Method for directed induction and differentiation of human induced pluripotent stem cell into IMRC and purification to obtain IMRC-ev

Abstract

Provided is a method for directed induction and differentiation of a human induced pluripotent stem cell into an IMRC and purification to obtain an IMRC-EV. A mesenchymal stem cell derived from the pediatric foreskin tissue is subjected to cell reprogramming to obtain a human induced pluripotent stem cell. The human induced pluripotent stem cell is prepared into an embryoid body, and an IMRC is prepared from the embryoid body. An iPSC-IMRC-EV is purified from the IMRC supernatant for use in the preparation of an immunomodulatory or tissue protection product.

Description

Methods for the directed differentiation of human induced pluripotent stem cells into IMRC and the purification of IMRC-EV

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202510113884.X, filed on January 24, 2025, entitled “Method for Directing Differentiation of Human Induced Pluripotent Stem Cells into IMRC and Purifying to Obtain IMRC-EV”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of stem cell technology, and in particular to a method for directed differentiation of human induced pluripotent stem cells into IMRC and purification to obtain IMRC-EV. Background Technology

[0004] IMRCs are a unique cell population derived from iPSCs or human embryonic stem cells (hESCs) that possess the ability to regulate immunity, reduce tissue damage, and promote repair and regeneration. While their surface markers and biological efficacy are similar to MSCs derived from adult tissues such as bone marrow, umbilical cord, and adipose tissue, they exhibit stronger characteristics and advantages in terms of cell stability, immune system regulation, and protective efficacy against damaged tissues.

[0005] Mesenchymal stem cells (MSCs) possess the capacity for differentiation, proliferation, and self-renewal. Studies have shown that MSCs exhibit various biological effects, including anti-inflammatory, antioxidant, and anti-apoptotic properties, participating in tissue repair, regeneration, and homeostasis maintenance within the body. Regarding the mechanism by which MSCs regulate tissue and cell damage repair and regeneration, research has found that after reaching the injury site, MSCs primarily exert their effects by secreting extracellular vesicles (EVs). MSC-EVs can produce biological effects similar to those of MSCs, mainly including influencing tissue responses to various injury stimuli, regulating cell-cell interactions, conducting cell signal transduction, and altering tissue and cellular metabolism. Furthermore, as an important mediator for MSCs to exert immune regulation and tissue protection, EVs are easier to store and transport than MSCs and have higher safety profiles, thus becoming a good alternative to MSC therapy.

[0006] Therefore, based on the aforementioned advantages of IMRC and the aforementioned application advantages of EV, it is evident that extracellular vesicles derived from mesenchymal stem cells and matrix regulatory cells (IMRC-EV) can become a highly promising cell therapy drug for the treatment of clinical diseases.

[0007] However, existing methods for inducing IMRCs primarily rely on hESCs (human embryonic stem cells), which are limited by the difficulty in obtaining embryos. Therefore, it is essential and urgent to develop a method for the directed induction and differentiation of IMRCs into EVs from iPSCs (human induced pluripotent stem cells).

[0008] In view of this, this disclosure is hereby made. Summary of the Invention

[0009] Unless otherwise specified, the terms and definitions used herein are those commonly used in the field. Specifically, the relevant technical terms or abbreviations are explained below:

[0010] MSC: Mesenchymal stem cell (MSC);

[0011] IMRC: Immunity and matrix regulatory cell (IMRC) formed by inducing differentiation from human embryonic stem cells or human induced pluripotent stem cells as seed cells, which have unlimited stable expansion and pluripotent differentiation capabilities.

[0012] iPSC: Induced pluripotent stem cell (iPSC);

[0013] iPSC-MSC: Induced pluripotent stem cell derived mesenchymal stem cell (iPSC-MSC);

[0014] EV: extracellular vesicle;

[0015] IMRC-EV: Mesenchymal stem cell-like immune and matrix regulatory cell-derived extracellular vesicle (IMRC-EV);

[0016] iPSC-IMRC-EV: IMRC-EV derived from human induced pluripotent stem cells;

[0017] hESC: Human embryonic stem cells;

[0018] hESC-IMRC-EV: IMRC-EV derived from human embryonic stem cells.

[0019] The purpose of this disclosure is to provide a method for the directed differentiation of human induced pluripotent stem cells into IMRC and the purification of IMRC-EV. Through experimental verification, the IMRC-EV is equivalent in characteristics and function to the IMRC-EV of human embryonic stem cells reported in the present invention, which effectively alleviates the problem of limited source when the existing IMRC-EV is usually obtained from human embryonic stem cells.

