Optimization method for in-vitro amplification culture of mesenchymal stem cells

Abstract

The invention discloses an optimization method for in-vitro amplification culture of mesenchymal stem cells, and belongs to the technical field of stem cell culture. The optimization method comprises the step of adding a substance for enhancing TLR3 gene expression in the in-vitro amplification culture process of the mesenchymal stem cells so as to improve the immunosuppression effect of the in-vitro cultured mesenchymal stem cells. In-vitro experiments show that through intervention of a TLR3 activator PIPC, the immunosuppression function of the P15MSC is successfully recovered, the immunosuppression effect of the P5MSC is further enhanced, an effective optimization strategy is provided for amplification and culture of the MSC before clinical application, the immunosuppression effect of the MSC is favorably maintained, the effectiveness of the MSC in treatment of GvHD is improved, and the immunosuppression effect of the P15MSC is improved. And a new solution is provided for treatment of immune diseases such as GvHD.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell culture, and in particular to an optimization method for in vitro expansion and culture of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSC) are a type of adult stem cell with self-renewal ability and multidirectional differentiation potential. They are widely found in various tissues, such as bone marrow, fat, umbilical cord, etc. MSC are clinically used to treat a variety of diseases, especially in immune diseases. In recent years, the application of MSC in the treatment of graft-versus-host disease (GvHD) has received widespread attention, especially in terms of immunomodulation and therapeutic effects of GvHD, MSC has shown good clinical prospects. MSC reduces T cell-mediated immune attacks by directly contacting T cells, secreting immunosuppressive factors (such as TGF-β, IL-10, etc.), and regulating the differentiation state of immune cells to inhibit the activation and proliferation of T cells. In addition, MSC can also promote the proliferation of regulatory T cells (Treg), further enhancing immune tolerance.

[0003] Although MSCs have demonstrated their immunosuppressive potential in multiple clinical studies, current research results indicate that there are certain individual differences in the immunosuppressive effects of MSCs, and in some cases, the immunosuppressive effects are not long-lasting. In particular, in the treatment of GvHD, the therapeutic effects of MSCs may weaken over time, leading to recurrence of GvHD symptoms. This instability of the effect may be closely related to the source of MSCs, culture conditions, and processing methods used in clinical applications. In clinical applications, MSCs need to be expanded in vitro to reach sufficient numbers. However, long-term in vitro expansion may lead to loss of MSC function or decreased differentiation potential, thereby affecting their immunosuppressive function.

[0004] In order to avoid the problem of functional loss and decreased differentiation potential during the in vitro expansion and culture of MSCs, currently commonly used measures include hypoxic culture (2-5% O2), reduced use of animal serum (animal serum-free culture volume), 3D culture and epigenetic regulation (such as HDAC inhibitors), etc. However, these methods have significant limitations: low-oxygen equipment is expensive; serum-free culture medium is expensive and affects biological function; small molecule regulators have genomic instability and the risk of inducing abnormal differentiation; and 3D culture operations are complex and difficult to standardize. In addition, although limiting the number of passages (usually ≤P5) can delay aging, it is difficult to meet large-scale clinical needs. Existing technologies generally face core problems such as high cost, difficulty in standardization, safety risks and low expansion efficiency. Therefore, there is an urgent need to develop a new method for in vitro expansion of MSCs that is economical, efficient, stable and safe, so as to achieve large-scale culture while maintaining the characteristics of stem cells, in order to promote their clinical application. Summary of the Invention

[0005] The present invention aims to provide an optimized method for the in vitro expansion and culture of mesenchymal stem cells to address the aforementioned problems with the prior art. In vitro experiments in the present invention demonstrated that intervention with the TLR3 activator PIPC successfully restored the immunosuppressive function of P15MSCs and further enhanced the immunosuppressive effect of P5MSCs, providing a novel solution for the future treatment of immune diseases such as GvHD.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an optimization method for in vitro expansion and culture of mesenchymal stem cells, comprising the steps of adding a substance that enhances TLR3 gene expression during the in vitro expansion and culture of the mesenchymal stem cells to improve the immunosuppressive effect of the in vitro cultured mesenchymal stem cells.

[0008] Optionally, the improving the immunosuppressive effect of mesenchymal stem cells cultured in vitro includes improving the immunosuppressive effect of mesenchymal stem cells obtained by early in vitro expansion and mesenchymal stem cells obtained by long-term in vitro expansion.

[0009] Optionally, the mesenchymal stem cells obtained by early in vitro expansion are mesenchymal stem cells that have been subcultured for less than 5 generations; and the mesenchymal stem cells obtained by long-term in vitro expansion are mesenchymal stem cells that have been subcultured for less than 5 generations.

[0010] Optionally, the substance that enhances TLR3 gene expression includes AMP-516, Rintatolimod, Ampligen, Hiltonol, BO-112 and PIPC.

[0011] Optionally, the substance that enhances TLR3 gene expression is PIPC.

[0012] Optionally, the concentration of PIPC is 1 mg / mL (final concentration is 10 μg / mL), and the action time is 24 hours.

[0013] The present invention also provides mesenchymal stem cells obtained by the optimization method.

