Ectodermal mesenchymal stem cells and methods for producing the same

Abstract

This invention provides ectodermal mesenchymal stem cells, a method for producing the same, and a method for screening substances that have pluripotent stem cell inducing activity. [Solution] The present inventors have found that ectodermal mesenchymal stem cells induced by necrotic tissue injury and circulating in the peripheral blood contribute to the regeneration of damaged tissue. Based on this discovery, the present inventors provide ectodermal mesenchymal stem cells and a method for producing them, as well as a method for screening substances that have pluripotent stem cell-inducing activity using cells in the peripheral blood induced by necrotic tissue injury as indicators.

Description

[Technical Field]

[0001] The present invention relates to ectodermal mesenchymal stem cells and a method for producing the same. The present invention also relates to a method for screening multipotent stem cell-inducing substances. [Background technology]

[0002] Mesenchymal stem cells (MSCs), found in bone marrow fluid and other tissues, possess the ability to differentiate into various tissues such as bone, cartilage, fat, muscle, nerve, and epithelium (multipotency). Therefore, in recent years, attempts to use MSCs in regenerative medicine (cell transplantation therapy) have become widespread. However, it is known that MSCs used in regenerative medicine so far gradually lose their proliferative capacity and multipotency when continuously subcultured outside the body. Therefore, there is a need to discover cells with a higher ability to promote tissue regeneration than typical MSCs, or substances that activate / induce such cells in vivo, in order to provide more effective treatment methods than conventional regenerative medicine. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] WO2012 / 147470 [Non-patent literature]

[0004] [Non-Patent Document 1] PNAS 2011 Apr 19;108(16):6609-14 [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide ectodermal mesenchymal stem cells and a method for producing the same. Another object of the present invention is to provide a method for screening pluripotent stem cell-inducing substances. [Means for solving the problem]

[0006] The inventors have discovered that ectomesenchymal stem cells (EMSCs), induced by necrotic tissue injury and circulating in the peripheral blood, contribute to the regeneration of damaged tissue. This is based on the discovery of a mechanism previously reported by the inventors, in which "HMGB1 released from necrotic tissue within the exfoliated epidermis in epidermal vesicles causes bone marrow mesenchymal stem cells to accumulate in the exfoliated epidermis via peripheral blood circulation, inducing the regeneration of damaged skin" (PNAS 2011 Apr 19;108(16):6609-14), as well as the results obtained this time, in which "when skin from an epidermal vesicle mouse is transplanted into one part of a parabiosis model and HMGB1 fragment peptide is administered to the other part, PDGFRα lineage-positive cells accumulate in the grafted area and regenerate the epidermis," and "when cartilage damage is created in mice and HMGB1 fragment peptide is administered, P0 lineage-positive cells accumulate in the cartilage damage site via peripheral blood circulation and regenerate cartilage tissue." Furthermore, experimental results were obtained suggesting that the source of EMSCs in peripheral blood may be specific PDGFRα-positive cells in the bone marrow, and that EMSCs in peripheral blood may have an embryological origin from ectomesenchyme cells arising from the epidermal side of the cranial neural fold. Based on these findings, the inventors have completed the invention of ectomesenchyme stem cells and a method for producing them, as well as a method for screening substances with pluripotent stem cell-inducing activity using cells in peripheral blood induced by necrotic tissue injury as indicators.

[0007] The inventors have previously discovered that a peptide consisting of the amino acid sequence (MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKK) at positions 1-44 of the N-terminus of the HMGB1 protein (hereinafter referred to as "HA1-44 peptide") mobilizes pluripotent stem cells, such as mesenchymal stem cells (MSCs), from the bone marrow into the peripheral blood, and exerts therapeutic effects in various disease models. Now, the inventors have newly discovered biological responses caused by the administration of HA1-44 peptide ((i) changes in the composition of the PDGFRα-positive cell population in the peripheral blood, (ii) an increase in the number of PDGFRα-positive cells in the peripheral blood, and (iii) changes in gene expression of PDGFRα-positive cells in the vertebral bone marrow). Using these responses as indicators, the inventors have discovered a method for screening substances with similar activity to HA1-44 peptide, i.e., pluripotent stem cell inducers, and have completed the present invention. In other words, the present invention provides a method for determining whether a test substance has similar activity to HA1-44 peptide (promoting induction and / or recruitment of pluripotent stem cells, and / or tissue regeneration) by administering the test substance to an animal or other subject and determining whether the same reaction as any of (i) to (iii) above occurs.

[0008] The inventors also conducted a detailed analysis of cells that are mobilized in peripheral blood and damaged tissue by the HA1-44 peptide and contribute to the regeneration of said tissue. As a result, they found that (i) the cells in peripheral blood that contribute to the promotion of tissue regeneration by the HA1-44 peptide are PDGFRα-positive cells derived from vertebral bone marrow, (ii) PDGFRα-positive cells derived from vertebral bone marrow have a higher differentiation potential into bone, cartilage, and / or fat than PDGFRα-positive cells derived from the bone marrow of other bones, and (iii) PDGFRα-positive cells of the same developmental lineage (Prx1 lineage negative) as PDGFRα-positive cells derived from vertebral bone marrow are also present in small quantities in the bone marrow of other bones. Based on these findings, the inventors have discovered that by culturing a population of cells derived from living tissues, including MSCs such as bone marrow and peripheral blood, on a dish to form colonies, subcloning each colony, and selecting cell clones that exhibit high differentiation potential into bone, cartilage, and / or fat, it is possible to obtain pluripotent stem cells with higher tissue regeneration-promoting ability than MSCs obtained by conventional methods (collecting bone marrow and culturing it on a dish), thus completing the present invention.

[0009] The present invention relates, in particular, to the following: A) Ectodermal mesenchymal stem cells. B) Method for producing ectodermal mesenchymal stem cells. C) Cells obtained by the manufacturing method described in item B). D) Cells or cell populations in the peripheral blood induced by MSC blood mobilization substances. E) Cells or cell populations in the vertebral bone marrow induced by a peptide consisting of the amino acid sequence (SEQ ID NO: 1) at positions 1-44 of the N-terminus of the A-box of the High-mobility group box 1 (HMGB1) protein. F) Methods for producing cells or cell populations as described in item D) or E). G) A method for obtaining, isolating, and / or concentrating cells from living tissue containing mesenchymal stem cells (MSCs) that have a high tissue regeneration-promoting capacity similar to that of PDGFRα-positive cells in vertebral bone marrow. H) Cells or cell populations obtained by the method described in item G). I) A composition containing ectodermal mesenchymal stem cells and used for promoting tissue regeneration. J) A method for screening substances having the inducing activity of pluripotent stem cells using cells in peripheral blood induced by necrotic tissue injury as an index. K) A method for screening substances having the inducing activity of pluripotent stem cells using the HA1-44 peptide as a positive control and the reaction of pluripotent stem cells contributing to tissue regeneration in vivo as an index. L) A method for determining the expected tissue regeneration promoting effect in a subject administered with an MSC blood mobilizing substance using colony-forming Pα cells in peripheral blood as an index.

[0010] The present invention more particularly relates to the following. a) Colony-forming PDGFR-positive cells having the following characteristics i) and ii) and / or iii): i) Having the ability to differentiate into osteoblasts, adipocytes, and chondrocytes; ii) Having the ability to differentiate into epidermal cells; iii) Being positive for the P0 lineage. b) The cells according to item a), which are PDGFRα-positive. c) Pα + 、Pα lin+ 、P0 lin+ 、Prx1 lin- 、Sox1 lin- 、LepR lin- 、CD34 + and Sca1 - The cells according to item a) or b) having one or more characteristics selected from the above. d) Cells derived from vertebral bone marrow, which are PDGFRα-positive, CD34-positive, and Sca1-negative. e) A method for producing colony-forming PDGFR-positive cells, comprising any one of the following steps 1) to 4): 1) A step of collecting peripheral blood from a subject having necrotic tissue injury and culturing it on a solid phase; 2) After collecting peripheral blood from a subject having necrotic tissue injury and culturing it on a solid phase, Pα + 、Pα lin+ 、P0 lin+ 、Prx1lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; 3) Collect peripheral blood from subjects with necrotic tissue damage, and extract Pα from the peripheral blood. + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; 4) Collect peripheral blood from subjects with necrotic tissue damage, and extract Pα from the peripheral blood. + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from the above, and culturing the cells on a solid phase. f) A method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) A step of culturing peripheral blood collected from a subject with necrotic tissue damage on a solid phase; 2) Peripheral blood collected from subjects with necrotic tissue damage is cultured on a solid phase, and then Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; 3) Pα obtained from peripheral blood collected from subjects with necrotic tissue injury + , Pα lin+ , P0 lin+ ,Prx1 lin-Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; 4) Pα obtained from peripheral blood collected from subjects with necrotic tissue injury + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from the above, and culturing the cells on a solid phase. g) A method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) The process of collecting vertebral bone marrow from the subject and culturing it on a solid phase; 2) Collect vertebral bone marrow from the subject, culture it on a solid phase, and then Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from; 3) Collect vertebral bone marrow from the subject, and extract Pα from the bone marrow. + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from; 4) Collect vertebral bone marrow from the subject, and extract Pα from the bone marrow. + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from the above, and culturing the cells on a solid phase. h) A method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) A step of culturing vertebral bone marrow collected from the subject on a solid phase; 2) After culturing the vertebral bone marrow collected from the subject on a solid phase, Pα+ , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from; 3) Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from; 4) Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from the above, and culturing the cells on a solid phase. i) A cell population obtained by administering a substance mobilized from MSC blood to a subject, collecting peripheral blood from the subject, and culturing the collected peripheral blood on a solid phase. j) Cell populations described in item i) in which the MSC blood mobilization substance is HA1-44 peptide. k) A method for producing cells, comprising the steps of administering a blood mobilization substance to a target, collecting peripheral blood from the target, and culturing the collected peripheral blood on a solid phase. l) A method for producing cells, comprising the step of culturing peripheral blood collected from a subject administered with an MSC blood mobilization substance on a solid phase. m) The method according to item k) or l), wherein the MSC blood mobilization substance is the HA1-44 peptide. n) A cell population obtained by 1) administering HA1-44 peptide to a subject, 2) collecting bone marrow from the vertebrae of the subject, and 3) culturing the collected bone marrow on a solid phase or sorting PDGFRα-positive cells from the collected bone marrow. o) A method for producing cells, comprising the steps of 1) administering HA1-44 peptide to a subject, 2) collecting bone marrow from the vertebrae of the subject, and 3) culturing the collected bone marrow on a solid phase or sorting PDGFRα-positive cells from the collected bone marrow. p) A method for producing cells, comprising culturing bone marrow of vertebra collected from a subject administered with HA1-44 peptide on a solid phase or sorting PDGFRα-positive cells from the collected bone marrow. q) A method for producing a cell population, comprising the following steps: 1) Culturing a cell population derived from a biological tissue containing MSC on a solid phase; 2) Subcloning the colonies obtained in step 1; 3) Culturing a part of the cells obtained by subcloning in a differentiation induction medium for bone, cartilage, and / or fat, and measuring the expression levels of differentiation markers for bone, cartilage, and / or fat; and 4) Selecting cell clones showing high expression levels by comparing with the expression levels of differentiation markers for bone, cartilage, and / or fat when MSC obtained by culturing bone marrow of femur on a solid phase is cultured in a differentiation induction medium for bone, cartilage, and / or fat. r) A method for producing a cell population, comprising the following steps: 1) Culturing a cell population derived from a biological tissue containing MSC on a solid phase; and 2) Selecting colonies having one or more characteristics selected from Pα + , Pα lin+ , P0 lin+ , Prx1 lin- , Sox1 lin- , LepR lin- , CD34 + , and Sca1 - . s) The method according to item r), wherein step 2 is a step of selecting Prx1 lineage-negative colonies. t) A method for producing a cell population, comprising selectively recovering cells having one or more characteristics selected from Pα + , Pα lin+ , P0 lin+ , Prx1 lin- , Sox1 lin- , LepR lin- , CD34 + , and Sca1 - from a cell population derived from a biological tissue containing MSC. u) A method for producing a cell population, comprising the following steps: 1) Selectively recovering cells having one or more characteristics selected from Pα + Pα lin+ P0 lin+ Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - from a cell population derived from a biological tissue containing MSCs; and 2) Culturing the cells recovered in step 1) on a solid phase. v) A cell or cell population obtained by the production method according to claims * to *. w) A composition for promoting tissue regeneration, containing colony-forming PDGFR-positive cells having the following characteristics of i) and ii) and / or iii): i) Having the ability to differentiate into osteoblasts, adipocytes, and chondrocytes; ii) Having the ability to differentiate into epidermal cells; iii) Being positive for the P0 lineage. x) The composition according to claim *, used for promoting the regeneration of tissues derived from mesoderm or ectoderm. y) A method for screening a pluripotent stem cell inducer, comprising the following steps: 1) Collecting peripheral blood from a subject and counting cells having one or more characteristics selected from Pα + Pα lin+ P0 lin+ Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - contained in the peripheral blood; 2) Collecting peripheral blood from a subject administered with a test substance and counting Pα + Pα lin+ P0 lin+ Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 -A step of counting cells having one or more characteristics selected from; and 3) If the number of cells counted in step 2) is greater than the number of cells counted in step 1), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity. z) A method for screening pluripotent stem cell inducers, comprising the following steps: 1) Pα contained in peripheral blood collected from the subject + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - A step of counting cells having one or more characteristics selected from; 2) Pα contained in peripheral blood collected from subjects who were administered the test substance. + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - A step of counting cells having one or more characteristics selected from; and 3) If the number of cells counted in step 2) is greater than the number of cells counted in step 1), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity. aa) A method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining an adherent cell population by collecting peripheral blood from a subject and culturing it on a solid phase; 2) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 1, either at the colony or single-cell level; 3) A step of administering a peptide (HA1-44 peptide) consisting of the amino acid sequence of SEQ ID NO: 1 to a target, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 4) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 3, either at the colony or single-cell level; 5) A step of administering the test substance to a subject, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 6) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 5, either at the colony or single-cell level; 7) A step of pooling the gene expression data obtained in steps 2 and 4 and performing clustering analysis; 8) A step of pooling the gene expression data obtained in steps 2 and 6 and performing clustering analysis; and 9) A step in which the analysis results of step 7 and step 8 are compared, and if the cell population obtained in step 5 (test substance administration group) has the same cluster composition as the cell population obtained in step 3 (HA1-44 peptide administration group), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity. ab) The method according to item aa), wherein the test substance is administered in place of the HA1-44 peptide in step 3, and the HA1-44 peptide is administered in place of the test substance in step 5. ac) The method described in item aa) or ab), wherein the comprehensive gene expression analysis is RNA sequencing (RNA-seq). ad) A method according to any one of items aa) to ac), wherein the clustering analysis is performed using the iterative clustering and guide-gene selection (ICGS) algorithm. ae) A method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject on a solid phase; 2) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 1) at the colony or single-cell level; 3) A step of obtaining an adherent cell population by culturing peripheral blood collected from subjects who have been administered a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (HA1-44 peptide) on a solid phase; 4) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 3) at the colony or single-cell level; 5) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject administered with the test substance on a solid phase; 6) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 5) at the colony or single-cell level; 7) A step of pooling the gene expression data obtained in steps 2) and 4) and performing clustering analysis; 8) A step of pooling the gene expression data obtained in steps 2) and 6) and performing clustering analysis; and 9) A step in which the analysis results from step 7) and step 8) are compared, and if the cell population obtained in step 5) has the same cluster structure as the cell population obtained in step 3), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity. af) A method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining an adherent cell population by collecting peripheral blood from a subject and culturing it on a solid phase; 2) A step to count the number of colonies obtained in step 1); 3) A step of administering the test substance to a subject, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 4) A step of counting the number of colonies obtained in step 3); and 5) A step in which, if the number of colonies counted in step 4) is greater than the number of colonies counted in step 2), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity. ag) A method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject on a solid phase; 2) A step to count the number of colonies obtained in step 1); 3) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject administered with the test substance on a solid phase; 4) A step of counting the number of colonies obtained in step 3); and 5) A step in which, if the number of colonies counted in step 4) is greater than the number of colonies counted in step 2), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity. ah) The colonies counted in steps 2) and 4) are Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - A screening method described in item ae) or af), wherein the colony has one or more characteristics selected from the above. ai) A method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of collecting bone marrow from the target vertebra and obtaining a population of PDGFRα-positive cells by culturing on a solid phase or cell sorting; 2) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 1, either at the colony or single-cell level; 3) A step of administering the test substance to the subject, collecting bone marrow from the vertebrae, and obtaining a population of PDGFRα-positive cells by culturing on a solid phase or cell sorting; 4) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 3, either at the colony or single-cell level; 5) A step of pooling the gene expression data obtained in steps 2 and 4 and performing pathway analysis; and 6) A step in which, as a result of the analysis in step 5, the cell population obtained in step 3 (test substance administered group) shows either (i) activation of pathways related to the regulation of EIF2 signaling, eIF4 and p70S6K signaling, and / or mTOR signaling, or (ii) suppression of the expression of cell death-related genes, compared to the cell population obtained in step 1 (untreated group), the test substance is selected as a candidate substance having pluripotent stem cell-inducing activity. aj) A method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining a population of PDGFRα-positive cells from bone marrow collected from the target vertebra by culture on a solid phase or cell sorting; 2) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 1) at the colony or single-cell level; 3) A step of obtaining a population of PDGFRα-positive cells from bone marrow collected from the vertebrae of subjects administered the test substance, by culturing on a solid phase or cell sorting; 4) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 3) at the colony or single-cell level; 5) A step of pooling the gene expression data obtained in steps 2) and 4) and performing pathway analysis; and 6) As a result of the analysis in step 5), if the cell population obtained in step 3) shows that (i) pathways related to the regulation of EIF2 signaling, eIF4 and p70S6K signaling, and / or mTOR signaling are activated, or (ii) the expression of cell death-related genes is suppressed, then the test substance is selected as a candidate substance having pluripotent stem cell-inducing activity. ak) The method described in item a) or aj) where comprehensive gene expression analysis is performed by RNA sequencing (RNA-seq). al) The following steps: 1) Pα contained in peripheral blood collected from subjects before administration of MSC blood mobilization substance + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - A step of counting cells having one or more characteristics selected from; and 2) Pα contained in peripheral blood collected from the subject after administration of MSC blood mobilization substance + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34+ and Sca1 - A step of counting cells having one or more characteristics selected from; A method for determining the tissue regeneration-promoting effect of MSC blood mobilization substances, wherein if the number of cells counted in step 2) is greater than the number of cells counted in step 1), it is suggested that tissue regeneration is promoted in the subject. [Effects of the Invention]

