Use of eutremicidin E in the preparation of mesenchymal stem cells with enhanced immunosuppressive activity

Abstract

The present application relates to the mesenchymal stem cell technical field, especially to the application of Eurotium cristatum E in the preparation of mesenchymal stem cell with enhanced immunosuppressive activity.The present application finds that Eurotium cristatum E can significantly improve the inhibition ability of iMSC to T cell proliferation, and then significantly improve the immunosuppressive activity of iMSC, and Eurotium cristatum E does not affect the cell properties of iMSC.Eurotium cristatum E can stably improve the immunosuppressive function of iMSC through simple co-culture process with iMSC, which provides an effective solution to the problems of insufficient immunosuppressive activity and unstable efficacy of existing iMSC, and has good application prospect in iMSC preparation.

Description

Technical Field

[0001] This invention relates to the field of mesenchymal stem cell technology, and more particularly to the application of coronavirus E in the preparation of mesenchymal stem cells with enhanced immunosuppressive activity. Background Technology

[0002] The core immunomodulatory function of mesenchymal stem cells (MSCs) is to inhibit abnormal T cell proliferation and activation, while simultaneously reshaping the balance of T cell subsets. Based on these immunomodulatory functions, MSCs have significant clinical translational value in areas such as autoimmune diseases, transplant rejection, inflammatory damage, and organ fibrosis, and can compensate for the shortcomings of traditional immunosuppressants, including significant side effects, easy drug resistance, and inability to repair tissue damage. Therefore, enhancing the ability of MSCs to inhibit T cell proliferation is expected to enable them to exert better clinical effects in the treatment of acute / chronic graft-versus-host disease (GVHD), systemic lupus erythematosus, Sjögren's syndrome, scleroderma, rheumatoid arthritis, and liver / lung / kidney fibrosis.

[0003] Induced pluripotent stem cells (iPSCs) can be induced to differentiate into mesenchymal-like cells with similar genotypes and phenotypes to primary MSCs, namely induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), which remain stable without chromosomal abnormalities during long-term passage. iMSCs overcome the challenges of obtaining a sufficient number of MSCs from a single donor, unsustainable cell expansion, and heterogeneity in cell source, laying an important foundation for the clinical translation and large-scale production of MSC therapy.

[0004] However, iMSCs have limited inherent immunosuppressive activity, typically requiring high-dose infusions to achieve therapeutic effects, significantly increasing treatment costs and becoming a core bottleneck limiting the widespread clinical application of MSCs. Current technologies employ several strategies to enhance the immunosuppressive activity of MSCs, but all have significant drawbacks: First, genetic engineering modification: overexpressing immunomodulatory molecules such as IDO1, HLA-G, PD-L1, and IL-10 in MSCs through viral vectors can enhance their immunosuppressive ability to a certain extent. However, there are problems such as vector genome integration and tumorigenicity risks, immunogenicity, complex preparation processes, and high costs, which significantly limit clinical translation.

[0005] Second, physical or technological methods, such as hypoxic culture, three-dimensional culture, and encapsulation with biomaterials, have limited effects on improving the immunosuppressive activity of MSCs and are often incompatible with existing large-scale, scalable cell preparation processes.

[0006] Therefore, developing methods to enhance the immunosuppressive activity of iMSCs that do not rely on gene editing and are more compatible with existing cell preparation processes is of great significance for the clinical application of MSCs. Summary of the Invention

[0007] This invention provides the use of coronavirusic cystin E in the preparation of mesenchymal stem cells with enhanced immunosuppressive activity.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides the use of coronavirusic acid E in the preparation of mesenchymal stem cells with enhanced immunosuppressive activity; wherein the mesenchymal stem cells are induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). The application includes treating mesenchymal stem cells with coronary cystin E; wherein the concentration of coronary cystin E is 0.1-1 µM.

[0010] Cristatumin E is a member of the cristatumin family and is a naturally occurring alkaloid with the molecular formula C2. 32 H 36 N6O4, CAS number 1459722-08-7, has the chemical structural formula shown in formula (1): Equation (1).

[0011] The effects of coronavirusic acid E on the immunomodulatory function of mesenchymal stem cells (MSCs) have not been reported. This invention unexpectedly discovered that coronavirusic acid E can significantly enhance the immunosuppressive function of MSCs. Adding coronavirusic acid E to the in vitro expansion culture of MSCs can prepare MSCs with significantly enhanced immunosuppressive function. Simultaneously, the MSCs retain their original cellular properties; their cell phenotype (e.g., expression of CD44, CD73, CD90, and CD105) and their ability to differentiate into osteoblasts, chondrocytes, and adipocytes remain unchanged.

