JP7304555B2 - Growth promoting composition for mesenchymal stem cells or bone marrow cells - Google Patents

Description

The present disclosure relates to compositions that promote proliferation of mesenchymal stem cells or bone marrow cells.

Anticancer drug therapy and radiation therapy are known to cause a side effect of decreasing bone marrow cells (myelosuppression). Such a decrease in bone marrow cells lowers the patient's immune function and hematopoietic function, and in some cases, makes it difficult to continue treatment or significantly worsens the patient's general condition. However, recovery from myelosuppression is limited to G-CSF preparations for neutrophils, and in many cases the condition is left to spontaneous recovery.

The inventors found that PDGFRα (platelet-derived growth factor receptor alpha)-positive cells (mesenchymal stem cells) in which HMGB1 (High mobility group box 1: high mobility group 1 protein) released from injured tissue is present in the bone marrow. are stimulated and mobilized from the bone marrow to the circulatory system, and the mobilized cells accumulate at the site of injury and induce tissue regeneration (Patent Documents 1 and 2). However, when the number of mesenchymal stem cells in the bone marrow is reduced due to some factor, tissue regeneration may be delayed or insufficient. In other words, in order for the in vivo tissue regeneration mechanism mediated by mesenchymal stem cells to function effectively, it is important that a required amount of mesenchymal stem cells be present in the bone marrow.

For these reasons, it is desired to maintain, restore, and proliferate the amount of cells in the bone marrow, and the development of new drugs having such effects is awaited.

WO/2008/053892 WO/2009/133939

One of the purposes of the present disclosure is to promote proliferation of mesenchymal stem cells, particularly mesenchymal stem cells in the bone marrow, and other cells in the bone marrow.

In one aspect, the present disclosure provides a composition comprising a substance that activates CD45-positive cells for promoting proliferation or inhibiting decline of mesenchymal stem cells in a subject.
In certain aspects, the present disclosure provides compositions comprising IL-33 for promoting proliferation or inhibiting loss of bone marrow cells in a subject.
In certain aspects, the disclosure provides compositions comprising IL-33 for preventing and/or treating myelosuppression in a subject.
In certain aspects, the present disclosure provides compositions comprising CD45-positive cells for promoting proliferation or inhibiting decline of mesenchymal stem cells in a subject.

The present disclosure enables promotion of proliferation or inhibition of decline of mesenchymal stem cells, particularly mesenchymal stem cells in the bone marrow, and bone marrow cells.

Fig. 10 is a photograph showing the results of solid-phase culture of bone marrow cells collected from mouse femurs and vertebrae in a medium containing mature IL-33 protein (10 days after initiation of culture). Fig. 3 is a graph showing the number of bone marrow-derived adherent cells on day 8 of culture. FIG. 10 shows the results of FACS analysis of bone marrow-derived adherent cells on day 8 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. Fig. 3 is a graph showing the numbers of PDGFRα ⁺ CD45 ^- cells and PDGFRα ^- CD45 ⁺ cells among bone marrow-derived adherent cells on day 8 of culture. Cell count (a), PDGFRα ⁺ CD45 ⁻ cell count (b), CD45 ⁺ cell count (c) and colony assay results per well of culture plate when bone marrow cells were cultured in IL-33-supplemented medium for 10 days. (d) is shown. The results of colony assay when PDGFRα ⁺ cells were sorted and cultured in IL-33 supplemented medium are shown. Fig. 7a shows that PDGFRα ⁺ cells (Pα ⁺ ) were sorted and then co-cultured with bone marrow cells in IL-33-supplemented medium, or bone marrow cells were sorted on Transwell and cultured in IL-33-supplemented medium. and the results of colony assay are shown. FIG. 7b shows colony assay results when PDGFRα ⁺ cells and CD45 ⁻ or CD45 ⁺ cells were co-cultured in IL-33-supplemented medium. FIG. 8a shows the results of a colony assay when myD88KO mouse bone marrow cells were cultured in IL-33-supplemented medium. FIG. 8b shows the results of FACS analysis when MyD88KO mouse bone marrow cells were cultured in IL-33-supplemented medium for 10 days. The results of a colony assay when bone marrow cells were cultured in IL-33/p38 MAPK inhibitor- or IL-33/NF-κB inhibitor-supplemented media are shown. The results (a) of a colony assay when bone marrow cells were cultured in an IL-33/DAPT-added medium are shown. FIG. 3 shows the number of PDGFRα ⁺ CD45 ⁻ cells (b) and CD45 ⁺ cells (c) analyzed by FACS when bone marrow cells were cultured in IL-33/DAPT-supplemented medium for 10 days. Concentrations of IL-33 in serum and bone marrow fluid are shown. N.D.; not detected. (a) shows an outline of the experimental method when 5-FU was administered. Bone marrow cells were collected and analyzed 5, 7, 10 and 14 days after the administration of 5-FU. Time-course changes in bone marrow cell count (b) and PDGFRα ⁺ CD45 ⁻ TER119 ⁻ cell count (c) after 5-FU administration are shown. (a) shows an outline of the experimental method when 5-FU/ST2 neutralizing antibody (ST2-Ab) was administered in a mouse myelosuppression model. 1, 3 and 5 days after administration of 5-FU, ST2-Ab was administered, and after 7 days bone marrow cells were collected and analyzed. Changes in the number of bone marrow cells after administration of 5-FU/ST2-Ab (b) and colony assay results of bone marrow cells after administration of 5-FU/ST2-Ab (c) are shown. Fig. 2 shows the results of a colony assay of bone marrow cells after administration of 5-FU/IL-33 in a mouse myelosuppression model.

In this disclosure, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" shall include "18-22". Numerical ranges include all numbers between and including the endpoints. "About" in reference to a range applies to both endpoints of that range. Thus, for example, "about 20 to 30" shall include "18 to 33."

Unless otherwise specified, terms used in this disclosure have meanings as commonly understood by those of ordinary skill in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, and the like. Listed below are definitions for some terms used in this disclosure, which definitions take precedence over general understanding in this disclosure.

