JPWO2020040293A1 - Method for producing mesenchymal stem cells - Google Patents

Abstract

The present disclosure comprises a method for producing or culturing a cell population containing mesenchymal stem cells, which comprises a step of culturing a cell population containing mesenchymal stem cells and CD45-positive cells; a medium or medium additive used in these methods; The present invention relates to a method for screening a substance that promotes the proliferation of mesenchymal stem cells; or a method for screening a substance that promotes the proliferation of CD45-positive cells.

Description

This patent application claims priority over Japanese patent applications 2018-157175 and 2019-068459, which are incorporated herein by reference in their entirety. do.
The present disclosure relates to a method for producing or culturing mesenchymal stem cells, a medium or medium additive used in these methods, or a method for screening a substance that promotes the proliferation of mesenchymal stem cells. The present disclosure also relates to a method for screening a substance that promotes the proliferation of CD45-positive cells.

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 (pluripotency). Attempts have been made to perform regenerative medicine (cell transplantation therapy) using foliar stem cells. Although a large amount of cells are required to use mesenchymal stem cells for treatment, the abundance of mesenchymal stem cells in vivo is extremely small, and the abundance frequency of mesenchymal stem cells also in the bone marrow, which is a typical source of mesenchymal stem cells. Is about 1 per 10,000 to 100,000 bone marrow cells. In addition, collecting bone marrow fluid imposes a heavy burden on the donor, so it cannot be collected in large quantities. Therefore, it is usual to obtain mesenchymal stem cells contained in bone marrow fluid, proliferate them by culturing, and then use them for treatment. However, mesenchymal stem cells gradually have proliferative ability and differentiation ability when subculture is continued in vitro. Is known to reduce. Against this background, the development of a method for efficiently proliferating mesenchymal stem cells is desired.

Interleukin-33 (IL-33) is a cytokine identified as a member of the IL-1 family and is released extracellularly in the event of cell injury or necrosis, activating immune cells and Th2. It induces the secretion of cytokines (IL-4, IL-5, IL-13) and / or inflammatory cytokines (TNF-α, IL-1, IL-6, IFN-γ). A method of adding a specific cytokine (bFGF) to the medium has been proposed for the purpose of improving the culture efficiency of mesenchymal stem cells (Patent Document 1), but for IL-33, the action on the proliferation of mesenchymal stem cells, etc. Is not known.

International Bulletin No. 02/22788 International Bulletin 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.

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

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

In certain embodiments, the present disclosure provides a method for producing a cell population containing mesenchymal stem cells, which comprises the step of culturing a cell population containing mesenchymal stem cells and CD45-positive cells.
In some embodiments, the present disclosure provides a method of culturing mesenchymal stem cells, comprising co-culturing mesenchymal stem cells with CD45-positive cells in the presence of IL-33.
In some embodiments, the present disclosure provides a medium or culture medium additive for use in culturing mesenchymal stem cells containing CD45-positive cells.
In some embodiments, the present disclosure provides a medium or culture medium additive containing IL-33 for use in co-culture of mesenchymal stem cells and CD45 positive cells.
In some embodiments, the present disclosure describes the following steps:
1) A step of co-culturing mesenchymal stem cells with CD45-positive cells using a control medium,
2) A step of co-culturing mesenchymal stem cells with CD45-positive cells using a medium obtained by adding a test substance 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 used as a substance that promotes the proliferation of mesenchymal stem cells. Process to select as a candidate for
Provided is a method for screening a substance that promotes the proliferation of mesenchymal stem cells, including.
In some embodiments, the present disclosure describes the following steps:
1) A step of 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 medium obtained by adding a test substance to a control medium, and
4) The number of CD45-positive cells obtained by step 2) is larger than the number of CD45-positive cells obtained by step 1), and the number of CD45-positive cells obtained by step 3) is the number of CD45-positive cells obtained by step 1). A step of selecting the test substance as a candidate for a substance that promotes the proliferation of CD45-positive cells when the number of CD45-positive cells is larger than the number of obtained CD45-positive cells.
Provided is a method for screening a substance that promotes the proliferation of CD45-positive cells, including.

The present disclosure makes it possible to promote the proliferation of mesenchymal stem cells in vitro.

3 is a photograph showing the results of solid-phase culture of bone marrow cells collected from the femur and vertebrae of mice in a medium containing mature IL-33 protein (10 days after the start of culture). It is a graph which shows the number of bone marrow-derived adherent cells on the 8th day from the start of culture. It is a figure which shows the FACS analysis result of the bone marrow-derived adherent cell on the 8th day from the start of culture. The horizontal axis represents the expression level of PDGFRα (detected by GFP fluorescence), and the vertical axis represents the expression level of CD45 (detected by APC (Allophycocyanin) -labeled anti-CD45 antibody). The numbers in the four quadrants indicate the percentage of cells corresponding to each quadrant in the total number of cells analyzed. It is a graph which shows the number of ^{PDGFRα +} CD45 ⁻ cells and PDGFRα ⁻ CD45 ⁺ cells among the bone marrow-derived adherent cells on the 8th day from the start of culture. ^{Cell count per well of culture plate (a), PDGFRα +} CD45 ^- cell count (b), CD45 ⁺ cell count (c) and colony assay results when bone marrow cells were cultured in IL-33-added medium for 10 days. (D) is shown. The results of the colony assay when PDGFRα ⁺ cells were separated and cultured in IL-33-added medium are shown. In FIG. 7a, PDGFRα ⁺ cells (Pα ⁺ ) were separated and then co-cultured with bone marrow cells in an IL-33-added medium, or PDGFRα ⁺ cells were collected on Transwell in the presence of bone marrow cells. The results of colony assay after culturing in IL-33-added medium are shown. FIG. 7b ^{shows the results of the colony assay when PDGFRα +} cells and CD45 ^- cells or CD45 ⁺ cells were co-cultured in IL-33-added medium. FIG. 8a shows the results of the colony assay when bone marrow cells of MyD88KO mice were cultured in IL-33-added medium. FIG. 8b shows the results of FACS analysis when bone marrow cells of MyD88KO mice were cultured in IL-33-added medium for 10 days. The results of the colony assay when bone marrow cells were cultured in IL-33 / p38MAPK inhibitor or IL-33 / NF-κB inhibitor-added medium are shown. FIG. 10a shows the results of the colony assay when bone marrow cells were cultured in IL-33 / DAPT-added medium. ^{10b and c show PDGFRα +} CD45 ^- cell count and CD45 ⁺ cell count analyzed by FACS when bone marrow cells were cultured in IL-33 / DAPT-added medium for 10 days.

In the present disclosure, when a number is accompanied by the term "about", it is intended to include a range of ± 10% of that value. For example, "about 20" is assumed to include "18-22". The range of numbers includes all numbers between the endpoints and the numbers at the endpoints. The "about" for a range applies to both ends of the range. Therefore, for example, "about 20 to 30" includes "18 to 33".

Unless otherwise specified, the terms used in this disclosure have meanings generally understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology and the like. The following are definitions of some terms used in this disclosure, but these definitions supersede the general understanding in this disclosure.

In the present disclosure, "cell" means one cell or multiple 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 a plurality of types of cells depending on the context.

In the present disclosure, "mesenchymal stem cells" are used without distinction from "mesenchymal stromal cells" and may be abbreviated as "MSC". In general, a stem cell means a cell that has 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 among mesenchymal tissues such as bone, cartilage, fat, and muscle. In addition to mesoderm mesenchymal tissue, mesenchymal stem cells may have the ability to differentiate into epithelial tissue, nervous tissue, and cardiovascular tissue beyond the embryo. In certain embodiments, mesenchymal stem cells have the ability to differentiate into multiple cells within osteoblasts, chondrocytes, muscle cells, and adipocytes. In certain embodiments, mesenchymal stem cells have the ability to differentiate into multiple cells within osteoblasts, chondrocytes, and adipocytes. In certain embodiments, mesenchymal stem cells have the ability to differentiate into osteoblasts and adipocytes. In certain embodiments, mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. In certain embodiments, 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 to mean a cell population, it may be a cell population composed only of mesenchymal stem cells, as long as the cell population exhibits the same differentiation ability as mesenchymal stem cells. , Mesenchymal stem cells and may be a heterogeneous cell population containing cells in the intermediate stage of differentiation between mesenchymal stem cells and mesenchymal tissue.

