Mesenchymal stem cell and application thereof

Abstract

The invention discloses a mesenchymal stem cell and application thereof. The MLPH + LPXN + MSCs disclosed by the invention can be used as a cell medicine for clinically treating GVHD and simultaneously promoting regeneration and reconstruction of hematopoietic stem cells after transplantation, can be preferentially applied to clinical treatment of graft hematopoietic malfunction, rejection of GVHD and the like, and provides a theoretical basis for targeted treatment of clinical diseases and selection of proper MSCs products. MLPH < + > and LPXN < + > are used as biomarkers of MSCs products in different culture stages, are used for treating different indications and diseases, provide reference for quality control standards and standardized production of MSCs cell drug preparation, and have important transformation application prospects in the aspect of precision medical treatment.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to mesenchymal stem cells and applications thereof. Background Art

[0002] Mesenchymal Stem Cells (MSCs) are cells with high differentiation potential and self-renewal ability. They have low immunogenicity, are relatively easy to obtain, and have strong proliferation ability. They are currently used to treat a variety of blood system diseases, nervous system diseases, cardiovascular diseases, autoimmune system diseases, etc. Based on their immunomodulation and tissue regeneration and repair capabilities, they have broad application prospects in the medical and health care fields and can bring huge health benefits. As an advanced health care method, it improves the quality of life from the root.

[0003] However, in clinical use, the number of mesenchymal stem cells, such as umbilical cord-derived mesenchymal stem cells (UC-MSCs), is often required to be very high, which requires the isolated MSCs to be continuously subcultured in vitro and a large number of large-scale expansions. However, the functional characteristics of MSCs after in vitro expansion will change, such as replicative senescence, reduced differentiation potential, etc., and MSCs and cells after subculture are a group of highly heterogeneous cell populations with great differences in their functions, which will directly lead to large differences in the therapeutic effects after clinical infusion. Therefore, how to prepare MSCs drugs with clear clinical therapeutic effects is the current technical bottleneck of MSCs products and a problem that needs to be solved urgently. Summary of the invention

[0004] Technical issues solved:

[0005] In view of the problems existing in the prior art: (1) there is a lack of MSCs drugs with clear clinical therapeutic effects; (2) the functional characteristics of MSCs will change after continuous in vitro subculture and large-scale expansion; (3) there is a lack of MSCs populations with functions such as hematopoietic support. One aspect of the present disclosure is to provide a new mesenchymal stem cell subset and its new applications in various aspects.

[0006] Specifically, the inventors performed single-cell transcriptome sequencing on UC-MSCs of different generations after in vitro expansion and culture, divided the heterogeneous populations of UC-MSCs of different generations into different subpopulations, analyzed the UC-MSCs subpopulations with both immunosuppressive and hematopoietic support effects, and screened out two effective molecular markers MLPH that can be used to enrich this subpopulation. + and LPXN + MLPH was obtained by flow cytometry sorting + LPXN +Double-positive MSCs and preparation of MLPH in vitro + LPXN + MSCs cell line. Using continuous infusion therapy, it was found that MLPH + LPXN + MSCs can be used to treat GVHD, while MSCs with negative expression of MLPH and LPXN are ineffective in treating GVHD. + LPXN + MSCs significantly support the proliferation of hematopoietic cells in vitro and significantly promote the implantation and hematopoietic reconstruction of hematopoietic stem / progenitor cells in vivo. The above creative invention solves the problems existing in the above-mentioned prior art.

[0007] Technical solution:

[0008] Use of mesenchymal stem cells in preparing products with hematopoietic support function, wherein the marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

[0009] Based on the research in the present disclosure, another aspect of the present disclosure is to provide a method for preparing mesenchymal stem cells to promote CD34 + Hematopoietic stem / progenitor cells and / or CD133 + Use of a product for the expansion of hematopoietic stem / progenitor cells, wherein the marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

[0010] In some embodiments of the present disclosure, the above-mentioned mesenchymal stem cells can promote CD34 + Hematopoietic stem / progenitor cells and / or CD133 + Expansion of hematopoietic stem / progenitor cells.

[0011] Based on the research in the present disclosure, another aspect of the present disclosure is to provide the use of mesenchymal stem cells in the preparation of a product that promotes hematopoietic reconstruction of human hematopoietic stem cells in vivo, wherein the marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

[0012] Based on the research in the present disclosure, another aspect of the present disclosure is to provide the use of mesenchymal stem cells in the preparation of a product that promotes the differentiation of human hematopoietic stem cells into megakaryocytes, wherein the marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

[0013] In some embodiments of the present disclosure, the above-mentioned mesenchymal stem cells can promote the differentiation of human hematopoietic stem cells into megakaryocytes in an in vitro or in vivo environment.

[0014] Based on the research in the present disclosure, another aspect of the present disclosure is to provide the use of mesenchymal stem cells in preparing a product with immunosuppressive function, wherein the marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

[0015] Based on the research in the present disclosure, another aspect of the present disclosure is to provide the use of mesenchymal stem cells in preparing a product for treating or assisting in treating graft-versus-host disease, wherein the marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

[0016] In some embodiments of the present disclosure, the above-mentioned graft-versus-host disease may be acute graft-versus-host disease (aGVHD) or chronic graft-versus-host disease (cGVHD).

[0017] Based on the research in the present disclosure, another aspect of the present disclosure is to provide the use of mesenchymal stem cells in preparing a product for treating or assisting in treating poor graft function (PGF), wherein the marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

[0018] In some embodiments of the present disclosure, the above-mentioned mesenchymal stem cells may come from, including but not limited to, umbilical cord blood, amniotic fluid, placenta, bone marrow, fat, synovium, bone, muscle, lung, liver or pancreas. They may be isolated from the above sources, or obtained by induced differentiation of ES cells or iPS cells using methods known in the prior art. They may be autologous cells or allogeneic cells (e.g., commercialized allogeneic mesenchymal stem cells).

