Composition and use thereof

Abstract

Cell-derived composition having physiological activity SOLUTION: The composition of the present invention contains a processed product of megakaryocytes or a culture thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition and its use. [Background technology]

[0002] Attempts have been made to develop cell preparations containing cells that promote tissue regeneration, such as mesenchymal stem cells, as therapeutic agents for regenerative medicine. Furthermore, the cells that promote tissue regeneration are thought to promote tissue regeneration by releasing physiologically active proteins, such as growth factors. Summary of the Invention [Problem to be solved by the invention]

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

[0004] To achieve the above object, the composition of the present invention comprises a processed product of megakaryocytes or a culture thereof.

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

[0006] The composition for promoting fibroblast function of the present invention includes the composition of the present invention.

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

[0008] The composition for promoting keratinocyte function of the present invention includes the composition of the present invention.

[0009] The composition for promoting the function of hair papilla cells of the present invention comprises the composition of the present invention.

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

[0011] According to the present invention, a composition having cell-derived physiological activity can be provided. [Brief explanation of the drawings]

[0012]

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[0013] <Composition> As described above, the composition of the present invention comprises a processed product of megakaryocytes or a culture thereof. As described above, the composition of the present invention is characterized by comprising a processed product of megakaryocytes or a culture thereof, and other components and conditions are not particularly limited. The composition of the present invention can provide, for example, a composition having cell-derived physiological activity. 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 suitable for use in promoting the healing of skin disorders such as skin ulcers, pressure sores, burns, scars, wounds, and skin aging, maintaining or improving the skin's barrier function, and promoting hair growth.

[0014] In the present invention, "megakaryocytes" are the largest cells present in the bone marrow in vivo, and refer to cells that release platelets and cells with equivalent functions. The cells with equivalent functions refer to cells capable of producing platelets. In the present invention, megakaryocytes may be megakaryocytes before multinucleation (polyploidization), i.e., immature megakaryocytes or megakaryocytes in the proliferative phase, or megakaryocytes after multinucleation (polynucleated megakaryocytes). Specific examples of megakaryocytes include promegakaryocytes, megakaryoblasts, promegakaryocytes, and mature megakaryocytes. The number of chromosome sets possessed by the megakaryocytes after multinucleation may be more than two sets, specifically 16 to 32 sets.

[0015] The origin of the megakaryocytes is not particularly limited, and examples thereof include humans and non-human animals. Examples of the 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 same applies to the origins of other cells hereinafter.

[0016] In the present invention, the megakaryocytes can be identified by cell surface markers. When the megakaryocytes are derived from humans, 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 derived from humans, the cell surface marker may be, for example, at least one selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c.

[0017] The megakaryocytes may be megakaryocytes isolated from a living body, or may be megakaryocytes induced from cells less differentiated than megakaryocytes, such as pluripotent cells (hereinafter also referred to as "progenitor cells"). The "cells less differentiated than megakaryocytes" refer to cells capable of differentiating into megakaryocytes.

[0018] When the megakaryocytes are isolated from a living body, the megakaryocytes can be isolated from bone marrow, for example, since they are present in bone marrow. In this case, the megakaryocytes may contain other cells derived from the living body.

[0019] When the megakaryocytes are megakaryocytes induced from precursor cells, the megakaryocytes are obtained by the following steps as described below: in vitro In this case, the megakaryocytes may contain the progenitor cells. Examples of the progenitor cells include hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, megakaryocyte-erythroid progenitor cells (MEPs), and megakaryocyte progenitor cells. The progenitor cells may be isolated from bone marrow, umbilical cord blood, peripheral blood, or the like, or may be induced from pluripotent cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), nuclear transfer ES cells (ntES cells), germ stem cells, somatic stem cells, and embryonic tumor cells.

[0020] When the megakaryocytes are megakaryocytes induced from precursor cells, the megakaryocytes are preferably immortalized megakaryocytes. The immortalized megakaryocytes have a higher degree of homogeneity in the differentiation stage of cells compared with, for example, megakaryocytes induced by other megakaryocyte induction methods, and therefore, variations in the component composition of the resulting processed product can be suppressed. The immortalized megakaryocytes are, for example, megakaryocytes induced by introducing an oncogene and a polycomb gene, or an oncogene, a polycomb gene, and an apoptosis-inhibiting gene, into the precursor cells, as described below.

[0021] The term "oncogene" refers to a gene capable of inducing canceration of cells in vivo, and examples thereof include MYC family genes such as c-MYC, N-MYC, and L-MYC, SRC family genes, RAS family genes, RAF family genes, and protein kinase family genes such as c-kit (CD117), PDGFR (platelet growth factor receptor), and Abl (Abelson murine leukemia viral oncogene homolog).

[0022] The "polycomb genes" refer to genes known to negatively regulate CDKN2a (cyclin-dependent kinase inhibitor 2A, INK4a / ARF) and function to prevent cellular senescence (see References 1 to 3 below). Specific examples of the polycomb genes include 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), and Dnmt (DNA (cytosine-5)-methyltransferase) 1 / 3a / 3b. Reference 1: Hideyuki Oguro et al., "Control of stem cell aging by polycomb group protein complexes," 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 "apoptosis-suppressing gene" refers to a gene that has the function of suppressing cellular apoptosis, and examples thereof 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. The term "exogenous" refers to a gene introduced into a cell from outside the cell. The exogenous gene may be present on the chromosome of the cell, or in the nucleus or cytoplasm. The exogenous gene can be detected, for example, by measuring the number of genes. When the gene is an autosomal gene, one copy of the gene is present on each autosome, resulting in two genes present in a single cell. Therefore, if the exogenous gene is absent, two copies of the gene are detected in a single cell. On the other hand, if the exogenous gene is present, three or more copies of the gene are detected in a single cell. In this case, the exogenous gene can be detected using, for example, PCR using primers, a probe, or a combination thereof. If the exogenous gene has a tag sequence or a selection marker, the exogenous gene can be detected by detecting the tag sequence or the selection marker. The exogenous gene can also be detected, for example, using an antibody against the protein translated from the gene.

[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 medium, as described below.

[0026] The megakaryocyte culture is obtained by culturing the megakaryocytes, and is therefore, for example, a mixture containing the megakaryocytes and platelets produced from the megakaryocytes as cellular components. The cellular components refer to cells and platelets. As described above, the megakaryocytes can be induced from cells less differentiated than the megakaryocytes. Therefore, when megakaryocytes for producing the megakaryocyte culture are induced from cells less differentiated than the megakaryocytes, the megakaryocyte culture may also contain cells less differentiated than the megakaryocytes.

[0027] In the present invention, the megakaryocyte culture may be the culture obtained by culturing the megakaryocytes, or may be a processed version of the culture. Examples of processing the culture include removing the liquid fraction, extracting a 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 "platelets" refers to a cellular component in blood that is positive for CD41a and CD42b. Platelets, for example, lack a nucleus and are smaller than megakaryocytes. Therefore, platelets can be distinguished from megakaryocytes by, for example, the presence or absence of a nucleus and / or their size. Platelets play an important role in thrombus formation and hemostasis, and are also known to be involved in the pathophysiology of tissue regeneration and inflammation after injury. Furthermore, when platelets are activated by bleeding or other causes, they express receptors for cell adhesion molecules, such as integrin αIIBβ3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61), on their membranes. Furthermore, upon activation, platelets aggregate, and various blood coagulation factors released from the platelets coagulate fibrin, forming a thrombus and promoting hemostasis. In the present invention, the platelets are derived from the same source as the megakaryocytes.

[0029] In the present invention, the processed product may be prepared from the megakaryocytes or from a culture of the megakaryocytes. Furthermore, when prepared from a culture of megakaryocytes, the megakaryocyte culture may be processed. Specifically, the processed product may be, for example, a processed product of a cell fraction or liquid fraction of the megakaryocytes or a culture thereof, or a processed product of the megakaryocytes or a culture thereof. The treatment used in preparing the processed product is not particularly limited, and examples thereof include treatments for changing the density of cellular components, such as concentration treatment, separation treatment, or purification treatment; extraction treatments for extracting cellular components, such as drying treatment, freezing treatment, freeze-drying treatment, solvent treatment, surfactant treatment, enzyme treatment, and protein fraction extraction treatment; and disruption treatments, such as grinding treatment and pulverization treatment. Specific examples of the processed product include extracts of the megakaryocytes or cultures thereof, such as concentrates, dried products, frozen products, lyophilized products, solvent-treated products, surfactant-treated products, enzyme-treated products, protein fractions, and ultrasonicated products; disrupted products such as grinding products and pulverized products; extracts of the megakaryocytes or cell fractions thereof, such as concentrates, dried products, frozen products, lyophilized products, solvent-treated products, surfactant-treated products, enzyme-treated products, protein fractions, and ultrasonicated products; and disrupted products such as grinding products and pulverized products. The processed product may consist of one type of processed product, or may be 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 contains, for example, at least one of one or more growth factors and growth factor receptors. The processed product also has physiological activity, such as cell proliferation-promoting activity. Furthermore, the processed product can be produced, for example, by treating the megakaryocytes or a culture thereof, as described above. Therefore, in the present invention, the processed product can be specified, for example, using the following conditions (1) to (3). The processed product may be specified, for example, by any one of the following conditions (1) to (3), or by a plurality of conditions, or by all of the conditions. Specifically, the processed product can be specified, for example, by a combination of conditions. (conditions) (1) growth factor and / or growth factor receptor content; (2) Physiological activity of the treated product; (3) Manufacturing method of processed products (Combination of conditions) Condition (1), (2), or (3); Conditions (1) and (2), conditions (1) and (3), or conditions (2) and (3); Conditions (1), (2), and (3)

[0031] (1) Condition (1) As described above, condition (1) is a condition regarding 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 may specify the content of the growth factors and the content of the growth factor receptors. Furthermore, the growth factors used in the definition of condition (1) may be one type or two or more types. The growth factor receptors used in the definition of condition (1) may be one type or two or more types.

[0032] In the condition (1), examples of the growth factor 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), and transforming growth factor β1 (TGFβ1).

[0033] In the condition (1), the growth factor receptor may be stem cell factor receptor (SCFR), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor 2 (VEGFR2), or the like.

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

[0035] The weight of the total protein can be determined, for example, by the BCA protein assay. The BCA protein assay is a protein assay method that utilizes the coordinate bond between a monovalent copper ion and two molecules of bicinchoninic acid. It is preferable that the sample subjected to the BCA protein assay does not contain, for example, a reducing agent and / or a copper ion chelating agent. The BCA protein assay can be performed according to Reference 4 below, and a commercially available kit may be used, for example. Examples of kits that can be used for the BCA protein assay include the Pierce™ BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Reference 4: Toshiharu Hase et al., "Protein Science Experiments Starting from Simple Principles 1: Protein Creation, Extraction, Purification and Synthesis," Kagaku Dojin, December 13, 2008

[0036] The total protein concentration in the processed product can be appropriately set, for example, depending on the number of cells subjected to the treatment and the volume of the solvent in the processed product.The total protein concentration in the processed product can be relatively increased, for example, by increasing the number of cells subjected to the treatment, reducing the volume of the solvent in the processed product, or extracting from the megakaryocytes.Furthermore, the total protein concentration in the processed product can be relatively decreased, for example, by reducing the number of cells subjected to the treatment, increasing the volume of the solvent in the processed product, or extracting from a culture of megakaryocytes.As a specific example, when the number of cells subjected to the treatment is 1 x 10 8 When the treated product is a cell and the volume of the 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 described below.