[0011] The present invention makes it possible to provide ectodermal mesenchymal stem cells and methods for producing them. It also makes it possible to provide a screening method for pluripotent stem cell-inducing substances. [Brief explanation of the drawing]

[0012] [Figure 1] This graph plots the number of Pα cells in peripheral blood and HMGB1 concentration for mice with and without skin flaps. [Figure 2] This image shows a photograph of a colony obtained by culturing peripheral blood from a mouse, and a graph showing the CFU activity converted to 1 mL of peripheral blood. [Figure 3] This photograph shows the results of differentiation induction of iCFPα cells obtained from mouse peripheral blood into osteoblasts, adipocytes, chondrocytes, and keratin 5-expressing cells. Osteoblasts were detected by ALP staining, adipocytes by Oil Red-O staining, chondrocytes by Toluidine blue staining, and keratin 5-expressing cells by fluorescence of the reporter protein tdTomato. [Figure 4] This figure shows the results of clustering analysis performed on iCFPα cells based on single-cell transcriptome analysis and the obtained data. The cells shown on the left are those predicted based on gene expression profiles (such as high expression of specific gene sets). [Figure 5]This figure shows the results of transcriptome analysis and clustering analysis performed on iCFPα cells at the colony level. The cells shown on the left are those predicted based on gene expression profiles (e.g., high expression of specific gene sets). Each column corresponds to one colony. [Figure 6] This is a photograph of colony-forming cells obtained by culturing peripheral blood from Pα-H2B-GFP mice. [Figure 7] This graph shows the results of examining the negative / positive rates of iCFPα cells for the Pα, P0, Prx1, Sox1, and LepR lineages. [Figure 8] This graph shows the results of evaluating the CFU activity of colony-forming cells obtained from peripheral blood of mice with and without skin flaps, categorized by Prx1 lineage. [Figure 9] This image shows the results of detecting Pα expression and Prx1 lineage in cells present in the bone marrow tissue of the femur, vertebrae, sternum, ilium, hip joint (femoral head and acetabulum), and skull of Pα-H2B-GFP::Prx1-Cre::Rosa26-tdTomato mice. [Figure 10] This figure shows the results of FACS analysis of bone marrow cells from the femur, vertebrae, sternum, and ilium of Pα-H2B-GFP::Prx1-Cre::Rosa26-tdTomato mice. [Figure 11] This graph shows the results of examining the P0, Prx1, Sox1, and LepR lineages of CFPα cells derived from vertebral bone marrow. [Figure 12] This graph shows the results of examining the P0, Prx1, Sox1, and LepR lineages of CFPα cells derived from femoral bone marrow. [Figure 13] For bone marrow-derived CFPα cells from the vertebrae and femur, (a) a photograph of the colony, (b) the number of colonies, and (c) the growth curve are shown. The horizontal axis of (c) indicates the passage number (P0 = primary culture). [Figure 14]This graph shows the results of culturing bone marrow-derived CFPα cells from the vertebrae and femur under conditions that induce differentiation into adipocytes, and examining the expression of adipocyte differentiation markers. [Figure 15] This graph shows the results of culturing bone marrow-derived CFPα cells from the vertebrae and femur under conditions that induce differentiation into osteoblasts, and examining the expression of osteoblast differentiation markers. [Figure 16] (a) A graph showing the results of culturing bone marrow-derived CFPα cells from vertebrae and femurs under conditions that induce differentiation into chondrocytes, and examining the expression of chondrocyte differentiation markers, as well as (b) a photograph of the formed cartilage pellets and (c) the weight of the cartilage pellets. [Figure 17] This is a photograph of K5-expressing cells obtained by culturing vertebral bone marrow-derived CFPα cells under conditions that induce differentiation into keratinocytes. [Figure 18] This is a photograph of K5-expressing cells obtained by culturing femoral bone marrow-derived CFPα cells under conditions that induce differentiation into keratinocytes. [Figure 19] This figure shows the results of clustering analysis performed on Pα cells in the bone marrow of the vertebrae and femur, following single-cell transcriptome analysis. [Figure 20] This graph shows the results of a CFU assay performed on Pα cells in the vertebral bone marrow, which were separated into four populations using FACS based on the expression levels of Sca1 and CD34. [Figure 21] This figure shows the results of clustering analysis of iCFPα cells in peripheral blood together with Pα cells from the vertebrae and femur. [Figure 22] This figure shows the expression of Procr in cells of the S34-MSC cluster in the bone marrow. [Figure 23] This graph shows the abundance of Sca1+CD34+ cells (percentage of PDGFRα+CD45- viable cells) in the bone marrow of the cervical, thoracic, lumbar, and femoral vertebrae of Pα-H2B-GFP mice. [Figure 24](a) is a photograph of colony-forming cells obtained by culturing peripheral blood, and (b) is a graph showing the CFU activity of these cells converted to 1 mL of peripheral blood. [Figure 25] Figure (a) shows a schematic of the parabiosis model, and figure (b) shows the results of observation of transplanted skin tissue after administration of HA1-44 peptide. Pα ​​cells were detected by YFP fluorescence, and type VII collagen was detected by antibody. [Figure 26] Figure (a) shows a schematic of the parabiosis model, a photograph (b) shows the results of observation of transplanted skin tissue after administration of HA1-44 peptide, and a graph (c) shows the percentage of PDGFRα+ cells in the transplanted skin tissue. PDGFRα expression was detected by GFP fluorescence, and the Prx1 lineage was detected by the fluorescence of the reporter protein tdTomato. [Figure 27] Figure (a) shows a schematic of the parabiosis model, and photograph (b) shows the tissue observation results of the knee cartilage injury site in the control group (administered with physiological saline) and the HA1-44 peptide administration group. P0lin+ cells were detected by fluorescence of the reporter protein tdTomato. [Figure 28] These photographs show the histological findings (safranin O staining) of the knee cartilage injury site in the control group (saline administration) and the HA1-44 peptide administration group at 2, 4, 8, and 12 weeks after the creation of the knee cartilage defect. The arrowheads indicate areas where hyaline cartilage regeneration was observed. [Figure 29] This figure shows the results of transcriptome analysis and clustering analysis performed on colonies of cells obtained by culturing peripheral blood from mice. The cells shown on the left are those predicted based on gene expression profiles (e.g., high expression of specific gene sets). Each column corresponds to one colony. A square is displayed below the column corresponding to the mouse colonies from the HA1-44 peptide administration group. [Figure 30] This is a simplified table showing the results of clustering analysis of mouse peripheral blood-derived colonies. [Figure 31]This graph shows the results of pathway analysis based on transcriptome analysis data of vertebral Pα cells in mice administered HA1-44 peptide and those administered physiological saline. [Figure 32] This graph shows the results of pathway analysis (function analysis) performed based on transcriptome analysis data of vertebral Pα cells in mice administered HA1-44 peptide and those administered physiological saline. [Figure 33] This graph shows the percentage of CD45-negative, TER-119-negative, and PDGFRβ-positive cells in human peripheral blood mononuclear cell fractions collected before, 8 hours after, and 24 hours after administration of HA1-44 peptide. [Modes for carrying out the invention]

[0013] In this application, "cell" means one cell or multiple cells, depending on the context. For example, a cell in this application may be a group of cells consisting of one type of cell, or a group of cells containing multiple types of cells. For example, the expression "cells having the ability to differentiate into osteoblasts, adipocytes, and chondrocytes" includes not only cases where one cell / one type of cell (or a homogeneous group of cells derived from it) has the ability to differentiate into these three cell types, but also cases where a group of cells containing multiple types of cells exhibits the ability to differentiate into these three cell types as a whole.

