Method for preparing extrahepatic vesicles / apoptosis vesicles by inducing fibroblasts to be directly reprogrammed into hepatocyte-like cells and application of extrahepatic vesicles / apoptosis vesicles

Abstract

The invention relates to the field of cell reprogramming, in particular to a method for preparing hepatic extracorporeal vesicles / hepatic apoptosis vesicles by inducing fibroblasts to be directly reprogrammed into hepatic-like cells by using a chemical small molecule composition without introducing any exogenous gene / transcription factor / MicroRNA gene, RNA, protein, polypeptide and other inducing factors and application of the hepatic extracorporeal vesicles / hepatic apoptosis vesicles. A cell reprogramming technology and an extracellular vesicle technology are combined, and liver apoptosis vesicles prepared from a hepatocyte-like cell source are directly reprogrammed through chemical induction to resist hepatic fibrosis. The small molecule composition can be added with normal saline, a buffer solution, a cell basal culture medium and the like to prepare a transdifferentiated hepatocyte-like cell reagent or culture medium; the obtained external hepatic vesicles / hepatic apoptosis vesicles can be used for preparing anti-hepatic fibrosis biological preparations for scientific research, medicine and clinic.

Description

Technical Field

[0001] The present invention relates to the interdisciplinary field of cell biology, stem cell biology (cell reprogramming), medicine, and pharmacy. Specifically, the present invention relates to a method for preparing extrahepatic vesicles / apoptotic vesicles by inducing direct reprogramming (also known as transdifferentiation) of fibroblasts into hepatoid cells using a chemical small molecule composition without introducing or using any exogenous genes / transcription factors / MicroRNA (miRNA) genes, or genes combined with vectors, or induction factors such as RNA, proteins, and polypeptides thereof, or adding or using any cytokines or growth factors. Background Art

[0002] The liver is one of the largest internal organs in the human body. It maintains the homeostasis of the human body's internal environment by participating in a variety of physiological processes such as metabolism, protein synthesis, detoxification, and secretion of cytokines and growth factors. Chronic liver damage caused by various causes leads to liver fibrosis, which can further develop into end-stage liver diseases such as cirrhosis, liver failure, and liver cancer, causing a serious health burden worldwide and resulting in approximately 2 million deaths each year.

[0003] Long-term persistence of fibrosis will develop into cirrhosis, and in the later stages of cirrhosis, liver failure and even liver cancer will occur, seriously endangering the patient's health. Decompensated cirrhosis is considered irreversible, but recent studies have found that liver fibrosis changes dynamically in both directions, and can develop and worsen, or reverse and regress. Early intervention in the process of liver fibrosis can effectively reverse liver fibrosis, so anti-fibrosis treatment is of great significance for delaying the progression of the disease and reducing or preventing the occurrence of cirrhosis, liver failure or liver cancer. Effective control of liver fibrosis has become a key link in the treatment of chronic liver diseases. Although people have a deeper understanding of the occurrence and development mechanism of liver fibrosis, there is still a lack of effective anti-liver fibrosis drugs and treatments in clinical practice, so finding new treatment methods has important clinical application value and significance.

[0004] Hepatocyte transplantation for the treatment of liver fibrosis is currently a hot topic of concern and research, but there is a lack of hepatocyte sources and defects or difficulties in the hepatocyte transplantation pathway. Therefore, exploring new hepatocyte sources and using stem cell extracellular vesicles or somatic cell extracellular vesicles to treat liver fibrosis has become an international research hotspot.

[0005] Technical methods for obtaining hepatocyte sources:

[0006] At present, there are mainly the following ways to obtain functional hepatocytes: 1) Isolation of primary hepatocytes from donor liver; 2) Directed differentiation of stem cells or induced pluripotent stem cells into hepatocytes; 3) Transdifferentiation of somatic cells such as fibroblasts into hepatocytes. The application of primary hepatocytes isolated from donor liver is limited due to the severe shortage of liver sources. Directed induction and differentiation of stem cells to obtain functional hepatocytes has become the most effective means and way to obtain a sufficient number of hepatocytes because of the unlimited proliferation potential of stem cells. However, the existing directed differentiation methods have one or more of the following defects: such as low differentiation efficiency, low conversion purity, lack of function of differentiated hepatocytes, possible immune rejection, incomplete differentiation of stem cells and potential carcinogenic risks, and high cost, which cannot meet clinical needs.

[0007] Direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes avoids the need for reprogramming of pluripotent stem cells into iPS cells (Induced pluripotent stem cell) and its potential risks of application, and has attracted attention. However, most of the existing methods for transdifferentiating fibroblasts into hepatocytes still use the introduction of exogenous genes / transcription factors / / MicroRNA (miRNA) genes, and other induction factors, combined with chemical small molecules, various cytokines or growth factors and other alternative factors, to form a variety of induction factor compositions to induce fibroblast transdifferentiation into hepatocytes. Because the introduction of exogenous genes or transcription factors destroys the original gene structure, there is a risk of carcinogenesis or pathogenicity, which makes it still difficult to apply clinically; and there are also defects such as complex operation, increased experimental links, difficult quality control, increased costs, and increased risks of clinical application. Therefore, how to avoid the introduction of induction factors such as exogenous genes / transcription factors / MicroRNA genes, only use chemical small molecules, chemically induce fibroblasts to directly reprogram into hepatocytes, and overcome the defects of the above-mentioned methods of introducing exogenous genes / transcription factors / MicroRNA genes to induce directed differentiation, iPS cell reprogramming, or transdifferentiation of hepatocytes, is a bottleneck problem that needs to be solved in the clinical application of hepatocytes. As for fibrotic diseases caused by abnormal fibroblasts or fibrocytes, such as cirrhosis (liver fibrosis), there is currently a lack of effective therapeutic drugs and methods in clinical practice, and there is an urgent need to develop effective new methods and new drugs.

[0008] Cell reprogramming is the conversion of cells from one type to another. It is a process of changing cell fate by targeted induction and regulation of specific cell signaling pathways or epigenetic modifications. Epigenetics refers to the fact that the DNA sequence / structure does not change, but gene expression undergoes heritable changes. Induced cell reprogramming includes: (1) reprogramming of induced pluripotent stem cells (iPS cells) to reverse differentiated cells and restore them to a pluripotent or totipotent state; (2) direct reprogramming of cells that directly transform from one differentiated cell type to another differentiated cell type without going through the pluripotent stem cell stage (also known as transdifferentiation / lineage reprogramming).

[0009] Chemically induced direct cell reprogramming (transdifferentiation) is a process in which one differentiated cell is directly transformed into another differentiated cell without introducing or using any exogenous genes / transcription factors / MicroRNA (miRNA) genes, and their induction factors such as RNA, proteins and peptides; only chemical small molecules are used to target and induce the regulation of cell signaling pathways and epigenetic modifications, so as to change their epigenetic modifications and thus change the gene expression profile of the cell.

[0010] Since Japanese scientist Shinya Yamanaka used viral vectors to introduce an induction combination of four transcription factors (genes) Oct4 (also known as Oct, Oct3, or OCT-3 polypeptide expression factor, etc.), Sox2, Klf4 and c-Myc into differentiated somatic cells in 2006 to obtain iPS cells through induced pluripotent stem cell reprogramming, the exogenous introduction of transcription factors / genes has the risk of destroying the stability of the original gene sequence of the cell, leading to the risk of mutation and carcinogenesis, as well as the defects of low cell reprogramming conversion rate, poor stability and complex operation. Therefore, the method of induced cell reprogramming has been continuously improved and has gradually been changed to non-integrated cell reprogramming. In addition to the introduction of only 1-2 exogenous transcription factors for induction, the combination of MicroRNA (miRNA) genes and their RNA, protein peptides, chemical small molecules, cytokines, growth factors and other alternative transcription factors constitutes a multi-factor induction composition to induce cell reprogramming; and the introduced exogenous transcription factors / genes must first be introduced into the induction starting cells using gene vectors during the reprogramming process, and other components that do not require the use of gene vectors, such as chemical small molecules, various growth factors, cytokines, etc., are induced at the same time and under the same culture conditions to reprogram the target cells. Therefore, the introduced exogenous induction transcription factors / genes, due to their irreplaceability, are still the necessary and most important induction factor components of the induction reprogramming composition.

[0011] Since chemical small molecules (referred to as small molecules) are targeted molecular compounds, they can target, induce and regulate specific signal pathways and epigenetics to transform one cell into another; and they have good drugability, low cost, good stability and simple operation, making them the best candidates for replacing induced transcription factors; therefore, obtaining transdifferentiated liver-like cell sources through direct chemical-induced cell reprogramming has become one of the important goals in the field of stem cell research.

[0012] In recent years, the research on the use of small molecule compounds to induce hepatocytes has made great progress. Studies have shown that mouse fibroblasts can be reprogrammed into hepatocytes by combining a single transcription factor (Foxa1, Foxa2, or Foxa3) with a combination of small molecule compounds (CHIR99021, RepSox, VPA, Parnate, TTNPB and Dznep). A recent study showed that mouse embryonic fibroblasts can be reprogrammed into hepatocytes by using only a combination of small molecule compounds. However, the conversion efficiency of this conversion scheme is low (0.2-15%), and it takes a long time (35-60 days). The cells obtained are mixed cell populations, among which the hepatocytes have the ability to proliferate in vitro, which is more in line with the biological behavior of hepatoblasts rather than mature hepatocytes.

[0013] Hepatic extracellular vesicles / hepatic apoptotic vesicles and fibrosis:

[0014] Extracellular vesicles (EVs) refer to various types of vesicles with membrane structures secreted by cells. Under the electron microscope, they are cup-shaped or spherical double-layer lipid membrane structures, which contain many bioactive components, such as proteins, nucleic acids, lipids, etc. More and more studies have found that EVs can carry and transmit information molecules to adjacent cells or distant cells, thereby regulating the physiological and pathological states of cells and participating in the occurrence and development of many important diseases. According to the formation process, biological characteristics and particle size, extracellular vesicles are mainly divided into three categories: apoptotic bodies (apoBDs), exosomes (Exosomes) and microvesicles (Microvesicles, MVs). Compared with cell transplantation, EVs infusion has the following advantages: 1) It is smaller than parent cells and easier to operate; 2) It is easy to produce and store; 3) It has low immunogenicity and can minimize the triggering of innate and adaptive immune responses; 4) It avoids the risk of tumor formation. Previous vesicle research has mostly focused on vesicles secreted by cells in the survival state, such as exosomes. Studies have found that more than billions of cells in the human body undergo apoptosis every day to maintain the body's physiological homeostasis. During apoptosis, the cell membrane shrinks and invaginates, encapsulating cytoplasm, organelles and nuclear fragments, and producing a large number of endogenous apoptotic vesicles (apoVs). Previous studies on apoVs have focused on apoBDs (1-5μm in diameter). More and more studies have found that smaller vesicles are produced during apoptosis, including apoptotic microvesicles (apoMVs) (0.1-1μm in diameter) and apoptotic exosomes (apoExos) (<150nm in diameter), which together constitute apoVs. In a variety of physiological and pathophysiological processes, apoVs are gradually being found to play an important role, such as regulating immune responses, stem cell-mediated tissue renewal and regenerative therapy.

[0015] At present, many advances have been made in the exploration of the treatment of EVs in liver fibrosis. Exosomes derived from cells such as induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and liver stem cells (LSCs) have been shown to have certain anti-liver fibrosis effects. Existing studies focus more on the therapeutic application of stem cell-derived EVs in liver fibrosis. Depending on the type of parental cells, EVs from different cell sources have similar physiological functions and characteristics as the parental cells. However, there is no literature report on the method of using chemically induced fibroblasts to efficiently transdifferentiate into hepatocytes to prepare liver-lineage apoptotic vesicles to fight liver fibrosis. Summary of the invention

[0016] The object of the present invention is to provide a method and application thereof for preparing extrahepatic extracellular vesicles / hepatic apoptotic vesicles by chemically inducing direct reprogramming (also known as transdifferentiation) of fibroblasts into hepatoid cells without introducing or using any exogenous genes / transcription factors / MicroRNA (miRNA) genes, and their RNA, protein or polypeptide induction factors; nor using any cytokines or growth factors, but only using an induction composition composed of chemical small molecule inhibitors: GSK3β inhibitors, BMP inhibitors, and Notch inhibitors.

