SASP-induced macrophage dysfunction may contribute to accelerated senescent fibroblast accumulation in the dermis.

Recently, increasing attention has been paid to senescence-associated secretory phenotype (SASP), a phenomenon that senescent cells secrete molecules such as inflammatory cytokines and matrix metalloproteinases (MMPs), due to its noxious effects on the surrounding tissue. Senescent cells in the blood and liver are known to be properly depleted by macrophages. In the dermis, accumulation of senescent cells has been reported and is thought to be involved with skin ageing. In this study, to elucidate the clearance mechanism of senescent cells in the dermis, we focused on macrophage functions. Our co-culture experiments of senescent fibroblasts and macrophages revealed a two-step clearance mechanism: first, TNF-α secreted from macrophages induces apoptosis in senescent fibroblasts, and then, dead cells are phagocytosed by macrophages. Furthermore, it was suggested that SASP factors suppress both of the two steps of the senescent cell clearance by macrophages. From these findings, normally senescent cells in the dermis are thought to be removed by macrophages, but when senescent cells are excessively accumulated owing to oxidative stress, ultraviolet (UV) ray or other reasons, SASP was suggested to suppress the macrophage-dependent clearance functions and thereby cause further accumulation of senescent cells.

UV irradiation-induced DNA hypomethylation around WNT1 gene: Implications for solar lentigines.

Wnt/ß-catenin signalling promotes melanogenesis in melanocytes and also induces melanocytogenesis from melanocyte stem cells (McSCs). Previous study reported that WNT1, a ligand which activates Wnt/ß-catenin signalling pathway, was more highly expressed in the epidermis at SLs than in normal skin areas, suggesting that WNT1 causes hyperpigmentation. To elucidate the mechanism by which WNT1 expression is increased in SLs, we examined the methylation of 5-carbon of cytosine (5mC), that is 5-methylcytosine (5mC) level, in a region within the WNT1 promoter; the methylation of the region was known to negatively regulate WNT1 gene expression. We used an immortalized cell line of human interfollicular epidermal stem cells to analyse the effect of UVB irradiation on DNA methylation level of WNT1 promoter and found that UVB irradiation caused demethylation of WNT1 promoter and promoted WNT1 mRNA expression. It was also found that UVB irradiation reduced the expression of DNA methyltransferase 1 (DNMT1), an enzyme responsible for maintaining methylation patterns during cell division. Pathological analysis of SLs and non-SL regions in the human skin revealed that both DNMT1 expression and 5mC level were decreased at SLs compared to non-SL skins. Furthermore, bisulphite sequencing showed that the methylated CpG level in WNT1 promoter was also lower at SLs than in non-SL skins. Thus, in the skin exposed to a high amount of UV rays, excessive expression of WNT1 is thought to be caused by the demethylation of WNT1 promoter, and the upregulated WNT1 promotes melanocytogenesis and melanogenesis, then resulting in SL formation.

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development.

Interstitial fluid flow-induced growth potential and hyaluronan synthesis of fibroblasts in a fibroblast-populated stretched collagen gel culture.

BACKGROUND: Tensioned collagen gels with dermal fibroblasts (DFs) as a dermis model are usually utilized in a static culture (SC) that lacks medium flowing. To make the model closer to its in vivo state, we created a device to perfuse the model with media flowing at a physiological velocity and examined the effects of medium flow (MF) on the cultures. METHODS: We constructed a medium perfusion device for human DF-embedded stretched collagen gels (human dermis model), exposed the model to media that flows upwardly at ~1mL/day, and examined water retention of the gels, cells' growth ability, metabolic activity, expression profiles of nine extracellular matrix (ECM)-related genes. The obtained data were compared with those from the model in SC. RESULTS: MF increases the gels' water retention and cells' growth potential but had little effect on their metabolic activities. MF robustly enhanced hyaluronan synthase 2 (HAS2) and matrix metalloprotease 1 (MMP1) gene expressions but not of the other genes (MMP2, HYAL1, HYAL2, HYAL3, COL1A1, COL3A1, and CD44). MF significantly increased the amounts of cellular hyaluronan and adenosine triphosphate. CONCLUSIONS: The MF at a physiological speed significantly influences the nature of ECMs and their resident fibroblasts and remodels ECMs by regulating hyaluronan metabolism. GENERAL SIGNIFICANCE: Fibroblasts in tensioned collagen gels altered their phenotypes in a MF rate-dependent manner. Collagen gel culture with tension and MF could be utilized as an appropriate in vitro model of interstitial connective tissues to evaluate the pathophysiological significance of mechanosignals generated by fluid flow and cellular/extracellular tension.

