Compositions and uses thereof

Abstract

Cell-derived compositions having physiological activity are provided. The compositions of the invention comprise a megakaryocyte or a treatment of a culture thereof.

Description

Technical Field

[0001] This invention relates to compositions and their uses. Background Technology

[0002] As a therapeutic agent for regenerative medicine, attempts have been made to develop cells that promote the regeneration of tissues such as mesenchymal stem cells as cell preparations. Furthermore, it is believed that cells that promote the regeneration of these tissues facilitate tissue regeneration by releasing physiologically active proteins such as growth factors. Summary of the Invention

[0003] The problem the invention aims to solve

[0004] Therefore, the object of the present invention is to provide a cell-derived composition with physiological activity.

[0005] Solution for solving the problem

[0006] To achieve the foregoing objectives, the compositions of the present invention comprise a treatment of megakaryocytes or their cultures.

[0007] The cell proliferation promoting composition of the present invention (hereinafter also referred to as "proliferation promoting composition") comprises the aforementioned composition of the present invention.

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

[0009] The composition of the present invention for promoting the healing of skin disorders comprises the aforementioned composition of the present invention.

[0010] The keratinocyte function-promoting composition of the present invention comprises the aforementioned composition of the present invention.

[0011] The hair papilla cell function-promoting composition of the present invention comprises the aforementioned composition of the present invention.

[0012] The hair growth promoting composition of the present invention comprises the aforementioned composition of the present invention.

[0013] The effects of the invention

[0014] According to the present invention, a cell-derived, physiologically active composition can be provided. Attached Figure Description

[0015] Figure 1 This is a schematic diagram showing the configuration of the concentration system in Example 1.

[0016] Figure 2 The graph shows the proliferation activity of cells in Example 1. (A) shows the relative value of proliferation activity, and (B) shows the doubling time.

[0017] Figure 3The graph shows the proliferation activity of cells in Example 2. (A) shows the relative value of proliferation activity, and (B) shows the doubling time.

[0018] Figure 4 This is a diagram showing the proliferative activity of fibroblasts in Example 3.

[0019] Figure 5 This is a graph showing the amount of type I collagen generated in Example 3.

[0020] Figure 6 This is a graph showing the amount of hyaluronic acid produced in Example 3.

[0021] Figure 7 This is a photograph showing the phase difference image of the cells in Example 4.

[0022] Figure 8 This is a diagram illustrating the proliferative activity of keratinocytes in Example 4.

[0023] Figure 9 This is a graph showing the expression level of the FLG gene in Example 4.

[0024] Figure 10 This is a graph showing the expression level of the SPTLC1 gene in Example 4.

[0025] Figure 11 This is a diagram illustrating the proliferative activity of dermal papilla cells in Example 5.

[0026] Figure 12 This is a graph showing the expression level of the FGF7 gene in Example 5.

[0027] Figure 13 This is a graph showing the expression level of the VEGFA gene in Example 5. Detailed Implementation

[0028] <Composition>

[0029] As previously stated, the compositions of the present invention comprise a treatment of megakaryocytes or their cultures. As previously stated, the compositions of the present invention are characterized by comprising a treatment of megakaryocytes or their cultures; other components and conditions are not particularly limited. According to the compositions of the present invention, for example, cell-derived physiologically active compositions can be provided. According to the compositions of the present invention, for example, they can promote the proliferation of cells such as mesenchymal cells, fibroblasts, keratinocytes, and dermal papilla cells; furthermore, they are expected to be suitable for promoting the healing of skin disorders such as ulcers, bedsores, burns, scars, trauma, and skin aging; maintaining or improving the skin barrier function; and promoting hair growth.

[0030] In this invention, "megakaryocyte" refers to the largest cells found in the bone marrow of an organism, encompassing cells that release platelets and cells with equivalent functions. The aforementioned cells with equivalent functions refer to cells capable of producing platelets. In this invention, megakaryocytes can be pre-polynuclearized (polyploidized) megakaryocytes, i.e., immature megakaryocytes or proliferating megakaryocytes, or post-polynuclearized megakaryocytes (multinucleated megakaryocytes). Specifically, the aforementioned megakaryocytes can be any of the following: megakaryocyte progenitor cells (Japanese: pre-megakaryocyte buds), promegakaryocytes, immature megakaryocytes, and mature megakaryocytes. The aforementioned post-polynuclearized megakaryocytes only need to possess more than two sets of chromosomes; specifically, 16 to 32 sets are used.

[0031] There are no particular restrictions on the source of the aforementioned megakaryocytes; for example, humans and non-human animals can be listed. Examples of non-human animals include primates such as monkeys, gorillas, chimpanzees, and marmosets, as well as mice, rats, dogs, cats, rabbits, sheep, horses, and guinea pigs. The sources of the other cells listed below are the same.

[0032] In this invention, the aforementioned megakaryocytes can be identified using cell surface markers. When the megakaryocytes are derived from humans, the aforementioned cell surface markers may include CD41a, CD42a, and CD42b. That is, the aforementioned megakaryocytes are cells that are positive for CD41a, CD42a, and CD42b. When the aforementioned megakaryocytes are derived from humans, the aforementioned cell surface markers 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.

[0033] The aforementioned megakaryocytes can be megakaryocytes isolated from an organism, or they can be megakaryocytes induced by pluripotent cells or other cells that are undifferentiated compared to megakaryocytes (hereinafter also referred to as "progenitor cells"). The aforementioned "cells that are undifferentiated compared to megakaryocytes" refer to cells that have the differentiation ability to differentiate into the aforementioned megakaryocytes.

[0034] When the aforementioned megakaryocytes are megakaryocytes isolated from an organism, these megakaryocytes may be present in, for example, bone marrow, and therefore can be isolated from the bone marrow. In this case, the aforementioned megakaryocytes may contain other cells derived from the organism.

[0035] When the aforementioned megakaryocytes are megakaryocytes induced by progenitor cells, as described later, these megakaryocytes can be induced in vitro. In this case, the aforementioned megakaryocytes may include the aforementioned progenitor cells. Examples of such progenitor cells include hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, megakaryocyte-erythroid progenitor (MEP), and megakaryocyte progenitor cells. These progenitor cells can be isolated from bone marrow, umbilical cord blood, peripheral blood, etc., and can also be induced from pluripotent cells such as ES cells (embryonic stem cells), induced pluripotent stem cells (iPS cells), nuclear transfer ES cells (ntES cells), germline stem cells, somatic stem cells, and embryonic cancer cells.

[0036] When the aforementioned megakaryocytes are megakaryocytes induced by progenitor cells, immortalized megakaryocytes are preferred. These immortalized megakaryocytes, for example, exhibit higher homogeneity in their cell differentiation stage compared to megakaryocytes induced using other methods, thus suppressing variations in the composition of the resulting processed material. As described later, these immortalized megakaryocytes are, for example, megakaryocytes induced by introducing oncogenes and polycomb genes, or by introducing oncogenes, polycomb genes, and apoptosis-inhibiting genes, into the aforementioned progenitor cells.

[0037] The aforementioned "oncogenes" refer to genes that can induce cancerous changes in cells within an organism. Examples include MYC family genes such as c-MYC, N-MYC, and L-MYC, SRC family genes, RAS family genes, RAF family genes, c-kit (CD117), PDGFR (platelet growth factor receptor), and Abl (Abelson murine leukemia viral oncogene homolog) and other protein kinase family genes.

[0038] The aforementioned "multicomb genes" refer to genes that are known to negatively regulate CDKN2a (cyclin-dependent kinase inhibitor 2A, INK4a / ARF) and thus play a role in preventing cellular aging (see references 1-3 below). As specific examples, the aforementioned multicomb genes include BMI1 (multicomb complex BMI-1, multicomb family ring finger protein 4 (PCGF4), ring finger protein 51 (RNF51)), Mel18 (multicomb family ring finger protein 2), Ring (ring finger protein) 1a / b, Phc (Polyhomeotichomolog) 1 / 2 / 3, Cbx (pigment box) 2 / 4 / 6 / 7 / 8, Ezh2 (enhancer of the second subunit of the Zeste 2 multicomb repression complex), Eed (embryonic ectoderm development protein), Suz12 (the second subunit of the SUZ12 multicomb repression complex), HADC (histone deacetylases), Dnmt (DNA (cytosine-5)-methyltransferase) 1 / 3a / 3b, etc.

[0039] Reference 1: Hideyuki Koguro et al., “Steel cell aging control based on polycom protein complex”, Regenerative Medicine, 2007, Vol. 6, No. 4, pp. 26-32

[0040] Reference 2: Jesus Gil et.al, "Regulation of the INK4b-ARF-INK4a tumoursuppressor locus: all for one or one for all", Nature Reviews Molecular CellBiology, 2007, vol.7, pages 667-677

[0041] Reference 3: Soo-Hyun Kim et al., “Absence of p16 INK4a and truncation ofARF tumor suppressors in chickens”,PNAS,2003,vol.100,No.1,pages 211-216

[0042] The aforementioned "apoptosis-inhibiting genes" refer to genes that have the function of inhibiting cell apoptosis, such as BCL2 (B-lymphoma-2), Bcl-xL (large B-cell lymphoma), Survivin (containing baculovirus IAP repeat sequence protein 5), MCL1 (BCL2 family apoptosis regulator), etc.

[0043] The aforementioned immortalized megakaryocytes are preferably megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes. "Exogenous" refers to genes introduced into the cell from outside. These exogenous genes can be present on the chromosomes of the aforementioned cells, or in the nucleus or cytoplasm. The exogenous genes can be detected, for example, by measuring their quantity. When the aforementioned gene is an autosomal gene, there is one of each autosome, therefore two genes are present in one cell. Therefore, if the aforementioned exogenous gene is absent, two of the aforementioned genes can be detected in one cell. On the other hand, when the aforementioned exogenous gene is present, three or more of the aforementioned genes can be detected in one cell. In this case, the exogenous genes can be detected, for example, using PCR with primers, probes, or combinations thereof. When the aforementioned exogenous gene has a tag sequence or a selection marker, the detection of the aforementioned exogenous gene can also be performed by detecting the aforementioned tag sequence or selection marker. Furthermore, for the aforementioned exogenous gene, for example, antibodies can be used to detect the proteins translated from the aforementioned gene.

[0044] The aforementioned megakaryocyte culture is, for example, a culture produced by culturing the aforementioned megakaryocytes. As described below, the aforementioned megakaryocyte culture can be carried out, for example, by culturing the aforementioned megakaryocytes in the presence of a culture medium.

[0045] The aforementioned megakaryocyte culture is a substance obtained by culturing the aforementioned megakaryocytes, and therefore, as a cellular component, it is a mixture containing the aforementioned megakaryocytes and platelets produced by the megakaryocytes. The aforementioned cellular component refers to cells and platelets. As previously mentioned, the aforementioned megakaryocytes can be induced from cells that are undifferentiated compared to the aforementioned megakaryocytes. Therefore, when the megakaryocytes used as a culture for producing the aforementioned megakaryocytes contain megakaryocytes induced from cells that are undifferentiated compared to the aforementioned megakaryocytes, the aforementioned megakaryocyte culture can contain cells that are undifferentiated compared to the aforementioned megakaryocytes.

[0046] In this invention, the aforementioned megakaryocyte culture can be the culture itself obtained by culturing the aforementioned megakaryocytes, or it can be a processed form of the aforementioned culture. Processing of the aforementioned culture may include, for example, the removal of liquid fractions, the extraction of cellular component fractions, and alteration of the composition of cellular components including platelets. Alteration of the aforementioned cellular component composition may include, for example, the removal of cells and / or platelets from the aforementioned mixture, the extraction of cells and / or platelets from the aforementioned mixture, and the addition of cells and / or platelets to the aforementioned mixture.

[0047] The aforementioned "platelets" are one of the cellular components of blood, referring to cellular components that are positive for CD41a and CD42b. For example, platelets lack a nucleus and are smaller in size compared to megakaryocytes. Therefore, platelets and megakaryocytes can be distinguished, for example, based on the presence and / or size of a nucleus. It is known that platelets play an important role in thrombosis and hemostasis, and also participate in tissue regeneration after injury and the pathophysiology of inflammation. Furthermore, it is known that when platelets are activated due to bleeding, they express receptors for cell adhesion factors such as integrin αIIBβ3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61) on their membranes. Additionally, if platelets are activated, they aggregate, and various coagulation factors released from the platelets cause fibrin coagulation, thereby forming a thrombus and achieving hemostasis. In this invention, the source of the aforementioned platelets is the same as the source of the aforementioned megakaryocytes.

[0048] In this invention, the aforementioned processed product can be prepared from the aforementioned megakaryocytes or from a culture of the aforementioned megakaryocytes. Furthermore, when prepared from a culture of the aforementioned megakaryocytes, the culture of the aforementioned megakaryocytes may have undergone processing. Specifically, the aforementioned processed product may be a cell fraction or liquid fraction of the aforementioned megakaryocytes or their culture, or a processed product derived from the aforementioned megakaryocytes or their culture. The processing in the preparation of the aforementioned processed product is not particularly limited; examples include processing that alters the density of cell components, such as concentration, separation, or purification; extraction processing that extracts cell components, such as drying, freezing, freeze-drying, solvent treatment, surfactant treatment, enzyme treatment, or protein fraction extraction; and crushing processing, such as grinding or pulverizing. Specific examples of the aforementioned processed materials include extracts such as concentrates, dried products, frozen products, freeze-dried products, solvent-treated products, surfactant-treated products, enzyme-treated products, protein fractions, and ultrasonically treated products of megakaryocytes or their cultures; ground products, pulverized products, and other fragmented products; extracts of cell fractions of megakaryocytes or their cultures such as concentrates, dried products, frozen products, freeze-dried products, solvent-treated products, surfactant-treated products, enzyme-treated products, protein fractions, and ultrasonically treated products; ground products, pulverized products, and other fragmented products; etc. The aforementioned processed materials may consist of one type of processed material or a mixture of two or more processed materials. There are no particular limitations on the aforementioned mixtures; they can be prepared as mixtures of any combination and ratio of processed materials.

[0049] The aforementioned processed material, for example, contains at least one of one or more growth factors and growth factor receptors. Furthermore, the aforementioned processed material, for example, possesses physiological activities such as cell proliferation-promoting activity. Moreover, as described above, the aforementioned processed material can be manufactured, for example, by treating the aforementioned megakaryocytes or their cultures. Therefore, in this invention, the aforementioned processed material can be specified using, for example, the following conditions (1) to (3). The aforementioned processed material can be specified using, for example, any one of the following conditions (1) to (3), multiple conditions, or all conditions. As a specific example, the aforementioned processed material can be specified using, for example, a combination of conditions.

[0050] (condition)

[0051] (1) The content of growth factors and / or growth factor receptors;

[0052] (2) Physiological activity of the treated organism;

[0053] (3) Method for manufacturing processed goods

[0054] (Combination of conditions)

[0055] Conditions (1), (2), or (3);

[0056] Conditions (1) and (2), conditions (1) and (3), or conditions (2) and (3);

[0057] Conditions (1), (2), and (3)

[0058] (1) Condition (1)

[0059] As mentioned above, condition (1) pertains to the content of growth factors and / or growth factor receptors. Condition (1) may specify the content of the aforementioned growth factor or the content of the aforementioned growth factor receptor, or it may specify both the content of the aforementioned growth factor and the content of the aforementioned growth factor receptor. Furthermore, the growth factor used in the specification of condition (1) may be one type or two or more types. The growth factor receptor used in the specification of condition (1) may be one type or two or more types.

[0060] In the above condition (1), examples of the growth factors include basic fibroblast growth factor (bFGF), insulin-like growth factor binding protein-1 (IGFBP-1), insulin-like growth factor binding protein-2 (IGFBP-2), insulin-like growth factor binding protein-3 (IGFBP-3), insulin-like growth factor binding protein-6 (IGFBP-6), placental growth factor (PIGF), vascular endothelial growth factor (VEGF), endocrine gland-derived vascular endothelial growth factor (EG-VEGF), growth differentiation factor-15 (GDF-15), amphiregulin (AR), osteogenic protein-5 (BMP-5), osteogenic protein-7 (BMP-7), hepatocyte growth factor (HGF), TGFβ1 (transforming growth factor β1), etc.

[0061] In the above condition (1), examples of the growth factor receptors include stem cell factor receptor (SCFR), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor 2 (VEGFR2), etc.

[0062] The above content can be, for example, the weight of each growth factor and each growth factor receptor in the above-mentioned treatment substance, or the weight of each growth factor and each growth factor receptor in the above-mentioned treatment substance relative to the weight of the total protein (content per unit total protein), but the latter is preferred.

[0063] The weight of the above total protein can be determined, for example, by the BCA protein quantification method. The BCA protein quantification method is a protein quantification method that utilizes the coordination binding of monovalent copper ions with two molecules of bicinchoninic acid. Samples for the above BCA protein quantification method preferably do not contain a reducing agent and / or a copper ion chelating agent, for example. The BCA protein quantification method can be carried out according to the following Reference 4. For example, commercially available kits can also be used. As the kit for the BCA protein quantification method, Pierce (trademark) BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) can be used, etc.

[0064] Reference 4: Hase Toshiharu et al., "Protein Science Experiment Methods Starting from Simple Principles 1 Protein Extraction, Purification, and Synthesis", Kagaku Dojin, December 13, 2008

[0065] The total protein concentration in the aforementioned treatment can be appropriately set, for example, based on the number of cells supplied for treatment and the volume of solvent in the treatment. The total protein concentration in the aforementioned treatment can be relatively increased, for example, by increasing the number of cells supplied for treatment, decreasing the volume of solvent in the aforementioned treatment, or by extracting from the aforementioned megakaryocytes. Conversely, the total protein concentration in the aforementioned treatment can be relatively decreased, for example, by decreasing the number of cells supplied for treatment, increasing the volume of solvent in the aforementioned treatment, or by extracting from the aforementioned megakaryocyte culture. As a specific example, the number of cells supplied for treatment is 1 × 10⁻⁶. 8 When the volume of the solvent in the aforementioned treatment is 100 μl, the total protein concentration in the aforementioned treatment is, for example, 0.1–200 mg / ml. The aforementioned solvent is, for example, an aqueous solvent described later.

[0066] The weights of the aforementioned growth factors and growth factor receptors can be determined, for example, using a sandwich ELISA method. This sandwich ELISA method can be performed according to reference 5 below, or, for example, using a commercially available kit. As a kit for the aforementioned sandwich ELISA method, Quantibody (registered trademark) Human Growth Factor Array 1 (manufactured by RayBiotech) can be used, etc.