[0020] In order to achieve the above-mentioned objectives of this disclosure, the following technical solution is adopted:

[0021] According to one aspect of this disclosure, a method for directed differentiation of human induced pluripotent stem cells into IMRC-EVs is provided, the method comprising:

[0022] S1: Human foreskin mesenchymal stem cells were prepared using pediatric foreskin tissue, and then human induced pluripotent stem cells were obtained through cell reprogramming;

[0023] S2: Prepare embryoids from human induced pluripotent stem cells from step S1, then culture them in differentiation medium to obtain primary IMRCs, and then expand and culture the primary IMRCs in expansion medium to obtain human induced pluripotent stem cell-derived IMRCs.

[0024] S3: After amplification culture, collect the cell supernatant to obtain a solution containing IMRC-EV.

[0025] This disclosure provides a method for the directed induction of human induced pluripotent stem cells into IMRC-EVs. This method uses mesenchymal stem cells derived from pediatric foreskin tissue as seed cells for inducing human induced pluripotent stem cells, thereby avoiding the problem of limited source of existing IMRC-EVs, which are usually obtained from human embryonic stem cells.

[0026] Meanwhile, this disclosure successfully developed a method for inducing human induced pluripotent stem cells to prepare IMRC-EVs by selecting differentiation and expansion media, and confirmed that iPSC-IMRC is equivalent to hESC-IMRC-EV in cell phenotype, EV yield, shape, immune regulation, and tissue protection function, thus providing a foundation for the research and development and clinical translation of iPSC-IMRC-EV drugs.

[0027] It should be noted that pediatric foreskin tissue, as a type of waste tissue from traditional surgery, is usually unusable. However, pediatric foreskin tissue often contains abundant epidermal, dermal fibroblasts, mesenchymal stem cells, and other cells. As an innovative source of iPSCs, it can avoid unnecessary damage to the donor during traditional skin or blood sample collection.

[0028] In a preferred embodiment of this disclosure, step S1 involves reprogramming human foreskin mesenchymal stem cells into human induced pluripotent stem cells via electroporation.

[0029] As a preferred embodiment, the human induced pluripotent stem cells (iPSCs) disclosed herein are prepared from pediatric foreskin tissue to produce human foreskin MSCs, which are then obtained through cell reprogramming. They are easier to obtain than embryonic stem cells and have the same quality and performance as embryonic stem cells.

[0030] In the preferred embodiment described above, the transfection solution contains the Sendai virus vector.

[0031] As a preferred embodiment, the Sendai virus vector used for the above-mentioned 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. This RNA vector requires only a single transfection step to reprogram somatic cells into highly efficient induced pluripotent stem cells in vitro.

[0032] In a preferred embodiment of this disclosure, the reprogrammed human induced pluripotent stem cells express positive expression of the surface markers Nanog, Sox2, and Oct4.

[0033] In a preferred embodiment of this disclosure, the differentiation culture medium in step S2 comprises: bFGF, TGF-β, γ-linolenic acid and PPARα antagonist GW9662.

[0034] In an optional embodiment, in step (1), the concentration of bFGF is 10 to 100 ng / mL, specifically any one or any two of 10, 20, 40, 60, 80 and 100 ng / mL.

[0035] In an optional embodiment, in step (1), the concentration of TGF-β is 3 to 100 ng / mL, specifically any one or any two of 3, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ng / mL.

[0036] In an optional embodiment, in step (1), the concentration of γ-linolenic acid is 1 to 50 μg / mL, specifically any one or any two of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg / mL.

[0037] In an optional embodiment, in step (1), the concentration of the PPARα antagonist GW9662 is 1 to 10 μM, specifically any one or any two of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 μM.

[0038] The amplification culture medium in step S2 contains: AMPK activator compound I-3-24, PPARα antagonist GW9662, and TNF-α.

[0039] In an optional embodiment, in step (2), the effective concentration of the AMPK activator compound I-3-24 is 1-10 μM, specifically any one or any two of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 μM.

[0040] In an optional embodiment, in step (2), the effective concentration of the PPARα antagonist GW9662 is 1-10 μM, specifically any one or any two of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 μM.

[0041] In an optional implementation, in step (2), the effective concentration of TNF-α is 10-100 ng / mL, specifically any one or any two of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ng / mL.

[0042] As a preferred embodiment, the IMRC of this application is a subtype of IMRC induced from pediatric foreskin tissue iPSCs by 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, and TNF-α. It has been verified to possess properties consistent with embryonic IMRCs, and the IMRC of this application exhibits stronger characteristics in regulating the immune system and modulating the extracellular matrix. Specifically:

[0043] 1. Adding TNF-α preactivation stimulates the resting IMRC to become an anti-inflammatory subtype, thereby upregulating immune regulation and the secretion level of regeneration-related factors;

[0044] 2. Adding the PPARα antagonist GW9662 and the AMPK activator compound I-3-24 can improve cell proliferation efficiency, reduce cell apoptosis, increase IMRC purity, increase EV secretion, and increase miR-21-5p expression level in EVs.

