A method for increasing the content of specific miRNA in extracellular vesicles derived from human cells

Abstract

The present invention provides a method for increasing the content of specific miRNA in extracellular membrane vesicles derived from human cells. Specifically, the method of the present invention includes: in the presence of immunomodulatory factors, inducing and culturing stem cells to obtain a fermentation culture fluid; and isolating the extracellular membrane vesicles from the fermentation culture fluid. In the extracellular membrane vesicles prepared by the method of the present invention, the content or expression of anti-inflammatory miRNA (such as miR-21-5p, miR146a-5p) is significantly increased, and the content of pro-inflammatory factors such as interleukin 1β (IL1β), interleukin 6 (IL6) and metalloproteinase 8 (MMP8) is significantly reduced, so the extracellular membrane vesicles of the present invention have better anti-inflammatory activity. The present invention also provides a pharmaceutical composition and application containing the extracellular membrane vesicles.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for increasing the content of specific miRNA in extracellular vesicles derived from human cells. Background Art

[0002] Extracellular vesicles (EVs) refer to substances with a phospholipid bilayer vesicle structure secreted by cells. According to their size and production mechanism, they can be divided into exosomes and microvesicles. Among them, exosomes are actively secreted by cells and are an important pathway for intercellular signal transduction. They are secreted outside the cell through the lysosomal pathway and after the multi-vesicle body fuses with the plasma membrane.

[0003] The main characteristics of extracellular vesicles include: 1) size is 30-150nm, 2) has a phospholipid bilayer, and appears as a cup-shaped sac structure under transmission electron microscopy, 3) has four transmembrane proteins such as CD63, CD9, CD81, and markers such as TSG101 and Alix. Exosome cargo is diverse, including proteins, small RNAs (including microRNA, lncRNA, circRNA, etc.) and a small amount of DNA.

[0004] MicroRNA plays an important role in cell activities such as proliferation and apoptosis. Studies have shown that they are closely related to the progression of lung inflammation and have great potential in the treatment of pulmonary fibrosis. Exosomes derived from mesenchymal stem cells are rich in microRNAs, which may play an anti-inflammatory role by regulating inflammatory pathways. Increasing the content of certain microRNAs in extracellular vesicles derived from human mesenchymal stem cells is of great significance in inflammatory diseases and fibrotic diseases. However, in the prior art, there is no particularly efficient method for increasing certain specific microRNAs in extracellular vesicles.

[0005] In summary, the present invention requires the development of a method for efficiently increasing the content of specific miRNA in extracellular vesicles derived from human cells. Summary of the invention

[0006] The object of the present invention is to provide a method for increasing the content of specific miRNA in extracellular vesicles derived from human cells.

[0007] In a first aspect of the present invention, a method for increasing the content of miRNA in extracellular vesicles is provided, comprising the steps of:

[0008] (a) providing a stem cell;

[0009] (b) pre-culturing the stem cells under appropriate culture conditions to obtain pre-cultured stem cells with a confluence degree P of 60-95%;

[0010] (c) inducing the cultured stem cells in the presence of immunomodulatory factors to obtain a fermentation broth, wherein the fermentation broth contains the induced cultured stem cells and the extracellular membrane vesicles produced by the induced cultured stem cells; wherein the immunomodulatory factors include interferon;

[0011] (d) isolating the extracellular membrane vesicles from the fermentation broth obtained in the previous step.

[0012] In another preferred embodiment, in the isolated extracellular vesicles, the content of one or more miRNAs selected from the following group is increased: miR-146a, miR-21, let-7a or a combination thereof.

[0013] In another preferred embodiment, the isolated extracellular membrane vesicles have one or more characteristics selected from the following group:

[0014] (m1) miR-21 (such as miR-21-5p) with increased relative expression level M1, preferably M1 ≥ 3, more preferably 3-6;

[0015] (m2) miR-146a (such as miR-146a-5p) with increased relative expression level M2, preferably M2 ≥ 3, more preferably 3-9;

[0016] (m3) let-7a (such as let-7a-5p) with increased relative expression level M3, preferably M3 ≥ 3, more preferably 3-6;

[0017] The relative expression level is the relative expression level obtained by comparing the expression level of the corresponding miRNA in the extracellular membrane vesicles (control) prepared under the same conditions but without the immunomodulatory factors (wherein the relative expression level of the control is set to 1).

[0018] In another preferred embodiment, the isolated extracellular membrane vesicles have one or more characteristics selected from the following group:

[0019] (f1) TGFβ1 with increased relative expression level E1, preferably E1 ≥ 1.5, preferably 1.5-2.0;

[0020] (f2) HGF with increased relative expression level E2, preferably E2 ≥ 1.5, preferably 1.5-2.0;

[0021] The relative expression level is the relative expression level obtained by comparing the expression level of TGFβ1 or HGF in the extracellular membrane vesicles (control) prepared under the same conditions but without immunomodulatory factors (wherein the relative expression level of the control is set to 1).

[0022] In another preferred embodiment, the isolated extracellular membrane vesicles have:

[0023] (f3) MMP8 with decreased relative expression level E3, preferably E3 ≤ 0.6, more preferably 0.4-0.6;

[0024] The relative expression level is the relative expression level obtained by comparing the expression level of MMP8 in extracellular membrane vesicles (control) prepared under the same conditions but without immunomodulatory factors (wherein the relative expression level of the control is set to 1).

[0025] In another preferred embodiment, the isolated extracellular membrane vesicles have one or more characteristics selected from the following group:

[0026] (f4) IL1β with similar relative expression level E4, wherein E4≤15, for example, 1-15, preferably 1-12;

[0027] (f5) IL6 with similar relative expression level E5, wherein E5≤10, for example, 1-10, preferably 1-6;

[0028] The relative expression level is the relative expression level obtained by comparing the expression level of IL1β or IL6 in the extracellular membrane vesicles (control) prepared under the same conditions but without immunomodulatory factors (wherein the relative expression level of the control is set to 1).

