A method for quantifying and distinguishing the immune activity of MSCs using CD82 and applications thereof

Abstract

The application discloses a method for quantifying and distinguishing MSC immunological activity by CD82 and application, collects cells, uses a fluorescently labeled antibody to mark a membrane protein CD82 on a surface of MSC, and uses a flow cytometer to detect a positive rate and MFI of the cell CD82, quantifies the MSC immunological activity by an expression level of the CD82, the immunological activity refers to an inhibition ability of the MSC on secretion of inflammatory cytokines TNF-alpha and IFN-gamma by activated PBMC, meanwhile, the MSC cell group with high immunological activity can be distinguished by detecting the expression level of the CD82 on the MSC, the MSC cell group with high immunological activity screened by using the method shows good curative effect in treating diseases related to inflammation and immunity, the detection method is simple, easy to operate, economical and efficient, and has wide application prospect in a stem cell drug market.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method and application for quantifying and differentiating MSC immune activity using CD82. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a population of cells derived from the mesoderm that can be isolated and expanded in large quantities in vitro. They are a group of sub-pluripotent stem cells with self-renewal and multi-lineage differentiation capabilities, possessing multiple biological functions such as self-renewal, multi-lineage differentiation, angiogenesis, hematopoietic support, promotion of tissue damage repair, and immune regulation. It is worth noting that MSCs are a heterogeneous cell population. The biological properties of MSCs isolated from different donors and tissues vary considerably (Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell Rev Rep. 2013 Feb;9(1):32-43). Even MSCs isolated from the same tissue contain several subpopulations (VCAM-1+ placenta chorionic villi-derived mesenchymal stem cells display potent pro-angiogenic activity. Stem Cell Res Ther. 2016Apr 4:7:49.;Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, and placenta. Stem Cell Res Ther. 2016 Nov). 10;7(1):163), therefore, finding a suitable quantitative method to assess the biological activity of MSCs and to find highly immune-active MSC populations is crucial.

[0003] Currently, as a Class I new stem cell drug, the main mechanisms of MSC drug treatment for diseases include two aspects: immune regulation and tissue damage repair. Immune regulation is the core mechanism for its treatment of various inflammatory and immune-related diseases. Although research on the immune regulation mechanism of MSCs has been ongoing for more than half a century, researchers from various countries have published many research results on the immunomodulatory effects of MSCs from the perspectives of contact inhibition and paracrine inhibition. For example, known soluble factors include TGF-β1, HGF, IDO, PGE2, TSG-6, TNFR1, etc., as well as surface antigen molecules such as VCAM-1, PD-L1, ICAM-1, etc. (Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases. Nat Rev Nephrol. 2018 Aug;14(8):493-507). However, because MSCs are a group of "living" cells with heterogeneous characteristics, their therapeutic mechanism remains a difficult problem that has troubled scientists from various countries. From a microscopic perspective, MSC cell lines are composed of multiple different cell subpopulations, with varying biological characteristics and functions among them. From a macroscopic perspective, cell lines exhibit heterogeneity due to differences in donor and tissue sources, isolation and preparation methods, resulting in significant variations in the biological properties of different batches of formulations. This makes it extremely difficult to determine standard products, and the mechanisms of action are complex. Currently, there is no universally recognized core potency molecule, making the quality control of MSC drugs, especially in terms of biological activity (also known as biological efficacy), a major challenge that restricts their clinical application and efficacy (MSC-based product characterization for clinical trials: an FDA perspective. Cell Stem Cell. 2014 Feb 6;14(2):141-5).

[0004] Current methods for evaluating the bioactivity of MSCs in quality control primarily assess their inhibitory effects on the proliferation of peripheral blood mononuclear cells (PBMCs) and the level of inhibition of their secretion of inflammatory factors. There are also evaluation systems targeting the effects of MSCs on specific lymphocyte subsets, such as Th1 / Treg / Th17. However, these methods are complex and costly. To reduce the cost of quality control, researchers are exploring alternative functional indicators to assess the bioactivity of MSCs. These alternative indicators often utilize key cytokines or proteins expressed by the MSCs themselves. Currently known methods for evaluating the biological activity of MSCs include ELISA to detect the expression of PGE2 (application number CN201811394547.9), TNFRI (application number CN201911149147.6), and cell membrane protein CD276 (patent number ZL202211276848.8) in MSC supernatant, and flow cytometry to detect MSC surface antigens CD155 (patent number ZL202211113993.4) ​​and CD273 (application number CN202410284307.2). Considering that ELISA detection of cytokines (such as PGE2 and TNFRI) in the supernatant requires cell culture and supernatant collection, which is time-consuming, and that cytokines are often unstable and easily degraded, and that storage methods (such as temperature and humidity) often exacerbate their degradation, leading to inaccurate measurement results, these methods are not feasible. While ELISA detection of cell membrane protein (CD276) is not as easily degraded as cytokines, it is still susceptible to storage conditions, often requiring immediate cell lysis and testing, which involves numerous steps. Flow cytometry detection of MSC surface antigens CD155 or CD273, on the other hand, eliminates the cumbersome cell culture and lysis steps, making it simpler and easier to perform. Therefore, compared to ELISA, flow cytometry will become a new method for analyzing and predicting MSC biological activity. Furthermore, since research on MSC activity markers is still in its early stages, more new and effective molecules and corresponding detection methods will be developed in the future.