[0019] Preferably, in some embodiments of the present disclosure, the above-mentioned mesenchymal stem cells are mesenchymal stem cells derived from human umbilical cord blood.

[0020] Another aspect of the present disclosure is to provide a pharmaceutical composition, wherein the pharmaceutical composition includes a marker molecule carried by MLPH + and LPXN + mesenchymal stem cells, and a pharmaceutically acceptable carrier.

[0021] In some embodiments of the present disclosure, the mesenchymal stem cells are mesenchymal stem cells derived from human umbilical cord blood.

[0022] In some embodiments of the present disclosure, the above-mentioned pharmaceutical composition may also include CD34 + of cells and / or accessory components.

[0023] In some embodiments of the present disclosure, the auxiliary components are one or more selected from cytokines, nutritional support substances, antioxidants, immunomodulators, anticoagulants or anti-inflammatory drugs.

[0024] In some embodiments of the present disclosure, the mesenchymal stem cells may be daughter cells after subculture. In other embodiments of the present disclosure, the daughter cells may be first generation cells, second generation cells, third generation cells, fourth generation cells, fifth generation cells, sixth generation cells, seventh generation cells, eighth generation cells, ninth generation cells, tenth generation cells, eleventh generation cells or twelfth generation cells. Preferably, in other embodiments of the present disclosure, the daughter cells may be sixth to ninth generation cells.

[0025] Another aspect of the present disclosure is to provide an application of the above-mentioned pharmaceutical composition, wherein the application is selected from:

[0026] (1) Preparation of products with hematopoietic support function; or

[0027] (2) Preparation of CD34-promoting + Hematopoietic stem / progenitor cells and / or CD133 + Products of expansion of hematopoietic stem / progenitor cells; or

[0028] (3) Preparation of products that promote hematopoietic reconstruction of human hematopoietic stem cells in vivo; or

[0029] (4) Preparation of products that promote the differentiation of human hematopoietic stem cells into megakaryocytes; or

[0030] (5) Preparation of products with immunosuppressive function; or

[0031] (6) Preparation of products for the treatment or adjunctive treatment of graft-versus-host disease; or

[0032] (7) Preparation of products for the treatment or auxiliary treatment of transplant hematopoietic dysfunction.

[0033] Another aspect of the present disclosure is to provide a method for preparing the following product:

[0034] (1) Products with hematopoietic support function; or

[0035] (2) Promote CD34 + Hematopoietic stem / progenitor cells and / or CD133 + Products of hematopoietic stem cell expansion; or

[0036] (3) Products that promote hematopoietic reconstruction of human hematopoietic stem cells in vivo; or

[0037] (4) Products that promote the differentiation of human hematopoietic stem cells into megakaryocytes; or

[0038] (5) Products with immunosuppressive function; or

[0039] (6) Products for the treatment or adjunctive treatment of graft-versus-host disease; or

[0040] (7) A product for treating or assisting in treating poor hematopoietic function of transplant recipients, wherein the method comprises obtaining a marker molecule carrying MLPH + and LPXN + Steps for mesenchymal stem cells.

[0041] In some embodiments of the present disclosure, the mesenchymal stem cells are mesenchymal stem cells derived from human umbilical cord blood.

[0042] Another aspect of the present disclosure is to provide a method for treating or assisting in treating graft-versus-host disease, which comprises administering an effective dose of the above product or the above pharmaceutical composition to a patient.

[0043] Another aspect of the present disclosure is to provide a method for treating or assisting in treating transplant hematopoietic dysfunction, which comprises administering an effective dose of the above product or the above pharmaceutical composition to a patient.

[0044] Beneficial effects:

[0045] MLPH disclosed in the present invention + LPXN + MSCs can be used as a cell drug for the clinical treatment of GVHD and the promotion of regeneration and reconstruction after hematopoietic stem cell transplantation. It can be preferentially used in the clinical treatment of transplant hematopoietic dysfunction and rejection such as GVHD, providing a theoretical basis for the targeted treatment of clinical diseases and the selection of appropriate MSCs products. + and LPXN + As a biomarker of MSCs products at different culture stages, it is used to treat diseases with different indications, and provides a reference for quality control standards and standardized production of MSCs cell drug preparation. It has important translational application prospects in precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a result diagram showing comparative analysis of UC-MSCs subpopulations at different stages of culture in the disclosed embodiments, and scoring of the hematopoietic support (left figure) function and immunosuppression (right figure) ability of UC-MSCs subpopulations;

[0047] Figure 2 This is a result diagram of analyzing the gene expression levels of M+L+UC-MSCs subpopulations cultured at different generations in the embodiments of the present disclosure;

[0048] Figure 3 MLPH in the embodiment of the present disclosure + LPXN + MSCs, MLPH - LPXN - MSCs, Mix MSCs and CB CD34 + Cells were co-cultured for 7 days and CB CD34 cells were cultured alone + CB CD34 cells for 7 days + Flow cytometry results of cells, where the left picture is CD34 + The right picture shows the results of hematopoietic stem / progenitor cells. The right picture shows CD133 + Results of hematopoietic stem cells;

[0049] Figure 4 MLPH in the embodiment of the present disclosure + LPXN + MSCs, MLPH - LPXN - MSCs, Mix MSCs and CB CD34 + Co-transplantation and single transplantation of CB CD34 + Cells, 8 weeks and 12 weeks after transplantation, flow cytometry results of human cell engraftment in the peripheral blood of NOG mice;

