng / mL to 150 ng / mL, dexamethasone with a final concentration of 0.05 μM to 1.2 μM and L-glutamine with a final concentration of 2 mM to 3 mM.

[0084] In a specific example, preparing a hepatocyte-like cell sheet derived from human embryonic stem cells using a temperature-responsive culture container includes:

[0085] The mature human embryonic stem cell-derived hepatocytes are digested into single cells, inoculated into a temperature-responsive culture container, and cultured for 3 to 4 days. After the human embryonic stem cell-derived hepatocytes in the container establish a connection, the cells automatically separate from the bottom of the container in the form of cell sheets, thereby preparing human embryonic stem cell-derived hepatocytes sheets.

[0086] Specifically, the induced hESC-HPLCs are digested into single cells, and then the number of live cells is counted, and then inoculated into a 35mm temperature-responsive culture dish. After culturing for 3 to 4 days, when the hESC-HPLCs in the dish are confluent and a tight connection is established, the dish is taken out and replaced with non-preheated IV stage culture medium, and placed at room temperature for 15min to 30min. The cells are automatically separated from the bottom of the dish in the form of cell sheets to obtain hESC-HPLCs sheets, and the hESC-HPLCs sheets are prepared in combination with cell sheet engineering technology and related identification is performed;

[0087] In a specific example, the preparation method has one or more of the following technical features:

[0088] (1) Replace the culture medium every 22 to 26 hours;

[0089] (2) When changing the culture medium, wash 1 to 2 times with RPIM-1640.

[0090] On the other hand, the present application provides a human embryonic stem cell-derived liver-like cell sheet prepared by the method for preparing the human embryonic stem cell-derived liver-like cell sheet.

[0091] On the other hand, the present application also provides the method for preparing the hepatic cell sheet derived from human embryonic stem cells, and the use of the hepatic cell sheet derived from human embryonic stem cells in preparing drugs for liver diseases;

[0092] Optionally, the liver disease is acute drug-induced liver failure.

[0093] In this application, "drug" includes any agent, compound, composition or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects of "drug" in vivo is not particularly limited, and it can be a systemic effect or only a local effect. The activity of the "drug" is not particularly limited, and it can be an active substance that can interact with other substances, or it can be an inert substance that does not interact.

[0094] In one example, the drug may be a liquid preparation or a solid preparation. Liquid preparations refer to preparations containing a liquid phase, non-limiting examples of which include solutions, suspensions, emulsions, etc. Non-limiting examples of solid preparations include tablets, capsules, granules, pills, etc.

[0095] In one example, depending on the administration method, it can be an oral preparation, an injection, a drop, a patch, a tube feeding preparation, etc.

[0096] In one example, the excipients selected may vary depending on the dosage form.

[0097] In the present application, "excipients" include but are not limited to mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium sodium EDTA, carbonates, acetates, phosphates or aqueous solutions of monovalent alkali metals, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, and magnesium stearate.

[0098] As used herein, "pharmaceutically acceptable" refers to those ligands, materials, compositions, and / or dosage forms that are suitable for administration to a patient within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0099] In this application, "pharmaceutically acceptable excipients" refer to pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. As used herein, the language "pharmaceutically acceptable carriers" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are compatible with drug administration. Each substance must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0100] In one example, the drug comprises an effective amount of the hepatoid cell sheet derived from human embryonic stem cells.

[0101] In the present application, "effective amount" refers to the dosage of the component corresponding to the term in a subject to achieve the treatment, prevention, alleviation and / or relief of a specific disease, disorder and / or symptom. In the present invention, unless otherwise specified, it refers to the dosage to achieve the treatment, prevention, alleviation and / or relief of liver diseases, disorders and / or symptoms.

[0102] In the present application, "subject" is an animal, preferably a mammal, more preferably a human, and the subject includes but is not limited to consumers of health products and patients with diseases, disorders and / or symptoms. The subject in the present invention is preferably a mammal. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0103] In one example, the subject is a mammal.

[0104] In one example, the subject is a human or a mouse.

[0105] In the present application, "patient" refers to an animal, preferably a mammal, more preferably a human. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0106] It is understood that the present application also provides a method for treating liver failure based on a hepatic cell sheet derived from human embryonic stem cells, comprising:

[0107] Establish an APAP-induced liver failure mouse model and clarify the mouse's tolerance to abdominal surgery; this application successfully constructed a DILF mouse model that matches the clinical mortality rate by gavage with 750 mg / kg APAP after fasting for 12-15 hours. The model mice tolerate abdominal surgery 6 hours after modeling, which is suitable for subsequent experimental needs to study the effectiveness of in situ transplantation of hESC-HPLCs sheets in the treatment of DILF.