[0014] In this application, ectodermal mesenchymal stem cells (EMSCs) refer to PDGFR-positive cells that possess colony-forming ability and the ability to differentiate into three mesenchymal lineages (osteoblasts, adipocytes, and chondrocytes), and that are suggested to be of ectoderm origin. The ectoderm origin of such cells is indicated, for example, by P0 lin+This suggests that, in one embodiment, EMSCs also have the ability to differentiate into epidermal cells (specifically, K5-positive keratinocytes). Epidermal cells that EMSCs can differentiate into include, but are not limited to, keratinocytes, cells expressing keratin 5 (K5) (K5-positive cells), and keratinocytes expressing K5 (K5-positive keratinocytes). For example, whether or not a cell has the ability to differentiate into K5-positive keratinocytes can be determined by whether or not it can differentiate into cells expressing K5 when cultured under keratinocyte differentiation induction conditions.

[0015] Markers that characterize EMSCs (including cell lineage markers) include Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - These are some examples.

[0016] Examples of EMSC include: (a) Colony-forming Pα cells (also known as iCFPα cells) whose abundance in the peripheral blood increases in response to necrotic tissue injury (such as skin flap), and (b) Colony-forming Pα cells contained in vertebral bone marrow (hereinafter also referred to as "vertebral CFPα cells" or "vertebral-derived CFPα cells") are an example.

[0017] iCFPα cells possess the ability to form colonies and to differentiate into osteoblasts, adipocytes, and chondrocytes. Therefore, iCFPα cells can be said to have the properties of mesenchymal stem cells. Furthermore, iCFPα cells have the ability to differentiate into epidermal cells (K5-positive keratinocytes) and P0 lin+ Therefore, iCFPα cells can be said to be ectodermal mesenchymal stem cells.

[0018] A marker that characterizes iCFPα cells is Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - These are some examples.

[0019] Vertebral-derived CFPα cells possess the following properties: i) colony-forming ability, and ii) differentiation ability into osteoblasts, adipocytes, and chondrocytes. Therefore, vertebral-derived CFPα cells can be said to possess the properties of mesenchymal stem cells. Furthermore, vertebral-derived CFPα cells possess the differentiation ability into epidermal cells (K5-positive keratinocytes), and P0 lin+ Therefore, vertebral-derived CFPα cells can be considered ectodermal mesenchymal stem cells.

[0020] Pα is a marker that characterizes vertebral-derived CFPα cells. + , P0 lin+ ,Prx1 lin- , and Sox1 lin- These include vertebral-derived CFPα cells, and LepR lin+ Cells and LepR lin- It contains cells. Among these, LepR lin- The cells are thought to exhibit properties closer to those of iCFPα cells in peripheral blood.

[0021] This application provides, as one embodiment of EMSC, colony-forming PDGFR-positive cells having the following features i) and ii) and / or iii): i) Possesses the ability to differentiate into osteoblasts, adipocytes, and chondrocytes; ii) Possesses the ability to differentiate into epidermal cells; iii) Positive for the P0 lineage. In one embodiment, the colony-forming PDGFR-positive cells are PDGFRα-positive cells. In another embodiment, the colony-forming PDGFR-positive cells are Pα + , Pα lin+ , P0lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - It is a cell that has one or more characteristics selected from the following.

[0022] Further characteristics of the colony-forming PDGFR-positive cells mentioned above include the following: ·Pα + is; • CD34 + is; ·Sca1 - is; • CD34 + , and Sca1 - is; • CD34 + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Having one or more characteristics selected from; ·Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Having one or more characteristics selected from; • CD34 + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Having one or more characteristics selected from; ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Having one or more characteristics selected from; ·P0 lin+ , and Prx1 lin- is; ·P0 lin+ ,Prx1 lin- , and Sox1 lin- is; ·P0 lin+ ,Prx1 lin- , and LepR lin- is; ·P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- is; ·Pα lin+ , P0 lin+ , and Prx1 lin- is; ·Pα lin+ , P0 lin+ ,Prx1 lin- , and Sox1 lin- is; ·Pα lin+ , P0 lin+ ,Prx1 lin- , and LepR lin- is; ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- is; ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and CD34 + is; ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and Sca1 - is; ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - is; ·Pα + , and CD34+ is; ·Pα + , and Sca1 - is; ·Pα + CD34 + , and Sca1 - is; ·Pα + , and CD34 + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Having one or more characteristics selected from; ·Pα + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Having one or more characteristics selected from; ·Pα + CD34 + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Having one or more characteristics selected from; ·Pα + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Having one or more characteristics selected from; ·Pα + , P0 lin+ , and Prx1 lin- is; ·Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- is; ·Pα + , P0 lin+ ,Prx1lin- , and LepR lin- is; ·Pα + , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- is; ·Pα + , Pα lin+ , P0 lin+ , and Prx1 lin- is; ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- , and Sox1 lin- is; ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- , and LepR lin- is; ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- is; ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and CD34 + is; ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and Sca1 - is; ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - That is the case.

[0023] In one embodiment, the present invention relates to vertebral bone marrow-derived cells that are PDGFRα-positive, CD34-positive, and Sca1-negative. These cells are presumed to be equivalent to iCFPα cells in peripheral blood, based on the commonality of the markers. Therefore, they are expected to exhibit similar properties to iCFPα cells. These vertebral bone marrow-derived cells are obtained, for example, by collecting vertebral bone marrow from a subject and pharmacokinetically ionizing (Pα) + CD34 + , and Sca1 - It can be obtained by selectively harvesting the cells.

[0024] Furthermore, this application relates to bone marrow-derived Pα + CD34 + Sca1 + The present invention provides cells. Furthermore, the present invention provides Pα from bone marrow. + CD34 + , and Sca1 + The present invention provides a method for producing cells, which includes a step of selectively recovering the cells. Pα + CD34 + Sca1 + Bone marrow that can be used as a source of cells includes bone marrow from the vertebrae (cervical, thoracic, and lumbar vertebrae) and the femur. In one embodiment, Pα + CD34 + Sca1 + The bone marrow that can be used as a source of cells is the bone marrow of the vertebra. In another embodiment, Pα + CD34 + Sca1 + The bone marrow that can be used as a source of cells is the bone marrow from the cervical vertebrae.

[0025] The inventors also, Pα + CD34 + Sca1 + We found that these cells are also abundant in the bone marrow of bones that have ectomesenchyme as their embryological origin. Therefore, this application relates to Pα derived from bone marrow of bones that have ectomesenchyme as their embryological origin. + CD34 + Sca1 +The present invention provides cells. Furthermore, the present invention provides Pα from bone marrow, which originates from ectomesenchyme embryologically. + CD34 + , and Sca1 + The present invention provides a method for producing cells, which includes a step of selectively recovering cells. Examples of bones that originate from ectomesenchyme include the frontal bone, nasal bone, zygomatic bone, maxilla, palatine bone, and mandible.

[0026] In one embodiment, the present invention relates to a method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) A step of collecting peripheral blood from a subject with necrotic tissue damage and culturing it on a solid phase; 2) Peripheral blood is collected from subjects with necrotic tissue damage, cultured on a solid phase, and then Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; 3) Collect peripheral blood from subjects with necrotic tissue damage, and extract Pα from the peripheral blood. + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; 4) Collect peripheral blood from subjects with necrotic tissue damage, and extract Pα from the peripheral blood. + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 -A step of selectively harvesting cells having one or more characteristics selected from the above, and culturing the cells on a solid phase.

[0027] In one embodiment, the present invention relates to a method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) A step of culturing peripheral blood collected from a subject with necrotic tissue damage on a solid phase; 2) Peripheral blood collected from subjects with necrotic tissue damage is cultured on a solid phase, and then Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; 3) Pα obtained from peripheral blood collected from subjects with necrotic tissue injury + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; 4) Pα obtained from peripheral blood collected from subjects with necrotic tissue injury + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from the above, and culturing the cells on a solid phase.

[0028] Necrotizing tissue injuries include, but are not limited to, skin flaps and epidermal detachment in epidermolysis blisters. In skin flaps, insufficient blood supply to the tip of the flap leads to ischemia, causing cell / tissue necrosis. In epidermolysis blisters, necrosis occurs in the detached epidermal tissue.

[0029] When culturing peripheral blood on a solid phase, red blood cells may be removed from the peripheral blood before culturing. Red blood cell removal can be performed by methods known to those skilled in the art, such as using hemolytic reagents, or by treating the peripheral blood with a hetastarch and recovering the supernatant containing nucleated cells.

[0030] Examples of cells selectively harvested in step 2), 3), or 4) of the above method for producing colony-forming PDGFR-positive cells include the following: ·Pα + cells • CD34 + cells ·Sca1 - cells • CD34 + , and Sca1 - cells • CD34 + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from • CD34 + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin-Cells having one or more characteristics selected from ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·P0 lin+ , and Prx1 lin- cells ·P0 lin+ ,Prx1 lin- , and Sox1 lin- cells ·P0 lin+ ,Prx1 lin- , and LepR lin- cells ·P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- cells ·Pα lin+ , P0 lin+ , and Prx1 lin- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- , and Sox1 lin- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- , and LepR lin- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and CD34 + cells ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and Sca1- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - cells ·Pα + , and CD34 + cells ·Pα + , and Sca1 - cells ·Pα + CD34 + , and Sca1 - cells ·Pα + , and CD34 + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα + CD34 + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα+ , P0 lin+ , and Prx1 lin- cells ·Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- cells ·Pα + , P0 lin+ ,Prx1 lin- , and LepR lin- cells ·Pα + , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- cells ·Pα + , Pα lin+ , P0 lin+ , and Prx1 lin- cells ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- , and Sox1 lin- cells ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- , and LepR lin- cells ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- cells ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and CD34 + cells ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and Sca1- cells ·Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - The cells.

[0031] In this application, examples of methods for "selectively recovering" cells include the following: (1) A method for "sorting" cells that express a desired marker molecule using a cell sorter, etc. (2) A method for “recovering,” “selecting,” “separating,” “isolating,” or “concentrating” cells / colonies that express a desired marker molecule, either by visual inspection or based on the results of gene expression analysis. Examples of marker molecules used here include surface markers (cell surface antigens) and reporter proteins for lineage marker genes.

[0032] In one embodiment, the present invention relates to a method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) The process of collecting vertebral bone marrow from the subject and culturing it on a solid phase; 2) Collect vertebral bone marrow from the subject, culture it on a solid phase, and then Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from; 3) Collect vertebral bone marrow from the subject, and extract Pα from the bone marrow. + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from; 4) Collect vertebral bone marrow from the subject, and extract Pα from the bone marrow. + , P0 lin+ ,Prx1 lin- , and Sox1 lin-A step of selectively harvesting cells having one or more characteristics selected from the above, and culturing the cells on a solid phase.

[0033] In one embodiment, the present invention relates to a method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) A step of culturing vertebral bone marrow collected from the subject on a solid phase; 2) After culturing the vertebral bone marrow collected from the subject on a solid phase, Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from; 3) Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from; 4) Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- A step of selectively harvesting cells having one or more characteristics selected from the above, and culturing the cells on a solid phase.

[0034] In one embodiment, in step 2), 3), or 4) of the method for producing the above-mentioned colony-forming PDGFR-positive cells, Pα + , P0 lin+ ,Prx1 lin- Sox1 lin- and LepR lin- Cells having one or more characteristics selected from the above may be selectively harvested.

[0035] Examples of cells selectively harvested in step 2), 3), or 4) of the above method for producing colony-forming PDGFR-positive cells include the following: ·P0 lin+ cells ·Prx1 lin- cells ·Sox1 lin- cells ·P0 lin+ , and Prx1 lin- cells ·P0 lin+ , and Sox1 lin- cells ·Prx1 lin- , and Sox1 lin- cells ·P0 lin+ ,Prx1 lin- , and Sox1 lin- cells ·Pα + , and P0 lin+ cells ·Pα + , and Prx1 lin- cells ·Pα + , and Sox1 lin- cells ·Pα + , P0 lin+ , and Prx1 lin- cells ·Pα + , P0 lin+ , and Sox1 lin- cells ·Pα + ,Prx1 lin- , and Sox1 lin- cells ·Pα + , P0 lin+ ,Prx1 lin- , and Sox1 lin- cells ·LepR lin- cells ·P0 lin+ , and LepR lin- cells ·Prx1 lin- , and LepR lin- cells ·Sox1 lin- , and LepR lin- cells ·P0 lin+ ,Prx1 lin- , and LepR lin- cells ·P0 lin+ Sox1lin- , and LepR lin- cells ·Prx1 lin- Sox1 lin- , and LepR lin- cells ·P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- cells ·Pα + , P0 lin+ , and LepR lin- cells ·Pα + ,Prx1 lin- , and LepR lin- cells ·Pα + Sox1 lin- , and LepR lin- cells ·Pα + , P0 lin+ ,Prx1 lin- , and LepR lin- cells ·Pα + , P0 lin+ Sox1 lin- , and LepR lin- cells ·Pα + ,Prx1 lin- Sox1 lin- , and LepR lin- cells ·Pα + , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- The cells.