[0037] The weights of the growth factors and growth factor receptors can be determined, for example, by sandwich ELISA. The sandwich ELISA can be performed according to the method described in Reference 5 below, and a commercially available kit may be used. Examples of kits that can be used for the sandwich ELISA include Quantibody (registered trademark) Human Growth Factor Array 1 (manufactured by RayBiotech). Reference 5: "Immunoassays: From Basics to Cutting Edge," edited by the Biochemical Measurement Research Group, Kodansha, December 20, 2014

[0038] The contents of the growth factors and the growth factor receptors in the treated material may be, for example, as follows:

[0039] When the growth factor is bFGF, the processed product contains, for example, 2,000 to 20,000 pg, 5,000 to 20,000 pg, or 10,000 to 20,000 pg of bFGF per 1 mg of total protein. By containing bFGF, the processed product exhibits cell proliferation-promoting activity, as described below.

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

[0041] When the growth factor is IGFBP-2, the treated 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 mg of total protein.

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

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

[0044] When the growth factor is GDF-15, the treated product contains 1000 to 10000 pg, 1000 to 5000 pg, or 2000 to 5000 pg of GDF-15 per 1 mg of total protein.

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

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

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

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

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

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

[0051] As described above, the condition (1) may be defined by the content of one or more growth factors, the content of one or more growth factor receptors, or any combination of these contents. In this case, the condition (1) is defined by, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 conditions selected from the group consisting of the following conditions (A1) to (A9) and (B1) to (B3). Specific examples of combinations of the content of growth factors and / or the content of growth factor receptors include the following combinations: (Combination of growth factor content and / or growth factor receptor content) Any one of the conditions (A1) to (A9) and (B1) to (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 conditions (A1) to (A9) and (B1) to (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 proteins other than the growth factors and growth factor receptors depending on the intended use of the composition of the present invention. Examples of the other proteins include proteins that do not affect the activity of the growth factors and growth factor receptors, such as serum albumins (e.g., human serum albumin) and gamma globulins (e.g., human gamma globulin). The proteins can be removed, for example, by using a column or an antibody.

[0053] (2) Condition (2) As described above, condition (2) is a condition regarding the physiological activity of the processed product. In condition (2), the physiological activity of the processed product refers to, for example, the activity of regulating the function of a cell, tissue, or organ. Examples of the cellular function include proliferation, differentiation, induction or suppression of gene expression, and induction or suppression of the expression of biopolymers such as proteins and sugar chains.

[0054] Examples of the physiological activity of the processed product include cell proliferation-promoting activity, fibroblast function-promoting activity, keratinocyte function-promoting activity, and dermal papilla cell function-promoting activity. In the cell proliferation-promoting activity, the cells are not particularly limited, and examples include mesenchymal stem cells, fibroblasts, keratinocytes such as epidermal keratinocytes, and dermal papilla cells such as scalp dermal papilla cells. The processed product may have, for example, 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 CD73, CD90, and CD105 positive, and CD14, CD34, and CD45 negative.

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

[0057] The keratinocytes are epidermal cells that have the ability to keratinize, divide in the basal layer of the epidermis, and contribute to epidermal formation. The keratinocytes can be identified by cell surface markers. The keratinocytes are, for example, androgen receptor- and cytokeratin-positive. The keratinocytes are preferably epidermal keratinocytes.

[0058] The dermal papilla cells are cells present in the dermal papilla at the base of the hair follicle and are important 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; SOX2 gene, WIF1 gene, Noggin gene, BMP4 gene, or VCAN gene-positive; and the dermal papilla cells are preferably scalp dermal papilla cells.

[0059] The proliferation-promoting activity of the cells may be, for example, an improvement in the proliferation ability of the cells compared to a control group that is otherwise identical except for the addition of the composition of the present invention; for example, the proliferation ability of the cells may be decreased from the start. In this case, the "proliferation-promoting activity" can also be expressed as, for example, suppression of a decrease in proliferation activity. As a specific example, the proliferation activity of the mesenchymal stem cells decreases with each passage. Since the composition of the present invention can suppress a decrease in the proliferation activity of the mesenchymal stem cells, it can be said that the composition of the present invention exhibits proliferation-promoting activity, for example. 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 depending on, for example, the type of the cells. As a specific example, when mesenchymal stem cells are used as the cells, the proliferation activity of the cells can be measured, for example, by culturing human-derived mesenchymal stem cells in the presence of a proliferation medium. in vitro The proliferation activity of the mesenchymal stem cells can be measured, for example, based on Example 1 described below. 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 proliferation medium. in vitro The proliferation activity of the fibroblasts can be measured, for example, based on 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 proliferation medium. in vitro The proliferation activity of the keratinocytes can be measured, for example, based on 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 proliferation medium. invitro 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 may be, for example, an improvement in fibroblast function compared to a control group that is otherwise identical except for the addition of the composition of the present invention. The fibroblast function may refer to, for example, either the proliferation of the fibroblasts or the production of extracellular matrix by the fibroblasts. Examples of the extracellular matrix include collagens such as type I collagen, fibrous substances such as elastin, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, proteoglycans, and matrix materials such as cell adhesion proteins such as integrins, fibronectin, and laminin. The fibroblast function can be measured, for example, according to Example 3 described below.

[0061] The keratinocyte function-promoting activity may be, for example, an improvement in keratinocyte function compared to a control group that is otherwise identical but does not contain the composition of the present invention. The keratinocyte function may refer to, for example, the proliferation of the keratinocytes, differentiation into epidermal cells, or induction of barrier function genes. The barrier function gene refers to a gene that functions to maintain the skin's barrier function, and specific examples include the profilaggrin gene (FLG) and the ceramide synthase gene (serine palmitoyltransferase long chain base subunit 1: SPTLC1). Examples of the profilaggrin gene include a polynucleotide consisting of the nucleotide sequence registered in GenBank under Accession No. NM_002016.2. Examples of the ceramide synthase gene include a polynucleotide consisting of the nucleotide sequence registered in GenBank under Accession No. NM_001281303.2. The keratinocyte function can be measured, for example, according to Example 4 described below.

[0062] The activity of promoting the function of dermal papilla cells may be, for example, an improvement in the function of dermal papilla cells compared to a control group that is otherwise identical but does not contain the composition of the present invention. The function of dermal papilla cells may refer to, for example, the proliferation of pre-dermal 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 hair maintenance, and specific examples include the FGF7 (fibroblast growth factor 7) gene and the vascular endothelial growth factor (VEGF) gene. The VEGF may be, for example, VEGFA. As the FGF7 gene, for example, a human proFGF7 gene may be a polynucleotide consisting of the nucleotide sequence registered in GenBank under Accession No. NM_002009.4. As the VEGFA gene, for example, a human VEGFA gene may be a polynucleotide consisting of the nucleotide sequence registered in GenBank under Accession No. NM_001025366.3. The function of the dermal papilla cells can be measured, for example, based on Example 5 described below.

[0063] (3) Conditions (3) As described above, the condition (3) is a condition related to the method for producing the treated product. The method for producing the treated product of the composition of the present invention, which will be described later, can be applied to the method for producing the treated product of the composition of the present invention.

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

[0065] The composition of the present invention can be used, for example, as a composition for promoting cell proliferation, a composition for promoting fibroblast function, a composition for promoting keratinocyte function, a composition for promoting dermal papilla cell function, etc., as described below. Furthermore, due to the above-described activity, the composition of the present invention is expected to be suitably used, for example, as a composition for promoting the healing of skin disorders and a composition for promoting hair growth. The explanations for the composition for promoting cell proliferation, the composition for promoting fibroblast function, the composition for promoting keratinocyte function, and the composition for promoting dermal papilla cell function described below can be applied to the method of use of the present invention. Furthermore, the composition of the present invention can be used, for example, for repairing knee joint injury, tendon injury, or ligament injury; treating ulcers, pressure ulcers, burns, scars, or wounds; improving skin texture; hair growth; and / or skin beautification.

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

[0067] In the production method of the present invention, the treatment target in the treatment step is megakaryocytes or a culture thereof. Therefore, the production method of the present invention may include, prior to the treatment step, a megakaryocyte induction step of inducing the megakaryocytes from cells less differentiated than megakaryocytes and / or a production step of producing a culture of megakaryocytes.

[0068] In the megakaryocyte induction step, the method for inducing megakaryocytes is not particularly limited and can be performed using known induction methods. Specific examples of the megakaryocyte induction method include those described in International Publication No. 2011 / 034073 (U.S. Patent Application Publication No. 2012 / 0238023), International Publication No. 2012 / 157586 (U.S. Patent Application Publication No. 2014 / 0127815), and International Publication No. 2014 / 123242 (U.S. Patent Application Publication No. 2016 / 0002599), as well as the megakaryocyte induction method described in Reference 6 below, which are incorporated herein by reference. Specific examples of the megakaryocyte induction step include forcibly expressing the oncogene and the polycomb gene in cells less differentiated than megakaryocytes. As a result, in the megakaryocyte induction step, for example, immortalized megakaryocytes that proliferate indefinitely can be obtained. Furthermore, for example, by canceling the forced expression of the immortalized megakaryocytes, the immortalized megakaryocytes can be induced to become multinucleated megakaryocytes, allowing platelets to be produced. Furthermore, in the megakaryocyte induction step, for example, the apoptosis-inhibiting gene can be forced to be expressed in the megakaryocyte progenitor cells. As a result, in the megakaryocyte induction step, the immortalized megakaryocytes can be obtained. Furthermore, for example, by canceling the forced expression of the immortalized megakaryocytes, the immortalized megakaryocytes can be induced to become multinucleated megakaryocytes, allowing platelets to be produced. 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-inhibiting gene may be forcedly expressed. In this case, the forced expression of the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be performed simultaneously or separately. Specifically, in the megakaryocyte induction step, the oncogene and the polycomb gene may be forcedly expressed, and then the apoptosis-inhibiting gene may be forcedly expressed, or the oncogene, the polycomb gene, and the apoptosis-inhibiting gene may be forcedly expressed, or the oncogene and the polycomb gene may be forcedly expressed, and then the apoptosis-inhibiting gene may be expressed. In this way, the megakaryocyte induction step can obtain the immortalized megakaryocytes. Furthermore, for example, by canceling the forced expression of the immortalized megakaryocytes, multinucleated megakaryocytes can be induced from the immortalized megakaryocytes, allowing platelets to be produced.

[0070] The megakaryocyte induction step preferably includes a first expression step of forcibly expressing an oncogene and a polycomb gene in cells less differentiated than the megakaryocytes, a second expression step of forcibly expressing an apoptosis-suppressing gene such as the Bcl-xL gene in the undifferentiated cells, and a release step of releasing all of the forced expression, since this can improve the efficiency of introducing each gene, for example.