[0108] The hESC-HPLCs sheet prepared above was transplanted on the surface of the liver of DILF mice, and the efficacy of hESC-HPLCs sheet transplantation on DILF was evaluated by comprehensive comparison with the status of mice in the sham operation group, N-acetyl-L-cysteine ​​(NAC), and suspension treatment groups, as well as histopathological changes. And through histopathological analysis, its ability to integrate with the host was preliminarily explored;

[0109] The therapeutic efficacy of hESC-HPLCs tablets was evaluated by comparing experimental methods such as mouse survival rate, behavioral analysis, liver tissue H&E staining, and TUNEL staining. To further explore the effectiveness of hESC-HPLCs tablets, serum biochemistry and immunohistochemistry were used to detect liver function, inflammatory cell infiltration in liver tissue, and hepatocyte proliferation in mice at different time points after treatment. RT-qPCR technology was used to detect cell proliferation and expression of inflammation-related genes in liver tissue of mice at different time points after treatment. GO and KEGG enrichment analysis was performed on the proteins of hESC-HPLCs secretome to screen out key effector proteins or pathways that may affect liver tissue regeneration and repair. RT-qPCR technology and Western blot experiments were used to explore the key signal regulation mechanism in hESC-HPLCs tablets. In addition, H&E staining, immunohistochemistry, and ELISA experiments were used to evaluate the long-term function of hESC-HPLCs tablets in orthotopic transplantation in the treatment of acute drug-induced liver failure in mice.

[0110] In some embodiments, the number of hepatoid cell sheets for treating each mouse is 1×10 6 The temperature control dish used is 35mm Nunc TM UpCellTM Dish, bottom area 8cm 2 .

[0111] In the above steps, single clones were selected for screening after viral transfection, the established cell lines were expanded and cultured, and the expression of the NERCe002-A-Luc-ZsGreen exogenous gene was detected.

[0112] The method provided in this application is to prepare hESC-HPLCs sheets by inducing differentiation of hepatic-like cells from human embryonic stem cells, and to treat DILF mice modeled by APAP. The method comprehensively evaluates the therapeutic effect of suspension injection and hESC-HPLCs sheet transplantation in treating DILF mice through mouse survival analysis, serum biochemical index analysis, liver tissue pathology analysis, etc. The results show that hESC-HPLCs sheet transplantation significantly improves the survival rate of mice; compared with the sham operation group and the suspension treatment group, hESC-HPLCs sheet transplantation can reduce the degree of liver damage in mice and accelerate the recovery of damaged tissues, thereby accelerating the recovery process. Through macroscopic observation of the liver of transplanted mice and HE staining of tissue sections, the present application found that the graft can spontaneously adhere to the surface of the liver without suturing and other measures, and quickly establish vascular connections and integrate with host tissues, which provides a new idea for the use of the hESC-HPLCs sheet of this application to treat genetic metabolic diseases.

[0113] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0114] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0115] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0116] Example 1

[0117] 1. Providing and preparing hESC-HPLC slices

[0118] Directed differentiation culture of liver cells derived from human embryonic stem cells

[0119] 1. Recovery of hES cells: Take out the cells from the liquid nitrogen tank, thaw them immediately in a 37°C constant temperature water bath, centrifuge and discard the supernatant to remove dimethyl sulfoxide, add ES culture medium to resuspend the cell pellet, and plant it on the prepared mouse embryonic fibroblast feeder cells (feeder). Change the medium once a day and subculture once a week.

[0120] 2. Cultivation of hES cells: After testing to confirm that there is no mycoplasma or bacterial contamination, select hES cell clones with good growth, manually cut and subculture them onto new feeder cells. After subculture, change the medium every 24 hours and observe the cell growth every day.

[0121] 3. Induced differentiation of hES cells: When hES cells are in the best growth state on the feeder, the cells are manually cut and subcultured in a culture dish pre-coated with Matrigel. Cultivate the cells in the mTESR culture system for 2-4 days, switch to mouse fibroblast conditioned medium and then replace the induction medium after one day of culture. The specific induction process is as follows:

[0122] The first stage (3-6 days) is the induction of hES cells into defined endoderm:

[0123] The induction medium on days 1 to 2 was prepared by adding human activin A and Wnt3a to the basic medium RPTM-1640, wherein the concentrations of human activin A and Wnt3a were 100 ng / mL and 25 ng / mL, respectively.

[0124] The culture medium for days 3 to 4 is the basic culture medium RPTM-1640 supplemented with human activin A and fetal bovine serum, wherein the final concentration of human activin A is 100 ng / mL, and the final concentration of fetal bovine serum is 1.5%; the culture medium for days 5 to 6 is the basic culture medium RPTM-1640 supplemented with human activin A and fetal bovine serum, wherein the final concentration of human activin A is 100 ng / mL, and the final concentration of fetal bovine serum is 1.5%; there are many dead cells in the first few days of induction. In order to avoid the dead cells affecting the induction process, when changing the culture medium, try to aspirate the culture medium as much as possible and wash it one to two times with RPIM-1640 before changing the culture medium.

