Compositions for treating liver fibrosis and methods of use thereof

Abstract

Provided are compositions for treating liver disease, injury, and damage, methods for making compositions, and methods for using compositions.The compositions can include chemically induced hepatic progenitor cells (CLiPs), and / or acellular materials formed from CLiPs, such as extracellular vesicles (EVs) (e.g., exosomes).In some embodiments, the compositions and / or methods are effective for reducing existing liver collagen or the formation of new liver collagen; and / or reducing the amount of existing fibrosis or the formation of new fibrosis in subjects who need to do the above.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 63 / 256,840, filed October 18, 2021, which is specifically incorporated by reference herein in its entirety.

[0002] Sequence Listing Reference The sequence listing submitted as an xml file named "EVIA102PCT.xml" (created on October 18, 2022, size: 18,496 bytes) is hereby incorporated by reference pursuant to 37 CFR 1.834(c)(1).

[0003] FIELD OF THEINVENTION The field of the invention relates generally to chemically induced hepatic progenitor cells, compositions made with and from the cells, and methods of using same to treat liver fibrosis. [Background technology]

[0004] 2. Background of the Invention Hepatocytes are considered the only effective cell source for cell transplantation to treat liver disease; however, their availability is limited due to donor shortage. Therefore, improved cell sources and alternative treatments must be developed. Results show that hepatic progenitor cells with repopulation capacity can be obtained from mature rodent hepatocytes and human infant hepatocytes using appropriate combinations of small molecule inhibitors (Katsuda et al., Cell Stem Cell 20, 41-55, (2017), dx.doi.org / 10.1016 / j.stem.2016.10.007, Katsuda et al., eLife 8:e47313, 31 pages, (2019) doi.org / 10.7554 / eLife.47313). However, the mechanisms underlying the ability of these cells to improve liver function and thus the scope of their use to treat liver diseases and disorders remained unclear. It is therefore an object of the present invention to provide insight into the mechanisms underlying the ability of chemically induced hepatic progenitor cells to improve liver function, as well as improved compositions developed based thereon and methods of use thereof. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Katsuda et al., Cell Stem Cell 20, 41-55, (2017), dx.doi.org / 10.1016 / j.stem.2016.10.007 [Non-Patent Document 2] Katsuda et al., eLife 8:e47313, 31 pages, (2019) doi.org / 10.7554 / eLife.47313 Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention Compositions, methods of making the compositions, and methods of using the compositions for the treatment of liver disease, disorders, and injury are provided. The compositions include chemically induced hepatic progenitor cells (CLiPs), and / or acellular material (e.g., extracellular vesicles (EVs) such as exosomes) formed from the CLiPs. In some embodiments, the compositions and / or methods reduce existing liver collagen or the formation of new liver collagen in a subject in need thereof; reduce the amount of existing fibrosis or the formation of new fibrosis; induce a change in the expression of one or more liver fibrosis-associated genes, optionally Mmp2, Mmp3, Mmp4, Mmp5, Mmp6, Mmp7, Mmp8, Mmp9, Mmp10, Mmp11, Mmp12, Mmp13, Mmp14, Mmp15, Mmp16, Mmp17, Mmp18, Mmp19, Mmp20, Mmp21, Mmp22, Mmp23, Mmp24, Mmp25, Mmp26, Mmp27, Mmp28, Mmp2 ...30, Mmp31, Mmp32, Mmp33, Mmp34, Mmp35, Mmp36, Mmp37, Mmp38, Mmp39, Mmp39, Mmp38, Mmp39, Mmp39, Mmp40, Mmp41, Mmp42, Mmp43, Mmp44, Mmp45, Mmp46, Mmp47, Mmp48, Mmp49, Mmp49, Mmp49, Mmp50, Mmp51, Mmp520, Mmp53, Mmp54, Mmp55, Mmp56, Mmp57, Mmp58, increase expression of Timp1, αSMA, and / or Col1a mRNA and / or protein, or any combination thereof; induce a decrease in expression of one or more markers of hepatic satellite cell activation, preferably in hepatic satellite cells (e.g., αSMA); induce a decrease in expression of one or more genes associated with cell cycle, autophagy, cell membrane fusion, and / or zinc finger proteins (optionally, the genes being Dmtf1, Zfp612, It the antibody is effective for inducing a change in expression of a cellular signaling pathway involved in the activation of hepatic satellite cells, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-35, IL-4, IL-56, IL-57, IL-68, IL-79, IL-80, IL-81, IL-82, IL-83, IL-84, IL-85, IL-86, IL-87, IL-89, IL-90, IL-91, IL-92, IL-93, IL-94, IL-95, IL-96, IL-97, IL-98, IL-99, IL-109, IL-1109, IL-120, IL-130, IL-140, IL-141, IL-142, IL-143, IL-144, IL-145, IL-146, IL-147, IL-148, IL-149, IL-150, IL-151, IL-152, IL-153, IL-154, IL-155, IL-156, IL-157, IL-168, IL-179, IL-189, IL-190, IL-201, IL-202, IL-2

[0007] Also provided is a method for producing EVs formed from CLiPs. The method typically includes culturing CLiPs and harvesting EVs secreted by the CLiPs. Typically, the cells are cultured with an inhibitor of TGFβ signaling (e.g., A83-01) at a concentration of, for example, about 1 μM to about 10 μM, or about 0.1 μM to about 10 μM, or about 0.5 μM. Typically, the cells are also cultured with a GSK3 inhibitor (e.g., CHIR99021) at a concentration of, for example, about 0.1 μM to about 20 μM, about 1 μM to about 10 μM, or about 3 μM.

[0008] Typically, the cells begin as hepatocytes isolated / purified from a mammalian liver.

[0009] In some embodiments, particularly when the starting cells are human hepatocytes, the cells are cultured with serum (e.g., fetal bovine serum (FBS)) at a concentration of, for example, 5-20% of the culture medium, or about 10% of the culture medium.

[0010] In some embodiments, particularly when the starting cells are rodent cells (e.g., derived from a mouse or rat), the cells are cultured with a ROCK inhibitor (e.g., Y-27632) at a concentration of, for example, about 1 μM to about 100 μM, or about 5 μM to about 25 μM, or about 10 μM. In some embodiments, particularly when the cells are human, a ROCK inhibitor can be omitted from the culturing.

[0011] The cells may be cultured with the inhibitor and / or serum for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days; or from about 5 days to about 25 days, or any subrange or integer number of days therebetween, as appropriate, from about 7 days to about 22 days, from about 5 days to about 25 days, or from about 10 days to about 20 days, or from about 12 days to about 17 days; or from about 13 days, 14 days, or 15 days.

[0012] Pharmaceutical compositions comprising an effective amount of CLiPs and / or EVs formed therefrom are also provided.

[0013] The compositions can be used in therapeutic and non-therapeutic methods of treating a subject in need thereof, typically comprising administering to the subject a pharmaceutical composition comprising an effective amount of CLiPs and / or EVs formed therefrom. In some embodiments, the methods are effective for treating a subject for liver fibrosis.

[0014] In some embodiments, the composition comprises EVs or CLiPs secreting EVs, wherein the EVs are expressed by one or more microRNAs (e.g., hsa-miR-103a-3p, hsa-miR-hsa-miR-122-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-1324, hsa-miR-1 42-3p, hsa-miR-151a-3p, hsa-miR-155-5p, hsa-miR-16-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-191 -5p, hsa-miR-192-5p, hsa-miR-21-5p, hsa-miR-221-3p, hsa-miR-224-5p, hsa-miR-23a-3p, hsa-miR-24-3p, hsa-miR-24-3p, hsa-miR-26a-3p, hsa-miR-28-3p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-30a-5p, hsa -miR-30d-5p, hsa-miR-30e-5p, hsa-miR-31-5p, hsa-miR-34a-5p, hsa-miR-3663-3p, hsa-miR-4435, hsa-miR and / or one or more of miR-4440, hsa-miR-5096, hsa-miR-510-3p, hsa-miR-92a-3p, hsa-miR-93-5p, and hsa-miR-99b-5p), and / or one or more cytokines (optionally wherein the cytokine is or includes TNFα), or any combination thereof.

[0015] In some embodiments, the method further comprises administering to the subject a second active agent. In some embodiments, the method comprises administering to the subject TNFα.

[0016] The methods may be used to treat a subject having a liver disease or disorder (e.g., an infection, optionally hepatitis A, hepatitis B, or hepatitis C; an immune system problem, optionally autoimmune hepatitis, primary biliary cholangitis, or primary sclerosing cholangitis; cancer, optionally liver cancer, cholangiocarcinoma, or hepatocellular adenoma; an inherited liver disorder, optionally hemochromatosis, hyperoxaluria, Wilson's disease, or Alpha-1 antitrypsin deficiency; damage from alcohol abuse and / or drug overdose; or nonalcoholic fatty liver disease). [Brief description of the drawings]

[0017] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figures 1A and 1B are line graphs showing blood biochemistry results in a mouse model of liver fibrosis. At the time of transplantation, total bilirubin levels were normal, and platelet counts were higher than baseline (Table 2). AST (Figure 1A) and ALT (Figure 1B) were monitored over time after transplantation, and no significant differences were found between transplanted and non-transplanted groups.

[0018] [Diagram 2] FIG. 2 is a plot showing collagen content in liver tissue with and without hCLiP transplantation.

[0019] [Diagram 3] Figure 3A is a plot showing Col1a positive areas in liver tissue with and without hCLiP transplantation, and Figure 3B is a plot showing changes in pathological liver fibrosis throughout hCLiP transplantation.

[0020] [Figure 4] 4A-4D are bar graphs showing changes in expression of liver fibrosis-related genes resulting from hCLiP transplantation: Mmp1 mRNA (FIG. 4A), Timp1 mRNA (FIG. 4B), Col1a mRNA (FIG. 4C), and αSMA mRNA (FIG. 4D).

[0021] [Diagram 5]Figure 5 is a plot showing the presence of hCLiPs in liver tissue. Total DNA was collected from frozen liver tissue and the copy numbers of each of them were measured using mouse Tfrc and human RNase P. 0-1% human cells were detected in the transplanted groups.

[0022] [Figure 6] 6 is a heat map showing changes in gene profiles resulting from hCLiP transplantation. hCLiP transplantation resulted in a significant decrease in the expression of 18 types of genes.

[0023] [Figure 7] FIG. 7 is a bar graph showing changes in hepatic satellite cell activation levels due to co-culture of hepatic satellite cells and hCLiPs.

[0024] [Figure 8] 8A to 8D are bar graphs showing CYP3A4 enzyme activity upon induction of hepatic differentiation of immortalized hCLiPs "A" to "D" in FIGS. 8A to 8D, respectively.

[0025] [Figure 9] FIG. 9 is a bar graph showing changes in hepatic satellite cell activation levels due to co-culture of hepatic satellite cells and immortalized hCLiPs.

[0026] [Figure 10] 10A-10D are bar graphs showing changes in gene expression in hepatic satellite cells resulting from coculture of hepatic satellite cells and hCLiPs: TNFα mRNA (FIG. 10A), TIMP3 mRNA (FIG. 10B), MMP13 mRNA (FIG. 10C), and MMP1 mRNA (FIG. 10D).

[0027] [Figure 11]11A-11C are bar graphs showing changes in hCLiPs gene expression resulting from co-culture of hepatic satellite cells and hCLiPs in the presence of TGF: MMP13 mRNA (FIG. 11A), TIMP3 mRNA (FIG. 11B), and TNFα mRNA (FIG. 11C).

[0028] [Figure 12] FIG. 12 is a bar graph showing the change in gene expression upon addition of 10 ng / ml, 20 ng / ml, or 50 ng / ml of TNFα to hepatic satellite cells: αSMA mRNA.

[0029] [Figure 13] Figures 13A and 13B are bar graphs showing changes in hepatic satellite cell activation caused by adding exosomes derived from hCLiP to hepatic satellite cells. Figure 13A shows the change in αSMA (a hepatic satellite cell activation marker) at the protein level in hepatic satellite cells with and without exosomes and with and without TGFβ. Figure 13B shows the levels of mRNA (MMP13, TIMP3, IL-13, and TNFα) in added exosomes.

