Composition and its uses

Abstract

Provided is a composition having cell-derived physiological activity. The composition according to the present invention includes a processed product of megakaryocytes or a cultured product thereof.

Description

[Technical Field]

[0001] This invention relates to a composition and its uses. [Background technology]

[0002] As therapeutic agents for regenerative medicine, attempts are being made to develop cells that promote tissue regeneration, such as mesenchymal stem cells, as cell therapies. Furthermore, it is believed that these tissue regeneration-promoting cells accelerate tissue regeneration by releasing physiologically active proteins such as growth factors. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] Therefore, the present invention aims to provide a composition having cell-derived physiological activity. [Means for solving the problem]

[0004] To achieve the aforementioned objective, the composition of the present invention comprises a processed product of megakaryocytes or their cultures.

[0005] The cell proliferation-promoting composition of the present invention (hereinafter also referred to as the "proliferation-promoting composition") includes the above-mentioned composition of the present invention.

[0006] The fibroblast function-promoting composition of the present invention comprises the composition of the present invention described above.

[0007] The skin disorder healing promoting composition of the present invention comprises the composition of the present invention.

[0008] The keratinocyte function-promoting composition of the present invention comprises the composition of the present invention.

[0009] The hair papilla cell function-promoting composition of the present invention comprises the composition of the present invention described above.

[0010] The hair growth promoting composition of the present invention comprises the composition of the present invention described above. [Effects of the Invention]

[0011] According to the present invention, a composition having a physiological activity derived from cells can be provided.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] <Composition> As described above, the composition of the present invention comprises a processed product of megakaryocytes or their culture. The composition of the present invention is characterized by comprising a processed product of megakaryocytes or their culture, as described above, and other components and conditions are not particularly limited. According to the composition of the present invention, for example, a composition having cell-derived physiological activity can be provided. According to the composition of the present invention, for example, cell proliferation of mesenchymal cells, fibroblasts, keratinocytes, dermal papilla cells, etc. can be promoted, and it is expected that it can be suitably used to promote the healing of skin disorders such as skin ulcers, pressure sores, burns, scars, wounds, and skin aging, maintain or improve the skin barrier function, and promote hair growth.

[0014] In this invention, "megakaryocyte" refers to the largest cell present in the bone marrow in living organisms, and means a cell that releases platelets or has equivalent functions. The aforementioned cell having equivalent functions means a cell that has the ability to produce platelets. In this invention, megakaryocytes may be megakaryocytes before multinucleation (polyploidization), i.e., immature megakaryocytes or megakaryocytes in the proliferative phase, or megakaryocytes after multinucleation (multinucleated megakaryocytes). Specifically, the megakaryocyte may be, for example, a promegakaryblast, megakaryblast, promegakaryocyte, or mature megakaryocyte. The number of chromosome sets in the post-multinucleated megakaryocyte should be greater than 2 sets, and specifically, 16 to 32 sets.

[0015] The origin of the megakaryocytes is not particularly limited, but examples include humans and non-human animals. Examples of non-human animals include primates such as monkeys, gorillas, chimpanzees, and marmosets, as well as mice, rats, dogs, cats, rabbits, sheep, horses, and guinea pigs. The origin of other cells is similar.

[0016] In the present invention, megakaryocytes can be identified by cell surface markers. When the megakaryocytes are of human origin, the cell surface markers include CD41a, CD42a, and CD42b. That is, the megakaryocytes are cells that are positive for CD41a, CD42a, and CD42b. When the megakaryocytes are of human origin, the cell surface markers may be at least one selected from the group consisting of, for example, CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c.

[0017] The megakaryocytes mentioned above may be megakaryocytes isolated from living organisms, or megakaryocytes induced from cells less differentiated than megakaryocytes, such as pluripotent cells (hereinafter also referred to as "progenitor cells"). The term "cells less differentiated than megakaryocytes" means cells that have the ability to differentiate into megakaryocytes.

[0018] If the megakaryocytes are megakaryocytes isolated from a living organism, they can be isolated from bone marrow, for example, since they are present in bone marrow. In this case, the megakaryocytes may also include other cells of living origin.

[0019] If the megakaryocyte is a megakaryocyte induced from a progenitor cell, then the megakaryocyte is as described below. in vitro They can be induced by [method]. In this case, the megakaryocytes may include the progenitor cells. Examples of the progenitor cells include hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, megakaryocyte-erythroid progenitor cells (MEPs), megakaryocyte progenitor cells, etc. The progenitor cells may be isolated from bone marrow, umbilical cord blood, peripheral blood, etc., or induced from pluripotent cells such as ES cells (embryonic stem cells), induced pluripotent stem cells (iPS cells), nuclear transplantation ES cells (ntES cells), germline stem cells, somatic stem cells, embryonic tumor cells, etc.

[0020] When the megakaryocytes are megakaryocytes induced from progenitor cells, immortalized megakaryocytes are preferred. Compared to megakaryocytes induced by other megakaryocyte induction methods, for example, immortalized megakaryocytes exhibit higher homogeneity in the differentiation stage of the cells, thus suppressing variations in the component composition of the resulting processed product. The immortalized megakaryocytes are, for example, megakaryocytes induced by introducing oncogenes and Polycomb genes, or oncogenes, Polycomb genes, and apoptosis suppressor genes, into the progenitor cells, as described later.

[0021] The aforementioned "oncogenes" refer to genes capable of inducing cell carcinogenesis in living organisms, and examples include MYC family genes such as c-MYC, N-MYC, and L-MYC, SRC family genes, RAS family genes, RAF family genes, protein kinase family genes such as c-kit (CD117), PDGFR (platelet growth factor receptor), and Abl (Abelson murine leukemia viral oncogene homolog).

[0022] The aforementioned "Polycomb genes" refer to genes known to negatively regulate CDKN2a (cyclin-dependent kinase inhibitor 2A, INK4a / ARF) and function to avoid cellular senescence (see references 1-3 below). Specific examples of Polycomb genes include, for instance, BMI1 (Polycomb complex protein BMI-1, polycomb group RING finger protein 4 (PCGF4), RING finger protein 51 (RNF51)), Mel18 (Polycomb group RING finger protein 2), Ring (Ring Finger Protein) 1a / b, Phc (Polyhomeotic Homolog) 1 / 2 / 3, Cbx (Chromobox) 2 / 4 / 6 / 7 / 8, Ezh2 (Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit), Eed (Embryonic Ectoderm Development), Suz12 (SUZ12 Polycomb Repressive Complex 2 Subunit), HADC (Histone deacetylases), Dnmt (DNA (cytosine-5)-methyltransferase) 1 / 3a / 3b, etc. Reference 1: Hideyuki Oguro et al., "Control of stem cell aging by Polycomb group protein complex," Regenerative Medicine, 2007, Vol. 6, No. 4, pp. 26-32. Reference 2: Jesus Gil et.al, “Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all”, Nature Reviews Molecular Cell Biology, 2007, vol.7, pages 667-677 Reference 3: Soo-Hyun Kim et.al., “Absence of p16 INK4aand truncation of ARF tumor suppressors in chickens”, PNAS, 2003, vol.100, No.1, pages 211-216

[0023] The aforementioned "apoptosis suppressor genes" refer to genes that have the function of suppressing apoptosis in cells, and examples include BCL2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), Survivin (Baculoviral IAP Repeat Containing 5), and MCL1 (BCL2 Family Apoptosis Regulator).

[0024] The immortalized megakaryocytes are preferably megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes. "Exogenous" means introduced into the cell from outside the cell. The exogenous genes may be located on the cell's chromosomes, or in the nucleus or cytoplasm. The exogenous genes can be detected, for example, by measuring their number. If the genes are on autosomes, there are two copies of the gene on each autosome, resulting in two copies in a single cell. Therefore, if the exogenous genes are absent, two copies of the gene will be detected in a single cell. On the other hand, if the exogenous genes are present, three or more copies will be detected in a single cell. In this case, the exogenous genes can be detected, for example, using PCR with primers, probes, or a combination thereof. If the exogenous genes have a tag sequence or a selection marker, detection of the exogenous genes may be performed by detecting the tag sequence or the selection marker. Furthermore, the exogenous genes can be detected, for example, against the protein translated from the gene using an antibody.

[0025] The megakaryocyte culture is, for example, a culture produced by culturing the megakaryocytes. The megakaryocyte culture can be carried out, for example, by culturing the megakaryocytes in the presence of a culture medium, as described later.

[0026] Since the megakaryocyte culture is obtained by culturing the megakaryocytes, it is, for example, a mixture containing the megakaryocytes and platelets produced from the megakaryocytes as cellular components. The cellular components mean cells and platelets. As mentioned above, the megakaryocytes can be induced from cells that are less differentiated than the megakaryocytes. Therefore, if the megakaryocytes used to produce the megakaryocyte culture include megakaryocytes induced from cells that are less differentiated than the megakaryocytes, the megakaryocyte culture may also contain cells that are less differentiated than the megakaryocytes.

[0027] In the present invention, the megakaryocyte culture may be the culture obtained by culturing the megakaryocytes itself, or it may be a processed version of the culture. Examples of processing the culture include removing the liquid fraction, extracting the cellular component fraction, and changing the composition of cellular components including platelets. Examples of changing the composition of cellular components include removing cells and / or platelets from the mixture, extracting cells and / or platelets from the mixture, and adding cells and / or platelets to the mixture.

[0028] The term "platelet" refers to a cellular component in the blood that is positive for CD41a and CD42b. For example, platelets lack a nucleus and are smaller in size compared to megakaryocytes. Therefore, platelets and megakaryocytes can be distinguished, for example, by the presence or absence of a nucleus and / or size. Platelets are known to play an important role in thrombus formation and hemostasis, and are also involved in tissue regeneration after injury and the pathophysiology of inflammation. Furthermore, when platelets are activated by bleeding or other factors, receptors for cell adhesion factors such as Integrin αIIBβ3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61) are expressed on their membranes. When platelets are activated, they aggregate, and fibrin coagulates due to various blood coagulation factors released from the platelets, forming a thrombus and promoting hemostasis. In this invention, the origin of the platelets is the same as that of the megakaryocytes.

[0029] In the present invention, the processed product may be prepared from megakaryocytes or from a culture of megakaryocytes. Furthermore, when prepared from a culture of megakaryocytes, the culture of megakaryocytes may be processed. Specifically, the processed product may be, for example, a processed product of the cell fraction or liquid fraction of the megakaryocytes or their culture, or a processed product of a processed product obtained by processing the megakaryocytes or their culture. The processing in the preparation of the processed product is not particularly limited and may include, for example, processing to change the density of cellular components such as concentration processing, separation processing or purification processing; extraction processing to extract cellular components such as drying processing, freezing processing, freeze-drying processing, solvent processing, surfactant processing, enzyme processing, protein fraction extraction processing; crushing processing such as grinding processing or pulverizing processing; and so on. Specific examples of the processed products include, for example, extracts from concentrates, dried products, frozen products, freeze-dried products, solvent-treated products, surfactant-treated products, enzyme-treated products, protein fractions, sonicated products, etc. of megakaryocytes or their cultures; crushed products such as ground products and pulverized products; extracts from concentrates, dried products, frozen products, freeze-dried products, solvent-treated products, surfactant-treated products, enzyme-treated products, protein fractions, sonicated products of the cell fractions of megakaryocytes or their cultures; crushed products such as ground products and pulverized products; and so on. The processed product may consist of one type of processed product or a mixture of two or more types of processed products. The mixture is not particularly limited and can be a mixture of any combination and ratio of processed products.

[0030] The processed product comprises, for example, at least one of one or more growth factors and growth factor receptors. Furthermore, the processed product has physiological activities such as cell proliferation-promoting activity. Moreover, the processed product can be produced, for example, by processing the megakaryocytes or their cultures, as described above. Therefore, in the present invention, the processed product can be defined, for example, using the following conditions (1) to (3). The processed product may be defined by any of the following conditions (1) to (3), by a combination of conditions, or by all of the conditions. As a specific example, the processed product can be defined, for example, by a combination of conditions. (conditions) (1) Content of growth factors and / or growth factor receptors; (2) The physiological activity of the treated product; (3) Method for manufacturing processed products (Combination of conditions) Condition (1), (2), or (3); Conditions (1) and (2), condition (1) and (3), or condition (2) and (3); Conditions (1), (2), and (3)

[0031] (1) Condition (1) Condition (1), as described above, is a condition relating to the content of growth factors and / or growth factor receptors. Condition (1) may specify the content of the growth factors or the content of the growth factor receptors, or it may specify the content of the growth factors and the content of the growth factor receptors. Furthermore, the growth factors used in the provision of condition (1) may be one type or two or more types. The growth factor receptors used in the provision of condition (1) may be one type or two or more types.

[0032] In the above condition (1), the growth factors include basic fibroblast growth factor (bFGF), insulin-like growth factor-binding protein-1 (IGFBP-1), insulin-like growth factor-binding protein-2 (IGFBP-2), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-binding protein-6 (IGFBP-6), placental growth factor (PIGF), vascular endothelial growth factor (VEGF), endocrine gland-derived vascular endothelial growth factor (EG-VEGF), differentiation growth factor-15 (GDF-15), amphiregulin (AR), bone morphogenetic protein-5 (BMP-5), bone morphogenetic protein-7 (BMP-7), hepatic growth factor (HGF), TGFβ1 (transforming growth factor β1), etc.

[0033] In the above condition (1), the growth factor receptors include stem cell factor receptor (SCFR), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor 2 (VEGFR2), etc.

[0034] The aforementioned content may be, for example, the weight of each growth factor and each growth factor receptor in the processed product, or the weight of each growth factor and each growth factor receptor relative to the total protein weight of the processed product (content per total protein), but the latter is preferred.

[0035] The total protein weight can be determined, for example, by the BCA protein quantification method. The BCA protein quantification method is a protein quantification method that utilizes the coordination bond between a monovalent copper ion and two molecules of bicinchoninic acid. The sample used for the BCA protein quantification method preferably does not contain, for example, reducing agents and / or copper ion chelating agents. The BCA protein quantification method can be carried out in accordance with Reference 4 below, and commercially available kits may be used, for example. As a kit for the BCA protein quantification method, the Pierce® BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific, Inc.) can be used. Reference 4: Shunji Hase et al., "Experimental Methods in Protein Science: Starting from Simple Principles 1 - Making Proteins: Extraction, Purification, and Synthesis," Kagaku Dojin, December 13, 2008.

[0036] The total protein concentration in the treated product can be appropriately set, for example, by the number of cells used in the treatment and the volume of solvent in the treated product. The total protein concentration in the treated product can be relatively increased, for example, by increasing the number of cells used in the treatment, decreasing the volume of solvent in the treated product, or by extracting from megakaryocytes. Conversely, the total protein concentration in the treated product can be relatively decreased, for example, by reducing the number of cells used in the treatment, increasing the volume of solvent in the treated product, or by extracting from a megakaryocyte culture. As a specific example, the number of cells used in the treatment is 1 × 10⁻⁶. 8 The sample is a cell, and when the volume of solvent in the treated product is 100 μl, the total protein concentration in the treated product is, for example, 0.1 to 200 mg / ml. The solvent is, for example, an aqueous solvent as described later.