[0012] Preferably, the treatment of mesenchymal stem cells with coronavirus E includes: performing in vitro expansion culture of mesenchymal stem cells using a mesenchymal stem cell expansion medium containing coronavirus E.

[0013] Preferably, the application includes: expanding and culturing mesenchymal stem cells using a mesenchymal stem cell expansion medium containing coronavirus E; wherein the concentration of coronavirus E in the mesenchymal stem cell expansion medium is 0.1-1 µM.

[0014] The amplification culture is performed by passage at least 1-3 times.

[0015] In this invention, the immunosuppressive effect of coronavirus E on mesenchymal stem cells does not depend on the specific composition of the mesenchymal stem cell expansion culture medium. Therefore, any culture medium suitable for mesenchymal stem cell expansion culture can be used.

[0016] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 45%-50% IMDM medium, 45%-50% Ham's F12, 2%-8% human serum albumin, 0.5%-2% insulin-transferrin-selenium additive, 8-12 ng / mL FGF-2, and 8-12 ng / mL EGF.

[0017] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 45%-50% IMDM medium, 45%-50% Ham's F12, 4%-6% human serum albumin, 0.8%-1.2% insulin-transferrin-selenium additive, 9-11 ng / mL FGF-2, and 9-11 ng / mL EGF.

[0018] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 47% IMDM medium, 47% Ham's F12, 5% human serum albumin, 1% insulin-transferrin-selenium additive, 10 ng / mL LFGF-2, and 10 ng / mL EGF.

[0019] Preferably, the enhanced immunosuppressive activity is manifested in the following way: compared with untreated mesenchymal stem cells, mesenchymal stem cells treated with coronavirus E show increased activity against CD4+. + The inhibitory activity of T cell proliferation was reduced, and the expression levels of the immunosuppressive molecule IDO1 and the anti-inflammatory factor TGFβ1 were increased in iMSCs.

[0020] In a second aspect, the present invention provides the use of coronary cystin E in the preparation of a formulation that enhances the immunosuppressive activity of mesenchymal stem cells; wherein the mesenchymal stem cells are induced pluripotent stem cell-derived mesenchymal stem cells.

[0021] Preferably, the formulation is used to prepare mesenchymal stem cells with enhanced immunosuppressive activity in vitro.

[0022] Preferably, the formulation enhances the activity of mesenchymal stem cells for CD4+. + The function of inhibiting T cell proliferation.

[0023] Thirdly, the present invention provides the use of coronary cystin E in the preparation of a formulation that enhances the inhibitory activity of mesenchymal stem cells on T cell proliferation; wherein the mesenchymal stem cells are induced pluripotent stem cell-derived mesenchymal stem cells.

[0024] Preferably, the T cells are CD4 cells. + T cells.

[0025] Preferably, the formulation is used to prepare CD4 with enhanced activity in vitro. + Mesenchymal stem cells with T-cell proliferation-inhibiting activity.

[0026] Fourthly, the present invention provides the application of coronavirusic acid E in enhancing the immunosuppressive activity of mesenchymal stem cells in vitro; wherein the mesenchymal stem cells are induced pluripotent stem cell-derived mesenchymal stem cells.

[0027] The application includes treating mesenchymal stem cells with coronary cystin E; wherein the concentration of coronary cystin E is 0.1-1 µM.

[0028] Fifthly, the present invention provides the application of coronavirusic acid E in enhancing the inhibitory activity of mesenchymal stem cells on T cell proliferation in vitro; wherein the mesenchymal stem cells are induced pluripotent stem cell-derived mesenchymal stem cells.

[0029] The application includes treating mesenchymal stem cells with coronary cystin E; wherein the concentration of coronary cystin E is 0.1-1 µM.

[0030] Preferably, the application includes: expanding and culturing mesenchymal stem cells using a mesenchymal stem cell expansion medium containing coronavirus E; wherein the concentration of coronavirus E in the mesenchymal stem cell expansion medium is 0.1-1 µM.

[0031] Preferably, the T cells are CD4 cells. + T cells.

[0032] In a sixth aspect, the present invention provides a method for enhancing the inhibitory activity of mesenchymal stem cells on T cell proliferation in vitro, the method comprising: treating mesenchymal stem cells with coronavirus E; wherein the concentration of coronavirus E in the treatment is 0.1-1 µM; and the mesenchymal stem cells are induced pluripotent stem cell-derived mesenchymal stem cells.

[0033] Preferably, the method includes: performing in vitro expansion culture of mesenchymal stem cells using a mesenchymal stem cell expansion medium containing coronavirus E; wherein the concentration of coronavirus E in the mesenchymal stem cell expansion medium is 0.1-1 µM.