[0014] The present invention also provides use of a substance that enhances TLR3 gene expression in preparing a MSC preparation for treating graft-versus-host disease. The substance that enhances TLR3 gene expression is used to enhance the immunosuppressive effect of mesenchymal stem cells in the MSC preparation.

[0015] Optionally, the substance that enhances TLR3 gene expression includes AMP-516, Rintatolimod, Ampligen, Hiltonol, BO-112 and PIPC.

[0016] Optionally, the substance that enhances TLR3 gene expression is PIPC.

[0017] The present invention discloses the following technical effects:

[0018] This study systematically analyzed changes in the immunosuppressive function of umbilical cord mesenchymal stem cells (MSCs) at different passages, combined with single-cell transcriptome sequencing and functional validation experiments, to propose an optimized strategy for in vitro expansion of MSCs using a TLR3 agonist to enhance their immunomodulatory capacity. In vitro experiments demonstrated that intervention with the TLR3 activator PIPC successfully restored the immunosuppressive function of P15 MSCs and further enhanced the immunosuppressive effect of P5 MSCs.

[0019] The method of the present invention provides an effective optimization strategy for the expansion and culture of MSCs prior to clinical application, helping to maintain their immunosuppressive effects and enhance the effectiveness of MSCs in treating GvHD. Furthermore, through in-depth gene and pathway analysis, the present invention reveals the key role of TLR3 in regulating the immunosuppressive function of MSCs, providing a potential target for future immunotherapy and offering a new solution for the treatment of immune diseases such as GvHD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1Cell type identification results of 52,153 cells, where 1 represents the Biofunctional MSC subpopulation, 2 represents the Active proliferation MSC subpopulation, and 3 represents the Multipotent progenitor MSC subpopulation;

[0022] Figure 2 represents the composition ratio of P5 and P15 MSC cell subsets, where 1 represents the Biofunctional MSC subset, 2 represents the Active proliferation MSC subset, and 3 represents the Multipotent progenitor MSC subset;

[0023] Figure 3 GO pathway enrichment analysis (A) and KEGG enrichment analysis (B) of differentially expressed genes between P15 and P5;

[0024] Figure 4 is the box plot of TLR3 expression levels of P5 and P15 MSCs;

[0025] Figure 5 Figure 2 Changes in TLR3 expression level (A), Jurkat cell number (B), apoptosis level (C), and IL-10 mRNA level (D) in Jurkat cells between the control group and the experimental group. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] This study systematically analyzed the changes in the immunosuppressive function of umbilical cord mesenchymal stem cells (MSCs) at different generations, combined with single-cell transcriptome sequencing and functional validation experiments, to propose an in vitro expansion optimization strategy for MSCs based on TLR3 agonists to enhance their immunoregulatory capacity, providing a new solution for the clinical treatment of immune diseases such as graft-versus-host disease (GvHD).

[0032] In the present invention, UMAP cluster analysis classified MSCs into three subpopulations: biofunctional MSCs (strong immunomodulatory function), active proliferation MSCs (high proliferation), and multipotent progenitor MSCs (maintaining stemness). The proportion of biofunctional MSCs decreased significantly in P15 MSCs, suggesting that long-term culture leads to a decline in immunosuppressive function.

[0033] In the present invention, GO / KEGG enrichment analysis revealed that differentially expressed genes were significantly enriched in the Toll-like receptor pathway (such as TLR3). TLR3 expression was reduced in P15 MSCs, which was associated with a weakened immunosuppressive function.

[0034] In the present invention, in vitro experiments confirmed that TLR3 agonist treatment can significantly enhance the inhibitory effect of P15 MSC on Jurkat cells (increased apoptosis rate and increased IL-10 secretion), and further enhance the immunosuppressive effect of P5 MSC.

[0035] Optionally, the TLR3 agonist includes poly(I:C) (polyinosinic-cytidylic acid), AMP-516, Rintatolimod, Ampligen, Hiltonol, BO-112 and PIPC.

[0036] In a specific embodiment of the present invention, the TLR3 agonist is PIPC.

[0037] Optionally, the concentration of PIPC is 1 mg / mL (final concentration is 10 μg / mL), and the treatment time is 24 h.

[0038] Example 1

[0039] 1. Acquisition and culture of MSC samples

[0040] First, umbilical cord MSCs were obtained from healthy clinical donors and primary cells were obtained using collagenase digestion. MSCs were then expanded in vitro using DMEM / DF-12 medium containing 10% fetal bovine serum (FBS). Culture conditions were controlled at 37°C, pH 7.2-7.4, and 5% CO2. Digestion and passage were performed when the cell confluence reached 80%-90%. The cells were cultured to passage 5 (P5, representing early stage) and passage 15 (P15, representing long-term expansion) for subsequent experiments.

[0041] 2. Single-cell transcriptome sequencing and subpopulation classification

[0042] P5 and P15 MSCs were isolated from single cells, and single cell suspensions were prepared. High-throughput sequencing was performed using single-cell transcriptome technology. After data quality control, low-quality cells were eliminated, and 52,153 high-quality cell samples were retained. The UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction analysis method was used to cluster single cells and divide the cells into three subpopulations: Biofunctional MSC, Active proliferation MSC, and Multipotent progenitor MSC. The cell type identification results of the 52,153 cells finally retained and the proportion of different cell subpopulations are shown below. Figure 1 shown.

