WO2020040293A1 - Production method for mesenchymal stem cells - Google Patents

Abstract

The present disclosure pertains to: a production method or culture method that is for a cell population including mesenchymal stem cells, and that comprises a step for culturing a cell population including mesenchymal stem cells and CD45-positive cells; a culture medium or a culture additive for use in these methods; a method for screening for a substance that promotes the growth of mesenchymal stem cells; a method for screening for a substance that promotes the growth of CD45-positive cells; or the like.

Description

Method for producing mesenchymal stem cells

This patent application claims priority to Japanese Patent Application Nos. 2018-157175 and 2019-684459, which are hereby incorporated by reference in their entirety. I do.
The present disclosure relates to a method for producing or culturing mesenchymal stem cells, a medium or a medium additive used in these methods, or a method for screening a substance that promotes the growth of mesenchymal stem cells. The present disclosure also relates to a method for screening a substance that promotes the proliferation of CD45-positive cells.

In recent years, mesenchymal stem cells contained in bone marrow fluid and the like have the ability to differentiate into various tissues such as bone, cartilage, fat, muscle, nerve, and epithelium (multipotency). Attempts to perform regenerative medicine (cell transplantation treatment) using lobe stem cells have been widely made. The use of mesenchymal stem cells for treatment requires a large amount of cells, but the abundance of mesenchymal stem cells in vivo is extremely small, and the frequency of occurrence in bone marrow, which is a typical source of mesenchymal stem cells, Is about one cell per 10,000 to 100,000 bone marrow cells. Further, collection of bone marrow fluid cannot be performed in a large amount because the burden on the donor is large. Therefore, it is usual to obtain mesenchymal stem cells contained in bone marrow fluid and expand them by culturing before using them for treatment.However, mesenchymal stem cells can be gradually expanded and differentiated when subcultured in vitro. Is known to reduce Against this background, development of a method for efficiently growing mesenchymal stem cells has been desired.

Interleukin-33 (interleukin-33, IL-33) is a cytokine identified as a member of the IL-1 family, which is released extracellularly when cell injury or necrosis occurs, activates immune cells and activates Th2 Induces secretion of cytokines (IL-4, IL-5, IL-13) and / or inflammatory cytokines (TNF-α, IL-1, IL-6, IFN-γ). A method has been proposed in which a specific cytokine (bFGF) is added to a medium for the purpose of improving the culture efficiency of mesenchymal stem cells (Patent Document 1). Is not known.

International Publication No. 02/22788 International Publication No. 2012/113927

Nat Rev Immunol. 2010 Feb; 10 (2): 103-10. Proc Natl Acad Sci U S A. 2012 Jan 31; 109 (5): 1673-8.

の One of the objects of the present disclosure is to promote the proliferation of mesenchymal stem cells in vitro.

The present inventors have found that the coculture of mesenchymal stem cells with CD45-positive cells promotes the proliferation of mesenchymal stem cells.

In one aspect, the present disclosure provides a method for producing a cell population containing mesenchymal stem cells, comprising culturing a cell population containing mesenchymal stem cells and CD45-positive cells.
In one aspect, the present disclosure provides a method for culturing mesenchymal stem cells, comprising co-culturing mesenchymal stem cells with CD45-positive cells in the presence of IL-33.
In certain aspects, the present disclosure provides a medium or medium additive for use in culturing mesenchymal stem cells, comprising CD45 positive cells.
In one aspect, the present disclosure provides a medium or medium additive for use in co-culturing mesenchymal stem cells and CD45-positive cells, comprising IL-33.
In one aspect, the disclosure includes the following steps:
1) co-culturing mesenchymal stem cells with CD45-positive cells using a control medium,
2) co-culturing mesenchymal stem cells with CD45-positive cells using a medium in which a test substance is added to a control medium; and
3) When the number of mesenchymal stem cells obtained in step 2) is larger than the number of mesenchymal stem cells obtained in step 1), the test substance is replaced with a substance that promotes the growth of mesenchymal stem cells. Selecting as a candidate for
A method for screening a substance that promotes the growth of mesenchymal stem cells, comprising:
In one aspect, the disclosure includes the following steps:
1) culturing CD45-positive cells using a control medium,
2) a step of culturing CD45-positive cells using a medium in which IL-33 is added to a control medium;
3) a step of culturing CD45-positive cells using a control medium supplemented with a test substance, and
4) The number of CD45-positive cells obtained in step 2) is greater than the number of CD45-positive cells obtained in step 1), and the number of CD45-positive cells obtained in step 3) is If the number of obtained CD45-positive cells is larger than the number of obtained CD45-positive cells, selecting the test substance as a candidate for a substance that promotes the proliferation of CD45-positive cells;
A method for screening a substance that promotes the growth of CD45-positive cells, comprising:

According to the present disclosure, it becomes possible to promote the proliferation of mesenchymal stem cells in vitro.

FIG. 2 is a photograph showing the results of solid-phase culturing of bone marrow cells collected from mouse femurs and vertebrae in a medium containing mature IL-33 protein (10 days after the start of culture). It is a graph which shows the number of the bone marrow-derived adherent cells on the 8th day of the culture start. It is a figure which shows the FACS analysis result of the bone marrow-derived adherent cell on the 8th day of culture start. The horizontal axis indicates the expression level of PDGFRα (detected by GFP fluorescence), and the vertical axis indicates the expression level of CD45 (detected with an anti-CD45 antibody labeled with APC (Allophycocyanin)). The numbers in the four quadrants indicate the percentage of cells corresponding to each quadrant in the total number of cells analyzed. Among bone marrow-derived adherent cells in culture after 8 days, PDGFR [alpha] ⁺ CD45 ^- is a graph showing the number of CD45 ⁺ cells ^- and cell, PDGFR [alpha]. Number of cells per well of culture plate (a), number of PDGFRα ⁺ CD45 ^- cells (b), number of CD45 ⁺ cells (c), and results of colony assay when bone marrow cells were cultured in IL-33 supplemented medium for 10 days (D) is shown. The results of the colony assay when PDGFRα ⁺ cells were sorted and cultured in a medium supplemented with IL-33 are shown. FIG. 7a shows that PDGFRα ⁺ cells (Pα ⁺ ) were sorted and then co-cultured with bone marrow cells in a medium supplemented with IL-33, or PDGFRα ⁺ cells were collected in the presence of bone marrow cells sorted on Transwell. The results obtained by culturing the cells in a medium supplemented with IL-33 and performing a colony assay are shown. FIG. 7b shows the results of a colony assay when PDGFRα ⁺ cells and CD45 ⁻ cells or CD45 ⁺ cells were co-cultured in a medium supplemented with IL-33. FIG. 8a shows the results of a colony assay when bone marrow cells of MyD88KO mice were cultured in a medium supplemented with IL-33. FIG. 8b shows the results of FACS analysis when bone marrow cells of MyD88KO mice were cultured in a medium supplemented with IL-33 for 10 days. The results of a colony assay when bone marrow cells were cultured in a medium supplemented with an IL-33 / p38 MAPK inhibitor or an IL-33 / NF-κB inhibitor are shown. FIG. 10a shows the results of a colony assay when bone marrow cells were cultured in a medium supplemented with IL-33 / DAPT. FIGS. 10 b and c show the number of PDGFRα ⁺ CD45 ⁻ cells and the number of CD45 ⁺ cells analyzed by FACS when bone marrow cells were cultured in IL-33 / DAPT-supplemented medium for 10 days.

In this disclosure, when a value is accompanied by the term “about”, it is intended to include the range of ± 10% of that value. For example, “about 20” includes “18 to 22”. Numeric ranges are inclusive of all numbers between the endpoints and the endpoints. “About” a range applies to both endpoints of the range. Therefore, for example, “about 20 to 30” includes “18 to 33”.

限 り Unless otherwise specified, terms used in the present disclosure have meanings as commonly understood by a person of ordinary skill in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, and the like. The following provides definitions for some terms used in the present disclosure, but these definitions supersede general understanding in the present disclosure.

に お い て In the present disclosure, “cell” means one cell or a plurality of cells depending on the context. A plurality of cells may be referred to as a “cell population”, and the cell population may be a cell population consisting of one type of cell or a cell population containing multiple types of cells, depending on the context.

に お い て In the present disclosure, “mesenchymal stem cell” is used without distinction from “mesenchymal stromal cell”, and may be abbreviated as “MSC”. Generally, a stem cell refers to a cell having both the ability to differentiate from a single cell into one or more tissues or cell types and the ability to self-renew. The mesenchymal stem cells of the present disclosure have the ability to differentiate into one or more mesenchymal tissues from among mesenchymal tissues such as bone, cartilage, fat, and muscle. Mesenchymal stem cells may have the ability to differentiate beyond the germ layer into epithelial tissue, neural tissue, and cardiac / vascular tissue in addition to mesodermal mesenchymal tissue. In certain embodiments, the mesenchymal stem cells have the ability to differentiate into a plurality of osteoblasts, chondrocytes, muscle cells, adipocytes. In certain embodiments, the mesenchymal stem cells have the ability to differentiate into a plurality of osteoblasts, chondrocytes, adipocytes. In certain embodiments, the mesenchymal stem cells have the ability to differentiate into osteoblasts and adipocytes. In certain embodiments, the mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. In certain embodiments, the mesenchymal stem cells have the ability to differentiate into at least one of osteoblasts, chondrocytes, and adipocytes.

When the term "mesenchymal stem cell" is used in the meaning of a cell population, it may be a cell population consisting only of mesenchymal stem cells, as long as the cell population shows the same differentiation ability as mesenchymal stem cells Alternatively, the cell population may be a heterogeneous cell population including mesenchymal stem cells and cells at an intermediate stage of differentiation between mesenchymal stem cells and mesenchymal tissue.

Mesenchymal stem cells can be obtained from tissues such as bone marrow, blood, for example, peripheral blood or umbilical cord blood, skin, fat, dental pulp and the like. Mesenchymal stem cells can be cultured and expanded as solid cells, for example, as adherent cells on a culture dish. Thus, in certain embodiments, mesenchymal stem cells can be obtained from adherent cells contained in bone marrow or blood. In one embodiment, mesenchymal stem cells can be distinguished by using the ability to form colonies as an index. Mesenchymal stem cells may be separated from tissues containing mesenchymal stem cells, for example, bone marrow, by cell sorting (FACS, MACS, etc.) using at least one mesenchymal stem cell marker as an index. Alternatively, mesenchymal stem cells differentiated from pluripotent stem cells or established mesenchymal stem cell lines may be used.

As markers for human mesenchymal stem cells, PDGFRα-positive, PDGFRβ-positive, Lin-negative, CD45-negative, CD44-positive, CD90-positive, CD29-positive, Flk-1 negative, CD105-positive, CD73-positive, CD90-positive, CD71-positive, Stro- Examples include 1 positive, CD106 positive, CD166 positive, CD31 negative, CD271 positive, and CD11b negative, but are not limited thereto. In one embodiment, the marker for human mesenchymal stem cells is PDGFRα positive. In certain embodiments, the marker for human mesenchymal stem cells is PDGFRα positive and CD45 negative.

As a marker for mouse mesenchymal stem cells, CD44-positive, PDGFRα-positive, PDGFRβ-positive, CD45-negative, Lin-negative, Sca-1 positive, c-kit negative, CD90 positive, CD105 positive, CD29 positive, Flk-1 negative, CD271 Positive and CD11b negative can be exemplified, but are not limited thereto. In certain embodiments, the mouse mesenchymal stem cell marker is PDGFRα positive. In certain embodiments, the marker for mouse mesenchymal stem cells is PDGFRα positive and CD45 negative.