[0036] Vertebrals that can be used as a source of colony-forming PDGFR-positive cells include the cervical, thoracic, and lumbar vertebrae. In one embodiment, the vertebra used as a source of colony-forming PDGFR-positive cells is the cervical vertebrae.

[0037] Furthermore, experiments conducted by the inventors have confirmed that all colonies obtained by culturing vertebral bone marrow on a solid phase are PDGFR-positive.

[0038] In this application, "bone marrow" collected from the subject means bone marrow tissue containing various types of bone marrow cells.

[0039] In one embodiment, the present invention relates to a method for screening substances that have pluripotent stem cell-inducing activity using cells in peripheral blood induced by necrotic tissue injury as indicators.

[0040] The inventors have shown that necrotic tissue injury (e.g., skin flap) increases iCFPα cells in peripheral blood, and that administration of HA1-44 peptide increases Pα cells in peripheral blood. + P0 lin+ Prx1 lin- We found that the number of cells (i.e., cell populations including iCFPα cells) increased. Therefore, by using the increase in iCFPα cells in peripheral blood as an indicator, it is possible to screen for substances that increase the abundance of pluripotent stem cells (e.g., MSCs) with proliferative capacity (colony-forming capacity) and multi-lineage differentiation potency in peripheral blood (hereinafter also referred to as pluripotent stem cell mobilizing substances or pluripotent stem cell inducing substances).

[0041] In one embodiment, the present invention relates to a method for screening pluripotent stem cell inducers, comprising the following steps: 1) Collect peripheral blood from the subject and collect Pα contained in the peripheral blood. + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - A step of counting cells having one or more characteristics selected from; 2) Peripheral blood is collected from subjects who have been administered the test substance, and Pα contained in the peripheral blood is collected. + , Pαlin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - A step of counting cells having one or more characteristics selected from; and 3) If the number of cells counted in step 2) is greater than the number of cells counted in step 1), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

[0042] In one embodiment, the present invention relates to a method for screening pluripotent stem cell inducers, comprising the following steps: 1) Pα contained in peripheral blood collected from the subject + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - A step of counting cells having one or more characteristics selected from; 2) Pα contained in peripheral blood collected from subjects who were administered the test substance. + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - A step of counting cells having one or more characteristics selected from; and 3) If the number of cells counted in step 2) is greater than the number of cells counted in step 1), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

[0043] Regarding the characteristics of cells to be counted in the above screening method, surface markers (Pα, CD34, Sca1) can be detected using antibodies, etc. Alternatively, if an experimental animal has a reporter gene incorporated downstream of the promoter of the surface marker gene, the product of the reporter gene (fluorescent protein, etc.) can be used as an indicator for detection. Lineage markers (Pα, P0, Prx1, Sox1, LepR) can be detected by using transgenic animals that have a DNA structure / construct (Cre-loxP system, etc.) that enables lineage tracing of the target gene.

[0044] Examples of cells to be counted in steps 1) and 2) of the above screening method include the following: ·Pα + cells • CD34 + cells ·Sca1 - cells • CD34 + , and Sca1 - cells • CD34 + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from • CD34 + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα lin+ , P0lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·P0 lin+ , and Prx1 lin- cells ·P0 lin+ ,Prx1 lin- , and Sox1 lin- cells ·P0 lin+ ,Prx1 lin- , and LepR lin- cells ·P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- cells ·Pα lin+ , P0 lin+ , and Prx1 lin- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- , and Sox1 lin- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- , and LepR lin- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- cells ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and CD34 + cells ·Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- , and Sca1 - cells ·Pα lin+ , P0 lin+,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - cells ·Pα + , and CD34 + cells ·Pα + , and Sca1 - cells ·Pα + CD34 + , and Sca1 - cells ·Pα + , and CD34 + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα + CD34 + , and Sca1 - And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα + And, Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- , and LepR lin- Cells having one or more characteristics selected from ·Pα + , P0 lin+ , and Prx1 lin- cells ·Pα + 、P0 lin+ 、Prx1 lin- 、and Sox1 lin- cells ·Pα + 、P0 lin+ 、Prx1 lin- 、and LepR lin- cells ·Pα + 、P0 lin+ 、Prx1 lin- 、Sox1 lin- 、and LepR lin- cells ·Pα + 、Pα lin+ 、P0 lin+ 、and Prx1 lin- cells ·Pα + 、Pα lin+ 、P0 lin+ 、Prx1 lin- 、and Sox1 lin- cells ·Pα + 、Pα lin+ 、P0 lin+ 、Prx1 lin- 、and LepR lin- cells ·Pα + 、Pα lin+ 、P0 lin+ 、Prx1 lin- 、Sox1 lin- 、and LepR lin- cells ·Pα + 、Pα lin+ 、P0 lin+ 、Prx1 lin- 、Sox1 lin- 、LepR lin- 、and CD34 + cells ·Pα + 、Pα lin+ 、P0 lin+ 、Prx1 lin- 、Sox1 lin- 、LepR lin- 、and Sca1 - cells ·Pα + 、Pα lin+, P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - The cells.

[0045] In one embodiment, the present invention relates to a method for screening substances that have pluripotent stem cell inducing activity, using HA1-44 peptide as a positive control and the response of pluripotent stem cells that contribute to tissue regeneration in vivo as an indicator.

[0046] In one embodiment, the present invention relates to a method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining an adherent cell population by collecting peripheral blood from a subject and culturing it on a solid phase; 2) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 1, either at the colony or single-cell level; 3) A step of administering a peptide (HA1-44 peptide) consisting of the amino acid sequence of SEQ ID NO: 1 to a target, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 4) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 3, either at the colony or single-cell level; 5) A step of administering the test substance to a subject, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 6) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 5, either at the colony or single-cell level; 7) A step of pooling the gene expression data obtained in steps 2 and 4 and performing clustering analysis; 8) A step of pooling the gene expression data obtained in steps 2 and 6 and performing clustering analysis; and 9) A step in which the analysis results of step 7 and step 8 are compared, and if the cell population obtained in step 5 (test substance administration group) has the same cluster composition as the cell population obtained in step 3 (HA1-44 peptide administration group), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

[0047] In other embodiments, the test substance is administered instead of the HA1-44 peptide in step 3, and the HA1-44 peptide is administered instead of the test substance in step 5. That is, either the HA1-44 peptide or the test substance may be administered to the subject first. The subjects in steps 1, 3, and 5 may be the same individual or different individuals. For example, three animals from the same strain may be prepared, one may not be administered the substance (or only the solvent), another may be administered the HA1-44 peptide, and the remaining one may be administered the test substance. Peripheral blood may then be collected from each individual to obtain adherent cell populations, perform comprehensive gene expression analysis, and perform clustering analysis. The subject in step 1 may be an individual that has been administered only the same solvent as the solvent used when administering the HA1-44 peptide and the test substance in steps 3 and 5, respectively.

[0048] In another embodiment, a variant, modified, or tagged HA1-44 peptide is used instead of the HA1-44 peptide. The variant has an amino acid sequence in which several amino acids, for example, 1 to 5, preferably 1 to 4, 1 to 3, more preferably 1 to 2, and more preferably 1 amino acid are substituted, inserted, deleted, and / or added to the amino acid sequence of the HA1-44 peptide. For example, the variant is a peptide having an amino acid sequence that exhibits 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, and particularly preferably 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%) homology to the amino acid sequence of the HA1-44 peptide when local alignment is performed. The homology of amino acid sequences can be measured using, for example, FASTA, BLAST, DNASIS (manufactured by Hitachi Software Engineering Co., Ltd.), or GENETYX (manufactured by Genetics Co., Ltd.). Alternatively, it can be simply calculated by comparing the sequences. The modified peptide has an amino acid sequence in which several amino acid residues, for example, 1 to 5, preferably 1 to 4, 1 to 3, more preferably 1 to 2, and more preferably 1 amino acid, are modified in the amino acid sequence of the HA1-44 peptide. When a tagged HA1-44 peptide is used, exemplary tags include, but are not limited to, the His6- tag, FLAG tag, myc tag, and GST tag. The tag may be attached to the N-terminus or the C-terminus of the amino acid sequence.

[0049] In this screening method, comprehensive gene expression analysis may be performed by RNA sequencing (RNA-seq). In this screening method, clustering analysis may be performed using the iterative clustering and guide-gene selection (ICGS) algorithm.

[0050] In another embodiment, the present invention relates to a method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject on a solid phase; 2) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 1) at the colony or single-cell level; 3) A step of obtaining an adherent cell population by culturing peripheral blood collected from subjects who have been administered a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (HA1-44 peptide) on a solid phase; 4) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 3) at the colony or single-cell level; 5) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject administered with the test substance on a solid phase; 6) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 5) at the colony or single-cell level; 7) A step of pooling the gene expression data obtained in steps 2) and 4) and performing clustering analysis; 8) A step of pooling the gene expression data obtained in steps 2) and 6) and performing clustering analysis; and 9) A step in which the analysis results from step 7) and step 8) are compared, and if the cell population obtained in step 5) has the same cluster structure as the cell population obtained in step 3), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

[0051] In another embodiment, the present invention relates to a method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining an adherent cell population by collecting peripheral blood from a subject and culturing it on a solid phase; 2) A step to count the number of colonies obtained in step 1); 3) A step of administering the test substance to a subject, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 4) A step of counting the number of colonies obtained in step 3); and 5) A step in which, if the number of colonies counted in step 4) is greater than the number of colonies counted in step 2), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity. The subjects in step 1) may be subjects that have been administered only with the same solvent used when administering the test substance in step 3). The colonies to be counted in steps 2) and 4) are Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 - The colony may have one or more characteristics selected from the following.

[0052] In another embodiment, the present invention relates to a method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject on a solid phase; 2) A step to count the number of colonies obtained in step 1); 3) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject administered with the test substance on a solid phase; 4) A step of counting the number of colonies obtained in step 3); and 5) A step in which, if the number of colonies counted in step 4) is greater than the number of colonies counted in step 2), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity. The subjects in step 1) may be subjects that have been administered only with the same solvent used when the test substance is administered in step 3). The colonies to be counted in steps 2) and 4) are Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + and Sca1 -The colony may have one or more characteristics selected from the following.

[0053] In another embodiment, the present invention relates to a method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of collecting bone marrow from the target vertebra and obtaining a population of PDGFRα-positive cells by culturing on a solid phase or cell sorting; 2) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 1, either at the colony or single-cell level; 3) A step of administering the test substance to the subject, collecting bone marrow from the vertebrae, and obtaining a population of PDGFRα-positive cells by culturing on a solid phase or cell sorting; 4) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 3, either at the colony or single-cell level; 5) A step of pooling the gene expression data obtained in steps 2 and 4 and performing pathway analysis; and 6) A step in which, as a result of the analysis in step 5, the cell population obtained in step 3 (test substance administered group) shows either (i) activation of pathways related to the regulation of EIF2 signaling, eIF4 and p70S6K signaling, and / or mTOR signaling, or (ii) suppression of the expression of cell death-related genes, compared to the cell population obtained in step 1 (untreated group), the test substance is selected as a candidate substance having pluripotent stem cell-inducing activity. The subjects in step 1 may be those to which only the same solvent used when administering the test substance in step 3 has been administered.

[0054] In this screening method, comprehensive gene expression analysis may be performed by RNA sequencing (RNA-seq). In this screening method, pathway analysis may be performed using Ingenuity Pathway Analysis (IPA) software (https: / / www.qiagenbioinformatics.com).

[0055] In another embodiment, the present invention relates to a method for screening pluripotent stem cell inducers, comprising the following steps: 1) A step of obtaining a population of PDGFRα-positive cells from bone marrow collected from the target vertebra by culture on a solid phase or cell sorting; 2) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 1) at the colony or single-cell level; 3) A step of obtaining a population of PDGFRα-positive cells from bone marrow collected from the vertebrae of subjects administered the test substance, by culturing on a solid phase or cell sorting; 4) A step in which comprehensive gene expression analysis is performed on the cell population obtained in step 3) at the colony or single-cell level; 5) A step of pooling the gene expression data obtained in steps 2) and 4) and performing pathway analysis; and 6) As a result of the analysis in step 5), if the cell population obtained in step 3) shows that (i) pathways related to the regulation of EIF2 signaling, eIF4 and p70S6K signaling, and / or mTOR signaling are activated, or (ii) the expression of cell death-related genes is suppressed, then the test substance is selected as a candidate substance having pluripotent stem cell-inducing activity.

[0056] In this screening method, comprehensive gene expression analysis may be performed by RNA sequencing (RNA-seq). In this screening method, pathway analysis may be performed using Ingenuity Pathway Analysis (IPA) software (https: / / www.qiagenbioinformatics.com).

