Pluripotent stem cells, pharmaceutical compositions, and methods for preparing them and their applications

Abstract

The present invention relates to the field of cell therapy, and more particularly to a method for producing a mesenchymal stem cell population, a mesenchymal stem cell population produced by the method and its culture supernatant, and a pharmaceutical composition containing such cells or its culture supernatant. The present invention further relates to the use of the mesenchymal stem cell population and its culture supernatant for the prevention and treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy. Specifically, the present invention relates to a method for producing a mesenchymal stem cell population, a mesenchymal stem cell population produced by the method and its culture supernatant, and a pharmaceutical composition containing such cells or its culture supernatant. The present invention also relates to the use of a mesenchymal stem cell population and its culture supernatant, and a pharmaceutical composition containing such cells or its culture supernatant for the prevention and / or treatment of diseases.

Background Art

[0002] Mesenchymal stem cells (MSCs) are adult stem cells with self-renewal ability and multi-directional differentiation ability. These are derived from a wide range of sources such as the brain, spleen, liver, kidney, lung, bone marrow, etc., and can be separated from almost all connective tissues. Over the past few decades, researchers have targeted MSCs for stem cell therapy research. MSCs can be easily obtained from various connective tissues and can be proliferated in large quantities to obtain a large number of cells. Embryonic stem cells (ESCs) / induced pluripotent stem cells (iPSCs) have limited clinical use due to their potential tumorigenicity and ethical issues. Although mesenchymal stem cells have multi-directional differentiation ability, immunomodulatory function, and low immunogenicity, they have not been found to have tumorigenicity, so they have become seed cells useful for clinical use related to stem cell therapy.

[0003] So far, most therapeutic applications have studied MSCs derived from adult bone marrow or umbilical cord. Although these can be easily separated from bone marrow and umbilical cord, problems such as limited sources and the decline of their proliferation ability and differentiation ability with the increase of in vitro culture time still exist. Therefore, the problem that the quantity and quality of cells are unstable affects the clinical use of MSCs derived from bone marrow sources.

[0004] To solve the problem of the source of MSCs, it is possible to explore new sources of MSCs. Since embryonic stem cells have the ability to proliferate indefinitely and the ability to differentiate into various cells and tissues of the mesoderm, endoderm, and ectoderm, they can be used as a new source of MSCs. In recent years, many studies have reported methods for inducing mesenchymal cells from human embryonic stem cells. However, there are still disadvantages such as low induction efficiency, complex induction processes, and long induction times, and most methods require the use of xenogeneic substances such as serum, so they cannot be used clinically.

Summary of the Invention

Problems to be Solved by the Invention

[0005] As a conclusion, considering the fact that the current methods for obtaining mesenchymal stem cells are limited by several disadvantages, it is highly necessary to find a method for producing mesenchymal stem cells with high purity, high yield, and in a short time for use in the clinical treatment and prevention of various diseases.

Means for Solving the Problems

[0006] Content of the Present Invention After many experiments and repeated investigations, the inventors of the present application obtained a method for manufacturing mesenchymal stem cells in vitro from stem cells (for example, totipotent stem cells or pluripotent stem cells). The mesenchymal stem cells obtained by this method significantly improved the cytokine secretion level, thus leading to the completion of the present invention. In this specification, the cells of the present invention or the cells obtained by the method of the present invention may be called M cells.

[0007] Mesenchymal stem cell population Accordingly, in a first aspect, the present invention provides a mesenchymal stem cell population having an average MMP1 expression level that is at least about 10-fold (e.g., at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 2000-fold, at least about 3000-fold, at least about 5000-fold, at least about 8000-fold, at least about 10000-fold, or at least about 12000-fold) higher than that of primary mesenchymal stem cells (e.g., in the absence of genetic modification), and / or an average PGE2 expression level that is at least about 10-fold (e.g., at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 60-fold, or at least about 80-fold) higher than that of primary mesenchymal stem cells (e.g., in the absence of genetic modification).

[0008] As used herein, the term "primary mesenchymal stem cells" refers to mesenchymal stem cells isolated from tissue directly removed from the body (e.g., adipose tissue, umbilical cord, bone marrow, or umbilical cord blood).

[0009] In certain embodiments, the MMP1 expression level of the mesenchymal stem cell population is at least about 10-fold (e.g., at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 2000-fold, at least about 3000-fold, at least about 5000-fold, at least about 8000-fold, at least about 10000-fold, or at least about 12000-fold) higher than that of the same amount of primary mesenchymal stem cells.

[0010] In certain embodiments, the PGE2 expression level of the mesenchymal stem cell population is at least about 10-fold (e.g., at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 60-fold, or at least about 80-fold) higher than that of the same amount of primary mesenchymal stem cells. In certain embodiments, the PGE2 expression level of the mesenchymal stem cell population is about 80-fold higher than that of the same amount of primary mesenchymal stem cells.

[0011] In certain embodiments, after stimulation with IFN-γ (e.g., 25 ng / ml to 100 ng / ml), the mesenchymal stem cell population has a higher average PD-L1 expression level (e.g., in the absence of genetic modification) than primary mesenchymal stem cells. In certain embodiments, after stimulation with IFN-γ (e.g., 25 ng / ml to 100 ng / ml), the average PD-L1 expression level of the mesenchymal stem cell population is at least about 2-fold (e.g., at least about 3-fold) higher than that of primary mesenchymal stem cells. In certain embodiments, after stimulation with 50 ng / ml to 100 ng / ml of IFN-γ, the average PD-L1 expression level of the mesenchymal stem cell population is about 3-fold higher than that of primary mesenchymal stem cells. In certain embodiments, after stimulation with IFN-γ (e.g., 25 ng / ml to 100 ng / ml), the average PD-L1 expression level of the mesenchymal stem cell population is at least about 2-fold (e.g., at least about 3-fold) higher than that of the same amount of primary mesenchymal stem cells. In certain embodiments, after stimulation with 50 ng / ml to 100 ng / ml of IFN-γ, the average PD-L1 expression level of the mesenchymal stem cell population is about 3-fold higher than that of the same amount of primary mesenchymal stem cells.

[0012] In certain embodiments, the average IDO expression level of the mesenchymal stem cell population (e.g., in the absence of genetic modification) is higher than that of primary mesenchymal stem cells. In certain embodiments, the average IDO expression level of the mesenchymal stem cell population is at least about 10-fold (e.g., at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 60-fold, at least about 80-fold, at least about 100-fold, or at least about 110-fold) higher than that of primary mesenchymal stem cells. In certain embodiments, the average IDO expression level of the mesenchymal stem cell population is about 110-fold higher than that of primary mesenchymal stem cells. In certain embodiments, the mesenchymal stem cell population has an IDO expression level that is at least about 10-fold (e.g., at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 60-fold, at least about 80-fold, at least about 100-fold, or at least about 110-fold) higher than that of the same amount of primary mesenchymal stem cells. In certain embodiments, the mesenchymal stem cell population has an IDO expression level that is at least about 110-fold higher than that of the same amount of primary mesenchymal stem cells.

[0013] As used herein, expression can be monitored by measuring the level of full-length mRNA of a gene, mRNA fragments, full-length protein, or protein fragments. Thus, in certain embodiments, the expression level is an mRNA level or a protein level.

[0014] In some embodiments, expression is evaluated by analyzing the expression of the mRNA transcript of the gene. For example, the expression of the above-described gene in a cell population is determined by determining the presence or content of the mRNA of IDO, MMP1, PDL1, or PGE2 in the cell population by RT-PCR.

[0015] In other embodiments, expression is evaluated by analyzing the expression of the protein product of the gene. For example, the expression of the above-described gene in a cell population is determined by determining the presence or content of IDO protein, MMP1 protein, PDL1 protein, or PGE2 protein in the culture supernatant of the cell population by immunological detection. Thus, in certain embodiments, the expression level of a gene (e.g., IDO, MMP1, PDL1, or PGE2) is evaluated by the level of the corresponding protein secreted in the culture supernatant.

[0016] In certain embodiments, the mesenchymal stem cell population has one or more of the above-described gene expression characteristics in the absence of genetic modification. Here, the term "in the absence of genetic modification" refers to not having undergone the process of introducing exogenous genetic material in the form of DNA or RNA into the total genetic material of the cell, and the term "exogenous genetic material" can refer to an artificially introduced nucleotide sequence that is exogenous to a non-genetically modified cell. The above "in the absence of genetic modification" is used only to explain the condition that the mesenchymal stem cell population of the present invention has one or more of the above-described gene expression characteristics, and it is easily understood that it is not used as a limitation that the mesenchymal stem cell population of the present invention must not contain genetic modification. Thus, in certain embodiments, the mesenchymal stem cell population of the present invention may contain one or more genetic modifications.

[0017] In certain embodiments, the mesenchymal stem cell population is produced from stem cells. In certain embodiments, the stem cells are totipotent stem cells or pluripotent stem cells. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells, haploid stem cells, induced pluripotent stem cells, or adult stem cells. In certain embodiments, the mesenchymal stem cell population is produced from embryonic stem cells or induced pluripotent stem cells.

[0018] In certain embodiments, the mesenchymal stem cell population is produced in vitro.

[0019] In certain embodiments, the mesenchymal stem cell population has the following characteristics: (1) It contains 80% or more (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) of cells expressing one or more selected from the group consisting of CD105, CD73, CD90, CD13, CD29, CD44, CD166, and HLA-ABC; (2) It contains 2% or less (e.g., 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, or 0.01% or less) of cells expressing one or more selected from the group consisting of CXCL1, CD34, CD45, CD133, FGFR2, CD271, Stro-1, and CXCR4; and further has:

[0020] In certain embodiments, the mesenchymal stem cell population further has the following properties: (3) Having cells expressing CD274; (4) Having cells expressing CD24; (5) Having cells expressing CD31; and has one or more of the above.

[0021] In certain embodiments, CD274 + cells have a proportion of 80% or more, e.g., 80% - 95%, e.g., about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%.

[0022] In certain embodiments, CD24 + cells have a proportion of 50% or more, e.g., 50% - 70%, e.g., about 50%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, or about 70%.

[0023] In certain embodiments, CD31 +The cells have a proportion of 5% or more, for example 5% - 20%, for example about 5%, about 10%, about 12%, about 15%, about 18%, or about 20%.

[0024] The mesenchymal stem cell population of the present invention can be formulated and administered as a pharmaceutical composition. Such a pharmaceutical composition can be in any form known in the medical field, preferably an injection (including injection solution, lyophilized powder). In certain embodiments, the pharmaceutical composition includes a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0025] Method for preparing a mesenchymal stem cell population In a second aspect, the present invention is a method for producing a mesenchymal stem cell population, or a method for producing the mesenchymal stem cell population described in the first aspect, comprising the following steps: (1) A step of culturing stem cells using a first culture medium to form embryoid bodies, wherein the first culture medium is a basal medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and bFGF; (2) A step of culturing the embryoid bodies using a second culture medium to induce their differentiation into mesenchymal stem cells, wherein the second culture medium is a basal medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and one or more growth factors; Provided is a method including

[0026] In certain embodiments, step (2) includes attaching the embryoid body to a culture vessel and culturing it using a second culture medium.

[0027] As used herein, the term "basal medium" refers to any culture medium that can support cell growth and typically contains inorganic salts, vitamins, glucose, a buffer system, and essential amino acids, and typically has an osmolarity of about 280 mOsmol to 330 mOsmol.

[0028] In certain embodiments, the stem cells described in step (1) are totipotent stem cells or pluripotent stem cells. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells, haploid stem cells, induced pluripotent stem cells, or adult stem cells.

[0029] In certain embodiments, the first culture medium has the following characteristics: (i) One or more serum replacements have a total content of 3% (v / v) to 30% (v / v), such as about 3% (v / v), about 5% (v / v), about 8% (v / v), about 10% (v / v), about 12% (v / v), about 15% (v / v), about 18% (v / v), about 20% (v / v), about 22% (v / v), about 25% (v / v), about 28% (v / v), or about 30% (v / v); (ii) Each of one or more non-essential amino acids has a content of 0.1 mM to 0.5 mM, such as about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, or about 0.5 mM; (iii) A stabilizing dipeptide of glutamine or L-alanyl-L-glutamine has a content of 1 mM to 5 mM, such as about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. (iv) bFGF has a content of 1 ng / ml to 100 ng / ml, for example 2 ng / ml to 100 ng / ml, 2 ng / ml to 50 ng / ml, 5 ng / ml to 100 ng / ml, 5 ng / ml to 50 ng / ml, or 5 ng / ml to 20 ng / ml, for example about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 5 ng / ml, about 8 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about 100 ng / ml, and has one or more of

[0030] In a certain specific embodiment, the first culture medium has the following characteristics: (a) The serum substitute is selected from the group consisting of KOSR, MSC serum-free supplement, Ultraser™ G, and any combination thereof, preferably the serum substitute is KnockOut™ SR (for example, Thermo: catalog number 10828028) (hereinafter referred to as KOSR), (b) The non-essential amino acids are selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and any combination thereof, (c) The basal medium is selected from the group consisting of KnockOut™ DMEM (for example, Gibco: catalog number 10829018) (hereinafter referred to as KO-DMEM), KnockOut™ DMEM / F-12 (for example, Gibco: catalog number 12660-012) (hereinafter referred to as KO-DMEM / F12), DMEM, α-MEM, F-12, MEM, BME, RPMI 1640, G-MEM, and any combination thereof, preferably the basal medium is selected from the group consisting of KO-DMEM, KO-DMEM / F12, DMEM, DMEM / F12, preferably the basal medium is KO-DMEM, having one or more of.

[0031] In certain embodiments, the first culture medium comprises KO-DMEM, KOSR, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, a stabilized dipeptide of L-alanyl-L-glutamine, and bFGF. In certain embodiments, the first culture medium comprises 3% (v / v) to 30% (v / v) KOSR, 1 mM to 5 mM of the stabilized dipeptide of L-alanyl-L-glutamine, 1 ng / ml to 100 ng / ml of bFGF, and the following amino acids at a concentration of about 0.1 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0032] In certain embodiments, the first culture medium further comprises β-mercaptoethanol. In certain embodiments, β-mercaptoethanol has a content of 0.1% (v / v) to 0.5% (v / v), such as about 0.1% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v), or about 0.5% (v / v).

[0033] In certain embodiments, the first culture medium consists essentially of the following components: KO-DMEM, KOSR, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, a stabilized dipeptide of L-alanyl-L-glutamine, bFGF, and β-mercaptoethanol.

[0034] In certain embodiments, the first culture medium comprises 3% (v / v) to 30% (v / v) KOSR, 1 mM to 5 mM of the stabilized dipeptide L-alanyl-L-glutamine, 1 ng / ml to 100 ng / ml bFGF, 0.1% (v / v) to 0.5% (v / v) β-mercaptoethanol, and the following amino acids at a concentration of about 0.1 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0035] In certain embodiments, the first culture medium comprises about 15% (v / v) KOSR, about 1 mM of the stabilized dipeptide L-alanyl-L-glutamine, about 8 ng / ml bFGF, about 0.1% (v / v) β-mercaptoethanol, and the following amino acids at a concentration of about 0.1 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0036] In certain embodiments, the first culture medium comprises about 18% (v / v) KOSR, about 1 mM of the stabilized dipeptide L-alanyl-L-glutamine, about 12 ng / ml bFGF, about 0.1% (v / v) β-mercaptoethanol, and the following amino acids at a concentration of about 0.1 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0037] In certain embodiments, the first culture medium comprises about 20% (v / v) KOSR, about 2 mM of the stabilized dipeptide L-alanyl-L-glutamine, about 10 ng / ml bFGF, about 0.1% (v / v) β-mercaptoethanol, and the following amino acids at a concentration of about 0.1 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0038] In certain embodiments, the first culture medium comprises about 22% (v / v) KOSR, about 2 mM of the stabilized dipeptide L-alanyl-L-glutamine, about 12 ng / ml bFGF, about 0.2% (v / v) β-mercaptoethanol, and the following amino acids at a concentration of about 0.1 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0039] In certain embodiments, the first culture medium comprises about 22% (v / v) KOSR, about 2 mM of the stabilized dipeptide L-alanyl-L-glutamine, about 12 ng / ml bFGF, about 0.1% (v / v) β-mercaptoethanol, and the following amino acids at a concentration of about 0.2 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0040] In certain exemplary embodiments, the first culture medium consists essentially of KO-DMEM (Gibco: catalog number 10829018), KOSR (Thermo: catalog number 10828028), NEAA (Gibco: catalog number 11140050), GlutaMAX (Gibco: catalog number A1286001), bFGF, and β-mercaptoethanol.

[0041] In certain exemplary embodiments, the first culture medium comprises 18% (v / v) - 22% (v / v) KOSR, 0.5% (v / v) - 1.5% (v / v) GlutaMAX, 1 ng / ml - 100 ng / ml bFGF, 0.1% (v / v) - 0.5% (v / v) β-mercaptoethanol, and 1% (v / v) - 2% (v / v) NEAA.

[0042] In certain exemplary embodiments, the first culture medium comprises about 15% (v / v) KOSR, about 0.5% (v / v) GlutaMAX, about 8 ng / ml bFGF, about 0.1% (v / v) β-mercaptoethanol, and about 1% (v / v) NEAA.

[0043] In certain exemplary embodiments, the first culture medium comprises about 18% (v / v) KOSR, about 0.5% (v / v) GlutaMAX, about 12 ng / ml bFGF, about 0.1% (v / v) β-mercaptoethanol, and about 1% (v / v) NEAA.

[0044] In certain exemplary embodiments, the first culture medium comprises about 20% (v / v) KOSR, about 1% (v / v) GlutaMAX, about 10 ng / ml bFGF, about 0.1% (v / v) β-mercaptoethanol, and about 1% (v / v) NEAA.

[0045] In certain exemplary embodiments, the first culture medium comprises about 22% (v / v) KOSR, about 1% (v / v) GlutaMAX, about 12 ng / ml bFGF, about 0.2% (v / v) β-mercaptoethanol, and about 1% (v / v) NEAA.

[0046] In certain exemplary embodiments, the first culture medium comprises about 22% (v / v) KOSR, about 1% (v / v) GlutaMAX, about 12 ng / ml bFGF, about 0.1% (v / v) β-mercaptoethanol, and about 1% (v / v) NEAA.

[0047] In certain embodiments, the second culture medium has the following properties: (i) One or more serum replacements have a total content of 1% (v / v) to 40% (v / v), such as 1% (v / v) to 35% (v / v), 1% (v / v) to 30% (v / v), 2% (v / v) to 30% (v / v), 5% (v / v) to 30% (v / v), 1% (v / v) to 20% (v / v), 2% (v / v) to 20% (v / v), 5% (v / v) to 20% (v / v), 1% (v / v) to 10% (v / v), 2% (v / v) to 10% (v / v), or 5% (v / v) to 10% (v / v), such as about 1% (v / v), about 2% (v / v), about 3% (v / v), about 5% (v / v), about 8% (v / v), about 10% (v / v), about 12% (v / v), about 15% (v / v), about 18% (v / v), about 20% (v / v), about 22% (v / v), about 25% (v / v), about 28% (v / v), or about 30% (v / v). (ii) Each of one or more non-essential amino acids has a content of 0.1 mM to 0.5 mM, such as 0.1 mM to 0.2 mM, such as about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, or about 0.5 mM. (iii) A stabilizing dipeptide of glutamine or L-alanyl-L-glutamine has a content of 1 mM to 5 mM, such as 1 mM to 3 mM, such as about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. (iv) Each of one or more growth factors has a content of 1 ng / ml to 100 ng / ml, such as about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 5 ng / ml, about 8 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, or about 100 ng / ml. Has one or more of the above.

[0048] In certain embodiments, the second culture medium has the following characteristics: (a) The serum replacement is selected from the group consisting of KOSR, MSC serum-free supplement, Ultroser™ G, and any combination thereof; (b) The non-essential amino acids are selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and any combination thereof; (c) The basal medium is selected from the group consisting of KO-DMEM, KO-DMEM / F-12, α-MEM, DMEM, F12, MEM, BME, RPMI 1640, G-MEM, and any combination thereof; preferably, the basal medium is selected from the group consisting of KO-DMEM, KO-DMEM / F12, α-MEM, DMEM, DMEM / F12; and has one or more of the above.

[0049] In certain embodiments, the serum replacement is a serum replacement selected from the group consisting of KOSR, and MSC serum-free supplement (e.g., TBD: catalog number SC2013-G-B) and Ultraser™ G (e.g., PALL: catalog number 15950-017) (hereinafter referred to as Ultroser G). In certain embodiments, the volume ratio of KOSR to MSC serum-free supplement or Ultroser G ranges from 2:1 to 150:1, such as about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 50:1, about 80:1, about 100:1, about 120:1, or about 150:1. In certain embodiments, the volume ratio of KOSR to MSC serum-free supplement or Ultroser G ranges from 10:1 to 1:2, such as about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, or about 1:2.

[0050] In certain embodiments, KOSR has a content of about 1% (v / v) to 30% (v / v), about 1% (v / v) to 20% (v / v), about 1% (v / v) to 10% (v / v), about 2% (v / v) to 10% (v / v), or about 5% (v / v) to 10% (v / v). In certain embodiments, MSC serum-free supplement or Ultraser G has a content of about 1% (v / v) to 10% (v / v), or about 1% (v / v) to 5% (v / v).

[0051] In certain embodiments, the second culture medium comprises KO-DMEM / F12, α-MEM, MSC serum-free supplement or Ultraser G, KOSR, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, a stabilized dipeptide of L-alanyl-L-glutamine, and one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF). In certain embodiments, the second culture medium comprises 1% (v / v) to 10% (v / v) of Ultraser G, 1% (v / v) to 20% (v / v) of KOSR, 1 mM to 5 mM of a stabilized dipeptide of L-alanyl-L-glutamine, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at a concentration of 1 ng / ml to 100 ng / ml each, and the following amino acids at a concentration of 0.1 mM to 0.5 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0052] In certain embodiments, the second culture medium further comprises ascorbic acid. In certain embodiments, ascorbic acid has a content of 1 μg / ml to 100 μg / ml, such as 1 μg / ml to 100 μg / ml, 1 μg / ml to 50 μg / ml, 1 μg / ml to 20 μg / ml, or 5 μg / ml to 20 μg / ml, such as about 1 μg / ml, about 10 μg / ml, about 100 μg / ml, about 500 μg / ml, or about 1000 μg / ml.

[0053] In certain embodiments, the second culture medium consists essentially of KO-DMEM / F12, α-MEM, MSC serum-free supplement or Ultroser G, KOSR, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, a stabilized dipeptide of L-alanyl-L-glutamine, ascorbic acid, and one or more growth factors (for example, one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF).

[0054] In certain embodiments, the second culture medium comprises 1% (v / v) to 5% (v / v) (such as about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v)) of Ultraser G, 2% (v / v) to 20% (v / v) (such as about 2% (v / v), about 4% (v / v), about 6% (v / v), about 8% (v / v), about 10% (v / v), about 12% (v / v), about 14% (v / v), about 16% (v / v), about 18% (v / v), or about 20% (v / v)) of KOSR, 1 mM to 5 mM of a stabilized dipeptide of L-alanyl-L-glutamine, 1 μg / ml to 1000 μg / ml of ascorbic acid, one or more growth factors (for example, one or more selected from VEGF, bFGF, EGF, TGFβ, PDGF) each at a concentration of 1 ng / ml to 100 ng / ml, and the following amino acids each at a concentration of 0.1 mM to 0.5 mM: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0055] In certain embodiments, the second culture medium comprises about 1% (v / v) Ultroser G, about 4% (v / v) KOSR, a stabilizing dipeptide of about 2 mM L-alanyl-L-glutamine, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) each at a concentration of about 1 ng / ml to 100 ng / ml, and the following amino acids each at a concentration of about 0.1 mM: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0056] In certain embodiments, the second culture medium comprises about 1% (v / v) Ultroser G, about 6% (v / v) KOSR, a stabilizing dipeptide of about 2 mM L-alanyl-L-glutamine, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) each at a concentration of about 1 ng / ml to 100 ng / ml, and the following amino acids each at a concentration of about 0.1 mM: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0057] In certain embodiments, the second culture medium comprises about 1% (v / v) Ultroser G, about 8% (v / v) KOSR, a stabilizing dipeptide of about 2 mM L-alanyl-L-glutamine, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) each at a concentration of about 1 ng / ml to 100 ng / ml, and the following amino acids each at a concentration of about 0.1 mM: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0058] In certain embodiments, the second culture medium comprises about 2% (v / v) Ultroser G, about 4% (v / v) KOSR, a stabilized dipeptide of about 1 mM L-alanyl-L-glutamine, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at respective concentrations of about 1 ng / ml to 100 ng / ml, and the following amino acids at respective concentrations of about 0.1 mM: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0059] In certain embodiments, the second culture medium comprises about 2% (v / v) Ultroser G, about 6% (v / v) KOSR, a stabilized dipeptide of about 1 mM L-alanyl-L-glutamine, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at respective concentrations of about 1 ng / ml to 100 ng / ml, and the following amino acids at respective concentrations of about 0.1 mM: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0060] In certain embodiments, the second culture medium comprises about 2% (v / v) Ultroser G, about 8% (v / v) KOSR, a stabilized dipeptide of about 1 mM L-alanyl-L-glutamine, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at respective concentrations of about 1 ng / ml to 100 ng / ml, and the following amino acids at respective concentrations of about 0.1 mM: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

[0061] In certain exemplary embodiments, the second culture medium consists essentially of the following components: KO-DMEM / F12 (Gibco: catalog number 12660-012), α-MEM (HyClone: catalog number SH30265.01B), Ultraser G (PALL: catalog number 15950-017), KOSR (Thermo: catalog number 10828028), NEAA (Gibco: catalog number 11140050), GlutaMAX (Gibco: catalog number A1286001), ascorbic acid, and one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF).