[0034] The amplification culture is performed by passage at least 1-3 times.

[0035] In this invention, the immunosuppressive effect of coronavirus E on mesenchymal stem cells does not depend on the specific composition of the mesenchymal stem cell expansion culture medium. Therefore, any culture medium suitable for mesenchymal stem cell expansion culture can be used.

[0036] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 45%-50% IMDM medium, 45%-50% Ham's F12, 2%-8% human serum albumin, 0.5%-2% insulin-transferrin-selenium additive, 8-12 ng / mL FGF-2, and 8-12 ng / mL EGF.

[0037] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 45%-50% IMDM medium, 45%-50% Ham's F12, 4%-6% human serum albumin, 0.8%-1.2% insulin-transferrin-selenium additive, 9-11 ng / mL FGF-2, and 9-11 ng / mL EGF.

[0038] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 47% IMDM medium, 47% Ham's F12, 5% human serum albumin, 1% insulin-transferrin-selenium additive, 10 ng / mL LFGF-2, and 10 ng / mL EGF.

[0039] The above methods are for non-disease treatment purposes.

[0040] In a seventh aspect, the present invention provides a method for preparing mesenchymal stem cells, the method comprising: performing in vitro expansion culture of mesenchymal stem cells using a mesenchymal stem cell expansion medium containing coronavirus E; wherein the concentration of coronavirus E in the mesenchymal stem cell expansion medium is 0.1-1 µM; and wherein the mesenchymal stem cells are induced pluripotent stem cell-derived mesenchymal stem cells.

[0041] In this invention, the immunosuppressive effect of coronavirus E on mesenchymal stem cells does not depend on the specific composition of the mesenchymal stem cell expansion culture medium. Therefore, any culture medium suitable for mesenchymal stem cell expansion culture can be used.

[0042] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 45%-50% IMDM medium, 45%-50% Ham's F12, 2%-8% human serum albumin, 0.5%-2% insulin-transferrin-selenium additive, 8-12 ng / mL FGF-2, and 8-12 ng / mL EGF.

[0043] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 45%-50% IMDM medium, 45%-50% Ham's F12, 4%-6% human serum albumin, 0.8%-1.2% insulin-transferrin-selenium additive, 9-11 ng / mL FGF-2, and 9-11 ng / mL EGF.

[0044] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises the following components: 47% IMDM medium, 47% Ham's F12, 5% human serum albumin, 1% insulin-transferrin-selenium additive, 10 ng / mL LFGF-2, and 10 ng / mL EGF.

[0045] Eighthly, the present invention provides a method for preparing mesenchymal stem cells with enhanced immunosuppressive activity, the method comprising: in vitro expansion culture of mesenchymal stem cells using a mesenchymal stem cell expansion medium containing coronavirus E; wherein the concentration of coronavirus E in the mesenchymal stem cell expansion medium is 0.1-1 µM; and wherein the mesenchymal stem cells are induced pluripotent stem cell-derived mesenchymal stem cells.

[0046] The beneficial effects of this invention include at least the following: This invention discovers that coronavirusic acid E can significantly enhance the inhibitory ability of iMSCs against T cell proliferation, thereby significantly improving the immunosuppressive activity of iMSCs, while coronavirusic acid E does not affect the cell properties of iMSCs. Verification showed that before and after coronavirusic acid E treatment, the expression of CD44, CD73, CD90, and CD105 on the surface of iMSCs was all above 95%, and the three-lineage differentiation ability did not show significant changes; coronavirusic acid E treatment increased the resistance of iMSCs to CD44... + The average T cell proliferation inhibition rate increased from 47.41% to over 65%, representing a 38.2% improvement in inhibitory effect. The method for preparing mesenchymal stem cells with enhanced immunosuppressive activity provided by this invention is simple, easy to implement, and inexpensive, making it suitable for practical applications in mesenchymal stem cell preparation. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 This is for the detection of iMSC cell viability and doubling time in Example 1 of the present invention; wherein, A: iMSC cells (untreated with coronavirus E); B1: iMSC cells treated with coronavirus E (100 nM); B2: iMSC cells treated with coronavirus E (1 µM).

[0049] Figure 2 The cell morphology of iMSCs in Example 2 of the present invention is shown below; wherein, A: iMSC cells (untreated with coronary cystin E); B1: iMSC cells treated with coronary cystin E (100 nM); B2: iMSC cells treated with coronary cystin E (1 µM).

[0050] Figure 3 The results of cell phenotype detection of iMSCs in Example 2 of the present invention are shown below; where A: iMSC cells (untreated with coronary cystin E); B1: iMSC cells treated with coronary cystin E (100 nM); B2: iMSC cells treated with coronary cystin E (1 µM).