[0071] Forced expression and deactivation of forced expression of each gene can be carried out by known methods, such as those described in International Publication Nos. 2011 / 034073, 2012 / 157586, and 2014 / 123242, or Reference 7 listed below, or by methods equivalent thereto, which are incorporated herein by reference. Specifically, forced expression and deactivation of forced expression of each gene can be carried out using, for example, a drug-responsive gene expression induction system. Examples of such gene expression induction systems include the Tet-on (registered trademark) system and the Tet-off (registered trademark) system. When using the Tet-on system, for example, the forced expression step involves culturing in the presence of a drug that induces gene expression, such as tetracycline or doxycycline, and the deactivation step involves culturing 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, in the production step, a culture of megakaryocytes is produced. The production step can be carried out, for example, by culturing the megakaryocytes in the presence of a medium. The megakaryocytes may be cultured, for example, on feeder cells or without feeder cells. The megakaryocytes can be cultured, for example, in suspension culture, and therefore can be cultured without feeder cells. The megakaryocyte culture contains, for example, the platelets.

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

[0074] The medium is not particularly limited and may be, for example, a known medium suitable for producing platelets from megakaryocytes or a medium similar thereto. Specifically, the medium may be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include 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, RPMI 1640 medium, Fischer's medium, and Neurobasal® Medium (manufactured by Thermo Fisher Scientific), as well as mixtures thereof. The medium may contain, for example, serum or plasma, or may be a serum-free medium that does not contain these. The serum and plasma are preferably derived from the same source as the megakaryocytes. Specifically, when the megakaryocytes are derived from humans, the serum and plasma are preferably derived from humans.

[0075] The medium may contain, for example, other components. The 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, pyruvic acid, buffers, inorganic salts, cytokines, and the like. The other components may be, for example, one type or two or more types. Cytokines are 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, and aryl hydrocarbon receptor (AhR) inhibitors. The medium is preferably an IMDM medium containing, for example, serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. The medium may further contain, for example, SCF, or 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, or 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, or about 50 ng / ml. The concentration of heparin is, for example, about 10 U / ml to about 100 U / ml, or about 25 U / ml. The medium may further contain, for example, a phorbol ester (for example, phorbol-12-myristate-13-acetate; PMA).

[0076] Next, in the treatment step, the megakaryocytes or a culture thereof are treated. In the treatment step, for example, the cell membranes of the cells contained in the megakaryocytes or a culture thereof are destroyed to extract proteins. Specific examples of the treatment in the treatment step are not particularly limited, and include treatments that change the density of cellular components, such as concentration treatments; extraction treatments that extract cellular components, such as drying treatments, freezing treatments, freeze-drying treatments, solvent treatments, surfactant treatments, enzyme treatments, protein fraction extraction treatments, and ultrasonic treatments; and disruption treatments, such as grinding treatments and pulverization treatments. The treatment step may involve one type of treatment or two or more types of treatments. Furthermore, in the treatment step, the treatment may be carried out once or two or more times.

[0077] The concentration treatment can be carried out, for example, by centrifuging the megakaryocytes or a culture thereof. 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 a culture thereof with a dry spray, a drum dryer, or the like. The freeze-drying treatment can be carried out, for example, using a freeze dryer. In the solvent treatment, the solvent can be, for example, an organic solvent such as phenol or chloroform; or an aqueous solvent such as water, physiological saline, or a 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 an ultrasonic treatment, which will be described later. The solvent treatment can be carried out, for example, by mixing the megakaryocytes or a culture thereof with the solvent. In the surfactant treatment, examples of the surfactant include ionic surfactants such as sodium lauryl sulfate; nonionic surfactants such as NP-40, Triton X-100, Tween 20, and n-Dodecyl-β-D-maltopyranoside; and zwitterionic surfactants such as CHAPS. The surfactant concentration is, for example, a concentration capable of disrupting the cell membrane of cellular components in the megakaryocytes or a culture thereof. The surfactant treatment can be carried out, for example, by contacting the megakaryocytes or a culture thereof with the surfactant in the presence of an aqueous solvent. The contact with the surfactant is carried out, for example, at about 0 to about 10°C. Examples of the aqueous solvent include water, physiological saline, and buffer solutions. Examples of the enzyme used in the enzyme treatment include peptidases and proteases. The enzyme treatment can be carried out, for example, by contacting the megakaryocytes or a culture thereof with the enzyme in the presence of the aqueous solvent. The enzyme treatment conditions are, for example, conditions under which the enzyme exhibits activity. The protein fraction extraction treatment can be carried out, for example, by subjecting the megakaryocytes or a culture thereof to osmotic shock, freeze-thawing, or the like. The ultrasonic treatment can be carried out, for example, using an ultrasonic generator. The ultrasonic treatment can be carried out under conditions that disrupt the cells, for example.

[0078] The treatment conditions and treatment times for various treatments can be appropriately determined depending on, for example, the type of treatment. In addition, in the treatment step, the total protein concentration in the treated product can be adjusted, for example, by adjusting the amount of the aqueous solvent.

[0079] The megakaryocytes or a culture thereof may be processed (pretreated) prior to the treatment. In this case, the megakaryocyte culture may be the culture obtained by culturing the megakaryocytes, or a processed mixture. Examples of processing the culture include removing the liquid fraction, extracting a 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] When the production method of the present invention includes the pretreatment, it may also include a removal step of removing platelets from the megakaryocytes or a culture thereof. In this case, the treatment step is performed using megakaryocytes or a culture thereof from which the platelets have been removed, or removed platelets, as the megakaryocytes or a culture thereof. Megakaryocytes after the release of platelets have a high bFGF content, for example. Therefore, the production method of the present invention can produce a composition with a high bFGF content, for example, by removing the platelets. Removal of the platelets allows separation of the platelets from other cellular fractions. Therefore, the removal step can also be referred to as, for example, a platelet separation step or a separation step of platelets from other cellular components. In the removal step, the method of separating platelets from the megakaryocyte culture can be performed using known methods, such as those described in International Publication No. 2017 / 065280 (U.S. Patent Application Publication No. 2018 / 282697), or methods equivalent thereto, and is incorporated herein by reference.

[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 to 90%.

[0082] The production method of the present invention may include a storage step of storing the megakaryocytes or a culture thereof, the platelet-removed megakaryocytes or a culture thereof, or the platelets-removed megakaryocytes. Examples of storage in the storage step include refrigerated storage (about 1 to about 10°C) and frozen storage (about -200 to about -4°C). The storage period in the storage step is not particularly limited. Frozen storage is preferred because the storage step can also serve as the freezing treatment in the treatment step.

[0083] The production method of the present invention can be carried out, for example, as follows. However, the present invention is not limited to the following example. First, a medium containing the megakaryocytes or a culture thereof is subjected to a concentration treatment by centrifugation to concentrate cellular components. The centrifugation treatment is carried out, for example, under conditions that result in the precipitation of the cellular components. Specific examples of the centrifugation treatment include centrifugation at 1000 to 3000 × g for 5 to 20 minutes. Next, the precipitate is recovered after centrifugation, and the resulting precipitate is rapidly frozen for a freezing treatment. The freezing treatment can be carried out, for example, by contacting the precipitate with a liquefied gas such as liquid nitrogen. Furthermore, the frozen precipitate is dissolved in an aqueous solvent containing the surfactant for a surfactant treatment. The resulting solution is centrifuged to precipitate impurities. The centrifugation treatment is carried out, for example, under conditions that result in the precipitation of impurities such as cell membranes. Specifically, the centrifugation can be carried out, for example, at 10,000 to 20,000 × g for 3 to 10 minutes. After the centrifugation, the protein is present in the supernatant, and the treated product can be obtained by collecting the supernatant as a protein fraction.

[0084] <Composition obtained by the production method> The composition of the present invention (hereinafter also referred to as "second composition") is obtained by the manufacturing method of the present invention. The second composition of the present invention is characterized by being obtained by the manufacturing method of the present invention, and other configurations and conditions are not particularly limited. For example, the second composition of the present invention can provide a composition having physiological activity. For example, the second composition of the present invention can promote cell proliferation. The explanations of the composition and manufacturing method of the present invention can be applied to the second composition of the present invention.

[0085] <Cell proliferation promoting composition> In another embodiment, the present invention provides a composition capable of promoting cell proliferation. As described above, the cell proliferation-promoting composition of the present invention is characterized by comprising the composition of the present invention. The proliferation-promoting composition of the present invention is characterized by comprising the composition of the present invention, and other configurations and conditions are not particularly limited. The proliferation-promoting composition of the present invention can promote the proliferation of cells, particularly mesenchymal stem cells. The proliferation-promoting composition of the present invention can be described in accordance with the above-mentioned descriptions of the composition and manufacturing method of the present invention.

[0086] The proliferation-promoting composition of the present invention comprises: in vitro You can also use in vivo It may also be used in

[0087] The growth-promoting composition of the present invention in vitro When used in the above, the administration target may be, for example, a cell, a tissue, an organ, etc., and the cell may be, for example, a cell collected from a living body, a cultured cell, etc.

[0088] The growth-promoting composition of the present invention in vivo When used in the above, the subject of administration can be, for example, a human or a non-human animal other than a human, such as a mouse, rat, rabbit, dog, sheep, horse, cat, goat, monkey, or guinea pig.

[0089] The conditions for use (administration conditions) of the proliferation-promoting composition of the present invention are not particularly limited, and the administration form, administration time, dosage, etc. can be appropriately determined depending on the type of subject to be administered, for example.

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

[0091] The growth-promoting composition of the present invention in vivo When used in this manner, the dosage can be appropriately determined depending on, for example, the type, symptoms, age, and administration method of the subject. Specifically, when administered to humans, the daily dosage of the proliferation-promoting composition is not particularly limited in terms of the total amount of protein derived from the proliferation-promoting composition, and can be appropriately determined depending on, for example, the intended use. The number of administrations per day is, for example, 1 to 5 times, 1 to 3 times, or 1 or 2 times.

[0092] The administration form of the proliferation-promoting composition of the present invention is not particularly limited. in vivo When the compound is administered intravenously, it may be administered orally or parenterally. Examples of parenteral administration include intravenous injection (intravenous administration), intramuscular injection (intramuscular administration), transdermal administration, subcutaneous administration, intradermal administration, enteral administration, rectal administration, vaginal administration, nasal administration, pulmonary administration, intraperitoneal administration, and topical administration.

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

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

[0095] <Fibroblast function-promoting composition> In another embodiment, the present invention provides a composition capable of promoting fibroblast function. As described above, the fibroblast function-promoting composition of the present invention comprises the composition of the present invention. The fibroblast function-promoting composition of the present invention is characterized by comprising the composition of the present invention, and other configurations and conditions are not particularly limited. The fibroblast function-promoting composition of the present invention can promote fibroblast function. The descriptions of the composition, manufacturing method, and proliferation-promoting composition of the present invention can be used for the fibroblast function-promoting composition of the present invention.

[0096] The composition for promoting fibroblast function of the present invention comprises: in vitro You can also use in vivo It may also be used in

[0097] The fibroblast function-promoting composition of the present invention in [[ID=4 When used in the above-described manner, the fibroblast function-promoting composition of the present invention can be used, for example, by adding it to a culture medium for the target fibroblasts. The fibroblast function-promoting composition of the present invention can promote the proliferation of the fibroblasts and / or the production of extracellular matrix by the fibroblasts, for example, by maintaining the fibroblasts in a culture medium containing the fibroblast function-promoting composition. The final concentration of total protein in the fibroblast function-promoting composition of the present invention in the culture medium is 10 to 1000 μg / ml, 10 to 500 μg / ml, or 10 to 300 μg / ml.

[0098] The subjects and conditions for administration of the composition for promoting fibroblast function of the present invention can be determined by reference to the explanations for the subjects and conditions for administration of the proliferation-promoting composition of the present invention described above.