[0037] The weights of the growth factors and growth factor receptors can be determined, for example, by the sandwich ELISA method. The sandwich ELISA method can be carried out in accordance with Reference 5 below, and commercially available kits may be used, for example. Examples of sandwich ELISA kits include Quantibody® Human Growth Factor Array 1 (manufactured by RayBiotech). Reference 5: Biochemical Measurement Research Group (ed.), "Immunoassays: From Basics to Advanced Techniques," Kodansha, December 20, 2014.

[0038] Examples of the content of the growth factor and the growth factor receptor in the treated product include the following:

[0039] When the growth factor is bFGF, the processed product contains, for example, 2000-20000 pg, 5000-20000 pg, or 10000-20000 pg of bFGF per 1 mg of total protein. The processed product, by containing bFGF, exhibits cell proliferation-promoting activity as described below.

[0040] If the growth factor is IGFBP-1, the processed product contains 0-200 pg, 0.01-200 pg, 0.01-100 pg, or 0.01-50 pg of IGFBP-1 per 1 mg of total protein.

[0041] If the growth factor is IGFBP-2, the processed product contains 8,000 to 80,000 pg, 10,000 to 80,000 pg, or 20,000 to 80,000 pg of IGFBP-2 per 1 mg of total protein.

[0042] If the growth factor is PIGF, the processed product contains 1 to 60 pg, 1 to 30 pg, or 1 to 20 pg of PIGF per 1 mg of total protein.

[0043] If the growth factor is VEGF, the processed product contains 20-800 pg, 20-600 pg, or 20-400 pg of VEGF per 1 mg of total protein.

[0044] If the growth factor is GDF-15, the processed product contains 1,000 to 10,000 pg, 1,000 to 5,000 pg, or 2,000 to 5,000 pg of GDF-15 per 1 mg of total protein.

[0045] If the growth factor is AR, the processed product contains 0-16 pg, 0.01-16 pg, 0.1-16 pg, or 1-16 pg of AR per 1 mg of total protein.

[0046] If the growth factor is HGF, the processed product contains 0-100 pg, 0.01-100 pg, 0.01-50 pg, or 0.01-30 pg of HGF per 1 mg of total protein.

[0047] When the growth factor is BMP-7, the processed product contains 0 to 1000 pg or 0.01 to 1000 pg of BMP-7 per 1 mg of total protein.

[0048] If the growth factor receptor is SCFR, the processed product contains 200-2000 pg, 300-1500 pg, or 400-1000 pg of SCFR per 1 mg of total protein.

[0049] If the growth factor receptor is EGFR, the processed product contains 0-60 pg, 0.01-60 pg, 1-50 pg, 1-45 pg, or 10-40 pg of EGFR per 1 mg of total protein.

[0050] When the growth factor receptor is VEGFR2, the processed product contains 20-400 pg, 50-350 pg, or 100-300 pg of VEGFR2 per 1 mg of total protein.

[0051] As described above, condition (1) may be defined by the content of one or more types of growth factors, or by the content of one or more types of growth factor receptors, or by any combination of these contents. In this case, condition (1) is defined by at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve conditions selected from the group consisting of the following conditions (A1) to (A9) and (B1) to (B3). As specific examples, the following combinations of growth factor content and / or growth factor receptor content can be exemplified. (Combination of growth factor content and / or growth factor receptor content) One of the following conditions applies: (A1)~(A9) and (B1)~(B3): (A1) bFGF content, (A2) IGFBP-1 content, (A3) IGFBP-2 content, (A4) PIGF content, (A6) VEGF content, (A6) GDF-15 content, (A7) AR content, (A8) HGF content, (A9) BMP-7 content, (B1) SCFR content, (B2) EGFR content, or (B3) VEGFR2 content; Any two of the following conditions: (A1)~(A9) and (B1)~(B3): (A1) and (A2), (A1) and (A3), (A1) and (A4), (A1) and (A5), (A1) and (A6), (A1) and (A7), (A1) and (A8), (A1) and (A9), (A1) and (B1), (A1) and (B2), (A1) and (B3), (A2) and (A3), (A2) and (A4), (A2) and (A5), (A2) and (A6), (A2) and (A7), (A2) and (A8), (A2) and (A9), (A2) and (B1), (A2) and (B2), (A2) and (B3), (A3) and (A4), (A3) and (A5), (A3) and (A6), (A3) and (A7), (A3) and (A8), (A3) and (A9), (A3) and (B1), (A3) and (B2), (A3) and (B3), (A4) and (A5), (A4) and (A6), (A4) and (A7), (A4) and (A8), (A4) and (A9), (A4) and (B1), (A4) and (B2), (A4) and (B3), (A5) and (A6), (A5) and (A7), (A5) and (A8), (A5) and (A9), (A5) and (B1), (A5) and (B2), (A5) and (B3), (A6) and (A7), (A6) and (A8), (A6) and (A9), (A6) and (B1), (A6) and (B2), (A6) and (B3), (A7) and (A8), (A7) and (A9), (A7) and (B1), (A7) and (B2), (A7) and (B3), (A8) and (A9), (A8) and (B1), (A8) and (B2), (A8) and (B3), (A9) and (B1), (A9) and (B2), (A9) and (B3), (B1) and (B2), (B1) and (B3), or (B2) and (B3).

[0052] The content per total protein may be adjusted, for example, by adding or removing other proteins other than the growth factor and growth factor receptor, depending on the intended use of the composition of the present invention. Examples of these other proteins include proteins that do not affect the activity of the growth factor and growth factor receptor, such as serum albumin such as human serum albumin and gamma globulin such as human gamma globulin. Examples of protein removal include removal using a column or removal using an antibody.

[0053] (2) Condition (2) Condition (2), as described above, is a condition relating to the physiological activity of the treated product. In condition (2), the physiological activity of the treated product means, for example, activity that regulates the function of cells, tissues, or organs. Examples of cell functions include proliferation, differentiation, induction or suppression of gene expression, induction or suppression of the expression of biomacromolecules such as proteins and glycans.

[0054] The physiological activity of the treated product may include, for example, cell proliferation-promoting activity, fibroblast function-promoting activity, keratinocyte function-promoting activity, and dermal papilla cell function-promoting activity. In the case of cell proliferation-promoting activity, the cells are not particularly limited and may include, for example, mesenchymal stem cells, fibroblasts, keratinocytes such as epidermal keratinocytes, and dermal papilla cells such as scalp dermal papilla cells. The treated product may have one activity or multiple activities.

[0055] The mesenchymal stem cells are cells that have the ability to self-renew and differentiate into bone, cartilage, and adipocytes. The mesenchymal stem cells can be identified by cell surface markers. For example, the mesenchymal stem cells are positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45.

[0056] The aforementioned fibroblasts are cells that constitute the connective tissue of organs such as the skin, lungs, and heart, and are cells that supply fibrous components (extracellular matrix components such as collagen, elastin, and hyaluronic acid). The aforementioned fibroblasts can be identified by cell surface markers. For example, the aforementioned fibroblasts are positive for vimentin, CD90, and TE-7 antibodies.

[0057] The keratinocytes are epidermal cells that possess keratinizing ability, divide in the basal layer of the epidermis, and contribute to epidermal formation. The keratinocytes can be identified by cell surface markers. For example, the keratinocytes are positive for androgen receptors and cytokeratin. Epidermal keratinocytes are preferred.

[0058] The dermal papilla cells are cells located within the dermal papilla at the base of the hair follicle and are important cells for inducing and maintaining hair growth. The dermal papilla cells can be identified by cell surface markers and / or gene expression. The dermal papilla cells are, for example, alkaline phosphatase-positive; positive for the SOX2 gene, WIF1 gene, Noggin gene, BMP4 gene, and VCAN gene. The dermal papilla cells are preferably scalp dermal papilla cells.

[0059] The proliferation-promoting activity of the cells may be such that, for example, the proliferative capacity of the cells is improved compared to a control group that is similar except that the composition of the present invention is not added, and for example, the proliferative capacity of the cells may be decreased from the start. In this case, the "proliferation-promoting activity" can also be said to be, for example, the suppression of the decrease in proliferation activity. As a specific example, the proliferative activity of the mesenchymal stem cells decreases with each passage. Since the decrease in the proliferative activity of the mesenchymal stem cells can be suppressed according to the composition of the present invention, for example, the composition of the present invention can be said to exhibit proliferation-promoting activity. The proliferation-promoting activity of the cells can be measured, for example, under culture conditions in which the target cells proliferate. The culture conditions can be appropriately set, for example, depending on the type of cell. As a specific example, when mesenchymal stem cells are used as the cells, the proliferative activity of the cells can be measured, for example, when human-derived mesenchymal stem cells are cultured in the presence of a growth medium. in vitro It can be measured by culturing in [a certain medium]. The proliferation activity of the mesenchymal stem cells can be measured, for example, according to Example 1 described below. Furthermore, when fibroblasts are used as the cells, the proliferation activity of the cells can be measured, for example, by culturing human-derived fibroblasts in the presence of a growth medium. in vitro It can be measured by culturing in [a certain manner]. The proliferation activity of the fibroblasts can be measured, for example, according to Example 3 described below. When keratinocytes are used as the cells, the proliferation activity of the cells can be measured, for example, by culturing human-derived epidermal keratinocytes in the presence of a growth medium. in vitro It can be measured by culturing in [a certain manner]. The proliferation activity of the keratinocytes can be measured, for example, according to Example 4 described below. When dermal papilla cells are used as the cells, the proliferation activity of the cells can be measured, for example, by culturing human-derived hair dermal papilla cells in the presence of a growth medium. invitro It can be measured by culturing. The proliferation activity of the hair papilla cells can be measured, for example, according to Example 5 described below.

[0060] The fibroblast function-promoting activity described above is sufficient if, for example, the function of the fibroblasts is improved compared to a control group that is similar except that the composition of the present invention is not added. The function of the fibroblasts may mean, for example, either the proliferation of the fibroblasts or the production of extracellular matrix by the fibroblasts. The extracellular matrix may include, for example, collagens such as type I collagen, fibrous substances such as elastin; substrate substances such as glycosaminoglycans and proteoglycans such as hyaluronic acid and chondroitin sulfate, integrins, fibronectin, laminin and other cell adhesion proteins; and so on. The function of the fibroblasts can be measured, for example, based on Example 3 described below.

[0061] The keratinocyte function-promoting activity is sufficient if, for example, the function of the keratinocytes is improved compared to a control group that is similar except that the composition of the present invention is not added. The function of the keratinocytes may mean, for example, proliferation of the keratinocytes, differentiation into epidermal cells, or induction of barrier function genes. The barrier function genes refer to genes that function to maintain the barrier function of the skin, for example, and specific examples include the profilaggrin gene (FLG) and the ceramide synthase gene (serine palmitoyltransferase long chain base subunit 1: SPTLC1). As the profilaggrin gene, a human-derived profilaggrin gene is a polynucleotide consisting of a base sequence registered in Genbank with Accession No.: NM_002016.2. As the ceramide synthase gene, a human-derived ceramide synthase gene is a polynucleotide consisting of a base sequence registered in Genbank with Accession No.: NM_001281303.2. The function of the keratinocytes can be measured, for example, based on Example 4 described below.

[0062] The dermal papilla cell function-promoting activity is sufficient if, for example, the function of the dermal papilla cells is improved compared to a control group that is similar except that the composition of the present invention is not added. The function of the dermal papilla cells may mean, for example, the proliferation of predermal papilla cells or the induction of hair growth-promoting genes. The hair growth-promoting genes refer to genes that function in hair growth, hair growth, or maintenance, and specific examples include the FGF7 (fibroblast growth factor 7) gene and the vascular endothelial growth factor (VEGF) gene. The VEGF may be VEGFA, for example. As the FGF7 gene, a human-derived pro-FGF7 gene is a polynucleotide consisting of a nucleotide sequence registered in Genbank with Accession No.: NM_002009.4, for example. As the VEGFA gene, a human-derived VEGFA gene is a polynucleotide consisting of a nucleotide sequence registered in Genbank with Accession No.: NM_001025366.3, for example. The function of the aforementioned hair papilla cells can be measured, for example, based on Example 5 described later.

[0063] (3) Conditions (3) Condition (3), as described above, is a condition relating to the method of manufacturing the treated product. For the method of manufacturing the treated product in the composition of the present invention, refer to the description of the method of manufacturing the composition of the present invention described later.

[0064] The composition of the present invention can be produced, for example, by the method for producing the composition of the present invention described later.

[0065] The compositions of the present invention can be used, for example, as described later, as cell proliferation promoting compositions, fibroblast function promoting compositions, keratinocyte function promoting compositions, dermal papilla cell function promoting compositions, etc. Furthermore, due to the above-described activity, the compositions of the present invention are expected to be suitably used, for example, as compositions that promote the healing of skin disorders and compositions that promote hair growth. For methods of using the present invention, refer to the descriptions of the cell proliferation promoting compositions, fibroblast function promoting compositions, keratinocyte function promoting compositions, and dermal papilla cell function promoting compositions described later. In addition, the compositions of the present invention can be used, for example, for repairing knee joint injuries, tendon injuries, or ligament injuries; treating ulcers, pressure sores, burns, scars, or wounds; improving skin texture; promoting hair growth; and / or beautifying skin; etc.

[0066] <Method for producing the composition> The method for producing the composition of the present invention (hereinafter also referred to as the "production method") includes a processing step for treating megakaryocytes or their cultures. The production method of the present invention is characterized by including the processing step, and other steps and conditions are not particularly limited. The composition of the present invention can be produced according to the production method of the present invention. The description of the composition of the present invention above can be incorporated into the production method of the present invention.

[0067] In the manufacturing method of the present invention, the object to be processed in the processing step is megakaryocytes or their cultures. For this reason, the manufacturing method of the present invention may include, prior to the processing step, a megakaryocyte induction step in which megakaryocytes are induced from cells that are less differentiated than megakaryocytes, and / or a production step in which cultures of megakaryocytes are produced.

[0068] In the megakaryocyte induction step, the method for inducing megakaryocytes is not particularly limited and can be carried out by known induction methods. For example, the method for inducing megakaryocytes can be, for example, the method for inducing immortalized megakaryocytes described in International Publication No. 2011 / 034073 (US Patent Application Publication No. 2012 / 0238023), International Publication No. 2012 / 157586 (US Patent Application Publication No. 2014 / 0127815), International Publication No. 2014 / 123242 (US Patent Application Publication No. 2016 / 0002599), etc.; the method for inducing megakaryocytes described in Reference 6 below; etc., which are incorporated herein by reference as constituting a part of this specification. For example, in the megakaryocyte induction step, for example, the oncogene and the Polycomb gene may be forced to be expressed in cells that are less differentiated than the megakaryocytes. As a result, in the megakaryocyte induction step, for example, immortalized megakaryocytes that proliferate indefinitely can be obtained. Furthermore, for example, by releasing the forced expression of the immortalized megakaryocytes, the immortalized megakaryocytes can be induced into multinucleated megakaryocytes and platelet production can be made. Alternatively, in the megakaryocyte induction step, for example, the apoptosis suppressor gene may be forced into the megakaryocyte precursor cells. As a result, in the megakaryocyte induction step, immortalized megakaryocytes can be obtained. Furthermore, for example, by releasing the forced expression of the immortalized megakaryocytes, multinucleated megakaryocytes can be induced from the immortalized megakaryocytes and platelet production can be made. Reference 6: Ann-Kathrin Borger et.al., “Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions”, Mol. Med., 2016, vol. 22, pages 274-288

[0069] In the megakaryocyte induction step, for example, the oncogene, the Polycomb gene, and the apoptosis suppressor gene may be forcibly expressed. In this case, the forcible expression of the oncogene, the Polycomb gene, and the apoptosis suppressor gene may be performed simultaneously or separately. Specifically, in the megakaryocyte induction step, after forcibly expressing the oncogene and the Polycomb gene, the forcible expression may be released, and then the apoptosis suppressor gene may be forcibly expressed; or the oncogene, the Polycomb gene, and the apoptosis suppressor gene may be forcibly expressed; or the oncogene and the Polycomb gene may be forcibly expressed, and then the apoptosis suppressor gene may be expressed. As a result, the megakaryocyte induction step can yield the immortalized megakaryocytes. Furthermore, for example, by releasing the forcible expression of the immortalized megakaryocytes, multinucleated megakaryocytes can be induced from the immortalized megakaryocytes, and platelets can be produced.