[0050] Figure 5 MLPH in the embodiment of the present disclosure + LPXN + MSCs, MLPH - LPXN - MSCs and CB CD34 + After cell co-culture, CB CD34 + Result graph showing the percentage of cells differentiating into megakaryocytes;

[0051] Figure 6 MLPH in the embodiment of the present disclosure + LPXN + MSCs, MLPH - LPXN - The results of T cell proliferation after MSCs co-cultured with T cells and T cells cultured alone for 5 days;

[0052] Figure 7 Inject MLPH into mice with aGVHD in the disclosed embodiments +LPXN + MSCs, MLPH - LPXN - Survival curve of mice after MSCs and MixMSCs. DETAILED DESCRIPTION

[0053] The present invention discloses a mesenchymal stem cell and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0054] In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "including" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The term "a", "an" and "the" include plural indicators. The term "multiple" refers to two or more. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0055] In the present disclosure, when a range of values ​​is provided, it should be understood that the endpoints are included in the range and each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the specified range and any value in the smaller range between the specified values ​​are included unless the context clearly dictates otherwise.

[0056] In this disclosure, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0057] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The definition of common terms in molecular biology can be found in Lewin's GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. The definition of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. The definition of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. The definition of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0058] Unless otherwise specified, the experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in standard books such as the following: Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0059] 1. Terminology:

[0060] The terms "marker molecule" in the present disclosure have the same meaning as "cell marker molecule", "marker", "biomarker" and "biomarker", which can be used interchangeably in the present disclosure, and mean that the presence of the molecule can be used to identify specific cells or cell types. These molecules can be biological macromolecules such as proteins, carbohydrates, lipids, or other molecules with specific biological functions. In some embodiments of the present disclosure, the marker molecule is a protein. Furthermore, in other embodiments of the present disclosure, the corresponding cell population or cell type can also be identified, identified or sorted based on the expression difference of the gene expressing the marker molecule of the present disclosure. In addition, the cell population carrying a specific marker molecule can be sorted using methods in the prior art. For example, in some embodiments of the present disclosure, the cells can be sorted using flow cytometry (FACS) or immunomagnetic bead sorting (MACS).

[0061] The term "support human hematopoiesis" in this disclosure refers to helping patients restore or maintain normal hematopoietic function through drugs, blood transfusions, growth factors or other medical means. This usually involves promoting the proliferation, differentiation and release of hematopoietic stem cells in the bone marrow, and improving the hematopoietic microenvironment. Hematopoietic support is very important in a variety of clinical situations, including but not limited to hematopoietic stem cell transplantation, bone marrow suppression, blood loss or anemia.

[0062] The term "immune depression" in the present disclosure refers to a state that causes temporary or permanent dysfunction of the immune response. Specifically, it refers to the use of immunosuppressant drugs to promote immune tolerance to foreign antigens such as foreign proteins, organ transplants, bone marrow and tissue transplants, thereby intentionally reducing the activation or efficacy of the host immune system. Examples of existing immunosuppressant drugs include cyclophosphamide, methotrexate, CD4 antibodies, cyclosporine, etc. Reducing the activation or efficacy of the host immune system can generally be reflected in the inhibition of immune cell proliferation. For example, in some embodiments of the present disclosure, CD3 + T cells, CD4 + T cells, CD8 + T cells, CD4 + CD25 + T cells and CD3 + CD25 + Proliferation of T cells.

[0063] The term "graft versus host disease" (GVHD) in the present disclosure is a specific immune phenomenon that mainly occurs after bone marrow transplantation (or other immunocompetent cell transplantation). It is caused by the reaction between the immunocompetent cells (mainly T lymphocytes) in the transplanted tissue and the tissue of the immunosuppressed, tissue-incompatible antigen recipient. The immunocompetent cells in the transplanted bone marrow recognize the different tissue compatibility antigens of the recipient and proliferate and differentiate. When these immunocompetent cells proliferate to a certain extent in the recipient, they will target certain tissues or organs of the recipient for immune attack, thereby causing damage. The clinical manifestations of GVHD are similar to multi-system autoimmune diseases, such as Sjögren's syndrome, systemic lupus erythematosus, scleroderma, etc. It can involve multiple organs and systems, including the skin, digestive system, liver, eyes, etc. GVHD can be divided into two types: acute and chronic. Acute GVHD often occurs within 100 days after transplantation, but can also develop into chronic damage. Chronic GVHD often occurs after 100 days, but can also occur before 100 days. Clinically, the incidence of acute graft-versus-host disease is approximately 35% to 64%, and the incidence of chronic graft-versus-host disease is approximately 30% to 70%.

[0064] The term "poor graft function" (PGF) in this disclosure is a serious complication of allogeneic stem cell transplantation (alloSCT) and is defined as the presence of multilineage cytopenia in the presence of 100% donor chimerism. Inducers or enhancers of alloimmunity, such as cytomegalovirus reactivation and graft-versus-host disease, have been associated with the development of PGF. The pathophysiology of PGF can be defined as dysfunction related to the number or productivity of stem cells, defects in bone marrow microenvironment components (such as mesenchymal stromal cells and endothelial cells), or immunosuppression of hematopoiesis after allogeneic stem cell transplantation.

[0065] 2. Pharmaceutical composition:

[0066] Based on the research disclosed in the present invention, a pharmaceutical composition can be provided, wherein the pharmaceutical composition includes a marker molecule carried by MLPH + and LPXN + mesenchymal stem cells, and a pharmaceutically acceptable carrier.