[0029] In another preferred embodiment, the method is used to increase the content of miRNA with anti-inflammatory activity (or anti-inflammatory miRNA) in extracellular vesicles.

[0030] In another preferred embodiment, the anti-inflammatory miRNA is selected from the following group: miR-146a-5p, miR-21-5p, or a combination thereof.

[0031] In another preferred embodiment, in step (d), comprising:

[0032] (d1) separating the fermentation broth to obtain a cell-free supernatant, wherein the supernatant contains the extracellular membrane vesicles;

[0033] (d2) incubating the supernatant in step (d1) with a polyethylene glycol (PEG) solution overnight;

[0034] (d3) separating the solution obtained in step (d2) to obtain a precipitate, wherein the precipitate contains the extracellular membrane vesicles.

[0035] In another preferred embodiment, the interferon is selected from the following group: IFN-γ, IFN-α, IFN-β, or a combination thereof.

[0036] In another preferred embodiment, the interferon is IFN-γ and does not contain α and / or β type IFN (such as IFN-α and IFN-β).

[0037] In another preferred embodiment, the immunomodulatory factor is a human immunomodulatory factor.

[0038] In another preferred embodiment, the immunomodulatory factor is a recombinant immunomodulatory factor.

[0039] In another preferred embodiment, the immunomodulatory factor is human IFN-γ.

[0040] In another preferred embodiment, the immunomodulatory factors include IFN and TNFα, wherein the weight ratio of TNFα to IFN is 0.01-1:1, preferably 0.01-0.2:1.

[0041] In another preferred embodiment, the stem cells are mesenchymal stem cells.

[0042] In another preferred embodiment, the mesenchymal stem cells are derived from mammals, preferably humans.

[0043] In another preferred embodiment, the mesenchymal stem cells are derived from human tissues, such as fat, bone marrow, placenta, or a combination thereof.

[0044] In another preferred embodiment, the stem cells are adipose-derived mesenchymal stem cells.

[0045] In another preferred embodiment, in step (b), the stem cell fusion degree P is 60-95%, preferably 70-90%, and more preferably 75-80%.

[0046] In another preferred embodiment, in step (c), the medium for induction culture is αMEM medium containing platelet lysate.

[0047] In another preferred embodiment, in step (c), the concentration of the platelet lysate is 2-15% (v / v), preferably, 5-10% (v / v).

[0048] In another preferred embodiment, in step (c), the induction culture time is 24-48 hours.

[0049] In another preferred embodiment, in step (c), the immunomodulatory factor is preferably IFN-γ (eg, SEQ INNO: 1).

[0050] In another preferred embodiment, in step (c), the final mass concentration of IFN-γ is 5 to 40 ng / ml, preferably 5 to 20 ng / ml; more preferably 9 to 11 ng / ml (based on the volume of the culture system).

[0051] In another preferred embodiment, in step (d1), the fermentation broth is centrifuged to obtain a cell-free supernatant.

[0052] In another preferred embodiment, in step (d1), the centrifugation condition is 3000-10,000 g differential centrifugation for 10-40 minutes.

[0053] In another preferred embodiment, in step (d2), the polyethylene glycol (PEG) is selected from PEG4000-PEG8000, preferably PEG6000.

[0054] In another preferred embodiment, in step (d2), the final concentration of the PEG6000 solution is 8-20% (w / v), preferably, 10-12% (w / v).

[0055] In another preferred embodiment, in step (d3), the fermentation broth is centrifuged to obtain a precipitate, wherein the precipitate contains the extracellular membrane vesicles.

[0056] In another preferred embodiment, in step (d3), the centrifugation condition is 3000-15,000 g differential centrifugation for 40-70 minutes.

[0057] In another preferred embodiment, the method further comprises step (e): detecting the miRNA content, cell activity and marker expression level of the extracellular membrane vesicles with increased miRNA content obtained in step (d).

[0058] In another preferred embodiment, the detection of the marker expression level includes detecting the gene expression level and / or protein expression level of the marker.

[0059] In another preferred embodiment, the markers include growth factors, pro-inflammatory factors, and extracellular vesicle-specific markers.

[0060] In another preferred embodiment, the growth factor is selected from the following group: transforming growth factor β1 (TGFβ1), hepatocyte growth factor (HGF), or a combination thereof.

[0061] In another preferred embodiment, the pro-inflammatory factor is selected from the following group: interleukin 1β (IL1β), interleukin 6 (IL6), metalloproteinase 8 (MMP8) or a combination thereof.

[0062] In another preferred embodiment, the extracellular vesicle-specific marker is selected from CD9, CD63 and CD81.

[0063] In another preferred embodiment, the miRNA includes miR-146a, miR-21, let-7a or a combination thereof.

[0064] In another preferred embodiment, the miR-146a is miR-146a-5p and / or miR-146a-3p.

[0065] In another preferred embodiment, the miR-21 is miR-21-5p and / or miR-21-3p.

[0066] In another preferred embodiment, the let-7a is let-7a-5p and / or let-7a-3p.

[0067] In a second aspect of the present invention, a cell membrane extracellular vesicle with increased miRNA content is provided, wherein the cell membrane extracellular vesicle is obtained by the method described in the first aspect of the present invention.

[0068] In another preferred example, the extracellular vesicles are derived from human cells.

[0069] In another preferred embodiment, the human cells are selected from induced stem cells and mesenchymal stem cells.

[0070] In another preferred embodiment, compared with the exosomes obtained in the negative control group, the exosomes have one or more characteristics selected from the following group:

[0071] (1) Increased levels of anti-inflammatory miRNAs and / or increased levels of miRNAs that inhibit epithelial-mesenchymal transition, among which;

[0072] (2) the mRNA and / or protein content of a growth factor is increased, wherein the growth factor is selected from the group consisting of TGFβ1, HGF, or a combination thereof;

[0073] (3) decreased mRNA and / or protein levels of the proinflammatory factor metalloproteinase 8 (MMP8);

[0074] (4) The expression of extracellular vesicle-specific markers is similar or increased.

