We have recently shown that the expression of nestin, the neural stem cell marker protein, is expressed in bulge-area stem cells of the hair follicle. We used transgenic mice with GFP expression driven by the nestin regulatory element [nestin-driven GFP (ND-GFP)]. The ND-GFP stem cells give rise to the outer-root sheath of the hair follicle as well as an ND-GFP interfollicular vascular network. In this study, we demonstrate that ND-GFP stem cells isolated from the hair-follicle bulge area that are negative for the keratinocyte marker keratin 15 can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. These pluripotent ND-GFP stem cells are positive for the stem cell marker CD34, as well as keratin 15-negative, suggesting their relatively undifferentiated state. The apparent primitive state of the ND-GFP stem cells is compatible with their pluripotency. Furthermore, we show that cells derived from ND-GFP stem cells can differentiate into neurons after transplantation to the subcutis of nude mice. These results suggest that hair-follicle bulge-area ND-GFP stem cells may provide an accessible, autologous source of undifferentiated multipotent stem cells for therapeutic application.bulge area ͉ GFP ͉ differentiation ͉ glial cell ͉ smooth muscle cell ͉ transgenic mice

green fluorescent protein ͉ nerve regeneration ͉ nestin ͉ glial cell ͉ bulge area

Besides forming hair shafts, the highly organized, metabolically vigorous hair follicle plays several crucial roles in skin architecture. The follicle contains a distinct population of presumptive follicular stem cells that express nestin, also a marker for neural stem cells. These nestin-expressing follicle cells are located principally in the follicular bulge region. Nestin-driven GFP (ND-GFP), transfected into mice, principally labels cells in the bulge region, which is consistent with the cells' being the stem cells of the hair follicle. We report here that ND-GFP also labels developing skin blood vessels that appear to originate from hair follicles and form a folliclelinking network. This is seen most clearly by transplanting ND-GFP-labeled vibrissa (whisker) hair follicles to unlabeled nude mice. New vessels grow from the transplanted follicle, and these vessels increase when the local recipient skin is wounded. The ND-GFPexpressing structures are blood vessels, because they display the characteristic endothelial-cell-specific markers CD31 and von Willebrand factor. This model displays very early events in skin angiogenesis and can serve for rapid antiangiogenesis drug screening.GFP ͉ skin angiogenesis ͉ interfollicle network ͉ wound healing ͉ stem cells H air growth is a unique cyclic regeneration phenomenon. The hair follicle undergoes repeated cycles of periods of growth (anagen), regression (catagen), and rest (telogen) throughout the life of mammals (1). The progenitor or stem cells for the outer-root sheath and possibly other structures of the hair follicle appear to reside in a permanent upper portion of the hair follicle, the so-called bulge area (2, 3). This region has been shown to contain the slow-cycling cells or label-retaining cells that mark a stem cell population. Taylor et al. (4) reported that, during the follicle growth cycle, bulge stem cells differentiate into the various cell types of the hair follicle and can, in addition, form a variety of epidermal cells. A similar result was obtained by Fuchs and coworkers (5), who engineered transgenic mice to express histone H2B-GFP controlled by a tetracyclineresponsive regulatory element as well as a keratin-5 promoter. Bulge cells behaved as label-retaining cells, consistent with a stem cell role. During anagen, newly formed GFP-positive populations, derived from the bulge stem cells, form the outerroot sheath hair matrix cells, hair, and inner-root sheath. Also, in response to wounding, some GFP-labeled stem cells exited the bulge, migrated, and proliferated to repopulate the infundibulum and epidermis (5). Other experiments (2) have shown that, in addition to the bulge area, the upper outer-root sheath of vibrissa (whisker) follicles of adult mice may contain stem cells. These can differentiate into hair-follicle matrix cells, sebaceous gland basal cells, and epidermis. Morris et al. (6) used the keratin-15 promoter to drive GFP in the hair-follicle bulge cells. They showed that bulge cells in adult mice generate all epithelial cell types...

The mouse hair follicle is an easily accessible source of actively growing, pluripotent adult stem cells. C57BL transgenic mice, labeled with the fluorescent protein GFP, afforded follicle stem cells whose fate could be followed when transferred to recipient animals. These cells appear to be relatively undifferentiated since they are positive for the stem cell markers nestin and CD34 but negative for the keratinocyte marker keratin 15. These hair follicle stem

We report here a novel transgenic nude mouse for the visualization of human tumor angiogenesis. We have recently shown that the neural stem cell marker nestin is expressed in hair follicle stem cells and blood vessel networks in the skin of C57/B6 transgenic mice with nestin regulatory elementdriven green fluorescent protein (ND-GFP). Others have shown ND-GFP is expressed in the brain, pancreas, and testes in these mice. In the present study, the nestin ND-GFP gene was crossed into nude mice on the C57/B6 background to obtain ND-GFP nude mice. ND-GFP was expressed in the brain, spinal cord, pancreas, stomach, esophagus, heart, lung, blood vessels of glomeruli, blood vessels of skeletal muscle, testes, hair follicles, and blood vessel network in the skin of ND-GFP nude mice. Human lung cancer, pancreatic cancer, and colon cancer cell lines as well as a murine melanoma cell line and breast cancer tumor cell line expressing red fluorescent protein were implanted orthotopically, and a red fluorescent proteinexpressing human fibrosarcoma was implanted s.c. in the ND-GFP nude mice. These tumors grew extensively in the ND-GFP mice. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, visualized by dual-color fluorescence imaging. Results of immunohistochemical staining showed that CD31 was expressed in the ND-GFP-expressing nascent blood vessels. The ND-GFP transgenic nude mouse model enables the visualization of nascent angiogenesis in human and mouse tumor progression. These results suggest that this model is useful for the imaging of the angiogenesis of human as well as rodent tumors and visualization of the efficacy of angiogenetic inhibitors. (Cancer Res 2005; 65(12): 5352-7)

The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral-, plasmid- or transposon-mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K-15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary-acidic-protein (GFAP)-positive Schwann cells and promoted the recovery of pre-existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells.

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The aim of the present study was to analyze the characteristics of atopic dermatitis (AD) in the senile phase. Subjects were comprised of 16 patients investigated for clinical features, serum immunoglobulin (Ig)E levels and skin manifestations. Mean age was 76.9 +/- 6.2 years (range, 68-87), with a man : woman ratio of 3:1. Mean age at onset was 67.7 +/- 15.7 years. Eight patients (50%) had personal histories of chronic eczema until the young adult phase and three patients (18.8%) showed the classic course of child AD. Eczematous erythroderma in 10 patients (62.5%) and unclassified chronic eczema in five patients (31.3%) were the predominant clinical presentations. Mean total IgE level in sera of the 16 patients was 8810 +/- 13 511 IU/mL (range, 5-53 605). Fourteen patients showed positive results for antigen-specific IgE antibodies, and the mean total IgE level for these patients was 10 056 +/- 14 044 IU/mL. Specific IgE to the main antigen, Dermatophagoides farinae, was observed in 12 patients (85.7%), representing the principal antibody in eight patients (57.1%). Eczematous dermatitis manifested predominantly in the face and neck, trunk and extensor and flexure sites of extremities, and less commonly in the antecubital and popliteal areas. Other stigmata of AD were observed as follows: red face in 10 patients (62.5%); Hertoghe's sign in six (37.5%); goose-skin in four (25%); facial pallor in three (18.8%); and dirty neck in one (6.3%). These results indicate that senile-type AD represents a characteristic subgroup of AD that appears in the last stage of life in AD patients.