[0062] In certain exemplary embodiments, the second culture medium contains 1% (v / v) to 2% (v / v) of Ultraser G, 4% (v / v) to 6% (v / v) of KOSR, 0.5% (v / v) to 1.5% (v / v) of GlutaMAX, 1 μg / ml to 1000 μg / ml of ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) each at a concentration of about 1 ng / ml to 100 ng / ml, and 1% (v / v) to 2% (v / v) of NEAA.

[0063] In certain exemplary embodiments, the second culture medium contains about 1% (v / v) of Ultroser G, about 4% (v / v) of KOSR, about 1% (v / v) of GlutaMAX, about 100 μg / ml of ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) each at a concentration of about 1 ng / ml to 10 ng / ml, and about 1% (v / v) of NEAA.

[0064] In certain exemplary embodiments, the second culture medium comprises about 1% (v / v) Ultroser G, about 6% (v / v) KOSR, about 1% (v / v) GlutaMAX, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at a concentration of about 1 ng / ml to 10 ng / ml each, and about 1% (v / v) NEAA.

[0065] In certain exemplary embodiments, the second culture medium comprises about 1% (v / v) Ultroser G, about 8% (v / v) KOSR, about 1% (v / v) GlutaMAX, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at a concentration of about 1 ng / ml to 10 ng / ml each, and about 1% (v / v) NEAA.

[0066] In certain exemplary embodiments, the second culture medium comprises about 2% (v / v) Ultroser G, about 4% (v / v) KOSR, about 0.5% (v / v) GlutaMAX, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at a concentration of about 1 ng / ml to 10 ng / ml each, and about 1% (v / v) NEAA.

[0067] In certain exemplary embodiments, the second culture medium comprises about 2% (v / v) Ultroser G, about 6% (v / v) KOSR, about 0.5% (v / v) GlutaMAX, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at a concentration of about 1 ng / ml to 10 ng / ml each, and about 1% (v / v) NEAA.

[0068] In certain exemplary embodiments, the second culture medium comprises about 2% (v / v) Ultroser G, about 8% (v / v) KOSR, about 0.5% (v / v) GlutaMAX, about 100 μg / ml ascorbic acid, one or more growth factors (e.g., one or more selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF) at a concentration of about 1 ng / ml to 10 ng / ml each, and about 1% (v / v) NEAA.

[0069] In certain embodiments, all components included in the first culture medium and the second culture medium are of cell therapy grade (CTS grade). In certain embodiments, the basal medium (e.g., KO-DMEM), serum replacement (e.g., KOSR), and stabilized dipeptide of L-alanyl-L-glutamine included in the first culture medium and the second culture medium are all of cell therapy grade (CTS grade).

[0070] In certain embodiments, step (1) includes culturing pluripotent stem cells in a low-attachment cell culture vessel.

[0071] As used herein, the term "low-attachment cell culture vessel" refers to a culture vessel having a coating on its surface that prevents protein adsorption on the surface of the culture vessel, thereby minimizing the attachment of monolayer cells to the culture vessel. Such cell culture vessels are known to those skilled in the art and include, but are not limited to, Corning's low-attachment dishes (catalog number 3262).

[0072] In certain embodiments, the culturing in step (1) involves a duration of 3 days to 14 days, such as about 3 days, about 4 days, about 5 days, about 7 days, about 10 days, or about 14 days.

[0073] In certain embodiments, the culturing in step (1) involves a duration of 4 days to 7 days, such as about 5 days.

[0074] In certain embodiments, step (2) includes inoculating the embryoid bodies from step (1) into a culture vessel at a density of about 1 embryoid body / cm 2 of the culture vessel.

[0075] In certain embodiments, step (2) includes culturing the embryoid bodies in a culture vessel coated with gelatin, collagen type I, collagen type IV, vitronectin, fibronectin, or polylysine.

[0076] In certain embodiments, step (2) includes culturing the embryoid bodies in a culture vessel coated with vitronectin.

[0077] In certain embodiments, the culturing in step (2) involves a duration of 10 to 21 days, such as about 10 days, about 14 days, or about 21 days.

[0078] In certain embodiments, the culturing in step (2) involves a duration of 10 to 14 days, such as about 14 days.

[0079] In certain embodiments, step (2) includes replacing the used culture medium with fresh second culture medium daily, or every 1 to 7 days (e.g., daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days). In certain embodiments, step (2) includes discarding the used culture medium and replacing it with fresh second culture medium daily, or every 1 to 7 days (e.g., daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days).

[0080] In certain embodiments, in steps (1) to (2), the culturing is carried out under conditions of 37°C and 5% CO2. In certain embodiments, in steps (1) to (2), the culturing is carried out in an incubator at 37°C and 5% CO2.

[0081] In some embodiments, the cells attached to the culture vessel in step (2) are mesenchymal stem cells at P0 (passage 0). In certain embodiments, when the cells attached to the culture vessel as described in step (2) have a confluence density of about 80% or more (e.g., about 85% or more, about 90% or more, or about 95% or more), the cells can be separated from the culture vessel to obtain mesenchymal stem cells at P0 (passage 0).

[0082] Thus, in certain embodiments, the method further includes obtaining mesenchymal stem cells by separating the cells attached to the culture vessel in step (2).

[0083] In certain embodiments, the method further includes passaging the mesenchymal stem cells of step (3).

[0084] In certain embodiments, the cells are passaged when the cells have a confluence of about 80% or more (e.g., about 85% or more, about 90% or more, or about 95% or more).

[0085] In certain embodiments, the mesenchymal stem cells are passaged for 1 passage, 2 passages, 3 passages, 4 passages, or 5 passages.

[0086] Methods for passaging cells are known to those skilled in the art. For example, the method can include separating the cells from the culture vessel, uniformly dispersing the cells in a culture medium, and then inoculating them into a culture vessel. After adding an appropriate amount of medium, an appropriate amount of fresh culture medium is exchanged at regular intervals (e.g., every 1 to 5 days) according to the cell growth state, and the passaging operation is repeated until the cells grow to reach a confluence of 70% to 100%. Each time the cells are passaged, the passage number increases by 1.

[0087] In certain embodiments, the passage is about 5×10 3 cells to 5×10 4 cells / cm 2 (e.g., about 5×10 3 cells / cm2 , about 1×10 4 cells / cm 2 , about 2×10 4 cells / cm 2 , about 3×10 4 cells / cm 2 , about 4×10 4 cells / cm 2 , or about 5×10 4 cells / cm 2 ) including performing passage at a cell density of

[0088] In certain embodiments, passage includes inoculating cells into a second culture medium and culturing them.

[0089] In certain embodiments, separation includes disrupting the attachment of mesenchymal stem cells to the culture vessel by the following methods: (i) contacting the culture with one or more enzymes selected from the group consisting of trypsin or its analogs, collagenase, dispase, papain, a mixture of collagenase and dispase, and a mixture of collagenase and trypsin or its analogs, (ii) performing mechanical separation by using a cell scraper or the like, or (iii) contacting the culture with EDTA or EGTA.

[0090] In certain embodiments, separation includes disrupting the attachment of mesenchymal stem cells to the culture vessel by enzymatic digestion.

[0091] In certain embodiments, the enzyme is trypsin (e.g., Gibco: catalog number 25200072).

[0092] Optionally, after obtaining umbilical cord mesenchymal stem cells by culture, the cell growth curve can be determined by methods such as the MTT method, WST method, DNA content detection method, ATP detection method, etc. to evaluate the growth activity of umbilical cord mesenchymal stem cells. Furthermore, the isolated and cultured umbilical cord mesenchymal stem cells can be identified by flow cytometry detection of cell surface markers, three-directional differentiation assay, and detection of cell-expressed genes by PCR.

[0093] Prior to step (1), stem cells (e.g., totipotent or pluripotent stem cells such as embryonic stem cells, haploid stem cells, induced pluripotent stem cells, or adult stem cells) can be expanded and maintained using any culture method known in the art. For example, embryonic stem cells can be cultured in the presence of feeder cells such as mouse cells (e.g., mouse embryonic fibroblasts (MEFs)), human feeder cells (e.g., adult skin cells, neonatal dermal fibroblasts (HNDF), etc.). For example, embryonic stem cells can be cultured in xenobiotic-free cultures and / or under feeder cell-free conditions. See Klimanskaya et al, Lancet. 2005 May 7 to 13; 365(9471):1636-41, Richards et al, stem cell Cells. 2003; 21(5): 546-56, U.S. Patent No. 7,410,798, Ilic et al., stem cells Dev., 2009 Nov; 18(9): 1343-5, Xu et al., Nat Biotechnol., 2001 Oct;19(10): 971-4 (each of these references is hereby incorporated by reference in its entirety). For example, embryonic stem cells can be cultured on a matrix, which can be selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, nestin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (a soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof.

[0094] In a third aspect, the invention also relates to a mesenchymal stem cell population produced by the method of the second aspect.

[0095] In certain embodiments, the mesenchymal stem cell population is as defined in the first aspect.

[0096] Kit In a fourth aspect, the present invention provides a kit comprising a first culture medium and a second culture medium prepared separately, wherein the first culture medium is a basal medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and bFGF; the second culture medium is a basal medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and one or more growth factors.

[0097] In certain embodiments, the first culture medium and the second culture medium are as defined in any one of the embodiments of the second aspect.

[0098] Culture and culture supernatant In a fifth aspect, the present invention provides a culture comprising a mesenchymal stem cell population of the first aspect or the third aspect and a culture medium.

[0099] The culture medium can be any culture medium used for culturing stem cells, examples of which include KO-DMEM, KO-DMEM / F12 (an equal mixture of KO-DMEM and F-12), α-MEM, DMEM / F-12 (an equal mixture of DMEM and F-12), DMEM, IMDM, F-12, RPMI1640, and mixed culture media formed by any combination of the above. The above culture media may optionally further contain supplementary substances such as serum (e.g., fetal bovine serum, human serum, goat serum, etc.), serum substitutes (e.g., KOSR, etc.), bovine serum albumin (BSA), antibiotics, vitamins, minerals, etc.

[0100] In certain embodiments, the culture medium is the second culture medium as defined in any one of the embodiments of the second aspect.

[0101] The culture of the present invention can be formulated and administered as a pharmaceutical composition. Such a pharmaceutical composition can be in any form known in the medical field, preferably injection (including injection solution and lyophilized powder). In certain preferred embodiments, the pharmaceutical composition includes a sterile, pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. The general principles regarding the formulation of such pharmaceutical compositions can be found in Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0102] In a sixth aspect, the present invention provides a culture supernatant that is the supernatant of the culture described in the fifth aspect, or a culture supernatant produced by culturing the mesenchymal stem cell population described in the first aspect or the third aspect in a culture medium.

[0103] In certain embodiments, the culture medium is the second culture medium defined in any one of the embodiments of the second aspect.

[0104] In certain embodiments, the culture supernatant does not contain a mesenchymal stem cell population.

[0105] The culture supernatant of the present invention can be formulated and administered as a pharmaceutical composition. Such a pharmaceutical composition can be in any form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), and other forms.

[0106] In a seventh aspect, the present invention also provides a method for preparing the culture supernatant described herein, the method comprising the following steps: (1) culturing a mesenchymal stem cell population of the first aspect or the third aspect; (2) recovering the supernatant of the culture obtained in step (1) (i.e., the culture supernatant); and relates to a method comprising these steps.

[0107] In certain embodiments, the method further comprises (3) subjecting the supernatant obtained in step (2) to a treatment, where the treatment is selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, and any combination thereof.

[0108] In the present invention, the mesenchymal stem cell population of the present invention can be cultured using any culture medium and culture conditions known in the art that can be used to culture stem cells. In certain embodiments, the mesenchymal stem cell population is cultured in step (1) using a second culture medium as defined in any one of the embodiments of the second aspect.

[0109] In certain embodiments, the culture supernatant of the present invention is serum-free for improved safety. Thus, in certain exemplary embodiments, in step (1), the mesenchymal stem cell population can be cultured using a serum-free culture medium (e.g., a basal medium or a serum-free medium), so that a serum-free culture supernatant is obtained. In such embodiments, the serum-free culture medium can be used throughout the culture process or in the culture in the last passage or the last few passages. In certain exemplary embodiments, a serum-free culture supernatant can also be obtained by subjecting the culture supernatant obtained in step (2) to dialysis or solvent replacement to remove serum.

[0110] Microcarriers and cryopreservation methods In an eighth aspect, the present invention also relates to a method of culturing a mesenchymal stem cell population of the first or third aspect, which includes using microcarriers. In certain embodiments, examples of microcarriers include carrier tables (e.g., TableTrix), carrier spheres (e.g., CultiSpher, Coring, Cytodex 1, Cytodex 2, Cytodex 3, Solohill, Cytopore, Cytoline), etc., or liquid microcarriers, macroporous gelatin microcarriers, polystyrene microcarriers, PHEMA microcarriers, chitin microcarriers, polyurethane foam microcarriers, alginate gel microcarriers, and magnetic microcarriers, etc.

[0111] In certain embodiments, the microcarriers of the present invention can improve the survival rate of the mesenchymal stem cell population after cryopreservation. In certain embodiments, the mesenchymal stem cell population and / or the mesenchymal stem cell population cultured on the microcarriers can recover at least 50%, 60%, 70%, 80%, 90% survival rate after cryopreservation for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 month, 2 months, 3 months, half a year, and 1 year within the range of 37°C ± 3°C, 37°C ± 2°C, or 37°C ± 1°C.

[0112] In certain embodiments, the microcarriers contain, consist of, or consist essentially of substances selected from the group consisting of proteins, cellulose, polyethylene, polystyrene, glass, dextran, diethylaminoethanol (DEAE)-dextran, collagen, collagen-glucose-amino glycans, gelatin, acrylamide (e.g., polyacrylamide), and any combination thereof.

[0113] In certain embodiments, the microcarriers do not have a matrix coating. In certain embodiments, the surface of the microcarriers is coated with a matrix.

[0114] In certain embodiments, examples of the matrix include the extracellular matrix. In certain embodiments, the matrix comprises one or more selected from the group consisting of Matrigel (trademark) (BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, collagen, elastin, heparan sulfate, dextran, dextran sulfate, and chondroitin sulfate.

[0115] In certain embodiments, the microcarriers are non-porous microcarriers. In certain embodiments, the microcarriers are porous microcarriers.

[0116] In certain embodiments, culturing on the microcarriers is performed under static culture conditions.

[0117] In certain embodiments, culturing on the microcarriers is performed under dynamic culture conditions.

[0118] In certain embodiments, culturing is performed by a method that includes culturing a mesenchymal stem cell population on microcarriers in a culture medium, where the growth culture medium is a basal medium containing the following substances: one or more serum replacements, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and one or more growth factors. In certain embodiments, the growth culture medium is the second culture medium described in any one of the embodiments of the second aspect.

[0119] Pharmaceutical composition In a ninth aspect, the present invention provides a pharmaceutical composition comprising at least one selected from the mesenchymal stem cell population described in the first aspect or the third aspect, the culture described in the fifth aspect, and the culture supernatant described in the sixth aspect. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, or other adjuvants that may be required.

[0120] As used herein, the term "adjuvant" refers to a substance other than the main drug required in the preparation or formulation of a pharmaceutical preparation. Generally, such substances are required to have no physiological activity and not to affect the effectiveness, determination of content, and stability of the drug in the pharmaceutical preparation. The main purpose of adding an adjuvant is to facilitate the preparation and clinical use of the preparation. Preferably, the adjuvant used in the pharmaceutical composition of the present invention is pharmaceutically acceptable and compatible with the active ingredient.

[0121] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the mesenchymal stem cell population and / or the culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0122] In some embodiments, the pharmaceutical composition includes, but is not limited to, collagen (e.g., collagen gel, collagen scaffold, gelatin microcarrier), gelatin (e.g., gelatin gel, gelatin electrospinning silk, gelatin scaffold, gelatin microcarrier, etc.), aminated gelatin (e.g., aminated gelatin gel, aminated gelatin electrospinning silk, aminated gelatin scaffold, aminated gelatin microcarrier, etc.), chitosan (e.g., chitosan gel, chitosan scaffold, etc.), decellularized scaffold (e.g., decellularized uterine scaffold, decellularized cardiac scaffold, etc.), skin repair membrane, bone repair membrane, oral repair membrane, cellulose, fibrin, polylactic acid, cellulose polylactic acid, polyurethane, tropoelastin, hyaluronic acid, sodium alginate, polyethylene oxide, polyethylene glycol, poly(lactic-co-glycolic acid), poly(ε-caprolactone), silicate, silicone rubber, extracellular matrix, or any combination thereof, etc., pharmaceutically acceptable biomaterials. In some embodiments, the pharmaceutical composition includes a sterilized pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion.

[0123] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In some preferred embodiments, the pharmaceutical composition is an injection (including injection solution, lyophilized powder) or an aerosol.

[0124] In some embodiments, the pharmaceutical composition is a spray that can be used for skin surface reconstruction or skin transplantation, etc.

[0125] In some embodiments, the pharmaceutical composition can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0126] General principles regarding the formulation of pharmaceutical compositions containing the mesenchymal stem cell population of the present invention can be found in Cell Therapy: stem cell Transplantation, Gene Therapy and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.In some embodiments, the pharmaceutical composition is an injection solution (including normal saline, Ringer's lactate solution, compound electrolyte injection solution, 5% glucose injection solution, 20% HSA injection solution, succinylated gelatin injection solution, succinylated gelatin MIX injection solution, MZJ injection solution 1, MZJ injection solution 2, MZJ injection solution 3, human serum protein injection solution, Plasmalyte-A, potassium chloride injection solution, magnesium sulfate injection solution, sodium bicarbonate injection solution, glucose sodium chloride injection solution, compound sodium chloride injection solution (Ringer's solution), dextran-20 glucose injection solution (small molecule), amino acid injection solution, hydroxyethyl starch-40 sodium chloride injection solution, hydroxyethyl starch-40 sodium chloride injection solution, hydroxyethyl starch-40 sodium chloride injection solution, low molecular weight heparin calcium for injection, heparin sodium injection solution, coenzyme A for injection, cytidine triphosphate disodium, lysine hydrochloride for injection, vitamin C injection solution, citicoline sodium chloride, fat-soluble vitamin II for injection, reduced glutathione for injection, brain protein hydrolysate for injection, deoxynucleotide sodium injection solution, multiple trace element injection solution II, mannitol injection solution, arginine hydrochloride injection solution, potassium chloride injection solution, cytidine triphosphate disodium for injection, ornithine aspartate for injection, etc.), and the following materials: propylene glycol, sodium bicarbonate, cholesterol, heparin, FBS, culture medium, dimethyl sulfoxide, sodium glycerophosphate solution, hydroxyethyl starch, mannitol solution, ethylene glycol, polyvinyl alcohol, trehalose, polyvinyl pyrrolidone, exosome solution derived from human umbilical cord mesenchymal stem cells, short peptide or polypeptide compound solution derived from human umbilical cord mesenchymal stem cells, sodium lactate, mannitol, dextran, potassium chloride, calcium chloride, azone, a mixture containing one or more of low molecular dextran. In some embodiments, the above cells can maintain a survival rate of at least 50%, 60%, 70%, 80%, or 90% at about 4°C within the range of 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day after non-freezing storage or freezing storage in the pharmaceutical composition.

[0127] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial (e.g., but not limited to, collagen scaffold, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid).

[0128] In some embodiments, the pharmaceutical composition comprises a sterile, pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion.

[0129] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In some preferred embodiments, the pharmaceutical composition is an injection (including injection solution, lyophilized powder) or an aerosol.

[0130] In some embodiments, the pharmaceutical composition is an injection such as a solution-type injection.

[0131] In some embodiments, the injection comprises one or more additives for injection selected from, for example, solubilizers, wetting agents, emulsifying agents, buffering agents, suspending agents, chelating agents, antioxidants, bacteriostatic agents, local anesthetics, isotonicity modifiers, fillers, protective agents, and any combination thereof.

[0132] In some embodiments, the injection comprises an isotonic solution or a hypertonic solution, and preferably, the solution is selected from NaCl injection solution (e.g., 0.9% - 2.7% NaCl injection solution), glucose injection solution (e.g., 4% - 5% glucose injection solution), sodium lactate Ringer's injection solution, compound electrolyte injection solution, HSA injection solution (e.g., 10% - 20% HSA injection solution), succinylated gelatin injection solution (e.g., 4% - 5% succinylated gelatin injection solution), and any combination thereof.

[0133] In some embodiments, the mesenchymal stem cells are 1×10 4 or more cells / ml (e.g., 1×10 4 or more cells / ml, 3×10 4 or more cells / ml, 5×10 4 or more cells / ml, 7×10 4 or more cells / ml, 1×10 5 or more cells / ml, 3×10 5 or more cells / ml, 5×10 5 or more cells / ml, 7×10 5 or more cells / ml, 1×10 6 or more cells / ml, 3×10 6 or more cells / ml, 5×10 6 or more cells / ml, 7×10 6 or more cells / ml, 1×10 7 or more cells / ml, 3×10 7 or more cells / ml, 5×10 7 or more cells / ml, 7×10 7 or more cells / ml, 1×10 8 or more cells / ml, 3×10 8 or more cells / ml, 5×10 8 or more cells / ml, 7×10 8 or more cells / ml, 1×10 9 or more cells / ml, 3×10 9 or more cells / ml, 5×10 9 or more cells / ml, 7×10 9 or more cells / ml, 1×10 10 or more cells / ml, 3×10 10 or more cells / ml, 5×10 10 or more cells / ml, or 7×10 10 or more cells / ml. As another example, 1×10 5 to 1×10 8 cells, 7×10 5 to 7×10 6 cells, 1×10 6 to 5×10 6 cells, preferably 1×10 6 cells, 3×10 6 cells, 5×106 cells / ml, more preferably 3×10 6 administered at a dose of cells / ml).

[0134] In some embodiments, the mesenchymal stem cells are 1×10 3 or more cells / kg (e.g., 1×10 3 or more cells / kg, 3×10 3 or more cells / kg, 5×10 3 or more cells / kg, 7×10 3 or more cells / kg, 1×10 4 or more cells / kg, 3×10 4 or more cells / kg, 5×10 4 or more cells / kg, 7×10 4 or more cells / kg, 1×10 5 or more cells / kg, 3×10 5 or more cells / kg, 5×10 5 or more cells / kg, 7×10 5 or more cells / kg, 1×10 6 or more cells / kg, 3×10 6 or more cells / kg, 5×10 6 or more cells / kg, 7×10 6 or more cells / kg, 1×10 7 or more cells / kg, 3×10 7 or more cells / kg, 5×10 7 or more cells / kg, 7×10 7 or more cells / kg, 1×10 8 or more cells / kg, 3×10 8 or more cells / kg, 5×10 8 or more cells / kg, 7×10 8 or more cells / kg, 1×10 9 or more cells / kg, 3×10 9 or more cells / kg, 5×10 9 or more cells / kg, 7×10 9 or more cells / kg, 1×10 10 or more cells / kg, 3×10 10 or more cells / kg, 5×10 10 or more cells / kg, or 7×1010 more than 1 cell / kg, as another example, 1×10 5 cells to 1×10 8 cells, 7×10 5 cells to 7×10 6 cells, 1×10 6 cells to 5×10 6 cells, preferably 1×10 6 cells, 3×10 6 cells, 5×10 6 cells / kg, more preferably 3×10 6 cells / kg) is administered at a dosage.

[0135] In some embodiments, the dosage to be administered is 1×10 4 or more cells per administration (e.g., 1×10 4 or more cells per administration, 3×10 4 or more cells per administration, 5×10 4 or more cells per administration, 7×10 4 or more cells per administration, 1×10 5 or more cells per administration, 3×10 5 or more cells per administration, 5×10 5 or more cells per administration, 7×10 5 or more cells per administration, 1×10 6 or more cells per administration, 3×10 6 or more cells per administration, 5×10 6 or more cells per administration, 7×10 6 or more cells per administration, 1×10 7 or more cells per administration, 3×10 7 or more cells per administration, 5×10 7 or more cells per administration, 7×10 7 or more cells per administration, 1×10 8 or more cells per administration, 3×10 8 or more cells per administration, 5×10 8 or more cells per administration, 7×10 8 or more cells per administration, 1×10 9 or more cells per administration, 3×10 9 or more cells per administration, 5×10 9 or more cells per administration, 7×10 9 or more cells per administration, 1×10 10or more cells per time, 3×10 10 or more cells per time, 5×10 10 or more cells per time, or 7×10 10 or more cells per time), preferably 3×10 6 cells to 6×10 6 cells per time.

[0136] In some embodiments, the injection contains the following functional components: (1) A component for maintaining the activity of mesenchymal stem cells, (2) A component for promoting the proliferation of mesenchymal stem cells, and / or (3) A component for promoting the differentiation of mesenchymal stem cells. It further contains.

[0137] In some embodiments, the functional component is selected from the group consisting of serum replacements, non-essential amino acids, glutamine, a stabilizing dipeptide of L-alanyl-L-glutamine, growth factors, and any combination thereof.

[0138] In some embodiments, the functional component is selected from the group consisting of KOSR, MSC serum-free supplement, Ultraser™ G, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, VEGF, bFGF, EGF, TGFβ, PDGF, and any combination thereof.

