Method for directing differentiation of human induced pluripotent stem cells into IMRC and purifying IMRC-EVs

Abstract

The present invention provides a method for directional induction and differentiation of human induced pluripotent stem cells into IMRC and purification to obtain IMRC-EV, which relates to the field of stem cell technology. The above-mentioned method for directional induction and differentiation of human induced pluripotent stem cells into IMRC and purification to obtain IMRC-EV uses mesenchymal stem cells derived from foreskin tissue of children as seed cells for induction into human induced pluripotent stem cells, thereby avoiding the problem of limited sources caused by the existing IMRC-EVs usually being obtained from human embryonic stem cells. At the same time, the present invention obtains a method for inducing the preparation of IMRC-EV by human induced pluripotent stem cells through the selection of differentiation medium and expansion medium, thereby providing a basis for the research and development and clinical transformation of iPSC-IMRC-EV drugs.

Description

Technical Field

[0001] The present invention relates to the field of stem cell technology, and in particular to a method for directed induction and differentiation of human induced pluripotent stem cells into IMRC and purification to obtain IMRC-EV. Background Art

[0002] IMRC is a unique cell population derived from iPSC or human embryonic stem cells (hESC) that has the ability to regulate immunity, reduce tissue damage, and promote its repair and regeneration. The surface markers and biological efficacy of this type of cell are similar to those of MSCs derived from adult tissues such as bone marrow, umbilical cord, and fat, but they show stronger characteristics and advantages in terms of their own cell stability, immune system regulation ability, and damaged tissue protection efficacy.

[0003] Mesenchymal stem cells (MSC) have the ability to differentiate, proliferate and self-renew. Studies have shown that MSC has a variety of biological effects such as anti-inflammatory, antioxidant, and anti-apoptotic, and participates in tissue repair, regeneration and homeostasis maintenance in the body. Regarding the mechanism by which MSC regulates tissue cell damage repair and regeneration, studies have found that after MSC reaches the site of injury, it mainly exerts its effects by secreting extracellular vesicles (EVs). MSC-EVs can produce similar biological effects to MSC, mainly including affecting the response of tissues to various damaging stimuli, regulating cell-to-cell interactions, conducting cell signal transduction, and changing the metabolism of tissues and cells. In addition, EVs, as an important medium for MSC to exert immune regulation and tissue protection, are easier to store and transport than MSCs, and have higher safety, thus becoming a good alternative to MSC therapy.

[0004] Therefore, based on the above-mentioned advantages of IMRC and the above-mentioned application advantages of EV, it can be seen that mesenchymal stem cell-like immune and matrix regulatory cell derived extracellular vesicles (IMRC-EV) can become a very promising cell therapy drug for the treatment of clinical diseases.

[0005] However, the existing IMRC induction preparation is mainly derived from hESC (human embryonic stem cells), which has the source limitation factor of difficult embryo acquisition. Therefore, it is necessary and urgent to develop a method for directed induction and differentiation of IMRC-EV from iPSC (human induced pluripotent stem cells).

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] Unless otherwise specified, the terms and definitions used in this article are applicable to common terms in the field. Specifically, the relevant technical terms or abbreviations are explained as follows:

[0008] MSC: Mesenchymal stem cell (MSC);

[0009] IMRC: Immunity and matrix regulatory cells (IMRC) formed by inducing differentiation of human embryonic stem cells or human induced pluripotent stem cells with unlimited stable proliferation and totipotent differentiation capabilities as seed cells;

[0010] iPSC: human induced pluripotent stem cell (iPSC);

[0011] iPSC-MSC: induced pluripotent stem cell derived mesenchymal stem cell (iPSC-MSC);

[0012] EV: extracellular vesicle (EV);

[0013] IMRC-EV: Immunity and matrix regulatory cell derived extracellular vesicles (IMRC-EV);

[0014] iPSC-IMRC-EV: human induced pluripotent stem cell-derived IMRC-EV;

[0015] hESC: human embryonic stem cell;

[0016] hESC-IMRC-EV: human embryonic stem cell-derived IMRC-EV.

[0017] The purpose of the present invention is to provide a method for inducing the differentiation of human induced pluripotent stem cells into IMRC and purifying IMRC-EVs, and verify through experiments that the IMRC-EVs are equivalent to the IMRC-EVs derived from human embryonic stem cells in properties and functions, effectively alleviating the problem of limited sources of existing IMRC-EVs that are usually obtained from human embryonic stem cells.