[0005] CD82 (also known as KAI1 or TSPAN27) is a member of the tetraspanin superfamily of glycoproteins. It is a cell membrane glycoprotein that mediates various functions, including cell migration, immune synapse formation, and secretion. Similar to CD276, CD155, and CD273 mentioned earlier, it is a transmembrane protein, but it also has unique characteristics; it can be found in extracellular vesicles (EVs). Studies have shown that EVs secreted by cells such as dendritic cells, macrophages, B cells, and endothelial cells are also rich in CD82 (The Many and Varied Roles of Tetraspanins in Immune Cell Recruitment and Migration. Front Immunol. 2018 Jul 18:9:1644). Furthermore, CD82 expression is negatively correlated with tumor invasion and metastasis, and strongly correlated with p53 expression (Prognostic role of CD82 / KAI1 in multiple human malignantneoplasms: a meta-analysis of 31 studies. Onco Targets Ther. 2017 Dec 6:10:5805-5816). Additionally, CD82 has been reported to be associated with decidualization during pregnancy (A positive COX-2 / IL-1β loop promotes decidualization by upregulating CD82. Reproduction. 2021 Aug 11;162(3):227-236) and viral infection (Tetraspanin Assemblies in Virus Infection. FrontImmunol. 2018 May 25:9:1140).

[0006] To date, there has been very little research on CD82 in the field of MSCs, mainly including the following aspects: ① Phenotypic detection: It can be detected on the surface of MSCs from various tissue sources, including bone marrow MSCs, adipose MSCs, adult dermal MSCs, neonatal foreskin MSCs, etc. (Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell Rev Rep. 2013 Feb;9(1):32-43); ② CD82 has been reported to be expressed on the exosome membrane of MSCs (Therapeutic Use of Mesenchymal Stem Cell-Derived Exosomes: From Basic Science to Clinics. Pharmaceutics. 2020 May 22;12(5):474; The potential use of mesenchymal stem cells and their exosomes in Parkinson's disease treatment. Stem Cell Res Ther. 2022 Jul 28;13(1):371). ③CD82 can be used to distinguish a group of cells with stronger chondrocyte differentiation potential, namely CD82 low CD59 + Mesenchymal progenitor cells (MPCs) are used for cartilage injury repair (Mesenchymal progenitor cells from non-inflamed versus inflamed synovium post-ACL injury present with distinct phenotypes and cartilage regeneration capacity. Stem Cell Res Ther. 2023 Jun25;14(1):168). However, no studies have reported the "role" of CD82 in MSC-mediated immunosuppression, nor have any studies reported whether CD82 can serve as an indicator of MSC immune activity.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a method for quantifying and differentiating the immune activity of MSCs using CD82. CD82 is a tetraspan membrane protein with a stable structure that is not easily degraded. The immune activity of MSCs can be determined by detecting the positive rate and mean fluorescence intensity (MFI) of CD82 on the MSC surface using flow cytometry. The detection method is simple, easy to operate, time-saving, economical and efficient, and the judgment results are accurate and reliable. It has broad application prospects and is conducive to its widespread application.

[0009] To achieve the above objectives, this invention provides a method for quantifying and differentiating the immune activity of MSCs using CD82. This involves collecting cells, labeling the membrane protein CD82 on the surface of MSCs with a fluorescently labeled antibody, and using flow cytometry to detect the CD82 positivity rate and MFI (membrane fibrillation factor). The immune activity of the cells is evaluated by detecting the expression level of CD82 on the MSC surface. Under the premise that the CD82 positivity rate and MFI on the MSC surface are positively correlated, when the CD82 positivity rate on the MSC surface is >54% and the MFI is >70, its inhibitory ability on the secretion of TNF-α and IFN-γ by activated PBMCs is greater than 50%. The immune activity of different MSC cell lines can be quantified by detecting the expression level of CD82 in MSCs from different donors using flow cytometry, thus distinguishing MSC cell lines with high immune activity. This immune activity refers to the ability to inhibit the secretion of inflammatory cytokines TNF-α and IFN-γ by activated PBMCs.

[0010] Preferably, the MSCs are derived from bone marrow, adipose tissue, perinatal tissue, or induced differentiation from pluripotent stem cells (iPSCs) / embryonic stem cells (ESCs), and the perinatal tissues include umbilical cord, placenta, amnion, or amniotic fluid.

[0011] Preferably, the specific steps include:

[0012] S1: Collecting cells to be tested: Digest and collect cultured MSCs, count the cells, and transfer them to flow cytometry tubes;

[0013] S2: Labeled flow cytometry antibody: Centrifuge the cell suspension, remove the supernatant, add fluorescently labeled antibodies to the cell pellet, vortex to mix, and incubate at room temperature in the dark or in a refrigerator at 4°C. Add flow cytometry washing buffer to wash away unbound antibodies, centrifuge, add flow cytometry washing buffer to resuspend the cells, filter out cell clumps using a filter membrane, and wait for detection.

[0014] S3: Flow cytometry detection: The positivity rate of CD82 on the surface of MSCs and MFI were detected by flow cytometry;

[0015] S4: Quantitative analysis of the immune activity of MSC cell population;

[0016] S5: Differentiate between highly active MSC cell populations: When the CD82 positivity rate on the surface of MSCs is higher than 70%, they are considered highly active MSC cell populations, while when the CD82 positivity rate is lower than 40%, they are considered low-activity MSC cell populations.

[0017] This invention also provides an application of the above-mentioned method for quantifying and differentiating MSC immune activity using CD82 in the formulation of quality testing standards for cell banks and cell injection solutions.

[0018] The present invention also provides an application of the above-mentioned method for quantifying and distinguishing MSC immune activity using CD82 in screening a seed bank of highly immune MSCs.