[0125] The second stage (6-12 days) is directed differentiation to the liver lineage: the culture medium used in the first 1-4 days of the second stage is the basic culture medium KO / DMEM with 50ng / mL keratinocyte growth factor and a final concentration of 2.5% fetal bovine serum added. During the second stage of culture, the number of cells has changed significantly compared with the previous few days. At this time, the culture medium should be added as appropriate according to the number of cells to ensure normal growth of the cells;

[0126] The culture medium for the next 5 to 12 days consisted of a basal medium KO / DMEM supplemented with serum replacement (SR), L-glutamine, non-essential amino acids, β-mercaptoethanol and dimethyl sulfoxide. The culture medium was changed every 24 hours, with the final concentration of serum replacement being 20%, the final concentration of L-glutamine being 2 mM, the final concentration of non-essential amino acids being 10%, the final concentration of β-mercaptoethanol being 0.1 mM, and the final concentration of dimethyl sulfoxide being 1%.

[0127] The third stage (6 to 10 days) is to promote the maturation of hepatocytes: the culture medium is the basic culture medium L-15 with bovine serum, hepatocyte growth factor, oncostatin, dexamethasone, and L-glutamine added, and the culture medium is replaced every 24 hours. The final concentration of fetal bovine serum is 10%, the final concentration of hepatocyte growth factor is 150 ng / mL, the final concentration of oncostatin is 75 ng / mL, the final concentration of dexamethasone is 0.8 μM, and the final concentration of L-glutamine is 2.5 mM.

[0128] The entire induction process lasted 15-28 days. NERCe002-A-Luc-ZsGreen then combined the harvested hESC-HPLCs with cell sheet engineering technology, prepared hESC-HPLCs sheets using the temperature-responsive culture dish NUNC UP Cell, and performed related identification such as frozen section HE staining and immunofluorescence.

[0129] Figure 1 These are images of hESCs under the feeder layer system and the cell morphology at various stages during the process of hESCs being induced to differentiate into hepatocytes (gray is light microscopy image, green is ZsGreen fluorescence image). Figure 1 A in the figure shows chHES-90-Luc-ZsGreen growing in oval-shaped colonies on the feeder layer (scale bar: 400 μm). Figure 1 B in the figure is NERCe002-A-Luc-ZsGreen, which is hESCs cultured in a feeder-free system; I: cell morphology of induction stage I (definitive endoderm); II: cell morphology of induction stage III (liver progenitor cells); III: cell morphology of induction stage IV (maturation) (scale bar: 200 μm);

[0130] 4. Establish an APAP-induced liver failure mouse model and determine the mouse's tolerance to abdominal surgery;

[0131] The dosage (35 μL / Kg) was calculated according to the weight of the mice. Different concentrations of drugs were set (Control group: DPBS containing 1.6% DMSO; low-dose group: 500 mg / kg APAP; medium-dose group: 750 mg / kg APAP; high-dose group: 1000 mg / kg APAP) and the drug was administered orally to model the disease.

[0132] The obtained hESC-HPLCs sheet was transplanted on the surface of the liver of DILF mice, and the efficacy of hESC-HPLCs sheet transplantation on DILF was evaluated by comprehensively comparing the mouse status and tissue pathological changes of the sham operation group, suspension treatment group, and N-acetyl-L-cysteine ​​(NAC) treatment group. And through tissue pathological analysis, its ability to integrate with the host was preliminarily explored;

[0133] Specifically, the mice were first anesthetized with isoflurane, and then the abdominal hair of the mice was removed; the body of the mice was wiped with alcohol gauze, and then placed on a fixed plate in a biosafety cabinet, with the abdomen of the mice facing up, the limbs fixed with tape, and the nose placed in a gas anesthesia mask. The abdomen of the mice was disinfected with alcohol, and an opening of about 1 cm was cut along the middle with ophthalmic scissors, and then the abdominal wall was cut along the linea alba in the middle of the abdomen to expose the abdominal cavity for relevant intraperitoneal operations; the abdominal wall of the mice was sutured with 5-0 sutures, and then the skin of the mice was sutured with 4-0 sutures; the abdomen of the mice was disinfected with iodine tincture, and then it was gently put back into the cage and observed until it woke up. The time of gavage of APAP to mice was recorded as 0h, and observation was performed 30min, 12h, 16h, 20h, 24h, 30h, 36h, 48h, 60h, and 72h before and after gavage. If any mice died, the time of death was accurately recorded. Mice that survived after 72h of observation were recorded as alive. Three mice surviving 72 hours after modeling were selected from different groups of mice for mouse tissue sampling, paraffin embedding, paraffin sectioning, and HE staining.