[0017] The present invention combines cell reprogramming technology with extracellular vesicle technology, and uses hepatic extracellular vesicles (ciHeps-EVs) / hepatic apoptotic vesicles (ciHeps-apoVs) prepared from hepatic cells (Chemical induced hepatocyte-like cells, ciHeps) directly reprogrammed by chemically induced fibroblasts to be applied to anti-liver fibrosis; the small molecule composition can be added with physiological saline / buffer / basic cell culture medium, etc., to prepare a transdifferentiation hepatic cell reagent or culture medium; each small molecule component of the small molecule composition does not have the function of inducing and regulating the transdifferentiation of fibroblasts into hepatic cells when it exists alone or partially, but they are effective when used in combination as a composition, and have the overall function of chemically inducing fibroblasts to transdifferentiate into hepatic cells. The obtained hepatic cells can be used as a source of hepatic cells; the obtained hepatic extracellular vesicles / hepatic apoptotic vesicles can be developed and prepared as biological preparations for anti-liver fibrosis for scientific research, medicine, and clinical applications.

[0018] In a first aspect, the present invention provides a chemical small molecule composition for inducing direct reprogramming of fibroblasts into hepatocytes to prepare extrahepatic vesicles / hepatic apoptotic vesicles, wherein the composition consists only of chemical small molecule inhibitors: GSK3β inhibitor, BMP inhibitor, Notch inhibitor;

[0019] Wherein, no exogenous gene / transcription factor / MicroRNA (miRNA) gene, and its RNA, protein or polypeptide and other induction factors are introduced into the fibroblasts; or, the chemical small molecule composition does not contain any exogenous gene / transcription factor / MicroRNA (miRNA) gene, or its transcribed RNA, or its translated protein and polypeptide and other induction factors.

[0020] Furthermore, in the chemical small molecule composition, the weight ratio of GSK3β inhibitor: BMP inhibitor: Notch inhibitor is (5-80): (0.1-50): (0.1-50); or the molar concentration ratio of GSK3β inhibitor: BMP inhibitor: Notch inhibitor in solution is (0.1-20): (0.01-20): (0.01-20).

[0021] Furthermore, the GSK3β inhibitor is a general term for chemical small molecule inhibitors that can target and inhibit the GSK3β signaling pathway, including but not limited to: CHIR-99021, LiCl, BIO, Ly2090314, IM-12, TWS119, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, SB216763, AZD1080, and the same class of GSK3β inhibitors or compounds with the same function or inducing inhibition of the same target of the GSK3β signaling pathway; preferably, the GSK3β inhibitors CHIR-99021, LY2090314, LiCl, BIO, Ly2090314, CHIR-98014.

[0022] Furthermore, the BMP inhibitor refers to a general term for chemical small molecule inhibitors that can target and inhibit the BMP signaling pathway, including but not limited to: BMP signaling agonist sb4, UK-383367 (UK 383367), LDN193189, BMT-124110, LDN-214117, SGC-AAK1-1, BMT-090605, BMT-090605hydrochloride, LP-922761, LP-922761hydrate, Asperosaponin VI, Elezanumab (ABT-555; AE12-1Y-QL), AZ12799734, DMH2, GW788388, ER-50891, LDN-212854, Dorsomorphin (Compound C) dihydrochloride, DMH-1, LDN193189Tetrahydrochloride, K02288, PD-161570, BMP inhibitors or small molecule compounds of the same type that have the same function or induce inhibition of the same BMP target; preferably LDN193189, AZ12799734, DMH-1, LDN-212854.

[0023] Furthermore, the Notch inhibitor refers to a general term for chemical small molecule inhibitors that can target and inhibit the Notch signaling pathway and γ-secretase, including but not limited to:

[0024] Notch inhibitor1, IMR-1, Tangeretin (Tangeritin), BMS-906024, Nirogacestat (PF-3084014), LY900009, Brontictuzumab (OMP 52M51), Nirogacestat dihydrobromide(PF-3084014dihydrobromide), Semagacestat, ZLDI-8, L-685458, BMS-983970, DAPT(GSI-IX), 3,5-Bis(4-nitrophenoxy)benzoic acid, FLI-06, Crenigacestat (LY3039478), BT-GSI, Z-Ile-Leu-aldehyde (Z-IL-CHO), Demcizumab (OMP 21M18), YO-01027 (Dibenzazepine; DBZ), Bruceine D, RO4929097 (RG-4733), MRK-560, Compound E, LY-411575, IMR-1A, DAPT (GSI-IX), Avagacestat (BMS-708163), Notch inhibitors or small molecule compounds of the same type that have the same function or induce inhibition of the same Notch target; preferably Crenigacestat, DAPT, Semagacestat, YO-01027.

[0025] Furthermore, in the chemical small molecule composition, the combined weight of the GSK3β inhibitor, BMP inhibitor, and Notch inhibitor accounts for 0.01-99.9% of the total weight of the composition; more preferably 50-99.9%; in solution state, it is 0.01-50%, such as 0.01%, 1%, 5%, 10%, 20%, 30%, 50%, etc.

[0026] The weight unit of the above-mentioned weight ratio can be any weight unit such as kilogram (kg), milligram (mg), microgram (μg), etc.; the molar unit of the molar concentration ratio can be any molar concentration unit such as mole (M), millimole (mM), micromole (μM), etc.

[0027] The second aspect of the present invention provides the use of the chemical small molecule composition as described above in inducing direct reprogramming of fibroblasts into hepatoid cells to prepare extrahepatic vesicles / hepatic apoptotic vesicles.

[0028] Furthermore, the chemical small molecule composition can be added with physiological saline / buffer / cell basal culture medium, etc. to prepare a transdifferentiated hepatocyte reagent or culture medium; the transdifferentiated hepatocytes can be added with physiological saline / buffer / serum-free culture medium to prepare a hepatocyte preparation; or the extrahepatic vesicles / hepatic apoptotic vesicles prepared from the transdifferentiated hepatocyte parents can be added with physiological saline, buffer, and a pharmaceutically acceptable carrier or excipient to prepare a biological preparation against liver fibrosis.

[0029] The third aspect of the present invention provides a method for preparing extrahepatic vesicles / hepatic apoptotic vesicles by inducing direct reprogramming of fibroblasts into hepatocytes using a small molecule chemical composition, comprising: (1) using the small molecule chemical composition as described above to induce transdifferentiation of fibroblasts into hepatocytes; (2) using the transdifferentiated hepatocytes to prepare extrahepatic vesicles / hepatic apoptotic vesicles.

[0030] Furthermore, the method for preparing extrahepatic vesicles / hepatic apoptotic vesicles by inducing direct reprogramming of fibroblasts into hepatoid cells using a chemical small molecule composition comprises the following steps:

[0031] (A) The method of using any of the above small molecule compositions to induce direct reprogramming of fibroblasts into hepatocytes comprises the following steps:

[0032] ① Preparation of concentrated liquid reagent: According to any of the small molecule compositions described in the present invention, each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrated liquid reagent; preferably, the organic solvent includes dimethyl sulfoxide; preferably, the aqueous solvent includes: water, physiological saline, phosphate buffer;

[0033] ② Obtaining a medium for inducing fibroblast transdifferentiation into hepatocytes: diluting the concentrated liquid reagent in step ① into a cell basal medium containing 5-20% calf serum (so that the concentration of each component meets the final concentration defined in any of the small molecule compositions described above) to obtain a medium for inducing fibroblast transdifferentiation into hepatocytes; wherein the percentage content of each component of the medium may also fluctuate by 50%; preferably by 30%; more preferably by 20%, such as 10%, 5%;

[0034] ③ Induce fibroblasts to transdifferentiate into hepatocytes: suspend and plate fibroblasts in a cell basal medium containing 5-20% calf serum or a serum-free medium containing various cytokines or growth factors, and replace with the transdifferentiation medium in step ② after the cells adhere to the wall, culture at 37°C, change the medium every 2-4 days; subculture once every 3-15 days;

[0035] ④ Induce fibroblasts to transdifferentiate into hepatocytes and subculture: discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, 37°C, 1-5 minutes, terminate cell digestion, centrifuge, discard the supernatant, resuspend the cell pellet, and subculture and plate at a ratio of 1:1-1:3; use the transdifferentiation medium in step ②, culture according to the above method, change the medium every 2-4 days; the digestion solution used includes trypsin, EDTA, Acutase, TrypleE, etc.; subculture once every 3-15 days;

[0036] ⑤ Harvesting of induced fibroblast transdifferentiation hepatocytes: After the above experimental steps ③ and ④ transdifferentiation hepatocyte culture and subculture for 2-4 weeks, the fibroblasts can be transdifferentiated into hepatocytes, and the transdifferentiated hepatocyte-like cells can be obtained.

[0037] (B) Isolation, extraction and identification of extrahepatic vesicles / apoptotic vesicles (apoVs)

[0038] ① Induce cell apoptosis by adding the apoptosis-inducing agent staurosporine (STS), and separate and extract apoptotic vesicles (apoVs).

[0039] ② Use transmission electron microscopy (TEM) to observe the morphology and size of apoVs, nanoparticle tracking analysis (NTA) to observe the diameter of apoVs particles, and use immunoglobulin blotting (Western Blot) to verify the molecular characteristics of apoVs.

[0040] The fourth aspect of the present invention provides a method for preparing extrahepatic vesicles / hepatic apoptotic vesicles by using the chemical small molecule composition as described above to induce direct reprogramming of fibroblasts into hepatic-like cells. The obtained transdifferentiated hepatic-like cells can be prepared as hepatic-like cell preparations; or prepared as extrahepatic vesicles / hepatic apoptotic vesicles.

[0041] Furthermore, the use of the chemical small molecule composition to induce fibroblasts to directly reprogram into hepatocytes to prepare extrahepatic vesicles / hepatic apoptotic vesicles is characterized in that the extrahepatic vesicles / hepatic apoptotic vesicles prepared by transdifferentiating hepatocytes can be added with physiological saline / buffer / pharmaceutically acceptable carriers or excipients to be further prepared as biological preparations against liver fibrosis.

[0042] A fifth aspect of the present invention provides a use of the transdifferentiated hepatoid cells described above in the preparation of hepatocyte transplantation preparations, bioartificial livers, and anti-liver fibrosis biological preparations.

[0043] The transdifferentiated hepatocytes can be added with physiological saline / buffer / serum-free culture medium to prepare hepatocyte preparations; the hepatocytes can be used for other scientific research experiments; new drug toxicity and efficacy testing and evaluation; to provide a source of hepatocytes for the construction of bioartificial livers, clinical cell transplantation, etc. The extrahepatic vesicles / hepatic apoptotic vesicles prepared from the transdifferentiated hepatocyte parents can be added with physiological saline / buffer / pharmaceutically acceptable carriers or excipients to prepare biological preparations against liver fibrosis.

[0044] In a sixth aspect, the present invention provides a method for preparing extrahepatic vesicles / hepatic apoptotic vesicles by inducing direct reprogramming of fibroblasts into hepatoid cells using the chemical small molecule composition as described above.

[0045] A seventh aspect of the present invention provides a use of the extrahepatic vesicles / hepatic apoptotic vesicles as described above in the preparation of biological agents for treating liver fibrosis diseases.