Mesenchymal stem cells can be obtained from tissues such as bone marrow, blood, such as peripheral or umbilical cord blood, skin, fat, pulp. Mesenchymal stem cells can be cultured and proliferated as solid-phase cells, for example, cells attached to a culture dish. Thus, in certain embodiments, mesenchymal stem cells can be obtained from adherent cells contained in bone marrow or blood. In certain embodiments, mesenchymal stem cells can be discriminated using the ability to form colonies as an index. Mesenchymal stem cells may be separated from a tissue 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, but are not limited to, 1 positive, CD106 positive, CD166 positive, CD31 negative, CD271 positive, and CD11b negative. In certain embodiments, the marker for human mesenchymal stem cells is PDGFRα positive. In certain embodiments, the markers for human mesenchymal stem cells are PDGFRα positive and CD45 negative.

As markers 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 the present invention is not limited to these. In certain embodiments, the marker for mouse mesenchymal stem cells is PDGFRα positive. In certain embodiments, the markers of mouse mesenchymal stem cells are PDGFRα positive and CD45 negative.

In the present disclosure, cells expressing CD45 are referred to as "CD45-positive cells". CD45 is a type I glycoprotein with a transmembrane domain, also known as a leukocyte common antigen, and is a tyrosine phosphatase expressed in many nucleated hematopoietic cells. CD45 is known as a signal transduction molecule that regulates various cellular processes such as cell proliferation, differentiation, mitosis, and carcinogenesis. The amino acid sequence of CD45 in various animal species is known.

Examples of CD45-positive cells include, but are not limited to, human leukocytes, that is, 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. CD45-positive cells can usually be cultured and proliferated as floating cells, but the present invention is not limited to this, and some CD45-positive cells can be cultured and proliferated as adherent cells. CD45-positive cells may be isolated from tissues containing CD45-positive cells, for example, bone marrow, by cell sorting (FACS, MACS, etc.) using CD45 as an index. Alternatively, CD45-positive cells induced to differentiate from pluripotent stem cells or established CD45-positive cell lines may be used.

A "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 cell in addition to these cells. .. In addition, 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.

Bone marrow-derived cells can be used as a cell population containing mesenchymal stem cells and CD45-positive cells. "Bone marrow-derived cells" means bone marrow cells (cell population existing in the bone marrow) extracted from the bone marrow to the outside of 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 taken from an animal, the bone can be crushed to recover the contents, or the cross section of the bone can be exposed and a syringe needle inserted into the bone marrow and extruded with a buffer. Is. In certain embodiments, the production methods of the present disclosure may include the step of collecting bone marrow cells from an animal.

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 to the solid phase (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 evidenced by the examples, bone marrow-derived adherent cells include mesenchymal stem cells and can include CD45-positive cells.

In the present disclosure, the solid phase means a solid support to which cells can adhere, and includes, for example, plastic or glass culture dishes, flasks, multi-well plates and the like. The solid phase may be coated, and examples of the coating substance include, but are not limited to, collagen I, laminin, vitronectin, fibronectin, poly-L-lysine, and poly-L-ornithine. In certain embodiments, the solid phase is coated with collagen I.

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, and guinea pigs. Examples of the type of bone from which bone marrow cells are collected include femur, vertebra, sternum, ilium, tibia, and the like, but are not particularly limited. In certain embodiments, bone marrow cells are harvested from the femur or vertebra.

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

In the present disclosure, "separating cells" means that the target cells in the cell population are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more. Means to operate on.

In the present disclosure, the "substance that activates a CD45-positive cell" means a substance that enhances signal transduction from a CD45-positive cell to another cell, and is a polypeptide, protein, amino acid, nucleic acid, lipid, sugar, or the like. It can be a small molecule compound or the like. The enhancement of signal transduction may be due to an increase in the signal transduction response, eg, the response in the p38MAPK signaling pathway, or may be due to an increase in the number of CD45-positive cells. That is, the substance that activates the CD45-positive cells includes a substance that promotes the proliferation of the CD45-positive cells. In addition, enhancement of signal transduction includes inducing and promoting signal transduction. As substances that activate CD45-positive cells, substances that promote the growth of CD45-positive cells, such as IL-5, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocytes, and the like. Macrophage colony stimulator (GM-CSF), lipopolysaccharide (LPS) or IL-33 can be used, but is not limited thereto. Further, 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 nucleoprotein consisting of an amino acid sequence having a total length of about 270 residues, although it varies depending on the species. IL-33 is released extracellularly in the event of cell damage or necrosis 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). The full-length IL-33 protein is also active, but 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.

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.

In the present disclosure, the species of origin of IL-33 includes, but is not limited to, humans and non-human animals such as mice, rats, monkeys, pigs, dogs, rabbits, hamsters and guinea pigs.

The full-length IL-33 protein means the IL-33 protein after being translated from the mRNA encoding IL-33 and before being processed by a protease. In the present disclosure, examples of the full-length IL-33 protein include 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). Not limited to these.

The mature IL-33 protein means a C-terminal polypeptide that has been confirmed or suggested to be produced by processing the 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 sequences (SEQ ID NO: 2) of the full-length mouse IL-33 protein, and the 109th to 266th amino acid sequences of the full-length mouse IL-33 protein. Polypeptide consisting of (SEQ ID NO: 3), polypeptide consisting of 95th to 270th amino acid sequences of full-length human IL-33 protein (SEQ ID NO: 5), 99th to 270th amino acid sequence of full-length human IL-33 protein The polypeptide consisting of (SEQ ID NO: 6), the polypeptide consisting of the 109th to 270th amino acid sequences of the full-length human IL-33 protein (SEQ ID NO: 7), and the 112th to 270th amino acids of the full-length human IL-33 protein. A polypeptide consisting of the sequence (SEQ ID NO: 8) is known. Any of these proteins can be used in the methods of the present disclosure and the like. In certain embodiments, the mature IL-33 protein has the amino acid sequence of SEQ ID NO: 3 or 8.

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

"Functionally equivalent" to a full-length or mature IL-33 protein means having the same biological activity as the protein, and "homogeneous" here means the same in qualitative evaluation. do. Biological activity of full-length or mature IL-33 proteins in the present disclosure includes, for example, activation of the p38MAPK signaling pathway, induction of Th2 cytokine and / or inflammatory cytokine secretion from immune cells, ST2 (reception 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 a) to g) below:
a) A polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein.
b) A polypeptide containing the amino acid sequence of the mature IL-33 protein and consisting of a partial amino acid sequence of the full-length IL-33 protein.
c) A polypeptide that contains or comprises a portion of the amino acid sequence of a full-length or mature IL-33 protein and is functionally equivalent to the full-length or mature IL-33 protein.
d) Substitution, deletion, insertion or addition of one or more, eg, 1-10, 1-5, 1-3 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 the amino acid sequence, which is functionally equivalent to the full-length or mature IL-33 protein.
e) Amino acid sequence of full-length or mature IL-33 protein and about 80% or more, for example 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 A polypeptide containing or consisting of an amino acid sequence having 99% or more sequence identity and functionally equivalent to a full-length or mature IL-33 protein.
f) A polypeptide encoded by a nucleic acid sequence encoding a full-length or mature IL-33 protein and a DNA that hybridizes under stringent conditions and functionally equivalent to the full-length or mature IL-33 protein, and g) full-length or About 70% or more, for example, 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% with 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 nucleic acid sequence means the degree of sequence matching between polypeptides or polynucleotides, and the optimum state (amino acid or nucleotide matching is maximum) over the region of the sequence to be compared. It is determined by comparing two sequences aligned to (the state of). The sequence identity number (%) determines the same amino acid or nucleotide present in both sequences to determine the number of matching sites, and then the number of matching sites is the amino acid or nucleotide within the sequence region to be compared. It is calculated by dividing by the total number of and multiplying the obtained numerical value by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those skilled in the art (eg, BLAST algorithm, FASTA algorithm, etc.). Sequence identity can be determined using, for example, sequence analysis software such as BLAST, FASTA.

With respect to "hybridizing under stringent conditions", the hybridization used herein is according to the usual method described in, for example, Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983). It can be carried out. Further, "stringent conditions" means, for example, 45 ° C. in a solution containing 6 × SSC (1.5 M NaCl, 0.15 M trisodium citrate is 10 × SSC) and 50% formamide. (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6) and equivalent conditions such as washing at 50 ° C. with 2 × SSC after forming a hybrid in The conditions that bring about stringency can be mentioned.