[0134] 2. Reagents and equipment used

[0135] hESC (human embryonic stem cell): select human embryonic stem cell NERCe002-A-Luc-ZsGreen from the stem cell bank of the National Engineering Research Center for Human Stem Cells.

[0136] Cell culture reagents:

[0137] Human activin A (R&D, 338-AC-050, R&D Systems, Catalog No. 338-AC-050, the same applies below), Wnt3a (R&D, 1324-WN-010), fetal bovine serum (Life, 10099141), keratinocyte growth factor (KGF) (R&D, 251-KG-050), serum replacement (Life, N10828-028), L-glutamine (Gibco, 250 30-081), non-essential amino acids (Gibco, 1140-050), β-mercaptoethanol (Gibco, 21985023), dimethyl sulfoxide (DMSO) (Sigma, D2650), hepatocyte growth factor (HGF) (R&D, 294-HG-025), oncostatin (OSM) (R&D, 295-OM-050), dexamethasone (Calbiochem, 265005), mTESR (Stem cell, 05850), RPTM-1640 (Life, 11875-085), KO / DMEM (Life, 10829-018), L-15 (Life, 21083-027), Matrigel (BD, 354234).

[0138] Experimental consumables:

[0139] Temperature-controlled dish, support film, suture needle with thread, syringe, gavage needle, surgical instruments, centrifuge tube, gauze, cotton swab, blade, slide, cover glass, tissue embedding box.

[0140] 1. Establishment of hESC lines stably expressing Luc and ZsGreen green fluorescent proteins

[0141] 1.1 Lentiviral packaging

[0142] (1) Subculture 293FT cells into cell culture dishes and start the experiment when the cell confluence reaches about 70%;

[0143] (2) Change the mTeSR1 medium 4 h before transfection;

[0144] (3) Add 6 μg of pCMV-8.91 plasmid, 2 μg of pMD2VSV-G plasmid, and 8 μg of the plasmid containing the target gene into a centrifuge tube containing 1.2 mL of Opti-MEM and mix well, labeled as tube 1;

[0145] (4) Add 48 μL of Lip-2000 to another centrifuge tube containing 1.2 mL of Opti-MEM, labeled as tube 2, mix gently, and let stand at room temperature for 5 min;

[0146] (5) Add tube 1 to tube 2, mix gently, and let stand for 20 minutes;

[0147] (6) Add the mixture to 293FT cells and culture at 37°C. Replace with 9 mL of fresh culture medium after 6 to 12 hours and continue culturing for 48 hours.

[0148] (7) Filter the supernatant using a 0.45 μm filter, aliquot and store at -80°C.

[0149] 1.2 Construction of hESCs lines stably expressing Luc

[0150] (1) Digest the cells into single cells using Accutase;

[0151] (2) Resuspend the cells in 0.5 mL of mTeSR1, add an equal amount of lentiviral supernatant containing Luc, mix well, and add polybrene to a final concentration of 8 μg / mL;

[0152] (3) The cell mixture was placed in a well of a Metrigel-coated dish and cultured for 7 days. The cells were screened with 2 μg / mL puromycin and a cell line was established and named chHES-90-Luc.

[0153] 1.3 Construction of hESC lines stably expressing Luc and ZsGreen green fluorescent proteins

[0154] (1) Use Accutase to digest chHES-90-Luc cells that are growing well at 37°C into single cells;

[0155] (2) Resuspend the cells in 0.5 mL of mTeSR1, add an equal amount of lentiviral supernatant containing ZsGreen, mix well, and add polybrene to a final concentration of 8 μg / mL;

[0156] (3) The cell mixture was placed in a well of a Metrigel plate and continued to be cultured. The cells were screened with 2 μg / mL puromycin for 7 days. Single clones with spontaneous ZsGreen fluorescence were selected under a fluorescence microscope for further expansion and culture. The cultured cells were re-established and named chHES-90-Luc-ZsGreen.

[0157] The hESC-HPLCs slices were successfully prepared and can be used for the next experiment after identification.