In this disclosure, "cell" means a single cell or a plurality of cells, depending on the context. A plurality of cells may be referred to as a "cell population", and the cell population may be a cell population consisting of one type of cell or a cell population containing multiple types of cells depending on the context.

Compositions of the present disclosure are administered to a subject to promote cell proliferation or inhibit cell loss in vivo. In the present disclosure, promoting cell proliferation includes increasing cell number in a subject, and inhibiting cell decline includes preventing and reducing cell number decline in a subject. For example. Proliferation or decrease of cells can be evaluated, for example, by cell sorting (FACS, MACS, etc.) using cell markers as indicators.

Subjects include, but are not limited to, humans and non-human animals such as mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, and the like. In some embodiments, the subject is human.

In certain embodiments, the compositions of the present disclosure promote proliferation or inhibit decline of mesenchymal stem cells. In the present disclosure, "mesenchymal stem cells" are used interchangeably with "mesenchymal stromal cells" and are sometimes abbreviated as "MSCs". Mesenchymal stem cells refer to cells that have the ability to differentiate into mesenchymal tissues such as bone, cartilage, fat, and muscle. Mesenchymal stem cells may further have the ability to differentiate into epithelial tissues and neural tissues. Mesenchymal stem cells are present in bone marrow, blood such as peripheral blood or umbilical cord blood, skin, fat, dental pulp, and the like. In certain embodiments, the mesenchymal stem cells are mesenchymal stem cells within the bone marrow.

Markers for human mesenchymal stem cells include 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- 1-positive, CD106-positive, CD166-positive, CD31-negative, CD271-positive, and CD11b-negative, all or part of which can be exemplified, but not limited to these. In certain embodiments, the human mesenchymal stem cells are PDGFRα positive. In certain embodiments, the human mesenchymal stem cells are PDGFRα positive and CD45 negative.

Mouse mesenchymal stem cell markers include 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. All or part of positive, CD11b negative can be exemplified, but not limited to these. In certain embodiments, the murine mesenchymal stem cells are PDGFRα positive. In certain embodiments, the murine mesenchymal stem cells are PDGFRα positive and CD45 negative.

In certain embodiments, mesenchymal stem cell proliferation or loss is assessed by changes in PDGFRα-positive (PDGFRα ⁺ ) cells, or PDGFRα-positive and CD45-negative (PDGFRα ⁺ CD45 ⁻ ) cells.

In some embodiments, the compositions of the present disclosure promote proliferation or inhibit depletion of bone marrow cells. In the present disclosure, "bone marrow cells" means a population of cells present within the bone marrow. Bone marrow cells include cells expressing CD45 (herein referred to as CD45-positive cells or CD45 ⁺ cells) and non-expressing cells (herein referred to as CD45-negative cells or ^CD45- cells). described) exists. CD45 is one of the negative selection markers for mesenchymal stem cells, and CD45-negative cells include mesenchymal stem cells. In certain embodiments, the compositions of the present disclosure promote proliferation or inhibit decline of CD45-positive cells and/or mesenchymal stem cells in bone marrow cells.

CD45 is a 180-240 kD single-chain type I membrane glycoprotein, also known as leukocyte common antigen, and is a tyrosine phosphatase expressed on the plasma membrane of cells of the hematopoietic lineage, excluding erythrocytes and platelets. CD45 is known as a signaling molecule that regulates various cellular processes such as cell proliferation, differentiation, cell cycle and malignant transformation. The amino acid sequences of CD45 in various animal species are known.

The compositions of the present disclosure may treat/or prevent myelosuppression by promoting proliferation or inhibiting bone marrow cell loss. Myelosuppression means a state of reduced bone marrow function, and a decrease in blood cell components such as leukocytes and neutrophils is observed. Treatment/or prevention of myelosuppression includes treatment and/or prevention of leukopenia or neutropenia. Loss of bone marrow cells or myelosuppression can result from, for example, but not limited to, drug administration, radiation therapy, and the like. Agents that can cause bone marrow cell loss or myelosuppression include, but are not limited to, so-called anti-cancer agents. Anticancer agents include, but are not limited to, antifolates, including methotrexate; purine antagonists, including cladribine, 6-mercaptopurine, nerarabine, pentostatin; pyrimidine antagonists, including capecitabine, 5-fluorouracil, gemcitabine; biological response modifiers, including alpha; DNA alkylating agents, including carmustine; DNA cross-linkers and alkylating agents, including bendamustine, ifosfamide, melphalan, dacarbazine, temozolomide, procarbazine; platinum complexes, including carboplatin, cisplatin, oxaliplatin. proteasome inhibitors, including bortezomib; spindle toxins, including taxanes (including docetaxel, paclitaxel (Abraxane)), vincristine, vinorelbine; anthracyclines (including daunorubicin, daunomycin, doxorubicin, epirubicin), camptothecins (irinotecan ), etoposide, topoisomerase inhibitors, including mitoxantrone; tyrosine kinase inhibitors, including erlotinib (Tarceva®), gefitinib, imatinib, lapatinib, pazopanib, sorafenib, sunitinib.

In the present disclosure, "a substance that activates CD45-positive cells" means a substance that enhances signal transduction from CD45-positive cells to other cells, and includes polypeptides, proteins, amino acids, nucleic acids, lipids, carbohydrates, It may be a low-molecular-weight compound or the like. Enhanced signaling may be due to increased signaling-related responses, eg, responses in the p38 MAPK signaling pathway, or increased signaling due to increased CD45-positive cells. That is, substances that activate CD45-positive cells include substances that proliferate CD45-positive cells. Enhancement of signal transduction includes initiation and promotion of signal transduction. Substances that activate CD45-positive cells include substances that proliferate CD45-positive cells, such as granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage colony-stimulating factor (GM -CSF), Lipopolysaccharide (LPS) or IL-33 may be used, but are not limited to these. In the present disclosure, "activate CD45-positive cells" and "activate CD45-positive cells" are used synonymously.