The method for obtaining the full-length or mature IL-33 protein or a polypeptide functionally equivalent thereto is not particularly limited, and for example, recombinant expression (mammalian cells, yeast, Escherichia coli, insect cells, etc.), synthesis using a cell-free system. In addition to being able to manufacture by means of, etc., it is also possible to purchase commercially available products.

Method for Producing a Cell Population Containing Mesenchymal Stem Cells The method for producing a cell population containing mesenchymal stem cells according to the present disclosure is characterized by culturing a cell population containing mesenchymal stem cells and CD45-positive cells. In this production method, mesenchymal stem cells contained in the cell population are proliferated by culturing a cell population containing mesenchymal stem cells and CD45-positive cells. 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). For example, MEM, MEMα, DMEM, GMEM, RPMI 1640, MesenCult ^TM (STEMCELL Technologies) can be mentioned.

The culturing method can be appropriately adjusted according to the purpose of use of the cells and the like. Culture conditions are not particularly limited as long as it allows the survival of mesenchymal stem cells, for example, in a typical incubator, "37 ℃, 5% O 2, 5% CO 2 ", "37 ° C. Under conditions such as "5% CO ₂ ", culture (adhesion culture) or suspension culture (for example, stirring culture or shaking culture) can be performed on a solid phase. Adhesive culture is preferably used.

The density of mesenchymal stem cells to be cultured is not particularly limited as long as it can be cultured, but for example, 1 to about 1 × 10 ¹⁰ cells / mL, 10 to about 1 × 10 ⁹ cells / mL, about 1 × It can 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 densities of CD45-positive cells to be cultured are, for example, about 1 × 10 ² cells to about 1 × 10 ¹⁰ cells / mL, about 1 × 10 ³ cells to about 1 × 10 ⁹ cells / mL, and about 1 × 10 ³ cells to about. It can be ^{1x10 8} pieces / mL, about 1x10 ⁴ pieces to about ^{1x10 8} pieces / mL, about 1x10 ⁴ pieces to about ^{1x10 7} pieces / 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 long enough to form colonies of cell populations containing mesenchymal stem cells, and may be several days to several months, for example, 3 days to 3 months, 4 days to 1 month. Between, 5 days to 3 weeks or 7 days to 14 days.

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

In certain embodiments, a cell population containing 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.

Cell populations containing mesenchymal stem cells and CD45 positive cells may be cultured in the presence of Notch activators. Examples of the Notch activator include delta-like 1, delta-like 3, delta-like 4, Jagged 1, Jagged 2, Dlk1 / Pref 1, Contactin 1, Contactin 6, and CCN3 / NO, which are described in Special Table 2013-518588. MAGP1 and MAGP2; Notch1 and Notch2 agonists described in Table 2017-516486; PDGF receptor kinase inhibitors described in Table 2009-502962 (eg, AG-370 or AG-1296 (6,7-dimethoxy-3)). -Phenyl kinaselin)), K ⁺ and H ⁺ ionophore (eg, Nigericin-Na), inhibitor of actin polymerization (eg, Cytokarasan D), inhibitor of sodium pump (Na ⁺ / K ⁺ ATPase) ( For example, vavine), inhibitors of oxidative phosphorylation by mitochondria (eg, FCCP (carbonylcyanide-4- (trifluoromethoxy) -phenylhydrazone), and c-Jun N-terminal kinase (JNK) inhibitors (eg, eg). SP600125), but is not limited to these. These Notch activators can be appropriately selected and used. These Notch activators are substances that activate CD45-positive cells, such as IL-. It may be used together with 33.

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

In certain embodiments, the medium is free of differentiation inducers. In other words, the medium is a medium used for cell proliferation or maintenance, not for differentiation purposes. In the present disclosure, the differentiation-inducing agent means a substance that induces differentiation into a specific cell type, and in particular, a substance that induces differentiation into a mesenchymal cell type. Examples of differentiation-inducing agents include dexamethasone, β-glycerophosphate, and 2-ascorbic acid 2-phosphate as differentiation-inducing agents for osteoblasts, and isobutylmethylxanthin (IBMX) as a differentiation-inducing agent for fat cells. ), Dexametazone, insulin, and indomethacin, and as a differentiation-inducing agent for cartilage cells, dexametazone, insulin, 2-phosphate 2-ascorbic acid, L-proline, pyruvate, transformation growth factor (TGF-b3) , Bone morphogenetic protein 2 (BMP-2), insulin-like growth factor (IGF). In addition, "not containing a differentiation inducer" means that the medium is substantially free of the differentiation inducer, that is, it is not contained at a level or amount that induces differentiation.

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

In the above production method, mesenchymal stem cells after culturing 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 separating mesenchymal stem cells, for example, one or more subcultures may be performed, and the medium after passage is a medium containing no substance that activates CD45-positive cells. May be. After completion of the culture, the cells adhering to the solid phase can be recovered as a cell population containing mesenchymal stem cells. For example, by removing the medium containing non-adherent cells, a cell population containing mesenchymal stem cells can be recovered. Further, the adherent cells may be detached from the solid phase by treatment with an enzyme such as trypsin.

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

Method for culturing mesenchymal stem cells The present disclosure also comprises a step of co-culturing mesenchymal stem cells with CD45-positive cells in the presence of a substance that activates CD45-positive cells (for example, IL-33). A method for culturing stem cells is provided. This culture method promotes the proliferation of mesenchymal stem cells. "Co-culture" means culturing cells in a state where mesenchymal stem cells and CD45-positive cells can be in cell-to-cell contact. Co-culture can be carried out according to the culture in the 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, which comprises the step of culturing CD45-positive cells in the presence of IL-33. This culture method promotes the proliferation of CD45-positive cells. This culturing method can be carried out by culturing the cell population containing CD45-positive cells in the presence of IL-33 in the same manner as in culturing the cell population containing mesenchymal stem cells and CD45-positive cells. Here, the cell population may or may not contain mesenchymal stem cells. In addition, a method for producing a cell population containing CD45-positive cells is provided, which comprises a step of proliferating CD45-positive cells by this culture method.

The method for culturing or producing CD45-positive cells is applied as a method for producing CD45-positive cells used as a medium or a medium additive for co-culturing with mesenchymal stem cells or culturing mesenchymal stem cells. It may be what is done.

Medium or Medium Additive In certain embodiments, the present disclosure provides a medium or medium additive that can be used in any of the above methods. For example, a medium or medium additive for use in culturing mesenchymal stem cells, which contains CD45-positive cells; mesenchymal, which contains CD45-positive cells and substances that activate CD45-positive cells (eg, IL-33). Medium or medium additive for use in culturing stem cells; medium or medium addition for use in co-culture of mesenchymal stem cells and CD45-positive cells containing a substance that activates CD45-positive cells (eg IL-33) Agents; media or medium additives for use in culturing CD45-positive cells containing IL-33 may be provided. In certain embodiments, the medium or medium additive is a medium or medium additive for promoting the proliferation of mesenchymal stem cells.

The medium is, for example, a medium for culturing general animal cells described above for a method for producing a cell population containing mesenchymal stem cells, to which a substance that activates CD45-positive cells and / or CD45-positive cells is added. Can be. The 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 (for example, 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 include, for example, a Notch activator. Further, for example, the medium may not contain a differentiation inducer. The medium may be provided in any form and may be provided, for example, as a ready-to-use liquid medium, a concentrate for dilution or dissolution and use, a solid or lyophilized product. The medium may be provided in any container.

The 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 containing mesenchymal stem cells, together with any component that is acceptable for culturing animal cells. obtain. The medium additive may further contain other components as long as it does not inhibit the proliferation of mesenchymal stem cells or the proliferation (activation) of CD45-positive cells, and may include, for example, a Notch activator. The media additives may be provided in any dosage form, for example solids such as granules, powders, tablets, liquids such as solutions, suspensions, emulsions, or capsules containing solids, semi-solids or liquids. Can be provided as, but is not limited to these. The medium additive may be provided in any container. The medium additive can be added to, for example, a medium for general animal cell culture described above for a method for producing a cell population containing mesenchymal stem cells.

Methods for Screening Substances That Promote the Proliferation of Mesenchymal Stem Cells In some embodiments, the present disclosure provides methods for screening substances that promote the proliferation of mesenchymal stem cells.
Specifically, in step 1), mesenchymal stem cells were co-cultured with CD45-positive cells using a control medium, and in step 2), a mesenchymal system was used in which a test substance was 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 applied to the mesenchymal stem cells. Select as a candidate for a substance that promotes growth.