に お い て In the present disclosure, cells expressing CD45 are referred to as “CD45-positive cells”. CD45 is a type I glycoprotein having a transmembrane domain, also known as a leukocyte common antigen, and is a tyrosine phosphatase expressed on many nucleated hematopoietic cells. CD45 is known as a signaling molecule that regulates various cellular processes such as cell proliferation, differentiation, mitosis, and canceration. The amino acid sequences of CD45 in various animal species are known.

Examples of CD45-positive cells include, but are not limited to, human leukocytes, ie, lymphocytes, monocytes, eosinophils, basophils, neutrophils, dendritic cells, and macrophages.

CD45-positive cells can be obtained from tissues such as bone marrow, blood, and lymph nodes. The CD45-positive cells can be normally cultured and expanded as floating cells, but are not limited thereto. Some CD45-positive cells can be cultured and expanded as adherent cells. CD45-positive cells may be separated from a tissue containing CD45-positive cells, such as bone marrow, by cell sorting (FACS, MACS, etc.) using CD45 as an index. Alternatively, CD45-positive cells differentiated from pluripotent stem cells or established CD45-positive cell lines may be used.

The “cell population containing mesenchymal stem cells and CD45-positive cells” is a cell population containing at least one mesenchymal stem cell and at least one CD45-positive cell, and may contain any cells in addition to these cells. . Further, the cell population may include only mesenchymal stem cells and CD45-positive cells. The cell population includes mesenchymal stem cells and CD45-positive cells in a contactable state. For example, the cell population may be a cell population collected from a tissue containing mesenchymal stem cells and CD45-positive cells, or a mixture of isolated mesenchymal stem cells and isolated CD45-positive cells. Good. Mesenchymal stem cells and CD45-positive cells may be derived from the same tissue or different tissues.

骨 髄 Bone marrow-derived cells can be used as a cell population containing mesenchymal stem cells and CD45-positive cells. The term “bone marrow-derived cells” refers to bone marrow cells (cell populations existing in bone marrow) removed from inside the bone marrow and outside the bone marrow. Bone marrow cells can be removed from a living body by a method such as bone marrow aspiration. If there is a bone sample removed from the animal, it is possible to crush the bone and collect the contents, or to expose the cross section of the bone and insert the syringe needle into the bone marrow and push it with the buffer It is. In one embodiment, the production method of the present disclosure may include a step of collecting bone marrow cells from an animal.

The bone marrow-derived cells include a population of cells that can adhere to the solid phase and proliferate (adherent cells) and a population of cells that cannot adhere (non-adherent cells). In the present disclosure, a cell population obtained by culturing bone marrow-derived cells on a solid phase is referred to as “bone marrow-derived adherent cells”. As demonstrated by the examples, bone marrow-derived adherent cells include mesenchymal stem cells and may include CD45-positive cells.

に 関 し て In the context of the present disclosure, a solid phase refers to a solid support to which cells can adhere, and includes, for example, plastic or glass culture dishes, flasks, multiwell plates, and the like. The solid phase may be coated, and examples of the coating material include, but are not limited to, collagen I, laminin, vitronectin, fibronectin, poly-L-lysine, poly-L-ornithine, and the like. In some embodiments, the solid phase is coated with collagen I.

生物 Species from which bone marrow cells are collected include, but are not limited to, humans and non-human animals such as mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, and the like. The types of bone from which bone marrow cells are collected include, but are not limited to, femur, vertebra, sternum, iliac bone, tibia, and the like. In certain embodiments, bone marrow cells are obtained from a femur or vertebra.

間 Mesenchymal stem cells and / or CD45-positive cells may be separated from bone marrow-derived cells and used. Separation can be performed, for example, by cell sorting (FACS, MACS, etc.) using the surface marker as an index.

In the present disclosure, “separating cells” means that cells of interest in a cell population are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more. Means to operate.

In the present disclosure, “a substance that activates CD45-positive cells” means a substance that enhances signal transduction from CD45-positive cells to other cells, and includes polypeptides, proteins, amino acids, nucleic acids, lipids, carbohydrates, It may be a low molecular compound or the like. The enhanced signaling may be due to an increase in a signal-related response, for example, a response in the p38 MAPK signaling pathway, or may be caused by an increase in the number of CD45-positive cells. That is, the substance that activates CD45-positive cells includes a substance that promotes the proliferation of CD45-positive cells. Further, the enhancement of signal transduction includes the initiation and promotion of signal transduction. As a substance that activates CD45-positive cells, a substance that promotes the proliferation of CD45-positive cells, such as IL-5, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), Macrophage colony stimulating factor (GM-CSF), lipopolysaccharide (LPS) or IL-33 can be used, but is not limited thereto. These may be used alone or in combination.

IL-33 is a cytokine identified as a member of the IL-1 family, and is a nuclear protein consisting of an amino acid sequence of about 270 residues in length, depending on the species. IL-33 is released extracellularly when cell injury or necrosis occurs and activates immune cells to activate Th2 cytokines (IL-4, IL-5, IL-13) and / or inflammatory cytokines (TNF-α). , IL-1, IL-6, IFN-γ). Although full-length IL-33 protein also has activity, the full-length IL-33 protein is cleaved in vivo by a protease (eg, cathepsin G (Accession No. P08311 etc.) or neutrophil elastase (Accession No. P08246 etc.)). It is known that the resulting C-terminal polypeptide fragment functions as a mature IL-33 protein.

に お い て In the present disclosure, “IL-33” can be a full-length IL-33 protein, a mature IL-33 protein, or a polypeptide functionally equivalent thereto.

に お い て In the present disclosure, the species from which IL-33 originates include, but are not limited to, humans and non-human animals such as mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, and the like.

The full-length IL-33 protein refers to an IL-33 protein that has been translated from mRNA encoding IL-33 and has not yet been processed by a protease. In the present disclosure, the full-length IL-33 protein includes a full-length mouse IL-33 protein (SEQ ID NO: 1, Accession No. Q8BVZ5) and a full-length human IL-33 protein (SEQ ID NO: 4, Accession No. O95760). It is not limited to these.

Mature IL-33 protein means a C-terminal polypeptide that has been confirmed or suggested to be produced by processing of full-length IL-33 protein by protease, and has biological activity.

Examples of the mature IL-33 protein include a polypeptide consisting of the 102nd to 266th amino acid sequence (SEQ ID NO: 2) of the full-length mouse IL-33 protein, and the 109th to 266th amino acid sequence of the full-length mouse IL-33 protein A polypeptide consisting of (SEQ ID NO: 3); a polypeptide consisting of the 95th to 270th amino acid sequence of the full-length human IL-33 protein (SEQ ID NO: 5); a 99th to 270th amino acid sequence of the full-length human IL-33 protein A polypeptide consisting of SEQ ID NO: 6; a polypeptide consisting of the amino acid sequence from position 109 to position 270 of the full-length human IL-33 protein (SEQ ID NO: 7); and an amino acid position 112 to position 270 of the full-length human IL-33 protein Polypeptide consisting of sequence (SEQ ID NO: 8) It is known. Any of these proteins can be used in the method of the present disclosure and the like. In one embodiment, the mature IL-33 protein has the amino acid sequence of SEQ ID NO: 3 or 8.

For example, it has been confirmed that a polypeptide consisting of the amino acid sequence from position 109 to position 266 of the full-length mouse IL-33 protein (SEQ ID NO: 3) has an activity of inducing the production of TNF-α (Bae et al, J Biol Chem. 2012 Mar 9; 287 (11): 8205-13). In addition, a polypeptide consisting of the amino acid sequence from position 112 to position 270 of the full-length human IL-33 protein (SEQ ID NO: 8) has a biological activity equivalent to that of the full-length human IL-33 protein (eg, through binding to ST2). NF-κB signaling pathway) (Cayrol and Girard, PNAS 2009 and Jun 2; 106 (22): 9021-6).

"Functionally equivalent" to the full-length or mature IL-33 protein means having the same biological activity as the protein, and "same quality" as used herein means being the same in qualitative evaluation. I do. Biological activities of the full-length or mature IL-33 protein in the present disclosure include, for example, activation of the p38 MAPK signaling pathway, induction of Th2 cytokine and / or inflammatory cytokine secretion from immune cells, ST2 (receptor of IL-33 Activation of the NF-κB signal transduction pathway through binding to the body), promotion of proliferation of bone marrow-derived adherent cells including mesenchymal stem cells, promotion of activation of CD45-positive cells, and the like. In one embodiment, the biological activity of the full-length or mature IL-33 protein is to activate CD45 positive cells.

For example, IL-33 can be a polypeptide selected from the following a) to g):
a) a polypeptide comprising or consisting of the amino acid sequence of full-length or mature IL-33 protein;
b) a polypeptide comprising the amino acid sequence of mature IL-33 protein and consisting of a partial amino acid sequence of full-length IL-33 protein;
c) a polypeptide comprising or consisting of a partial amino acid sequence of the full-length or mature IL-33 protein, and functionally equivalent to the full-length or mature IL-33 protein;
d) Substitution, deletion, insertion or addition of one or more, for example, 1 to 10, 1 to 5, 1 to 3, or 1 or 2 amino acids in the amino acid sequence of the full-length or mature IL-33 protein A polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or functionally equivalent to the full-length or mature IL-33 protein;
e) the amino acid sequence of the full-length or mature IL-33 protein is at least about 80%, such as at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about A polypeptide comprising or consisting of an amino acid sequence having 99% or more sequence identity, and functionally equivalent to the full-length or mature IL-33 protein;
f) a polypeptide encoded by a DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein and functionally equivalent to the full-length or mature IL-33 protein; About 70% or more, such as about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more of the nucleic acid sequence encoding the mature IL-33 protein. As described above, a polypeptide encoded by a nucleic acid sequence having about 98% or more or about 99% or more sequence identity and functionally equivalent to a full-length or mature IL-33 protein.

In the present disclosure, the identity of an amino acid sequence or a nucleic acid sequence refers to the degree of sequence identity between polypeptides or polynucleotides, and is the optimal condition over the region of the sequence to be compared (maximum amino acid or nucleotide identity). Is determined by comparing the two sequences aligned to The sequence identity numbers (%) determine the number of identical amino acids or nucleotides present in both sequences to determine the number of matching sites, and then determine the number of matching sites for amino acids or nucleotides in the sequence region to be compared. Is calculated by dividing by the total number and multiplying the obtained numerical value by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms routinely available to those of skill in the art (eg, the BLAST algorithm, the FASTA algorithm, etc.). Sequence identity can be determined using sequence analysis software such as, for example, BLAST, FASTA.

Regarding "hybridize under stringent conditions", the hybridization used herein may be carried out, for example, according to a usual method described in Molecular Cloning, T. Maniatis et al., CSH Laboratory or the like (1983). It can be carried out. Further, “under stringent conditions” refers to, for example, 45 ° C. in a solution containing 6 × SSC (a solution containing 1.5 M NaCl and 0.15 M trisodium citrate is called 10 × SSC) and 50% formamide. After forming a hybrid at 50 ° C. with 2 × SSC (Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6), and Conditions that provide equivalent stringency can be mentioned.

The method of obtaining the full-length or mature IL-33 protein or a polypeptide functionally equivalent thereto is not particularly limited. For example, recombinant expression (mammalian cells, yeast, E. coli, insect cells, etc.), synthesis using a cell-free system Etc., and it is also possible to purchase a commercial product.