[0070] The megakaryocyte induction step preferably includes, for example, a first expression step in which oncogenes and Polycomb genes are forcibly expressed in cells less differentiated than megakaryocytes, a second expression step in which apoptosis-suppressing genes such as the Bcl-xL gene are forcibly expressed in the undifferentiated cells, and a deactivation step in which all of the forcible expression is deactivated, since this can improve the efficiency of introducing each gene.

[0071] The forced expression and de-expression of each gene can be carried out by known methods, such as those described in International Publication No. 2011 / 034073, International Publication No. 2012 / 157586, International Publication No. 2014 / 123242, or Reference 7 below, or by similar methods, which are incorporated herein by reference as part of this specification. Specifically, the forced expression and de-expression of each gene can be carried out, for example, using a drug-responsive gene expression induction system. Examples of such gene expression induction systems include the Tet-on® system and the Tet-off® system. When using the Tet-on system, for example, in the forced expression step, culture is carried out in the presence of a gene expression-inducing drug such as tetracycline or doxycycline, and in the de-expression step, culture is carried out in the absence of the drug. Reference 7: Nakamura S et al, “Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells.”, Cell Stem Cell, 2014, vol.14, No.4, pages 535-548

[0072] Next, the production process involves producing a culture of megakaryocytes. This production process can be carried out, for example, by culturing the megakaryocytes in the presence of a culture medium. The culture of megakaryocytes may be carried out, for example, on feeder cells or without feeder cells. The megakaryocytes can be cultured without feeder cells, for example, because they can be cultured in suspension. The megakaryocyte culture includes, for example, the platelets.

[0073] The culture conditions for the megakaryocytes are not particularly limited, and the usual culture conditions for megakaryocytes can be used. For example, the culture temperature is, for instance, about 35 to 42°C, about 36 to 40°C, or about 37 to 39°C. The CO2 concentration is, for example, about 5 to 15%. The O2 concentration is, for example, about 15 to 25% or about 20%. The culture period is not particularly limited, for example, about 1 day to 2 weeks or about 4 to 8 days.

[0074] The culture medium is not particularly limited, and examples include known media suitable for platelet production from megakaryocytes and similar media. Specifically, the culture medium can be prepared using, for example, a culture medium used for animal cell culture as a base medium. Examples of the base medium include single media or mixtures thereof such as IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI1640 medium, Fischer's medium, Neurobasal® Medium (manufactured by Thermo Fisher Scientific). The culture medium may contain, for example, serum or plasma, or it may be a serum-free medium that does not contain these. Preferably, the serum and plasma are of the same origin as the megakaryocytes. Specifically, if the megakaryocytes are of human origin, it is preferable that the serum and plasma are also of human origin.

[0075] The culture medium may contain, for example, other components. These other components are not particularly limited and include, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines, etc. These other components may be, for example, one type or two or more types. The cytokines are, for example, substances that promote the differentiation of hematopoietic cells, and specific examples include vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like substances, stem cell factor (SCF), ITS (insulin-transferrin-selenite) supplements, ADAM inhibitors, FLT inhibitors, WNT inhibitors, ROCK inhibitors, aromatic hydrocarbon receptor (AhR) inhibitors, etc. The culture medium is preferably an IMDM medium containing, for example, serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. The culture medium may further contain, for example, SCF, and may further contain heparin. The concentrations of the other components are not particularly limited. The concentration of TPO is, for example, about 10 ng / ml to about 200 ng / ml, and about 50 ng / ml to about 100 ng / ml. The concentration of SCF is, for example, about 10 ng / ml to about 200 ng / ml, and about 50 ng / ml. The concentration of heparin is, for example, about 10 U / ml to about 100 U / ml, and about 25 U / ml. The culture medium may further contain, for example, phorbol ester (e.g., phorbol-12-myristo-13-acetate; PMA).

[0076] Next, the processing step involves processing the megakaryocytes or their culture. In this processing step, proteins are extracted, for example, by disrupting the cell membranes of the cells contained in the megakaryocytes or their culture. Specific examples of the processing in this step are not particularly limited and include, for example, processing to change the density of cellular components, such as concentration; extraction processing to extract cellular components, such as drying, freezing, freeze-drying, solvent processing, surfactant processing, enzyme processing, protein fraction extraction, and sonication; and crushing processing, such as grinding and pulverization. The processing in this step may be one type or two or more types. Furthermore, the processing in this step may be performed once or two or more times.

[0077] The concentration treatment can be carried out, for example, by centrifugation of the megakaryocytes or their culture. The centrifugation conditions can be, for example, conditions that precipitate cells or platelets. The drying treatment can be carried out, for example, by drying the megakaryocytes or their culture with a dry spray, drum dryer, etc. The freeze-drying treatment can be carried out, for example, using a freeze-dryer. In the solvent treatment, the solvent is, for example, an organic solvent such as phenol or chloroform; or an aqueous solvent such as water, physiological saline, or buffer solution. When an aqueous solvent is used as the solvent, the solvent treatment is preferably carried out in combination with, for example, a surfactant treatment, an enzyme treatment, and / or sonication treatment described later. The solvent treatment can be carried out, for example, by mixing the megakaryocytes or their culture with the solvent. In the surfactant treatment, the surfactant may be, for example, an ionic surfactant such as sodium lauryl sulfate; a nonionic surfactant such as NP-40, Triton X-100, Tween 20, or n-Dodecyl-β-D-maltopyranoside; or an amphoteric surfactant such as CHAPS. The concentration of the surfactant may be, for example, a concentration capable of disrupting the cell membranes of the cellular components in the megakaryocyte or its culture. The surfactant treatment can be carried out, for example, by contacting the megakaryocyte or its culture with the surfactant in the presence of an aqueous solvent. The contact with the surfactant may be carried out, for example, at a temperature of about 0 to about 10°C. The aqueous solvent may be, for example, water, physiological saline, or a buffer solution. The enzyme in the enzyme treatment may be, for example, a peptidase or a protease. The enzyme treatment can be carried out, for example, by contacting the megakaryocyte or its culture with the enzyme in the presence of the aqueous solvent. The conditions for the enzyme treatment may be, for example, conditions under which the enzyme exhibits activity. The protein fraction extraction process can be carried out, for example, by applying osmotic shock, freeze-thaw cycles, etc., to the megakaryocytes or their culture. The sonication can be carried out, for example, using an ultrasonic generator. The conditions for the sonication can be, for example, conditions that disrupt cells.

[0078] The processing conditions and processing time in each processing step can be appropriately determined, for example, depending on the type of processing. Furthermore, in the processing step, the total protein concentration in the processed product can be adjusted, for example, by adjusting the amount of aqueous solvent.

[0079] Prior to the above treatment, the megakaryocytes or their culture may be processed (pre-treated). In this case, the megakaryocyte culture may be the culture obtained by culturing the megakaryocytes itself, or a processed mixture may be used. Examples of processing the culture include removing the liquid fraction, extracting the cellular component fraction, and changing the composition of cellular components including platelets. Examples of changing the composition of cellular components include removing cells and / or platelets from the mixture, extracting cells and / or platelets from the mixture, and adding cells and / or platelets to the mixture.

[0080] If the manufacturing method of the present invention includes the above-mentioned pretreatment, the manufacturing method of the present invention may also include a removal step of removing platelets from the megakaryocytes or their culture. In this case, the processing step is carried out using megakaryocytes or their culture from which the platelets have been removed, or the removed platelets, as the megakaryocytes or their culture. Megakaryocytes after platelet release have, for example, a high bFGF content. Therefore, the manufacturing method of the present invention can produce a composition with a high bFGF content by, for example, removing the platelets. By removing the platelets, the platelets can be separated from other cell fractions. Therefore, the removal step can also be called, for example, a platelet separation step or a platelet-to-other-cell component separation step. In the removal step, the method for separating platelets from the megakaryocyte culture can be carried out by known methods such as the method described in, for example, International Publication No. 2017 / 065280 (US Patent Application Publication No. 2018 / 282697), or by similar methods, which are incorporated herein by reference as constituting a part of this specification.

[0081] The platelet removal rate (separation rate) in the removal step is, for example, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The platelet removal rate is, for example, 60-90%.

[0082] The manufacturing method of the present invention may include a storage step for storing the megakaryocytes or their culture, the megakaryocytes or their culture from which the platelets have been removed, or the removed platelets. Examples of storage in the storage step include refrigeration (about 1 to about 10°C) and freezing (about -200 to about -4°C). The storage period in the storage step is not particularly limited. Freezing is preferred because the storage step can, for example, also serve as the freezing treatment in the processing step.

[0083] The manufacturing method of the present invention can be carried out as follows, for example. However, the present invention is not limited in any way to the following examples. First, the culture medium containing the megakaryocytes or their culture is concentrated by centrifugation to concentrate the cellular components. The centrifugation treatment is, for example, under conditions in which the cellular components precipitate. Specifically, the centrifugation treatment can be carried out by centrifugation at 1000 to 3000 × g for 5 to 20 minutes. Next, the precipitate is collected after centrifugation and the obtained precipitate is subjected to a freezing treatment by rapid freezing. The freezing treatment can be carried out, for example, by contacting the precipitate with a liquefied gas such as liquid nitrogen. Furthermore, the precipitate after freezing is subjected to a surfactant treatment by dissolving it in an aqueous solvent containing the surfactant. The obtained solution is centrifuged to precipitate impurities. The centrifugation treatment is, for example, under conditions in which impurities such as cell membranes precipitate. As a specific example, the centrifugation process can be carried out by centrifugation at 10,000 to 20,000 × g for 3 to 10 minutes. After centrifugation, the protein is present in the supernatant, and the processed product can be obtained by recovering the supernatant as a protein fraction.

[0084] <Composition obtained by the manufacturing method> The composition of the present invention (hereinafter also referred to as the "second composition") is obtained by the manufacturing method of the present invention described above. The second composition of the present invention is characterized by being obtainable by the manufacturing method of the present invention described above, and other configurations and conditions are not particularly limited. According to the second composition of the present invention, for example, a composition having physiological activity can be provided. According to the second composition of the present invention, for example, cell proliferation can be promoted. The description of the composition and manufacturing method of the present invention described above can be used to describe the second composition of the present invention.

[0085] <Cell proliferation promoting composition> The present invention provides compositions capable of promoting cell proliferation in other examples. The cell proliferation-promoting compositions of the present invention are characterized by comprising the compositions of the present invention as described above. The proliferation-promoting compositions of the present invention are characterized by comprising the compositions of the present invention, and other configurations and conditions are not particularly limited. According to the proliferation-promoting compositions of the present invention, the proliferation of cells, in particular mesenchymal stem cells, can be promoted. The proliferation-promoting compositions of the present invention can be described by reference to the above description of the compositions and methods of production of the present invention.

[0086] The growth-promoting composition of the present invention is in vitro You can use it as well, in vivo It can be used in this way.

[0087] The growth-promoting composition of the present invention in vitro When used in this manner, the target of administration may be, for example, cells, tissues, organs, etc., and the cells may be, for example, cells collected from living organisms, cultured cells, etc.

[0088] The growth-promoting composition of the present invention in vivo When used in this manner, the target of administration may be, for example, humans or non-human animals other than humans. Examples of non-human animals include mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, guinea pigs, etc.

[0089] The conditions for use (administration conditions) of the growth-promoting composition of the present invention are not particularly limited, and the administration form, timing of administration, dosage, etc. can be appropriately set depending on the type of target organism, etc.

[0090] The growth-promoting composition of the present invention in vitro When used in the present invention, the growth-promoting composition can be used, for example, by adding it to the culture medium of the target cells. The final concentration of total protein derived from the growth-promoting composition of the present invention in the culture medium is 10-1000 μg / ml, 10-500 μg / ml, 10-320 μg / ml, 10-300 μg / ml, or 20-300 μg / ml.

[0091] The growth-promoting composition of the present invention in vivo When used in this context, the dosage can be appropriately determined based on factors such as the type of target, symptoms, age, and method of administration. For example, when administered to humans, the daily dose of the growth-promoting composition, and the total amount of protein derived from the growth-promoting composition, are not particularly limited and can be appropriately set according to their intended use. The number of daily doses can be, for example, 1 to 5 times, 1 to 3 times, 1 time, or 2 times.

[0092] The administration form of the growth-promoting composition of the present invention is not particularly limited. in vivo When administering the drug, it may be administered orally or parenterally. Parenteral administration may include, for example, intravenous injection, intramuscular injection, transdermal administration, subcutaneous administration, intradermal administration, enteral administration, rectal administration, vaginal administration, nasal administration, pulmonary administration, intraperitoneal administration, local administration, etc.

[0093] The dosage form of the growth-promoting composition of the present invention is not particularly limited and can be appropriately determined, for example, depending on the administration method. The dosage form may be, for example, a liquid or a solid.

[0094] The growth-promoting composition of the present invention may, for example, optionally contain an additive. The additive is preferably a pharmaceutically acceptable additive or a pharmaceutically acceptable carrier.

[0095] <Composition to promote the function of fibroblasts> The present invention provides compositions capable of promoting fibroblast function in other examples. The fibroblast function-promoting compositions of the present invention comprise the compositions of the present invention as described above. The fibroblast function-promoting compositions of the present invention are characterized by comprising the compositions of the present invention, and other components and conditions are not particularly limited. The fibroblast function-promoting compositions of the present invention can promote fibroblast function. The fibroblast function-promoting compositions of the present invention can be described by reference to the descriptions of the compositions, methods of production, and proliferation-promoting compositions of the present invention.

[0096] The fibroblast function-promoting composition of the present invention is in vitro You can use it as well, in vivo It can be used in this way.

[0097] The fibroblast function-promoting composition of the present invention in vitro When used in a culture medium, the fibroblast function-enhancing composition of the present invention can be used, for example, by adding it to the culture medium of the target fibroblasts. According to the fibroblast function-enhancing composition of the present invention, for example, by maintaining the fibroblasts in a culture medium containing the fibroblast function-enhancing composition, the proliferation of the fibroblasts and / or the production of extracellular matrix by the fibroblasts can be promoted. The final concentration of total protein of the fibroblast function-enhancing composition of the present invention in the culture medium is 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml.