Steps 1) and 2) of the screening method of the present disclosure can be carried out with reference to the above-mentioned method for producing and culturing a cell population containing mesenchymal stem cells.
The "test substance" includes all substances such as polypeptides, proteins, amino acids, nucleic acids, lipids, sugars and small molecule compounds. The test substance can typically be purified and isolated, but may be an unpurified or unisolated crude product or a living 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 for screening a substance that promotes the proliferation 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 the cells are cultured on the solid phase, the cells may be counted while they are attached to the solid phase, or the cells may be detached from the solid phase by enzyme treatment or the like and the number of cells may be counted. A method well known to those skilled in the art can be used for counting the number of cells. The "number of mesenchymal stem cells" can be determined, for example, by FACS analysis using the expression of typical surface markers of mesenchymal stem cells (for example, PDGFRα positive and CD45 negative) as an index. Alternatively, the number of mesenchymal stem cells may be counted by performing immunofluorescent staining or the like and counting the cells expressing the surface marker of the mesenchymal stem cells in a state of being attached to the solid phase. Here, "the number of cells is large" means that the number of cells is at least 3%, 5%, or 10% higher than that of the comparison target, and is preferably at least 10% higher.

In the screening method of the present disclosure, IL-33 may be used as a positive control. If the proliferation of mesenchymal stem cells is promoted in the medium supplemented with IL-33, it is ensured that the experimental system is functioning. Therefore, the screening method of the present disclosure may further include 1') a step of co-culturing mesenchymal stem cells with CD45-positive cells using a medium obtained by adding IL-33 to a control medium, and in step 3), step 2 The number of mesenchymal stem cells obtained by step 1) is larger than the number of mesenchymal stem cells obtained by step 1), and the number of mesenchymal stem cells obtained by step 1') is the number of mesenchymal stem cells obtained by step 1). When the number of mesenchymal stem cells is larger than the number obtained, the test substance can be selected as a candidate for a substance that promotes the proliferation of mesenchymal stem cells.

Thus, in certain embodiments, a method of screening for a substance that promotes mesenchymal stem cell proliferation using a positive control is provided, including the following steps:
1) A step of 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 medium obtained by adding IL-33 to a control medium.
2) A step of co-culturing mesenchymal stem cells with CD45-positive cells using a medium obtained by adding a test substance to a control medium, and
3) The number of mesenchymal stem cells obtained by step 2) is larger than the number of mesenchymal stem cells obtained by step 1), and the number of mesenchymal stem cells obtained by step 1') is higher. A step of selecting the test substance as a candidate for a substance that promotes the proliferation of mesenchymal stem cells when the number is larger than the number of mesenchymal stem cells obtained in step 1).

In some embodiments, a method of screening for a substance that promotes the growth of CD45-positive cells is provided, which comprises the following steps:
1) A step of 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 obtained by adding the test substance to the control medium, and 4) the number of CD45-positive cells obtained by step 2) is the number of CD45-positive cells obtained by step 1). When the number of CD45-positive cells obtained by step 3) is larger than the number of CD45-positive cells obtained by step 1), the test substance promotes the proliferation of CD45-positive cells. The process of selecting as a candidate for a substance.
This method can be performed according to the method of screening for substances that promote the proliferation of mesenchymal stem cells.

The present disclosure provides, for example, the following embodiments:
[1] A method for producing a cell population containing mesenchymal stem cells, which comprises 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, which comprises 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 proliferation of mesenchymal stem cells, which comprises 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 mesenchymal stem cells derived from bone marrow.
[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 a step of separating mesenchymal stem cells from a cell population containing the obtained mesenchymal stem cells.
[8] The method according to item 7, wherein the step of separating mesenchymal stem cells includes a) one or more subcultures and / or b) cell sorting targeting 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 paragraph 9 or 10, wherein the solid phase is coated.
[12] The method according to any one of paragraphs 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 one of Items 9 to 12, wherein the solid phase is coated with collagen I.
[14] The method according to any one of Items 9 to 13, wherein the cell population containing the mesenchymal stem cells obtained is a bone marrow-derived adherent cell containing the 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 paragraph 15, wherein the substance that activates CD45-positive cells is IL-33.
[17] A method for culturing mesenchymal stem cells, which comprises a step of co-culturing mesenchymal stem cells with CD45-positive cells in the presence of IL-33.
[18] A method for promoting proliferation of mesenchymal stem cells, which comprises a 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, which comprises a step of culturing CD45-positive cells in the presence of IL-33.

[20] IL-33
a) A polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein.
b) A polypeptide containing the amino acid sequence of the mature IL-33 protein and consisting of a partial amino acid sequence of the full-length IL-33 protein.
c) A polypeptide that contains or comprises a portion of the amino acid sequence of a full-length or mature IL-33 protein and is functionally equivalent to the full-length or mature IL-33 protein.
d) Substitution, deletion, insertion or addition of one or more, eg, 1-10, 1-5, 1-3 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 the amino acid sequence, which is functionally equivalent to the full-length or mature IL-33 protein.
e) Amino acid sequence of full-length or mature IL-33 protein and about 80% or more, for example 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 A polypeptide containing or consisting of an amino acid sequence having 99% or more sequence identity and functionally equivalent to a full-length or mature IL-33 protein.
f) A polypeptide encoded by a nucleic acid sequence encoding a full-length or mature IL-33 protein and a DNA that hybridizes under stringent conditions and functionally equivalent to the full-length or mature IL-33 protein, and g) full-length or About 70% or more, for example, 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% with the nucleic acid sequence encoding the mature IL-33 protein. Item 16 to above, selected from the group consisting of polypeptides encoded by a nucleic acid sequence having about 98% or more or about 99% or more sequence identity and functionally equivalent to the full-length or mature IL-33 protein. The method according to any of paragraph 19.

[21] IL-33
a) A polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein.
b) A polypeptide containing the amino acid sequence of the mature IL-33 protein and consisting of a partial amino acid sequence of the full-length IL-33 protein.
c) A polypeptide that contains or consists of a portion of the amino acid sequence of a full-length or mature IL-33 protein and activates CD45-positive cells.
d) In the amino acid sequence of the full-length or mature IL-33 protein, 1 to 10 amino acids contain or consist of an amino acid sequence substituted, deleted, inserted or added to activate CD45-positive cells. Polypeptide,
e) A polypeptide containing or consisting of an amino acid sequence having about 90% or more sequence identity with the amino acid sequence of a full-length or mature IL-33 protein that activates CD45-positive cells.
f) A polypeptide encoded by a nucleic acid sequence encoding a full-length or mature IL-33 protein and DNA that hybridizes under stringent conditions to activate CD45-positive cells, and g) a full-length or mature IL-33 protein. 16. the method of.

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

[37] A medium or medium additive for use in culturing mesenchymal stem cells containing CD45-positive cells.
[38] A medium or medium additive for promoting the proliferation of mesenchymal stem cells, which contains CD45-positive cells.
[39] The medium or culture medium additive according to claim 37 or 38, further comprising IL-33.
[40] A medium or medium additive containing IL-33 for use in co-culture of mesenchymal stem cells and CD45-positive cells.
[41] A medium or medium additive for use in culturing CD45-positive cells containing IL-33.
[42] IL-33
a) Polypeptide containing the amino acid sequence of the full-length or mature IL-33 protein or consisting of the amino acid sequence b) The amino acid sequence of the mature IL-33 protein and a part of the amino acid sequence of the full-length IL-33 protein Polypeptide consisting of c) full-length or mature IL-33 protein containing or consisting of a partial amino acid sequence, or consisting of the amino acid sequence and functionally equivalent to full-length or mature IL-33 protein d) full-length or mature An amino acid sequence in which one or more, for example, 1-10, 1-5, 1-3 or 1 or 2 amino acids have been substituted, deleted, inserted or added in the amino acid sequence of the IL-33 protein. Polypeptide containing or consisting of the amino acid sequence and functionally equivalent to the full-length or mature IL-33 protein e) Approximately 80% or more, eg, about 85% or more, about the amino acid sequence of the full-length or mature IL-33 protein. Contains or consists of an amino acid sequence having 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, and is full-length or mature. Polypeptide functionally equivalent to IL-33 protein f) Encoded by DNA that hybridizes to a nucleic acid sequence encoding a full-length or mature IL-33 protein under stringent conditions and functions with a full-length or mature IL-33 protein Approximately 70% or more, eg, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 70% or more with a nucleic acid sequence encoding a full-length or mature IL-33 protein. A polypeptide encoded by a nucleic acid sequence having 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity and functionally equivalent to a full-length or mature IL-33 protein. The medium or medium additive according to any one of Items 39 to 41, selected from the group consisting of.

