Method for identifying dermal fibroblast of aging subpopulation

Abstract

The purpose of the present invention is to identify a dermal fibroblast aging subpopulation. The present invention has reclassified dermal fibroblasts using a single-cell analysis technique, and upon doing so, has discovered a novel subpopulation that has not previously been classified, on the basis of differences in the gene expression of dermal fibroblasts which were isolated from tissue. This subpopulation is contained in a large amount in the skin of an elderly subject, and is also contained in the skin of a young subject. The subpopulation is identified as an aging subpopulation associated with the aging of the skin.

Description

Method for determining dermal fibroblasts in an aging subpopulation

[0001] Relates to a method for determining subpopulations of dermal fibroblasts and a method for isolating dermal fibroblasts of a determined specific subpopulation.

[0002] Skin is histologically classified into the epidermis, dermis, and subcutaneous tissue. Each tissue contains various cells, and the epidermis is mainly composed of keratinocytes. Keratinocytes with proliferative activity present in the basal layer stop proliferating and differentiate outward into the spinous layer, granular layer, and stratum corneum to form the epidermis, which serves as the boundary with the external environment. Thus, even one cell within a single tissue forms subpopulations with different differentiation states. Compared to a cell population that proliferates only in the basal layer and does not proliferate after differentiation, such as keratinocytes, dermal cells repeat differentiation and proliferation, making it difficult to identify subpopulations, and research focusing on cells belonging to specific subpopulations has lagged behind that of keratinocytes. On the other hand, with the establishment of recent single-cell focused analysis techniques, dermal fibroblasts have been analyzed by dividing them into subpopulations. As an example, they are classified into a subpopulation that constitutes the papillary superficial layer and a subpopulation of the reticular layer that extends to the subcutaneous tissue, and differences in morphology, proliferative ability, culture matrix production ability, growth factor production ability, and response ability to growth factors have been reported (Patent Document 1: JP-A-2024-3103). Conventional subpopulations of dermal fibroblasts were mainly classified based on their localization.

[0003] JP-A-2024-3103

[0004] Han et al., Nature, 2020

[0005] Analysis of subpopulations in dermal fibroblasts has been carried out based on their morphological characteristics and distribution. On the other hand, the present inventors have found that dermal fibroblasts at the same location also have different properties, and have found the need for a more detailed classification of dermal fibroblasts.

[0006] The inventors reclassified dermal fibroblasts using a single-cell analysis method and discovered a new subpopulation that had not been previously classified, based on differences in gene expression in dermal fibroblasts isolated from tissue. This subpopulation was found to be abundant in the skin of elderly subjects, but also in the skin of young subjects, and was identified as an aging subpopulation involved in skin aging, leading to the present invention. The present invention relates to the following: [1] A method for determining dermal fibroblasts of an aging subpopulation, comprising the steps of: determining the gene profile of selected dermal fibroblasts; and determining dermal fibroblasts with high expression of aging subpopulation-related genes by comparing them with the average gene profile of all dermal fibroblasts as dermal fibroblasts of the aging subpopulation. [2] The method according to item 1, wherein the gene profile is determined by single-cell analysis. [3] A method for determining dermal fibroblasts of an aging subpopulation, comprising the steps of determining the gene expression of an aging subpopulation-related gene group in selected dermal fibroblasts, and determining cells in which the gene expression of the aging subpopulation-related gene group is higher than a predetermined threshold as dermal fibroblasts of an aging subpopulation. [4] The method according to any one of items 1 to 3, wherein the aging subpopulation-related gene group is one gene or combination of genes selected from the group consisting of SLPI, PI16, IGFBP6, ACKR3, CD55, FBN1, MFAP4, SEMA3C, TNXB, CHRDL1, CFD, CD34, PCOLCE2, ADH1B, FSTL1, COL12A1, EFEMP1, SCARA5, CLDN11, FBLN1, MEDAG, DCN, SFRP4, MMP2, CCN5, SFRP2, IGSF10, IGFBP5, CST3, SERPINF1, C3, ADAMTS5, CD248, FBLN2, CREB5, SCN2A, AOX1, and ALDH1A1. [5] A method for selecting a cell population containing dermal fibroblasts of an aging subpopulation, further comprising the step of separating cells determined to be cells of an aging subpopulation by the method described in any one of items 1 to 4. [6] A skin cell culture containing dermal fibroblasts of an aging subpopulation selected by the method described in item 5.[7] A method for screening dermal cell anti-aging agents, comprising the steps of: culturing a skin cell culture described in item 6 in a medium containing a candidate drug; determining the proliferation activity or gene expression activity of dermal fibroblasts of an aging subpopulation; and comparing the activity with a control to select a candidate drug as a dermal cell anti-aging agent. [8] The method according to item 7, wherein the gene expression activity of a dermal fibroblast culture of an aging subpopulation is selected from the expression activity of at least one gene selected from the group consisting of BMP2, BMP4, BMP7, TGFβ1, TGFβ2, and TGFβ3. [9] The method according to item 7, wherein the proliferation activity of a dermal fibroblast culture of an aging subpopulation is determined by a change in the proportion of the aging subpopulation.