[0051] Figure 4 The results of the three-lineage differentiation ability detection of iMSCs in Example 3 of the present invention are shown; wherein, A: iMSC cells (untreated with coronary cystin E); B1: iMSC cells treated with coronary cystin E (100 nM); B2: iMSC cells treated with coronary cystin E (1 µM).

[0052] Figure 5 The images show cells co-cultured with iMSCs and PBMCs in Example 4 of this invention; where A: iMSC cells; B1: iMSC cells treated with 100 nM of argentin E; B2: iMSC cells treated with 1 µM of argentin E; C: control group.

[0053] Figure 6 In Example 4 of this invention, iMSC inhibits CD4 in PBMCs. +Flow cytometry image of T cell proliferation; where A: iMSC cells (untreated with coronary cystin E); B1: iMSC cells treated with coronary cystin E (100 nM); B2: iMSC cells treated with coronary cystin E (1 µM); C: control group.

[0054] Figure 7 In Example 4 of this invention, iMSC inhibits CD4 in PBMCs. + Flow cytometry statistics of T cell proliferation; where A: iMSC cells (untreated with coronary cystin E); B1: iMSC cells treated with coronary cystin E (100 nM); B2: iMSC cells treated with coronary cystin E (1 µM).

[0055] Figure 8 This invention describes the detection of cytokine release in iMSC cells treated with coronavirus cytokine E (100 nM) in Example 5 of this invention; wherein, A: iMSC cells (untreated with coronavirus cytokine E); B1: iMSC cells treated with coronavirus cytokine E (100 nM).

[0056] Figure 9 The results of an animal experiment in Example 6 of this invention, involving intra-articular injection of iMSC cells treated with coronary cystin E (100 nM) to treat OA in rats, are shown. A: Animal experimental design; B: Rat motor function, with the number of electric shocks from left to right showing the rats' motor function 1 month after the first cell injection and 1 month after the second cell injection.

[0057] Figure 10 The image shows the pathological staining results of rats treated with coronary cystin E (100 nM) intra-articular injection in Example 6 of this invention, and one month after the second cell injection. Detailed Implementation

[0058] In a specific embodiment of the present invention, a method for preparing mesenchymal stem cells with enhanced immunosuppressive activity is provided, comprising the step of in vitro expansion culture of mesenchymal stem cells using a mesenchymal stem cell expansion medium containing coronavirus E; wherein the mesenchymal stem cells are iMSCs.

[0059] For the iMSCs obtained from the above-mentioned expansion culture, the expression of CD44, CD73, CD90, and CD105 on the surface of iMSCs was detected by flow cytometry. iMSCs were cultured in their respective culture media to induce osteogenic, chondrogenic, and adipogenic differentiation, and their trilineal differentiation capacity was assessed after staining with Alizarin Red, Safranin O, and Oil Red O. iMSCs were also cultured in vitro with peripheral blood mononuclear cells (PBMCs) at different effector-target ratios, and the effect of iMSCs on CD44, CD73, CD90, and CD105 was assessed by flow cytometry using a CFSE fluorescent labeling system. + Inhibition of T cell subset expansion. iMSCs before treatment with coronavirusic strobilurin E were used as a control.

[0060] The results showed that the expression of CD44, CD73, CD90, and CD105 on the surface of iMSCs was above 95% before and after treatment with coronavirusicin E, and the differentiation ability of the three lineages did not change significantly. Coronavirusicin E treatment induced a decrease in the expression of CD44, CD73, CD90, and CD105 in iMSCs. + The average T cell proliferation inhibition rate increased from 47.41% to 65.52%, representing a 38.2% improvement in inhibition effect.

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0062] Example 1: Treatment of iMSC cells with Coronavirus E Digestion and inoculation of iPSCs: After diluting the vilinkin (VTN) stock solution and coating the culture plate, select iPSCs with a confluence of 80%-90%, wash with DPBS, digest with TrypLE Select, collect the cell pellet by centrifugation, resuspend in E8 complete medium containing Y-27632, dilute and inoculate into VTN-coated plates for static culture.