[0098] The target cells and administration conditions for the fibroblast function-promoting composition of the present invention can be described by referring to the description of the target cells and administration conditions for the proliferation-promoting composition of the present invention.

[0099] <Composition to promote the healing of skin disorders> The present invention provides compositions that can be used to promote the healing of skin disorders in other examples. The present invention provides a composition for promoting the healing of skin disorders (hereinafter also referred to as the "healing-promoting composition") which comprises the composition of the present invention as described above. The healing-promoting composition of the present invention is characterized by comprising the composition of the present invention, and other components and conditions are not particularly limited. The healing-promoting composition of the present invention is expected to promote the healing of skin disorders. The healing-promoting composition of the present invention can be described by reference to the description of the composition, method of production, and growth-promoting composition of the present invention described above.

[0100] In the present invention, the skin disorder refers to a condition in which wounds, damage, etc., occur to the skin, that is, a condition in which the structure of normal skin tissue is impaired or destroyed. Specific examples include skin ulcers, pressure sores, burns, scars, wounds, and skin aging.

[0101] The healing-promoting composition of the present invention, in vitro You can use it as well, in vivo It can be used in this way.

[0102] The healing-promoting composition of the present invention in vitro When used in the present invention, the healing-promoting composition can be used, for example, by adding it to the culture medium of the target cells. The final concentration of total protein of the healing-promoting composition in the culture medium is 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml.

[0103] The target population and administration conditions for the healing-promoting composition of the present invention can be described by referring to the description of the target population and administration conditions for the proliferation-promoting composition of the present invention. The healing-promoting composition of the present invention is preferably used in a dosage form that can be administered subcutaneously or transdermally.

[0104] <Composition to promote the function of keratinocytes> The present invention provides compositions capable of promoting the function of keratinocytes in other examples. The keratinocyte function-promoting compositions of the present invention include the compositions of the present invention as described above. The keratinocyte function-promoting compositions of the present invention are characterized by including the compositions of the present invention, and other components and conditions are not particularly limited. The keratinocyte function-promoting compositions of the present invention can promote the function of keratinocytes. The keratinocyte function-promoting compositions of the present invention can be described by reference to the descriptions of the compositions, methods of production, and proliferation-promoting compositions of the present invention.

[0105] The keratinocyte function-promoting composition of the present invention, in vitro You can use it as well, in vivo It can be used in this way.

[0106] The keratinocyte function-promoting composition of the present invention in vitro When used, the keratinocyte function-enhancing composition of the present invention can be used, for example, by adding it to the culture medium of the target keratinocytes. The keratinocyte function-enhancing composition of the present invention may be added, for example, to the maintenance medium used to maintain the keratinocytes, or to the differentiation medium used to differentiate the keratinocytes into epithelial cells such as epidermal cells, or to both the maintenance medium and the differentiation medium. According to the keratinocyte function-enhancing composition of the present invention, for example, by maintaining the keratinocytes in a culture medium containing the keratinocyte function-enhancing composition, the proliferation of the keratinocytes, differentiation into epidermal cells, and / or induction of barrier function genes can be promoted. The final concentration of total protein of the keratinocyte function-enhancing composition of the present invention in the culture medium is 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml.

[0107] The target population and administration conditions for the keratinocyte function-promoting composition of the present invention can be described by referring to the description of the target population and administration conditions for the proliferation-promoting composition of the present invention.

[0108] As described above, the keratinocyte function-promoting composition of the present invention promotes the differentiation of keratinocytes into epithelial cells such as epidermal cells. For this reason, the keratinocyte function-promoting composition of the present invention is expected to be suitably used, for example, as an additive in artificial skin culture. Furthermore, as described above, the keratinocyte function-promoting composition of the present invention exhibits fibroblast function-promoting activity. For this reason, the keratinocyte function-promoting composition of the present invention is expected to be suitably used in the regeneration of aging skin by promoting skin tissue turnover through its keratinocyte and fibroblast function-promoting activity. Accordingly, the keratinocyte function-promoting composition of the present invention is expected to be suitably used, for example, as a topical skin composition such as cosmetics, or as an injectable preparation for subcutaneous administration.

[0109] <Composition to promote the function of hair papilla cells> The present invention provides compositions capable of promoting the function of dermal papilla cells in other examples. The dermal papilla cell function-promoting composition of the present invention comprises the composition of the present invention as described above. The dermal papilla cell function-promoting composition of the present invention is characterized by comprising the composition of the present invention, and other components and conditions are not particularly limited. The dermal papilla cell function-promoting composition of the present invention can promote the function of keratinocytes. The dermal papilla cell function-promoting composition of the present invention can be described by reference to the description of the composition, method of production, and proliferation-promoting composition of the present invention described above.

[0110] The present invention's function-promoting composition for hair papilla cells, in vitro You can use it as well, in vivo It can be used in this way.

[0111] The present invention provides a function-promoting composition for hair papilla cells. in vitroWhen used in a culture medium, the dermal papilla cell function-promoting composition of the present invention can be used, for example, by adding it to the culture medium of the target dermal papilla cells. According to the dermal papilla cell function-promoting composition of the present invention, for example, by maintaining the dermal papilla cells in a culture medium containing the dermal papilla cell function-promoting composition, the proliferation of the dermal papilla cells and / or the induction of hair growth-promoting genes can be promoted. The final concentration of total protein of the dermal papilla cell function-promoting composition of the present invention in the culture medium is 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml.

[0112] The target population and administration conditions for the hair papilla cell function-promoting composition of the present invention can be described by referring to the description of the target population and administration conditions for the proliferation-promoting composition of the present invention.

[0113] <Hair growth promoting composition> The present invention provides compositions that can promote hair growth in other examples. The hair growth promoting compositions of the present invention include the compositions of the present invention as described above. The hair growth promoting compositions of the present invention are characterized by including the compositions of the present invention, and other components and conditions are not particularly limited. According to the hair growth promoting compositions of the present invention, hair growth is expected to be promoted because the function of dermal papilla cells is promoted. The dermal papilla cell function promoting compositions of the present invention can be described by reference to the descriptions of the compositions, methods of production, and proliferation promoting compositions of the present invention.

[0114] In the present invention, "hair growth promotion" means enhancing (increasing, raising) the possibility of hair growing from a hairless state, promoting hair growth (e.g., growth in hair length and / or thickness), and / or reducing (suppressing, lowering) hair loss.

[0115] The hair growth promoting composition of the present invention may be used, for example, to prevent, suppress, or stop hair loss. In this case, the hair growth promoting composition of the present invention can also be called, for example, a hair loss prevention, suppression, or stop composition.

[0116] The hair growth promoting composition of the present invention, in vitro You can use it as well, invivo It can be used in this way.

[0117] The hair growth promoting composition of the present invention in vitro When used in the present invention, the hair growth promoting composition can be used, for example, by adding it to the culture medium of target dermal papilla cells. According to the hair growth promoting composition of the present invention, for example, by maintaining the dermal papilla cells in a culture medium containing the composition of the present invention, the proliferation of the dermal papilla cells and / or the induction of hair growth promoting genes can be promoted, thereby promoting hair growth. The final concentration of total protein of the hair growth promoting composition of the present invention in the culture medium is 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml.

[0118] The target population and administration conditions for the hair growth promoting composition of the present invention can be determined by referring to the descriptions of the target population and administration conditions for the hair growth promoting composition of the present invention and the hair growth promoting method described later.

[0119] <Methods for promoting cell proliferation> The cell proliferation promotion method of the present invention (hereinafter also referred to as the "proliferation promotion method") uses the cell proliferation promotion composition of the present invention. The proliferation promotion method of the present invention is characterized by the use of the proliferation promotion composition of the present invention, and other steps and conditions are not particularly limited. According to the proliferation promotion method of the present invention, the proliferation of cells, in particular mesenchymal stem cells, can be promoted. The proliferation promotion method of the present invention can be described by reference to the descriptions of the composition, manufacturing method, proliferation promotion composition, and differentiation promotion composition of the present invention.

[0120] The present invention's method for promoting growth includes, for example, in vitro You can do it this way, in vivo It may be done in this way.

[0121] The present invention's method for promoting proliferation in vitroWhen implemented, the growth-promoting method of the present invention includes, for example, a culture step of culturing cells in the presence of the growth-promoting composition. The target and administration conditions of the growth-promoting composition of the present invention can be described, for example, by referring to the description of the target and administration conditions for the growth-promoting composition of the present invention.

[0122] <Methods for promoting fibroblast function> The present invention provides a method for promoting the function of fibroblasts, in other examples. The method for promoting the function of fibroblasts of the present invention uses the composition of the present invention. The method for promoting the function of fibroblasts of the present invention is characterized by the use of the composition of the present invention, and other steps and conditions are not particularly limited. According to the method for promoting the function of fibroblasts of the present invention, the function of fibroblasts can be promoted. The method for promoting the function of fibroblasts of the present invention can be described by reference to the description of the composition of the present invention, the method of production, the proliferation-promoting composition, and the fibroblast function-promoting composition.

[0123] The present invention provides a method for promoting the function of fibroblasts, for example, in vitro You can do it this way, in vivo It may be done in this way.

[0124] The present invention provides a method for promoting the function of fibroblasts. [[ID=...]] in When implemented, the method for promoting fibroblast function of the present invention includes, for example, a culture step of culturing fibroblasts in the presence of the composition. The target and administration conditions of the composition of the present invention can be described, for example, by referring to the description of the target and administration conditions for the proliferation-promoting composition of the present invention.

[0125] <Methods to promote healing of skin disorders> The present invention provides a method that can be used in other examples to promote the healing of skin disorders. The method for promoting the healing of skin disorders of the present invention (hereinafter also referred to as the "healing promotion method") uses the composition of the present invention. The healing promotion method of the present invention is characterized by the use of the composition of the present invention, and other steps and conditions are not particularly limited. According to the healing promotion method of the present invention, it is expected that the healing of skin disorders can be promoted. The healing promotion method of the present invention can be described by reference to the description of the composition of the present invention, the method of production, the growth-promoting composition, and the skin disorder healing promotion composition.

[0126] The present invention's method for promoting the healing of skin disorders can also be described, for example, as a method for treating or managing skin disorders.

[0127] The healing promotion method of the present invention may include, for example, an administration step of administering the composition of the present invention to a subject. The subject may be, for example, a patient with a skin disorder, a patient at risk of developing a skin disorder, and so on.

[0128] The healing promotion method of the present invention is, for example, vitro in You can do it this way, vitro in It may be done in this way.

[0129] The present invention's method for promoting healing vivo in When implemented, the healing promotion method of the present invention includes, for example, a culture step of culturing skin-related cells such as keratinocytes and fibroblasts in the presence of the composition. The target and administration conditions of the composition of the present invention can be described, for example, by referring to the description of the target and administration conditions for the proliferation-promoting composition of the present invention.

[0130] <Methods for promoting the function of keratinocytes> The present invention provides a method capable of promoting the function of keratinocytes in other examples. The method for promoting the function of keratinocytes of the present invention uses the composition of the present invention. The method for promoting the function of keratinocytes of the present invention is characterized by the use of the composition of the present invention, and other steps and conditions are not particularly limited. According to the method for promoting the function of keratinocytes of the present invention, the function of keratinocytes can be promoted. The method for promoting the function of keratinocytes of the present invention can be described by reference to the description of the composition, manufacturing method, proliferation-promoting composition, and keratinocyte function-promoting composition of the present invention.

[0131] The present invention provides a method for promoting the function of keratinocytes, for example. vitro in You can do it this way, vitro in It may be done in this way.

[0132] The present invention provides a method for promoting the function of keratinocytes. vivo in When implemented, the method for promoting the function of keratinocytes according to the present invention includes, for example, a culture step of culturing keratinocytes in the presence of the composition. The target and administration conditions of the composition of the present invention can be described, for example, by referring to the description of the target and administration conditions for the proliferation-promoting composition of the present invention.

[0133] <Methods for promoting the function of hair papilla cells> The present invention provides a method capable of promoting the function of dermal papilla cells in other examples. The method for promoting the function of dermal papilla cells of the present invention uses the composition of the present invention. The method for promoting the function of dermal papilla cells of the present invention is characterized by the use of the composition of the present invention, and other steps and conditions are not particularly limited. According to the method for promoting the function of dermal papilla cells of the present invention, the function of dermal papilla cells can be promoted. The method for promoting the function of dermal papilla cells of the present invention can be described by reference to the description of the composition of the present invention, the method of production, the proliferation-promoting composition, and the dermal papilla cell function-promoting composition.

[0134] The present invention provides a method for promoting the function of hair papilla cells, for example, vitro in You can do it this way, vitro [[ID=9...]] It may be done in this way.

[0135] The present invention provides a method for promoting the function of hair papilla cells. in vivo When implemented, the method for promoting the function of dermal papilla cells of the present invention includes, for example, a culture step of culturing dermal papilla cells in the presence of the composition. The target and administration conditions of the composition of the present invention can be described, for example, by referring to the description of the target and administration conditions for the proliferation-promoting composition of the present invention.

[0136] <Methods to promote hair growth> The present invention provides, in other examples, a method that can promote hair growth. The hair growth promoting method of the present invention uses the composition of the present invention. The hair growth promoting method of the present invention is characterized by the use of the composition of the present invention, and other steps and conditions are not particularly limited. According to the hair growth promoting method of the present invention, it is expected that hair growth can be promoted. The hair growth promoting method of the present invention can be described by reference to the description of the composition of the present invention, the method of production, the proliferation promoting composition, and the hair growth promoting composition.

[0137] The hair growth promoting method of the present invention can also be described, for example, as a treatment or procedure for alopecia or thinning hair. Furthermore, the hair growth promoting method of the present invention may be used, for example, to prevent, suppress, or stop hair loss. In this case, the hair growth promoting method of the present invention can also be described, for example, as a method for preventing, suppressing, or stopping hair loss.

[0138] The hair growth promoting method of the present invention may include, for example, an administration step of administering the composition of the present invention to a subject. The subject may be, for example, a patient who has alopecia, a patient who may have alopecia, etc. Patients with alopecia may be, for example, patients with male pattern baldness, female pattern baldness, or alopecia after anticancer drug treatment. Patients who may have alopecia may be, for example, patients before anticancer drug treatment, patients with genetic alopecia who have not yet experienced hair loss, etc.

[0139] The hair growth promotion method of the present invention is, for example, in vitro You can do it this way, in vitro It may be done in this way.

[0140] The present invention's method for promoting hair growth in vivo When implemented, the hair growth promoting method of the present invention includes, for example, a culture step of culturing hair growth-related cells such as dermal papilla cells in the presence of the composition. The target and administration conditions of the composition of the present invention can be described, for example, by referring to the description of the target and administration conditions for the proliferation-promoting composition of the present invention.