[0045] 3. Adding PPARα antagonist GW9662 and γ-linolenic acid during EB differentiation into IMRC can effectively improve the differentiation efficiency and purity of IMRC.

[0046] In a preferred embodiment of this disclosure, the method further includes: S4: centrifuging the IMRC-EV-containing solution obtained in step S3 to remove dead cells and debris, and then purifying it to obtain IMRC-EV.

[0047] According to one aspect of this disclosure, this disclosure provides an IMRC-EV prepared by the above-described method for the directed differentiation of human induced pluripotent stem cells into IMRC-EV.

[0048] In a preferred embodiment of this disclosure, the IMRC-EV has a particle size range of 30–150 nm, an average particle size of 100 nm, and is positive for expression of protein markers CD9, CD63, and HSP70.

[0049] According to one aspect of this disclosure, the above-described IMRC-EV is used in the preparation of immunomodulatory and tissue protection products.

[0050] The IMRC-EV disclosed herein can be widely used in the preparation of immunomodulatory and tissue protection products.

[0051] This application also provides the use of the method described in any of the foregoing embodiments or the IMRC-EV described in any of the foregoing embodiments in the preparation of products with at least one of the following effects: immunomodulation, anti-inflammation, anti-oxidative damage, anti-apoptosis, regulation of cell viability, tissue protection, and reduction of tissue damage.

[0052] This application also provides a method for immune regulation, anti-inflammation, anti-oxidative damage, anti-apoptosis, regulating cell viability, tissue protection, or reducing tissue damage, comprising: applying a product obtained by the method described in any of the foregoing embodiments.

[0053] Compared with the prior art, the beneficial effects of this disclosure are as follows:

[0054] This disclosure provides a method for the directed induction of human induced pluripotent stem cells (iPSCs) into IMRC-EVs. This method uses mesenchymal stem cells derived from pediatric foreskin tissue as seed cells for inducing iPSCs, thus avoiding the limited source of existing IMRC-EVs, which are typically obtained from human embryonic stem cells. Furthermore, this disclosure successfully developed a method for inducing IMRC-EVs from human induced pluripotent stem cells by selecting appropriate differentiation and expansion media. It also confirmed that iPSC-IMRCs are equivalent to human embryonic stem cell-derived IMRC-EVs in cell phenotype, EV yield, morphology, immunomodulation, and tissue protection functions, thus providing a foundation for the research and clinical translation of iPSC-IMRC-EV drugs. Attached Figure Description

[0055] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0056] Figure 1a is a flow cytometry diagram of Nanog, a surface marker of human induced pluripotent stem cells (iPSCs) provided in Embodiment 1 of this disclosure.

[0057] Figure 1b is a flow cytometry diagram of Sox2, a surface marker of human induced pluripotent stem cells (iPSCs) provided in Embodiment 1 of this disclosure;

[0058] Figure 1c is a flow cytometry diagram of Oct4, a surface marker of human induced pluripotent stem cells (iPSCs) provided in Embodiment 1 of this disclosure.

[0059] Figure 2a is a flow cytometry diagram of Nanog, a surface marker of human embryonic stem cells (ESCs) provided in Embodiment 1 of this disclosure.

[0060] Figure 2b is a flow cytometry diagram of Sox2, a surface marker of human embryonic stem cells (ESCs) provided in Embodiment 1 of this disclosure;

[0061] Figure 2c is a flow cytometry diagram of Oct4, a surface marker of human embryonic stem cells (ESCs) provided in Embodiment 1 of this disclosure.

[0062] Figure 3a is a flow cytometry diagram of the surface protein CD90 of iPSC-IMRC and hESC-IMRC provided in Embodiment 2 of this disclosure;

[0063] Figure 3b is a flow cytometry diagram of the surface protein CD105 of iPSC-IMRC and hESC-IMRC provided in Embodiment 2 of this disclosure;

[0064] Figure 3c is a flow cytometry diagram of the surface protein CD73 of iPSC-IMRC and hESC-IMRC provided in Embodiment 2 of this disclosure;

[0065] Figure 3d is a flow cytometry diagram of the surface protein CD34 of iPSC-IMRC and hESC-IMRC provided in Embodiment 2 of this disclosure;

[0066] Figure 3e is a flow cytometry diagram of HLA-DR of iPSC-IMRC and hESC-IMRC provided in Embodiment 2 of this disclosure;

[0067] Figure 3f is a flow cytometry diagram of the surface protein CD79a of iPSC-IMRC and hESC-IMRC provided in Embodiment 2 of this disclosure;