[0046] Furthermore, the extrahepatic vesicles / hepatic apoptotic vesicles can also be added with pharmaceutically acceptable carriers or excipients to prepare as biological preparations; preferably, the carriers or excipients include one or more selected from the following groups: water, saline, phosphate buffer or other aqueous solvents; DMSO, glycerol and ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymer surfactants; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, pH buffer substances, adhesives, fillers, lubricants or other drug excipients; or colloidal drug delivery systems, polymer drug delivery systems.

[0047] Furthermore, the dosage form of the biological agent includes but is not limited to liquid dosage form, gaseous dosage form, or semi-solid dosage form and solid dosage form; the liquid dosage form includes injection, infusion, suspension, or other liquid dosage forms.

[0048] In an eighth aspect of the present invention, a method for preparing extrahepatic vesicles / hepatic apoptotic vesicles by inducing direct reprogramming of fibroblasts into hepatoid cells using a chemical small molecule composition, wherein the chemical small molecule composition is composed only of chemical small molecule inhibitors: GSK3β inhibitor, BMP inhibitor, Notch inhibitor;

[0049] In the process of inducing fibroblasts to directly reprogram into hepatocytes to prepare extrahepatic vesicles / hepatic apoptotic vesicles, no exogenous genes / transcription factors / MicroRNA genes, and their RNA, proteins, polypeptides; and exogenous cytokines or growth factors are introduced or used.

[0050] In the ninth aspect of the present invention, a medicine kit or reagent kit is provided for preparing extrahepatic vesicles by inducing direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes using a chemical small molecule composition, wherein the medicine kit or reagent kit comprises any of the chemical small molecule compositions described above; or a transdifferentiation hepatocyte culture medium or reagent prepared by adding an organic solvent / physiological saline / buffer / cell basal culture medium; or a hepatocyte preparation prepared based on the obtained transdifferentiated hepatocytes; or a biological preparation prepared based on the obtained extrahepatic vesicles / hepatic apoptotic vesicles.

[0051] As in any of the foregoing aspects, the fibroblasts include but are not limited to: fibroblasts of mice or mammals; preferably, include but are not limited to: embryonic fibroblasts, skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts or fibroblasts of other tissues or organs of the body; more preferably, embryonic fibroblasts or skin fibroblasts.

[0052] As in any of the above aspects, the extrahepatic vesicles are hepatic apoptotic vesicles prepared by transdifferentiating hepatoid cells.

[0053] After in-depth research, the present invention discloses a method and application thereof for preparing hepatic extracellular vesicles / hepatic apoptotic vesicles by inducing fibroblasts to directly reprogram (transdifferentiate) into hepatoid cells using only a chemical small molecule composition, without introducing or using any exogenous gene / transcription factor / MicroRNA (miRNA) gene, and its RNA, protein or polypeptide induction factors; or, in a chemical small molecule composition / transdifferentiation medium, without containing or using any exogenous gene / transcription factor / MicroRNA (miRNA) gene, or its transcribed RNA, or its translated protein and polypeptide induction factors; and without adding or using any cytokine or growth factor. The invention combines cell reprogramming technology with extracellular vesicle technology, and prepares hepatic apoptotic vesicles (ciHeps-apoVs) from hepatocytes (ciHeps) by chemically inducing direct reprogramming of hepatocytes, which are used to fight liver fibrosis. The small molecule composition can be added with physiological saline / buffer / cell basal culture medium to be prepared as a transdifferentiated hepatocyte reagent or culture medium. The obtained transdifferentiated hepatocytes can be used as a source of hepatocytes for scientific research, medical and clinical applications. The obtained extracellular vesicles / hepatic apoptotic vesicles can be used to prepare biological preparations for fighting liver fibrosis for scientific research, medicine and clinical applications.

[0054] Basic mechanism of the invention

[0055] Chemically induced direct cell reprogramming (also known as transdifferentiation / lineage reprogramming) is a process in which one differentiated cell is directly transformed into another differentiated cell without introducing or using any exogenous genes / transcription factors / MicroRNA (miRNA) genes, and their induction factors such as RNA, proteins and peptides; only chemical small molecules are used to target and induce the regulation of cell signaling pathways and epigenetic modifications, so as to change their epigenetic modifications and thus change the gene expression profile of the cell.

[0056] The present invention only uses a small molecule cocktail (SMC) composed of chemical small molecules: GSK3β inhibitor, BMP inhibitor, Notch inhibitor, to chemically induce and regulate the fibroblast signaling pathway and epigenetic modification, so that its epigenetic modification changes, so as to change the gene expression profile of the cells, thereby directly reprogramming / transdifferentiating into hepatocytes (ciHeps), and its conversion rate or conversion purity is high (up to 88% or more), and no hepatocyte purification link is required. Then, the apoptosis inducing agent staurosporine (STS) is added to induce apoptosis of ciHeps, and apoptotic vesicles of hepatocytes (ciHeps-apoVs) are separated and extracted. The ciHeps-apoVs are prepared into a biological preparation and applied to anti-liver fibrosis. A large number of experimental results have shown that ciHep-apoVs has multiple biological functions in vitro and has regulatory effects on aHSCs, macrophages, and hepatocytes; ciHep-apoVs is mainly enriched in the liver after intravenous infusion and is mainly taken up by MFs and macrophages; ciHep-apoVs has anti-liver fibrosis effects in vivo, can reduce liver fibrosis and inflammatory response, and improve liver function; ciHep-apoVs inhibits the activity of aHSCs by inhibiting aerobic glycolysis, PI3K / Akt / mTOR pathway, Snail1 and other pathways; ciHep-apoVs targets multiple links of liver fibrosis by inhibiting aHSCs, regulating liver inflammatory response, etc.

[0057] The aforementioned small molecule inhibitors have the function of specifically inhibiting or blocking specific signaling pathways or epigenetic enzyme activities. Other targeted small molecule inhibitors under each category play an effective regulatory function according to the specific cell signaling pathways or enzyme activities that they induce, regulate or inhibit, and belong to functionally classified small molecule compounds (this classification work is completed jointly by chemical small molecule inventors and biomedical researchers); for example, all small molecules in the GSK3β inhibitor category have the common functional characteristics of targeted inhibition of the GSK3β signaling pathway. Among the small molecule inhibitors in the same category, there are only differences in effective dose, activity size, and strength of effect, but there is no essential difference in the function of inducing and regulating specific signaling pathways or epigenetic enzyme activities. Therefore, due to the particularity that small molecule inhibitors of the same category can target, induce and regulate the same specific cell signaling pathway or its specific target of epigenetic enzymes, the same category of small molecule inhibitors have basically the same efficacy as a single component; as a participating component of the composition, the efficacy they play in the organic whole of the composition is also basically the same, and there will be no qualitative difference. Similarly, the same is true for other small molecule inhibitors or small molecule compounds that target specific signaling pathways or specific epigenetic targets of induced cells, which is common knowledge known to those skilled in the art.

[0058] Therefore, GSK3β inhibitors, BMP inhibitors, and Notch inhibitors respectively include small molecule compounds in their respective categories that induce and regulate the same specific cell signaling pathway or epigenetic modification enzyme, and have the same functional activity, effect, and effect; the different combinations formed can induce fibroblasts to transdifferentiate into hepatocytes to varying degrees. Therefore, small molecule compounds in the same category that have the same function or the same induction target, or have the same functional effect on the same signaling pathway or epigenetic enzyme, and the small molecule combinations that can induce and regulate the transdifferentiation of fibroblasts into hepatocytes are all within the scope of protection of the present invention.

[0059] On the other hand, fibroblasts, also known as fibroblasts, are the main cell components of loose connective tissue, which are differentiated from mesenchymal cells in the embryonic period. Fibroblasts can be divided into fibroblasts and fibrocytes according to their different functional activity states; fibroblasts have vigorous functional activities, weakly alkaline cytoplasm, and obvious protein synthesis and secretion activities; those in the mature stage or quiescent state are called fibrocytes; under certain conditions, the two can achieve mutual transformation. Fibroblasts have different types and exist in various tissues or organs in the body; they have different names and characteristics in different tissues or organs, including: skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung fibroblasts, pancreatic fibroblasts and fibroblasts in other tissues or organs.

[0060] The hepatocytes obtained by the method of the present invention, and the extrahepatic vesicles prepared by using them as parent cells, including but not limited to apoptotic bodies (apoBDs), exosomes (Exosomes) and microvesicles (MVs) and other derivative products, all fall within the scope of this protection.

[0061] Invention method and its application

[0062] The present invention proposes a method and application thereof for preparing extrahepatic extracellular vesicles / hepatic apoptotic vesicles by chemically inducing fibroblasts to directly reprogram into hepatoid cells without introducing or using any exogenous genes / transcription factors / MicroRNA (miRNA) genes, or RNA transcribed therefrom, or proteins and polypeptides translated therefrom, or adding or using any cytokines or growth factors. Only a small molecule composition composed of chemical small molecule inhibitors: GSK3β inhibitor, BMP inhibitor, Notch inhibitor is used.

[0063] The small molecule composition can be added with physiological saline / buffer / cell basal culture medium to prepare a transdifferentiated hepatocyte reagent or culture medium; the method can be used to prepare transdifferentiated hepatocytes, and to prepare extrahepatic vesicles / hepatic apoptotic vesicles using transdifferentiated hepatocytes as parental sources, for preparing biological preparations against liver fibrosis.

[0064] Therefore, the above-mentioned applications or uses should also be included in the present invention.

[0065] It should be understood that small molecule inhibitors of the same category differ only in effective dose, activity, and effect, but there is no essential difference in their function of inducing inhibition of specific signaling pathways or epigenetic enzymes. In addition to the specific and representative chemical small molecule GSK3β inhibitors listed in the embodiments of the present invention, other GSK3β small molecule inhibitors that can target and inhibit the GSK3β cell signaling pathway can also achieve the same technical effects and should also be included in the present invention;

[0066] In addition to the specific and representative BMP inhibitors listed in the embodiments of the present invention, other chemical small molecule BMP inhibitors that target the induction and inhibition of BMP can also achieve the same technical effects and should also be included in the present invention;

[0067] In addition to the specific and representative Notch inhibitors listed in the embodiments of the present invention, other chemical small molecule Notch inhibitors that target and induce inhibition of the Notch signaling pathway can also achieve the same technical effects and should also be included in the present invention;

[0068] As used herein, the terms "comprising" or "including" include "comprising," "consisting essentially of, and "consisting of.

[0069] As used herein, the term "consisting essentially of..." means that in addition to the essential ingredients or components, the composition may also contain a small amount of minor ingredients and / or impurities that do not affect the effective ingredients. For example, the chemical small molecule composition can be added with physiological saline / buffer / basal cell culture medium, etc. to prepare a transdifferentiated hepatocyte reagent or culture medium; the transdifferentiated hepatocytes can be added with physiological saline / buffer / serum-free culture medium to prepare a hepatocyte preparation; the extrahepatic vesicles / hepatic apoptotic vesicles prepared from the transdifferentiated hepatocyte parents can be added with physiological saline / buffer / pharmaceutically acceptable carriers or excipients to prepare a biological preparation against liver fibrosis.

[0070] As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in mice and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio; such as pharmaceutical carriers or excipients commonly used in the art.

[0071] As used herein, the term "effective amount" refers to an amount that can produce a function or activity on mice and / or mammals and can be accepted by mice and / or mammals.

[0072] As used herein, the term "pharmaceutically acceptable carrier or excipient", wherein the carrier refers to a system that can change the way a drug enters the body and its distribution in the body, control the release rate of the drug and deliver the drug to the target organ; the drug carrier itself is not a necessary active ingredient and is not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art, and include, but are not limited to: water, saline, phosphate buffer and other aqueous solvents; DMSO (dimethyl sulfoxide), glycerol and ethanol and other organic solvents; microspheres, liposomes, microemulsions, polymer surfactants; colloidal drug delivery systems, new polymer drug delivery systems, new drug carriers and other pharmaceutical carriers; wherein the excipient refers to an additive other than the main drug in a pharmaceutical preparation, which may also be called an excipient. For example, adhesives, fillers, disintegrants, and lubricants in tablets; wine, vinegar, and medicinal juice in Chinese medicine pills; the matrix part in semi-solid preparations such as ointments and creams; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.