[0067] Reference 5: Edited by the Biochemical Assay Research Association, "From Immunoassay to Advanced Methods", Kodansha, December 20, 2014

[0068] Examples of the content of the aforementioned growth factors and growth factor receptors in the aforementioned treatments include the following.

[0069] When the aforementioned growth factor is bFGF, the aforementioned treatment contains, for example, 2000–20000 pg, 5000–20000 pg, or 10000–20000 pg of bFGF per 1 mg of total protein. By including the aforementioned treatment in bFGF, cell proliferation-promoting activity is exhibited as described below.

[0070] When the aforementioned growth factor is IGFBP-1, the aforementioned treatment contains 0–200 pg, 0.01–200 pg, 0.01–100 pg, or 0.01–50 pg of IGFBP-1 relative to 1 mg of total protein.

[0071] When the aforementioned growth factor is IGFBP-2, the aforementioned treatment contains 8000-80000 pg, 10000-80000 pg, or 20000-80000 pg of IGFBP-2 relative to 1 mg of total protein.

[0072] When the aforementioned growth factor is PIGF, the aforementioned treatment contains PIGF at a concentration of 1–60 pg, 1–30 pg, or 1–20 pg relative to 1 mg of total protein.

[0073] When the aforementioned growth factor is VEGF, the aforementioned treatment contains VEGF at a concentration of 20–800 pg, 20–600 pg, or 20–400 pg relative to 1 mg of total protein.

[0074] When the aforementioned growth factor is GDF-15, the aforementioned treatment contains 1000-10000 pg, 1000-5000 pg, or 2000-5000 pg of GDF-15 relative to 1 mg of total protein.

[0075] When the aforementioned growth factor is AR, the aforementioned treatment contains AR at a concentration of 0–16 pg, 0.01–16 pg, 0.1–16 pg, or 1–16 pg relative to 1 mg of total protein.

[0076] When the aforementioned growth factor is HGF, the aforementioned treatment contains HGF at a concentration of 0–100 pg, 0.01–100 pg, 0.01–50 pg, or 0.01–30 pg relative to 1 mg of total protein.

[0077] When the aforementioned growth factor is BMP-7, the aforementioned treatment contains 0 to 1000 pg or 0.01 to 1000 pg of BMP-7 relative to 1 mg of total protein.

[0078] In the case where the aforementioned growth factor receptor is SCFR, the aforementioned treatment contains 200–2000 pg, 300–1500 pg, or 400–1000 pg of SCFR relative to 1 mg of total protein.

[0079] In the case where the aforementioned growth factor receptor is EGFR, the aforementioned treatment contains EGFR in amounts of 0–60 pg, 0.01–60 pg, 1–50 pg, 1–45 pg, or 10–40 pg relative to 1 mg of total protein.

[0080] In the case where the aforementioned growth factor receptor is VEGFR2, the aforementioned treatment contains 20–400 pg, 50–350 pg, or 100–300 pg of VEGFR2 relative to 1 mg of total protein.

[0081] As mentioned above, the aforementioned condition (1) can be specified by the content of one or more growth factors, or by the content of one or more growth factor receptors, or by any combination thereof. In this case, the aforementioned condition (1) can be specified, for example, by selecting at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve conditions from the group consisting of conditions (A1) to (A9) and (B1) to (B3). As a specific example, the following combinations can be exemplified as combinations of the aforementioned growth factor content and / or growth factor receptor content.

[0082] (A combination of growth factor levels and / or growth factor receptor levels)

[0083] Any one of the following conditions: (A1)~(A9) and (B1)~(B3)

[0084] (A1) content of bFGF, (A2) content of IGFBP-1, (A3) content of IGFBP-2, (A4) content of PIGF, (A5) content of VEGF, (A6) content of GDF-15, (A7) content of AR, (A8) content of HGF, (A9) content of BMP-7, (B1) content of SCFR, (B2) content of EGFR, or (B3) content of VEGFR2;

[0085] Any two of the following conditions: (A1)~(A9) and (B1)~(B3)

[0086] (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)

[0087] (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)

[0088] (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)

[0089] (A4) and (A5), (A4) and (A6), (A4) and (A7), (A4) and (A8), (A4) and (A9), (A4) and (B1), (A4) and (B2), (A4) and (B3)

[0090] (A5) and (A6), (A5) and (A7), (A5) and (A8), (A5) and (A9), (A5) and (B1), (A5) and (B2), (A5) and (B3)

[0091] (A6) and (A7), (A6) and (A8), (A6) and (A9), (A6) and (B1), (A6) and (B2), (A6) and (B3)

[0092] (A7) and (A8), (A7) and (A9), (A7) and (B1), (A7) and (B2), (A7) and (B3)

[0093] (A8) and (A9), (A8) and (B1), (A8) and (B2), (A8) and (B3)

[0094] (A9) and (B1), (A9) and (B2), (A9) and (B3)

[0095] (B1) and (B2), (B1) and (B3), or

[0096] (B2) and (B3).

[0097] The content of the aforementioned total protein in the unit can be adjusted, for example, according to the use of the composition according to the invention, by adding or removing other proteins besides the aforementioned growth factors and growth factor receptors. The aforementioned other proteins may include, for example, proteins that do not affect the activity of the aforementioned growth factors and growth factor receptors; specific examples include serum albumin such as human serum albumin, gamma globulin such as human gamma globulin, etc. The removal of the aforementioned proteins may include, for example, removal using a column or removal using an antibody.

[0098] (2) Condition (2)

[0099] As mentioned above, condition (2) is a condition relating to the physiological activity of the treated substance. In condition (2), the physiological activity of the treated substance refers, for example, to the activity of regulating the function of cells, tissues, or organs. Examples of such cellular functions include, for example, proliferation, differentiation, induction or inhibition of gene expression, and induction or inhibition of the expression of macromolecules such as proteins and glycans.

[0100] The physiological activities of the aforementioned treated substances may include, for example, cell proliferation-promoting activity, fibroblast function-promoting activity, keratinocyte function-promoting activity, and dermal papilla cell function-promoting activity. In the aforementioned cell proliferation-promoting activity, there are no particular limitations on the cells mentioned; for example, mesenchymal stem cells, fibroblasts, keratinocytes such as epidermal keratinocytes, and dermal papilla cells such as scalp dermal papilla cells may be included. The aforementioned treated substances may possess one activity or multiple activities.

[0101] The aforementioned mesenchymal stem cells are cells capable of self-replication and differentiation into bone, cartilage, and adipocytes. These mesenchymal stem cells can be identified using cell surface markers. For example, they are positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45.

[0102] The aforementioned fibroblasts are cells that make up the connective tissue of organs such as the skin, lungs, and heart. They are cells that supply fibrous components (extracellular matrix components such as collagen, elastin, and hyaluronic acid). These fibroblasts can be identified using cell surface markers. For example, they may be positive for vimentin, CD90, and TE-7 antibodies.

[0103] The aforementioned keratinocytes are epidermal cells capable of keratinization; they divide in the basal layer of the epidermis and contribute to epidermal formation. These keratinocytes can be identified by cell surface markers. For example, they may be positive for androgen receptors or cytokeratin proteins. Epidermal keratinocytes are preferred.

[0104] The aforementioned dermal papilla cells are cells located within the dermal papilla at the base of the hair follicle and are crucial for inducing and maintaining hair growth. These dermal papilla cells can be identified by cell surface markers and / or gene expression. For example, they may be alkaline phosphatase positive; positive for SOX2, WIF1, Noggin, BMP4, and VCAN genes. Scalp dermal papilla cells are preferred.

[0105] The aforementioned cell proliferation-promoting activity can be achieved, for example, simply by improving the cell proliferation capacity compared to the same control group except for the one without the composition of the present invention; for example, the cell proliferation capacity may be reduced from the beginning. In this case, the aforementioned "proliferation-promoting activity" can also be referred to as inhibition of the reduction in proliferation activity. As a specific example, the proliferation activity of the aforementioned mesenchymal stem cells decreases with each passage. According to the composition of the present invention, since the reduction in the proliferation activity of the aforementioned mesenchymal stem cells can be inhibited, it can be said, for example, that the composition of the present invention exhibits proliferation-promoting activity. The aforementioned cell proliferation-promoting activity can be measured, for example, under the culture conditions for the proliferation of the target cells. The aforementioned culture conditions can be appropriately set, for example, according to the type of the aforementioned cells. As a specific example, when mesenchymal stem cells are used as the aforementioned cells, the proliferation activity of the aforementioned cells can be measured, for example, by culturing human-derived mesenchymal stem cells in vitro in the presence of a proliferation medium. The proliferation activity of the aforementioned mesenchymal stem cells can be measured, for example, based on Example 1 described later. In addition, when fibroblasts are used as the aforementioned cells, the proliferation activity of the aforementioned cells can be measured, for example, by culturing human-derived fibroblasts in vitro in the presence of a proliferation medium. The proliferation activity of the aforementioned fibroblasts can be measured, for example, based on Example 3 described later. When keratinocytes are used as the aforementioned cells, their proliferative activity can be measured, for example, by culturing human-derived epidermal keratinocytes in vitro in the presence of a proliferation medium. The proliferative activity of the aforementioned keratinocytes can be measured, for example, based on Example 4 described later. When dermal papilla cells are used as the aforementioned cells, their proliferative activity can be measured, for example, by culturing human-derived hair dermal papilla cells in vitro in the presence of a proliferation medium. The proliferative activity of the aforementioned dermal papilla cells can be measured, for example, based on Example 5 described later.

[0106] The aforementioned fibroblast function-promoting activity is simply defined as an improvement in fibroblast function compared to the same control group except for the one without the composition of the present invention. The aforementioned fibroblast function can be, for example, any meaning of the aforementioned fibroblast proliferation and the production of extracellular matrix by fibroblasts. The aforementioned extracellular matrix can include, for example, collagen-like substances such as type I collagen, fibrous substances such as elastin; matrix substances such as glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, proteoglycans, integrins, fibronectin, laminins, etc., which are cell adhesion proteins; etc. The aforementioned fibroblast function can be measured, for example, based on Example 3 described later.

[0107] The aforementioned keratinocyte function-promoting activity is simply defined as an improvement in keratinocyte function compared to the same control group except for the one without the composition of the present invention. The aforementioned keratinocyte function can be any of the following: keratinocyte proliferation, differentiation into epidermal cells, and induction of barrier function genes. The aforementioned barrier function genes refer to genes that maintain the barrier function of the skin; specific examples include the filaggrinogen gene (FLG) and the ceramide synthase gene (serine palmitoyltransferase long chain basesubunit 1: SPTLC1). As for the aforementioned filaggrinogen gene, human-derived filaggrinogen genes can be listed, for example, a polynucleotide sequence registered in GenBank with Accession No.: NM_002016.2. As for the aforementioned ceramide synthase gene, human-derived ceramide synthase genes can be listed, for example, a polynucleotide sequence registered in GenBank with Accession No.: NM_001281303.2. The aforementioned keratinocyte function can be measured, for example, based on Example 4 described later.

[0108] The aforementioned dermal papilla cell function-promoting activity is simply defined as an improvement in dermal papilla cell function compared to the same control group except for the one without the composition of the present invention. The aforementioned dermal papilla cell function can be any meaning within the context of pre-dermal papilla cell proliferation and the induction of hair growth-promoting genes. The aforementioned hair growth-promoting genes can refer to genes with functions of hair growth, hair growth, or maintenance; specific examples include the FGF7 (fibroblast growth factor 7) gene and the vascular endothelial growth factor (VEGF) gene. The aforementioned VEGF can be exemplified by VEGFA. As for the aforementioned FGF7 gene, human-derived pre-FGF7 genes can be exemplified by polynucleotides in GenBank consisting of a base sequence registered with Accession No.: NM_002009.4. As for the aforementioned VEGFA gene, human-derived VEGFA genes can be exemplified by polynucleotides in GenBank consisting of a base sequence registered with Accession No.: NM_001025366.3. The aforementioned dermal papilla cell function can be measured, for example, based on Example 5 described later.

[0109] (3) Condition (3)

[0110] As mentioned above, condition (3) pertains to the method of manufacturing the treated article. For the method of manufacturing the treated article in the composition of the present invention, the description of the method of manufacturing the composition of the present invention, which will be described later, can be referenced.

[0111] The compositions of the present invention can be manufactured, for example, using the methods for manufacturing the compositions of the present invention described later.

[0112] As described below, the compositions of the present invention can be used, for example, as cell proliferation promoting compositions, fibroblast function promoting compositions, keratinocyte function promoting compositions, and dermal papilla cell function promoting compositions. Furthermore, due to the aforementioned activities, the compositions of the present invention can be suitably used, for example, as compositions promoting the healing of skin disorders and hair growth promoting compositions. For the methods of using the present invention, the descriptions of the cell proliferation promoting compositions, fibroblast function promoting compositions, keratinocyte function promoting compositions, and dermal papilla cell function promoting compositions described below can be referenced. Additionally, the compositions of the present invention can be used, for example, for the repair of knee joint injuries, tendon injuries, or ligament injuries; the treatment of ulcers, bedsores, burns, scars, or trauma; skin texture improvement; hair growth; and / or skin beautification; etc.

[0113] <Method for manufacturing the composition>

[0114] The method for manufacturing the composition of the present invention (hereinafter also referred to as the "manufacturing method") includes a treatment step of processing megakaryocytes or cultures thereof. The manufacturing method of the present invention is characterized by including the aforementioned treatment step; other steps and conditions are not particularly limited. The composition of the present invention can be manufactured according to the manufacturing method of the present invention. The manufacturing method of the present invention can be referenced from the foregoing description of the composition of the present invention.

[0115] In the manufacturing method of the present invention, the object of treatment in the aforementioned processing steps is megakaryocytes or their cultures. Therefore, the manufacturing method of the present invention may include, prior to the aforementioned processing steps, a megakaryocyte induction step of inducing the aforementioned megakaryocytes from cells that are undifferentiated compared to megakaryocytes and / or a production step of producing the aforementioned megakaryocyte cultures.

[0116] In the aforementioned megakaryocyte induction process, the megakaryocyte induction method is not particularly limited and can be implemented using known induction methods. Specifically, the aforementioned megakaryocyte induction method may refer to methods for inducing immortalized megakaryocytes such as International Patent Publication No. 2011 / 034073 (US Patent Application Publication No. 2012 / 0238023), International Patent Publication No. 2012 / 157586 (US Patent Application Publication No. 2014 / 0127815), and International Patent Publication No. 2014 / 123242 (US Patent Application Publication No. 2016 / 0002599); the megakaryocyte induction method described in Reference 6 below; etc., which are incorporated herein by reference as part of this specification. Specifically, in the aforementioned megakaryocyte induction process, for example, cells that are undifferentiated compared to the aforementioned megakaryocytes may be forced to express the aforementioned oncogenes and the aforementioned polycomb genes. Thus, in the aforementioned megakaryocyte induction process, for example, immortalized megakaryocytes with unlimited proliferation can be obtained. Furthermore, for example, by deactivating the aforementioned forced expression of the immortalized megakaryocytes, the immortalized megakaryocytes can be induced into multinucleated giant cells, which then produce platelets. Additionally, in the aforementioned megakaryocyte induction process, for example, the aforementioned megakaryocyte progenitor cells can be forced to express the aforementioned apoptosis-inhibiting gene. Thus, in the aforementioned megakaryocyte induction process, the aforementioned immortalized megakaryocytes can be obtained. Furthermore, for example, by deactivating the aforementioned forced expression of the immortalized megakaryocytes, multinucleated giant cells can be induced from the aforementioned immortalized megakaryocytes, which then produce platelets.

[0117] Reference 6: Ann-Kathrin Borger et.al., "Generation of HLA-UniversaliPSC-Derived Megakaryocytes and Platelets for Survival Under RefractorinessConditions", Mol. Med., 2016, vol.22, pages 274-288

[0118] In the aforementioned megakaryocyte induction process, for example, the aforementioned oncogene, the aforementioned polycomb gene, and the aforementioned apoptosis-inhibiting gene can be forcibly expressed. In this case, the forced expression of the aforementioned oncogene, the aforementioned polycomb gene, and the aforementioned apoptosis-inhibiting gene can be performed simultaneously or independently. As a specific example, in the aforementioned megakaryocyte induction process, after the forced expression of the aforementioned oncogene and the aforementioned polycomb gene is deactivated, the aforementioned apoptosis-inhibiting gene is then forcibly expressed. Alternatively, the aforementioned oncogene, the aforementioned polycomb gene, and the aforementioned apoptosis-inhibiting gene can be forcibly expressed, or the aforementioned oncogene and the aforementioned polycomb gene can be forcibly expressed, followed by the expression of the aforementioned apoptosis-inhibiting gene. Thus, in the aforementioned megakaryocyte induction process, the aforementioned immortalized megakaryocytes can be obtained. Furthermore, for example, by deactivating the aforementioned forced expression of the aforementioned immortalized megakaryocytes, multinucleated megakaryocytes can be induced from the aforementioned immortalized megakaryocytes, and platelets can be produced.

[0119] For the aforementioned megakaryocyte induction process, for example, from the perspective of improving the efficiency of gene introduction, it is preferable to include the following steps: a first expression step, in which undifferentiated cells compared with the aforementioned megakaryocytes are forced to express oncogenes and polycomb genes; a second expression step, in which the aforementioned undifferentiated cells are forced to express apoptosis-inhibiting genes such as Bcl-xL gene; and a de-expression step, in which all the aforementioned forced expression is de-expressed.

[0120] The forced expression and de-expression of each gene can be carried out, for example, using known methods such as those described in International Publication No. 2011 / 034073, International Publication No. 2012 / 157586, International Publication No. 2014 / 123242, or Reference 7 below, and are incorporated herein by reference as part of this description. Specifically, the forced expression and de-expression of each gene can be carried out, for example, using a drug-responsive gene expression induction system. Examples of such gene expression induction systems include the Tet-on (registered trademark) system and the Tet-off (registered trademark) system. When using the Tet-on system, for example, in the forced expression step, culture is performed in the presence of a gene-expression-inducing agent such as a tetracycline antibiotic or doxycycline; in the de-expression step, the culture is performed in the absence of the aforementioned agent.

