The adult hair follicle dermal papilla (DP) and dermal sheath (DS) cells are developmentally active cell populations with a proven role in adult hair follicle-cycling activity and unique inductive powers. In stem cell biology, the hair follicle epithelium has recently been the subject of a great deal of investigation, but up to now, the follicle dermis has been largely overlooked as a source of stem cells. Following the sporadic appearance of muscle, lipid and bone-type cells in discretely isolated follicle DP and DS cell primary cultures, we demonstrated that cultured papilla and sheath cell lines were capable of being directed to lipid and bone differentiation. Subsequently, for the first time, we produced clonal DP and DS lines that had extended proliferative capabilities. Dye exclusion has been reported to be an identifying feature of stem cells; therefore, clonal papilla and sheath lines with differing capacity to exclude rhodamine 123 were cultured in medium known to induce adipocyte and osteocyte differentiation. Both DS- and DP-derived clones showed the capacity to make lipid and to produce calcified material; however, different clones had varied behaviour and there was no obvious correlation between their stem cell capabilities and dye exclusion or selected gene expression markers. As a highly accessible source, capable of being discretely isolated, the follicle has important potentially as a stem cell source for tissue engineering and cell therapy purposes. It will also be interesting to compare follicle dermal stem cell properties with the broader stem cell capabilities discovered in skin dermis and investigate whether, as we believe, the follicle is a key dermal stem cell niche. Finally, the discovery of stem cells in the dermis may have implications for certain pathologies in which abnormal differentiation occurs in the skin.

Summary A trophoblast cell surface antigen has been characterised by a monoclonal antibody (mAb) 5T4, raised following immunisation with solubilised wheat germ agglutinin binding glycoproteins from human syncytiotrophoblast plasma membrane (StMPM). The expression of the 72 kDa glycoprotein was assessed on cryostat sections of a range of neoplastic and non-neoplastic tissues, using an avidin-biotin immunoperoxidase technique. In products of conception, intense reactions were noted with villous syncytiotrophoblast membrane in normal early and term placenta, with weaker positivity of placental site trophoblast. Most normal or non-neoplastic tissues were negative, including liver, kidney, spleen, small intestine, ovary Specifically, sections were washed in two changes of tris buffered saline (TBS) pH 7.6 and then covered with 10% normal horse serum in TBS for 20 min. After draining, the slides were incubated with neat culture supernatant for 30 min in a moist chamber. Following three washes in TBS (5 min each), biotinylated anti-mouse Ig (Vector Laboratories) diluted 1/250 in TBS containing 10% normal human serum was applied. After 30 min incubation in the moist chamber, the slides were washed three times and incubated with the avidin-biotin peroxidase complexes reagent (Vector Laboratories) for 50 min. After three washes in TBS, peroxidase was visualised using a freshly prepared and filtered solution of diaminobenzidine tetrahydrochloride (DAB-Sigma) in TBS containing 0.03% hydrogen peroxide (6 min). Sections were washed in tap water and counterstained in Coles' haematoxylin, dehydrated, cleared and mounted (Ralmount-R.A. Lamb). The immunohistochemical results were interpreted with reference to a set of controls run in parallel with each test. These included sections treated with DAB only to show endogenous peroxidase, omission of the primary antibody and replacement of the primary antibody with one of the same class but of unrelated specificity. Reactivity of mAb 5T4 with fixed and paraffin wax embedded sections of term placental trophoblast was also assessed by immunoperoxidase. 5T4 mAb reactivity was assessed by P.J.S. and G.M.B. and the intensity of staining was scored on an arbitrary scale ( + to + + + ). Very weak or equivocal reactions were scored + /-.Flow cytometric analyses of human bone marrow cells labelled in PBS, 1% BSA 0. 1% azide with 5T4 mAb followed by 1/20 rabbit anti-mouse Ig-FITC (Serotec, Bicester, UK) was performed on a Becton-Dickenson FACS analyser. The purified mAb 5T4 at 10 ig ml-' was tested for interference with the growth of human pluripotential haematopoietic colonies as described by Welte et al. (1985). RadioimmunoassaySyncytiotrophoblast microvillous plasma membranes (StMPM) were prepared as previously described (Hole &

There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with longterm repopulating ability, but these have problems in terms of yield, complexity, or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences, Oxford, U.K.

Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of ''stemness'' have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP ؉ BM cells showed longterm, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues.intestinal stem cell ͉ telomerase ͉ tissue stem cells

Embryonic stem cells (ESCs) are capable of extended selfrenewal and maintenance of pluripotency even after many population doublings. This is supported by high levels of telomerase activity and enhanced antioxidant protection in ESCs, both of which are downregulated during differentiation. To examine the role of telomerase for ESC selfrenewal and differentiation, we overexpressed the reverse transcriptase subunit (Tert) of murine telomerase in ESCs. Increased telomerase activity enhances the self-renewal ability of the Tert-overexpressing ESCs, improves their resistance to apoptosis, and increases their proliferation. The differentiated progeny of wild-type ESCs express little Tert and show shortening of telomeric overhangs. In contrast, the progeny of Tert-overexpressing ESCs maintain high telomerase activity, as well as the length of G-rich overhangs. In addition, these cells accumulate lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Finally, differentiation toward hematopoietic lineages is more efficient as a result of the continued expression of Tert. Microarray analysis revealed that overexpression of Tert altered expression of a variety of genes required for extended self-renewal and lifespan. Our results suggest that telomerase functions as a "survival enzyme" in ESCs and its differentiated progeny by protecting the telomere cap and by influencing the expression patterns of stress response and defense genes. This results in improved proliferation of ESCs and more efficient differentiation, and these results might have profound consequences for stem cell-replacement therapies. Stem Cells 2005;23:516-529