[0073] The general requirements for excipients are that they are stable in nature, have no incompatibility with the main drug, have no side effects, do not affect the efficacy, are not easily deformed, cracked, moldy, or worm-eaten at room temperature, are harmless to the body, have no physiological effects, do not produce chemical or physical effects with the main drug, and do not affect the content determination of the main drug, etc. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0074] As used herein, "parts by weight" or "parts by weight" are used interchangeably, and the parts by weight may be any fixed weight expressed in micrograms, milligrams, grams or kilograms (such as 1ug, 1mg, 1g, 2g, 5g, or kg, etc.). For example, a composition consisting of 1 part by weight of component a and 9 parts by weight of component b may be a composition consisting of 1 gram of component a + 9 grams of component b, or 10 grams of component a + 90 grams of component b, etc. In the composition, the percentage content of a component = (parts by weight of the component / sum of the parts by weight of all components) × 100%. Therefore, in a composition consisting of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and that of component b is 90%.

[0075] In addition, in the solution state, the above "weight parts" can also be converted into "moles"; "weight parts ratio" can also be converted into "molar concentration ratio". The weight unit of the weight parts ratio can be any weight unit such as kilogram (kg), milligram (mg), microgram (ug); the molar unit of the molar concentration ratio can be any molar concentration unit such as mole (M), millimole (mM), micromole (uM);

[0076] In the chemical small molecule composition, the small molecule inhibitor: GSK3β inhibitor: BMP inhibitor: Notch inhibitor are present in a weight ratio of (5-80): (0.1-50): (0.1-50); preferably, (10-70): (0.5-40): (0.5-40); or in a solution state, the molar concentration ratio is (0.1-20): (0.01-20): (0.01-20); preferably, (0.5-10): (0.05-10): (0.05-10).

[0077] For example, the composition includes components and weight ratios as shown in Table 1 or molar concentrations as shown in Table 2 (solution state).

[0078] Table 1. Weight ratio (weight unit: kg, mg, μg...)

[0079] Components Optimal amount Better quantity example GSK3β inhibitors 5-80 10-70 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 BMP inhibitors 0.1-50 0.5-40 0.8、1、1.5、2、4、6、8、10、15、20、25、30、35 Notch inhibitors 0.1-50 0.5-40 0.8、1、1.5、2、4、6、8、10、15、20、25、30、35

[0080] Table 2. Molar concentration ratio (molar unit: M, mM, μM...)

[0081]

[0082] The formulation ranges in Tables 1 and 2 can be used as reference guidance. However, it should be understood that when used to prepare scientific research reagents, the effective dose of the composition used may vary with the mode of administration and the weight of the animal body. Moreover, when used in vivo, "weight / kg (body weight)" is usually used as the dosage unit; when the small molecule composition is applied to large animals, the effective dosage for large animals (including solid or solution dosage conversion) converted according to the dosage for small animals through the corresponding professional conversion formula also falls within the scope of protection of the present invention.

[0083] It should also be understood that since each category of chemical small molecules has a large number of small molecule members, and the effective doses and activity sizes of each small molecule member vary greatly, although the present invention has long studied chemical small molecules to induce direct cell reprogramming and has conducted a large number of experiments, it is impossible to list all the embodiments in the specification. The embodiments are only for the small molecule induced regulation combination components, and the representative small molecules of each category (such as GSK3β inhibitors, representatively demonstrated by several small molecule inhibitors such as CHIR-99021, LiCl, CHIR-98014) are used in the experiment at the concentration; therefore, the reasonable concentration range summarized in the claims of the present patent application naturally includes, but is not limited to, the concentration range used in the experiments of specific representative small molecules of each category in the embodiments; this basic and simple truth is well known to professionals in the field and should not be confused.

[0084] The present invention also includes compounds, pharmaceutical preparations, analogs and / or salts, hydrates or precursors equivalent to the above-mentioned small molecule inhibitors; and also includes naturally occurring and artificially synthesized compounds thereof.

[0085] Analogs of the small molecule compounds include, but are not limited to, isomers and racemates of the small molecule compounds. The compounds have one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis or trans isomers.

[0086] The "salts" include, but are not limited to: (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine, etc. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium), etc.

[0087] The "precursor of a compound" refers to a compound that can be converted into any of the above compounds, or a salt or solution composed of any of the above compounds in culture medium, or in an animal or organism after being applied or treated by an appropriate method.

[0088] The small molecule composition of the present invention can be added with physiological saline / buffer / cell basal culture medium, etc. to prepare a transdifferentiation hepatocyte reagent or culture medium; the method for preparing the reagent is determined according to the dosage form to be prepared and the route of administration of the reagent. After referring to the combination and ratio provided by the present invention, those skilled in the art can prepare the transdifferentiation reagent or culture medium of the present invention by using conventional reagent or culture medium preparation methods.

[0089] It should be understood that although the present invention lists several small molecule combination forms in specific embodiments, those skilled in the art can also deduce from this that any other small molecule combination form of the present invention also has the same outstanding effect.

[0090] Preparation of transdifferentiated hepatocyte culture medium and reagents:

[0091] The present invention also provides a small molecule composition that can be added with an organic solvent / physiological saline / buffer / cell basal culture medium and other carriers / excipients / basal nutrient solution to prepare a chemically induced fibroblast direct reprogramming (transdifferentiation) into a transdifferentiation medium or reagent for hepatocytes; a transdifferentiation medium for inducing fibroblast direct reprogramming (transdifferentiation) into hepatocytes (hereinafter referred to as: Transdifferentiation Hepatoid Cell Culture Medium ).

[0092] Method for preparing transdifferentiation hepatocyte culture medium: According to the final concentration formula of the chemical small molecule composition provided by the present invention, a small molecule composition with a specific final concentration is selected for preparation. As a preferred embodiment of the present invention, different components in the specific small molecule composition are dissolved in DMSO (dimethyl sulfoxide) or other organic solvents or aqueous solvents according to the different properties and solubilities of their solutes to prepare a concentrated liquid reagent (ranging from 150-110,000); then, according to the final concentration requirement of the specific small molecule composition, each small molecule organic solution concentrated liquid reagent is diluted and added into a cell basal culture medium containing 10% calf serum to obtain the transdifferentiation hepatocyte culture medium. Among them, the percentage content of each component of the culture medium can also fluctuate up and down by 50%; preferably, it fluctuates up and down by 30%; more preferably, it fluctuates up and down by 20%, such as 10%, 5%. Unless otherwise specified, percentages are measured in v / v.

[0093] As a preferred embodiment of the present invention, the cell basal culture medium includes but is not limited to: DMEM / F12, MEM, DMEM, F12, IMDM, RPMI1640, Neuronal basal or Fischers, etc., all of which are commercially available.

[0094] As a preferred mode of the present invention, the aforementioned transdifferentiation medium can also be prepared using serum-free medium under special conditions. The "serum-free medium" refers to a cell culture medium that does not contain serum but contains a variety of nutrients (such as growth factors, tissue extracts, etc.) that support cell proliferation and biological reactions. That is, a cell culture medium composed of various cytokines or growth factors other than serum, and other additives added to the cell basal culture medium.

[0095] As a preferred embodiment of the present invention, the serum-free culture medium containing various cytokines or growth factors includes but is not limited to: ITS, N2, B27, etc., all of which can be prepared by oneself or commercially available.

[0096] It should be understood that those skilled in the art are familiar with the preparation or purchase routes of the cell basal culture medium or serum-free culture medium. Therefore, the cell basal culture medium or serum-free culture medium is not limited to those exemplified in the present invention.

[0097] As a preferred embodiment of the present invention, the "transdifferentiation medium" is specifically prepared or formulated as follows:

[0098] (1) Mix ① GSK3β inhibitor (such as CHIR99021) with a final concentration of 0.1μM-20mM; preferably 0.5μM-10mM; ② BMP inhibitor (such as LDN193189) with a final concentration of 0.01-20μM; preferably 0.05-10μM; ③ Notch inhibitor (such as DAPT) with a final concentration of 0.01-20μM; preferably 0.05-10μM to obtain the small molecule composition of the present invention for chemically inducing direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes.

[0099] (2) The above-mentioned small molecules are respectively dissolved to prepare a concentrated solution → diluted into a cell basal culture medium → mixed to prepare a "transdifferentiation hepatocyte culture medium".

[0100] A method for the culture and transformation of fibroblasts directly reprogrammed into hepatocytes by using a small molecule chemical composition:

[0101] The present invention also discloses a method for preparing transformed hepatocytes by inducing direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes using a chemical small molecule composition, the method comprising the following steps:

[0102] (1) Preparation of concentrated liquid reagent: According to any of the compositions described in the present invention, each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrated liquid reagent; preferably, the organic solvent includes dimethyl sulfoxide; preferably, the aqueous solvent includes: water, physiological saline, phosphate buffer;

[0103] (2) Obtaining a transdifferentiated hepatocyte culture medium: diluting the concentrated liquid reagents in step (1) into a cell basal culture medium containing 5-20% calf serum (so that the concentration of each component meets the concentration specified in the composition described above) to obtain a transdifferentiated hepatocyte culture medium;

[0104] The percentage of each component of the culture medium may fluctuate by 50%; preferably by 30%; more preferably by 20%, such as 10% or 5%.

[0105] (3) Inducing fibroblasts to transdifferentiate into hepatocytes: Fibroblasts are suspended and plated in a cell basal medium containing 5-20% calf serum or a serum-free medium. After the cells adhere, the original medium is discarded and replaced with the medium for transdifferentiating fibroblasts into hepatocytes in step (2) at 37°C. The medium is changed every 2-4 days and the cells are subcultured every 3-15 days.

[0106] (4) Subculture to induce fibroblast transdifferentiation: discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, 37°C, 1-5 minutes, terminate cell digestion, centrifuge, discard the supernatant, resuspend the cell pellet, and subculture and plate at a ratio of 1:1-1:3. Culture according to the experimental steps (2) and (3), and change the medium every 2-4 days. The digestion solution used includes trypsin, EDTA, Acutase, TrypleE, etc. Subculture once every 3-15 days.

[0107] (5) Inducing fibroblast transdifferentiation and obtaining transdifferentiated hepatocytes: After the above experimental steps (3) and (4) transdifferentiation culture and subculture of fibroblasts for 2-4 weeks, transdifferentiated hepatocytes can be obtained.

[0108] The transdifferentiated hepatocytes can be added with physiological saline / buffer / serum-free culture medium to prepare hepatocyte transplant preparations; the hepatocytes can be used for other scientific research experiments; new drug toxicity and efficacy testing and evaluation; to provide a source of hepatocytes for the construction of bioartificial livers, clinical cell transplantation, etc. The extrahepatic vesicles / hepatic apoptotic vesicles prepared from the transdifferentiated hepatocyte parents can be added with physiological saline / buffer / pharmaceutically acceptable carriers or excipients to prepare biological preparations against liver fibrosis.

[0109] Methods for Isolation and Identification of Apoptotic Vesicles from Hepatocytes:

[0110] ①ciHeps were obtained by the above-mentioned small molecule compound-induced MEFs direct reprogramming culture transformation method.