[0139] In some embodiments, the pharmaceutical composition is a spray that can be used for skin surface reconstruction or skin transplantation, etc.

[0140] In some embodiments, the pharmaceutical composition can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0141] General principles regarding the formulation of pharmaceutical compositions containing the mesenchymal stem cell population of the present invention can be referred to Cell Therapy: stem cell Transplantation, Gene Therapy and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.In some embodiments, the pharmaceutical composition is an injection solution (including normal saline, Ringer's lactate solution, compound electrolyte injection solution, 5% glucose injection solution, 20% HSA injection solution, succinylated gelatin injection solution, succinylated gelatin MIX injection solution, MZJ injection solution 1, MZJ injection solution 2, MZJ injection solution 3, human serum protein injection solution, Plasmalyte-A, potassium chloride injection solution, magnesium sulfate injection solution, sodium bicarbonate injection solution, glucose sodium chloride injection solution, compound sodium chloride injection solution (Ringer's solution), dextran-20 glucose injection solution (small molecule), amino acid injection solution, hydroxyethyl starch-40 sodium chloride injection solution, hydroxyethyl starch-40 sodium chloride injection solution, hydroxyethyl starch-40 sodium chloride injection solution, low molecular weight heparin calcium for injection, heparin sodium injection solution, coenzyme A for injection, disodium cytidine triphosphate, lysine hydrochloride for injection, vitamin C injection solution, citicoline sodium chloride, fat-soluble vitamin II for injection, reduced glutathione for injection, brain protein hydrolysate for injection, deoxynucleotide sodium injection solution, multiple trace element injection solution II, mannitol injection solution, arginine hydrochloride injection solution, potassium chloride injection solution, disodium cytidine triphosphate for injection, ornithine aspartate for injection, etc.), and the following materials: propylene glycol, sodium bicarbonate, cholesterol, heparin, FBS, culture medium, dimethyl sulfoxide, sodium glycerophosphate solution, hydroxyethyl starch, mannitol solution, ethylene glycol, polyvinyl alcohol, trehalose, polyvinylpyrrolidone, exosome solution derived from human umbilical cord mesenchymal stem cells, short peptide or polypeptide compound solution derived from human umbilical cord mesenchymal stem cells, sodium lactate, mannitol, dextran, potassium chloride, calcium chloride, azone, a mixture containing one or more of low molecular dextran. In some embodiments, the above cells can maintain a survival rate of at least 50%, 60%, 70%, 80%, or 90% at about 4°C within the range of 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day after non-freezing storage or freezing storage in the pharmaceutical composition.

[0142] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial (e.g., but not limited to, collagen scaffold, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid).

[0143] Biological scaffold In one aspect, the present invention provides an article comprising a mesenchymal stem cell population and a biological scaffold, wherein the mesenchymal stem cell population has an average MMP1 expression level that is at least about 10 times that of primary mesenchymal stem cells, and / or the mesenchymal stem cell population has an average PGE2 expression level that is at least about 10 times that of primary mesenchymal stem cells.

[0144] In another aspect, the present invention provides an article comprising a mesenchymal stem cell population and a biological scaffold, wherein the mesenchymal stem cell population has an average MMP1 expression level that is at least about 10 times that of primary mesenchymal stem cells, and optionally, the mesenchymal stem cell population has an average PGE2 expression level that is at least about 10 times that of primary mesenchymal stem cells.

[0145] In certain embodiments, the mesenchymal stem cell population is prepared in vitro.

[0146] In certain embodiments, the mesenchymal stem cell population is as defined or described above.

[0147] In certain embodiments, the mesenchymal stem cell population is partially or fully loaded onto the biological scaffold.

[0148] In certain embodiments, the biological scaffold is prepared using a biodegradable or non-biodegradable material.

[0149] In certain embodiments, the biological scaffold is prepared using a naturally occurring material, an artificially synthesized material, a recombinantly produced material, a modified material, or any combination thereof.

[0150] In certain embodiments, the naturally occurring materials are selected from the group consisting of collagen (e.g., type I collagen, type II collagen, type III collagen), fibrin, silk protein, cellulose, chitosan, alginate (e.g., sodium alginate), starch, hyaluronic acid, laminin, elastin, agarose, gelatin, dextran, extracellular matrix, silicate, or any combination thereof.

[0151] In certain embodiments, the artificially synthesized materials are selected from the group consisting of polyphosphazene, polyacrylic acid and its derivatives (e.g., polymethacrylic acid, copolymers of acrylic acid and methacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA), copolymer of polylactic acid - glycolic acid (PLGA), polyorthoester (POE), polycaprolactone (PCL), polyhydroxybutyrate (PHB), polyamino acids (e.g., polylysine), polyurethane, polyethylene oxide, polyethylene glycol, polylactic acid - glycolic acid, silicone rubber, decellularized scaffold, or any combination thereof.

[0152] In certain embodiments, the modified materials are selected from the group consisting of modified alginate, modified gelatin, or combinations thereof, preferably, the modified alginate is oxidized alginate (e.g., sodium oxidized alginate), and preferably, the modified gelatin is aminated gelatin.

[0153] In certain embodiments, the materials used to prepare the biological scaffold are selected from the group consisting of collagen, aminated gelatin, chitosan, or any combination thereof.

[0154] In certain embodiments, the biological scaffold is solid or semi - solid (e.g., gel).

[0155] In certain embodiments, the biological scaffold has a layered structure (e.g., single layer, bilayer, or multilayer, such as a biofilm, skin repair membrane), or a sheet-like structure (e.g., a sheet-like structure in a rectangular, square, circular, elliptical, hexagonal, or irregular shape), or a hollow tubular structure or a hollow annular (e.g., circular ring-shaped) structure, or a hollow three-dimensional structure (e.g., a hollow cube, hollow sphere, hollow rectangular column, hollow cylinder, or hollow irregular three-dimensional structure), or a solid three-dimensional structure (e.g., a solid cube, solid sphere, solid rectangular column, solid cylinder, or solid irregular three-dimensional structure), or any combination thereof.

[0156] In certain embodiments, the biological scaffold mimics the shape of a natural tissue or organ.

[0157] In certain embodiments, the biological scaffold has a sheet-like structure, a layered structure, or a hollow annular (e.g., circular ring-shaped) structure.

[0158] In certain embodiments, the biological scaffold is selected from the group consisting of a collagen scaffold, a skin repair membrane, a gelatin scaffold, an aminated gelatin scaffold, and a chitosan scaffold.

[0159] In certain embodiments, the biological scaffold contains 1×10 4 or more cells / ml (e.g., 1×10 4 or more cells / ml, 3×10 4 or more cells / ml, 5×10 4 or more cells / ml, 7×10 4 or more cells / ml, 1×10 5 or more cells / ml, 3×10 5 or more cells / ml, 5×10 5 or more cells / ml, 7×10 5 or more cells / ml, 1×10 6 or more cells / ml, 3×10 6 or more cells / ml, 5×10 6 or more cells / ml, 7×10 6 or more cells / ml, 1×10 7 or more cells / ml, 3×10 7 or more cells / ml, 5×10 7cells / ml or more, 7×10 7 cells / ml or more, 1×10 8 cells / ml or more, 3×10 8 cells / ml or more, 5×10 8 cells / ml or more, 7×10 8 cells / ml or more, 1×10 9 cells / ml or more, 3×10 9 cells / ml or more, 5×10 9 cells / ml or more, 7×10 9 cells / ml or more, 1×10 10 cells / ml or more, 3×10 10 cells / ml or more, 5×10 10 cells / ml or more, or 7×10 10 cells / ml or more), the mesenchymal stem cells are loaded in an amount of.

[0160] In certain embodiments, the article further comprises an additional active ingredient.

[0161] In certain embodiments, the additional active ingredient is partially or entirely loaded onto the biological scaffold.

[0162] In certain embodiments, the additional active ingredient is selected from the group consisting of a drug for treating osteoarthritis (e.g., meniscus injury, bone injury), a drug for treating heart disease (e.g., myocardial infarction), a drug for treating nervous system diseases (e.g., spinal cord injury), a drug for treating skin diseases (e.g., skin injury, burn, scald), a drug for treating eye diseases (e.g., corneal alkali burn), or any combination thereof.

[0163] In certain embodiments, the drug for treating osteoarthritis is selected from glucosamine sulfate capsules, aminated chondroitin sulfate, sodium hyaluronate injection, anti-osteoporosis tablets, ossotide tablets, Henggu wound healing agent, Gentongping granules, non-steroidal anti-inflammatory drugs (e.g., loxoprofen sodium tablets, diclofenac sodium sustained release tablets, celecoxib, meloxicam, indomethacin tablets, voltaren ointment), or any combination thereof.

[0164] In certain embodiments, the drug for treating heart disease is selected from the group consisting of aspirin, clopidogrel, ticagrelor, ACE1, ARBs, beta blockers, calcium antagonists, nitrate vasodilators, trimetazidine hydrochloride, nicorandil, lidocaine, amiodarone, quinidine, or any combination thereof.

[0165] In certain embodiments, the drug for treating nervous system diseases is selected from the group consisting of carbamazepine, phenobarbital, phenytoin, sodium valproate, clonazepam, lamotrigine, oxcarbazepine, donepezil, memantine, vitamin B1, vaccinia vaccination of rabbits inflammation of the skin extract, alteplase, aspirin, clopidogrel, low molecular weight heparin, edaravone, urinary kallidinogenase, butylphthalide, injectable gamma globulin, pyridostigmine, glucocorticoid, or any combination thereof.

[0166] In certain embodiments, the drug for treating skin diseases is selected from the group consisting of ebastine tablets, loratadine tablets, cetirizine tablets, mometasone furoate ointment, halometasone ointment, mupirocin ointment, fusidic acid ointment, cefixime tablets, roxithromycin tablets, naftifine ketoconazole ointment, sertaconazole ointment, itraconazole tablets, terbinafine tablets, acyclovir tablets, valacyclovir tablets, penciclovir ointment, interferon gel, or any combination thereof.

[0167] In certain embodiments, the drug for treating eye diseases is an antibacterial and anti-inflammatory drug.

[0168] In certain embodiments, the article further comprises a component for culturing / differentiating cells.

[0169] In certain embodiments, the components for culturing / differentiating cells are selected from the group consisting of serum replacements, non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, growth factors, or any combination thereof.

[0170] In certain embodiments, the serum replacement is selected from the group consisting of KOSR, MSC serum-free supplement, Ultroser(™)G, or any combination thereof.

[0171] In certain embodiments, the non-essential amino acids are selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, or any combination thereof.

[0172] In certain embodiments, the growth factors are selected from the group consisting of VEGF, bFGF, EGF, TGFβ, PDGF, or any combination thereof.

[0173] In another aspect, the present invention provides the use of the above-described article in the manufacture of a medicament for treating and / or preventing osteoarthritis (e.g., meniscus injury, bone injury), heart disease (e.g., myocardial infarction), nervous system diseases (e.g., spinal cord injury), skin diseases (e.g., skin injury, burns, scalds), eye diseases (e.g., corneal alkali burn), or any combination thereof in a subject.

[0174] In certain embodiments, the medicament is used for the treatment and / or prevention of spinal cord injury, skin injury, corneal alkali burn, or any combination thereof in a subject.

[0175] In another aspect, the present invention provides a method for treating and / or preventing a disease in a subject, which comprises administering (e.g., transplanting or adhering, preferably transplanting) the above-described article to a subject in need thereof, wherein the disease is selected from the group consisting of osteoarthritis (e.g., meniscus injury, bone injury), heart disease (e.g., myocardial infarction), nervous system disease (e.g., spinal cord injury), skin disease (e.g., skin injury, burn, scald), eye disease (e.g., corneal alkali burn), or any combination thereof.

[0176] In certain embodiments, the disease is selected from the group consisting of spinal cord injury, skin injury, corneal alkali burn, or any combination thereof.

[0177] Use for the prevention and / or treatment of a disease In a tenth aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein for the prevention and / or treatment of a disease in a subject, or the use for the manufacture of a medicament for preventing and / or treating a disease in a subject, and a method for preventing and / or treating a disease in a subject, which comprises administering to a subject in need thereof the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition of the present invention.

[0178] In certain embodiments, the subject is a mammal such as a human.

[0179] In certain embodiments, the disease is selected from the group consisting of osteoarthrosis (e.g., meniscus injury, osteoarthritis, or bone injury, etc.), genital system diseases (e.g., ovarian aging, ovarian insufficiency, endometrial injury, uterine trauma, intrauterine adhesions, or uterine thinning, etc.), heart diseases (e.g., myocardial infarction, etc.), lung diseases (e.g., idiopathic pulmonary fibrosis, acute respiratory distress syndrome, pneumoconiosis, or pneumonia, etc.), skin diseases (e.g., psoriasis, skin lesions, pressure ulcers, decubitus ulcers, or burns, etc.), eye diseases (e.g., corneal injury, etc.), nervous system diseases (e.g., spinal cord injury, cerebral palsy, stroke, Alzheimer's disease, senile dementia, or neuropathic pain, etc.), digestive system diseases (e.g., inflammatory bowel disease, colitis, Crohn's disease, or irritable bowel syndrome, etc.), kidney diseases (e.g., anti-glomerular basement membrane disease, diabetic nephropathy, lupus nephritis, or acute nephritis, etc.), liver diseases (liver injury, liver fibrosis, hepatitis, cirrhosis, or liver failure, etc.), autoimmune diseases (e.g., scleroderma, lupus erythematosus, or multiple sclerosis, etc.), transplant rejection reactions (e.g., graft-versus-host disease, etc.), and metabolic diseases (e.g., diabetes, etc.).

[0180] In the present invention, the mesenchymal stem cell population or culture described herein, or a pharmaceutical composition containing the mesenchymal stem cell population or culture, can be administered to a subject by various suitable means. In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory pathway transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0181] In certain embodiments, the pharmaceutical composition contains a therapeutically effective amount of the culture supernatant described herein. The culture supernatant of the present invention can be formulated and administered as a drug. Such a pharmaceutical composition can contain a therapeutically effective amount of the culture supernatant.

[0182] In the present invention, the culture supernatant of the present invention, or a pharmaceutical composition containing the culture supernatant, can be administered to a subject in various appropriate manners. In certain preferred embodiments, the culture supernatant of the present invention, or a pharmaceutical composition containing the culture supernatant, can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, or the like.

[0183] Female genital system diseases In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing, treating, delaying, and / or alleviating female genital system diseases. Alternatively, the present invention provides a method for preventing, treating, delaying, and / or alleviating diseases of the female genital system, which includes administering a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant to a subject in need thereof.

[0184] In certain specific embodiments, female genital system diseases include: (1) gynecological inflammations such as vulvitis, vaginitis, cervicitis, endometritis, metritis, and pelvic inflammatory disease, adnexitis, etc.; (2) gynecological tumors such as benign tumors and malignant tumors; (3) menstrual disorders such as dysmenorrhea, increased menstrual bleeding, decreased menstrual bleeding, etc.; (4) infertility caused by ovulatory dysfunction, infertility caused by fallopian tube obstruction, immune infertility, etc. In certain specific embodiments, female genital system diseases are selected from the group consisting of ovarian aging, ovarian insufficiency, endometrial injury, uterine trauma, intrauterine adhesions, and uterine thinning.

[0185] In certain specific embodiments, endometrial injury is mainly damage to the basal layer of the endometrium manifested by irregular menstrual cycles, or less overall menstrual bleeding and shortened menstrual bleeding periods. Generally, most endometrial injuries occur after abortion, including induced abortion or medical abortion. Additionally, related intrauterine surgeries can also cause endometrial injury.

[0186] In certain specific embodiments, endometritis is an inflammation of the endometrium. Endometritis can be divided into acute endometritis and chronic endometritis according to the duration of the disease.

[0187] In certain embodiments, "intrauterine adhesions" is a type of uterine disease belonging to female genital system diseases, caused by various factors including intrauterine trauma during pregnancy and non-pregnancy, resulting in damage to the basal layer of the endometrium, so that the uterine cavity and / or the cervical canal are partially or completely blocked, the uterine walls adhere to each other, thus referring to endometrial damage that causes abnormal menstruation, infertility, or recurrent miscarriage, etc., which usually has no typical symptoms. Its nature is endometrial fibrosis. Examples include endometrial lesions, intrauterine foreign bodies, benign endometrial lesions, uterine malformations, malignant endometrial lesions, etc.

[0188] In certain embodiments, since the mesenchymal stem cell population of the present invention is administered in combination with an additional active ingredient, the medicament may contain an additional active ingredient. In certain embodiments, the mesenchymal stem cells are administered simultaneously, separately, or sequentially with an additional therapeutic agent. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation.

[0189] In certain embodiments, the additional active ingredient is selected from the group consisting of estrogen, anti-estrogen or selective estrogen receptor modulator, androgen, anti-androgen, or progestin.

[0190] In certain embodiments, the medicament is in unit dosage form, and the unit dose of the medicament is 1×10 4 or more cells / ml (for example, 1×10 4 or more cells / ml, 3×10 4 or more cells / ml, 5×10 4 or more cells / ml, 7×10 4 or more cells / ml, 1×10 5 or more cells / ml, 3×10 5 or more cells / ml, 5×10 5 or more cells / ml, 7×10 5 or more cells / ml, 1×10 6cells / ml or more, 3×10 6 cells / ml or more, 5×10 6 cells / ml or more, 7×10 6 cells / ml or more, 1×10 7 cells / ml or more, 3×10 7 cells / ml or more, 5×10 7 cells / ml or more, 7×10 7 cells / ml or more, 1×10 8 cells / ml or more, 3×10 8 cells / ml or more, 5×10 8 cells / ml or more, 7×10 8 cells / ml or more, 1×10 9 cells / ml or more, 3×10 9 cells / ml or more, 5×10 9 cells / ml or more, 7×10 9 cells / ml or more, 1×10 10 cells / ml or more, 3×10 10 cells / ml or more, 5×10 10 cells / ml or more, or 7×10 10 cells / ml) and is contained in an amount of. In certain embodiments, the unit dose of the medicament is 1×10 4 cells to 1×10 10 cells (for example, 1×10 6 cells to 1×10 8 cells, 1×10 6 cells to 1×10 7 cells, or 1×10 6 cells to 5×10 6 cells) and is contained in an amount of.

[0191] In another aspect, the present invention also provides a product for preventing, treating, delaying, and / or alleviating female genital system diseases, which comprises the mesenchymal stem cell population of the present invention. In certain embodiments, the product further comprises an additional active ingredient as defined above. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation. In certain embodiments, the product is an injection, microinjection, mucosal patch, enema, suppository, gel, oral preparation, aerosol, drop, ointment, implant, or capsule, preferably an injection. In certain embodiments, the product is an implant.

[0192] Intrauterine adhesion Intrauterine adhesions (including partial or complete obstruction of the uterine cavity caused by intrauterine fibrosis, endometrial injury, and later inducing oligomenorrhea, amenorrhea, infertility, or recurrent miscarriage, etc.). In recent years, due to the frequent operations in the uterine cavity and the popularization of hysteroscopic surgery, the incidence rate and detection rate of intrauterine adhesions have gradually increased, the onset age has become younger, and it has become the second main cause of secondary infertility in women. Clinicians have been continuously exploring new treatment options, but the cure rate and pregnancy rate have not been much improved, and its recurrence rate is high (the recurrence rate of mild patients after treatment is high, and the recurrence rate of severe patients after treatment is even higher), which thereby causes obstetric complications such as infertility, recurrent miscarriage, premature birth, placenta previa, placental adhesion, or placental implantation, etc., which poses a serious threat to women's reproductive health. Its high incidence rate and the resulting damage to women's reproductive function have become an urgent clinical problem to be solved. The current clinical treatment aims to restore the shape of the uterine cavity, prevent the recurrence of adhesions, promote the repair and regeneration of the damaged endometrium, and restore normal reproductive function. The treatment process includes hysteroscopic separation of intrauterine adhesions, intraoperative placement of intrauterine devices, and postoperative administration of estrogen and progesterone, but there are problems such as a long treatment cycle, a low cure rate, easy recurrence of adhesions, a low pregnancy rate, and the application of high-dose estrogen that increases the risk of breast tumors and endometrial tumors in patients. In the case of severe muscle tissue or connective tissue adhesions, the basal layer of the endometrium has been damaged and may not respond sufficiently to estrogen and progesterone.

[0193] So far, many studies on the etiology of this disease have been carried out by scholars at home and abroad, and it has been agreed that the disorder of endometrial repair may be the main mechanism of formation. For example, after abortion or other uterine cavity operations, due to several pathological factors, the repair of the endometrium is hindered, resulting in scar formation and adhesions.

[0194] In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing, treating, delaying, and / or reducing intrauterine adhesions. Alternatively, the present invention provides a method for preventing, treating, delaying, and / or reducing intrauterine adhesions, the method comprising administering to a subject in need thereof a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant.

[0195] In certain embodiments, the mesenchymal stem cell population of the present invention can thicken the endometrium, increase blood vessels, glands, and cells, improve intrauterine adhesions, restore the shape of the uterine cavity, and / or reduce uterine exudate.

[0196] In certain embodiments, the mesenchymal stem cell population of the present invention can prevent or treat the symptoms of uterine adhesions, for example, reduce the accumulation of uterine exudate.

[0197] In certain embodiments, examples of intrauterine adhesions include uterine adhesions in infertile patients or uterine adhesions caused by abortion.

[0198] In certain embodiments, the mesenchymal stem cell population of the present invention can improve the uterine morphology and suppress intrauterine adhesions.

[0199] In certain embodiments, the mesenchymal stem cell population of the present invention can promote the repair and regeneration of the endometrium, such as the repair and regeneration of damaged endometrium, and enhance the ability to reproduce offspring.

[0200] In certain embodiments, the mesenchymal stem cell population of the present invention can restore uterine scars and prevent or treat intrauterine adhesions.

[0201] In certain embodiments, the subject is a mammal such as a human.

[0202] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art are aware of methods for selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0203] In certain preferred embodiments, the mesenchymal stem cell population of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc.

[0204] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0205] In certain embodiments, the medicament comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion.

[0206] In certain embodiments, the pharmaceutical composition includes a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteogen, hyaluronic acid, and the like.

[0207] In certain embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0208] In certain embodiments, the mesenchymal stem cell population of the present invention is administered in combination with an additional active ingredient, so the medicine may contain an additional active ingredient. In certain embodiments, the mesenchymal stem cells are administered simultaneously, separately, or sequentially with an additional therapeutic agent. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation.

[0209] In certain embodiments, the additional active ingredient is selected from the group consisting of estrogen, anti-estrogen or selective estrogen receptor modulator, androgen, anti-androgen, or progestogen.

[0210] In certain embodiments, the medicine is in unit dosage form, and the unit dose of the medicine is 1×10 4 or more cells / ml (for example, 1×10 4 or more cells / ml, 3×10 4 or more cells / ml, 5×10 4 or more cells / ml, 7×10 4 or more cells / ml, 1×10 5 or more cells / ml, 3×10 5 or more cells / ml, 5×10 5 or more cells / ml, 7×10 5 or more cells / ml, 1×10 6 or more cells / ml, 3×10 6cells / ml or more, 5×10 6 cells / ml or more, 7×10 6 cells / ml or more, 1×10 7 cells / ml or more, 3×10 7 cells / ml or more, 5×10 7 cells / ml or more, 7×10 7 cells / ml or more, 1×10 8 cells / ml or more, 3×10 8 cells / ml or more, 5×10 8 cells / ml or more, 7×10 8 cells / ml or more, 1×10 9 cells / ml or more, 3×10 9 cells / ml or more, 5×10 9 cells / ml or more, 7×10 9 cells / ml or more, 1×10 10 cells / ml or more, 3×10 10 cells / ml or more, 5×10 10 cells / ml or more, or 7×10 10 cells / ml) and is contained in an amount of. In certain embodiments, the unit dose of the medicament is 1×10 4 cells to 1×10 10 cells (for example, 1×10 6 cells to 1×10 8 cells, 1×10 6 cells to 1×10 7 cells, or 1×10 6 cells to 5×10 6 cells) and is contained in an amount of.

[0211] Primary ovarian insufficiency Primary ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40. In the 2015 ESHER guidelines, it is defined as follows: (1) amenorrhea / oligomenorrhea for at least 4 months, (2) blood FSH exceeding 25 U / L twice (the monitoring time interval is at least 4 weeks). POI is characterized by menstrual disorders (amenorrhea or oligomenorrhea), elevated gonadotropins, and low estrogen levels (hot flashes, sweating, facial flushing, low libido, etc.). The incidence of POI is approximately 1%, and the incidence varies slightly among different ethnic groups. The incidence of POI in patients with premature amenorrhea is 10% - 28%, and the incidence of POI in patients with secondary amenorrhea is 4% - 18%.

[0212] The causes of POI include genetic causes, immune causes, iatrogenic causes (radiation therapy, chemotherapy, immunosuppressive therapy, and surgical treatment, etc.), and other causes, but the causes of most POI are unknown. POI may be associated with various endocrine disorders including hypoparathyroidism and adrenal insufficiency. Pelvic surgery may also lead to ovarian function disorders. Since adrenal antibodies or ovarian antibodies are present in approximately 4% of patients with POI, it is suggested that this disease is autoimmune. In many cases, the mechanism is unknown. POI causes the loss of female fertility and may increase the risk of osteoporosis, lipid metabolism disorders, and cardiovascular diseases. Early amenorrhea and the loss of fertility during the reproductive period increase the psychological burden on women and reduce the quality of married life, thus bringing about a series of serious psychological and social problems.