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

In the present disclosure, "IL-33" can be full-length IL-33 protein, mature IL-33 protein, or polypeptides functionally equivalent thereto.

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

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

The mature IL-33 protein means a C-terminal polypeptide that is confirmed or suggested to be produced by protease processing of the full-length IL-33 protein and has biological activity.

Examples of the mature IL-33 protein include a polypeptide consisting of the 102nd to 266th amino acid sequence of the full-length mouse IL-33 protein (SEQ ID NO: 2), and the 109th to 266th amino acid sequence of the full-length mouse IL-33 protein. (SEQ ID NO: 3), a polypeptide consisting of the 95th to 270th amino acid sequence of the full-length human IL-33 protein (SEQ ID NO: 5), the 99th to 270th amino acid sequence of the full-length human IL-33 protein (SEQ ID NO: 6), a polypeptide consisting of the 109th to 270th amino acid sequence of full-length human IL-33 protein (SEQ ID NO: 7), and 112th to 270th amino acids of 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 compositions and the like of this disclosure. In some embodiments, the mature IL-33 protein has the amino acid sequence of SEQ ID NO:3 or 8.

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

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

For example, IL-33 can be a polypeptide selected from a)-g) of:
a) a polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein;
b) a polypeptide comprising 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 comprising or consisting of a partial amino acid sequence of a full-length or mature IL-33 protein and functionally equivalent to the full-length or mature IL-33 protein;
d) 1 or more, for example 1-10, 1-5, 1-3 or 1 or 2 amino acid substitutions, deletions, insertions or additions in the amino acid sequence of the full-length or mature IL-33 protein a polypeptide comprising, or consisting of, an amino acid sequence described herein and functionally equivalent to a full-length or mature IL-33 protein;
e) about 80% or more, such as about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about a polypeptide comprising or consisting of an amino acid sequence with a sequence identity of 99% or greater and functionally equivalent to a full-length or mature IL-33 protein;
f) a polypeptide encoded by DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein and which is functionally equivalent to a full-length or mature IL-33 protein; and g) a full-length or mature IL-33 protein. about 70% or more, such as about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% A polypeptide functionally equivalent to a full-length or mature IL-33 protein encoded by a nucleic acid sequence having about 98% or greater or about 99% or greater sequence identity.

In the present disclosure, amino acid or nucleic acid sequence identity refers to the degree of sequence identity between polypeptides or polynucleotides, and is optimal (maximum amino acid or nucleotide identity) over the region of the sequences being compared. is determined by comparing two sequences that are aligned to each other. A sequence identity number (%) is determined by determining the identical amino acids or nucleotides present in both sequences to determine the number of matching sites, and then comparing this number of matching sites to the number of amino acids or nucleotides in the sequence region being compared. It is calculated by dividing by the total number and multiplying the resulting number by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those of skill in the art (eg, BLAST algorithms, FASTA algorithms, etc.). Sequence identity can be determined, for example, using sequence analysis software such as BLAST, FASTA.

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

Methods for obtaining full-length or mature IL-33 proteins or polypeptides functionally equivalent thereto are not particularly limited, and examples include recombinant expression (mammalian cells, yeast, E. coli, insect cells, etc.), synthesis using cell-free systems. etc., or it is possible to purchase a commercially available product.

In some embodiments, compositions of the present disclosure comprise CD45 positive cells. CD45-positive cells are present in bone marrow, blood, lymph nodes, etc., and can be obtained from these tissues. CD45-positive cells can usually be cultured/proliferated as floating cells, but are not limited to this, and some CD45-positive cells can be cultured/proliferated as adherent cells. CD45-positive cells may be separated from tissues containing CD45-positive cells, such as 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.

In certain embodiments, compositions of the present disclosure are substantially free of cells other than CD45 positive cells. The expression "substantially free of cells other than CD45-positive cells" means that only cells obtained by a method for obtaining CD45-positive cells and methods commonly understood by those skilled in the art are included. For example, in compositions of the present disclosure, CD45-positive cells account for 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98 or 99% or more of the cells contained in the composition . The number of cells included in the composition of the present disclosure is not limited, but is for example 1 cell to 1×10 ¹⁰ cells, 1×10 cells to 1×10 ⁹ cells, 1×10 ² cells to 1×10 ⁸ cells. , 1×10 ³ to 1×10 ⁸ cells, 1×10 ⁴ to 1×10 ⁸ cells, 1×10 ⁵ to 1×10 ⁸ cells, 1×10 ⁶ to 1×10 ⁸ cells, or 1×10 ⁵ to 1×10 ⁷ cells.

In some embodiments, compositions of the present disclosure comprise a Notch activator. Any known Notch activator can be used as appropriate.

The composition of the present disclosure is administered in an amount appropriately determined according to the age, weight, health condition, etc. of the subject. For example, in the case of IL-33, the dosage can be selected in the range of 0.0000001 mg to 1000 mg per kg body weight per administration. Alternatively, for example, the dosage can be selected in the range of 0.00001 to 100000 mg/body per patient. However, it is not limited to these doses. In the case of CD45-positive cells, the number of cells to be administered at one time is not limited, but is, for example, 1 cell to 1×10 ¹⁰ cells, 1×10 cells to 1×10 ⁹ cells, 1×10 ² cells to 1×10 ⁸ cells, 1×10 ³ cells to 1×10 ⁸ cells, 1×10 ⁴ cells to 1×10 ⁸ cells, 1×10 ⁵ cells to 1×10 ⁸ cells, 1×10 ⁶ cells to 1× 10 ⁸ cells, or 1×10 ⁵ to 1×10 ⁷ cells. Also, per 1 kg of body weight, for example, 1 cell to 1 x 10 ⁹ cells, 1 x 10 cells to 1 x 10 ⁸ cells, 1 x 10 ² cells to 1 x 10 ⁷ cells, 1 x 10 ³ cells to 1 x 10 ^{6 cells} cells, 1×10 ⁴ to 1×10 ⁶ cells, or 1×10 ⁵ to 1×10 ⁷ cells. Compositions of the present disclosure may be administered once daily or in multiple (e.g. 2, 3 or 4) divided doses for one or several days (e.g. 2, 3, 4, 5 or 6). days), at intervals of one week or several weeks (eg 2, 3, 4, 5 or 6 weeks), one month or several months (eg 2, 3, 4, 5 or 6 months). . The period during which administration is continued is not particularly limited, either for one day or several days (eg 2, 3, 4, 5 or 6 days), 1 week or several weeks (eg 2, 3, 4, 5 or 6 weeks), 1 It can be months or several months (eg 2, 3, 4, 5 or 6 months).

