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

stem cells ͉ hair cycle ͉ neurological ͉ GFP ͉ imaging 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. The progenitor or stem cells for the outer-root sheath of the hair follicle were thought to reside in the permanent upper portion of the hair follicle, the so-called bulge area (1, 2).Recently Taylor et al. (3) reported that hair follicle bulge stem cells are potentially bipotent because they can give rise to not only cells of the hair follicle but also to epidermal cells. Other experiments (1) also have provided new evidence that the upper outer-root sheath of vibrissal (whisker) follicles of adult mice contains multipotent stem cells, which can differentiate into hair follicle matrix cells, sebaceous gland basal cells, and epidermis. Recently, Toma et al. (4) reported that multipotent adult stem cells isolated from mammalian skin dermis, termed skin-derived precursors, can proliferate and differentiate in culture to produce neurons, glia, smooth muscle cells, and adipocytes. However, the exact location of these stem cells in skin is unknown, and their functions are still unclear.We report here the expression of nestin, a marker for neural progenitor cells, in the cells of the follicle bulge. Nestin was linked to GFP, allowing us to observe that the nestin-containing cells formed the major part of the hair follicle each cycle. This expression of the neural stem cell protein nestin in hair follicle stem cells suggests a possible relation. Materials and Methods Nestin-GFP Transgenic Mice. Nestin is an intermediate filament (IF) gene that is a marker for CNS progenitor cells and neuroepithelial stem cells (5). Enhanced GFP (EGFP) transgenic mice carrying EGFP under the control of the nestin second-intron enhancer are used for studying and visualizing the self-renewal and multipotency of CNS stem cells (5-7). Here we report that hair follicle stem cells strongly express nestin as evidenced by nestin-regulated EGFP expression.Induction of Anagen. Nestin-regulated GFP transgenic mice, 6 -8 weeks old, in the telogen phase of hair growth were depilated by a hot mixture of rosin and beeswax. Samples (5 ϫ 5 mm 2 ) were excised from the dorsal skin right before depilation (telogen) and at days 1-5 (early anagen), days 8 and 10 (middle anagen), days 14 and 15 (late anagen), and days 19 and 20 (catagen) after depilation. The skin samples were divided into two parts, one for f luorescence microscopy and the other for frozen sections. Brief ly, the skin samples were embedded in tissue-freezing embedding medium and frozen at Ϫ80°C overnight. Sections 8 m thick were cut with a Leica CM1850 cryostat. The frozen sections were air-dried and counterstained with propidium iodide for f luorescence microscopy.Fluorescence and Confocal Microscopy. The nestin-GFP skin samples, after dissecting out the s.c. tissue, were directly observed with dermis up and epidermis down under a N...

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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...

We have developed a simple yet powerful technique for delineating the morphological events of tumor-induced angiogenesis and other tumor-induced host processes with dual-color fluorescence. The method clearly images implanted tumors and adjacent stroma, distinguishing unambiguously the host and tumor-specific components of the malignancy. The dual-color fluorescence imaging is effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice. This model shows with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis and tumor-infiltrating lymphocytes. The GFP-expressing tumor vasculature, both nascent and mature, could be readily distinguished interacting with the RFP-expressing tumor cells. GFP-expressing dendritic cells were observed contacting RFP-expressing tumor cells with their dendrites. GFP-expressing macrophages were observed engulfing RFP-expressing cancer cells. GFP lymphocytes were seen surrounding cells of the RFP tumor, which eventually regressed. Dual-color fluorescence imaging visualizes the tumor-host interaction by whole-body imaging and at the cellular level in fresh tissues, dramatically expanding previous studies in fixed and stained preparations.GFP mouse-RFP tumor ͉ host vessels ͉ tumor-infiltrating lymphocytes ͉ macrophages ͉ dendritic cells T he current interest in angiogenesis is the most recent development in the ongoing study of tumor-host interactions. Although much recent research has focused on the genetic makeup of the tumors themselves, it has long been apparent that host tissues also participate in the phenomena of malignancy. Studies pioneered by Judah Folkman showed that the development of tumor-induced vasculature was essential for tumor growth beyond an initial small size (1). This remarkable finding afforded important new insights into the mechanisms regulating tumor growth and, perhaps most importantly, indicated that newly induced blood vessels offered promising new therapeutic targets. The findings that tumor-induced angiogenesis is a critical determinant of tumor growth and that newly formed vessels offer an especially promising target for chemotherapy have greatly increased the importance understanding the tumor-host interaction.One of the earliest indications of the importance of host tissue to tumor growth was the selectivity of metastatic seeding. Target tissues most often were characteristic of the originating tumor. Such metastasis was described in the ''seed and soil'' hypothesis by Paget (2) more than 100 years ago. Paget (2) proposed that tumor cells, or ''seeds,'' were randomly disseminated by vascular routes, but that metastatic deposits grew only on permissive organs, i.e., the ''soil.'' Paget hypothesized that tumors act together with the distant organ to effect tumor metastases. Fidler (3-6) developed the concept of the tumor microenvironment in the host tissue necessary for growth promotion. The metastatic host microenvironment consists of critical host endothelial cells...

Purpose: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, whereas in a tumor Ang2 antagonizes Ang1's function and promotes the initiation of the angiogenic switch. Specifically targeting Ang2 is a promising anticancer strategy. Here we describe the development and characterization of a new class of biotherapeutics referred to as CovX-Bodies, which are created by chemical fusion of a peptide and a carrier antibody scaffold.Experimental Design: Various linker tethering sites on peptides were examined for their effect on CovXBody in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nmol/L IC 50 s and significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in combination with standard of care agents. Tumor samples were analyzed for target engagement, via Ang2 protein level, CD31-positive tumor vasculature, and Tie2 expressing monocyte penetration.Results: Bivalent Ang2 CovX-Bodies selectively block the Ang2-Tie2 interaction (IC 50 < 1 nmol/L) with dramatically improved pharmacokinetics (T ½ > 100 hours). Using a staged Colo-205 xenograft model, significant tumor growth inhibition (TGI) was observed (40%-63%, P < 0.01). Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), whereas tumor microvessel density (P < 0.01) and intratumor proangiogenic Tie2 CD11bþ cells (P < 0.05) were significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang2 CovX-Bodies produced even greater efficacy ($80% TGI, P < 0.01). Conclusion: CovX-Bodies provide an elegant solution to overcome the pharmacokinetic-pharmacodynamic problems of peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in combination with standard-of-care agents.

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