[0068] Figure 3g is a flow cytometry diagram of the surface protein CD45 of iPSC-IMRC and hESC-IMRC provided in Example 2 of this disclosure;

[0069] Figure 3h is a flow cytometry diagram of the surface protein CD14 of iPSC-IMRC and hESC-IMRC provided in Embodiment 2 of this disclosure;

[0070] Figure 4a is an electron microscope image of the iPSC-IMRC-EV provided in Embodiment 2 of this disclosure;

[0071] Figure 4b is an NTA diagram of the iPSC-IMRC-EV provided in Embodiment 2 of this disclosure;

[0072] Figure 4c is an immunoblot map of iPSC-IMRC-EV provided in Embodiment 2 of this disclosure;

[0073] Figure 5a is an electron microscope image of hESC-IMRC-EV provided in Embodiment 2 of this disclosure;

[0074] Figure 5b is the NTA diagram of hESC-IMRC-EV provided in Embodiment 2 of this disclosure;

[0075] Figure 5c is an immunoblot map of hESC-IMRC-EV provided in Example 2 of this disclosure;

[0076] Figure 6a is a flow cytometry diagram showing the inhibition of peripheral blood mononuclear cell proliferation by iPSC-IMRC-EV and hESC-IMRC-EV provided in Embodiment 3 of this disclosure.

[0077] Figure 6b is another flow cytometry diagram showing the inhibition of peripheral blood mononuclear cell proliferation by iPSC-IMRC-EV and hESC-IMRC-EV provided in Embodiment 3 of this disclosure;

[0078] Figure 6c is a bar chart showing the inhibition of peripheral blood mononuclear cell proliferation by iPSC-IMRC-EV and hESC-IMRC-EV provided in Embodiment 3 of this disclosure;

[0079] Figure 7 is an experimental diagram showing the inhibition of pro-inflammatory factors in peripheral blood mononuclear cells by iPSC-IMRC-EV and hESC-IMRC-EV provided in Example 3 of this disclosure;

[0080] Figure 8 is a bar chart comparing the antioxidant damage of iPSC-IMRC-EV to cardiomyocytes (AC16) and hippocampal neurons (HT22) provided in Embodiment 3 of this disclosure;

[0081] Figure 9 is a bar chart comparing the anti-apoptotic effects of iPSC-IMRC-EV on cardiomyocytes (AC16) and hippocampal neurons (HT22) provided in Embodiment 3 of this disclosure;

[0082] Figure 10 is a bar chart comparing the regulatory cell viability of iPSC-IMRC-EV on cardiomyocytes (AC16) and hippocampal neurons (HT22) provided in Embodiment 3 of this disclosure;

[0083] Figure 11 shows the proportions of interleukin-6, interleukin-10, neutrophils, and macrophages in mouse lung tissue after iPSC-IMRC-EV injection, as provided in Example 4 of this disclosure. Detailed Implementation

[0084] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0085] The technical solutions of this disclosure will be further described below with reference to the embodiments.

[0086] Example 1: Preparation of human induced pluripotent stem cells derived from pediatric foreskin tissue

[0087] (I) Preparation of MSCs derived from pediatric foreskin tissue:

[0088] 1. Prepare serum-free MSC culture medium (Hangzhou Luyuan Biotechnology Co., Ltd., LYMSC-1001): basal medium (DMEM) + additives (bFGF, recombinant human platelet-derived growth factor-BB (PDGF-BB), recombinant human insulin, ascorbic acid, transferrin, etc.).

[0089] 2. Place 0.5-1cm 2 The pediatric foreskin tissue was washed 2-3 times with DPBS, and the tissue block was cut into 0.5-1 mm pieces with scissors. 2 Small pieces.

[0090] 3. Place the tissue block from step 2 into a cell culture dish, add the serum-free MSC-specific medium from step 1, incubate the dish in a 37°C CO2 (5%) incubator, change the medium, and passage the cells after they have emerged to obtain human foreskin mesenchymal stem cells (MSCs).

[0091] (II) MSC reprogrammed to iPSC:

[0092] 1. Take the MSCs that are in good condition after the above culture and perform transfection, add transfection solution (CTS) TM CytoTune TM A34546: Contains Sendai virus, a single-stranded RNA vector containing five reprogramming factors: OCT4, KLF-4, SOX2, GLIS1 and c-MYC, as well as a puromycin resistance gene) and transfected after mixing.

[0093] 2. Add the cells to the well plates coated with matrix gel, add serum-free MSC medium and culture for 1 day, then transfer to complete iPSC cell culture medium.

[0094] 3. Observe daily, iPSCs form clones, select them for passage, expansion and cryopreservation to obtain human induced pluripotent stem cells (iPSCs) derived from pediatric foreskin tissue.