[0099] <Composition for promoting healing of skin disorders> In another embodiment, the present invention provides a composition that can be used to promote the healing of skin disorders. The present invention provides a composition for promoting the healing of skin disorders (hereinafter also referred to as "healing-promoting composition"), as described above, which comprises the composition of the present invention. The healing-promoting composition of the present invention is characterized by comprising the composition of the present invention, and other configurations and conditions are not particularly limited. It is expected that the healing-promoting composition of the present invention can promote the healing of skin disorders. The explanations of the composition, manufacturing method, and proliferation-promoting composition of the present invention can be applied to the healing-promoting composition of the present invention.

[0100] In the present invention, the skin disorder refers to, for example, a state in which wounds, damage, etc. have occurred to the skin, i.e., a state in which the structure of normal skin tissue has been impaired or destroyed, and specific examples include skin ulcers, bedsores, burns, scars, wounds, skin aging, etc.

[0101] The healing promoting composition of the present invention comprises: ​ ​ You can also use ​ ​ It may also be used in

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

[0103] The administration target and administration conditions of the healing-promoting composition of the present invention can be determined by referring to the explanation of the administration target and administration conditions of 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] <Keratinocyte function-promoting composition> In another embodiment, the present invention provides a composition capable of promoting keratinocyte function. The keratinocyte function-promoting composition of the present invention comprises the composition of the present invention, as described above. The keratinocyte function-promoting composition of the present invention is characterized by comprising the composition of the present invention, and other configurations and conditions are not particularly limited. The keratinocyte function-promoting composition of the present invention can promote keratinocyte function. The explanations of the composition, manufacturing method, and proliferation-promoting composition of the present invention can be used for the keratinocyte function-promoting composition of the present invention.

[0105] The composition for promoting keratinocyte function of the present invention comprises: ​ ​ You can also use ​ ​ It may also be used in

[0106] The keratinocyte function-promoting composition of the present invention ​ ​ When used in the above-described manner, the keratinocyte function-promoting composition of the present invention can be used, for example, by adding it to a culture medium for the target keratinocytes. The keratinocyte function-promoting composition of the present invention may be added, for example, to a maintenance medium used to maintain the keratinocytes, or to a differentiation medium for differentiating the keratinocytes into epithelial cells such as epidermal cells, or may be added to both the maintenance medium and the differentiation medium. The keratinocyte function-promoting composition of the present invention can promote the proliferation of the keratinocytes, their differentiation into epidermal cells, and / or the induction of barrier function genes by, for example, maintaining the keratinocytes in a medium containing the keratinocyte function-promoting composition. The final concentration of total protein in the keratinocyte function-promoting composition of the present invention in the medium is 10 to 1,000 μg / ml, 10 to 500 μg / ml, or 10 to 300 μg / ml.

[0107] The subjects and conditions for administration of the composition for promoting keratinocyte function of the present invention can be determined by reference to the explanations for the subjects and conditions for administration of the proliferation-promoting composition of the present invention described above.

[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. Therefore, the keratinocyte function-promoting composition of the present invention is expected to be suitable for use, 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. Therefore, the keratinocyte function-promoting composition of the present invention is expected to be suitable for use, for example, in the regeneration of aged skin by promoting the turnover of skin tissue through its keratinocyte and fibroblast function-promoting activity. Therefore, the keratinocyte function-promoting composition of the present invention is expected to be suitable for use, for example, as a topical skin composition such as a cosmetic, an injectable solution for subcutaneous administration, etc.

[0109] <Composition for promoting the function of dermal papilla cells> In another embodiment, the present invention provides a composition capable of promoting the function of dermal papilla cells. As described above, the composition for promoting dermal papilla cell function of the present invention comprises the composition of the present invention. The composition for promoting dermal papilla cell function of the present invention is characterized by comprising the composition of the present invention, and other configurations and conditions are not particularly limited. The composition for promoting dermal papilla cell function of the present invention can promote keratinocyte function. The explanations of the composition, production method, and proliferation-promoting composition of the present invention can be applied to the composition for promoting dermal papilla cell function of the present invention.

[0110] The composition for promoting the function of hair papilla cells of the present invention comprises: ​ ​ You can also use ​ ​ It may also be used in

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

[0112] The subjects and conditions for administration of the composition for promoting the function of dermal papilla cells of the present invention can be determined by reference to the explanations for the subjects and conditions for administration of the proliferation-promoting composition of the present invention described above.

[0113] <Hair growth promoting composition> In another embodiment, the present invention provides a composition capable of promoting hair growth. As described above, the hair growth-promoting composition of the present invention comprises the composition of the present invention. The hair growth-promoting composition of the present invention is characterized by comprising the composition of the present invention, and other configurations and conditions are not particularly limited. The hair growth-promoting composition of the present invention is expected to promote hair growth by promoting the function of dermal papilla cells. The description of the composition, production method, and proliferation-promoting composition of the present invention can be applied to the composition for promoting dermal papilla cell function of the present invention.

[0114] In the present invention, "promoting hair growth" means enhancing (increasing, raising) the likelihood of hair growth from a hairless state, promoting hair growth (e.g., growth in hair length and / or thickness), and / or reducing (inhibiting, slowing down) hair loss.

[0115] The hair growth-promoting composition of the present invention may be used, for example, to prevent, suppress, or prevent hair loss. In this case, the hair growth-promoting composition of the present invention can also be referred to as, for example, a composition for preventing, suppressing, or preventing hair loss.

[0116] The hair growth promoting composition of the present invention comprises: ​ ​ You can also use ​​ It may also be used in

[0117] The hair growth promoting composition of the present invention ​ ​ When used in the above manner, the hair growth-promoting composition of the present invention can be used, for example, by adding it to a culture medium for target dermal papilla cells. The hair growth-promoting composition of the present invention can promote the proliferation of dermal papilla cells and / or the induction of hair growth-promoting genes by, for example, maintaining the dermal papilla cells in a culture medium containing the composition of the present invention, which is expected to promote hair growth. The final concentration of total protein in the hair growth-promoting composition of the present invention in the culture medium is 10 to 1,000 μg / ml, 10 to 500 μg / ml, or 10 to 300 μg / ml.

[0118] The subjects and conditions for administering the hair growth-promoting composition of the present invention can be determined by reference to the explanations for the subjects and conditions for administering the growth-promoting composition of the present invention described above and the hair growth-promoting method described below.

[0119] <Method for promoting cell proliferation> The cell proliferation promoting method of the present invention (hereinafter also referred to as "proliferation promoting method") uses the cell proliferation promoting composition of the present invention. The proliferation promoting method of the present invention is characterized by using the proliferation promoting composition of the present invention, and other steps and conditions are not particularly limited. The proliferation promoting method of the present invention can promote the proliferation of cells, particularly mesenchymal stem cells. The proliferation promoting method of the present invention can be applied to the explanations of the composition, production method, proliferation promoting composition, and differentiation promoting composition of the present invention.

[0120] The proliferation promoting method of the present invention can be carried out, for example, by ​ ​ It may be implemented by ​ ​ It may also be implemented in

[0121] The method for promoting proliferation of the present invention ​ ​When the proliferation-promoting method of the present invention is carried out in the above manner, it includes, for example, a culturing step of culturing cells in the presence of the proliferation-promoting composition. The subjects and administration conditions of the proliferation-promoting composition of the present invention can be determined by referring to the descriptions of the subjects and administration conditions of the proliferation-promoting composition of the present invention.

[0122] <Method for promoting fibroblast function> In another embodiment, the present invention provides a method capable of promoting fibroblast function. The method for promoting fibroblast function of the present invention uses the composition of the present invention. The method for promoting fibroblast function of the present invention is characterized by using the composition of the present invention, and other steps and conditions are not particularly limited. According to the method for promoting fibroblast function of the present invention, fibroblast function can be promoted. The explanations of the composition, production method, proliferation-promoting composition, and fibroblast function-promoting composition of the present invention can be used for the method for promoting fibroblast function of the present invention.

[0123] The method for promoting fibroblast function of the present invention includes, for example, ​ ​ It may be implemented by ​ ​ It may also be implemented in

[0124] The method for promoting fibroblast function of the present invention ​ ​ When the method for promoting fibroblast function of the present invention is carried out in the above manner, 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 subjects and administration conditions of the composition of the present invention can be determined by referring to the descriptions of the subjects and administration conditions of the proliferation-promoting composition of the present invention.

[0125] <Method for promoting healing of skin disorders> In another example, the present invention provides a method that can be used 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 "method for promoting healing") uses the composition of the present invention. The method for promoting healing of the present invention is characterized by using the composition of the present invention, and other steps and conditions are not particularly limited. It is expected that the method for promoting healing of skin disorders can be promoted by using the description of the composition, production method, growth-promoting composition, and composition for promoting healing of skin disorders of the present invention. The method for promoting healing of the present invention can be applied to the method for promoting healing of the present invention.

[0126] The method of the present invention for promoting healing of skin disorders can also be referred to as, for example, a method for treating or curing skin disorders.

[0127] The healing promotion method of the present invention may include, for example, a step of administering the composition of the present invention to a subject. The subject may be, for example, a patient with a skin disorder or a patient who may have a skin disorder.

[0128] The healing promotion method of the present invention includes, for example, ​ ​ It may be implemented by ​ ​ It may also be implemented in

[0129] The method for promoting healing of the present invention ​ ​ When carried out in the above manner, the healing-promoting 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 subjects and administration conditions of the composition of the present invention can be determined, for example, from the descriptions of the subjects and administration conditions of the proliferation-promoting composition of the present invention.

[0130] <Method for promoting keratinocyte function> In another embodiment, the present invention provides a method capable of promoting keratinocyte function. The method for promoting keratinocyte function of the present invention uses the composition of the present invention. The method for promoting keratinocyte function of the present invention is characterized by using the composition of the present invention, and other steps and conditions are not particularly limited. According to the method for promoting keratinocyte function of the present invention, keratinocyte function can be promoted. The explanations of the composition, manufacturing method, proliferation-promoting composition, and keratinocyte function-promoting composition of the present invention can be used for the method for promoting keratinocyte function of the present invention.

[0131] The method for promoting keratinocyte function of the present invention includes, for example, ​ ​ It may be implemented by ​ ​ It may also be implemented in

[0132] The method for promoting keratinocyte function of the present invention ​ ​ When the method for promoting keratinocyte function of the present invention is carried out in the above manner, the method for promoting keratinocyte function of the present invention includes, for example, a culture step of culturing keratinocytes in the presence of the composition. The subjects and administration conditions for the composition of the present invention can be determined by referring to the descriptions of the subjects and administration conditions for the proliferation-promoting composition of the present invention.

[0133] <Method for promoting the function of dermal papilla cells> In another embodiment, the present invention provides a method capable of promoting the function of dermal papilla cells. The method for promoting dermal papilla cell function of the present invention uses the composition of the present invention. The method for promoting dermal papilla cell function of the present invention is characterized by using the composition of the present invention, and other steps and conditions are not particularly limited. According to the method for promoting dermal papilla cell function of the present invention, the function of dermal papilla cells can be promoted. The explanations of the composition, production method, proliferation-promoting composition, and dermal papilla cell function-promoting composition of the present invention can be used for the method for promoting dermal papilla cell function of the present invention.