[0141] <Use of composition> The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in promoting cell proliferation. Furthermore, the present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in promoting fibroblast function. The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in promoting the healing of skin disorders. The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in promoting keratinocyte function. The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in promoting dermal papilla cell function. The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in promoting hair growth. The present invention relates to the use of a composition containing a processed megakaryocyte or culture thereof as an active ingredient for producing a cell proliferation-promoting composition. Furthermore, the present invention relates to the use of a composition containing a processed megakaryocyte or culture thereof as an active ingredient for producing a fibroblast function-promoting composition. The present invention relates to the use of a composition containing a processed megakaryocyte or culture thereof as an active ingredient for producing a composition that promotes the healing of skin disorders. The present invention relates to the use of a composition containing a processed megakaryocyte or culture thereof as an active ingredient for producing a composition that promotes the function of keratinocytes. The present invention relates to the use of a composition containing a processed megakaryocyte or culture thereof as an active ingredient for producing a composition that promotes the function of dermal papilla cells. The present invention relates to the use of a composition containing a processed megakaryocyte or culture thereof as an active ingredient for producing a composition that promotes hair growth. [Examples]

[0142] The present invention will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples.

[0143] [Example 1] The composition of the present invention was prepared and confirmed to contain growth factors and growth factor receptors, and to have cell proliferation-promoting activity.

[0144] (1) Production of immortalized megakaryocyte cells Immortalized megakaryocytes were prepared using the following procedure.

[0145] (1-1) Preparation of hematopoietic progenitor cells from iPS cells Human iPS cells (TKDN SeV2 and NIH5: human fetal dermal fibroblast-derived iPS cells established using Sendai virus) were differentiated into hematopoietic cells according to the method described in Reference 8 below. Specifically, human ES / iPS cell colonies were co-cultured with C3H10T1 / 2 feeder cells for 14 days in the presence of 20 ng / ml VEGF (R&D SYSTEMS) to produce hematopoietic progenitor cells (HPCs). The culture conditions were 37°C, 20% O2, and 5% CO2 (unless otherwise specified, the same conditions were used hereafter). Reference 8: Takayama N. et al., “Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells”, J. Exp. Med., 2010, vo.13, pages 2817-2830

[0146] (1-2) Gene transfer systems The gene transfer system utilized a lentiviral vector system. The lentiviral vector is a tetracycline-regulated Tet-on® gene expression induction system vector. It was prepared by rearranging the mOKS cassette of LV-TRE-mOKS-Ubc-tTA-I2G (see reference 9 below) with c-MYC, BMI1, or BCL-xL. The vectors into which c-MYC, BMI1, or BCL-xL were introduced were named LV-TRE-c-Myc-Ubc-tTA-I2G, LVTRE-BMI1-Ubc-tTA-I2G, and LV-TRE-BCL-xL-Ubc-tTA-I2G, respectively. The c-MYC, BMI1, and BCL-xL viruses were prepared by gene transfer into 293T cells using the aforementioned lentiviral vector. By infecting target cells with the obtained virus, the c-MYC, BMI1, and BCL-xL genes are introduced into the target cells' genome sequences. These genes, stably introduced into the genome sequence, can be forced to be expressed by adding doxycycline (clontech#631311) to the culture medium. Reference 9: Kobayashi, T. et al., “Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.”, Cell, 2010, vol.142, No.5, pages 787-799

[0147] (1-3) Infection of hematopoietic progenitor cells with cMYC and BMI1 virus On a 6-well plate pre-seed with C3H10T1 / 2 feeder cells, 5 × 10⁻¹⁵ HPC obtained by the method described in (1-1) above are placed. 4Cells were seeded to a cell / well ratio, and c-MYC and BMI1 were forced to express using lentiviral assays with BMI1 and c-MYC viruses. Six wells were used for each cell line. Specifically, virus particles were added to the culture medium to achieve an MOI (multiplicity of infection) of 20, and infection was carried out by spin infection (centrifugation at 32°C, 900 rpm, 60 minutes). This spin infection was performed twice, 12 hours apart. The culture medium was prepared by adding 50 ng / ml Human thrombopoietin (TPO) (R&D SYSTEMS), 50 ng / ml Human Stem Cell Factor (SCF) (R&D SYSTEMS), and 2 μg / ml Doxycycline (Dox, clontech #631311) to a base medium (IMDM (Iscove's Modified Dulbecco's Medium) (Sigma-Aldrich) containing 15% Fetal Bovine Serum (GIBCO), 1% Penicillin-Streptomycin-Glutamine (GIBCO), 1% Insulin, Transferrin, Selenium Solution (ITS-G) (GIBCO), 0.45 mmol / l 1-Thioglycerol (Sigma-Aldrich), and 50 μg / ml L-Ascorbic Acid (Sigma-Aldrich)). This resulted in a culture medium (hereinafter referred to as differentiation medium), to which Protamine was further added to reach a final concentration of 10 A culture medium containing the additive at a concentration of μg / ml was used.

[0148] (1-4) Preparation and maintenance culture of megakaryocyte autoproliferating strains Using the methods described in (1-3) above, the day on which infection with c-MYC and BMI1 viruses was carried out was designated as infection day 0. Megakaryocyte autoproliferating strains were then created by culturing HPCs into which the c-MYC gene and the BMI1 gene had been introduced, as follows. Forced expression of the c-MYC gene and the BMI1 gene was performed by adding DOX to the culture medium to a concentration of 1 μg / ml DOX.

[0149] • Day 2 to Day 11 of infection On day 2 of infection, virus-infected hematopoietic cells obtained by the above method were collected by pipetting, centrifuged at 1200 rpm for 5 minutes to remove the supernatant, and then resuspended in fresh differentiation medium and seeded on fresh C3H10T1 / 2 feeder cells (6-well plate). Passaging was performed on day 9 of infection by the same procedure. At the time of reseeding, the cell count was measured and then 1 × 10⁶ cells were seeded. 5 C3H10T1 / 2 feeder cells were seeded at a rate of cells / 2ml / well (6-well plate).

[0150] • Day 12 to Day 13 of infection The same procedure as on the second day of infection was performed. After counting the number of cells, 3 × 10⁻⁶ cells were collected. 5 C3H10T1 / 2 feeder cells were seeded on a 100mm dish so that the cell density was 10ml / 100mm dish.

[0151] • Day 14 of infection Virus-infected blood cells were collected, and the cells were 1.0 × 10⁶ 5 For each cell, 2 μl, 1 μl, and 1 μl of anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), and anti-human CD235ab-pacific blue (BioLegend) antibody were used to react the hematopoietic cells with the antibodies. After the reaction, the cells were analyzed using FACS Verse® (BD Biosciences). Cells with a CD41a positivity rate of 50% or higher on day 14 of infection were designated as megakaryocyte autoproliferating cells.

[0152] (1-5) BCL-xL virus infection of megakaryocyte autoproliferating strains On the 14th day after infection of the megakaryocyte self-proliferating strain, BCL-xL was introduced by the lentiviral method using the BCL-xL virus. Virus particles were added to the medium to achieve an MOI of 10, and the cells were infected by spin infection (centrifugation at 32°C, 900 rpm for 60 minutes). The forced expression of the BCL-xL gene was carried out by adding DOX to the medium to a concentration of 1 μg / ml DOX.

[0153] (1-6) Creation and maintenance culture of immortalized megakaryocyte strains · From the 14th day to the 18th day after infection The megakaryocyte self-proliferating strain transfected with the BCL-xL gene obtained by the method of (1-5) above was collected and centrifuged at 1200 rpm for 5 minutes. After the centrifugation, the precipitated cells were suspended in a new differentiation medium and then seeded onto new C3H10T1 / 2 feeder cells at a density of 2×10 5 cells / 2ml / well (6-well plate).

[0154] · 18th day after infection: Subculture The megakaryocyte self-proliferating strain after transfection with the BCL-xL gene was collected. After counting the cell number, it was seeded at a density of 3×10 5 cells / 10ml / 100mm dish.

[0155] · 24th day after infection: Subculture The megakaryocyte self-proliferating strain after transfection with the BCL-xL gene was collected. After counting the cell number, it was seeded at a density of 1×10 5 cells / 10ml / 100mm dish. Thereafter, subculture was carried out in the same manner every 4 - 7 days to perform maintenance culture. At the time of subculture, after suspending in a new differentiation medium, it was seeded.

[0156] On the 24th day after infection, the megakaryocyte self-proliferating strain transfected with BCL-xL was collected, and 1.0×10 5Immunostaining was performed on each cell using 2 μl, 1 μl, and 1 μl of anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), and anti-human CD235ab-Pacific Blue (Anti-CD235ab-PB; BioLegend) antibody, respectively, followed by analysis using FACS Verse®. Cells with a CD41a positivity rate of 50% or higher on day 24 after infection were designated as immortalized megakaryocyte cell lines. These cells that were able to proliferate for more than 24 days after infection were designated as immortalized megakaryocyte cell lines SeV2-MKCL and NIH5-MKCL.

[0157] The obtained SeV2-MKCL and NIH5-MKCL were cultured statically in 10cm dishes (10ml / dish). The culture medium consisted of IMDM as the base medium, with the following components added (concentrations are final concentrations). The culture conditions were 37°C and 5% CO2. FBS (Sigma #172012 lot.12E261) 15% L-Glutamin (Gibco #25030-081) 2mmol / l ITS (Gibco #41400-045) 100x dilution MTG (monothioglycerol, sigma #M6145-25ML) 450μmol / l Ascorbic acid (sigma #A4544) 50 μg / ml Puromycin (sigma #P8833-100MG) 2μg / ml SCF (Wako Pure Chemical #193-15513) 50ng / ml TPO-like active substance 200 ng / ml

[0158] (2) Production of megakaryocyte cultures Forced expression was removed by culturing in a DOX-free medium. Specifically, the immortalized megakaryocyte cell lines (SeV2-MKCL and NIH5-MKCL) obtained by the method described in (1) above were washed twice with PBS(-) and suspended in the platelet production medium described below. The cell seeding density was 1.0 × 10⁻⁶. 5 The value was set to cells / ml.

[0159] The aforementioned platelet production medium was prepared by adding the following components to IMDM as the base medium (concentrations are final concentrations). human plasma A6% L-Glutamin (Gibco #25030-081) 4mmol / l ITS (Gibco #41400-045) 100x dilution MTG (monothioglycerol, sigma #M6145-25ML) 450μmol / l Ascorbic acid (sigma #A4544) 50 μg / ml SCF (Wako Pure Chemical #193-15513) 50ng / ml TPO-like active substance 200 ng / ml ADAM inhibitor 15 μmol / l GNF351(Calbiochem #182707)500nmol / LY39983(Chemscene LLC #CS-0096)500nmol / l Urokinase 5U / ml Low molecular weight heparin (SANOFI, Clexane) 1U / ml

[0160] Then, by culturing the cells in the aforementioned platelet production medium for 6 days to induce platelet production, megakaryocyte cultures were produced.

[0161] (3) Production of purified platelets Platelets were produced (purified) from the megakaryocyte culture obtained in (2) above using the following procedure. The same purification process was performed twice.

[0162] (3-1) Concentration of megakaryocyte cultures The megakaryocyte culture obtained in (2) above was introduced into a culture bag. The culture bag was then connected to a concentration system as shown in Figure 1. In Figure 1, washing and preservation solution bags 1 and 2 contain the washing and preservation solution. The washing and preservation solution used was bicarbonate infusion (bicarbon infusion, manufactured by Otsuka Pharmaceutical Co., Ltd.) to which 20% ACD and 2.5% human serum albumin were added, and the pH was adjusted to 7.2 with NaOH. Then, according to Table 1 below, the megakaryocyte culture was concentrated using a hollow fiber membrane (Plasma Flow OP, manufactured by Asahi Kasei Medical Co., Ltd.), and the resulting concentrated megakaryocyte culture was collected in a storage bag.

[0163] [Table 1]

[0164] (3-2) Centrifugation of platelets First, using a sterile joining device, the waste liquid bag of the ACP215 disposable set was replaced with a collection bag. The collection bag used was a Highcalic IVH bag (Terumo HC-B3006A). Next, 10% of ACD-A solution (Terumo Corporation) was added to the concentrated megakaryocyte culture. After the addition, the concentrated solution with ACD-A solution added was injected into a cell bag. The cell bag used was a Highcalic IVH bag (Terumo HC-B3006A).

[0165] Next, using a sterile joining device, the cell bag containing the culture with ACD-A solution added was joined to the ACP215 disposable set. Then, the ACP215 was started in service mode and the rotation speed was set to 2500 rpm (350 × g). The ACP215 was started and the culture from the cell bag was introduced into the separation bowl at approximately 100 ml / min. The liquid components flowing out of the separation bowl were collected in a recovery bag. After introducing the entire amount of culture from the cell bag into the separation bowl, another 500 ml of washing and preservation solution was introduced into the separation bowl. After introducing the washing and preservation solution into the separation bowl, centrifugation was stopped and the recovery bag containing the recovered solution (the recovered liquid components including platelets) was detached using a tube sealer.

[0166] A new ACP215 disposable set was attached to a collection bag containing the recovery solution (including platelets) using the sterile joining device described above. The ACP215 was started in normal mode. The program setting was set to WPC, and the ACP215 disposable set with the attached collection bag was set up according to the instrument's instructions. The collection bag containing the recovery solution was placed on the stand.

[0167] Next, the centrifugal speed of the ACP215 was changed to 5000 rpm (1398.8 × g), and centrifugation was started. When the recovered liquid began to be introduced into the separation bowl, the injection method was changed from automatic to manual. Specifically, the recovered liquid was introduced into the separation bowl at an introduction rate of approximately 100 ml / min. After the entire amount of recovered liquid was added to the separation bowl, an additional 500 ml of washing and storage solution was added.

[0168] (3-3) Washing of platelets The washing was performed using 2000 ml of the aforementioned washing and storage solution, according to the ACP215 program.

[0169] (3-4) Platelet recovery Following the ACP215 program, 200 ml of washed platelets were collected into a platelet preparation bag.

[0170] (3-5) Separation of platelets Platelets were separated from the platelet preparation bag using the hollow fiber membrane by a conventional method and collected in a recovery bag.

[0171] (4) Production of extracts As the megakaryocytes or their cultures, the immortalized megakaryocyte cell line obtained by the method in (1) above, platelets collected in the platelet preparation bag obtained in (3-5) above, and megakaryocyte cultures from which platelets have been removed collected in the drainage bag (hereinafter collectively referred to as "raw materials") were used. For the megakaryocyte cultures from which platelets have been removed, four separately prepared samples were used (platelet-removed megakaryocyte cultures 1-4).

[0172] Each raw material was washed twice with a washing solution. The washing solution used was prepared by adding ACD-A solution to bicarnate infusion to a concentration of approximately 20 (v / v)%, then adding NaOH to adjust the pH to a range of 7.0-7.4. After washing, each raw material was centrifuged at 2000 × g for 10 minutes at room temperature (approximately 25°C). After centrifugation, the precipitate was collected and frozen using liquid nitrogen. Next, cell lysis buffer was added to the frozen precipitate and dissolved by shaking at 50 rpm at 4°C for 30 minutes. The cell lysis buffer used was a commercially available cell lysis buffer (2× Cell Lysis Buffer, RayBiotech, Cat. No.: AA-LYS) with a protease inhibitor cocktail (Protease Inhibitor Cocktail, RayBiotech, Cat. No.: AA-PI) added.