[10] A method for screening dermal cell anti-aging agents, comprising: a step of determining the proportion of dermal fibroblasts of an aging subpopulation in a culture containing dermal fibroblasts; a step of culturing a culture containing dermal fibroblasts in a medium containing a candidate drug; a step of determining the proportion of dermal fibroblasts of an aging subpopulation after culturing; and a step of selecting a candidate drug that reduces the proportion of dermal fibroblasts of an aging subpopulation as a dermal cell anti-aging agent.

[11] A method for evaluating skin aging, comprising the steps of determining the proportion of dermal fibroblasts of an aging subpopulation in a skin sample obtained from a subject, and determining the skin aging state by comparing the proportion of dermal fibroblasts of an aging subpopulation with a predetermined threshold or correspondence.

[12] The method according to item 11, wherein the dermal fibroblasts of the aging subpopulation are a subpopulation with high expression of aging subpopulation-related gene groups when compared with the gene profile of all fibroblasts.

[13] The method according to item 11, wherein the aging subpopulation-related gene group is one gene or combination of genes selected from the group consisting of SLPI, PI16, IGFBP6, ACKR3, CD55, FBN1, MFAP4, SEMA3C, TNXB, CHRDL1, CFD, CD34, PCOLCE2, ADH1B, FSTL1, COL12A1, EFEMP1, SCARA5, CLDN11, FBLN1, MEDAG, DCN, SFRP4, MMP2, CCN5, SFRP2, IGSF10, IGFBP5, CST3, SERPINF1, C3, ADAMTS5, CD248, FBLN2, CREB5, SCN2A, AOX1, and ALDH1A1.

[0007] By identifying aging subpopulations involved in aging, the degree of skin aging can be determined. Furthermore, by isolating these aging subpopulations, cultures rich in dermal fibroblasts from these subpopulations can be prepared. This allows for the use of cultures rich in dermal fibroblasts from aging subpopulations, contributing to aging research and enabling the screening of drugs highly effective against the cells of the target subpopulation.

[0008] Figure 1A shows cell subpopulations classified by single-cell cluster analysis of cells contained in the dermis. Fibroblasts were classified into cell subpopulations FB1 to FB7. Figure 1B shows characteristic gene expression in each subpopulation of fibroblasts. Figure 1C shows the classification of genes expressed in each subpopulation of fibroblasts (FB1 to FB7). Figure 2 shows the distribution of each subpopulation of fibroblasts within the dermis. Figure 3A shows the results of calculating a simulated time flow using Monocle 3 for subpopulations of dermal fibroblasts, along with the annotated cell types. Figure 3B shows whether each cell belongs to young or old skin during the simulated time flow in Figure 3A. Figure 4 shows the ratio of old cells to young cells in each cell cluster and fibroblast subpopulation contained in the dermis. Figure 5 shows the distribution of the FB2 subpopulation in the skin of young (A) and elderly (B) individuals. α6 integrin indicates the basement membrane and blood vessels, and PDGFRα indicates all fibroblasts. Arrows indicate cells that are double-positive for the FB2 marker and PDGFRα. In the skin of elderly individuals, FB2 is abundant and present from the papillary layer to the subcutaneous junctional layer. Figure 5 shows the distribution of the FB2 subpopulation in the skin of young individuals (A) and elderly individuals (B). α6 integrin indicates the basement membrane and blood vessels, and PDGFRα indicates all fibroblasts. Arrows indicate cells that are double-positive for the FB2 marker and PDGFRα. In the skin of elderly individuals, FB2 is abundant and present from the papillary layer to the subcutaneous junctional layer. Figure 6 shows a fluorescence immunostaining image in which 8-hydroxyguanosine (8-OHdG) is stained in addition to the α6 integrin and FB2 markers from Figure 5. 8-OHdG is a cell damage marker, and arrows indicate cells co-stained with FB2. Figure 7 shows the results of NicheNet analysis on receptors in each fibroblast subpopulation (FB1, FB3, FB6) and other cell types (PC, SMC, macrophage, LEC) that can respond to ligands expressed by FB2.