[0030] [Figure 14-1] 14A-14C are bar graphs showing mRNAs in exosomes derived from hCLiP in the presence or absence of TGFβ: MMP13 (FIG. 14A), TIMP3 (FIG. 14B), and TNFα (FIG. 14C). n=1. [Figure 14-2] 14A-14C are bar graphs showing mRNAs in exosomes derived from hCLiP in the presence or absence of TGFβ: MMP13 (FIG. 14A), TIMP3 (FIG. 14B), and TNFα (FIG. 14C). n=1.

[0031] [Figure 15]Figure 15 is a model illustrating a proposed mechanism of action explaining hCLiPs-induced improvement in liver fibrosis. The proposed role of TNFα, a cytokine derived from hCLiPs, and hCLiP-derived exosomes is shown.

[0032] [Figure 16] Figure 16 is a bar graph showing the relative levels of various microRNAs detected in hCLiP EVs. Circular miRNAs represent those that may be particularly impactful in inhibiting liver fibrosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description of the Invention I. Definition As used herein, the term "carrier" or "excipient" refers to an inactive ingredient, organic or inorganic, natural or synthetic, in a formulation with which one or more active ingredients are combined.

[0034] As used herein, the term "pharmacologically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

[0035] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water and emulsions (e.g., oil / water or water / oil emulsions), as well as various types of wetting agents.

[0036] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to alleviate one or more symptoms of the disorder, disease, or condition being treated, or to otherwise provide the desired pharmacological and / or physiological effect. The exact dosage will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, and the route of administration and pharmacokinetics of the agent being administered.

[0037] As used herein, the term "prevention" or "preventing" means administering a composition to a subject or system at risk for or predisposed to one or more symptoms caused by a disease or disorder, to cause the arrest of a particular symptom of said disease or disorder, the reduction or prevention of one or more symptoms of said disease or disorder, the reduction of the severity of said disease or disorder, the complete elimination of said disease or disorder, or the stabilization or delay of the onset or progression of said disease or disorder.

[0038] As used herein, the terms "subject", "individual" and "patient" refer to any individual who is a target of treatment using the compositions of the present disclosure. The subject may be a vertebrate, e.g., a mammal. Thus, the subject may be a human. The subject may be symptomatic or asymptomatic. The terms do not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. The subject may include a control subject or a test subject.

[0039] As used herein, "substantially altered" means at least, e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100% or more altered compared to a control.

[0040] As used herein, the terms "purified," "isolated," and the like refer to the isolation of a molecule or compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) of other components that are normally associated with the molecule or compound in its natural environment.

[0041] As used herein, "treatment" refers to the medical management of a patient with the intent of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at removing the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathological condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the occurrence of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific treatment aimed at improving the associated disease, pathological condition, or disorder.

[0042] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually set forth herein.

[0043] Use of the term "about" is intended to describe values ​​that are either above or below the stated value in a range of approximately ±10%; in other forms, values ​​can range between values ​​that are either above or below the stated value in a range of approximately ±5%; in other forms, values ​​can range between values ​​that are either above or below the stated value in a range of approximately ±2%; in other forms, values ​​can range between values ​​that are either above or below the stated value in a range of approximately ±1%. The above ranges are intended to be clear by context, and no further limitation is implied.

[0044] Ranges may be expressed herein as "about" one particular value and / or to "about" another particular value. When such a range is expressed, the range from the one particular value and / or to the other particular value is also considered to be specifically contemplated and disclosed, unless the context specifically dictates otherwise. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another specifically contemplated embodiment that should be considered disclosed, unless the context specifically dictates otherwise. It is further understood that the endpoints of each of the above ranges are significant both relative to the other endpoint and independently of the other endpoint, unless the context specifically dictates otherwise. It should be understood that all individual values ​​and subranges of values ​​included within the explicitly disclosed ranges are also to be considered to be specifically contemplated and disclosed, unless the context specifically dictates otherwise. Finally, it should be understood that all ranges refer to both the ranges described and to a collection of individual numbers from a first endpoint (inclusive) to a second endpoint (inclusive). In the latter case, it should be understood that any of the individual numbers can be selected as one form of the amount, value, or characteristic to which the range refers. In this way, a range describes a set of numbers or values ​​from a first endpoint (inclusive) to a second endpoint (inclusive), from which a single member of the set (i.e., a single numerical value) can be selected as the amount, value, or characteristic to which the range refers. The above applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular case.

[0045] Any compound disclosed herein is intended to be specifically disclosed herein and should be considered to be specifically disclosed herein.Furthermore, any subrange that may be specified within this disclosure is intended to be specifically disclosed herein and should be considered to be specifically disclosed herein.As a result, it is specifically contemplated that any compound, or subgroup of compounds, may be either specifically included or excluded for use, or included or excluded in a list of compounds.

[0046] Disclosed are the components to be used to prepare the compositions of the present disclosure, and the compositions themselves to be used within the methods disclosed herein. When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even though specific reference to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed. For example, when a particular polypeptide is disclosed and discussed, and many modifications that can be made to many polypeptides are discussed, each and every combination and permutation of the polypeptides, and possible modifications, are specifically contemplated, unless specifically indicated to the contrary. Thus, when a class of molecules A, B, and C is disclosed, as is a class of molecules D, E, and F, and an example of a combination, A-D, is disclosed, each is individually and specifically contemplated, even if each is not individually described. That is, the combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups AE, BF, and CE are considered to be disclosed.This concept applies to all aspects of this application, including but not limited to the steps in the method of making and using the disclosed compositions.Thus, when there are various additional steps to be performed, it is understood that each of these additional steps can be performed in any specific embodiment or combination of embodiments of the disclosed method.

[0047] II. Composition Disclosed herein are compositions and methods for treating liver disease, disorders and injuries.The compositions can include and / or be formed by chemically induced hepatic progenitor cells (CLiPs).The compositions can be cell-based compositions or cell-free compositions.Methods for producing CLiPs are also provided.

[0048] A. Chemically induced hepatic progenitor cells (CLiPs) The compositions and methods of the present disclosure typically consist of or are formed from chemically induced hepatic progenitor cells (CLiPs), preferably human chemically induced hepatic progenitor cells (hCLiPs). The cells are preferably not intentionally genetically modified, for example, by recombinant gene technology, directional gene editing, etc. However, genetically modified cells are also contemplated. Examples include, but are not limited to, immortalized CLiPs (e.g., those having CDK4, CCND1 (cyclin D1), and / or TERT, typically under the control of conditionally or constitutively active promoters) (see, for example, the following examples).

[0049] 1. Source of starting hepatocytes Liver cells (also called hepatocytes) used as starting material for chemical induction typically contain at least one type of hepatocyte marker genes (e.g., albumin (ALB), transthyretin (TTR), glucose-6-phosphatase (G6PC), tyrosine aminotransferase (TAT), tryptophan-2,3-dioxygenase (TDO2), cytochrome P450 (CYP), miR-122, etc.), preferably two or more types, more preferably three or more types, even more preferably four or more types, particularly preferably five or more types, and most preferably all six types selected from ALB, TTR, G6PC, TAT, TDO2 and CYP. Preferably, the hepatocytes are functional. A functional hepatocyte refers to a hepatocyte that retains one or more, preferably two or more, more preferably three or more, even more preferably four or more, and most preferably all, of the following functions selected from: (i) having bile canalicular structures and accumulating drug metabolites in the canaliculi; (ii) expressing ABC transporters (e.g., MDR1, MRP, etc.) in the cell membrane; (iii) secreting and expressing ALB; (iv) accumulating glycogen; and (v) having activity as a drug metabolizing enzyme (e.g., CYP1A1, CYP1A2, etc.).

[0050] As long as the hepatocytes are hepatocytes, for example, they are characterized by the expression of the hepatocyte marker genes, and can be provided from any source.For example, the hepatocytes can be obtained from mammals, such as humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys, etc., preferably humans, rats or mice.The hepatocytes can be obtained from embryonic stem cells (ES cells) or pluripotent stem cells (for example, iPS cells by differentiation induction method), or from fibroblasts by direct reprogramming.In some embodiments, the hepatocytes are not genetically modified.

[0051] An exemplary source is hepatocytes isolated / purified from mammalian liver. For example, in the case of non-human mammals, the liver can be removed. For humans, adult liver tissue pieces can be removed by surgery or from recently ill donors (which can be adults or juveniles). Livers removed from aborted fetuses can also be used. Cells can be freshly isolated or cryopreserved cells that have previously been removed from the liver and isolated / purified hepatocytes. The liver can be a healthy liver. In some embodiments, the liver cells are autologous to the subject to be treated.

[0052] Hepatocytes can be purified from mammalian liver or tissue fragments thereof by perfusion methods (e.g., "Handbook of Cultured Cell Experiments" (Yodosha, 2004)). Specifically, after preperfusion with EGTA solution via the portal vein, the liver can be digested by perfusion with an enzyme solution such as collagenase or dispase (e.g., Hank's solution), and the hepatocytes can be purified by removing cell debris and non-parenchymal cells by filtration, low-speed centrifugation, etc.

[0053] 2. Inhibitors, serum, and other factors To form CLiPs, the hepatocytes are typically contacted with one or more inhibitors of TGF-β receptor and one or more inhibitors of GSK3. In some embodiments, the cells are also contacted with one or more ROCK inhibitors and / or serum. The contacting typically occurs in vitro / ex vivo.

[0054] Each of the above inhibitors (discussed in more detail below) can be a protein, nucleic acid, small molecule, antibody or other agent that reduces or prevents expression of a target molecule or signaling pathway (e.g., TGF-β, GSK3, ROCK, etc.).

[0055] Inhibitors can directly or indirectly inhibit or otherwise reduce the expression or activity of the target molecule.For example, negative regulators of ROCK activation include small GTP-binding proteins (e.g., Gem, RhoE, and Rad), which can attenuate ROCK activity.The autoinhibitory activity of ROCK has also been demonstrated by reducing kinase activity with its carboxy terminus and its kinase domain interaction.

[0056] The inhibitors may be, but are not limited to, small molecules, antibodies, antisense compounds, and negative regulators. Preferably, one or more or all of the inhibitors are low molecular weight compounds (e.g., small molecules).

[0057] In other examples, the inhibitor is an antisense compound. In general, the principle behind antisense technology is that antisense compounds hybridize to target nucleic acids, resulting in modulation of gene expression activity, or function (e.g., transcription, translation, or splicing). Modulation of gene expression can be achieved, for example, by target RNA degradation or occupancy-based inhibition. An example of modulation of target RNA function by degradation is RNase H-based degradation of target RNA upon hybridization with a DNA-like antisense compound (e.g., an antisense oligonucleotide). Antisense oligonucleotides can also be used to modulate gene expression, such as splicing, by occupancy-based inhibition (e.g., by blocking access to splice sites).

[0058] Antisense compounds include, but are not limited to, antisense oligonucleotides, siRNA, miRNA, shRNA and ribozymes.Antisense compounds can specifically target the nucleic acid that codes for the target for inhibition.Each of the above antisense compounds provides sequence-specific target gene regulation.This sequence specificity makes antisense compounds an effective tool for selectively modulating the target nucleic acid of interest.The method of designing, preparing and using antisense compounds that specifically target nucleic acid is within the capabilities of those skilled in the art.

[0059] In another embodiment, the inhibitor may be a function-blocking antibody.

[0060] a. TGF-β receptor inhibitors Hepatocytes are typically contacted in vitro with one or more low molecular weight signal transduction pathway inhibitors, including TGF-β receptor inhibitors.The TGF-β receptor inhibitors used for the present invention can be any inhibitor as long as they inhibit the function of transforming growth factor (TGF)-β receptor, including TGF-β / Smad signal transduction inhibitors, such as small molecules, antibodies, antisense compounds, and negative regulators of TGF-β / Smad signal transduction molecules.Antibodies, antisense compounds, and negative regulators can be designed to target TGF-β signal transduction molecules (e.g., ALK4, 5, and / or 7).