[0018] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0019] According to one aspect of the present invention, the present invention provides a method for inducing the differentiation of human induced pluripotent stem cells into IMRC-EVs, the method comprising:

[0020] S1: Human foreskin mesenchymal stem cells were prepared from pediatric foreskin tissue, and then human induced pluripotent stem cells were obtained through cell reprogramming;

[0021] S2: preparing human induced pluripotent stem cells in step S1 into embryoid bodies, then culturing with differentiation medium to obtain primary IMRCs, and amplifying and culturing the primary IMRCs with expansion medium to obtain human induced pluripotent stem cell-derived IMRCs;

[0022] S3: After expansion culture, the cell supernatant is collected to obtain a solution containing IMRC-EVs.

[0023] The present invention provides a method for directed induction and differentiation of human induced pluripotent stem cells into IMRC-EVs. In this method, mesenchymal stem cells derived from foreskin tissue of children are used as seed cells for inducing human induced pluripotent stem cells, thereby avoiding the problem of limited sources caused by the fact that existing IMRC-EVs are usually obtained from human embryonic stem cells.

[0024] At the same time, the present invention successfully developed a method for inducing the preparation of IMRC-EVs from human induced pluripotent stem cells through the selection of differentiation medium and expansion medium, and confirmed that iPSC-IMRC is equivalent to hESC-IMRC-EV in terms of cell phenotype, EV production, shape, immune regulation and tissue protection functions of the IMRC-EV, thereby providing a basis for the research and development and clinical transformation of iPSC-IMRC-EV drugs.

[0025] It should be noted that pediatric foreskin tissue, as a type of waste tissue from traditional surgery, is usually not used. However, pediatric foreskin tissue often contains rich epidermal and dermal fibroblasts, mesenchymal stem cells and other cells. As an innovative source of iPSCs, it can avoid the unnecessary damage to donors caused by traditional skin or blood sample collection.

[0026] In a preferred embodiment of the present invention, the step S1 reprograms human foreskin mesenchymal stem cells into human induced pluripotent stem cells by electroporation.

[0027] As a preferred embodiment, the human induced pluripotent stem cells (iPSCs) of the present invention are prepared from human foreskin MSCs from foreskin tissue of children and then obtained through cell reprogramming. They are easier to obtain than embryonic sources and have the same quality and performance as embryonic stem cells.

[0028] In the above preferred embodiment, the transfection solution for transfection contains Sendai virus vector.

[0029] As a preferred embodiment, the Sendai virus vector used in the above cell reprogramming is a single-stranded RNA vector containing five reprogramming factors: OCT4, KLF-4, SOX2, GLIS1 and c-MYC, as well as a puromycin resistance gene. The above RNA vector only requires one transfection step to reprogram somatic cells into highly efficient induced pluripotent stem cells in vitro.

[0030] In a preferred embodiment of the present invention, the human induced pluripotent stem cells after cell reprogramming express positive surface markers Nanog, Sox2, and Oct4.

[0031] In a preferred embodiment of the present invention, the differentiation culture medium in step S2 comprises: bFGF, TGF-β, γ-linolenic acid and PPARα antagonist GW9662.

[0032] The expansion culture medium in step S2 contains: AMPK activator compound I-3-24, PPARα antagonist GW9662, and TNF-α.

[0033] As a preferred embodiment, the IMRC of the present application is a subtype of IMRC induced by iPSC derived from pediatric foreskin tissue through specific inducers such as basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), γ-linolenic acid, PPARα antagonist GW9662, AMPK activator compound I-3-24, TNF-α, etc. It has been verified that it has the same performance as the IMRC formed by the embryo, and the IMRC of the present application shows stronger characteristics in regulating the immune system and regulating the extracellular matrix. Specifically:

[0034] 1. Adding TNF-α for pre-activation stimulates quiescent IMRC to the anti-inflammatory subtype, thereby increasing the immunomodulatory ability and secretion level of regeneration-related factors;

[0035] 2. Adding PPARα antagonist GW9662 and AMPK activator compound I-3-24 can improve cell proliferation efficiency, reduce cell apoptosis, improve IMRC purity, increase the number of EV secretion and increase the expression level of miR-21-5p in EV.