[0121] Reference 7: Nakamura S et al, "Expandable megakaryocyte cell linesenable clinically applicable generation of platelets from human induced pluripotent stem cells.", Cell Stem Cell, 2014, vol.14, No.4, pages 535-548

[0122] Next, in the aforementioned production process, a culture of the aforementioned megakaryocytes is produced. This production process can be carried out, for example, by culturing the aforementioned megakaryocytes in the presence of a culture medium. The culture of the aforementioned megakaryocytes can be carried out, for example, on feeder cells, or without feeder cells. The aforementioned megakaryocytes can be cultured in suspension, thus allowing culture without the aforementioned feeder cells. The culture of the aforementioned megakaryocytes may include, for example, the aforementioned platelets.

[0123] There are no particular restrictions on the culture conditions for the aforementioned megakaryocytes; the usual culture conditions for megakaryocytes can be used. For specific examples, the culture temperature may be approximately 35–42°C, approximately 36–40°C, or approximately 37–39°C. The CO2 concentration may be approximately 5–15%. The O2 concentration may be approximately 15–25% or approximately 20%. There are no particular restrictions on the culture time; for example, it may be approximately 1 day to approximately 2 weeks or approximately 4 days to approximately 8 days.

[0124] The aforementioned culture medium is not particularly limited; for example, known culture media suitable for platelet production from the aforementioned megakaryocytes and culture media based thereon can be listed. As a specific example, the aforementioned culture medium can be prepared using a culture medium used in animal cell culture as a basal medium. Examples of such basal media include, for example, IMDM medium, Medium 199 medium, EMEM (Emergency Essential Medium), αMEM medium, DMEM (Durbeco Modified IgM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal (registered trademark) medium (manufactured by Thermo Fisher Scientific), and other single or mixed media. The aforementioned culture medium can, for example, contain serum or plasma, or be a serum-free medium without these. The source of the aforementioned serum and plasma is preferably the same as the source of the aforementioned megakaryocytes. As a specific example, when the aforementioned megakaryocytes are derived from humans, the aforementioned serum and plasma are preferably derived from humans, respectively.

[0125] The aforementioned culture medium may contain other components, for example. There are no particular limitations on these other components; examples include albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and cytokines. These other components may be one or more. The aforementioned cytokines are substances that promote the differentiation of hematologic cells; 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 aromatic hydrocarbon receptor (AhR) inhibitors. The aforementioned culture medium preferably contains, for example, serum, insulin, transferrin, serine, thioglycerol, ascorbic acid, or IMDM medium containing TPO. The aforementioned culture medium may further contain, for example, SCF, or heparin. The concentrations of the aforementioned other components are not particularly limited. The concentration of the aforementioned 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 the aforementioned SCF is, for example, about 10 ng / ml to about 200 ng / ml, or about 50 ng / ml. The concentration of the aforementioned heparin is, for example, about 10 U / ml to about 100 U / ml, or about 25 U / ml. The aforementioned culture medium may further contain, for example, phorbol esters (e.g., phorbol-12-myristate-13-acetate; PMA).

[0126] Next, in the aforementioned processing steps, the aforementioned megakaryocytes or their cultures are treated. In these processing steps, for example, proteins are extracted by disrupting the cell membranes of the cells contained in the aforementioned megakaryocytes or their cultures. Specifically, the treatments in the aforementioned processing steps are not particularly limited, and examples include: treatments that alter the density of cell components, such as concentration treatments; extraction treatments that extract cell components, such as drying treatments, freezing treatments, freeze-drying treatments, solvent treatments, surfactant treatments, enzyme treatments, protein fractionation extraction treatments, and ultrasonic treatments; and crushing treatments, such as grinding and pulverizing treatments. The aforementioned processing steps may involve one or more treatments. Furthermore, the aforementioned processing steps may be performed once or twice or more.

[0127] The aforementioned concentration process can be performed, for example, by centrifuging the aforementioned megakaryocytes or their cultures. The centrifugation conditions can, for example, be conditions that cause cell or platelet precipitation. The aforementioned drying process can, for example, be performed by drying the aforementioned megakaryocytes or their cultures using a dry sprayer, drum dryer, or the like. The aforementioned freeze-drying process can, for example, be performed using a freeze dryer. In the aforementioned solvent treatment, the aforementioned solvent is, for example, an organic solvent such as phenol or chloroform; or an aqueous solvent such as water, physiological saline, or buffer solution. When using an aqueous solvent as the aforementioned solvent, the aforementioned solvent treatment preferably combines, for example, the surfactant treatment, enzyme treatment, and / or ultrasonic treatment described later. The aforementioned solvent treatment can, for example, be performed by mixing the aforementioned megakaryocytes or their cultures with the aforementioned solvent. In the aforementioned surfactant treatment, the aforementioned surfactants can, for example, include ionic surfactants such as sodium lauryl sulfate; nonionic surfactants such as NP-40, Triton X-100, Tween20, and n-dodecyl-β-D-maltodextrin; and amphoteric surfactants such as CHAPS; etc. The concentration of the aforementioned surfactant is, for example, a concentration capable of disrupting the cell membrane of the aforementioned megakaryocytes or their cultures. The aforementioned surfactant treatment can be performed, for example, by contacting the aforementioned megakaryocytes or their cultures with the aforementioned surfactant in the presence of an aqueous solvent. Contact with the aforementioned surfactant is performed, for example, at approximately 0 to approximately 10°C. Examples of the aforementioned aqueous solvent include water, physiological saline, and buffer solutions. Examples of the aforementioned enzymes in the enzyme treatment include peptidases and proteases. The aforementioned enzyme treatment can be performed, for example, by contacting the aforementioned megakaryocytes or their cultures with the aforementioned enzyme in the presence of the aforementioned aqueous solvent. The conditions for the aforementioned enzyme treatment are, for example, conditions under which the aforementioned enzyme exhibits activity. The aforementioned protein fractionation extraction treatment can be performed, for example, by subjecting the aforementioned megakaryocytes or their cultures to osmotic pressure shock, freeze-thaw cycles, etc. The aforementioned ultrasonic treatment can be performed, for example, using an ultrasonic generator. The conditions for the aforementioned ultrasonic treatment can, for example, utilize conditions that disrupt cells.

[0128] The processing conditions and processing time in various processes can be appropriately determined, for example, according to the type of processing described above. Furthermore, in the aforementioned processing steps, for example, the concentration of total protein in the processed product can be adjusted by adjusting the amount of the aforementioned aqueous solvent.

[0129] The aforementioned megakaryocytes or their cultures may be processed (pretreated) prior to the aforementioned treatment. In this case, the culture of the aforementioned megakaryocytes may be the culture itself obtained from culturing the aforementioned megakaryocytes, or it may be the culture obtained by processing the aforementioned mixture. Examples of processing of the aforementioned culture include, for example, removal of liquid fractions, extraction of cellular component fractions, and alteration of the composition of cellular components, including platelets. Examples of alteration of the composition of the aforementioned cellular components include, for example, removal of cells and / or platelets from the aforementioned mixture, extraction of cells and / or platelets from the aforementioned mixture, and addition of cells and / or platelets to the aforementioned mixture.

[0130] When the manufacturing method of the present invention includes the aforementioned pretreatment, the manufacturing method of the present invention may include a platelet removal step from the aforementioned megakaryocytes or their cultures. In this case, in the aforementioned treatment step, the megakaryocytes or their cultures from which the aforementioned platelets have been removed are used as the aforementioned megakaryocytes or their cultures, or the removed platelets are used to perform the treatment. The megakaryocytes after the release of the aforementioned platelets have, for example, a high content of bFGF. Therefore, the manufacturing method of the present invention can, for example, produce a composition with a high content of bFGF by removing the aforementioned platelets. By removing the aforementioned platelets, the aforementioned platelets can be separated from other cell fractions. Therefore, the aforementioned removal step may also be referred to as a platelet separation step or a platelet separation step from other cellular components. The method for separating platelets from the aforementioned megakaryocyte culture in the aforementioned removal process, for example, can be carried out using known methods such as those described in International Publication No. 2017 / 065280 (U.S. Patent Application Publication No. 2018 / 282697), and is incorporated herein by reference as part of this description.

[0131] The platelet removal rate (separation rate) in the aforementioned removal process 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 aforementioned platelet removal rate is, for example, 60-90%.

[0132] The manufacturing method of the present invention may include a preservation step of preserving the aforementioned megakaryocytes or their cultures, megakaryocytes or their cultures from which the aforementioned platelets have been removed, or platelets that have been removed. Examples of preservation steps include refrigeration (approximately 1 to approximately 10°C) and freezing (approximately -200 to approximately -4°C). There is no particular limitation on the preservation time in the aforementioned preservation steps. For example, considering that freezing is also a part of the aforementioned processing steps, freezing is preferred in the aforementioned preservation steps.

[0133] As an example, the manufacturing method of the present invention can be implemented as follows. However, the present invention is not limited to any of the following examples. First, the culture medium containing the aforementioned megakaryocytes or their cultures is concentrated by centrifugation to concentrate the cell components. The aforementioned centrifugation is, for example, the condition for precipitation of the aforementioned cell components. As a specific example, the aforementioned centrifugation can be performed, for example, by centrifuging at 1000–3000 × g for 5–20 minutes. Next, the precipitate is recovered after centrifugation, and the obtained precipitate is rapidly frozen, thereby performing a freezing treatment. The aforementioned freezing treatment can be performed, for example, by contacting the aforementioned precipitate with a liquefied gas such as liquid nitrogen. Furthermore, the frozen precipitate is dissolved in an aqueous solvent containing the aforementioned surfactant, thereby performing a surfactant treatment. The resulting solution is centrifuged to precipitate impurities. The aforementioned centrifugation is, for example, the condition for precipitation of impurities such as cell membranes. As a specific example, the aforementioned centrifugation can be performed, for example, by centrifuging at 10000–20000 × g for 3–10 minutes. After centrifugation, the protein remains in the supernatant. Therefore, by recovering the supernatant as a protein fraction, the aforementioned processed product can be obtained.

[0134] <Composition obtained in the manufacturing method>

[0135] The composition of the present invention (hereinafter also referred to as "the second composition") can be obtained using the aforementioned manufacturing method of the present invention. The second composition of the present invention is characterized by being obtained using the aforementioned manufacturing method of the present invention; other components and conditions are not particularly limited. According to the second composition of the present invention, for example, a physiologically active composition can be provided. According to the second composition of the present invention, for example, it can promote cell proliferation. The second composition of the present invention can be understood by referring to the description of the compositions and manufacturing methods of the present invention described above.

[0136] <Cell proliferation promoting composition>

[0137] In another example, the present invention provides a composition capable of promoting cell proliferation. As previously described, 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, with no particular limitation on other components and conditions. The proliferation-promoting composition according to the present invention can promote the proliferation of cells, particularly mesenchymal stem cells. The proliferation-promoting composition of the present invention can be described using the foregoing description of the composition and manufacturing method of the present invention.

[0138] The proliferation-promoting composition of the present invention can be used in vitro or in vivo.

[0139] When the proliferation-promoting composition of the present invention is used in vitro, the aforementioned target of administration may include, for example, cells, tissues, organs, etc., and the aforementioned cells may include, for example, cells collected from organisms, cultured cells, etc.

[0140] When the proliferation-promoting composition of the present invention is used in vivo, the aforementioned subjects may include, for example, humans, or non-human animals other than humans. Examples of such non-human animals include, for example, mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, guinea pigs, etc.

[0141] The conditions for use (administration conditions) of the proliferation-promoting composition of the present invention are not particularly limited. For example, the administration form, administration period, administration amount, etc. can be appropriately set according to the type of drug target.

[0142] When using the proliferation-promoting composition of the present invention in vitro, the proliferation-promoting composition of the present invention can be used, for example, by adding it to a culture medium containing the cells to be targeted. The final concentration of the total protein derived from the proliferation-promoting composition of the present invention in the aforementioned culture medium is 10–1000 μg / ml, 10–500 μg / ml, 10–320 μg / ml, 10–300 μg / ml, or 20–300 μg / ml.

[0143] When using the proliferation-promoting composition of the present invention in vivo, the dosage can be appropriately determined based on factors such as the type of recipient, symptoms, age, and method of administration. Specifically, when administering the composition to a human, there is no particular limitation on the total amount of protein derived from the proliferation-promoting composition for each day's dosage; it can be appropriately set according to its intended use. The number of times the composition is administered each day can be, for example, 1 to 5 times, 1 to 3 times, once, or twice.

[0144] There are no particular limitations on the administration method of the proliferation-promoting composition of the present invention. When administering the proliferation-promoting composition of the present invention in vivo, it can be administered orally or non-orally. Examples of non-oral administration include intravenous injection, intramuscular injection, transdermal administration, subcutaneous administration, intradermal administration, enteral administration, rectal administration, vaginal administration, nasal administration, pulmonary administration, intraperitoneal administration, and local administration.

[0145] The dosage form of the proliferation-promoting composition of the present invention is not particularly limited, and can be appropriately determined according to the aforementioned administration form. Examples of such dosage forms include liquids and solids.

[0146] The proliferation-promoting composition of the present invention may, for example, contain additives as needed. The aforementioned additives are preferably pharmaceutically acceptable additives or pharmaceutically acceptable carriers.

[0147] <Fibroblast Function Promoting Composition>

[0148] In another example, the present invention provides a composition capable of promoting fibroblast function. As previously described, the fibroblast function promoting composition of the present invention comprises the aforementioned composition of the present invention. The fibroblast function promoting composition of the present invention is characterized by comprising the aforementioned composition of the present invention, while other components and conditions are not particularly limited. The fibroblast function promoting composition according to the present invention is capable of promoting fibroblast function. The fibroblast function promoting composition of the present invention can be referenced from the descriptions of the aforementioned compositions, manufacturing methods, and proliferation promoting compositions of the present invention.

[0149] The fibroblast function-promoting composition of the present invention can be used in vitro or in vivo.

[0150] When using the fibroblast function-promoting composition of the present invention in vitro, the fibroblast function-promoting composition of the present invention can be used, for example, by adding it to a culture medium containing the fibroblasts to which the target cells are located. According to the fibroblast function-promoting composition of the present invention, for example, by maintaining the aforementioned fibroblasts in a culture medium containing the aforementioned fibroblast function-promoting composition, it is possible to promote the proliferation of the aforementioned fibroblasts and / or the production of extracellular matrix by the fibroblasts. The final concentration of total protein of the fibroblast function-promoting composition of the present invention in the aforementioned culture medium is 10–1000 μg / ml, 10–500 μg / ml, or 10–300 μg / ml.

[0151] The target population and administration conditions of the fibroblast function promoting composition of the present invention can be referred to the description of the target population and administration conditions of the proliferation promoting composition of the present invention described above.

[0152] Compositions that promote the healing of skin disorders

[0153] In another example, the present invention provides a composition capable of promoting the healing of skin disorders. In this invention, as previously described, the composition for promoting the healing of skin disorders (hereinafter also referred to as the "healing-promoting composition") comprises the aforementioned composition of the present invention. The healing-promoting composition of the present invention is characterized by comprising the aforementioned composition of the present invention, with no particular limitation on other components and conditions. According to the healing-promoting composition of the present invention, it is expected to promote the healing of skin disorders. The description of the healing-promoting composition of the present invention, the manufacturing method, and the proliferation-promoting composition of the present invention can be referenced.

[0154] In this invention, the aforementioned skin disorder refers to a state in which damage or injury to the skin has occurred, that is, a state in which the structure of normal skin tissue is damaged or destroyed. Specific examples include skin ulcers, bedsores, burns, scars, trauma, and skin aging.

[0155] The healing-promoting composition of the present invention can be used in vitro or in vivo.

[0156] When using the healing-promoting composition of the present invention in vitro, the healing-promoting composition of the present invention can be used, for example, by adding it to a culture medium of the cells to which the object is located. The final concentration of total protein of the healing-promoting composition of the present invention in the aforementioned culture medium is 10–1000 μg / ml, 10–500 μg / ml, or 10–300 μg / ml.

[0157] The target population and administration conditions for the healing-promoting composition of the present invention can be found in the description of the target population and administration conditions for the proliferation-promoting composition of the present invention described above. The healing-promoting composition of the present invention can be used in dosage forms that can be administered subcutaneously or transdermally.

[0158] <Keratinocyte Function-Enhancing Composition>

[0159] In another example, the present invention provides a composition that promotes keratinocyte function. As previously described, the keratinocyte function promoting composition of the present invention comprises the aforementioned composition of the present invention. The keratinocyte function promoting composition of the present invention is characterized by comprising the aforementioned composition of the present invention, while other components and conditions are not particularly limited. The keratinocyte function promoting composition according to the present invention is capable of promoting keratinocyte function. The keratinocyte function promoting composition of the present invention can be referenced from the descriptions of the aforementioned compositions, manufacturing methods, and proliferation-promoting compositions of the present invention.

[0160] The keratinocyte function-promoting composition of the present invention can be used in vitro or in vivo.

[0161] When using the keratinocyte function-promoting composition of the present invention in vitro, it can be used, for example, by adding it to a culture medium containing the keratinocytes to be used. The keratinocyte function-promoting composition of the present invention can be added, for example, to a maintenance culture medium used to maintain the keratinocytes, to a differentiation culture medium that causes the keratinocytes to differentiate into epithelial cells such as epidermal cells, or to both the maintenance culture medium and the differentiation culture medium. According to the keratinocyte function-promoting composition of the present invention, for example, by maintaining the keratinocytes in a culture medium containing the keratinocyte function-promoting composition, the proliferation of the keratinocytes, their differentiation into epidermal cells, and / or the induction of barrier function genes can be promoted. The final concentration of total protein of the keratinocyte function-promoting composition of the present invention in the aforementioned culture medium is 10–1000 μg / ml, 10–500 μg / ml, or 10–300 μg / ml.

[0162] The target population and administration conditions of the keratinocyte function promoting composition of the present invention can be referred to the description of the target population and administration conditions of the proliferation promoting composition of the present invention described above.

[0163] As previously stated, 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 the artificial culture of skin. Furthermore, as previously stated, the keratinocyte function-promoting composition of the present invention exhibits fibroblast function-promoting activity. Therefore, the keratinocyte function-promoting composition of the present invention promotes the renewal of skin tissue, for example, through keratinocyte and fibroblast function-promoting activity, and is expected to be suitable for the regeneration of aging skin. 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 cosmetics, or as an injectable preparation for subcutaneous administration.

[0164] <Composition for Promoting Hair Papill Cell Function>

[0165] In another example, the present invention provides a composition that promotes dermal papilla cell function. As previously described, the dermal papilla cell function promoting composition of the present invention comprises the aforementioned composition of the present invention. The dermal papilla cell function promoting composition of the present invention is characterized by comprising the aforementioned composition of the present invention, while other components and conditions are not particularly limited. The dermal papilla cell function promoting composition according to the present invention can promote keratinocyte function. The dermal papilla cell function promoting composition of the present invention can be referenced from the descriptions of the aforementioned compositions, manufacturing methods, and proliferation-promoting compositions of the present invention.