[0061] Exemplary small molecule inhibitors of TGF-β / Smad signaling include A83-01, SB431542, LDN-193189, galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox (E-616452), LDN-193189 2HCl, K02288, BIBF-0775, TP0427736 HCl, LDN-214117, SD-208, bactosertib (TEW-7197), ML347, LDN-212854, DMH1, dorsomorphin (compound C), 2HCl, pirfenidone (S-7701), sulfasalazine (NSC 667219), AUDA, PD 169316, TA-02, ITD-1, LY 3200882, alantolactone, halofuginone, SIS3 HCl, dorsomorphin (compound C), and hesperetin.

[0062] Other examples include, but are not limited to, 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole (A-83-01), 2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-ylamine (SD-208), 3-(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all from Merck) and SB431542 (Sigma Aldrich).

[0063] A preferred example is A-83-01 (also referred to herein as "A"). Typically, for example, the inhibitor, A83-01, is used at a concentration of about 0.1 μM to about 10 μM, or about 0.5 μM.

[0064] b. GSK3 inhibitors Hepatocytes are also typically contacted in vitro with one or more inhibitors of GSK3 inhibitors. The GSK3 inhibitor can be any GSK3 inhibitor as long as it inhibits the function of glycogen synthase kinase (GSK) 3. Examples include SB216763 (Selleck), CHIR98014, CHIR99021 (all from Axon medchem), SB415286 (Tocris Bioscience), and Kenpaullone (Cosmo Bio). A preferred example is CHIR99021 (also referred to herein as "C"). Typically, for example, the inhibitor CHIR99021 is used at a concentration of about 0.1 μM to about 20 μM, about 1 μM to about 10 μM, or about 3 μM.

[0065] c. ROCK inhibitor In some embodiments, the hepatocytes are also contacted with one or more inhibitors of ROCK. In some embodiments, for example, when the hepatocytes are human cells, the ROCK inhibitors can be omitted.

[0066] Rho-associated kinases (also known and / or referred to herein as ROCK, Rock, Rho-associated coiled-coil kinase, and Rho kinase) include ROCK1 (also referred to as ROKβ or p160ROCK) and ROCK2 (also referred to as ROKα). ROCK proteins are serine-threonine kinases that interact with Rho GTPases. Preferably, the ROCK inhibitors are small molecules. Exemplary small molecule ROCK inhibitors include Y-27632 (U.S. Pat. No. 4,997,834) and fasudil (also known as HA 1077; Asano et al., J. Pharmacol. Exp. Ther. 241:1033-1040, 1987). These inhibitors bind to the kinase domain and inhibit ROCK enzyme activity. Other small molecules that have been reported to specifically inhibit ROCK include H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine, Ikenoya et al., J. Neurochem. 81:9, 2002; Sasaki et al., Pharmacol. Ther. 93:225, 2002); N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea (Takami et al., Bioorg. Med. Chem. 12:2115, 2004); and 3-(4-pyridyl)-1H-indole (Yarrow et al., Chem. Biol. 12:385, 2005), GSK269962A (Axon medchem), and fasudil hydrochloride (Tocris Bioscience).

[0067] Additional small molecule Rho kinase inhibitors include those described in PCT Publication Nos. WO 03 / 059913, WO 03 / 064397, WO 05 / 003101, WO 04 / 112719, WO 03 / 062225 and WO 03 / 062227; U.S. Patent Nos. 7,217,722 and 7,199,147; and U.S. Patent Application Publication Nos. 2003 / 0220357, 2006 / 0241127, 2005 / 0182040 and 2005 / 0197328.

[0068] In a particularly preferred embodiment, the ROCK inhibitor is Y-27632 (also referred to herein as "Y"). Y-27632, also known as (+ / -)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, is a small molecule inhibitor that specifically inhibits the activity of Rho-associated kinase. Y-27632 is disclosed in U.S. Pat. No. 4,997,834 and PCT Publication No. WO 98 / 06433. In some embodiments, when the ROCK inhibitor is Y-27632, the effective amount of the ROCK inhibitor is about 1 to about 100 μM, or about 5 to about 25 μM, or about 10 μM.

[0069] The GSK3 inhibitor and the ROCK inhibitor individually do not induce hepatic stem / progenitor cells, whereas the efficiency of inducing hepatic stem / progenitor cells (also called "reprogramming efficiency") is significantly increased when the GSK3 inhibitor is contacted with hepatic cells together with the TGF-β receptor inhibitor, compared to when only the TGF-β receptor inhibitor is contacted with hepatic cells. Furthermore, the reprogramming efficiency of rat and mouse cells is also increased when the ROCK inhibitor is contacted with hepatic cells together with the TGF-β receptor inhibitor, compared to when only the TGF-β receptor inhibitor is contacted with hepatic cells (Katsuda et al., Cell Stem Cell 20, 41-55, (2017), dx.doi.org / 10.1016 / j.stem.2016.10.007, which is incorporated herein by reference in its entirety). Thus, in some embodiments, a GSK3 inhibitor and / or a ROCK inhibitor is contacted with hepatocytes in addition to a TGF-β receptor inhibitor.

[0070] d. Serum and other factors The results also indicate that when some human hepatocytes (e.g., primary infant human hepatocytes (IPHHs)) are used, the cells are preferably not contacted with a ROCK inhibitor, and are additionally or alternatively contacted with serum (e.g., fetal bovine serum) (Katsuda et al., eLife 8:e47313, 31 pages, (2019) doi.org / 10.7554 / eLife.47313, which is specifically incorporated by reference herein in its entirety). Thus, in some embodiments, a GSK3 inhibitor and / or serum is contacted with the hepatocytes in addition to a TGF-β receptor inhibitor.

[0071] Examples of serum include those derived from mammals, including but not limited to cows, humans, horses, goats, rabbits, sheep, pigs, rats, and mice. In certain embodiments, the serum is fetal bovine serum (FBS), fetal or neonatal calf serum (FCS), adult bovine serum (ABS), and human serum. When present, serum typically comprises about 5-20% of the culture medium. In certain embodiments, the serum is 10% FBS.

[0072] In the case of serum-free media, serum substitutes (BSA, HAS, KSR, etc.) may be added.

[0073] Generally, factors such as growth factors, cytokines, or hormones are further added.Examples of such factors include, but are not limited to, one or more of epidermal growth factor (EGF), insulin, transferrin, hepatocyte growth factor (HGF), oncostatin M (OsM), hydrocortisone 21-hemisuccinate or its salts, and dexamethasone (Dex).

[0074] e. MEK inhibitors Other low molecular weight signal transduction pathway inhibitors than GSK3 inhibitors and ROCK inhibitors can also be combined with TGF-β receptor inhibitors. Examples of such inhibitors include, but are not limited to, MEK inhibitors. The above MEK inhibitors are not particularly limited, and any inhibitor can be used as long as it inhibits the function of MEK (MAP kinase-ERK kinase). Examples here include AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY869766), SL327, U0126 (all from Selleck), PD98059, U0124 and U0125 (all from Cosmo Bio).

[0075] f. Exemplary Preferred Embodiments In particular, it is preferred to contact said cells with at least: A-83-01 (A) as a TGF-β receptor inhibitor in combination with CHIR99021 (C) as a GSK3 inhibitor (AC), optionally further combined with serum (e.g., FBS) (FAC); A-83-01 (A) as a TGF-β receptor inhibitor in combination with Y-27632 (Y) as a ROCK inhibitor (YA), optionally further combined with serum (e.g., FBS) (FYA); A-83-01 (A) as a TGF-β receptor inhibitor in combination with CHIR99021 (C) as a GSK3 inhibitor and Y-27632 (Y) as a ROCK inhibitor (YAC), optionally further combined with serum (e.g., FBS) (FYAC).

[0076] A preferred formulation for culturing mouse and rat hepatocytes is the YAC.

[0077] A preferred preparation for culturing IPHHs is FAC.

[0078] In certain embodiments, the concentration of the TGF-β receptor inhibitor added to the medium may be appropriately selected, for example, in the range of 0.01 to 10 μM, and preferably 0.1 to 1 μM; the concentration of the GSK3 inhibitor added to the medium may be appropriately selected, for example, in the range of 0.01 to 100 μM, and preferably 1 to 10 μM; the concentration of the ROCK inhibitor added to the medium may be appropriately selected, for example, in the range of 0.0001 to 500 μM, and preferably 1 to 50 μM; and the concentration of serum added to the medium may be appropriately selected, for example, in the range of 5% to 20%, preferably 8% to 12%, for example, 10%.

[0079] Methods of making the inhibitors and / or CLiPs are also described in one or more of WO 2020 / 080550, WO 2017 / 119512, U.S. Pat. No. 10,961,507, U.S. Patent Application No. 17 / 285,038, Katsuda et al., Cell Stem Cell 20, 41-55, (2017), dx.doi.org / 10.1016 / j.stem.2016.10.007, and Katsuda et al., eLife 8:e47313, 31 pages, (2019) doi.org / 10.7554 / eLife.47313, each of which is specifically incorporated by reference herein in its entirety.

[0080] 3. Culturing and Selection Guidelines The contact between the hepatocytes and the inhibitors and optional serum can be performed by culturing the hepatocytes in the presence of these substances. Specifically, these inhibitors and, if necessary, serum are added to the medium at effective concentrations for culturing. Examples of suitable media include, but are not limited to, basal media. Commercially available basal media can also be used. Examples include, but are not limited to, Minimum Essential Medium (MEM), Dulbecco's Modified Minimum Essential Medium (DMEM), RPMI1640 medium, 199 medium, Ham's F12 medium, and William's E medium, which may be used alone or in combination of two or more of them. Examples of additives to the medium include various amino acids (e.g., L-glutamine, L-proline, etc.), various inorganic salts (salts of selenite, NaHCO 3 etc.), various vitamins (nicotinamide, ascorbic acid derivatives, etc.), various antibiotics (e.g., penicillin, streptomycin, etc.), antifungal agents (e.g., amphotericin, etc.), and buffers (HEPES, etc.).

[0081] If these inhibitors are water-insoluble or poorly water-soluble compounds, they may be dissolved in a small amount of a low-toxicity organic solvent (such as DMSO) and the resultant may then be added to the medium to give the final concentrations described above.

[0082] The culture vessel used for this culture is not particularly limited as long as it is suitable for adhesion culture, and examples thereof include dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, and culture bags. The culture vessel used may have its inner surface coated with a cell support substrate in order to enhance adhesion to the cells. Examples of such cell support substrates include collagen, gelatin, Matrigel, poly-L-lysine, laminin, and fibronectin, and preferably collagen and / or Matrigel.

[0083] Hepatocytes were cultured at 10 2 ~10 6 cells / cm 2 , and preferably 10 3 ~10 5 cells / cm 2 The cells can be seeded onto the culture medium at a cell density of 100-200 μg / ml. 2 In an incubator, keep at 1-10%, preferably 2-5%, and more preferably about 5% CO 2 The culture may be performed in an atmosphere of high concentration at 30 to 40° C., preferably 35 to 37.5° C., and more preferably about 37° C. The culture period may be, for example, 1 to 4 weeks, preferably 1 to 3 weeks, and more preferably about 2 weeks. The medium may be replaced with a fresh one every 1 to 3 days.

[0084] In this way, hepatocytes are contacted with the TGF-β receptor inhibitor, and optionally the GSK3 inhibitor and / or the ROCK inhibitor and / or serum, so as to reprogram the hepatocytes into hepatic stem / progenitor cells. Mature hepatocytes are generally considered not to proliferate in vitro, but as described in Katsuda et al., Cell Stem Cell 20, 41-55, (2017), dx.doi.org / 10.1016 / j.stem.2016.10.007, they were found to proliferate approximately 15-fold by 2 weeks of culture in YAC. Similarly, IPHHs proliferated efficiently and became the predominant population when cultured for 2 weeks in FAC (Katsuda et al., eLife 8:e47313, 31 pages, (2019) doi.org / 10.7554 / eLife.47313).

[0085] In a preferred embodiment, the CLiPs have: (a) the ability to self-regenerate; and (b) Bipotential ability to differentiate into both hepatocytes and biliary epithelial cells. As used herein, the term "biliary epithelial cells" (also referred to as "BECs") refers to cells that express cytokeratin 19 (CK19) and GRHL2 as BEC markers. The CLiPs may also include fetal hepatoblasts and oval cells that appear upon liver injury.