[0067] In the above-mentioned pharmaceutical composition, the mesenchymal stem cells are a kind of multipotent stem cells. Mesenchymal stem cells were first found in the bone marrow, and then scientists found that they are widely present in various tissues of the human body, such as fat, placenta, umbilical cord blood, etc. They are non-terminal differentiated cells, and have the characteristics of mesenchymal cells, endothelial cells and epithelial cells. Mesenchymal stem cells have strong proliferation ability and multidirectional differentiation potential, immunomodulatory function, and are easy to separate, culture, amplify and purify.

[0068] In some embodiments of the present disclosure, the mesenchymal stem cells may come from, including but not limited to, umbilical cord blood, amniotic fluid, placenta, bone marrow, fat, synovium, bone, muscle, lung, liver or pancreas. They may be isolated from the above sources, or obtained by induced differentiation of ES cells or iPS cells using methods known in the prior art. They may be autologous cells or allogeneic cells (e.g., commercial allogeneic mesenchymal stem cells).

[0069] In some embodiments of the present disclosure, the mesenchymal stem cells can be cultured using methods known in the prior art. For example, α-MEM or DMEM culture medium containing or not containing 10% fetal bovine serum (FBS) is used for culture under the conditions of temperature (37°C), humidity (95%) and carbon dioxide concentration (5%). Mesenchymal stem cells from different sources may have different culture conditions. When MSCs grow to a certain density in a culture bottle, they need to be subcultured. During subculture, digestive enzymes such as trypsin or collagenase are used to digest the cells from the bottle wall, and then subcultured in an appropriate proportion. In some embodiments of the present disclosure, the inventors studied MSCs of different culture generations and found that the highest expression was found in MSCs of culture P6 and P9 generations used in clinical infusion therapy. Therefore, the mesenchymal stem cells in the pharmaceutical composition are preferably daughter cells after subculture, more preferably, sixth to ninth generation cells. The generation of the above cells is calculated from the primary cells. Generally, the cell generation number can be calculated and determined using methods known in the prior art, for example, the cell population doubling number (PDL) method is recorded and calculated using the formula: PDL = X + 3.322 (logY-logI), where X is the generation number before subculture, I is the number of cells before cell inoculation, and Y is the total number of cells after culture. This formula can be used to calculate the cell generation number after subculture. For another example, the cell density can be estimated by observing it in conventional culture. Under conventional culture conditions, if a subculture ratio of 1:2 is used, the generation number is increased by 1 for each subculture. If a subculture ratio of 1:3 is used, the generation number is increased by 1.5 for each subculture. In other embodiments of the present disclosure, in order to more accurately achieve the effect of the pharmaceutical composition, the marker molecule MLPH carried by mesenchymal stem cells can be detected. + and LPXN+ The cell population was sorted based on the expression level of

[0070] In some embodiments of the present disclosure, the pharmaceutical composition is a cell preparation, which further includes a pharmaceutically acceptable carrier known in the prior art. The carrier is a substance that does not affect the function of the mesenchymal stem cells and has no effect on the patient's physical condition. For example, in some embodiments of the present disclosure, the carrier can be physiological saline.

[0071] In addition, in order to achieve better effects, the above-mentioned pharmaceutical composition includes, in addition to the marker molecule carried as an effective ingredient, MLPH + and LPXN + In addition to mesenchymal stem cells, other auxiliary molecules may also be included in other embodiments of the present disclosure. These auxiliary molecules may include, but are not limited to, cytokines (in order to promote the proliferation, differentiation and migration of stem cells, improve the survival rate of stem cells, and enhance their therapeutic effects in vivo, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), etc.), nutritional support substances (in order to ensure their normal growth and differentiation during storage or culture, such as glucose, amino acids, vitamins, inorganic salts, etc.), antioxidants (in order to remove free radicals, reduce the damage of oxidative stress to stem cells, thereby improving the survival rate and therapeutic effect of stem cells, such as vitamin C, vitamin E, glutathione, etc.), immunomodulators, anticoagulants, anti-inflammatory drugs, etc., and combinations thereof. In addition, other known additives may also be included, such as pH regulators, stabilizers, preservatives, etc.

[0072] In addition, in some embodiments of the present disclosure, CD34 + The cells are used as part of a pharmaceutical composition to enhance the therapeutic effect.

[0073] The subject of administration of the pharmaceutical composition described in the present disclosure is a human or other mammal. The administration method can be intravenous injection, local subcutaneous injection, arterial injection, intranasal instillation, inhalation, etc. The number of administrations can be one or more. The dosage can be adjusted according to the conditions of the subject (e.g., age, weight, health status, diet, severity of the disease). Generally, the dosage of mesenchymal stem cells in the pharmaceutical composition is 10 5 ~10 6 Pieces / times.

[0074] 3. Embodiment:

[0075] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0076] Example 1: Marker screening and enrichment of UC-MSCs subpopulations with significant hematopoietic support and immunosuppressive functions

[0077] (1) Wash the human umbilical cord twice with PBS, cut it into small segments of 2-3 cm, cut the umbilical cord longitudinally, remove one umbilical vein and two umbilical arteries, and peel off the Wharton's jelly.

[0078] (2) Cut the Wharton's jelly into pieces and place them in a 15 cm culture dish. Add 5 ml of clinical-grade mesenchymal stem cell serum-free culture medium. When the cells grow to 80%-90% confluence, digest them with stem cell mild digestion enzymes, subculture them, and subculture them until P6 for use.

[0079] (3) Aspirate the old culture medium of P6 UC-MSCs into a 50 ml centrifuge tube and add 10 ml PBS to wash once.

[0080] (4) Add 5 ml of stem cell mild digestion enzyme and digest for 3 minutes. Add old UC-MSCs culture medium to terminate digestion.