Method for Producing Cell Population Containing Mesenchymal Stem Cells The method for producing a cell population containing mesenchymal stem cells of the present disclosure is characterized by culturing a cell population containing mesenchymal stem cells and CD45-positive cells. In this production method, a cell population containing mesenchymal stem cells and CD45-positive cells is cultured to proliferate the mesenchymal stem cells contained in the cell population. The medium for culturing these cells is not particularly limited as long as it can be used for culturing animal cells (for example, mesenchymal stem cells). Examples include MEM, MEMα, DMEM, GMEM, RPMI 1640, MesenCult ^™ (STEMCELL Technologies).

The culture method can be appropriately adjusted depending on the purpose of use of the cells and the like. The culture conditions are not particularly limited as long as the mesenchymal stem cells can survive. For example, in a general incubator, “37 ° C., 5% O ₂ , 5% CO ₂ ”, “37 ° C.” , 5% CO ₂ ”, etc., on a solid phase (adhesive culture) or suspension culture (eg, stirring culture or shaking culture). Preferably, adhesion culture is used.

The density of the mesenchymal stem cells to be cultured is not particularly limited as long as it can be cultured. For example, 1 to about 1 × 10 ¹⁰ cells / mL, 10 to about 1 × 10 ⁹ cells / mL, and about 1 × It may be 10 ² to about 1 × 10 ⁸ / mL, about 1 × 10 ³ to about 1 × 10 ⁷ / mL, about 1 × 10 ⁴ to about 1 × 10 ⁷ / mL. The density of CD45-positive cells to be cultured is, for example, about 1 × 10 ² to about 1 × 10 ¹⁰ cells / mL, about 1 × 10 ³ to about 1 × 10 ⁹ cells / mL, about 1 × 10 ³ to about 1 × 10 ³ cells / mL. It can be 1 × 10 ⁸ / mL, about 1 × 10 ⁴ to about 1 × 10 ⁸ / mL, about 1 × 10 ⁴ to about 1 × 10 ⁷ / mL. The medium may be changed typically once every 1 to 7 days, for example, once every 4 or 5 days. The culture period may be a period sufficient to form a colony of a cell population containing mesenchymal stem cells, and may be several days to several months, for example, three days to three months, four days to one month. For 5 days to 3 weeks or 7 days to 14 days.

The medium may further contain other components as long as the growth of mesenchymal stem cells or the growth (activation) of CD45-positive cells is not inhibited. In certain embodiments, a cell population comprising mesenchymal stem cells and CD45 positive cells is cultured in the presence of a substance that activates CD45 positive cells. The substance may be added to or contained in the culture medium.

In one embodiment, a cell population comprising mesenchymal stem cells and CD45 positive cells is cultured in the presence of IL-33. The cell population may be cultured in a medium containing IL-33, or IL-33 may be added to the medium in which the cell population is being cultured. The concentration of IL-33 in the medium can be, for example, about 0.01-100 ng / mL, about 0.1-10 ng / mL, or about 0.5-5 ng / mL.

A cell population comprising mesenchymal stem cells and CD45 positive cells may be cultured in the presence of a Notch activator. Examples of the Notch activator include, for example, delta-like 1, delta-like 3, delta-like 4, Jagged 1, Jugged 2, Dlk1 / Pref1, Contactin1, Contactin6, CCN3 / NO4 described in JP-A-2013-518588. MAGP1 and MAGP2; Notch1 agonist and Notch2 agonist described in JP-T-2017-516486; PDGF receptor kinase inhibitors described in JP-T-2009-502962 (eg, AG-370 or AG-1296 (6,7-dimethoxy-3) - phenylquinoxaline)), ^{K +} and ^{H +} ionophore (e.g., nigericin-Na), an inhibitor of actin polymerization (e.g., site Kara San D), sodium pump (Na ⁺ K ⁺ ATP-ase) inhibitor (e.g., Ouabain), an inhibitor of oxidative phosphorylation by mitochondria (e.g., FCCP (carbonyl cyanide-4- (trifluoromethoxy) - phenyl hydrazone), and c-Jun N-terminal Kinase (JNK) inhibitors (eg, SP600125) include, but are not limited to, such Notch activators that can be appropriately selected and used.These Notch activators include CD45-positive cells. May be used together with a substance that activates, for example, IL-33.

After activating CD45-positive cells with a substance that activates CD45-positive cells, the activated CD45-positive cells and mesenchymal stem cells may be co-cultured. Here, after activating the CD45-positive cells, the substance may be separated from the CD45-positive cells, and the CD45-positive cells may be co-cultured with the mesenchymal stem cells. When the substance that activates CD45-positive cells is a substance that promotes the proliferation of CD45-positive cells, co-culture of the activated CD45-positive cells and mesenchymal stem cells is performed by using the expanded CD45-positive cells and mesenchymal stem cells. Means co-culture.

In certain embodiments, the medium does not contain a differentiation inducing agent. In other words, the medium is not used for differentiation but for growth or maintenance of cells. In the present disclosure, a differentiation inducing agent means a substance that induces differentiation into a specific cell type, and particularly a substance that induces differentiation into a mesenchymal cell type. Examples of the differentiation inducer include dexamethasone, β-glycerophosphate, and L-ascorbic acid 2-phosphate as differentiation inducers for osteoblasts, and isobutyl methylxanthine (IBMX) as a differentiation inducer for adipocytes. ), Dexamethasone, insulin, indomethacin, and as a chondrocyte differentiation inducer, dexamethasone, insulin, L-ascorbic acid 2-phosphate, L-proline, pyruvate, transforming growth factor (TGF-b3) , Bone morphogenetic protein 2 (BMP-2), and insulin-like growth factor (IGF). The expression "not containing a differentiation-inducing agent" means that the culture medium does not substantially contain a differentiation-inducing agent, that is, does not contain a differentiation-inducing agent at a level or an amount that induces differentiation.

細胞 The cell population containing mesenchymal stem cells obtained by the above production method may contain any other cells other than mesenchymal stem cells and CD45-positive cells, and examples thereof include non-stem cells.

に お い て In the above production method, mesenchymal stem cells after culture may be separated. For example, mesenchymal stem cells can be isolated by culturing a cell population containing mesenchymal stem cells and CD45-positive cells on a solid phase. In the culture on a solid phase for isolating mesenchymal stem cells, for example, one or more subcultures may be performed, and the subcultured medium is a medium containing no substance that activates CD45-positive cells. It may be. After completion of the culture, the cells attached to the solid phase can be collected as a cell population including mesenchymal stem cells. For example, by removing the medium containing non-adherent cells, a cell population containing mesenchymal stem cells can be recovered. Furthermore, the adherent cells may be detached from the solid phase by treatment with an enzyme such as trypsin.

Alternatively, mesenchymal stem cells may be separated by cell sorting (FACS, MACS, etc.) targeting mesenchymal stem cells. Cell sorting targeting mesenchymal stem cells can be performed, for example, using an antibody against a surface marker of mesenchymal stem cells. By culturing the obtained cell population containing mesenchymal stem cells in the presence of an agent (for example, Mesenpure (STEMCELL Technologies)) that suppresses the survival or proliferation of other cells, for example, CD45-positive cells, Stem cells may be separated (purified).
When mesenchymal stem cells are separated, only one of subculture and cell sorting may be performed, or both may be performed.

Method of culturing mesenchymal stem cells The present disclosure also includes a step of co-culturing mesenchymal stem cells with CD45-positive cells in the presence of a substance that activates CD45-positive cells (eg, IL-33). A method for culturing a stem cell is provided. This culture method promotes the growth of mesenchymal stem cells. “Co-culture” means that cells are cultured in a state where cell-to-cell contact between mesenchymal stem cells and CD45-positive cells is possible. Co-culture can be performed according to the culture in the above-described method for producing a cell population containing mesenchymal stem cells.

Method for culturing or producing CD45-positive cells The present disclosure also provides a method for culturing CD45-positive cells, comprising culturing CD45-positive cells in the presence of IL-33. This culture method promotes the growth of CD45 positive cells. This culture method can be carried out by culturing a cell population containing CD45-positive cells in the presence of IL-33 according to the culture of a cell population containing mesenchymal stem cells and CD45-positive cells described above. Here, the cell population may or may not include mesenchymal stem cells. Also provided is a method for producing a cell population containing CD45-positive cells, comprising a step of expanding CD45-positive cells by this culture method.

The method for culturing or producing CD45-positive cells is applicable as a method for producing CD45-positive cells used for co-culture with mesenchymal stem cells or a medium or a medium additive for culturing mesenchymal stem cells. May be performed.

Media or Media Additives In certain embodiments, the present disclosure provides media or media additives that can be used in any of the methods described above. For example, a medium or a medium additive for use in culturing mesenchymal stem cells containing CD45-positive cells; a mesenchymal system containing CD45-positive cells and a substance that activates CD45-positive cells (eg, IL-33) A medium or medium additive for use in culturing stem cells; a medium or medium supplement containing a substance for activating CD45-positive cells (eg, IL-33) for use in co-culture of mesenchymal stem cells and CD45-positive cells An agent; a medium or a medium additive for use in culturing CD45-positive cells, which contains IL-33, may be provided. In certain embodiments, the media or media additive is a media or media additive for promoting the growth of mesenchymal stem cells.

The medium is, for example, a medium obtained by adding a substance that activates CD45-positive cells and / or CD45-positive cells to the general medium for animal cell culture described above for the method for producing a cell population containing mesenchymal stem cells Can be The component composition of the medium other than the CD45-positive cells and / or the substance that activates the CD45-positive cells is not particularly limited as long as the medium can be used for culturing animal cells (eg, mesenchymal stem cells). . The medium may further contain other components as long as it does not inhibit the growth of mesenchymal stem cells or CD45-positive cells, and may contain, for example, a Notch activator. Further, for example, the medium may not contain a differentiation inducer. The medium may be provided in any form, for example, a liquid medium that can be used as it is, a concentrate for use by diluting or dissolving, a solid, or a lyophilized product. The medium may be provided in any container.

The culture medium additive contains the CD45-positive cells and / or the substance that activates the CD45-positive cells described above for the method for producing a cell population including mesenchymal stem cells, together with any components that are acceptable for culturing animal cells. obtain. The culture medium additive may further contain other components as long as it does not inhibit the growth of mesenchymal stem cells or the growth (activation) of CD45-positive cells, and may include, for example, a Notch activator. The medium additive may be provided in any dosage form, for example, a solid such as granules, powders and tablets, a liquid such as a solution, a suspension or an emulsion, or a capsule encapsulating a solid, semi-solid or liquid. , But is not limited thereto. The media additive may be provided in any container. The culture medium additive can be added to, for example, a general culture medium for animal cells described above for the method for producing a cell population containing mesenchymal stem cells.

Method for Screening a Substance that Promotes Proliferation of Mesenchymal Stem Cells In one aspect, the present disclosure provides a method for screening a substance that promotes proliferation of mesenchymal stem cells.
Specifically, in step 1), mesenchymal stem cells are co-cultured with CD45-positive cells using a control medium, and in step 2), mesenchymal stem cells are cultured using a medium in which a test substance is added to the control medium. Stem cells are co-cultured with CD45 positive cells. Then, in step 3), when the number of mesenchymal stem cells obtained in step 2) is larger than the number of mesenchymal stem cells obtained in step 1), the test substance is replaced with mesenchymal stem cells. It is selected as a candidate for a substance that promotes growth.

Steps 1) and 2) of the screening method of the present disclosure can be performed with reference to the method for producing and culturing a cell population containing mesenchymal stem cells described above.
The “test substance” includes all substances such as polypeptides, proteins, amino acids, nucleic acids, lipids, carbohydrates, low molecular weight compounds and the like. The test substance may typically be purified and isolated, but may be an unpurified or unisolated crude product, or a live cell that produces the test substance. The test substance may be provided in the form of a compound library, a nucleic acid library, a random peptide library, or the like, or may be provided as a natural product.