[0057] In other embodiments, the present invention relates to cells or cell populations in peripheral blood induced by MSC blood mobilization substances. The present invention also relates to cells or cell populations in vertebral bone marrow induced by HA1-44 peptide.

[0058] In this application, "MSC blood mobilization substance" means a substance that has the activity to mobilize mesenchymal stem cells (MSCs) into peripheral blood, or the activity to increase the amount of MSCs present in peripheral blood. Examples of MSC blood mobilization substances include, but are not limited to, HMGB1 protein, HMGB2 protein, and HMGB3 protein (for example, described in WO2008 / 053892 and WO2009 / 133939), S100A8 protein and S100A9 protein (for example, described in WO2009 / 133940 and WO2011 / 052668), and various HMGB1 peptides described in the inventors' international application WO2012 / 147470 (for example, a peptide consisting of amino acid residues 1-44 of HMGB1 protein (HA1-44 peptide in this application)).

[0059] In one embodiment, the present invention relates to a cell population obtained by administering an MSC blood mobilization substance to a subject, collecting peripheral blood from the subject, and culturing the collected peripheral blood on a solid phase. In one embodiment, the MSC blood mobilization substance may be HA1-44 peptide.

[0060] In other embodiments, the present invention relates to a cell population obtained by 1) administering HA1-44 peptide to a subject, 2) collecting vertebral bone marrow from the subject, and 3) culturing the collected bone marrow on a solid phase or sorting PDGFRα-positive cells from the collected bone marrow.

[0061] In another embodiment, the present invention relates to a method for producing the above-mentioned cells or cell populations.

[0062] In one embodiment, the present invention relates to a method for producing cells, comprising the steps of administering an MSC blood mobilization substance to a subject, collecting peripheral blood from the subject, and culturing the collected peripheral blood on a solid phase. In another embodiment, the present invention relates to a method for producing cells, comprising the step of culturing peripheral blood collected from a subject administered with an MSC blood mobilization substance on a solid phase. In one embodiment of these production methods, the MSC blood mobilization substance may be HA1-44 peptide.

[0063] In other embodiments, the present invention relates to a method for producing cells, comprising the steps of 1) administering HA1-44 peptide to a subject, 2) collecting vertebral bone marrow from the subject, and 3) culturing the collected bone marrow on a solid phase or sorting PDGFRα-positive cells from the collected bone marrow.

[0064] In yet another embodiment, the present invention relates to a method for obtaining, isolating, and / or concentrating cells from living tissue containing mesenchymal stem cells (MSCs) that have a high tissue regeneration-promoting capacity similar to that of PDGFRα-positive cells in vertebral bone marrow. In yet another embodiment, the present invention relates to cells or cell populations obtained by the above-described method of obtaining, isolating, and / or concentrating.

[0065] In one embodiment, the present invention relates to a method for producing a cell population, comprising the following steps: 1) A step of culturing a cell population derived from biological tissue, including mesenchymal stem cells (MSCs), on a solid phase; 2) A step of subcloning the colonies obtained in step 1; 3) A step of culturing a portion of the cells obtained by subcloning in a differentiation-inducing medium for bone, cartilage, and / or adipose tissue, and measuring the expression levels of differentiation markers for bone, cartilage, and / or adipose tissue; and 4) A step of selecting cell clones that show high expression levels when MSCs obtained by culturing femoral bone marrow on a solid phase are compared with the expression levels of bone, cartilage, and / or adipose differentiation markers when cultured in a differentiation induction medium for bone, cartilage, and / or adipose.

[0066] In other embodiments, the present invention relates to a method for producing a cell population, comprising the following steps: 1) A step of culturing a cell population derived from biological tissue, including MSCs, on a solid phase; and 2) Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1- A step of selecting colonies having one or more characteristics selected from. In one embodiment, step 2 may be a step of selecting Prx1 lineage-negative colonies.

[0067] In another embodiment, the present invention provides Pα from a cell population derived from biological tissue, including MSCs. + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - The present invention relates to a method for producing a cell population, comprising the step of selectively recovering cells having one or more characteristics selected from a given set.

[0068] In other embodiments, the present invention relates to a method for producing a cell population, comprising the following steps: 1) From a population of cells derived from living tissue, including MSCs, Pα + , Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- CD34 + , and Sca1 - A step of selectively harvesting cells having one or more characteristics selected from; and 2) A step in which the cells recovered in step 1) are cultured on a solid phase.

[0069] In the method for producing a cell population of the present invention, the biological tissue containing MSCs includes, but is not limited to, bone marrow, umbilical cord, umbilical cord blood, placenta, adipose tissue, dental pulp, periosteum, synovial membrane, amniotic membrane, peripheral blood, etc. The bone marrow includes, but is not limited to, bone marrow from the femur, vertebrae, sternum, ilium, skull, etc.

[0070] In yet another embodiment, the present invention relates to a composition for promoting tissue regeneration, comprising ectodermal mesenchymal stem cells. In one embodiment, the present invention relates to a composition for promoting tissue regeneration, comprising colony-forming PDGFR-positive cells having the following characteristics: i) and ii) and / or iii): i) Possesses the ability to differentiate into osteoblasts, adipocytes, and chondrocytes; ii) Possesses the ability to differentiate into epidermal cells; iii) Positive for the P0 lineage. In one embodiment, the composition is used to promote the regeneration of tissues derived from the mesoderm or ectoderm. Examples of tissues derived from the mesoderm include, but are not limited to, bone, cartilage, muscle, and vascular endothelium. Examples of tissues derived from the ectoderm include, but are not limited to, epithelial tissue (e.g., epidermis) and nerve tissue.

[0071] The composition used for promoting tissue regeneration in the present invention may contain pharmaceutically acceptable carriers, diluents, and / or excipients. The amount of ectodermal mesenchymal stem cells contained in the composition used for promoting tissue regeneration in the present invention, the dosage form of the composition, the frequency of administration, etc., can be appropriately selected according to conditions such as the type of tissue to be regenerated and / or the condition of the recipient.

[0072] In yet another embodiment, the present invention relates to a method for promoting tissue regeneration in a target, comprising administering ectodermal mesenchymal stem cells. In one embodiment, the present invention relates to a method for promoting tissue regeneration in a target, comprising administering colony-forming PDGFR-positive cells having the following characteristics: i) and ii) and / or iii): i) Possesses the ability to differentiate into osteoblasts, adipocytes, and chondrocytes; ii) Possesses the ability to differentiate into epidermal cells; iii) Positive for the P0 lineage. The above-described method of administering the cells can be appropriately selected according to conditions such as the type of tissue to promote regeneration and / or the state of the subject to be administered. Examples of such administration methods include intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, nasal administration, oral administration, suppositories, etc., but are not limited thereto.

[0073] In yet another aspect, the present invention relates to ectodermal mesenchymal stem cells for use in promoting tissue regeneration in a subject.

[0074] In yet another aspect, the present invention relates to the use of ectodermal mesenchymal stem cells for the manufacture of a medicament for promoting tissue regeneration in a subject.

[0075] iCFPα cells in peripheral blood are considered to be a useful biomarker for evaluating tissue regeneration activity mediated by EMSC in cases where necrotic tissue damage has occurred. Therefore, the present application provides a method for determining the expected tissue regeneration promoting effect in a subject administered with an MSC blood mobilizing substance using iCFPα cells in peripheral blood as an index.

[0076] In one embodiment, the present invention comprises the following steps: 1) Counting cells having one or more characteristics selected from Pα + , Pα lin+ , P0 lin+ , Prx1 lin- , Sox1 lin- , LepR lin- , CD34 + and Sca1 - contained in peripheral blood collected from a subject before administration of an MSC blood mobilizing substance; and 2) Pα + , Pα lin+ , P0 lin+ , Prx1 lin- , Sox1 lin- , LepR lin- , CD34 + and Sca1 -A step of counting cells having one or more characteristics selected from; The present invention relates to a method for determining the tissue regeneration-promoting effect of MSC blood mobilization substances, wherein if the number of cells counted in step 2) is greater than the number of cells counted in step 1), it is suggested that tissue regeneration is promoted in the subject.

[0077] In this application, the "subject" may be either a human or a non-human animal. In one embodiment, the subject is a non-human animal. Examples of non-human animals include, but are not limited to, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, and sheep.

[0078] With regard to the method for producing cells (or cell populations), the screening method, and the method for determining the tissue regeneration promoting effect of MSC blood mobilization substances provided in this application, the timing of collecting peripheral blood from the subject is not particularly limited. When artificially creating necrotic tissue injury or administering MSC blood mobilization substances, for example, peripheral blood can be collected 2 to 24 hours after the creation of necrotic tissue injury or administration of MSC blood mobilization substances. In one embodiment, the timing of collecting peripheral blood from the subject may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after the creation of necrotic tissue injury or administration of MSC blood mobilization substances. In another embodiment, the timing of collecting peripheral blood from the subject may be 4–24 hours, 8–24 hours, 8–16 hours, or 10–14 hours after the creation of necrotizing tissue injury or administration of MSC blood mobilization substance.

[0079] In one embodiment of the cells, method for producing cells (or cell populations), composition containing cells, and screening method provided in this application, PDGFR-positive cells are PDGFRα-positive cells.

[0080] All prior art documents cited herein are incorporated herein by reference. This application claims priority under U.S. Provisional Patent Application No. 62 / 593310, filed with the U.S. Patent and Trademark Office on December 1, 2017, the contents of which are incorporated herein by reference.

[0081] The present invention will be described in more detail below. While the present invention will be described in more detail below, it is not limited to the embodiments described below. [Examples]

[0082] material and method 1. Mouse We used Pα-H2B-GFP mice (The Jackson Laboratory, Stock No: 007669), in which PDGFRα expression can be confirmed by GFP fluorescence, and various cell lineage tracking mice utilizing the Cre-loxP system in our experiments. Cell lineage tracking mice utilizing the Cre-loxP system can be created by crossing Cre driver mice with Cre reporter mice. Cre driver mice are transgenic mice that have a DNA structure in which the coding sequence of Cre recombinase is introduced downstream of the promoter sequence of the desired gene. Cre reporter mice are transgenic mice in which a DNA sequence with the structure "promoter (CAG promoter, etc.)-loxP-stop cassette-loxP-desired reporter gene (EYFP or tdTomato in the examples of this application)" is introduced at a gene locus such as ROSA26.

[0083] In the embodiment of this invention, the following mouse was prepared as the Cre driver mouse. • Pα-Cre mouse (The Jackson Laboratory, Stock No: 013148) • P0-Cre mouse (The Jackson Laboratory, Stock No: 017927) • Prx1-Cre mouse (The Jackson Laboratory, Stock No: 005584) • Sox1-Cre mouse (RIKEN BioResource Research Center, Accession No. CDB0525K) • LepR-Cre mouse (The Jackson Laboratory, Stock No: 008320) • Krt5-Cre mouse (MGI ID: 1926815, K5 Cre transgenic mouse described in Proc Natl Acad Sci US A. 1997 Jul 8;94(14):7400-5)

[0084] The following mice were prepared as Cre reporter mice. • Rosa26-EYFP mouse (The Jackson Laboratory, Stock No: 006148) • Rosa26-tdTomato mouse (The Jackson Laboratory, Stock No: 007909)

[0085] By crossing the six driver mice mentioned above with Rosa26-tdTomato reporter mice, the following cell lineage tracing mice were created. Pα-Cre::Rosa26-tdTomato mouse • P0-Cre::Rosa26-tdTomato Mouse • Prx1-Cre::Rosa26-tdTomato Mouse • Sox1-Cre::Rosa26-tdTomato Mouse • LepR-Cre::Rosa26-tdTomato Mouse • Krt5-Cre::Rosa26-tdTomato Mouse

[0086] Furthermore, Pα-Cre::Rosa26-EYFP mice were created by crossing Pα-Cre driver mice with Rosa26-EYFP reporter mice. The Pα-H2B-GFP mouse is a mouse in which a sequence encoding a histone H2B-eGFP fusion protein is knocked in downstream of the promoter of the PDGFRα gene. By crossing this Pα-H2B-GFP mouse with the Prx1-Cre::Rosa26-tdTomato mouse, the Pα-H2B-GFP::Prx1-Cre::Rosa26-tdTomato mouse was created.

[0087] 2. Creating a Skin flap In this embodiment, a skin flap was created as a method to induce necrotic tissue damage. The specific method for creating the skin flap is as follows. Male mice (20-25g) 8-10 weeks old were shaved on the back under 1.5-2.0% (v / v) isoflurane inhalation anesthesia. Using scissors, a 2.0 x 4.0 cm skin flap was created in the center of the back, maintaining skin continuity only on the caudal side (this caused ischemia at the tip of the flap away from the base, resulting in necrotic damage to the skin tissue). The affected area was protected with a sufficiently large bandage after skin flap creation. Twelve hours after skin flap creation, cardiac blood was collected, cells were separated from the bone marrow of the vertebrae and femur, and frozen sections were prepared of the vertebrae and femur.

