Currently, human-skin derived cell culture is a basic technique essential for dermatological research, cellular engineering research, drug development, and cosmetic development. But the number of ...donors is limited, and primary cell function reduces through cell passage. In particular, since adult stem cells are present in a small amount in living tissues, it has been difficult to obtain a large amount of stem cells and to stably culture them. In this study, skin derived cells were isolated from the epidermis, dermis, and adipose tissue collected from single donor, and immortalization was induced through gene transfer. Subsequently, cell lines that could be used as stem cell models were selected using the differentiation potential and the expression of stem cell markers as indices, and it was confirmed that these could be stably cultured. The immortalized cell lines established in this study have the potential to be applied not only to basic dermatological research but also to a wide range of fields such as drug screening and cell engineering.
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.
The self-duplication and differentiation of dermal stem cells are essential for the maintenance of dermal homeostasis. Fibroblasts are derived from dermal stem cells and produce components of ...connective tissue, such as collagen, which maintains the structure of the dermis. Cell–cell communication is required for the maintenance of tissue homeostasis, and the role of exosomes in this process has recently been attracting increasing attention. Dermal stem cells and fibroblasts have been suggested to communicate with each other in the dermis; however, the underlying mechanisms remain unclear. In the present study, we investigated communication between dermal stem/progenitor cells (DSPCs) and fibroblasts via exosomes. We collected exosomes from DSPCs and added them to a culture of fibroblasts. With the exosomes, COL1A1 mRNA expression was up-regulated and dependent on the Akt phosphorylation. Exosomes collected from fibroblasts did not show the significant up-regulation of COL1A1 mRNA expression. We then performed a proteomic analysis and detected 74 proteins specific to DSPC-derived exosomes, including ANP32B related to Akt phosphorylation. We added exosomes in which ANP32B was knocked down to a fibroblast culture and observed neither Akt phosphorylation nor enhanced type I collagen synthesis. Additionally, an immunohistochemical analysis of skin tissues revealed that ANP32B expression levels in CD271-positive dermal stem cells were lower in old subjects than in young subjects. These results suggest that DSPCs promote type I collagen synthesis in fibroblasts by secreting exosomes containing ANP32B, which may contribute to the maintenance of skin homeostasis; however, this function of DSPCs may decrease with aging.
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.
•Capillary structural abnormalities on sun-exposed skin with skin redness unevenness.•Capillary structural abnormalities include dilation and disappearance.•Capillaries are excessively dilated by ...increased ETBR expression on HUVECs by UVA.•Capillary migration disappears under decreased BMPR2 expression on HUVECs by UVA.
Capillary structural abnormalities cause skin disorders. Mottled redness, i.e., skin redness unevenness, may appear on the sun-exposed skin, suggesting capillary structural abnormalities, although its mechanism remains unclear.
To observe the capillary structures in the sun-exposed skin where skin redness unevenness is likely to occur, and clarify the mechanism of capillary structural abnormalities.
The tissue structures in the skin with skin redness unevenness were observed by LC-OCT. Subsequently, immunostaining of the sun-exposed skin where skin redness unevenness is often observed, was performed. Vascular endothelial cells were UV-irradiated to analyze the expression and functions of genes involved in the capillary structures and morphogenesis.
The skin with skin redness unevenness exhibited scattering of dilated tubular tissue and disturbance of distribution uniformity. Immunostaining of the sun-exposed skin that were more likely to be exposed to UV rays also revealed similarly disorder of capillary structures. In addition, UVA-irradiated vascular endothelial cells exhibited increased expression of ETBR, involved in telangiectasia, decreased expression of BMPR2, involved in the morphogenesis and maintenance of the blood vessels, and reduced migration of the capillaries.
UV rays alter ETBR and BMPR2 expression in the skin capillaries, and cause partial dilation and decreased migration, resulting in capillary structural abnormalities and causing skin redness unevenness.