Dermal CD271+ Cells are Closely Associated with Regeneration of the Dermis in the Wound Healing Process.

Stem cells have recently been shown to play important roles in wound healing. The aim of this study was to investigate the role of dermal CD271+ cells in wound healing. Full-thickness wounds were produced on the backs of 5-year-old and 24-week-old mice, and time-course of wound closure, CD271+ cell counts, and gene expression levels were compared. Delayed wound healing was observed in 24-week-old mice. The peak of CD271+ cell increase was delayed in 24-week-old mice, and gene expression levels of growth factors in wounded tissue were significantly increased in 5-year-old mice. Dermal CD271+ cells purified by fluorescence-activated cell sorting (FACS) expressed higher growth factors than CD271- cells, suggesting that CD271+ cells play important roles by producing growth factors. This study also investigated dermal CD271+ cells in patients with chronic skin ulcers. Dermal CD271+ cells in patients were significantly reduced compared with in healthy controls. Thus, dermal CD271+ cells are closely associated with wound healing.

Morphological change of skin fibroblasts induced by UV Irradiation is involved in photoaging.

Human dermal fibroblasts (HDFs) are typically flattened or extensible shaped and play a critical role in the metabolism of extracellular matrix components. As the properties of fibroblasts in the dermis are considered to be influenced by their morphology, we investigated the morphological changes induced in fibroblasts by ultraviolet (UV) irradiation as well as the relationship between these changes and collagen metabolism. In this study, we showed that UVA exposure induced morphological changes and reduced collagen contents in HDFs. These morphological changes were accompanied a reduction in actin filaments and upregulation of the actin filament polymerization inhibitor, capping protein muscle Z-line ɑ1 (CAPZA1). External actin filament growth inhibitors also affected the shape of HDFs and reduced collagen levels. These results suggest that UVA exposure may inhibit the polymerization of actin filaments and induce morphological changes in skin fibroblasts. These morphological changes in fibroblasts may accelerate reductions in collagen synthesis. This mechanism may be one of the processes responsible for collagen reductions observed in photoaged skin. When natural materials that suppress these morphological changes in HDFs were evaluated, we found that an extract of Lilium 'Casa Blanca' (LCB) suppressed UVA-induced alterations in the shape of HDFs, which are typically followed by inhibition of collagen reduction. An analysis of the active compounds in LCB extract led to the identification of regaloside I, which had a structure of phenylpropanoid glycerol glucoside, as the active compound inhibiting the upregulation of CAPZA1. Therefore, inhibition of UVA-induced morphological changes in HDFs is considered to be promising way for the suppression of collagen reduction in photoaging.

Inhibitory effect of Phalaenopsis orchid extract on WNT1-induced immature melanocyte precursor differentiation in a novel in vitro solar lentigo model.