In step 3) of the method of screening for a substance that promotes the growth of mesenchymal stem cells of the present disclosure, the number of colonies may be counted and regarded as the number of cells, or the number of cells may be counted. When cells are cultured on a solid phase, the cells may be counted while they are still attached to the solid phase, or the cells may be separated from the solid phase by enzyme treatment or the like, and the number of cells may be counted. For the counting of the number of cells, a method well known to those skilled in the art can be used. The “number of mesenchymal stem cells” can be determined by, for example, FACS analysis using the expression of a representative surface marker of mesenchymal stem cells (eg, PDGFRα positive and CD45 negative) as an index. Alternatively, immunofluorescent staining or the like may be performed, and cells expressing the surface marker of the mesenchymal stem cells may be counted while remaining attached to the solid phase, and regarded as the number of mesenchymal stem cells. Here, “the number of cells is large” means that the number of cells is at least 3%, 5% or 10% more than that of the comparison target, and preferably at least 10% more.

で は In the screening method of the present disclosure, IL-33 may be used as a positive control. If the growth of mesenchymal stem cells is promoted in a medium supplemented with IL-33, it is assured that the experimental system is functioning. Therefore, the screening method of the present disclosure may further include 1 ′) co-culturing mesenchymal stem cells with CD45-positive cells using a medium supplemented with IL-33 in a control medium. The number of mesenchymal stem cells obtained by step 1) is greater than the number of mesenchymal stem cells obtained by step 1), and the number of mesenchymal stem cells obtained by step 1 ′) is When the number is larger than the obtained number of mesenchymal stem cells, the test substance can be selected as a candidate for a substance that promotes the proliferation of mesenchymal stem cells.

Accordingly, in one embodiment, there is provided a method of screening for a substance that promotes the proliferation of mesenchymal stem cells using a positive control, comprising the steps of:
1) co-culturing mesenchymal stem cells with CD45-positive cells using a control medium,
1 ′) a step of co-culturing mesenchymal stem cells with CD45-positive cells using a control medium supplemented with IL-33,
2) co-culturing mesenchymal stem cells with CD45-positive cells using a medium in which a test substance is added to a control medium; and
3) The number of mesenchymal stem cells obtained in step 2) is greater than the number of mesenchymal stem cells obtained in step 1), and the number of mesenchymal stem cells obtained in step 1 ′) is A step of selecting the test substance as a candidate for a substance that promotes the growth of mesenchymal stem cells when the number is greater than the number of mesenchymal stem cells obtained in step 1).

In one aspect, there is provided a method for screening a substance that promotes the growth of CD45-positive cells, comprising the following steps:
1) culturing CD45-positive cells using a control medium,
2) a step of culturing CD45-positive cells using a medium in which IL-33 is added to a control medium;
3) the step of culturing CD45-positive cells using a medium in which a test substance is added to a control medium; and 4) the number of CD45-positive cells obtained in step 2) is the number of CD45-positive cells obtained in step 1) If the number of CD45-positive cells obtained in step 3) is greater than the number of CD45-positive cells obtained in step 1), the test substance is used to promote the proliferation of CD45-positive cells. A step of selecting as a substance candidate;
This method can be performed according to the method of screening for a substance that promotes the growth of mesenchymal stem cells.

According to the present disclosure, for example, the following embodiments are provided.
[1] A method for producing a cell population containing mesenchymal stem cells, comprising a step of culturing a cell population containing mesenchymal stem cells and CD45-positive cells.
[2] A method for producing a cell population containing mesenchymal stem cells, comprising a step of culturing a cell population containing mesenchymal stem cells and CD45-positive cells in order to promote the proliferation of mesenchymal stem cells.
[3] A method for promoting the proliferation of mesenchymal stem cells, comprising a step of culturing a cell population containing mesenchymal stem cells and CD45-positive cells.
[4] The method according to any one of items 1 to 3, wherein the cell population containing mesenchymal stem cells and CD45-positive cells contains bone marrow-derived mesenchymal stem cells.
[5] The method according to any one of items 1 to 4, wherein the cell population containing mesenchymal stem cells and CD45-positive cells contains bone marrow-derived CD45-positive cells.
[6] The method according to any one of items 1 to 5, wherein the cell population containing mesenchymal stem cells and CD45-positive cells is bone marrow-derived cells.
[7] The method according to any one of items 1 to 6, further comprising the step of separating mesenchymal stem cells from the obtained cell population containing mesenchymal stem cells.
[8] The method according to item 7, wherein the step of isolating the mesenchymal stem cells comprises a) one or more subcultures and / or b) cell sorting targeting the mesenchymal stem cells.

[9] The method according to any one of items 1 to 8, wherein a cell population containing mesenchymal stem cells and CD45-positive cells is cultured on a solid phase.
[10] The method according to item 9, further comprising a step of collecting cells adhering to the solid phase after culturing as a cell population containing mesenchymal stem cells.
[11] The method according to item 9 or 10, wherein the solid phase is coated.
[12] The method according to any one of items 9 to 11, wherein the solid phase is coated with collagen I, laminin, vitronectin, fibronectin, poly L-lysine or poly L-ornithine.
[13] The method according to any of items 9 to 12, wherein the solid phase is coated with collagen I.
[14] The method according to any of items 9 to 13, wherein the obtained cell population containing mesenchymal stem cells is bone marrow-derived adherent cells containing mesenchymal stem cells.

[15] The method according to any one of items 1 to 14, wherein a cell population containing mesenchymal stem cells and CD45-positive cells is cultured in the presence of a substance that activates CD45-positive cells.
[16] The method according to item 15, wherein the substance that activates CD45-positive cells is IL-33.
[17] A method for culturing mesenchymal stem cells, comprising a step of co-culturing mesenchymal stem cells with CD45-positive cells in the presence of IL-33.
[18] A method for promoting the growth of mesenchymal stem cells, comprising the step of co-culturing mesenchymal stem cells with CD45-positive cells in the presence of IL-33.
[19] A method for culturing CD45-positive cells, comprising culturing CD45-positive cells in the presence of IL-33.

[20] IL-33 is
a) a polypeptide comprising or consisting of the amino acid sequence of full-length or mature IL-33 protein;
b) a polypeptide comprising the amino acid sequence of mature IL-33 protein and consisting of a partial amino acid sequence of full-length IL-33 protein;
c) a polypeptide comprising or consisting of a partial amino acid sequence of the full-length or mature IL-33 protein, and functionally equivalent to the full-length or mature IL-33 protein;
d) Substitution, deletion, insertion or addition of one or more, for example, 1 to 10, 1 to 5, 1 to 3, or 1 or 2 amino acids in the amino acid sequence of the full-length or mature IL-33 protein A polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or functionally equivalent to the full-length or mature IL-33 protein;
e) the amino acid sequence of the full-length or mature IL-33 protein is at least about 80%, such as at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about A polypeptide comprising or consisting of an amino acid sequence having 99% or more sequence identity, and functionally equivalent to the full-length or mature IL-33 protein;
f) a polypeptide encoded by a DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein and functionally equivalent to the full-length or mature IL-33 protein; About 70% or more, such as about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more of the nucleic acid sequence encoding the mature IL-33 protein. As set forth in any one of claims 16 to 16, selected from the group consisting of polypeptides encoded by nucleic acid sequences having about 98% or more or about 99% or more sequence identity and functionally equivalent to the full-length or mature IL-33 protein 20. The method according to any of paragraphs 19 to 19.

[21] IL-33 is
a) a polypeptide comprising or consisting of the amino acid sequence of full-length or mature IL-33 protein;
b) a polypeptide comprising the amino acid sequence of mature IL-33 protein and consisting of a partial amino acid sequence of full-length IL-33 protein;
c) a polypeptide that comprises or consists of the amino acid sequence of a part of the full-length or mature IL-33 protein and activates CD45-positive cells;
d) Activating CD45-positive cells, comprising or consisting of an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted, inserted or added in the amino acid sequence of the full-length or mature IL-33 protein Polypeptide,
e) a polypeptide which comprises or consists of an amino acid sequence having about 90% or more sequence identity with the amino acid sequence of the full-length or mature IL-33 protein, and which activates CD45-positive cells;
f) a polypeptide encoded by DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein and activating CD45-positive cells; and g) a full-length or mature IL-33 protein. 21. Any of paragraphs 16 to 20, encoded by a nucleic acid sequence having at least about 90% sequence identity with the encoding nucleic acid sequence, selected from the group consisting of polypeptides that activate CD45-positive cells. the method of.

[22] The method according to any of paragraphs 16 to 21, wherein IL-33 is a polypeptide comprising the amino acid sequence of full-length or mature IL-33 protein.
[23] The method according to any one of Items 16 to 22, wherein IL-33 is a polypeptide consisting of an amino acid sequence of a full-length or mature IL-33 protein.
[24] The method according to any one of paragraphs 16 to 23, wherein IL-33 is a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 8.
[25] The method according to any of paragraphs 16 to 24, wherein IL-33 is a polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 8.
[26] The method according to any of paragraphs 16 to 25, wherein the nucleic acid sequence encoding IL-33 comprises the nucleic acid sequence of any of SEQ ID NOs: 9 to 16.
[27] The method according to any of paragraphs 16 to 26, wherein IL-33 is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 8.
[28] The method according to any of paragraphs 16 to 27, wherein IL-33 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or 8.
[29] The method according to any of paragraphs 16 to 28, wherein the nucleic acid sequence encoding IL-33 comprises the nucleic acid sequence of SEQ ID NO: 11 or 16.
[30] The method of any one of paragraphs 16 to 29, wherein IL-33 is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.
[31] The method according to any of paragraphs 16 to 30, wherein IL-33 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.
[32] The method of any of paragraphs 16 through 31, wherein the nucleic acid sequence encoding IL-33 comprises the nucleic acid sequence of SEQ ID NO: 11.
[33] The method according to any of paragraphs 16 to 32, wherein the concentration of IL-33 is about 0.01 to 100 ng / mL.
[34] The method according to any one of paragraphs 16 to 33, wherein the concentration of IL-33 is about 0.1 to 10 ng / mL.
[35] The method according to any of paragraphs 16 to 34, wherein the concentration of IL-33 is about 0.5 to 5 ng / mL.
[36] A method for producing bone marrow-derived adherent cells including mesenchymal stem cells, comprising a step of culturing bone marrow-derived cells on a solid phase in the presence of IL-33.

[37] A medium or a medium additive for use in culturing mesenchymal stem cells, which contains CD45-positive cells.
[38] A medium or medium additive for promoting the growth of mesenchymal stem cells, comprising a CD45-positive cell.
[39] The medium or medium additive according to paragraph 37 or 38, further comprising IL-33.
[40] A medium or medium additive for use in co-culture of mesenchymal stem cells and CD45-positive cells, comprising IL-33.
[41] A medium or a medium additive for use in culturing CD45-positive cells, comprising IL-33.
[42] IL-33 is
a) a polypeptide comprising or consisting of the amino acid sequence of the full-length or mature IL-33 protein b) an amino acid sequence comprising the amino acid sequence of the mature IL-33 protein and part of the full-length IL-33 protein C) a polypeptide comprising or consisting of the amino acid sequence of a part of the full-length or mature IL-33 protein and functionally equivalent to the full-length or mature IL-33 protein d) full-length or mature An amino acid sequence in which one or more, for example, 1 to 10, 1 to 5, 1 to 3, or 1 or 2 amino acids has been substituted, deleted, inserted or added in the amino acid sequence of IL-33 protein. A full-length or mature IL-33 protein comprising or consisting of the amino acid sequence Functionally equivalent polypeptides e) about 80% or more, such as about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more of the amino acid sequence of the full-length or mature IL-33 protein A) a polypeptide comprising or consisting of an amino acid sequence having at least about 98% or more than about 99% sequence identity, or comprising a functional equivalent of a full-length or mature IL-33 protein f) full-length or mature IL A polypeptide encoded by DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein, and g) a full-length or mature IL-33 protein. About 70% or more, such as about 75% or more, about 80% or more, about 85% or more Encoded by a nucleic acid sequence having a sequence identity of about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more, and functions with full-length or mature IL-33 protein 42. The medium or medium additive according to any of paragraphs 39 to 41, which is selected from the group consisting of polypeptides that are equivalent to each other.