[43] IL-33
a) A polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein.
b) A polypeptide containing the amino acid sequence of the mature IL-33 protein and consisting of a partial amino acid sequence of the full-length IL-33 protein.
c) A polypeptide that contains or consists of a portion of the amino acid sequence of a full-length or mature IL-33 protein and activates CD45-positive cells.
d) In the amino acid sequence of the full-length or mature IL-33 protein, 1 to 10 amino acids contain or consist of an amino acid sequence substituted, deleted, inserted or added to activate CD45-positive cells. Polypeptide,
e) A polypeptide containing or consisting of an amino acid sequence having about 90% or more sequence identity with the amino acid sequence of a full-length or mature IL-33 protein that activates CD45-positive cells.
f) A polypeptide encoded by a nucleic acid sequence encoding a full-length or mature IL-33 protein and DNA that hybridizes under stringent conditions to activate CD45-positive cells, and g) a full-length or mature IL-33 protein. 39-42, which is selected from the group consisting of polypeptides that are encoded by a nucleic acid sequence having about 90% or more sequence identity with the encoding nucleic acid sequence and activate CD45-positive cells. Nucleic acid or media additive.

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

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

[61] A method for screening a substance that promotes the proliferation of CD45-positive cells, which comprises the following steps:
1) A step of culturing CD45-positive cells using a control medium,
2) A step of culturing CD45-positive cells using a medium in which the medium additive according to any one of Items 39 to 57 is added to a control medium.
3) A step of culturing CD45-positive cells using a medium obtained by adding a test substance to a control medium, and
4) The number of CD45-positive cells obtained by step 2) is larger than the number of CD45-positive cells obtained by step 1), and the number of CD45-positive cells obtained by step 3) is the number of CD45-positive cells obtained by step 1). A step of selecting the test substance as a candidate for a substance that promotes the proliferation of CD45-positive cells when the number of CD45-positive cells is larger than the number obtained.
[62] When the number of CD45-positive cells obtained in step 3) is at least 10% higher than the number of CD45-positive cells obtained in step 1), the test substance is a substance that promotes the proliferation of CD45-positive cells. The screening method according to paragraph 61, which is selected as a candidate for.

Methods for Producing Bone Marrow-Derived Adhesive Cells Containing Mesenchymal Stem Cells In some embodiments, the present disclosure comprises culturing bone marrow-derived cells on a solid phase in the presence of IL-33. A method for producing derived adherent cells is provided.

In this method, bone marrow-derived adherent cells can be efficiently obtained. Bone marrow-derived adherent cells include mesenchymal stem cells and adherent cells other than mesenchymal stem cells, but by solid-phase culture in the presence of IL-33, other than mesenchymal stem cells and mesenchymal stem cells Adhesive cells, both proliferative. According to this method, adherent cells containing mesenchymal stem cells can be obtained from a small amount of myeloid tissue with high efficiency. In addition, since adherent cells containing many mesenchymal stem cells are obtained at the stage of primary culture, it is expected that the target number of cells will be achieved in a short culture period and the decrease in mesenchymal stem cell capacity due to culture will be avoided. Will be done.

In this method, bone marrow-derived cells may be cultured in a medium containing IL-33, or IL-33 may be added to the medium in which 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). For example, MEM, MEMα, DMEM, GMEM, RPMI 1640, MesenCult ^TM (STEMCELL Technologies) can be mentioned. The medium may further contain other components as long as it does 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 culture conditions and period can be appropriately adjusted according to the purpose of use of the cells and the like. Culture conditions are not particularly limited as long as it allows the survival of bone marrow-derived adherent cells or mesenchymal stem cells, for example, in a typical incubator, "37 ℃, 5% O 2, 5% CO _It can be carried out under conditions such as "2", "37 ° C., 5% CO _2". The density of bone marrow-derived cells to be cultured can be, for example, about 1 × 10 ⁴ cells to about 1 × 10 ⁷ cells / 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 for the formation of colonies of bone marrow-derived adherent cells including mesenchymal stem cells, and may be several days to several months, for example, 3 days to 3 months, 4 days. It can be ~ 1 month, 5 days ~ 3 weeks or 7 ~ 14 days.

After completion of the culture, the cells adhering to the solid phase can be recovered as bone marrow-derived adherent cells containing mesenchymal stem cells. For example, bone marrow-derived adherent cells containing mesenchymal stem cells can be recovered by removing the medium containing non-adherent cells. Furthermore, adherent cells may be separated from the solid phase by enzymatic treatment such as trypsin.

In the above production method, mesenchymal stem cells after culturing may be separated. Separation of mesenchymal stem cells may be performed by, for example, 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 using, for example, an antibody against a surface marker of mesenchymal stem cells. By culturing the obtained cell population containing mesenchymal stem cells in the presence of a drug that suppresses the survival or proliferation of other cells, for example, CD45-positive cells (eg, Mesenpure (STEMCELL Technologies)), the mesenchymal system Stem cells may be separated (purified).

Methods for Culturing Bone Marrow-Derived Adhesive Cells Containing Mesenchymal Stem Cells In some embodiments, the present disclosure comprises culturing bone marrow-derived cells on a solid phase in the presence of IL-33, a bone marrow containing mesenchymal stem cells. A method for culturing derived adherent cells is provided. This culturing method can be carried out according to the above-mentioned method for producing bone marrow-derived adherent cells containing mesenchymal stem cells. 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 mesenchymal stem cells contained in bone marrow-derived adherent cells and adherent cells other than mesenchymal stem cells can be promoted together.

Medium for use in culturing bone marrow-derived adherent cells containing mesenchymal stem cells In some embodiments, the present disclosure of bone marrow-derived adherent cells containing mesenchymal stem cells, characterized by containing IL-33. A medium for use in culturing is provided. The medium may be, for example, a medium obtained by adding IL-33 to a general animal cell culture medium described above for a method for producing bone marrow-derived adherent cells containing mesenchymal stem cells.

The composition of the components of the medium other than IL-33 is not particularly limited as long as the medium can be used for culturing animal cells (for example, mesenchymal stem cells). The medium preferably does not contain components that inhibit the growth of bone marrow-derived adherent cells or mesenchymal stem cells. The medium may be provided in any form and may be provided, for example, as a ready-to-use liquid medium, a concentrate for dilution or dissolution and use, a solid or lyophilized product. The medium may be provided in any container.

Medium Additives for Use in Culturing Bone Marrow-Derived Adhesive Cells Containing Mesenchymal Stem Cells In some embodiments, the present disclosure is characterized by containing IL-33, bone marrow-derived adherent cells containing mesenchymal stem cells. A medium additive for use in cell culture is provided.

The medium additive may contain IL-33 with any component that is acceptable for culturing animal cells. The medium additive preferably does not contain a component that inhibits the growth of bone marrow-derived adherent cells or mesenchymal stem cells. The media additives may be provided in any dosage form, for example solids such as granules, powders, tablets, liquids such as solutions, suspensions, emulsions, or capsules containing solids, semi-solids or liquids. Can be provided as, but is not limited to these. The medium additive may be provided in any container. The medium additive is added to the medium for general animal cell culture described above for the method for producing bone marrow-derived adherent cells containing mesenchymal stem cells, so that the bone marrow-derived adherent cells containing mesenchymal stem cells are attached. It can be used for culturing sex cells.

Methods for Screening Substances That Promote the Proliferation of Bone Marrow-Derived Adhesive Cells or Mesenchymal Stem Cells In some embodiments, the present disclosure comprises substances that promote the proliferation of bone marrow-derived adherent cells, including mesenchymal stem cells. Provides a screening method for:
1) A step of 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) The steps of counting the number of adherent cells obtained by steps 1) to 3), and 5) the number of adherent cells obtained by step 2) is the number of adherent cells obtained by step 1). When the number of adherent cells is larger than the number and the number of adherent cells obtained by step 3) is larger than the number of adherent cells obtained by step 1), the test substance is applied to the bone marrow containing mesenchymal stem cells. Origin The step of selecting as a candidate for a substance that promotes the growth of adherent cells.