[0063] iPSC-induced differentiation to prepare iMSCs: After the iPSC density reached a suitable level, the culture medium was changed to a differentiation medium containing 50% IMDM (Thermo Fisher, 12440053), 50% Ham's F-12 Nutrient Mix (Thermo Fisher, 11765054), 20 ng / mL BMP4 (Peprotech, 120-05), and 20 ng / mL Activin A (Peprotech, AF-120-14E-1MG). After 1 week of culture, the cells were transferred to a medium containing 25% Stem-Span... TM Serum-free amplification medium (Stemcell, 09650), 50% ES-Cult TM Mesenchymal stem cell colony-forming semi-solid medium containing M3120 (Stemcell, 03120), 25% ESFM human endothelial serum-free medium (Thermo Fisher, 11111044), and 10 ng / mL FGF-2 (Stemimmune, HST-F2-1000) was used for further culture until spherical mesenchymal vascular cell colonies formed. Cell colonies were collected through a 100 μM cell sieve and then resuspended in serum-free mesenchymal stem cell medium containing 80% StemLine II (Sigma, S0192), 20% human ESFM, and 10 ng / mL FGF-2. The cells were then seeded into VTN-coated culture flasks. After confluence, iMSC P0 generation cells were obtained and passaged for expansion.

[0064] During subculture amplification, iMSCs were divided into three groups: A, B1, and B2. Group A was amplified using MSC maintenance medium, Group B1 was amplified using MSC maintenance medium supplemented with coronavirus E (the final concentration of coronavirus E in the medium was 100 nM), and Group B2 was amplified using MSC maintenance medium supplemented with coronavirus E (the final concentration of coronavirus E in the medium was 1 μM). The subcultures were repeated three times. The MSC maintenance medium consisted of the following: 47% IMDM (gibco, 21056-023), 47% Ham's F12 (gibco, 31765035), 5% human serum albumin (Ambroxol, Jetbelin Biopharmaceuticals, S20190035), 1% insulin-transferrin-selenium supplement (ITS-G, Shanghai Yuanpei, S450J7), 10 ng / mL FGF-2 (Stemimmune, HST-F2-1000), and 10 ng / mL EGF (Stemimmune, EME-EF-1000).

[0065] Cell viability and viable cell count were recorded using a K2 cell counter during iMSC cell passage. The results showed that ( Figure 1 Treatment with coronary cystin E had no significant effect on the viability and doubling time of iMSC cells.

[0066] Example 2: Phenotypic detection of iMSC The iMSCs prepared in Example 1 were subjected to morphological and phenotypic analysis.

[0067] iPSCs were directed to differentiate into iMSCs under the synergistic effect of different small molecules. The cell morphology results of iMSCs prepared without and after treatment with coronary cystin E are shown in the figure. Figure 2 .

[0068] For iMSC phenotypic detection, the cell suspension was first centrifuged at 400g for 5 min, the supernatant was discarded, and the pellet was resuspended in cell staining buffer and centrifuged again. The mixture was then diluted to a final volume and aliquoted into 7 flow cytometry tubes, labeled NC, FITC IgG1 ISO, FITC-CD44, FITC-CD90, PE IgG1 ISO, PE-CD73, and PE-CD105, respectively. Except for the NC tube, the corresponding antibody was added to each tube and incubated at 4℃ for 30 min. After washing and centrifuging twice with cell staining buffer, the cells were resuspended and finally analyzed using a flow cytometer to obtain data.

[0069] Flow cytometry was used to analyze and compare the cell phenotypes of iMSCs amplified by conventional culture and those amplified by adding coronary stromalin E. The results are as follows: Figure 3 As shown, the expression rates of cell surface marker antigens CD44, CD73, CD90, and CD105 in iMSCs prepared without and after treatment with coronavirus E were all above 95%, indicating that iMSCs treated with coronavirus E still have obvious mesenchymal stem cell surface markers, and that coronavirus E treatment has no significant effect on the cell phenotype of iMSCs.

[0070] Example 3: Detection of osteogenic, chondrogenic, and adipogenic differentiation of iMSCs During iMSC trilineage differentiation assays, the digested cell suspension was induced at 2-3 × 10⁻³ cells per cell line for osteogenic, chondrogenic, and adipogenic induction. 5The dose of MSCs was seeded into 6-well plates, and 2 mL of MSC maintenance medium (same as in Example 1) was added to each well. After labeling the relevant information, the plates were placed in an incubator for culture. The day after plating, the old medium was aspirated and the corresponding induction differentiation medium (Cyagen Biosciences, catalog numbers HUXUC-90021, HUXUC-90031, HUXUC-90041) was added. The medium was completely changed every 3 days. After differentiation, the cells were washed twice with PBS, fixed with 10% formalin at room temperature for 30 min and washed again. Then, osteogenic, chondrogenic and adipogenic differentiation were stained with Alizarin Red, Safranin O and Oil Red O staining solutions at room temperature, respectively. After staining, the cells were washed, and PBS was added. The staining effect was observed and photographed under an inverted microscope.

