Immunosuppressed mesenchymal stem cell subpopulation and application thereof

Abstract

The invention relates to an immunosuppressive mesenchymal stem cell subset and application thereof, and relates to the technical field of biological medicines. According to the immunosuppressed mesenchymal stem cell subgroup, a marker expressed by the mesenchymal stem cell subgroup comprises a CD63 negative marker or a CD63 negative marker and a CD317 positive marker. The mesenchymal stem cell subgroup (CD63-MSC or CD317 +, CD63-MSC) provided by the invention has a relatively strong immunosuppression function, and has a treatment effect of improving autoimmune diseases and / or inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an immunosuppressive mesenchymal stem cell subpopulation and application thereof. Background Art

[0002] Mesenchymal stem cells (MSC) are adult stem cells with multiple differentiation and self-renewal abilities. Due to their low immunogenicity and immunoregulatory abilities, they are widely used in regenerative medicine and disease treatment related research. However, the effect and stability of MSC treatment need to be improved, so the present invention develops an immunosuppressive mesenchymal stem cell subpopulation and its application. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide an immunosuppressive mesenchymal stem cell subpopulation and application thereof.

[0004] The technical solution of the present invention to solve the above technical problems is as follows:

[0005] In a first aspect, an immunosuppressive mesenchymal stem cell subpopulation is provided, wherein the markers expressed by the mesenchymal stem cell subpopulation include CD63 negative, or CD63 negative and CD317 positive.

[0006] In a second aspect, a method for preparing an immunosuppressive mesenchymal stem cell subpopulation is provided, characterized in that it comprises the following steps: using anti-CD63-FITC antibody or IgG-FITC antibody to sort mesenchymal stem cells to obtain a CD63-negative mesenchymal stem cell subpopulation.

[0007] Furthermore, the mesenchymal stem cells are any one of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and dental pulp mesenchymal stem cells.

[0008] Furthermore, the sorting is specifically carried out using a cell sorter.

[0009] In a third aspect, an immunosuppressive mesenchymal stem cell subpopulation is used to prepare a drug for negative immune regulation.

[0010] Furthermore, the immunosuppressive mesenchymal stem cell subset is used to prepare a drug for treating autoimmune diseases and / or inflammatory diseases.

[0011] Furthermore, the autoimmune disease includes at least one of SLE, RA or immune rejection caused by cell transplantation or organ transplantation, and the inflammatory disease includes inflammatory bowel disease or pneumonia.

[0012] In a fourth aspect, a negative immune regulatory drug comprises the immunosuppressive mesenchymal stem cell subset.

[0013] Furthermore, the drug dosage form is an injection or an oral targeted drug.

[0014] Furthermore, the medicine also includes a pharmaceutically acceptable carrier and / or excipient.

[0015] The present invention has carried out the following verification experiment:

[0016] (1) The present invention utilizes the obtained mesenchymal stem cell subpopulation CD63 - MSCs were used to treat acute inflammation in mice, and the clinical symptoms of the mice were improved after treatment. The treatment effect was better than that of CD63 + MSC subpopulations were evident.

[0017] (2) The present invention converts CD317 + 、CD63 - MSCs were co-cultured with PBMCs to detect their inhibitory effect on PBMC proliferation, which was used to indicate the immunosuppressive function of MSCs. + 、CD63 - MSCs have strong immunosuppressive properties.

[0018] (3) The present invention used CD317+CD63-MSC to treat an acute pneumonia mouse model and found that the serum inflammatory factor concentrations were negatively correlated, indicating that it can improve the therapeutic effect of inflammatory diseases.

[0019] Beneficial effects of the present invention:

[0020] (1) The markers expressed by the mesenchymal stem cell subpopulations of the present invention include CD63 negative, or CD63 negative and CD317 positive, and CD63 and CD317 are used as surface marker molecules for flow cytometry sorting of mesenchymal stem cell subpopulations.