[0158] 3. Specific verification is as follows:

[0159] 1.1 hESC-HPLC gene expression detection

[0160] (1) Cell RNA extraction

[0161] Collect the corresponding cells, add 1mL of TRIZOL, and lyse the cells. Add 0.2mL of chloroform to every 1mL of TRIZOL and incubate at room temperature for 10 minutes. Centrifuge at 13000×g for 15 minutes at 4℃. After centrifugation, the mixture is divided into three layers, and RNA is present in the water layer. Transfer the water layer to a clean test tube. Add the same volume of isopropanol as the water phase and mix thoroughly. Incubate the mixed sample at room temperature for 5 minutes and centrifuge at 8000rpm for 5 minutes at 4℃. Remove the upper suspension. Wash the RNA precipitate once with 75v / v% (i.e., the volume fraction of ethanol is 75%, the same applies below) ethanol, and centrifuge at 10000rpm for 3 minutes at 2℃~8℃. Simply dry the RNA precipitate (air dry for 5-10 minutes). Dissolve it with RNase-free DEPC water, and detect the RNA concentration by UV spectrophotometer after complete dissolution.

[0162] (2) cDNA reverse transcription

[0163] Take 1μg total RNA, 1μL oligo(d T)primer, add RNase-free water to make the final reaction volume 10μL in an EP tube, mix well and centrifuge, place in a PCR instrument for the first step of reaction: 65℃, 10min, then quickly cool on ice. Then add 4μL 5X reverse transcription reaction buffer, 2μL 10mM dNTP, 0.5μL RNase inhibitor, 0.5μL Transcriptor Reverse Transcriptase, H 2 O 3μL, gently mix, quickly centrifuge, and place in a PCR instrument at 55℃ for 1h, 85℃ for 5min, and 4℃+∞. The obtained cDNA is stored at -20℃ or continued to PCR amplify the target gene.

[0164] Then RT-PCR was performed using cDNA as a template to detect the expression of related genes OCT4, NANOG, HNF4α, AFP, RBP4, and ALB. PCR reaction conditions: 94°C pre-denaturation for 5 min; 40 cycles (95°C for 10 seconds, 58°C for 10 seconds, and 72°C for 10 seconds). Specific primers are shown in Table 1:

[0165] Table 1

[0166]

[0167]

[0168] Figure 2 This is the expression of related genes after hESCs were induced into hepatocytes and hepatocyte sheets were prepared in the examples of this application.

[0169] 1.2 hESC-HPLC protein expression detection

[0170] The hESC-HPLCs induced by differentiation were identified by protein fluorescence and antigen staining, including HNF4α, AFP, and ALB.

[0171] Immunofluorescence staining steps:

[0172] (1) Observe the cell morphology under a microscope. If the cell morphology is uniform and in good condition, you can start the subsequent experiments.

[0173] (2) Remove the original culture medium in the culture dish to be tested and add 1 mL of DPBS to quickly wash the cells twice;

[0174] (3) Add 0.5 mL of 4% PFA to each dish and fix at room temperature for 15 min;

[0175] (4) Discard the paraformaldehyde fixative and quickly wash the cells twice with DPBS;

[0176] (5) Add 0.5 mL of 0.1% Triton-X-100 permeabilization solution and incubate at room temperature for 10 min;

[0177] (6) Discard the permeabilization solution and rinse the cells twice with DPBS, each wash for 3 min;

[0178] (7) Add 5% donkey serum blocking buffer and block at room temperature for 60 min;

[0179] (8) Discard the blocking solution and add the primary antibody working solution prepared with donkey serum blocking solution (1:200 for HNF4α, 1:200 for AFP, and 1:100 for ALB) and incubate at 4°C overnight.

[0180] (9) After rewarming, discard the primary antibody and wash with DPBS three times for 5 min;

[0181] (10) All subsequent steps must be performed in the dark. Add the secondary antibody working solution (1:1000) prepared with donkey serum blocking solution and incubate at room temperature for 60 min.

[0182] (11) Discard the secondary antibody and wash with DPBS three times, 5 min each time;

[0183] (12) Add DAPI working solution (1:1000) to stain the cell nucleus and incubate at room temperature in the dark for 5 min;

[0184] (13) Discard DAPI and rinse with DPBS three times for 3 min;

[0185] (14) Finally, add 1 mL of DPBS and observe under a fluorescence microscope.

[0186] Figure 3 The expression of hepatocyte-related marker proteins ALB, AFP, and HNF4α in hESC-HPLCs of the present application example.

[0187] 1.3 Functional identification of hESC-HPLCs

[0188] The present application performs glycogen staining on the induced hESC-HPLCs to detect their ability to store glycogen; performs oil red staining to detect their ability to store lipids; and also performs ICG experiments to detect their ability to metabolize exogenous substances. Figure 4 This is the identification of hepatocyte-related functions in hESC-HPLCs in the examples of this application.

[0189] 1.4 Preparation of hESC-HPLC slices

[0190] When the hESC-HPLCs seeded on the temperature-responsive culture dish were confluent and established tight cell-cell connections, the old culture medium at 37°C was replaced with the culture medium at 20°C, and then the process of cells on the temperature-responsive culture dish falling off in the form of cell sheets and being harvested after cooling was observed and recorded. Figure 5 This is the preparation and harvesting process of hESC-HPLCs slices in the present application example. The scale bar is 1 mm.