[0009] One aspect of the present invention relates to a method for determining a subpopulation of dermal fibroblasts. More specifically, the method includes the steps of: determining the gene profile of selected dermal fibroblasts; and determining dermal fibroblasts with high expression of aging subpopulation-related genes by comparing them with the average gene profile of all dermal fibroblasts as aging subpopulation dermal fibroblasts. By including these steps, a subpopulation can be determined in a skin sample, particularly a dermal sample. The subpopulation may be determined as individual cells in the dermal sample, or as a proportion or number of cells. In particular, an aging-related subpopulation can be determined within the subpopulation. The method of the present invention may be in vivo, in vitro, or ex vivo, but from the viewpoint of using a skin sample, the in vitro and ex vivo methods are preferred. The determination of subpopulations according to the present invention is performed for medical or non-medical purposes, and is particularly preferably performed for non-medical purposes such as cosmetic or research.

[0010] The selected dermal fibroblasts may be single cells, homogeneous cells such as cultured cells, or heterogeneous dermal fibroblasts extracted from tissue. For single cells or heterogeneous dermal fibroblasts extracted from tissue, the gene profile is determined by single-cell analysis. In single-cell analysis, a unique tag is added to all cDNA obtained by reverse transcription of mRNA expressed by a single cell, and the gene profile can be determined by performing analysis using a next-generation sequencer. In this invention, the dermal fibroblasts may be dermal fibroblasts derived from any animal, but it is particularly preferable to use human-derived dermal fibroblasts.

[0011] Whole dermal fibroblasts refer to the entire collection of dermal fibroblasts (hereinafter referred to as the whole collection of dermal cells), without considering subpopulations of dermal fibroblasts. Although this can vary depending on how the sample is obtained, if the sample is of a certain size and taken from the dermis layer from the papillary layer to the subcutaneous junctional layer, all subpopulations of cells will be included on average, and the collection of dermal fibroblasts contained in such a sample can be called whole dermal fibroblasts. Cells other than dermal fibroblasts can be removed from the dermal sample. Cells other than fibroblasts may include pericytes, vascular endothelial cells, T cells, lymphatic endothelial cells, sweat gland epithelial cells, macrophages, and melanocytes. These cells can be identified from fibroblasts and removed through the expression of cell-specific genes, such as ACTA2 and RGS5 (pericytes), CLDN5 (vascular and lymphatic endothelial cells), CD3D, TPSB2, HDC (T cells), LYVE1 (lymphatic endothelial cells), KRT5, and AQP5 (sweat gland epithelial cells), LYZ (macrophages), and MLANA (melanocytes).

[0012] A gene expression profile refers to a set of gene expression levels for one or more genes. The average gene profile for the entire set of dermal fibroblasts may be determined for each sample or may be predetermined. A gene profile can be determined by comprehensively measuring the amount of genes expressed in a specific tissue or specific cell. Preferably, all genes are included, but specific genes may be selected for ease of experimental setup. Gene expression may be determined by any method, and the gene expression of individual genes may be determined by real-time PCR, but analysis using microarrays or RNA sequencing or similar techniques is preferred.

[0013] The average gene expression profile refers to the gene expression profile determined in all dermal fibroblasts without distinguishing between subpopulations. The average gene expression profile may be determined by mixing dermal fibroblasts contained in the dermis, or by averaging gene expression profiles obtained by single-cell analysis. Each subpopulation has characteristic gene expression, and therefore has a characteristic gene expression profile that differs from the average gene expression profile. Genes that exhibit characteristic gene expression are called subpopulation-related genes, and subpopulations can be determined by identifying cells with high expression of such subpopulation-related genes.

[0014] In this invention, dermal fibroblasts refer to fibroblasts originating from the dermis. Dermal fibroblasts can be characterized by gene expression, such as PDGFRA and DCN. The dermis is the region sandwiched between the epidermis and the subcutaneous tissue, and is classified into the papillary layer, subpapillary layer, reticular layer, and subcutaneous junctional layer directly below the basement membrane, with dermal fibroblasts distributed in each of these layers. Organs such as hair follicles and sweat glands can extend from the dermis to the subcutaneous tissue. Blood vessels ascend from the subcutaneous tissue, fold back directly below the basement membrane, and descend. Dermal fibroblasts can also be found near these hair follicles and blood vessels. Further gene expression profile analysis of cells identified as fibroblasts allows for the classification of dermal fibroblasts into seven subpopulations according to their profiles (Figure 1A-C). Based on the characteristic gene expression of each classified subpopulation, the localization of each subpopulation can be investigated (Figure 2).

[0015] The FB1 subpopulation (hereinafter simply referred to as FB1) is a subpopulation characterized by high expression of APOE, C7, CXCL12, CCL2, APOC1, and PTGDS (Figure 1B). FB1 expresses proteins involved in the immune and inflammatory systems, particularly proteins related to cytoplasmic translation (Figure 1C). FB1 is present in the reticular layer and subcutaneous junctional layer and has a flat, elongated shape (Figure 2).