[0111] ② Ordinary fetal bovine serum (FBS) was centrifuged at 4°C, 100,000g for 18h to remove exosomes and obtain exosome-free FBS. After washing ciHeps / hepatoid cells three times with PBS buffer, the transdifferentiation culture medium / culture fluid was replaced with DMEM medium containing 10% exosome-free FBS and STS, and a final concentration of 0.1-10μM apoptosis-inducing agent staurosporine (STS) was added to induce cell apoptosis. After 12h of culture, the supernatant of apoptotic cells was collected and centrifuged at 800g for 10min. The supernatant after centrifugation was filtered through a 5μm filter membrane and then centrifuged at 16,000g at 4°C for 30min. The apoVs precipitate was washed twice with sterile saline and finally stored in a device at -80°C.

[0112] ③ Observe the morphology of apoVs using transmission electron microscopy (TEM). Professional technicians of TEM will observe the morphology of apoVs according to the routine operation of TEM and take photos for preservation.

[0113] ④ Detection of apoVs marker protein by routine Western Blot

[0114] Western Blot results showed that compared with the parental cells in the control group, the expression of apoptosis marker protein Cleaved Caspase-3 in apoVs derived from cells in the STS treatment group was significantly upregulated. β-actin, as a non-vesicular marker, was significantly downregulated in apoVs of each group.

[0115] The innovative advantages of the present invention are:

[0116] (1) Small molecule compounds are easy to synthesize and store, and have lower costs; (2) They avoid the potential risks of gene mutation and tumorigenesis during genetic manipulation; (3) The effect can be optimized by regulating the concentration, combination, and duration of action of the compounds, thereby improving the reprogramming efficiency; (4) The ciHeps conversion rate is much higher than that of the small molecule chemical-induced transdifferentiation of hepatocytes disclosed in previous literature, and the purity of ciHeps can be as high as 88%, eliminating the purification process of hepatocytes. (5) It is the first to combine chemically induced cell direct reprogramming technology with extracellular vesicle technology; it brings new perspectives and visions to the treatment of liver fibrosis; and it provides reference for the treatment of other liver diseases or other organ fibrosis. (6) According to the characteristics of the parental cells, apoVs produced by different cell types can have completely different and unique biological functions. (7) Non-cellular biological products derived from liver functional cells are used to fight liver fibrosis. They are easy to produce and store, easy to operate, convenient to administer, have good tissue compatibility, and low immunogenicity. (8) The method of directly reprogramming fibroblasts into ciHeps with small molecule compounds overcomes the shortcomings of previous methods. The use of apoVs as a therapeutic vector overcomes the shortcomings of using cells as a therapeutic vector. It also overcomes the defects of using exosomes and other cells in a viable state, such as the small amount of vesicles secreted and the difficulty in collecting and purifying them. (9) It is the first to use apoVs derived from transformed liver functional cells (ciHeps) for anti-liver fibrosis.

[0117] After extensive research, the present invention proposes for the first time a method and application of preparing extrahepatic vesicles / apoptotic vesicles by inducing fibroblasts to directly reprogram into hepatoid cells using a chemical small molecule composition without introducing any exogenous genes / transcription factors / MicroRNA genes, and induction factors such as RNA, proteins and polypeptides. The method combines cell reprogramming technology with extracellular vesicle technology, and prepares hepatic apoptotic vesicles (apoVs) from chemically induced direct reprogramming of hepatoid cells (ciHeps) to resist liver fibrosis; the small molecule composition can be added with physiological saline / buffer / basal cell culture medium to prepare a transdifferentiation hepatoid cell reagent or culture medium. The obtained hepatoid cells can be used as a cell model for liver disease research, pharmacological and toxicological safety testing; functional testing, preclinical research, etc. can be continued. The obtained extrahepatic vesicles / hepatic apoptotic vesicles can be added with physiological saline / buffer / pharmaceutically acceptable carriers or excipients to further prepare biological preparations for anti-liver fibrosis for scientific research, medicine, and clinical practice.

[0118] The present invention not only opens up a new way for the research and development of biopharmaceutical preparations for the prevention and treatment of liver fibrosis / cirrhosis and their mechanism research, but also provides a new liver cell source for the medical application, clinical application and scientific research application of transdifferentiated hepatocytes; it opens up a new field for the anti-fibrosis research of exosome-derived products prepared from various transdifferentiated functional cell parents. It has a wide range of application prospects; it also enriches the theory of stem cell reprogramming and expands its application range. It has important scientific significance and great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 . Transdifferentiation Hepatocyte-like Cell Culture Medium 1 The morphological structure of chemically induced embryonic fibroblasts (MEFs) transdifferentiated into hepatocyte-like cells (ciHep): the cell volume increased, and the cells were round or irregular polygonal, with abundant cytoplasm, and some cells had binucleate; the cell boundaries were clear, and the bile duct structure with high refractive index, which was unique to hepatocytes, appeared. It was obviously different from the original long spindle shape and less cytoplasm of MEFs; the morphological structure characteristics were the same or similar to those of mouse primary hepatocytes (MPHs).

[0120] Figure 2 .The morphological structure of mouse skin fibroblasts (MAF) transdifferentiated into hepatocytes (ciHep) induced by transdifferentiation hepatocyte-like cell culture medium 2: ciHep cells became larger in size, round or irregular polygonal in shape, with abundant cytoplasm, and some cells had binucleate; they were significantly different from the original long spindle-shaped and less cytoplasmic morphological characteristics of MAF; and they were the same or similar to the morphological structural characteristics of mouse primary hepatocytes (MPH).

[0121] Figure 3 The hepatocytes ciHep obtained by transdifferentiation culture were positive for glycogen staining, which was the same or similar to the positive MPH in the right figure, but significantly different from the negative MEFs in the left figure. This proves that the transdifferentiated hepatocytes obtained by the method of the present invention have the glycogen storage activity unique to hepatocytes.

[0122] Figure 4 The hepatocytes ciHep obtained by transdifferentiation culture were positive for oil-red staining, which was the same or similar to the positive MPH in the right figure, but significantly different from the negative oil-red staining of MEFs in the left figure. This proves that the transdifferentiated hepatocytes ciHep obtained by the method of the present invention have the unique fat metabolism function of hepatocytes.

[0123] Figure 5.Compared with MEFs, the expression of hepatocyte-specific genes in ciHeps was significantly increased, among which the expression of HNF4α, HNF1α, and CYP1A2 was about 50% of the expression of MPHs in the positive control group, which was 73 times, 5 times, and 952 times higher than that of MEFs in the negative control group, respectively. The expression of CYP1A1 reached the expression level of MPHs, which was 56 times higher than that of MEFs in the negative control group. The expression of ALB was about 12% of the expression of MPHs, which was 306 times higher than that of MEFs in the negative control group. After fibroblasts were transdifferentiated into hepatocytes, hepatocyte-related genes were significantly highly expressed (* indicates P < 0.05, *** indicates P < 0.001), including HNF4α, HNF1α, ALB, CYP1A1, and CYP1A2. This indicates that fibroblasts have been transdifferentiated into hepatocytes.

[0124] Figure 6 .Under transmission electron microscopy, apoVs derived from cells in each group were biconcave disc-shaped with a relatively complete double-layer membrane structure, which was consistent with the conventional morphology of extracellular vesicles.

[0125] Figure 7 .Western Blot results showed that compared with the parental cells, the expression of apoptosis marker protein Cleaved Caspase-3 in apoVs derived from each group of cells was significantly upregulated. β-actin, as a non-vesicular marker, was significantly downregulated in apoVs of each group ( Figure 7 ). This is consistent with the characteristics of apoptotic vesicles.

[0126] Figure 8 .The degree of liver tissue fibrosis and collagen accumulation were detected by Sirius red staining. It can be seen that compared with the Control group, the area of ​​collagen positive staining in the ciHep-apoVs group was significantly reduced, and the results were similar to those in the MPH-apoVs group, while MEF-apoVs had no effect on collagen accumulation; the expression level of αSMA, a marker protein of MFs, in liver tissue was detected by immunohistochemistry. The results showed that the ciHep-apoVs group significantly downregulated the area of ​​αSMA positive staining in liver tissue, indicating that the number of MFs was significantly reduced, and the results were similar to those in the MPH-apoVs group, while MEF-apoVs had no effect on αSMA expression. The statistical chart on the right side of the figure shows that the results of Sirius red (SR) staining showed that ciHep-apoVs significantly reduced the area of ​​SR positive staining in liver tissue. The results of immunohistochemical staining showed that ciHep-apoVs significantly reduced the area of ​​αSMA positive staining in liver tissue. Scale bar 100μm. (*** indicates P < 0.001). The experimental results showed that ciHep-apoVs can reduce collagen deposition in liver tissue in the mouse liver fibrosis model and has the effect of improving mouse liver fibrosis.

[0127] Fig. 9 Compared with the Control group, the expression of fibrosis genes ACTA2, COL1a1, and COL3a1 in the ciHep-apoVs group was downregulated, and the results were similar to those in the MPH-apoVs group, while MEF-apoVs had no effect on the expression of fibrosis genes. The results showed that ciHep-apoVs reduced the expression level of fibrosis genes in liver tissue.

[0128] (* indicates P<0.05).

[0129] The experimental results showed that ciHep-apoVs reduced the expression level of liver tissue fibrosis genes and had the effect of improving liver fibrosis in mice.

[0130] Fig.10 .By detecting the ALT and AST levels in the serum of each group of mice, the results showed that the ALT and AST levels in the ciHep-apoVs group were significantly reduced compared with the negative control group. The results of blood biochemistry tests showed that ciHep-apoVs reduced the ALT and AST levels in the serum of mice and improved the liver function of mice. (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001).

[0131] Fig.11 .ciHeps highly expressed ALB and CYP1A2, of which about 83% of cells were ALB-positive cells and 88% of cells were CYP1A2-positive cells. The proportion of ALB and CYP1A2-positive cells in ciHeps was similar to that of MPHs, indicating that ciHeps had the characteristics of mature hepatocytes and that SMC-induced direct cell reprogramming was efficient and uniform. The experimental results showed that ciHeps highly expressed hepatocyte-specific markers ALB and CYP1A2, with positive expression rates of 83% and 88%, respectively. Scale bar 100μm. (*** indicates P < 0.001). It shows that the small molecule composition SMC chemically induced fibroblasts to directly reprogram into hepatocyte-like cells with high efficiency and uniformity.

[0132] Fig.12 . Transdifferentiation hepatoid cell culture medium 2 The hepatoid cells ciHep obtained by chemically inducing transdifferentiation culture of adult skin fibroblasts (MAFs) were positive for glycogen staining, which was the same or similar to the positive MPH in the right figure; and significantly different from the negative glycogen staining of MAFs in the left figure. It was proved that the transdifferentiation hepatoid cells obtained by the method of the present invention had the glycogen storage activity unique to hepatocytes.

[0133] Fig.13The hepatocytes ciHep obtained by transdifferentiation culture were positive for oil-red staining, which was the same or similar to the positive MPH in the right figure, but significantly different from the negative oil-red staining of MAFs in the left figure. This proves that the transdifferentiated hepatocytes ciHep obtained by the method of the present invention have the unique fat metabolism function of hepatocytes.

[0134] Fig.14 Compared with MAFs, the expression of hepatocyte-specific genes in ciHeps was significantly increased, among which the expression of HNF4α and CYP1A1 reached about 80% of the expression of MPHs in the positive control group, which was 77 times and 79 times higher than that of MAFs in the negative control group, respectively. The expression of CYP27A1 reached the expression level of MPHs, which was 59 times higher than that of MAFs in the negative control group. The expression of ALB reached about 10% of the expression of MPHs, which was 330 times higher than that of MAFs in the negative control group. After adult skin fibroblasts (MAF) were transdifferentiated into hepatocytes, the hepatocyte-related genes were significantly highly expressed (*** indicates P < 0.001), including HNF4α, ALB, CYP1A1, CYP7A1, and CYP27A1. This shows that MAF has been transdifferentiated into hepatocytes.