[43] IL-33 is
a) a polypeptide comprising or consisting of the amino acid sequence of full-length or mature IL-33 protein;
b) a polypeptide comprising the amino acid sequence of mature IL-33 protein and consisting of a partial amino acid sequence of full-length IL-33 protein;
c) a polypeptide that comprises or consists of the amino acid sequence of a part of the full-length or mature IL-33 protein and activates CD45-positive cells;
d) Activating CD45-positive cells, comprising or consisting of an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted, inserted or added in the amino acid sequence of the full-length or mature IL-33 protein Polypeptide,
e) a polypeptide which comprises or consists of an amino acid sequence having about 90% or more sequence identity with the amino acid sequence of the full-length or mature IL-33 protein, and which activates CD45-positive cells;
f) a polypeptide encoded by DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein and activating CD45-positive cells; and g) a full-length or mature IL-33 protein. 43. The method according to any of paragraphs 39 to 42, wherein the nucleic acid sequence is encoded by a nucleic acid sequence having about 90% or more sequence identity with the encoding nucleic acid sequence and is selected from the group consisting of polypeptides that activate CD45-positive cells. Medium or medium additive.

[44] The medium or the medium additive according to any of items 39 to 43, wherein the IL-33 is a polypeptide comprising the amino acid sequence of the full-length or mature IL-33 protein.
[45] The medium or medium additive according to any one of items 39 to 44, wherein IL-33 is a polypeptide consisting of an amino acid sequence of a full-length or mature IL-33 protein.
[46] The medium or the medium additive according to any one of items 39 to 45, wherein IL-33 is a polypeptide comprising any one of the amino acid sequences of SEQ ID NOS: 1 to 8.
[47] The medium or the medium additive according to any of items 39 to 46, wherein IL-33 is a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8.
[48] The medium or the medium additive according to any of items 39 to 47, wherein the nucleic acid sequence encoding IL-33 comprises any one of the nucleic acid sequences of SEQ ID NOS: 9 to 16.
[49] The medium or the medium additive according to any of items 39 to 48, wherein IL-33 is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 8.
[50] The medium or the medium additive according to any one of items 39 to 49, wherein IL-33 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or 8.
[51] The medium or the medium additive according to any of items 39 to 50, wherein the nucleic acid sequence encoding IL-33 comprises the nucleic acid sequence of SEQ ID NO: 11 or 16.
[52] The medium or medium additive according to any of items 39 to 51, wherein IL-33 is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.
[53] The medium or the medium additive according to any of paragraphs 39 to 52, wherein IL-33 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.
[54] The culture medium or culture medium additive according to any of paragraphs 39 to 53, wherein the nucleic acid sequence encoding IL-33 comprises the nucleic acid sequence of SEQ ID NO: 11.
[55] The medium or the medium additive according to any one of items 39 to 54, wherein the concentration of IL-33 in the medium is about 0.01 to 100 ng / mL.
[56] The medium or the medium additive according to any one of items 39 to 55, wherein the concentration of IL-33 in the medium is about 0.1 to 10 ng / mL.
[57] The medium or the medium additive according to any of items 39 to 56, wherein the concentration of IL-33 in the medium is about 0.5 to 5 ng / mL.

[58] A method for screening a substance that promotes the growth of mesenchymal stem cells, comprising the following steps:
1) co-culturing mesenchymal stem cells with CD45-positive cells using a control medium,
2) co-culturing mesenchymal stem cells with CD45-positive cells using a medium in which a test substance is added to a control medium; and
3) When the number of mesenchymal stem cells obtained in step 2) is larger than the number of mesenchymal stem cells obtained in step 1), the test substance is replaced with a substance that promotes the growth of mesenchymal stem cells. Selecting as candidates.
[59] When the number of mesenchymal stem cells obtained in step 2) is at least 10% greater than the number of mesenchymal stem cells obtained in step 1), the test substance is used to proliferate the mesenchymal stem cells. 59. The screening method according to item 58, wherein the method is selected as a candidate for a substance to be promoted.
[60] 1 ′) further comprising a step of co-culturing mesenchymal stem cells with CD45-positive cells using a medium obtained by adding the medium additive according to any one of items 39 to 57 to a control medium, In 3), the number of mesenchymal stem cells obtained in step 2) is larger than the number of mesenchymal stem cells obtained in step 1), and the number of mesenchymal stem cells obtained in step 1 ′) is If the number is greater than the number of mesenchymal stem cells obtained in step 1), select the test substance as a candidate for a substance that promotes the proliferation of mesenchymal stem cells, according to paragraph 58 or 59. The screening method as described above.

[61] A method for screening a substance that promotes the growth of CD45-positive cells, comprising the following steps:
1) culturing CD45-positive cells using a control medium,
2) culturing CD45-positive cells using a medium in which the medium additive according to any one of paragraphs 39 to 57 is added to a control medium;
3) a step of culturing CD45-positive cells using a control medium supplemented with a test substance, and
4) The number of CD45-positive cells obtained in step 2) is greater than the number of CD45-positive cells obtained in step 1), and the number of CD45-positive cells obtained in step 3) is A step of selecting the test substance as a candidate for a substance that promotes the proliferation of CD45-positive cells when the number is larger than the number of obtained CD45-positive cells.
[62] When the number of CD45-positive cells obtained in step 3) is at least 10% larger than the number of CD45-positive cells obtained in step 1), the test substance is used as a substance that promotes the proliferation of CD45-positive cells. 62. The screening method according to claim 61, wherein the screening method is selected as a candidate.

Methods for Producing Bone Marrow-Derived Adherent Cells Comprising Mesenchymal Stem Cells In one aspect, the present disclosure provides a method for producing bone marrow-derived adherent cells comprising solid phase in the presence of IL-33. A method for producing a derived adherent cell is provided.

骨 髄 In this method, adherent cells derived from bone marrow can be obtained efficiently. Bone marrow-derived adherent cells include mesenchymal stem cells and adherent cells other than mesenchymal stem cells, but solid phase culture in the presence of IL-33 results in non-mesenchymal stem cells and mesenchymal stem cells. Adherent cells, both promote proliferation. According to this method, adherent cells including mesenchymal stem cells can be obtained from a small amount of bone marrow tissue with high efficiency. In addition, since many adherent cells including mesenchymal stem cells can be obtained at the stage of primary culture, it is expected that the target cell number can be achieved in a short culture period, and that the ability of mesenchymal stem cells to be reduced due to culture is avoided. Is done.

In this method, the bone marrow-derived cells may be cultured in a medium containing IL-33, or IL-33 may be added to the medium in which the bone marrow-derived cells are being cultured. The concentration of IL-33 in the medium can be, for example, about 0.01-100 ng / mL, about 0.1-10 ng / mL, or about 0.5-5 ng / mL.

The medium for culturing bone marrow-derived cells is not particularly limited as long as it can be used for culturing animal cells (for example, mesenchymal stem cells). Examples include MEM, MEMα, DMEM, GMEM, RPMI 1640, and MesenCult ^™ (STEMCELL Technologies). The medium may further contain other components as long as they do not inhibit the growth of bone marrow-derived adherent cells or mesenchymal stem cells. Further, for example, the medium may not contain a differentiation inducer.

The conditions and period of the culture can be appropriately adjusted depending on the purpose of use of the cells and the like. The culture conditions are not particularly limited as long as the bone marrow-derived adherent cells or mesenchymal stem cells can survive. For example, in a general incubator, “37 ° C., 5% O ₂ , 5% CO 2 ₂ "," 37 ° C., 5% CO ₂ "and the like. The density of the bone marrow-derived cells to be cultured can be, for example, about 1 × 10 ⁴ to about 1 × 10 ⁷ / ml. The medium may be changed typically once every 1 to 7 days, for example, once every 4 or 5 days. The culture period may be a period sufficient to form colonies of bone marrow-derived adherent cells including mesenchymal stem cells, and may be several days to several months, for example, three days to three months, four days It can be for 5 months to 3 weeks or 7 days to 14 days for up to 1 month.

後 に After completion of the culture, the cells adhered to the solid phase can be collected as adherent cells derived from bone marrow including mesenchymal stem cells. For example, bone marrow-derived adherent cells including mesenchymal stem cells can be recovered by removing the medium containing non-adherent cells. Furthermore, the adherent cells may be separated from the solid phase by treatment with an enzyme such as trypsin.

に お い て In the above production method, mesenchymal stem cells after culture may be separated. Isolation of mesenchymal stem cells may be performed, for example, by a) one or more subcultures and / or b) cell sorting (FACS, MACS, etc.) targeting mesenchymal stem cells. Cell sorting targeting mesenchymal stem cells can be performed, for example, using an antibody against a surface marker of mesenchymal stem cells. By culturing the obtained cell population containing mesenchymal stem cells in the presence of an agent that suppresses the survival or proliferation of other cells, for example, CD45-positive cells (eg, Mesenpure (STEMCELL Technologies)), the mesenchymal stem cell is cultured. Stem cells may be separated (purified).

Methods for Culturing Bone Marrow-Derived Adherent Cells Containing Mesenchymal Stem Cells In one aspect, the present disclosure provides a method of culturing bone marrow-derived cells on a solid phase in the presence of IL-33, the method comprising: Provided is a method for culturing derived adherent cells. This culture method can be carried out according to the method for producing bone marrow-derived adherent cells containing mesenchymal stem cells described above. According to the culture method, bone marrow-derived adherent cells including mesenchymal stem cells can be efficiently proliferated. According to the culture method, the proliferation of both mesenchymal stem cells contained in the bone marrow-derived adherent cells and adherent cells other than mesenchymal stem cells can be promoted.

Media for Use in Culture of Bone Marrow-Derived Adherent Cells Containing Mesenchymal Stem Cells In certain embodiments, the present disclosure provides a method of producing bone marrow-derived adherent cells comprising mesenchymal stem cells, comprising IL-33. A medium for use in culturing is provided. The medium may be, for example, a medium in which IL-33 is added to a general medium for animal cell culture described above for the method for producing bone marrow-derived adherent cells including mesenchymal stem cells.

成分 The component composition of the medium other than IL-33 is not particularly limited as long as the medium can be used for culturing animal cells (eg, mesenchymal stem cells). The medium preferably does not contain any component that inhibits the growth of bone marrow-derived adherent cells or mesenchymal stem cells. The medium may be provided in any form, for example, a liquid medium that can be used as it is, a concentrate for use by diluting or dissolving, a solid, or a lyophilized product. The medium may be provided in any container.

Media Additives for Use in Culturing Bone Marrow-Derived Adherent Cells Including Mesenchymal Stem Cells In certain embodiments, the present disclosure provides a bone marrow-derived adherent cell comprising mesenchymal stem cells, comprising IL-33. A medium additive for use in culturing cells is provided.

