Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.

Cutaneous stem cells and wound healing.

In the last two decades, there has been a tremendous increase in the understanding of stem cell biology, including the field of cutaneous stem cells. Extensive stem cell research and potential clinical applications have provided new perspectives in the use of stem cells in the treatment of human skin disorders such as severe burns and wounds, as well as skin cancer and alopecia. Adult, tissue-specific stem cells are required for tissue homeostasis as well as for the ability to respond to insults such as during wound healing. Fetal wounds can heal rapidly without scars, while in adults wound healing decreases with aging, and this likely represents changes in the functional status of stem cells. In this review, we summarize the main characteristics of cutaneous stem cells in general and present the most recent knowledge in our understanding of these stem cells. We also address the difference in tissue regeneration between fetal and adult tissues in the aspect of stem cell biology.

In vitro differences between keratinocyte stem cells and transit-amplifying cells of the human hair follicle.

Epithelial stem cells within the human hair follicle are critical for hair development, hair cycling, wound healing, and tumorigenesis. We and others have previously shown that the hair follicle bulge area contains keratinocyte stem cells, whereas the hair matrix represents the proliferating and differentiating transit-amplifying (TA) cell compartment. In order to better characterize the phenotypic differences between human keratinocyte stem cells and their daughter TA cells, we compared the in vitro properties of cell adhesion, cell migration, clonogenicity, and in vitro life span. Epithelial outgrowths from the hair matrix appeared within 2 d of explant, whereas stem cell outgrowths appeared between 7 and 10 d after explant. Both populations form colonies; however, stem cells from telogen follicles formed more total colonies, and more colonies greater than 3 mm. Upon subculture, stem cells formed colonies until passage 6 and terminally differentiated at passage 7, whereas TA cells only formed colonies until passage 2. Stem cells express more beta1 integrin and adhere more rapidly to collagen IV. Most strikingly, TA cells showed a 7-fold greater mobility on migration assays than stem cells (0.704 vs 0.102 microm per min). These results help define the human hair follicle stem cell and TA cell phenotypes and correlate with the in vivo properties of these compartments.

Dermal papilla-induced hair differentiation of adult epithelial stem cells from human skin.

The epithelial-mesenchymal interactions between keratinocyte stem cells and dermal papilla (DP) cells are crucial for normal development of the hair follicle as well as during hair cycling. During the cyclical regrowth of a new lower follicle, the multipotent hair follicle stem cells are stimulated to proliferate and differentiate through interactions with the underlying mesenchymal DP cells. To characterize the events occurring during the process of epithelial stem cell fate determination, we utilized a coculture system by incubating human hair follicle keratinocyte stem cells with DP cells. Using GeneChip microarrays, we analyzed changes in gene expression within the stem cells upon coculture with the DP over a 5-day time course. A number of important signaling pathways and growth factors were regulated. The hair-specific keratin 6hf (K6hf) gene proved a particularly good marker of hair differentiation, with a 7.9-fold increase in mRNA and resulting increased protein levels. The high expression of K6hf was unique to DP-induced keratinocyte differentiation, since expression of K6hf was not induced by high calcium. Since the beta-catenin signaling pathway has been implicated in hair follicle development, we examined the role of beta-catenin in our system and demonstrated that beta-catenin/lef-1 signaling is required for DP-induced hair differentiation.

Nutrient-sensing mTOR-mediated pathway regulates leptin production in isolated rat adipocytes.

Leptin biosynthesis in adipose cells in vivo is increased by food intake and decreased by food deprivation. However, the mechanism that couples leptin production to food intake remains unknown. We found that addition of leucine to isolated rat adipocytes significantly increased leptin production by these cells, suggesting that postprandial leptin levels may be directly regulated by dietary leucine. The effect of leucine was inhibited by rapamycin and not by actinomycin D. Besides, leucine administration did not increase the amount of leptin mRNA in adipocytes. Therefore, we concluded that leucine activates leptin expression in adipose cells at the level of translation via a mammalian target of rapamycin (mTOR)-mediated pathway. Because leptin is a secreted protein, its biosynthesis is compartmentalized on the endoplasmic reticulum. To analyze mTOR signaling in this subcellular fraction, we separated adipose cells by centrifugation into a heavy membrane fraction that includes virtually all endoplasmic reticulum and the cytosolic extract. Phosphorylation of the major mTOR targets, the ribosomal protein S6 and the translational inhibitor 4E-binding protein (BP)/phosphorylated heat- and acid-stable protein (PHAS)-1, was stimulated by leucine in the cytosolic extract, whereas, in the heavy fraction, S6 was constitutively phosphorylated and leucine only induced phosphorylation of 4E-BP/PHAS-1. We also found that 60-70% of leptin mRNA was stably associated with the heavy fraction, and leucine administration did not change the ratio between compartmentalized and free cytoplasmic leptin mRNA. We suggest that, in adipose cells, a predominant part of leptin mRNA is compartmentalized on the endoplasmic reticulum, and leucine activates translation of these messages via the mTOR/4E-BP/PHAS-1-mediated signaling pathway.