[0166] The hair papilla cell function promoting composition of the present invention can be used in vitro or in vivo.

[0167] When using the dermal papilla cell function-promoting composition of the present invention in vitro, the composition can be used, for example, by adding it to a culture medium containing the dermal papilla cells to be used. According to the dermal papilla cell function-promoting composition of the present invention, for example, by maintaining the dermal papilla cells in a culture medium containing the aforementioned dermal papilla cell function-promoting composition, the proliferation of the aforementioned dermal papilla cells and / or the induction of hair growth-promoting genes can be promoted. The final concentration of total protein of the dermal papilla cell function-promoting composition of the present invention in the aforementioned culture medium is 10–1000 μg / ml, 10–500 μg / ml, or 10–300 μg / ml.

[0168] The target population and administration conditions of the hair papilla cell function promoting composition of the present invention can be referred to the description of the target population and administration conditions of the proliferation promoting composition of the present invention described above.

[0169] Hair growth promoting composition

[0170] In another example, the present invention provides a composition that promotes hair growth. As previously described, the hair growth promoting composition of the present invention comprises the aforementioned composition of the present invention. The hair growth promoting composition of the present invention is characterized by comprising the aforementioned composition of the present invention, while other components and conditions are not particularly limited. According to the hair growth promoting composition of the present invention, hair growth is expected to be promoted because the function of dermal papilla cells is enhanced. The description of the dermal papilla cell function promoting composition of the present invention can be referenced from the aforementioned description of the compositions, manufacturing methods, and proliferation promoting compositions of the present invention.

[0171] In this invention, "hair growth promotion" means: increasing the likelihood of hair growing from a hairless state; promoting hair growth (e.g., growth in hair length and / or thickness); and / or reducing hair loss (inhibiting or decreasing).

[0172] The hair growth promoting composition of the present invention can be used, for example, for the prevention, inhibition, or prevention of hair loss. In this case, the hair growth promoting composition of the present invention can also be used, for example, as a hair loss prevention, inhibition, or prevention composition.

[0173] The hair growth promoting composition of the present invention can be used in vitro or in vivo.

[0174] When the hair growth promoting composition of the present invention is used in vitro, it can be used, for example, by adding it to a culture medium containing dermal papilla cells. According to the hair growth promoting composition of the present invention, by maintaining the dermal papilla cells in a culture medium containing the aforementioned composition, the proliferation of the dermal papilla cells and / or the induction of hair growth promoting genes can be promoted, thereby potentially promoting hair growth. The final concentration of total protein of the hair growth promoting composition of the present invention in the aforementioned culture medium is 10–1000 μg / ml, 10–500 μg / ml, or 10–300 μg / ml.

[0175] The subjects and administration conditions of the hair growth promoting composition of the present invention can be referred to the descriptions of the subjects and administration conditions in the aforementioned hair growth promoting composition of the present invention and the hair growth promoting method described below.

[0176] <Methods to Promote Cell Proliferation>

[0177] The cell proliferation promotion method of the present invention (hereinafter also referred to as the "proliferation promotion method") uses the aforementioned cell proliferation promotion composition of the present invention. The proliferation promotion method of the present invention is characterized by the use of the aforementioned proliferation promotion composition of the present invention; other steps and conditions are not particularly limited. According to the proliferation promotion method of the present invention, the proliferation of cells, particularly mesenchymal stem cells, can be promoted. The proliferation promotion method of the present invention can be referenced from the descriptions of the aforementioned compositions, manufacturing methods, proliferation promotion compositions, and differentiation promotion compositions of the present invention.

[0178] The proliferation-promoting method of the present invention can be implemented, for example, in vitro or in vivo.

[0179] When the proliferation-promoting method of the present invention is implemented in vitro, the proliferation-promoting method of the present invention includes, for example, a cell culture step in the presence of the aforementioned proliferation-promoting composition. The target population and administration conditions of the proliferation-promoting composition of the present invention can be found, for example, in the description of the target population and administration conditions of the proliferation-promoting composition of the present invention.

[0180] <Methods to Promote Fibroblast Function>

[0181] In another example, the present invention provides a method for promoting fibroblast function. The method for promoting fibroblast function of the present invention uses the aforementioned composition of the present invention. The method for promoting fibroblast function of the present invention is characterized by the use of the aforementioned composition of the present invention; other steps and conditions are not particularly limited. The method for promoting fibroblast function according to the present invention is capable of promoting fibroblast function. The method for promoting fibroblast function of the present invention can be referenced from the descriptions of the aforementioned compositions, manufacturing methods, proliferation-promoting compositions, and fibroblast function-promoting compositions of the present invention.

[0182] The fibroblast function promotion method of the present invention can be implemented, for example, in vitro or in vivo.

[0183] When the fibroblast function promotion method of the present invention is implemented in vitro, the fibroblast function promotion method of the present invention includes, for example, a culture step of culturing fibroblasts in the presence of the aforementioned composition. The target population and administration conditions of the aforementioned composition of the present invention can be referenced, for example, from the description of the target population and administration conditions of the proliferation-promoting composition of the present invention.

[0184] Methods to promote the healing of skin disorders

[0185] In another example, the present invention provides a method for promoting the healing of skin disorders. The method for promoting the healing of skin disorders of the present invention (hereinafter also referred to as the "healing promotion method") uses the aforementioned composition of the present invention. The healing promotion method of the present invention is characterized by the use of the aforementioned composition of the present invention; other steps and conditions are not particularly limited. According to the healing promotion method of the present invention, it is expected to promote the healing of skin disorders. The healing promotion method of the present invention can be referenced by the description of the aforementioned compositions, manufacturing methods, proliferation-promoting compositions, and compositions for promoting the healing of skin disorders of the present invention.

[0186] The method of promoting the healing of skin disorders according to the present invention may also be referred to as a treatment method or treatment method for skin disorders.

[0187] The healing-promoting method of the present invention may include, for example, a drug administration step of administering the aforementioned composition of the present invention to a subject. Examples of such subjects include patients who have developed skin disorders, patients who are at risk of developing skin disorders, etc.

[0188] The healing-promoting method of the present invention can be implemented, for example, in vitro or in vivo.

[0189] When the healing-promoting method of the present invention is implemented in vitro, 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 aforementioned composition. The target population and administration conditions of the aforementioned composition of the present invention can be referenced, for example, from the description of the target population and administration conditions of the proliferation-promoting composition of the present invention.

[0190] <Methods to Promote Keratinocyte Function>

[0191] In another example, the present invention provides a method for promoting keratinocyte function. The keratinocyte function promotion method of the present invention uses the aforementioned composition of the present invention. The keratinocyte function promotion method of the present invention is characterized by the use of the aforementioned composition of the present invention; other steps and conditions are not particularly limited. According to the keratinocyte function promotion method of the present invention, keratinocyte function can be promoted. The keratinocyte function promotion method of the present invention can be referenced from the descriptions of the aforementioned compositions, manufacturing methods, proliferation-promoting compositions, and keratinocyte function-promoting compositions of the present invention.

[0192] The method for promoting keratinocyte function of the present invention can be implemented, for example, in vitro or in vivo.

[0193] When implementing the keratinocyte function promotion method of the present invention in vitro, the keratinocyte function promotion method of the present invention includes, for example, a culturing step of culturing keratinocytes in the presence of the aforementioned composition. The target population and administration conditions of the aforementioned composition of the present invention can be referenced, for example, from the description of the target population and administration conditions of the proliferation-promoting composition of the present invention.

[0194] <Methods to Promote Hair Papill Cell Function>

[0195] In another example, the present invention provides a method for promoting dermal papilla cell function. The method for promoting dermal papilla cell function of the present invention uses the aforementioned composition of the present invention. The method for promoting dermal papilla cell function of the present invention is characterized by the use of the aforementioned composition of the present invention; other steps and conditions are not particularly limited. The method for promoting dermal papilla cell function according to the present invention is capable of promoting dermal papilla cell function. The method for promoting dermal papilla cell function of the present invention can be referenced from the descriptions of the aforementioned compositions, manufacturing methods, proliferation-promoting compositions, and dermal papilla cell function-promoting compositions of the present invention.

[0196] The method for promoting dermal papilla cell function of the present invention can be implemented, for example, in vitro or in vivo.

[0197] When implementing the dermal papilla cell function promotion method of the present invention in vitro, the method includes, for example, a culturing step of culturing dermal papilla cells in the presence of the aforementioned composition. The target population and administration conditions of the aforementioned composition of the present invention can be referenced, for example, from the description of the target population and administration conditions of the proliferation-promoting composition of the present invention.

[0198] <Methods to Promote Hair Growth>

[0199] In another example, the present invention provides a method for promoting hair growth. The hair growth promotion method of the present invention uses the aforementioned composition of the present invention. The hair growth promotion method of the present invention is characterized by the use of the aforementioned composition of the present invention; other steps and conditions are not particularly limited. According to the hair growth promotion method of the present invention, it is expected to promote hair growth. The hair growth promotion method of the present invention can be referenced from the descriptions of the aforementioned compositions, manufacturing methods, proliferation-promoting compositions, and hair growth-promoting compositions of the present invention.

[0200] The hair growth promotion method of the present invention may also be referred to as a treatment method or management method for alopecia or thinning hair. Furthermore, the hair growth promotion method of the present invention may be used for the prevention, inhibition, or prevention of hair loss. In this case, the hair growth promotion method of the present invention may also be referred to as a method for the prevention, inhibition, or prevention of hair loss.

[0201] The hair growth promotion method of the present invention may include, for example, a drug administration step of administering the aforementioned composition of the present invention to a subject. The aforementioned subject may include, for example, patients who have experienced hair loss, patients who are likely to experience hair loss, etc. The aforementioned patients with hair loss may include, for example, patients with male pattern baldness, female pattern baldness, and patients with hair loss following anticancer drug treatment. The aforementioned patients who are likely to experience hair loss may include, for example, patients before anticancer drug treatment, and patients with hereditary hair loss before the onset of hair loss, etc.

[0202] The hair growth promotion method of the present invention can be implemented, for example, in vitro or in vivo.

[0203] When the hair growth promotion method of the present invention is implemented in vitro, the hair growth promotion method of the present invention includes, for example, a culture step of culturing hair papilla cells and other hair growth-related cells in the presence of the aforementioned composition. The target population and administration conditions of the aforementioned composition of the present invention can be referenced, for example, from the description of the target population and administration conditions of the proliferation-promoting composition of the present invention.

[0204] <Application of the Composition>

[0205] This invention relates to compositions or their applications, wherein the compositions are used to promote cell proliferation and contain a treatment of megakaryocytes or their cultures as an active ingredient. Additionally, this invention relates to compositions or their applications, wherein the compositions are used to promote fibroblast function and contain a treatment of megakaryocytes or their cultures as an active ingredient. This invention relates to compositions or their applications, wherein the compositions are used to promote the healing of skin disorders and contain a treatment of megakaryocytes or their cultures as an active ingredient. This invention relates to compositions or their applications, wherein the compositions are used to promote keratinocyte function and contain a treatment of megakaryocytes or their cultures as an active ingredient. This invention relates to compositions or their applications, wherein the compositions are used to promote dermal papilla cell function and contain a treatment of megakaryocytes or their cultures as an active ingredient. This invention relates to compositions or their applications, wherein the compositions are used to promote hair growth and contain a treatment of megakaryocytes or their cultures as an active ingredient. This invention relates to the application of compositions containing a treatment of megakaryocytes or their cultures as an active ingredient in the manufacture of cell proliferation promoting compositions. Additionally, this invention relates to the application of compositions containing a treatment of megakaryocytes or their cultures as an active ingredient in the manufacture of fibroblast function promoting compositions. This invention relates to the use of compositions containing a treatment of megakaryocytes or their cultures as an active ingredient in the manufacture of compositions promoting the healing of skin disorders. This invention also relates to the use of compositions containing a treatment of megakaryocytes or their cultures as an active ingredient in the manufacture of compositions promoting keratinocyte function. Furthermore, this invention relates to the use of compositions containing a treatment of megakaryocytes or their cultures as an active ingredient in the manufacture of compositions promoting dermal papilla cell function. Finally, this invention relates to the use of compositions containing a treatment of megakaryocytes or their cultures as an active ingredient in the manufacture of compositions promoting hair growth.

[0206] Example

[0207] The present invention will be described in detail below using examples, but the present invention is not limited to the methods described in the examples.

[0208] [Example 1]

[0209] The composition of the present invention was manufactured and confirmed to contain growth factors and growth factor receptors and to have cell proliferation promoting activity.

[0210] (1) Production of immortalized megakaryocytes

[0211] Immortalized megakaryocytes are made in the following order.

[0212] (1-1) Preparation of hematopoietic progenitor cells from iPS cells

[0213] Following the method described in Reference 8 below, differentiation culture of human iPS cells (TKDN SeV2 and NIH5: human embryonic skin fibroblast-derived iPS cells established using Sendai virus) into hematopoietic progenitor cells was performed. Specifically, hematopoietic progenitor cells (HPCs) were produced by co-culturing human ES / iPS cell colonies with C3H10T1 / 2 feeder cells for 14 days in the presence of 20 ng / ml VEGF (manufactured by R&D Systems). Culture conditions were 37°C, 20% O2, and 5% CO2 (unless otherwise specified, the same conditions apply hereinafter).

[0214] Reference 8: Takayama N.et al., "Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotentstem cells", J.Exp.Med., 2010, vol.13, pages 2817-2830

[0215] (1-2) Gene delivery system

[0216] The gene delivery system utilizes a lentiviral vector system. The lentiviral vector is a tetracycline-regulated Tet-on (trademarked) gene expression induction system vector. It can be prepared by replacing the mOKS box in LV-TRE-mOKS-Ubc-tTA-I2G (see reference 9 below) with c-MYC, BMI1, or BCL-xL. The vectors containing c-MYC, BMI1, or BCL-xL are designated LV-TRE-c-Myc-Ubc-tTA-I2G, LV-TRE-BMI1-Ubc-tTA-I2G, and LV-TRE-BCL-xL-Ubc-tTA-I2G, respectively. c-MYC, BMI1, and BCL-xL viruses are prepared by introducing the aforementioned lentiviral vector gene into 293T cells. By infecting the target cells with the obtained virus, the c-MYC, BMI1, and BCL-xL genes are introduced into the genomic sequence of the target cells. These genes, which are stably introduced into the genome sequence, can be forced to express by adding doxycycline (clontech #631311) to the culture medium.

[0217] 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

[0218] (1-3) Infect hematopoietic progenitor cells with c-MYC and BMI1 viruses.

[0219] In 6-well plates pre-inoculated with C3H10T1 / 2 feeder cells, to become 5 × 10⁶ 4 HPC cells obtained using the method described in (1-1) were seeded at a cell / well ratio. c-MYC and BMI1 were forcibly expressed using a lentiviral method employing BMI1 and c-MYC viruses. Six wells were used for each cell line. Specifically, viral particles were added to the culture medium at an MOI (multiplicity of infection) of 20 and infected using spin infection (centrifugation at 32°C, 900 rpm, 60 minutes). The spin infection was performed twice at 12-hour intervals. The following culture medium was used: In IMDM (Iscove's Modified Durbeco Medium) containing basal medium (15% fetal bovine serum (GIBCO), 1% penicillin-streptomycin-glutamine (GIBCO), 1% insulin-transferrin-selenium solution (ITS-G) (GIBCO), 0.45 mmol / L 1-thioglycerol (Sigma-Aldrich), 50 μg / ml L-ascorbic acid (Sigma-Aldrich), and ISBC (Sigma-Aldrich), each substance was added in a manner that resulted in 50 ng / ml human thrombopoietin (TPO) (R&D SYSTEMS), 50 ng / ml human stem cell growth factor (SCF) (R&D SYSTEMS), and 2 μg / ml doxycycline (Dox, clontech #631311) (hereinafter referred to as differentiation medium), protamine was further added to a final concentration of 10 μg / ml.

[0220] (1-4) Preparation and maintenance culture of megakaryocyte self-proliferating lines

[0221] The day on which c-MYC and BMI1 viruses were infected using the methods described in (1-3) was designated as day 0 of infection. As described below, megakaryocyte self-proliferating lines were created by culturing HPC cells infused with the c-MYC and BMI1 genes, respectively. Forced expression of the c-MYC and BMI1 genes was achieved by adding DOX to the culture medium at a concentration of 1 μg / ml.

[0222] Day 2 to Day 11 of infection

[0223] On day 2 of infection, virus-infected blood cells obtained using the above method were recovered by pipetting, centrifuged at 1200 rpm for 5 minutes to remove the supernatant, resuspended in fresh differentiation medium, and seeded onto fresh C3H10T1 / 2 feeder cells (6-well plates). The same procedure was performed on day 9 of infection, thus performing passage. Upon re-seeding, the cell count was determined to be 1 × 10⁻⁶ cells. 5 The cells were seeded onto C3H10T1 / 2 feeder cells at a rate of 1 cell / 2 ml / well (6-well plate).

[0224] Day 12 to Day 13 of infection

[0225] Perform the same procedures as on day 2 of infection. After counting the cells, make sure the count is 3 × 10⁻⁶. 5 The cells were seeded onto C3H10T1 / 2 feeder cells (100mm culture dish) at a rate of 1 cell / 10ml / 100mm culture dish.

[0226] Day 14 of infection

[0227] Recover virus-infected blood cells, relative to 1.0 × 10⁻⁶. 5 Cells were used to react 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 antibody (BioLegend), respectively. The reactions were then analyzed using FACS Verse (trademark) (BD Biosciences). On day 14 after infection, cells with a CD41a positivity rate of 50% or higher were designated as megakaryocyte self-proliferating lines.

[0228] (1-5) Infect megakaryocyte autoproliferating strains with BCL-xL virus.

[0229] The BCL-xL gene was introduced into the megakaryocyte self-proliferating line on day 14 of infection using a lentiviral method employing the BCL-xL virus. Viral particles were added to the culture medium at an MOI of 10, and infection was achieved by spin infection (centrifugation at 32°C, 900 rpm, 60 minutes). Forced expression of the BCL-xL gene was implemented by adding DOX to the culture medium at a concentration of 1 μg / ml.