Compositions of the present disclosure can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Sciences, latest edition, Mark Publishing Company, Easton, U.S.A.), and in addition to active ingredients, may contain pharmaceutically acceptable carriers. . Pharmaceutically acceptable carriers include surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants. , fluidity promoters, corrigents and the like. For example, pharmaceutically acceptable carriers include light anhydrous silicic acid, lactose, microcrystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, Medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like. If the composition comprises cells, pharmaceutically acceptable carriers are isotonic solutions including water, media, saline, glucose, D-sorbitol, D-mannose, or D-mannitol, phosphate-buffered saline, and the like. It may be water (PBS) or the like. The dosage form of the composition is, but is not limited to, an oral or parenteral administration formulation such as an injection. Injections include solution injections, suspension injections, emulsion injections, and ready-to-use injections. The compositions may be frozen and may contain cryoprotectants such as DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose and the like.

The compositions of this disclosure can be administered systemically or locally. Administration methods include oral administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intrathecal administration. In certain embodiments, the compositions of this disclosure are administered intrathecally.

Illustrative embodiments of the invention are described below.

[1]
A composition comprising a substance that activates CD45-positive cells for promoting proliferation of mesenchymal stem cells in a subject.
[2]
A composition containing a substance that activates CD45-positive cells for suppressing a decrease in mesenchymal stem cells in a subject.
[3]
3. The composition of paragraph 1 or paragraph 2, wherein the mesenchymal stem cells are intramedullary mesenchymal stem cells.
[4]
4. The composition according to any one of items 1 to 3, wherein the substance that activates CD45-positive cells is IL-33.
[5]
5. The composition according to any one of items 2 to 4, wherein the decrease in mesenchymal stem cells is caused by administration of an anticancer agent.
[6]
A composition comprising IL-33 for promoting proliferation of bone marrow cells in a subject.
[7]
7. The composition of paragraph 6, which promotes proliferation of mesenchymal stem cells or CD45-positive cells in the bone marrow.
[8]
A composition comprising IL-33 for inhibiting myeloid cell depletion in a subject.
[9]
9. The composition of paragraph 8, wherein the bone marrow cell depletion is due to anticancer drug administration.
[10]
A composition comprising IL-33 for preventing and/or treating myelosuppression in a subject.
[11]
11. The composition of paragraph 10, wherein the myelosuppression results from anticancer drug administration.
[12]
IL-33 is
a) a polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein;
b) a polypeptide comprising 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 comprising or consisting of a partial amino acid sequence of a full-length or mature IL-33 protein and functionally equivalent to the full-length or mature IL-33 protein;
d) 1 or more, for example 1-10, 1-5, 1-3 or 1 or 2 amino acid substitutions, deletions, insertions or additions in the amino acid sequence of the full-length or mature IL-33 protein a polypeptide comprising, or consisting of, an amino acid sequence described herein and functionally equivalent to a full-length or mature IL-33 protein;
e) about 80% or more, such as about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about A polypeptide comprising or consisting of an amino acid sequence having a sequence identity of 99% or more and functionally equivalent to a full-length or mature IL-33 protein f) a nucleic acid encoding a full-length or mature IL-33 protein a polypeptide that is functionally equivalent to full-length or mature IL-33 protein encoded by DNA that hybridizes to the sequence under stringent conditions; and g) a nucleic acid sequence that encodes full-length or mature IL-33 protein, and about 70 % or more, such as about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more or about 99% or more of the sequence 12. The composition of any of paragraphs 4-11, selected from the group consisting of polypeptides encoded by nucleic acid sequences of identity and functionally equivalent to full-length or mature IL-33 protein.
[13]
IL-33 is
a) a polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein b) an amino acid sequence comprising an amino acid sequence of a mature IL-33 protein and a portion of the full-length IL-33 protein a polypeptide consisting of
c) a polypeptide comprising or consisting of an amino acid sequence of a full-length or partial mature IL-33 protein and which activates CD45-positive cells;
d) comprises or consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, inserted or added in the amino acid sequence of the full-length or mature IL-33 protein, and activates CD45-positive cells polypeptide,
e) a polypeptide comprising 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 and which activates CD45-positive cells;
f) a polypeptide encoded by DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein and that activates CD45-positive cells; and g) a full-length or mature IL-33 protein. 13. Any one of paragraphs 4 to 12, selected from the group consisting of a polypeptide that activates CD45 positive cells, encoded by a nucleic acid sequence having about 90% or more sequence identity with the encoding nucleic acid sequence. composition.
[14]
14. The composition of any of paragraphs 4-13, wherein IL-33 is a polypeptide comprising the amino acid sequence of full-length or mature IL-33 protein.
[15]
15. The composition according to any one of items 4 to 14, wherein IL-33 is a polypeptide consisting of the amino acid sequence of full-length or mature IL-33 protein.
[16]
16. The composition of paragraphs 4-15, wherein IL-33 is a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 1-8.
[17]
17. The composition according to any one of items 4 to 16, wherein IL-33 is a polypeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1-8.
[18]
18. The composition of any of paragraphs 4-17, wherein the nucleic acid sequence encoding IL-33 comprises the nucleic acid sequence of any of SEQ ID NOS:9-16.
[19]
19. The composition of any of paragraphs 4-18, wherein IL-33 is a polypeptide comprising the amino acid sequence of SEQ ID NO:3 or 8.
[20]
20. The composition of any one of items 4-19, wherein IL-33 is a polypeptide consisting of the amino acid sequence of SEQ ID NO:3 or 8.
[21]
21. The composition of any of paragraphs 4-20, wherein the nucleic acid sequence encoding IL-33 comprises the nucleic acid sequence of SEQ ID NO: 11 or 16.
[22]
22. The composition of any of paragraphs 4-21, wherein IL-33 is a polypeptide comprising the amino acid sequence of SEQ ID NO:3.
[23]
Item 23. The composition according to any one of items 4 to 22, wherein IL-33 is a polypeptide consisting of the amino acid sequence of SEQ ID NO:3.
[24]
24. The composition of any of paragraphs 4-23, wherein the nucleic acid sequence encoding IL-33 comprises the nucleic acid sequence of SEQ ID NO:11.