[0135] Fig.15 .ciHeps highly expressed ALB and CYP1A2, of which about 81% of cells were ALB-positive cells and 85% of cells were CYP1A2-positive cells. The proportion of ALB and CYP1A2-positive cells in ciHeps was similar to that of MPHs, indicating that ciHeps had the characteristics of mature hepatocytes and that SMC-induced direct cell reprogramming was efficient and uniform. The experimental results showed that ciHeps highly expressed hepatocyte-specific markers ALB and CYP1A2, with positive expression rates of 81% and 85%, respectively. Scale bar 100μm. (*** indicates P < 0.001). It shows that the small molecule composition SMC chemically induced direct reprogramming of adult skin fibroblasts into hepatocyte-like cells with high efficiency and uniformity. DETAILED DESCRIPTION

[0136] The specific embodiments provided by the present invention are described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as the conditions described in Short Protocols in Cell Biology (Science Press, 2007) by JS Bonifacion et al., translated by Zhang Jingbo, Fang Jin, Wang Haijie et al., or according to the conditions recommended by the manufacturer.

[0137] Example 1: Small molecule composition for inducing direct reprogramming (transdifferentiation) of fibroblasts into hepatocytes, and preparation of culture medium and reagents for transdifferentiation of hepatocytes

[0138] The following composition or transdifferentiation medium is designed and can be prepared in molar concentration or weight concentration:

[0139] 1. Formula of small molecule composition for fibroblast transdifferentiation into hepatocytes

[0140] (1) Reprogramming small molecule composition 1

[0141] GSK3β inhibitor CHIR-99021: final concentration 6 μM;

[0142] BMP inhibitor LDN193189: final concentration 3 μM;

[0143] Notch inhibitor DAPT: final concentration 5 μM;

[0144] (2) Reprogramming small molecule composition 2

[0145] GSK3β inhibitor BIO: final concentration 2 μM;

[0146] BMP inhibitor AZ12799734: final concentration 5 μM;

[0147] Notch inhibitor Crenigacestat: final concentration 2 μM;

[0148] (3) Reprogramming small molecule composition 3

[0149] GSK3β inhibitor Ly2090314: final concentration 1 μM;

[0150] BMP inhibitor DMH-1: final concentration 2.5 μM;

[0151] Notch inhibitor Semagacestat: final concentration 3 μM;

[0152] (4) Reprogramming small molecule composition 4

[0153] GSK3β inhibitor LiCl: final concentration 4 mM;

[0154] BMP inhibitor LDN-212854: final concentration 2 μM;

[0155] Notch inhibitor YO-01027: final concentration 2 μM;

[0156] (5) Reprogramming small molecule composition 5

[0157] GSK3β inhibitor CHIR-98014: final concentration 0.5 μM;

[0158] BMP inhibitor LDN193189: final concentration 5 μM;

[0159] Notch inhibitor Crenigacestat: final concentration 2 μM;

[0160] (6) Reprogramming small molecule composition 6

[0161] GSK3β inhibitor CHIR99021: final concentration 3 μM;

[0162] BMP inhibitor AZ12799734: final concentration 2 μM;

[0163] Notch inhibitor DAPT: final concentration 2.5 μM;

[0164] (7) Reprogramming small molecule composition 7

[0165] GSK3β inhibitor Ly2090314: final concentration 1.5 μM;

[0166] BMP inhibitor DMH-1: final concentration 0.5 μM;

[0167] Notch inhibitor Semagacestat: final concentration 5 μM;

[0168] (8) Reprogramming small molecule composition 8

[0169] GSK3β inhibitor CHIR-98014: 1 mg / kg;

[0170] BMP inhibitor LDN-212854: 0.3 mg / kg;

[0171] Notch inhibitor YO-01027: 1 mg / kg;

[0172] (9) Reprogramming small molecule composition 9

[0173] GSK3β inhibitor BIO: 0.5 mg / kg;

[0174] BMP inhibitor AZ12799734: 0.5 mg / kg;

[0175] Notch inhibitor Crenigacestat: 0.5 mg / kg;

[0176] (10) Reprogramming small molecule composition 10

[0177] GSK3β inhibitor LiCl: final concentration 15 mM;

[0178] BMP inhibitor DMH-1: final concentration 10 μM;

[0179] Notch inhibitor DAPT: final concentration 5 μM.

[0180] Each specific small molecule composition is first dissolved in DMSO according to step ① of the aforementioned "culture transformation method" to prepare a concentrated liquid reagent.

[0181] 2. Preparation of Transdifferentiation Hepatocyte Culture Medium

[0182] The DMSO concentrated solution reagents of the components of the fibroblast transdifferentiation hepatocyte composition 1 to 10 prepared in the above experimental step 1 were prepared according to the aforementioned "method for preparing transdifferentiation hepatocyte culture medium" steps, and the selected cell basal culture medium was DMEM supplemented with 10% calf serum to obtain transdifferentiation hepatocyte culture medium 1 to 10 (i.e., the final concentrations of the compounds in culture medium 1 and composition 1 were the same, the final concentrations of the compounds in culture medium 2 and composition 2 were the same, ..., the final concentrations of the compounds in culture medium 10 and composition 10 were the same).

[0183] Example 2: Comparative experiment on chemically induced transdifferentiation of embryonic fibroblasts (MEFs) into hepatocytes by transdifferentiation hepatocyte-like cells medium 1

[0184] 1. Fibroblast-to-hepatocyte transdifferentiation culture

[0185] Mouse embryonic fibroblasts (MEFs) were suspended in DMEM containing 10% calf serum, plated, and cultured at 37°C. A control group and a treatment group were set up;

[0186] After embryonic fibroblasts (MEFs) adhered to the wall, the original culture medium was discarded and the treated groups were replaced with differentiation hepatoid cell culture medium 1, cultured at 37°C, with the medium changed every 3 days; and passaged every 3-7 days.

[0187] 2. Subculture of embryonic fibroblasts (MEFs) into hepatocytes

[0188] Subculture steps: discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, 37℃, 3 minutes, terminate cell digestion, centrifuge, discard the supernatant, resuspend the cell pellet, and subculture at a ratio of 1:2. The treatment group is cultured in the transdifferentiation hepatocyte culture medium 1, and the medium is changed once every 3 days. The digestion solution used is trypsin (EDTA, Acutase, TrypleE can also be used). Subculture once every 3-7 days.

[0189] 3. Harvesting of transdifferentiated hepatocytes (ciHep)

[0190] After the fibroblasts were transdifferentiated into hepatocytes in the above experimental steps 1-2 and subcultured for 2-4 weeks, transdifferentiated hepatocytes (ciHep) were obtained after culturing for 2 weeks in this experiment, and they can be used for further experiments.

[0191] 4. Comparison of morphology between embryonic fibroblasts (MEFs), transdifferentiated hepatocytes (ciHep), and mouse primary hepatocytes (MPHs)

[0192] The morphology of embryonic fibroblasts (MEFs) in the negative control group was compared with the hepatocytes (ciHep) derived from the transdifferentiation of the treatment group, and the mouse primary hepatocytes (MPHs) in the positive control group. Figure 1 .

[0193] Figure 1 The experimental results showed that the morphological structure of hepatocyte-like cells (ciHep) transdifferentiated from embryonic fibroblasts (MEFs) induced by transdifferentiation hepatocyte-like cell culture medium 1 was as follows: the cells were enlarged, round or irregularly polygonal, with abundant cytoplasm, and some cells had binucleate; the cell boundaries were clear, and the bile duct structure with high refractive index, which was unique to hepatocytes, appeared. It was significantly different from the original long spindle-shaped and less cytoplasmic morphological characteristics of MEFs; and the morphological and structural characteristics were the same or similar to those of mouse primary hepatocytes (MPHs).

[0194] Example 3: Comparison of morphological structures of mouse adult skin fibroblasts (MAF) transdifferentiated into hepatoid cells induced by transdifferentiation hepatoid cell culture medium 2

[0195] Transdifferentiation hepatocyte medium 2, the method steps of chemically inducing mouse adult skin fibroblasts (MAF) to transdifferentiate into hepatocytes are the same as those in Example 2. The experimental results are shown in Figure 2 .

[0196] Figure 2 The results showed that the morphological structure of mouse adult skin fibroblasts (MAF) transdifferentiated into hepatocytes (ciHep) induced by transdifferentiation hepatocyte-like cell medium 2 was as follows: the cells became larger in size, round or irregular polygonal in shape, with abundant cytoplasm, and some cells had binucleate; these cells were significantly different from the original long spindle-shaped and less cytoplasmic morphological characteristics of MAF; and their morphological and structural characteristics were the same or similar to those of mouse primary hepatocytes (MPH).

[0197] Example 4: Comparison of glycogen staining of transdifferentiation of embryonic fibroblasts (MEFs) into hepatocytes (ciHep) using transdifferentiation hepatocyte-like cell culture medium 3 and mouse primary hepatocytes (MPHs)

[0198] Transdifferentiation hepatocyte culture medium 3, the method steps for chemically inducing mouse embryonic fibroblasts (MEFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0199] MEFs, their transdifferentiated hepatocytes (ciHep), and mouse primary hepatocytes (MPHs) were stained for glycogen. The depth of staining reflects the ability of hepatocytes to store glycogen. The Schiff method was used for glycogen staining. The specific method is as follows: (1) discard the cell culture medium and rinse once with PBS; (2) fix with 4% paraformaldehyde for 10 minutes, and rinse with PBS for 5 minutes × 3 times; (3) add PAS-I solution for 10 minutes and rinse with running water; (4) add PAS-II solution for 1-2 minutes and rinse with running water; (5) take pictures under a microscope. The experimental results are shown in Figure 3 .

[0200] Depend on Figure 3 The experimental results show that the hepatocytes ciHep obtained by transdifferentiation culture in the figure are positive for glycogen staining, which is the same or similar to the positive MPH in the right figure, and significantly different from the negative glycogen staining of MEFs in the left figure. This proves that the transdifferentiated hepatocytes obtained by the method of the present invention have the glycogen storage activity unique to hepatocytes.

[0201] Example 5: Transdifferentiation of embryonic fibroblasts (MEFs) into hepatocytes (ciHep) induced by transdifferentiation hepatocyte-like cell culture medium 4, and oil-red staining of mouse primary hepatocytes (MPHs)

[0202] Transdifferentiation hepatocyte culture medium 4: The method steps for chemically inducing mouse embryonic fibroblasts (MEFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0203] MEFs, transdifferentiated hepatocytes (ciHep), and mouse primary hepatocytes (MPHs) were stained using a conventional oil-red staining kit. The method and steps are shown in the kit. The staining results are shown in Figure 4 .

[0204] Figure 4 The experimental results show that the hepatocytes ciHep obtained by transdifferentiation culture in the figure are positive for oil-red staining, which is the same or similar to the positive MPH in the right figure, and significantly different from the negative oil-red staining of MEFs in the left figure. This proves that the transdifferentiated hepatocytes ciHep obtained by the method of the present invention have the unique fat metabolism function of hepatocytes.

[0205] Example 6: Comparison of hepatocyte-related gene detection in embryonic fibroblasts (MEFs) induced by transdifferentiation hepatocyte-like cell medium 5 into hepatocyte-like cells (ciHep) and mouse primary hepatocytes (MPHs)

[0206] Transdifferentiation hepatocyte culture medium 5: The method steps for chemically inducing mouse embryonic fibroblasts (MEFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0207] Total RNA from the control group MEFs, transdifferentiation group ciHep, and primary hepatocytes (MPHs) were collected for RT-qPCR analysis to analyze the relative expression levels of hepatocyte-specific genes HNF4α, HNF1α, ALB, CYP1A1, and CYP1A2. Figure 5 .