[0230] (1-6) Creation and maintenance culture of megakaryocyte immortalized strains

[0231] Day 14 to Day 18 of infection

[0232] The megakaryocyte self-proliferating lines introduced with the BCL-xL gene obtained by the methods described above (1-5) were recovered and centrifuged at 1200 rpm for 5 minutes. After centrifugation, the precipitated cells were resuspended in fresh differentiation medium and then cultured on fresh C3H10T1 / 2 feeder cells at a density of 2 × 10⁻⁶ cells. 5 Seeds were generated at a rate of 1 cell / 2 ml / well (6-well plate).

[0233] Day 18 of infection: Transmission

[0234] Megakaryotic cell lines that had undergone BCL-xL gene induction were recovered and self-proliferated. Cell counts were performed to ensure a cell count of 3 × 10⁻⁶ cells. 5 Inoculate cells at a rate of 10 cells / 10ml / 100mm culture dish.

[0235] Day 24 of infection: Transmission

[0236] Megakaryotic cell lines that had undergone BCL-xL gene induction were recovered and self-proliferated. Cell counts were performed to ensure a cell count of 1×10⁻⁶. 5 10 cells / 10ml / 100mm culture dish were seeded. Subculture was then performed every 4-7 days for maintenance culture. It should be noted that during subculturing, cells were suspended on fresh differentiation medium before seeding.

[0237] Megakaryotic cell line with BCL-xL gene regeneration was recovered on day 24 after infection, relative to 1.0 × 10⁻⁶ cells. 5Cells were immunostained 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 (Anti-CD235ab-PB; BioLegend) antibody, respectively, and analyzed using FACS Verse (trademark). Furthermore, on day 24 after infection, cell lines with a CD41a positivity rate of 50% or higher were designated as immortalized megakaryocyte lines. These cells, capable of proliferating for more than 24 days post-infection, were designated as immortalized megakaryocyte lines SeV2-MKCL and NIH5-MKCL.

[0238] The obtained SeV2-MKCL and NIH5-MKCL were statically cultured in 10cm petri dishes (10ml / dish). Regarding the culture medium, IMDM was used as the basal medium with the following components added (final concentrations). The culture conditions were 37℃ and 5% CO2.

[0239] FBS(Sigma#172012lot.12E261)15%

[0240] L-Glutamine (Gibco#25030-081) 2 mmol / L

[0241] ITS (Gibco #41400-045) 100x dilution

[0242] MTG (monothioglycerol, Sigma#M6145-25ML) 450 μmol / L

[0243] Ascorbic acid (sigma#A4544) 50μg / ml

[0244] Puromycin (sigma#P8833-100MG) 2μg / ml

[0245] SCF (Wako Pure Chemicals #193-15513) 50ng / ml

[0246] TPO-like active substance 200 ng / ml

[0247] (2) Production of megakaryocyte cultures

[0248] Forced expression was deactivated by culturing in a DOX-free medium. Specifically, immortalized megakaryocyte lines (SeV2-MKCL and NIH5-MKCL) obtained using the method described above (1) were washed twice with PBS(-) and suspended in the platelet production medium described below. The cell seeding density was 1.0 × 10⁶ cells / year. 5 Cells / ml

[0249] Regarding the aforementioned platelet production culture medium, IMDM was used as the basal medium and the following components were added (concentration is final).

[0250] Human plasma A6%

[0251] L-Glutamine (Gibco#25030-081) 4 mmol / L

[0252] ITS (Gibco #41400-045) 100x dilution

[0253] MTG (monothioglycerol, Sigma#M6145-25ML) 450 μmol / L

[0254] Ascorbic acid (sigma#A4544) 50μg / ml

[0255] SCF (Wako Pure Chemicals #193-15513) 50ng / ml

[0256] TPO-like active substance 200 ng / ml

[0257] ADAM inhibitor 15 μmol / L

[0258] GNF351(Calbiochem#182707)500nmol / LY39983(Chemscene LLC#CS-0096)500nmol / l

[0259] Urokinase 5U / ml

[0260] Low molecular weight heparin (SANOFI, Clexane) 1U / ml

[0261] In addition, platelets were produced by culturing the cells in the aforementioned platelet production medium for 6 days, thereby producing a culture of megakaryocytes.

[0262] (3) Production of purified platelets

[0263] For the megakaryocyte culture obtained in (2) above, platelets were produced (purified) in the following order. It should be noted that the same purification was performed twice.

[0264] (3-1) Concentration of megakaryocyte culture

[0265] The megakaryocyte culture obtained in (2) above is transferred into a culture bag. Furthermore, for the aforementioned culture bag, such as... Figure 1 As shown, connect to the concentration system. Figure 1In this embodiment, cleaning and preservation solution bags 1 and 2 contain cleaning and preservation solution. The aforementioned cleaning and preservation solution is obtained by adding 20% ​​ACD and 2.5% human serum albumin to BICANATE infusion solution (BICARBON infusion solution, manufactured by Otsuka Pharmaceutical Co., Ltd.) and adjusting the pH to 7.2 with NaOH. Furthermore, according to Table 1 below, the aforementioned megakaryocyte culture was concentrated using a hollow fiber membrane (Plasmaflo.OP, manufactured by Asahi Kasei Medical Co., Ltd.), and the resulting concentrated megakaryocyte culture was recovered into the storage bag.

[0266] [Table 1]

[0267]

[0268] (3-2) Centrifugation of platelets

[0269] First, using a sterile connection device, the waste bag from the ACP215 disposable kit was replaced with a recycling bag. The recycling bag used was a HICALIQ IVH bag (Terumo HC-B3006A). Next, 10% ACD-A solution (manufactured by Terumo) was added to the concentrated culture medium of the aforementioned megakaryocytes. After this addition, the concentrated medium with added ACD-A solution was injected into the cell bag. The cell bag used was a HICALIQ IVH bag (Terumo HC-B3006A).

[0270] Next, using a sterile connection device, the cell bag containing the culture with added ACD-A fluid was connected to the ACP215 disposable kit. The ACP215 was then started in service mode, with the rotation speed set to 2500 rpm (350 × g). The ACP215 was started, and the culture from the aforementioned cell bag was transferred to a separation bowl at approximately 100 ml / min. The liquid components flowing out of the separation bowl were recovered into a recovery bag. After the total volume of the culture from the aforementioned cell bag was transferred to the separation bowl, 500 ml of washing and preservation solution was then transferred into the separation bowl. After the washing and preservation solution was transferred into the separation bowl, centrifugation was stopped, and the recovery bag containing the recovery solution (containing platelet-containing recovery liquid components) was separated using a tubing sealer.

[0271] Using the aforementioned aseptic connection device, connect the recovery bag containing the recovered fluid (including platelets) to the new ACP215 disposable kit. Start the ACP215 in normal mode. Select WPC for the program setting and, following the device's instructions, set up the ACP215 disposable kit with the aforementioned recovery bag connected. It should be noted that the recovery bag containing the recovered fluid is positioned on the support.

[0272] Next, the centrifugation speed of the ACP215 was changed to 5000 rpm (1398.8 × g), and centrifugation was started. When starting to introduce the aforementioned recovered solution into the aforementioned separation bowl, the automatic injection was changed to manual injection. Specifically, the aforementioned recovered solution was introduced into the aforementioned separation bowl at an injection rate of approximately 100 ml / min. After the total amount of the aforementioned recovered solution was added to the separation bowl, 500 ml of washing and preservation solution was added.

[0273] (3-3) Platelet washing

[0274] For cleaning, follow the ACP215 procedure and clean with 2000 ml of the aforementioned cleaning and preservation solution.

[0275] (3-4) Platelet recycling

[0276] Following the ACP215 procedure, 200 ml of washed platelets were collected into a platelet preparation bag.

[0277] (3-5) Platelet separation

[0278] For the aforementioned platelet preparation bags, the aforementioned hollow fiber membrane is used to separate platelets using conventional methods and recycle them into recycling bags.

[0279] (4) Preparation of extracts

[0280] As the aforementioned megakaryocytes or their cultures, the immortalized megakaryocyte line obtained using the method described in (1) above, the platelets recovered into the platelet preparation bag obtained in (3-5) above, and the platelet-free megakaryocyte cultures recovered into the drainage bag (hereinafter collectively referred to as "raw materials") were used. For the platelet-free megakaryocyte cultures, four samples prepared separately (platelet-free megakaryocyte cultures 1-4) were used.

[0281] Each raw material was washed twice with a washing solution. The washing solution was prepared by adding ACD-A liquid to BICANATE infusion at approximately 20% (v / v), followed by the addition of NaOH to adjust the pH to 7.0–7.4. After the washing, each raw material was centrifuged at 2000×g for 10 minutes at room temperature (approximately 25°C). After centrifugation, the precipitate was recovered and frozen using liquid nitrogen. Then, cell lysis buffer was added to the frozen precipitate, and the mixture was shaken at 50 rpm and 4°C for 30 minutes to dissolve it. The cell lysis buffer was prepared by adding a protease inhibitor cocktail (RayBiotech, Cat. No.: AA-LYS) to a commercially available cell lysis buffer (2×Cell Lysis Buffer, Cat. No.: AA-PI).

[0282] The resulting solution was centrifuged at 14000×g for 5 minutes at 4°C. After centrifugation, the supernatant was recovered as the treated product for this example. The total protein concentration of the treated products obtained from each raw material was determined using the Pierce (trademark) BCA protein assay kit (Thermo Fisher Scientific). The results showed that, when using an immortalized megakaryocyte line, the total protein concentration was 2.2×10⁻⁶. 7 2.604 mg of total protein was extracted from each cell. Additionally, when platelets were used, the total protein content was 2.5 × 10⁻⁶ mg. 8 1.187 mg of total protein was extracted from each cell. Furthermore, when platelets were used to remove megakaryocyte cultures 1–4, the total protein content was 1.97 × 10⁻⁴ mg. 8 5.25 × 10 cells 8 3.64 × 10 cells 8 6.22 × 10 cells 8 Total protein was extracted from each cell at concentrations of 2.8, 5.944, 3.6, and 4.496 mg. Furthermore, after adjusting the total protein concentration to 5 mg / ml, the concentrations of growth factors (bFGF, IGFBP-1, IGFBP-2, PIGF, VEGF, GDF-15, AR, BMP-7, HGF) and growth factor receptors (SCFR, EGFR, VEGFR2) were determined for each treatment using a Quantibody Human Growth Factor Array 1 (Ray Biotech). These results are shown in Table 2 below. It should be noted that Table 2 can also be referred to as the concentration per 5 mg of total protein.

[0283] [Table 2]

[0284]

[0285] unit: p g / mL

[0286] (5) Confirmation of cell proliferation-promoting activity

[0287] Human adipose tissue-derived mesenchymal stem cells were prepared at a size of 2.4–4.8 × 10⁻⁶. 5 Cells were seeded in 10cm culture dishes at a rate of 10 cells / 10ml of culture medium / dish. It should be noted that the aforementioned mesenchymal stem cells were obtained by passaged commercially available human adipose tissue-derived mesenchymal stem cells (manufactured by Takara Bio, Cat. No.: C-12977) twice and then recovered. The aforementioned culture medium was prepared by freezing and thawing the composition from platelet-depleted megakaryocyte culture 2 into Mesenchymal Stem Cell Growth Medium 2 (manufactured by Takara Bio, Cat. No.: C-28009). The aforementioned composition was added at a specified concentration (0, 125, 250, or 500 μg / ml) of total protein derived from the aforementioned composition in the culture medium. It should be noted that the aforementioned culture medium is a maintenance medium.

[0288] Following the aforementioned inoculation, the aforementioned mesenchymal stem cells were cultured for 3 days at 37°C and 5% CO2. After the aforementioned culture, the aforementioned mesenchymal stem cells were recovered, inoculated under the same conditions, and cultured again at 37°C and 5% CO2 for 3 days. Next, the cultured mesenchymal cells were recovered, and the cell count was counted. Furthermore, the relative value of the cell count at recovery (based on the cell count at the time of inoculation) was calculated and used as the relative value of proliferative activity. In addition, based on the cell count at the time of inoculation and the cell count at recovery, the time required for one cell proliferation (doubling time) was calculated. These results are presented in... Figure 2 .

[0289] Figure 2 This is a diagram illustrating the proliferative activity of cells. Figure 2 In the diagram, (A) shows the proliferation activity, and (B) shows the doubling time. Figure 2 In (A), the horizontal axis represents the total protein concentration from the aforementioned composition, and the vertical axis represents the relative value of proliferative activity. Figure 2 In (B), the horizontal axis represents the total protein concentration from the aforementioned composition, and the vertical axis represents the doubling time. The values ​​in the figure represent the doubling time. Figure 2As shown in (A), when the composition of the present invention is added, the proliferative activity of mesenchymal stem cells increases dependently on the concentration of total protein derived from the aforementioned composition. Furthermore, although not illustrated, in mesenchymal stem cells without the aforementioned composition, the doubling time after the third passage is 21 hours. Figure 2 As shown in (B), without the addition of the composition of the present invention, the doubling time of mesenchymal stem cells is extended to approximately 1.5 times. In contrast, as... Figure 2 As shown in (B), when the composition of the present invention is added, the time required for mesenchymal stem cells to proliferate once is shortened, depending on the total protein concentration derived from the aforementioned composition. Furthermore, when cultured in a medium without the composition of the present invention, the doubling time of mesenchymal stem cells at the passage number is prolonged. In contrast, when the composition of the present invention is added, the aforementioned prolongation of doubling time is substantially suppressed. Therefore, it can be seen that the composition of the present invention exhibits cell proliferation-promoting activity and is able to inhibit the reduction of proliferation activity.

[0290] [Example 2]

[0291] The composition of the present invention has been confirmed to have cell proliferation promoting activity.

[0292] As the aforementioned mesenchymal stem cells, after two passage cultures, they were recovered and cryopreserved. Cells that had been thawed and passaged once were added to the maintenance medium to achieve a specified total protein concentration (0, 0.2, 1.3, 31.3, 62.5, or 125 μg / ml). Otherwise, as in Example 1(5), proliferation activity and doubling time were calculated. These results are presented in… Figure 3 .

[0293] Figure 3 This is a diagram illustrating the proliferative activity of cells. Figure 3 In the diagram, (A) shows the proliferation activity, and (B) shows the doubling time. Figure 3 In (A), the horizontal axis represents the total protein concentration from the aforementioned composition, and the vertical axis represents the relative value of proliferative activity. Figure 3 In (B), the horizontal axis represents the total protein concentration from the aforementioned composition, and the vertical axis represents the doubling time. For example... Figure 3 As shown in (A), when the composition of the present invention is added, the proliferative activity of mesenchymal stem cells increases with increasing total protein concentration derived from the aforementioned composition; in particular, when the total protein concentration is 31.3 μg / ml or higher, the proliferative activity of mesenchymal stem cells increases significantly. Furthermore, although not illustrated, in mesenchymal stem cells without the aforementioned composition, the doubling time after the third passage is 17 hours. Figure 3As shown in (B), without the addition of the composition of the present invention, the doubling time of mesenchymal stem cells is extended to approximately 1.5 times. In contrast, as... Figure 3 As shown in (B), when the composition of the present invention is added, the time required for mesenchymal stem cells to proliferate once is shortened depending on the total protein concentration from which the composition is derived. In particular, when the total protein concentration is 31.3 μg / ml or higher, the time required for mesenchymal stem cells to proliferate once is significantly shortened. Furthermore, when cultured in a medium without the composition of the present invention, the doubling time of mesenchymal stem cells at passage number is prolonged. In contrast, when the composition of the present invention is added, the aforementioned prolongation of doubling time is substantially suppressed, and this effect is significant when the total protein concentration from which the composition is derived is 31.3 μg / ml or higher. Therefore, it can be seen that the composition of the present invention exhibits cell proliferation-promoting activity and is able to inhibit the reduction of proliferation activity.

[0294] [Example 3]

[0295] The composition of the present invention has been confirmed to have fibroblast function-promoting activity.

[0296] (1) Culture of fibroblasts

[0297] Normal human skin fibroblasts (nHDF, KURABO, Cat. No.: KF-4109) were awakened in T-75 flasks (Sumilon) using proliferation medium and cultured in a CO2 incubator under specified conditions (5% CO2, 37°C, humid conditions, as described below). The aforementioned proliferation medium was DMEM medium (Nacalai Tesque, Cat. No.: 08456-65) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, Cat. No.: 172012) and 1% penicillin / streptomycin (Thermo Fisher Scientific, Cat. No.: 15140-122). The medium was changed every 1–2 days during the culture. At approximately 80% confluence, the cells were harvested for subsequent experiments. The cells were passaged as described below. First, the cells were washed with phosphate-buffered saline (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95), and then dissected using a stripping 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. Next, the cell suspension was transferred to 15 ml centrifuge tubes and centrifuged (room temperature, 1000 rpm, 5 minutes) using a multi-functional chilled centrifuge (TOMY, Cat. No.: CAX-571). After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were agitated. The viable cell count was performed using the trypan blue assay. The cells were then adjusted to the target cell concentration using the aforementioned growth medium and seeded in the culture vessels used in subsequent experiments.

[0298] (2) Confirmation of the cell proliferation-promoting activity of fibroblasts

[0299] For the aforementioned cells, at 5 × 10 3Cells were seeded at a density of 0.1 ml / well in 96-well plates (Sumilon, Cat. No.: MS-8096F) and cultured for 1 day under the aforementioned conditions. After the aforementioned culture, the culture medium was replaced with DMEM medium containing a composition prepared from the aforementioned platelet-depleted megakaryocyte culture (MDF) or platelet (PLT) at the specified concentrations (total protein concentration of 5 mg / ml, as in Examples 4 and 5). Thus, the concentrations of total protein derived from the aforementioned composition in the 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). It should be noted that the composition prepared from platelet-depleted megakaryocyte culture uses two types (iMDF1 and iMDF2). After the aforementioned culture medium was replaced, the culture was incubated for 48 hours.

[0300] Following the aforementioned culture, the proliferation activity of cells in each well was determined by measuring the number of viable cells using the WST-8 assay. First, the culture medium was replaced with DMEM containing 10% chromogenic reagent (Cell Count Reagent SF, Nacalai Tesque, Cat. No.: 07553-15), and incubation was performed under the aforementioned conditions. Changes in absorbance (450 nm) were measured using a microplate reader (Varioskan Flash, Thermo Fisher Scientific, Cat. No.: 5250040) over a 60-minute period from 30 to 90 minutes after the start of incubation (n=3 for each group). The negative control (NC) was performed in the same manner except that the aforementioned composition was not added. For the positive control (FBS), FBS was added at 1% or 10% instead of the aforementioned composition, and the same procedure was followed. Furthermore, in the reference examples (ASA, Mes), instead of the aforementioned compositions, L-ascorbic acid (L-Ascorbic Acid (ASA), manufactured by Fujifilm-Wako, Cat. No.: 013-19641), which has collagen production-promoting activity, was added at a concentration of 2 mmol / L, or N-methyl-L-serine (N-Methyl-L-serine (MeS), manufactured by Sigma-Aldrich, Cat. No.: 73156), which has hyaluronic acid production-promoting activity, was added at a concentration of 10 mmol / L, and the same procedures were followed otherwise. Additionally, the cell survival rate in the negative control was set to 100%, and the cell survival rate of each sample was set as a relative proliferation activity value. Furthermore, a p-value less than 0.05 in the Student's T-test (two-tailed, unpaired test) compared to the negative control was considered significant. These results are presented below... Figure 4 .