[0088] 3. Creation of parabiosis Based on Kamran P et al. J Vis Exp. 2013 Oct 6;(80), a parabiosis model was created using 6-week-old male wild-type mice and 6-week-old male cell lineage tracing mice. Both mice were simultaneously induced with isoflurane for general anesthesia and positioned on a heat pad. The opposing sides of the two mice were shaved, and a 5mm wide strip of skin was excised from the elbow joint to the knee joint. The dorsal skin was sutured with 5-0 Vicryl. The opposing elbow and knee joints were sutured with 3-0 Vicryl. Similarly, the ventral skin was sutured with 5-0 Vicryl. The mice were allowed to recover from anesthesia on the heat pad. Due to the limited activity, only one pair was kept per cage, and feeding was provided.

[0089] 4. Tissue staining and observation After harvesting the vertebrae and femurs, frozen blocks were prepared using Super Cryoembedding Medium (SCEM; Leica) after fixation with 4% paraformaldehyde, decalcification with 0.5M EDTA solution, and replacement with 30% sucrose solution. Sections were made to a thickness of 10 μm using Cryofilm type 2C(9) (Leica), and immunostaining was performed using various antibodies (primary antibody overnight at 4°C, secondary antibody for 1 hour at 4°C). Tissue observation was performed using a confocal microscope.

[0090] 5. Acquisition and culture of peripheral blood, vertebral, and femoral cells. Cell collection from peripheral blood was performed using the following method: Approximately 800-1000 μL of peripheral blood was collected from the heart under general anesthesia (using a 1 mL syringe containing heparin). To remove red blood cells, an equal volume of Hetasep (STEMCELL Technologies, Cat No. ST-07906) was added to the collected blood, the mixture was centrifuged at 100G for 2 minutes, incubated at room temperature for 15 minutes, and the supernatant was collected. This supernatant was used as a sample containing nucleated cells from peripheral blood for the following experiments. Cells were collected from the bone marrow using the following method: Vertebral bones and femurs, collected under general anesthesia, were immersed in 0.2% collagenase A solution (Sigma, Cat#10103578001) and incubated at 37°C for 1 hour. Bone marrow cells were extruded using a mortar and pestle, collected by pipetting, and passed through a 40 μm cell strainer. The cells were centrifuged at 300 G for 5 minutes, the supernatant was removed, and hemolysis with RBC Lysis buffer (BioLegend, cat#420301) was performed to remove red blood cells. These bone marrow cells were then used in the following experiments.

[0091] 6. Peripheral blood colony assay The supernatant (peripheral blood sample containing nucleated cells) obtained by the above procedure was seeded into a collagen I-coated 6-well plate (Corning, Cat No. 356400), and cultured for 10 days at 37°C, 5% CO2, and 5% O2 using an Expansion Medium prepared according to the manual of the MesenCult Expansion Kit (STEMCELL Technologies, Cat No. ST-05513) containing 1% L-glutamine (Nacalai Tesque), 10 μM ROCK inhibitor (Y27632, Tocris Bioscience), and 1% penicillin / streptomycin (Nacalai Tesque) (all values ​​are final concentrations). The culture medium was changed to fresh medium twice a week during the culture period. On day 10 of culture, cells on the plate were stained using the Differential Quik Stain Kit (Sysmex Corporation, Cat No. 16920), and the number of colonies containing 50 or more cells was counted.

[0092] 7. Differentiation into osteoblasts / adipocytes / chondrocytes Live cells from Passages 3-5 were seeded at 50,000 cells / well in 12-well plates and cultured in 10% FBS / DMEM until subconfluent. Subsequently, adipocyte differentiation was induced for 14 days using 10% FBS / DMEM containing 100 nM dexamethasone (Sigma), 0.5 mM isobutylmethylxanthine (Sigma), 50 mM indomethacin (Wako), and 10 μg / ml insulin (Sigma). Osteoblast differentiation was induced for 21 days using 10% FBS / DMEM containing 1 nM dexamethasone, 20 mM β-glycerol phosphate (Wako), and 50 μg / ml ascorbate-2-phosphate (Sigma-Aldrich Corp.). After each differentiation induction, cells were fixed with 4% PFA, adipocytes were stained with oil red-O, and osteoblasts were stained with ALP using the ALP activity assay kit (TAKARA BIO Inc., Kusatsu, Japan). Both cells were then observed under a microscope. For chondrogenic differentiation induction, 300,000 cells were first centrifuged in a 15 ml tube at 300 g for 5 minutes. Chondrogenic differentiation was induced for 21 days in 10% FBS / DMEM containing 40 ng / ml proline (Sigma), 50 μg / ml ascorbic acid 2-phosphate, x100 ITS mix (BD Bioscience), 2 μg / ml fluocinolone (Tokyo Chemistry Industry, Tokyo, Japan), 5 ng / ml transforming growth factor-b3 (R&D Systems, Minneapolis, MN), and 100 nM dexamethasone (Sigma). The completed cartilage pellet was embedded in paraffin, sectioned to a thickness of 6 μm, and stained with toluidine blue.

[0093] 8. Induction of differentiation into keratinocytes ·animal Krt5-Cre::Rosa26-tdTomato mice were reared until 8-10 weeks of age and used in the experiment. ·Collecting materials A skin flap was created on the back of each mouse the day before tissue collection. Peripheral blood was collected by cardiac aspiration under anesthesia 12 hours after the flap was created. Peripheral blood was collected similarly from mice that did not have a skin flap. After blood collection, the femur and vertebrae were collected. ·Cell adjustment Peripheral blood was deerythrocyte-removed using HetaSep, and the blood from one animal was seeded into one well of a 6-well collagen I-coated plate. The cells were cultured under conditions of 5% O2, 5% CO2, and 37°C. The culture medium used was either MesenCult or 20% FBS / MEMα (both containing Rock inhibitor and 1% Penicillin-Streptomycin). The collected femurs were cut in half lengthwise after both ends were severed, and the vertebrae were also cut in half lengthwise. They were then treated with 0.2% Collagenase A / DMEM (10 mM HEPES, 1% Penicillin-Streptomycin) (37°C water bath, 1 hour). After Collagenase A treatment, bone marrow cells were collected using a mortar and pestle, dispersed into single cells using a 40 μm cell strainer, hemolyzed with 1 × RBC Lysis solution, and then seeded. The cells were cultured under conditions of 5% O2, 5% CO2, and 37°C. The culture medium used was MesenCult or 20% FBS / MEMα (both containing Rock inhibitor and 1% Penicillin-Streptomycin). ·Induction of differentiation After confirming colony formation, culture medium containing 1 μM retinoic acid (SIGMA) and 25 ng / mL BMP4 (R&D) was added to the wells, and the cells were cultured at 5% CO2 and 37°C to induce differentiation into keratinocytes. Tomato-positive / negative cells were observed using an all-in-one microscope (KEYENCE).

[0094] 9. Transcriptome analysis (RNA-seq of cell populations) RNA was extracted from a cell population, and an RNA-seq library was prepared according to the Smart-seq2 protocol (Nature Protocols 9, 171-181 (2014) doi:10.1038 / nprot.2014.006). The resulting library was sequenced using a Nextseq 500 (Illumina) with a Nextseq high output kit (37bp paired-end reads). Base calls were converted to fastq format and demultiplexed using Illumina's bcl2fastq v2.17.1.14 with default parameters and the --no-lane-splitting option. Reads were trimmed using TrimGalore (http: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ), and mapping and counting (quantification) were performed using RSEM (Li et al., BMC Bioinformatics 12, 323 (2011); version STAR-2.5.2b). Differential expression analysis between samples was performed using DESeq2 (Love et al., Genome Biol. 15, 550 (2014)). Differentially expressed genes (DEGs) were uploaded to Ingenuity Pathway Analysis (IPA) software (https: / / www.qiagenbioinformatics.com) to extract the biological pathways and functions most closely associated with the DEGs. Clustering analysis was performed using the ICGS algorithm of AltAnalyze_v.2.1.0-Py, based on the value obtained by adding 1 to the TPM (Transcripts Per kilobase Million) count obtained from RSEM and performing a log2 transformation (log2(TPM+1)).

[0095] 10. Transcriptome analysis (single-cell RNA-seq) Single-cell suspensions were prepared, and cell viability was assessed using an automated cell counter TC20 (BioRad). Single-cell RNA-seq libraries were prepared using the ddSEQ Single-Cell Isolator and SureCell WTA 3' Library prep kit according to the manufacturer's protocol. The resulting libraries were sequenced using a Nextseq 500 (Illumina) with a Nextseq high output kit (Read1: 68 bp, Read2: 75 bp). Base calls were converted to fastq format and demultiplexed using Illumina's bcl2fastq v2.17.1.14 with default parameters and the --no-lane-splitting option. Potential synthetic and sequencing errors in the cell barcode region were corrected with an Edit distance (ED) < 2. Reads were analyzed (mapped and quantified) using Drop-Seq Tools v1.12 (STAR ​​version STAR-2.5.2b) to generate a digital expression matrix. The resulting matrix was standardized using voom (limma 3.32.10). Based on this standardized matrix, clustering analysis was performed using the ICGS algorithm with AltAnalyze_v.2.1.0-Py with default settings. Furthermore, dimensionality reduction using the tSNE algorithm (using Rtsne) and clustering analysis using hclust (method=ward.D2) were performed based on this standardized matrix, and the results were plotted using ggplot2 or plot.ly. DESingle was used for differential expression analysis.

[0096] 11.FACS analysis FACS analysis was performed on bone marrow cells collected from vertebrae and femurs using fluorescently labeled antibodies against various surface molecules. The entire process, including fluorescence detection and sorting, was carried out using the BD FACS Aria III system, and the resulting data was analyzed using FlowJo software Ver. 6.3.3 (Tree Star, Ashland, OR).

[0097] 12. Parabiosis and cartilage defect models Parabiosis models were created using 6-week-old male wild-type mice and 6-week-old male P0-Cre::Rosa26-tdTomato mice using the method described in section 3 above. After blood chimeras were completed 4 weeks post-surgery, a 0.5 x 0.5 x 0.5 mm cartilage defect was created in the knee joint of wild-type mice using a 0.5 mm diameter hand-cranked drill (MEISINGER). Immediately after creating the cartilage defect, 100 μg of HA1-44 peptide diluted in 100 μL of physiological saline was administered via tail vein, and the same dose was administered twice a week until 4 weeks post-surgery. The control group was administered 100 μL of physiological saline per mouse via tail vein according to the same schedule as the HA1-44 peptide administration group. Twelve weeks after the creation of a knee cartilage defect, the knee joint was harvested and frozen into a block after fixation with 4% paraformaldehyde (overnight), decalcification with 0.5M EDTA solution (3 days), and substitution with 30% sucrose (1 day). Sections were made to a thickness of 10 μm using a cryostat, and the distribution of tomato-positive cells was analyzed using a confocal microscope. In addition, cartilage defects were created in the knee joint of 8-week-old male wild-type mice in the same manner as above, and HA1-44 peptide was administered at the same dose and schedule as above. Knee joints were harvested 2, 4, 8, and 12 weeks after the creation of the knee cartilage defect, and tissue sections were stained with safranin O.

[0098] Abbreviation Regarding the marker (XX, YY are the names of the desired genes / proteins) XX + : XX positive XX - : XX negative YYlin+ : YY lineage positive YY lin- : YY lineage negative PDGFR: platelet-derived growth factor receptor Pα: platelet-derived growth factor receptor alpha (PDGFRα) Pα cells: PDGFRα positive cells MSC: Mesenchymal stem cells EMSC: ectomesenchymal stem cell CFPα cells: colony-forming Pα cells iCFPα cells: Necrotic injury-induced colony-forming Pα cells. CFU: Colony-forming unit LepR: Leptin receptor [Examples]

[0099] Example 1 Properties of iCFPα cells in peripheral blood Skin flaps were created on the backs of Pα-H2B-GFP mice, and the number of Pα cells in peripheral blood collected 12 hours later was examined. The results showed a significant increase in Pα cells in the skin flap group compared to the control group without skin flaps, and this increase in Pα cells correlated with an increase in HMGB1 concentration in peripheral blood (Figure 1). Furthermore, colony assays performed on peripheral blood cultures of Pα-H2B-GFP mice yielded significantly more colonies (all Pα-positive cells) in the skin flap group than in the control group, indicating higher CFU activity (Figure 2). These results suggest that necrotic tissue injury increases the number of colony-forming Pα cells in peripheral blood. Furthermore, colony-forming Pα cells (i.e., iCFPα cells) obtained by culturing peripheral blood from mice that had produced skin flaps showed differentiation potential into osteoblasts, adipocytes, and chondrocytes, and also demonstrated the ability to differentiate into cells expressing keratin 5 (Krt5, K5) under conditions that induced differentiation into keratinocytes (Figure 3).