In another aspect, the disclosure provides a method of screening for substances that promote mesenchymal stem cell proliferation, including the following steps:
1) A step of 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) The steps of counting the number of mesenchymal stem cells contained in the adherent cells obtained in steps 1) to 3), and 5) the mesenchymal stem cells contained in the adherent cells obtained in steps 2). The number of mesenchymal stem cells contained in the adherent cells obtained in step 1) is larger 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 the number of mesenchymal stem cells. ), When the number of mesenchymal stem cells contained in the adherent cells is larger than the number of mesenchymal stem cells, the test substance is selected as a candidate for a substance that promotes the proliferation of mesenchymal stem cells.

In general, the activity of a test substance by comparing the proliferation of bone marrow-derived adherent cells or mesenchymal stem cells in a control medium with the proliferation of bone marrow-derived adherent cells or mesenchymal stem cells in a medium supplemented with the test substance. It is possible to evaluate the presence or absence of. However, if the growth of bone marrow-derived adherent cells or mesenchymal stem cells is not promoted in the medium to which the test substance is added, it may be that the test substance is inactive, but it is actually active, but the cell condition, experimental conditions, etc. It is not possible to determine if there is a problem with the activity and the activity cannot be detected. This is due to the absence of a positive control substance.

Both of the above screening methods use IL-33 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 ensured that the experimental system is functioning. Therefore, the accuracy and efficiency of screening should be improved by evaluating the medium to which the test substance is added after confirming the promotion of proliferation of bone marrow-derived adherent cells or mesenchymal stem cells by IL-33, which is a positive control. Can be done.

Steps 1), 2) and 3) of both screening methods of the present disclosure can be carried out with reference to the above-mentioned method for producing or culturing bone marrow-derived adherent cells containing mesenchymal stem cells. "Control medium" means a medium that does not contain IL-33.

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

In step 4) of the method for screening a substance that promotes the proliferation of mesenchymal stem cells of the present disclosure, "the number of mesenchymal stem cells contained in adherent cells" is, for example, a representative surface marker of mesenchymal stem cells. It can be determined by FACS analysis or the like using the expression (for example, PDGFRα positive and CD45 negative) as an index. Alternatively, the number of mesenchymal stem cells may be counted by performing immunofluorescent staining or the like and counting the cells expressing the surface marker of the mesenchymal stem cells in a state of being attached to the solid phase.

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% higher than that of the comparison target, and is at least 10% higher. Is preferable.

The present disclosure provides, for example, the following embodiments:
(1) A medium additive for use in culturing bone marrow-derived adherent cells containing mesenchymal stem cells containing IL-33.
(2) A medium containing IL-33 for use in culturing bone marrow-derived adherent cells containing mesenchymal stem cells.
(3) IL-33
a) Polypeptide containing the amino acid sequence of the full-length or mature IL-33 protein b) Polypeptide containing or consisting of a partial amino acid sequence of the full-length or mature IL-33 protein c) Mature IL-33 protein A polypeptide containing the amino acid sequence of the above and consisting of a partial amino acid sequence of the full-length IL-33 protein d) One or more in the amino acid sequence of the full-length or mature IL-33 protein, for example, 1 to 10 or 1 Polypeptides containing, deleted, inserted or added amino acid sequences of 5, 1-3 or 1 or 2 amino acids e) Full-length or mature IL-33 protein amino acids Amino acid sequence having about 80% or more, for example, 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 with the sequence. F) Full-length or mature Polypeptide encoding a nucleic acid sequence encoding an IL-33 protein and a polypeptide encoded by DNA that hybridizes under stringent conditions, and g) Full-length or mature About 70% or more, for example, 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 with the nucleic acid sequence encoding the IL-33 protein. , Which is 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 has an activity of promoting the proliferation of bone marrow-derived adherent cells or mesenchymal stem cells. The medium additive according to item 1 or the medium according to item 2, which is a protein having.
(4) IL-33
a) Polypeptide containing the amino acid sequence of the full-length or mature IL-33 protein b) Polypeptide containing or consisting of a partial amino acid sequence of the full-length or mature IL-33 protein c) Mature IL-33 protein D) One or more amino acids are substituted, deleted, inserted or inserted in the amino acid sequence of the full-length or mature IL-33 protein. A polypeptide containing an added amino acid sequence or consisting of the amino acid sequence e) Contains an amino acid sequence having approximately 90% or more sequence identity with the amino acid sequence of a full-length or mature IL-33 protein, or the amino acid sequence. A polypeptide consisting of f) a polypeptide encoded by a DNA that hybridizes with a nucleic acid sequence encoding a full-length or mature IL-33 protein under stringent conditions, and g) a nucleic acid encoding a full-length or mature IL-33 protein. A polypeptide selected from the group consisting of polypeptides encoded by a nucleic acid sequence having about 90% or more sequence identity with the sequence and having 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 items 1 to 3.
(5) The medium additive or medium according to any one of items 1 to 4, wherein IL-33 is a polypeptide containing the amino acid sequence of the 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 medium additive or medium according to any one of Items 3 to 6, wherein the amino acid sequence of the full-length or mature IL-33 protein is any of the amino acid sequences of SEQ ID NOs: 1 to 8.
(8) The medium additive or medium according to any one of Items 3 to 7, wherein the nucleic acid sequence encoding the full-length or mature IL-33 protein is the nucleic acid sequence of any of SEQ ID NOs: 9 to 16.
(9) The medium additive or medium according to any one of Items 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 medium additive or medium according to any one of Items 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 one 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 one of Items 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 one of Items 2 to 12, wherein the concentration of IL-33 is about 0.01 to 100 ng / mL.
(14) The medium according to any one of Items 2 to 13, wherein the concentration of IL-33 is about 0.1 to 10 ng / mL.
(15) The medium according to any one of Items 2 to 14, wherein the concentration of IL-33 is about 0.5 to 5 ng / mL.
(16) The medium or medium additive according to any one of items 1 to 15 for proliferating mesenchymal stem cells.

(17) A method for producing bone marrow-derived adherent cells containing mesenchymal stem cells, which comprises a step of culturing bone marrow-derived cells on a solid phase in the medium according to any one of Items 2 to 16.
(18) Mesenchymal stem cells, which comprises a step of culturing bone marrow-derived cells on a solid phase in the medium according to any one of Items 2 to 16 in order to promote the proliferation of mesenchymal stem cells. A method for producing bone marrow-derived adherent cells including.
(19) A method for promoting the proliferation of mesenchymal stem cells, which comprises a step of culturing bone marrow-derived cells on a solid phase in the medium according to any one of Items 2 to 16.
(20) The method according to any one of Items 17 to 19, further comprising a step of recovering cells adhering to the solid phase after culturing as bone marrow-derived adherent cells containing mesenchymal stem cells.
(21) 1) For the adherent cells obtained by the step of culturing the bone marrow-derived cells on a solid phase in the medium according to any one of Items 2 to 16 and 2) Step 1). A method for producing mesenchymal stem cells, which comprises a step of performing one or more subcultures and / or b) cell sorting targeting mesenchymal stem cells to separate the mesenchymal stem cells.
(22) A method for culturing bone marrow-derived adherent cells including mesenchymal stem cells, which comprises a step of culturing bone marrow-derived cells on a solid phase in the medium according to any one 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 one of 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 one 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, which comprises the following steps:
1) A step of 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 to which the medium additive according to any one of Items 1 and 3 to 11 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) The steps of counting the number of adherent cells obtained by steps 1) to 3), and 5) the number of adherent cells obtained by step 2) is the number of adherent cells obtained by step 1). When the number of adherent cells is larger than the number and the number of adherent cells obtained by step 3) is larger than the number of adherent cells obtained by step 1), the test substance is applied to the bone marrow containing mesenchymal stem cells. Origin The step of selecting as a candidate for a substance that promotes the growth of 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 adhered to the bone marrow containing mesenchymal stem cells. 26. The screening method according to paragraph 26, which 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 proliferation of mesenchymal stem cells, which comprises the following steps:
1) A step of 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 to which the medium additive according to any one of Items 1 and 3 to 11 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) The steps of counting the number of mesenchymal stem cells contained in the adherent cells obtained in steps 1) to 3), and 5) the mesenchymal stem cells contained in the adherent cells obtained in steps 2). The number of mesenchymal stem cells contained in the adherent cells obtained in step 1) is larger 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 the number of mesenchymal stem cells. ), When the number of mesenchymal stem cells contained in the adherent cells is larger than the number of mesenchymal stem cells, the test substance is selected as a candidate for a substance that promotes the proliferation of mesenchymal stem cells.
(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). 28. The screening method according to Item 28, wherein the test substance is selected as a candidate for a substance that promotes the proliferation of mesenchymal stem cells.