The medium additive may contain IL-33 together with any components that are acceptable for culturing animal cells. The culture medium additive preferably does not contain a component that inhibits the growth of bone marrow-derived adherent cells or mesenchymal stem cells. The medium additive may be provided in any dosage form, for example, a solid such as granules, powders and tablets, a liquid such as a solution, a suspension or an emulsion, or a capsule encapsulating a solid, semi-solid or liquid. , But is not limited thereto. The media additive may be provided in any container. The medium additive is added to the general medium for animal cell culture described above for the method for producing bone marrow-derived adherent cells containing mesenchymal stem cells, for example, whereby the bone marrow-derived adherent cells containing mesenchymal stem cells are added. It can be used for culturing sex cells.

Screening method for substance that promotes proliferation of bone marrow-derived adherent cells or mesenchymal stem cells In one aspect, the present disclosure provides a substance that promotes proliferation of bone marrow-derived adherent cells including mesenchymal stem cells, comprising the following steps: Provide screening methods for:
1) culturing bone marrow-derived cells on a solid phase using a control medium,
2) a step of culturing bone marrow-derived cells on a solid phase using a medium in which IL-33 is added to a control medium;
3) a step of culturing bone marrow-derived cells on a solid phase using a medium in which a test substance is added to a control medium;
4) counting the number of adherent cells obtained in steps 1) to 3), and 5) counting the number of adherent cells obtained in step 2) of the adherent cells obtained in step 1). If the number is larger than the number and the number of adherent cells obtained in step 3) is larger than the number of adherent cells obtained in step 1), the test substance is treated with bone marrow containing mesenchymal stem cells. Selecting as a candidate for a substance that promotes the proliferation of the derived adherent cells.

In another aspect, the present disclosure provides a method for screening a substance that promotes the growth of mesenchymal stem cells, comprising the following steps:
1) culturing bone marrow-derived cells on a solid phase using a control medium,
2) a step of culturing bone marrow-derived cells on a solid phase using a medium in which IL-33 is added to a control medium;
3) a step of culturing bone marrow-derived cells on a solid phase using a medium in which a test substance is added to a control medium;
4) counting the number of mesenchymal stem cells contained in the adherent cells obtained in steps 1) to 3), and 5) mesenchymal stem cells contained in the adherent cells obtained in step 2) Is greater than the number of mesenchymal stem cells contained in the adherent cells obtained in step 1), and the number of mesenchymal stem cells contained in the adherent cells obtained in step 3) is A) selecting the test substance as a candidate for a substance that promotes the proliferation of mesenchymal stem cells when the number of mesenchymal stem cells is larger than the number of mesenchymal stem cells contained in the adherent cells obtained in the above step).

Generally, the activity of a test substance is determined by comparing the growth of bone marrow-derived adherent cells or mesenchymal stem cells in a control medium with the growth of bone marrow-derived adherent cells or mesenchymal stem cells in a medium to which the test substance is added. It is possible to evaluate the presence or absence of However, if growth of bone marrow-derived adherent cells or mesenchymal stem cells is not promoted in the medium to which the test substance is added, the test substance is not active, or it is actually active, but the state of the cells, experimental conditions, etc. Cannot determine whether the activity cannot be detected. This is because no positive control substance was used.

ＩＬ In both of the above screening methods, IL-33 is used as a positive control. If the proliferation of bone marrow-derived adherent cells or mesenchymal stem cells is promoted in the medium supplemented with IL-33, it is assured that the experimental system is functioning. Therefore, the accuracy and efficiency of screening can be improved by confirming the promotion of proliferation of bone marrow-derived adherent cells or mesenchymal stem cells by the positive control, IL-33, and then evaluating the medium supplemented with the test substance. Can be.

工程 Steps 1), 2) and 3) of both screening methods of the present disclosure can be performed with reference to the method for producing or culturing bone marrow-derived adherent cells including mesenchymal stem cells. "Control medium" means a medium without IL-33.

In step 4) of the method for screening a substance that promotes the growth of adherent cells derived from bone marrow according to the present disclosure, the number of colonies may be counted and regarded as the number of cells, and the cells contained in the colonies may be left attached to the solid phase. Counting may be performed in a state, or cells may be separated from the solid phase by enzyme treatment or the like, and the number of cells may be counted. For the counting of the number of cells, a method well known to those skilled in the art can be used.

In step 4) of the method for screening for a substance that promotes the growth of mesenchymal stem cells of the present disclosure, the “number of mesenchymal stem cells contained in the adherent cells” may be, for example, a typical surface marker of mesenchymal stem cells. It can be determined by FACS analysis using expression (eg, PDGFRα positive and CD45 negative) as an index. Alternatively, immunofluorescent staining or the like may be performed, and cells expressing the surface marker of the mesenchymal stem cells may be counted while remaining attached to the solid phase, and regarded as the number of mesenchymal stem cells.

In step 5) of both screening methods of the present disclosure, "large number of cells" means that the number of cells is at least 3%, 5% or 10% more than the control, and at least 10% more. Is preferred.

According to the present disclosure, for example, the following embodiments are provided.
(1) A medium additive containing IL-33 for use in culturing adherent cells derived from bone marrow including mesenchymal stem cells.
(2) A medium containing IL-33 for culturing bone marrow-derived adherent cells including mesenchymal stem cells.
(3) IL-33 is
a) a polypeptide comprising the amino acid sequence of the full-length or mature IL-33 protein b) a polypeptide comprising or consisting of a partial amino acid sequence of the full-length or mature IL-33 protein c) the mature IL-33 protein And d) one or more amino acids in the full-length or mature IL-33 protein, for example, 1 to 10, 1 to 10 in the amino acid sequence of the full-length or mature IL-33 protein. A polypeptide comprising or consisting of an amino acid sequence in which five, one to three or one or two amino acids have been substituted, deleted, inserted or added, or e) amino acids of the full-length or mature IL-33 protein About 80% or more, such as about 85% or more, about 90% or more, about 95% or more A polypeptide comprising or consisting of about 96% or more, about 97% or more, about 98% or more or about 99% or more sequence identity f) full-length or mature IL-33 protein A polypeptide encoded by DNA that hybridizes under stringent conditions to the encoding nucleic acid sequence; and g) a nucleic acid sequence encoding the full-length or mature IL-33 protein, at least about 70%, such as at least about 75%, Encoded by a nucleic acid sequence having 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more or about 99% or more sequence identity. Polypep selected from the group consisting of polypeptides and having an activity of promoting proliferation of bone marrow-derived adherent cells or mesenchymal stem cells A de culture medium according to the medium additives or second term according to paragraph 1.
(4) IL-33 is
a) a polypeptide comprising the amino acid sequence of the full-length or mature IL-33 protein b) a polypeptide comprising or consisting of a partial amino acid sequence of the full-length or mature IL-33 protein c) the mature IL-33 protein And d) one or more amino acids are substituted, deleted, inserted, or substituted in the amino acid sequence of the full-length or mature IL-33 protein, comprising a partial amino acid sequence of the full-length IL-33 protein. A polypeptide comprising or consisting of an added amino acid sequence e) comprising or having an amino acid sequence having about 90% or more sequence identity with the amino acid sequence of the full-length or mature IL-33 protein F) full length or mature I A polypeptide encoded by DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a -33 protein, and g) at least about 90% sequence identity to a nucleic acid sequence encoding a full-length or mature IL-33 protein 4. The polypeptide according to any one of items 1 to 3, wherein the polypeptide is selected from the group consisting of polypeptides encoded by the nucleic acid sequence having, and has an activity of promoting the proliferation of bone marrow-derived adherent cells or mesenchymal stem cells. The medium additive or medium according to any one of the above.
(5) The culture medium additive or culture medium according to any one of (1) to (4), wherein IL-33 is a polypeptide containing the amino acid sequence of full-length or mature IL-33 protein.
(6) The medium additive or medium according to any one of items 1 to 5, wherein IL-33 is a polypeptide consisting of an amino acid sequence of a full-length or mature IL-33 protein.

(7) The culture medium additive or medium according to any of (3) to (6), wherein the amino acid sequence of the full-length or mature IL-33 protein is any of SEQ ID NOS: 1 to 8.
(8) The culture medium additive or culture medium according to any of (3) to (7), wherein the nucleic acid sequence encoding the full-length or mature IL-33 protein is any of SEQ ID NOS: 9 to 16.
(9) The culture medium additive or medium according to any of (3) to (8), wherein the amino acid sequence of the full-length or mature IL-33 protein is the amino acid sequence of SEQ ID NO: 3 or 8.
(10) The culture medium additive or medium according to any of (3) to (9), wherein the nucleic acid sequence encoding the full-length or mature IL-33 protein is the nucleic acid sequence of SEQ ID NO: 11 or 16.
(11) The medium additive or medium according to any of items 3 to 10, wherein the amino acid sequence of the full-length or mature IL-33 protein is the amino acid sequence of SEQ ID NO: 3.
(12) The medium additive or medium according to any of (3) to (11), wherein the nucleic acid sequence encoding the full-length or mature IL-33 protein is the nucleic acid sequence of SEQ ID NO: 11.

(13) The medium according to any of (2) to (12), wherein the concentration of IL-33 is about 0.01 to 100 ng / mL.
(14) The medium according to any of (2) to (13), wherein the concentration of IL-33 is about 0.1 to 10 ng / mL.
(15) The medium according to any of (2) to (14), wherein the concentration of IL-33 is about 0.5 to 5 ng / mL.
(16) The medium or the medium additive according to any one of (1) to (15) for growing mesenchymal stem cells.

(17) A method for producing adherent cells derived from bone marrow containing mesenchymal stem cells, comprising the step of culturing bone marrow-derived cells on a solid phase in the medium according to any of items 2 to 16.
(18) A mesenchymal stem cell comprising a step of culturing a bone marrow-derived cell on a solid phase in the medium according to any one of (2) to (16) in order to promote proliferation of the mesenchymal stem cell A method for producing bone marrow-derived adherent cells, comprising:
(19) A method for promoting the growth of mesenchymal stem cells, comprising the step of culturing bone marrow-derived cells on a solid phase in the medium according to any of items 2 to 16.
(20) The method according to any of (17) to (19), further comprising a step of collecting cells adhered to the solid phase after culture as bone marrow-derived adherent cells including mesenchymal stem cells.
(21) 1) a step of culturing the bone marrow-derived cells on a solid phase in the medium according to any of the above items 2 to 16, and 2) a step of culturing the adherent cells obtained in step 1) A method for producing mesenchymal stem cells, comprising the steps of: a) performing one or more subcultures and / or b) performing cell sorting targeting mesenchymal stem cells to separate mesenchymal stem cells.
(22) A method for culturing adherent cells derived from bone marrow, including mesenchymal stem cells, comprising a step of culturing bone marrow-derived cells on a solid phase in the medium according to any of items 2 to 16.
(23) The method according to any one of Items 17 to 22, wherein the solid phase is coated.
(24) The method according to any of paragraphs 17 to 23, wherein the solid phase is coated with collagen I, laminin, vitronectin, fibronectin, poly L-lysine or poly L-ornithine.
(25) The method according to any of items 17 to 24, wherein the solid phase is coated with collagen I.

(26) A method for screening a substance that promotes the proliferation of bone marrow-derived adherent cells including mesenchymal stem cells, comprising the following steps:
1) culturing bone marrow-derived cells on a solid phase using a control medium,
2) a step of culturing the bone marrow-derived cells on a solid phase using a medium obtained by adding the medium additive according to any one of items 1 and 3 to a control medium;
3) a step of culturing bone marrow-derived cells on a solid phase using a medium in which a test substance is added to a control medium;
4) counting the number of adherent cells obtained in steps 1) to 3), and 5) counting the number of adherent cells obtained in step 2) of the adherent cells obtained in step 1). If the number is larger than the number and the number of adherent cells obtained in step 3) is larger than the number of adherent cells obtained in step 1), the test substance is treated with bone marrow containing mesenchymal stem cells. Selecting as a candidate for a substance that promotes the proliferation of the derived adherent cells.
(27) When the number of adherent cells obtained in step 3) is at least 10% larger than the number of adherent cells obtained in step 1), the test substance is attached to bone marrow-derived adherent cells including mesenchymal stem cells. 27. The screening method according to paragraph 26, wherein the method is selected as a candidate for a substance that promotes the proliferation of sex cells.