Recently, it has been reported that increased expression of WNT1 accelerates the differentiation of melanocyte stem cells (McSCs) in solar lentigines (SLs), hyperpigmented maculae commonly seen on sun-exposed areas of the skin. In this study, to establish an in vitro SL model, human epidermal squamous carcinoma cell line HSC-1, which expresses higher levels of WNT1 than normal human epidermal keratinocytes, was co-cultured with early passage normal human epidermal melanocytes (NHEMs) as an in vitro McSC model. As a result, mRNA expression levels of melanocyte differentiation-related genes MITF and TYR in NHEMs were significantly increased by co-culturing with HSC-1 cells. Furthermore, Phalaenopsis orchid extract (Phex) inhibited McSCs differentiation by suppressing WNT1 expression via down-regulation of DLX2, a transcriptional activator of WNT1, in HSC-1 cells. Therefore, our finding suggested that extracts such as Phex, which suppresses WNT1 expression, may be useful as a novel treatment of SLs.

ZIP2 protein, a zinc transporter, is associated with keratinocyte differentiation.

Zinc is essential for the proper functioning of various enzymes and transcription factors, and its homeostasis is rigorously controlled by zinc transporters (SLC39/ZIP, importers; SLC30/ZnT, exporters). Skin disease is commonly caused by a zinc deficiency. Dietary and inherited zinc deficiencies are known to cause alopecia and the development of vesicular or pustular dermatitis. A previous study demonstrated that zinc played crucial roles in the survival of keratinocytes and their unique functions. High levels of zinc have been detected in the epidermis. Epidermal layers are considered to use a mechanism that preferentially takes in zinc, which is involved with the unique functions of keratinocytes. However, few studies have investigated the ZIP (Zrt- and Irt-like protein) proteins specifically expressed in keratinocytes and their functions. We explored the ZIP proteins specifically expressed in the epidermis and analyzed their functions. Gene expression analysis showed that the expression of ZIP2 was consistently higher in the epidermis than in the dermis. Immunohistochemistry analysis confirmed the expression of ZIP2 in differentiating keratinocytes. The expression of ZIP2 was found to be up-regulated by the differentiation induction of cultured keratinocytes. Intracellular zinc levels were decreased in keratinocytes when ZIP2 was knocked down by siRNA, and this subsequently inhibited the differentiation of keratinocytes. Moreover, we demonstrated that ZIP2 knockdown inhibited the normal formation of a three-dimensional cultured epidermis. Taken together, the results of this study suggest that ZIP2, a zinc transporter expressed specifically in the epidermis, and zinc taken up by ZIP2 are necessary for the differentiation of keratinocytes.

Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis.

During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.

Increase of stratifin triggered by ultraviolet irradiation is possibly related to premature aging of human skin.

Although ultraviolet (UV) rays cause premature aging of human skin, which is called photoaging, its detailed mechanisms are not known. Stratifin (SFN), a member of the 14-3-3 protein family, is secreted by keratinocytes on human skin, and has an effect on gene expression in other cells. In this study, the association of SFN with the mechanism of photoaging was investigated. The effect of UVB irradiation on SFN expression in epidermal keratinocytes was examined by in vitro and in vivo studies. In addition, the effects of SFN on epidermal keratinocytes and dermal fibroblasts were examined. SFN mRNA expression and protein levels increased significantly in UVB-irradiated keratinocytes. SFN significantly decreased filaggrin and serine palmitoyltransferase mRNA expression in epidermal keratinocytes and hyaluronan synthase 2 mRNA expression in dermal fibroblasts. In addition, it was reconfirmed that SFN induces the downregulation of collagen content through changes of COL-1, MMP-1 and MMP-2 mRNA expressions. Furthermore, the expression level of SFN mRNA was significantly higher in sun-exposed compared with that in sun-shielded skin. These results suggest that SFN affects the water-holding capacity, barrier function and dermal matrix components in photoaging skin. An increase of SFN triggered by UVB irradiation may be one of the causes of alterations observed in photoaging skin.

Accelerated differentiation of melanocyte stem cells contributes to the formation of hyperpigmented maculae.