[0081] (5) Pipette the liquid in the culture dish into a 50ml centrifuge tube and add 10ml PBS to wash once.

[0082] (6) Centrifuge at 1200 rpm, 4°C, for 5 min, discard the supernatant, and obtain P6 UC-MSCs.

[0083] (7) Resuspend the cells in 1 ml of mesenchymal stem cell serum-free culture medium (containing 1% additive 1), blow evenly after passing through the membrane, and count.

[0084] (8) Resuspend the cells in culture medium according to the counting results at a density of 2x10 6 / ml, add MLPH-FITC and LPXN-APC antibodies at a ratio of 1:100, label for 2h at 4℃, and mix every 20min. Make all negative tubes and FMO tubes at the same time.

[0085] (9) Add 2 ml of culture medium to the FMO tube to wash the antibody, and add 20 ml of culture medium to the all-positive tube to wash the antibody. Centrifuge at 1200 rpm for 5 minutes at 4°C, discard the supernatant, resuspend with culture medium, and adjust the cell density to 5x10 6 / ml, add DAPI, and sort MLPH on the machine (flow cytometer) + LPXN + Cells, MLPH - LPXN -The cells of DAPI- and DAPI- cells (i.e., Mix group) were placed in low DNA-binding EP tubes.

[0086] (10) Blow the cells in the EP tube evenly, inoculate them into a 10 cm culture dish, add 10 ml of mesenchymal stem cell serum-free culture medium (containing 1% additive 1), and place it in a 37°C, 5% CO2 incubator for culture and subculture.

[0087] (11) Single-cell transcriptome sequencing was performed on hUC-MSCs of different culture generations. The raw data of single-cell transcriptome sequencing was decoupled, unevenly distributed bases were filtered, and reads containing too many N were removed. The Cell Ranger count command was run to complete the quantification of cells and genes and output the gene expression matrix. After the run, the gene expression digital matrix, cell annotation file and gene annotation file of the sample were output. Single-sample Seurat data quality control screening, loading the Seurat package, removing low-frequency genes, removing low-frequency genes, and removing cells containing too few genes. Multi-sample integration and batch clustering were performed, and UMAP visualization was performed.

[0088] (12) The various subpopulations of UC-MSCs were analyzed and a subpopulation with both significant hematopoietic support and immunosuppressive functions was selected, which was named the M+L+ subpopulation (e.g. Figure 1 Analysis of the genes in the M+L+ subgroup revealed that the M+L+ subgroup significantly overexpressed MLPH. + and LPXN + The two proteins were expressed at the highest levels in UC-MSCs cultured from P6 to P9, which are used in clinical infusion therapy (e.g. Figure 2 as shown).

[0089] Example 2: MLPH + LPXN + MSCs cells have significant hematopoietic support function

[0090] (1) Using the flow sorted MLPH obtained in Example 1 + LPXN + MLPH - LPXN - UC-MSCs were cultured in vitro to obtain MLPH + LPXN + MSCs and MLPH - LPXN - MSCs cell line, unsorted cells are Mix MSCs line.

[0091] (2) Digestion of MLPH from P6 to P9 + LPXN + MSCs, MLPH- LPXN - MSCs, Mix MSCs, aspirate the old culture medium of MSCs into a 50ml centrifuge tube, add 10ml PBS to wash and discard, add 5ml stem cell mild digestion enzyme, digest at room temperature for 3 minutes, add the old culture medium of MSCs to terminate digestion, centrifuge at 1200rpm, 4℃, 5 minutes, discard the supernatant, add 2ml mesenchymal stem cell serum-free culture medium (containing 1% additive 1), blow evenly, and count the cells.

[0092] (3) MLPH + LPXN + MSCs, MLPH - LPXN - MSCs, Mix MSCs and resuspend at 3x10 4 Add 1 ml of cell suspension to each well of a 24-well plate.

[0093] (4) Place in a 37°C, 5% CO2 incubator and culture overnight.

[0094] (5) Aspirate the culture medium of MSCs and add CB CD34 + Cell expansion medium, 1 ml / well: SFEMII + 100 ng / ml SCF + 10 ng / ml TPO + 10 ng / ml Flt3-L + 1% PS + 1% L-Glu.

[0095] (6) 1x10 sorting per well 4 Lin - CD34 + CB CD34 + The cells were placed in a 37°C, 5% CO2 incubator for 7 days.

[0096] (7) On the 7th day of culture, collect the suspended cells in each well: first gently shake the culture medium and gently aspirate it, then add 1 ml of PBE to each well, gently shake to wash and aspirate it, and repeat 3 times.

[0097] (8) Centrifuge the cells at 1500 rpm, 4°C for 5 min, discard the supernatant, resuspend in 1 ml PBE, take 100 μl from each well, add DAPI, and count the cells using a flow cytometer.

[0098] (9) Centrifuge the remaining 900 μl cells in each well at 1500 rpm, 4°C, for 5 min and discard the supernatant.

[0099] (10) Add antibody, 4°C, 30 min.

[0100] (11) Add 2 ml of PBE to wash away unbound antibodies, centrifuge at 1500 rpm, 4°C, for 5 min, and discard the supernatant.

[0101] (12) Resuspend the cells in 100ul PBE, add DAPI, and detect by flow cytometry. The color scheme of the detection is as shown in Table 1:

[0102] Table 1

[0103] antigen aisle volume hCD34 FITC 1μl hCD90 Percp_Cy5.5 1μl hCD133 APC 1μl hCD201 PE 1μl hCD45RA APC-Cy7 1μl hCD45 PE-Cy7 1μl

[0104] Results: The results showed that, compared with MLPH - LPXN - Compared with MSCs co-culture, MLPH + LPXN + MSCs can significantly promote CD34 + Expansion of hematopoietic stem / progenitor cells (eg Figure 3 as shown in the left figure) and CD133 + Expansion of hematopoietic stem cells (eg Figure 3 ).