[0016] The FB2 subpopulation (hereinafter simply referred to as FB2) is a subpopulation characterized by high expression of SLPI, PI16, ACKR3, and / or IGFBP6 (Figure 1B). FB2 expresses proteins involved in proteolysis and extracellular matrix (ECM) construction. In particular, it expresses regulatory proteins such as those for proteolysis and peptidase activity, and proteins that regulate the organization of the extracellular matrix (Figure 1C). FB2 is widely distributed from the papillary layer to the subcutaneous junctional layer, but is especially abundant in the deep subcutaneous junctional layer (Figure 2). The FB2 subpopulation is found in the skin of elderly people and can be called an age-related subpopulation.

[0017] The FB3 subpopulation (hereinafter simply referred to as FB3) is a subpopulation characterized by high expression of STC1, LEPR, THSD4, and / or COMP (Figure 1B). FB3 expresses proteins involved in the construction of the extracellular matrix (ECM). In particular, it expresses extracellular proteins and proteins that regulate the organization of the extracellular matrix (Figure 1C). FB3 is present in the papillary layer and subpapillary layer (Figure 2).

[0018] The FB4 subpopulation (hereinafter simply referred to as FB4) is a subpopulation characterized by high expression of COL11A1, EDNRA, MFAP5, and / or CCN2 (Figure 1B). FB4 expresses proteins involved in development, particularly genes involved in cytoplasmic translation and ossification (Figure 1C). FB4 is present around hair follicles (Figure 2), especially in hair root sheath cells.

[0019] The FB5 subpopulation (hereinafter simply referred to as FB5) is a subpopulation characterized by high expression of IGFBP3, INHBA, CENPW, and / or SERPINE2 (Figure 1B). FB5 expresses proteins involved in development, particularly genes involved in the regeneration of tissues and structures (Figure 1C). FB5 is present around the hair follicle and in the dermal papilla (Figure 2), especially in hair root sheath cells or dermal papilla cells.

[0020] The FB6 subpopulation (hereinafter simply referred to as FB6) is a subpopulation characterized by high expression of COCH, SPARCL1, ASPN, and / or CRABP1 (Figure 1B). FB6 expresses proteins involved in development and genes involved in the formation of the extracellular matrix. In particular, it expresses genes involved in ossification and connective tissue development (Figure 1C). FB6 is located in the papillary layer (Figure 2).

[0021] The FB7 subpopulation (hereinafter simply referred to as FB7) is a subpopulation characterized by high expression of CLDN1, TAGLN, NR2F2, and / or APOD (Figure 1B). FB7 expresses proteins involved in RNA modification, particularly genes involved in RNA splicing and mRNA metabolism (Figure 1C).

[0022] In the case of FB1, the subpopulation-related gene group consists of at least one gene, or a combination thereof, selected from the group comprising APOE, C7, CXCL12, CCL2, and APOC1. As a subpopulation-related gene group, in the case of FB2, at least one gene or a combination thereof is used, selected from the group consisting of SLPI, PI16, IGFBP6, ACKR3, CD55, FBN1, MFAP4, SEMA3C, TNXB, CHRDL1, CFD, CD34, PCOLCE2, ADH1B, FSTL1, COL12A1, EFEMP1, SCARA5, CLDN11, FBLN1, MEDAG, DCN, SFRP4, MMP2, CCN5, SFRP2, IGSF10, IGFBP5, CST3, SERPINF1, C3, ADAMTS5, CD248, FBLN2, CREB5, SCN2A, AOX1, and ALDH1A1. For FB3, the subpopulation-related gene group consists of at least one gene, or a combination thereof, selected from the group consisting of STC1, LEPR, THSD4, and / or COMP. For FB4, the subpopulation-related gene group consists of at least one gene, or a combination thereof, selected from the group consisting of COL11A1, EDNRA, MFAP5, and / or CCN2. For FB5, the subpopulation-related gene group consists of at least one gene, or a combination thereof, selected from the group consisting of IGFBP3, INHBA, CENPW, and / or SERPINE2. For FB6, the subpopulation-related gene group consists of at least one gene, or a combination thereof, selected from the group consisting of COCH, SPARCL1, ASPN, and / or CRABP1. In the case of FB7, the subpopulation-related gene group consists of at least one gene, or a combination thereof, selected from the group comprising CLDN1, TAGLN, NR2F2, and / or APOD.