[0036] 3. Adding PPARα antagonist GW9662 and γ-linolenic acid during EB differentiation into IMRC can effectively improve the differentiation efficiency and purity of IMRC.

[0037] In a preferred embodiment of the present invention, the method further comprises: S4: the IMRC-EV-containing solution obtained in step S3 is centrifuged to remove dead cells and debris and then purified to obtain IMRC-EV.

[0038] According to one aspect of the present invention, the present invention provides an IMRC-EV prepared by the above-mentioned method of inducing differentiation of human induced pluripotent stem cells into IMRC-EV.

[0039] In a preferred embodiment of the present invention, the particle size of the IMRC-EV ranges from 30 to 150 nm, the average particle size is 100 nm, and the protein markers CD9, CD63, and HSP70 are positively expressed.

[0040] According to one aspect of the present invention, a use of the above-mentioned IMRC-EV in the preparation of immunomodulatory and tissue protection products.

[0041] The IMRC-EV provided by the present invention can be widely used in the preparation process of immunomodulatory and tissue protection products.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention provides a method for directional induction and differentiation of human induced pluripotent stem cells into IMRC-EVs, in which mesenchymal stem cells derived from pediatric foreskin tissue are used as seed cells for induction into human induced pluripotent stem cells, thereby avoiding the problem of limited sources caused by the conventional IMRC-EVs usually being obtained from stem cells derived from human embryos. At the same time, the present invention successfully developed a method for inducing the preparation of IMRC-EVs by inducing human induced pluripotent stem cells through the selection of differentiation medium and expansion medium, and confirmed that iPSC-IMRC is equivalent to IMRC-EVs derived from human embryonic stem cells in terms of cell phenotype, EV yield, shape, immune regulation, and tissue protection function of the IMRC-EVs, thereby providing a basis for the research and development and clinical transformation of iPSC-IMRC-EV drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1a This is a flow cytometry identification diagram of Nanog, a surface marker of human induced pluripotent stem cells iPSC provided in Example 1 of the present invention;

[0046] Figure 1b This is a flow cytometry identification diagram of Sox2, a surface marker of human induced pluripotent stem cells (iPSCs) provided in Example 1 of the present invention;

[0047] Figure 1c This is a flow cytometry identification diagram of Oct4, a surface marker of human induced pluripotent stem cells iPSC provided in Example 1 of the present invention;

[0048] Figure 2a This is a flow cytometric identification diagram of Nanog, a surface marker of human embryonic stem cells (ESC) provided in Example 1 of the present invention;

[0049] Figure 2b This is a flow cytometry identification diagram of Sox2, a surface marker of human embryonic stem cells (ESC) provided in Example 1 of the present invention;

[0050] Figure 2c This is a flow cytometry identification diagram of Oct4, a surface marker of human embryonic stem cells (ESC) provided in Example 1 of the present invention;

[0051] Figure 3a This is a flow cytometric identification diagram of the surface protein CD90 of iPSC-IMRC and hESC-IMRC provided in Example 2 of the present invention;

[0052] Figure 3b This is a flow cytometric identification diagram of the surface protein CD105 of iPSC-IMRC and hESC-IMRC provided in Example 2 of the present invention;

[0053] Figure 3c This is a flow cytometric identification diagram of the surface protein CD73 of iPSC-IMRC and hESC-IMRC provided in Example 2 of the present invention;

[0054] Figure 3d This is a flow cytometric identification diagram of the surface protein CD34 of iPSC-IMRC and hESC-IMRC provided in Example 2 of the present invention;

[0055] Figure 3e This is a flow cytometric identification diagram of HLA-DR of iPSC-IMRC and hESC-IMRC provided in Example 2 of the present invention;

[0056] Figure 3f This is a flow cytometric identification diagram of the surface protein CD79a of iPSC-IMRC and hESC-IMRC provided in Example 2 of the present invention;

[0057] Figure 3g This is a flow cytometric identification diagram of the surface protein CD45 of iPSC-IMRC and hESC-IMRC provided in Example 2 of the present invention;

[0058] Figure 3h This is a flow cytometric identification diagram of the surface protein CD14 of iPSC-IMRC and hESC-IMRC provided in Example 2 of the present invention;