[0095] The ratio of surface markers Nanog, Sox2, and Oct4 in the harvested iPSCs was measured, and the purity was greater than 95%.

[0096] Figure 1a is a flow cytometry diagram of Nanog, a surface marker of human induced pluripotent stem cells (iPSCs) provided in this embodiment. Figure 1b is a flow cytometry diagram of Sox2, a surface marker of human induced pluripotent stem cells (iPSCs) provided in this embodiment. Figure 1c is a flow cytometry diagram of Oct4, a surface marker of human induced pluripotent stem cells (iPSCs) provided in this embodiment.

[0097] As shown in Figures 1a to 1c, the activity of the iPSCs prepared in this application is 95%; Nanog 99.6%, Sox2 98.8%, and Oct4 99.3%.

[0098] Figure 2a shows the flow cytometry identification of Nanog, a surface marker of human embryonic stem cells (ESCs). Figure 2b shows the flow cytometry identification of Sox2, a surface marker of human embryonic stem cells (ESCs). Figure 2c shows the flow cytometry identification of Oct4, a surface marker of human embryonic stem cells (ESCs).

[0099] As shown in Figures 2a-2c, the activity rate of hESC was 96%; Nanog was 99.85%, Sox2 was 99.21%, and Oct4 was 99.62%.

[0100] As can be seen from the above, the iPSC and hESC flow cytometry were used to identify the relevant surface markers. The surface markers Nanog, Sox2, and Oct4 were positively expressed with a purity greater than 95%, and the cell quality was consistent.

[0101] Example 2: Preparation of iPSC-IMRC-EV

[0102] (I) iPSC fabrication of embryoid bodies (EB)

[0103] 1. 5×10 5 One iPSC was seeded into a pre-coated plate. 2 μl of ROCK inhibitor y27632 (Sigma, Y0503) was added, along with 1 ml of EB medium (NutriStem, 06-5100-01-1A). The plates were continuously incubated at 37°C with 5% CO2, and the medium was changed frequently.

[0104] (II) Differentiation and Amplification of EB-IMRC

[0105] 1. Fifty EB cells were seeded in cell culture dishes and cultured in IMRC differentiation medium (Hangzhou Luyuan Biotechnology Co., Ltd., LYIMRC-4001). The differentiation medium was specifically supplemented with bFGF (50 ng / mL) and TGF-β (20 ng / mL), and γ-linolenic acid (10 μg / mL) and PPARα antagonist GW9662 (2 μM) were added for continuous culture.

[0106] 2. Once a large number of IMRCs have emerged from around the EB and the confluence of cells in the culture dish reaches more than 80%, the IMRCs are passaged into culture flasks. IMRCs amplification culture medium (Hangzhou Luyuan Biotechnology Co., Ltd., LYIMRC-4002) is added, along with AMPK activator compound I-3-24 (2μM), PPARα antagonist GW9662 (1μM), and TNF-α (20ng / mL) to enhance IMRC proliferation and induce IMRC polarization towards the anti-inflammatory subtype. Continuous culture and passage yield iPSC-IMRC-EV.

[0107] 3. Target passage IMRC, and detect the ratio of positive surface proteins CD73, CD90, CD105 and negative surface proteins CD14, CD34, CD45, CD79a and HLA-DR in IMRC, with a purity greater than 95%.

[0108] Figure 3a shows the flow cytometry identification of the surface protein CD90 of iPSC-IMRC and hESC-IMRC provided in this embodiment. In Figure 3a(a), the flow cytometry identification of the surface protein CD90 of iPSC-IMRC is shown; in Figure 3a(b), the flow cytometry identification of the surface protein CD90 of hESC-IMRC is shown.

[0109] Figure 3b shows the flow cytometry identification of the surface protein CD105 of iPSC-IMRC and hESC-IMRC provided in this embodiment. In Figure 3b(a), the flow cytometry identification of the surface protein CD105 of iPSC-IMRC is shown; in Figure 3b(b), the flow cytometry identification of the surface protein CD105 of hESC-IMRC is shown.

[0110] Figure 3c shows the flow cytometry identification of the surface protein CD73 of iPSC-IMRC provided in this embodiment. In Figure 3c(a), the flow cytometry identification of the surface protein CD73 of iPSC-IMRC is shown; in Figure 3c(b), the flow cytometry identification of the surface protein CD73 of hESC-IMRC is shown.

[0111] Figure 3d shows the flow cytometry identification of the surface protein CD34 of iPSC-IMRC provided in this embodiment. In Figure 3d, (a) is the flow cytometry identification of the surface protein CD34 of iPSC-IMRC; in Figure 3d, (b) is the flow cytometry identification of the surface protein CD34 of hESC-IMRC.