[0071] The results are as follows Figure 4 As shown, iMSCs possess the ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro. After approximately two weeks of differentiation in osteogenic induction medium, significant calcium precipitation was observed under a microscope, and distinct red calcium nodules were visible after Alizarin Red staining. After approximately two weeks of differentiation in chondrocyte differentiation medium, most cells could be stained with Safranin O. After approximately two weeks of differentiation in adipocyte differentiation medium, distinct lipid droplets were observed under a high-power microscope after Oil Red O staining. The results indicate that treatment with Coronavirus E did not significantly affect the osteogenic, chondrogenic, and adipocyte differentiation abilities of iMSCs, and iMSCs treated with Coronavirus E still exhibited good trilineage differentiation capabilities.

[0072] Example 4: Detection of the inhibitory effect of iMSCs on T cell proliferation in vitro iMSC treatment and plating: 2 hours prior to plating, 24-well plates were coated with VTN. iMSCs with 80-90% confluence (as in Example 1) were collected, the culture medium was discarded, and the cells were washed three times with DPBS to thoroughly remove the medium. After Tryple digestion and centrifugation, the cells were resuspended in MSC maintenance medium (same as in Example 1) without coronary cystin E. Approximately 8 × 10⁶ cells were then collected. 6 One iMSC cell suspension was added, and MSC maintenance medium (same as in Example 1) without coronary cystin E was added to bring the volume to 2 mL. Mitomycin stock solution was added to bring the final concentration of mitomycin to 10 μg / mL. After incubation for 40 min, normal PBMC medium was added. The cells were centrifuged at 400g for 5 min, the supernatant was discarded, the cells were resuspended, counted, and the cell density was adjusted to 2 × 10⁶ cells / mL. 5 / mL, 1×10 5 / mL; finally, aspirate the VTN coating solution, and add 0.5mL of a 2×10⁻⁶ m³ / mL solution to each well in the 1:5 group. 5 For the 1:10 group, add 0.5 mL of cell suspension at a density of 1×10⁶ cells / mL to each well. 5 / mL of cell suspension, 0.5mL of PBMC normal culture medium was added to each well of the NC and PC groups to the 24-well plate, and the culture plate was placed in a cell incubator for incubation.

[0073] PBMC cell plating: First, PBMC cells were revived in a 37°C water bath, centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were resuspended and counted. Then, an appropriate amount of cell suspension was taken and the volume was adjusted to 5 mL, carboxyfluorescein diacetate succinimide (CFSE) stock solution was added, and the cells were incubated in the dark for 10 min. The cells were terminated with pre-cooled FBS and centrifuged. After resuspending and counting, the cell density was adjusted to 1×10⁻⁶ cells / mL. 6 / mL; Finally, take out the 24-well cell culture plate, add 0.5mL of PBMC cell suspension to each well according to the group, except for the NC group, add 10μg / mL of phytohemagglutinin (PHA) and 10ng / mL of IL-2 to each well of the PC group and different effector-target ratio groups, and incubate the culture plate under suitable conditions for about 96h.

[0074] Sample collection and flow cytometry: Cell status was first observed and photographed using an inverted microscope. Cells were then collected by pipetting from the wells, washed with cell staining buffer, and centrifuged at 500g to obtain a cell pellet. Antibody working solutions containing CD45, CD3, and CD4 were prepared according to the specified ratio, added to the cell pellet, and incubated at 4°C in the dark for 30 min for staining. After two washes and centrifugations, the cells were resuspended. CD3+ was then detected using flow cytometry. + CD4 + In the cell population, CFSE - The proportion of proliferating cell populations was determined, and the values ​​of each group were compared.

[0075] Flow cytometry data analysis: The standard for PBMC samples is defined as 1 lymphocyte and CD3+. + CD4 + Relative cell proliferation rate = CD3 + CD4 + Cell relative proportion × CFSE - Cell ratio; CD3 + CD4 + Cell proliferation inhibition rate = (1 - experimental group CD3) + CD4 + Relative cell proliferation rate / CD3 in the proliferation group + CD4 + Relative cell proliferation rate (×100%); Immunosuppressive efficacy (IPA) assay CD4 =CD3 in each experimental group + CD4 + Mean cell proliferation inhibition rate.

[0076] T cells are important effector cells in the adaptive immune response. Mesenchymal stem cells (MSCs) can influence T cell differentiation and cytokine secretion through various mechanisms, thereby exerting their immunomodulatory function. In this example, iMSCs before and after treatment with coronavirusesin E were co-cultured with PBMCs in vitro. T cell expansion was stimulated by the addition of PHA and IL-2, and the effect of adding different amounts of iMSCs on T cell expansion was observed. Cell images (Results) Figure 5 The results showed that, compared with the positive control group (PC), different numbers of iMSCs inhibited the in vitro expansion of T cells to varying degrees.