[0059] Figure 4a This is an electron micrograph of iPSC-IMRC-EV provided in Example 2 of the present invention;

[0060] Figure 4b This is the NTA diagram of iPSC-IMRC-EV provided in Example 2 of the present invention;

[0061] Figure 4c This is the immunoblot image of iPSC-IMRC-EV provided in Example 2 of the present invention;

[0062] Figure 5a This is an electron micrograph of hESC-IMRC-EV provided in Example 2 of the present invention;

[0063] Figure 5b This is the NTA diagram of hESC-IMRC-EV provided in Example 2 of the present invention;

[0064] Figure 5c This is the immunoblot image of hESC-IMRC-EV provided in Example 2 of the present invention;

[0065] Figure 6a This is a flow cytometric identification diagram of the inhibition of proliferation of peripheral blood mononuclear cells stimulated by iPSC-IMRC-EV and hESC-IMRC-EV provided in Example 3 of the present invention;

[0066] Figure 6b Another flow cytometry identification diagram of the inhibition of proliferation of peripheral blood mononuclear cells stimulated by iPSC-IMRC-EV and hESC-IMRC-EV provided in Example 3 of the present invention;

[0067] Figure 6c A bar chart showing the inhibition of proliferation of peripheral blood mononuclear cells stimulated by iPSC-IMRC-EV and hESC-IMRC-EV provided in Example 3 of the present invention;

[0068] Figure 7 This is an experimental diagram of the inhibition of pro-inflammatory factors of peripheral blood mononuclear cells by iPSC-IMRC-EV and hESC-IMRC-EV provided in Example 3 of the present invention;

[0069] Figure 8 A bar graph comparing the antioxidant damage of iPSC-IMRC-EV to cardiomyocytes (AC16) and hippocampal neuron cells (HT22) provided in Example 3 of the present invention;

[0070] Fig. 9 This is a bar graph comparing the anti-apoptosis effects of iPSC-IMRC-EV on cardiomyocytes (AC16) and hippocampal neuron cells (HT22) provided in Example 3 of the present invention;

[0071] Fig.10 A bar graph comparing the regulation of cell viability of cardiomyocytes (AC16) and hippocampal neuron cells (HT22) by iPSC-IMRC-EVs provided in Example 3 of the present invention;

[0072] Fig.11 This is a graph of interleukin-6, interleukin-10, neutrophil ratio, and macrophage ratio in mouse lung tissue after iPSC-IMRC-EV injection provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0073] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0074] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0075] Example 1 Preparation of human induced pluripotent stem cells from foreskin tissue of children

[0076] 1. Preparation of MSCs from foreskin tissue of children:

[0077] 1. Prepare MSC-specific serum-free culture medium (LYMSC-1001, Hangzhou Luyuan Biotechnology Co., Ltd.): basal culture medium (DMEM) + additives (bFGF, recombinant human platelet-derived growth factor-BB (PDGF-BB), recombinant human insulin, ascorbic acid, transferrin, etc.).

[0078] 2. 0.5~1cm 2 Wash the foreskin tissue of children with DPBS 2-3 times and cut the tissue into pieces of 0.5-1 mm with scissors. 2 of small pieces.

[0079] 3. Place the tissue block from step 2 in a cell culture dish, add the MSC-specific serum-free culture medium from step 1, and place the culture dish in a 37°CO2 (5%) incubator for incubation. Change the medium and wait for the cells to crawl out and then passage to obtain human foreskin mesenchymal stem cells (MSC).

[0080] (II) Reprogramming of MSCs into iPSCs:

[0081] 1. Take the MSCs in good condition after culture for transfection, add transfection solution (CTS™ CytoTune™, A34546: containing Sendai virus, a single-stranded RNA vector, containing five reprogramming factors: OCT4, KLF-4, SOX2, GLIS1 and c-MYC, and a puromycin resistance gene) and mix well before transfection.

[0082] 2. Add the cells to the well plate coated with matrix gel, add MSC-specific serum-free medium and culture stably for 1 day, then switch to iPSC complete medium.

[0083] 3. Observe every day to allow iPSCs to form clones, pick out clones for passage, expand and freeze, and obtain human induced pluripotent stem cells (iPSCs) derived from pediatric foreskin tissue.