[25]
A composition comprising CD45-positive cells for promoting proliferation of mesenchymal stem cells in a subject.
[26]
A composition comprising CD45-positive cells for suppressing a decrease in mesenchymal stem cells in a subject.
[27]
27. The composition of paragraph 25 or paragraph 26, wherein the mesenchymal stem cells are intramedullary mesenchymal stem cells.
[28]
28. The composition of paragraph 26 or 27, wherein the decrease in mesenchymal stem cells is due to anticancer drug administration.
[29]
29. The composition of any of paragraphs 25-28, which is substantially free of cells other than CD45 positive cells.

[30]
A method for promoting proliferation of mesenchymal stem cells, comprising administering to a subject a composition containing a substance that activates CD45-positive cells.
[31]
A method for suppressing a decrease in mesenchymal stem cells, the method comprising administering to a subject a composition containing a substance that activates CD45-positive cells.
[32]
32. The method according to item 30 or 31, wherein the substance that activates CD45-positive cells is IL-33.
[33]
A method of promoting proliferation of bone marrow cells comprising administering to a subject a composition comprising IL-33.
[34]
A method of inhibiting bone marrow cell depletion, the method comprising administering a composition comprising IL-33 to a subject.
[35]
A method for preventing and/or treating myelosuppression comprising administering to a subject a composition comprising IL-33.

[36]
A method for promoting proliferation of mesenchymal stem cells, the method comprising administering a composition comprising CD45-positive cells to a subject.
[37]
A method for suppressing a decrease in mesenchymal stem cells, the method comprising administering a composition containing CD45-positive cells to a subject.

[38]
Use of a substance that activates CD45-positive cells for the manufacture of a medicament for promoting proliferation of mesenchymal stem cells in a subject.
[39]
Use of a substance that activates CD45-positive cells for the manufacture of a medicament for suppressing reduction of mesenchymal stem cells in a subject.
[40]
40. Use according to item 38 or 39, wherein the substance that activates CD45-positive cells is IL-33.
[41]
Use of IL-33 for the manufacture of a medicament for promoting bone marrow cell proliferation in a subject.
[42]
Use of IL-33 for the manufacture of a medicament for inhibiting bone marrow cell loss in a subject.
[43]
Use of IL-33 for the manufacture of a medicament for preventing and/or treating myelosuppression in a subject.

[44]
Use of CD45 positive cells for the manufacture of a medicament for promoting proliferation of mesenchymal stem cells in a subject.
[45]
Use of CD45-positive cells for the manufacture of a medicament for suppressing mesenchymal stem cell decline in a subject.

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

Example 1
Proliferation of Mesenchymal Stem Cells by IL-33-Containing Medium (1) Materials and Methods Femurs and vertebrae were dissected from C57BL/6 mice (6-7 weeks old, female), and other attached tissues were removed. After that, the bone was finely crushed in a mortar, and PBS (containing 2% FBS) was added to collect the cells. Then, it was passed through a 40 μm nylon mesh to remove cell clumps and muscle tissue. After centrifugation at 300 g for 5 minutes, the supernatant was discarded, and the precipitated cells were suspended in RBC Lysis buffer (Biolegend) and reacted at room temperature for 5 minutes to lyse red blood cells (hemolysis). After stopping the reaction by adding PBS (containing 2% FBS) in an amount equal to the RBC Lysis buffer, the mixture was centrifuged at 300 g for 5 minutes. The supernatant was discarded, the precipitated cells were suspended in the following medium A or B, and placed on a collagen I-coated plastic 6-well plate (Corning Inc., Cat No. 356400) at 1 x ¹⁰⁶ cells/well. The cells were seeded at a high density and cultured for 10 days under conditions of 37° C., 5% O ₂ , 5% CO ₂ (changed to fresh medium of the same composition once on the 4th or 5th day of culture).

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

(2) Results Bone marrow cells collected from both the femur and the vertebrae were cultured in a medium containing IL-33, resulting in a marked increase in the number of cells obtained as colonies on the plate (Fig. 1). . Also, similar results were obtained when the experiment was conducted in the same manner and under the same conditions as above except that a 6-well plate without collagen I coating (costar, Cat No. 3516) was used for culturing bone marrow cells. rice field.

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 and form colonies. Therefore, the experimental results in this example show that culture in the presence of IL-33 promotes 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) Jun;23(11):4013-25), the femur and vertebrae were cut out, and other attached tissues were removed. Cells were harvested. Then, it was passed through a 40 μm nylon mesh to remove cell clumps and muscle tissue. After centrifugation at 300 g for 5 minutes, the supernatant was discarded, and the precipitated cells were suspended in RBC Lysis buffer (Biolegend) and reacted at room temperature for 5 minutes to lyse red blood cells (hemolysis). After adding PBS (containing 2% FBS) in the same amount as the RBC Lysis buffer to stop the hemolytic reaction, the mixture was centrifuged at 300 g for 5 minutes. The supernatant was discarded, and the precipitated cells were suspended in the following medium C (control) or medium D (containing IL-33) and placed on a collagen I-coated plastic 6-well plate (Corning Inc., Cat No. 356400). ) at a density of 1×10 ⁶ cells/well and cultured for 8 days under conditions of 37° C., 5% O ₂ , 5% CO ₂ (once on the 4th or 5th day of culture, fresh cells of the same composition were added). medium).