[0213] In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing, treating, delaying, and / or alleviating ovarian insufficiency (e.g., primary ovarian insufficiency), or the present invention provides a method for preventing, treating, delaying, and / or alleviating ovarian insufficiency (e.g., primary ovarian insufficiency), which comprises administering a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant to a subject in need thereof.

[0214] In certain embodiments, the mesenchymal stem cell population of the present invention can improve blood sex hormone (e.g., FSH and E2) levels, increase the number of follicles, improve body weight and ovarian weight, restore ovulation levels, and / or improve fertility levels.

[0215] In certain embodiments, the subject is a mammal such as a human.

[0216] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intraarterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0217] In certain preferred embodiments, the mesenchymal stem cell population of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In certain embodiments, the administration is performed by abdominal injection or intravenous injection.

[0218] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a sterile, pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of such pharmaceutical compositions can be found in Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0219] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastinogen, hyaluronic acid, etc.

[0220] In certain embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0221] In certain embodiments, the mesenchymal stem cell population of the present invention is administered in combination with an additional active ingredient, so the medicament may contain an additional active ingredient. In certain embodiments, the mesenchymal stem cells are administered simultaneously, separately, or sequentially with an additional therapeutic agent. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation.

[0222] In certain embodiments, the additional active ingredient is selected from the group consisting of estrogen, anti-estrogen or selective estrogen receptor modulator, androgen, anti-androgen, or progestogen.

[0223] In certain embodiments, the medicament is in unit dosage form, and the unit dose of the medicament is 1×10 4 or more cells / ml (for example, 1×10 4 or more cells / ml, 3×10 4 or more cells / ml, 5×10 4 or more cells / ml, 7×10 4 or more cells / ml, 1×10 5 or more cells / ml, 3×10 5 or more cells / ml, 5×10 5 or more cells / ml, 7×10 5 or more cells / ml, 1×10 6 or more cells / ml, 3×10 6 or more cells / ml, 5×10 6 or more cells / ml, 7×10 6 or more cells / ml, 1×10 7 or more cells / ml, 3×10 7 or more cells / ml, 5×10 7 or more cells / ml, 7×10 7 or more cells / ml, 1×10 8 or more cells / ml, 3×10 8 or more cells / ml, 5×10 8 or more cells / ml, 7×10 8 or more cells / ml, 1×10 9cells / ml or more, 3×10 9 cells / ml or more, 5×10 9 cells / ml or more, 7×10 9 cells / ml or more, 1×10 10 cells / ml or more, 3×10 10 cells / ml or more, 5×10 10 cells / ml or more, or 7×10 10 cells / ml or more) and is contained in such an amount. In certain embodiments, the unit dose of the medicament contains mesenchymal stem cells in an amount of 1×10 4 cells to 1×10 10 cells (for example, 1×10 6 cells to 1×10 8 cells, 1×10 6 cells to 1×10 7 cells, or 1×10 6 cells to 5×10 6 cells).

[0224] Male genital system diseases Male genital system diseases include abnormal urination, pyuria, abnormal urethral discharge, pain, tumors, sexual dysfunction, and male infertility related to urinary system diseases, mainly including inflammations of the urinary system such as cystitis, urethritis, urinary incontinence, and urinary retention, inflammations of the genital system such as epididymitis, seminal vesiculitis, and prostatitis, genital tuberculosis such as testicular epididymal tuberculosis and seminal vesicle tuberculosis, injuries of the genital system ducts such as testicular contusion, penile fracture, and urethral rupture, male infertility diseases such as varicocele, asthenospermia, congenital ejaculatory duct obstruction, and absence of ejaculatory duct, and male sexual dysfunction diseases such as male erectile dysfunction, premature ejaculation, hypaphrodisia, anejaculation, and delayed ejaculation. In this document, the term "male infertility" refers to infertility caused by male factors. Generally, a woman has not become pregnant after marriage, and at that time, contraceptive measures have not been taken over a cohabitation period exceeding two years. Male infertility includes testicular atrophy, testicular hypoplasia, oligospermia, spermatogenesis failure, azoospermia, obstructive azoospermia, asthenospermia, Klinefelter syndrome, XYY syndrome, Kallmann syndrome, selective LH deficiency and FSH deficiency, adrenocortical hyperplasia, hyperprolactinemia, varicocele, sperm malformation, etc. As used herein, the term "oligospermia" refers to a condition in which the sperm count in semen is less than that in a normal healthy fertile male, including oligospermia caused by endocrine dysfunction, genital system infections, varicocele, anti-sperm antibodies, retained testis, edema, malnutrition, chemotherapy, radiotherapy, obesity, etc.

[0225] In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing, treating, delaying, and / or alleviating male genital system diseases. Alternatively, the present invention provides a method for preventing, treating, delaying, and / or alleviating male genital system diseases, which includes administering a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant to a subject in need thereof.

[0226] In certain embodiments, the male genital system disorder is azoospermia or oligospermia. In certain embodiments, the mesenchymal stem cell population of the present invention can be used in azoospermia to increase sperm concentration, increase sperm motility, restore the testis, and / or restore seminal vesicle function.

[0227] In certain embodiments, the subject is a mammal such as a human.

[0228] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to the subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory pathway transplantation (e.g., intravenous injection transplantation or intraarterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art are aware of methods for selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0229] In certain preferred embodiments, the mesenchymal stem cell population of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In certain embodiments, the administration is performed by testicular injection or seminiferous tubule injection or intravenous injection.

[0230] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition includes a sterile, pharmaceutically acceptable, isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0231] In certain embodiments, the pharmaceutical composition includes a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteogen, hyaluronic acid, etc.

[0232] In certain embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0233] In certain embodiments, the mesenchymal stem cell population of the present invention is administered in combination with an additional active ingredient, so the medicament may contain an additional active ingredient. In certain embodiments, the mesenchymal stem cells are administered simultaneously, separately, or sequentially with an additional therapeutic agent. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation.

[0234] In certain embodiments, the additional active ingredient is selected from the group consisting of estrogen, anti-estrogen or selective estrogen receptor modulator, androgen, and anti-androgen.

[0235] In certain embodiments, the medicament is in unit dosage form, and the unit dose of the medicament is 1×10 4 or more cells / ml (e.g., 1×10 4 or more cells / ml, 3×10 4 or more cells / ml, 5×10 4 or more cells / ml, 7×10 4 or more cells / ml, 1×10 5 or more cells / ml, 3×10 5 or more cells / ml, 5×10 5 or more cells / ml, 7×10 5 or more cells / ml, 1×10 6 or more cells / ml, 3×10 6 or more cells / ml, 5×10 6 or more cells / ml, 7×10 6 or more cells / ml, 1×10 7 or more cells / ml, 3×10 7 or more cells / ml, 5×10 7 or more cells / ml, 7×10 7 or more cells / ml, 1×10 8 or more cells / ml, 3×10 8 or more cells / ml, 5×10 8 or more cells / ml, 7×10 8 or more cells / ml, 1×10 9 or more cells / ml, 3×109 cells / ml or more, 5×10 9 cells / ml or more, 7×10 9 cells / ml or more, 1×10 10 cells / ml or more, 3×10 10 cells / ml or more, 5×10 10 cells / ml or more, or 7×10 10 cells / ml or more) in an amount. In certain embodiments, the unit dose of the medicament contains mesenchymal stem cells in an amount of 1×10 4 cells to 1×10 10 cells (for example, 1×10 6 cells to 1×10 8 cells, 1×10 6 cells to 1×10 7 cells, or 1×10 6 cells to 5×10 6 cells).

[0236] In another aspect, the present invention also provides a product for preventing, treating, delaying, and / or alleviating male genital system diseases, which comprises the mesenchymal stem cell population of the present invention. In certain embodiments, the product further comprises an additional active ingredient, and the additional active ingredient is as defined above. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation. In certain embodiments, the product is an injection, microinjection, mucosal patch, enema, suppository, gel, oral preparation, aerosol, drop, ointment, implant, or capsule. In certain embodiments, the product is an implant.

[0237] Digestive system diseases In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing and / or treating digestive system diseases, or the present invention provides a method for preventing and / or treating digestive system diseases, which comprises administering to a subject in need thereof a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant.

[0238] In certain embodiments, the gastrointestinal disease is selected from the group consisting of esophageal diseases, gastric diseases, intestinal diseases, liver diseases, gallbladder diseases, pancreatic diseases, or any combination thereof.

[0239] Intestinal diseases are selected from the group consisting of duodenal ulcers, functional colonic diseases, intestinal polyps, colon cancer, rectal cancer, inflammatory bowel disease (IBD), colitis, proctitis, irritable bowel syndrome, indigestion, functional constipation, gastroesophageal reflux disease, esophagitis, peritonitis, autoimmune liver diseases, gastritis, tuberculous intestinal diseases, or any combination thereof.

[0240] In certain embodiments, the intestinal disease is an intestinal inflammatory disease.

[0241] In certain embodiments, the intestinal inflammatory disease is selected from the group consisting of inflammatory bowel disease (IBD), colitis, proctitis, or any combination thereof.

[0242] In certain embodiments, the intestinal inflammatory disease is inflammatory bowel disease (IBD).

[0243] Gastrointestinal diseases (gastric diseases, intestinal diseases) mainly refer to common inflammatory gastrointestinal diseases (such as acute gastritis or chronic gastritis, acute appendicitis or chronic appendicitis, etc.), peptic ulcers, gastric cancer, esophageal cancer, colorectal cancer, and irritable bowel syndrome, etc. Inflammatory bowel disease is a disease in the gastrointestinal tract.

[0244] Liver diseases are a general term for all diseases occurring in the liver. Depending on various causes of liver damage, they can be divided into viral liver diseases including hepatitis A, hepatitis B, hepatitis C, hepatitis E, etc., as well as abnormal metabolic liver diseases, alcoholic liver diseases, drug-induced liver diseases or toxic liver diseases, and non-alcoholic fatty liver diseases, autoimmune liver diseases, etc. Liver diseases can be divided into chronic liver diseases and acute liver diseases according to the onset rate.

[0245] Liver diseases are selected from the group consisting of liver injury, liver fibrosis, hepatitis (e.g., viral hepatitis A, viral hepatitis B, viral hepatitis C), cirrhosis, renal insufficiency, liver abscess, liver cyst, intrahepatic hemangioma, liver cancer, intrahepatic bile duct stones, liver fluke disease, hepatic echinococcosis, alcoholic liver disease, non-alcoholic fatty liver disease, or any combination thereof.

[0246] Examples of liver diseases include fatty liver. As used herein, the term "fatty liver" refers to a pathological change caused by excessive accumulation of fat in liver cells due to various causes, which is a common pathological change of the liver rather than an independent disease. Fatty liver is generally divided into two categories: alcoholic fatty liver and non-alcoholic fatty liver.

[0247] Examples of liver diseases include fatty hepatitis. As used herein, the term "fatty hepatitis" is a type of fatty liver. Mild fatty liver generally has no obvious liver injury and obvious symptoms. Moderate to severe fatty liver generally involves liver cell injury, is called fatty hepatitis, and has corresponding clinical symptoms.

[0248] Examples of liver diseases include non-alcoholic fatty liver disease. In this context, the term "non-alcoholic fatty liver disease" refers to a clinicopathological syndrome characterized mainly by excessive deposition of fat in liver cells and caused by factors other than alcohol and other definite liver injury factors, which is an acquired metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility. This includes simple fatty liver, non-alcoholic fatty hepatitis, and related cirrhosis.

[0249] Liver diseases include non-alcoholic steatohepatitis. As used herein, the term "non-alcoholic steatohepatitis" refers to an inflammatory subtype of non-alcoholic fatty liver disease with fatty liver, evidence of hepatocyte injury (ballooning change), and inflammation, with or without liver fibrosis. Over time, non-alcoholic steatohepatitis may progress to liver fibrosis, cirrhosis, end-stage liver disease, or require liver transplantation.

[0250] Liver diseases include fatty liver. As used herein, the term "fatty liver" refers to the presence of fat droplets in the cytoplasm of hepatocytes. When fatty liver occurs, in mild cases, no obvious abnormalities are visible by macroscopic observation. In severe cases, the liver is enlarged, the texture is soft, the color is light yellow to yellowish-brown, the structure of the cut surface is fluffy and greasy. Microscopically, vacuoles of various sizes appear in the degenerated liver cytoplasm, and in severe cases, these may fuse into large vacuoles resembling adipocytes.

[0251] Liver diseases include liver injury. Liver injury is selected from the group consisting of acute liver injury, chronic liver injury, chemical liver injury, physical liver injury, or any combination thereof. As used herein, the term "liver injury" is the pathological consequence of various liver diseases. Liver injuries caused by various harmful factors mainly include viral liver injury, alcohol-induced liver injury, drug-induced liver injury, etc. In certain embodiments, the liver injury is acute liver injury.

[0252] In certain embodiments, the medicament further comprises a carrier or an excipient.

[0253] In certain embodiments, the carrier is selected from the group consisting of gelatin, chitosan, sodium alginate, collagen, silk protein, cellulose, fibrin, polylactic acid, polyurethane, polyethylene oxide, polyethylene glycol, poly(lactic-co-glycolic acid), poly(ε-caprolactone), silicate, silicone rubber, extracellular matrix, decellularized scaffold, or any combination thereof.

[0254] In certain embodiments, the carrier is selected from the group consisting of gelatin, collagen, or any combination thereof.

[0255] In certain embodiments, the medicament further comprises a second active ingredient.

[0256] In certain embodiments, the second active ingredient is selected from the group consisting of diisopropylamine ascorbate, choline chloride, inositol, dehydrocholic acid, magnesium sulfate, polyene phosphatidylcholine (Essentiale), glucuronolactone (glucuron), glutathione (glutathione, Atmolan), tiopronin (Capen), glycyrrhizin preparation, adenosylmethionine (Transmetil), hepatocyte growth promoting factor, Schisandra chinensis (biphenyldiester), silymarin (silibinin, Legalon, Baoganning), oleanolic acid (Ganshu tablet), Yinzhihuang preparation, Herba Artemisiae Capillariae, or any combination thereof.

[0257] In certain embodiments, the second active ingredient is selected from the group consisting of aminosalicylate drugs, corticosteroid drugs, immunosuppressive agents, biological agents, or any combination thereof. In certain embodiments, the aminosalicylate drug is 5-aminosalicylic acid. In certain embodiments, the corticosteroid drug is a glucocorticoid. In certain embodiments, the immunosuppressive agent is selected from azathioprine, 6-mercaptopurine, methotrexate, cyclosporine A, tacrolimus, or any combination thereof. In certain embodiments, the biological agent is a TNF antagonist.

[0258] In certain embodiments, the medicament is in unit dosage form, and the unit dosage of the medicament is mesenchymal stem cells at 1×10 4 cells or more per ml (e.g., 1×10 4 cells or more per ml, 3×10 4 cells or more per ml, 5×10 4 cells or more per ml, 7×10 4 cells or more per ml, 1×10 5 cells or more per ml, 3×10 5 cells or more per ml, 5×10 5 cells or more per ml, 7×10 5 cells or more per ml, 1×10 6 cells or more per ml, 3×10 6 cells or more per ml, 5×10 6 cells or more per ml, 7×10 6 cells or more per ml, 1×10 7 cells or more per ml, 3×10 7 cells or more per ml, 5×10 7 cells or more per ml, 7×10 7 cells or more per ml, 1×10 8 cells or more per ml, 3×10 8 cells or more per ml, 5×10 8 cells or more per ml, 7×10 8 cells or more per ml, 1×10 9 cells or more per ml, 3×10 9 cells or more per ml, 5×10 9 cells or more per ml, 7×10 9 cells or more per ml, 1×1010 more than / ml, 3×10 10 more than / ml, 5×10 10 more than / ml, or 7×10 10 contained in an amount of / ml). In certain embodiments, the unit dose of the medicament is 1×10 mesenchymal stem cells 4 to 1×10 10 (for example, 1×10 6 to 1×10 8 or 1×10 6 to 1×10 7 or 1×10 6 to 5×10 6 ).

[0259] In certain embodiments, the administration route of the mesenchymal stem cells is selected from the group consisting of injection, topical application, adhesion, enema, perfusion, rectal administration, and oral administration.

[0260] In certain embodiments, the method further comprises administering to a subject in need thereof a second active ingredient as previously described or defined.

[0261] In certain embodiments, the subject is a mammal such as a human.

[0262] In another aspect, the present invention provides a product for treating a digestive system disease, comprising a mesenchymal stem cell population as a first active ingredient.

[0263] In certain embodiments, the mesenchymal stem cell population is as previously described or defined.

[0264] In certain embodiments, the digestive disease is as previously described or defined.

[0265] In certain embodiments, the product further comprises a second active ingredient.

[0266] In certain specific embodiments, the second active ingredient is as described or defined above.

[0267] In certain specific embodiments, the first active ingredient and the second active ingredient are present alone or in combination.

[0268] In certain specific embodiments, the first active ingredient is administered in combination with a second active ingredient selected from those described above.

[0269] In certain specific embodiments, the product is an implant, and preferably, the implant is used to improve the microenvironment and suppress the immune rejection reaction.

[0270] In certain specific embodiments, the subject is a mammal such as a human.

[0271] Acute liver injury In this document, the term "acute liver injury" refers to the acute injury or necrosis of liver cells that occurs in a short period with abnormal liver function, and in some patients, liver failure. The main causes of acute liver injury include mainly viral infections, inappropriate drug use, food additives, excessive ethanol intake, accidental ingestion of toxic foods, radiation damage, etc. Acute liver injuries include viral liver injury, chemical liver injury, and drug-induced liver injury.

[0272] As used herein, the term "chemical acute liver injury" refers to liver injury caused by chemical hepatotoxic substances. These chemical substances include alcohol, chemical poisons in the environment (such as carbon tetrachloride), and certain specific drugs.

[0273] The liver has strong defensive and reparative capabilities. When liver injury is caused by various reasons, hepatocyte regeneration can be relied on to reconstruct the liver structure and restore liver function. Acute liver injury is a kind of disease characterized by short-term liver failure caused by viruses, drugs, alcohol, autoimmune abnormalities, and other factors. The main pathological changes are extensive necrosis and apoptosis of liver cells, whereby the liver can no longer perform normal synthetic and metabolic functions. If the progression of the disease is not intervened in a short period, the disease will rapidly deteriorate, causing coagulation dysfunction, jaundice, ascites, and hepatic encephalopathy, and may further lead to multiple organ failure. As it progresses to liver failure, it progresses rapidly and is difficult to treat, so the overall prognosis is extremely poor. Autologous liver transplantation is the most effective method for treating severe liver injury, but due to various risks such as a shortage of liver donors, high surgical costs, postoperative complications, and immune rejection reactions, the application of liver transplantation is limited. Considering the high mortality rate of acute liver injury and the limitations of liver transplantation, stem cell therapy shows great potential and advantages in the treatment of acute liver diseases and chronic liver diseases.

[0274] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein for preventing and / or treating acute liver injury in a subject or for manufacturing a medicament for preventing and / or treating acute liver injury in a subject. The above use can protect the liver and maintain and / or extend and / or improve liver function.

[0275] More specifically, in a certain specific embodiment, the pharmaceutical composition can suppress or reduce the weight loss rate of patients with acute liver injury and reduce the mortality rate of patients with acute liver injury.

[0276] In a certain specific embodiment, the pharmaceutical composition can reduce the content of transaminase and / or alkaline phosphatase in the serum.

[0277] In certain embodiments, the pharmaceutical composition can prevent inflammatory cell infiltration.

[0278] In certain embodiments, the subject is a mammal such as a human.

[0279] In certain embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0280] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), blood circulation pathway transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0281] In certain embodiments, the culture supernatant of the present invention, or a pharmaceutical composition containing the culture supernatant, can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc.

[0282] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous solution or sterile isotonic non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0283] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial, such as the above-described biological scaffold, or including, but not limited to, collagen scaffold, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteogen, hyaluronic acid, etc.

[0284] In certain embodiments, the medicament can be transplanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0285] Non-alcoholic steatohepatitis Non-alcoholic steatohepatitis (NASH), also known as metabolic steatohepatitis, is a progressive non-alcoholic fatty liver disease defined as the presence of 5% or more fatty liver with inflammation and hepatocyte injury (e.g., ballooning changes), with or without liver fibrosis. NASH is prone to develop into cirrhosis, liver cancer, and other diseases. There are 3% - 5% NASH patients worldwide. In China, there are about 1.09 million patients with cirrhosis, and it is expected to increase to 2.32 million in 2030. The onset of NASH is closely related to genetics (polymorphism of PNPLA3), lifestyle (host eating habits, meal frequency, sleep-wake cycle, etc.), obesity, metabolic syndrome, etc. Common symptoms of NASH include loss of appetite, fatigue, abdominal distension, nausea and vomiting, dull pain in the liver area, and hepatomegaly. Environmental factors, metabolic factors, and genetic factors lead to the accumulation of free fatty acids in the liver, which further causes a series of cell damage. Currently, NASH treatment includes non-clinical treatment and clinical treatment. Non-clinical treatment includes lifestyle changes to improve the course of the disease, while clinical treatment includes liver transplantation, surgery, and investigational drugs for treating the disease. For adjuvant therapy, a treatment strategy that progresses to a healthy lifestyle is more appropriate. Liver transplantation is costly and there is a shortage of donors. Surgical treatment requires the patient to meet eligibility criteria and has limitations. Currently, there are no drugs approved by the FDA for the treatment of NASH. Therefore, the treatment of NASH still remains in an urgent shortage state.

[0286] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating non-alcoholic steatohepatitis in a subject, or for preventing non-alcoholic steatohepatitis in a subject, or delaying or reducing non-alcoholic steatohepatitis, or preventing / mitigating non-alcoholic steatohepatitis.

[0287] In certain embodiments, the pharmaceutical composition can reduce liver weight, suppress fat accumulation in the liver, and / or alleviate fatty liver.

[0288] In certain embodiments, the pharmaceutical composition can reduce the content of transaminase and improve liver function.

[0289] In certain embodiments, the pharmaceutical composition can suppress fibrosis and / or suppress inflammation.

[0290] In certain embodiments, the subject is a mammal such as a human.

[0291] In certain embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0292] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), blood circulation pathway transplantation (e.g., intravenous injection transplantation or intraarterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art recognize methods for selecting an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0293] In certain preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In certain embodiments, the administration is performed by intravenous injection.

[0294] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic solution or sterile isotonic non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0295] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial, such as the above-described biological scaffold or, but not limited to, collagen scaffold, Matrigel, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose poly(lactic acid), elastic proteoglycan, hyaluronic acid, etc.

[0296] In certain embodiments, the medicament can be transplanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0297] Inflammatory bowel disease Inflammatory bowel disease (IBD) is a typical chronic relapsing disease associated with dysregulation of the mucosal immune system and commensal ecosystem that embodies the interaction between host genetics, host immunology, microbiota, and environmental exposure.

[0298] IBD presents as two major disease types: Crohn's disease (CD) and ulcerative colitis (UC). UC affects the colon, while CD can affect any area of the gastrointestinal tract, but mainly occurs at the terminal ileum of the small intestine.

[0299] Intestinal complications of IBD include abscess, intestinal obstruction, intestinal perforation, colon cancer, anal fissure, fistula, worsening of menstrual symptoms, and toxic megacolon. Specific complications of IBD (Crohn's disease and ulcerative colitis) can be life-threatening and require prompt treatment to prevent more serious diseases.

[0300] Abscess: An abscess, which is more common in Crohn's disease than in ulcerative colitis, is the accumulation of pus at the site of infection. Abscesses can occur invisibly inside the body, such as on the inner or outer intestinal wall, or inside the skin, etc. Internal abscesses can be treated with antibiotics, but if they cannot be resolved, drainage is required. This can be done by inserting a catheter through the skin into the abscess site. In other methods, such as passing through the stomach wall, a catheter can also be inserted. Surgery may be required for drainage in some cases.

[0301] Intestinal obstruction: Intestinal obstruction refers to a situation where a part of the small intestine or large intestine is partially or completely blocked, thereby preventing the body from secreting excreta. Intestinal obstruction is usually accompanied by severe pain, vomiting, and constipation. In some cases, a nasogastric tube may help relieve the symptoms, but surgery may be required to remove the obstruction in some cases.

[0302] Intestinal perforation: The risk of intestinal perforation (hole) is rare but is a potentially fatal complication of IBD. Intestinal perforation is most common during the first episode of ulcerative colitis in people with very thin intestinal walls due to severe disease. Intestinal perforation is almost always surgically treated to repair the hole or even remove a part of the intestine.

[0303] Colorectal cancer: Patients with IBD, especially those who have had ulcerative colitis affecting the entire colon for 8 to 10 years, have an increased risk of developing colorectal cancer. Patients with Crohn's disease are also at risk, but there is little information about the degree of risk. People with IBD, especially those at highest risk, should be closely monitored for colorectal cancer by colonoscopy.

[0304] Anal fissure: An anal fissure is a painful tear in the anal canal that can cause bleeding. Most tears heal without surgery, and treatment using local creams etc. can ensure soft, painless bowel movements. Surgery may be necessary for tears that do not heal and become chronic.