[0112] Figure 3e shows the flow cytometry identification of HLA-DR in iPSC-IMRC provided in this embodiment. In Figure 3e(a), the flow cytometry identification of HLA-DR in iPSC-IMRC is shown; in Figure 3e(b), the flow cytometry identification of HLA-DR in hESC-IMRC is shown.

[0113] Figure 3f is a flow cytometry diagram of the surface protein CD79a of iPSC-IMRC provided in this embodiment; Figure 3f(a) is a flow cytometry diagram of the surface protein CD79a of iPSC-IMRC; Figure 3f(b) is a flow cytometry diagram of the surface protein CD79a of hESC-IMRC.

[0114] Figure 3g is a flow cytometry diagram of the surface protein CD45 of iPSC-IMRC provided in this embodiment; Figure 3g(a) is a flow cytometry diagram of the surface protein CD45 of iPSC-IMRC; Figure 3g(b) is a flow cytometry diagram of the surface protein CD45 of hESC-IMRC.

[0115] Figure 3h shows the flow cytometry identification of the surface protein CD14 of iPSC-IMRC provided in this embodiment. In Figure 3h, (a) is the flow cytometry identification of the surface protein CD14 of iPSC-IMRC; in Figure 3h, (b) is the flow cytometry identification of the surface protein CD14 of hESC-IMRC.

[0116] It should be noted that in Figures 3a to 3h, red represents the expression level of the target protein labeled with fluorescent antibody, and green represents the same type of cells, but without fluorescent antibody labeling of the target protein as a control.

[0117] If the red and green peaks can be clearly separated, it indicates that this type of protein can be labeled with a specific fluorescent antibody, meaning that the protein is expressed positively in cells (positive marker).

[0118] If the red and green peaks overlap, it indicates that this type of protein has not been labeled with a specific fluorescent antibody, meaning that the protein is negatively expressed in cells (negative marker).

[0119] As can be seen from the above, the identification of relevant surface protein markers by iPSC-IMRC and hESC-IMRC flow cytometry showed that the ratios of positive surface proteins CD73, CD90, and CD105 and negative surface proteins CD14, CD34, CD45, CD79a, and HLA-DR were generally consistent, and the purity of all of them was greater than 95%.

[0120] (III) Isolation, purification and identification of iPSC-IMRC-EV

[0121] 1) Collection of IMRC culture supernatant: When continuously amplifying cultured cells using IMRC-specific amplification medium (Nextexo, NE002023), collect the cell supernatant when the cell confluence is greater than 80%.

[0122] 2) Centrifugation supernatant removal of dead cell debris: Centrifuge the IMRC cell culture supernatant (fresh or -20℃ thawed) at 3000g for 20 minutes to remove dead cells and debris, and transfer the supernatant.

[0123] 3) Purification of EV: The supernatant was filtered through a 0.45 μm membrane to remove large particle impurities, then concentrated and removed by ultrafiltration through a 100 kD tangential flow membrane to remove small particle impurities. The concentrate was filtered through a 0.22 μm membrane, and then subjected to 35 nm size exclusion chromatography to remove impurities such as proteins and small particles. Finally, the EV was obtained by sterilization filtration through a 0.22 μm membrane. The collected EV was stored in a -80℃ freezer.

[0124] The iPSC-IMRC-EV prepared above was subjected to electron microscopy, NTA, and immunoblotting to identify its morphology, particle size, and surface protein markers.

[0125] Figure 4a is an electron microscope image of the iPSC-IMRC-EV provided in this embodiment.

[0126] Figure 4b is the NTA diagram of the iPSC-IMRC-EV provided in this embodiment.

[0127] Figure 4c is an immunoprotein imprint of iPSC-IMRC-EV provided in this embodiment.

[0128] Figure 5a is an electron microscope image of hESC-IMRC-EV provided in this embodiment.

[0129] Figure 5b is the NTA diagram of hESC-IMRC-EV provided in this embodiment.

[0130] Figure 5c is an immunoglobulin imprint of hESC-IMRC-EV provided in this embodiment.

[0131] Example 3

[0132] (I) Peripheral blood mononuclear cell (PBMC) stimulation and proliferation inhibition experiment:

[0133] Peripheral blood mononuclear cells (PBMCs) were isolated and labeled with a live-cell fluorescent tracer probe (CFSE). Cell division could be observed by the fluorescence intensity of CFSE. After cell division, the fluorescence intensity of CFSE was reduced. Then, 5 μg of phytohemagglutinin (PHA) was added and cultured for 5 days. Flow cytometry analysis of CFSE fluorescence intensity showed that PBMCs proliferated significantly.