[0105] Example 3: MLPH + LPXN + MSCs cells significantly promote hematopoietic reconstruction of human hematopoietic stem cells in vivo

[0106] (1) Eight-week-old female NOG mice were irradiated with 1.5 Gy and transplanted 6 hours after irradiation.

[0107] (2) Enrichment of fresh cord blood CB CD34 using Miltenyi's CD34 MicroBeads Kit + The cells were then counted.

[0108] (3) Digestion of MLPH obtained in Example 1 of P3-P6 generations + LPXN + MSCs, MLPH - LPXN - MSCs, MixMSCs: Aspirate the old culture medium of MSCs into a 50ml centrifuge tube, add 10ml PBS to wash and discard, add 5ml stem cell mild digestion enzyme, digest at room temperature for 3 minutes, add the old culture medium of MSCs to terminate digestion, centrifuge at 1200rpm, 4℃, 5 minutes, discard the supernatant, add 2ml mesenchymal stem cell serum-free culture medium (containing 1% additive 1), blow evenly, and count the cells.

[0109] (4) Transplantation: 2x10 UC-MSCs with different markers were transplanted into each mouse. 5 Combined CB CD34 +5x10 4 CB CD34 + The cell group only transplanted CB CD34+ cells 5x10 4 indivual.

[0110] (5) At 8 and 12 weeks after transplantation, 20 μl of blood was collected from each mouse, 2 ml of red blood cell lysis liquid was added to lyse the red blood cells at room temperature for 10 minutes, the blood was centrifuged at 1500 rpm, 4°C, for 5 minutes, the supernatant was discarded, 2 ml of PBE was added to wash away ACS, the blood was centrifuged at 1500 rpm, 4°C, for 5 minutes, and the supernatant was discarded.

[0111] (6) Add antibody, 4°C, 30 min.

[0112] (7) Add 2 ml of PBE to wash away unbound antibodies, centrifuge at 1500 rpm, 4°C, for 5 min, and discard the supernatant.

[0113] (8) Resuspend the cells in 100ul PBE, add DAPI, and detect by flow cytometry. The color scheme of the detection is as shown in Table 2:

[0114] Table 2

[0115] antigen aisle volume mCD45 Percp-Cy5.5 0.5μl hCD45 APC-Cy7 1μl hCD19 PE 1μl hCD235a PE 1μl hCD33 APC 1μl hCD3 FITC 1μl hCD61 PE-Cy7 1μl

[0116] Results: The results showed that, compared with MLPH - LPXN - Compared with the MSCs group, MLPH + LPXN + Human CD45 in MSCs co-transplantation group + The percentage of cells increased significantly (e.g. Figure 4 ), MLPH + LPXN + MSCs infusion significantly promoted the expression of CD34 + In vivo hematopoietic reconstitution by hematopoietic stem cells.

[0117] Example 4: MLPH + LPXN + MSCs cells significantly promote the differentiation of human hematopoietic stem cells into megakaryocytes

[0118] (1) Digestion of MLPH obtained in Example 1 of P2-P6 generations + LPXN + MSCs, MLPH - LPXN -MSCs, aspirate the old culture medium of MSCs into a 50ml centrifuge tube, add 10ml PBS to wash and discard, add 5ml stem cell mild digestion enzyme, digest at room temperature for 3 minutes, add the old culture medium of MSCs to terminate the digestion, centrifuge at 1200rpm, 4℃, 5 minutes, discard the supernatant, add 2ml mesenchymal stem cell serum-free culture medium (containing 1% additive 1), blow evenly, and count the cells.

[0119] (2) MLPH + LPXN + MSCs, MLPH - LPXN - MSCs were resuspended at 3x10 4 Add 1 ml of cell suspension to each well of a 24-well plate.

[0120] (3) Place in a 37°C, 5% CO2 incubator and culture overnight.

[0121] (4) Aspirate the MSCs culture medium and add megakaryocyte differentiation medium, 1 ml / well: SFEMII + 25 ng / ml SCF + 10 ng / ml TPO + 10 ng / ml Flt3-L + 1% PS + 1% L-Glu + 2.5 ng / ml IL3 + 2.5 ng / ml IL6.

[0122] (5) 1x10 sorting per well 4 Lin - CD34 + CB CD34 + The cells were placed in a 37°C, 5% CO2 incubator for 14 days, with half the medium replaced weekly.

[0123] (6) On the 14th day of culture, collect the suspended cells in each well: first gently shake the culture medium and gently aspirate it, then add 1 ml of PBE to each well, gently shake to wash and aspirate it, and repeat 3 times.

[0124] (7) Centrifuge the cells at 1500 rpm, 4°C, for 5 min and discard the supernatant.

[0125] (8) Add antibody, 4°C, 30 min.

[0126] (9) Add 2 ml of PBE to wash away unbound antibodies, centrifuge at 1500 rpm, 4°C, for 5 min, and discard the supernatant.

[0127] (10) Resuspend the cells in 100ul PBE, add DAPI, and detect by flow cytometry. The color scheme of the detection is as shown in Table 3:

[0128] Table 3

[0129] antigen aisle volume hCD45 APC-Cy7 1μl hCD14 PE-Cy7 1μl hCD15 FITC 1μl hCD235a PE 1μl hCD56 Percp-cy5.5 1μl hCD41a APC 1μl

[0130] Results: The results showed that, compared with MLPH - LPXN - Compared with MSCs, MLPH + LPXN + MSCs and CD34 from cord blood + After cell co-culture, CD34 + The percentage of hematopoietic stem / progenitor cells that differentiate into megakaryocytes (e.g. Figure 5 as shown).