[0305] Fistula: A fistula is an abnormal connection between two body cavities or between a body cavity and the skin. Fistulas are more common in Crohn's disease than in ulcerative colitis, and in fact, about 25 percent of patients with Crohn's disease may develop fistulas at some point during the course of their disease. Some fistulas can be treated with drug therapy, but the more severe or extensive they are, the more likely they are to require surgery.

[0306] Premenstrual syndrome: Some women with IBD notice that their symptoms worsen during menstruation. Diarrhea and pain may increase premenstrually and during menstruation. The cause of these symptoms may be an increase in hormones during the menstrual cycle.

[0307] Toxic megacolon: Toxic megacolon is rare but life-threatening. Left untreated, toxic megacolon can cause shock, perforation, or an infection of the abdomen or blood. In some cases, it may be treated with medication, but surgery may be necessary in severe cases.

[0308] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, and / or pharmaceutical composition described herein for preventing and / or treating inflammatory bowel disease in a subject or for manufacturing a medicament for preventing and / or treating inflammatory bowel disease in a subject.

[0309] The above use can prevent and treat the infiltration of inflammatory cells, protect the colon, reduce inflammatory factors (such as IFN-γ, IL-6, TFN-α, iNOS, etc.), increase or upregulate anti-inflammatory factors (such as IL10, etc.), suppress the onset of inflammation, secrete trophic factors (such as VEGF, HGF, SDF-1a, etc.), promote the repair of colon tissue, etc., and realize functions such as suppressing inflammation and promoting tissue repair in the prevention and / or treatment of inflammatory bowel disease, thereby protecting the colon tissue, protecting the intestine, and improving the tissue repair ability.

[0310] In certain embodiments, the pharmaceutical composition can treat Crohn's disease (CD) and / or ulcerative colitis (UC).

[0311] In certain embodiments, the subject is a mammal such as a human.

[0312] In certain embodiments, the medicament comprises a therapeutically effective amount of the mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0313] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to the subject by local injection transplantation (such as stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), blood circulation pathway transplantation (such as intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid pathway transplantation (such as lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0314] In certain embodiments, the culture supernatant of the present invention, or a pharmaceutical composition containing the culture supernatant, can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, or the like. In certain embodiments, the pharmaceutical composition is administered by intravenous injection or intraperitoneal injection.

[0315] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), or the like. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic solution or sterile isotonic non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of such pharmaceutical compositions can be found in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0316] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including a biological scaffold, such as, but not limited to, a collagen scaffold, a skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid, and the like.

[0317] In certain embodiments, the medicament can be transplanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0318] In certain specific embodiments, the pharmaceutical composition may also include, but is not limited to, another pharmaceutical combination comprising 5-aminosalicylic acid, NF-κB and activator protein-1 (activator protein-1, AP1), immunosuppressive agents (azathioprine), 6-mercaptopurine, methotrexate, cyclosporin A, tacrolimus, tumor necrosis factor (TNF), antagonists, etc.

[0319] Nervous system diseases In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for the prevention and / or treatment of nervous system diseases. Alternatively, the present invention provides a method for preventing and / or treating nervous system diseases, which comprises administering a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant to a subject in need thereof.

[0320] Nervous system diseases are mainly diseases manifested by nervous system dysfunction, and their symptoms mainly include abnormal mental behavior, amnesia, insomnia, mood changes, and intellectual changes. Nervous system diseases include cerebrovascular diseases, neurodegenerative diseases, infectious diseases of the central nervous system, demyelinating diseases of the central nervous system, movement disorders, epilepsy, spinal cord diseases, genetic diseases of the nervous system, nervous system dysplasia, systemic poisoning diseases of the central nervous system, tumor diseases of the central nervous system, immune diseases of the central nervous system, etc. Examples include cerebrovascular diseases, periodic paralysis, progressive muscular dystrophy, myotonic dystrophy, ataxia, insomnia, neurasthenia, epilepsy, trigeminal neuralgia, neurodegenerative diseases, neurogenic headache (e.g., migraine, etc.), and neuropathy, etc.

[0321] Neuropathy Neuropathy is a disease accompanied by clinical symptoms of sensory dysfunction, motor dysfunction, consciousness dysfunction, and autonomic nerve dysfunction of the central nervous system, peripheral nervous system, and autonomic nervous system.

[0322] Neurodegenerative diseases Neurodegenerative diseases are diseases of dysfunction caused by neurons gradually losing their structure or function, or even dying, including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, epilepsy, Huntington's disease, and spinal muscular atrophy, brain injury, various types of spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, transmissible spongiform encephalopathy, primary lateral sclerosis, multiple sclerosis, cardiovascular dementia and cerebrovascular dementia, neuropathic pain, glaucoma, traumatic spinal cord injury, multiple system atrophy, etc.

[0323] (1) Amyotrophic lateral sclerosis (ALS): Amyotrophic lateral sclerosis (ALS), commonly known as ALS, is a sporadic and fatal neurodegenerative disease that affects upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord. The loss of a large number of motor neurons leads to muscle wasting and causes spontaneous contractions and spasms. ALS is divided into two categories: familial ALS (FALS) and sporadic ALS (SALS), with familial ALS accounting for 10% and sporadic ALS accounting for 90%. The onset age of ALS patients is usually after 40 years old. The high incidence rates of FALS and SALS are 47 to 52 years old and 58 to 63 years old respectively. The incidence rate decreases after the age of 80, and men are more likely to develop this disease than women. Patients generally survive 3 to 5 years from the onset of the disease. Various factors such as genetic nature, occupation, lifestyle, and age are closely related to the incidence rate of ALS. On the one hand, the cause of ALS is that astrocytes cannot recover the glutamate accumulated in the synapse in time, causing glutamate excitotoxicity. On the other hand, mutant genes including SOD1, UBQLN2, OPTN, VCP, TDP43, FUS, and C9ORF72 lead to the production of misfolded protein conformational multimers that cause toxicity and the production of toxic RNA species that exacerbate the damage of motor neurons, causing synaptic retraction, being unable to bind to the postsynaptic membrane receptors, resulting in the failure of complete electrical signal transmission, and finally clinical symptoms appear. Drug treatment is available for the treatment of ALS. Currently, only two neuroprotective drugs approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) can extend the lifespan of some patients by several months: riluzole, which can block excessive glutamine neurotransmission, and edaravone, which can prevent damage caused by oxidative stress. Surgical treatment: When the patient has difficulty swallowing or chewing, nasogastric nutrition or gastrostomy may be performed. When the respiratory muscles are paralyzed, tracheotomy should be performed as soon as possible and ventilation should be used to maintain breathing. Adjuvant therapy: There is also rehabilitation training. Clinically, there are specific treatment methods for specific symptoms. The above-mentioned treatment methods can only extend the patient's survival period by several months, but do not significantly improve the patient's quality of life.Therefore, more effective treatments are still urgently needed. In addition to the above-mentioned treatment methods, gene editing is currently a topic of interest in preclinical research. For example, direct editing of SOD1 via the CRISPR / Cas9 gene editing system has been used in vitro and in transgenic mice for the treatment of amyotrophic lateral sclerosis. However, there are still many uncertainties in gene editing.

[0324] In some embodiments, the medicament of the present invention can be administered by intravenous injection or intracerebral injection in addition to the above-described administration methods.

[0325] In certain embodiments, the medicament delays the onset of the disease.

[0326] In certain embodiments, the medicament can enhance reaction capabilities such as limb coordination, motor ability, grip strength, etc.

[0327] In certain embodiments, the medicament can improve muscle strength and reduce damage to motor neurons in amyotrophic lateral sclerosis.

[0328] In certain embodiments, the medicament can improve motor neurons, reduce microglia and astrocytes, and alleviate the progression of the disease in amyotrophic lateral sclerosis.

[0329] In some embodiments, the subject is a mammal such as a human.

[0330] In some embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0331] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art recognize methods for selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0332] In some embodiments, the culture supernatant of the present invention, or a pharmaceutical composition containing the culture supernatant, can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the pharmaceutical composition is administered by intravenous injection or intracerebral tissue injection.

[0333] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In some embodiments, the pharmaceutical composition contains a pharmaceutically acceptable sterile isotonic aqueous solution or sterile isotonic non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0334] In some embodiments, the pharmaceutical composition includes pharmaceutically acceptable biomaterials including, but not limited to, collagen scaffolds, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastinogen, hyaluronic acid, and the like.

[0335] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, a gel, a colloid, a serous fluid, or a mixture.

[0336] (2) Epilepsy Epilepsy is a chronic brain disease caused by various etiologies, characterized by sudden recurrent and transient central nervous system dysfunction caused by excessive firing of brain neurons. Epilepsy includes idiopathic epilepsy syndrome, symptomatic epilepsy syndrome, epilepsy syndrome with possible symptoms or epilepsy of unknown cause, reflex epilepsy syndrome, benign epilepsy syndrome, and epileptic encephalopathy.

[0337] Epilepsy is a chronic brain disorder characterized by the recurrence of seizures that occur suddenly for no obvious reason, and is the second most common neurological disorder after stroke. "Abnormal firing" of neurons in the brain causes repeated and short-term epileptic seizures. More than 70 million people worldwide suffer from epilepsy, and the incidence in the Chinese population is between 5‰ and 7‰, with 6.5 million to 9.1 million patients nationwide. Some cerebrovascular complications, head injuries, central nervous system infections, etc. may cause secondary epilepsy, and sleep, age, and genetic nature are closely related to idiopathic epilepsy. In the treatment of epilepsy, antiepileptic drugs are most widely used. However, despite the existence of 30 antiepileptic drugs (AEDs) with different molecular targets, there are still many problems in the drug treatment of epilepsy, such as drug resistance, side effects, toxicity related to frequent dependence toxicity, and memory impairment. Furthermore, brain surgery is the most important alternative treatment, but registration eligibility, as well as risks and costs, must be considered. Currently, clinical trials are mainly drug treatments, and more than 200 are in Phase III.

[0338] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating epilepsy, delaying or alleviating epileptic seizures, or preventing epileptic seizures, or alternatively, the present invention relates to a method for preventing and / or treating epilepsy, delaying or alleviating epileptic seizures, or preventing epileptic seizures, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0339] The mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition of the present invention can prevent epilepsy, or delay or alleviate epileptic seizures, or prevent epileptic seizures.

[0340] In some preferred embodiments, the administration is performed by intracerebral injection or intravenous injection.

[0341] In some embodiments, the pharmaceutical composition can increase the number of GABAergic neurons in the brain, or activate GABAergic neurons, or decrease the number of microglia, or reconstruct and repair the neural circuit in which GABAergic neurons are deficient in the model, or promote the ability of endogenous stem cells to differentiate into the GABAergic lineage, or suppress the inflammatory response.

[0342] In some embodiments, the pharmaceutical composition can improve the memory and learning abilities in model animals.

[0343] In some embodiments, the pharmaceutical composition can provide the brain endocrine level of a trophic molecule (e.g., GDNF) to protect neurological function.

[0344] In some embodiments, the subject is a mammal such as a human.

[0345] In some embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0346] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory pathway transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art recognize how to select an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0347] In some preferred embodiments, the culture supernatant of the present invention, or a pharmaceutical composition containing the culture supernatant, can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the pharmaceutical composition is administered by intracerebral injection or intravenous injection.

[0348] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In some embodiments, the pharmaceutical composition includes a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0349] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastinogen, hyaluronic acid, etc.

[0350] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0351] (3) Alzheimer's disease (AD) Alzheimer's disease (AD) is the most common form of senile dementia and one of the most common chronic diseases in old age, accounting for about 50% - 70% of senile dementia. More than 35 million people worldwide are suffering from AD. The clinical symptoms of AD are progressive memory loss and cognitive dysfunction. Alzheimer's disease is associated with two pathogenic features, namely extracellular amyloid-beta (Aβ) deposition and intracellular neurofibrillary changes (neurofibrillary tangles, NFT), accompanied by neuroinflammation and extensive neuron and synapse deficits, causing progressive memory loss and cognitive dysfunction. Currently, there is no specific drug that can cure Alzheimer's disease or effectively reverse the disease process. The combination of drug therapy, non-drug therapy, and intensive care can alleviate and delay the onset of the disease. Therefore, it is important to develop an effective treatment strategy that can cure AD or reverse AD. Currently, clinical trials are mainly conducted on drug research, and there are more than 200 clinical trials. There are 10 clinical trials on mesenchymal stem cells (MSCs), which are in Phase I and Phase II of clinical trials.

[0352] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating Alzheimer's disease or cerebrovascular disease, delaying or alleviating Alzheimer's disease or cerebrovascular disease, or preventing and alleviating Alzheimer's disease or cerebrovascular disease, or to a method for preventing and / or treating Alzheimer's disease or cerebrovascular disease, delaying or alleviating Alzheimer's disease, or preventing or alleviating Alzheimer's disease or cerebrovascular disease, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0353] In some embodiments, the medicament can improve learning ability and memory ability and improve memory impairment and cognitive impairment.

[0354] In some embodiments, the medicament can reduce the accumulation of amyloid deposits in the brain and reduce the adverse effects of amyloid deposits on nerves.

[0355] In some embodiments, the medicament can inhibit the conversion of microglia into an inflammatory form and suppress the excessive activation and dysfunction of microglia.

[0356] In some embodiments, the medicament can increase the phagocytic ability of microglia, remove amyloid deposits and apoptotic cell debris, inhibit the production of A1 astrocytes in the inflammatory environment of the AD brain, and inhibit overactivated immunity.

[0357] In some embodiments, the medicament can increase nerve cell survival and improve cognition and memory.

[0358] In some embodiments, the subject is a mammal such as a human.

[0359] In some embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0360] In some embodiments, the medicament is in unit dosage form, and the unit dosage of the medicament is 1×10 4 or more cells / ml (for example, 1×10 4 or more cells / ml, 3×10 4 or more cells / ml, 5×10 4 or more cells / ml, 7×10 4 or more cells / ml, 1×10 5 or more cells / ml, 3×10 5 or more cells / ml, 5×10 5 or more cells / ml, 7×10 5 or more cells / ml, 1×10 6 or more cells / ml, 3×10 6 or more cells / ml, 5×106 cells / ml or more, 7×10 6 cells / ml or more, 1×10 7 cells / ml or more, 3×10 7 cells / ml or more, 5×10 7 cells / ml or more, 7×10 7 cells / ml or more, 1×10 8 cells / ml or more, 3×10 8 cells / ml or more, 5×10 8 cells / ml or more, 7×10 8 cells / ml or more, 1×10 9 cells / ml or more, 3×10 9 cells / ml or more, 5×10 9 cells / ml or more, 7×10 9 cells / ml or more, 1×10 10 cells / ml or more, 3×10 10 cells / ml or more, 5×10 10 cells / ml or more, or 7×10 10 cells / ml or more, preferably 3×10 6 cells to 6×10 6 contained in an amount of).

[0361] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier or excipient, preferably, the carrier is selected from the group consisting of gelatin, chitosan, sodium alginate, collagen, silk protein, cellulose, fibrous protein, polylactic acid, polyurethane, polyethylene oxide, polyethylene glycol, poly(lactic-co-glycolic acid), poly(ε-caprolactone), silicate, silicone rubber, extracellular matrix, decellularized scaffold, and any combination thereof, preferably, the carrier is selected from the group consisting of gelatin, collagen, and any combination thereof, preferably, the medicament is an injection, microinjection, mucosal patch, enema, suppository, gel, oral preparation, aerosol, drops, ointment, implant, or capsule, preferably an injection, preferably, the medicament further comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution, dispersion, suspension, or emulsion.

[0362] In some embodiments, the medicament further comprises an additional active ingredient, which is, for example, a β-secretase inhibitor (e.g., OM99-2), a γ-secretase inhibitor (e.g., R-flurbiprofen), a cholinesterase inhibitor (e.g., donepezil, donepezil hydrochloride, rivastigmine, fuperzine A, tacrine, galantamine, or galantamine hydrobromide), an M cholinergic receptor agonist or antagonist (e.g., M1 cholinergic receptor agonists including xanomeline, saccomeline, nefiracetam, AF-102B, and SR-46559A, M2 cholinergic receptor antagonists including BIBN-99 and AF-DX11, or N-cholinergic receptor agonists including nicotine and ABT-418), a glutamate receptor antagonist (e.g., memantine, memantine hydrochloride, or riluzole), a calcium ion antagonist (e.g., nimodipine or flunarizine), an antioxidant (e.g., vitamin E, L-deprenyl, melatonin, deferoxamine, idebenone, or tiritazad mesylate), an Aβ inhibitor (e.g., estrogen, chloroquine, congo red, or phenyl aminophenyl acetate), a dopamine substitute (e.g., levodopa), a peripheral decarboxylase inhibitor (e.g., carbidopa or benserazide), a dopamine D receptor agonist (e.g., bromocriptine, pergolide, apomorphine, pramipexole, or ropinirole), a nerve differentiation inducer (e.g., piperacetam, aniracetam, oxiracetam, pramiracetam, or nefiracetam), an anticholinergic agent (e.g., trihexyphenidyl hydrochloride, procyclidine, biperiden, or benzotropine), an antidepressant (e.g., amitriptyline, phenelzine, tranylcypromine, isocarboxazid, or istradefylline), a 5-hydroxytryptamine agonist (e.g., sarizotan or buspirone), an MAO-B inhibitor (e.g., selegiline or rasagiline), a dopamine β-hydroxylase inhibitor (e.g., fusaric acid), a COMT inhibitor (e.g., entacapone or tolcapone), an immunosuppressant (e.g., azathioprine, 6-mercaptopurine, methotrexate, cyclosporin A, or tacrolimus),selected from the group consisting of and any combination thereof, preferably, the mesenchymal stem cell population and the additional active ingredient are present alone or in combination.

[0363] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intraarterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize how to select an appropriate cell transplantation route according to the location and nature of the lesion.

[0364] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some preferred embodiments, the administration is performed by intracerebral injection or intravenous injection.

[0365] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0366] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastinogen, hyaluronic acid, etc.

[0367] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0368] In some embodiments, the dosage to be administered is 1×10 4 or more cells / ml (e.g., 1×10 4 or more cells / ml, 3×10 4 or more cells / ml, 5×10 4 or more cells / ml, 7×10 4cells / ml or more, 1×10 5 cells / ml or more, 3×10 5 cells / ml or more, 5×10 5 cells / ml or more, 7×10 5 cells / ml or more, 1×10 6 cells / ml or more, 3×10 6 cells / ml or more, 5×10 6 cells / ml or more, 7×10 6 cells / ml or more, 1×10 7 cells / ml or more, 3×10 7 cells / ml or more, 5×10 7 cells / ml or more, 7×10 7 cells / ml or more, 1×10 8 cells / ml or more, 3×10 8 cells / ml or more, 5×10 8 cells / ml or more, 7×10 8 cells / ml or more, 1×10 9 cells / ml or more, 3×10 9 cells / ml or more, 5×10 9 cells / ml or more, 7×10 9 cells / ml or more, 1×10 10 cells / ml or more, 3×10 10 cells / ml or more, 5×10 10 cells / ml or more, or 7×10 10 cells / ml or more).

[0369] In some embodiments, a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population is administered to a subject by injection, mucosal administration, cavity administration, oral administration, airway administration, or topical administration.

[0370] In some embodiments, the method further comprises administering to the subject a prophylactically effective amount and / or a therapeutically effective amount of an additional active ingredient simultaneously, sequentially, or alternately, wherein the additional active ingredient is, for example, a β-secretase inhibitor (e.g., OM99-2), a γ-secretase inhibitor (e.g., R-flurbiprofen), a cholinesterase inhibitor (e.g., donepezil, donepezil hydrochloride, rivastigmine, fuperzine A, tacrine, galantamine, or galantamine hydrobromide), an M cholinergic receptor agonist or antagonist (e.g., M1 cholinergic receptor agonists including xanomeline, saccopine, nepicastam, AF-102B, and SR-46559A, M2 cholinergic receptor antagonists including BIBN-99 and AF-DX11, or N-cholinergic receptor agonists including nicotine and ABT-418), a glutamate receptor antagonist (e.g., memantine, memantine hydrochloride, or riluzole), a calcium ion antagonist (e.g., nimodipine or flunarizine), an antioxidant (e.g., vitamin E, L-deprenyl, melatonin, deferoxamine, idebenone, or tirilazad mesylate), an Aβ inhibitor (e.g., estrogen, chloroquine, congo red, or phenylaminophenyl acetic acid), a dopamine replacement (e.g., levodopa), a peripheral decarboxylase inhibitor (e.g., carbidopa or benserazide), a dopamine D receptor agonist (e.g., bromocriptine, pergolide, apomorphine, pramipexole, or ropinirole), a nerve differentiation inducer (e.g., piracetam, aniracetam, oxiracetam, pramiracetam, or nepicastam), an anticholinergic agent (e.g., trihexyphenidyl hydrochloride, procyclidine, biperiden, or benzotropine), an antidepressant (e.g., amitriptyline, phenelzine, tranylcypromine, isocarboxazid, or istradefylline), a 5-hydroxytryptamine agonist (e.g., sarizotan or buspirone), an MAO-B inhibitor (e.g., selegiline or rasagiline), a dopamine β-hydroxylase inhibitor (e.g., fusaric acid), a COMT inhibitor (e.g., entacapone or tolcapone), an immunosuppressant (e.g., azathioprine, 6-mercaptopurine, methotrexate, cyclosporin A,selected from the group consisting of rapamycin (sirolimus), everolimus, temsirolimus, zotarolimus, or tacrolimus, and any combination thereof, and preferably, the mesenchymal stem cell population and the additional active ingredient are present alone or in combination.,

[0371] (4) Extrapyramidal disorders and movement disorders Extrapyramidal disorders and movement disorders, including Parkinson's disease, secondary Parkinson's disease, Parkinson's disease caused by other classified diseases, other degenerative diseases of the basal ganglia, dystonia, other extrapyramidal disorders and movement disorders, and extrapyramidal disorders and movement disorders caused by other classified diseases.

[0372] Movement disorders, also known as extrapyramidal diseases, are mainly characterized by dysfunction of voluntary movement regulation that does not affect muscle strength, sensory function, and cerebellar function. Diseases in this group are caused by dysfunction of the basal ganglia and are usually divided into two categories: hypertonia - decreased movement and hypotonia - excessive movement. Hypertonia is characterized by lack of movement, and hypotonia is mainly characterized by abnormal involuntary movements.

[0373] Parkinson's disease (PD) is a movement disorder and a chronic neurodegenerative disease that affects the central nervous system, mainly the motor nervous system. Its symptoms usually appear slowly over time, and the most obvious initial symptoms are tremors, limb rigidity, decreased motor function, and abnormal gait. There may also be problems with cognition and behavior. Dementia is very common in patients with severe diseases, but major depressive disorder and anxiety disorder also occur in more than one - third of cases. Other possible symptoms include perceptual problems, sleep problems, and emotional problems. The main motor symptoms associated with Parkinson's disease are generally known as the Parkinsonian syndrome.

[0374] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating Parkinson's disease, delaying or alleviating Parkinson's disease, or preventing or alleviating Parkinson's disease, or to a method for preventing and / or treating Parkinson's disease, delaying or alleviating Parkinson's disease, or preventing or alleviating Parkinson's disease, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0375] In some embodiments, the medicament can reduce dopaminergic denervation and / or alleviate the progression of Parkinson's disease.

[0376] In some embodiments, the medicament can improve limb rigidity and / or enhance motor ability.

[0377] In some embodiments, the medicament can protect neurons, reduce neuronal damage and death, have trophic and synaptogenic effects on neurons, reduce inflammation in the brain, and / or improve the microenvironment in the brain.

[0378] In some embodiments, the subject is a mammal such as a human.

[0379] In some embodiments, the medicament comprises a therapeutically effective amount of the mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0380] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intraarterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize how to select an appropriate cell transplantation route according to the location and nature of the lesion.

[0381] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the administration is performed by intravenous injection or intracerebral injection.

[0382] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For the general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0383] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastinogen, hyaluronic acid, and the like.

[0384] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0385] Spinal cord injury (SCI) "Spinal cord injury" refers to a transverse injury to the structure and function of the spinal cord caused by various pathogenic factors (trauma, inflammation, tumor, etc.), which results in impairment of spinal cord nerve functions (motor function, sensory function, sphincter function, and autonomic nerve function) below the level of the damaged segment, which is divided into primary spinal cord injury and secondary spinal cord injury. Here, primary spinal cord injury includes traumatic spinal cord injury, and secondary spinal cord injury includes spinal tuberculosis, spinal pyogenic infection, transverse myelitis, spinal degenerative diseases, congenital scoliosis, and tethered cord syndrome.

[0386] Spinal cord injury (SCI) is a traumatic spinal surgical disease caused by trauma, which appears as sensory dysfunction, motor dysfunction, and autonomic nerve dysfunction below the damaged segment. According to foreign epidemiological surveys, there are 130,000 new spinal cord injury patients worldwide every year, and more than 2.5 million patients are suffering from various degrees of spinal cord injury sequelae. The annual medical expenses of these SCI patients exceed $6 billion, imposing a heavy burden on families and society.

[0387] The two main common outcomes of primary spinal cord injury are spinal cord contusion and spinal cord compression (external force or internal force). Secondary injury refers to the secondary injury of the spinal cord caused by spinal cord edema induced by external force, hematoma formed by bleeding of small blood vessels in the spinal canal, compressive fracture, and spinal cord compression caused by destruction of intervertebral disc tissue.