[0134] The method for promoting the function of dermal papilla cells of the present invention includes, for example, ​ ​ It may be implemented by ​ ​ It may also be implemented in

[0135] The method for promoting the function of dermal papilla cells of the present invention ​ ​ When the method for promoting the function of dermal papilla cells of the present invention is carried out in the above manner, 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 subjects and administration conditions for the composition of the present invention can be determined by reference to the descriptions of the subjects and administration conditions for the proliferation-promoting composition of the present invention.

[0136] <Hair growth promotion method> In another embodiment, the present invention provides a method for promoting 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 using the composition of the present invention, and other steps and conditions are not particularly limited. It is expected that hair growth can be promoted according to the hair growth promoting method of the present invention. The hair growth promoting method of the present invention can refer to the descriptions of the composition, manufacturing method, growth promoting composition, and hair growth promoting composition of the present invention.

[0137] The hair growth promoting method of the present invention can also be referred to as, for example, a method for treating or treating alopecia or thinning hair.The hair growth promoting method of the present invention can also be used, for example, to prevent, suppress, or prevent hair loss.In this case, the hair growth promoting method of the present invention can also be referred to as, for example, a method for preventing, suppressing, or preventing hair loss.

[0138] The hair growth promoting method of the present invention may, for example, comprise the administration step of administering the composition of the present invention to a subject.The subject may, for example, be a patient who has alopecia, a patient who may have alopecia, etc.The patient who has alopecia may, for example, be a patient who has androgenetic alopecia, female pattern alopecia, or alopecia after anticancer drug treatment.The patient who may have alopecia may, for example, be a patient who has not yet undergone anticancer drug treatment, or a patient who has genetic alopecia and has not yet begun to have alopecia, etc.

[0139] The hair growth promoting method of the present invention includes, for example, ​ ​ It may be implemented by ​ ​ It may also be implemented in

[0140] The method for promoting hair growth of the present invention ​ ​ When carried out in the above manner, the hair growth-promoting method of the present invention includes, for example, a culture step of culturing cells associated with hair growth, such as dermal papilla cells, in the presence of the composition. The subjects and administration conditions for the composition of the present invention can be determined by reference to the descriptions of the subjects and administration conditions for the proliferation-promoting composition of the present invention.

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

[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) Preparation of immortalized megakaryocytes Immortalized megakaryocytes were prepared by the following procedure.

[0145] (1-1) Preparation of hematopoietic progenitor cells from iPS cells Human iPS cells (TKDN SeV2 and NIH5: iPS cells derived from human fetal skin fibroblasts established using Sendai virus) were cultured to differentiate into blood cells according to the method described in Reference 8 below. Specifically, human ES / iPS cell colonies were co-cultured with C3H10T1 / 2 feeder cells in the presence of 20 ng / ml VEGF (R&D SYSTEMS) for 14 days to generate hematopoietic progenitor cells (HPCs). Culture conditions were 37°C, 20% O2, and 5% CO2 (the same conditions apply hereinafter unless otherwise noted). 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 system The gene transfer system used was a lentiviral vector system. The lentiviral vector was a tetracycline-regulated Tet-on® gene expression induction system vector. It was generated by recombining the mOKS cassette of LV-TRE-mOKS-Ubc-tTA-I2G (Reference 9 below) with c-MYC, BMI1, or BCL-xL. The vectors carrying c-MYC, BMI1, or BCL-xL were designated 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 generated by transfecting 293T cells with the lentiviral vectors. By infecting cells of interest with the resulting virus, the c-MYC, BMI1, and BCL-xL genes are introduced into the genomic sequence of the cells of interest. These genes, once stably introduced into the genomic sequence, can be forced to express 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) c-MYC and BMI1 virus infection of hematopoietic progenitor cells On a 6-well plate previously seeded with C3H10T1 / 2 feeder cells, 5 × 10 HPCs obtained by the method described in (1-1) were added. 4Cells were seeded at 1000 cells / well, and c-MYC and BMI1 were forced to express by lentiviral methods using BMI1 and c-MYC viruses. Six wells were used for each cell line. Specifically, viral particles were added to the medium to give an MOI (multiplicity of infection) of 20, and infection was carried out by spin infection (centrifugation at 32°C, 900 rpm, 60 minutes). The spin infection was carried out twice, with an interval of 12 hours. The 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 basal 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 l-thioglycerol (Sigma-Aldrich), and 50 μg / ml L-ascorbic acid (Sigma-Aldrich) (hereafter referred to as differentiation medium). The medium was supplemented with ATP so that the concentration was μg / ml.

[0148] (1-4) Creation and maintenance of megakaryocyte self-renewing line The day of infection with the c-MYC and BMI1 viruses as described in (1-3) above was designated as day 0. HPCs transfected with the c-MYC and BMI1 genes were cultured as follows to generate megakaryocyte self-renewing lines. Forced expression of the c-MYC and BMI1 genes was achieved by adding DOX to the culture medium at 1 μg / ml DOX.

[0149] Day 2 to Day 11 of infection On the second day after infection, the virus-infected hemocytes obtained by the above method were collected by pipetting, centrifuged at 1200 rpm for 5 minutes to remove the supernatant, and then suspended in fresh differentiation medium and seeded onto fresh C3H10T1 / 2 feeder cells (6-well plate). Subculture was carried out by repeating the same procedure on the ninth day after infection. At the time of reseeding, the cells were counted and then 1 x 10 5 The cells were seeded onto C3H10T1 / 2 feeder cells at 2 ml / well (6-well plate).

[0150] Day 12 to Day 13 of infection The same procedure as on day 2 of infection was carried out. After counting the number of cells, 3 × 10 5 The cells were seeded onto C3H10T1 / 2 feeder cells (100 mm dish) at 10 ml / 100 mm dish.

[0151] Day 14 of infection Virus-infected blood cells were collected and 1.0 x 10 cells were collected. 5 The blood cells were reacted with 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) antibodies per cell. After the reaction, the cells were analyzed using a FACS Verse™ (BD Biosciences). On day 14 of infection, cells with a CD41a positivity rate of 50% or higher were identified as megakaryocyte self-renewing lines.

[0152] (1-5) BCL-xL virus infection of megakaryocyte autologous proliferation line The megakaryocyte self-renewing line on day 14 of infection was transduced with BCL-xL using the lentiviral method using the BCL-xL virus. Viral particles were added to the medium at an MOI of 10, and the cells were infected by spin infection (centrifugation at 32°C, 900 rpm, 60 minutes). Forced expression of the BCL-xL gene was achieved by adding DOX to the medium at 1 μg / ml DOX.

[0153] (1-6) Creation and maintenance of immortalized megakaryocyte lines Days 14 to 18 of infection The megakaryocyte self-proliferating line transfected with the BCL-xL gene obtained by the method (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 plated on new C3H10T1 / 2 feeder cells at a concentration of 2 × 10 5 The cells were seeded at 2 ml / well (6-well plate).

[0154] Day 18 of infection: Passage After transfection with the BCL-xL gene, the megakaryocyte self-renewing line was collected and the cell number was counted. 5 The cells were seeded at 10 ml per 100 mm dish.

[0155] Day 24 of infection: Passage After transfection with the BCL-xL gene, the megakaryocyte self-renewing line was collected, the cell number was counted, and the cell number was 1 × 10 5 The cells were seeded at 10 ml per 100 mm dish. Subculture was carried out every 4-7 days in the same manner for maintenance. When subculturing, the cells were suspended in new differentiation medium and seeded.

[0156] On day 24 after infection, BCL-xL-transduced megakaryocyte self-renewing cells were collected and 1.0 × 10 cells were cultured. 5Immunostaining was performed 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 per cell, respectively, followed by analysis using a FACS Verse™. Cells with a CD41a positivity rate of 50% or higher on day 24 postinfection were designated as immortalized megakaryocytic cell lines. Cells that were able to proliferate for more than 24 days postinfection were designated as immortalized megakaryocytic cell lines, SeV2-MKCL and NIH5-MKCL.

[0157] The resulting SeV2-MKCL and NIH5-MKCL were statically cultured in 10 cm dishes (10 ml / dish). IMDM was used as the basal medium, and the following components were added (final concentrations). 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) 50μg / ml Puromycin (sigma #P8833-100MG) 2μg / ml SCF (Wako Pure Chemical #193-15513) 50ng / ml TPO-like substance 200ng / ml

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

[0159] The platelet production medium was prepared by adding the following components to IMDM as a basal medium (concentrations are final concentrations). human plasm 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) 50μg / ml SCF (Wako Pure Chemical #193-15513) 50ng / ml TPO-like substance 200ng / 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, the cells were cultured in the presence of the platelet production medium for 6 days to produce platelets, thereby producing a megakaryocyte culture.

[0161] (3) Manufacturing of purified platelets Platelets were produced (purified) from the megakaryocyte culture obtained in (2) above by the following procedure. The same purification was carried out twice.

[0162] (3-1) Enrichment 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, cleaning and preservation solution bags 1 and 2 contain cleaning and preservation solutions. The cleaning and preservation solution used was a bicanate infusion (Bicarbon Infusion, manufactured by Otsuka Pharmaceutical Co., Ltd.) supplemented with 20% ACD and 2.5% human serum albumin and adjusted to pH 7.2 with NaOH. The megakaryocyte culture was then concentrated using a hollow fiber membrane (Plasma Flow OP, manufactured by Asahi Kasei Medical Co., Ltd.) according to Table 1 below, and the resulting megakaryocyte culture concentrate was collected in a storage bag.

[0163] [Table 1]

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

[0165] Next, using a sterile connecting device, the cell bag containing the culture medium supplemented with ACD-A solution was connected to the ACP215 disposable set. The ACP215 was then started in service mode and the rotation speed was set to 2500 rpm (350 × g). The ACP215 was started, and the culture medium in 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 collection bag. After the entire culture medium in the cell bag was introduced into the separation bowl, an additional 500 ml of washing preservation solution was introduced into the separation bowl. After the washing preservation solution was introduced into the separation bowl, the centrifuge was stopped, and the collection bag containing the recovery solution (recovered liquid components including platelets) was separated using a tube sealer.

[0166] A collection bag containing the collection fluid (including platelets) was attached to a new ACP215 disposable set using the sterile attachment device. The ACP215 was started in normal mode. WPC was selected as the program setting, and the ACP215 disposable set with the attached collection bag was set according to the device's instructions. The collection bag containing the collection fluid was placed on the stand.

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

[0168] (3-3) Washing of platelets Washing was carried out using 2000 ml of the above-mentioned washing storage solution according to the ACP215 program.

[0169] (3-4) Platelet collection According to the ACP215 program, 200 ml of washed platelets were collected into a platelet product bag.

[0170] (3-5) Platelet isolation Platelets were separated from the platelet preparation bag using the hollow fiber membrane in a conventional manner and collected in a collection bag.

[0171] (4) Extract manufacturing The megakaryocytes or cultures thereof used were the immortalized megakaryocyte cell line obtained by the method in (1) above, the platelets collected in the platelet preparation bag obtained in (3-5) above, and the platelet-removed megakaryocyte culture collected in the drainage bag (hereinafter collectively referred to as "raw material"). Four separately prepared samples of platelet-removed megakaryocyte cultures were used (Platelet-removed megakaryocyte cultures 1 to 4).