[0191] (II) Identification of hESC-HPLCs

[0192] The harvested hESC-HPLCs slices were cryosectioned, and the structure of the longitudinal section of the hESC-HPLCs slices was observed by HE staining of the cryosections. The hESC-HPLCs slices were then further identified by immunofluorescence staining of hepatocyte surface markers on the cryosections. Figure 6 This is the immunofluorescence identification of ALB, AAT, and HNF4α of the hESC-HPLCs slice in the present application example, scale bar 100 μm;

[0193] The results showed that the harvested cell sheets showed a uniform and consistent white opaque disc structure. HE staining showed that the hESC-HPLCs cell sheets showed a single layer structure. Immunofluorescence analysis further confirmed that the mature hepatocyte markers ALB and AAT were positively expressed in these cell sheets. This indicates that the hESC-HPLCs cell sheets we prepared have the ability to synthesize ALB and AAT.

[0194] (III) APAP gavage in mice to establish an acute drug-induced liver failure model and perform hESC-HPLCs sheet transplantation

[0195] The specific operation method is as follows:

[0196] 1. Oral administration of mice:

[0197] The mice were fasted the night before gavage. The mice were weighed the next day. The gavage dose (30 μL / Kg) was calculated based on the weighing results. The prepared mouse gavage needle was installed on a 1 mL syringe to extract the completely dissolved APAP for later use.

[0198] Place the mouse on an appropriate pad or cage to ensure its safety and comfort. Use a syringe equipped with a gavage needle to insert the needle from the corner of the mouse's mouth. After lifting the tongue, insert the needle downward along the posterior wall of the upper palate. Carefully insert the gavage needle into the mouse's mouth and guide it toward the stomach. Confirm that APAP is completely injected into the mouse's stomach. If there is no air reflux during withdrawal, the injection resistance is small, and the mouse swallows, it proves that the drug solution has been successfully injected. Slowly remove the gavage needle, then observe the mouse's reaction and behavior, pay attention to whether the mouse has adverse reactions or abnormal symptoms such as shortness of breath, limb twitching, foaming at the mouth, etc., and record the gavage time, dose, and the mouse's state and reaction. Figure 7 This is a comparison of the survival rates of modeled mice in different APAP dosage groups in the examples of this application.

[0199] The results showed that the mice in the solvent group (n=11) and the low-dose group (500 mg / kg, n=6) all survived, with a survival rate of 100%; the survival rate of the medium-dose group (750 mg / kg, n=8) was 62.5%; the survival rate of the high-dose group (1000 mg / kg, n=9) was 33.3%. Log-rank (Mantel-Cox) test showed that there was a significant statistical difference in the survival rate of each group (P=0.0015).

[0200] 2. Surgical transplantation of hESC-HPLCs sheets:

[0201] The time of intragastric administration was defined as 0 h, and the corresponding surgical procedures were performed on the mice in the sham operation group and the ZS-hESC-HPLCs sheet transplantation group at 4 to 6 h.

[0202] Transplantation method

[0203] (1) The hESC-HPLC slices were harvested by cooling the mouse to 20°C. The mouse was anesthetized with isoflurane and the skin of the surgical site was prepared. After disinfection, an opening with a diameter of 1 cm was made in the mouse abdomen with ophthalmic scissors to expose part of the liver.

[0204] (2) Use the supporting membrane provided with the dish to stick the hESC-HPLCs sheet in the dish, and stick it to the liver surface of the mice in the hESC-HPLCs sheet transplantation group and leave it for 5-10 minutes, then carefully peel off the supporting membrane, leaving the hESC-HPLCs sheet adhered to the liver surface. In the sham operation group, stick the supporting membrane without the cell sheet to the mouse liver surface, leave it for 5-10 minutes, then peel off the supporting membrane; perform the operation carefully, move gently and avoid excessive pulling;

[0205] (3) After the operation, the peritoneum was sutured with a 5-0 suture needle and the skin was sutured with a 4-0 suture needle. Simple suture was used to ensure that the suture was firm.

[0206] (4) Transfer the mouse to a quiet, warm recovery box and continue to monitor the mouse's breathing, body temperature, and recovery.