[0208] Figure 5 The results showed that compared with MEFs, the expression of hepatocyte-specific genes in ciHeps was significantly increased, among which the expression levels of HNF4α, HNF1α, and CYP1A2 were about 50% of the expression levels of MPHs in the positive control group, which were 73 times, 5 times, and 952 times higher than those in the negative control group MEFs, respectively. The expression level of CYP1A1 reached the expression level of MPHs, which was 56 times higher than that in the negative control group MEFs. The expression level of ALB was about 12% of the expression level of MPHs, which was 306 times higher than that in the negative control group MEFs.

[0209] After fibroblasts were transdifferentiated into hepatocytes, hepatocyte-related genes were significantly overexpressed (* indicates P < 0.05, *** indicates P < 0.001), including HNF4α, HNF1α, ALB, CYP1A1, and CYP1A2, indicating that fibroblasts had been transdifferentiated into hepatocytes.

[0210] Example 7: Isolation and identification of apoptotic vesicles from embryonic fibroblasts (MEFs) transdifferentiated into hepatic-like cells (ciHep) induced by transdifferentiation hepatic-like cell medium 6

[0211] Experimental steps:

[0212] (1) Transdifferentiation Hepatocyte Culture Medium 6: The method steps for chemically inducing mouse embryonic fibroblasts (MEFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0213] (2) According to the operation steps of "Isolation and Identification of Apoptotic Vesicles in Hepatocytes", apoVs were successfully isolated and extracted from MPHs, MEFs, and ciHeps using gradient centrifugation in the last step. The morphological structure was observed by transmission electron microscopy. Figure 6 .

[0214] Under transmission electron microscopy, apoVs derived from cells in each group were biconcave disc-shaped with a relatively complete double-layer membrane structure, which was consistent with the conventional morphology of extracellular vesicles ( Figure 6 ).

[0215] (3) According to the operation steps of “Isolation and identification of apoptotic vesicles of hepatoid cells”, Western Blot was used to detect apoVs marker protein to identify apoptotic vesicles. Figure 7 .

[0216] Figure 7 Western Blot results showed that the expression of apoptosis marker protein Cleaved Caspase-3 in apoVs derived from each group of cells was significantly upregulated compared with that of the parental cells. β-actin, as a non-vesicular marker, was significantly downregulated in apoVs of each group ( Figure 7 ). This is consistent with the characteristics of apoptotic vesicles.

[0217] Example 8: Therapeutic effect of apoVs on mouse liver tissue fibrosis induced by transdifferentiation of embryonic fibroblasts (MEFs) into hepatic-like cells (ciHep) by transdifferentiation hepatic-like cell culture medium 7

[0218] (1) Transdifferentiation Hepatocyte Culture Medium 6: The method steps for chemically inducing mouse embryonic fibroblasts (MEFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0219] (2) Following the procedures of “Isolation and Identification of Apoptotic Vesicles in Hepatocytes”, apoVs were isolated and extracted from MPHs, MEFs, and ciHeps using gradient centrifugation in the last step.

[0220] (3) Induction of CCl4 liver fibrosis model in mice and apoVs treatment

[0221] 8-week-old C57BL / 6J mice were used to construct a liver fibrosis model. Mice were intraperitoneally injected with CCl4 at a dose of 0.25 μl / g (body weight), dissolved in olive oil, three times a week for 8 weeks. After 8 weeks, the CCl4-treated mice were randomly divided into 4 groups, control group (normal saline group), MPH-apoVs group, MEF-apoVs group, ciHep-apoVs group, and normal saline or different groups of apoVs were injected through the tail vein at a dose of 30 μg protein content / mouse, dissolved in 100 μl normal saline. After 4 weeks, liver samples were taken for corresponding analysis.

[0222] (4) Histology and immunohistochemistry

[0223] The liver tissue was fixed with 10% formalin and then embedded in paraffin. The tissue wax block was cut into 5 μm thick paraffin sections using a microtome for subsequent histological and immunohistochemical detection. For Sirius red staining, the nucleus was stained with hematoxylin for 10 minutes, differentiated with hydrochloric acid and ethanol, and washed with running water for anti-blueing. Then, Sirius red staining was performed using the Sirius red staining kit according to the kit instructions. For immunohistochemical staining, after dewaxing to water, 3% H2O2 was used to treat at room temperature for 20 minutes to inactivate endogenous peroxidase. Antigen retrieval was performed using a citric acid repair solution with a pH of 6, and then blocked with 1% BSA at 37°C for 30 minutes, and the corresponding primary antibody was added and incubated overnight at 4°C. The next day, the corresponding secondary antibody was incubated at 37°C for 30 minutes, and then DAB was used for color development. After hematoxylin staining of the nucleus, it was dehydrated and transparent, and the slides were sealed with neutral resin. The experimental results are shown in Figure 8 .

[0224] Figure 8 The results showed that the degree of liver tissue fibrosis and collagen accumulation were detected by Sirius red staining. It can be seen that compared with the Control group, the area of ​​collagen positive staining in the ciHep-apoVs group was significantly reduced, and the results were similar to those in the MPH-apoVs group, while MEF-apoVs had no effect on collagen accumulation;

[0225] The expression level of αSMA, a marker protein of MFs, in liver tissue was detected by immunohistochemistry. The results showed that the ciHep-apoVs group significantly downregulated the area of ​​αSMA positive staining in liver tissue, indicating that the number of MFs was significantly reduced, and the results were similar to those of the MPH-apoVs group, while MEF-apoVs had no effect on αSMA expression.

[0226] The statistical graph on the right side of the figure shows that Sirius red (SR) staining results show that ciHep-apoVs significantly reduced the SR positive staining area in liver tissue. Immunohistochemical staining results show that ciHep-apoVs significantly reduced the αSMA positive staining area in liver tissue. Scale bar 100μm. (*** indicates P < 0.001).

[0227] The experimental results showed that ciHep-apoVs can reduce collagen deposition in liver tissue in the mouse liver fibrosis model and has the effect of improving mouse liver fibrosis.

[0228] Example 9: Effect of apoVs of hepatic embryonic fibroblasts (MEFs) transdifferentiated into hepatic hepatic cells (ciHep) induced by transdifferentiation hepatic cell culture medium 8 on the expression of fibrotic genes in mouse liver tissue

[0229] The experimental operation steps (1) to (3) are the same as those in Example 8 (1) to (3).

[0230] (4) Detection of gene expression in liver tissue fibrosis.

[0231] ① Extraction of total RNA from liver tissue: Take an appropriate volume of the obtained mouse liver tissue and place it in a 2ml centrifuge tube, add 500μl Trizol and two steel balls, and grind it in an automatic oscillating grinder at a frequency of 60HZ for 60s. After grinding, transfer the solution to a new 1.5ml centrifuge tube with a pipette, add 100μl chloroform, shake vigorously for 30s to fully mix, and let it stand at room temperature for 5min. After centrifugation at 4℃, 12000g for 15min, carefully pipette the supernatant into a new 1.5ml centrifuge tube. Add an equal volume of isopropanol, mix it upside down, let it stand at room temperature for 10min, and then centrifuge it at 4℃, 12000g for 10min. Discard the supernatant and wash the precipitate with pre-cooled 75% ethanol. Centrifuge at 4℃, 7500g for 5min, and discard the supernatant. Drain for 5-10min, add an appropriate amount of water to dissolve, and store at -80℃ for later use.

[0232] ② The expression of fibrosis genes ACTA2, COL1a1, and COL3a1 in liver tissue was detected by conventional RT-qPCR method. Fig. 9 .

[0233] Fig. 9 The results showed that compared with the Control group, the expression of fibrosis genes ACTA2, COL1a1, and COL3a1 in the ciHep-apoVs group was downregulated, and the results were similar to those in the MPH-apoVs group, while MEF-apoVs had no effect on the expression of fibrosis genes. The results showed that ciHep-apoVs reduced the expression level of fibrosis genes in liver tissue. (* indicates P < 0.05)

[0234] The experimental results showed that ciHep-apoVs reduced the expression level of liver tissue fibrosis genes and had the effect of improving liver fibrosis in mice.

[0235] Example 10: Effect of apoVs on mouse liver function induced by transdifferentiation of embryonic fibroblasts (MEFs) into hepatic-like cells (ciHep) induced by transdifferentiation hepatic-like cell culture medium 9

[0236] The experimental operation steps (1) to (3) were the same as those in Example 8 (1) to (3). After 4 weeks, serum samples were taken for corresponding analysis. The ALT and AST levels in the serum of each group of mice were detected. The results showed that the ALT and AST levels in the ciHep-apoVs group were significantly reduced compared with those in the negative control group. Fig.10 .

[0237] Fig.10The results showed that by detecting the ALT and AST levels in the serum of each group of mice, the ALT and AST levels in the ciHep-apoVs group were significantly reduced compared with the negative control group. The results of blood biochemistry tests showed that ciHep-apoVs reduced the ALT and AST levels in the serum of mice and improved the liver function of mice. (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001)

[0238] Example 11: Transdifferentiation of embryonic fibroblasts (MEFs) into hepatocyte-like cells (ciHep) induced by transdifferentiation hepatocyte-like cell culture medium 10, and detection of hepatocyte-specific markers of mouse primary hepatocytes (MPHs)

[0239] Transdifferentiation hepatocyte culture medium 10, the method steps for chemically inducing mouse embryonic fibroblasts (MEFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0240] After the cells in each group were fixed with 4% paraformaldehyde, immunofluorescence staining was performed to detect the expression of hepatocyte-specific proteins ALB and CYP1A2 in the cells. Fig.11 .

[0241] Fig.11 The results showed that ciHeps highly expressed ALB and CYP1A2, of which about 83% of cells were ALB-positive cells and 88% of cells were CYP1A2-positive cells. The proportion of ALB and CYP1A2-positive cells in ciHeps was similar to that in MPHs, indicating that ciHeps had the characteristics of mature hepatocytes and that SMC-induced direct cell reprogramming was highly efficient and uniform.

[0242] The experimental results showed that ciHeps highly expressed hepatocyte-specific markers ALB and CYP1A2, with positive expression rates of 83% and 88%, respectively. Scale bar 100 μm. (*** indicates P < 0.001). This indicates that the small molecule composition SMC chemically induced fibroblasts to directly reprogram into hepatocyte-like cells with high efficiency and uniformity.

[0243] Example 12: Chemically induced transdifferentiation of adult skin fibroblasts (MAFs) into hepatocytes (ciHep) by transdifferentiation hepatocyte-like cell culture medium 2, and comparison of glycogen staining of mouse primary hepatocytes (MPHs)

[0244] Transdifferentiation hepatocyte culture medium 2: The method steps for chemically inducing mouse adult skin fibroblasts (MAFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0245] MAFs, their transdifferentiated hepatocytes (ciHep), and mouse primary hepatocytes (MPHs) were stained for glycogen. The depth of staining reflects the ability of hepatocytes to store glycogen. The Schiff method was used for liver glycogen staining. The specific method is as follows: (1) discard the cell culture medium and rinse once with PBS; (2) fix with 4% paraformaldehyde for 10 minutes, and then rinse with PBS for 5 minutes × 3 times; (3) add PAS-I solution for 10 minutes and rinse with running water; (4) add PAS-II solution for 1-2 minutes and rinse with running water; (5) take pictures under a microscope. The experimental results are shown in Fig.12 .

[0246] Depend on Fig.12 The experimental results show that the hepatic glycogen staining of the hepatic fibroblasts (MAFs) transdifferentiation cultured by chemical induction in the transdifferentiation hepatic fibroblast culture medium 2 in the figure is positive, which is the same or similar to the positive MPH in the right figure; and significantly different from the negative glycogen staining of MAFs in the left figure. This proves that the transdifferentiated hepatic fibroblasts obtained by the method of the present invention have the glycogen storage activity unique to hepatocytes.