[0021] (2) The mesenchymal stem cell subsets of the present invention (CD63 - MSC or CD317 + 、CD63 - MSCs) have strong immunosuppressive function and have therapeutic effects on improving autoimmune diseases and / or inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the results of single-cell RNA-seq sequencing and nonlinear dimensionality reduction analysis using UMAP;

[0023] Figure 2 CD63 -Proliferation analysis of MSC subsets co-cultured with PBMC and analysis of therapeutic effects in acute pneumonia mouse model; A is the expression of CD63 under the condition of 20ng / ml IFN-γ stimulation for 48 hours + MSCs or CD63 - Analysis of PBMC proliferation after MSCs co-culture (n=3); B is the detection of CD45 in the lung by flow cytometry 7 days after LPS lipopolysaccharide induced acute pneumonia mouse model + cells (n=8); C is the number of neutrophils (CD45 + CD11b + Ly-6G + Ly-6C med )(n=8); D is the MPO activity measured 24 hours after LPS lipopolysaccharide induced acute pneumonia mouse model (n=8);

[0024] Figure 3 CD317 of the present invention + 、CD63 - The inhibitory effect of MSCs on PBMC proliferation was detected after co-incubation with MSCs.

[0025] Figure 4 CD317 of the present invention + 、CD63 - MSCs transplantation was used to treat LPS-induced acute pneumonia mouse model to evaluate the therapeutic effect of MSCs. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0027] Example

[0028] 1. Experimental methods

[0029] (1) Isolation, expansion and identification of human mesenchymal stem cells

[0030] Human umbilical cord tissue was minced and digested with 1 mg / mL collagenase B (STEMCELL Technologies), cultured with a fully synthetic culture system (NBVbe, disclosed in the literature Xu J, Lian W, Chen J, Li W, Li L, Huang Z. Chemical-defined medium supporting the expansion of human mesenchymal stem cells. StemCell Res Ther. 2020 Mar 19; 11 (1): 125. doi: 10.1186 / s13287-020-01641-7. PMID: 32192530; PMCID: PMC7083066.), and cultured with TrypLE (Thermo The MSCs were passaged by using 20 ng / ml of IFN-γ. For details, please refer to the patent application number CN117660320A applied by the applicant, and the patent name is a mesenchymal stem cell CD317_subpopulation, its preparation and its application.

[0031] (2) Single-cell RNA-seq sequencing and analysis

[0032] The single-cell RNA sequencing (scRNA-seq) was performed using human umbilical cord MSCs. Specifically, MSCs were isolated using TrypLE and resuspended in HBSS (Hank's Balanced Salt Solution) containing 0.04% BSA (1×10 6 Cells / mL). The library was constructed using the 10×Genomics Chromium platform, and sequencing was performed using the Illumina NovaSeq 6000 system (paired-end sequencing mode). Data were processed using the 10xGenomics pipeline Cell Ranger (v2.1.0) and analyzed using the Seurat package in R (v4.0.0).

[0033] (3)CD63 + / CD63 - Screening of MSCs

[0034] MSCs were derived from human umbilical cord. MSCs were digested with TrypLE and labeled with CD63-FITC antibody (purchased from Thermo Fisher Scientific) or IgG-FITC (Thermo Fisher Scientific). + and CD63 - MSCs were sorted using BD FACSAria SORP (BD Biosciences) cell sorter.

[0035] (4) MSC-PBMC co-culture

[0036] Human peripheral blood mononuclear cells (PBMCs) were isolated by EasySep TM Direct Human PBMC Isolation Kit (STEMCELL167 Technologies) was used to extract the PBMCs were stimulated with Human T-Activator CD3 / CD28 (Thermo Fisher Scientific) for 24 hours and then incubated with selected CD63 + MSCs or CD63 - MSCs (20×10 4 PBMCs vs 5×10 4 The cell proliferation was evaluated by Cell Proliferation KitI (Roche) and quantified by an automatic microplate reader (Bio-Rad) at a wavelength of 570 nm.

[0037] (5) Mouse acute inflammation model and cell transplantation

[0038] Mice (C57BL / 6J, female, 8 weeks old) were purchased from Guangdong Medical Experimental Animal Center and raised under sterile conditions. This experiment used lipopolysaccharide (LPS, Sigma) to induce an acute inflammatory model in mice. LPS was injected into the mouse intraperitoneally (20 mg / kg), and after waiting for ten minutes, PBS (PBS refers to phosphate buffered saline at pH 6.8, and its preparation method is: take 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution, add 118 mL of 0.2 mol / L sodium hydroxide solution, dilute with water to 1000 mL, shake well, and obtain CD63 + MSCs (1x10 6 cells / mouse) and CD63 - MSCs (1x10 6 Cells / mouse) were transplanted into the peritoneal cavity of the mouse model, n=8.

