Project description:We sequenced mRNA from FACS purified hair follicle bulge stem cells from 21 d old control and ILK-deficient mice, 3 biological replicates each Examination of mRNA levels in control and ILK-deficient hair follicle bulge stem cells
Project description:We sequenced mRNA from FACS purified hair follicle bulge stem cells from 21 d old control and ILK-deficient mice, 3 biological replicates each
Project description:The human hair follicle bulge is an important niche for keratinocyte stem cells (KSCs). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell-surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSCs. In this study, we determined the distribution of label-retaining cells to define the human anagen bulge. Using navigated laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and activin/bone morphogenic protein signaling were overrepresented in the bulge, while genes responsible for cell proliferation were underrepresented, consistent with the existence of quiescent noncycling KSCs in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1, while CD24, CD34, CD71, and CD146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hiCD24loCD34loCD71loCD146lo) obtained from hair follicle suspensions demonstrated high colony-forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. The stem cell behavior of enriched bulge cells and their utility for gene therapy and hair regeneration will need to be assessed in in vivo assays.
Project description:Hair follicle (HF) regeneration begins when communication between quiescent epithelial stem cells (SCs) and underlying mesenchymal dermal papillae (DP) generates sufficient activating cues to overcome repressive BMP signals from surrounding niche cells. We uncovered a hitherto unrecognized DP transmitter, TGFβ2, which activates Smad2/3 transiently in HFSCs concomitant with entry into tissue regeneration. We used microarrays to detect the genes specifically affected by TGFß receptor II-deficient mice upon HFSC activation. Hair follicle stem cells (HFSCs) of hair gem (HG) and bulge, and total skin keratinocytes were FACS-purified from the mouse back skin at 2nd telogen-to-anagen transition stages.
Project description:Cyclosporine A (CSA) leads to the precocious onset of hair follicle growth, which is driven by premature activation and proliferation of hair follicle stem cells. Here, we identify gene expression changes associated with CSA treatment in hair follicle stem cells prior to the onset of proliferation as a readout for the early events in stem cell actvation. Hair follicle bulge stem cells were FACS-isolated from mouse skin during the 2nd telogen. Two biological replicated were performed.
Project description:Mouse back skin was disassociated to single cells, sorted by cell surface markers and tested by microarrray To compare the gene expression of mouse bulge (CD34+CD200+CD49+) versus secondary hair germ (CD34-CD200+CD49+) versus interfollicular epidermis (CD34-CD200-CD49+) xx Bald scalp retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells Androgenetic alopecia (AGA) or common baldness results from a marked decrease in hair follicle size. This miniaturization may be related to loss of hair follicle stem or progenitor cells. To test this hypothesis, we analyzed bald and non-bald scalp from the same individuals for the presence of hair follicle stem and progenitor cells using flow cytometry to quantitate cells expressing CYTOKERATIN 15 (KRT15), CD200, CD34 and ALPHA-6-INTEGRIN (ITGA6). High levels of KRT15 expression correlated with stem cell properties of small cell size and quiescence. Cells with the highest level of KRT15 expression were maintained in bald scalp; however, distinct populations of CD200high ITGA6high cells and CD34-positive cells were markedly diminished. Consistent with a progenitor cell phenotype, the diminished populations localized closely to the stem-cell rich bulge area but were larger and more proliferative than the bulge stem cells. In functional assays, analogous CD200 high /Itga6 high cells from murine hair follicles were multipotent and generated new hair follicles in skin reconstitution assays. These findings suggest that a defect in stem cell activation plays a role in the pathogenesis of AGA. 4 independent biologic replicates (each pooled from 3 distinct mice) were sorted for Mouse bulge (CD34+CD200+CD49+) versus secondary hair germ (CD34-CD200+CD49+) versus interfollicular epidermis (CD34-CD200-CD49+)
Project description:Mammalian epidermis consists of three self-renewing compartments: the hair follicle, sebaceous gland and interfollicular epidermis. We generated knock-in alleles of murine Lgr6, a close relative to the Lgr5 stem cell gene. Lgr6 was expressed in the earliest embryonic hair placodes. In adult hair follicles, Lgr6+ cells resided in a previously uncharacterized region directly above the follicle bulge. They expressed none of the known bulge stem cell markers. Prenatal Lgr6+ cells established the hair follicle, sebaceous gland and interfollicular epidermis. Postnatally, Lgr6+ cells generated sebaceous gland and interfollicular epidermis, while contribution to hair lineages gradually diminished with age. Adult Lgr6+ cells executed long-term wound repair, including the formation of new hair follicles. We conclude that Lgr6 marks the most primitive epidermal stem cell. For the Lgr5 and Lgr6 stem cell comparison RNA was isolated from sorted GFPhi cell fractions of dorsal skin from Lgr5-EGFP-ires-CreERT2 mice and Lgr6-EGFP-ires-CreERT2, respectively (3 mice per group per sort).
Project description:The human hair follicle bulge is an important niche for keratinocyte stem cells (KSC). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSC. In this study, we determined the distribution of label-retaining cells to carefully define the human anagen bulge. Using navigated-laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and Activin/BMP signaling were over-represented in the bulge while genes responsible for cell proliferation were under-represented, consistent with quiescent non-cycling KSC in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1 while CD24, 34, 71 and 146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hi24lo34lo71lo146lo) obtained from hair follicle suspensions demonstrated high colony forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. Keywords: Affymetrix micrarray analysis of human hair follicles
Project description:Mammalian epidermis consists of three self-renewing compartments: the hair follicle, sebaceous gland and interfollicular epidermis. We generated knock-in alleles of murine Lgr6, a close relative to the Lgr5 stem cell gene. Lgr6 was expressed in the earliest embryonic hair placodes. In adult hair follicles, Lgr6+ cells resided in a previously uncharacterized region directly above the follicle bulge. They expressed none of the known bulge stem cell markers. Prenatal Lgr6+ cells established the hair follicle, sebaceous gland and interfollicular epidermis. Postnatally, Lgr6+ cells generated sebaceous gland and interfollicular epidermis, while contribution to hair lineages gradually diminished with age. Adult Lgr6+ cells executed long-term wound repair, including the formation of new hair follicles. We conclude that Lgr6 marks the most primitive epidermal stem cell.