[0084] The ratio of Nanog, Sox2, and Oct4 surface markers of the harvested iPSCs was detected, and the purity was greater than 95%.

[0085] Figure 1a This is a flow cytometric identification diagram of Nanog, a surface marker of human induced pluripotent stem cells (iPSCs) provided in this example. Figure 1b This is a flow cytometric identification diagram of Sox2, a surface marker of human induced pluripotent stem cells (iPSCs) provided in this example. Figure 1c This is a flow cytometric identification diagram of Oct4, a surface marker of human induced pluripotent stem cells (iPSCs) provided in this example.

[0086] Depend on Figure 1a~Figure 1c It can be seen that the viability of iPSCs prepared in the present application is 95%; Nanog 99.6%, Sox2 98.8%, Oct4 99.3%.

[0087] Figure 2a Flow cytometric identification of Nanog, a surface marker of human embryonic stem cells (ESC). Figure 2b Flow cytometric identification of Sox2, a surface marker of human embryonic stem cells (ESCs). Figure 2c Flow cytometric identification of Oct4, a surface marker of human embryonic stem cells (ESCs).

[0088] Depend on Figure 2a~Figure 2c It can be seen that the viability of hESC is 96%; Nanog 99.85%, Sox2 99.21%, Oct4 99.62%.

[0089] From the above, we can see that the identification of relevant surface markers detected by flow cytometry of iPSC and hESC showed that the surface markers Nanog, Sox2, and Oct4 were positively expressed, the purity was greater than 95%, and the cell quality was consistent.

[0090] Example 2 Preparation of iPSC-IMRC-EV

[0091] 1. iPSC production of embryoid bodies (EBs)

[0092] 1. Inoculate iPSCs into pre-coated well plates. Add ROCK inhibitor y27632 (Sigma, Y0503) and EB culture medium (NutriStem, 06-5100-01-1A) for continuous culture and medium change.

[0093] EB-IMRC differentiation and expansion

[0094] 1. EBs were seeded in a cell culture dish and cultured using IMRC differentiation medium (Hangzhou Luyuan Biotechnology Co., Ltd., LYIMRC-4001). The differentiation medium specifically added bFGF (10-100 ng / mL) and TGF-β (3-100 ng / mL), and γ-linolenic acid (1-50 μg / mL) and PPARα antagonist GW9662 (1-10 μM) were added for continuous culture.

[0095] 2. When a large number of IMRCs crawl out from around the EB and the confluence of the culture dish reaches more than 80%, the IMRCs cells are subcultured into culture flasks, and IMRCs expansion culture medium (Hangzhou Luyuan Biotechnology Co., Ltd., LYIMRC-4002) is added, and AMPK activator compound I-3-24 (1-10μM), PPARα antagonist GW9662 (1-10μM), and TNF-α (10-100ng / mL) are added to enhance the proliferation capacity of IMRCs and induce IMRC polarization to the anti-inflammatory subtype. After continuous culture and subculture, iPSC-IMRC-EVs are obtained.

[0096] 3. IMRC of target generation, and detect the ratio of IMRC's positive surface proteins CD73, CD90, CD105 and negative surface proteins CD14, CD34, CD45, CD79a and HLA-DR. The purity should be greater than 95%.

[0097] Figure 3a This is a flow cytometric identification diagram of the surface protein CD90 of iPSC-IMRC and hESC-IMRC provided in this example. Figure 3a (a) is a flow cytometry analysis of the surface protein CD90 of iPSC-IMRC; Figure 3a Middle (b) is a flow cytometric analysis of the surface protein CD90 of hESC-IMRC.

[0098] Figure 3bThis is a flow cytometric identification diagram of the surface protein CD105 of iPSC-IMRC and hESC-IMRC provided in this example. Figure 3b (a) is a flow cytometry analysis of the surface protein CD105 of iPSC-IMRC; Figure 3b Middle (b) is a flow cytometric identification of the surface protein CD105 of hESC-IMRC.

[0099] Figure 3c This is a flow cytometric identification diagram of the surface protein CD73 of iPSC-IMRC provided in this example. Figure 3c (a) is a flow cytometry analysis of the surface protein CD73 of iPSC-IMRC; Figure 3c Middle (b) is a flow cytometric identification of the surface protein CD73 of hESC-IMRC.