[0301] Figure 4 This is a diagram illustrating the proliferative activity of fibroblasts. Figure 4 In the diagram, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents cell viability (proliferative activity). For example... Figure 4 As shown, a concentration-dependent cell proliferation-promoting effect was observed in all compositions containing either the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT, PLTMax Human Platelet Lysate, manufactured by EMD Millipore, Cat. No.: SCM141). Furthermore, it is believed that the cell proliferation-promoting activity of the compositions of the present invention is higher than that of FBS at the same concentration.

[0302] (3) Confirmation of the activity promoting the production of extracellular matrix

[0303] After 48 hours of incubation in Example 3(2), the culture supernatant of each well was recovered and stored at -80°C for use in the type I collagen and hyaluronic acid production promotion test described later.

[0304] Next, the amount of type I collagen in the aforementioned culture supernatant (n=3 for each group) was measured using a type I collagen assay kit (Human Collagen type I, ELISA kit (without pepsin), manufactured by ACEL, Cat. No.: EC1-E105). Additionally, the amount of hyaluronic acid in the aforementioned culture supernatant was measured using a hyaluronic acid assay kit (Hyaluronan DuoSet ELISA, manufactured by R&D Systems, Cat. No.: DY3614). The assay methods for each kit were performed according to the accompanying protocol. The negative control (NC) was performed in the same manner except that the aforementioned composition was not added. For the FBS addition group (FBS), the procedure was performed in the same manner as in Example 3(2). In the positive controls (ASA or Mes), for the samples used in the type I collagen assay, L-ascorbic acid (L-Ascorbic Acid (ASA), manufactured by Fujifilm-Wako, Cat. No.: 013-19641), which promotes collagen production, was added at a concentration of 2 mmol / L instead of the aforementioned composition. For the samples used in the hyaluronic acid assay, N-methyl-L-serine (N-Methyl-L-serine (MeS), manufactured by Sigma-Aldrich, Cat. No.: 73156), which promotes hyaluronic acid production, was added at a concentration of 10 mmol / L instead of the aforementioned composition. Otherwise, the procedure was performed in the same manner. Furthermore, a p-value less than 0.05 in the Student's t-test (two-tailed, unpaired) compared to the negative control was considered significant. These results are presented in... Figure 5 and Figure 6 .

[0305] Figure 5 This is a graph showing the amount of type I collagen produced. Figure 5 In the graph, the horizontal axis represents the type of sample, and the vertical axis represents the amount of type I collagen produced. It should be noted that the ASA values ​​in the graph represent the collagen production amount (μg / ml) in the reference example (ASA). Figure 5As shown, in any case of platelet-derived megakaryocyte culture or platelet-prepared composition, a concentration-dependent increase in type I collagen production was observed. It is speculated that the aforementioned type I collagen production-promoting activity, compared to the cell proliferation-promoting activity results of Example 3(2), is due to cell proliferation-promoting activity.

[0306] then, Figure 6 This is a graph showing the amount of hyaluronic acid produced. Figure 6 In the diagram, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents the amount of hyaluronic acid produced. For example... Figure 6 As shown, in any of the aforementioned compositions prepared by removing megakaryocyte cultures from platelets or platelets, a concentration-dependent increase in hyaluronic acid production was observed. It is speculated that, compared to the aforementioned cell proliferation-promoting activity, the aforementioned hyaluronic acid production-promoting activity is an activity independent of cell proliferation-promoting activity; that is, the compositions of the present invention act on fibroblasts and promote hyaluronic acid production.

[0307] The results above indicate that the composition of the present invention possesses fibroblast proliferation-promoting activity and activity promoting the production of extracellular matrix components such as type I collagen and hyaluronic acid. In cases of skin or other skin disorders, the aforementioned fibroblasts migrate to the site of the disorder and contribute to its healing through cell division and secretion of extracellular matrix components. The composition of the present invention promotes these functions of fibroblasts, and therefore is expected to promote the healing of skin disorders.

[0308] [Example 4]

[0309] The compositions of the present invention have been confirmed to have keratinocyte function-promoting activity.

[0310] (1) Culture of keratinocytes

[0311] Normal human epidermal keratinocytes (nHEK, KURABO, Cat. No.: KK-4109) were awakened in T-75 flasks (Sumilon) using proliferation medium and cultured in a CO2 incubator under specified conditions (5% CO2, 37°C, humid conditions, as described below). The proliferation medium used was Humedia-KG2 medium (KURABO, Cat. No.: KK-2150S). The medium was changed every 1–2 days. At approximately 80% confluence, the cells were harvested for subsequent experiments. Cell passage was performed as described below. First, the cells were washed with phosphate-buffered saline (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95), and then dissected using a stripping 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 trypsin. Next, the cell suspension was transferred to 15 ml centrifuge tubes and centrifuged (room temperature, 1000 rpm, 5 minutes) using a multi-functional chilled centrifuge (TOMY, Cat. No.: CAX-571). After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were agitated. The viable cell count was performed using the trypan blue assay. The cells were then adjusted to the target cell concentration using the aforementioned growth medium and seeded in the culture vessels used in subsequent experiments.

[0312] (2) Confirmation of cell proliferation-promoting activity of keratinocytes

[0313] For the aforementioned cells, at 1×10 3 Cells were seeded at a density of 0.1 ml / well in 96-well plates (Sumilon, Cat. No.: MS-8096F) and cultured for 1 day under the aforementioned conditions. After the above culture, the medium was replaced with maintenance medium (Humedia-KB2, KURABO, Cat. No.: KK-2350S) containing a composition prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) at the specified concentrations (treatment concentrations for each test substance: 0.5%, 1%, 2.5%, 5%, 10%) to achieve the specified concentrations. It should be noted that two compositions were used to prepare the platelet-depleted megakaryocyte cultures. After the medium replacement, the cells were cultured for 48 hours.

[0314] After the aforementioned culture, the morphology of each cell in each well was observed using a phase-contrast microscope (OLYMPUS, Cat. No.: CKX53). Additionally, the change in absorbance (450 nm) was measured for each cell in each well, similar to Example 3(2) above (n=3 for each group). The negative control (NC) was performed in the same manner except that the aforementioned composition was not added. For the positive control (additive), the aforementioned proliferation medium was used instead of the maintenance medium containing the aforementioned composition, and the procedure was performed in the same manner. Furthermore, for the reference examples (JTC or NA), instead of the aforementioned composition, JTC-801 (JTC, Sigma-Aldrich, Cat. No.: J3955, final concentration 100 nmol / l) with FLG gene expression-promoting activity or nicotinamide (NA, Sigma-Aldrich, Cat. No.: N0636-100G, final concentration 30 μmol / l) with SPTLC1 gene expression-promoting activity was added, and the procedure was performed in the same manner. In addition, cell survival rate in the negative control was set to 100%, and cell survival rate in each sample was set as a relative proliferation activity value. Furthermore, a p-value less than 0.05 in the Student's T-test (two-tailed, unpaired) compared to the negative control was considered significant. These results are presented in... Figure 7 and Figure 8 .

[0315] Figure 7 These are photographs showing the phase difference images of the cells in each well. It should be noted that, for groups supplemented with compositions prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT), an example of the 10% supplementation group is shown as a representative example. Figure 7 As shown, in the negative control, the boundaries of keratinocytes were well-defined, and no differentiation into cells (epidermal cells) reaching the epithelial (epidermal) layer was observed. On the other hand, in the compositions prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT), keratinocytes extended on the well plate, and the cell boundaries became unclear. This is due to the differentiation of keratinocytes into cells (epidermal cells) of the epithelial (epidermal) layer. Furthermore, in the cases of compositions prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT), the proportion of cells in the epithelial (epidermal) layer increased in a concentration-dependent manner; in particular, a significant increase was observed when using the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2). These results indicate that the compositions of the present invention promote the differentiation of keratinocytes into cells of the epithelial layer.

[0316] Figure 8 This is a diagram illustrating the proliferative activity of keratinocytes. Figure 8 In the diagram, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents cell viability (proliferative activity). For example... Figure 8 As shown, cell proliferation promotion was observed when any of the compositions prepared by adding platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) was added. Furthermore, it is believed that the cell proliferation promotion activity of the compositions of the present invention is higher than that of FBS at the same concentration.

[0317] (3) Confirmation of the induction-promoting activity of barrier function genes

[0318] With 5×10 4 Keratinocytes were seeded at a density of 0.5 ml / well in 24-well plates (Sumilon, Cat. No.: MS-8024) and cultured otherwise in the same manner as in Example 4(2). 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 extraction was performed according to the protocol provided with the kit. The concentration of the purified RNA was determined using a spectrophotometer (NanoDrop One, Thermo Fisher Scientific, Cat. No.: ND-ONE-W), and purity was confirmed by A260 / A280. The purified RNA was stored at -80°C until used for reverse transcription.

[0319] 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 obtained cDNA, primer pairs for the GAPDH, FLG, or SPTLC1 genes described below, and a qPCR kit (TB Green Premix Ex Taq II (Tli RNaseH Plus), Takara, Cat. No.: RR820A). The PCR reaction was performed by heating at 95°C for 30 seconds, followed by 40 cycles of 95°C, 5 seconds, 60°C, and 30 seconds as one cycle, to dissociate the PCR product. The qPCR was performed using a qPCR device (LightCycler96 Instrument, Roche, Cat. No.: 05815916001). The negative control (NC) was performed in the same manner except that the aforementioned composition was not added. In the positive control (JTC or NA), the sample used for the FLG gene expression assay was replaced with JTC-801 (JTC, Sigma-Aldrich, Cat. No.: J3955), which has FLG gene expression-promoting activity, at a concentration of 100 nmol / L, instead of the aforementioned composition. The sample used for the SPTLC1 gene expression assay was replaced with nicotinamide (NA, Sigma-Aldrich, Cat. No.: N0636-100G), which has SPTLC1 gene expression-promoting activity, at a concentration of 30 μmol / L, instead of the aforementioned composition. All other procedures were performed in the same manner. Additionally, groups with added additives were prepared for both the negative and positive controls and performed similarly. The expression levels of each gene, corrected for GAPDH gene expression, were calculated using the ΔΔCt method from the obtained assay data. Furthermore, the relative expression level with the negative control expression level set to 1 was calculated. A p-value less than 0.05 in Student's t-test (two-sided, unpaired) compared to the negative control was considered significant. These results are presented in... Figure 9 and Figure 10 .

[0320] • GAPDH gene primer pair

[0321] Forward primer (serial number 1)

[0322] 5'-CATCCCTGCCTCTACTGGCGCTGCC-3'

[0323] Reverse primer (serial number 2)

[0324] 5'-CCAGGATGCCCTTGAGGGGGCCCTC-3'

[0325] FLG gene primer pair

[0326] Forward primer (serial number 3)

[0327] 5'-TCGGCAAATCCTGAAGAATCCAGA-3'

[0328] Reverse primer (serial number 4)

[0329] 5'-GCTTGAGCCAACTTGAATACCATCAG-3'

[0330] Primer pairs for the SPTLC1 gene

[0331] Forward primer (serial number 5)

[0332] 5'-ACAAAGCAAGAATCTTCCTGGAGGAAAGCC-3'

[0333] Reverse primer (serial number 6)

[0334] 5'-AAACCTCCAATAGAAGCAAGTGCATTCTCC-3'

[0335] Figure 9 This is a graph showing the expression levels of the FLG gene. Figure 9 In the diagram, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents the expression level of the FLG gene. For example... Figure 9 As shown, when any of the compositions prepared by adding platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) was added, a promoting effect on FLG gene expression was observed. Furthermore, it is believed that the FLG gene expression-promoting activity of the compositions of the present invention is higher than that of JTC801 as a positive control at the same concentration.

[0336] then, Figure 10 This is a graph showing the expression level of the SPTLC1 gene. Figure 10 In the diagram, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents the expression level of the SPTLC1 gene. For example... Figure 10 As shown, when any of the compositions prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) were added, a promoting effect on SPTLC1 gene expression was observed. Furthermore, it is believed that the SPTLC1 gene expression-promoting activity of the compositions of the present invention is higher than that of nicotinamide (NA) as a positive control at the same concentration.

[0337] The results above indicate that the composition of the present invention possesses keratinocyte proliferation-promoting activity, promotes differentiation into epidermal cells, and promotes the expression of barrier function genes such as FLG and SPTLC1. It is known that the aforementioned keratinocyte proliferation, differentiation into epidermal cells, and induction of barrier function gene expression are important for skin tissue and the maintenance of its barrier function. Therefore, since the composition of the present invention promotes these functions of keratinocytes, it is expected to be used for maintaining skin function, maintaining skin barrier function, and other skin conditioning purposes.

[0338] [Example 5]

[0339] The composition of the present invention has been confirmed to have dermal papilla cell function-promoting activity.

[0340] (1) Culture of dermal papilla cells

[0341] Human hair dermal papilla cells (HFDPC, TOYOBO, Cat. No.: CA60205a) were awakened in T-75 flasks (Sumilon) using proliferation medium and cultured in a CO2 incubator under specified conditions (5% CO2, 37°C, humid conditions, as described below). The aforementioned proliferation medium was prepared by adding accompanying additives to dermal papilla cell proliferation medium (PCGM, TOYOBO, Cat. No.: TMTPGM-250S). The medium was changed every 1–2 days. At approximately 80% confluence, the cells were harvested for subsequent experiments. The cells were passaged as described below. First, the cells were washed with phosphate-buffered saline (PBS) (- / -) (Nacalai Tesque, Cat. No.: 14249-95), and then dissected using a stripping 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. Next, the cell suspension was transferred to 15 ml centrifuge tubes and centrifuged (room temperature, 1000 rpm, 5 minutes) using a multi-functional chilled centrifuge (TOMY, Cat. No.: CAX-571). After centrifugation, the supernatant was removed, fresh growth medium was added, and the cells were agitated. The viable cell count was performed using the trypan blue assay. The cells were then adjusted to the target cell concentration using the aforementioned growth medium and seeded in the culture vessels used in subsequent experiments.

[0342] (2) Confirmation of cell proliferation-promoting activity of dermal papilla cells

[0343] For the aforementioned cells, at 5 × 10 3 0.1 ml of cells per well was seeded in 96-well plates (Sumilon, Cat. No.: MS-8096F) and cultured for 1 day under the aforementioned conditions. After the culture, the medium was replaced with dermal papilla cell proliferation medium containing the composition prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) at the specified concentrations (treatment concentrations for each test substance: 0.5%, 1%, 2.5%, 5%, 10%). It should be noted that two compositions were used from the platelet-depleted megakaryocyte cultures. After the medium replacement, the cells were cultured for 48 hours.

[0344] Following the aforementioned culture, for each well, the change in absorbance (450 nm) was measured, similarly to (2) of Example 3 above (n=3 for each group). The negative control (NC) was performed in the same manner except that the aforementioned composition was not added. For the positive control (additive), a dermal papilla cell proliferation medium containing the aforementioned additive was used, and the procedure was performed in the same manner otherwise. In the reference examples (Mx or Ad), minoxidil (Minoxidil (Mx), manufactured by Sigma-Aldrich, Cat. No.: M4145, final concentration 30 μmol / l) or adenosine (Ad), manufactured by Sigma-Aldrich, Cat. No.: A9251, final concentration 100 μmol / l), known as hair growth agents, was added instead of the aforementioned composition, and the procedure was performed in the same manner otherwise. In addition, the cell viability in the negative control was set to 100%, and the cell viability of each sample was set as a relative proliferation activity value. Furthermore, a p-value less than 0.05 in the Student's T-test (two-tailed, unpaired) compared to the negative control will be considered significant. These results are presented in... Figure 11 .

[0345] Figure 11 This is a diagram showing the proliferative activity of dermal papilla cells. Figure 11 In the diagram, the horizontal axis represents the type and concentration of the sample, and the vertical axis represents cell viability (proliferative activity). For example... Figure 11 As shown, cell proliferation promotion was observed when any of the compositions prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) was added. Furthermore, a recommended addition amount of 2.5–5% was suggested.

[0346] (3) Confirmation of the induction and promotion activity of hair growth promoting genes

[0347] With 2.5×10 4The dermal papilla cells were seeded at a density of 0.5 ml / well in 24-well plates (Sumilon, Cat. No.: MS-8024), and otherwise cultured in the same manner as in Example 5 (2). After the culture, cDNA was synthesized in the same manner as in Example 4 (3).

[0348] Next, qPCR was performed using primer pairs for the aforementioned GAPDH gene, the FGF7 gene described below, or the VEGFA gene, in the same manner as in Example 4 (3) above. The negative control was performed in the same manner except that the aforementioned composition was not added. In the reference example, minoxidil (manufactured by Sigma-Aldrich, Cat. No.: M4145, final concentration 30 μmol / l) or adenosine (manufactured by Sigma-Aldrich, Cat. No.: A9251, final concentration 100 μmol / l), known as hair growth agents, was added instead of the aforementioned composition, and the procedure was performed in the same manner. The expression levels of each gene, corrected for the expression level of the GAPDH gene, were calculated using the ΔΔCt method from the obtained assay data, and then the relative expression level with the negative control expression level set to 1 was calculated. A p-value less than 0.05 in the Student's T-test (two-sided, unpaired) compared to the negative control was considered significant. These results are presented in… Figure 12 and Figure 13 .