[0019] The present invention also provides an application of the above-mentioned method for quantifying and distinguishing MSC immune activity using CD82 in the preparation of highly immune-active cell injections for the treatment of inflammation and immune diseases, such as acute respiratory distress syndrome (ARDS), diabetic nephropathy (DKD), acute-on-chronic liver failure (ACLF), and hormone-resistant graft-versus-host disease (GvHD).

[0020] The present invention provides a method and application for quantifying and differentiating MSC immune activity using CD82, which has the following beneficial effects.

[0021] Compared to ① culturing MSCs, collecting the supernatant and then using ELISA to detect the cytokine content in the supernatant; or ② directly lysing MSCs, collecting the lysed supernatant and then using ELISA to detect the protein content; or ③ in vitro culture and detecting the effect of MSCs on the proliferation level of PBMCs; or ④ in vitro culture and detecting the effect of MSCs on the level of inflammatory cytokines (TNF-α and IFN-γ) secreted by PBMCs, this invention uses flow cytometry to detect the level of CD82 on the cell surface to determine its biological activity. The operation is simple and can avoid potential human contamination and operational errors, saving time and effort. At the same time, CD82 is a transmembrane glycoprotein with a more stable structure, which makes the detection results more accurate.

[0022] The inventors serendipitously discovered the gene through transcriptome sequencing, immunological activity detection, and correlation analysis of MSCs from multiple donors. CD82This may be closely related to the level of MSC immune activity. Flow cytometry was used to detect the expression level (positivity rate and MFI) of CD82 on the surface of MSCs from multiple batches and multiple donors. Simultaneously, in vitro cell experiments were conducted to detect its immune activity, revealing a significant positive correlation between the two. Furthermore, it was found that when the positivity rate of CD82 on the MSC surface was >54% and the MFI was >70, the immunosuppressive level against the secretion of inflammatory factors by activated PBMCs exceeded 50%. In addition, it was found that when the positivity rate of CD82 on the MSC surface exceeded 70%, its immune activity was high and it could be considered a highly active MSC subset, while below 40% showed low immune activity and could be considered a low-activity MSC subset. Furthermore, the inventors found that knocking down the expression of CD82 in cells using siRNA interference significantly downregulated the immune activity of MSCs. At the same time, stimulation of MSCs with the cytokine IL-1β significantly upregulated the expression level of CD82 and the secretion level of PGE2, and the two showed a significant positive correlation. Notably, the inventors screened for one high-activity and one low-activity MSC cell line using CD82, and conducted in vivo animal experiments to treat ARDS mice and DKD rats. They found that compared to the low-activity MSC cell line, the high-activity MSC cell line significantly improved the survival rate of ARDS mice and the proteinuria in DKD rats. Compared to other inventions, this invention not only develops an innovative and stable protein indicator, CD82, to indicate the immune activity of MSCs, but also uses this indicator to distinguish MSC cell populations with different immune activities, and its effectiveness has been demonstrated in animal experiments. Attached Figure Description

[0023] Figure 1 Immunological activity and genetic information of umbilical cord MSCs from multiple samples and multiple donors. CD82 Correlation analysis of transcriptional levels (FPKM values);

[0024] Figure 2 The expression of CD82 on the surface of umbilical cord MSCs from multiple samples and multiple donors was shown by histograms of fluorescence intensity from flow cytometry.

[0025] Figure 3 The expression level of CD82 on the surface of umbilical cord MSCs from multiple samples and multiple donors was analyzed, including the positive rate and MFI, as well as the correlation between the two.

[0026] Figure 4 The expression level (positive rate and MFI) of CD82 on the surface of umbilical cord MSCs from multiple samples and multiple donors showed a significant positive correlation with their immune activity level. This can be used to quantify the immune activity level of different MSC cell lines and to distinguish MSC cell populations with different immune activities.

[0027] Figure 5To knock down the expression level of CD82 in MSCs using siRNA interference technology and to detect its effect on MSC immune activity;

[0028] Figure 6 To investigate the effects of IL-1β stimulation on the expression level of CD82 on the surface of umbilical cord MSCs in multiple samples and its secretion of PGE2, and to analyze the correlation between the two.

[0029] Figure 7 The effects of single tail vein administration of different doses of cell therapy to mice with acute respiratory distress syndrome (ARDS) were investigated based on the high and low activity MSC cell lines screened in this invention.

[0030] Figure 8 To investigate the effects of repeated tail vein injections of high- and low-activity MSC cell lines selected based on this invention on the urinary protein levels of diabetic nephropathy (DKD) rats. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments and accompanying drawings to help understand the content of the present invention.

[0032] This invention provides a method and application for quantifying and differentiating the immune activity of MSCs using CD82. Cells are collected, and the membrane protein CD82 on the surface of MSCs is labeled with a fluorescently labeled antibody. Flow cytometry is used to detect the positivity rate of CD82 and the mean fluorescence intensity (MFI). The immune activity of MSCs is quantified by detecting the expression level of CD82 on the MSC surface. Under the premise that the positivity rate of CD82 on the MSC surface and MFI are positively correlated, when the positivity rate of CD82 on the MSC surface is >54% and the MFI is >70, its inhibitory ability on the secretion of TNF-α and IFN-γ by activated PBMCs is greater than 50%. The immune activity of different cells can be quantified and different MSC cell populations with different immune activities can be distinguished by detecting the expression level of CD82 in MSCs from different donors using flow cytometry. The immune activity refers to its ability to inhibit the secretion of inflammatory cytokines TNF-α and IFN-γ by activated PBMCs. The MSCs are derived from bone marrow, adipose tissue, perinatal tissue, or induced differentiation from iPSCs / ESCs. The perinatal tissue includes the umbilical cord, placenta, amnion, or amniotic fluid.