[0100] Single-cell transcriptome analysis of peripheral blood-derived CFPα cells (iCFPα cells) after skin flap creation revealed that most cells showed high expression of gene groups corresponding to cell types such as MSCs (Figure 4). Cells expressing genes characteristic of epidermal cells, such as Krt8 and Krt18, were also included. Furthermore, transcriptome analysis of peripheral blood-derived CFPα cells from the control group and the skin flap group was performed at the colony level, and clustering analysis using the ICGS algorithm showed that clusters predominantly composed of CFPα cells after skin flap creation exhibited high expression of gene groups characteristic of cell types such as bone marrow stem cells and MSCs, with particularly high expression of the HoxA2 gene (Figure 5). [Examples]

[0101] Example 2 Surface markers of iCFPα cells in peripheral blood In both the control group and the skin flap group, all colonies obtained by culturing peripheral blood were Pα-positive (Figure 6). Furthermore, single-cell transcriptome analysis of peripheral blood CFPα cells (iCFPα cells) from the skin flap group revealed that they were CD34-positive and Sca1-negative. [Examples]

[0102] Example 3 Cell lineage markers of iCFPα cells in peripheral blood When peripheral blood-derived CFPα cells (iCFPα cells) from skin flap-generating mice were traced for the Pα, P0, Prx1, Sox1, and LepR lineages using transgenic mice with the Cre-loxP lineage, all iCFPα cells were positive for the Pα lineage, positive for the P0 lineage, negative for the Sox1 lineage, and negative for the LepR lineage (Figure 7). For the Prx1 lineage, 93% of iCFPα cells were negative (Figure 7). Furthermore, while small amounts of Prx1 lineage-positive and negative colony-forming cells are present in normal blood, the increase in skin flap was exclusively due to Prx1 lineage-negative cells (Figure 8). Therefore, iCFPα cells can be defined as Prx1 lineage-negative. iCFPα cells in peripheral blood are P0 lin+ And Prx1 lin- Therefore, it is thought to be of ectoderm origin, and furthermore, Sox1 lin- Considering this characteristic and the high expression of the HoxA2 gene, it is suggested that these cells originate from the cranial nerve folds. [Examples]

[0103] Example 4 Search for ectoderm-derived mesenchymal cells When bone marrow tissue from the femur, vertebrae, sternum, ilium, hip joint (femoral head and acetabulum), and skull of Pα-H2B-GFP::Prx1-Cre::Rosa26-tdTomato mice was examined, Pα + And Prx1 lin- These cells were found specifically in the vertebrae (within the scope of the study) (Figures 9 and 10). [Examples]

[0104] Example 5 Lineage markers of CFPα cells in the vertebrae and femur CFPα cells obtained by culturing bone marrow from vertebrae and femurs were examined for P0, Prx1, Sox1, and LepR lineages using cell lineage tracing mice. The results showed that CFPα cells from vertebrae were P0 lineage. lin+,Prx1 lin- , and Sox1 lin- So, LepR lin Regarding this, approximately 60% were negative (Figure 11). CFPα cells in the femur are P0 lin+ ,Prx1 lin+ , and Sox1 lin- So, LepR lin Regarding this, approximately 80% were positive (Figure 12). From these results, it was concluded that peripheral blood iCFPα cells (Pα lin+ , P0 lin+ ,Prx1 lin- Sox1 lin- LepR lin- This suggests that the source of ) is present in the vertebrae. [Examples]

[0105] Example 6 Properties of CFPα cells in the vertebrae and femur When CFPα cells obtained by culturing bone marrow from the vertebrae and femur were compared in terms of colony formation ability and differentiation ability into osteoblasts, adipocytes, and chondrocytes, vertebral CFPα cells showed higher ability in all aspects (Figures 13-16). Furthermore, when CFPα cells from the vertebrae and femur were differentiated using all-trans retinoic acid (ATRA) and BMP-4, colonies expressing keratin 5 were observed (Figures 17 and 18), confirming that CFPα cells from the vertebrae and femur contain cells capable of differentiating into K5-positive cells. [Examples]

[0106] Example 7 Transcriptome analysis of Pα cells in the vertebrae and femur Pα cells were sorted from bone marrow cells of the vertebrae and femurs, and single-cell transcriptome analysis was performed. Clustering analysis revealed that bone marrow Pα cells were divided into six clusters (Figure 19). These six clusters were defined based on the genes specifically expressed by the cells in each cluster: (1) S34-MSC (Sca1 and CD34-expressing), (2) osteoprogenitor (Osteomodulin and Wnt16-expressing), (3) osteoblast (osterix and osteocalcin-expressing), (4) osteoocyte (PHEX and DMP1-expressing), (5) CAR cell (CXCL12 and LepR-expressing), and (6) CD45-expressing cell. Comparing the vertebrae and femurs, it can be seen that the vertebrae had a large number of S34-MSC cluster cells and a small number of CAR cluster cells, while the femurs had a large number of CAR cluster cells and a small number of S34-MSC cluster cells. In addition, within the S34-MSC cluster, there is Sca1 + CD34 + , Sca1 + CD34 - , and Sca1 - CD34 + The cells included [this]. Therefore, Pα cells from the vertebra were isolated by FACS using Sca1 and CD34 expression as indicators, and a CFU assay was performed. As a result, the CFU activity was [this] + CD34 + Cell>Sca1 + CD34 - Cells and Sca1 - CD34 + Cell>Sca1 - CD34 - The cells were the most abundant (Figure 20). [Examples]

[0107] Example 8 Correspondence between Pα cells in peripheral blood and Pα cells in the vertebrae Based on transcriptome analysis data of iCFPα cells in peripheral blood, clustering analysis was performed together with Pα cells from the vertebrae and femur. As a result, the peripheral blood iCFPα cells were located near the S34-MSC cluster (Figure 21). Furthermore, these peripheral blood iCFPα cells were found to be CD34 + Sca1 - This result, when considered in conjunction with lineage markers, suggests that peripheral blood iCFPα cells are CD34 cells included in the S34-MSC cluster of the vertebra. + Sca1 - This suggests that it corresponds to a cell. [Examples]

[0108] Example 9 Intramedullary Sca1 + CD34 + cell Sca1 contained in the S34-MSC cluster of bone marrow + CD34 + The cells specifically expressed Procr (Figure 22). Therefore, Procr is thought to be the cell with the highest proliferative capacity and the highest hierarchy among bone marrow Pα cells, Sca1 + CD34 + It can serve as a cellular marker. Also, Sca1 in the cervical, thoracic, lumbar, and femoral vertebrae. + CD34 + When the abundance of cells was examined, it was found to be highest in the cervical vertebrae, followed by the thoracic vertebrae, then the lumbar vertebrae, and finally the femur (Figure 23). [Examples]

[0109] Example 10 Contribution of HMGB1 administration to tissue regeneration of circulating cells in the blood. The inventors have previously identified a peptide (HA1-44 peptide) consisting of the 1st to 44th amino acid sequence (SEQ ID NO: 1) at the N-terminus of the HMGB1 protein as a domain with activity to recruit bone marrow-derived Pα-positive mesenchymal stem cells into peripheral blood. This time, the following experimental results were obtained regarding cells in peripheral blood induced by the administration of the HA1-44 peptide. (1) When peripheral blood of lineage-tracking mice administered with HA1-44 peptide was cultured, more colonies were obtained than in the peripheral blood of the control group (administered with physiological saline), and all of these colonies were Pα-positive, with the majority being Prx1 lineage-negative (Figure 24). (2) Parabiosis models were created using Pα-Cre::Rosa26-EYFP mice and wild-type (WT) mice. After transplanting skin from epidermal vesicle mice into wild-type mice, HA1-44 peptide was administered to the Pα-Cre::Rosa26-EYFP mice. As a result, Pα expressing type VII collagen was found in the regenerated epithelial tissue within the skin grafts. lin+ The presence of these cells was confirmed (Figure 25). (3) A parabiosis model was created using Pα-H2B-GFP::Prx1-Cre::Rosa26-tdTomato mice and wild-type mice. After transplanting skin from newborn wild-type mice onto the backs of wild-type mice, HA1-44 peptide was administered to the Pα-H2B-GFP::Prx1-Cre::Rosa26-tdTomato mice. As a result, Pα was found in the skin grafts. + And Prx1 lin- The presence of these cells was confirmed (Figure 26). (4) Parabiosis models were created using P0-Cre::Rosa26-tdTomato mice and wild-type mice. After inducing cartilage damage in the knee joint of wild-type mice, HA1-44 peptide was administered to the P0-Cre::Rosa26-tdTomato mice. As a result, in the HA1-44 peptide administration group, P0 lin+ Cell accumulation was observed in the control group (administered with physiological saline), while P0 lin+ No cell accumulation was observed (Figure 27). Furthermore, after inducing cartilage damage in the knee joint of a single wild-type mouse (not a parabiosis model), administration of either HA1-44 peptide or saline solution resulted in the regeneration of hyaline cartilage at the cartilage damage site in the HA1-44 peptide administration group, whereas only fibrocartilage was observed at the cartilage damage site in the saline solution administration group (Figure 28). Based on the above results, Pα cells in peripheral blood induced by HA1-44 peptide (Pα + P0 lin+Prx1 lin- These cells are thought to be the same as, or at least contain, iCFPα cells induced by necrotic tissue injury, and to play a role in repairing tissue damage such as epidermis and cartilage. [Examples]

[0110] Example 11 Cellular changes induced by HMGB1 administration (1) Peripheral blood was collected from mice administered with HA1-44 peptide and mice administered with physiological saline, and cultured on plastic plates to obtain colonies of adherent cells. Transcriptome analysis was performed on the colony level of these cells, and clustering was performed using the ICGS algorithm based on the obtained data, resulting in the results shown in Figure 29. A simplified representation of the clustering results is shown in Figure 30. In the screening method of this application, substances that produce results similar to those of HA1-44 peptide, for example, clusters characterized by predicted cell types (and corresponding gene set expression) similar to those in Figure 30, and in which the number of colonies belonging to each cluster is "Cluster 1: Saline group ≈ Test substance group, Cluster 2: Saline group < Test substance group, Cluster 3: Saline group > Test substance group, Cluster 4: Saline group > Test substance group", can be evaluated as candidates for substances with pluripotent stem cell inducing activity. (2) Transcriptome analysis was performed on Pα cells from the vertebrae of mice administered with HA1-44 peptide and mice administered with physiological saline, and pathway analysis was performed using IPA based on the obtained data. As a result, pathways related to the regulation of EIF2 signaling, eIF4 and p70S6K signaling, and mTOR signaling were activated in vertebral Pα cells of the HA1-44 peptide-administered group compared with the control group (Figure 31). In addition, the expression of cell death-related genes was suppressed in vertebral Pα cells of the HA1-44 peptide-administered group compared with the control group (Figure 32). [Examples]

[0111] Example 12 Activity of HMGB1 peptide in the human body In a Phase I clinical trial, intravenous administration of HA1-44 peptide was shown to increase CD45-negative, TER-119-negative, and PDGFRβ-positive cells in the circulating blood (Figure 33). Since PDGFRβ is a marker for human mesenchymal stem cells, it is considered that replacing "PDGFRα" with "PDGFRβ" in the markers (including combinations of multiple markers) that define iCFPα cells in peripheral blood and vertebral CFPα cells described herein will also result in markers (including combinations of multiple markers) that define EMSCs (colony-forming PDGFR-positive cells in peripheral blood or vertebral cells) in humans. [Industrial applicability]

[0112] The ectodermal mesenchymal stem cells in peripheral blood according to the present invention have superior proliferative and pluripotent capacities compared to bone marrow-derived mesenchymal stem cells conventionally used in regenerative medicine. Furthermore, they can be obtained through peripheral blood collection, a less invasive method than bone marrow aspiration, making them suitable for use in cell transplantation therapy and the like. In addition, by clarifying the characteristics (markers, etc.) of ectodermal mesenchymal stem cells in peripheral blood that contribute to the regeneration of damaged tissue, it becomes possible to efficiently screen for substances that have the activity to induce pluripotent stem cells in the body using these cells as indicators.

Claims

1. Colony-forming PDGFR-positive cells having the following characteristics: i) and ii) and / or iii): i) Having the ability to differentiate into osteoblasts, adipocytes, and chondrocytes; ii) Possesses the ability to differentiate into epidermal cells; iii) P0 lineage positive.

2. The cells according to claim 1, which are PDGFRα positive.

3. The cell according to claim 1 or 2, having one or more characteristics selected from PDGFRα-positive, PDGFRα lineage-positive, P0 lineage-positive, Prx1 lineage-negative, Sox1 lineage-negative, LepR lineage-negative, CD34-positive, and Sca1-negative.

4. Vertebral bone marrow-derived cells that are PDGFRα-positive, CD34-positive, and Sca1-negative.

5. A method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) A step of collecting peripheral blood from a subject with necrotic tissue damage and culturing it on a solid phase; 2) A step of collecting peripheral blood from subjects with necrotic tissue damage, culturing it on a solid phase, and then selectively recovering cells that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative; 3) A step of collecting peripheral blood from a subject with necrotic tissue damage, and selectively recovering cells from the peripheral blood that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative; 4) A step of collecting peripheral blood from a subject with necrotic tissue damage, selectively recovering cells from the peripheral blood that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative, and culturing the cells on a solid phase.