[0388] In one aspect, the present invention relates to the use of the mesenchymal stem cell population or its culture supernatant described herein, or a pharmaceutical composition, in the manufacture of a medicament for preventing and / or treating spinal cord injury, delaying or alleviating spinal cord injury, or preventing and alleviating spinal cord injury. Alternatively, the present invention relates to a method for preventing and / or treating spinal cord injury, delaying or alleviating spinal cord injury, or preventing and alleviating spinal cord injury, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0389] In some embodiments, the medicament can improve motor ability.

[0390] In some embodiments, the medicament can reduce bladder outlet resistance and detrusor overactivity and improve micturition function.

[0391] In some embodiments, the medicament can promote cell survival, enhance axonal regeneration, suppress glial cell activation and antifibrosis, and reduce the inflammatory response at the injury site.

[0392] In some embodiments, the subject is a mammal such as a human.

[0393] In some embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0394] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to the subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art are aware of methods for selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0395] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the administration is performed by intravenous injection.

[0396] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions and lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For the general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0397] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffold, Matrigel, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid, etc.

[0398] In some embodiments, the medicament can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0399] In some embodiments, the medicament further comprises prostacyclin, an endothelin-1 receptor antagonist, and / or a phosphodiesterase type 5 inhibitor in addition to the above-described drug.

[0400] Cerebrovascular disease "Cerebrovascular disease" refers to a group of diseases occurring in the cerebrovascular system, related to brain tissue damage and brain dysfunction caused by intracranial blood circulation disorders, including stroke, cerebral palsy, cerebral atherosclerotic arteriosclerosis, cerebral arteritis, cerebral artery injury, cerebral aneurysm, intracranial vascular malformation, cerebral arteriovenous fistula, cerebrovascular attack, cerebrovascular spasm, etc.

[0401] (1) Stroke Stroke refers to a group of diseases related to brain tissue damage caused by sudden rupture of blood vessels in the brain or occlusion of blood vessels that prevent blood from flowing into the brain, including two categories: ischemic and hemorrhagic. Ischemic stroke includes cerebral thrombosis, cerebral embolism, and cerebral infarction, while hemorrhagic stroke includes subarachnoid hemorrhage, hypertensive cerebral hemorrhage, etc.

[0402] Stroke is a type of cerebrovascular disease in which blood vessels in the brain narrow, become blocked, or rupture, leading to ischemia or bleeding in brain tissue and causing necrosis of brain cells and brain tissue. Stroke can be divided into ischemic stroke (also known as cerebral infarction) and hemorrhagic stroke (including intracerebral hemorrhage, ventricular hemorrhage, and subarachnoid hemorrhage). The incidence rates of ischemic stroke in men and women are 212 / 100,000 and 170 / 100,000 respectively, and the incidence rate of hemorrhagic stroke is 12 - 15 / 100,000. However, people with lifestyles such as smoking, poor diet, inactivity, and those with complications including hypertension, diabetes, hyperlipidemia, obesity, etc. are often prone to having strokes. Currently, the most widely used treatment for stroke is the thrombolytic agent tissue plasminogen activator (t-PA). For the application of t-PA treatment, patients need to meet the eligibility criteria, so t-PA is for specific stroke patients, and the treatment time frame is short, limited to 4.5 hours. Furthermore, endovascular therapy is also a major treatment strategy. However, there are also disadvantages. Endovascular stents are only suitable for solving the problem of blocked blood flow in large blood vessels. Although the incidence rate of stroke has been reduced by the use of drug therapy and preventive measures including a healthy lifestyle and aerobic exercise, the high recurrence rate remains a headache. Therefore, the treatment of stroke is still a major problem. Existing clinical trials mainly focus on some electronic technology products or software systems to assist the recovery of stroke patients, improvement of behavior and lifestyle, drug therapy and cell therapy for the recovery of stroke patients. In clinical trials, mesenchymal stem cells (MSCs) are basically in Phase I and Phase II of clinical trials.

[0403] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating stroke, delaying or alleviating stroke, or preventing stroke, or the present invention relates to a method for preventing and / or treating stroke, delaying or alleviating stroke, or preventing stroke, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0404] In some embodiments, the medicament can reduce the degree of cerebral infarction, decrease the infarct size of brain tissue, lower the water content of brain tissue, increase the utilization rate of the lateral forelimb, increase the exercise time, and attenuate the degree of neuronal damage caused by stroke.

[0405] In some embodiments, the medicament can promote neuron regeneration, reduce neuron damage and death, provide nutrition to neurons, and promote synapse regeneration.

[0406] In some embodiments, the medicament can attenuate the inflammatory response in the brain and improve the microenvironment in the brain.

[0407] In some embodiments, the subject is a mammal such as a human.

[0408] In some embodiments, the medicament comprises a therapeutically effective amount of the mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0409] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intraarterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0410] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the administration is performed by intravenous injection or brain tissue injection.

[0411] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For the general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0412] In some embodiments, the pharmaceutical composition includes pharmaceutically acceptable biomaterials including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastinogen, hyaluronic acid, and the like.

[0413] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0414] Neuropathic pain Neuropathic pain refers to pain induced or caused by a primary lesion or dysfunction such as a disease that affects the peripheral nerves, nervous system, sensory nerves, etc. of the body, and is characterized by spontaneous pain, allodynia, and hyperalgesia. This disease can be caused by damage to the peripheral nerves, dorsal roots of the spinal cord, spinal cord, and a part of the central nervous system induced by trauma and / or disease. Examples include hemifacial spasm, painful diabetic neuropathy, fascial disease, central neuropathic pain, peripheral neuropathic pain, radicular pain, post-surgical back syndrome, chronic regional pain syndrome and peripheral nerve injury, ischemic pain (e.g., peripheral vascular disease and angina), epileptic seizures, movement disorders associated with Parkinson's syndrome (e.g., tremors, paralysis, rigidity, and abnormal movements), neuropathic pain associated with spinal cord injury, pain caused by intervertebral discs, greater occipital neuralgia, sciatica, intercostal neuralgia, cerebrovascular disease, epilepsy, cerebral edema, hydrocephalus, cancerous neuropathic pain, encephalitis, meningitis, neurodermatitis, neuropathic headache, and other neuropathic pains.

[0415] Neuropathic pain is a difficult-to-treat pain condition caused by damage or abnormality of the nervous system, and is pain that occurs in nerve tissues such as neuritis. This type of pain is mainly characterized by neuropathy and neuralgia, and is somewhat paroxysmal. Local pain may be felt, but there is no pain when pressed. This is all about neuropathic pain. The general treatment for neuralgia is mainly to use drugs that nourish the nerves and drugs that relieve pain, preferably under the guidance of a doctor.

[0416] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating neuropathic pain-induced injury or associated neuropathic pain, or for preventing neuropathic pain, or for delaying or reducing neuropathic pain, or for preventing or alleviating neuropathic pain. Alternatively, the present invention relates to a method for preventing and / or treating neuropathic pain-induced injury or associated neuropathic pain, or for preventing neuropathic pain, or for delaying or reducing neuropathic pain, or for preventing or alleviating neuropathic pain, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0417] In some embodiments, the pharmaceutical composition can improve the subject's tolerance to pain and allodynia and promote motor coordination.

[0418] In some embodiments, the pharmaceutical composition can reduce inflammatory cytokines (IL-1β, IL-6, and IL-17) and inhibit the inflammatory response.

[0419] In some embodiments, the subject is a mammal such as a human.

[0420] In some embodiments, the medicament comprises a therapeutically effective amount of the mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0421] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0422] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the administration is performed by intravenous injection.

[0423] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For the general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0424] In some embodiments, the pharmaceutical composition includes pharmaceutically acceptable biomaterials including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastinogen, hyaluronic acid, etc.

[0425] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0426] Demyelinating disease Demyelinating diseases are a group of acquired diseases with various etiologies and various clinical symptoms but having similar characteristics, and their characteristic pathological change is demyelination of nerve fibers having relatively intact nerve cells. Since the function of the myelin sheath is to protect neurons and rapidly transmit nerve impulses on neurons, the loss of the myelin sheath affects the transmission of nerve impulses.

[0427] Demyelinating diseases in the central nervous system include multiple sclerosis, other acute disseminated demyelination, and other demyelinating diseases of the central nervous system.

[0428] Multiple sclerosis Multiple sclerosis: Multiple sclerosis (MS) is a demyelinating neuropathy where the insulating substance (i.e., myelin) on the surface of nerve cells in the patient's brain or spinal cord is damaged, impairing information transmission in the nervous system, resulting in various symptoms that can affect the patient's activities, mental state, and even psychological state. These symptoms can include double vision, unilateral visual impairment, muscle weakness, abnormal sensations, or impaired coordination. Multiple sclerosis is in various states, and patients may experience recurrence of episodes or worsening of symptoms. Symptoms may disappear completely during episodes, but in particularly severe patients, permanent nerve damage remains.

[0429] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating multiple sclerosis, or for preventing multiple sclerosis, or for delaying or reducing multiple sclerosis, or for preventing or alleviating multiple sclerosis, or the present invention relates to a method for preventing and / or treating multiple sclerosis, or for preventing multiple sclerosis, or for delaying or reducing multiple sclerosis, or for preventing or alleviating multiple sclerosis, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0430] In some embodiments, the medicament can reduce spinal cord demyelination.

[0431] In some embodiments, the medicament can suppress inflammation at the spinal cord site, reduce the number of astrocytes, protect oligodendrocytes, and / or reduce inflammation in the spinal cord segment.

[0432] In some embodiments, the medicament can reduce the content of pro-inflammatory factors (e.g., IFN-γ, IL-17, TNF-α, IL-2), increase the content of anti-inflammatory factors (e.g., IL-10), and suppress inflammation.

[0433] In some embodiments, the subject is a mammal such as a human.

[0434] In some embodiments, the medicament comprises a therapeutically effective amount of the mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0435] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0436] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the administration is performed by intravenous injection.

[0437] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For the general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0438] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid, and the like.

[0439] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0440] Neuroinflammation Neuroinflammation is an inflammatory and degenerative disease of the peripheral nerves caused by traumatic brain injury, stroke, cerebral hemorrhage, and various neurodegenerative diseases. In a normal state, neuroinflammation maintains homeostasis and promotes tissue repair. However, uncontrolled neuroinflammation can be harmful to the brain. Therefore, the control of harmful inflammatory responses is a promising therapeutic approach for neurological disorders.

[0441] "Neuroinflammation" refers to inflammation caused by degeneration or deterioration of nerves or nerve groups due to various reasons including central nervous system inflammation and peripheral nerve inflammation caused by poisoning, infectious diseases, nutritional disorders and metabolic disorders, immune abnormalities, aging, genetic mutations, and the like.

[0442] "Central nervous system infectious diseases" refer to acute or chronic inflammatory (or non-inflammatory) diseases caused by various biological pathogens (including viruses, bacteria, rickettsia, spirochetes, parasites, prion proteins, etc.) that invade the parenchyma, meninges, and blood vessels of the central nervous system, such as encephalitis, cerebellitis, diencephalitis, brain stem encephalitis, encephalomyelitis, and meningoencephalitis caused by viral infection, bacterial infection, fungal infection, and parasitic infection.

[0443] As used herein, the term "bacterial meningitis" refers to inflammation of the pia mater and brain parenchyma caused by bacterial infection, including meningitis, encephalitis, or meningoencephalitis caused by streptococcus, staphylococcus, pneumococcus, diplococcus, pasteurella multocida, bacillus pyogenes, necrobacillus, proteusbacillus, corynebacterium pyogenes, Listeria monocytogenes, etc., as well as bacterial meningitis, encephalitis, or meningoencephalitis caused by traumatic brain injury, otitis media, nasopharyngitis, other local inflammations of the head, and the migration of lymphatic or blood-borne emboli after rupture of the focus of infection.

[0444] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating neuroinflammation, delaying or reducing neuroinflammation, or preventing or alleviating neuroinflammation, or to a method for preventing and / or treating neuroinflammation, delaying or reducing neuroinflammation, or preventing or alleviating neuroinflammation, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0445] In some embodiments, the medicament can reduce inflammatory inducing factors (e.g., IL-1β, IL-6), increase anti-inflammatory factors (e.g., IL-10), and reduce inflammation.

[0446] In some embodiments, the medicament can promote neuron regeneration, reduce neuron damage and death, reduce neuroinflammatory responses, provide nutrition to neurons, and promote synapse regeneration.

[0447] In some embodiments, the medicament can attenuate the inflammatory response and improve the microenvironment of the nervous system.

[0448] In some embodiments, the medicament can improve contextual memory.

[0449] In some embodiments, the subject is a mammal such as a human.

[0450] In some embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0451] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to the subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0452] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the administration is performed by intravenous injection.

[0453] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For the general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0454] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose poly(lactic acid), elastic proteoglycan, hyaluronic acid, etc.

[0455] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0456] Mental disorder Mental disorder is a general term for disorders in brain function activities that cause various degrees of disorders in mental activities such as cognition, emotion, behavior, and will. Common mental disorders include affective disorders and organic mental disorders. The pathogenic factors are multifaceted: congenital inheritance, personality traits and physical factors, organic factors, social environmental factors, etc. Mental disorders include schizophrenia, bipolar disorder, delusional disorder (delusions, hallucinations), phobic disorder (phobia, anxiety disorder), behavioral volitional disorder (obsessive-compulsive disorder), postpartum mental disorder (postpartum psychosis, postpartum depression, maternal depression), climacteric disorder, paranoid mental disorder, and various mental disorders caused by organic lesions (delirium, amnestic syndrome, dementia, bulimia nervosa / psychogenic anorexia nervosa, post-traumatic stress disorder).

[0457] (1) Mood disorder Mood disorder, also known as affective disorder, refers to a group of disorders characterized by significant and long-term emotional or mood changes caused by various reasons. Clinically, mood disorder mainly presents as mood elevation or mood depression, accompanied by corresponding cognitive changes, behavioral changes, and psychotic symptoms such as hallucinations and delusions. Mood disorders include depression, mania, bipolar disorder, persistent mood disorder, and cyclothymia.

[0458] (2) Depression Depression, also known as depressive disorder, is the main type of mood disorder characterized by significant and persistent mood swings.

[0459] The main clinical symptoms are depression, slow thinking, decreased volitional activity, cognitive impairment, sleep disorder, and other physical symptoms.

[0460] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating depression, or delaying or reducing depression, or preventing or alleviating depression, or alternatively, the present invention relates to a method for preventing and / or treating depression, or delaying or reducing depression, or preventing or alleviating depression, which comprises administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0461] In some embodiments, the medicament can promote nerve growth and nerve development.

[0462] In some embodiments, the subject is a mammal such as a human.

[0463] In some embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or a culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0464] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to the subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory pathway transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art are aware of the method of selecting an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0465] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some embodiments, the administration is performed by intravenous injection or intracerebral injection.

[0466] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For the general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0467] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid, etc.

[0468] In some embodiments, the mesenchymal stem cell population can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0469] In another aspect, the present invention also provides a product for preventing, treating, delaying, and / or alleviating a nervous system disease, which comprises the mesenchymal stem cell population of the present invention. In some embodiments, the product further comprises an additional active ingredient, and the additional active ingredient is as defined above. In some embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation. In some embodiments, the product is an injection, a microinjection, a mucosal patch, an enema, a suppository, a gel, an oral preparation, an aerosol, a drop, an ointment, an implant, or a capsule. In certain embodiments, the product is an implant.

[0470] Skin diseases In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing and / or treating skin diseases. Alternatively, the present invention provides a method for preventing and / or treating skin diseases, which comprises administering a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant to a subject in need thereof.

[0471] The skin is the organ with the largest surface area in the human body. The skin is an important structure in protecting internal tissues from the effects of mechanical damage, microbial infection, ultraviolet rays, and extreme temperatures.

[0472] Skin diseases include viral skin diseases, bacterial skin diseases, fungal skin diseases, animal skin diseases, physical skin diseases, dermatitis, eczema, drug eruptions, urticarial skin diseases, pruritic skin diseases, erythematosquamous skin diseases, connective tissue diseases, vesicular skin diseases, vasculitic skin diseases, skin appendage diseases, pigmentary disorder skin diseases, hereditary skin diseases, skin tumors, and sexually transmitted infections.

[0473] Scaly skin diseases are selected from the group consisting of psoriasis, psoriasiform dermatitis, erythema multiforme, erythema annulare, pityriasis simplex, pityriasis rosea, pityriasis circinata, pityriasis amiantacea, lichen planus, lichen nitidus, lichen ruber moniliformis, lichen sclerosus et atrophicus, lichen striatus, exfoliative dermatitis, or any combination thereof.

[0474] Psoriasis is a type of erythematous scaly skin disease.

[0475] Dermatitis is a skin inflammatory disease.

[0476] Dermatitis mostly occurs at the back of the neck or on both sides thereof, in the cubital fossa, popliteal fossa, forearm, thigh, calf, and lumbosacral region, etc., and often has obvious skin lesions such as thin plate, triangular or polygonal flat upper papules, thickened skin, raised skin ridges, deepened skin grooves, lichenoid shapes, often appearing in a banded reddish-brown or light brown color. Dermatitis is characterized by phenomena such as skin peeling, flaky peeling, thickening, discoloration, and itching when touched. Dermatitis includes the following.

[0477] Neurodermatitis: It is more common in young and middle-aged people. There is initially severe itching, followed by skin lesions. The rash is flat papules, lichenoid and without exudate. The rash is more common on the extensor sides of the neck, extremities, lumbosacral region, popliteal fossa, and vulva. Its course is chronic and often recurs.

[0478] Atopic dermatitis: The initial lesions are mainly on the cheeks. The initial lesions are scattered or aggregated small red papules or erythema, which gradually increase, with small blisters, yellowish to white scaly crusts, and exudation, erosion, and secondary infections may be seen. There is severe itching. The chronic symptoms are dry, larger raised banded brownish-red papules, and thick scaly light brown lichenoid changes, which may fuse into fragments. After scratching, there is often a little exudation, desquamation, and scratches.

[0479] Summer dermatitis: Initially, the skin lesions are erythema and papules the size of small dots. However, after scratching due to itching, scratches, blood scabs, skin thickening, and hyperpigmentation may appear. Erosion and exudation are not seen, and it tends to occur on the extensor limbs of adults. When the temperature drops, the condition improves significantly and may heal naturally, with an obvious relationship between the condition and the climate.

[0480] Seborrheic dermatitis: Its rash begins as small red papules around the hair follicle openings, gradually develops, and fuses into yellow to red patches covered with greasy scales or crusts. Due to different lesion locations, the clinical symptoms vary slightly.

[0481] Infantile seborrheic dermatitis usually occurs at 1 to 3 months after birth. The top of the head or the entire scalp may be covered with greasy crusts of various thicknesses, ranging from grayish-yellow to yellow or yellowish-brown, which may include the eyebrow area, nasolabial folds, behind the ears, etc., and is accompanied by slight itching. Infantile seborrheic dermatitis usually heals within 3 to 4 weeks. If it persists without healing, it often complicates with infectious diseases or atopic dermatitis.

[0482] Solar dermatitis: It is a delayed photoallergic skin disease induced by sunlight. The clinical symptoms are polymorphic rashes accompanied by erythema, papules, blisters, erosion, scales, and lichenification, and a certain specific rash often predominates.

[0483] Candidal dermatitis mostly occurs in skin folds such as the groin, perianal gluteal cleft, armpits, and the skin under the breasts of women, and may also occur on the glans prepuce, labia majora and minora, nail grooves, and corners of the mouth. Its rash is mainly local skin flushing, slightly swollen, accompanied by surface erosion and foul-smelling secretions. Sometimes it may dry and desquamate. Pediatric candidal dermatitis also often affects the skin of the trunk and neck, showing extensive and dense erythematous papular rashes that look like erythema of prickly heat. Candidal dermatitis may also affect the oral or vulvar mucosa, often accompanied by cheesy secretions that are often pseudomembranous.

[0484] Mosquito Bite Dermatitis: Mosquito bite dermatitis is a type of dermatitis caused by insect bites, insect venom, or contact with powdery hairs. Common pests include fleas, lice, mosquitoes, centipedes, moths, mosquitoes, bedbugs, bees, etc. Symptoms such as erythema, papules, and wheals may appear. In severe cases, edema or blisters may appear, and petechiae or edema may be seen at the site of the bite.

[0485] Hormone - dependent Dermatitis: Hormone - dependent dermatitis is a type of dermatitis caused by the repeated and inappropriate use of topical hormones over a long period. After topical use of high - efficiency corticosteroids at the same site for more than 3 weeks, symptoms such as erythema, papules, dry desquamation, atrophy, linear atrophoderma, telangiectasia, purpura, excoriation, abnormal pigmentation, rosacea - like dermatitis, perioral dermatitis, photosensitivity, alopecia areata incognita, ichthyosis - like changes, and other secondary symptoms may appear on the skin, and local obvious pruritus or a burning sensation may occur.

[0486] In certain embodiments, the medicament further comprises a carrier or an excipient.

[0487] In certain embodiments, the carrier is selected from the group consisting of gelatin, chitosan, sodium alginate, collagen, silk protein, cellulose, fibrin, polylactic acid, polyurethane, polyethylene oxide, polyethylene glycol, poly(lactic - co - glycolic acid), poly(ε - caprolactone), silicate, silicone rubber, extracellular matrix, decellularized scaffold, or any combination thereof.

[0488] In certain embodiments, the carrier is selected from the group consisting of gelatin, collagen, or any combination thereof.

[0489] In certain embodiments, the medicament further comprises a second active ingredient.

[0490] In certain specific embodiments, the second active ingredient is selected from the group consisting of ebastine tablets, loratadine tablets, cetirizine tablets, mometasone furoate ointment, halometasone ointment, mupirocin ointment, fusidic acid ointment, cefixime tablets, roxithromycin tablets, naftifine ketoconazole ointment, sertaconazole ointment, itraconazole tablets, terbinafine tablets, acyclovir tablets, valacyclovir tablets, penciclovir ointment, interferon gel, or any combination thereof.

[0491] In certain specific embodiments, the medicament exists in unit dosage form, and the unit dosage of the medicament contains mesenchymal stem cells at 1×10 4 or more (e.g., 1×10 4 or more, 3×10 4 or more, 5×10 4 or more, 7×10 4 or more, 1×10 5 or more, 3×10 5 or more, 5×10 5 or more, 7×10 5 or more, 1×10 6 or more, 3×10 6 or more, 5×10 6 or more, 7×10 6 or more, 1×10 7 or more, 3×10 7 or more, 5×10 7 or more, 7×10 7 or more, 1×10 8 or more, 3×10 8 or more, 5×10 8 or more, 7×10 8 or more, 1×10 9 or more, 3×10 9 or more, 5×10 9 or more, 7×10 9 or more, 1×10 10 or more, 3×10 10 or more, 5×10 10 or more, or 7×10 10 or more, preferably 1×10 6 or 3×10 6 or 5×10 6 or more, more preferably 3×10 6It is contained in an amount of (pieces).

[0492] In a certain specific embodiment, the dosage of the mesenchymal stem cell administered is 1×10 4 pieces or more / ml (for example, 1×10 4 pieces or more / ml, 3×10 4 pieces or more / ml, 5×10 4 pieces or more / ml, 7×10 4 pieces or more / ml, 1×10 5 pieces or more / ml, 3×10 5 pieces or more / ml, 5×10 5 pieces or more / ml, 7×10 5 pieces or more / ml, 1×10 6 pieces or more / ml, 3×10 6 pieces or more / ml, 5×10 6 pieces or more / ml, 7×10 6 pieces or more / ml, 1×10 7 pieces or more / ml, 3×10 7 pieces or more / ml, 5×10 7 pieces or more / ml, 7×10 7 pieces or more / ml, 1×10 8 pieces or more / ml, 3×10 8 pieces or more / ml, 5×10 8 pieces or more / ml, 7×10 8 pieces or more / ml, 1×10 9 pieces or more / ml, 3×10 9 pieces or more / ml, 5×10 9 pieces or more / ml, 7×10 9 pieces or more / ml, 1×10 10 pieces or more / ml, 3×10 10 pieces or more / ml, 5×10 10 pieces or more / ml, or 7×10 10 pieces or more / ml, preferably 1×10 6 pieces / ml, 3×10 6 pieces / ml, 5×10 6 pieces / ml, more preferably 3×10 6 pieces / ml).

[0493] In certain embodiments, the administration route of the mesenchymal stem cells is selected from the group consisting of injection administration, topical administration, adhesion administration, enema administration, perfusion administration, rectal administration, and oral administration.

[0494] In certain embodiments, the method further comprises administering to a subject in need thereof a second active ingredient as described or defined above.

[0495] In certain embodiments, the subject is a mammal such as a human.

[0496] In another aspect, the present invention provides a product for treating skin diseases, comprising a mesenchymal stem cell population as a first active ingredient.

[0497] In certain embodiments, the mesenchymal stem cell population is as described or defined above.

[0498] In certain embodiments, the skin disease is as described or defined above.

[0499] In certain embodiments, the product further comprises a second active ingredient.

[0500] In certain embodiments, the second active ingredient is as defined or described above.

[0501] In certain embodiments, the first active ingredient and the second active ingredient are present alone or in combination.

[0502] In certain embodiments, the first active ingredient is administered in combination with a second active ingredient selected from those described above.

[0503] In certain embodiments, the product is an implant, and preferably, the implant is used to improve the microenvironment and suppress the immune rejection reaction.

[0504] In certain embodiments, the subject is a mammal such as a human.