[0023] The aging subpopulation (FB2) related gene group includes one or more genes selected from the group consisting of SLPI, PI16, IGFBP6, ACKR3, CD55, FBN1, MFAP4, SEMA3C, TNXB, CHRDL1, CFD, CD34, PCOLCE2, ADH1B, FSTL1, COL12A1, EFEMP1, SCARA5, CLDN11, FBLN1, MEDAG, DCN, SFRP4, MMP2, CCN5, SFRP2, IGSF10, IGFBP5, CST3, SERPINF1, C3, ADAMTS5, CD248, FBLN2, CREB5, SCN2A, AOX1, and ALDH1A1. These genes are those whose expression is more than twice as high compared to the average of fibroblasts. Any number of genes can be selected from the aging subpopulation-related gene group. For example, 10, 9, 8, 7, 6, 5, 4, 3, or 2 genes can be selected. For example, genes with high expression compared to the average of fibroblasts, selected in descending order, include SLPI, PI16, IGFBP6, ACKR3, CFD, and CD34. The expression of the selected genes can be used as an indicator, for example, the total or average expression of the genes, and compared with the expression of the subpopulation-related gene group in all dermal fibroblasts. In another embodiment, the number of genes with high expression compared to the expression of the subpopulation-related gene group in all dermal fibroblasts can also be used as an indicator. Cells with high expression of one or more genes included in the aging subpopulation-related gene group can be determined to be cells included in the aging subpopulation.

[0024] High expression of a subpopulation-related gene group means that it is statistically significantly higher than the expression of the subpopulation-related gene group in all dermal fibroblasts. Cluster analysis to compare gene expression profiles can be performed using programs well known in this art. For example, it is performed by analysis using the R package Seurat. Preferably, it means that the expression level is at least 1.2 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, or at least 5 times higher than the expression level in all dermal fibroblasts.

[0025] In another aspect of the present invention, a method for determining aging subpopulation dermal fibroblasts can also be used to determine a subpopulation by focusing only on the expression of subpopulation-related gene groups, without focusing on the average gene profile. More specifically, a method for determining aging subpopulation dermal fibroblasts includes the following steps: determining the gene expression of aging subpopulation-related gene groups in selected dermal fibroblasts, and determining cells in which the gene expression of aging subpopulation-related gene groups is higher than a predetermined threshold as aging subpopulation dermal fibroblasts. Such a method may be a simpler method that can determine aging subpopulations without determining gene profiles. The aging subpopulation-related gene group may be at least one, two, three, four, five, six, seven, eight, nine, ten, or any combination of eleven or more genes. The expression of control genes may also be determined. For example, PDGFRA, DCN, and other housekeeping genes, which are expressed in all dermal fibroblasts, may be used as control genes, and the expression of aging subpopulation-related genes may be evaluated in comparison to these control genes. If the expression is higher than or at the same level as the control genes, it may be determined that the expression of aging subpopulation-related genes is high. Since control genes are usually highly expressed in fibroblasts, even if the expression is lower than that of the control genes, if it is higher than a predetermined expression level, such as 1 / 2 or 1 / 3, it may be determined that the expression of aging subpopulation-related genes is high.

[0026] When each subpopulation is classified into elderly and young, FB2 can be classified as elderly. Furthermore, when observing the pseudo-time course, it transitions from fibroblasts in the hair papilla, dermal root sheath, deep skin, and papillary layer (Figure 3), indicating that the FB2 subpopulation is a group associated with aging. The proportion of the FB2 subpopulation increases in the elderly (Figure 4), so it can also be called an aging-related subpopulation. When skin samples from young and elderly individuals were stained with an FB2 marker that can specifically identify the FB2 subpopulation, the FB2 subpopulation increased with age (Figure 5). The FB2 subpopulation has been shown to co-localize with 8-hydroxy-2'-deoxyguanosine (8-OHdG) (Figure 6). 8-OHdG is a marker that measures cell damage and accumulates in cells subjected to oxidative stress when the 8th position of deoxyguanosine is hydroxylated. Cells in the FB2 subpopulation are shown to be senescent cells that have accumulated cell damage. When the ligands released by the FB2 subpopulation were investigated for their effects on other cells, it was found that they can act on other cells via ligands from the TGFβ family and the BMP family (Figure 7). TGF-β and BMP are related to the properties and differentiation of mesenchymal cells such as fibroblasts.

[0027] If a subpopulation of aging dermal fibroblasts is determined, the process may include a step to further isolate these determined cells. Cell isolation may be performed using a cell sorter or the like. By culturing the selected dermal fibroblasts of the aging subpopulation in a medium for dermal fibroblast proliferation, a dermal fibroblast culture of the aging subpopulation can be obtained. Dermal fibroblast cultures of the aging subpopulation are useful when conducting experiments that mimic aged skin. The isolated dermal fibroblast culture of the aging subpopulation can also be appropriately mixed with dermal fibroblast cultures of other subpopulations to obtain a dermal fibroblast culture that mimics a desired aging state. Such a dermal fibroblast culture may be a three-dimensional skin construct. By using dermal fibroblast cultures of the aging subpopulation, it is possible to elucidate the mechanisms of aging in the skin. Furthermore, by using dermal fibroblast cultures of the aging subpopulation, it becomes possible to screen for drugs that act on the aging subpopulation.