[0086] In a preferred embodiment, similar to the above characteristics (a) and (b) and previously known liver stem cells (LSCs), the CLiPs obtained by the reprogramming method of the present disclosure have: (c) express epithelial cell adhesion molecule (EpCAM) as a surface antigen marker, but do not express delta homolog 1 (Dlk1), which is expressed by other known LSCs. Additionally, according to some embodiments, CLiPs do not express leucine-rich repeat-containing G protein-coupled receptor 5 (LGRS) and FoxL1, which are known LSC markers. CLiPs may also have one or more of the following features: (d) the apparent growth rate does not slow over at least 10 passages, preferably 20 or more passages; (e) the ability to differentiate into hepatocytes and BECs is retained for at least 10 passages, preferably 20 passages or more; (f) the nuclear-cytoplasmic (N / C) ratio is higher than that of hepatocytes; (g) Expression of one or more LSC marker genes selected from the group consisting of alpha-fetoprotein (AFP), SRY-box (Sox) 9, EpCAM, Thy-1 / CD90, hepatocyte nuclear factor 1 homeobox B (HNF1-β), forkhead box J1 (FoxJ1), HNF6 / one cut-1 (OC1), CD44, integrin alpha 6 (A6) and CK19 genes is increased compared to hepatocytes. (h) expression of one or more proteins selected from the group consisting of AFP, CD44, EpCAM, CK19, Sox9, A6 and CD90 is increased compared to hepatocytes.

[0087] In some embodiments, the CLiPs have all of the above features (d)-(h).

[0088] Thus, CLiPs can most typically be derived from hepatocytes in vitro or ex vivo by contacting the hepatocytes with a TGF-β receptor inhibitor, and preferably also with a GSK3 inhibitor and / or a ROCK inhibitor and / or serum, in an effective amount and under conditions suitable to induce cells having one or more, preferably most or all, of the characteristics discussed above.

[0089] 4. Maintenance / Proliferation of CLiPs The above CLiPs are, for example, (i) on collagen or Matrigel-coated culture vessels for passages 1 through 4; and (ii) on Matrigel-coated culture vessels for passage 5 or more; They can be efficiently maintained / grown by passaging them in the presence of inhibitor(s) and optionally serum.

[0090] As the culture vessel, a culture vessel similar to that used for inducing CLiPs from hepatocytes can be used. The culture vessel used from the first to fourth passages is coated with collagen or Matrigel.

[0091] Once the primary CLiPs obtained as described above reached 70–100% confluency, they were cultured on collagen- or Matrigel-coated culture vessels at 10 3 ~10 5 cells / cm 2 The medium may be similarly used as the medium described for the induction culture of CLiPs. The concentrations of the inhibitor and, if necessary, serum to be added may also be appropriately selected from the concentration ranges described above for the induction culture of CLiPs. Culture temperature and CO 2 The concentration also follows the conditions for the induction culture of CLiPs. Once reaching 70-100% confluency, the cells can be dissociated by treatment with trypsin and passaged.

[0092] For the fifth passage, etc., culture vessels coated with Matrigel are preferably used. Stable CLiPs can be obtained after about 5-8 passages. After 10 passages or even more, cloning can be performed by routine procedures.

[0093] As mentioned above, the inhibitors and optionally serum can be added to the culture medium not only for the induction of CLiPs but also for the maintenance / expansion cultures.

[0094] 5. Redifferentiation of CLiPs into hepatocytes In some embodiments, the CLiPs are utilized as CLiPs. In other embodiments, the CLiPs are redifferentiated into hepatocytes. The induction of CLiPs to redifferentiate into hepatocytes can be performed by any known method. Such a method can be, for example, a method of culturing in a culture vessel supplemented with oncostatin M (OsM), dexamethasone (Dex), hepatocyte growth factor (HGF), or the like (Journal of Cellular Physiology, Vol. 227(5), p. 2051-2058 (2012); Hepatology, Vol. 45(5), p. 1229-1239 (2007)), or a method combined with a matrigel overlay method (Hepatology 35, 1351-1359 (2002)). The medium for inducing differentiation into hepatocytes may or may not be supplemented with inhibitor(s) and serum as necessary, but is preferably supplemented.

[0095] Hepatocytes obtained by inducing differentiation of CLiPs can have bile canaliculus-like structures typical of mature hepatocytes, and can therefore accumulate drug metabolites in their canaliculi. In addition, they can express ABC transporters (e.g., MRP2 protein) in the cell membrane. Furthermore, they can exert a series of hepatic functions (e.g., secretory expression of albumin, glycogen accumulation, and cytochrome p450 (CYP) drug-metabolizing enzyme activity). Specifically, CLiPs can be redifferentiated into functional hepatocytes.

[0096] 6. Inducing Differentiation of CLiPs into BECs The induction of differentiation of CLiPs into BECs can be carried out by any known method, such as a method in which a collagen gel is used for culturing in a medium containing EGF and insulin-like growth factor 2 (IGF2).

[0097] In some embodiments, the differentiated CLiPs can form bile duct-like structures. In certain embodiments, the BEC induction method comprises the following steps: (i) culturing CLiPs on feeder cells at low density in the presence of inhibitor(s) and optionally serum; and (ii) further culturing the cells obtained in step (i) in a medium containing Matrigel.

[0098] The feeder cells used in step (i) are not particularly limited, and any cells generally used for the purpose of supporting maintenance and culture can be used.For example, they can be mouse embryo fibroblasts (MEF) and STO cells (ATCC, CRL-1503), preferably MEF.

[0099] Low density refers to a cell density lower than that typically used for maintenance and supporting culture purposes, for example, 1×10 3 ~5×10 4 cells / cm 2 , and preferably 5 × 10 3 ~3×10 4 cells / cm 2 The cell density is in the range of 10 to 15%. Culture vessels for seeding feeder cells can be coated with cell support substrates such as collagen and gelatin. Primary or passaged CLiPs can be treated with trypsin to dissociate and resuspended in medium containing inhibitor(s) and optionally serum, and cultured at 10 4 ~10 5 cells / cm 2 The cells may be seeded onto the feeder cells at a cell density of 100-200 μg / ml. If necessary, the medium may be supplemented with serum.

[0100] The next day, the medium is replaced with pluripotent stem cells (e.g., mTeSR TM(Stemcell Technologies)) and subjected to culture in the presence of inhibitor(s) and serum as required for 3-10 days, preferably 4-8 days. The medium may be replaced with a fresh one every 1-3 days. Subsequently, the medium may be replaced with a medium containing Matrigel and subjected to further culture for 3-10 days, preferably 4-8 days. The medium may be replaced with a fresh one every 1-3 days. The concentration of Matrigel added to the medium may be appropriately selected in the range of 1-5%, preferably 1-3%. After a total of about 1-3 weeks of culture, bile duct-like structures are formed, in which the cells express high levels of CK19 and GRHL2 as BEC markers. In addition, gene and protein expression of aquaporins (e.g., AQP1 and AQP9) and ion channels (e.g., CFTR and AE2) is increased. Furthermore, strong expression of ZO-1 as a tight junction marker is observed in the lumen of the ductal structures. Furthermore, since these cells have the ability to transport water and to transport and accumulate drug metabolites in the lumen, the LSCs of the present invention can be differentiated into functional BECs.

[0101] B. Cell-free materials Generally, cell-based therapy may have limitations such as uncontrolled differentiation, side effects, tumor formation, and incompatibility with allogeneic use.On the contrary, therapeutic and non-therapeutic use of extracellular vesicles (EVs) derived from CLiPs has the potential to overcome such shortcomings.Therefore, acellular compositions derived from CLiPs are also provided.

[0102] A cell-free composition comprising EVs and its method of use are provided. The EVs can be a part of a heterogeneous mixture of factors (e.g., conditioned medium) or a fraction isolated therefrom. In other embodiments, the EVs, or one or more subtypes thereof, are isolated or otherwise collected from the conditioned medium of CLiPs. The EVs, or one or more subtypes thereof, can be suspended in a pharma- ceutically acceptable composition (e.g., carrier or matrix or depot) before administration to the subject.

[0103] 1. Extracellular vesicles The compositions of the present disclosure may be or include extracellular vesicles derived from CLiPs, or isolated or fractionated subtype(s) thereof. Extracellular vesicles are lipid bilayer bounded particles that are naturally released from cells and, unlike cells, cannot replicate. EVs range in diameter from sizes approaching the smallest physically possible unilamellar liposomes (approximately 20-30 nanometers) to 10 microns or larger, although the vast majority of EVs are smaller than 200 nm.

[0104] Various EV subtypes have been proposed, including ectosomes, microvesicles (MVs), microparticles, exosomes, oncosomes, apoptotic bodies (ABs), and tunneling nanotubes (TNTs) (Yanez-Mo, et al., J Extracell Vesicles. 4: 27066 (2015) doi:10.3402 / jev.v4.27066. PMC 4433489). These EV subtypes are defined by various, often overlapping, definitions, mostly based on biogenesis (cellular pathway, cellular or tissue entity, state of origin) (Thery, et al., J Extracell Vesicles. 7 (1): 1535750 (2018). doi:10.1080 / 20013078.2018.1535750). However, EV subtypes can also be defined by size, constituent molecules, function, or isolation method. As discussed in Thery et al., subtypes of EVs can be defined by: a) physical characteristics of EVs (e.g., size ("small EVs" (sEVs) and "medium / large EVs" (m / lEVs), ranges are defined (e.g., <100 nm or <200 nm [small], or >200 nm [large and / or medium], respectively) or density (low, medium, high, ranges are defined for each); b) biochemical composition (e.g., CD9+ / CD63+ / CD81+- EVs, annexin A5 staining EVs); or c) Description of state or cell of origin (podocyte EVs, hypoxic EVs, large oncosomes, apoptotic bodies).

[0105] Thus, in some embodiments, the composition is or comprises one or more EV subtypes defined according to (a), (b), or (c) as discussed above.

[0106] In some embodiments, the vesicles are or comprise exosomes. Exosomes have surface proteins that facilitate endocytosis, they have the ability to deliver macromolecules, and when the exosomes are obtained from the same individual to whom they are delivered, the exosomes are immune tolerant.

[0107] Exosomes are vesicles with sizes of 30-150 nm, often 40-100 nm, that are observed in most cell types. Exosomes are often similar to MVs, but with an important difference: instead of originating directly from the plasma membrane, they are generated by inward budding into multivesicular bodies (MVBs). The formation of exosomes involves three distinct steps: (1) the formation of endocytic vesicles from the plasma membrane, (2) the inward budding of the endosomal vesicle membrane resulting in MVBs composed of intraluminal vesicles (ILVs), and (3) the fusion of these MVBs with the plasma membrane, which releases the vesicle contents (known as exosomes).

[0108] Exosomes have a lipid bilayer with an average thickness of about 5 nm (see, e.g., Li, Theranostics, 7(3):789-804 (2017) doi: 10.7150 / thno.18133). The lipid components of exosomes include ceramides (sometimes used to distinguish exosomes from lysosomes), cholesterol, sphingolipids, and phosphoglycerides with long and saturated fatty-acyl chains. The outer surface of exosomes is typically rich in saccharide chains (e.g., mannose, polylactosamine, α-2,6 sialic acid, and N-linked glycans).

[0109] Many exosomes contain proteins such as platelet-derived growth factor receptor, lactadherin, transmembrane and lysosomal-associated membrane protein-2B, membrane trafficking and fusion proteins (annexins, flotillins, GTPases, heat shock proteins, tetraspanins, etc.), proteins involved in multivesicular body biogenesis, as well as lipid-associated proteins and phospholipases. Thus, these characteristic proteins serve as good biomarkers for the isolation and quantification of exosomes. Another important cargo carried by exosomes is nucleic acid, including coding and non-coding ribonucleic acid (RNA), such as deoxynucleic acid (DNA), messenger RNA (mRNA) and microRNA (miRNA).

[0110] In some embodiments, the vesicles include or are one or more alternative extracellular vesicles (e.g., ABs, MVs, TNTs, or others discussed herein or elsewhere).

[0111] ABs are heterogeneous in size and originate from the plasma membrane. They can be released from all cell types and are approximately 1-5 μm in size.

[0112] MVs, with sizes ranging from 20 nm to 1 μm, are formed due to blebbing involving the incorporation of cytosolic proteins. In contrast to ABs, MVs are uniform in shape. They arise from the plasma membrane and are observed in most cell types.