(28) A method for screening a substance that promotes the growth of mesenchymal stem cells, comprising the following steps:
1) culturing bone marrow-derived cells on a solid phase using a control medium,
2) a step of culturing the bone marrow-derived cells on a solid phase using a medium obtained by adding the medium additive according to any one of items 1 and 3 to a control medium;
3) a step of culturing bone marrow-derived cells on a solid phase using a medium in which a test substance is added to a control medium;
4) counting the number of mesenchymal stem cells contained in the adherent cells obtained in steps 1) to 3), and 5) mesenchymal stem cells contained in the adherent cells obtained in step 2) Is greater than the number of mesenchymal stem cells contained in the adherent cells obtained in step 1), and the number of mesenchymal stem cells contained in the adherent cells obtained in step 3) is A) selecting the test substance as a candidate for a substance that promotes the proliferation of mesenchymal stem cells when the number of mesenchymal stem cells is larger than the number of mesenchymal stem cells contained in the adherent cells obtained in the above step).
(29) When the number of mesenchymal stem cells contained in the adherent cells obtained in step 3) is at least 10% larger than the number of mesenchymal stem cells contained in the adherent cells obtained in step 1) 29. The screening method according to claim 28, wherein the test substance is selected as a candidate for a substance that promotes the proliferation of mesenchymal stem cells.

All documents cited in this disclosure are hereby incorporated by reference.
All of the above description is non-limiting and can be modified without departing from the scope of the invention as defined in the appended claims. Further, the following examples are all non-limiting examples, which are only provided to illustrate the invention.

Example 1
Promotion of Mesenchymal Stem Cell Proliferation by IL-33-containing Medium (1) Materials and Methods Femurs and vertebrae were cut out from C57BL / 6 mice (6-7 weeks old, female), and other tissues attached to the periphery were removed. Thereafter, the bone was finely crushed in a mortar, and cells were recovered by adding PBS (containing 2% FBS). Next, the cells were passed through a 40 μm nylon mesh to remove cell clumps and muscle tissue. The mixture was centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the precipitated cells were suspended in RBC Lysis buffer (Biolegend) and reacted at room temperature for 5 minutes to lyse (lyse) red blood cells. The reaction was stopped by adding an equal amount of PBS (containing 2% FBS) to the RBC Lysis buffer, followed by centrifugation at 300 g for 5 minutes. The supernatant was discarded, and the precipitated cells were obtained as bone marrow-derived cells. Bone marrow-derived cells are suspended in the following medium A or B and seeded at a density of 1 × 10 ⁶ cells / well in a plastic 6-well plate (Corning Inc., Cat No. 356400) coated with collagen I. The cells were cultured in 3 mL of medium at 37 ° C., 5% O ₂ , and 5% CO ₂ for 10 days (on the fourth or fifth day of culture, the medium was replaced with a fresh medium having the same composition).

Medium A (control): MEMα (Gibco) (containing 15% FBS, 1% penicillin-streptomycin; L-Glutamine was added to a final concentration of 4 mM, and mature mouse IL- 33 with the same volume of PBS as the protein solution)
Medium B (containing IL-33): MEMα (Gibco) (containing 15% FBS, 1% penicillin-streptomycin; L-Glutamine was added to a final concentration of 4 mM, and mature mouse IL-33 protein (sequence No. 3) added to a final concentration of 2 ng / mL)
On the 10th day from the start of the culture, the medium was removed, and the cells adhering to the plate were stained with Diff Quick (Sysmex) and observed.

(2) Results Regarding bone marrow-derived cells collected from both femurs and vertebrae, the number of cells obtained as colonies on the plate was significantly increased by culturing using a medium containing IL-33 (FIG. 1). ). Similar results were obtained when the experiment was performed under the same method and conditions as described above except that a 6-well plate (costar, Cat No. 3516) without collagen I coating was used for culturing bone marrow-derived cells. Was done.

It is well known that when bone marrow-derived cells are cultured on a solid phase, adherent cells including mesenchymal stem cells adhere to the solid phase to form colonies. Therefore, the experimental results in this example show that culture in the presence of IL-33 promotes the proliferation of bone marrow-derived adherent cells including mesenchymal stem cells.

Example 2
Analysis of Bone Marrow-Derived Cells Cultured Using IL-33-Containing Medium (1) Materials and Methods Mice (Hamilton TG, Mol Cell Biol. 2003) in which the gene (cDNA) of the H2B-GFP fusion protein was knocked in downstream of the PDGFRα promoter. Jun; 23 (11): 4013-25), the femur and the vertebra were cut out, and other tissues attached to the periphery were removed. Then, the bone was finely crushed in a mortar, and PBS (containing 2% FBS) was added. Cells were collected. Next, the cells were passed through a 40 μm nylon mesh to remove cell clumps and muscle tissue. The mixture was centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the precipitated cells were suspended in RBC Lysis buffer (Biolegend) and reacted at room temperature for 5 minutes to lyse (lyse) red blood cells. The hemolysis reaction was stopped by adding PBS (containing 2% FBS) in the same amount as the RBC Lysis buffer, followed by centrifugation at 300 g for 5 minutes. The supernatant was discarded, and the precipitated cells were obtained as bone marrow-derived cells. Bone marrow-derived cells were suspended in the following medium C (control) or medium D (containing IL-33) and placed in a plastic 6-well plate (Corning Inc., Cat No. 356400) coated with collagen I at 1 × 10 6 ^The cells were seeded at a density of ⁶ cells / well and cultured in 3 mL of medium at 37 ° C., 5% O ₂ , and 5% CO ₂ for 8 days (on the fourth or fifth day of culture, fresh fresh cells of the same composition were used). Medium was replaced).

Medium C (control): MEMα (Gibco) (containing 15% FBS, 1% penicillin-streptomycin; L-Glutamine was added to a final concentration of 4 mM, and mature mouse IL- 33 with the same volume of PBS as the protein solution)
Medium D (containing IL-33): MEMα (Gibco) (containing 15% FBS, 1% penicillin-streptomycin; L-Glutamine was added to a final concentration of 4 mM, and mature mouse IL-33 protein (sequence No. 3) added to a final concentration of 1 ng / mL)

On the 8th day from the start of the culture, the cells adhered to the plate were detached by trypsin treatment and collected, and the expression of PDGFRα, a typical positive marker of mesenchymal stem cells, and CD45, a typical negative marker, was determined by FACS. Analyzed.

(2) Results Regarding bone marrow-derived cells collected from both the femur and the vertebra, the number of adherent cells forming colonies on the plate was significantly increased by culturing using a medium containing IL-33 ( (Fig. 2). When the number of cells was examined for each expression pattern of the surface marker, the number of PDGFRα-positive CD45-negative (PDGFRα ⁺ CD45 ⁻ ) cells and the number of PDGFRα-negative CD45-positive (PDGFRα ⁻ CD45 ⁺ ) cells were significantly increased (FIG. 3 and FIG. 3). 4). The culture results in the presence of IL-33 significantly increased the number of adherent cells of “PDGFRα ⁺ CD45 ⁻ ”, which is a typical marker expression pattern of mesenchymal stem cells. Is considered to indicate that the action of has promoted the proliferation of mesenchymal stem cells.

Example 3
Materials and methods
Mouse In this example, C57BL / 6jjcl (CLEA Japan) was used as a wild-type mouse. B6.129S4-Pdgfratm11 ^{(EGFP) Sor} (Jackson Laboratory, 007669) was used as PDGFRαGFP knock-in mouse. MyD88 knockout mouse (C57BL / 6) (Adachi, O. et al. Targeted Disruption of the MyD88 Gene Results in Loss of IL-1- and IL-18-Mediated Function. Immunity 9, 143-150, doi: 10.1016 / S1074 -7613 (00) 80596-8 (1998)) was purchased from Oriental Bio Services. The mice were housed in an animal breeding room maintained on light and dark for 12 hours each with solid feed under automatic watering.

Isolation of bone marrow-derived cells The femur was collected from the mouse, and the cells were removed using a mortar and pestle in PBS supplemented with 2% fetal bovine serum (FBS) (PBS / 2% FBS). The cell suspension was passed through a 40 μm Cell Strainer (Falcon) to remove aggregates such as bone. After centrifugation, erythrocytes were removed by hemolysis with an RBC lysis buffer (Biolegend) to obtain bone marrow-derived cells.

Culture of bone marrow-derived cells Bone marrow-derived cells were cultured on a collagen-coated 6-well plate (Corning Inc.) using 15% FBS, 1 × GlutaMAX ^™ Supplement (Gibco, Cat No. 35050061), 1 × MEM non-essential amino acid solution (Nacalai Tesque) The cells were cultured in αMEM (3 mL / well) supplemented with 10 mM HEPES buffer (Nacalai Tesque), 1 × monothioglycerol solution (Wako), and 1% penicillin-streptomycin mixed solution using a hypoxic incubator. When culturing whole bone marrow-derived cells without sorting, the cells were cultured at 5 × 10 ⁵ cells / well. When mesenchymal stromal cells (MSCs) were sorted from bone marrow-derived cells using FACS, PDGFRα ⁺ cells were sorted at 800 cells / well. In the co-culture experiment, 5 × 10 ⁵ cells / well of bone marrow-derived cells, 4.75 × 10 ⁵ cells / well of CD45 ⁺ cells, and 2.5 × 10 ³ cells / well of CD45 ⁻ cells were further collected. Each was co-cultured with MSC. At the time of culture using Transwell (Corning Inc.), 800 PDGFRα ⁺ cells collected on a collagen-coated 6-well plate were seeded, and 5 × 10 ⁵ bone marrow-derived cells were collected and seeded on Transwell. Cultured. IL-33 (Biolegend) (SEQ ID NO: 3) was added at 1 ng / mL, and an equal volume of PBS was added to the control. DAPT (TCI) was added at a final concentration of 1 μM or 10 μM, and an equivalent amount of DMSO (dimethyl sulfoxide) was added to the control. A p38 MAPK inhibitor (SB203580, AdipoGen) and an NK-κB inhibitor (BAY-11-7082, Cayman Chemical) were added at a final concentration of 5 μM, and an equivalent amount of DMSO was added to the control. The medium was changed every four days. Cells not sorted were cultured for 10 days, and cells sorted by FACS were cultured for 12 days, and analyzed by FACS or stained with Diff-Quick solution (Sysmex) to analyze the number of colonies.

Cell analysis by FACS, cell sorting Cell analysis by FACS, cell sorting are performed using FACSAria ^™ IIIμ (BD Bioscience) or FACSCanto ^™ II (BD Bioscience), and data analysis is performed using FlowJo software (Tree Star). Was. PDGFRα GFP knock-in mice were used for PDGFRα expression analysis. Accutase ^™ (Nacalai Tesque) was used to detach cells from the culture plate when analyzing the cultured cells. At the time of addition of the antibody, Trustain FcX ^™ (Biolegend) was added to the cell suspension to inhibit non-specific binding to the Fc receptor, and the mixture was incubated at 4 ° C. for 5 minutes, and then an APC-binding CD45 antibody (Biolegend, 103112) , PerCP / cy5.5-conjugated CD45 antibody (Biolegend, 103132) and PerCP / cy5.5-conjugated TER119 antibody (Biolegend, 116228) were added and incubated at 4 ° C. for 20 minutes. Before FACS analysis, the cells were stained with SYTOX ^™ Blue Dead Cell Stain (Thermo Fisher Scientific) to remove dead cells. At the time of cell sorting, the cells were sorted into αMEM supplemented with 15% FBS, 1 × GlutaMAX ^™ , 1 × MEM non-essential amino acid solution, 10 mM HEPES buffer, 1 × monothioglycerol solution, and 1% penicillin-streptomycin mixed solution.