[0349] ·FGF7 gene primer pair

[0350] Forward primer (serial number 7)

[0351] 5'-TCTGTCGAACACAGTGGTACCTGAG-3'

[0352] Reverse primer (serial number 8)

[0353] 5'-GCCACTGTCCTGATTTCCATGA-3'

[0354] ·VEGFA gene primer pair

[0355] Forward primer (serial number 9)

[0356] 5'-AAAGCATTTGTTTGTACAAGATCCG-3'

[0357] Reverse primer (serial number 10)

[0358] 5'-CTTGTCACATCTGCAAGTACGTTCG-3'

[0359] Figure 12This is a graph showing the expression level of the FGF7 gene. Figure 12 In the diagram, the horizontal axis represents the type of sample, and the vertical axis represents the expression level of the FGF7 gene. For example... Figure 12 As shown, when the compositions prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) were added, an FGF7 gene expression-promoting effect was observed. Furthermore, it is believed that the FGF7 gene expression-promoting activity of the compositions of the present invention, at the same concentration, is comparable to that of known hair growth agents.

[0360] then, Figure 13 This is a graph showing the expression levels of the VEGFA gene. Figure 13 In the diagram, the horizontal axis represents the type of sample, and the vertical axis represents the expression level of the VEGFA gene. For example... Figure 13 As shown, when the compositions prepared from the aforementioned platelet-depleted megakaryocyte cultures (iMDF1, iMDF2) or platelets (PLT) were added, a VEGFA gene expression-promoting effect was observed. Furthermore, it is believed that the VEGFA gene expression-promoting activity of the compositions of the present invention, at the same concentration, is comparable to that of known hair growth agents.

[0361] The results above indicate that the composition of the present invention possesses dermal papilla cell proliferation-promoting activity and expression-promoting activity of hair growth genes such as FGF7 and VEGFA. It is known that the aforementioned induction of dermal papilla cell proliferation and expression of hair growth genes is important for hair growth and maintenance. Therefore, since the composition of the present invention promotes these functions of dermal papilla cells, it is expected to be used for hair growth.

[0362] The present invention has been described above with reference to the embodiments and examples, but the present invention is not limited to the above embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the structure and details of the present invention within the scope of this application.

[0363] The application claims priority based on Japanese Special Application No. 2019-225959, filed on December 13, 2019, the entire contents of which are hereby incorporated.

[0364] <Appendix>

[0365] Some or all of the above-described implementation methods and embodiments may be as described in the following appendix, but are not limited thereto.

[0366] <Composition>

[0367] (Appendix 1)

[0368] A composition comprising a treatment of megakaryocytes or their cultures.

[0369] (Appendix 2)

[0370] According to the composition described in Appendix 1, wherein the aforementioned treated material is an extract of megakaryocytes or cell fractions of their cultures.

[0371] (Appendix 3)

[0372] The composition according to Appendix 1 or 2, wherein the aforementioned treated material contains basic fibroblast growth factor (bFGF) at a concentration of 2000 to 20000 pg relative to 1 mg of total protein.

[0373] (Appendix 4)

[0374] The composition according to any one of Appendices 1 to 3, wherein the aforementioned treated material contains insulin-like growth factor binding protein-2 (IGFBP-2) at a concentration of 8,000 to 80,000 pg relative to 1 mg of total protein.

[0375] (Appendix 5)

[0376] The composition according to any one of Appendices 1 to 4, wherein the aforementioned treated product contains placental growth factor (PIGF) at a concentration of 1 to 60 pg relative to 1 mg of total protein.

[0377] (Appendix 6)

[0378] The composition according to any one of Appendices 1 to 5, wherein the aforementioned treated material contains 200 to 2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein.

[0379] (Appendix 7)

[0380] The composition according to any one of Appendices 1 to 6, wherein the aforementioned treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20 to 800 pg relative to 1 mg of total protein.

[0381] (Appendix 8)

[0382] The composition according to any one of Appendices 1 to 7, wherein the aforementioned treated material comprises vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20 to 400 pg relative to 1 mg of total protein.

[0383] (Appendix 9)

[0384] The composition according to any one of Appendices 1 to 8, wherein the aforementioned treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000 to 10000 pg relative to 1 mg of total protein.

[0385] (Appendix 10)

[0386] The composition according to any one of Appendices 1 to 9, wherein the aforementioned treated material comprises osteogenic protein-7 (BMP-7) in a concentration of 0 to 1000 pg relative to 1 mg of total protein.

[0387] (Appendix 11)

[0388] The composition according to any one of Appendices 1 to 10, wherein the aforementioned treated material comprises abimodulin (AR) at a concentration of 0 to 16 pg relative to 1 mg of total protein.

[0389] (Appendix 12)

[0390] The composition according to any one of Appendices 1 to 11, wherein the aforementioned treated material comprises epidermal growth factor receptor (EGFR) at a concentration of 0 to 60 pg relative to 1 mg of total protein.

[0391] (Appendix 13)

[0392] The composition according to any one of Appendices 1 to 12, wherein the aforementioned treated material contains hepatocyte growth factor (HGF) in the form of 0 to 100 pg relative to 1 mg of total protein.

[0393] (Appendix 14)

[0394] The composition according to any one of Appendices 1 to 13, wherein the aforementioned treated material comprises insulin-like growth factor binding protein-1 (IGFBP-1) in a concentration of 0 to 200 pg relative to 1 mg of total protein.

[0395] (Appendix 15)

[0396] The composition according to any one of Appendices 1 to 14 has cell proliferation promoting activity.

[0397] (Appendix 16)

[0398] The composition according to Appendix 15, wherein the aforementioned cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells.

[0399] (Appendix 17)

[0400] The composition according to any one of Appendices 1 to 16 has fibroblast function promoting activity.

[0401] (Appendix 18)

[0402] According to the composition described in Appendix 17, the aforementioned fibroblast function is the proliferation of fibroblasts and / or the production of extracellular matrix by fibroblasts.

[0403] (Appendix 19)

[0404] The composition according to Appendix 18, wherein the aforementioned extracellular matrix comprises collagen and / or hyaluronic acid.

[0405] (Appendix 20)

[0406] The composition according to any one of Appendices 1 to 19 has keratinocyte function promoting activity.

[0407] (Appendix 21)

[0408] According to the composition described in Appendix 20, the aforementioned keratinocyte function is the proliferation of keratinocytes, differentiation into epidermal cells, and / or induction of barrier function genes.

[0409] (Appendix 22)

[0410] According to the composition described in Appendix 21, wherein the aforementioned barrier function gene comprises a filaggrinogen gene and / or a ceramide synthase gene.

[0411] (Appendix 23)

[0412] The composition according to any one of Appendices 1 to 22 has dermal papilla cell function promoting activity.

[0413] (Appendix 24)

[0414] According to the composition described in Appendix 23, the aforementioned dermal papilla cell function is the induction of dermal papilla cell proliferation and / or hair growth promoting genes.

[0415] (Appendix 25)

[0416] The composition according to Appendix 24, wherein the aforementioned hair growth gene is the FGF7 gene and / or the VEGF gene.

[0417] (Appendix 26)

[0418] The composition according to any one of Appendices 1 to 25, wherein the aforementioned megakaryocyte culture is a platelet-free culture.

[0419] (Appendix 27)

[0420] The composition according to any one of Appendices 1 to 26, wherein the aforementioned megakaryocytes are immortalized megakaryocytes.

[0421] (Appendix 28)

[0422] According to the composition described in Appendix 27, the aforementioned immortalized megakaryocytes are megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes.

[0423] (Appendix 29)

[0424] The composition according to any one of Appendices 1 to 28, wherein the aforementioned megakaryocytes are in vitro induced megakaryocytes.

[0425] (Appendix 30)

[0426] The composition according to any one of Appendices 1 to 29, wherein the aforementioned megakaryocytes are of pluripotent origin.

[0427] (Appendix 31)

[0428] The composition according to Appendix 30, wherein the aforementioned pluripotent cells are induced pluripotent stem (iPS) cells.

[0429] (Appendix 32)

[0430] The composition according to any one of Appendices 1 to 31, wherein,

[0431] The aforementioned processed items are:

[0432] Treatment of megakaryocytes or their cultures

[0433] The aforementioned treatments include concentration treatment, drying treatment, freezing treatment, freeze-drying treatment, solvent treatment, surfactant treatment, enzyme treatment, protein fractionation extraction treatment, ultrasonic treatment, and / or crushing treatment.

[0434] (Appendix 33)

[0435] According to the composition described in Appendix 32, wherein,

[0436] The aforementioned processed items are:

[0437] Platelets were removed from the aforementioned megakaryocytes or their cultures.

[0438] Megakaryocytes or their cultures from which the aforementioned platelets have been removed were processed.

[0439] (Appendix 34)

[0440] According to the composition described in Appendix 33, wherein...

[0441] The aforementioned processed items are:

[0442] Preserve megakaryocytes or their cultures after removing the aforementioned platelets.

[0443] The aforementioned preserved megakaryocytes or their cultures were processed.

[0444] (Appendix 35)

[0445] According to the composition described in Appendix 34, wherein...

[0446] The aforementioned processed items are:

[0447] Preserve the aforementioned megakaryocytes or their cultures.

[0448] The aforementioned preserved megakaryocytes or their cultures were disrupted.

[0449] <Method for manufacturing the composition>

[0450] (Appendix 36)

[0451] A method for manufacturing a composition, comprising a processing step of treating megakaryocytes or cultures thereof.

[0452] The aforementioned processing steps include concentration, drying, freezing, freeze-drying, solvent treatment, surfactant treatment, enzyme treatment, protein fractionation and extraction, ultrasonic treatment, and / or crushing.

[0453] (Appendix 37)

[0454] The manufacturing method according to Appendix 36 includes a platelet removal step of removing platelets from the aforementioned megakaryocytes or their cultures.

[0455] In the aforementioned processing steps, megakaryocytes or their cultures from which the aforementioned platelets have been removed are processed.

[0456] (Appendix 38)

[0457] The manufacturing method described in Appendix 37 includes a preservation step of preserving megakaryocytes or their cultures after the aforementioned platelets have been removed.

[0458] In the aforementioned processing steps, the preserved megakaryocytes or their cultures are processed.

[0459] (Appendix 39)

[0460] The manufacturing method described in Appendix 38 includes a preservation step for storing the aforementioned megakaryocytes or their cultures.

[0461] In the aforementioned processing steps, the preserved megakaryocytes or their cultures are subjected to a disruption process.

[0462] (Appendix 40)

[0463] A composition obtained by any one of the manufacturing methods in Appendices 36 to 39.

[0464] <Cell proliferation promoting composition>

[0465] (Appendix 41)

[0466] A cell proliferation promoting composition comprising any one of the compositions listed in Appendices 1 to 35.

[0467] (Appendix 42)

[0468] The cell proliferation promoting composition according to Appendix 41, wherein the aforementioned cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells.

[0469] <Methods to Promote Cell Proliferation>

[0470] (Appendix 43)

[0471] A method for promoting cell proliferation, which uses the cell proliferation promoting composition described in Appendix 41 or 42.

[0472] (Appendix 44)

[0473] According to the cell proliferation promotion method described in Appendix 43, the aforementioned cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells.

[0474] (Appendix 45)

[0475] The cell proliferation promotion method according to Appendix 43 or 44, wherein the aforementioned cell proliferation promotion composition is used in vitro or in vivo.

[0476] <Fibroblast Function Promoting Composition>

[0477] (Appendix 46)

[0478] A fibroblast function-promoting composition comprising any one of the compositions listed in Appendices 1 to 35.

[0479] (Appendix 47)

[0480] According to the functional enhancement composition described in Appendix 46, the aforementioned fibroblast function is the proliferation of fibroblasts and / or the production of extracellular matrix by fibroblasts.

[0481] (Appendix 48)

[0482] The functional enhancement composition according to Appendix 47, wherein the aforementioned extracellular matrix comprises collagen and / or hyaluronic acid.

[0483] <Methods to Promote Fibroblast Function>

[0484] (Appendix 49)

[0485] A method for promoting fibroblast function, comprising using the fibroblast function promoting composition described in any one of Appendices 46 to 48.

[0486] (Appendix 50)

[0487] According to the fibroblast function promotion method described in Appendix 49, the aforementioned fibroblast function promotion composition is used in vitro or in vivo.

[0488] Compositions that promote the healing of skin disorders

[0489] (Appendix 51)

[0490] A composition for promoting the healing of skin disorders, comprising any one of the compositions in Annexes 1 to 35.

[0491] (Appendix 52)

[0492] According to the healing-promoting composition described in Appendix 51, the aforementioned skin disorder is a skin ulcer, bedsore, burn, scar, and / or trauma.

[0493] Methods to promote the healing of skin disorders

[0494] (Appendix 53)

[0495] A method for promoting the healing of skin disorders, which uses the composition for promoting the healing of skin disorders described in Appendix 51 or 52.

[0496] (Appendix 54)

[0497] The method for promoting the healing of skin disorders according to Appendix 53 involves using the aforementioned composition for promoting the healing of skin disorders either in vitro or in vivo.

[0498] <Keratinocyte Function-Enhancing Composition>

[0499] (Appendix 55)

[0500] A keratinocyte function-promoting composition comprising any one of the compositions listed in Annexes 1 to 35.

[0501] (Appendix 56)

[0502] According to the functional promoting composition described in Appendix 55, the aforementioned keratinocyte function is the proliferation of keratinocytes, differentiation into epidermal cells, and / or induction of barrier function genes.

[0503] (Appendix 57)

[0504] According to the functional enhancement composition described in Appendix 56, the aforementioned barrier function genes include the filaggrinogen gene and / or the ceramide synthase gene.

[0505] <Methods to Promote Keratinocyte Function>

[0506] (Appendix 58)

[0507] A method for promoting keratinocyte function, comprising using the keratinocyte function promoting composition described in any one of Appendices 55 to 57.

[0508] (Appendix 59)

[0509] According to the keratinocyte function promotion method described in Appendix 58, the aforementioned keratinocyte function promotion composition is used in vitro or in vivo.

[0510] <Composition for Promoting Hair Papill Cell Function>

[0511] (Appendix 60)

[0512] A hair papilla cell function-enhancing composition comprising any one of the compositions listed in Appendices 1 to 35.

[0513] (Appendix 61)

[0514] According to the functional promoting composition described in Appendix 60, the aforementioned dermal papilla cell function is the induction of dermal papilla cell proliferation and / or hair growth promoting genes.

[0515] (Appendix 62)

[0516] According to the functional enhancement composition described in Appendix 61, the aforementioned barrier function genes include the filaggrinogen gene and / or the ceramide synthase gene.

[0517] <Methods to Promote Hair Papill Cell Function>

[0518] (Appendix 63)

[0519] A method for promoting dermal papilla cell function, comprising using the dermal papilla cell function promoting composition as described in any one of Appendices 60 to 62.

[0520] (Appendix 64)

[0521] According to the method for promoting dermal papilla cell function as described in Appendix 63, the aforementioned dermal papilla cell function promoting composition is used in vitro or in vivo.

[0522] Hair growth promoting composition

[0523] (Appendix 65)

[0524] A hair growth promoting composition comprising any one of the compositions listed in Appendices 1 to 35.

[0525] <Methods to Promote Hair Growth>

[0526] (Appendix 66)

[0527] A method for promoting hair growth, which uses the hair growth promoting composition described in Appendix 65.

[0528] (Appendix 67)

[0529] According to the hair growth promotion method described in Appendix 66, the aforementioned hair growth promotion composition is used in vitro or in vivo.

[0530] <Application of the Composition>

[0531] (Appendix 68)

[0532] A composition for promoting cell proliferation, wherein a treatment of megakaryocytes or their cultures is used as the active ingredient.

[0533] (Appendix 69)

[0534] A composition for promoting fibroblast function, using a treatment of megakaryocytes or their cultures as the active ingredient.

[0535] (Appendix 70)

[0536] A composition for promoting the healing of skin disorders, using a treatment of megakaryocytes or their cultures as the active ingredient.

[0537] (Appendix 71)

[0538] A composition for promoting keratinocyte function, using a treatment of megakaryocytes or their cultures as the active ingredient.

[0539] (Appendix 72)

[0540] A composition for promoting the function of dermal papilla cells, using a treatment of megakaryocytes or their cultures as the active ingredient.

[0541] (Appendix 73)

[0542] A composition for promoting hair growth, wherein a treatment of megakaryocytes or their cultures is used as the active ingredient.

[0543] Industrial availability

[0544] As described above, according to the present invention, physiologically active cell-derived compositions can be provided. Furthermore, the compositions of the present invention are expected to promote cell proliferation, for example, mesenchymal cells, fibroblasts, keratinocytes, and dermal papilla cells, and are also expected to appropriately promote the healing of skin disorders such as ulcers, bedsores, burns, scars, and trauma; maintain or improve the skin barrier function; and promote hair growth. Therefore, the present invention is extremely useful in the fields of medicine and regenerative medicine. sequence list <110> MEGAKARYON CORPORATION, Inc. <120> Compositions and their uses <130> TF19291WO <150> JP2019-225959 <151> 2019-12-13 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primers for the GAPDH gene <400> 1 catccctgcc tctactggcg ctgcc 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH gene <400> 2 ccaggatgcc cttgagggggccctc 25 <210> 3 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FLG gene <400> 3 tcggcaaatc ctgaagaatc caga 24 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the FLG gene <400> 4 gcttgagcca acttgaatac catcag 26 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primers for the SPTLC1 gene <400> 5 acaaagcaag aatcttcctg gaggaaagcc 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the SPTLC1 gene <400> 6 aaacctccaa tagaagcaag tgcattctcc 30 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primers for the FGF7 gene <400> 7 tctgtcgaac acagtggtac ctgag 25 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FGF7 gene <400> 8 gccactgtcc tgatttccat ga 22 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for the VEGFA gene <400> 9 aaagcatttg tttgtacaag atccg 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the VEGFA gene <400> 10 cttgtcacat ctgcaagtac gttcg 25

Claims

1. A composition comprising a treatment of megakaryocytes or a treatment of a culture of megakaryocytes; The treatment is defined by the content of growth factors and / or growth factor receptors, the content of which is selected from the group consisting of: The treated material contains 2000–20000 pg of basic fibroblast growth factor (bFGF) relative to 1 mg of total protein. The treated material contains 8000–80000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) relative to 1 mg of total protein. The treated material contains placental growth factor (PIGF) at a concentration of 1–60 pg relative to 1 mg of total protein. The treated material contains 200–2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20–800 pg relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20–400 pg relative to 1 mg of total protein. The treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000–10000 pg relative to 1 mg of total protein. The treated material contains osteogenic protein-7 (BMP-7) at a concentration of 0–1000 pg relative to 1 mg of total protein. The treated material contains abimodulin (AR) at a concentration of 0–16 pg relative to 1 mg of total protein. The treated material contains epidermal growth factor receptor (EGFR) at a concentration of 0–60 pg relative to 1 mg of total protein. The treated material contains hepatocyte growth factor (HGF) at a concentration of 0–100 pg relative to 1 mg of total protein, or, The treated material contains insulin-like growth factor binding protein-1 (IGFBP-1) at a concentration of 0–200 pg relative to 1 mg of total protein; in, The processed product is: Platelets are removed from the megakaryocytes or their cultures. The megakaryocytes or their cultures from which the platelets have been removed are treated; The processing includes concentration, drying, freezing, solvent treatment, surfactant treatment, enzyme treatment, and / or crushing. The megakaryocytes mentioned above are immortalized megakaryocytes; The immortalized megakaryocytes are in vitro induced megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the megakaryocytes are derived from induced pluripotent stem cells.