[0172] Each raw material was washed twice with a washing solution. The washing solution used was prepared by adding ACD-A solution to Bicanate infusion to a concentration of approximately 20% (v / v), followed by adding NaOH to adjust the pH to 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. Cell lysis buffer was then added to the frozen precipitate, and the mixture was lysed by shaking at 50 rpm for 30 minutes at 4°C. The cell lysis buffer used was a commercially available cell lysis buffer (2× Cell Lysis Buffer, RayBiotech, Cat. No.: AA-LYS) supplemented with a protease inhibitor cocktail (Protease Inhibitor Cocktail, RayBiotech, Cat. No.: AA-PI).

[0173] The resulting lysate was centrifuged at 14,000 × g for 5 minutes at 4°C. After centrifugation, the supernatant was collected as the processed product of the present example. The total protein concentration of the processed product obtained from each raw material was measured using a Pierce™ BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). As a result, when an immortalized megakaryocytic cell line was used, 2.2 × 10 7 A total of 2.604 mg of protein was extracted from cells. 8 A total of 1.187 mg of protein was extracted from the cells. Furthermore, when platelet-depleted megakaryocyte cultures 1 to 4 were used, 1.97 × 10 8 cells, 5.25×10 8 cells, 3.64×10 8 cells, 6.22×10 82.8, 5.944, 3.6, and 4.496 mg of total protein were extracted from the cells, respectively. The total protein concentration was adjusted to 5 mg / ml, and 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 treatment using Quantibody® Human Growth Factor Array 1 (RayBiotech). The results are shown in Table 2 below. The values ​​in Table 2 can also be expressed as 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 were cultured in a 10-cm dish at 2.4–4.8 × 10 5 The mesenchymal stem cells were seeded at a ratio of 10 cells / 10 ml medium / dish. The mesenchymal stem cells used were cells recovered after two passages of commercially available human adipose tissue-derived mesenchymal stem cells (Takara Bio, Cat. No.: C-12977). The medium used was Mesenchymal Stem Cell Growth Medium 2 (Takara Bio, Cat. No.: C-28009), to which a frozen and reconstituted composition prepared from platelet-depleted megakaryocyte culture 2 was added. The composition was added so that the total protein concentration in the medium was a predetermined concentration (0, 125, 250, or 500 μg / ml). The medium used was a maintenance medium.

[0176] After seeding, the mesenchymal stem cells were cultured for 3 days at 37°C in a humidified atmosphere of 5% CO2. After the culture, the mesenchymal stem cells were recovered, seeded under the same conditions, and cultured again for 3 days at 37°C in a humidified atmosphere of 5% CO2. Next, the mesenchymal cells after the culture were recovered and the number of cells was counted. The relative value of the number of cells at the time of recovery, with the number of cells at the time of seeding as the reference (1), was calculated and used as the relative value of proliferation activity. Furthermore, the time required for one cell proliferation (doubling time) was calculated based on the number of cells at the time of seeding and the number of cells at the time of recovery. These results are shown in Figure 2.

[0177] FIG. 2 is a graph showing the proliferation activity of cells. In FIG. 2, (A) shows proliferation activity, and (B) shows doubling time. In FIG. 2(A), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the relative value of proliferation activity. In FIG. 2(B), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows doubling time, with the numerical values ​​in the graph indicating doubling time. As shown in FIG. 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. Furthermore, although not shown, the doubling time after the third passage of mesenchymal stem cells to which the composition of the present invention had not been added was 21 hours. As shown in FIG. 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 FIG. 2(B), when the composition of the present invention was added, the time required for mesenchymal stem cells to undergo a single proliferation cycle 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 increasing passage number. 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] It was confirmed that the composition of the present invention has cell proliferation-promoting activity.

[0179] The mesenchymal stem cells were collected and cryopreserved after two passages. The cryopreserved cells were thawed and then passaged once. 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 adjusted to a predetermined concentration (0, 0.2, 1.3, 31.3, 62.5, or 125 μg / ml). The results are shown in Figure 3.

[0180] Figure 3 is a graph showing the proliferation activity of cells. In Figure 3, (A) shows proliferation activity, and (B) shows 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 proliferation activity. In Figure 3(B), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows 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 depending on the total protein concentration derived from the composition. In particular, when the total protein concentration was 31.3 μg / ml or higher, the proliferation activity of mesenchymal stem cells increased significantly. Furthermore, 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 mesenchymal stem cells to undergo a single proliferation cycle was shortened, depending on the concentration of total protein derived from the composition. In particular, when the total protein concentration was 31.3 μg / ml or higher, the time required for mesenchymal stem cells to undergo a single proliferation cycle was significantly shortened. Furthermore, when mesenchymal stem cells were cultured in a medium without the composition of the present invention, the doubling time increased with each passage. In contrast, when the composition of the present invention was added, the increase in doubling time was almost completely 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 a decrease in proliferation activity.

[0181] [Example 3] It was confirmed that the composition of the present invention has the activity of promoting fibroblast function.

[0182] (1) Fibroblast culture Normal human dermal fibroblasts (nHDF, KURABO, Cat. No. KF-4109) were cultured in a T-75 flask (Sumilon) in growth medium under the specified conditions (5% CO2, 37°C, humidified conditions; the same applies below). The growth medium was DMEM (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). The medium was changed every 1–2 days. When the cells reached approximately 80% confluence, they were harvested and used for subsequent experiments. The cells were passaged as follows: First, the cells were washed with phosphate buffered saline (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95) and 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). Growth medium was then added to neutralize the trypsin. The cell suspension was then collected in a 15 ml centrifuge tube and centrifuged at room temperature at 1,000 rpm for 5 minutes using a multipurpose refrigerated centrifuge (TOMY, Cat. No.: CAX-571). After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were stirred. The number of viable cells was then counted using the trypan blue method. The growth medium was used to adjust the cell concentration to the desired level and seed the cells into the culture vessels used in the subsequent tests.

[0183] (2) Confirmation of cell proliferation promoting activity in fibroblasts The cells were 5 x 10 3Cells were seeded at a density of 0.1 ml / well into a 96-well plate (Sumilon, Cat. No. MS-8096F) and cultured for 1 day under the aforementioned conditions. After the culture, the medium was replaced with DMEM medium containing a composition prepared from the platelet-depleted megakaryocyte culture (MDF) or platelets (PLT) at a predetermined concentration (treatment concentration of each test substance: 0.5%, 1%, 2.5%, 5%, or 10%) (total protein concentration: 5 mg / ml; the same applies to Examples 4 and 5). Thus, the total protein concentrations derived from the composition 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%) (hereinafter the same). Two types of compositions prepared from platelet-depleted megakaryocyte culture were used (iMDF1 and iMDF2). After the medium change, the cells were cultured for 48 hours.

[0184] After the incubation, the proliferation activity of the cells in each well was measured by counting the viable cell count using the WST-8 method. First, the medium was replaced with DMEM medium containing 10% colorimetric reagent (Cell Count Reagent SF, manufactured by Nacalai Tesque, Cat. No.: 07553-15), and the cells were incubated under the above incubation conditions. The change in absorbance (450 nm) over 60 minutes, from 30 to 90 minutes after the start of incubation, was measured using a plate reader (Varioskan Flash, manufactured by Thermo Fisher Scientific, Cat. No.: 5250040) (n=3 per group). A negative control (NC) was performed in the same manner except that the composition was not added. A positive control (FBS) was performed in the same manner except that FBS was added to the medium to a concentration of 1% or 10% instead of the composition. Furthermore, in the reference examples (ASA, Mes), the above-mentioned composition was replaced with L-ascorbic acid (ASA, Fujifilm-Wako Co., Ltd., Cat. No.: 013-19641), which has collagen production-promoting activity, at a concentration of 2 mmol / L, or N-methyl-L-serine (MeS, Sigma-Aldrich Co., Ltd., Cat. No.: 73156), which has hyaluronic acid production-promoting activity, at a concentration of 10 mmol / L. The cell viability of each sample was used as a relative proliferation activity value, with the cell viability of the negative control being defined as 100%. A p-value of less than 0.05 was determined to be significant in a Student's T-test (two-tailed, unpaired) comparing the negative control. These results are shown in Figure 4.

[0185] Figure 4 is a graph showing the proliferation activity of fibroblasts. In Figure 4, the horizontal axis indicates the type and concentration of the sample, and the vertical axis indicates cell viability (proliferation activity). As shown in Figure 4, a concentration-dependent cell proliferation-promoting effect was observed when either the platelet-depleted megakaryocyte cultures (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 extracellular matrix production-promoting activity After 48 hours of culture as in Example 3(2) above, 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 test described below.

[0187] Next, the amount of type I collagen in the culture supernatant (n=3 per 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 measured using a hyaluronic acid measurement kit (Hyaluronan DuoSet ELISA, R&D Systems, Cat. No.: DY3614). The measurement method using each kit followed the attached protocol. The negative control (NC) was performed in the same manner except that the composition was not added. The FBS-added group (FBS) was performed in the same manner as in Example 3(2). For the positive control (ASA or Mes), the samples used for type I collagen measurement were prepared in the same manner except that, instead of the composition, L-ascorbic acid (ASA, Fujifilm-Wako Co., Ltd., Cat. No. 013-19641), which has collagen production-promoting activity, was added to a concentration of 2 mmol / L. For the samples used for hyaluronic acid measurement, N-methyl-L-serine (MeS, Sigma-Aldrich Co., Ltd., Cat. No. 73156), which has hyaluronic acid production-promoting activity, was added to a concentration of 10 mmol / L. Furthermore, in a Student's T-test (two-tailed, unpaired) comparing the results with the negative control, a p-value of less than 0.05 was determined to be significant. These results are shown in Figures 5 and 6.

[0188] FIG. 5 is a graph showing the amount of type I collagen produced. In FIG. 5, the horizontal axis indicates the type of sample, and the vertical axis indicates the amount of type I collagen produced. The ASA values ​​in the graph indicate the amount of collagen produced (μg / ml) in the Reference Example (ASA). As shown in FIG. 5, a concentration-dependent increase in the amount of type I collagen produced was observed regardless of whether the platelet-depleted megakaryocyte culture or the composition prepared from platelets was added. When compared with the results of the cell proliferation-promoting activity in Example 3(2), the type I collagen production-promoting activity was presumed to be due to cell proliferation-promoting activity.

[0189] Next, Figure 6 is a graph showing the amount of hyaluronic acid produced. In Figure 6, the horizontal axis indicates the type and concentration of sample, and the vertical axis indicates the amount of hyaluronic acid produced. As shown in Figure 6, whether the platelet-removed megakaryocyte culture or the composition prepared from platelets was added, the amount of hyaluronic acid produced was observed to increase in a concentration-dependent manner. Compared with the cell proliferation-promoting activity, the hyaluronic acid production-promoting activity was an activity independent of the cell proliferation-promoting activity, that is, it was presumed 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 the activity of promoting fibroblast proliferation and the activity of promoting the production of extracellular matrix components such as type I collagen and hyaluronic acid. When an injury occurs in the skin, the fibroblasts migrate to the injury site and contribute to the healing of the injury site through cell division and secretion of extracellular matrix components. It was expected that the composition of the present invention can promote these functions in fibroblasts and therefore promote the healing of skin injuries.