[0033] Specifically, the steps include the following:

[0034] S1: Collecting cells to be tested: Digest and collect cultured MSCs, count the cells, and transfer them to flow cytometry tubes;

[0035] S2: Labeled flow cytometry antibody: Centrifuge the cell suspension, remove the supernatant, add fluorescently labeled antibodies to the cell pellet, vortex to mix, and incubate at room temperature in the dark or in a refrigerator at 4°C. Add flow cytometry washing buffer to wash away unbound antibodies, centrifuge, add flow cytometry washing buffer to resuspend the cells, filter out cell clumps using a filter membrane, and wait for detection.

[0036] S3: Flow cytometry detection: The positivity rate of CD82 on the surface of MSCs and MFI were detected by flow cytometry;

[0037] S4: Quantitative analysis of the immune activity of MSC cell population;

[0038] S5: Differentiate between highly active MSC cell populations: When the CD82 positivity rate on the surface of MSCs is higher than 70%, they are considered highly active MSC cell populations, while when the CD82 positivity rate is lower than 40%, they are considered low-activity MSC cell populations.

[0039] This invention also provides an application of the above-mentioned method for quantifying and differentiating MSC immune activity using CD82 in the formulation of quality testing standards for cell banks and cell injection solutions.

[0040] The present invention also provides an application of the above-mentioned method for quantifying and distinguishing MSC immune activity using CD82 in screening a seed bank of highly immune MSCs.

[0041] This invention also provides an application of the above-described method for quantifying and differentiating MSC immune activity using CD82 in the preparation of highly immunologically active cell injections for the treatment of inflammation and immune diseases, such as acute respiratory distress syndrome (ARDS), diabetic nephropathy (DKD), acute-on-chronic liver failure (ACLF), and hormone-resistant graft-versus-host disease (GvHD).

[0042] Example 1: Transcriptome sequencing and immunological activity analysis of umbilical cord MSCs from multiple samples and multiple donors under different treatment conditions. The specific implementation method is as follows:

[0043] Conditioned medium (CM): 20 umbilical cord MSCs from multiple donors and multiple samples were seeded into T75 flasks. After adherence, a control group and an inflammatory factor stimulation group were established, allowing the cells to continue culturing under different treatment conditions for 24 hours. The culture supernatant (CM) was collected, centrifuged at 2000 rpm for 10 minutes to remove cell debris, aliquoted, and stored at -80°C.

[0044] Immunomodulatory activity analysis: After thawing CM cells under different treatment conditions, 100 μl of each cell was added to a 96-well plate, followed by 100 μl of PBMC suspension containing phytohemagglutinin (PHA) stimulation, ensuring a PBMC cell count of 1 × 10⁶ cells per well. 5After thorough mixing, continue culturing for 24 hours. A negative control group consisted of PBMC wells without PHA stimulation, and a positive control group consisted of PBMC wells with PHA stimulation. The 24-hour culture supernatant was collected, centrifuged at 2000 rpm for 10 minutes, and the supernatant was carefully aspirated, avoiding cell debris. The supernatant was then aliquoted and stored at -80°C. The IFN-γ content in the supernatant was then detected by ELISA, and the IFN-γ inhibition rate was calculated using the following formula.

[0045] IFN-γ inhibition rate (%) = [1 - IFN-γ （24h-CM） / IFN-γ （阳性组） ] × 100%

[0046] Transcriptome sequencing: 2×10⁻⁶ samples were collected. 6 The MSCs treated with different stimuli were washed with DPBS, centrifuged, and the supernatant was carefully discarded. The cell pellets were lysed with Trizol, mixed, and then placed on dry ice for sequencing.

[0047] Immunological activity and genetic makeup of multiple MSCs treated with different stimuli CD82 Correlation analysis was performed on the transcriptional levels (FPKM values), and the results are as follows: Figure 1 As shown.

[0048] Depend on Figure 1 It can be seen that the analysis of the immune activity levels of umbilical cord MSCs from multiple samples and multiple donors after untreated and inflammatory factor treatment (n=40) shows that... Figure 1 As shown in A, genes CD82 Correlation analysis of transcriptome levels (FPKM value) with their immune activity, such as... Figure 1 As shown in B, genes were discovered. CD82 The transcriptional level (FPKM value) was significantly positively correlated with the level of inhibition of the secretion of the inflammatory cytokine IFNγ by activated PBMCs (r=0.46, **p<0.01). This result infers gene expression from the transcriptional level. CD82 It may be a potential indicator for assessing the immune activity of MSCs.

[0049] Example 2: Flow cytometry analysis of CD82 expression levels in umbilical cord MSCs from multiple samples and multiple donors (n=18). The specific implementation steps are as follows:

[0050] The expression level of CD82 on the surface of 18 umbilical cord MSCs from different donors was detected by flow cytometry, including the positive rate and MFI. Umbilical cord MSCs were first collected by digestion and then processed at a ratio of 2 × 10⁻⁶. 5Two tubes of each cell type were transferred into flow cytometry tubes. After centrifugation at 2000 rpm for 5 min, the cell pellet was collected and washed with flow cytometry washing buffer (i.e., DPBS solution containing 2% fetal bovine serum). The cells were centrifuged again to remove the supernatant, and PE-IgG antibody and PE-CD82 antibody (BioLegend) were added separately and vortexed to mix. After incubation at room temperature in the dark for 20 min, the cells were washed with flow cytometry washing buffer, centrifuged at 2000 rpm for 5 min, and the supernatant was carefully discarded, retaining the cell pellet. The cell pellet was resuspended with flow cytometry washing buffer, and after filtering to remove cell clumps, the positivity rate of CD82 and MFI were detected by flow cytometry.