[0505] Atopic dermatitis (AD) Atopic dermatitis (AD) is a chronic, relapsing, pruritic, and inflammatory skin disease. AD has become a major public health problem, with a prevalence of up to 20% in children and 3% - 10% in adults. The etiology of AD is complex, involving many factors such as genetics, immunity, and environment. Among them, abnormal immune function, especially the immune response effect of immune cells, plays an important role in the onset of AD.

[0506] Currently, the treatment of AD usually requires the topical and / or systemic use of glucocorticoids and immunosuppressants. However, the topical use of glucocorticoids shows limited effects in moderate to severe AD patients. On the other hand, the systemic use of immunomodulators is associated with risks such as bone marrow suppression and increased risk of infection. New biologics such as dupilumab, an anti-interleukin (IL)-4R monoclonal antibody, and omalizumab, an anti-immunoglobulin IgE monoclonal antibody, show limited research results and significant differences. Therefore, there is a need to develop new, safe, and effective methods for treating AD.

[0507] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein for the prevention and / or treatment of atopic dermatitis in a subject, or for the prevention of atopic dermatitis in a subject, or for delaying or reducing atopic dermatitis, or for preventing and alleviating atopic dermatitis, in the manufacture of a medicament.

[0508] The present invention provides a treatment for atopic dermatitis using M cells. M cell treatment can improve the microenvironment of the skin of mice and suppress inflammation. Since there are more skin appendages in the M cell group than in the OVA group, it indicates that M cells can protect skin appendages and achieve a very excellent therapeutic effect against atopic dermatitis.

[0509] In certain embodiments, the pharmaceutical composition can alleviate erythematous rash, reduce the phenotype of atopic dermatitis, reduce AD-like lesions, reduce the thickness of the adipose layer, and / or reduce the thickness of the stratum corneum.

[0510] In certain embodiments, the pharmaceutical composition can reduce the degree of itching, protect the skin appendages, reduce the proliferation of mast cells, mediate the imbalance of Th1 / Th2 cells, reduce the intensity of IgE expression of CD19-positive cells, improve allergic diseases, and / or suppress the inflammatory reaction.

[0511] In certain embodiments, the subject is a mammal such as a human.

[0512] In certain embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0513] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), blood circulation route transplantation (e.g., intravenous injection transplantation or intraarterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0514] In certain preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In certain embodiments, the administration is performed by subcutaneous injection or subcutaneous spot injection.

[0515] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), etc. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic solution or sterile isotonic non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0516] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial, such as the above-described biological scaffold or, but not limited to, collagen scaffold, Matrigel, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid, etc.

[0517] In certain embodiments, the medicament can be implanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0518] In certain embodiments, the pharmaceutical composition also comprises biological antibodies (e.g., but not limited to, dupilumab, omalizumab).

[0519] Burn or scald Burn: A burn generally refers to tissue damage caused by heat, including high-temperature liquids (such as water, soup, oil, etc.), steam, high-temperature gases, flames, high-temperature liquid or solid metals (such as molten steel, steel ingots), etc., mainly related to the skin and / or mucous membranes. In severe cases, subcutaneous tissue and / or submucosal tissue such as muscles, bones, joints, and even organs may be damaged. A thermal burn is tissue damage caused by high-temperature liquids, steam, etc., and is a type of thermal injury.

[0520] Scald: A scald is tissue damage caused by high-temperature liquids without flames (such as boiling water, heated oil, molten steel), high-temperature solids (such as heated metals), or high-temperature steam. Low-heat scalds are common and are also known as low-temperature burns, which are burns caused by exposing the skin to low-heat objects higher than body temperature for a long time.

[0521] Burns often cause large-scale skin damage, resulting in the loss of skin barrier function and disruption of the internal environment balance, and wound healing takes a long time. Clinical treatment often requires large-area skin grafting, but the skin of burn patients is limited, there are secondary injuries during skin extraction, and wound infections can cause various complications such as difficult wound healing, septic shock, and progressive deepening of infected necrotic wounds. Contracture deformities are caused by scar healing after wound healing, which may thus lead to an unsightly appearance and functional impairment. The prognosis of patients is poor, the functional recovery is poor, and subsequent rehabilitation treatment increases the psychological and economic burden on patients. Therefore, finding a method that can more quickly promote wound healing and better restore the appearance and function of the skin has become an issue to be solved in the field of burns.

[0522] So far, skin injuries have been treated by autologous skin transplantation or artificial skin transplantation, but they are still insufficient in the case of large-area burns and scalds. The emergence of mesenchymal stem cells and combination treatment with materials has brought some hope for skin injuries.

[0523] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein for the prevention and / or treatment of burns or scalds in a subject, or for the manufacture of a medicament for preventing and / or treating burns or scalds in a subject.

[0524] In certain embodiments, the pharmaceutical composition can be used to address issues such as lack of functional recovery after skin injury, limited sources of skin grafts, and limited autologous skin.

[0525] In certain embodiments, the pharmaceutical composition can regenerate appendages, accelerate wound healing, reduce fibrosis after skin injury, thereby restoring skin function, reducing the wound area, treating skin injury, and protecting the skin.

[0526] In certain embodiments, the pharmaceutical composition can reduce inflammation at the wound site after burns, thereby suppressing inflammation.

[0527] In certain embodiments, the pharmaceutical composition can promote angiogenesis in skin wounds.

[0528] In certain embodiments, the pharmaceutical composition can promote hair follicle regeneration and upregulate factors such as β-catenin, CD133, and Ki67.

[0529] In certain embodiments, the pharmaceutical composition can reduce collagen deposition, thereby treating skin injury.

[0530] In certain embodiments, the subject is a mammal such as a human.

[0531] In certain embodiments, the medicament comprises a therapeutically effective amount of the mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0532] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), blood circulation pathway transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art are aware of methods for selecting an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0533] In certain embodiments, the culture supernatant of the present invention, or a pharmaceutical composition containing the culture supernatant, can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some preferred embodiments, the pharmaceutical composition is administered by topical application, surface transplantation or surface injection, or surface spray.

[0534] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic solution or sterile isotonic non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0535] In certain specific embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial, such as the aforementioned biological scaffold, or without limitation, a collagen scaffold, Matrigel, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteogen, hyaluronic acid, etc.

[0536] In certain specific embodiments, the medicine can be implanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0537] Refractory skin injury Refractory skin injury is a phenomenon of skin injury caused by various diseases or injuries, manifested by repeated skin ulcer formation, partial loss of skin function, and easy formation of scars and other skin hyperplastic tissues. Common causes of refractory skin injury include burns, diabetes (leading to diabetic foot), lupus erythematosus, and psoriasis.

[0538] Diabetic foot: The main symptoms of diabetic foot disease are lower limb pain and skin ulcers. Diabetic foot ulcers and gangrene are the main causes of clinical non-traumatic amputations and also seriously endanger the working ability and quality of life of diabetic patients. Diabetic foot is usually the result of a combination of lower limb neuropathy, vascular disease, and infections.

[0539] Diabetic complications 1. Diabetic nephropathy Diabetic nephropathy is one of the most important co-existing diseases for diabetic patients. The incidence of diabetic nephropathy is also increasing in China and is the second leading cause of end-stage renal disease after various glomerulonephritides. Due to its complex metabolic disorders, when diabetic nephropathy progresses to end-stage renal disease, it is often more difficult to treat than other kidney diseases. However, active and appropriate intervention measures can significantly reduce and delay the occurrence of diabetic nephropathy, especially in the early stages of the disease process.

[0540] 2. Diabetic eye complications (1) Diabetic retinopathy is the most important symptom of diabetic microangiopathy. It is an eye disease accompanied by specific changes and is one of the severe complications of diabetes. Clinically, depending on the presence or absence of retinal neovascularization, diabetic retinopathy without retinal neovascularization is called non-proliferative diabetic retinopathy (or simple or background type), and diabetic retinopathy with retinal neovascularization is called proliferative diabetic retinopathy.

[0541] (2) Diabetes-related uveitis generally includes the following four states: (i) uveitis related to diabetes itself, (ii) infectious uveitis in diabetic patients, where the possibility of endogenous infectious endophthalmitis is significantly higher than that in normal people, (iii) uveitis with some specific types, two of which are coincidental or have an inherent relationship, (iv) infectious or aseptic endophthalmitis after intraocular surgery. Diabetes-related uveitis mainly occurs in middle-aged and elderly patients with diabetes.

[0542] (3) Diabetic cataract occurs in young diabetic patients with poorly controlled blood sugar levels. Diabetic cataract almost always occurs in both eyes, develops rapidly, and may even develop into complete opacity within a few days, weeks, or months.

[0543] 3. Diabetic foot The foot is a complex target organ of diabetes, a multi-system disease. Due to the combination of peripheral neuropathy and peripheral vascular disease in diabetic patients, after excessive mechanical pressure causes damage and deformation of the soft tissues and osteoarticular system of the foot, a series of foot problems can occur, ranging from mild neurological symptoms to severe ulcers, infections, vascular diseases, Charcot arthropathy, and neuropathic fractures. In fact, although similar pathological changes may also occur in the upper extremities, face, and trunk, the incidence of diabetic foot is significantly higher than that in other parts.

[0544] 4. Diabetic cardiovascular complications Diabetic cardiovascular complications include microvascular diseases, cardiomyopathy, and cardiac autonomic neuropathy in the heart and large blood vessels, which are the main causes of death in diabetic patients. Coronary heart disease is the main macrovascular complication of diabetes. According to research, the risk of death due to coronary heart disease in diabetic patients has been shown to be 3 to 5 times higher than that in non-diabetic patients. The pathological mechanism is atherosclerosis, and hyperglycemia, high systolic blood pressure, high cholesterol, increased low-density lipoprotein, decreased high-density lipoprotein, age, gender, smoking, and family history are all risk factors for its development.

[0545] 5. Diabetic Cerebrovascular Diseases Diabetic cerebrovascular diseases refer to the lesions of large and small blood vessels in the skull caused by diabetes. According to statistics, 20% to 40% of patients with type 2 diabetes will develop cerebrovascular diseases mainly manifested as arteriosclerosis, ischemic cerebrovascular diseases, cerebral hemorrhage, brain atrophy, etc., so it is one of the main causes of death in patients with diabetes.

[0546] 6. Diabetic Neuropathy The most common type of diabetic neuropathy is chronic distal symmetric sensorimotor polyneuropathy, that is, diabetic peripheral neuropathy, which has a high incidence. Some patients already have peripheral neuropathy when newly diagnosed with diabetes. Unfortunately, especially regarding the treatment for the radical cure of diabetic neuropathy, radical cure is very difficult, so the focus is on preventing its occurrence and controlling its onset.

[0547] Refractory skin injury is not a disease but a phenomenon of skin injury caused by various diseases or injuries, which is manifested by easy repeated skin ulcer formation, partial loss of skin function, easy generation of scars and other hyperplastic skin tissues. Common factors leading to refractory skin injury include burns and scalds, diabetes, lupus erythematosus, and psoriasis. Currently, there is no general solution to these problems. This is because such injuries are often accompanied by complex immune disorders and tissue regeneration disorders, and a single treatment plan cannot solve all problems.

[0548] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating refractory skin injury in a subject, or for preventing refractory skin injury in a subject, or for delaying or alleviating or preventing scleroderma, or for alleviating the symptoms of refractory skin injury. In some cases, the refractory skin injury is caused by these factors (e.g., but not limited to, burns and scalds, diabetes, lupus erythematosus, psoriasis, etc.).

[0549] In certain preferred embodiments, administration can be effected by subcutaneous injection.

[0550] In certain embodiments, the pharmaceutical composition can accelerate the wound healing rate, reduce the wound area, promote angiogenesis in skin wounds, and regenerate the skin after injury during the treatment of refractory skin injury.

[0551] In certain embodiments, the pharmaceutical composition can reduce the expression of the genes or proteins of CD3, F4 / 80, MPO and suppress inflammation during the treatment of refractory skin injury.

[0552] In certain embodiments, the pharmaceutical composition can increase the expression of the genes or proteins of β-catenin, CD133, Ki67, CD31 during the treatment of refractory skin injury.

[0553] In certain embodiments, the pharmaceutical composition can promote hair follicle regeneration.

[0554] In certain embodiments, the pharmaceutical composition can reduce the expression of the inflammatory-inducing factors IL-1β, IL-6, and TNFα in diabetic nephropathy, reduce mesangial hyperplasia and macrophage infiltration, alleviate diabetic-induced glomerulopathy, and increase kidney weight, kidney, and body mass index in rats, so it may have a good therapeutic effect on diabetic nephropathy.

[0555] In certain embodiments, the pharmaceutical composition can accelerate the healing of diabetic foot, reduce the inflammation of skin wounds, promote the regeneration of blood vessels and hair follicles, reduce the deposition of collagen, and inhibit the occurrence of fibrosis in diabetic foot, so it can effectively treat skin damage.

[0556] In certain embodiments, the pharmaceutical composition can lower blood glucose in diabetic complications, regulate the inflammatory response, significantly reduce fasting blood glucose and HbA1c levels, and improve visual function and macular edema to a certain extent, so it can sufficiently treat diabetic complications.

[0557] In certain embodiments, the pharmaceutical composition can inhibit vascular calcification in vascular calcification complicated with diabetes, so it has a good therapeutic effect on vascular calcification diseases in complications.

[0558] In certain embodiments, the pharmaceutical composition enhances the ability of astrocytes to resist oxidative stress in diabetic neuropathy, removes glutamate in the brain, and enhances their ability to maintain the K + balance in the brain, thereby promoting neuron function, brain homeostasis, and synapse formation, and improving cognitive impairment caused by diabetes.

[0559] In certain embodiments, the subject is a mammal such as a human.

[0560] In certain embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0561] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to the subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), blood circulation pathway transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0562] In certain preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In certain preferred embodiments, the administration is performed by subcutaneous injection.

[0563] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), and the like. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic solution or sterile isotonic non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of this pharmaceutical composition can be found in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0564] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including the above-described biological scaffolds, or without limitation, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose poly(lactic acid), elastic proteoglycan, hyaluronic acid, and the like.

[0565] In certain embodiments, the medicament can be transplanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0566] Psoriasis Psoriasis is a common chronic inflammatory skin disease with intractable and recurrent characteristics. The cause of psoriasis is unknown, but it is currently considered to be a disease caused by the interaction of genetic factors, environmental factors, and other factors.

[0567] Clinically, it is divided into four common types: vulgaris, pustular, arthritic, and erythrodermic. Its skin lesions are initially erythematous papules, covered by a layer of silvery-white scales on their surface, dry skin, desquamation, and crusts, with several skin symptoms connected like a map, and may be accompanied by itching, pus and water, and unbearable blood streaks.

[0568] Vulgaris psoriasis is very obvious because the red papules the size of mung beans caused by dermatitis then slowly grow to form silvery-white dry scales. In severe cases, large white scales cover the body, especially looking terrifying. Vulgaris psoriasis may be accompanied by bleeding, but this is unacceptable.

[0569] Pustular type: Very dense pustules of various sizes can be seen. As the disease worsens, the pustules continue to grow and eventually form erythema. This symptom is an emergency and occurs suddenly. People with this type of psoriasis have fever, joint pain and swelling.

[0570] Erythrodermic psoriasis: It appears as a diffuse flushing, infiltration, and swelling of the whole body, with a large number of bran-like scales, and there is normal skin in flakes in between, and may be accompanied by systemic symptoms such as fever and superficial lymph node swelling. The process of this disease is long and it is prone to relapse.

[0571] Arthritic psoriasis: In addition to skin lesions, joint lesions may occur, and any joint including the elbows, large knee joints, joints of the little finger and toes, spine and sacroiliac joints may be affected. Arthritic psoriasis may appear as joint swelling and pain, limited movement, joint deformation in severe cases, and progressive development, but the rheumatoid factor test is often negative.

[0572] Psoriasis (commonly known as tinea) is a known skin disease. When psoriasis occurs, red papules or plaques may be seen on the skin, which are covered with multiple layers of silvery white scales. Psoriasis tends to occur on the limbs, head and back, and even throughout the body, and may last almost a lifetime. Currently, there is no effective treatment. This disease mainly affects young and middle-aged people, greatly affecting the physical and mental health of patients and causing a heavy burden on society and the economy. Epidemiological surveys have shown that there are currently about 6.5 million psoriasis patients in China, and the incidence rate is 0.47%.

[0573] Currently, psoriasis is considered an autoimmune skin disease caused by the predominance of dendritic cells (DCs) and T lymphocytes, the involvement of innate and adaptive immunity, and the interaction of genetic background and environmental factors. Characteristic lesions of psoriasis include excessive proliferation of keratinocytes caused by inflammatory conditions. Although antagonistic biologics targeting the major cytokines (TFN-α, IL-12, IL-23, IL-17) in the etiology of psoriasis are extremely effective in clinical treatment, the high cost for maintaining long-term treatment and potential serious adverse reactions limit the widespread application of such biologics.

[0574] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating psoriasis in a subject, or for preventing psoriasis in a subject, or delaying or alleviating psoriasis, or preventing and alleviating psoriasis.

[0575] In certain embodiments, the pharmaceutical composition can relieve erythematous eruptions, relieve scales, relieve infiltration, reduce the phenotype of psoriatic dermatitis, reduce psoriatic lesions, reduce the spinous layer of the epidermis, or reduce the thickness of the stratum corneum.

[0576] In certain embodiments, the pharmaceutical composition can reduce ROS levels, reduce the recruitment of splenic neutrophil cells and dendritic cells, reduce inflammatory infiltrating cells, and / or regulate immune function.

[0577] In certain embodiments, the subject is a mammal such as a human.

[0578] In certain embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0579] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), blood circulation pathway transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid pathway transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation pathway according to the location and nature of the lesion.

[0580] In certain preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In certain preferred embodiments, the administration is performed by dorsal injection or intravenous injection.

[0581] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), and the like. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic solution or sterile isotonic non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of such pharmaceutical compositions can be found in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0582] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial, such as the above-described biological scaffold or, but not limited to, collagen scaffold, Matrigel, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose poly(lactic acid), elastinogen, hyaluronic acid, and the like.

[0583] In certain embodiments, the medicament can be transplanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0584] Autoimmune disease An immune system disease refers to a pathological reaction caused by damage to the immune system. Immune system diseases mainly include infectious diseases, allergic diseases, autoimmune diseases, immunoproliferative diseases, immunodeficiency diseases, and immune-related diseases. As used herein, the term "autoimmune disease" refers to a disease in which the body attacks its own tissues due to immune system dysfunction. Common autoimmune diseases are often related to multiple systems and organs (e.g., skin, bone, muscle, internal organs, etc.), and thus form systemic autoimmune diseases such as systemic lupus erythematosus, ankylosing spondylitis, rheumatoid arthritis, psoriasis, erythroderma, glomerulonephritis, ANCA-related vasculitis, scleroderma, primary systemic amyloidosis, autoimmune hepatitis, autoimmune pancreatitis, autoimmune gastritis, Crohn's disease, ulcerative colitis, erythema nodosum, Hashimoto's thyroiditis, alopecia areata, eczema, and type 1 diabetes.

[0585] In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing, treating, delaying, and / or alleviating an autoimmune disease, or the present invention provides a method for preventing, treating, delaying, and / or alleviating an autoimmune disease, which comprises administering a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant to a subject in need thereof.

[0586] In certain embodiments, the autoimmune disease is selected from the group consisting of scleroderma, lupus (e.g., systemic lupus erythematosus), psoriasis, rheumatoid arthritis, dermatomyositis, multiple sclerosis, myasthenia gravis, polymyositis, inflammatory bowel disease (e.g., ulcerative colitis (UC), Crohn's disease (CD)), Sjogren's syndrome, vasculitis (e.g., systemic vasculitis), adult Still's disease, or any combination thereof.

[0587] In certain embodiments, the mesenchymal stem cell population of the present invention is administered in combination with an additional active ingredient, so the medicament may contain an additional active ingredient. In certain embodiments, the mesenchymal stem cells are administered simultaneously, separately, or sequentially with an additional therapeutic agent. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation.

[0588] In certain embodiments, the additional active ingredient is selected from the group consisting of anti-inflammatory drugs or immunosuppressive agents. In certain embodiments, the additional active ingredient is a non-steroidal anti-inflammatory drug (e.g., ibuprofen, diclofenac, naproxen, indomethacin, piroxicam, meloxicam, nabumetone, or nimesulide), a steroidal anti-inflammatory drug (e.g., prednisone, dexamethasone, or hydrocortisone), an antibody or antagonist of an inflammation-inducing cytokine (e.g., an antibody or receptor antagonist of TNFα, IL-1, IL-6, IL-8, GM-CSF, or PAF), an anti-inflammatory cytokine (e.g., IL-10, IL-4, IL-11, IL-13, or TGFβ), an anti-proliferative / antimetabolite (e.g., cyclophosphamide, methotrexate, azathioprine, leflunomide), a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), or any combination thereof.

[0589] In certain embodiments, the medicament is in unit dosage form, and the unit dose of the medicament is 1×10 4 or more cells (e.g., 1×10 4 or more cells, 3×10 4 or more cells, 5×10 4 or more cells, 7×10 4 or more cells, 1×10 5 or more cells, 3×10 5 or more cells, 5×10 5 or more cells, 7×10 5 or more cells, 1×10 6 or more cells, 3×10 6 or more cells, 5×106 more than one cell, 7×10 6 more than one cell, 1×10 7 more than one cell, 3×10 7 more than one cell, 5×10 7 more than one cell, 7×10 7 more than one cell, 1×10 8 more than one cell, 3×10 8 more than one cell, 5×10 8 more than one cell, 7×10 8 more than one cell, 1×10 9 more than one cell, 3×10 9 more than one cell, 5×10 9 more than one cell, 7×10 9 more than one cell, 1×10 10 more than one cell, 3×10 10 more than one cell, 5×10 10 more than one cell, or 7×10 10 more than one cell) in an amount. In certain embodiments, the unit dose of the medicament is 1×10 5 cells to 1×10 8 cells (e.g., 1×10 6 cells to 1×10 8 cells, 1×10 6 cells to 1×10 7 cells, or 1×10 6 cells to 5×10 6 cells) in an amount.

[0590] In another aspect, the present invention also provides a product for preventing, treating, delaying, and / or alleviating an autoimmune disease, which comprises the mesenchymal stem cell population of the present invention. In certain embodiments, the product further comprises an additional active ingredient as defined above. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation. In certain embodiments, the product is an injection, microinjection, mucosal patch, enema, suppository, gel, oral preparation, aerosol, drop, ointment, implant, or capsule. In certain embodiments, the product is an implant.

[0591] Eritematodes Eritematodes is a typical autoimmune connective tissue disease that is more common in women aged 15 to 40. Eritematodes is a spectrum disease that can be divided into several subtypes such as discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), systemic lupus erythematosus (SLE), lupus erythematosus profundus (LEP), neonatal lupus erythematosus (NLE), and drug-induced lupus erythematosus (DIL). The term "systemic lupus erythematosus" refers to an autoimmune disease that is slow to develop, has a insidious onset, and has a variety of clinical symptoms, and is related to many systems and organs in which a large number of autoantibodies are produced due to cellular immunodeficiency and humoral immunodeficiency. Systemic lupus erythematosus can affect the skin, serosa, joints, kidneys, and central nervous system and is characterized by autoimmunity. Patients have various autoantibodies, which affect not only humoral immunity but also cellular immunity, and the complement system also changes.

[0592] In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing, treating, delaying, and / or alleviating lupus erythematosus (e.g., systemic lupus erythematosus), or alternatively, the present invention provides a method for preventing, treating, delaying, and / or alleviating lupus erythematosus (e.g., systemic lupus erythematosus), which comprises administering a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant to a subject in need thereof.

[0593] In certain embodiments, the mesenchymal stem cell population of the present invention can delay the systemic pathogenic process by reducing anti-double-stranded DNA antibodies.

[0594] In certain embodiments, the mesenchymal stem cell population of the present invention can delay the pathogenic process of lupus erythematosus by avoiding, preventing, or suppressing the enlargement of the spleen and cervical lymph nodes.

[0595] In certain embodiments, the mesenchymal stem cell population of the present invention can promote the formation of glomeruli.

[0596] In certain embodiments, the mesenchymal stem cell population of the present invention can inhibit inflammatory factors.

[0597] In certain embodiments, the mesenchymal stem cell population of the present invention can reduce the number of T cell populations (e.g., CD3 + T cells, CD4 + T cells, and CD4 + T cells) in the spleen.

[0598] In certain embodiments, the subject is a mammal such as a human.

[0599] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0600]

[0601] In certain preferred embodiments, the mesenchymal stem cell population of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In certain preferred embodiments, the administration is performed by intravenous injection.In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), and the like. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In certain embodiments, the pharmaceutical composition includes a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of such pharmaceutical compositions can be found in Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0602] In certain embodiments, the pharmaceutical composition includes a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteogen, hyaluronic acid, and the like.

[0603] In certain embodiments, the medicament can be implanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0604] In certain embodiments, the mesenchymal stem cell population of the present invention is administered in combination with an additional active ingredient, so the medicament may contain an additional active ingredient. In certain embodiments, the mesenchymal stem cells are administered simultaneously, separately, or sequentially with an additional therapeutic agent. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation.