[0100] Figure 3d This is a flow cytometric identification diagram of the surface protein CD34 of iPSC-IMRC provided in this example. Figure 3d (a) is a flow cytometry analysis of the surface protein CD34 of iPSC-IMRC; Figure 3d Middle (b) is a flow cytometric analysis of CD34, a surface protein of hESC-IMRC.

[0101] Figure 3e This is a flow cytometric identification diagram of HLA-DR of iPSC-IMRC provided in this example. Figure 3e (a) is the flow cytometry identification of HLA-DR of iPSC-IMRC; Figure 3e (b) is a flow cytometric analysis of HLA-DR in hESC-IMRC.

[0102] Figure 3f This is a flow cytometry identification diagram of the surface protein CD79a of iPSC-IMRC provided in this example; Figure 3f (a) is a flow cytometry analysis of the surface protein CD79a of iPSC-IMRC; Figure 3f Middle (b) is a flow cytometric identification of the surface protein CD79a of hESC-IMRC.

[0103] Figure 3g This is a flow cytometry identification diagram of the surface protein CD45 of iPSC-IMRC provided in this example; Figure 3g (a) is a flow cytometry analysis of the surface protein CD45 of iPSC-IMRC; Figure 3g Middle (b) is a flow cytometric analysis of CD45, a surface protein of hESC-IMRC.

[0104] Figure 3hThis is a flow cytometric identification diagram of the surface protein CD14 of iPSC-IMRC provided in this example. Figure 3h (a) is a flow cytometry image of the surface protein CD14 of iPSC-IMRC; Figure 3h Middle (b) is a flow cytometric identification of the surface protein CD14 of hESC-IMRC.

[0105] It should be noted that Figure 3a~Figure 3h The red color in the middle represents the expression level of the target protein labeled with a fluorescent antibody, and the green color represents the same type of cells, but the target protein is not labeled with a fluorescent antibody as a control.

[0106] If the red and green peaks can be clearly separated, it means that this type of protein can be labeled with a specific fluorescent antibody, indicating that the protein is positively expressed in the cell (positive marker).

[0107] If the red and green peaks overlap, it means that this type of protein is not labeled by the specific fluorescent antibody, indicating that the protein is negatively expressed in the cell (negative marker).

[0108] From the above, we can see that the identification of relevant surface protein markers detected by flow cytometry between iPSC-IMRC and hESC-IMRC showed that the ratios of positive surface proteins CD73, CD90, CD105 and negative surface proteins CD14, CD34, CD45, CD79a and HLA-DR were roughly consistent, and the purity was greater than 95%.

[0109] 3. Isolation, purification and identification of iPSC-IMRC-EVs

[0110] 1) Collection of IMRC culture supernatant: When cells are continuously expanded using IMRC-specific expansion medium, collect the cell supernatant when the cell confluence is greater than 80%.

[0111] 2) Centrifuge the supernatant to remove dead cell debris: Centrifuge the IMRC cell culture supernatant (fresh or rewarmed at -20℃) at 3000g for 20 min to remove dead cells and debris, and transfer the supernatant.

[0112] 3) EV purification: The supernatant was filtered through a 0.45 μm membrane to remove large particle impurities, and then concentrated and removed small particle impurities through ultrafiltration using a 100 kd tangential flow membrane package. The concentrate was filtered through a 0.22 μm membrane, and then subjected to 35 nm molecular exclusion chromatography to remove impurities such as proteins and small particle impurities. Finally, EVs were obtained by sterilization filtration using a 0.22 μm membrane. The collected EVs were stored in a -80°C refrigerator.

[0113] The iPSC-IMRC-EVs prepared as above were subjected to electron microscopy, NTA, and immunoblotting to identify their morphology, particle size, and surface protein markers, respectively.

[0114] Figure 4a This is an electron micrograph of the iPSC-IMRC-EV provided in this example.

[0115] Figure 4b This is the NTA diagram of iPSC-IMRC-EV provided in this example.

[0116] Figure 4c This is the immunoblot image of iPSC-IMRC-EV provided in this example.

[0117] Figure 5a This is an electron micrograph of hESC-IMRC-EV provided in this example.

[0118] Figure 5b NTA diagram of hESC-IMRC-EV provided for this example.

[0119] Figure 5c This is the immunoblot image of hESC-IMRC-EV provided in this example.