[0075] In another preferred embodiment, the miRNA includes miR-146a-5p, miR-21-5p, and let-7a-5p.

[0076] In another preferred embodiment, the growth factors include transforming growth factor β1 (TGFβ1) and hepatocyte growth factor (HGF).

[0077] In another preferred embodiment, the mRNA and / or protein content of interleukin 1β (IL1β) and interleukin 6 (IL6) in the extracellular vesicles is similar to that of the control.

[0078] In another preferred embodiment, the extracellular vesicle-specific protein markers include CD9, CD63 and CD81.

[0079] In another preferred embodiment, in the "negative control group", stem cells are induced and cultured under the same culture conditions but in the absence of immunomodulatory factors, and extracellular vesicles are isolated.

[0080] In a third aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0081] (Z1) the extracellular vesicle as described in the second aspect of the present invention;

[0082] (Z2) a pharmaceutically acceptable carrier.

[0083] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid dosage form, solid dosage form (such as lyophilized dosage form).

[0084] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating inflammatory diseases or fibrotic diseases.

[0085] In another preferred embodiment, the fibrotic disease includes pulmonary fibrosis.

[0086] In another preferred embodiment, the pharmaceutical composition includes injection and aerosol inhalation preparation.

[0087] In the fourth aspect of the present invention, a medicine kit is provided, wherein the medicine kit comprises the pharmaceutical composition as described in the third aspect of the present invention.

[0088] In another preferred embodiment, the medicine kit further comprises other drugs for treating inflammatory diseases or fibrotic diseases.

[0089] In the fifth aspect of the present invention, a method for treating a disease is provided, comprising the steps of administering the extracellular membrane vesicles described in the second aspect of the present invention to a subject in need thereof.

[0090] In another preferred embodiment, the disease is selected from the following group: inflammatory diseases, fibrotic diseases, or a combination thereof.

[0091] In another preferred embodiment, the disease is selected from the following group: pulmonary fibrosis, pneumonia, respiratory inflammation, ARDS, COPD.

[0092] In another preferred embodiment, the pharmaceutical composition is used to treat diseases caused by viruses.

[0093] In another preferred embodiment, the virus is selected from the group consisting of influenza virus, SARS coronavirus, SARS coronavirus 2, and MERS coronavirus infection.

[0094] In another preferred embodiment, the inflammation is selected from the group consisting of viral infection inflammation, bacterial infection inflammation, fungal infection inflammation, autoimmune reaction inflammation, or a combination thereof;

[0095] The injury is selected from the group consisting of ischemic injury, hypoxic injury, chemical injury, physical injury, or a combination thereof.

[0096] In another preferred embodiment, the drug is used to treat diseases caused by viruses.

[0097] In another preferred embodiment, the virus is selected from the group consisting of influenza virus, SARS coronavirus, SARS coronavirus 2, and MERS coronavirus infection.

[0098] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 The effects of different inflammatory factors on the activity of P4 generation adipose-derived mesenchymal stem cells are shown. Among them, control is the negative control group; TNFα is tumor necrosis factor; IFNγ is interferon γ; TNFα+IFNγ is the combination of the two factors. All groups were statistically analyzed with the negative control. The CCK8 kit was used to detect the activity of cells after incubating the cells with B solution containing the factors for 48 hours. It was found that the activity of cells in the TNFα and IFNγ groups was not significantly reduced compared with the negative control group; the activity of cells in the TNFα+IFNγ combined group was partially reduced compared with the negative control group.

[0100] Figure 2 The results show the expression of cell mRNA levels after pretreatment of P4 adipose-derived mesenchymal stem cells with different inflammatory factors. Figure 2 A, Expression of transforming growth factor β1 (TGFβ1) mRNA level; Figure 2 B, Expression of hepatocyte growth factor (HGF) mRNA level; Figure 2 C metalloproteinase 8 (MMP8) mRNA level expression; Figure 2D, Expression of interleukin 6 (IL6) mRNA level; Figure 2 E, Expression of interleukin 1β (IL1β) mRNA level. Control is the negative control group; TNFα is tumor necrosis factor; IFNγ is interferon γ; TNFα+IFNγ is the combination of the two factors. All groups were statistically analyzed with the negative control, *P<0.05, **P<0.001, ***P<0.0001; Error bars, SD

[0101] Figure 3 The miRNA expression of extracellular vesicles of adipose-derived mesenchymal stem cells at generation 4 pretreated with different inflammatory factors is shown. Figure 3 A is a comparison chart of miR-21-5p expression; Figure 3 B is a comparison of the expression of let-7a-5p; Figure 3 C is a comparison chart of miR-146a-5p expression. Control is the negative control group; TNFα is tumor necrosis factor; IFNγ is interferon γ; TNFα+IFNγ is the combination of the two factors. All groups were statistically analyzed with the negative control, *P<0.05, **P<0.001, ***P<0.0001; Error bars, SD

[0102] Figure 4 The NTA detection shows the particle concentration and particle size of extracellular vesicles after pretreatment with different inflammatory factors. (A) is the particle size distribution diagram of the negative control group; (B) is the particle size distribution diagram of the TNFα group; (C) is the particle size distribution diagram of the IFNγ group; (D) is the particle size distribution diagram of the TNFα+IFNγ group; (E) is the particle concentration of extracellular vesicles after pretreatment with different inflammatory factors; (F) is the particle size of extracellular vesicles after pretreatment with different inflammatory factors. All groups were statistically analyzed with the negative control, *P<0.05, **P<0.001, ***P<0.0001; Error bars, SD