6. A method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) A step of culturing peripheral blood collected from a subject with necrotic tissue damage on a solid phase; 2) A step of culturing peripheral blood collected from a subject with necrotic tissue damage on a solid phase, and then selectively recovering cells having one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative; 3) A step of selectively recovering cells from peripheral blood collected from subjects with necrotic tissue damage that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative; 4) A step of selectively recovering cells from peripheral blood collected from subjects with necrotic tissue damage that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative, and culturing said cells on a solid phase.

7. A method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) The process of collecting vertebral bone marrow from the subject and culturing it on a solid phase; 2) A step of collecting vertebral bone marrow from the subject, culturing it on a solid phase, and then selectively recovering cells that have one or more characteristics selected from PDGFRα positivity, P0 lineage positivity, Prx1 lineage negative, and Sox1 lineage negative; 3) A step of collecting vertebral bone marrow from the subject and selectively recovering cells from the bone marrow that have one or more characteristics selected from PDGFRα positivity, P0 lineage positivity, Prx1 lineage negativity, and Sox1 lineage negativity; 4) A step of collecting vertebral bone marrow from the subject, selectively recovering cells from the bone marrow that have one or more characteristics selected from PDGFRα positivity, P0 lineage positivity, Prx1 lineage negative, and Sox1 lineage negative, and culturing the cells on a solid phase.

8. A method for producing colony-forming PDGFR-positive cells, comprising any of the following steps 1) to 4): 1) A step of culturing vertebral bone marrow collected from the subject on a solid phase; 2) A step of culturing vertebral bone marrow collected from the subject on a solid phase, and then selectively recovering cells having one or more characteristics selected from PDGFRα positivity, P0 lineage positivity, Prx1 lineage negativity, and Sox1 lineage negativity; 3) A step of selectively recovering cells from vertebral bone marrow collected from the subject that have one or more characteristics selected from PDGFRα positivity, P0 lineage positivity, Prx1 lineage negativity, and Sox1 lineage negativity; 4) A step of selectively recovering cells from vertebral bone marrow collected from the subject that have one or more characteristics selected from PDGFRα positivity, P0 lineage positivity, Prx1 lineage negativity, and Sox1 lineage negativity, and culturing said cells on a solid phase.

9. A cell population obtained by administering a substance mobilized from MSC blood to a target, collecting peripheral blood from the target, and culturing the collected peripheral blood on a solid phase.

10. The cell population according to claim 9, wherein the MSC blood mobilization substance is a peptide consisting of the amino acid sequence of SEQ ID NO:

1.

11. A method for producing cells, comprising the steps of administering a substance mobilized from MSC blood to a target, collecting peripheral blood from the target, and culturing the collected peripheral blood on a solid phase.

12. A method for producing cells, comprising the step of culturing peripheral blood collected from a subject administered with an MSC blood mobilization substance on a solid phase.

13. The method according to claim 11 or 12, wherein the MSC blood mobilization substance is a peptide consisting of the amino acid sequence of SEQ ID NO:

1.

14. 1) A cell population obtained by administering a peptide consisting of the amino acid sequence of Sequence ID No. 1 to a target, 2) collecting bone marrow from the vertebrae of the target, and 3) culturing the collected bone marrow on a solid phase or sorting PDGFRα-positive cells from the collected bone marrow.

15. A method for producing cells, comprising the steps of: 1) administering a peptide consisting of the amino acid sequence of Sequence ID No. 1 to a target; 2) collecting bone marrow from the vertebrae of the target; and 3) culturing the collected bone marrow on a solid phase or sorting PDGFRα-positive cells from the collected bone marrow.

16. A method for producing cells, comprising the steps of culturing bone marrow from a vertebra collected from a subject administered with a peptide consisting of the amino acid sequence of SEQ ID NO: 1 on a solid phase, or sorting PDGFRα-positive cells from the collected bone marrow.

17. A method for producing a cell population, including the following steps: 1) A step of culturing a cell population derived from biological tissue, including mesenchymal stem cells, on a solid phase; 2) A step of subcloning the colonies obtained in step 1); 3) A step of culturing a portion of the cells obtained by subcloning in a differentiation-inducing medium for bone, cartilage, and / or adipose tissue, and measuring the expression levels of differentiation markers for bone, cartilage, and / or adipose tissue; and 4) A step of selecting cell clones that show high expression levels when mesenchymal stem cells obtained by culturing bone marrow from the femur on a solid phase are cultured in differentiation induction medium for bone, cartilage, and / or adipose tissue, compared with the expression levels of differentiation markers for bone, cartilage, and / or adipose tissue.

18. A method for producing a cell population, including the following steps: 1) A step of culturing a cell population derived from biological tissue, including mesenchymal stem cells, on a solid phase; and 2) A step of selecting colonies that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative.

19. The method according to claim 18, wherein step 2) is a step of selecting colonies that are negative for the Prx1 lineage.

20. A method for producing a cell population, comprising the step of selectively recovering cells from a cell population derived from living tissue, including mesenchymal stem cells, that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative.

21. A method for producing a cell population, including the following steps: 1) A step of selectively recovering cells from a cell population derived from living tissue, including mesenchymal stem cells, that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative; and 2) A step in which the cells recovered in step 1) are cultured on a solid phase.

22. Cells or cell populations obtained by the manufacturing method described in any one of claims 5 to 8, 11 to 13, and 15 to 21.

23. A composition used to promote tissue regeneration, containing colony-forming PDGFR-positive cells having the following characteristics: i) and ii) and / or iii): i) Having the ability to differentiate into osteoblasts, adipocytes, and chondrocytes; ii) Possesses the ability to differentiate into epidermal cells; iii) P0 lineage positive.

24. The composition according to claim 23, which is used to promote the regeneration of tissue derived from mesoderm or ectoderm.

25. A method for screening pluripotent stem cell inducers, including the following steps: 1) A step of collecting peripheral blood from the subject and counting the number of cells in the peripheral blood that have one or more characteristics selected from PDGFRα-positive, PDGFRα lineage-positive, P0 lineage-positive, Prx1 lineage-negative, Sox1 lineage-negative, LepR lineage-negative, CD34-positive, and Sca1-negative; 2) A step of collecting peripheral blood from subjects who have been administered the test substance, and counting the number of cells in the peripheral blood that have one or more characteristics selected from PDGFRα-positive, PDGFRα lineage-positive, P0 lineage-positive, Prx1 lineage-negative, Sox1 lineage-negative, LepR lineage-negative, CD34-positive, and Sca1-negative; and 3) If the number of cells counted in step 2) is greater than the number of cells counted in step 1), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

26. A method for screening pluripotent stem cell inducers, including the following steps: 1) A step of counting cells in peripheral blood collected from a subject that have one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative; 2) A step of counting cells in peripheral blood collected from subjects administered the test substance that have one or more characteristics selected from PDGFRα-positive, PDGFRα lineage-positive, P0 lineage-positive, Prx1 lineage-negative, Sox1 lineage-negative, LepR lineage-negative, CD34-positive, and Sca1-negative; and 3) If the number of cells counted in step 2) is greater than the number of cells counted in step 1), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

27. A method for screening pluripotent stem cell inducers, including the following steps: 1) A step of obtaining an adherent cell population by collecting peripheral blood from a subject and culturing it on a solid phase; 2) A step of performing comprehensive gene expression analysis on the cell population obtained in step 1) at the colony or single-cell level; 3) A step of administering a peptide consisting of the amino acid sequence of Sequence ID No. 1 to a target, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 4) A step of performing comprehensive gene expression analysis on the cell population obtained in step 3) at the colony or single-cell level; 5) A step of administering the test substance to a subject, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 6) A step of performing comprehensive gene expression analysis on the cell population obtained in step 5) at the colony or single cell level; 7) A step of pooling the gene expression data obtained in steps 2) and 4) and performing clustering analysis; 8) A step of pooling the gene expression data obtained in steps 2) and 6) and performing clustering analysis; and 9) A step in which the analysis results from step 7) and step 8) are compared, and if the cell population obtained in step 5) has the same cluster structure as the cell population obtained in step 3), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

28. A method for screening pluripotent stem cell inducers, including the following steps: 1) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject on a solid phase; 2) A step of performing comprehensive gene expression analysis on the cell population obtained in step 1) at the colony or single-cell level; 3) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject that has been administered a peptide consisting of the amino acid sequence of SEQ ID NO: 1 on a solid phase; 4) A step of performing comprehensive gene expression analysis on the cell population obtained in step 3) at the colony or single-cell level; 5) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject administered with the test substance on a solid phase; 6) A step of performing comprehensive gene expression analysis on the cell population obtained in step 5) at the colony or single cell level; 7) A step of pooling the gene expression data obtained in steps 2) and 4) and performing clustering analysis; 8) A step of pooling the gene expression data obtained in steps 2) and 6) and performing clustering analysis; and 9) A step in which the analysis results from step 7) and step 8) are compared, and if the cell population obtained in step 5) has the same cluster structure as the cell population obtained in step 3), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

29. A method for screening pluripotent stem cell inducers, including the following steps: 1) A step of obtaining an adherent cell population by collecting peripheral blood from a subject and culturing it on a solid phase; 2) A step of counting the number of colonies obtained in step 1); 3) A step of administering the test substance to a subject, collecting peripheral blood, and culturing it on a solid phase to obtain an adherent cell population; 4) A step of counting the number of colonies obtained in step 3); and 5) If the number of colonies counted in step 4) is greater than the number of colonies counted in step 2), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

30. A method for screening pluripotent stem cell inducers, including the following steps: 1) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject on a solid phase; 2) A step of counting the number of colonies obtained in step 1); 3) A step of obtaining an adherent cell population by culturing peripheral blood collected from a subject administered with the test substance on a solid phase; 4) A step of counting the number of colonies obtained in step 3); and 5) If the number of colonies counted in step 4) is greater than the number of colonies counted in step 2), the test substance is selected as a candidate substance having pluripotent stem cell inducing activity.

31. The screening method according to claim 29 or 30, wherein the colonies counted in steps 2) and 4) are colonies having one or more characteristics selected from PDGFRα positivity, PDGFRα lineage positivity, P0 lineage positivity, Prx1 lineage negative, Sox1 lineage negative, LepR lineage negative, CD34 positivity, and Sca1 negative.

32. A method for screening pluripotent stem cell inducers, including the following steps: 1) A step of collecting bone marrow from the target vertebra and obtaining a population of PDGFRα-positive cells by culturing on a solid phase or cell sorting; 2) A step of performing comprehensive gene expression analysis on the cell population obtained in step 1) at the colony or single-cell level; 3) A step of administering the test substance to a subject, collecting bone marrow from the vertebrae, and obtaining a population of PDGFRα-positive cells by culturing on a solid phase or cell sorting; 4) A step of performing comprehensive gene expression analysis on the cell population obtained in step 3) at the colony or single-cell level; 5) A step of pooling the gene expression data obtained in steps 2) and 4) and performing pathway analysis; and 6) As a result of the analysis in step 5), if the cell population obtained in step 3) shows that (i) pathways related to the regulation of EIF2 signaling, eIF4 and p70S6K signaling, and / or mTOR signaling are activated, or (ii) the expression of cell death-related genes is suppressed, then the test substance is selected as a candidate substance having pluripotent stem cell-inducing activity.

33. A method for screening pluripotent stem cell inducers, including the following steps: 1) A step of obtaining a population of PDGFRα-positive cells from bone marrow collected from the target vertebra by solid-phase culture or cell sorting; 2) A step of performing comprehensive gene expression analysis on the cell population obtained in step 1) at the colony or single-cell level; 3) A step of obtaining a population of PDGFRα-positive cells from bone marrow collected from the vertebrae of subjects administered the test substance, by culturing on a solid phase or cell sorting; 4) A step of performing comprehensive gene expression analysis on the cell population obtained in step 3) at the colony or single-cell level; 5) A step of pooling the gene expression data obtained in steps 2) and 4) and performing pathway analysis; and 6) As a result of the analysis in step 5), if the cell population obtained in step 3) shows that (i) pathways related to the regulation of EIF2 signaling, eIF4 and p70S6K signaling, and / or mTOR signaling are activated, or (ii) the expression of cell death-related genes is suppressed, then the test substance is selected as a candidate substance having pluripotent stem cell-inducing activity.

34. The following steps: 1) A step of counting cells having one or more characteristics selected from PDGFRα-positive, PDGFRα lineage-positive, P0 lineage-positive, Prx1 lineage-negative, Sox1 lineage-negative, LepR lineage-negative, CD34-positive, and Sca1-negative, contained in peripheral blood collected from subjects before administration of MSC blood mobilization substance; and 2) A step of counting cells having one or more characteristics selected from PDGFRα-positive, PDGFRα lineage-positive, P0 lineage-positive, Prx1 lineage-negative, Sox1 lineage-negative, LepR lineage-negative, CD34-positive, and Sca1-negative, contained in peripheral blood collected from a subject after administration of an MSC blood mobilization substance. A method for determining the tissue regeneration-promoting effect of MSC blood mobilization substances, wherein if the number of cells counted in step 2) is greater than the number of cells counted in step 1), it is suggested that tissue regeneration is promoted in the subject.

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