[0113] TNTs are thin (e.g., 50-700 nm) and up to 100 μm long actin-containing tubes that form from the plasma membrane.

[0114] In some embodiments, the EVs are between about 20 nm and about 500 nm, In some embodiments, the EVs are between about 20 nm and about 250 nm or 200 nm or 150 nm or 100 nm.

[0115] The following example shows that EVs isolated from CLiPs contain many miRNAs and cytokines. The following example shows that EVs isolated from CLiPs contain the following: hsa-miR-103a-3p, hsa-miR-122-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-1324, hsa-miR-142-3p, hsa-miR-151a-3p, hsa-miR-155-5p, hsa-mi R-16-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa -miR-21-5p, hsa-miR-221-3p, hsa-miR-224-5p, hsa-miR-23a-3p, hsa-miR-24-3p, hs a-miR-24-3p, hsa-miR-26a-3p, hsa-miR-28-3p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-30a-5p, hsa-miR-30d-5p, hsa-miR-30e-5p, hsa-miR-31-5p, hsa-miR-34a-5p, hsa-miR-3663-3p, hsa-miR-4435, hsa-miR-4440, hsa-miR-5096, hsa-miR-510-3p, hsa-miR-92a-3p, hsa-miR-93-5p, and hsa-miR-99b-5p (see, e.g., Figure 16), and TNFα.

[0116] Thus, in some embodiments, the EVs comprise one or more of the following: hsa-miR-103a-3p, hsa-miR-122-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-1324, hsa-miR-142-3p, hsa-miR-151a-3p, hsa-miR-155-5p, hsa-miR-16-5 p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-21-5 p, hsa-miR-221-3p, hsa-miR-224-5p, hsa-miR-23a-3p, hsa-miR-24-3p, hsa-miR-24-3p, hsa-miR-26a-3p, hsa-miR-28-3p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-30a-5p, hsa-miR-30d-5p, hsa-miR-30e-5p, hsa-miR-31-5p, hsa-miR-34a-5p, hsa-miR-3663-3p, hsa-miR-4435, hsa-miR-4440, hsa-miR-5096, hsa-miR-510-3p, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-99b-5p, and / or one or more cytokines (e.g., TNFα), or any combination thereof.

[0117] 2. Method for producing extracellular vesicles a. Cell source for production Extracellular vesicles As used herein, EVs (including AB, MV, exosomes, and TNT) refer to lipid vesicles typically formed by cells or tissues. They can be isolated directly from tissues, cells, and / or fluids, including cultured and uncultured tissues, cells, or fluids, and fluids derived from or conditioned by cultured cells (e.g., conditioned medium), from subjects. For example, exosomes are present in physiological fluids (e.g., plasma, lymph, malignant pleural effusion, amniotic fluid, milk, semen, saliva, and urine) and are secreted into the medium of cultured cells.

[0118] The EVs of the compositions of the present disclosure are typically formed from CLiPs. The CLiPs can be prepared and maintained as discussed herein above and below, or elsewhere (e.g., Katsuda et al., Cell Stem Cell 20, 41-55, (2017), dx.doi.org / 10.1016 / j.stem.2016.10.007, Katsuda et al., eLife 8:e47313, 31 pages, (2019) doi.org / 10.7554 / eLife.47313, and U.S. Patent No. 10,961,507, each of which is specifically incorporated herein by reference in its entirety).

[0119] Methods for isolating extracellular vesicles directly from tissues, cells, and fluids, including cultured and uncultured tissues, cells, or fluids, as well as fluids derived from or conditioned by cultured cells (e.g., conditioned medium), from a subject are known in the art.

[0120] For example, Li, Thernaostics, 7(3):789-804 (2017) doi: 10.7150 / thno.18133, Ha, et al., Acta Pharmaceutica Sinica B, 6(4):287-296 (2016) doi: 10.1016 / j.apsb.2016.02.001, Skotland, et al., Progress in Lipid Research, 66:30-41 (2017) doi: 10.1016 / j.plipres.2017.03.001, Phinney and Pittenger, Stem Cells, 35:851-858 (2017) doi: 10.1002 / stem.2575, each of which is specifically incorporated by reference and describes isolating extracellular vesicles, particularly exosomes.

[0121] The EVs can be collected from primary cells or tissues or fluids. In some embodiments, the vesicles are isolated from the cells, tissues, or fluids of the subject to be treated. The advantages of using EVs isolated from natural sources include avoiding the immunogenicity that may be associated with artificially produced lipid vesicles.

[0122] The EVs can also be collected from cell lines or tissues. Exemplary cell lines are commercially available and include various sources thereof, including human bone marrow, human umbilical cord, human embryonic tissue, and human adipose (including those derived from lipoaspirates or dedifferentiated from mature adipocytes).

[0123] b. How to collect extracellular vesicles Extracellular vesicles (including exosomes) can be isolated using differential centrifugation, buoyant density gradient centrifugation, filtration, high performance liquid chromatography, and immunoaffinity capture.

[0124] For example, one of the most common isolation techniques for isolating exosomes from cell cultures is differential centrifugation, whereby large particles and cell debris in the culture medium are separated using centrifugal forces between 200 and 100,000 × g, and the exosomes are separated from the supernatant by sedimenting the exosomes at approximately 100,000 × g. However, purity can be improved by centrifuging the sample using buoyant density gradient centrifugation on sucrose or Optiprep. Tangential flow filtration combined with deuterium / sucrose-based density gradient ultracentrifugation has been used to isolate therapeutic exosomes for clinical trials.

[0125] In the following examples, hCLiPs were suspended, seeded, and cultured in SHM + FAC for 4 days. The final medium change was serum-free. The culture supernatant was collected and centrifuged at 20000g. The supernatant was filtered and ultracentrifuged at 35000 rpm. After ultracentrifugation, the supernatant was discarded and exosomes were formed into a pellet (ultracentrifugation can be repeated depending on the amount of culture supernatant). PBS was added to the pellet, the mixture was ultracentrifuged again, the supernatant was discarded, and the resulting product was washed. The pellet was dissolved in a very small amount of PBS (about 100 μL) remaining in the tube to prepare an exosome solution.

[0126] Ultrafiltration and high performance liquid chromatography (HPLC) are further methods to isolate EVs based on their size differences. EVs prepared by HPLC are highly purified.

[0127] Hydrostatic diafiltration has been used to isolate extracellular vesicles from urine.

[0128] Other common techniques for EV collection involve positive and / or negative selection using affinity-based methodologies. Antibodies can be immobilized in different media conditions and combined with magnetic beads, chromatography matrices, plates, and microfluidic devices for separation. For example, antibodies against exosome-associated antigens (e.g., cluster of differentiation (CD) molecules, CD63, CD81, CD82, CD9, epithelial cell adhesion molecule (EpCAM), and Ras-associated protein (Rab5)) can be used for affinity-based separation of exosomes. Non-exosomal vesicles carrying these or different antigens can also be isolated in a similar manner.

[0129] Microfluidic-based devices have also been used to rapidly and efficiently isolate EVs (e.g., exosomes), taking advantage of both the physical and biochemical properties of exosomes at the microscale. In addition to size, density, and immunoaffinity, sorting techniques (e.g., acoustic, electrophoretic, and electromagnetic manipulation) can be implemented.

[0130] Methods for characterizing EVs, including exosomes, are also known in the art. Exosomes can be characterized based on their size, protein content, and lipid content. Exosomes are spherical structures with sizes between 40-100 nm, much smaller compared to other systems (e.g., microvesicles with a size range of 100-500 nm). Several methods can be used to characterize EVs, including flow cytometry, nanoparticle tracking analysis, dynamic light scattering, Western blotting, mass spectrometry, and microscopy techniques. EVs can also be characterized and scored based on their protein composition. For example, integrins and tetraspanins are two of the most abundant proteins found in exosomes. Other protein markers include TSG101, ALG-2 interacting protein X (ALIX), flotillin 1, and cell adhesion molecules. Like proteins, lipids are major constituents of EVs and can be utilized to characterize them.

[0131] C. Pharmaceutical Compositions Also provided are pharmaceutical compositions comprising one or more of the CLiPs, EVs, and other molecules described herein for modulating liver function (e.g., miRNAs or cytokines, or nucleic acids encoding same, etc.). The pharmaceutical compositions can be administered parenterally (e.g., intramuscularly (IM), intraperitoneally (IP), intravenously (IV), subcutaneously (SubQ), or subdermal injection or infusion), transdermally (passively or using iontophoresis or electroporation), or by any other suitable means, and can be formulated in dosage forms suitable for each administration route.

[0132] In some embodiments, the compositions are administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective for delivery of the composition to targeted cells.

[0133] In some embodiments, the composition is administered locally, for example, by injection directly at or near the site to be treated. In some embodiments, such as for liver treatment, the composition is injected or otherwise administered directly into the liver or an area adjacent thereto, although other sites are also contemplated. For example, orthotopic liver transplantation is a therapeutic response for the treatment of some liver diseases, such as cirrhosis, fulminant hepatitis, and some fatal inherited enzyme deficiencies (Sharma, et al., Toxicologic Pathology, 40(1):83-92 (2012). doi:10.1177 / 0192623311425061). Although the procedure is now routine, it is not without its drawbacks, including post-transplant complications and donor shortages. Thus, alternative procedures are being investigated to support liver function. Among these procedures is the transplantation of isolated hepatocytes into various systemic sites. Many sites have been tested, including fat pads, muscles, subcutaneous tissue, peritoneum, lungs, kidneys, liver, and spleen. Thus, in some embodiments, compositions of the present disclosure, including cells and / or acellular materials, are administered locally to one or more of the above sites.

[0134] In some embodiments, local injection causes an increase in the local concentration of the composition greater than can be achieved by systemic administration.

[0135] In some embodiments, the composition is delivered locally to the appropriate location by using a catheter or syringe.Other means of delivering such compositions locally include using an infusion pump (e.g., Alza Corporation, Palo Alto, Calif.) or incorporating the composition into a polymeric implant (e.g., see P. Johnson and JG Lloyd-Jones, eds., Drug Delivery Systems: Fundamentals and Techniques (Chichester, England: Ellis Horwood Ltd., 1988 ISBN-10: 0895735806), which can provide sustained release of material to the area immediately adjacent to the implant.

[0136] The composition can be provided to the cells either directly (e.g., by contacting it with the cells) or indirectly (e.g., through the action of any biological process). For example, the vesicles can be formulated in a physiologically acceptable carrier and injected into tissues or fluids surrounding the cells.

[0137] Exemplary dosages for in vivo methods are discussed in the following experiments. As further tests are conducted, information regarding dosage levels suitable for treating various conditions in various patients will become clear, and those skilled in the art can ascertain appropriate administration, taking into account the recipient's treatment status, age, and general health. The dosage selected depends on the desired therapeutic effect, the route of administration, and the desired duration of treatment.

[0138] Generally, for local injection or infusion, the dosage may be low. In general, the total amount of active agent administered to an individual using the vesicles of the present disclosure may be smaller than the amount of active agent that must be administered for the same desired or intended effect and / or may exhibit reduced toxicity.

[0139] In a preferred embodiment, the compositions are administered in an aqueous solution by parenteral injection (eg, intramuscular, intraperitoneal, intravenous, subcutaneous, subdermal, etc.).

[0140] The formulations may be in the form of suspensions or emulsions. In general, pharmaceutical compositions are provided that contain an effective amount of one or more active agents, and optionally contain pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions may contain diluents, sterile water, buffered salt solutions of various buffer contents at various pH and ionic strengths (e.g., Tris-HCl, acetate, phosphate); and, optionally, additives (e.g., detergents and solubilizers (e.g., TWEEN® 20, TWEEN® 80 (also known as polysorbate 20 or 80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), as well as preservatives (e.g., thimerosal, benzyl alcohol) and bulking substances (e.g., lactose, The non-aqueous solvent or vehicle may include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). The formulation may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0141] Transdermal formulations can also be prepared. These are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations can include penetration enhancers. Chemical enhancers and physical methods, including electroporation and microneedles, can work with this method.