[0051] Depend on Figure 2 Flow cytometry histograms showed that the expression levels of CD82 varied significantly among the 18 MSCs from different donors. Statistical analysis of the positivity rate of CD82 on the surface of umbilical cord MSCs from different donors and MFI results are as follows. Figure 3 As shown in Figure A, the mean positivity rate of CD82 was 49.09% ± 4.60, and the mean mean molecular weight fraction (MFI) was 69.01 ± 4.62. Pearson correlation analysis was performed on the positivity rate of CD82 and MFI on the surface of umbilical cord MSCs from different donors, and the results are as follows: Figure 3 As shown in B, the two are significantly positively correlated (r = 0.7586, ***p < 0.001).

[0052] Example 3: Immunological activity of umbilical cord MSCs from multiple donors and samples was detected, and their correlation with CD82 expression levels was analyzed. To quantify the immunological activity of MSCs from different donors, the preferred criteria were a CD82 positivity rate >54% and an MFI >70, ensuring that the inhibition levels of inflammatory factors (TNF-α and IFN-γ) secreted by PBMCs were both greater than 50%. To differentiate between MSC cell populations with different immunological activities, the preferred criteria were a CD82 positivity rate higher than 70% for highly active MSCs and lower than 40% for low-activity MSCs. The specific implementation method is as follows:

[0053] Flow cytometry analysis of CD82 expression levels on the surface of MSCs from multiple samples and multiple donors: Umbilical cord MSCs (n=18) were digested and collected, and then analyzed at a ratio of 2×10⁻⁶. 5 Cells / tubes were transferred to flow cytometry tubes, with two tubes prepared for each cell type. After centrifugation at 2000 rpm for 5 min, cells were washed with flow cytometry washing buffer (DPBS + 2% FBS), centrifuged again, and then PE-IgG antibody or PE-CD82 antibody was added. The mixture was vortexed and incubated at 4°C in the dark for 30 min. After washing the cells again, the cells were centrifuged, resuspended in flow cytometry washing buffer, and the cell clumps were filtered through a filter membrane. The positivity rate of CD82 on the surface of umbilical cord MSCs and the MFI were detected by flow cytometry.

[0054] Immunological activity assay: umbilical cord MSCs were digested and counted, and resuspended in complete culture medium to a final concentration of 1 × 10⁻⁶ mcg / mL. 5 Cells were seeded at 100 μL per well in 96-well plates, mixed in a crosswise pattern, and then a PBMC suspension containing PHA stimulation was added to each well, resulting in a PHA-to-PBMC ratio of 1:10. The cells were thoroughly mixed and incubated for 72 h. A positive control group (PBMCs containing PHA stimulation) and a negative control group (PBMCs without PHA stimulation) were also established. The cell supernatant after 72 hours of culture was collected, centrifuged, aliquoted, and stored at -80°C. The levels of inflammatory cytokines TNF-α and IFN-γ in the culture supernatant were detected by ELISA, and the inhibition rate (%) was calculated using the following formula:

[0055] TNF-α inhibition rate (%) = [1-TNF-α] （MSC组） / TNF-α （阳性组） ]×100%

[0056] IFN-γ inhibition rate (%) = [1-IFN-γ （MSC组） / IFN-γ （阳性组） ]×100%

[0057] Depend on Figure 4 It can be seen that the expression levels of CD82 in MSCs from 18 different donors varied, with positive rates and MFI as follows: Figure 4 As shown in Figure A. MSCs from 18 different donors were co-cultured with PBMCs for 72 h, and their inhibition rates on the secretion of inflammatory factors TNF-α and IFN-γ by PBMCs were calculated. The results are shown in Figure A. Figure 4 As shown in B and 4C, the inhibitory levels of MSCs from different donors on inflammatory factors secreted by PBMCs varied: the highest inhibition rate of TNF-α reached 70.99%, while the lowest was 0. Figure 4 B); the maximum IFN-γ inhibition rate reached 85.64%, and the minimum was 30.16% ( Figure 4 C). The expression level of CD82 was correlated with the TNF-α inhibition rate ( Figure 4 B) Correlation analysis revealed that both the positive rate of CD82 and MFI were significantly positively correlated with the TNF-α inhibition rate, with correlation coefficients of 0.8390 (p<0.0001). Figure 4 D) and 0.6429 (**p<0.01, Figure 4 D). Similarly, it was found that the CD82 positivity rate and MFI were related to the IFN-γ inhibition rate ( Figure 4 C) showed a significant positive correlation, with a correlation coefficient of 0.7957 (p<0.0001). Figure 4 E) and 0.6821 (**p<0.01, Figure 4E). When the preferred conditions are met, namely CD82 positivity rate > 54% and MFI > 70, the inhibition rate of MSCs on the secretion of TNF-α and IFN-γ by activated PBMCs can be guaranteed to exceed 50%. However, when the CD82 positivity rate is > 70% or < 40%, significant differences in CD82 expression levels, including positivity rate % and MFI, are observed among MSCs from different donor sources meeting the conditions (*p < 0.05). Figure 4 F). Cell populations with high CD82 expression (i.e., CD82 positivity >70%, abbreviated as CD82) High ) and low expression cell populations (i.e., CD82 positivity rate <40%, abbreviated as CD82) Low Analysis of the immune activity of the two cell populations revealed a significant difference in the inhibitory levels of activated PBMCs on the secretion of inflammatory factors (*p<0.05). Figure 4 G). CD82 High vs CD82 Low The cell population showed an inhibition rate of 70.25% ± 0.60% against 32.10% ± 6.14% (*p < 0.05). Figure 4 The inhibition rate of IFN-γ (G) was 75.70%±2.53% vs 53.06%±6.41% (*p<0.05). Figure 4 G). The above results suggest that the expression level of CD82 can be used to measure the level of MSC immune activity and can distinguish MSC cell populations with different immune activities.