[0103] Figure 5 The expression of exosomes of P4 adipose-derived mesenchymal stem cells pretreated with different inflammatory factors (same volume loading) is shown. Ctrl is the negative control group; TNF 10 is TNFα10ng / ml; IFN10 is IFNγ10ng / ml; T+I10 is TNFα10ng / ml+IFNγ10ng / ml. DETAILED DESCRIPTION

[0104] After extensive and in-depth research and a large amount of screening, the inventors have developed for the first time a method for increasing certain specific miRNAs in extracellular membrane vesicles derived from human cells in vitro. Specifically, by inducing the culture of pre-cultured stem cells with specific immunomodulatory factors (such as interferon γ) to obtain a fermentation culture broth, and then separating from the fermentation culture broth, extracellular membrane vesicles with increased content of certain specific miRNAs and / or reduced content of pro-inflammatory factors can be obtained. Experiments show that by culturing mesenchymal stem cells by the method of the present invention, under conditions that do not affect the activity of mesenchymal stem cells, the content of miR-146a, miR-21, and let-7a in extracellular membrane vesicles is greatly increased, while the content of pro-inflammatory factors such as interleukin 1β (IL1β), interleukin 6 (IL6) and metalloproteinase 8 (MMP8) is significantly reduced. The present invention was completed on this basis.

[0105] Extracellular vesicles

[0106] There are three main types of extracellular vesicles (EVs): exosomes, microvesicles, and apoptotic bodies. All three major types of EVs are encapsulated by a lipid bilayer and have a diameter ranging from 30 to 2000 nm.

[0107] The term exosomes refers to a subset of EVs with a diameter of 50–100 nm that are derived from endosomes and are the major components of paracrine secretions of various cell types, including mesenchymal stem cells (MSCs).

[0108] MSCs exosomes are a type of MSCs-derived EVs with a diameter ranging from 50-100 nm and a complete lipid bilayer membrane structure.

[0109] Exosomes are a type of cargo-rich carrier that functions mainly through the continuous transport of microRNAs (miRNAs) and proteins. More than 150 miRNAs and more than 850 unique proteins have been identified in MSCs-derived exosomes, which alter various activities of target cells through different pathways. MSC exosomes are involved in physiological and pathological processes such as body development, epigenetic regulation, immune regulation (miR-155 and miR-146), tumorigenesis and tumor progression (miR-23b, miR-451, miR-223, miR-24, miR-125b, miR-31, miR-214 and miR-122). According to ExoCarta, more than 900 proteins have been collected from MSCs exosomes. Some studies have shown that MSC exosomes carry some cytokines and growth factors, such as TGFβ1, interleukin-6 (IL-6), IL-10 and hepatocyte growth factor (HGF), which have been shown to contribute to immune regulation. Vascular endothelial growth factor (VEGF), extracellular matrix metalloproteinase inducer (EMMPRIN) and MMP-9 have all been reported in MSC exosomes. These three proteins play an important role in stimulating angiogenesis and may be the basis for the tissue repair effect of exosomes.

[0110] MicroRNA

[0111] MicroRNA is a non-coding RNA of about 22 nt. It is a post-transcriptional regulation mode of cells and plays an important role in cell activities such as proliferation and apoptosis. Studies have shown that they are closely related to the progression of lung inflammation. For example, LPS stimulation can cause upregulation of miR-21, and its upregulation plays an anti-inflammatory role through the NF-κB pathway. TNF-α stimulation can cause upregulation of miR-146, and overexpression of miR-146 in cells can downregulate the expression of pro-inflammatory factors such as IL-6 and IL-8, indicating that they play an important feedback regulatory role in the progression of inflammation. In in vivo and in vitro idiopathic pulmonary fibrosis experiments, the microRNA of the let-7 family was inhibited by high expression of transforming growth factor TGF-β and HGMA2 in alveolar epithelial cells, thereby regulating epithelial-mesenchymal transition (EMT). This shows that microRNA of the let-7 family has great potential in the treatment of pulmonary fibrosis. Mesenchymal stem cell-derived exosomes contain abundant microRNAs, including miR-21 and miR146, which may play an anti-inflammatory role by regulating inflammatory pathways.

[0112] Therefore, increasing miR-21, miR-146 and let-7a in extracellular vesicles derived from human mesenchymal stem cells is of great significance in inflammatory diseases and fibrotic diseases (such as pulmonary fibrosis).

[0113] Immunomodulatory factors

[0114] As used herein, the immunomodulatory factor is selected from the group consisting of interferons (such as IFN-γ, IFN-α, IFN-β), or a combination thereof.

[0115] As used herein, the terms "IFNγ" and "interferon γ" are used interchangeably and refer to an immunomodulatory factor that can effectively increase certain miRNAs in extracellular vesicles derived from human cells in vitro in the method of the present invention. IFNγ is an important immunomodulatory factor in vivo that can promote antigen processing and presentation of MHC class I and class II. IFNγ mainly induces virus-infected cells to produce a variety of antiviral proteins by binding to cell surface receptors.

[0116] Molecular weight: 16.8 kDa

[0117] A representative amino acid sequence of human recombinant interferon gamma is shown in SEQ ID NO: 1:

[0118] MQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFQGRRASQ(SEQ ID No:1)

[0119] It should be understood that there may be an optional tag sequence (such as a 6His tag for easy purification) at the N-terminus or C-terminus of the interferon. In addition, the immunomodulatory inhibitor (such as interferon) of the present invention may include wild-type and mutant types, wherein the mutant type retains at least 30%, preferably at least 50% of the corresponding biological activity of the wild type.

[0120] In the present invention, the concentration of immunomodulatory factors is not particularly limited, as long as the concentration of immunomodulatory factors can promote the increase of miRNA content in cells and / or reduce proinflammatory factors. Typically, the concentration of immunomodulatory factors is 0.5-200ng / ml, preferably 1-100ng / ml, such as 1ng / ml, 2ng / ml, 3ng / ml, 4ng / ml, 5ng / ml, 7ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 30ng / ml, 40ng / ml, 50ng / ml, 60ng / ml, 80ng / ml, 100ng / ml. Preferably about 10±5ng / ml.