All references cited in this disclosure are part of this disclosure by explicit source.
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. In addition, the examples below are all non-limiting examples and are provided solely to illustrate the invention.

Example 1
Promotion of mesenchymal stem cell proliferation with IL-33-containing medium (1) Materials and methods Femurs and vertebrae were excised from C57BL / 6 mice (6-7 weeks old, female) and other tissues adhering to the surroundings were removed. After that, the bone was finely crushed in a dairy pot, and PBS (containing 2% FBS) was added to collect the cells. Then, it was passed through a 40 μm nylon mesh to remove cell mass and muscle tissue. The cells were centrifuged at 300 g for 5 minutes, the supernatant was discarded, the precipitated cells were suspended in an RBC Lysis buffer (Biolegend), and the cells were reacted at room temperature for 5 minutes to lyse (hemolyze) erythrocytes. After stopping the reaction by adding an equal amount of PBS (containing 2% FBS) to RBC Lysis buffer, the mixture was centrifuged 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 medium A or B below and seeded on a collagen I-coated plastic 6-well plate (Corning Inc., Cat No. 356400) at ^{a density of 1 × 10 6 cells / well.} , Cultured in 3 mL of medium at 37 ° C., 5% O ₂ , 5% CO ₂ for 10 days (once on the 4th or 5th day of culture, replaced with fresh medium of the same composition).

Medium A (control): MEMα (Gibco) (15% FBS, 1% penicillin-containing streptomycin; mature mouse IL- added to L-Glutamine to a final concentration of 4 mM and added to medium B below. 33 The same volume of PBS as the protein solution was added)
-Medium B (containing IL-33): MEMα (Gibco) (15% FBS, containing 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 after the start of culturing, the medium was removed, and the cells adhering to the plate were stained with Diff-Quik (Sysmex) and observed.

(2) Results For bone marrow-derived cells collected from either the femur or vertebra, the number of cells obtained as colonies on the plate was significantly increased by culturing in a medium containing IL-33 (Fig. 1). ). Further, the same results were obtained when the experiment was carried out under the same method and conditions as 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 in which the H2B-GFP fusion protein gene (cDNA) was knocked in downstream of the PDGFRα promoter (Hamilton TG, Mol Cell Biol. 2003) After cutting out the femoral bone and vertebral bone from Jun; 23 (11): 4013-25) and removing other tissues adhering to the surroundings, the bone was crushed into small pieces in a dairy pot and PBS (containing 2% FBS) was added. The cells were collected. Then, it was passed through a 40 μm nylon mesh to remove cell mass and muscle tissue. The cells were centrifuged at 300 g for 5 minutes, the supernatant was discarded, the precipitated cells were suspended in an RBC Lysis buffer (Biolegend), and the cells were reacted at room temperature for 5 minutes to lyse (hemolyze) erythrocytes. After stopping the hemolytic reaction by adding an equal amount of PBS (containing 2% FBS) to RBC Lysis buffer, the mixture was centrifuged 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 medium C (control) or medium D (containing IL-33) below and 1 × 10 on a plastic 6-well plate (Corning Inc., Cat No. 356400) coated with collagen I. ^{Seed at a density of 6} cells / well and cultured in 3 mL of medium _{under the conditions of 37 ° C., 5% O 2} , 5% CO ₂ for 8 days (once on the 4th or 5th day of culture, fresh of the same composition. Replaced with medium).

Medium C (control): MEMα (Gibco) (15% FBS, 1% penicillin-containing streptomycin; mature mouse IL- added to L-Glutamine to a final concentration of 4 mM and added to medium D below. 33 The same volume of PBS as the protein solution was added)
-Medium D (containing IL-33): MEMα (Gibco) (15% FBS, containing 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 after the start of culture, the cells attached to the plate were peeled off by trypsin treatment and collected, and the expression of PDGFRα, which is a typical positive marker of mesenchymal stem cells, and CD45, which is a typical negative marker, was expressed by FACS. Analyzed.

(2) Results For bone marrow-derived cells collected from either the femur or vertebra, the number of adherent cells forming colonies on the plate was significantly increased by culturing in a medium containing IL-33 (). Figure 2). We also examined the number of cells by the expression pattern of surface markers, PDGFR [alpha] positive CD45-negative (PDGFR [alpha] ⁺ CD45 ^-) and cells, PDGFR [alpha] negative CD45-positive (PDGFR [alpha] ^- CD45 ⁺⁾ cells was significantly increased (FIG. 3 and 4). The culture in IL-33 the presence of a representative marker expression pattern of mesenchymal stem cells "PDGFR [alpha] ⁺ CD45 ^-" because it adherent cells is increasing remarkably, this experimental result, IL-33 It is considered that this action 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-Pdgfra tm11 (EGFP) Sor} (Jackson Laboratory, 007669) was used as a 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 Bioservices. Mice were bred in an animal breeding room maintained at 12 hours each in light and dark using solid feed under automatic water supply.

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

Culture of bone marrow-derived cells Bone marrow-derived cells were prepared in a collagen-coated 6-well plate (Corning Inc.) with 15% FBS, 1 × GlutaMAX ^TM Supplement (Gibco, Cat No. 35050061), and 1 × MEM non-essential amino acid solution (Nacalai Tesque). The cells were cultured in an αMEM (3 mL / well) supplemented with 10, 10 mM HEPES buffer (Nacalai Tesque), 1 × monothioglycerol solution (Wako), and 1% penicillin-streptomycin mixed solution using a low oxygen incubator. When whole bone marrow-derived cells were cultured without fractionation, they were cultured at 5 × 10 ⁵ cells / well. When mesenchymal stromal cells (MSCs) were fractionated from bone marrow-derived cells using FACS, PDGFRα ⁺ cells were fractionated at 800 cells / well. In coculture experiments, further bone marrow-derived cells of 5 × 10 ⁵ cells / well, CD45 ⁺ cells 4.75 × 10 ⁵ cells / well, CD45 ^- Shi collected cells at 2.5 × 10 ³ cells / well , Each was co-cultured with MSC. ^{When culturing using Transwell (Corning Inc.), 800 PDGFRα +} cells separated on a collagen-coated 6-well plate were seeded, and 5 × 10 ⁵ bone marrow-derived cells were seeded on Transwell. It was 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 equal amount of DMSO (dimethyl sulfoxide) was added to the control. A p38MAPK inhibitor (SB203580, AdipoGen) and an NK-κB inhibitor (BAY-11-7082, Cayman Chemical) were added at a final concentration of 5 μM, and an equal amount of DMSO was added to the control. The medium was changed every 4 days. The cells that had not been sorted were cultured for 10 days, and the cells sorted by FACS were cultured for 12 days, and the number of colonies was analyzed by analysis with FACS or staining with Diff-Quick solution (Sysmex).

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

Example 3-1: IL-33 enhances the colonization activity of bone marrow-derived MSC Bone marrow-derived cells prepared from PDGFRαGFP knock-in mouse femur are seeded in each well of a 6-well plate ^{with 5 × 10 5 cells each, and 3 mL of medium is used.} In the middle, the cells were cultured in IL-33-added medium for 10 days and analyzed by flow cytometry (FACS). As a result, a significant increase in bone marrow-derived cells due to IL-33 addition was confirmed (Fig. 5a). Since it is known that MSCs express PDGFRα and not CD45, which is a marker of blood cell lineage, PDGFRα ⁺ CD45 ⁻ cells were used as the MSC fraction in this example. FACS revealed that IL-33 significantly increased bone marrow-derived MSC (Fig. 5b). At the same time, IL-33 ^{significantly increased not only MSCs but also CD45 +} cells (Fig. 5c), suggesting that IL-33 may act on multiple cell populations in the bone marrow.

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

Example 3-2: IL-33 enhances CFU-F activity of bone marrow-derived MSCs through cell-cell contact between ^{CD45 + cells and MSCs.} It was investigated whether -33 acts directly on bone marrow-derived MSCs to enhance their CFU-F activity or to mediate signals from other cells. ^{MSCs (PDGFRα +} cells) were collected by FACS from bone marrow-derived cells prepared from PDGFRαGFP knock-in mouse femur and cultured in IL-33-added medium. However, under the culture conditions of MSC alone, IL-33 did not change the CFU-F activity (Fig. 6). This suggests that the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 may be indirectly regulated by cells other than MSCs present in the bone marrow.