[0134] Add iPSC-IMRC-EV or hESC-IMRC-EV (1*10) 11 After co-culturing iPSC-IMRC-EV (200 μl / ml) particles for 24 hours, it significantly inhibited PHA-induced PBMC proliferation. This indicates that iPSC-IMRC-EV and hESC-IMRC-EV have the same immunomodulatory capabilities.

[0135] Figure 6a is a flow cytometry diagram of peripheral blood mononuclear cell stimulation and proliferation inhibition experiment provided in this embodiment.

[0136] Figure 6b is another flow cytometry diagram showing the inhibition of peripheral blood mononuclear cell proliferation by iPSC-IMRC-EV and hESC-IMRC-EV provided in this embodiment;

[0137] Figure 6c is a bar chart of the peripheral blood mononuclear cell stimulation and proliferation inhibition experiment provided in this embodiment.

[0138] (II) ELISA analysis showed that PBMCs secreted relatively low levels of the pro-inflammatory factor tumor necrosis factor-α (TNF-α) at ​​rest; however, the addition of 5 μg PHA to PBMC culture stimulated the secretion of large amounts of TNF-α; however, the addition of iPSC-IMRC-EV or hESC-IMRC-EV (1*10) further increased the levels of TNF-α. 11 After administration of 200 μl ( / ml), it significantly inhibited the amount of TNF-α secreted by PBMCs after PHA induction. This indicates that both iPSC-IMRC-EV and hESC-IMRC-EV have good anti-inflammatory capabilities.

[0139] Note: Here, EV originates from the secretion of iPSC-IMRC or hESC-IMRC.

[0140] Figure 7 shows the experimental diagram of inhibition of pro-inflammatory factors in peripheral blood mononuclear cells provided in this embodiment.

[0141] (III) iPSC-IMRC-EV Antioxidant Damage Function:

[0142] Using cardiomyocytes (AC16) and hippocampal neurons (HT22), cellular hypoxia-reoxygenation (HR) models were established, respectively. Then, EVs derived from iPSC-IMRC (1*10) were administered. 11Intervention with 200 μl / ml showed that both AC16 and HT22 cells underwent oxidative damage after HR stimulation. However, the application of EV significantly reduced the degree of oxidative damage to AC16 and HT22 cells caused by HR stimulation. This indicates that iPSC-IMRC-EV has the ability to resist cellular oxidative damage, thereby exerting a protective function for tissue cells. Note: Here, EV is produced by the secretion of iPSC-IMRC.

[0143] Figure 8 is a bar chart comparing the antioxidant damage of iPSC-IMRC-EV to cardiomyocytes (AC16) and hippocampal neurons (HT22).

[0144] (iv) Anti-apoptotic function of iPSC-IMRC-EV:

[0145] Human renal failure (HR) models were established using cardiomyocytes (AC16) and hippocampal neurons (HT22), respectively, and apoptosis was observed. Adding EVs to unmodeled cell lines did not induce apoptosis in normally cultured cells. However, adding 1*102 EVs to HR-modeled cell lines did not. 11 At 200 μl EV, the apoptosis level was significantly reduced, indicating that iPSC-IMRC-EV has anti-apoptotic ability and can thus play a role in protecting tissue cells.

[0146] Figure 9 is a bar chart comparing the anti-apoptotic effects of iPSC-IMRC-EV on cardiomyocytes (AC16) and hippocampal neurons (HT22) disclosed in this paper.

[0147] (V) iPSC-IMRC-EV regulates cell viability:

[0148] HR (human risk factor) models were established using cardiomyocytes (AC16) and hippocampal neurons (HT22), respectively, and decreased cell viability was observed. Adding EVs to unmodeled cell lines did not cause a decrease in cell viability in normally cultured cells. However, adding 1*102 EVs to cell lines with HR modeling did not. 11 At 200 μl EV, cell viability was significantly restored, indicating that iPSC-IMRC-EV has the ability to restore cell viability, thereby exerting its tissue cell protection function.

[0149] Figure 10 is a bar chart comparing the regulatory cell viability of iPSC-IMRC-EV on cardiomyocytes (AC16) and hippocampal neurons (HT22) according to this disclosure.

[0150] Example 4: Animal experimental verification of the immunomodulatory and tissue protection functions of iPSC-IMRC-EV:

[0151] A lung inflammation model was simulated in mice by intratracheal infusion of lipopolysaccharide (LPS). After modeling, the control group received intravenous infusion of PBS, while the experimental group received infusion of iPSC-IMRC-EV (2.5*10). 10 Lung tissues from mice were collected at 24h and 48h for pathological sections, and bronchoalveolar lavage fluid was collected at 24h to analyze interleukin-6, interleukin-10, neutrophil ratio, and macrophage ratio.