[0043] 3. Differential analysis and functional enrichment

[0044] The composition ratio of P5 and P15 MSC cell subsets is as follows Figure 2 As shown in Figure 2, the differences in the Biofunctional MSC population between P5 and P15 generations of MSC were compared, and it was found that the proportion of this population decreased significantly in P15 generation. To further analyze the molecular mechanism behind this, the present invention extracted the differentially expressed genes in the Biofunctional MSC population and performed enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The results are shown in Figure 2. Figure 3 As shown, Figure 3It showed that multiple immune-related pathways were enriched, among which the Toll-like receptor pathway was significantly enriched. Further screening of key genes in this pathway revealed that Toll-like receptor 3 (TLR3) was expressed significantly differently in P15 and P5 MSCs ( Figure 4 ).

[0045] According to the results of differential analysis, the proportion of Biofunctional MSCs in the P15 generation was significantly reduced, and the expression of TLR3 was significantly downregulated, suggesting that TLR3 may play a key role in the immunosuppressive function of MSCs.

[0046] 4. TLR3 activation experiment

[0047] To verify the role of TLR3 in immunosuppressive function, the present invention designed an experiment to activate TLR3. In the in vitro experiment, the TLR3 agonist PIPC was used to stimulate P5 and P15 MSCs, and its effect on immunosuppressive function was detected.

[0048] Activated Jurkat cells (human T lymphocyte line) were selected as the test immune cell line to observe their immunosuppressive effect under MSC treatment.

[0049] (1) MSCs of different passages (P5 MSCs and P15 MSCs) were seeded into 6-well plates (density 7×10 4 / mL) and were randomly divided into a control group and an experimental group. The experimental group was stimulated with the TLR3-specific agonist PIPC (10μg / mL), while the control group was not treated and cultured alone for 24 hours. At the same time, Jurkat cells were activated with 10ng / mL phorbol myristate (PMA) and 1ug / mL phytohemagglutinin (PHA) and cultured for 24 hours. After 24 hours, Jurkat cells were inoculated at a ratio of MSC:Jurkat cells = 1:10, and MSCs and Jurkat cells were co-cultured in 3mL normal DMEM / F-12 for 24 hours;

[0050] (2) The absolute number and apoptosis rate of Jurkat cells were analyzed by flow cytometry, and the IL-10 level in the supernatant was detected by qPCR. Figure 5 shown.

[0051] Depend on Figure 5It can be seen that compared with the control group, the expression level of TLR3 in P5 and P15 MSCs was significantly upregulated after treatment with 1 mg / mL PIPC, indicating that TLR3 was activated. The number of Jurkat cells in P5 and P15 MSCs treated with PIPC was significantly decreased, and the apoptosis rate was significantly increased. In addition, the secretion level of the immunosuppressive factor (IL-10) was also significantly increased, indicating that TLR3 agonists can restore the immunosuppressive effect of P15 MSCs and further enhance the immunosuppressive function of P5 MSCs.

[0052] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for optimizing the in vitro expansion and culture of mesenchymal stem cells, characterized in that: The method comprises the steps of adding a substance that enhances TLR3 gene expression during the in vitro expansion and culture process of the mesenchymal stem cells to improve the immunosuppressive effect of the in vitro cultured mesenchymal stem cells.

2. The optimization method according to claim 1, characterized in that The method of improving the immunosuppressive effect of mesenchymal stem cells cultured in vitro includes improving the immunosuppressive effect of mesenchymal stem cells obtained by early in vitro expansion and mesenchymal stem cells obtained by long-term in vitro expansion.

3. The optimization method according to claim 2, characterized in that The mesenchymal stem cells obtained by early in vitro expansion are mesenchymal stem cells that have been subcultured for less than 5 generations; the mesenchymal stem cells obtained by long-term in vitro expansion are mesenchymal stem cells that have been subcultured for less than 5 generations.

4. The optimization method according to claim 1, characterized in that The substances that enhance TLR3 gene expression include AMP-516, Rintatolimod, Ampligen, Hiltonol, BO-112 and PIPC.

5. The optimization method according to claim 4, characterized in that: The substance that enhances TLR3 gene expression is PIPC.

6. The optimization method according to claim 5, characterized in that: The concentration of PIPC is 1 mg / mL, and the action time is 24 hours.

7. Mesenchymal stem cells obtained by the optimization method according to any one of claims 1 to 6.

8. Use of a substance that enhances TLR3 gene expression in the preparation of a MSC preparation for treating graft-versus-host disease, characterized in that: The substance that enhances TLR3 gene expression is used to enhance the immunosuppressive effect of mesenchymal stem cells in the MSC preparation.

9. The use according to claim 8, characterized in that The substances that enhance TLR3 gene expression include AMP-516, Rintatolimod, Ampligen, Hiltonol, BO-112 and PIPC.

10. The use according to claim 9, characterized in that The substance that enhances TLR3 gene expression is PIPC.

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