[0207] 3. Analysis of mouse survival rate

[0208] Modeling survival rate + survival status score:

[0209] (1) The end of intragastric administration was taken as the time point, recorded as 0 h, and the time point for observing the behavioral status was determined: the status of the mice was recorded before and after APAP intragastric administration, and the behavioral status of the mice was recorded at specific time points within three days after intragastric administration;

[0210] (2) Ask an observer who is unaware of the experimental groups to evaluate the state of the mice at each time point according to the state scoring table in the literature, and record the following conditions: the degree of pain in the expression, the bulge of the nose, whether the eyes are closed, whether the ears are erect, and the degree of activity;

[0211] (3) Record the time of mouse death and record the survival status of mice after 72 hours of observation. Figure 7 This is a comparison of the survival rates of modeled mice in different APAP dosage groups in the examples of this application. Figure 8 The observation scores of the survival status of mice in different dosage groups in the examples of this application;

[0212] 4. Pathological analysis of mouse liver tissue:

[0213] After 72 h of observation, liver tissues of mice in each group were taken for histopathological section analysis. Fig. 9 This is a graph showing the results of mouse liver tissue pathology analysis 72 hours after modeling in the embodiment of this application. Fig.10 This is an analysis of the survival rate in the tolerance assessment of mouse surgery in the examples of this application. Fig.11 This is the mouse status score in the surgical tolerance assessment of the present application example, Fig.12 This is a survival rate analysis in the effectiveness evaluation of hESC-HPLCs sheets in treating APAP-induced DILF mice in the present application example.

[0214] The results showed: By analyzing the liver tissue pathology of each group of mice after 72 hours, we found that the degree of liver damage increased significantly with the increase in dose. The low-dose group mainly showed punctate and focal necrosis, the medium-dose group showed sheet and band necrosis and mild hydropic degeneration, and the high-dose group showed central necrosis and extensive ballooning degeneration. This is consistent with the differences in survival rates of mice in each group. The survival rate and mouse status scores in the tolerance assessment also indicate that the trends of status changes of the two groups of mice are consistent and there is no significant difference. This shows that the model mice tolerate abdominal surgery 4-6 hours after modeling, and are suitable for subsequent experimental needs for studying the effectiveness of hESC-HPLCs tablets in treating DILF. Fig.12 The analysis results of the survival rates of each treatment group showed that transplantation of hepatoid cell sheets could significantly improve the survival rate of mice.

[0215] (IV) Analysis of therapeutic efficacy of hESC-HPLCs tablets

[0216] Mouse liver morphology observation, blood collection and tissue sampling:

[0217] Taking oral administration as 0h, the morphology of mice in each group was observed at 0h, 4-6h, 12h, 24h, 48h, 72h, 7D, and 14D, and various tissues were collected. Blood samples of mice in each group were collected at 0h, 4-6h, 12h, 24h, 72h, and 7D:

[0218] (1) At each time point, at least three mice were randomly selected from each group of surviving mice and anesthetized by intraperitoneal injection of 0.2 mL of 4% chloral hydrate.

[0219] (2) Cut open the abdomen of the mouse (near the diaphragm) to fully expose the liver, and quickly observe and photograph its morphology.

[0220] (3) The stomach and intestine tissues of the mice were opened to expose the right kidney and the vein near it. Blood samples were collected from each group of mice using a procoagulant tube. The samples were allowed to stand at room temperature for 30 to 60 minutes and then centrifuged at 13,000 rpm for 15 minutes. Part of the supernatant was collected and sent for liver function testing or frozen at -80°C for later use.

[0221] (4) The heart, liver, spleen, lung, kidney, large intestine, small intestine, stomach, and brain tissues of each group of mice were quickly and gently collected in triplicate under light-proof conditions. One portion was fixed with 4% PFA (24 h), and the remaining two portions were sealed and frozen at -80°C for later use.

[0222] Fig.13 The gross changes in the liver of mice in different treatment groups in the examples of this application; Fig.14 The pathological results of the liver of mice in different treatment groups in the examples of this application; Fig.15 This is the TUNEL staining analysis of the liver tissues of mice in the different treatment groups in the examples of this application; Fig.16Analysis of serum biochemical changes in mice in different treatment groups in the examples of this application; Fig.17 qPCR detection of PI3K-AKT-mTOR signaling pathway in liver tissue of mice in different treatment groups in the examples of this application; Fig.18 Western blot was used to detect the PI3K-AKT-mTOR signaling pathway in the liver tissues of mice in different treatment groups in the examples of this application; Fig.19 This is the H&E staining of the mouse liver + hESC-HPLCs slice 30 days after the transplantation of the hepatoid cell slice in the example of this application. Fig. 20 This is a graph showing the hALB immunohistochemical staining results of mouse liver + hESC-HPLCs slice 14 days after the transplantation of the hepatoid cell sheet in the example of this application.

[0223] The results showed that hESC-HPLCs sheets may continue to differentiate into mature functional cells to replace damaged cells or play a compensatory role after in situ transplantation through cell replacement. In addition, they can also activate the PI3K-AKT-mTOR signaling pathway through paracrine secretion of multiple bioactive factors, thereby promoting the regeneration of damaged liver tissue, while inhibiting the function of inflammatory cells in serum, inhibiting the attack of immune cells on liver cells, preventing continuous liver damage, and promoting liver function repair.