[0058] Example 4: Using siRNA to interfere with CD82 expression in MSCs and detecting its effect on MSC immune activity. Specific implementation method:

[0059] After digestion, umbilical cord MSCs were seeded into six-well plates. Transfection was performed when the cell confluence reached approximately 50-70%. Transfection complexes, consisting of control NC-siRNA oligo (NC-siRNA) and CD82 siRNA oligo (CD82-siRNA), were prepared in advance and incubated at room temperature. After replacing the preheated OPTI-MEM medium with the culture medium in the six-well plates, the prepared transfection complexes were added to the corresponding wells, gently mixed, and incubated in a CO2 incubator for 24 hours. The culture medium was then replaced with complete medium, and the cells were cultured for another 72 hours. Untransfected cells (Ctrl) were used as a control to observe the effect of the transfection reagents on the cells. 72 hours after transfection, the cells were digested and collected, and labeled with antibodies PE-CD82 and PE-IgG, respectively. After incubation at room temperature in the dark for 30 minutes, the positivity rate of CD82 on the cell surface and the mean fibroblast indicative cytometry (MFI) were detected. The remaining cells were co-cultured with PHA-activated PBMCs at a ratio of 1:10 for 48 h and 72 h. The co-culture supernatant was collected, and the levels of TNF-α and IFN-γ in the supernatant were detected by ELISA. A positive control group consisted of PHA-activated PBMCs, and a negative control group consisted of PBMCs. Based on the TNF-α and IFN-γ levels measured by ELISA, the effect of CD82-siRNA interference on MSC immune activity was calculated. The inhibition rate of PBMC secretion of inflammatory factors (TNF-α and IFN-γ) was calculated using the same method as before.

[0060] Depend on Figure 5 It can be seen that: siRNA transfection was performed on umbilical cord MSCs from three different donors, and flow cytometry was used to detect the expression of CD82 in untransfected cells (Ctrl) and cells transfected with NC-siRNA and CD82-siRNA. Figure 5 A). Statistical analysis showed that after transfection with CD82-siRNA, the positive rate of CD82 on the cell surface (**p<0.01) and MFI (***p<0.001) were both significantly reduced. Figure 5 B), while after transfection with NC-siRNA, no significant changes were observed in the positivity rate of CD82 on the cell surface and MFI. Figure 5 B). Next, untransfected MSCs and MSCs transfected with two different siRNAs were co-cultured with PBMCs for 48 h and 72 h, respectively. The results showed that after 48 h of co-culture, MSCs transfected with CD82-siRNA significantly reduced the inhibitory levels of inflammatory factors TNF-α (*p<0.05, **p<0.01) and IFN-γ (*p<0.05) secreted by PBMCs compared to Ctrl and NC-siRNA-transfected cells. Figure 5C); After co-culturing for 72 h, MSCs transfected with CD82-siRNA showed a significantly lower TNF-α inhibition rate compared to Ctrl and NC-siRNA transfected cells (*p<0.05), while the IFN-γ inhibition rate showed a decreasing trend ( Figure 5 D). The above results suggest that knocking down the expression level of CD82 in MSCs can significantly reduce their immune activity.

[0061] Example 5: Detection of the effects of IL-1β stimulation on the expression level of CD82 on the surface of multiple umbilical cord MSCs and its secreted PGE2 level, and the correlation between the two. The specific experimental methods are as follows:

[0062] Five different donor-derived umbilical cord MSCs were digested and seeded into six-well plates. After the cells adhered to the plates to 50%-70%, the medium was replaced with complete medium containing or without 10 ng / ml IL-1β. The cells were cultured for 24 h and 48 h, and the culture supernatant was collected to detect PGE2 (pg / ml). At the same time, the expression level of CD82 on the cell surface was also collected.

[0063] Depend on Figure 6 As shown: After stimulation with 10 ng / ml IL-1β for 24 h and 48 h from multiple different donor umbilical cord MSCs, the CD82 positivity rate on the surface of umbilical cord MSCs was significantly increased compared with unstimulated (Ctrl) cells (*p<0.05). Figure 6 A), and at the same time, the PGE2 content in the culture supernatant was also significantly increased (**p<0.01, Figure 6 B). Correlation analysis was performed on the CD82 expression level and PGE2 content in the supernatant of umbilical cord MSCs from different donors under different treatment conditions. The results showed that regardless of whether stimulation was performed for 24 h or 48 h, the positive rate of CD82 expression in umbilical cord MSCs from different donors was significantly positively correlated with the PGE2 level in their supernatant, with correlation coefficients of 0.7688 (**p<0.01). Figure 6 C) and 0.8061 (**p<0.01, Figure 6(D) This suggests that the expression level of CD82 on the surface of MSCs can indicate the level of PGE2 secretion to a certain extent. PGE2 is widely known to be involved in MSC-mediated immunosuppression (The Role of COX-2 and PGE2 in the Regulation of Immunomodulation and Other Functions of Mesenchymal Stromal Cells. Biomedicines. 2023 Feb 3;11(2):445; Thesecretion profile of mesenchymal stem cells and potential applications intreating human diseases. Signal Transduct Target Ther. 2022 Mar 21;7(1):92), and is one of the core molecules for MSCs to exert their immunosuppressive function. The high correlation between CD82 expression level and PGE2 further proves that CD82 can serve as an important indicator of MSC immune activity.