[0191] [Example 4] It was confirmed that the composition of the present invention has an activity of promoting keratinocyte function. (1) Keratinocyte culture Normal human epidermal keratinocytes (nHEK, KURABO, Cat. No. KK-4109) were placed in a T-75 flask (Sumilon) using growth medium and cultured in a CO2 incubator under the specified culture conditions (5% CO2, 37°C, humidified conditions; the same applies below). The growth medium 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 experiments. The cells were passaged as follows. First, the cells were washed with phosphate buffered saline (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95) and 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). Growth medium was then added to neutralize the trypsin. The cell suspension was then collected in a 15 ml centrifuge tube and centrifuged at room temperature at 1,000 rpm for 5 minutes using a multipurpose refrigerated centrifuge (TOMY, Cat. No.: CAX-571). After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were stirred. The number of viable cells was then counted using the trypan blue method. The growth medium was used to adjust the cell concentration to the desired level and seed the cells into the culture vessels used in the subsequent tests.

[0192] (2) Confirmation of cell proliferation promoting activity of keratinocytes The cells were 1 x 10 3The cells were seeded into a 96-well plate (Sumilon, Cat. No.: MS-8096F) at a density of 0.1 ml / well and cultured for 1 day under the aforementioned culture conditions. After the culture, the medium was replaced with a maintenance medium (Humedia-KB2, KURABO, Cat. No.: KK-2350S) containing a composition prepared from the platelet-depleted megakaryocyte culture (iMDF1, iMDF2) or platelets (PLT) at a predetermined concentration (treatment concentration of each test substance: 0.5%, 1%, 2.5%, 5%, 10%). Two types of compositions prepared from the platelet-depleted megakaryocyte culture were used. After the medium replacement, the cells were cultured for 48 hours.

[0193] After the incubation, the morphology of the cells in each well was observed using a phase-contrast microscope (OLYMPUS, Cat. No.: CKX53). The change in absorbance (450 nm) of the cells in each well was measured in the same manner as in Example 3(2) above (n=3 per group). A negative control (NC) was performed in the same manner except that the composition was not added. A positive control (additive) was performed in the same manner except that the growth medium was used instead of the maintenance medium containing the composition. Furthermore, reference examples (JTC or NA) were performed 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. The cell viability of the negative control was set at 100%, and the cell viability of each sample was used as a relative proliferation activity value. A Student's T-test (two-tailed, unpaired) comparing the negative control was performed, and a p-value of less than 0.05 was considered significant. These results are shown in Figures 7 and 8.

[0194] Figure 7 shows phase-contrast images of cells in each well. For the groups containing the platelet-removed megakaryocyte cultures (iMDF1, iMDF2) or compositions prepared from platelets (PLT), the 10% addition 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 cells of the epithelial (epidermal) layer (epidermal cells) was not observed. On the other hand, in the case of the compositions prepared from the platelet-removed megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT), the keratinocytes spread out on the plate, and the cell boundaries became unclear. This is due to the differentiation of the keratinocytes into cells of the epithelial (epidermal) layer (epidermal cells). Furthermore, the compositions prepared from the platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) increased the proportion of cells in the epithelial (epidermal) layer in a concentration-dependent manner, and a particularly significant increase was observed when the platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) were used. These results demonstrate that the compositions of the present invention promote the differentiation of keratinocytes into the epithelial layer.

[0195] Figure 8 is a graph showing the proliferation activity of keratinocytes. In Figure 8, the horizontal axis indicates the type and concentration of the sample, and the vertical axis indicates the cell viability (proliferation activity). As shown in Figure 8, a cell proliferation-promoting effect was observed when either the platelet-depleted megakaryocyte cultures (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 the induction and promotion activity of barrier function genes 5×10 4Keratinocytes were cultured in the same manner as in Example 4(2) above, except that the cells were seeded in a 24-well plate (Sumilon, Cat. No. MS-8024) at a density of 0.5 ml / well. After the culture, total RNA was recovered and purified from the cells using an RNA extraction kit (RNeasy 96 Kit, QIAGEN, Cat. No. 74181). RNA was extracted according to the protocol provided with the kit. The concentration of the purified RNA obtained was measured using a spectrophotometer (NanoDrop One, Thermo Fisher Scientific, Cat. No. ND-ONE-W), and the purity was confirmed by A260 / A280. The RNA 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 resulting cDNA was stored at -30°C. Quantitative PCR was then performed using the resulting cDNA, the following primer sets 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 consisted of a 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 resulting 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 composition was not added. For the positive control (JTC or NA), the samples used for measuring FLG gene expression were supplemented with JTC-801 (JTC, Sigma-Aldrich, Cat. No. J3955), which has FLG gene expression-promoting activity, at 100 nmol / L, instead of the composition. For the samples used for measuring SPTLC1 gene expression, the composition was replaced with nicotinamide (NA, Sigma-Aldrich, Cat. No. N0636-100G), which has SPTLC1 gene expression-promoting activity, at 30 μmol / L, instead of the composition. For the negative and positive controls, groups containing additives were also prepared and performed in the same manner. The expression levels of each gene, normalized by the expression level of the GAPDH gene, were calculated from the obtained measurement data using the ΔΔCt method. Furthermore, the relative expression levels were calculated by setting the expression level of the negative control to 1. Compared with the negative control, a p-value of less than 0.05 in Student's T-test (two-tailed, unpaired) was determined to be significant. The results are shown in Figures 9 and 10.

[0198] Primer set for GAPDH gene Forward primer (SEQ ID NO: 1) 5'-CATCCCTGCCTCTACTGGGCTGCC-3' Reverse primer (SEQ ID NO: 2) 5'-CCAGGATGCCCTTGAGGGGGCCCTC-3' Primer set for 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 indicates the type and concentration of the sample, and the vertical axis indicates the expression level of the FLG gene. As shown in Figure 9, the effect of promoting FLG gene expression was observed when either the platelet-depleted megakaryocyte cultures (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, the effect of promoting SPTLC1 gene expression was observed when either the platelet-depleted megakaryocyte cultures (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 the activity of promoting keratinocyte proliferation, promoting differentiation into epidermal cells, and promoting 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 the expression of barrier function genes are known to be important for maintaining skin tissue and its barrier function. Therefore, since the composition of the present invention promotes these functions in keratinocytes, it is expected that it can be used to maintain skin condition, such as maintaining skin function and skin barrier function.

[0202] [Example 5] It was confirmed that the composition of the present invention has the activity of promoting the function of dermal papilla cells.

[0203] (1) Cultivation of dermal papilla cells Human hair dermal papilla cells (HFDPC, TOYOBO, Cat. No.: CA60205a) were cultured in a T-75 flask (Sumilon) using growth medium and cultured under the specified culture conditions (5% CO, 37°C, humid conditions; the same applies below) in a CO incubator. The growth medium was a dermal papilla cell growth medium (PCGM, TOYOBO, Cat. No.: TMTPGM-250S) supplemented with the included additives. 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 passaged as follows. First, the cells were washed with phosphate buffered saline (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95) and 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). Growth medium was then added to neutralize the trypsin. The cell suspension was then collected in a 15 ml centrifuge tube and centrifuged at room temperature at 1,000 rpm for 5 minutes using a multipurpose refrigerated centrifuge (TOMY, Cat. No.: CAX-571). After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were stirred. The number of viable cells was then counted using the trypan blue method. The growth medium was used to adjust the cell concentration to the desired level and seed the cells into the culture vessels used in the subsequent tests.

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

[0205] After the culture, the change in absorbance (450 nm) of the cells in each well was measured in the same manner as in Example 3(2) above (n=3 per group). A negative control (NC) was performed in the same manner, except that the composition was not added. A positive control (additive) was performed in the same manner, except that a dermal papilla cell growth medium containing the additive was used. Reference examples (Mx or Ad) were performed in the same manner, except that minoxidil (Mx), Sigma-Aldrich, Cat. No.: M4145, final concentration 30 μmol / L) or adenosine (Ad), Sigma-Aldrich, Cat. No.: A9251, final concentration 100 μmol / L), known as a hair growth agent, was added instead of the composition. The cell viability in the negative control was set to 100%, and the cell viability of each sample was used as a relative proliferation activity value. In a Student's T-test (two-tailed, unpaired) comparing with the negative control, a p-value of less than 0.05 was determined to be significant. These results are shown in FIG.

[0206] Figure 11 is a graph showing the proliferation activity of dermal papilla cells. In Figure 11, the horizontal axis indicates the type and concentration of the sample, and the vertical axis indicates the cell viability (proliferation activity). As shown in Figure 11, a cell proliferation-promoting effect was observed when either the platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or the composition prepared from platelets (PLT) were added. It was also suggested that an appropriate amount to add was 2.5 to 5%.

[0207] (3) Confirmation of induction and promotion activity of hair growth promoting genes 2.5×10 4 Dermal papilla cells were cultured in the same manner as in Example 5(2) above, except that they were seeded in a 24-well plate (manufactured by 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) above.

[0208] Next, qPCR was performed in the same manner as in Example 4(3), except that the primer sets used were the GAPDH gene and the primer sets for the FGF7 gene or VEGFA gene described below. A negative control was performed in the same manner except that the composition was not added. A reference example was performed in the same manner except that minoxidil (Sigma-Aldrich, Cat. No. M4145, final concentration 30 μmol / L) or adenosine (Sigma-Aldrich, Cat. No. A9251, final concentration 100 μmol / L), known as a hair growth agent, was added instead of the composition. From the obtained measurement data, the expression level of each gene was calculated using the ΔΔCt method, corrected for the expression level of the GAPDH gene. Furthermore, the relative expression level was calculated by setting the expression level of the negative control to 1. Compared to the negative control, a p-value of less than 0.05 in Student's T-test (two-tailed, unpaired) was determined to be significant. These results are shown in Figures 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 indicates the type of sample, and the vertical axis indicates the expression level of the FGF7 gene. As shown in Figure 12, when a composition prepared from the platelet-depleted 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 at the same level as 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 indicates the type of sample, and the vertical axis indicates the expression level of the VEGFA gene. As shown in Figure 13, when a composition prepared from the platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) was added, an effect of promoting VEGFA gene expression was observed. Furthermore, the VEGFA gene expression-promoting activity of the composition of the present invention was considered to be at the same level as 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 the activity of promoting the proliferation of dermal papilla cells and the activity of promoting the expression of hair growth genes such as the FGF7 gene and the VEGFA gene. The proliferation of dermal papilla cells and the induction of hair growth gene expression are known to be important for hair growth, hair growth, and hair maintenance. Therefore, it was expected that the composition of the present invention could be used for hair growth because it promotes these functions in dermal papilla cells.