[0131] Example 5: MLPH + LPXN + MSCs have significant immunosuppressive effects

[0132] (1) Digestion of MLPH obtained in Example 1 + LPXN + MSCs, MLPH - LPXN - MSCs, aspirate the old culture medium of MSCs into a 50ml centrifuge tube, add 10ml PBS to wash and discard, add 5ml stem cell mild digestion enzyme, digest at room temperature for 3 minutes, add the old culture medium of MSCs to terminate the digestion, centrifuge at 1200rpm, 4℃, 5 minutes, discard the supernatant, add 2ml mesenchymal stem cell serum-free culture medium (containing 1% additive 1), blow evenly, and count the cells.

[0133] (2) MLPH + LPXN + MSCs, MLPH - LPXN - MSCs were resuspended at 1x10 6 100ul of cell suspension (containing 1x10 5 UC-MSCs cells), and then 900 μl of mesenchymal stem cell serum-free culture medium (containing 1% additive 1) was added, and only 1 ml of mesenchymal stem cell serum-free culture medium (containing 1% additive 1) was added to the positive control group.

[0134] (3) Place in a 37°C, 5% CO2 incubator and culture overnight.

[0135] (4) Use Miltenyi CD3 MicroBeads, human-lyophilized, to enrich T cells in PB according to the procedures described on the official website.

[0136] (5) Centrifuge the enriched T cell suspension at 1500 rpm, 4°C for 5 minutes, discard the supernatant, add 500 μl PBS and mix well to make a single cell suspension, and place on ice until needed.

[0137] (6) Preparation of RPMI 1640 complete medium: RPMI 1640 basal medium + 10% FBS + 1% Glutamax + 1% Penicillin-Streptomycin (PS) solution. All subsequent steps should be carried out in the dark.

[0138] (7) Preparation of CFSE working solution: 1 μl CFSE + 500 μl PBS per aliquot, pipette thoroughly to mix.

[0139] (8) Add one portion of CFSE working solution (1 μl CFSE + 500 μl PBS in each portion) to the resuspended single-cell suspension (500 μl PBS resuspended single-cell suspension), pipette thoroughly to mix, and let stand at room temperature for 10 minutes.

[0140] (9) Quickly add 1 ml of pre-cooled 50% FBS to each tube and mix thoroughly by pipetting to terminate staining. Place the tube on ice for 2 minutes and centrifuge at 300 g, 4°C, for 10 minutes. Discard the supernatant.

[0141] (10) Add 2 ml of 1640 complete medium to each tube and mix thoroughly by pipetting. Centrifuge at 300 g, 4°C, for 10 min and discard the supernatant.

[0142] (11) Repeat step 10.

[0143] (12) Add 1640 complete medium to resuspend the cell pellet and adjust the concentration to 1×10 6 Pieces / ml.

[0144] (13) Aspirate the serum-free medium of mesenchymal stem cells (containing 1% additive 1) in the 48-well plate, add 400 μl of 1640 complete medium, and then add 100 μl of CFSE-labeled T cell suspension (containing 1x10 5 T cells), that is, 500 μl of culture medium was added to each well, and CD3 / 28 antibody was added to make the final concentration of CD3 / 28 antibody 10 μl / ml.

[0145] (14) Culture in a 37°C, 5% CO2 incubator for 5 days.

[0146] (15) On the fifth day of culture, collect the suspended cells in each well: first gently shake the culture medium and aspirate it, then add 1 ml of PBE to each well, shake it gently to wash, and aspirate it, and repeat three times.

[0147] (16) Centrifuge the cells at 1500 rpm, 4°C, for 5 min and discard the supernatant.

[0148] (17) Add antibody, 4°C, 30 min.

[0149] (18) Add 2 ml of PBE to wash away unbound antibodies, centrifuge at 1500 rpm, 4°C, for 5 min, and discard the supernatant.

[0150] (19) Resuspend the cells in 100 μl PBE, add DAPI, and detect by flow cytometry. The color scheme of the detection is as shown in Table 4:

[0151] Table 4

[0152] antigen aisle volume CD4 APC 0.5ul CD8 PE-Cy7 0.5ul CD25 APC-Cy7 0.5ul CD69 PE 0.5ul

[0153] Results: The results showed that, compared with MLPH - LPXN - Compared with MSCs, MLPH + LPXN + MSCs significantly inhibited the expression of CD3 + T cells, CD4 + T cells, CD8 + T cells, CD4 + CD25 + T cells and CD3 + CD25 + T cell proliferation (eg Figure 6 as shown).

[0154] Example 6: MLPH + LPXN + MSCs cell infusion for GVHD treatment

[0155] (1) Before the construction of the half-matched aGVHD model, UC-MSCs with three different markers were transplanted on day 1: MLPH obtained in Example 1 at the P6 generation was digested + LPXN + MSCs, MLPH - LPXN - MSCs, Mix MSCs, aspirate the old culture medium of MSCs into a 50ml centrifuge tube, add 10ml PBS to wash and discard, add 5ml stem cell mild digestion enzyme, digest at room temperature for 3 minutes, add the old culture medium of MSCs to terminate digestion, centrifuge at 1200rpm, 4℃, 5 minutes, discard the supernatant, add 2ml mesenchymal stem cell serum-free culture medium (containing 1% additive 1), blow evenly, and count cells. MLPH + LPXN + MSCs group, MLPH - LPXN- MSCs group and Mix MSCs group were transplanted with 2x10 5 UC-MSCs with corresponding markers.