Summary Background Embryonic stem (ES) cells, bone marrow, adipose tissue or other genetically modified stem cells are being widely used in basic research in the field of regenerative medicine. ...However, there is no specific surface antigen that can be used as a marker of multipotent stem cells. Objective We tried to isolate and collect putative multipotent stem cells from mouse subcutaneous adipose tissue using the p75 neurotrophin receptor (p75NTR) as a marker. Methods Adipose tissue was processed for immunostaining using antibodies anti-CD90, anti-CD105 and anti-Sca-1 as general mesenchymal stem cell (MSC) markers, and anti-p75NTR, an epithelial stem cell and MSC marker. Subsequently, the expression of cell surface markers in adipose tissue-derived stromal vascular fraction culture cells (ADSVF cells) was examined by flow cytometry (fluorescence-activated cell sorting: FACS). Finally, ADSVF cells positive for p75NTR were sorted and cultured to induce their differentiation into adipocytes, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells. Results Cells positive for several of these markers were found in the deep layers of adipose tissue. Among them, those positive for p75NTR differentiated into adipocytes, osteoblasts, chondrocytes, smooth muscle cells and neuronal cells. The rate of differentiation into adipocytes, osteoblasts and neuronal cells was higher for p75NTR-positive cells than for p75NTR-negative cells. Conclusions p75NTR proved to be a useful marker to isolate adipose tissue-derived stem cells (ASCs).
Background
A molecular budding signature (MBS), which consists of seven tumor budding-related genes, was recently presented as a prominent prognostic indicator in colon cancer (CC) using microarray ...data acquired from frozen specimens. This study aimed to confirm the predictive power of MBS for recurrence risk based on formalin-fixed, paraffin-embedded (FFPE) materials.
Methods
This research utilized the same microarray data from a prior multicenter study using FFPE whole tissue sections, which retrospectively reviewed 232 stage II CC patients without adjuvant chemotherapy and 302 stage III CC patients with adjuvant chemotherapy. All patients underwent upfront curative surgery without neoadjuvant therapy between 2009 and 2012. An MBS score was calculated using the mean of log2 each signal of seven genes (
MSLN
,
SLC4A11
,
WNT11
,
SCEL
,
RUNX2
,
MGAT3
, and
FOXC1
) as described before.
Results
The MBS-low group exhibited a better relapse-free survival (RFS) than the MBS-high group in stage II (
P
= 0.0077) and in stage III CC patients (
P
= 0.0003). Multivariate analyses revealed that the MBS score was an independent prognostic factor in both stage II (
P
= 0.0257) and stage III patients (
P
= 0.0022). Especially among T4, N2, or both (high-risk) stage III patients, the MBS-low group demonstrated markedly better RFS compared with the MBS-high group (
P
= 0.0013).
Conclusions
This study confirmed the predictive power of the MBS for recurrence risk by employing FFPE materials in stage II/III CC patients.
Age-related thinning and reduced cell proliferation in the human epidermis are associated with the accumulation of senescent cells and decreases in the number and function of epidermal stem cells.
...This study examined the expression of INHBA/Activin-A in human epidermis and expression differences with age, and the effect of Activin-A on epidermal stem/progenitor cells.
Immunohistochemical staining was used to analyze age-related changes in the expression of INHBA/Activin-A in the epidermal tissue of young and old subjects. Epidermal INHBA/Activin-A expression levels, epidermal morphology, and the number of epidermal stem/progenitor cells or proliferating cells were investigated using older abdominal skin samples. The effects of Activin-A on the development of a three-dimensional (3D) reconstructed epidermis and cell proliferation were also assessed.
INHBA/Activin-A expression levels in the human epidermis increased with age, although they varied among individuals. In the epidermis of older abdominal skin samples, INHBA/Activin-A expression levels negatively correlated with epidermal thickness, the rete ridge depth and the interdigitation index. The proportion of epidermal stem/progenitor cells and proliferating cells decreased with increases in INHBA/Activin-A expression levels. Activin-A had no effect on the differentiation of keratinocytes in the 3D-reconstructed epidermis; however, thinning of the 3D epidermis was noted. Moreover, the addition of Activin-A inhibited the proliferation of epidermal stem/progenitor cells in a concentration-dependent manner.
Age-related increased in INHBA/Activin-A expression levels were observed in the human epidermis, and may contribute to epidermal thinning and decreases in the number of epidermal stem/progenitor cells and proliferative activity.
•The expression of INHBA/Activin-A in human epidermis increased with age.•Reduced epidermal stem/progenitor cells in human epidermis highly expressing INHBA.•Reduced proliferating cells in human epidermis highly expressing INHBA.•Activin-A suppresses the proliferation of epidermal stem/progenitor cells.
Wnt/β‐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/β‐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.
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