[0173] The resulting solution was centrifuged at 14000 × g for 5 minutes at 4°C. After centrifugation, the supernatant was collected as the processed material for the example. The total protein concentration of the processed materials obtained from each raw material was measured using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific). As a result, when using an immortalized megakaryocyte cell line, the concentration was 2.2 × 10⁻⁶. 7 2.604 mg of total protein was extracted from the cells. In addition, when using platelets, 2.5 × 10⁻⁶ mg was extracted. 8 1.187 mg of total protein was extracted from the cells. Furthermore, when using platelet-free megakaryocyte cultures 1-4, 1.97 × 10⁶ was extracted. 8 cells, 5.25×10 8 cells, 3.64×10 8 cells, 6.22×10 8Total protein was extracted from the cells in amounts of 2.8, 5.944, 3.6, and 4.496 mg, respectively. After adjusting the total protein concentration to 5 mg / ml, the concentrations of growth factors (bFGF, IGFBP-1, IGFBP-2, PIGF, VEGF, GDF-15, AR, BMP-7, HGF) and growth factor receptors (SCFR, EGFR, VEGFR2) were measured for each treated sample using the Quantibody® Human Growth Factor Array 1 (RayBiotech). These results are shown in Table 2 below. Note that Table 2 also represents concentrations per 5 mg of total protein.

[0174] [Table 2]

[0175] (5) Confirmation of cell proliferation-promoting activity Human adipose tissue-derived mesenchymal stem cells are placed in a 10cm dish, measuring 2.4-4.8 x 10 cm. 5 Cells were seeded in a 10ml culture medium per dish. The mesenchymal stem cells used were commercially available human adipose tissue-derived mesenchymal stem cells (Takara Bio, Cat. No.: C-12977) that had been subcultured twice and then harvested. The culture medium was prepared by adding a composition prepared from platelet-free megakaryocyte culture 2 (Mesenchymal Stem Cell Growth Medium 2, Takara Bio, Cat. No.: C-28009) after freezing and thawing. The composition was added to the culture medium so that the total protein concentration derived from the composition in the medium reached a predetermined concentration (0, 125, 250, or 500 μg / ml). This medium served as a maintenance medium.

[0176] After seeding, the mesenchymal stem cells were cultured for 3 days at 37°C under humid conditions of 5% CO2. After this culture, the mesenchymal stem cells were harvested, seeded under the same conditions, and cultured again for 3 days at 37°C under humid conditions of 5% CO2. Next, the mesenchymal cells were harvested after the culture and the number of cells was counted. The relative value of the number of cells at harvest was calculated using the number of cells at seeding as the baseline (1), and this was used as the relative value of the proliferation activity. In addition, the time required for the cells to proliferate once (doubling time) was calculated based on the number of cells at seeding and the number of cells at harvest. These results are shown in Figure 2.

[0177] Figure 2 is a graph showing the proliferation activity of cells. In Figure 2, (A) shows the proliferation activity, and (B) shows the doubling time. In Figure 2(A), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the relative value of the proliferation activity. In Figure 2(B), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the doubling time, with the numerical values ​​in the figure indicating the doubling time. As shown in Figure 2(A), when the composition of the present invention was added, the proliferation activity of mesenchymal stem cells increased in a manner dependent on the total protein concentration derived from the composition. Although not shown, the doubling time after the third passage of mesenchymal stem cells without the addition of the composition was 21 hours. As shown in Figure 2(B), when the composition of the present invention was not added, the doubling time of mesenchymal stem cells was extended by approximately 1.5 times. In contrast, as shown in Figure 2(B), when the composition of the present invention was added, the time required for mesenchymal stem cells to proliferate once was shortened in a manner dependent on the total protein concentration derived from the composition. Furthermore, when mesenchymal stem cells were cultured in a medium without the composition of the present invention, the doubling time increased with the number of passages. In contrast, when the composition of the present invention was added, the increase in the doubling time was almost suppressed. Therefore, it was found that the composition of the present invention exhibits cell proliferation-promoting activity and can suppress the decrease in proliferation activity.

[0178] [Example 2] We have confirmed that the composition of the present invention has cell proliferation-promoting activity.

[0179] The mesenchymal stem cells were cultured twice, then harvested and cryopreserved. After thawing the cryopreserved cells, the cells were cultured once, and the proliferation activity and doubling time were calculated in the same manner as in Example 1(5), except that the total protein concentration derived from the composition in the maintenance medium was added to a predetermined concentration (0, 0.2, 1.3, 31.3, 62.5, or 125 μg / ml). These results are shown in Figure 3.

[0180] Figure 3 is a graph showing the proliferation activity of cells. In Figure 3, (A) shows the proliferation activity, and (B) shows the doubling time. In Figure 3(A), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the relative value of the proliferation activity. In Figure 3(B), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the doubling time. As shown in Figure 3(A), when the composition of the present invention was added, the proliferation activity of mesenchymal stem cells increased in a manner dependent on the total protein concentration derived from the composition, and in particular, when the total protein concentration was 31.3 μg / ml or higher, the proliferation activity of mesenchymal stem cells increased significantly. Although not shown, the doubling time after the third passage of mesenchymal stem cells without the addition of the composition was 17 hours. As shown in Figure 3(B), when the composition of the present invention was not added, the doubling time of mesenchymal stem cells was extended by approximately 1.5 times. In contrast, as shown in Figure 3(B), when the composition of the present invention was added, the time required for a single proliferation of mesenchymal stem cells was shortened in a manner dependent on the total protein concentration derived from the composition. In particular, when the total protein concentration was 31.3 μg / ml or higher, the time required for a single proliferation of mesenchymal stem cells was significantly shortened. Furthermore, when cultured in a medium without the composition of the present invention, the doubling time of mesenchymal stem cells increased as the number of passages increased. In contrast, when the composition of the present invention was added, the extension of the doubling time was almost suppressed, and this effect was particularly pronounced when the total protein concentration derived from the composition was 31.3 μg / ml or higher. Therefore, it was found that the composition of the present invention exhibits cell proliferation-promoting activity and can suppress the decrease in proliferation activity.

[0181] [Example 3] We have confirmed that the composition of the present invention has fibroblast function-promoting activity.

[0182] (1) Culture of fibroblasts Normal human dermal fibroblasts (nHDF, KURABO, Cat. No.: KF-4109) were induced in a T-75 flask (Sumilon) using growth medium and cultured in a CO2 incubator under specified culture conditions (5% CO2, 37°C, humid conditions; the same applies hereafter). The growth medium was DMEM medium (nacalai tesque, Cat. No.: 08456-65) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, Cat. No.: 172012) and 1% penicillin streptomycin (Thermo Fisher Scientific, Cat. No.: 15140-122). During the culture, the medium was changed every 1-2 days. When the cells reached approximately 80% confluence, they were harvested and used for subsequent tests. The cells were subculturised as follows. First, the cells were washed with phosphate buffer (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95), then detached using a detachment solution (2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution, with Phenol Red (0.25% Trypsin-EDTA), Thermo Fisher Scientific, Cat. No.: 32777-44), and the trypsin was neutralized by adding growth medium. Next, the cell suspension was collected in a 15 ml centrifuge tube and centrifuged using a multi-purpose refrigerated centrifuge (TOMY, Cat. No.: CAX-571) at room temperature, 1000 rpm, for 5 minutes. After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were mixed. The number of viable cells was then counted using the trypan blue method. The growth medium was adjusted to the desired cell concentration, and the cells were seeded into the incubator to be used in subsequent tests.

[0183] (2) Confirmation of the activity that promotes cell proliferation of fibroblasts The aforementioned cells are 5 × 10 3Cells were seeded at a density of 0.1 ml / well in a 96-well plate (Sumilon, Cat. No.: MS-8096F) and cultured for 1 day under the culture conditions described above. After the culture, the culture medium was changed to DMEM medium containing the compositions prepared from the platelet-depleted megakaryocyte culture (MDF) or platelets (PLT) (total protein concentration of 5 mg / ml, the same applies to Examples 4 and 5) to predetermined concentrations (concentrations of each test substance: 0.5%, 1%, 2.5%, 5%, 10%). As a result, the total protein concentrations derived from the compositions in each culture medium were 25 μg / ml (0.5%), 50 μg / ml (1%), 125 μg / ml (2.5%), 250 μg / ml (5%), and 500 μg / ml (10%) (the same applies hereafter). Two types of compositions prepared from platelet-depleted megakaryocyte culture were used (iMDF1, iMDF2). After the aforementioned culture medium change, the cells were incubated for 48 hours.

[0184] After the aforementioned culture, the proliferation activity of cells in each well was measured by the WST-8 method to determine the number of viable cells. First, the culture medium was changed to DMEM medium containing 10% chromogenic reagent (Cell Count Reagent SF, nacalai tesque, Cat. No.: 07553-15), and incubated under the aforementioned culture conditions. The change in absorbance (450 nm) over a 60-minute period from 30 to 90 minutes after the start of incubation was measured using a plate reader (Varioskan Flash, Thermo Fisher Scientific, Cat. No.: 5250040) (n=3 for each group). The negative control (NC) was performed in the same manner except that the aforementioned composition was not added. The positive control (FBS) was performed in the same manner except that FBS was added in the same manner as the aforementioned composition, at a concentration of 1% or 10%. Furthermore, the reference examples (ASA, Mes) were carried out in the same manner as above, except that L-ascorbic acid (ASA), which has collagen production promoting activity, was added to a concentration of 2 mmol / l, or N-methyl-L-serine (MeS), which has hyaluronic acid production promoting activity, was added to a concentration of 10 mmol / l. The cell viability of each sample was set to 100% as the cell viability of the negative control, and the cell viability of each sample was used as the relative proliferative activity value. Then, in the Student's T-test (two-tailed, unpaired) compared with the negative control, a p-value of less than 0.05 was judged to be statistically significant. These results are shown in Figure 4.

[0185] Figure 4 is a graph showing the proliferative activity of fibroblasts. In Figure 4, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents cell viability (proliferative activity). As shown in Figure 4, a concentration-dependent cell proliferation-promoting effect was observed when either the platelet-removed megakaryocyte culture (iMDF1, iMDF2) or the composition prepared from platelets (PLT, PLTMax Human Platelet Lysate, EMD Millipore, Cat. No.: SCM141) was added. Furthermore, the cell proliferation-promoting activity of the composition of the present invention was considered to be higher than that of FBS at the same concentration.

[0186] (3) Confirmation of activity that promotes the production of extracellular matrix After 48 hours of incubation in Example 3(2), the culture supernatant from each well was collected and stored at -80°C until used in the type I collagen and hyaluronic acid production promotion tests described later.

[0187] Next, the amount of type I collagen in the culture supernatant (n=3 in each group) was measured using a type I collagen measurement kit (Human Collagen type I, ELISA kit (without pepsin), ACEL, Cat. No.: EC1-E105). The amount of hyaluronic acid in the culture supernatant was also measured using a hyaluronic acid measurement kit (Hyaluronan DuoSet ELISA, R&D Systems, Cat. No.: DY3614). The measurement methods using each kit followed the attached protocols. The negative control (NC) was performed in the same manner except that the aforementioned composition was not added. The FBS-added group (FBS) was performed in the same manner as in Example 3(2). The positive control (ASA or Mes) was prepared in the same manner as above, except that for the sample used to measure type I collagen, L-ascorbic acid (ASA, Fujifilm-Wako, Cat. No.: 013-19641), which has collagen production promoting activity, was added to a concentration of 2 mmol / l instead of the aforementioned composition, and for the sample used to measure hyaluronic acid, N-methyl-L-serine (MeS, Sigma-Aldrich, Cat. No.: 73156), which has hyaluronic acid production promoting activity, was added to a concentration of 10 mmol / l instead of the aforementioned composition. Then, in the Student's T-test (two-tailed, unpaired) compared with the negative control, a p-value of less than 0.05 was considered statistically significant. These results are shown in Figures 5 and 6.

[0188] Figure 5 is a graph showing the production of type I collagen. In Figure 5, the horizontal axis represents the type of sample, and the vertical axis represents the production amount of type I collagen. The ASA value in the figure represents the collagen production amount (μg / ml) in the reference example (ASA). As shown in Figure 5, a concentration-dependent increase in type I collagen production was observed when either the platelet-removed megakaryocyte culture or the composition prepared from platelets was added. Compared with the results of the cell proliferation-promoting activity in Example 3(2), it was estimated that the type I collagen production-promoting activity was due to the cell proliferation-promoting activity.

[0189] Next, Figure 6 is a graph showing the amount of hyaluronic acid produced. In Figure 6, the horizontal axis shows the type and concentration of the sample, and the vertical axis shows the amount of hyaluronic acid produced. As shown in Figure 6, a concentration-dependent increase in hyaluronic acid production was observed when either the platelet-removed megakaryocyte culture or the composition prepared from platelets was added. Compared to the cell proliferation-promoting activity, the hyaluronic acid production-promoting activity was found to be an activity independent of the cell proliferation-promoting activity, meaning that the composition of the present invention acts on fibroblasts and promotes the production of hyaluronic acid.

[0190] From the above, it was found that the composition of the present invention has fibroblast proliferation-promoting activity and activity to promote the production of extracellular matrix components such as type I collagen and hyaluronic acid. When damage occurs to the skin or other tissues, these fibroblasts migrate to the site of the damage and contribute to the healing of the site through cell division and secretion of extracellular matrix components. Since the composition of the present invention promotes these functions in fibroblasts, it is expected to be able to promote the healing of skin damage.

[0191] [Example 4] We have confirmed that the composition of the present invention has keratinocyte function-promoting activity. (1) Culture of keratinocytes Normal human epidermal keratinocytes (nHEK, KURABO, Cat. No.: KK-4109) were induced in a T-75 flask (Sumilon) using growth medium and cultured in a CO2 incubator under specified culture conditions (5% CO2, 37°C, humid conditions; the same applies hereafter). The growth medium used was Humedia-KG2 medium (KURABO, Cat. No.: KK-2150S). During the culture, the medium was changed every 1-2 days. When the cells reached approximately 80% confluence, they were harvested and used for subsequent tests. The cells were subculturised as follows. First, the cells were washed with phosphate buffer (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95), then detached using a detachment solution (2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution, with Phenol Red (0.25% Trypsin-EDTA), Thermo Fisher Scientific, Cat. No.: 32777-44), and the trypsin was neutralized by adding growth medium. Next, the cell suspension was collected in a 15 ml centrifuge tube and centrifuged using a multi-purpose refrigerated centrifuge (TOMY, Cat. No.: CAX-571) at room temperature, 1000 rpm, for 5 minutes. After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were mixed. The number of viable cells was then counted using the trypan blue method. The growth medium was adjusted to the desired cell concentration, and the cells were seeded into the incubator to be used in subsequent tests.

[0192] (2) Confirmation of the activity that promotes the proliferation of keratinocytes The aforementioned cells are 1 × 10 3Cells were seeded at a density of 0.1 ml / well in a 96-well plate (Sumilon, Cat. No.: MS-8096F) and cultured for 1 day under the culture conditions described above. After the culture, the culture medium was changed to maintenance medium (Humedia-KB2, KURABO, Cat. No.: KK-2350S) containing compositions prepared from platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) to the predetermined concentrations (concentrations of each test substance: 0.5%, 1%, 2.5%, 5%, 10%). Two types of compositions prepared from platelet-depleted megakaryocyte cultures were used. After the medium change, the cells were cultured for 48 hours.