[0156] (2) Eight-week-old female CB6F1 mice were irradiated with 8 Gy (4 Gy + 4 Gy).

[0157] (3) 4 hours after irradiation, each CB6F1 mouse was transplanted with 5x10 6 Female B6 mouse BM cells combined with 9x10 7 Spleen cells from female C57 mice.

[0158] (4) After the half-matched aGVHD model was established, UC-MSCs with three different markers were transplanted on day 3: the MLPH obtained in Example 1 at P6 was digested + LPXN + MSCs, MLPH - LPXN - MSCs, Mix MSCs, aspirate the old culture medium of MSCs into a 50ml centrifuge tube, add 10ml PBS to wash and discard, add 5ml stem cell mild digestion enzyme, digest at room temperature for 3 minutes, add the old culture medium of MSCs to terminate digestion, centrifuge at 1200rpm, 4℃, 5 minutes, discard the supernatant, add 2ml mesenchymal stem cell serum-free culture medium (containing 1% additive 1), blow evenly, and count cells. MLPH + LPXN + MSCs group, MLPH - LPXN - MSCs group and Mix MSCs group were transplanted with 2x10 5 UC-MSCs with corresponding markers.

[0159] (5) After the construction of the half-matched aGVHD model, UC-MSCs with three different markers were transplanted on day 12: the MLPH obtained in Example 1 at P6 was digested + LPXN + MSCs, MLPH - LPXN - MSCs, Mix MSCs, aspirate the old culture medium of MSCs into a 50ml centrifuge tube, add 10ml PBS to wash and discard, add 5ml stem cell mild digestion enzyme, digest at room temperature for 3 minutes, add the old culture medium of MSCs to terminate digestion, centrifuge at 1200rpm, 4℃, 5 minutes, discard the supernatant, add 2ml mesenchymal stem cell serum-free culture medium (containing 1% additive 1), blow evenly, and count cells. MLPH + LPXN+ MSCs group, MLPH - LPXN - MSCs group and Mix MSCs group were transplanted with 2x10 5 UC-MSCs with corresponding markers.

[0160] (6) After the half-matched aGVHD model was established, the survival status of mice in each group was observed and recorded every day.

[0161] Results: The results showed that compared with infusion of MLPH - LPXN - Compared with mice infused with MLPH, mice infused with MSCs showed + LPXN + The GVHD symptoms of mice treated with MSCs were significantly alleviated and their survival was significantly prolonged (e.g. Figure 7 ), MLPH + LPXN + MSCs have the effect of treating GVHD.

[0162] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of mesenchymal stem cells in the preparation of a product with hematopoietic support function, characterized in that: The marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

2. Preparation of Mesenchymal Stem Cells to Promote CD34 + Hematopoietic stem / progenitor cells and / or CD133 + Use of a product for the expansion of hematopoietic stem / progenitor cells, characterized in that The marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

3. Use of mesenchymal stem cells in the preparation of a product that promotes hematopoietic reconstruction of human hematopoietic stem / progenitor cells in vivo, characterized in that: The marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

4. Use of mesenchymal stem cells in the preparation of a product for promoting differentiation of human hematopoietic stem cells into megakaryocytes, characterized in that: The marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

5. Use of mesenchymal stem cells in the preparation of a product with immunosuppressive function, characterized in that: The marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

6. Use of mesenchymal stem cells in the preparation of a product for treating or assisting in treating graft-versus-host disease, characterized in that: The marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

7. Use of mesenchymal stem cells in the preparation of a product for treating or assisting in the treatment of transplant hematopoietic dysfunction, characterized in that: The marker molecule carried by the mesenchymal stem cells is MLPH + and LPXN + .

8. A pharmaceutical composition, characterized in that The pharmaceutical composition includes a marker molecule MLPH + and LPXN + mesenchymal stem cells, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also includes CD34 + of cells and / or accessory components.

10. The pharmaceutical composition according to claim 9, characterized in that The auxiliary components are one or more selected from cytokines, nutritional support substances, antioxidants, immunomodulators, anticoagulants or anti-inflammatory drugs.

11. The pharmaceutical composition according to any one of claims 8 to 10, characterized in that The mesenchymal stem cells are daughter cells after subculture.

12. The use of the pharmaceutical composition according to any one of claims 8 to 11, characterized in that: The application is selected from: (1) Preparation of products with hematopoietic support function; or (2) Preparation of CD34-promoting + Hematopoietic stem / progenitor cells and / or CD133 + Products of expansion of hematopoietic stem / progenitor cells; or (3) Preparation of products that promote hematopoietic reconstruction of human hematopoietic stem cells in vivo; or (4) Preparation of products that promote the differentiation of human hematopoietic stem cells into megakaryocytes; or (5) Preparation of products with immunosuppressive function; or (6) Preparation of products for the treatment or adjunctive treatment of graft-versus-host disease; or (7) Preparation of products for the treatment or auxiliary treatment of transplant hematopoietic dysfunction.

13. A method for preparing the following products: (1) a product having hematopoietic support function; or (2) a product promoting CD34 + Hematopoietic stem / progenitor cells and / or CD133 + or (3) products for the expansion of hematopoietic stem / progenitor cells; or (4) products for promoting the differentiation of human hematopoietic stem cells into megakaryocytes; or (5) products with immunosuppressive function; or (6) products for the treatment or adjuvant treatment of graft-versus-host disease; or (7) products for the treatment or adjuvant treatment of poor hematopoietic function of transplanted tissues, characterized in that: The method includes obtaining a marker molecule carried as MLPH + and LPXN + Steps for mesenchymal stem cells.

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