[0064] Example 6: Comparison of the therapeutic effects of MSC cell lines expressing different levels of CD82 in a mouse model of acute respiratory distress syndrome (ARDS). Specific implementation method:

[0065] One CD82-positive MSC line from each of several different donor umbilical cord MSCs was selected by flow cytometry (as shown in Table 1). The selection criteria were: high-activity MSCs with a CD82 positivity rate >70% and low-activity MSCs with a CD82 positivity rate <40%.

[0066] Establishment of an ARDS mouse model: SPF-grade male BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were purchased. After anesthesia with isoflurane, the mice were fixed on a self-made suspension frame, and the skin in the midline of the neck was cut to expose the trachea. LPS was injected into the trachea using a 1 ml syringe to establish the model. The mice were then placed in a vertical position and rotated for 30 seconds to ensure that the LPS was evenly dispersed in the lungs. In the sham-operated group, only the trachea was exposed, and no LPS was injected.

[0067] ARDS mouse treatment: 4–6 hours after modeling, mice were randomly divided into 5 groups of 12 mice each: model group, treatment group 1 (low dose), treatment group 1 (high dose), treatment group 2 (low dose), and treatment group 2 (high dose). The low-dose group was given 0.5 × 10⁻⁶ cells. 6 / cell, high-dose group was given 1.0 × 10⁶ cells / cell. 6 / mouse. The model group was given an equal volume of cell solvent, while treatment groups 1 and 2 were treated with low-activity and high-activity MSC cell lines, respectively, all administered intravenously once. The day of model establishment and administration was designated as D0, and the experimental endpoint was defined as D10. Mice survival was observed and survival rate was calculated. Survival rate was assessed using the chi-square test for four-fold contingency data, with p < 0.05 considered statistically significant.

[0068] Table 1. CD82 expression of one high-activity and one low-activity MSC cell line selected from the screening.

[0069]

[0070] Depend on Figure 7 As shown, the mortality rates of mice in each group varied after LPS modeling, and the mortality rate tended to stabilize starting from day 4. Compared with the model group, treatment group 1 (low dose) showed a trend of improving the survival rate of ARDS mice (p>0.05), treatment group 1 (high dose) significantly improved the survival rate from day 4 to day 10 after drug administration (*p<0.05), treatment group 2 (low dose) significantly improved the survival rate from day 4 to day 10 after drug administration (*p<0.05), and treatment group 2 (high dose) significantly improved the survival rate from day 2 to day 10 after drug administration (*p<0.05). At the experimental endpoint, Day 10 after drug administration, 4 mice survived in the model group, with a survival rate of 33.3%; in treatment group 1, 8 mice survived at low dose (66.7%) and 9 mice survived at high dose (75%); in treatment group 2, 9 mice survived at low dose (75%) and 11 mice survived at high dose (91.7%). These results show that treatment group 1 was slightly less effective than treatment group 2, suggesting that highly active MSCs can significantly increase the survival rate of ARDS mice compared to low-activity MSCs, and have a better therapeutic effect.

[0071] Example 7: Comparison of the therapeutic effects of MSC cell lines expressing different levels of CD82 in a rat model of diabetic nephropathy (DKD). Specific implementation method:

[0072] Establishment of a DKD rat model: SPF-grade SD rats were purchased and DKD rats were established by a single intraperitoneal injection of streptozotocin STZ (Sigma-Aldrich, S0130 solution) at a concentration of 60 mg / kg. The solvent for preparing the STZ solution was sodium citrate buffer (0.1 M, pH 4.5). An equal volume of sodium citrate buffer was injected in the same manner to establish a control group of rats. Urine samples were collected from rats in metabolic cages after 24 hours, and urinary protein levels were measured using a urine protein quantification kit (CBB method) (Nanjing Jiancheng Bioengineering Institute, C035-2-1). A 24-hour urinary protein level greater than or equal to 30 mg was considered a successful DKD rat model.

[0073] Treatment of DKD rats: DKD model rats were randomly divided into three groups: a model group, treatment group 1 (low-activity MSCs), and treatment group 2 (high-activity MSCs), with 6–8 animals in each group. Treatment group rats were administered medication at a dose of 1 × 10⁻⁶ mg / L according to their body weight. 7 Cell therapy was administered via tail vein injection at a rate of cells / kg, once weekly for three consecutive weeks. Low- and high-activity MSCs were selected using cell lines screened in Example 6. The model group and control group were injected with equal volumes of cell solvent.

[0074] Detection indicators: Before and after drug administration, 24-hour urine samples were collected from rats using metabolic cages at fixed time points each week to determine the urinary protein content.

[0075] Depend on Figure 8 As shown, compared with the control group, the urinary protein level in rats with DKD was significantly increased after modeling (****p<0.0001). Compared with the model group, treatment group 1 (low-activity MSCs) significantly reduced urinary protein levels in DKD rats 3 weeks after treatment (*p<0.05); while treatment group 2 (high-activity MSCs) showed efficacy as early as 2 weeks after treatment, i.e., a significant reduction in urinary protein (*p<0.05), and a significant reduction in urinary protein levels 3 weeks after treatment (***p<0.001). These results also suggest that there is a significant difference in efficacy between high- and low-activity MSCs in treating DKD rats, with high-activity MSCs showing efficacy in the early stages of treatment.