It has been reported that the abnormal regulation of melanocyte stem cells (McSCs) causes hair greying; however, little is known about the role of McSCs in skin hyperpigmentation such as solar lentigines (SLs). To investigate the involvement of McSCs in SLs, the canonical Wnt signalling pathway that triggers the differentiation of McSCs was analysed in UVB-induced delayed hyperpigmented maculae in mice and human SL lesions. After inducing hyperpigmented maculae on dorsal skin of F1 mice of HR-1× HR/De, which was formed long after repeated UVB irradiation, the epidermal Wnt1 expression and the number of nuclear ß-catenin-positive McSCs were increased as compared to non-irradiated control mice. Furthermore, the expression of dopachrome tautomerase (Dct), a downstream target of ß-catenin, was significantly upregulated in McSCs of UVB-irradiated mice. The Wnt1 expression and the number of nuclear ß-catenin-positive McSCs were also higher in human SL lesions than in normal skin. Recombinant Wnt1 protein induced melanocyte-related genes including Dct in early-passage normal human melanocytes (NHEMs), an in vitro McSC model. These results demonstrate that the canonical Wnt signalling pathway is activated in SL lesions and strongly suggest that the accelerated differentiation of McSCs is involved in SL pathogenesis.

Analysis of facial skin-resident microbiota in Japanese acne patients.

OBJECTIVES: We investigated the facial skin microbiota of Japanese acne patients. METHODS: Skin swab samples were obtained from 100 acne patients and 28 healthy controls to evaluate Propionibacterium and Staphylococcus spp. using a culture method. Malassezia spp. were evaluated using a nonculture method. Antibiotic resistance of Propionibacterium spp. was also examined. RESULTS: Acne patients and controls did not show significant differences in Propionibacterium and Staphylococcus spp. populations. However, the number of Malassezia globosa from patients was greater than that from controls. Moreover, the number of Propionibacterium spp. from patients carrying antibiotic-resistant strains was significantly greater than that from patients not carrying them. CONCLUSIONS: The present study characterized the facial skin microbiota of Japanese acne patients, suggesting a correlation between acne and quantitative differences in Malassezia microbiota. It was also found that the antibiotic resistance of Propionibacterium spp. may affect its abundance in the skin.

Bleomycin inhibits adipogenesis and accelerates fibrosis in the subcutaneous adipose layer through TGF-ß1.

Systemic sclerosis [scleroderma (SSc)]-associated skin fibrosis is characterized by increased fibrosis in the dermis and a reduction in the thickness of the subcutaneous adipose tissue layer. Although many studies have examined fibrosis in SSc, only a few studies have focused on the associated reduction in the thickness of the subcutaneous adipose tissue layer. In this study, we investigated the effects of SSc-induced fibrosis on adipose tissue. We found that bleomycin suppresses adipogenesis in adipose-derived stem cells (ASCs) and stimulates ASCs to express transforming growth factor ß1 (TGF-ß1), which suppresses adipogenesis and promotes fibrosis. Furthermore, we found that adipocyte-conditioned medium suppressed collagen synthesis by fibroblasts in fibrosis-like conditions. We concluded that in the skin affected by bleomycin-induced fibrosis, increased TGF-ß1 expression suppresses adipogenesis and promotes adipocyte fibrosis. It was also suggested that adipocytes have an inhibitory effect on the progression of fibrosis.

Analysis of the effects of hydroquinone and arbutin on the differentiation of melanocytes.

Hydroquinone (HQ) is a chemical compound that inhibits the functions of melanocytes and has long been known for its skin-whitening effect. According to previous studies, the Tyrosinase (Tyr) activity inhibitory effect and melanocyte-specific cell toxicity are known depigmenting mechanisms; however, details of the underlying mechanisms are unknown. Arbutin (Arb) is also known for its Tyr activity inhibitory effect and is commonly used as a skin-whitening agent. However, the detailed depigmenting mechanism of Arb is also not yet fully understood. Few studies have attempted to elucidate the effects of HQ and Arb on undifferentiated melanocytes. In this study, we examined the effects of HQ and Arb throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell (ESC) culture system to induce melanocytes. The results showed that HQ in particular downregulated the early stage of differentiation, in which neural crest cells were generated, and the late stage of differentiation, in which melanogenesis became active. On the other hand, Arb had no effect on the differentiation of melanocytes, and only suppressed melanogenesis by specifically suppressing elevations in Tyr expression in the late stage of differentiation.