[0028] One aspect of the present invention relates to a screening method for dermal cell anti-aging agents using dermal fibroblast cultures of an aging subpopulation. More specifically, the method includes the following steps: culturing dermal fibroblast cultures of an aging subpopulation in a culture medium containing a candidate drug; determining the proliferation activity or gene expression activity of the dermal fibroblasts of the aging subpopulation; and comparing the activity with a control to select a candidate drug as a dermal cell anti-aging agent. In this screening method, proliferation activity can be determined by counting the number of cells after culturing. Gene expression activity can be determined by examining the expression of a specific gene, for example, at least one gene selected from the group consisting of BMP2, BMP4, BMP7, TGFβ1, TGFβ2, and TGFβ3. These genes are related to the properties and differentiation of mesenchymal cells, and suppressing their expression means suppressing changes in the properties of fibroblasts. The control shows the results of experiments conducted under the same conditions except that no candidate drug was added. Using dermal fibroblast cultures from an aging subpopulation makes it possible to screen for candidate drugs that can directly act on aging skin.

[0029] The dermal cell anti-aging agents screened by the screening method of the present invention can suppress the cellular activity of dermal fibroblasts, particularly dermal fibroblasts of the aging subpopulation. By suppressing the dermal fibroblast activity of the aging subpopulation, changes in the properties of fibroblasts can be suppressed, and skin elasticity can be increased. Since dermal fibroblasts of the aging subpopulation increase with age and affect the properties of other fibroblasts, agents that suppress the activity of these cells can be said to be agents that suppress the influence of cells of the aging subpopulation on dermal fibroblasts. By maintaining a normal number of dermal fibroblasts or suppressing changes in their properties, the extracellular matrix produced is also maintained, and the decrease in skin elasticity can be suppressed. Therefore, dermal fibroblast inhibitors of the aging subpopulation can also be called skin elasticity improving agents, wrinkle and sagging improving agents, and anti-skin aging agents.

[0030] Another aspect of the present invention relates to a method for screening skin elasticity enhancers based on cells of an aging subpopulation in a culture containing fibroblasts. Specifically, the method includes: determining the proportion of dermal fibroblasts of an aging subpopulation in a culture containing dermal fibroblasts; culturing the culture containing dermal fibroblasts in a medium containing a candidate drug; determining the proportion of dermal fibroblasts of an aging subpopulation after culturing; and selecting a candidate drug that reduces the proportion of dermal fibroblasts of an aging subpopulation as a dermal cell activator. Preferably, the culture containing fibroblasts contains cells of multiple subpopulations. By reducing the cells of an aging subpopulation or their proportion, the candidate drug can relatively increase cell activity. This can increase the amount of extracellular matrix produced and increase skin elasticity. The proportion of dermal fibroblasts of an aging subpopulation can be determined using the methods described herein. For example, it may be determined based on a gene profile or based on the expression of aging subpopulation-related genes.

[0031] Any culture medium and conditions can be used as long as they are capable of culturing dermal fibroblasts. For example, DMEM medium, RPMI medium, F12 medium, etc. can be used at 37°C and 5% CO2. ２ The cells can be cultured in a humidified atmosphere. After seeding, cells that have reached confluence or subconfluence can be used. Culturing in a medium containing the candidate drug can typically be for any period between a few hours, overnight, or 1 to 10 days, for example, 1, 2, 3, or 4 days.

[0032] The candidate components used in the screening method of the present invention can be any library, such as cosmetic materials, food materials, or pharmaceutical materials. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or synthesized compounds and prepared extracts.

[0033] In yet another aspect of the present invention, the present invention may relate to a method for evaluating the state of skin aging. Specifically, the present invention includes the steps of: determining the proportion of dermal fibroblasts of an aging subpopulation in a skin sample obtained from a subject; and determining the state of skin aging by comparing the proportion of dermal fibroblasts of the aging subpopulation with a predetermined threshold or correspondence. Determining the proportion of dermal fibroblasts of an aging subpopulation in a skin sample makes it possible to evaluate the state of skin aging. Alternatively, the state of skin aging can be evaluated based on the expression levels of aging subpopulation-related gene groups instead of the proportion of dermal fibroblasts. The threshold or correspondence can be determined by determining the proportion of dermal fibroblasts of an aging subpopulation in a control of a predetermined age and skin age. Methods for determining thresholds or correspondences are well known in the art.

[0034] All references made herein are incorporated herein by citation in their entirety.

[0035] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention.