[0193] After the culture, the morphology of each cell in each well was observed using a phase-contrast microscope (OLYMPUS, Cat. No.: CKX53). The change in absorbance (450 nm) was also measured for each cell in each well in the same manner as in Example 3(2) (n=3 for each group). The negative control (NC) was carried out in the same manner except that the composition was not added. The positive control (additive) was carried out in the same manner except that the growth medium was used instead of the maintenance medium containing the composition. Furthermore, the reference example (JTC or NA) was carried out in the same manner except that JTC-801 (JTC, Sigma-Aldrich, Cat. No.: J3955, final concentration 100 nmol / l), which has FLG gene expression-promoting activity, or Nicotinamide (NA, Sigma-Aldrich, Cat. No.: N0636-100G, final concentration 30 μmol / l), which has SPTLC1 gene expression-promoting activity, was added instead of the composition. Furthermore, the cell viability of each sample was set to 100% as the negative control, and the cell viability of each sample was used as the relative proliferative activity value. Then, in the Student's T-test (two-tailed, unpaired) compared with the negative control, a p-value of less than 0.05 was considered statistically significant. These results are shown in Figures 7 and 8.

[0194] Figure 7 shows a phase-contrast image of the cells in each well. For the groups treated with the thrombolytic megakaryocyte cultures (iMDF1, iMDF2) or the compositions prepared from platelets (PLT), the 10% treatment group is shown as a representative example. As shown in Figure 7, in the negative control, the boundaries of keratinocytes were clear, and differentiation into epithelial (epidermal) cells was not observed. On the other hand, in the groups treated with the thrombolytic megakaryocyte cultures (iMDF1, iMDF2) or the compositions prepared from platelets (PLT), keratinocytes spread on the plate, and cell boundaries became blurred. This is due to the differentiation of keratinocytes into epithelial (epidermal) cells. Furthermore, in the compositions prepared from the platelet-removed megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT), the proportion of epithelial (epidermal) layer cells increased in a concentration-dependent manner, and a particularly significant increase was observed when the platelet-removed megakaryocyte cultures (iMDF1, iMDF2) were used. These results indicate that the compositions of the present invention promote the cell differentiation of keratinocytes into the epithelial layer.

[0195] Figure 8 is a graph showing the proliferative activity of keratinocytes. In Figure 8, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents the cell viability (proliferative activity). As shown in Figure 8, a cell proliferation-promoting effect was observed when either the platelet-removed megakaryocyte culture (iMDF1, iMDF2) or the composition prepared from platelets (PLT) was added. Furthermore, the cell proliferation-promoting activity of the composition of the present invention was considered to be higher than that of FBS at the same concentration.

[0196] (3) Confirmation of activity that promotes induction of barrier function genes 5 x 10 4Keratinocytes were cultured in the same manner as in Example 4(2), except that they were seeded at a density of 0.5 ml / well in a 24-well plate (Sumilon, Cat. No.: MS-8024). After culturing, total RNA was recovered and purified from the cells using an RNA extraction kit (RNeasy 96 Kit, QIAGEN, Cat. No.: 74181). RNA extraction was performed according to the protocol provided with the kit. The obtained purified RNA was measured for concentration using a spectrophotometer (NanoDrop One, Thermo Fisher Scientific, Cat. No.: ND-ONE-W), and its purity was confirmed using A260 / A280. It was then stored at -80°C until use in the reverse transcription reaction.

[0197] cDNA was synthesized from the purified RNA using a reverse transcription kit (QuantiTect Reverse Transcription Kit, QIAGEN). The reverse transcription reaction was performed according to the protocol provided with the kit. The obtained cDNA was stored at -30°C. Next, quantitative PCR was performed using the obtained cDNA, a primer set for the GAPDH gene, FLG gene, or SPTLC1 gene, and a qPCR kit (TB Green Premix Ex Taq II (Tli RNaseH Plus), Takara, Cat. No.: RR820A). The PCR reaction was performed by heat treatment at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds, after which the obtained PCR product was dissociated. The qPCR was performed using a qPCR instrument (LightCycler 96 Instrument, Roche, Cat. No.: 05815916001). The negative control (NC) was performed in the same manner except that the aforementioned composition was not added. The positive control (JTC or NA) was performed in the same manner except that, for the sample used to measure the expression of the FLG gene, JTC-801 (JTC, Sigma-Aldrich, Cat. No.: J3955), which has FLG gene expression-promoting activity, was added to a concentration of 100 nmol / l instead of the aforementioned composition, and for the sample used to measure the expression of the SPTLC1 gene, Nicotinamide (NA, Sigma-Aldrich, Cat. No.: N0636-100G), which has SPTLC1 gene expression-promoting activity, was added to a concentration of 30 μmol / l instead of the aforementioned composition. In addition, groups with additives were prepared for both the negative control and the positive control and performed in the same manner. From the obtained measurement data, the expression levels of each gene corrected by the expression level of the GAPDH gene were calculated using the ΔΔCt method, and further, the relative expression levels were calculated with the expression level of the negative control set to 1. Compared to the negative control, a p-value of less than 0.05 in Student's T-test (two-tailed, unpaired) was considered statistically significant. These results are shown in Figures 9 and 10.

[0198] GAPDH gene primer set Forward primer (SEQ ID NO: 1) 5'-CATCCCTGCCTCTACTGGGCTGCC-3' Reverse primer (SEQ ID NO: 2) 5'-CCAGGATGCCCTTGAGGGGGCCCTC-3' • Primer set for the FLG gene Forward primer (SEQ ID NO: 3) 5'-TCGGCAAATCCTGAAGAATCCAGA-3' Reverse primer (SEQ ID NO: 4) 5'-GCTTGAGCCAACTTGAATACCATCAG-3' • Primer set for the SPTLC1 gene Forward primer (SEQ ID NO: 5) 5'-ACAAAGCAAGAATCTTCCTGGAGGAAAGCC-3' Reverse primer (SEQ ID NO: 6) 5'-AAACCTCCAATAGAAGCAAGTGCATTCTCC-3'

[0199] Figure 9 is a graph showing the expression level of the FLG gene. In Figure 9, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents the expression level of the FLG gene. As shown in Figure 9, an effect of promoting FLG gene expression was observed when either the platelet-removed megakaryocyte culture (iMDF1, iMDF2) or the composition prepared from platelets (PLT) was added. Furthermore, the FLG gene expression promoting activity of the composition of the present invention was considered to be higher than that of the positive control JTC801 at the same concentration.

[0200] Next, Figure 10 is a graph showing the expression level of the SPTLC1 gene. In Figure 10, the horizontal axis shows the type and concentration of the sample, and the vertical axis shows the expression level of the SPTLC1 gene. As shown in Figure 10, an SPTLC1 gene expression-promoting effect was observed when either the platelet-removed megakaryocyte culture (iMDF1, iMDF2) or the composition prepared from platelets (PLT) was added. Furthermore, the SPTLC1 gene expression-promoting activity of the composition of the present invention was considered to be higher than that of the positive control nicotinamide (NA) at the same concentration.

[0201] From the above, it was found that the composition of the present invention has activity to promote the proliferation of keratinocytes, promote differentiation into epidermal cells, and promote the expression of barrier function genes such as the FLG gene and the SPTLC1 gene. The proliferation of keratinocytes, differentiation into epidermal cells, and induction of barrier function gene expression are known to be important in maintaining skin tissue and its barrier function. Therefore, since the composition of the present invention promotes these functions in keratinocytes, it was expected that it could be used to maintain skin function, maintain the skin's barrier function, and thus maintain healthy skin.

[0202] [Example 5] We have confirmed that the composition of the present invention has activity that promotes the function of hair papilla cells.

[0203] (1) Culture of hair papilla cells Human scalp hair papilla cells (HFDPC, TOYOBO, Cat. No.: CA60205a) were induced in a T-75 flask (Sumilon) using growth medium and cultured in a CO2 incubator under specified culture conditions (5% CO2, 37°C, humid conditions; the same applies hereafter). The growth medium was dermal papilla cell growth medium (PCGM, TOYOBO, Cat. No.: TMTPGM-250S) with the attached additives added. During the culture, the medium was changed every 1-2 days. When the cells reached approximately 80% confluence, they were harvested and used for subsequent tests. The cells were subculturised as follows. First, the cells were washed with phosphate buffer (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95), then detached using a detachment solution (2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution, with Phenol Red (0.25% Trypsin-EDTA), Thermo Fisher Scientific, Cat. No.: 32777-44), and the trypsin was neutralized by adding growth medium. Next, the cell suspension was collected in a 15 ml centrifuge tube and centrifuged using a multi-purpose refrigerated centrifuge (TOMY, Cat. No.: CAX-571) at room temperature, 1000 rpm, for 5 minutes. After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were mixed. The number of viable cells was then counted using the trypan blue method. The growth medium was adjusted to the desired cell concentration, and the cells were seeded into the incubator to be used in subsequent tests.

[0204] (2) Confirmation of cell proliferation-promoting activity of dermal papilla cells The aforementioned cells are 5 × 10 3Cells were seeded at a density of cells / 0.1 ml / well in a 96-well plate (Sumilon, Cat. No.: MS-8096F) and cultured for 1 day under the aforementioned culture conditions. After the culture, the medium was exchanged with a dermal papilla cell growth medium containing a composition prepared from the platelet-depleted megakaryocyte culture (iMDF1, iMDF2) or platelets (PLT) so as to reach a predetermined concentration (each test substance treatment concentration: 0.5%, 1%, 2.5%, 5%, 10%). In addition, two types of compositions prepared from the platelet-depleted megakaryocyte culture were used. After the medium exchange, the cells were cultured for 48 hours.

[0205] After the culture, for the cells in each well, the change in absorbance (450 nm) was measured in the same manner as in Example 3(2) (n = 3 for each group). The negative control (NC) was carried out in the same manner except that the composition was not added. The positive control (additive) was carried out in the same manner except that a dermal papilla cell growth medium containing the additive was used. The reference example (Mx or Ad) was carried out in the same manner except that minoxidil (Minoxidil (Mx), manufactured by Sigma-Aldrich, Cat. No.: M4145, final concentration 30 μmol / l) or adenosine (Adenosine (Ad), manufactured by Sigma-Aldrich, Cat. No.: A9251, final concentration 100 μmol / l), which are known hair growth agents, was added instead of the composition. Also, taking the cell viability in the negative control as 100%, the cell viability of each sample was used as a relative growth activity value. And in the Student's T-test (two-sided test, unpaired) compared with the negative control, those with a p-value less than 0.05 were determined to be significant. These results are shown in Fig. 11.

[0206] Fig. 11 is a graph showing the growth activity of dermal papilla cells. In Fig. 11, the horizontal axis indicates the type and concentration of the sample, and the vertical axis indicates the cell viability (growth activity). As shown in Fig. 11, a cell growth promoting effect was observed when any of the compositions prepared from the platelet-depleted megakaryocyte culture (iMDF1, iMDF2) or platelets (PLT) was added. Also, it was suggested that 2.5 - 5% is indicated as the addition amount.

[0207] (3) Confirmation of the promoting activity of the hair growth-promoting gene 2.5×10 4 Hair papilla cells were cultured in the same manner as in Example 5(2) except that they were seeded in a 24-well plate (Sumilon, Cat. No.: MS-8024) at a density of cells / 0.5 ml / well. After the culture, cDNA was synthesized in the same manner as in Example 4(3).

[0208] Next, qPCR was performed in the same manner as in Example 4(3) except that a primer set for the GAPDH gene, the following FGF7 gene or VEGFA gene was used as the primer set. The negative control was performed in the same manner except that the composition was not added. The reference example was performed in the same manner except that minoxidil (Minoxidil, manufactured by Sigma-Aldrich, Cat. No.: M4145, final concentration 30 μmol / l) or adenosine (Adenosin, manufactured by Sigma-Aldrich, Cat. No.: A9251, final concentration 100 μmol / l), which are known hair growth stimulants, was added instead of the composition. From the obtained measurement data, the expression level of each gene corrected by the expression level of the GAPDH gene was calculated by the ΔΔCt method, and further, the relative expression level with the expression level of the negative control set to 1 was calculated. Compared with the negative control, those with a p-value less than 0.05 in the Student's T-test (two-sided test, unpaired) were determined to be significant. These results are shown in FIGS. 12 and 13.

[0209] · Primer set for FGF7 gene Forward primer (SEQ ID NO: 7) 5'-TCTGTCGAACACAGTGGTACCTGAG-3' Reverse primer (SEQ ID NO: 8) 5'-GCCACTGTCCTGATTTCCATGA-3' · Primer set for VEGFA gene Forward primer (SEQ ID NO: 9) 5'-AAAGCATTTGTTTGTACAAGATCCG-3' Reverse primer (SEQ ID NO: 10) 5'-CTTGTCACATCTGCAAGTACGTTCG-3'

[0210] Figure 12 is a graph showing the expression level of the FGF7 gene. In Figure 12, the horizontal axis represents the type of sample, and the vertical axis represents the expression level of the FGF7 gene. As shown in Figure 12, when the composition prepared from the platelet-removed megakaryocyte cultures (iMDF1, iMDF2) was added, an effect of promoting FGF7 gene expression was observed. Furthermore, the FGF7 gene expression promoting activity of the composition of the present invention was considered to be comparable to that of known hair growth agents at the same concentration.

[0211] Next, Figure 13 is a graph showing the expression level of the VEGFA gene. In Figure 13, the horizontal axis represents the type of sample, and the vertical axis represents the expression level of the VEGFA gene. As shown in Figure 13, when the composition prepared from the platelet-removed megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) was added, a VEGFA gene expression-promoting effect was observed. Furthermore, the VEGFA gene expression-promoting activity of the composition of the present invention was considered to be comparable to that of known hair growth agents at the same concentration.

[0212] From the above, it was found that the composition of the present invention has proliferation-promoting activity of dermal papilla cells and expression-promoting activity of hair growth genes such as the FGF7 gene and the VEGFA gene. The proliferation of dermal papilla cells and induction of hair growth gene expression are known to be important for hair growth, hair development, and maintenance. Therefore, since the composition of the present invention promotes these functions in dermal papilla cells, it was expected to be usable for hair growth.

[0213] Although the present invention has been described above with reference to embodiments and examples, the present invention is not limited to the above embodiments and examples. Various modifications to the configuration and details of the present invention can be understood by those skilled in the art within the scope of the present invention.

[0214] This application claims priority based on Japanese Patent Application No. 2019-225959, filed on 13 December 2019, and incorporates all of its disclosures herein.