[0142] III. Method The compositions of the present disclosure can be used for the treatment of liver disease and injury and damage.The method typically comprises administering to the subject who needs it one or more of the compositions of the present disclosure in an amount effective to reduce or reverse one or more symptoms of liver disease or injury or liver damage.

[0143] Liver diseases and disorders include, but are not limited to, infectious diseases (e.g., hepatitis A, hepatitis B, and hepatitis C); immune system problems (e.g., autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis); cancer (e.g., liver cancer, cholangiocarcinoma, and hepatocellular adenoma); inherited liver disorders (e.g., hemochromatosis, hyperoxaluria, Wilson's disease, and alpha-1 antitrypsin deficiency); damage from alcohol abuse and / or drug overdose; and nonalcoholic fatty liver disease. Devastating complications of liver disease include acute liver failure and cirrhosis. In some embodiments, the liver disease or disorder is or includes liver fibrosis.

[0144] In some embodiments, the composition reduces existing liver fibrosis and / or the formation of new fibrosis. In some embodiments, the composition reduces the amount of existing liver collagen or the formation of new liver collagen, for example, as measured by the amount of hydroxyproline. In some embodiments, the composition reduces the amount of existing fibrosis or the formation of new fibrosis, as detected by staining with anti-Col1a antibody, changes in expression of one or more liver fibrosis-related genes (e.g., increased expression of Mmp2 mRNA, decreased expression of Timp1, αSMA, and / or Col1a mRNA and / or protein, or any combination thereof).

[0145] In a preferred embodiment, the composition induces a reduction in the expression of one or more markers of hepatic satellite cell activation, such as αSMA, preferably in hepatic satellite cells.

[0146] In some embodiments, the composition induces changes in expression of one or more genes associated with cell cycle, autophagy, cell membrane fusion, and / or zinc finger proteins (e.g., Dmtf1, Zfp612, Itga6, Trim24, Eaf2, Zfp119a, Dido1, Masp2, Sgk1, Sm11567, Eml5, Srsf5, Rab35, Fam206a, Zfp131, Zkscan14, Insc, and Ntn3) (see, e.g., FIG. 6).

[0147] In some embodiments, the composition induces an increase in MMP1 and / or MMP13 mRNA and / or protein, and / or a decrease in TNFα mRNA and / or protein in hepatic satellite cells.

[0148] The following experimental results show that TNFα can reduce αSMA mRNA expression in hepatic satellite cells. Thus, in some embodiments, TNFα is included in the composition or otherwise co-administered with the composition of the present disclosure.

[0149] The composition comprises CliPs, substances formed therefrom (e.g., exosomes), and their active components (microRNAs (e.g., hsa-miR-103a-3p, hsa-miR-122-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-1324, hsa-miR-142-3p, hsa-miR-142-4p, hsa-miR-142-5p, hsa-miR-142-6p, hsa-miR-142-7p, hsa-miR-142-8p, hsa-miR-142-9p, hsa-miR-142-10 ... R-151a-3p, hsa-miR-155-5p, hsa-miR-16-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-191-5p, h sa-miR-192-5p, hsa-miR-21-5p, hsa-miR-221-3p, hsa-miR-224-5p, hsa-miR-23a-3p, hsa-miR-24-3 p, hsa-miR-24-3p, hsa-miR-26a-3p, hsa-miR-28-3p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-30 a-5p, hsa-miR-30d-5p, hsa-miR-30e-5p, hsa-miR-31-5p, hsa-miR-34a-5p, hsa-miR-3663-3p, hsa-m The present invention relates to a method for the treatment of various cancers, including but not limited to, one or more cytokines such as, but not limited to, miR-4435, hsa-miR-4440, hsa-miR-5096, hsa-miR-510-3p, hsa-miR-92a-3p, hsa-miR-93-5p, and / or hsa-miR-99b-5p), and / or TNFα.

[0150] In the examples below, some of the microRNAs identified as present in exosomes produced by hCLiPs are categorized by their potential function / activity. Thus, in some embodiments, exosomes selected for a particular use may have one or more of the functions / activities desired for the treatment of the target disease or disorder as outlined above:

[0151] The detected miRNAs were also classified according to their potential contribution to exosome function / activity: 1) MicroRNAs that act to suppress fibrosis ·miR-29b-3p: miR-29b-3p / HMGB1 / TLR4 / NF-κB signaling, aSMA↓ ·miR-24, miR-27b: TGFbeta signaling↓ ·miR-192-5p: Zeb1 and Zeb2 associated with TGFbeta signaling; Inhibition of EMT 2) MicroRNAs that act for liver regeneration: ·miR-24: inhibiting cell growth and migration and promoting differentiation; Inhibition of TGFβ signaling; 3) MicroRNAs with anti-inflammatory effects: ·miR-16: TNF↑; anti-apoptosis 4) MicroRNAs with therapeutic effects against NASH: miR-182-5p; miR-183-5p 5) MicroRNAs that suppress hepatocellular carcinoma: ·miR-23a; miR-27b, miR-31-5p; miR-182-5p; miR-183-5p

[0152] The composition may, for example, be suspended in an appropriate isotonic buffer (eg, PBS). Some embodiments further comprise a pharma- ceutically acceptable excipient.

[0153] For example, the suspension of cells or EVs may vary depending on the type of liver disease, severity of liver injury, etc., but in the case of adults, for example, 10 8 ~10 11 The cells may be transplanted by intraportal administration, intrasplenic administration, and the like.

[0154] Combination therapy is also contemplated.Thus, in some embodiments, CLiPs and / or EVs formed from CLiPs are co-administered with a second active agent to a subject that requires co-administration.The second active agent can be in the same or different mixture as the CLiPs and / or EVs, and can be administered at the same time or at different times.In some embodiments, the additional active agent is a conventional treatment for the disease or disorder (e.g., liver disease or disorder) that the subject suffers from. EXAMPLES

[0155] Working Example Example 1: Liver fibrosis is ameliorated by hCLiP transplantation material and method Cells used Primary human hepatocytes (lot: FCL) were purchased from Veritas Corporation (Tokyo, Japan). Human hepatic satellite cells (Science Cell Research Laboratories) were purchased as hepatic satellite cells.

[0156] Media composition SHM was used as the basal medium for primary human hepatocytes. SHM was prepared by adding 2.4 g / l NaHCO 3 and L-glutamine, containing 5 mM HEPES (Sigma, MO), 30 mg / l L-proline (Sigma), 0.05% bovine serum albumin (Sigma), 10 ng / ml epidermal growth factor (Sigma), insulin-transferrin-serine-X (Life Technologies), 10 -7The basal medium SHM was prepared by adding 10 mM dexamethasone (Sigma), 10 mM nicotinamide (Sigma), 1 mM ascorbic acid-2 phosphate (Wako, Osaka, Japan), and antibiotic / antimycotic solution (Life Technologies). SHM+AC+10% FBS (SHM+FAC) was prepared by adding 10% FBS (Life Technologies), 0.5 μM A-83-01 (Wako), and 3 μM CHIR99021 (Axon Medchem, Reston, VA) to the basal medium SHM and was used to culture hCLiPs. Stellate Cell Growth Supplement, 2% FBS, and P / S were added to Stellate Cell Medium (Science cell research laboratories), respectively, and used as the basal medium for hepatic satellite cells depending on the experiment.

[0157] Generation of hCLiPs from primary human hepatocytes Approximately half of the cryopreserved primary human hepatocytes were thawed in a 37°C water bath and dissolved in 10 ml Leibovitz's L-15 Medium (Life Technologies) supplemented with Glutamax (Life Technologies) and antibiotic / antimycotic solution. After centrifugation of the mixture at 50 g for 5 min, the cell pellet was diluted with 10% FBS, GlutaMAX, antibiotic / antimycotic solution, and 10 ml Leibovitz's L-15 Medium (Life Technologies). -7 The cells were resuspended in William's E medium supplemented with M insulin (Sigma). Trypan blue (Life Technologies) was used to measure viable cell counts. Primary human pediatric hepatocytes (lot: FCL) were plated at 2 × 10 4 live cells / cm 2 After 3-6 hours, the medium was replaced with SHM+FAC. The medium was subsequently replaced every 2-3 days and the cells were cultured for 14 days.

[0158] Subculture of hCLiPs 70–100% confluent hCLiPs were detached from culture dishes using TrypLE Express (Life Technologies, MA) and plated at 1 × 10 5 Re-seeded cells / dish.

[0159] Generation of model mice with liver fibrosis Carbon tetrachloride (0.5 ml / kg) was dissolved in olive oil in a 1:4 ratio and administered intraperitoneally to 8-week-old NOD-SCID mice (which are immunodeficient mice) twice a week for 8 weeks, thereby inducing liver fibrosis.

[0160] Transplantation of hCLiPs into a mouse model with liver fibrosis The prepared hCLiPs were formed into a cell pellet by using TrypLE Express (Life Technologies, MA), which was then suspended in DMEM. A model mouse with liver fibrosis anesthetized with isoflurane was subjected to laparotomy, the spleen of the mouse was exposed, and the cell solution was added at 5 × 10 5 or 1×10 6 Cells / mouse were injected, thereby engrafting the cells into the spleen. Two weeks after engraftment, the mice were sacrificed and the extent of liver fibrosis was assessed.

[0161] RNA extraction Total RNA was extracted by using the miRNeasy Mini Kit (QIAGEN, Venlo, The Netherlands).

[0162] Reverse transcription For reverse transcription, a High-Capacity cDNA Reverse Transcription Kit (Life Technologies) was used.

[0163] Real-time PCR For cDNA, real-time PCR was performed using Platinum SYBR Green qPCR SuperMix UDG (Lifetechnologies) or TaqMan TM Universal PCR Master Mix, no AmpErase TM The PCR was performed using UNG (Applied Biosystems). Changes in gene expression were examined by using ACTB as an internal standard. The primers used are shown in Table 1 below. [Table 1] MMP1:Gene Exp Mmp1 Hs00899658 M1(Thermo Fisher) β-actin: Gene Exp Actb Hs03023880 G1 (Thermo Fisher)

[0164] Tissue immunostaining The antibodies used for tissue immunostaining are as follows. Paraffin block samples were prepared after fixation with formalin. After defatting with ethanol and xylene, antigen retrieval was performed for 45 minutes at 98°C using a solution prepared by diluting ImmunoSaver (Nissin EM, Tokyo, Japan) 200 times. 0.3% H 2 O 2 Endogenous peroxidase was inactivated by immersion in methanol containing 0.1% Triton® X-100 for 30 minutes at room temperature. Blocking was performed using Blocking One solution for 30 minutes at 4°C after infiltration with PBS containing 0.1% Triton® X-100. Primary antibodies were then incubated for 1 hour at room temperature or overnight at 4°C. The samples were stained using ImmPRESS IgG-Peroxidase Kit (Vector Labs, Burlingame, CA) and Metal Enhanced DAB Substrate Kit (Life Technologies). Finally, the samples were immersed in hematoxylin solution and a cover glass was placed on top to observe the samples. [Table 3]

[0165] Digital PCR Total DNA was collected from frozen liver tissue by using the DNeasy Blood & Tissue Kit (QIAGEN). Total DNA was used to perform quantification of 100% chromatin by using the Taqman Copy Number Reference Assay probe, Mousae Tfrc (VIC), and the Taqman RNase P Detection Reagents Kit (FAM). TM Human cells were detected in the livers of mice after transplantation of hCLiPs using 3D Digital PCR Master Mix v2 and the Quant Studio 3D Digital PCR System (Thermo Fisher Scientific).

[0166] Hydroxyproline quantification Hydroxyproline was quantified using liver tissue using a Hydroxyproline Assay Kit (Bio Vision).

[0167] result Generation of a mouse model with liver fibrosis by intraperitoneal administration of carbon tetrachloride Blood was collected by cutting the tail, and the degree of fibrosis was monitored using AST and ALT, and the condition was examined over time after administration of carbon tetrachloride. Regarding the dosage, 100-400 mg / kg was administered twice a week as a standard to properly examine the condition. Referring to the previous test, 200 mg / kg of carbon tetrachloride was administered intraperitoneally to 6-week-old mice, and one-third of the mice died within 10 days. All of the mice that died were either originally underweight or had lost weight significantly after administration. In light of these facts, it was considered too early to start administration at 6-week-old mice. As a result, the administration was changed to 8-week-old mice and the test was conducted again. When administration was started at 8-week-old mice, only one mouse died, and stable production of mice with fibrosis was successful. The degree of fibrosis was observed pathologically by Sirius red staining or immunostaining using Col1a antibody.