[0152] Figure 11 shows the proportions of interleukin-6, interleukin-10, neutrophils, and macrophages in the bronchoalveolar lavage fluid of mice after iPSC-IMRC-EV infusion according to this application.

[0153] Pathology: After establishing the lung inflammation model, mild inflammatory infiltration was observed in the lungs of both the experimental and control groups at 24 hours. However, at 48 hours, the control group mice showed severe inflammatory cell infiltration and altered alveolar structure, while the experimental group exhibited reduced levels of inflammatory cell infiltration and better preservation of alveolar structure. This indicates that iPSC-IMRC-EV possesses good anti-inflammatory and tissue-protective functions.

[0154] Bronchoalveolar 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 macrophages with immunomodulatory capabilities in the alveoli.

[0155] The iPSC-IMRC-EV used in Examples 3 and 4 is the iPSC-IMRC-EV prepared in Example 2.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure. Industrial applicability

[0157] This disclosure provides a method for the directed induction of human induced pluripotent stem cells (iPSCs) into IMRC-EVs. This method uses mesenchymal stem cells derived from pediatric foreskin tissue as seed cells for inducing iPSCs, thus avoiding the limited source of existing IMRC-EVs, which are typically obtained from human embryonic stem cells. Furthermore, this disclosure successfully developed a method for inducing IMRC-EVs from human induced pluripotent stem cells by selecting appropriate differentiation and expansion media. It also confirmed that iPSC-IMRCs are equivalent to human embryonic stem cell-derived IMRC-EVs in cell phenotype, EV yield, morphology, immunomodulation, and tissue protection functions, thus providing a foundation for the research and clinical translation of iPSC-IMRC-EV drugs.

Claims

1. A method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs, characterized in that, The method includes: S1: Human foreskin mesenchymal stem cells were prepared using pediatric foreskin tissue, and then human induced pluripotent stem cells were obtained through cell reprogramming; S2: The human induced pluripotent stem cells from step S1 were prepared into embryoid bodies, and then cultured in differentiation medium to obtain primary IMRCs. The primary IMRCs were then expanded and cultured in amplification medium. The specific method is as follows: (1) EB was seeded in cell culture dishes and IMRC differentiation culture medium was used. bFGF and TGF-β were specifically added to the differentiation culture medium, and γ-linolenic acid and PPARα antagonist GW9662 were 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 passaged into a culture flask, IMRCs expansion culture medium is added, and 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 towards the anti-inflammatory subtype. After continuous culture and passage, iPSC-IMRC-EV is obtained. S3: After amplification culture, collect the cell supernatant to obtain a solution containing iPSC-IMRC-EV.

2. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain 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 via electroporation.

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

4. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 2, characterized in that, The reprogrammed human induced pluripotent stem cells expressed positive expression of the surface markers Nanog, Sox2, and Oct4.

5. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 1, characterized in that, The method further includes: S4: After centrifuging to remove dead cells and debris, the iPSC-IMRC-EV solution obtained in step S3 is purified to obtain iPSC-IMRC-EV.

6. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 5, characterized in that, The exosome purification method of iPSC-IMRC-EV includes any one of ultracentrifugation, ultrafiltration, immunoaffinity assay, and PEG chromatography.

7. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 1, characterized in that, In step (1), the concentration of bFGF is 10 to 100 ng / mL.

8. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 1, characterized in that, In step (1), the concentration of TGF-β is 3 to 100 ng / mL.

9. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 1, characterized in that, In step (1), the concentration of γ-linolenic acid is 1 to 50 μg / mL.

10. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 1, characterized in that, In step (1), the concentration of the PPARα antagonist GW9662 is 1–10 μM.

11. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 1, characterized in that, In step (2), the concentration of the AMPK activator compound I-3-24 is 1-10 μM.

12. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 1, characterized in that, In step (2), the effective concentration of the PPARα antagonist GW9662 is 1-10 μM.

13. The method for directed differentiation of human induced pluripotent stem cells into IMRCs and purification to obtain IMRC-EVs according to claim 1, characterized in that, In step (2), the effective concentration of TNF-α is 10-100 ng / mL.

14. An IMRC-EV prepared by the method according to any one of claims 1 to 13.

15. The use of the method according to any one of claims 1 to 13 or the IMRC-EV according to claim 14 in the preparation of products with at least one of the following effects: immunomodulation, anti-inflammatory, anti-oxidative damage, anti-apoptosis, regulation of cell viability, tissue protection, and reduction of tissue damage.

16. A method for immunomodulation, anti-inflammation, anti-oxidative damage, anti-apoptosis, regulation of cell viability, tissue protection, or reduction of tissue damage, comprising: The product obtained by applying the method as described in any one of claims 1 to 13.

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