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

On day 8 of culture, the cells adhering to the plate were detached and collected by trypsin treatment, and the expression of PDGFRα, a typical positive marker for mesenchymal stem cells, and CD45, a typical negative marker, was examined by FACS. Analyzed.

(2) Results Bone marrow cells collected from both the femur and vertebrae were cultured in a medium containing IL-33, resulting in a marked increase in the number of adherent cells forming colonies on the plate (Fig. 2). In addition, when the number of cells was examined according to the expression patterns of surface markers, PDGFRα ⁺ CD45 ⁻ cells and PDGFRα ⁻ CD45 ⁺ cells were significantly increased (FIGS. 3 and 4). By culturing in the presence of IL-33, the number of adherent cells of "PDGFRα ⁺ CD45 ^- ", which is a representative marker expression pattern of mesenchymal stem cells, was remarkably increased. It is thought that this indicates that the action of β-glucose stimulated the proliferation of mesenchymal stem cells.

Example 3
Materials and methods
Mouse C57BL/6jjcl (CLEA Japan) was used as a wild-type mouse in this example. B6.129S4-Pdgfra ^{tm11 (EGFP) Sor} (Jackson Laboratory, 007669) was used as a PDGFRαGFP knock-in mouse, and C57BL/6-Tg (CAG-EGFP) 1310sb/LeySopJ (Jackson Laboratory, 006567) was used as a GFP transgenic 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 housed in an animal room maintained on chow with automatic watering and 12 hours of light and darkness.

Isolation of Bone Marrow Cells Femoral bones were collected from mice, and bone marrow cells were removed using a mortar and pestle in PBS containing 2% fetal bovine serum (FBS) (PBS/2% FBS). The cell suspension was passed through a 40-μM cells strainer (Falcon) to remove aggregates such as bones. After centrifugation, hemolysis was performed with RBC lysis buffer (Biolegend) to remove erythrocytes.

Bone marrow cell culture Bone marrow cells were cultured in collagen-coated 6-well plates (Corning Inc.) in 15% FBS, 1x GlutaMAX ^™ Supplement (Gibco, Cat No. 35050061), 1x MEM non-essential amino acid solution (Nacalai Tesque), 10 mM. The cells were cultured in αMEM supplemented with HEPES buffer (Nacalai Tesque), 1× monothioglycerol solution (Wako), and 1% penicillin-streptomycin mixed solution using a hypoxic incubator. When culturing whole bone marrow cells without sorting, the cells were cultured at 5×10 ⁵ cells/well. When isolating mesenchymal stromal cells (MSCs) using FACS, PDGFRα ⁺ cells were sorted at 800/well. In the co-culture experiment, 5×10 ⁵ bone marrow cells/well, 4.75×10 ⁵ CD45 ⁺ cells/well, and 2.5×10 ³ CD45 ⁻ cells/well were sorted, co-cultured with MSCs. When culturing using Transwell (Corning Inc.), 800 PDGFRα ⁺ cells were collected on a collagen-coated 6-well plate and 5×10 ⁵ bone marrow cells were collected on Transwell and cultured. IL-33 (Biolegend) (SEQ ID NO: 3) was added at 1 ng/ml and an equal volume of PBS was added to controls. DAPT (TCI) was added at a final concentration of 1 µM or 10 µM, and an equal volume of DMSO was added to controls. A p38 MAPK inhibitor (SB203580, AdipoGen) and an NK-κB inhibitor (BAY-11-7082, Cayman Chemical) were added at a final concentration of 5 μM, and an equal volume of DMSO was added to controls. Medium was changed every 4 days. Cells that were not sorted were cultured for 10 days, and cells that were sorted by FACS were cultured for 12 days, followed by FACS analysis or staining with Diff-Quick solution (Sysmex) to analyze the number of colonies.

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

ELISA
Peripheral blood was collected from the mouse heart, and serum was collected using Capiject (Terumo Corporation) according to the manual. Samples were stored at -80°C until use. Bone marrow fluid was obtained by crushing the bones on a 0.6 ml microtube pierced with a 20 gauge needle, centrifuging (1000 g, 5 minutes) with a 1.5 ml microtube underneath, and transferring to a 1.5 ml microtube. The bone marrow fluid that could be collected in 1 was collected. Further, the bone marrow fluid was diluted 1/100 with PBS and then centrifuged again (20,000 g, 5 minutes) to collect only the supernatant and remove the cells. Samples were stored at -80°C until use. For quantification of IL-33, Mouse IL-33 DuoSet ELISA (R&D Systems) was used according to the manual and absorbance at 450 nm was measured with a TriStar 2 LB 942 Multimode Reader.

In vivo experiments When administering 5-fluorouracil (5-FU), 5-FU (Kyowa Hakko Kirin Co.) was diluted with PBS and intraperitoneally administered to mice at 150 mg/kg. For inhibition of the IL-33 signaling pathway, 50 μg/shot Ultra-LEAF ^™ Purified anti-mouse IL-33Rα (ST2) Antibody (Biolegend) was intraperitoneally administered on days 1, 3 and 5 after administration of 5-FU. Controls were intraperitoneally administered with an equivalent amount of Ultra-LEAF ^™ Purified Rat IgG1, κIsotype Ctrl Antibody (Biolegend). For administration of IL-33, mice (n=6) were administered 5-FU, and on days 1, 3, and 5 after 5-FU administration, 3 mice (n=3) were challenged with IL-33 ( Recombinant Mouse IL-33, BioLegend) 1 μg diluted in 100 μl PBS was administered intraperitoneally, and for comparison another 3 mice (n=3) received 100 μl PBS without IL-33 intraperitoneally. dosed.