[0168] Blood biochemistry test of a mouse model with liver fibrosis At the time of transplantation, blood was collected by tail clipping, serum was separated, and AST, ALT, total bilirubin, and platelet counts were measured. [Table 2]

[0169] Total bilirubin showed normal values ​​(Table 2). AST and ALT were measured over time twice a week, and after a significant increase at the first administration, they stabilized and were higher than the baseline values. These facts indicate that a model with a mild level of liver fibrosis was created. After transplantation, the above model was compared with the non-transplant group in terms of AST / ALT, and no significant differences were found (Figures 1A-1B).

[0170] Reduced collagen content in liver tissue due to hCLiP transplantation Hydroxyproline is one type of amino acid that constitutes collagen. Two weeks after hCLiP transplantation, the animals were dissected and the amount of hydroxyproline in the liver tissue was quantified. In the group transplanted with hCLiPs, the amount of hydroxyproline was significantly decreased (Figure 2). This suggests that hCLiP transplantation may suppress collagen production or degrade collagen.

[0171] Amelioration of pathological liver fibrosis by hCLiP transplantation The degree of fibrosis was pathologically evaluated by Sirius red staining or immunostaining using type 1a collagen (Col1a) antibody. In the group transplanted with hCLiPs, the Col1a-positive area was significantly reduced (Figure 3A). Sirius red staining showed a tendency to be improved by hCLiP transplantation. Furthermore, it was revealed that the amount of Sirius red staining and hydroxyproline showed a positive correlation (Figure 3B).

[0172] Altered expression of liver fibrosis-related genes resulting from hCLiP transplantation Changes in the expression of genes associated with liver fibrosis (Mmp2, Timp1, αSMA, and Col1a) were observed by real-time PCR. In the group transplanted with hCLiPs, the expression level of Mmp2 mRNA was significantly increased, whereas the expression levels of Timp1, αSMA, and Col1a mRNA were significantly decreased (Figures 4A-4D). As a result of hCLiP transplantation, an increase in the expression of collagen-degrading genes and a decrease in the expression of collagen-producing genes were observed in livers with fibrosis.

[0173] Presence of hCLiPs in liver tissue To test where the transplanted hCLiPs from the spleen resided, liver tissue slices were used to perform immunostaining with human mitochondrial antibodies. However, detection failed. Therefore, total DNA was collected from frozen liver tissue and the copy numbers of each of them were measured using mouse Tfrc and human RNase P. 0-1% human cells were detected in the transplanted group (Figure 5). 5 × 10 5There are approximately 1 x 10 transplanted cells. 8 Since there were 10000 human cells, the number of transplanted hCLiPs / cell number in the mouse liver was 0.5%. At 2 weeks after transplantation, the percentage of human cells present in the mouse liver was up to 1%, indicating that hCLiPs probably proliferated in the mouse liver after transplantation.

[0174] Altered gene profiles resulting from hCLiP transplantation Microarray analysis was performed using RNA from the non-transplanted and transplanted groups. hCLiP transplantation caused a significant decrease in the expression of 18 types of genes (Dmtf1, Zfp612, Itga6, Trim24, Eaf2, Zfp119a, Dido1, Masp2, Sgk1, Sm11567, Eml5, Srsf5, Rab35, Fam206a, Zfp131, Zkscan14, Insc, and Ntn3) (Figure 6). These genes were related to cell cycle, autophagy, cell membrane fusion, or zinc finger proteins, and their gene profiles were significantly changed by hCLiP transplantation.

[0175] Example 2: Co-culture with hepatic satellite cells reveals therapeutic mechanisms of hCLiPs material and method Co-culture using hepatic satellite cells and hCLiPs Hepatic satellite cells were suspended in medium prepared by adding Stellate Cell Growth Supplement, 2% FBS, and P / S to Stellate Cell Medium (Science Cell Research Laboratories) at a concentration of 1 × 10 4 live cells / cm 2 After overnight, the medium was replaced with medium prepared by adding TGFβ and P / S to Stellate Cell Medium. After 24 h of incubation, hCLiPs were suspended in SHM medium and co-cultured at 1 × 10 cells / well using Transwell-COL inserts (Corning). 5Viable cells / well for 48 hours.

[0176] Addition of TNFα to hepatic satellite cells Hepatic satellite cells were suspended in medium prepared by adding Stellate Cell Growth Supplement, 2% FBS, and P / S to Stellate Cell Medium (Science Cell Research Laboratories) at 1 × 10 4 live cells / cm 2 After overnight, the medium was replaced with medium prepared by adding TGFβ and P / S to Stellate Cell Medium. After 24 hours of incubation, 5ng / ml, 10ng / ml, 20ng / ml, and 50ng / ml of TNFα were added and exposed for 24 hours.

[0177] Exosome collection hCLiPs were suspended in SHM + FAC at 2 × 10 3 live cells / cm 2 The cells were seeded with 1000μL of SHM + AC. The medium was changed every 2 days, and the medium was replaced with SHM + AC on the 4th day of culture. After 24 hours of culture, the culture supernatant was collected. The collected culture supernatant was centrifuged at 20000g and 4℃ for 10 minutes. The supernatant was filtered with Stericup Quick Release-GP Sterile Vacuum Filtration System (Millipore). The above treated culture supernatant was ultracentrifuged at 35000rpm and 4℃ for 1 hour and 10 minutes. Immediately after ultracentrifugation, the supernatant was discarded, and exosomes were formed into a pellet (ultracentrifugation was repeated 2 to 5 times depending on the amount of culture supernatant). PBS was added to the pellet, the mixture was ultracentrifuged again, the supernatant was discarded, and the resulting product was washed. The pellet was dissolved in a small amount of PBS (about 100μL) remaining in the tube to prepare an exosome solution.

[0178] Analysis of miRNAs in hCLiP-derived exosomes The collected exosomes were analyzed for the presence of miRNAs. MiRNAs were purified from CLiP EVs by using the Qiagen microRNAeasy kit. The purified microRNAs were submitted to a global miRNA expression analysis and analyzed using 3D-Gene. (登録商標) The miRNA Labeling kit and 3D-Gene, designed to detect 2,588 miRNA sequences registered in the miRBase release 21 database (http: / / www.mirbase.org / ), were used. (登録商標) The microarray experiments were performed by Kamakura Techno-Science Inc. Signal intensity >2 6 were considered as detected miRNAs.

[0179] Addition of hCLiP-derived exosomes to hepatic satellite cells Hepatic satellite cells were suspended in medium prepared by adding Stellate Cell Growth Supplement, 2% FBS, and P / S to Stellate Cell Medium (Science Cell Research Laboratories) at a concentration of 1 × 10 4 live cells / cm 2 After overnight incubation, the medium was replaced with medium prepared by adding TGFβ and P / S to Stellate Cell Medium. After 24 hours of incubation, 10 μg / mL of hCLiP-derived exosome solution was added, and the mixture was cultured for 48 hours.

[0180] Generation of immortalized hCLiPs Three genes, CDK4, CCND1 (cyclin D1), and TERT, were introduced, and four types of cells (A to D) were generated according to the differences in the promoters. [Table 4]

[0181] Induction of hepatic differentiation hCLiPs were plated at 5 × 10 4 Cells / well (2.5 x 10 4 cells / cm 2 ) at a seeding density of 1000 x 1000. When the cells reached 50-80% confluence, the medium was replaced with SHM containing 2% FBS, 0.5 mM A-83-01, and 3 mM CHIR99021. For the differentiation-inducing group (Hep-i(+)), 5 ng / ml human OSM (R&D) and 10 -6 M dexamethasone was added. The cells were cultured for 6 days, where the medium was changed every 2 days. On day 6, a mixture of Matrigel (Corning, Corning, NY) and the above medium in a ratio of 1:7 was poured onto the differentiation-inducing group (Hep-i(+)) instead of the medium. On day 8, the gel was aspirated and Ca 2+ and Mg 2+ The cells were washed with Hanks' balanced salt solution (Life Technologies) supplemented with 100% sodium chloride.

[0182] Measurement of CYP activity For the measurement of CYP activity, SHM containing 2% FBS was used as the basal medium. CYP3A4 was induced with 10 μM rifampicin or 1 mM phenobarbital. CYP1A2 was induced with 50 μM omeprazole. The medium containing CYP inducers was changed every day. After 3 days, P450-Glo was detected. TM CYP activity was measured using the CYP3A4 Assay System (Promega).

[0183] Protein extraction Cells were then treated with M-PER TMThe solution was thoroughly pipetted with Mammalian Protein Extraction Reagent and dissolved. The solution was centrifuged at 15000g for 10 minutes at 4°C, and the supernatant was used as the protein solution. The protein concentration was measured using Qubit® 2.0 Fluorometer.

[0184] Western blotting The protein solution was mixed with 4×SDS sample buffer (Merck), and the mixture was incubated at 95° C. for 5 minutes to prepare the migration sample. Precision Plus Protein TM Dual Color Standards (BIORAD) were used as molecular weight markers. 4-20% Mini-PROTEAN® TGX TM Precast Protein Gels (BIORAD) were placed in a transfer tank and the specimens and molecular weight markers were loaded. 100 ml 10x Tris / glycine / SDS was diluted with 900 ml miliQ and used as running buffer, and transfer was performed at 100 V for 1 h 10 min. For transfer, 80 ml 10x Tris / glycine was diluted with 720 ml miliQ, 200 ml methanol was added and used as transfer buffer, and transfer to immobilon-P membrane (Merck) was performed at 100 V for 1 h. Blocking was performed with Blocking One solution for 1 h at room temperature, the primary antibody was diluted in TBS-T supplemented with 10% Blocking One solution, and they were left overnight at 4 °C. The resulting product was washed three times with TBS-T, after which the secondary antibody was diluted in TBS-T and incubated at room temperature for 1 h. The resulting products were washed again three times with TBS-T and stained with ImmunoStar LD (Wako, Japan), and detection was performed with a Molecular Imager ChemiDoc XRS System (BIORAD). [Table 5]

[0185] statistical analysis Statistical analysis was performed using SPSS. Student's t-test and Dunnett's test were performed. Annotation for p<0.05: * , p<0,01: ** , p<0.001: * is used hereinafter.

[0186] result Co-culture of hepatic satellite cells and hCLiPs reduces the level of hepatic satellite cell activation Hepatic satellite cells play a central role in the progression pathophysiology of liver fibrosis. Upon activation of hepatic satellite cells, they produce extracellular matrix material, which plays a central role in liver fibrosis. Therefore, an experiment was designed to investigate the effect of their co-culture with hCLiPs on hepatic satellite cell activation. Hepatic satellite cells were seeded and after overnight, their medium was replaced with medium prepared by adding TGFβ and P / S to Stellate Cell Medium. After 24 hours of incubation, hCLiPs were co-cultured for 48 hours using Transwell-COL inserts. As a result of co-culture of hepatic satellite cells and hCLiPs, the expression of αSMA, which is a hepatic satellite cell activation marker, was significantly decreased at the mRNA and protein levels in hepatic satellite cells (Figure 7).

[0187] Co-culture of hepatic satellite cells and immortalized hCLiPs reduces the level of hepatic satellite cell activation Although hCLiPs have a significantly higher proliferation capacity, the population of non-parenchymal cells increases after repeated passage due to contamination with non-parenchymal cells. Therefore, it is difficult to accurately evaluate the function and therapeutic effect of hCLiPs after multiple passages. Therefore, immortalized hCLiPs were generated to evaluate whether they have the same function as that of hCLiPs. First, four types (A-D) of immortalized hCLiPs were generated according to differences in promoters, etc. To test whether immortalized hCLiPs have the same function as that of hCLiPs, immortalized hCLiPs were subjected to induction of differentiation and CYP enzyme activity was measured. In A and D, CYP enzyme activity increased due to induction of differentiation (Figures 8A-8D), whereas in B and C, induction of differentiation did not cause any change and enzyme activity was low. In view of these results, it is possible that other types of mixed cells, such as bile duct epithelial cells, were immortalized instead of hepatic progenitor cells in the immortalization. Therefore, A and D were used as immortalized hCLiPs. Next, hepatic satellite cells and immortalized hCLiPs were co-cultured. As a result of co-culture of hepatic satellite cells and immortalized hCLiPs, the expression of αSMA mRNA (which is a hepatic satellite cell activation marker) was significantly decreased in hepatic satellite cells ( FIG. 9 ).