Example 3-1: IL-33 enhances the colony forming activity of bone marrow-derived MSCs Bone marrow-derived cells prepared from PDGFRαGFP knock-in mouse femur were seeded at 5 × 10 ⁵ cells per well of a 6-well plate, and 3 mL of medium was added. After culturing in a medium supplemented with IL-33 for 10 days and analyzing by flow cytometry (FACS), a significant increase in bone marrow-derived cells due to the addition of IL-33 was confirmed (FIG. 5a). Since it is known that MSCs express PDGFRα and do not express CD45 which is a marker of blood cells, in this example, PDGFRα ⁺ CD45 ⁻ cells were used as MSC fractions. FACS revealed that IL-33 significantly increased bone marrow-derived MSCs (FIG. 5b). At the same time, IL-33 significantly increased not only MSC but also CD45 ⁺ cells (FIG. 5c), suggesting that IL-33 may act on multiple cell populations in bone marrow.

Since bone marrow-derived MSCs form fibroblast-like colonies in culture, the abundance and proliferation rate of MSCs are measured by measuring fibroblast-like colony forming units (Colony forming unit-fibroblast; CFU-F). Can be evaluated. When a colony assay was performed after culturing in a medium supplemented with IL-33 for 10 days, the number of colonies was significantly increased about 7-fold by addition of IL-33 (FIG. 5d), and IL-33 enhanced the colony forming activity of bone marrow-derived MSCs. It was shown to be.

Example 3-2: IL-33 enhances CFU-F activity of bone marrow-derived MSCs through cell-to-cell contact between CD45 ⁺ cells and MSCs. It was examined whether -33 acts directly on bone marrow-derived MSCs to enhance its CFU-F activity or through signals of other cells. MSCs (PDGFRα ⁺ cells) were fractionated by FACS from bone marrow-derived cells prepared from PDGFRαGFP knock-in mouse femurs, and cultured in a medium supplemented with IL-33. However, under the culture condition of MSC alone, CFU-F activity was not changed by IL-33 (FIG. 6). This suggests that the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 may be indirectly controlled by cells other than MSCs present in bone marrow.

It was examined whether the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 relies on cell-cell contact between MSCs and other bone marrow-derived cells. Using Transwell, culture conditions were established in which bone marrow-derived cells and bone marrow-derived MSCs share a medium but cells did not contact each other, and the CFU-F activity of bone marrow-derived MSCs was investigated. Co-culture of bone marrow-derived MSCs with bone marrow-derived cells increased CFU-F activity by IL-33, but under conditions (Transwell) in which cell-cell contact between bone marrow-derived MSCs and bone marrow-derived cells was suppressed (Transwell). No change was observed in CFU-F activity by -33, indicating that cell-to-cell contact with other cells other than MSC and MSC is essential for enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33. (FIG. 7a). In addition, it was revealed that culturing bone marrow-derived cells in the presence of IL-33 promotes secretion of CCL2, CCL22, and osteopontin as humoral factors (data not disclosed). Bone marrow-derived cells were cultured with the addition, but CFU-F activity was not changed by the addition of any substance (data not disclosed).

Further, it was examined whether contact with any cell present in bone marrow enhances CFU-F activity of bone marrow-derived MSCs. In Example 3-1, since IL-33 increased CD45 ⁺ (CD45 ⁺ ) cells in bone marrow-derived cells (FIG. 5c), it was predicted that cell-to-cell contact with CD45 + cells was necessary, CD45-positive cells and CD-negative (CD45 ⁻ ) cells were separately collected by FACS, and co-cultured with bone marrow-derived MSCs in the presence of IL-33. CD45 ^- cells included PDGFRα ⁺ cells, but in negligible amounts. The enhancement of CFU-F activity of bone marrow-derived MSCs can be confirmed only by co-culture with CD45 ⁺ cells, and the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 is induced through intercellular contact with CD45 ⁺ cells (FIG. 7b).

Example 3-3: Enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 is dependent on MyD88 signal pathway The molecular mechanism of enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 was examined in further detail. When IL-33 binds to the ST2 / IL-1 receptor accessory protein (IL-1RAP) complex, which is a cell membrane receptor, Myeloid differentiation primary response (MyD88) 88, which is an adapter protein, is bound to this receptor. ) Is induced, and MyD88 is known to regulate the activity of a target gene through the activation of NF-κB, p38, ERK and JNK. Therefore, to examine the effect of the MyD88 signaling pathway on the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33, bone marrow-derived cells prepared from MyD88 knockout (KO) mouse femur were cultured in a medium supplemented with IL-33. . In the bone marrow-derived cells of MyD88KO mice, no change was observed in the CFU-F activity due to the addition of IL-33, indicating that the MyD88 signal pathway is essential for enhancing the CFU-F activity of bone marrow-derived MSCs by IL-33. (FIG. 8a). Furthermore, in the bone marrow-derived cells of MyD88KO mice, the increase of CD45 ⁺ cells by IL-33 was not observed (FIG. 8b), and the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 was caused by the MyD88 signal pathway in CD45 ⁺ cells. It was suggested that it might depend on.

Furthermore, the involvement of the p38 MAPK pathway and the NF-κB pathway, which are known to work downstream of MyD88, was examined. In bone marrow-derived cells prepared from PDGFRαGFP knock-in mouse femurs, IL-33 enhanced CFU-F activity in the DMSO-added control group and the NF-κB inhibitor (BAY-11-7082) -added group, but p38MAPK. In the group added with the inhibitor (SB203580), no change in CFU-F activity due to IL-33 was observed (FIG. 9). These results revealed that the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 was dependent on the MyD88 signal pathway and was mediated by the p38 MAPK pathway.

CD45 ⁺ cells activated in a MyD88 signaling pathway-dependent manner by IL-33 are important in the growth and maintenance of MSCs in order to investigate the molecular mechanism that induces the enhancement of CFU-F activity of bone marrow-derived MSCs. The effect of the Notch signaling pathway that has been performed was examined. Specifically, in bone marrow-derived cells prepared from PDGFRαGFP knock-in mouse femur, DAPT ((3,5-difluorophenylacetyl) -L-alanyl-L-, which inhibits γ-secretase which is a part of the Notch signal pathway, is used. The effect of 2-phenylglycine tert-butyl ester) on MSC proliferation by the addition of IL-33 was examined. As a result, in the presence of DAPT, the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 was suppressed (FIG. 10a). Furthermore, when the bone marrow-derived cells cultured in the medium supplemented with DAPT / IL-33 were analyzed by FACS, the number of MSCs decreased in a DAPT concentration-dependent manner (FIG. 10b). On the other hand, the addition of DAPT / IL-33 did not suppress the increase in the number of CD45 ⁺ cells (FIG. 10c).

According to the present disclosure, it is possible to improve the efficiency of obtaining mesenchymal stem cells, reduce the burden on donors, reduce the cost of regenerative medicine using mesenchymal stem cells, promote basic research using mesenchymal stem cells, and promote drug development, etc. There is expected.

Claims (16)

方法 A method for producing a cell population containing mesenchymal stem cells, comprising a step of culturing a cell population containing mesenchymal stem cells and CD45-positive cells. The method according to claim 1, wherein the cell population containing mesenchymal stem cells and CD45-positive cells comprises bone marrow-derived mesenchymal stem cells and / or CD45-positive cells. 方法 The method according to claim 1 or 2, wherein the cell population containing mesenchymal stem cells and CD45-positive cells is cultured in the presence of a substance that activates CD45-positive cells. 4. The method according to claim 3, wherein the substance that activates CD45-positive cells is IL-33. IL-33,
a) a polypeptide comprising or consisting of the amino acid sequence of full-length or mature IL-33 protein;
b) a polypeptide comprising the amino acid sequence of mature IL-33 protein and consisting of a partial amino acid sequence of full-length IL-33 protein;
c) a polypeptide that comprises or consists of the amino acid sequence of a part of the full-length or mature IL-33 protein and activates CD45-positive cells;
d) Activating CD45-positive cells, comprising or consisting of an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted, inserted or added in the amino acid sequence of the full-length or mature IL-33 protein Polypeptide,
e) a polypeptide which comprises or consists of an amino acid sequence having about 90% or more sequence identity with the amino acid sequence of the full-length or mature IL-33 protein, and which activates CD45-positive cells;
f) a polypeptide encoded by DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein and activating CD45-positive cells; and g) a full-length or mature IL-33 protein. 5. The method of claim 4, wherein the method is selected from the group consisting of a polypeptide encoded by a nucleic acid sequence having about 90% or more sequence identity to the encoding nucleic acid sequence and activating CD45 positive cells. The method according to claim 5, wherein IL-33 is a polypeptide comprising or consisting of the amino acid sequence of the full-length or mature IL-33 protein. The method according to claim 6, wherein IL-33 is a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 1 to 8, or a polypeptide consisting of the amino acid sequence. 8. The method according to any one of claims 1 to 7, further comprising a step of separating mesenchymal stem cells. 9. The method according to claim 8, wherein the step of separating the mesenchymal stem cells comprises a) one or more subcultures and / or b) cell sorting targeting the mesenchymal stem cells. (4) A method for culturing mesenchymal stem cells, comprising a step of co-culturing mesenchymal stem cells with CD45-positive cells in the presence of IL-33. 培 地 A medium or a medium additive for use in culturing mesenchymal stem cells, containing CD45-positive cells. The medium or medium additive according to claim 11, further comprising ΔIL-33. 培 地 A medium or medium additive for use in co-culture of mesenchymal stem cells and CD45-positive cells, comprising IL-33. A method for screening a substance that promotes the growth of mesenchymal stem cells, comprising the following steps:
1) co-culturing mesenchymal stem cells with CD45-positive cells using a control medium,
2) co-culturing mesenchymal stem cells with CD45-positive cells using a medium in which a test substance is added to a control medium; and
3) When the number of mesenchymal stem cells obtained in step 2) is larger than the number of mesenchymal stem cells obtained in step 1), the test substance is replaced with a substance that promotes the growth of mesenchymal stem cells. Selecting as candidates. 1 ′) further comprising a step of co-culturing mesenchymal stem cells with CD45-positive cells using a medium supplemented with IL-33 as a control medium, wherein in step 3) the mesenchymal stem cells obtained in step 2) The number is greater than the number of mesenchymal stem cells obtained in step 1), and the number of mesenchymal stem cells obtained in step 1 ′) is greater than the number of mesenchymal stem cells obtained in step 1). 15. The screening method according to claim 14, wherein the test substance is selected as a candidate for a substance that promotes the proliferation of mesenchymal stem cells when the number is large. A method for screening a substance that promotes the growth of CD45-positive cells, comprising the following steps:
1) culturing CD45-positive cells using a control medium,
2) a step of culturing CD45-positive cells using a medium in which IL-33 is added to a control medium;
3) a step of culturing CD45-positive cells using a control medium supplemented with a test substance, and
4) The number of CD45-positive cells obtained in step 2) is greater than the number of CD45-positive cells obtained in step 1), and the number of CD45-positive cells obtained in step 3) is A step of selecting the test substance as a candidate for a substance that promotes the proliferation of CD45-positive cells when the number is larger than the number of obtained CD45-positive cells.

Images