[0121] Pharmaceutical composition

[0122] The present invention also provides a pharmaceutical composition, which contains (a) the extracellular membrane vesicles prepared by the method described in the first aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0123] Since the extracellular membrane vesicles of the present invention have significantly increased content of specific miRNAs (such as miR-146a, miR-21, let-7a), and the content of pro-inflammatory factors such as interleukin 1β (IL1β), interleukin 6 (IL6) and metalloproteinase 8 (MMP8) is significantly reduced, they are more suitable for treating various diseases such as inflammation, fibrosis, and infectious diseases.

[0124] As used herein, the components of a "pharmaceutically acceptable carrier" are substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., have a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0125] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration. The dosage form of the pharmaceutical composition of the present invention is an injection, a lyophilized preparation, a stem cell preparation, and an aerosol inhalation preparation. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0126] The effective amount of the active ingredient of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg / kg animal body weight (preferably 0.0001 mg-10 mg / kg animal body weight) per day, a satisfactory effect can be obtained. For example, due to the urgent requirements of the treatment condition, several divided doses may be administered per day, or the dose may be reduced proportionally. Generally, the pharmaceutical composition of the present invention can be administered by local injection, aerosol inhalation, etc.

[0127] The pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

[0128] Preparation method

[0129] The present invention provides a method for preparing extracellular membrane vesicles as described in the first aspect of the present invention.

[0130] In another preferred embodiment, the present invention provides a method for increasing miR-146a and miR-21 in extracellular vesicles derived from human cells in vitro, comprising the steps of:

[0131] (1) Culture P2-P4 adipose-derived mesenchymal stem cells in a medium containing platelet lysate at a cell seeding density of 10,000-15,000 cells / cm 2 , the cells reached 70% to 90% confluence.

[0132] (2) After incubating the cells in step (1) with solution B containing immunomodulatory factors (such as recombinant human interferon γ) for 24 to 48 hours, the conditioned medium is collected.

[0133] (3) The conditioned medium obtained in step (2) is subjected to differential centrifugation at 3000-10,000 g to obtain the supernatant.

[0134] (4) After incubating the supernatant obtained in step (3) with solution C overnight, differential centrifugation is performed at 3000-15,000 g to obtain a precipitate, which is the extracellular vesicles derived from human cells.

[0135] The method of the present invention can unexpectedly and significantly increase the content or expression of certain specific miRNAs (such as miR-146a and miR-21) in extracellular vesicles, and has no effect on the particle size of the extracellular vesicles and the expression of specific proteins.

[0136] The present invention provides a method for increasing certain miRNAs in extracellular vesicles derived from human cells in vitro, comprising the steps of: (1) culturing P2-P4 adipose-derived mesenchymal stem cells in a culture medium containing platelet lysate at a cell seeding density of 10,000-15,000 cells / cm 2 , the cells reach a confluence of 70% to 90%; and (2) isolating the extracellular vesicles.

[0137] In another preferred embodiment, the mesenchymal stem cells are mesenchymal stem cells derived from human tissue, and the cells are selected from generation P2, generation P3, generation P4, generation P5, and generation P6. The mesenchymal stem cells are mesenchymal stem cells derived from human tissue, and the cells are selected from generation P4.

[0138] In another preferred embodiment, the seeding density of the mesenchymal stem cells is 10,000 to 15,000 cells / cm 2 The seeding density of the mesenchymal stem cells is 10,000 cells / cm 2 .

[0139] In another preferred embodiment, the platelet lysate is used at a final concentration of 2%, 5%, or 10%, and the optimal final concentration of the platelet lysate is 5%, and more preferably 10%.

[0140] In another preferred embodiment, the fusion degree of the mesenchymal stem cells reaches any concentration in the range of 70% to 90%, and the optimal concentration of the fusion degree of the mesenchymal stem cells is 80%, preferably 70%.

[0141] The present invention provides a kit (step (2)), wherein the kit contains a culture medium that can be used to increase the content of certain miRNAs in extracellular vesicles derived from human cells, wherein the cell culture medium is composed of a solution B containing a compound of formula I at a concentration of 10-40 ng / ml;

[0142] In another preferred example, the concentration of the compound in the cell culture medium is 10 ng / ml.

[0143] In another preferred embodiment, the B solution is an αMEM culture medium containing 5% to 10% (preferable concentration is 10%, and the optimal concentration is 5%) of platelet lysate, which is obtained by ultracentrifugation at 100,000 to 150,000 g for 6 to 24 hours to obtain the supernatant.

[0144] In another preferred embodiment, the liquid B is subjected to ultracentrifugation at 120,000 g for 6 hours to obtain the supernatant.

[0145] Preferably, the C solution contains PEG4000-8000 with a final concentration of 8%-20%.

[0146] The present invention also provides a method for isolating extracellular vesicles derived from human cells:

[0147] Step (3) subjecting the conditioned medium obtained in step (2) to differential centrifugation at 3000-10,000 g to obtain a supernatant; Step (4) co-incubating the supernatant obtained in step (3) with liquid C overnight, and then subjecting the supernatant to differential centrifugation at 3000-15,000 g to obtain a precipitate, which is the extracellular vesicles derived from human cells.

[0148] In another preferred embodiment, the conditioned medium obtained in step (2) is centrifuged at 3000 g for 15 minutes.

[0149] In another preferred embodiment, the conditioned medium obtained in step (2) is centrifuged at 10,000 g for 30 minutes.

[0150] In another preferred embodiment, the supernatant obtained in step (3) is incubated with liquid C overnight, and liquid C contains PEG4000-8000 with a final concentration of 8%-20%.

[0151] In another preferred embodiment, the C solution contains PEG6000 with a final concentration of 12%.

