Adult bone tissue marrow and skeletal muscle have been shown to contain a subpopulation of cells called side EN-7 population (SP) cells that can be isolated with the fluorescence-activated cell sorter. Adult stem cells with the potential to give rise to progenitor cells of multiple lineages have been described in the hematopoietic system (4-8) as well as in tissues where the demand for renewal is not as heavy such as skeletal muscle (5-8) and the central nervous system (9 10 The ability of these adult stem cells to differentiate and be integrated into multiple tissues offers new insights into cellular plasticity commitment and differentiation and opens the door for cell-based therapy. The skin is a particularly attractive source of stem cells to address both developmental and therapeutic questions. Its structure and cells are well characterized and stem cells have been found to be present in both the epidermis and dermis (11-14). Methods for the isolation of specific cell types have been developed and media formulations for expansion of both mouse and human skin cells are available. Finally skin is BMS-690514 easily accessible for transplant and gene therapy in humans. However purification of skin stem cells has BMS-690514 proven difficult. It often requires an culture period (13 15 16 with BMS-690514 unknown effects on stem cell function or relies on differences in the levels of expression BMS-690514 of surface markers also found on more differentiated cells (17). A technique based on the exclusion of the DNA binding dye Hoechst 33342 has been used to identify and purify using a fluorescence-activated cell sorter (FACS) a subpopulation of cells termed side population or SP cells from both adult bone marrow (5 18 and skeletal muscle (5). When introduced into the circulation of lethally irradiated mice an animal model for Duchenne muscular dystrophy (19) these cells have been shown to be able to regenerate the entire hematopoietic compartment and engraft in muscle by fusing with dystrophic muscle fibers (5). We therefore asked whether the same methodology could be used to purify SP cells from the adult mouse skin and whether they might be able to engraft in muscle without additional irradiation damage. Materials and Methods BMS-690514 Isolation of SP Cells. Male C57BL/6 and C57BL/10SnJ mice (6-24 weeks old; The Jackson Laboratory) were used. Bone marrow and muscle SP cells were isolated as described (5 18 For the isolation of skin SP cells mice were shaved and the dorsal and ventral skin was removed. Subcutaneous (s.c.) fat and muscle tissues were removed by scraping. The skin was minced and digested for 45 min at 37°C in 2 mg/ml collagenase IV and 1.2 units/ml dispase (Worthington). Trypsin was not used because most cell surface markers studied here were sensitive to trypsin digestion possibly explaining previously reported data (20). Cells were resuspended at 106 cells per ml in PBS with 0.5% BSA (Sigma) and incubated in 12.5 μg/ml Hoechst dye 33342 (Sigma) for 1 h at 37°C. An aliquot was stained with Hoechst in the presence of 100 μM verapamil. Propidium iodide (2 μg/ml) was added before FACS analysis. For injection into female mice (The Jackson Laboratory). Three months later muscles were harvested and fresh-frozen in liquid nitrogen. Preparation of the Y-chromosome probe (a kind gift of E. Snyder Harvard Medical School) immunohistochemistry for dystrophin with the 6 antibody and hybridization were performed as described (5). Control male muscle sections were also hybridized to determine probe efficiency defined as the percentage of BMS-690514 Y-chromosome-positive nuclei for 200-400 total male nuclei. Probes that were 230-310 bp long had an efficiency of hybridization >48% and showed a high signal with minimal background were used. Specificity of the signal observed on a 4 6 (DAPI)-labeled nucleus was first assayed by ensuring that signal could be detected only in the rhodamine filter and not in the fluorescein filter. Signals that were scored as specific were confirmed by using the more sensitive charge-coupled device (CCD) camera. To test whether the signal coincided with the DAPI-labeled DNA pictures were taken in sequential focal planes. Any signal that was not in the same plane of focus as the DAPI-labeled DNA was discarded. When this screening method was used no FISH signal was detected in control noninjected female mice. Consecutive sections were examined under a Zeiss Axioplan microscope and images were collected by using a CCD camera. For total fiber counts two additional consecutive sections were stained.