[0036] Example 1: Single-cell analysis of dermal fibroblasts. The epidermis was exfoliated from human skin using dispase, and dermal tissue was separated into cells using collagenase. A single-cell RNA sequencing library was prepared using the Chromium single-cell analyzer and the Chromium Single Cell 3' v3 Reagent kit (10x Genomics). The library was sequenced using Illumina HiSeq 2500. The obtained sequencing data was analyzed using Cell Ranger, and clustering analysis was performed using the Seurat package. Dermal cells were classified into clusters based on their gene profiles (Figure 1A). Furthermore, focusing on fibroblasts, they could be classified into seven subpopulations (Figures 1A and 1B). Figure 1B shows the results of analyzing the types of genes characteristically expressed in each cluster using Seurat's FindMarkers. Additionally, GO analysis was performed using genes characteristic of each cluster, and the results are shown in Figure 1C.

[0037] Example 2: Distribution of subpopulations Skin sections were prepared, fixed with cold methanol, and immunofluorescence staining was performed using antibodies specific to each subpopulation. Anti-APOC1 antibody (Atlas Antibodies) was used as the primary antibody for FB1, anti-IGFBP6 antibody (Atlas Antibodies) for FB2, anti-NKD2 antibody (Atlas Antibodies) for FB3, anti-MME antibody (Atlas Antibodies) for FB4, anti-IGFBP3 antibody (Sigma Aldrich) for FB5, and anti-SLC22A16 antibody (Atlas Antibodies) for FB6, all at 50- to 200-fold dilutions. In addition, anti-integrin α6 antibody (Santa Cruz Biotechnology) was used to stain the basement membrane and blood vessels, and anti-PDGFRα antibody (R & D systems) was used to stain fibroblasts at 200-fold and 50-fold dilutions, respectively. Further staining was performed using DAPI to stain the nuclei. The reaction was carried out using a fluorescently labeled (Alexa488, Alexa568, or Alexa647) conjugated secondary antibody corresponding to each primary antibody, and images were taken using a confocal laser microscope (Carl Zeiss) (Figure 2). (i) is the region from the epidermis to the dermal papillary layer and the region immediately below the papillary layer, (ii) is the region of the reticular layer directly below the papillary layer, (iii) is the region around the hair follicle in the reticular layer, (iv) is the region of the reticular layer near the boundary between the dermis and subcutaneous tissue, and (v) is the region of the subcutaneous tissue. FB1 was distributed from the reticular layer to the subcutaneous tissue. FB2 was distributed from the papillary layer to the deep subcutaneous junctional layer. FB3 was distributed in the papillary layer. FB4 was distributed around the hair follicle, especially in the hair root sheath cells. FB5 was distributed around the hair root, especially in the dermal papilla. FB6 was distributed in the papillary layer.

[0038] Example 3: Pseudotime analysis in each subpopulation To estimate the pseudotime pathway of fibroblast subpopulations, Monocle3 (https: / / cole-trapnell-lab.github.io / monocle3 / ) was used to perform pseudotime analysis (Figure 3A, B). The pseudotime pathway of the subpopulations started from FB5 around the hair root, proceeded to FB4 around the hair follicle, branched from FB1 to FB2 and FB7, then to FB3, and further proceeded to FB6, FB1, and then to FB2 (Figure 3A). When distinguishing between young and old cells, the FB2 region, which is the terminal point, was classified as old (Figure 3B).

[0039] Example 4: Prevalence of each subpopulation in the skin of elderly and young individuals. Dermal samples obtained from the skin of elderly individuals aged 73 and 79 years, and young individuals aged 25, 27, and 30 years were subjected to single-cell analysis to determine the prevalence of each subpopulation (Figure 4).

[0040] Example 5: Distribution of FB2 subpopulation cells Skin sections were prepared from the skin of an 84-year-old elderly person and a 27-year-old young person, fixed with cold methanol, and subjected to immunofluorescence staining using antibodies specific to each subpopulation. For FB2, anti-SCARA5 antibody (distributor: Atlas Antibodies) was used as the primary antibody at a 50-fold dilution. Furthermore, anti-integrin α6 antibody (distributor: Santa Cruz Biotechnology) was used to stain the basement membrane and blood vessels, and anti-PDGFRα antibody (distributor: R & D systems) was used to stain fibroblasts at 200-fold and 50-fold dilutions, respectively. The samples were reacted with fluorescently labeled (Alexa488, Alexa568, or Alexa647) conjugated secondary antibodies corresponding to each primary antibody, and photographs were taken using a confocal laser microscope (distributor: Carl Zeiss) (Figure 5A: young person, B: elderly person). Furthermore, Figure 6 shows the results of staining the skin of elderly individuals with 8-hydroxyguanosine instead of PDGFRα using the 8-oxo-dG antibody (distributor: R & D systems).