Increased hydrophobicity in Malassezia species correlates with increased proinflammatory cytokine expression in human keratinocytes.

Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and ß-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not ß-1,3-glucan expression.

Melanocyte stem cells express receptors for canonical Wnt-signaling pathway on their surface.

It has been reported that melanocytes play important roles in skin and hair pigmentation and are differentiated from melanocyte stem cells (MSCs) residing in the bulge area of hair follicles. Recently, interest has been growing in MSCs because regulation of the upstream of differentiated melanocytes is essential for the determination of skin and hair pigmentation; however, their precise characteristics remain to be elucidated. The aim of this study is to explore cell-surface markers expressed on MSCs in order to understand their characteristics. To explore genes specifically expressed in the bulge region, we classified a hair follicle into four areas, hair bulb, hair bulb to bulge (lower bulge), bulge, and epidermis to bulge (upper bulge), and collected these areas from back skin sections of C57BL/6 mice by laser microdissection. Real-time RT-PCR performed on these areas revealed that Frizzled (Fzd)-4, Fzd7, low density lipoprotein receptor-related protein 5 (Lrp5), and Lrp6, receptors for Wnt molecules, were expressed higher in the bulge area than other areas. Furthermore, FACS analysis showed that populations of Fzd4(+) cells and Fzd7(+) cells were different from those of Kit(+) cells (precursor of melanocytes: melanoblasts). Fzd4(+) and Fzd7(+) cells isolated by FACS required a longer culture period to differentiate into mature melanocytes than Kit(+) cells. Up-regulation of mRNA expressions of melanocyte markers (dopa chrometautomerase: Dct, tyrosinase: Tyr, tyrosinase-related protein 1: Tyrp1) was observed in Fzd4(+) and Fzd7(+) cells following Kit(+) cells during differentiation. These results suggested that Fzd4(+) and Fzd7(+) cells were more immature than melanoblasts, therefore raising the possibility that Fzd4(+) and Fzd7(+) cells are MSCs.

Cutaneous Malassezia microbiota in atopic dermatitis patients differ by gender and body part.

BACKGROUND: Malassezia is a particularly important factor in the occurrence of atopic dermatitis (AD). AIM: The aim of this study was to quantitatively clarify the Malassezia species isolated from AD patients by gender, body part and analytical method in detail. METHODS: The subjects were 20 AD males and 47 AD females. Samples were collected from lesion and nonlesion areas on the face and upper trunk of AD patients. Malassezia DNA was analyzed using a real-time PCR system. RESULTS: The cutaneous Malassezia microbiota in AD patients differed by gender, body part and analytical method. CONCLUSIONS: The present results indicate the possibility that the influence of Malassezia antigens is different according to gender and body part.

Involvement of a forkhead transcription factor, FOXO1A, in UV-induced changes of collagen metabolism.

Transcription factors belonging to the forkhead box gene, group O (FOXOs) family have been found to be crucial in downstream suppression of life-shortening effects of the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway, which accelerates aging by suppressing FOXOs. Thus, FOXOs could hold the key for counteracting aging. Although FOXOs may play a critical role in aging, the effects of FOXOs on UV-induced changes of collagen metabolism by dermal fibroblasts are unknown. In this study, UV-induced changes in FOXO1a expression and the roles of FOXO1a in the regulation of collagen synthesis and matrix metalloproteinase (MMPs) expression in human dermal fibroblasts were investigated. In UVA- or UVB-irradiated fibroblasts, the expression of FOXO1A mRNA decreased significantly. The expression of type I collagen (COLIAI) also decreased. On the other hand, MMP-1 and MMP-2 mRNA levels increased. FOXO1A-small interfering RNA transfection induced the downregulation of FOXO1A expression, it also induced a decrease in COLIAI expression, and it increased MMP-1 and MMP-2 expression. These changes are similar to those observed in UV-irradiated fibroblasts. Furthermore, FOXO1a-peptide induced opposite changes in COLIAI, MMP-1, and MMP-2 expression. Therefore, FOXO1a is involved in the UV-induced changes of type I collagen and MMPs expression.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 60-62; doi:10.1038/jidsymp.2009.2.