[0120] Example 3

[0121] 1. Peripheral blood mononuclear cell (PBMC) stimulation proliferation inhibition experiment:

[0122] PBMCs were separated from peripheral blood and labeled with a live cell fluorescent tracer probe (CFSE). The cell division could be observed by the CFSE fluorescence intensity. After cell division, the CFSE fluorescence intensity was reduced. Then, the stimulator - phytohemagglutinin (PHA) was added and cultured for 5 days. Flow cytometry examination of the CFSE fluorescence intensity showed significant proliferation of PBMCs.

[0123] After co-culture with iPSC-IMRC-EV or hESC-IMRC-EV, it can significantly inhibit the proliferation of PBMC caused by PHA, which indicates that iPSC-IMRC-EV and hESC-IMRC-EV have the same immunomodulatory ability.

[0124] Figure 6a This is a flow cytometric identification diagram of the peripheral blood mononuclear cell stimulation proliferation inhibition experiment provided in this example.

[0125] Figure 6b Another flow cytometry identification diagram of the inhibition of proliferation of peripheral blood mononuclear cells stimulated by iPSC-IMRC-EV and hESC-IMRC-EV provided in this example;

[0126] Figure 6cThis is a bar chart of the peripheral blood mononuclear cell stimulation proliferation inhibition experiment provided in this example.

[0127] (II) Elisa detection showed that PBMCs secreted less pro-inflammatory factor-tumor necrosis factor-a (TNF-a) in the resting state; after PHA was added to PBMC culture, it was found that it stimulated PBMCs to secrete a large amount of TNF-a; however, after adding iPSC-IMRC-EV or hESC-IMRC-EV, it could significantly inhibit the amount of TNF-a secreted by PBMCs after PHA induction. This shows that both iPSC-IMRC-EV and hESC-IMRC-EV have good anti-inflammatory ability.

[0128] NOTE: Here EVs are derived from secretion from iPSC-IMRC or hESC-IMRC.

[0129] Figure 7 This is a diagram of the peripheral blood mononuclear cell pro-inflammatory factor inhibition experiment provided in this example.

[0130] 3. iPSC-IMRC-EV anti-oxidative damage function:

[0131] Using cardiomyocytes (AC16) and hippocampal neuron cells (HT22), we established cell hypoxia-reoxygenation (HR) models, and then treated them with EVs from iPSC-IMRC. The results showed that both AC16 and HT22 cells showed oxidative damage after HR stimulation, but the application of EVs could significantly reduce the degree of oxidative damage to AC16 and HT22 cells caused by HR stimulation, indicating that iPSC-IMRC-EVs have the ability to resist cell oxidative damage, thus playing a protective role in tissue cells. Note: EVs here are secreted from iPSC-IMRCs.

[0132] Figure 8 This is a bar graph comparing the antioxidant damage of the iPSC-IMRC-EV of the present invention to cardiomyocytes (AC16) and hippocampal neuron cells (HT22).

[0133] (IV) Anti-apoptosis function of iPSC-IMRC-EV:

[0134] Cell HR models were established using cardiomyocytes (AC16) and hippocampal neuron cells (HT22), respectively, and cell apoptosis was observed. When EVs were added to unmodeled cell lines, they did not cause apoptosis in normally cultured cells. However, when EVs were added to HR-modeled cell lines, the level of apoptosis was significantly reduced, indicating that iPSC-IMRC-EVs have anti-apoptosis capabilities, thereby playing a protective role in tissue cells.

[0135] Fig. 9This is a bar graph comparing the anti-apoptosis effects of the iPSC-IMRC-EV of the present invention on cardiomyocytes (AC16) and hippocampal neuron cells (HT22).

[0136] (V) iPSC-IMRC-EV regulates cell viability function:

[0137] Using cardiomyocytes (AC16) and hippocampal neuron cells (HT22), cell HR models were established, and cell viability decreased. Adding EVs to unmodeled cell lines did not cause a decrease in cell viability in normally cultured cells. However, when EVs were added to cell lines after HR modeling, cell viability was significantly restored, indicating that iPSC-IMRC-EVs have the ability to restore cell viability, thereby exerting a tissue cell protection function.