[0215] <Note> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. <Composition> (Note 1) A composition comprising a processed product of megakaryocytes or their cultures. (Note 2) The composition according to Appendix 1, wherein the processed product is an extract of the cell fraction of megakaryocytes or their cultures. (Note 3) The processed product is the composition described in Appendix 1 or 2, comprising 2,000 to 20,000 pg of basic fibroblast growth factor (bFGF) per 1 mg of total protein. (Note 4) The processed product is a composition according to any one of the appendices 1 to 3, containing 8,000 to 80,000 pg of insulin-like growth factor-binding protein-2 (IGFBP-2) per 1 mg of total protein. (Note 5) The processed product is a composition according to any one of the appendices 1 to 4, containing 1 to 60 pg of placental growth factor (PIGF) per 1 mg of total protein. (Note 6) The processed product is a composition according to any one of the appendices 1 to 5, containing 200 to 2000 pg of stem cell factor receptors (SCFRs) per 1 mg of total protein. (Note 7) The processed product is a composition according to any one of the appendices 1 to 6, containing 20 to 800 pg of vascular endothelial growth factor (VEGF) per 1 mg of total protein. (Note 8) The processed product is a composition according to any one of the appendices 1 to 7, containing 20 to 400 pg of vascular endothelial growth factor receptor 2 (VEGFR2) per 1 mg of total protein. (Note 9) The processed product is the composition according to any one of Appendices 1 to 8, containing 1000 - 10000 pg of growth differentiation factor - 15 (GDF - 15) per 1 mg of total protein. (Appendix 10) The processed product is the composition according to any one of Appendices 1 to 9, containing 0 - 1000 pg of bone morphogenetic protein - 7 (BMP - 7) per 1 mg of total protein. (Appendix 11) The processed product is the composition according to any one of Appendices 1 to 10, containing 0 - 16 pg of amphiregulin (AR) per 1 mg of total protein. (Appendix 12) The processed product is the composition according to any one of Appendices 1 to 11, containing 0 - 60 pg of epidermal growth factor receptor (EGFR) per 1 mg of total protein. (Appendix 13) The processed product is the composition according to any one of Appendices 1 to 12, containing 0 - 100 pg of hepatocyte growth factor (HGF) per 1 mg of total protein. (Appendix 14) The processed product is the composition according to any one of Appendices 1 to 13, containing 0 - 200 pg of insulin - like growth factor - binding protein - 1 (IGFBP - 1) per 1 mg of total protein. (Appendix 15) The composition according to any one of Appendices 1 to 14, having cell growth - promoting activity. (Appendix 16) The composition according to Appendix 15, wherein the cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells. (Appendix 17) The composition according to any one of Appendices 1 to 16, having fibroblast function - promoting activity. (Appendix 18) The composition according to Appendix 17, wherein the function of the fibroblasts is the proliferation of fibroblasts and / or the production of extracellular matrix by fibroblasts. (Appendix

[19] ) The composition according to Appendix 18, wherein the extracellular matrix contains collagen and / or hyaluronic acid. (Appendix 20) A composition according to any one of the appendices 1 to 19, which has keratinocyte function-promoting activity. (Note 21) The composition according to Appendix 20, wherein the function of the keratinocytes is the proliferation of keratinocytes, differentiation into epidermal cells, and / or induction of barrier function genes. (Note 22) The composition according to Appendix 21, wherein the barrier function gene comprises a profilaggrin gene and / or a ceramide synthase gene. (Note 23) A composition according to any one of the appendices 1 to 22, which has activity to promote the function of hair papilla cells. (Note 24) The composition according to Appendix 23, wherein the function of the dermal papilla cells is the proliferation of dermal papilla cells and / or the induction of hair growth promoting genes. (Note 25) The composition according to Appendix 24, wherein the hair growth gene is the FGF7 gene and / or the VEGF gene. (Note 26) The composition according to any one of the appendices 1 to 25, wherein the megakaryocyte culture is a culture from which platelets have been removed. (Note 27) The composition according to any one of the appendices 1 to 26, wherein the megakaryocytes are immortalized megakaryocytes. (Note 28) The composition described in Appendix 27, wherein the immortalized megakaryocytes are megakaryocytes containing exogenous BMI1 genes, MYC genes, and Bcl-xL genes. (Note 29) The megakaryocytes mentioned above are in vitro A composition according to any one of the appendices 1 to 28, wherein megakaryocytes are induced by [the specified method]. (Note 30) The megakaryocytes are derived from pluripotent cells, as described in any of Appendix 1 to 29. (Note 31) The composition described in Appendix 30, wherein the pluripotent cells are induced pluripotent stem (iPS) cells. (Note 32) The processed material is Process megakaryocytes or their cultures, The composition according to any one of the appendices 1 to 31, wherein the treatment is a concentration treatment, a drying treatment, a freeze treatment, a freeze-drying treatment, a solvent treatment, a surfactant treatment, an enzyme treatment, a protein fractionation and extraction treatment, an ultrasonic treatment, and / or a crushing treatment. (Note 33) The processed material is Platelets are removed from the megakaryocytes or their cultures. The composition according to Appendix 32, which is used to treat megakaryocytes or cultures from which the platelets have been removed. (Note 34) The processed material is The megakaryocytes or cultures from which the platelets have been removed are stored. The composition according to Appendix 33 for processing the stored megakaryocytes or their cultures. (Note 35) The processed material is The megakaryocyte or its culture is stored, The composition according to Appendix 34, wherein the stored megakaryocytes or their cultures are subjected to a crushing treatment. <Method for producing the composition> (Note 36) The process includes a step for processing megakaryocytes or their cultures, A method for producing a composition, wherein the processing steps include concentration, drying, freezing, freeze-drying, solvent treatment, surfactant treatment, enzyme treatment, protein fractionation and extraction, sonication, and / or crushing. (Note 37) The process includes removing platelets from the megakaryocytes or their culture, The manufacturing method according to Appendix 36, wherein the processing step involves processing the megakaryocytes or cultures from which the platelets have been removed. (Note 38) The process includes a preservation step of storing megakaryocytes or cultures from which the platelets have been removed, The manufacturing method according to Appendix 37, wherein the processing step involves processing the stored megakaryocytes or their cultures. (Note 39) The process includes a preservation step of storing the megakaryocyte or its culture, The manufacturing method according to Appendix 38, wherein the processing step involves crushing the stored megakaryocytes or their cultures. (Note 40) A composition obtained by the manufacturing method described in any of the appendices 36 to 39. <Cell proliferation promoting composition> (Note 41) A cell proliferation-promoting composition comprising any of the compositions described in Appendix 1 to 35. (Note 42) The cell proliferation-promoting composition according to Appendix 42, wherein the cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells. <Methods for promoting cell proliferation> (Note 43) A method for promoting cell proliferation using the cell proliferation-promoting composition described in Appendix 41 or 42. (Note 44) The method for promoting cell proliferation according to Appendix 43, wherein the cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells. (Note 45) The cell proliferation promoting composition in vitro or in vitro A method for promoting cell proliferation as described in Appendix 43 or 44, used in [the specified application]. <Composition to promote the function of fibroblasts> (Note 46) A fibroblast function-promoting composition comprising any of the compositions described in Appendix 1 to 35. (Note 47) The function-promoting composition according to Appendix 46, wherein the function of the fibroblasts is the proliferation of fibroblasts and / or the production of extracellular matrix by fibroblasts. (Note 48) The extracellular matrix is ​​the function-enhancing composition described in Appendix 47, comprising collagen and / or hyaluronic acid. <Methods for promoting fibroblast function> (Note 49) A method for promoting the function of fibroblasts, using a fibroblast function-promoting composition described in any of Appendix 46 to 48. (Note 50) The fibroblast function-promoting composition is in vivo or in vitro A method for promoting the function of fibroblasts, as described in Appendix 50, for use in this context. <Composition to promote the healing of skin disorders> (Note 51) A composition for promoting the healing of skin disorders, comprising any of the compositions described in Appendix 1 to 35. (Note 52) The healing-promoting composition according to Appendix 51, wherein the skin disorder is a skin ulcer, pressure ulcer, burn, scar, and / or wound. <Methods to promote healing of skin disorders> (Note 53) A method for promoting the healing of skin disorders, using the skin disorder healing-promoting composition described in Appendix 51 or 52. (Note 54) The aforementioned composition for promoting the healing of skin disorders, in vivo or in vitro A method for promoting the healing of skin disorders as described in Appendix 53, to be used in [the specified context]. <Composition to promote the function of keratinocytes> (Note 55) A composition that promotes the function of keratinocytes, comprising any of the compositions described in Appendix 1 to 35. (Note 56) The function-enhancing composition according to Appendix 55, wherein the function of the keratinocytes is the proliferation of keratinocytes, differentiation into epidermal cells, and / or induction of barrier function genes. (Note 57) The barrier function gene is a profilaggrin gene and / or a ceramide synthase gene, as described in Appendix 56 of the function-enhancing composition. <Methods for promoting the function of keratinocytes> (Note 58) A method for promoting the function of keratinocytes, using a keratinocyte function-promoting composition described in any of appendices 55 to 57. (Note 59) The keratinocyte function-promoting composition is in vivo or invitro A method for promoting the function of keratinocytes as described in Appendix 58, used for this purpose. <Composition to promote the function of hair papilla cells> (Note 60) A composition that promotes the function of hair papilla cells, comprising any of the compositions described in Appendix 1 to 35. (Note 61) The function-enhancing composition according to Appendix 56, wherein the function of the dermal papilla cells is the proliferation of dermal papilla cells and / or the induction of hair growth-promoting genes. (Note 62) The barrier function gene is a profilaggrin gene and / or a ceramide synthase gene, as described in Appendix 57 of the function-enhancing composition. <Methods for promoting the function of hair papilla cells> (Note 63) A method for promoting the function of dermal papilla cells, using a dermal papilla cell function-promoting composition described in any of appendices 60 to 62. (Note 64) The aforementioned hair papilla cell function-promoting composition, in vivo or in vitro A method for promoting the function of hair papilla cells, as described in Appendix 63, used in [the specified context]. <Hair growth promoting composition> (Note 65) A hair growth promoting composition comprising any of the compositions described in Appendix 1 to 35. <Methods to promote hair growth> (Note 66) A method for promoting hair growth using the hair growth promoting composition described in Appendix 65. (Note 67) The hair growth promoting composition is in vivo or in vitro in vivo The hair growth promotion method described in Appendix 66, to be used. <Use of composition> (Note 68) A composition containing megakaryocytes or a treated culture thereof as an active ingredient for use in promoting cell proliferation. (Note 69) A composition comprising megakaryocytes or a treated culture thereof as an active ingredient for use in promoting the function of fibroblasts. (Note 70) A composition containing a processed product of megakaryocytes or their cultures as an active ingredient for use in promoting the healing of skin disorders. (Note 71) A composition comprising, as an active ingredient, a processed product of megakaryocytes or their cultures for use in promoting the function of keratinocytes. (Note 72) A composition containing megakaryocytes or a treated culture thereof as an active ingredient for use in promoting the function of dermal papilla cells. (Note 73) A composition containing megakaryocytes or a treated culture thereof as an active ingredient for use in promoting hair growth. [Industrial applicability]

[0216] As described above, the present invention provides a composition having cell-derived physiological activity. Furthermore, the composition of the present invention can promote the proliferation of cells such as mesenchymal cells, fibroblasts, keratinocytes, and dermal papilla cells, and is expected to be suitably used to promote the healing of skin disorders such as skin ulcers, pressure sores, burns, scars, and wounds, maintain or improve the skin's barrier function, and promote hair growth. For this reason, the present invention is extremely useful in the pharmaceutical field, the field of regenerative medicine, and the like.

Claims

1. Contains an extract of megakaryocytes after platelet release. The megakaryocytes after platelet release are obtained by culturing the megakaryocytes for 4 days to 2 weeks to obtain a mixture containing platelets, and then removing the platelets from the mixture.

2. The composition according to claim 1, wherein the extract contains 2,000 to 20,000 pg of basic fibroblast growth factor (bFGF) per 1 mg of total protein.

3. The composition according to claim 1 or 2, wherein the extract contains 8,000 to 80,000 pg of insulin-like growth factor-binding protein-2 (IGFB-2) per 1 mg of total protein.

4. The composition according to any one of claims 1 to 3, wherein the extract contains 1 to 60 pg of placental growth factor (PIGF) per 1 mg of total protein.

5. The composition according to any one of claims 1 to 4, wherein the extract contains 200 to 2000 pg of stem cell factor receptors (SCFRs) per 1 mg of total protein.

6. The composition according to any one of claims 1 to 5, wherein the extract contains 20 to 800 pg of vascular endothelial growth factor (VEGF) per 1 mg of total protein.

7. The composition according to any one of claims 1 to 6, wherein the extract contains 20 to 400 pg of vascular endothelial growth factor receptor 2 (VEGFR2) per 1 mg of total protein.

8. The composition according to any one of claims 1 to 7, wherein the extract contains 1,000 to 10,000 pg of differentiation and growth factor-15 (GDF-15) per 1 mg of total protein.

9. The composition according to any one of claims 1 to 8, wherein the extract contains 0 to 16 pg of amphiregulin (AR) per 1 mg of total protein.

10. The composition according to any one of claims 1 to 9, wherein the extract contains 0 to 60 pg of epidermal growth factor receptor (EGFR) per 1 mg of total protein.

11. The composition according to any one of claims 1 to 10, wherein the extract contains 0 to 100 pg of liver growth factor (HGF) per 1 mg of total protein.

12. The composition according to any one of claims 1 to 11, wherein the extract contains 0 to 200 pg of insulin-like growth factor-binding protein-1 (IGFB-1) per 1 mg of total protein.

13. A composition according to any one of claims 1 to 12, having cell proliferation-promoting activity.

14. The composition according to claim 13, wherein the cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells.

15. A composition according to any one of claims 1 to 14, having fibroblast function-promoting activity.

16. The composition according to claim 15, wherein the function of the fibroblasts is the proliferation of fibroblasts and / or the production of extracellular matrix by fibroblasts.

17. A composition according to any one of claims 1 to 16, having keratinocyte function-promoting activity.

18. The composition according to claim 17, wherein the function of the keratinocytes is proliferation of keratinocytes, differentiation into epidermal cells, and / or induction of barrier function genes.

19. A composition according to any one of claims 1 to 18, having activity to promote the function of hair papilla cells.

20. The composition according to claim 19, wherein the function of the dermal papilla cells is the proliferation of dermal papilla cells and / or the induction of hair growth promoting genes.

21. The composition according to any one of claims 1 to 20, wherein the megakaryocytes are immortalized megakaryocytes.

22. The composition according to any one of claims 1 to 21, wherein the megakaryocytes are megakaryocytes induced in vitro.

23. A composition for promoting cell proliferation, comprising the composition according to any one of claims 1 to 22.

24. The composition for promoting cell proliferation according to claim 23, wherein the cells are mesenchymal stem cells.

25. A composition for promoting the function of fibroblasts, comprising the composition according to any one of claims 1 to 22.

26. The composition for promoting the function of the fibroblasts according to claim 25, wherein the function of the fibroblasts is the proliferation of fibroblasts and / or the production of extracellular matrix by fibroblasts.

27. A composition for use in promoting the healing of skin disorders, comprising the composition according to any one of claims 1 to 22.

28. The composition for promoting healing according to claim 27, wherein the skin disorder is a skin ulcer, pressure ulcer, burn, scar, and / or wound.

29. A composition for use in promoting the function of keratinocytes, comprising the composition according to any one of claims 1 to 22.

30. The composition for promoting the function of keratinocytes according to claim 29, wherein the function of the keratinocytes is the proliferation of keratinocytes, differentiation into epidermal cells, and / or induction of barrier function genes.

31. A composition for use in promoting the function of hair papilla cells, comprising the composition according to any one of claims 1 to 22.

32. The composition for promoting the function of the dermal papilla cells according to claim 31, wherein the function of the dermal papilla cells is the proliferation of dermal papilla cells and / or the induction of hair growth promoting genes.

33. A composition for promoting hair growth, comprising the composition described in any one of claims 1 to 22.

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