[0039] CD45 + Lymphocytes and neutrophils (CD45 + CD11b + Ly-6G + Ly-6C med ) was determined by flow cytometry. The activity of myeloperoxidase (MPO) was determined using MPO Activity Assay Kit (Abcam).

[0040] 2. Experimental results

[0041] The experimental results are as follows Figures 1 to 4 shown.

[0042] Figure 1 Single-cell RNA-seq sequencing and UMAP nonlinear dimensionality reduction analysis results show that CD63 has differential expression distribution.

[0043] Figure 2 CD63 of the present invention - Proliferation analysis of MSC subsets co-cultured with PBMC and analysis of therapeutic effects in acute pneumonia mouse model; A is the expression of CD63 under the condition of 20ng / ml IFN-γ stimulation for 48 hours + MSCs or CD63 - Analysis of PBMC proliferation after MSCs co-culture (n=3); B is the detection of CD45 in the lung by flow cytometry 7 days after LPS lipopolysaccharide induced acute pneumonia mouse model + cells (n=8); C is the number of neutrophils (CD45 + CD11b + Ly-6G + Ly-6C med )(n=8); D is the MPO activity measured in the LPS lipopolysaccharide induced acute pneumonia mouse model 24 hours later (n=8); the results showed that CD63 - MSC subsets have strong immunosuppressive function.

[0044] After MSCs were co-cultured with PBMCs, their inhibitory effect on PBMC proliferation was detected to indicate the immunosuppressive function of MSCs. Figure 3 CD317 of the present invention + 、CD63 - After MSCs and PBMC cells were co-cultured, the inhibitory effect on PBMC proliferation was detected; it can be seen that CD317 + CD63 - The proportion of MSCs in MSCs is positively correlated with the immunosuppressive function of MSCs.

[0045] MSC transplantation was used to treat LPS-induced acute pneumonia mouse model to evaluate the therapeutic effect of MSC. Figure 4 For CD317 + 、CD63 - The proportion of MSCs in MSCs was negatively correlated with the concentration of serum inflammatory factors in the acute pneumonia mouse model.

[0046] In summary, the mesenchymal stem cell subsets (CD63 - MSC or CD317 + 、CD63 - MSCs) have strong immunosuppressive function and have therapeutic effects on improving autoimmune diseases and / or inflammatory diseases.

[0047] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An immunosuppressive mesenchymal stem cell subset, characterized in that: The markers expressed by the mesenchymal stem cell subpopulation include CD63 negative, or CD63 negative and CD317 positive.

2. A method for preparing an immunosuppressive mesenchymal stem cell subset according to claim 1, characterized in that: The method comprises the following steps: using anti-CD63-FITC antibody or IgG-FITC antibody to sort mesenchymal stem cells to obtain CD63 negative mesenchymal stem cell subpopulation.

3. The method for preparing an immunosuppressive mesenchymal stem cell subpopulation according to claim 2, characterized in that: The mesenchymal stem cells are any one of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and dental pulp mesenchymal stem cells.

4. The method for preparing an immunosuppressive mesenchymal stem cell subpopulation according to claim 2, characterized in that: The sorting is specifically carried out using a cell sorter.

5. A use of an immunosuppressive mesenchymal stem cell subset, characterized in that: The immunosuppressive mesenchymal stem cell subset described in claim 1 is used to prepare immune negative regulation drugs.

6. The use of an immunosuppressive mesenchymal stem cell subset according to claim 5, characterized in that: The immunosuppressive mesenchymal stem cell subpopulation is used for preparing medicines for treating autoimmune diseases and / or inflammatory diseases.

7. The use of an immunosuppressive mesenchymal stem cell subset according to claim 6, characterized in that: The autoimmune disease includes at least one of SLE, RA, or immune rejection caused by cell transplantation or organ transplantation, and the inflammatory disease includes inflammatory bowel disease or pneumonia.

8. A drug for negative immune regulation, characterized in that: Comprising the immunosuppressive mesenchymal stem cell subset as described in claim 1.

9. The immune negative regulation drug according to claim 8, characterized in that: The drug dosage form is an injection or an oral targeted drug.

10. The immune negative regulation drug according to claim 8, characterized in that: The medicine also includes a pharmaceutically acceptable carrier and / or excipient.

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