[0077] To further clarify the effect of iMSC cells on CD4 + The extent of T cell subset expansion was assessed using the CFSE fluorescent labeling system. Results were as follows: Figure 6 , Figure 7 As shown in Table 1, where A: iMSC cells (untreated with coronavirus E); B1: iMSC cells treated with coronavirus E (100 nM); B2: iMSC cells treated with coronavirus E (1 µM); C: control group. The results indicate that iMSCs untreated with coronavirus E have an effect on CD4+. + The average T cell proliferation inhibition rate was 47.41%, and iMSCs treated with coronavirus E showed a significant inhibitory effect on CD4+. + The average T cell proliferation inhibition rate increased to 65.52%, and treatment with coronavirusine E increased the inhibition rate of iMSCs against CD4+. + The T cell proliferation inhibition effect was increased by 38.2%, indicating that coronavirus E treatment can significantly improve the inhibition of CD4+ by iMSCs. + The ability of T cell subsets to inhibit proliferation.

[0078] Table 1

[0079] In conclusion, treatment with coronavirusic acid E does not affect the cell properties of iMSCs, while significantly improving iMSCs' resistance to CD4+. + The ability of T cell subsets to inhibit proliferation.

[0080] Example 5: Detection of Cytokine Release After discarding the culture medium from iMSC cells prepared in groups A and B1 of Example 1, wash three times with an appropriate amount of DPBS to completely remove the culture medium. After digestion with Tryple and centrifugation, resuspend the cells in MSC maintenance medium (same as in Example 1) without coronary cystin E. Terminate mitosis with mitomycin (10 μg / mL) and repeat at 1×10⁻⁶. 5Cells were seeded per well in cell culture plates. After cell attachment, they were stimulated for 72 hours with MSC maintenance medium (same as in Example 1) containing 50 ng / mL interferon-γ (IFN-γ, Peprotech, 300-02-20 μg). The cell supernatant was harvested, and the release levels of indoleamine 2,3-dioxygenase 1 (IDO1, Absin, catalog number abs551862) and transforming growth factor-β1 (TGFβ1, Boster, catalog number EK1218) cytokines were measured. The results are as follows: Figure 8 As shown, under the stimulation of inflammatory factors, treatment with coronary cystin E resulted in iMSCs expressing higher levels of the immunosuppressive molecule IDO1, while also increasing the expression of the anti-inflammatory factor TGFβ1.

[0081] Example 6: iMSC transplantation for the treatment of osteoarthritis (OA) in rats Eighteen SPF-grade male Wistar rats, 8 weeks old (weighing 230-250 g), were purchased from Speysford (Beijing) Biotechnology Co., Ltd. After one week of rearing, the animals were randomly divided into three groups of six rats each: a sham-operated group, a model control group, and an iMSC cell therapy group (the iMSCs used were those prepared from the group treated with 100 nM of coronary cystin E as described in Example 1). The rats were anesthetized with sodium phenobarbital (37.8 mg / kg) via intramuscular injection. Bilateral knee modeling was performed as follows: The knee joint was routinely prepared and disinfected. A medial parapatellar incision was made to expose the knee joint. After exposing the patella, it was dislocated laterally. The knee joint was flexed as much as possible to expose the anterior cruciate ligament (ACL). The ACL was cut under direct vision, and a drawer test was performed to confirm complete ACL transection. Hemostasis was achieved, the patella was repositioned, and the joint cavity was closed layer by layer with No. 1 silk sutures. The injured limb was not immobilized, and free movement was allowed. Postoperative anti-infection and analgesia were administered. Running ability tests and CT scans were performed one and two months after modeling to confirm the occurrence of osteoarthritis, thus establishing a successful rat OA model. After successful establishment of the rat OA model, rats were anesthetized with sodium phenobarbital. The sham-operated group and the model control group were each injected with 100 μL of PBS, while the iMSC cell therapy group was injected with 100 μL of iMSC cell suspension in PBS (containing 5 × 10⁻⁶ cells). 6 (iMSC cells), after injection, the rats were not restrained and were allowed to move freely in their cages. Animals in the iMSC cell therapy group were injected with iMSC cells again one month later, and after two cell injections, they were continued to be housed and observed for another month. The following tests were performed on the experimental rats: Treadmill exercise test: Rats were trained in running for 2 consecutive days, starting at a speed of 5 m / min for 5 minutes, then accelerating to 30 m / min with an acceleration of 1 m / min. 2The rats were trained for 25 minutes. After 2 days of training, they were tested once a day for 3 consecutive days, and the number of electrical stimulations within 30 minutes was recorded. The average number of electrical stimulations detected over 3 days was recorded and statistically analyzed.