[0138] Fig.10 This is a bar graph comparing the effects of iPSC-IMRC-EV of the present invention on regulating cell viability of cardiomyocytes (AC16) and hippocampal neuron cells (HT22).

[0139] Example 4 Animal experiment verification of the immunomodulatory and tissue protection functions of iPSC-IMRC-EVs:

[0140] Mice were instilled with lipopolysaccharide (LPS) through the airway to simulate the lung inflammation model. After modeling, the control group was intravenously infused with PBS, and the experimental group was infused with iPSC-IMRC-EV. The mouse lung tissues were collected for pathological sections at 24h and 48h, and the alveolar lavage fluid of the mice was collected at 24h to analyze interleukin-6, interleukin-10, neutrophil ratio, and macrophage ratio.

[0141] Fig.11 This is a graph of interleukin-6, interleukin-10, neutrophil ratio, and macrophage ratio in mouse bronchoalveolar lavage fluid after iPSC-IMRC-EV infusion for this application.

[0142] Pathology: After the establishment of the lung inflammation model, mild inflammatory infiltration was found in the lungs of mice in both the experimental and control groups at 24 hours. At 48 hours, the lungs of mice in the control group showed severe inflammatory cell infiltration and changes in alveolar structure, while the level of inflammatory cell infiltration in the experimental group was reduced, and the alveolar structure was well preserved. This indicates that iPSC-IMRC-EV has good anti-inflammatory and tissue protection functions.

[0143] Alveolar lavage fluid: Compared with the control group, iPSC-IMRC-EV can significantly inhibit the secretion of the pro-inflammatory factor interleukin-6, while promoting the secretion of the anti-inflammatory factor interleukin-10. It can also inhibit the recruitment of inflammation-related neutrophils into the alveoli and protect the macrophages with immune regulatory ability in the alveoli.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for inducing differentiation of human induced pluripotent stem cells into IMRCs and purifying IMRC-EVs, characterized in that: The method comprises: S1: Human foreskin mesenchymal stem cells were prepared from pediatric foreskin tissue, and then human induced pluripotent stem cells were obtained through cell reprogramming; S2: preparing human induced pluripotent stem cells from step S1 into embryoid bodies, then culturing with differentiation medium to obtain primary IMRCs, and amplifying and culturing the primary IMRCs with expansion medium; The specific method is as follows: (1) EBs were inoculated into a cell culture dish and cultured in IMRC differentiation medium. bFGF and TGF-β were specifically added to the differentiation medium. γ-linolenic acid and PPARα antagonist GW9662 were also added for continuous culture. (2) When a large number of IMRCs crawl out from around the EB and the confluence of the culture dish reaches more than 80%, the IMRCs cells are subcultured into culture flasks, and IMRCs expansion medium is added. AMPK activator compound I-3-24, PPARα antagonist GW9662, and TNF-α are added to enhance the proliferation capacity of IMRCs and induce IMRCs to polarize to anti-inflammatory subtypes. After continuous culture and subculture, iPSC-IMRC-EVs are obtained. S3: After expansion culture, the cell supernatant is collected to obtain a solution containing iPSC-IMRC-EVs.

2. The method for directing induction and differentiation of human induced pluripotent stem cells into IMRC and purifying IMRC-EVs according to claim 1, characterized in that: In step S1, human foreskin mesenchymal stem cells are reprogrammed into human induced pluripotent stem cells by electroporation.

3. The method for directing induction and differentiation of human induced pluripotent stem cells into IMRC and purifying IMRC-EVs according to claim 2, characterized in that: The electroporation solution for electroporation contains Sendai virus vector or plasmid vector.

4. The method for directed induction and differentiation of human induced pluripotent stem cells into IMRC and purification to obtain IMRC-EV according to claim 2, characterized in that: The human induced pluripotent stem cells after cell reprogramming express positive surface markers Nanog, Sox2, and Oct4.

5. The method for directed induction and differentiation of human induced pluripotent stem cells into IMRC and purification to obtain IMRC-EV according to claim 1, characterized in that: The method further comprises: S4: The iPSC-IMRC-EV solution obtained in step S3 is centrifuged to remove dead cells and debris, and then purified to obtain iPSC-IMRC-EVs; And / or, the exosome purification method of the iPSC-IMRC-EV comprises any one of ultracentrifugation, ultrafiltration, immunoaffinity method, and PEG chromatography.

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