[0041] Example 6: NicheNet Analysis of Ligand-Receptor Targets Using the ligand-receptor interaction tool NicheNet via the nichenetr package in R, target genes with differing expression levels between young and elderly individuals were calculated using Seurat's 'FindMarkers' function, based on the criteria of p-value < 0.05, log2 fold change > 1, and expression in 25% or more cells. Based on the target genes, the NicheNet ligand-receptor network was applied to "sender cells" (vertical axis) and "receiver cells" (FB1, 3, 6, pericytes, SMCs, macrophages, LECs) (Figure 7). Among the factors secreted by sender cells, TGFβ is indicated by a black arrow and BMP by a white arrow.

Claims

1. A method for determining dermal fibroblasts of an aging subpopulation, comprising the steps of: determining the gene profile of selected dermal fibroblasts; and determining dermal fibroblasts with high expression of aging subpopulation-related genes by comparing them with the average gene profile of all dermal fibroblasts as dermal fibroblasts of the aging subpopulation.

2. The method according to claim 1, wherein the gene profile is determined by single-cell analysis.

3. A method for determining dermal fibroblasts of an aging subpopulation, comprising the steps of: determining the gene expression of a group of genes related to an aging subpopulation in selected dermal fibroblasts; and determining cells in which the gene expression of the group of genes related to an aging subpopulation is higher than a predetermined threshold as dermal fibroblasts of an aging subpopulation.

4. The method according to claim 1 or 3, wherein the aging subpopulation-associated gene group is one gene or a combination of genes selected from the group consisting of SLPI, PI16, IGFBP6, ACKR3, CD55, FBN1, MFAP4, SEMA3C, TNXB, CHRDL1, CFD, CD34, PCOLCE2, ADH1B, FSTL1, COL12A1, EFEMP1, SCARA5, CLDN11, FBLN1, MEDAG, DCN, SFRP4, MMP2, CCN5, SFRP2, IGSF10, IGFBP5, CST3, SERPINF1, C3, ADAMTS5, CD248, FBLN2, CREB5, SCN2A, AOX1, and ALDH1A1.

5. A method for selecting a cell population including dermal fibroblasts of an aging subpopulation, further comprising the step of separating cells determined to be cells of an aging subpopulation by the method of claim 1 or 3.

6. A skin cell culture comprising dermal fibroblasts of an aging subpopulation selected by the method of claim 5.

7. A method for screening dermal cell anti-aging agents, comprising the steps of: culturing a skin cell culture according to claim 6 in a culture medium containing a candidate drug; determining the proliferation activity or gene expression activity of dermal fibroblasts of an aging subpopulation; and comparing the proliferation activity or gene expression activity with a control to select a candidate drug as a dermal cell anti-aging agent.

8. The method according to claim 7, wherein the gene expression activity of a dermal fibroblast culture of an aging subpopulation is selected from the expression activity of at least one gene selected from the group consisting of BMP2, BMP4, BMP7, TGFβ1, TGFβ2, and TGFβ3.

9. The method according to claim 7, wherein the proliferation activity of a dermal fibroblast culture of an aging subpopulation is determined by the change in the proportion of the aging subpopulation.

10. A method for screening dermal cell anti-aging agents, comprising: a step of determining the proportion of dermal fibroblasts of an aging subpopulation in a culture containing dermal fibroblasts; a step of culturing a culture containing dermal fibroblasts in a medium containing a candidate drug; a step of determining the proportion of dermal fibroblasts of an aging subpopulation after culturing; and a step of selecting a candidate drug that reduces the proportion of dermal fibroblasts of an aging subpopulation as a dermal cell anti-aging agent.

11. A method for evaluating skin aging, comprising the steps of determining the proportion of dermal fibroblasts of an aging subpopulation in a skin sample obtained from a subject, and determining the skin aging state by comparing the proportion of dermal fibroblasts of an aging subpopulation with a predetermined threshold or correspondence.

12. The method according to claim 11, wherein the dermal fibroblasts of the aging subpopulation are a subpopulation in which the expression of aging subpopulation-related genes is high when compared with the gene profile of all fibroblasts.

13. The method according to claim 11, wherein the aging subpopulation-associated gene group is one gene or a combination of genes selected from the group consisting of SLPI, PI16, IGFBP6, ACKR3, CD55, FBN1, MFAP4, SEMA3C, TNXB, CHRDL1, CFD, CD34, PCOLCE2, ADH1B, FSTL1, COL12A1, EFEMP1, SCARA5, CLDN11, FBLN1, MEDAG, DCN, SFRP4, MMP2, CCN5, SFRP2, IGSF10, IGFBP5, CST3, SERPINF1, C3, ADAMTS5, CD248, FBLN2, CREB5, SCN2A, AOX1, and ALDH1A1.

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