Malassezia folliculitis is caused by cutaneous resident Malassezia species.

Malassezia folliculitis [MF] is caused by the invasion of hair follicles by large numbers of Malassezia cells, but it remains unclear which Malassezia species are involved in the disease. To clarify this situation, Malassezia species isolated from lesions of MF patients were analyzed by both culture and non-culture methods. In addition, Malassezia species recovered from the non-lesion areas of the skin of MF patients and skin samples of healthy subjects were included in this study. The test population consisted of 32 MF patients and 40 healthy individuals. The lesions were obtained using a comedone extractor, while swabs were employed to obtain skin samples from non-lesion areas of the patients and healthy subjects. Malassezia DNA was analyzed using a real-time PCR technique. The detection limit of the culture method was 5 CFU/cm(2) as opposes 50 cells/cm(2) with non-culture procedures. The predominant species recovered from MF lesions were M. globosa and M. sympodialis by culture method analysis, and M. restricta, M. globosa, and M. sympodialis with non-culture methods. These results were in agreement with those found with samples from non-lesion skin areas of MF patients and healthy subjects. This study clarified that MF is caused by Malassezia species that are part of the cutaneous microflora and not by exogenous species.

Increase of gremlin 2 with age in human adipose-derived stromal/stem cells and its inhibitory effect on adipogenesis.

INTRODUCTION: Adipose-derived stromal/stem cells (ASCs) have attracted attention as a promising material for regenerative medicine. Previously, we reported an age-related decrease in the adipogenic potential of ASCs from human subjects and found that the individual difference in this potential increased with age, although the mechanisms remain unclear. Recently, other groups demonstrated that a secreted antagonist of bone morphogenetic protein (BMP) signaling, Gremlin 2 (GREM2), inhibits the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts and the adipogenesis of 3T3-L1 cell. Here, we examined the effects of GREM2 on the differentiation of ASCs into adipocytes. METHODS: To examine changes in GREM2 expression levels with age, immunohistochemistry was performed on subcutaneous adipose tissues from subjects 12-97 years of age. Next, GREM2 gene expression levels in ASCs collected from subjects 5-90 years of age were examined by RT-PCR, and the change with age and correlation between the expression level and the adipogenic potential of ASCs were analyzed. In addition, to assess whether GREM2 affects adipogenesis, ASCs (purchased from a vendor) were cultured to induce adipogenesis with recombinant GREM2 protein, and siRNA-induced GREM2 knockdown experiment was also performed using aged ASCs. RESULTS: In adipose tissues, GREM2 expression was observed in cells, including ASCs, but not in mature adipocytes, and the expression level per cell increased with age. GREM2 expression levels in ASCs cultured in vitro also increased with age, and the individual differences in the level increased with age. Of note, partial correlation analysis controlled for age revealed that the adipogenic potential of ASCs and the GREM2 gene expression level were negatively correlated. Furthermore, based on a GREM2 addition experiment, GREM2 has inhibitory effects on the adipogenesis of ASCs through activation of Wnt/ß-catenin signaling. On the other hand, GREM2 knockdown in aged ASCs promoted adipogenesis. CONCLUSIONS: The GREM2 expression level was confirmed to play a role in the age-related decrease in adipogenic potential observed in ASCs isolated from adipose tissues as well as in the enhancement of the individual difference, which increased with age. GREM2 in adipose tissues increased with age, which suggested that GREM2 functions as an inhibitory factor of adipogenesis in ASCs.