[0224] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for preparing a hepatoid cell sheet derived from human embryonic stem cells, characterized in that: include: Providing human embryonic stem cells, inducing the human embryonic stem cells to differentiate into defined endoderm, and collecting the induced cells; Then, directing differentiation of the induced cells into hepatic cells; and, The cells after directed differentiation are cultured until maturity, and a hepatocyte-like cell sheet derived from human embryonic stem cells is prepared using a temperature-responsive culture container.

2. The preparation method according to claim 1, characterized in that: Inducing differentiation of the human embryonic stem cells into defined endoderm comprises: The human embryonic stem cells are first cultured in a first culture medium for 1 to 2 days, and then cultured in a second culture medium for 2 to 4 days; The first culture medium comprises RPMI-1640 culture medium, human activin A and Wnt3a; The second culture medium comprises RPMI-1640 culture medium, human activin A and fetal bovine serum.

3. The preparation method according to claim 2, characterized in that: The first culture medium comprises RPMI-1640 culture medium, human activin A with a final concentration of 5 ng / mL to 300 ng / mL, and Wnt3a with a final concentration of 20 ng / mL to 100 ng / mL; and / or, The second culture medium comprises RPMI-1640 culture medium, human activin A with a final concentration of 10 ng / mL to 200 ng / mL, and fetal bovine serum with a final concentration of 0.2% to 2%.

4. The preparation method according to claim 1, characterized in that: Directing differentiation of the induced cells into hepatic cells comprises: The induced cells are first cultured in the third culture medium for 6 to 12 days, and then cultured in the fourth culture medium for 5 to 12 days; The third culture medium comprises KO / DMEM culture medium, keratinocyte growth factor and fetal bovine serum; The fourth culture medium comprises KO / DMEM medium, as well as serum replacement (SR), L-glutamine, non-essential amino acids, β-mercaptoethanol, and dimethyl sulfoxide.

5. The preparation method according to claim 4, characterized in that: The third culture medium comprises KO / DMEM culture medium, keratinocyte growth factor with a final concentration of 15 ng / mL to 100 ng / mL, and fetal bovine serum with a final concentration of 2% to 3%; The fourth culture medium comprises KO / DMEM culture medium, and serum replacement (SR) with a final concentration of 18% to 22%, L-glutamine with a final concentration of 1mM to 3mM, non-essential amino acids with a final concentration of 0.1% to 15%, β-mercaptoethanol with a final concentration of 0.1mM to 0.12mM, and dimethyl sulfoxide with a final concentration of 0.2% to 2%.

6. The preparation method according to any one of claims 1 to 5, characterized in that: Cultivating the directed differentiated cells to maturity comprises culturing in the fifth culture medium for 6 to 10 days; The fifth culture medium comprises L-15 culture medium, bovine serum, hepatocyte growth factor, oncostatin, dexamethasone and L-glutamine; Optionally, the fifth culture medium comprises L-15 culture medium and fetal bovine serum with a final concentration of 8% to 12%, hepatocyte growth factor with a final concentration of 10 ng / mL to 300 ng / mL, oncostatin with a final concentration of 5 ng / mL to 150 ng / mL, dexamethasone with a final concentration of 0.05 μM to 1.2 μM and L-glutamine with a final concentration of 2 mM to 3 mM.

7. The preparation method according to any one of claims 1 to 5, characterized in that: Preparation of human embryonic stem cell-derived hepatocyte-like cell sheets using a temperature-responsive culture vessel includes: The mature human embryonic stem cell-derived hepatocytes are digested into single cells, inoculated into a temperature-responsive culture container, and cultured for 3 to 4 days. After the human embryonic stem cell-derived hepatocytes in the temperature-responsive culture container establish a connection, the cells automatically separate from the bottom of the container in the form of cell sheets, thereby preparing human embryonic stem cell-derived hepatocytes sheets.

8. The preparation method according to any one of claims 1 to 5, characterized in that: The preparation method has one or more of the following technical features: (1) Replace the culture medium every 22 to 26 hours; (2) When changing the culture medium, wash 1 to 2 times with RPIM-1640. 9 . A hepatoid cell sheet derived from human embryonic stem cells prepared by the method for preparing a hepatoid cell sheet derived from human embryonic stem cells according to any one of claims 1 to 8 .

10. Use of the hepatic cell sheet derived from human embryonic stem cells prepared by the method for preparing the hepatic cell sheet derived from human embryonic stem cells according to any one of claims 1 to 8 in preparing a drug for preventing and treating liver diseases; Optionally, the drug for preventing or treating liver diseases prevents or treats acute drug-induced liver failure.

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