[0082] Histopathological staining: After two intra-articular injections of iMSC cells, rats were euthanized by CO2 asphyxiation, and bilateral knee joint specimens were collected. The tissues were immersed in 4% paraformaldehyde solution for paraffin section preparation. The tissues were removed from the 4% paraformaldehyde solution and placed in a decalcification solution, which was changed every 3 days. The knee cartilage tissue was routinely dehydrated, embedded in paraffin, and serially sectioned at a thickness of 5 μm per section, with approximately 20 sections per specimen. One section was taken every 5 sections for staining. For hematoxylin-eosin (HE) staining (Soleb, G1120), sections were dewaxed in xylene, rehydrated in a gradient of ethanol (100%, 100%, 95%, 80%, 70%), and cultured in hematoxylin solution until the desired staining intensity was achieved. The sections were then rinsed under running water to remove excess hematoxylin, differentiated in 1% acidic ethanol for 1 second, and then rinsed under running water for 1 minute. Finally, the sections were stained with eosin to the desired pink color and dehydrated in a gradient of ethanol and xylene. Special staining was performed according to the instructions of the modified safranin O-Fix Green (Solepro, G1371), toluidine blue (Solepro, G3668), and alexandrite blue (Solepro, G1562) staining kits. Experimental images were observed and acquired using a fluorescence microscope (Keyence, BZ-X800).

[0083] Statistical analysis: SPSS 11.0 statistical software was used to analyze the changes in relevant indicators of the control group and the experimental group. Experimental data are expressed as mean ± standard deviation (±S). The t-test was used to compare the two groups: P < 0.05 was considered statistically significant.

[0084] Results and Conclusions: To analyze the safety and efficacy of coronary cystin E-treated iMSCs in an animal model of osteoarthritis, a rat osteoarthritis model was first established by severing the anterior cruciate ligament. Then, coronary cystin E-treated (100 nM) iMSCs were transplanted into the joint cavity of the osteoarthritis rats in two separate procedures. Figure 9 (A). One month after the first and second iMSC cell injections, the rats' motor function was assessed. After only one intra-articular injection of iMSC cells, the pain caused by osteoarthritis (OA) in rats was not effectively relieved. One month after two iMSC cell transplants, the iMSC cell treatment group significantly reduced joint pain caused by OA in rats compared to the model control group. Figure 9HE staining results showed that in the model control group, chondrocytes were randomly distributed in the joint cavity, with significant loss of extracellular matrix and incomplete hyaline cartilage structure. Compared with the model control group, osteoarthritis was significantly relieved after iMSC transplantation, with intact cartilage structure, uniform cell distribution, and cells in the regenerated tissue exhibiting the same morphology as natural chondrocytes. Clear and complete tidal lines were also observed, and the cartilage repair effect was essentially consistent with the sham-operated group. Figure 10 The results of Alixin Blue, Safranin O, Fast Green, and Toluidine Blue staining showed increased expression of acidic mucopolysaccharides in the joint cavity, and the regenerated cartilage tissue was hyaline cartilage tissue rich in extracellular matrix. This indicates that iMSCs have a significant protective effect on cartilage or promote articular cartilage regeneration in a mild OA model in rats with transection of the anterior cruciate ligament. Figure 10 ).

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of coronavirusic acid E in the preparation of mesenchymal stem cells with enhanced immunosuppressive activity; among which, The mesenchymal stem cells mentioned are induced pluripotent stem cell-derived mesenchymal stem cells; The application includes treating mesenchymal stem cells with coronary cystin E; wherein the concentration of coronary cystin E is 0.1-1 µM.

2. The application according to claim 1, characterized in that, The enhanced immunosuppressive activity is manifested in the following way: compared with untreated mesenchymal stem cells, mesenchymal stem cells treated with coronavirus E show increased activity against CD4+. + Increased inhibitory activity against T cell proliferation.

3. The application according to claim 1, characterized in that, The treatment of mesenchymal stem cells with coronavirus E includes: performing in vitro expansion culture of mesenchymal stem cells using a mesenchymal stem cell expansion medium containing coronavirus E.

4. Application of coronavirusic acid E in the preparation of formulations that enhance the inhibitory activity of mesenchymal stem cells against T cell proliferation; among which, The mesenchymal stem cells mentioned are induced pluripotent stem cell-derived mesenchymal stem cells.

5. The application according to claim 4, characterized in that, The T cells are CD4. + T cells.

6. A method for enhancing the inhibitory activity of mesenchymal stem cells against T cell proliferation in vitro, characterized in that, The method includes: treating mesenchymal stem cells with coronary cystin E; In the treatment, the concentration of coronavirusic acid E is 0.1-1 µM; The mesenchymal stem cells mentioned are induced pluripotent stem cell-derived mesenchymal stem cells.

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