[0152] In another preferred embodiment, the supernatant obtained in step (3) is incubated with solution C overnight and then subjected to differential centrifugation at 3000-15,000 g (3000 g for 1 hour, or 120,000 g for 70 minutes).

[0153] In another preferred embodiment, the method further comprises the step of: (5) detecting cell activity, detecting gene expression level and / or protein expression level of a marker.

[0154] In another preferred embodiment, in step (5), the monitoring includes detecting the gene and / or protein expression level of a marker, and the marker includes a positive marker and / or a negative marker.

[0155] In another preferred embodiment, the positive marker refers to a gene expression level that is positively correlated with the ability to increase miR-146a, miR-21 and let-7a in extracellular membrane vesicles derived from human cells, and the negative marker refers to a gene expression level that is negatively correlated with the ability to increase miR-146a, miR-21 and let-7a in extracellular membrane vesicles derived from human cells.

[0156] In another preferred embodiment, the positive marker is selected from the following group: transforming growth factor β1 (TGFβ1) and hepatocyte growth factor (HGF).

[0157] In another preferred embodiment, the negative marker is selected from the group consisting of interleukin 1β (IL1β), interleukin 6 (IL6) and metalloproteinase 8 (MMP8).

[0158] In another preferred embodiment, in step (5), the monitoring includes detecting the relative expression levels of miR-146a, miR-21 and let-7a in extracellular vesicles derived from human cells by fluorescence quantitative PCR.

[0159] In another preferred embodiment, in step (5), the monitoring includes using a western blot method to detect human cell-derived extracellular vesicle-specific protein markers: CD9, CD63 and CD81. The markers are positively correlated with the expression level of extracellular vesicle-specific proteins.

[0160] In another preferred embodiment, in step (5), the monitoring includes using the NTA method to detect the concentration and particle size of human cell-derived extracellular vesicle particles.

[0161] The main advantages of the present invention include

[0162] 1. The present invention proves for the first time that IFNγ can effectively increase the expression of anti-inflammatory miRNAs (such as miR-21-5p, miR146a-5p) and let-7a-5p that inhibits epithelial-mesenchymal transition activity in human cell-derived extracellular vesicles in vitro, and significantly reduce the content of metalloproteinase 8 (MMP8) pro-inflammatory factor. Therefore, the extracellular vesicles of the present invention have better anti-inflammatory activity.

[0163] 2. The content (mRNA content or protein content) of growth factors (TGFβ1 and HGF) in the extracellular membrane vesicles of the present invention is increased, which helps to improve the therapeutic effect.

[0164] 3. The expression levels of the specific proteins CD9, CD63 and CD81 of the extracellular membrane vesicles obtained by the present invention are further improved, which indicates that the quality of the extracellular membrane vesicles of the present invention is high.

[0165] 4. Experiments at the gene level, protein level and miRNA level showed that the use of culture medium containing specific immunomodulatory factors (such as IFNγ) has no effect on cell activity and has little toxic side effects on cells.

[0166] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0167] Experimental methods

[0168] (1) Culture of human adipose-derived mesenchymal stem cells

[0169] P4 human adipose-derived mesenchymal stem cells were collected at 1×10 4 Pieces / cm 2 After inoculation, when the cells grew to 80%, the original culture medium was discarded. Liquid B (negative control group), liquid B containing 10 ng / ml IFNγ, liquid B containing 10 ng / ml TNFα, and liquid B containing 10 ng / ml TNFα + 10 ng / ml IFNγ were added, and cultured at 37°C and 5% CO2 for 48 hours.

[0170] (2) Isolation of extracellular vesicles derived from human adipose-derived mesenchymal stem cells

[0171] Collect the cell culture supernatant, centrifuge at 3000g for 15min, aspirate the supernatant, and remove the precipitate; centrifuge at 10,000g for 0.5h, aspirate the supernatant, and remove the cell debris. Add 24% PEG6000 to the culture medium after centrifugation and keep at 4℃ overnight; centrifuge at 3200g, 4℃ for 1h, discard the supernatant, and resuspend the precipitate with PBS; centrifuge at 120,000g for 70min, discard the supernatant, and resuspend the precipitate at the bottom of the centrifuge tube with PBS to collect the precipitate.

[0172] (3) Cell activity assay

[0173] The CCK8 kit was used to test the metabolic values ​​of cells after culturing B solution containing different factors for 48 hours.

[0174] (4) Detection of gene expression by quantitative PCR (qPCR)

[0175] Cell RNA was extracted using an RNA extraction kit and RevertAid TM First Strand cDNASynthesis Kit reverse transcribes 1 μg RNA. According to the experimental requirements, use The probes in the Gene Expression assays were used as primers, and fluorescence quantitative PCR was used to detect the mRNA expression of related transcription factors. GAPDH was used as an internal reference, and the experiment was repeated 3 times. After the PCR was completed, the Ct value was collected. This experiment used 2 -△△CT Method to analyze the experimental data.

[0176] Table 1 Fluorescence quantitative PCR primer design (mRNA)

[0177] Gene Product ID Number source Dye IL1β Hs01555410_m1 Human FAM-MGB IL 6 Hs00174131_m1 Human FAM-MGB TGFβ1 Hs00998133_m1 Human FAM-MGB IL10 Hs00961622_m1 Human FAM-MGB HGF Hs00300159_m1 Human FAM-MGB GAPDH Hs02786624_g1 Human VIC-MGB

[0178] Table Notes: qPCR analysis was performed using TaqMan Gene Expression assays to evaluate the mRNA expression levels of relevant transcription factors.

[0179] (5) Detection of miRNA expression by quantitative PCR (qPCR)

[0180] Extracellular vesicles were taken and miRNA was isolated using an RNA extraction kit. The miRNA was quantified using NanoDrop, with a uniform dosage of 10 ng for subsequent reversal experiments. Advanced miRNA Assays were reverse transcribed into cDNA. Using miR-191-5p as internal reference, the experiment was repeated 3 times. After PCR, the Ct value was collected. This experiment used 2 -△△CT Method to analyze the experimental data.