[0605] In certain embodiments, the additional active ingredient is selected from the group consisting of anti-inflammatory drugs or immunosuppressive agents. In certain embodiments, the additional active ingredient is a non-steroidal anti-inflammatory drug (e.g., ibuprofen, diclofenac, naproxen, indomethacin, piroxicam, meloxicam, nabumetone, or nimesulide), a steroidal anti-inflammatory drug (e.g., prednisone, dexamethasone, or hydrocortisone), an antibody or antagonist of an inflammation-inducing cytokine (e.g., an antibody or receptor antagonist of TNFα, IL-1, IL-6, IL-8, GM-CSF, or PAF), an anti-inflammatory cytokine (e.g., IL-10, IL-4, IL-11, IL-13, or TGFβ), an anti-proliferative / antimetabolite (e.g., cyclophosphamide, methotrexate, azathioprine, leflunomide), a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), or any combination thereof.

[0606] In certain embodiments, the medicament is in unit dosage form, and the unit dose of the medicament is 1×10 4 or more cells (e.g., 1×10 4 or more cells, 3×10 4 or more cells, 5×10 4 or more cells, 7×10 4 or more cells, 1×10 5 or more cells, 3×10 5 or more cells, 5×10 5 or more cells, 7×10 5 or more cells, 1×10 6 or more cells, 3×10 6 or more cells, 5×106 or more cells, 7×10 6 or more cells, 1×10 7 or more cells, 3×10 7 or more cells, 5×10 7 or more cells, 7×10 7 or more cells, 1×10 8 or more cells, 3×10 8 or more cells, 5×10 8 or more cells, 7×10 8 or more cells, 1×10 9 or more cells, 3×10 9 or more cells, 5×10 9 or more cells, 7×10 9 or more cells, 1×10 10 or more cells, 3×10 10 or more cells, 5×10 10 or more cells, or 7×10 10 or more cells) and is contained in an amount of. In certain embodiments, the unit dose of the medicament is 1×10 4 cells to 1×10 10 cells (for example, 1×10 6 cells to 1×10 8 cells, 1×10 6 cells to 1×10 7 cells, or 1×10 6 cells to 5×10 6 cells) and is contained in an amount of.

[0607] Scleroderma The skin is the organ with the largest surface area in the human body. The skin is an important structure in protecting internal tissues from mechanical damage, microbial infection, ultraviolet rays, and extreme temperatures. Skin diseases include viral skin diseases, bacterial skin diseases, fungal skin diseases, animal skin diseases, physical skin diseases, dermatitis, eczema, drug eruptions, urticarial skin diseases, pruritic skin diseases, erythematous scaly skin diseases, connective tissue diseases, vesicular skin diseases, vasculitic skin diseases, skin appendage diseases, skin pigmentation disorders, hereditary skin diseases, skin tumors, and sexually transmitted infections.

[0608] Dermatomyositis is a type of connective tissue disease of the skin. Dermatomyositis or systemic sclerosis (SSC) is a progressive debilitating autoimmune disease also known as cutaneous fibrosis, characterized by excessive protein deposition in the extracellular matrix by skin fibroblasts. Typical skin lesions sequentially go through three stages: swelling, infiltration, and atrophy. The lesions are symmetrical, and most of them spread from the fingers to the proximal end and are related to the connective tissue of internal organs such as the heart, lungs, kidneys, and digestive tract.

[0609] Dermatomyositis is an autoimmune disease that may affect the lungs, kidneys, liver, heart, and other organs, characterized by thickening of the skin and focal or diffuse fibrosis. Its etiology is unknown. Current research has found that this disease mainly includes three aspects: small blood vessel disease, fibrosis caused by excessive accumulation of extracellular matrix, and immune abnormalities. Inflammatory cell infiltration is a major feature in the initial stage of dermatomyositis, mainly T lymphocyte infiltration. According to research, it has been shown that T lymphocytes can release various cytokines, cause inflammation and vascular lesions, activate fibroblasts, and promote the synthesis of collagen fibers. Currently, immunosuppressants and symptomatic treatments are mainly used for dermatomyositis, but the treatment effect is not ideal and there are many side effects, so it is necessary to find more effective treatment methods.

[0610] In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing, treating, delaying, and / or alleviating dermatomyositis. Alternatively, the present invention provides a method for preventing, treating, delaying, and / or alleviating dermatomyositis, which includes administering a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant to a subject in need thereof.

[0611] In a certain specific embodiment, the mesenchymal stem cell population can significantly thin the dermis and reduce the accumulation of collagen fibers in dermatomyositis.

[0612] In certain embodiments, the mesenchymal stem cell population can reduce the occurrence of skin sclerosis and thickening in scleroderma and has an effective therapeutic effect on scleroderma.

[0613] In certain embodiments, the mesenchymal stem cell population can increase the number of hair follicles and reduce the dermal thickness in scleroderma.

[0614] In certain embodiments, the mesenchymal stem cell population can prevent the significant thinning of the fat layer and prevent the reduction of skin appendages in scleroderma.

[0615] In certain embodiments, the mesenchymal stem cell population can inhibit inflammatory factors (e.g., IL-17, IL-6, TNF), suppress the expression level of inflammatory factors, and / or increase the expression level of anti-inflammatory factors (e.g., IL10), increase the expression level of MMP1 protein, and reduce or suppress the expression of smooth muscle actin (α-SMA) in scleroderma.

[0616] In certain embodiments, the subject is a mammal such as a human.

[0617] In certain embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to the subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0618] In certain preferred embodiments, the mesenchymal stem cell population of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In certain preferred embodiments, the administration is performed by subcutaneous injection.

[0619] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), and the like. In certain embodiments, the pharmaceutical composition is an injection (including injection solutions and lyophilized powders). In certain embodiments, the pharmaceutical composition comprises a sterile, pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles regarding the formulation of such pharmaceutical compositions can be found in Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0620] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including, but not limited to, collagen scaffolds, Matrigel, skin repair membranes, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid, and the like.

[0621] In certain embodiments, the medicament can be implanted in the form of a suspension, gel, colloid, slurry, or mixture.

[0622] In certain embodiments, the mesenchymal stem cell population of the present invention is administered in combination with an additional active ingredient, and thus the medicament may contain an additional active ingredient. In certain embodiments, the mesenchymal stem cells are administered simultaneously, separately, or sequentially with an additional therapeutic agent. In certain embodiments, the mesenchymal stem cell population and the additional active ingredient exist as separate components or as a single formulation.

[0623] In certain embodiments, the additional active ingredient is selected from the group consisting of anti-inflammatory drugs or immunosuppressive agents. In certain embodiments, the additional active ingredient is a non-steroidal anti-inflammatory drug (e.g., ibuprofen, diclofenac, naproxen, indomethacin, piroxicam, meloxicam, nabumetone, or nimesulide), a steroidal anti-inflammatory drug (e.g., prednisone, dexamethasone, or hydrocortisone), an antibody or antagonist of an inflammation-inducing cytokine (e.g., an antibody or receptor antagonist of TNFα, IL-1, IL-6, IL-8, GM-CSF, or PAF), an anti-inflammatory cytokine (e.g., IL-10, IL-4, IL-11, IL-13, or TGFβ), an anti-proliferative / antimetabolite (e.g., cyclophosphamide, methotrexate, azathioprine, leflunomide), a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), or any combination thereof.

[0624] In certain embodiments, the medicament is in unit dosage form, and the unit dose of the medicament is 1×10 5 or more cells (e.g., 1×10 5 or more cells, 5×10 5 or more cells, 1×10 6 or more cells, 2×10 6 or more cells, 3×10 6 or more cells, 4×10 6 or more cells, 5×10 6 or more cells, 6×10 6 or more cells, 7×10 6 or more cells, 8×10 6 or more cells, 9×106 one or more cells, 1×10 7 one or more cells, 3×10 7 one or more cells, 5×10 7 one or more cells, 7×10 7 one or more cells, 1×10 8 one or more cells, 3×10 8 one or more cells, 5×10 8 one or more cells, 7×10 8 contained in an amount of (one or more cells). In certain embodiments, the unit dose of the medicament is 1×10 5 cells to 1×10 8 cells (e.g., 1×10 6 cells to 1×10 8 cells, 1×10 6 cells to 1×10 7 cells, or 1×10 6 cells to 5×10 6 cells).

[0625] Respiratory diseases In one aspect, the present invention provides the use of a mesenchymal stem cell population or its culture supernatant in the manufacture of a medicament for preventing and / or treating respiratory diseases. Alternatively, the present invention provides a method for preventing and / or treating respiratory diseases, comprising administering to a subject in need thereof a prophylactically effective amount and / or a therapeutically effective amount of a mesenchymal stem cell population or its culture supernatant.

[0626] The respiratory system is a general term for a series of organs related to gas exchange between the human body and the outside air, including tissues such as the nose, pharynx, larynx, trachea, bronchi, and the lungs composed of numerous alveoli, blood vessels, lymphatic vessels, nerves, and pleura. Clinically, the nose, pharynx, and larynx are often referred to as the upper airway, and a part of the gas passage below the trachea (including all levels of bronchi in the lungs) is called the lower airway.

[0627] Lung diseases refer to diseases of the lungs themselves or pulmonary symptoms of systemic diseases. Lung diseases mainly include infectious lung diseases, air pollution and smoking-related lung diseases, occupation-related lung diseases, immune-related lung diseases, genetic-related lung diseases, and lung diseases of unknown cause. In some embodiments, the lung disease is selected from the group consisting of pulmonary vascular diseases, idiopathic pulmonary fibrosis, acute respiratory distress, pneumoconiosis, and pneumonia. In some embodiments, the pulmonary vascular disease is selected from the group consisting of pulmonary hypertension, cor pulmonale, pulmonary embolism, pulmonary vasculitis, chronic obstructive pulmonary disease, and interstitial lung disease. In some embodiments, the lung disease is pulmonary arterial hypertension (PAH).

[0628] Occupation-related lung diseases: Occupation-related lung diseases refer to lung diseases such as pneumoconiosis caused by lung damage caused by inhaling harmful dust, smoke, or poisons in a specific occupation. Hazardous dusts include silica (i.e., quartz), silicates, coal, iron, and tin. Smokes include sulfur dioxide, nitrogen dioxide, ammonium, hydrochloric acid, chlorine, phosgene, and smokes of other harmful gases and strong acids. Toxic substances include uranium, nickel, chromate, asbestos, dichloromethyl ether, etc.

[0629] Pneumoconiosis: Pneumoconiosis is a systemic disease mainly manifested by diffuse fibrosis (scarring) of lung tissue induced by long-term inhalation and retention in the lungs of productive dust (ash) during occupational activities. Pneumoconiosis can be divided into inorganic pneumoconiosis and organic pneumoconiosis according to the type of inhaled dust. Pneumoconiosis caused by inhalation of inorganic dust in productive labor is called inorganic pneumoconiosis. Most pneumoconiosis is inorganic pneumoconiosis. Pneumoconiosis caused by inhalation of organic dust such as byssinosis and farmer's lung is called organic pneumoconiosis.

[0630] Pneumoconiosis is a progressive chronic disease. Different from acute infectious diseases or other chronic diseases (such as tuberculosis, hypertension, diabetes, etc.) for which obvious therapeutic effects can be recognized in a short period, pneumoconiosis generally requires long-term treatment for several years to obtain a more obvious curative effect.

[0631] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating pneumoconiosis, or delaying or reducing or preventing or alleviating pneumoconiosis, or alternatively, the present invention relates to a method for preventing and / or treating pneumoconiosis, or delaying or reducing or preventing or alleviating pneumoconiosis, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0632] In some embodiments, the medicament of the present invention can reduce the level of inflammatory factors in the serum, improve lung function, reduce the lung compact area, and / or reduce fibrosis formation.

[0633] In some embodiments, the subject is a mammal such as a human.

[0634] In some embodiments, the medicament comprises a therapeutically effective amount of a mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0635] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to the subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subdural injection transplantation). Those skilled in the art are aware of the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0636] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some preferred embodiments, the administration is performed by intravenous injection.

[0637] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For general principles regarding the formulation of such pharmaceutical compositions, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0638] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial, such as the above-mentioned biological scaffold, or without limitation, collagen scaffold, Matrigel, skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastic proteoglycan, hyaluronic acid, etc.

[0639] In some embodiments, the medicine can be transplanted in the form of a suspension, gel, colloid, serous fluid, or mixture.

[0640] Pneumonia: Pneumonia refers to infectious inflammation of the alveoli, distal airways, and lung interstitium that can be caused by bacterial pathogens, viral pathogens, and other pathogens. Among them, bacterial pneumonia and viral pneumonia are the most common. In a broad sense, pneumonia may be caused by pathogenic microorganisms, physicochemical factors, immune damage, allergies, and drugs. Patients often exhibit typical symptoms such as fever, cough, and dyspnea.

[0641] Emphysema: Emphysema is a pathological condition in which the airway elasticity of the distal terminal bronchi decreases, overinflates, expands, and the lung volume increases, or is accompanied by destruction of the airway wall. According to its etiology, emphysema includes the following types: senile emphysema, compensatory emphysema, interstitial emphysema, localized emphysema, paraseptal emphysema, and obstructive emphysema.

[0642] Bronchitis: Bronchitis is a chronic non-specific inflammation of the trachea, bronchial mucosa, and its surrounding tissues. The main cause of bronchitis is chronic non-specific inflammation of the bronchi due to repeated infections by viruses and bacteria. Bronchitis mainly includes acute bronchitis and chronic bronchitis.

[0643] Chronic bronchitis: Chronic bronchitis is a chronic non-specific inflammation of the trachea, bronchial mucosa, and its surrounding tissues. The main symptoms are cough, sputum production, or wheezing.

[0644] Chronic obstructive pulmonary disease: Chronic obstructive pulmonary disease is chronic bronchitis and / or emphysema characterized by airflow obstruction, which may further develop into common chronic diseases such as cor pulmonale and respiratory failure. Chronic obstructive pulmonary disease is related to abnormal inflammatory responses to harmful gases and harmful particles, and has a high morbidity and mortality rate.

[0645] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating chronic obstructive pulmonary disease, or delaying or reducing chronic obstructive pulmonary disease, or preventing or alleviating chronic obstructive pulmonary disease, or the present invention relates to a method for preventing and / or treating chronic obstructive pulmonary disease, or delaying or reducing or preventing or alleviating chronic obstructive pulmonary disease, the method comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0646] In some embodiments, the medicament of the present invention can reduce the lung dense area, improve lung function including vital capacity, improve maximal ventilation, reduce airway resistance, reduce mean alveolar diameter, maintain the integrity of the lung structure, and / or increase the partial pressure of oxygen in arterial blood.

[0647] In some embodiments, the medicament can reduce the level of inflammation-inducing factors, increase the level of anti-inflammatory factors, and suppress inflammation.

[0648] In some embodiments, the medicament can reduce the expression levels of collagen I and α-SMA protein in the lung and suppress the occurrence of fibrosis.

[0649] In some embodiments, the subject is a mammal such as a human.

[0650] In some embodiments, the medicament comprises a therapeutically effective amount of the mesenchymal stem cell population and / or culture, and / or a therapeutically effective amount of the culture supernatant described herein.

[0651] In some embodiments, the mesenchymal stem cell population or pharmaceutical composition described herein is administered to a subject by local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation), circulatory route transplantation (e.g., intravenous injection transplantation or intraarterial injection transplantation), or cerebrospinal fluid route transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art recognize the method of selecting an appropriate cell transplantation route according to the location and nature of the lesion.

[0652] In some preferred embodiments, the medicament of the present invention can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, etc. In some preferred embodiments, the administration is performed by intravenous injection.

[0653] In some embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the pharmaceutical composition is an injection (including injection solutions, lyophilized powders). In some embodiments, the pharmaceutical composition comprises a sterile pharmaceutically acceptable isotonic aqueous or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. For the general principles regarding the formulation of this pharmaceutical composition, reference can be made to Cell Therapy: stem cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996, and Hematopoietic stem cell treatment, E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

[0654] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial including the above-described biological scaffold, or without limitation, a collagen scaffold, Matrigel, a skin repair membrane, aminated gelatin, chitosan, silk fibroin, cellulose polylactic acid, elastinogen, hyaluronic acid, and the like.

[0655] In some embodiments, the medicament can be implanted in the form of a suspension, a gel, a colloid, a serous fluid, or a mixture.

[0656] Immune-related lung diseases: Immune-related lung diseases refer to the defensive immune response and allergic response in the lungs when the lungs are attacked by external allergens. Immune-related lung diseases mainly appear in acute, subacute, or chronic interstitial pneumonia.

[0657] Infectious lung diseases: Infectious pulmonary diseases refer to diseases caused by pathogenic microorganism infections in the lungs. Infectious pulmonary diseases are mainly classified into bacterial pneumonia, viral pneumonia, mycoplasma pneumonia, fungal pneumonia, and tuberculosis. Bacterial pneumonia is pneumonia caused by bacterial infections, including pneumonia caused by Streptococcus pneumoniae, Staphylococcus aureus, Gram-negative bacteria, and other infectious diseases. Viral pneumonia is pneumonia caused by viral infections in the upper respiratory tract, mainly including influenza, pharyngitis, atypical pneumonia (SARS), Middle East Respiratory Syndrome (MERS), and coronavirus disease 2019 (COVID-19), which are caused by influenza virus, parainfluenza virus, cytomegalovirus, adenovirus, rhinovirus, coronavirus (SARS virus, MERS virus, and novel coronavirus), and some enteroviruses, etc. Mycoplasma pneumonia is pneumonia caused by mycoplasma pneumoniae pneumonia. Fungal pneumonia includes pneumonia caused by fungi such as Aspergillus. Tuberculosis is a lung disease caused by infection with Mycobacterium tuberculosis.

[0658] Acute respiratory failure: Acute respiratory failure is acute respiratory failure caused by pulmonary ventilation dysfunction and / or ventilation dysfunction caused by hypoventilation induced by respiratory diseases such as severe respiratory infections, acute obstructive respiratory diseases, severe or critical asthma, acute pulmonary edema of various causes, pulmonary vascular diseases, chest trauma or surgical diseases, etc., spontaneous pneumothorax and sudden increase in pleural effusion, acute intracranial infections, craniocerebral trauma, cerebrovascular diseases that directly or indirectly inhibit the respiratory center, etc., poliomyelitis, myasthenia gravis, organophosphorus poisoning, cervical spine trauma that damages the neuromuscular transmission system, etc.

[0659] Respiratory distress syndrome (ARDS): Respiratory distress syndrome is a type of acute respiratory failure caused by various reasons, in which the fluid exchange function of pulmonary vascular tissue leads to an increase in pulmonary water content, a decrease in pulmonary compliance, alveolar collapse, and an imbalance in ventilation-perfusion ratio. Typical symptoms are severe hypoxemia and extreme respiratory distress. Respiratory distress syndrome is mainly caused by internal factors and external factors such as severe infections, trauma, and shock. Respiratory distress syndromes include acute respiratory distress syndrome and neonatal respiratory distress syndrome.

[0660] Pneumonia "COVID-19" caused by the infection of the novel coronavirus "SARS-CoV-2" has a long incubation period, high infectivity, and high harmfulness. So far, there is no effective treatment for COVID-19, but severely ill patients with COVID-19 have a poor prognosis, a high mortality rate, and their clinical treatment is particularly urgently needed.

[0661] According to the latest epidemiological data, some patients with COVID-19 develop acute respiratory distress syndrome (ARDS), which leads to respiratory failure, and even the functions of other organs are affected and may even result in death. ARDS appears as a clinical syndrome of rapidly progressive dyspnea, hypoxemia, diffuse pulmonary infiltration, and respiratory failure. The current treatment options for ARDS are limited to symptomatic treatments such as basic medical care and supportive ventilation strategies, and it is still unable to reverse the disease process, improve the quality of life of patients, and reduce the mortality rate. Mechanical ventilation is the main treatment for patients with acute respiratory distress syndrome. During the process of mechanical ventilation, complications such as ventilator-associated pneumonia, ventilator-associated lung injury, deep vein thrombosis, difficulty in weaning from mechanical ventilation, and pulmonary fibrosis often occur. Drug treatment methods include corticosteroids, statins, aspirin, β-2 receptor agonists, surfactants, and inhaled NO, but none of them have shown significant effectiveness. The above two treatment methods, even together with auxiliary methods such as blood purification treatment, nutritional intervention, and fluid management, cannot cope with the treatment of ARDS caused by COVID-19.

[0662] In one aspect, the present invention relates to the use of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein in the manufacture of a medicament for preventing and / or treating respiratory distress syndrome, or delaying or reducing respiratory distress syndrome, or alternatively, the present invention relates to a method for preventing and / or treating respiratory distress syndrome, or delaying or reducing or preventing or alleviating respiratory distress syndrome, comprising administering to a subject in need thereof an effective amount of the mesenchymal stem cell population, culture, culture supernatant, or pharmaceutical composition described herein.

[0663] In some embodiments, the medicament can relieve asthma, promote absorption and improvement at the lesion site, suppress inflammation, and / or restore lung function.

[0664] In some embodiments, the medicament can reduce inflammatory cytokines (e.g., IL-1α, IL-1β, IL-5, IL-8, IL-25, and CXCL10 / IP-10) and increase the levels of anti-inflammatory cytokines (e.g., IL-1RA, RANTES).

[0665] In some embodiments, the subject is a mammal such as a human.

[0666] In some embod...

Claims

1. A method for producing a mesenchymal stem cell population, comprising: (1) a step of culturing stem cells using a first culture medium to form embryoid bodies, wherein the first culture medium is a basal medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and bFGF; (2) a step of culturing the embryoid bodies using a second culture medium to induce their differentiation into mesenchymal stem cells, wherein the second culture medium is a basal medium supplemented with the following substances: 1% (v / v) to 5% (v / v) Ultroser (trademark) G, 2% (v / v) to 20% (v / v) KOSR, 1 mM to 5 mM of a stabilized dipeptide of L-alanyl-L-glutamine, 1 μg / ml to 100 μg / ml of ascorbic acid, one or more growth factors selected from the group consisting of VEGF, bFGF, EGF, TGFβ, and PDGF at a concentration of 1 ng / ml to 100 ng / ml each, and the following amino acids at a concentration of 0.1 mM to 0.5 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine; A method comprising the above steps.

2. The method according to claim 1, wherein the stem cells are totipotent stem cells or pluripotent stem cells.

3. The first culture medium has the following characteristics: (i) the one or more serum substitutes have a total content of 3% (v / v) to 30% (v / v); (ii) the one or more non-essential amino acids each have a content of 0.1 mM to 0.5 mM; (iii) the stabilized dipeptide of glutamine or L-alanyl-L-glutamine has a content of 1 mM to 5 mM; (iv) the bFGF has a content of 1 ng / ml to 100 ng / ml. The method according to claim 1 or 2, having one or more of the above characteristics.

4. The first culture medium has the following characteristics: (a) the serum substitute is selected from the group consisting of KOSR, MSC Serum-Free Supplement, Ultroser (trademark) G, and any combination thereof; (b) the non-essential amino acid is selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and combinations thereof; (c) the basal medium is selected from the group consisting of KO-DMEM, KO-DMEM / F12, DMEM, α-MEM, F-12, MEM, BME, RPMI 1640, G-MEM, and any combination thereof; The method according to any one of claims 1 to 3, having one or more of the above.

5. The first culture medium according to any one of claims 1 to 4, comprising KO-DMEM, KOSR, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, a stabilized dipeptide of L-alanyl-L-glutamine, and bFGF.

6. The first culture medium according to any one of claims 1 to 4, comprising 3% (v / v) to 30% (v / v) of KOSR, 1 mM to 5 mM of a stabilized dipeptide of L-alanyl-L-glutamine, 1 ng / ml to 100 ng / ml of bFGF, and the following amino acids at a concentration of 0.1 mM to 0.5 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

7. The method according to any one of claims 1 to 6, wherein the first culture medium further comprises β-mercaptoethanol.

8. The method according to any one of claims 1 to 6, wherein the β-mercaptoethanol has a content of 0.1% (v / v) to 0.5% (v / v).

9. The second culture medium has the following characteristics: The method according to any one of claims 1 to 8, wherein the basal medium is selected from the group consisting of KO-DMEM, KO-DMEM / F12, α-MEM, DMEM, F12, MEM, BME, RPMI 1640, G-MEM, and any combination thereof.

10. The method according to any one of claims 1 to 9, wherein step (1) comprises culturing the stem cells in a low-attachment cell culture vessel.

11. The method according to any one of claims 1 to 10, wherein step (2) comprises culturing the embryoid body in a culture dish coated with gelatin, collagen type I, collagen type IV, vitronectin, fibronectin, or polylysine.

12. The method according to any one of claims 1 to 11, further comprising obtaining mesenchymal stem cells by separating the cells attached to the culture vessel in step (3).

13. The method further comprises subculturing the mesenchymal stem cells of step (3). The method according to claim 12.

14. A kit comprising a first culture medium and a second culture medium, wherein the first culture medium is a basal medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and bFGF. The second culture medium is a basal medium supplemented with the following substances: 1% (v / v) to 5% (v / v) of Ultroser (trademark) G, 2% (v / v) to 20% (v / v) of KOSR, 1 mM to 5 mM of a stabilized dipeptide of L-alanyl-L-glutamine, 1 μg / ml to 100 μg / ml of ascorbic acid, one or more growth factors selected from the group consisting of VEGF, bFGF, EGF, TGFβ, and PDGF at a concentration of 1 ng / ml to 100 ng / ml each, and the following amino acids at a concentration of 0.1 mM to 0.5 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine.

15. The kit according to claim 14, wherein the first culture medium is as defined in any one of claims 3 to 8.

16. The kit according to claim 14 or 15, wherein the second culture medium is as defined in claim 9.

17. The kit according to any one of claims 14 to 16, wherein the first culture medium and the second culture medium are prepared separately.

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