[0076] During the development of biological efficacy methodologies for MSCs from multiple batches and multiple donors, the inventors incidentally discovered, through transcriptome sequencing, flow cytometry, immunomodulation, and correlation analysis, that the level of CD82 expression in cells was significantly positively correlated with its inhibitory effect on the secretion of inflammatory factors TNFα and IFN-γ by activated PBMCs. Simultaneously, the level of CD82 expression was significantly upregulated upon IL-1β stimulation, and similarly, the level of PGE2 secretion was also significantly upregulated, with a significant positive correlation between the two. This invention also employed siRNA interference to find that reducing CD82 expression significantly downregulated the immunomodulatory activity of MSCs. Furthermore, the inventors screened cells with high and low CD82 expression and conducted animal experiments, administering different cell therapies via tail vein injection to ARDS and DKD model mice, respectively, and found that highly active MSCs showed better therapeutic effects than low-active MSCs.

[0077] (1) By detecting multiple MSC samples from different donors by flow cytometry, it was found that the average expression level of CD82 on the surface of MSCs from different donors was about 50%, with a large range, making it easier to distinguish the differences in biological attributes of different MSC cell lines and to identify the heterogeneity level of MSCs themselves.

[0078] (2) Flow cytometry was used to detect the CD82 positivity rate and MFI, and the correlation between these two parameters and their inhibitory level on the secretion of inflammatory cytokines by activated PBMCs was analyzed. The results showed that both the CD82 positivity rate and MFI were significantly positively correlated with the inhibition rate of TNFα and IFNγ. When the CD82 positivity rate of MSCs was >54% and the MFI was >70, the immunosuppression level mediated by the MSCs could reach more than 50%. When the CD82 positivity rate on the surface of MSCs exceeded 70%, highly active MSC cell populations could be distinguished, while when the CD82 positivity rate was less than 40%, low-activity MSC cell populations could be distinguished.

[0079] (3) By interfering with the expression of CD82 in MSCs through transfection with siRNA, it was found that the level of inhibition of inflammatory cytokines secreted by PBMCs by transfected MSCs was significantly reduced.

[0080] (4) By stimulating MSCs with the addition of the cytokine IL-1β, it was found that the expression level of CD82 and the secretion level of PGE2 in MSCs were significantly upregulated, and the two were significantly positively correlated.

[0081] (5) Through animal experiments, MSCs expressing different levels of CD82 were administered via tail vein infusion to treat ARDS mice and DKD rats. The results showed that MSCs with high CD82 expression had better therapeutic effects than those with low expression.

[0082] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. A method for quantifying and differentiating MSC immune activity using CD82, characterized in that, Cells were collected, and the membrane protein CD82 on the surface of MSCs was labeled with a fluorescently labeled antibody. The positivity rate of CD82 and the molecular weight fraction (MFI) were detected by flow cytometry. The immunogenicity of MSCs was quantified by detecting the expression level of CD82 on the MSC surface. Under the premise that the positivity rate of CD82 on the MSC surface and MFI are positively correlated, when the positivity rate of CD82 on the MSC surface is >54% and the MFI is >70, the inhibitory ability of MSCs on the secretion of TNF-α and IFN-γ by activated PBMCs is greater than 50%. The immunogenicity of different MSC cell populations can be quantified by detecting the expression level of CD82 in MSCs from different donors by flow cytometry, and MSC cell populations with high immunogenicity can be distinguished. The immunogenicity refers to its ability to inhibit the secretion of inflammatory cytokines TNF-α and IFN-γ by activated PBMCs.

2. The method for quantifying and differentiating MSC immune activity using CD82 according to claim 1, characterized in that, The MSCs are derived from bone marrow, adipose tissue, perinatal tissue, or induced differentiation from iPSCs / ESCs. The perinatal tissues include the umbilical cord, placenta, amnion, or amniotic fluid.

3. The method for quantifying and differentiating MSC immune activity using CD82 according to claim 2, characterized in that, Specifically, the steps include the following: S1: Collecting cells to be tested: Digest and collect cultured MSCs, count the cells, and transfer them to flow cytometry tubes; S2: Labeled flow cytometry antibody: Centrifuge the cell suspension, remove the supernatant, add fluorescently labeled antibodies to the cell pellet, vortex to mix, and incubate at room temperature in the dark or in a refrigerator at 4°C. Add flow cytometry washing buffer to wash away unbound antibodies, centrifuge, add flow cytometry washing buffer to resuspend the cells, filter out cell clumps using a filter membrane, and wait for detection. S3: Flow cytometry detection: The positivity rate of CD82 on the surface of MSCs and MFI were detected by flow cytometry; S4: Quantitative analysis of the immune activity of MSC cell population; S5: Differentiate between highly active MSC cell populations: When the CD82 positivity rate on the surface of MSCs is higher than 70%, they are considered highly active MSC cell populations, while when the CD82 positivity rate is lower than 40%, they are considered low-activity MSC cell populations.

4. The application of the method for quantifying and distinguishing MSC immune activity using CD82 as described in any one of claims 1-3 in the formulation of quality testing standards for cell banks and cell injection solutions.

5. The application of the method for quantifying and distinguishing MSC immune activity using CD82 as described in any one of claims 1-3 in screening a seed bank of highly immune-active MSCs.

6. The application of the method for quantifying and distinguishing the immune activity of MSCs using CD82 as described in any one of claims 1-3 in the preparation of a highly immune-active cell injection for the treatment of inflammation and immune diseases.

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