Seven days after the administration of 5-FU, femoral bones were collected from each mouse, and cell culture was performed for each mouse. Specifically, after putting 1 ml of PBS (2% FBS) in a mortar, the collected femur was put into the mortar and ground. Subsequently, the pestle was washed in a mortar with 2 ml of PBS (2% FBS), and the suspension was thoroughly pipetted in the mortar, and the resulting suspension was filtered using a cellstrainer with a mesh size of 40 μm. , the filtrate was collected in a tube. Put 2 ml PBS (2% FBS) in a mortar, pipette well, filter through a cell strainer in the same manner, transfer the bones onto the cell strainer, and strain the bones and the cell strainer with 5 ml PBS (2% FBS). was washed and the filtrate was recovered. After centrifuging the filtrate stored in the tube (300 g, 4° C., 5 minutes), a hemolysis reagent (1×RBC lysis buffer) was added to the precipitate, and hemolysis was performed at room temperature for 5 minutes. Then, after adding an equal volume of PBS (2% FBS), the precipitated cells were collected by centrifugation. Collected cells were washed with 10 ml PBS (2% FBS) and counted by TC20 Automated cell counter (BioRad). 2×10 ⁶ cells were taken out, suspended in 1 ml of aMEM (2% FBS, SIGMA), and centrifuged to collect the cells. The collected cells were suspended in 3 ml of medium, and 2×10 ⁶ total cells were seeded on a plate (Collagen coated 6-well dish, Corning). Cell-seeded plates were cultured for 10 days at 37° C., 5% O ₂ , 5% CO ₂ . The number of generated colonies was counted.

Example 3-1: IL-33 enhances the colony-forming activity of bone marrow MSC Bone marrow cells prepared from femurs of PDGFRαGFP-knock-in mice were seeded in 6-well plates at 5×10 ⁵ cells and placed in IL-33-supplemented medium for 10 days. When the cells cultured in were analyzed by flow cytometry (FACS), a significant increase in bone marrow cells due to the addition of IL-33 was confirmed (Fig. 5a). Since it is known that MSCs express PDGFRα, a stem cell marker, but do not express CD45, a blood cell marker, PDGFRα ⁺ CD45 ⁻ cells were used as the MSC fraction in this example. FACS revealed that IL-33 significantly increased MSCs (Fig. 5b). At the same time, IL-33 significantly increased not only MSCs but also CD45 ⁺ cells, suggesting the possibility that IL-33 acts on multiple cell populations in the bone marrow (Fig. 5c). Since bone marrow MSCs form fibroblast-like colonies in culture, the abundance and proliferation rate of MSCs can be evaluated by measuring the colony forming unit-fibroblast (CFU-F). . Colony assay was performed after 10 days of culture in IL-33-supplemented medium, and the addition of IL-33 significantly increased the number of colonies about 7-fold (Fig. 5d), indicating that IL-33 enhances bone marrow CFU-F activity. became clear. Thus, IL-33 was shown to enhance the colony-forming activity of intramedullary MSCs.

Example 3-2: IL-33 enhances CFU-F activity of bone marrow MSCs through intercellular contact between CD45 ⁺ cells and MSCs The mechanism by which IL-33 enhances CFU-F activity of bone marrow MSCs was investigated. We investigated whether IL-33 acts directly on MSCs to enhance CFU-F activity of MSCs, or whether it is mediated by signals from other cells. MSCs (PDGFRα ⁺ cells) were sorted by FACS from bone marrow cells prepared from PDGFRαGFP knock-in mouse femurs and cultured in IL-33-added medium. However, under MSC-only culture conditions, IL-33 did not alter CFU-F activity (FIG. 6). This suggested the possibility that the enhancement of CFU-F activity in bone marrow MSCs by IL-33 is indirectly controlled by cells other than MSCs present in the bone marrow.

We examined whether the enhancement of CFU-F activity in bone marrow MSCs by IL-33 depends on cell-to-cell contact between MSCs and bone marrow cells. By using Transwell, culture conditions were created in which bone marrow cells and MSCs share a medium but cell-to-cell contact does not occur, and CFU-F activity was investigated. When MSCs and bone marrow cells were co-cultured (Co-culture), CFU-F activity was increased by IL-33. No change in activity was observed, indicating that cell-to-cell contact is essential for IL-33 to enhance CFU-F activity of MSCs (Fig. 7a). Although not shown, it was found that culture of bone marrow cells in the presence of IL-33 promotes the secretion of CCL2, CCL22, and osteopontin as humoral factors. When bone marrow cells were cultured by adding to the medium, the addition of any substance did not change the CFU-F activity.

Furthermore, it was examined which cells in the bone marrow would enhance the CFU-F activity of MSCs. Since IL-33 increases CD45-positive (CD45 ⁺ ) cells in the bone marrow (Fig. 5c), we speculated that cell-to-cell contact with CD45-positive cells might be necessary, and CD45-positive and CD45-negative (CD45 ⁻ ) cells were each sorted by FACS and co-cultured with MSCs in the presence of IL-33. CD45 ⁻ cells contained PDGFRα ⁺ cells, but in negligible amounts. Enhancement of CFU-F activity could be confirmed only in co-culture with CD45 ⁺ cells, indicating that enhancement of CFU-F activity of MSCs by IL-33 is induced by intercellular contact with CD45 ⁺ cells (Fig. 7b).

Example 3-3: Enhancement of CFU-F activity in bone marrow MSCs by IL-33 is dependent on the MyD88 signaling pathway The molecular mechanism of IL-33's enhancement of CFU-F activity in bone marrow MSCs was examined in more detail. When IL-33 binds to the cell membrane receptor, the ST2/IL-1 receptor accessory protein (IL-1RAP) complex, this receptor binds to the adapter protein Myeloid differentiation primary response 88 (MyD88). ) is induced. MyD88 is known to regulate target gene activity 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 MSCs by IL-33, bone marrow cells prepared from MyD88 knockout (KO) mouse femurs were cultured in IL-33-supplemented medium. In the bone marrow cells of MyD88KO mice, no change in CFU-F activity was observed due to the addition of IL-33, indicating that the MyD88 signaling pathway is essential for the enhancement of MSC CFU-F activity by IL-33 (Fig. 8a). Furthermore, IL-33-induced increase in CD45 ⁺ cells was not observed in myeloid cells from MyD88KO mice (Fig. 8b), suggesting that IL-33-induced enhancement of CFU-F activity depends on the MyD88 signaling pathway in CD45 ⁺ cells. It was suggested.