2. The composition according to claim 1, wherein, The processed product is: Preserve megakaryocytes or cultures thereof from which the platelets have been removed. The preserved megakaryocytes or their cultures are then processed.

3. The composition according to claim 2, wherein, The processed product is: Preserve the megakaryocytes or their cultures. The preserved megakaryocytes or their cultures are then disrupted.

4. The composition according to claim 1, wherein, The freezing process is freeze-drying; the crushing process is ultrasonic treatment.

5. A method for manufacturing a composition, comprising a processing step of treating megakaryocytes or cultures thereof. The processing steps include concentration, drying, freezing, solvent treatment, surfactant treatment, enzyme treatment, and / or crushing. The megakaryocytes or their cultures were treated to obtain the treated product. The treatment is defined by the content of growth factors and / or growth factor receptors, the content of which is selected from the group consisting of: The treated material contains 2000–20000 pg of basic fibroblast growth factor (bFGF) relative to 1 mg of total protein. The treated material contains 8000–80000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) relative to 1 mg of total protein. The treated material contains placental growth factor (PIGF) at a concentration of 1–60 pg relative to 1 mg of total protein. The treated material contains 200–2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20–800 pg relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20–400 pg relative to 1 mg of total protein. The treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000–10000 pg relative to 1 mg of total protein. The treated material contains osteogenic protein-7 (BMP-7) at a concentration of 0–1000 pg relative to 1 mg of total protein. The treated material contains abimodulin (AR) at a concentration of 0–16 pg relative to 1 mg of total protein. The treated material contains epidermal growth factor receptor (EGFR) at a concentration of 0–60 pg relative to 1 mg of total protein. The treated material contains hepatocyte growth factor (HGF) at a concentration of 0–100 pg relative to 1 mg of total protein, or, The treated material contains insulin-like growth factor binding protein-1 (IGFBP-1) at a concentration of 0–200 pg relative to 1 mg of total protein; It further includes a platelet removal step of removing platelets from the megakaryocytes or their cultures. In the processing step, megakaryocytes or their cultures after the platelets have been removed are treated. in, The megakaryocytes are immortalized megakaryocytes; The immortalized megakaryocytes are in vitro induced megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the megakaryocytes are derived from induced pluripotent stem cells.

6. The manufacturing method according to claim 5, further comprising a preservation step of preserving megakaryocytes or cultures thereof from which the platelets have been removed. In the processing step, the preserved megakaryocytes or their cultures are processed.

7. The manufacturing method according to claim 6, further comprising a preservation step of preserving the megakaryocytes or their culture. in, In the processing step, the preserved megakaryocytes or their cultures are broken up.

8. The manufacturing method according to claim 5, wherein, The freezing process is freeze-drying; the crushing process is ultrasonic treatment.

9. A cell proliferation promoting composition comprising the composition of any one of claims 1 to 4.

10. The cell proliferation promoting composition according to claim 9, wherein, The cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells.

11. A method for promoting cell proliferation for non-disease treatment purposes, comprising using the cell proliferation promoting composition of claim 9 or 10; in, The cells are mesenchymal stem cells, fibroblasts, keratinocytes, and / or dermal papilla cells; The megakaryocytes contained in the cell proliferation promoting composition are in vitro induced, immortalized megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the culture of the megakaryocytes is a platelet-free culture.

12. The method for promoting cell proliferation for non-disease treatment purposes according to claim 11, wherein, The cell proliferation promoting composition may be used in vitro or in vivo.

13. A fibroblast function-promoting composition comprising the composition of any one of claims 1 to 4.

14. The function-enhancing composition according to claim 13, wherein, The fibroblast function is the proliferation of fibroblasts and / or the production of extracellular matrix by fibroblasts.

15. The function-enhancing composition according to claim 14, wherein, The extracellular matrix contains collagen and / or hyaluronic acid.

16. A method for promoting fibroblast proliferation or extracellular matrix production for non-disease treatment purposes, using the fibroblast function-promoting composition according to any one of claims 13 to 15.

17. The method for promoting fibroblast proliferation or extracellular matrix production for non-disease treatment purposes according to claim 16, wherein the fibroblast function promoting composition is used in vitro or in vivo.

18. A composition for promoting the healing of skin disorders, comprising the composition of any one of claims 1 to 4.

19. The composition for promoting the healing of skin disorders according to claim 18, wherein, The skin disorders are skin ulcers, bedsores, burns, scars, and / or trauma.

20. Use of the composition of claim 18 or 19 for promoting the healing of skin disorders in the preparation of a medicament for promoting the healing of skin disorders.

21. The use according to claim 20, wherein the composition promoting the healing of skin disorders is used in vitro or in vivo.

22. A keratinocyte function promoting composition comprising the composition of any one of claims 1 to 4.

23. The function-enhancing composition according to claim 22, wherein, The keratinocyte functions mentioned are keratinocyte proliferation, differentiation into epidermal cells, and / or induction of barrier function genes.

24. The function-enhancing composition according to claim 23, wherein, The barrier function genes include the filaggrinogen gene and / or the ceramide synthase gene.

25. A method for promoting keratinocyte proliferation, differentiation into epidermal cells, and expression of FLG and SPTLC1 genes for non-disease treatment purposes, wherein the keratinocyte function promoting composition according to any one of claims 22 to 24 is used.

26. The method for promoting keratinocyte proliferation, differentiation into epidermal cells, and expression of FLG and SPTLC1 genes for non-disease treatment purposes according to claim 25, wherein the keratinocyte function promoting composition is used in vitro or in vivo.

27. A hair papilla cell function promoting composition comprising the composition of any one of claims 1 to 4.

28. The function-enhancing composition according to claim 27, wherein, The function of the dermal papilla cells is the proliferation of dermal papilla cells and / or the induction of hair growth promoting genes.

29. The function-enhancing composition according to claim 28, wherein, The hair growth promoting gene includes the fibroblast growth factor 7 (FGF7) gene and / or the vascular endothelial growth factor A (VEGFA) gene.

30. A method for promoting the proliferation of dermal papilla cells and the expression of FGF7 and VEGFA genes for non-disease treatment purposes, wherein the dermal papilla cell function promoting composition according to any one of claims 27 to 29 is used.

31. The method for promoting dermal papilla cell proliferation and FGF7 and VEGFA gene expression for non-disease treatment purposes according to claim 30, wherein the dermal papilla cell function promoting composition is used in vitro or in vivo.

32. A hair growth promoting composition comprising the composition according to any one of claims 1 to 4.

33. A method for promoting hair growth for non-disease treatment purposes, comprising using the hair growth promoting composition of claim 32.

34. The hair growth promotion method for non-disease treatment purposes according to claim 33, wherein the hair growth promotion composition is used in vitro or in vivo.

35. A composition for promoting cell proliferation, wherein a megakaryocyte treatment or a megakaryocyte culture treatment is used as the active ingredient; The treatment is defined by the content of growth factors and / or growth factor receptors, the content of which is selected from the group consisting of: The treated material contains 2000–20000 pg of basic fibroblast growth factor (bFGF) relative to 1 mg of total protein. The treated material contains 8000–80000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) relative to 1 mg of total protein. The treated material contains placental growth factor (PIGF) at a concentration of 1–60 pg relative to 1 mg of total protein. The treated material contains 200–2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20–800 pg relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20–400 pg relative to 1 mg of total protein. The treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000–10000 pg relative to 1 mg of total protein. The treated material contains osteogenic protein-7 (BMP-7) at a concentration of 0–1000 pg relative to 1 mg of total protein. The treated material contains abimodulin (AR) at a concentration of 0–16 pg relative to 1 mg of total protein. The treated material contains epidermal growth factor receptor (EGFR) at a concentration of 0–60 pg relative to 1 mg of total protein. The treated material contains hepatocyte growth factor (HGF) at a concentration of 0–100 pg relative to 1 mg of total protein, or, The treated material contains insulin-like growth factor binding protein-1 (IGFBP-1) at a concentration of 0–200 pg relative to 1 mg of total protein; in, The processed product is: Platelets are removed from the megakaryocytes or their cultures. The megakaryocytes or their cultures from which the platelets have been removed are treated; The processing includes concentration, drying, freezing, solvent treatment, surfactant treatment, enzyme treatment, and / or crushing. The megakaryocytes mentioned above are immortalized megakaryocytes; The immortalized megakaryocytes are in vitro induced megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the megakaryocytes are derived from induced pluripotent stem cells.

36. The composition according to claim 35, wherein, The freezing process is freeze-drying; the crushing process is ultrasonic treatment.

37. A composition for promoting fibroblast function, wherein a megakaryocyte treatment or a megakaryocyte culture treatment is used as an active ingredient; The treatment is defined by the content of growth factors and / or growth factor receptors, the content of which is selected from the group consisting of: The treated material contains 2000–20000 pg of basic fibroblast growth factor (bFGF) relative to 1 mg of total protein. The treated material contains 8000–80000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) relative to 1 mg of total protein. The treated material contains placental growth factor (PIGF) at a concentration of 1–60 pg relative to 1 mg of total protein. The treated material contains 200–2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20–800 pg relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20–400 pg relative to 1 mg of total protein. The treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000–10000 pg relative to 1 mg of total protein. The treated material contains osteogenic protein-7 (BMP-7) at a concentration of 0–1000 pg relative to 1 mg of total protein. The treated material contains abimodulin (AR) at a concentration of 0–16 pg relative to 1 mg of total protein. The treated material contains epidermal growth factor receptor (EGFR) at a concentration of 0–60 pg relative to 1 mg of total protein. The treated material contains hepatocyte growth factor (HGF) at a concentration of 0–100 pg relative to 1 mg of total protein, or, The treated material contains insulin-like growth factor binding protein-1 (IGFBP-1) at a concentration of 0–200 pg relative to 1 mg of total protein; in, The processed product is: Platelets are removed from the megakaryocytes or their cultures. The megakaryocytes or their cultures from which the platelets have been removed are treated; The processing includes concentration, drying, freezing, solvent treatment, surfactant treatment, enzyme treatment, and / or crushing. The megakaryocytes mentioned above are immortalized megakaryocytes; The immortalized megakaryocytes are in vitro induced megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the megakaryocytes are derived from induced pluripotent stem cells.

38. The composition according to claim 37, wherein, The freezing process is freeze-drying; the crushing process is ultrasonic treatment.

39. A composition for promoting the healing of skin disorders, wherein a megakaryocyte treatment or a megakaryocyte culture treatment is used as an active ingredient; The treatment is defined by the content of growth factors and / or growth factor receptors, the content of which is selected from the group consisting of: The treated material contains 2000–20000 pg of basic fibroblast growth factor (bFGF) relative to 1 mg of total protein. The treated material contains 8000–80000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) relative to 1 mg of total protein. The treated material contains placental growth factor (PIGF) at a concentration of 1–60 pg relative to 1 mg of total protein. The treated material contains 200–2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20–800 pg relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20–400 pg relative to 1 mg of total protein. The treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000–10000 pg relative to 1 mg of total protein. The treated material contains osteogenic protein-7 (BMP-7) at a concentration of 0–1000 pg relative to 1 mg of total protein. The treated material contains abimodulin (AR) at a concentration of 0–16 pg relative to 1 mg of total protein. The treated material contains epidermal growth factor receptor (EGFR) at a concentration of 0–60 pg relative to 1 mg of total protein. The treated material contains hepatocyte growth factor (HGF) at a concentration of 0–100 pg relative to 1 mg of total protein, or, The treated material contains insulin-like growth factor binding protein-1 (IGFBP-1) at a concentration of 0–200 pg relative to 1 mg of total protein; in, The processed product is: Platelets are removed from the megakaryocytes or their cultures. The megakaryocytes or their cultures from which the platelets have been removed are treated; The processing includes concentration, drying, freezing, solvent treatment, surfactant treatment, enzyme treatment, and / or crushing. The megakaryocytes mentioned above are immortalized megakaryocytes; The immortalized megakaryocytes are in vitro induced megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the megakaryocytes are derived from induced pluripotent stem cells.

40. The composition according to claim 39, wherein, The freezing process is freeze-drying; the crushing process is ultrasonic treatment.

41. A composition for promoting keratinocyte function, wherein a megakaryocyte treatment or a megakaryocyte culture treatment is used as an active ingredient; The treatment is defined by the content of growth factors and / or growth factor receptors, the content of which is selected from the group consisting of: The treated material contains 2000–20000 pg of basic fibroblast growth factor (bFGF) relative to 1 mg of total protein. The treated material contains 8000–80000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) relative to 1 mg of total protein. The treated material contains placental growth factor (PIGF) at a concentration of 1–60 pg relative to 1 mg of total protein. The treated material contains 200–2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20–800 pg relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20–400 pg relative to 1 mg of total protein. The treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000–10000 pg relative to 1 mg of total protein. The treated material contains osteogenic protein-7 (BMP-7) at a concentration of 0–1000 pg relative to 1 mg of total protein. The treated material contains abimodulin (AR) at a concentration of 0–16 pg relative to 1 mg of total protein. The treated material contains epidermal growth factor receptor (EGFR) at a concentration of 0–60 pg relative to 1 mg of total protein. The treated material contains hepatocyte growth factor (HGF) at a concentration of 0–100 pg relative to 1 mg of total protein, or, The treated material contains insulin-like growth factor binding protein-1 (IGFBP-1) at a concentration of 0–200 pg relative to 1 mg of total protein; in, The processed product is: Platelets are removed from the megakaryocytes or their cultures. The megakaryocytes or their cultures from which the platelets have been removed are treated; The processing includes concentration, drying, freezing, solvent treatment, surfactant treatment, enzyme treatment, and / or crushing. The megakaryocytes mentioned above are immortalized megakaryocytes; The immortalized megakaryocytes are in vitro induced megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the megakaryocytes are derived from induced pluripotent stem cells.

42. The composition according to claim 41, wherein, The freezing process is freeze-drying; the crushing process is ultrasonic treatment.

43. A composition for promoting the function of dermal papilla cells, wherein a megakaryocyte treatment or a megakaryocyte culture treatment is used as the active ingredient; The treatment is defined by the content of growth factors and / or growth factor receptors, the content of which is selected from the group consisting of: The treated material contains 2000–20000 pg of basic fibroblast growth factor (bFGF) relative to 1 mg of total protein. The treated material contains 8000–80000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) relative to 1 mg of total protein. The treated material contains placental growth factor (PIGF) at a concentration of 1–60 pg relative to 1 mg of total protein. The treated material contains 200–2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20–800 pg relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20–400 pg relative to 1 mg of total protein. The treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000–10000 pg relative to 1 mg of total protein. The treated material contains osteogenic protein-7 (BMP-7) at a concentration of 0–1000 pg relative to 1 mg of total protein. The treated material contains abimodulin (AR) at a concentration of 0–16 pg relative to 1 mg of total protein. The treated material contains epidermal growth factor receptor (EGFR) at a concentration of 0–60 pg relative to 1 mg of total protein. The treated material contains hepatocyte growth factor (HGF) at a concentration of 0–100 pg relative to 1 mg of total protein, or, The treated material contains insulin-like growth factor binding protein-1 (IGFBP-1) at a concentration of 0–200 pg relative to 1 mg of total protein; in, The processed product is: Platelets are removed from the megakaryocytes or their cultures. The megakaryocytes or their cultures from which the platelets have been removed are treated; The processing includes concentration, drying, freezing, solvent treatment, surfactant treatment, enzyme treatment, and / or crushing. The megakaryocytes mentioned above are immortalized megakaryocytes; The immortalized megakaryocytes are in vitro induced megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the megakaryocytes are derived from induced pluripotent stem cells.

44. The composition according to claim 43, wherein, The freezing process is freeze-drying; the crushing process is ultrasonic treatment.

45. A composition for promoting hair growth, wherein a megakaryocyte treatment or a megakaryocyte culture treatment is used as an active ingredient; The treatment is defined by the content of growth factors and / or growth factor receptors, the content of which is selected from the group consisting of: The treated material contains 2000–20000 pg of basic fibroblast growth factor (bFGF) relative to 1 mg of total protein. The treated material contains 8000–80000 pg of insulin-like growth factor binding protein-2 (IGFBP-2) relative to 1 mg of total protein. The treated material contains placental growth factor (PIGF) at a concentration of 1–60 pg relative to 1 mg of total protein. The treated material contains 200–2000 pg of stem cell factor receptor (SCFR) relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor (VEGF) at a concentration of 20–800 pg relative to 1 mg of total protein. The treated material contains vascular endothelial growth factor receptor 2 (VEGFR2) at a concentration of 20–400 pg relative to 1 mg of total protein. The treated material contains differentiation growth factor-15 (GDF-15) at a concentration of 1000–10000 pg relative to 1 mg of total protein. The treated material contains osteogenic protein-7 (BMP-7) at a concentration of 0–1000 pg relative to 1 mg of total protein. The treated material contains abimodulin (AR) at a concentration of 0–16 pg relative to 1 mg of total protein. The treated material contains epidermal growth factor receptor (EGFR) at a concentration of 0–60 pg relative to 1 mg of total protein. The treated material contains hepatocyte growth factor (HGF) at a concentration of 0–100 pg relative to 1 mg of total protein, or, The treated material contains insulin-like growth factor binding protein-1 (IGFBP-1) at a concentration of 0–200 pg relative to 1 mg of total protein; in, The processed product is: Platelets are removed from the megakaryocytes or their cultures. The megakaryocytes or their cultures from which the platelets have been removed are treated; The processing includes concentration, drying, freezing, solvent treatment, surfactant treatment, enzyme treatment, and / or crushing. The megakaryocytes mentioned above are immortalized megakaryocytes; The immortalized megakaryocytes are in vitro induced megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes, and the megakaryocytes are derived from induced pluripotent stem cells.

46. ​​The composition according to claim 45, wherein, The freezing process is freeze-drying; the crushing process is ultrasonic treatment.

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