It was investigated whether the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 depended on cell-cell contact between MSCs and other bone marrow-derived cells. Transwell was used to create culture conditions in which bone marrow-derived cells and bone marrow-derived MSCs share a medium but cell-to-cell contact does not occur, and the CFU-F activity of bone marrow-derived MSCs was investigated. When bone marrow-derived MSCs were co-cultured with bone marrow-derived cells, IL-33 increased CFU-F activity, but under conditions (Transwell) in which cell-cell contact between bone marrow-derived MSCs and bone marrow-derived cells was suppressed, IL. No change was observed in CFU-F activity by -33, indicating that cell-cell contact between MSC and other cells other than MSC is essential for enhancing CFU-F activity of bone marrow-derived MSC by IL-33. (Fig. 7a). Since it was revealed that culturing bone marrow-derived cells in the presence of IL-33 promotes the secretion of CCL2, CCL22, and osteopontin as humoral factors (data not disclosed), these substances were placed in the medium. Bone marrow-derived cells were cultured after the addition, but the CFU-F activity did not change with the addition of any substance (data not disclosed).

Furthermore, it was investigated which cells existing in the bone marrow contacted to enhance the CFU-F activity of bone marrow-derived MSCs. ^{Since IL-33 increased CD45-positive (CD45 +} ) cells in bone marrow-derived cells in Example 3-1 (Fig. 5c), it was predicted that cell-cell contact with CD45-positive cells might be necessary. CD45-positive cells and CD-negative (CD45 ^-) were collected by a cell each FACS min, were co-cultured with bone marrow-derived MSC in the presence IL-33. CD45 ^- cells ^{contained PDGFRα +} cells, but in negligible amounts. Enhancement of CFU-F activity of bone marrow-derived MSC can be confirmed only by co-culture with CD45 ⁺ cells, and enhancement of CFU-F activity of bone marrow-derived MSC by IL-33 is induced through cell-cell contact with ^{CD45 + cells.} It was shown to be done (Fig. 7b).

Example 3-3: The enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 is dependent on the MyD88 signal pathway. The molecular mechanism of enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 was investigated in more detail. When IL-33 binds to the ST2 / IL-1 receptor accessory protein (IL-1RAP) complex, which is a cell membrane receptor, it binds to this receptor as an adapter protein, Myeloid differentiation primary response 88 (MyD88). ) Is induced, and MyD88 is known to regulate the activity of the target gene through activation of NF-κB, p38, ERK and JNK. Therefore, in order to investigate the effect of the MyD88 signaling pathway on the enhancement of CFU-F activity of bone marrow-derived MSC by IL-33, bone marrow-derived cells prepared from the femur of MyD88 knockout (KO) mouse were cultured in IL-33-added medium. .. In the bone marrow-derived cells of MyD88KO mice, no change was observed in CFU-F activity due to the addition of IL-33, indicating that the MyD88 signaling pathway is essential for enhancing CFU-F activity of bone marrow-derived MSCs by IL-33. (Fig. 8a). Furthermore, in the bone marrow-derived cells of MyD88KO mice, ^{no increase in CD45 +} cells by IL-33 was observed (Fig. 8b), and the enhancement of CFU-F activity of bone marrow-derived MSC by IL-33 was caused by the MyD88 signaling pathway in ^{CD45 + cells.} It was suggested that it may depend on.

Furthermore, the involvement of the p38MAPK pathway and the NF-κB pathway, which are known to work downstream of MyD88, was investigated. In bone marrow-derived cells prepared from PDGFRαGFP knock-in mouse femur, enhancement of CFU-F activity by IL-33 was observed in the DMSO-added control group and the NF-κB inhibitor (BAY-11-7082) -added group, but p38MAPK. No change in CFU-F activity by IL-33 was observed in the inhibitor (SB203580) -added group (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 signaling pathway and further mediated by the p38MAPK pathway.

^{It has been reported that CD45 +} cells activated by IL-33 in a MyD88 signaling pathway-dependent manner are important in the proliferation and maintenance of MSCs in order to investigate the molecular mechanism that induces the enhancement of CFU-F activity in bone marrow-derived MSCs. The effect of the Notch signaling pathway was investigated. 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 signaling pathway. The effect of 2-phenylglycine tert-butyl ester) on MSC growth due to the addition of IL-33 was investigated. As a result, the enhancement of CFU-F activity of bone marrow-derived MSCs by IL-33 was suppressed in the presence of DAPT (Fig. 10a). Furthermore, when bone marrow-derived cells cultured in DAPT / IL-33-added medium 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 CD45 +} cell number (Fig. 10c).

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

Claims (16)

A method for producing a cell population containing mesenchymal stem cells, which comprises a step of culturing a cell population containing mesenchymal stem cells and CD45-positive cells. The method of claim 1, wherein the cell population comprising mesenchymal stem cells and CD45 positive cells comprises bone marrow-derived mesenchymal stem cells and / or CD45 positive cells. The method according to claim 1 or 2, 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. The method of claim 3, wherein the substance that activates CD45-positive cells is IL-33. IL-33
a) A polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein.
b) A polypeptide containing the amino acid sequence of the mature IL-33 protein and consisting of a partial amino acid sequence of the full-length IL-33 protein.
c) A polypeptide that contains or consists of a portion of the amino acid sequence of a full-length or mature IL-33 protein and activates CD45-positive cells.
d) In the amino acid sequence of the full-length or mature IL-33 protein, 1 to 10 amino acids contain or consist of an amino acid sequence substituted, deleted, inserted or added to activate CD45-positive cells. Polypeptide,
e) A polypeptide containing or consisting of an amino acid sequence having about 90% or more sequence identity with the amino acid sequence of a full-length or mature IL-33 protein that activates CD45-positive cells.
f) A polypeptide encoded by a nucleic acid sequence encoding a full-length or mature IL-33 protein and DNA that hybridizes under stringent conditions to activate CD45-positive cells, and g) a full-length or mature IL-33 protein. The method of claim 4, wherein the method is selected from the group consisting of polypeptides encoded by a nucleic acid sequence having about 90% or more sequence identity with the encoding nucleic acid sequence and activating CD45-positive cells. The method of claim 5, wherein IL-33 is a polypeptide comprising or consisting of the amino acid sequence of a full-length or mature IL-33 protein. The method of claim 6, wherein IL-33 is a polypeptide comprising or consisting of any of the amino acid sequences of SEQ ID NOs: 1-8. The method according to any one of claims 1 to 7, further comprising a step of separating mesenchymal stem cells. The method of claim 8, wherein the step of separating mesenchymal stem cells comprises a) one or more subcultures and / or b) cell sorting targeting mesenchymal stem cells. A method for culturing mesenchymal stem cells, which comprises a step of co-culturing mesenchymal stem cells with CD45-positive cells in the presence of IL-33. A medium or medium additive for use in culturing mesenchymal stem cells containing CD45-positive cells. The medium or medium additive according to claim 11, further comprising IL-33. A medium or medium additive containing IL-33 for use in co-culture of mesenchymal stem cells and CD45-positive cells. Screening method for substances that promote the proliferation of mesenchymal stem cells, including the following steps:
1) A step of co-culturing mesenchymal stem cells with CD45-positive cells using a control medium,
2) A step of co-culturing mesenchymal stem cells with CD45-positive cells using a medium obtained by adding a test substance 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 used as a substance that promotes the proliferation of mesenchymal stem cells. The process of selecting as a candidate for. 1') Further including a step of co-culturing mesenchymal stem cells with CD45-positive cells using a medium obtained by adding IL-33 to a control medium, and in step 3), the mesenchymal stem cells obtained by step 2). The number 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 greater than the number of mesenchymal stem cells obtained by step 1). The screening method according to claim 14, wherein when there are many cases, the test substance is selected as a candidate for a substance that promotes the proliferation of mesenchymal stem cells. A method for screening a substance that promotes the proliferation of CD45-positive cells, including the following steps:
1) A step of 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 medium obtained by adding a test substance to a control medium, and
4) The number of CD45-positive cells obtained by step 2) is larger than the number of CD45-positive cells obtained by step 1), and the number of CD45-positive cells obtained by step 3) is the number of CD45-positive cells obtained by step 1). A step of selecting the test substance as a candidate for a substance that promotes the proliferation of CD45-positive cells when the number of CD45-positive cells is larger than the number obtained.

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