Furthermore, the involvement of the p38 MAPK pathway and NF-κB pathway, which are known to work downstream of MyD88, was examined. In bone marrow cells prepared from PDGFRαGFP knock-in mouse femurs, 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. No change in CFU-F activity due to IL-33 was observed in the agent (SB203580)-added group (FIG. 9). These results revealed that IL-33 enhances CFU-F activity of MSCs depending on the MyD88 signal pathway and further through the p38 MAPK pathway.

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

Example 3-4: IL-33 is involved in maintaining the homeostasis of bone marrow cells in vivo The functions and roles of IL-33 in vivo were examined. First, the concentrations of IL-33 in peripheral blood and bone marrow were measured by ELISA (Fig. 11). As a result, it was revealed that the bone marrow contains IL-33 at a significantly higher concentration than the peripheral blood, suggesting that IL-33 also functions in the living bone marrow. Therefore, using a reversible mouse myelosuppression model induced by the anticancer drug 5-FU, the effect of IL-33 on myeloid cell proliferation (recovery) was examined (Fig. 12a). On the 5th day after administration of 5-FU, the number of bone marrow cells decreased rapidly, and on the 14th day after that, it was confirmed that the number of bone marrow cells recovered to the normal level, and it was confirmed that a myelosuppression model could be prepared. (Fig. 12b). PDGFRα ⁺ CD45 ⁻ TER119 ⁻ cells, including bone marrow MSCs (Morikawa, S. et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. Journal of Experimental Medicine 206, 2483-2496, doi:10.1084 /jem.20091046 (2009)) decreased after 5-FU administration, but recovered after 10 days (Fig. 12c).

To investigate the involvement of IL-33 in recovery from myelosuppression, neutralizing antibody (ST2-Ab) against IL-33 receptor ST2 was administered on days 1, 3, and 5 after administration of 5-FU (Fig. 13a). . Seven days after 5-FU administration, the number of bone marrow cells tended to decrease in the ST2-Ab administration group compared to the control group, indicating the possibility that the IL-33 signaling pathway contributes to recovery from myelosuppression. (Fig. 13b). Furthermore, when bone marrow cells were cultured after administration of 5-FU/ST2-Ab, a decrease in CFU-F activity was shown in the ST2-Ab administration group (Fig. 13c), suggesting that IL-33 signals increased MSCs during myelosuppression. It was found to be important for recovery of function.

Furthermore, the effect of IL-33 on reversing myelosuppression was investigated. Mice (n=6) were administered 5-FU, and on days 1, 3, and 5 post-administration, 3 mice (n=3) were administered PBS containing IL-33, and for comparison 3 mice (n=3) were administered PBS without IL-33. Bone marrow cells collected 7 days after 5-FU administration were cultured and CFU-F activity was measured. The results are shown in FIG. CFU-F activity was higher in the IL-33 administration group. This result indicates that IL-33 restores MSC function during myelosuppression and is effective in treating or preventing myelosuppression.

Claims (15)

A composition comprising IL-33 for promoting proliferation of mesenchymal stem cells in a tissue in which CD45-positive cells are present in a subject. A composition containing IL-33 for suppressing the decrease of mesenchymal stem cells in a tissue in which CD45-positive cells are present in a subject. 3. The composition according to claim 1 or 2, wherein the tissue in which the CD45-positive cells are present is bone marrow, blood or lymph node. The composition according to any one of claims 1 to 3, wherein the tissue in which the CD45-positive cells are present is bone marrow . A composition comprising IL-33 for promoting proliferation of bone marrow cells in a subject. 6. The composition of claim 5, which promotes proliferation of mesenchymal stem cells or CD45-positive cells in the bone marrow. A composition comprising IL-33 for inhibiting myeloid cell depletion in a subject. A composition comprising IL-33 for preventing and/or treating myelosuppression in a subject. 9. The composition of Claim 8, wherein said myelosuppression results from anticancer drug administration. IL-33 is
a) a polypeptide comprising or consisting of an amino acid sequence of a full-length or mature IL-33 protein;
b) a polypeptide comprising 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 comprising or consisting of an amino acid sequence of a full-length or partial mature IL-33 protein and which activates CD45-positive cells;
d) comprises or consists of an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, inserted or added in the amino acid sequence of the full-length or mature IL-33 protein, and activates CD45-positive cells polypeptide,
e) a polypeptide comprising 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 and which activates CD45-positive cells;
f) a polypeptide encoded by DNA that hybridizes under stringent conditions to a nucleic acid sequence encoding a full-length or mature IL-33 protein and that activates CD45-positive cells; and g) a full-length or mature IL-33 protein. 10. The composition of any one of claims 1 to 9, selected from the group consisting of polypeptides that activate CD45-positive cells, encoded by a nucleic acid sequence having about 90% or more sequence identity with the encoding nucleic acid sequence. thing. 11. The composition of any of claims 1-10 , wherein IL-33 is a polypeptide comprising the amino acid sequence of full-length or mature IL-33 protein. 12. The composition of any of claims 1-11 , wherein IL-33 is a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 1-8. A composition comprising IL-33 and CD45 positive cells for promoting proliferation of mesenchymal stem cells in a subject. A composition comprising IL-33 and CD45 positive cells for suppressing mesenchymal stem cell depletion in a subject. 15. The composition of claim 13 or 14, wherein the mesenchymal stem cells are intramedullary mesenchymal stem cells.

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