[0181] Table 2 Fluorescence quantitative PCR primer design (miRNA)

[0182] Gene Product ID Number source Dye hsa-miR-146a-5p 478399_mir Human FAM-MGB hsa-miR-21-5p 477975_mir Human FAM-MGB hsa-miR-191-5p 477952_mir Human FAM-MGB hsa-let-7a-5p 478575_mir Human FAM-MGB

[0183] Table Note: Use Advanced miRNA Assays were used for qPCR analysis to evaluate the expression levels of relevant miRNAs.

[0184] (6) Western blot detection of cell membrane extracellular vesicle-specific protein expression

[0185] Exosomes were lysed using NP-40 with protease inhibitors. The exosome protein concentration was detected using a BCA kit, and the remaining samples were added with 4× loading buffer and boiled at 95°C for 5 min. The samples were loaded onto protein precast gels with the same volume, run at 80V for 0.5h and 120V for 1h. After running the gel, transfer to PVDF membrane, constant current 250mA, transfer for 1.5h. After the transfer, block for 0.5h. Use primary antibodies (CD9, CD63, CD81) (1:1000) added to the blocking solution and incubate at 4°C overnight. Use TBST to wash the membrane, 10min / time, wash 3 times, use the corresponding species secondary antibody (1:3000), incubate at room temperature for 3h. Use TBST to wash the membrane, 10min / time, wash 3 times, and HRP color development.

[0186] (7) NTA detection of extracellular vesicle particle concentration and particle size

[0187] The ZetaView Nanoparticle Tracking Analyzer sample cell was washed with deionized water. The instrument was calibrated using polystyrene microspheres. The sample cell was washed with PBS. The samples were diluted with PBS and tested.

[0188] Example 1: CCK8 detection of the effects of different inflammatory factors on the activity of P4 adipose-derived mesenchymal stem cells

[0189] After the cells were treated with 10 ng / ml factor, CCK8 was used to detect the activity of the cells.

[0190] like Figure 1 As shown, the activity of cells treated with TNFα and IFNγ was not significantly affected compared with the negative control group; while the activity of cells treated with TNFα+IFNγ was partially reduced compared with the negative control group.

[0191] Example 2: Cell mRNA expression after pretreatment with different inflammatory factors

[0192] After the cells were treated with 10 ng / ml factor, fluorescence quantitative PCR was used to detect the expression of different mRNAs in the cells.

[0193] The results are as follows Figure 2 ,in,

[0194] (a) After INFγ treatment, the mRNA expression levels of transforming growth factor β1 (TGFβ1) and hepatocyte growth factor (HGF) were significantly increased compared with the negative control group; the mRNA expression level of pro-inflammatory cytokine MMP8 was significantly decreased compared with the negative control group; the mRNA expression levels of IL1β and IL6 increased to a certain extent (about several times) compared with the negative control group, but the increase was much smaller than the increase when TNFα was added (tens to hundreds of times).

[0195] (b) Compared with the negative control group, the mRNA expression levels of TGFβ1 and HGF in cells treated with TNFα and TNFα+INFγ did not increase significantly; however, the mRNA expression levels of IL1β, IL6, and MMP8 related to inflammatory factors increased significantly, with the increase ranging from dozens to hundreds of times.

[0196] The above results suggest that after treatment with specific immunomodulatory factors (especially IFNγ), the mRNA expression of TGFβ1 and HGF in cells increases, which leads to increased synthesis of TGFβ1 and HGF, and then leads to an increase in the mRNA and / or protein content of the corresponding factors in the extracellular vesicles of the cell membrane.

[0197] Correspondingly, after treatment with specific immunomodulatory factors (especially IFNγ alone), the mRNA expression of pro-inflammatory-related MMP8 in cells decreased, which led to a decrease in the synthesis of MMP8, and then to a decrease in the mRNA and / or protein content of this factor in the cell membrane extracellular vesicles.

[0198] Similarly, after treatment with specific immunomodulatory factors (especially IFNγ alone), the mRNA expression levels of pro-inflammatory related IL1β and IL6 in the cells are similar, which leads to similar (or slightly increased) mRNA and / or protein levels of IL1β and IL6 in the cell membrane extracellular vesicles.

[0199] Example 3: Expression of extracellular vesicle miRNA after pretreatment with different inflammatory factors

[0200] After the cells were treated with 10 ng / ml factor, the extracellular vesicles produced were used to extract RNA using an RNA extraction kit and then converted into cDNA. The expression levels of miR-21-5p, let-7a-5p and miR-146a-5p were detected using qPCR.

[0201] like Figure 3 As shown, the expression levels of miR-21-5p and miR-146a-5p, which are related to anti-inflammatory effects, in the cells treated with IFNγ group were significantly increased compared with those in the negative control group; the expression level of let-7a-5p, which is related to the regulation of epithelial-mesenchymal transition (EMT), was also significantly increased compared with that in the negative control group.

[0202] Example 4: NTA detection of particle concentration and particle size of extracellular vesicles after pretreatment with different inflammatory factors

[0203] ZetaView was used to detect the particle concentration and size of the extracellular vesicles produced after the cells were treated with each group of factors.

[0204] like Figure 4As shown, the particle concentration of extracellular vesicles treated with factors was significantly lower than that of the negative control group; there was no significant difference in the particle size of extracellular vesicles treated with TNFα and IFNγ compared with the negative control group; the particle size of extracellular vesicles treated with TNFα+IFNγ was significantly larger than that of the negative control group.

[0205] Example 5: Expression of cell membrane extracellular vesicle-specific markers after pretreatment with different inflammatory factors

[0206] Western Blot was used to detect the expression of specific markers of extracellular vesicles produced after cells were treated with each group of factors.

[0207] like Figure 5 As shown, the expression levels of CD9, CD63 and CD81 in the extracellular vesicles of the IFNγ group were the highest.