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

[0214] This application claims priority based on Japanese Patent Application No. 2019-225959, filed December 13, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0215] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Composition> (Appendix 1) A composition comprising a processed megakaryocyte or a culture thereof. (Appendix 2) The composition of claim 1, wherein the processed material is an extract of a cell fraction of megakaryocytes or a culture thereof. (Appendix 3) 3. The composition according to claim 1 or 2, wherein the treated product contains 2,000 to 20,000 pg of basic fibroblast growth factor (bFGF) per 1 mg of total protein. (Appendix 4) 4. The composition according to any one of Appendices 1 to 3, wherein the treated product contains 8,000 to 80,000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) per 1 mg of total protein. (Appendix 5) 5. The composition according to any one of claims 1 to 4, wherein the treated product contains 1 to 60 pg of placental growth factor (PIGF) per mg of total protein. (Appendix 6) 6. The composition according to any one of Appendices 1 to 5, wherein the treated product contains 200 to 2000 pg of stem cell factor receptor (SCFR) per 1 mg of total protein. (Appendix 7) 7. The composition according to any one of Appendices 1 to 6, wherein the treated product contains 20 to 800 pg of vascular endothelial growth factor (VEGF) per 1 mg of total protein. (Appendix 8) 8. The composition according to any one of Appendices 1 to 7, wherein the treated product contains 20 to 400 pg of vascular endothelial growth factor receptor 2 (VEGFR2) per 1 mg of total protein. (Appendix 9) 9. The composition according to any one of Appendices 1 to 8, wherein the treated product contains 1,000 to 10,000 pg of growth factor-15 (GDF-15) per 1 mg of total protein. (Appendix 10) 10. The composition according to any one of Appendices 1 to 9, wherein the treated product contains 0 to 1000 pg of bone morphogenetic protein-7 (BMP-7) per 1 mg of total protein. (Appendix 11) 11. The composition according to any one of Appendices 1 to 10, wherein the treated product contains 0 to 16 pg of amphiregulin (AR) per 1 mg of total protein. (Appendix 12) 12. The composition according to any one of Appendices 1 to 11, wherein the treated product contains 0 to 60 pg of epidermal growth factor receptor (EGFR) per 1 mg of total protein. (Appendix 13) 13. The composition according to any one of Appendices 1 to 12, wherein the treated product contains 0 to 100 pg of hepatic growth factor (HGF) per 1 mg of total protein. (Appendix 14) 14. The composition according to any one of Appendices 1 to 13, wherein the treated product contains 0 to 200 pg of insulin-like growth factor binding protein-1 (IGFBP-1) per 1 mg of total protein. (Appendix 15) A composition described in any one of Appendices 1 to 14, having cell proliferation-promoting activity. (Appendix 16) 16. The composition of claim 15, wherein the cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells. (Appendix 17) A composition described in any one of appendices 1 to 16, having fibroblast function-promoting activity. (Appendix 18) 18. The composition of claim 17, wherein the function of the fibroblasts is proliferation of the fibroblasts and / or production of extracellular matrix by the fibroblasts. (Appendix 19) 19. The composition of claim 18, wherein the extracellular matrix comprises collagen and / or hyaluronic acid. (Appendix 20) 20. A composition described in any one of appendices 1 to 19, having keratinocyte function-promoting activity. (Appendix 21) The composition of claim 20, wherein the function of the keratinocyte is proliferation of the keratinocyte, differentiation into an epidermal cell, and / or induction of a barrier function gene. (Appendix 22) 22. The composition of claim 21, wherein the barrier function gene comprises a profilaggrin gene and / or a ceramide synthase gene. (Appendix 23) 23. A composition according to any one of claims 1 to 22, having an activity of promoting the function of hair follicle papilla cells. (Appendix 24) The composition according to claim 23, wherein the function of the dermal papilla cells is proliferation of dermal papilla cells and / or induction of hair growth-promoting genes. (Appendix 25) 25. The composition of claim 24, wherein the hair growth gene is an FGF7 gene and / or a VEGF gene. (Appendix 26) 26. The composition of any of claims 1 to 25, wherein the megakaryocyte culture is a platelet-depleted culture. (Appendix 27) 27. The composition of any of claims 1 to 26, wherein the megakaryocytes are immortalized megakaryocytes. (Appendix 28) 28. The composition of claim 27, wherein the immortalized megakaryocytes are megakaryocytes comprising exogenous BMI1, MYC, and Bcl-xL genes. (Appendix 29) The megakaryocytes are ​ ​ 29. The composition of any of appendices 1 to 28, wherein the megakaryocytes are induced with (Appendix 30) 30. The composition of any of claims 1 to 29, wherein the megakaryocytes are derived from pluripotent cells. (Appendix 31) 31. The composition of claim 30, wherein the pluripotent cells are induced pluripotent stem (iPS) cells. (Appendix 32) The processed material is Treating megakaryocytes or cultures thereof; 32. The composition of any one of claims 1 to 31, wherein the treatment is a concentration treatment, a drying treatment, a freezing treatment, a lyophilization treatment, a solvent treatment, a surfactant treatment, an enzyme treatment, a protein fraction extraction treatment, an ultrasonic treatment, and / or a disruption treatment. (Appendix 33) The processed material is removing platelets from said megakaryocytes or cultures thereof; 33. The composition of claim 32, wherein the platelet-depleted megakaryocytes or cultures thereof are treated. (Appendix 34) The processed material is storing the platelet-depleted megakaryocytes or a culture thereof; 34. The composition of claim 33, for treating the preserved megakaryocytes or cultures thereof. (Appendix 35) The processed material is storing the megakaryocytes or a culture thereof; The composition according to claim 34, wherein the preserved megakaryocytes or cultures thereof are disrupted. <Method of producing the composition> (Appendix 36) a processing step of processing megakaryocytes or a culture thereof, A method for producing a composition, wherein the treatment in the treatment step is a concentration treatment, a drying treatment, a freezing treatment, a freeze-drying treatment, a solvent treatment, a surfactant treatment, an enzyme treatment, a protein fraction extraction treatment, an ultrasonic treatment, and / or a disruption treatment. (Appendix 37) a removal step of removing platelets from the megakaryocytes or the culture thereof, 37. The method according to claim 36, wherein the treatment step treats the platelet-removed megakaryocytes or a culture thereof. (Appendix 38) a preservation step of preserving the platelet-removed megakaryocytes or a culture thereof, The method according to claim 37, wherein the processing step processes the preserved megakaryocytes or a culture thereof. (Appendix 39) a preservation step of preserving the megakaryocytes or a culture thereof, The method according to claim 38, wherein the treatment step comprises disrupting the preserved megakaryocytes or a culture thereof. (Appendix 40) A composition obtainable by the method of any one of Appendices 36 to 39. <Cell proliferation promoting composition> (Appendix 41) A cell proliferation-promoting composition comprising a composition described in any one of appendices 1 to 35. (Appendix 42) 43. The cell proliferation-promoting composition according to claim 42, wherein the cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or hair papilla cells. <Method for promoting cell proliferation> (Appendix 43) A method for promoting cell proliferation, using the cell proliferation-promoting composition described in Appendix 41 or 42. (Appendix 44) 44. The method for promoting cell proliferation according to claim 43, wherein the cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or hair papilla cells. (Appendix 45) The cell proliferation-promoting composition, ​ ​ or ​ ​ 45. A method for promoting cell proliferation according to claim 43 or 44, wherein the method is used in the step (a). <Fibroblast function-promoting composition> (Appendix 46) A composition for promoting fibroblast function, comprising a composition described in any one of appendices 1 to 35. (Appendix 47) A function-promoting composition described in Appendix 46, wherein the function of fibroblasts is fibroblast proliferation and / or production of extracellular matrix by fibroblasts. (Appendix 48) 48. The function-promoting composition of claim 47, wherein the extracellular matrix comprises collagen and / or hyaluronic acid. <Method for promoting fibroblast function> (Appendix 49) A method for promoting fibroblast function using a composition for promoting fibroblast function described in any one of Appendices 46 to 48. (Appendix 50) The composition for promoting fibroblast function, ​ ​ or ​ ​ 51. A method for promoting fibroblast function according to claim 50, wherein the method is used in the step (a). <Composition for promoting healing of skin disorders> (Appendix 51) A composition for promoting healing of skin disorders, comprising the composition of any one of appendices 1 to 35. (Appendix 52) 52. The healing-promoting composition of claim 51, wherein the skin disorder is a skin ulcer, a bedsore, a burn, a scar, and / or a wound. <Method for promoting healing of skin disorders> (Appendix 53) A method for promoting the healing of skin disorders, using a composition for promoting the healing of skin disorders described in Appendix 51 or 52. (Appendix 54) The composition for promoting healing of skin disorders, ​ ​ or ​ ​ 54. A method for promoting healing of skin disorders according to claim 53, wherein the method comprises: <Keratinocyte function-promoting composition> (Appendix 55) A composition for promoting keratinocyte function, comprising the composition of any one of appendices 1 to 35. (Appendix 56) A function-promoting composition according to Appendix 55, wherein the function of keratinocytes is proliferation of keratinocytes, differentiation into epidermal cells, and / or induction of barrier function genes. (Appendix 57) 57. The function-promoting composition according to claim 56, wherein the barrier function gene comprises a profilaggrin gene and / or a ceramide synthase gene. <Method for promoting keratinocyte function> (Appendix 58) A method for promoting keratinocyte function using a composition for promoting keratinocyte function described in any one of Appendices 55 to 57. (Appendix 59) The keratinocyte function-promoting composition, ​ ​ or ​​ 59. A method for promoting keratinocyte function according to claim 58, wherein the method is used in the step (a). <Composition for promoting the function of dermal papilla cells> (Appendix 60) A composition for promoting the function of hair follicle papilla cells, comprising the composition described in any one of appendices 1 to 35. (Appendix 61) 57. A function-promoting composition according to claim 56, wherein the function of the hair papilla cells is proliferation of hair papilla cells and / or induction of hair growth-promoting genes. (Appendix 62) 58. The function-promoting composition according to claim 57, wherein the barrier function gene comprises a profilaggrin gene and / or a ceramide synthase gene. <Method for promoting the function of dermal papilla cells> (Appendix 63) A method for promoting the function of hair papilla cells, using a composition for promoting the function of hair papilla cells described in any one of Appendices 60 to 62. (Appendix 64) The composition for promoting the function of hair papilla cells, ​ ​ or ​ ​ 64. The method for promoting the function of dermal papilla cells according to claim 63, wherein the method is used in the step (a). <Hair growth promoting composition> (Appendix 65) A hair growth promoting composition comprising the composition of any one of Appendices 1 to 35. <Hair growth promotion method> (Appendix 66) A method for promoting hair growth using the hair growth promoting composition described in Appendix 65. (Appendix 67) The hair growth promoting composition ​ ​ or ​ ​ 67. The method for promoting hair growth according to claim 66, wherein <Use of the composition> (Appendix 68) A composition for use in promoting cell proliferation, comprising as an active ingredient a processed megakaryocyte or a culture thereof. (Appendix 69) A composition for use in promoting the function of fibroblasts, comprising as an active ingredient a processed product of megakaryocytes or a culture thereof. (Appendix 70) A composition for use in promoting the healing of skin disorders, comprising as an active ingredient a processed megakaryocyte or a culture thereof. (Appendix 71) A composition for use in promoting the function of keratinocytes, comprising as an active ingredient a processed product of megakaryocytes or a culture thereof. (Appendix 72) A composition for use in promoting the function of dermal papilla cells, comprising as an active ingredient a processed product of megakaryocytes or a culture thereof. (Appendix 73) A composition for use in promoting hair growth, comprising as an active ingredient a processed product of megakaryocytes or a culture thereof. [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 cell proliferation, for example, of mesenchymal cells, fibroblasts, keratinocytes, and dermal papilla cells, and is expected to be useful for promoting the healing of skin disorders such as skin ulcers, pressure sores, burns, scars, and wounds, maintaining or improving the skin's barrier function, and promoting hair growth. Therefore, the present invention is extremely useful in the fields of medicine, regenerative medicine, and the like.

Claims

[Claim 1] A composition comprising a processed megakaryocyte or a culture thereof.

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