[0247] Example 13: Transdifferentiation of adult skin fibroblasts (MAFs) into hepatocytes (ciHep) induced by transdifferentiation hepatocyte-like cell culture medium 3, and oil-red staining experiment of mouse primary hepatocytes (MPHs)

[0248] Transdifferentiation hepatocyte culture medium 3, the method steps for chemically inducing mouse adult skin fibroblasts (MAFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0249] MAFs, transdifferentiated hepatocytes (ciHep), and mouse primary hepatocytes (MPHs) were stained using a conventional oil-red staining kit. The method and steps are shown in the kit. The staining results are shown in Fig.13 .

[0250] Fig.13 The experimental results show that the hepatocytes ciHep obtained by transdifferentiation culture in the figure are positive for oil-red staining, which is the same or similar to the positive MPH in the right figure, and significantly different from the negative oil-red staining of MAFs in the left figure. This proves that the transdifferentiated hepatocytes ciHep obtained by the method of the present invention have the unique fat metabolism function of hepatocytes.

[0251] Example 14: Comparison of hepatocyte-related gene detection in mouse adult skin fibroblasts (MAFs) induced by transdifferentiation hepatocyte-like cell medium 4 into hepatocyte-like cells (ciHep) and mouse primary hepatocytes (MPHs)

[0252] Transdifferentiation hepatocyte culture medium 4, the method steps for chemically inducing mouse skin fibroblasts (MAFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0253] Total RNA from the control group MAFs, transdifferentiation group ciHep, and primary hepatocytes (MPHs) were collected for RT-qPCR analysis to analyze the relative expression levels of hepatocyte-specific genes HNF4α, ALB, CYP1A1, CYP7A1, and CYP27A1. Fig.14 .

[0254] Fig.14 The results showed that compared with MAFs, the expression of liver cell-specific genes in ciHeps was significantly increased, among which the expression levels of HNF4α and CYP1A1 were about 80% of the expression levels of MPHs in the positive control group, which were 77 times and 79 times higher than those in the negative control group MAFs, respectively. The expression level of CYP27A1 reached the expression level of MPHs, which was 59 times higher than that in the negative control group MAFs. The expression level of ALB was about 10% of the expression level of MPHs, which was 330 times higher than that in the negative control group MAFs.

[0255] After fibroblasts were transdifferentiated into hepatocytes, hepatocyte-related genes were significantly overexpressed (*** indicates P < 0.001), including HNF4α, ALB, CYP1A1, CYP7A1, and CYP27A1, indicating that fibroblasts had been transdifferentiated into hepatocytes.

[0256] Example 15: Transdifferentiation of skin fibroblasts (MAFs) into hepatocyte-like cells (ciHep) induced by transdifferentiation hepatocyte-like cell culture medium 1, and detection of hepatocyte-specific markers of mouse primary hepatocytes (MPHs)

[0257] Transdifferentiation hepatocyte culture medium 1, the method steps for chemically inducing mouse skin fibroblasts (MAFs) to transdifferentiate into hepatocyte-like cells (ciHep) are the same as those in Example 2.

[0258] After the cells in each group were fixed with 4% paraformaldehyde, immunofluorescence staining was performed to detect the expression of hepatocyte-specific proteins ALB and CYP1A2 in the cells. Fig.15 .

[0259] Fig.15 The results showed that ciHeps highly expressed ALB and CYP1A2, of which about 81% of cells were ALB-positive cells and 85% of cells were CYP1A2-positive cells. The proportion of ALB and CYP1A2-positive cells in ciHeps was similar to that in MPHs, indicating that ciHeps had the characteristics of mature hepatocytes and that SMC-induced direct cell reprogramming was highly efficient and uniform.

[0260] The experimental results showed that ciHeps highly expressed hepatocyte-specific markers ALB and CYP1A2, with positive expression rates of 81% and 85%, respectively. Scale bar 100μm. (*** indicates P < 0.001). This indicates that the small molecule composition SMC chemically induced fibroblasts to directly reprogram into hepatocyte-like cells with high efficiency and uniformity.

[0261] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A chemical small molecule composition for inducing direct reprogramming of fibroblasts into hepatoid cells to prepare extrahepatic vesicles / hepatic apoptotic vesicles, characterized in that: The chemical small molecule composition consists only of chemical small molecule inhibitors: GSK3β inhibitor, BMP inhibitor, Notch inhibitor; Wherein, no exogenous genes / transcription factors / MicroRNA genes, and their RNA, proteins or polypeptides and other induction factors are introduced into the fibroblasts; or, the chemical small molecule composition does not contain any exogenous genes / transcription factors / MicroRNA genes, or their transcribed RNA, or their translated proteins and polypeptides and other induction factors.

2. The chemical small molecule composition according to claim 1, characterized in that: The weight ratio of GSK3β inhibitor: BMP inhibitor: Notch inhibitor is (5-80): (0.1-50): (0.1-50); or the molar concentration ratio of GSK3β inhibitor: BMP inhibitor: Notch inhibitor in solution is (0.1-20): (0.01-20): (0.01-20).

3. The chemical small molecule composition according to claim 1, characterized in that: The GSK3β inhibitor is a general term for chemical small molecule inhibitors that can target and inhibit the GSK3β signaling pathway, including but not limited to: CHIR-99021, LiCl, BIO, Ly2090314, IM-12, TWS119, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, SB216763, AZD1080, and the same class of GSK3β inhibitors or compounds that have the same function or induce inhibition of the same target of the GSK3β signaling pathway; The BMP inhibitor refers to a general term for chemical small molecule inhibitors that can target and inhibit the BMP signaling pathway, including but not limited to: BMP signaling agonist sb4, UK-383367 (UK 383367), LDN193189, BMT-124110, LDN-214117, SGC-AAK1-1, BMT-090605, BMT-090605hydrochloride, LP-922761, LP-922761hydrate, Asperosaponin VI, Elezanumab (ABT-555; AE12-1Y-QL), AZ12799734, DMH2, GW788388, ER-50891, LDN-212854, Dorsomorphin (Compound C) dihydrochloride, DMH-1, LDN193189Tetrahydrochloride, K02288, PD-161570, BMP inhibitors or small molecule compounds of the same class that have the same function or induce inhibition of the same BMP target; The Notch inhibitor refers to a general term for chemical small molecule inhibitors that can target and inhibit the Notch signaling pathway and γ-secretase, including but not limited to: Notch inhibitor1, IMR-1, Tangeretin (Tangeritin), BMS-906024, Nirogacestat (PF-3084014), LY900009, Brontictuzumab (OMP 52M51), Nirogacestat dihydrobromide (PF-3084014dihydrobromide), Semagacestat, ZLDI-8, L-685458, BMS-983970, DAPT (GSI-IX), 3,5-Bis (4-nitrophenoxy) benzoic acid, FLI-06, Crenigacestat (LY3039478), BT-GSI, Z-Ile-Leu-aldehyde (Z-IL-CHO), Demcizumab (OMP21M18), YO-01027 (Dibenzazepine; DBZ), Bruceine D, RO4929097 (RG-4733), MRK-560, Compound E, LY-411575, IMR-1A, DAPT (GSI-IX), Avagacestat (BMS-708163), Notch inhibitors or small molecule compounds of the same class that have the same function or induce inhibition of the same Notch target.

4. Use of the chemical small molecule composition according to any one of claims 1 to 3 in inducing direct reprogramming of fibroblasts into hepatoid cells to prepare extrahepatic vesicles / hepatic apoptotic vesicles, characterized in that: The chemical small molecule composition is added with physiological saline / buffer / basic cell culture medium to prepare a transdifferentiation hepatocyte reagent or culture medium.

5. A method for preparing extrahepatic vesicles / hepatic apoptotic vesicles by inducing direct reprogramming of fibroblasts into hepatoid cells using a chemical small molecule composition, characterized in that: The method comprises the following steps: (1) Using the chemical small molecule composition described in any one of claims 1 to 3 to induce fibroblasts to directly reprogram into hepatocytes: ① Preparation of concentrated liquid reagent: According to any one of the small molecule compositions described in claims 1-3, each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrated liquid reagent; the organic solvent includes dimethyl sulfoxide; the aqueous solvent includes: water, physiological saline, phosphate buffer; ② Obtaining a medium for inducing fibroblast transdifferentiation into hepatocytes: diluting the concentrated liquid reagents in step ① into a cell basal medium containing 5-20% calf serum, so that the concentration of each component meets the final concentration defined in any of the aforementioned small molecule compositions, to obtain a medium for inducing fibroblast transdifferentiation into hepatocytes; ③ Induce fibroblasts to transdifferentiate into hepatocytes: suspend and plate fibroblasts in a cell basal medium containing 5-20% calf serum or a serum-free medium containing various cytokines or growth factors, and replace with the transdifferentiation medium in step ② after the cells adhere to the wall, culture at 37°C, change the medium every 2-4 days; subculture once every 3-15 days; ④ Induce fibroblasts to transdifferentiate into hepatocytes and subculture: discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, incubate at 37°C for 1-5 minutes, terminate cell digestion, centrifuge, discard the supernatant, resuspend the cell pellet, and subculture and plate at a ratio of 1:1-1:3; use the transdifferentiation medium in step ②, culture according to the above method, and change the medium every 2-4 days; the digestion solution used includes trypsin, EDTA, Acutase, TrypleE; subculture once every 3-15 days; ⑤ Harvesting of inducing fibroblasts to transdifferentiate into hepatocytes: After the above experimental steps ③ and ④, transdifferentiated hepatocytes culture and subculture for 2-4 weeks, fibroblasts can be transdifferentiated into hepatocytes, and the transdifferentiated hepatocytes can be obtained; (2) Isolation, extraction and identification of hepatic apoptotic vesicles ① Induce cell apoptosis by adding apoptosis-inducing agent staurosporine, and separate and extract apoptotic vesicles; ② The morphology and size of hepatic apoptotic vesicles were observed by transmission electron microscopy, the particle diameter of hepatic apoptotic vesicles was observed by nanoparticle tracking analysis, and the molecular characteristics of hepatic apoptotic vesicles were verified by immunoblotting.

6. A method for preparing extrahepatic vesicles / hepatic apoptotic vesicles by inducing direct reprogramming of fibroblasts into hepatoid cells using the chemical small molecule composition according to any one of claims 1 to 3, characterized in that: The obtained transdifferentiated hepatoid cells are prepared as hepatoid cell preparations; or as extrahepatic vesicles / hepatic apoptotic vesicles.

7. The use according to claim 6, characterized in that The extrahepatic vesicles / hepatic apoptotic vesicles prepared by transdifferentiating hepatic-like cells are further prepared into anti-liver fibrosis biological preparations by adding physiological saline / buffer / pharmaceutically acceptable carriers or excipients.

8. A drug kit or test kit for preparing extrahepatic vesicles / hepatic apoptotic vesicles by inducing direct reprogramming of fibroblasts into hepatoid cells using a chemical small molecule composition, characterized in that: The medicine box or test kit includes the chemical small molecule composition described in any one of claims 1-3; or a transdifferentiation hepatocyte culture medium or reagent prepared by adding an organic solvent / physiological saline / buffer / cell basal culture medium to the chemical small molecule composition described in any one of claims 1-3; or a hepatocyte preparation prepared based on the transdifferentiation hepatocytes obtained in claims 6 and 7; or a biological preparation prepared based on the extrahepatic vesicles / hepatic apoptotic vesicles obtained in claims 6 and 7.

9. The chemical small molecule composition according to any one of claims 1 to 3, the method according to claim 5, the use according to any one of claims 4, 6 to 7, the drug box or the test kit according to claim 8, characterized in that: The fibroblasts include but are not limited to: fibroblasts from mice or mammals.

10. The small molecule composition according to any one of claims 1 to 3, the method according to claim 5, the use according to any one of claims 4, 6 to 7, the kit or reagent kit according to claim 8, characterized in that: The extrahepatic vesicles are hepatic apoptotic vesicles prepared by transdifferentiating hepatoid cells.

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