[0208] discuss

[0209] Different culture conditions and induction conditions will have different effects on the production of EVs.

[0210] The research of the present invention shows that in the presence of specific immunomodulatory factors (such as IFNγ), the expression levels of miR-146a-5p, miR-21-5p and let-7a-5p in the extracellular vesicles obtained by cultured cells are unexpectedly significantly increased, showing a significant difference compared with the negative control group.

[0211] miR-146a-5p and miR-21-5p have anti-inflammatory activity. let-7a-5p has the activity of inhibiting epithelial-mesenchymal transition. Therefore, the EV of the present invention has better anti-inflammatory activity and / or the ability to inhibit epithelial-mesenchymal transition (EMT), which is very beneficial for the treatment of diseases such as inflammatory diseases or fibrotic diseases.

[0212] In addition, after adipose-derived mesenchymal stem cells were treated with IFNγ, the mRNA expression of TGFβ1 and HGF in the cells was significantly increased, which in turn led to an increase in the mRNA and / or protein content of TGFβ1 and HGF in the cell extracellular vesicles.

[0213] In addition, the mRNA expression level of the pro-inflammatory factor MMP8 also decreased significantly, while the mRNA content of IL1β and IL6 did not increase significantly, which also helps the EV of the present invention to be used to treat diseases such as inflammatory diseases or fibrotic diseases.

[0214] In addition, Western blot detection of extracellular vesicle-specific markers CD9, CD63, and CD81 revealed that the expression levels of CD9, CD63, and CD81 produced by exosomes secreted by cells treated with IFNγ were the highest, suggesting that the quality of the EVs of the present invention is qualified or better.

[0215] In addition, IFNγ induction can more effectively increase the content of anti-inflammatory miRNAs (miR-146a-5p, miR-21-5p) and let-7a-5p with epithelial-mesenchymal transition inhibitory activity in extracellular vesicles compared with other induction conditions (such as TNFα, TNFα+IFNγ), and obtain a favorable cytokine expression profile, thereby helping the multiple factors (such as TGFβ1 and HGF) and multiple active molecules (miRNA, mRNA, protein) in the EV of the present invention to work together and synergistically.

[0216] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Cellbio Biotech (Shanghai) Co., Ltd. <120> A method for increasing the content of specific miRNA in extracellular vesicles derived from human cells <130> P2020-2422 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 144 <212> PRT <213> Homo sapiens <400> 1 Met Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe 1 5 10 15 Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly 20 25 30 Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser 35 40 45 Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp 50 55 60 Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val 65 70 75 80 Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu 85 90 95 Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His 100 105 110 Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly 115 120 125 Lys Arg Lys Arg Ser Gln Met Leu Phe Gln Gly Arg Arg Ala Ser Gln 130 135 140

Claims

1. A method for producing extracellular vesicles enriched with at least one anti-inflammatory miRNA, the method comprising the steps of: (a) culturing human adipose stem cells in a cell culture medium for 24-48 hours to allow the release of extracellular vesicles, thereby obtaining a fermentation broth containing the cultured stem cells and the extracellular vesicles produced by the cultured stem cells, in, The culture medium is an αMEM culture medium containing platelet lysate at a concentration of 2-15% v / v, and is subjected to ultracentrifugation at 100,000-150,000 g for 6-24 hours to obtain a supernatant, and contains interferon γ at a concentration ranging from 1 ng / ml to 50 ng / ml, which can effectively induce the release of extracellular vesicles rich in at least one anti-inflammatory miRNA, wherein the at least one anti-inflammatory miRNA comprises hsa-miR-146a-5p, and the level of hsa-miR-146a-5p is increased by at least 2 times compared with the level of hsa-miR-146a-5p in control stem cells not treated with interferon γ, and the extracellular vesicles, compared with the extracellular vesicles produced by control stem cells not treated with interferon γ, also have one or more characteristics selected from the following group: (1) the mRNA and / or protein content of a growth factor is increased, wherein the growth factor is selected from the group consisting of TGFβ1, HGF, or a combination thereof, (2) decreased mRNA and / or protein levels of pro-inflammatory factor metalloproteinase 8, and (3) similar or increased expression of extracellular vesicle-specific markers; and (b) isolating the extracellular vesicles from the fermentation broth, the separation comprising: subjecting the fermentation broth to separation by differential centrifugation at 3000-10,000 g for 10-40 minutes to obtain a cell-free supernatant, incubating the supernatant with a solution of polyethylene glycol selected from PEG4000-PEG8000 overnight, and then separating the supernatant by differential centrifugation at 3000-15,000 g for 40-70 minutes to obtain a precipitate, wherein the precipitate contains the extracellular vesicles, Before step (a), the adipose-derived stem cells are pre-cultured until the degree of confluence is in the range of 60% to 95%, and then the original culture medium is discarded, wherein the adipose-derived stem cells are adipose-derived mesenchymal stem cells passaged to generation P2, P3, P4, P5 or P6.

2. The method according to claim 1, wherein the at least one anti-inflammatory miRNA further comprises miR-21, let-7a, or a combination thereof.

3. The method according to claim 1, wherein the interferon gamma concentration is 10 ng / ml.

4. The method according to claim 1, wherein the extracellular vesicles comprise exosomes.

5. The method according to claim 1, wherein the diameter of the extracellular vesicles ranges from 50 nm to 150 nm. The method according to claim 1 , wherein the adipose stem cells are adipose mesenchymal stem cells passaged to P4.

7. The method according to claim 1, wherein the level of the at least one anti-inflammatory miRNA is increased 2-fold to 5-fold compared to the level of the at least one anti-inflammatory miRNA in control stem cells not treated with interferon gamma.

8. The method according to claim 1, wherein the level of the at least one anti-inflammatory miRNA is increased 2-fold to 8-fold compared to the level of the at least one anti-inflammatory miRNA in control stem cells not treated with interferon gamma.

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