[0188] Changes in gene expression in hepatic satellite cells resulting from co-culture of hepatic satellite cells and hCLiPs Hepatic satellite cells and hCLiPs were co-cultured to confirm changes in the expression of signaling genes involved in hepatic satellite cell activation and degradation of collagen fibrosis. As a result of co-culture of hepatic satellite cells and hCLiPs, the expression of MMP1 and MMP13 mRNA increased in hepatic satellite cells. As a result of the addition of TGFβ, the expression of TNFα mRNA decreased (Figures 10A-10D).

[0189] Changes in hCLiPs gene expression resulting from co-culture of hepatic satellite cells and hCLiPs We co-cultured hepatic satellite cells and hCLiPs to confirm the changes in the expression of signal genes involved in hepatic satellite cell activation and collagen fibrosis degradation in the presence and absence of TGFβ. As a result of co-culture of hepatic satellite cells and hCLiPs in the presence of TGFβ, the expression of MMP13 mRNA in hCLiPs was significantly increased. The expression of TNFα mRNA was increased, whereas the expression of TIMP3 mRNA was decreased (Figures 11A-11C).

[0190] Changes in gene expression upon addition of TNFα to hepatic satellite cells. Co-culture of hepatic satellite cells and hCLiPs in the presence of TGFβ resulted in increased expression of TNFα mRNA in hCLiPs, suggesting that the secretion of TNFα was increased from hCLiPs as a cytokine. Therefore, TNFα (which is a type of cytokine) was added to activated hepatic satellite cells. The addition of TNFα resulted in a significant decrease in αSMA mRNA expression in the 10ng / ml, 20ng / ml, and 50ng / ml groups (Figure 12).

[0191] Addition of hCLiP-derived exosomes to hepatic satellite cells reduces the activation level of hepatic satellite cells Co-culture of hepatic satellite cells and hCLiPs shows a decrease in the expression of αSMA due to secretions from hCLiPs. There are various cell-derived secretions (e.g., cytokines or exosomes). Exosomes are stable and easy to use for cell-free therapy. Therefore, hCLiP-derived exosomes were collected, and the exosome solution was added to hepatic satellite cells to observe the change in expression. Hepatic satellite cells were seeded overnight, and the medium was replaced with a medium prepared by adding TGFβ and P / S to Stellate Cell Medium. After 24 hours of incubation, 10 μg / mL of hCLiP-derived exosome solution was added, and the mixture was cultured for 48 hours. Addition of hCLiP-derived exosomes to hepatic satellite cells caused a decrease in the expression of αSMA (which is a hepatic satellite cell activation marker) at the protein level in hepatic satellite cells. Furthermore, gene expression of mRNA in the added exosomes was confirmed, and it was found that they contained a large amount of TNFα mRNA (Figures 13A to 13B).

[0192] mRNA in exosomes derived from hCLiP in the presence of TGFβ Exosomes were collected in the presence and absence of TGFβ, and changes in gene expression in exosomes were observed. In the presence of TGFβ, the expression levels of MMP13 and TIMP3 mRNA in exosomes were decreased. Furthermore, the expression level of TNFα in exosomes was increased (Figures 14A-14C).

[0193] miRNAs in hCLiP-derived exosomes The collected exosomes were also analyzed for the presence of miRNAs, and the results are shown in Figure 16. Circular miRNAs are believed to contribute to the inhibition of fibrosis.

[0194] The detected miRNAs were also classified according to their potential contribution to exosome function / activity: 1) MicroRNAs that act to suppress fibrosis ·miR-29b-3p: miR-29b-3p / HMGB1 / TLR4 / NF-κB signaling, aSMA↓ ·miR-24, miR-27b: TGFbeta signaling↓ ·miR-192-5p: Zeb1 and Zeb2 associated with TGFbeta signaling; Inhibition of EMT 2) MicroRNAs that act for liver regeneration: ·miR-24: inhibits cell growth & migration, and promotes differentiation; Inhibition of TGFβ signaling; 3) MicroRNAs with anti-inflammatory effects: ·miR-16: TNF↑; anti-apoptosis 4) MicroRNAs with therapeutic effects against NASH: miR-182-5p; miR-183-5p 5) MicroRNAs that suppress hepatocellular carcinoma: ·miR-23a; miR-27b, miR-31-5p; miR-182-5p; miR-183-5p

[0195] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0196] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method for producing extracellular vesicles (EVs), the method comprising culturing chemically induced hepatic progenitor cells (CLiPs) and harvesting EVs secreted by the CLiPs.

2. 2. The method of claim 1, wherein the CLiPs are formed by a method comprising culturing hepatocytes with an inhibitor of TGFβ signaling.

3. The method of claim 2, wherein the inhibitor of TGFβ signaling is A83-01.

4. 4. The method of claim 3, wherein the A83-01 is at a concentration of about 1 μM to about 10 μM, or about 0.1 μM to about 10 μM, or about 0.5 μM.

5. 2. The method of claim 1, wherein the CLiPs are formed by a method comprising culturing hepatocytes with a GSK3 inhibitor.

6. 6. The method of claim 5, wherein the GSK3 inhibitor is CHIR99021.

7. 7. The method of claim 6, wherein the CHIR99021 is at a concentration of about 0.1 μM to about 20 μM, about 1 μM to about 10 μM, or about 3 μM.

8. 2. The method of claim 1, wherein the CLiPs are formed by a method comprising culturing hepatocytes with serum.

9. 9. The method of claim 8, wherein the serum is fetal bovine serum (FBS).

10. 9. The method of claim 8, wherein the serum is about 5-20% of the culture medium, or about 10% of the culture medium.

11. 2. The method of claim 1, wherein the CLIPs are formed by a method comprising culturing hepatocytes with a ROCK inhibitor.

12. The method of claim 11, wherein the ROCK inhibitor is Y-27632.

13. 13. The method of claim 12, wherein the Y-27632 is at a concentration of about 1 μM to about 100 μM, or about 5 μM to about 25 μM, or about 10 μM.

14. 10. The method of claim 1, wherein the cells originate as hepatocytes isolated / purified from a mammalian liver.

15. 2. The method of claim 1, wherein the cells are cultured with the inhibitor and / or serum for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days, or about 5 days to about 25 days, or any subrange or integer number of days therebetween, optionally about 7 days to about 22 days, about 5 days to about 25 days, or about 10 days to about 20 days, or about 12 days to about 17 days; or about 13 days, 14 days, or 15 days.

16. The method of claim 1, wherein the EVs comprise or consist of exosomes.

17. 2. The method of claim 1, wherein the cells are human cells and the culturing comprises a TGBβ inhibitor, a GSK3 inhibitor, and serum, and optionally excludes a ROCK inhibitor.

18. 2. The method of claim 1, wherein the cells are mouse or rat cells, and the culturing comprises a TGBβ inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and optionally, serum-deprivation.

19. Extracellular vesicles (EVs) produced according to the method of any one of claims 1 to 18.

20. A pharmaceutical composition comprising an effective amount of the EVs described in claim 19.

21. 21. The pharmaceutical composition of claim 20 for use in a therapeutic or non-therapeutic method of treating a subject.

22. 21. A pharmaceutical composition comprising an effective amount of CLIPs for treating a subject for liver fibrosis or the pharmaceutical composition of claim 20.

23. 23. The pharmaceutical composition of claim 21 or 22, wherein the CLiPs are formed from human cells.

24. 22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition comprises CLiPs.

25. The CLiPs include hsa-miR-103a-3p, hsa-miR-122-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, and hsa-miR-126-3p. , hsa-miR-1324, hsa-miR-142-3p, hsa-miR-151a-3p, hsa-miR-155-5p, hsa-miR-16-5p, hsa-miR-182- 5p, hsa-miR-183-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-21-5p, hsa-miR-221-3p, hsa-miR-2 24-5p, hsa-miR-23a-3p, hsa-miR-24-3p, hsa-miR-24-3p, hsa-miR-26a-3p, hsa-miR-28-3p, hsa-miR-2 9a-3p, hsa-miR-29b-3p, hsa-miR-30a-5p, hsa-miR-30d-5p, hsa-miR-30e-5p, hsa-miR-31-5p, hsa-mi R-34a-5p, hsa-miR-3663-3p, hsa-miR-4435, hsa-miR-4440, hsa-miR-5096, hsa-miR-510-3p, hsa-miR 25. The pharmaceutical composition of claim 24, wherein the EVs secrete EVs comprising one or more of miR-92a-3p, miR-93-5p, and miR-99b-5p, and / or one or more cytokines, optionally wherein the cytokine is or comprises TNFα, or any combination thereof.

26. 22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is cell-free.

27. The pharmaceutical composition contains hsa-miR-103a-3p, hsa-miR-122-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-1324, hsa-miR-142-3p, hsa-miR-151a-3p, hsa-miR-155-5p, hsa-miR-16-5p, hsa-miR-182- 5p, hsa-miR-183-5p, hsa-miR-191-5p, hsa-miR-192-5p, hsa-miR-21-5p, hsa-miR-221-3p, hsa-miR-2 24-5p, hsa-miR-23a-3p, hsa-miR-24-3p, hsa-miR-24-3p, hsa-miR-26a-3p, hsa-miR-28-3p, hsa-miR- 29a-3p, hsa-miR-29b-3p, hsa-miR-30a-5p, hsa-miR-30d-5p, hsa-miR-30e-5p, hsa-miR-31-5p, hsa-m iR-34a-5p, hsa-miR-3663-3p, hsa-miR-4435, hsa-miR-4440, hsa-miR-5096, hsa-miR-510-3p, hsa-mi 22. The pharmaceutical composition of claim 21, comprising EVs comprising one or more of miR-92a-3p, hsa-miR-93-5p, and hsa-miR-99b-5p, and / or one or more cytokines, optionally wherein the cytokine is or comprises TNFα, or any combination thereof.

28. The pharmaceutical composition described in claim 20, characterized in that the pharmaceutical composition is administered to the subject in combination with TNFα.

29. 21. The pharmaceutical composition of claim 20, wherein the subject has a liver disease or disorder.

30. 30. The pharmaceutical composition of claim 29, wherein the liver disease or disorder is selected from an infection, optionally hepatitis A, hepatitis B, or hepatitis C; an immune system problem, optionally autoimmune hepatitis, primary biliary cholangitis, or primary sclerosing cholangitis; cancer, optionally liver cancer, cholangiocarcinoma, or hepatocellular adenoma; a genetic liver disorder, optionally hemochromatosis, hyperoxaluria, Wilson's disease, or alpha-1 antitrypsin deficiency; damage from alcohol abuse and / or drug overdose; or non-alcoholic fatty liver disease.

31. The CLIPs or EVs reduce the amount of existing liver collagen or the formation of new liver collagen; reduce the amount of existing fibrosis or the formation of new fibrosis; induce a change in the expression of one or more liver fibrosis-associated genes, optionally increasing the expression of Mmp2 mRNA, reducing the expression of Timp1, αSMA, and / or Col1a mRNA and / or protein, or any combination thereof; induce a decrease in the expression of one or more markers of hepatic satellite cell activation (e.g., αSMA), preferably in hepatic satellite cells; induce a decrease in the expression of one or more genes associated with cell cycle, autophagy, cell membrane fusion, and / or zinc finger proteins (optionally, the genes are Dmtf1, Zfp612, Itga6, Trim24, The composition or method of claim 21, which is capable of inducing changes in the expression of a protein (e.g., Eaf2, Zfp119a, Dido1, Masp2, Sgk1, Sm11567, Em15, Srsf5, Rab35, Fam206a, Zfp131, Zkscan14, Insc, Ntn3, or a combination thereof); inducing an increase in MMP1 and / or MMP13 mRNA and / or protein in hepatic satellite cells; and / or inducing a decrease in TNFα mRNA and / or protein in hepatic satellite cells.

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