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A Functional Role of RB-Dependent Pathway in the Control of Quiescence in Adult Epidermal Stem Cells Revealed by Genomic Profiling

ABSTRACT: Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis. Total RNA was prepared from CD34+ and CD34- keratinocytes obtained from pools of at least six mice per MACS isolation procedure. A total of three isolation procedures were performed. RNA extracted from each fraction (CD34+ / CD34-) was assessed for its quality using Agilent 2100 Bioanalyzer (Agilent Technologies) and hybridized to two Affymetrix Mouse 430 2.0 arrays, to ensure reproducibility of the process. This resulted in a total of 12 chips (6 CD34+ / 6 CD34-).

ORGANISM(S): Mus musculus

SUBMITTER: Marta DueÃ±as

PROVIDER: E-GEOD-19448 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A Functional Role of RB-Dependent Pathway in the Control of Quiescence in Adult Epidermal Stem Cells Revealed by Genomic Profiling

Project description:Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis.

2010-03-20 | GSE19448 | GEO

Mouse bulge (CD34+CD200+CD49+) versus secondary hair germ (CD34-CD200+CD49+) versus interfollicular epidermis (CD34-CD200-CD49+)

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+)

2011-01-12 | E-GEOD-21568 | biostudies-arrayexpress

Mouse bulge (CD34+CD200+CD49+) versus secondary hair germ (CD34-CD200+CD49+) versus interfollicular epidermis (CD34-CD200-CD49+)

2011-01-12 | GSE21568 | GEO

Lgr6 Marks Stem Cells in the Hair Follicle that Generate all Cell Lineages of the Skin

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.

2010-03-26 | GSE20269 | GEO

Lgr6 Marks Stem Cells in the Hair Follicle that Generate all Cell Lineages of the Skin

2010-03-25 | E-GEOD-20269 | biostudies-arrayexpress

Transcription profiling of mouse epidermal cells sorted into three populations based on alpha 6 integrin, CD34 or Sca-1 surface marker expression

Project description:Epidermal keratinocytes were isolated from adult murine skin and FACS sorted into three populations based on their surface maker expression of alpha 6 integrin, CD34 and Sca-1. <br>- Alpha 6 positive cells constitute the entire population of basal or proliferative keratinocytes from the interfollicular epidermis and hair follicle. Both BIB and Bulge cells are contained within the total population of alpha 6 positive cells. <br>- BIB cells reside in the hair follicle in a region between the infundibulum and bulge and these cells are characterized by low alpha 6 integrin surface levels and are negative for CD34 and Sca-1 surface proteins. <br>- Bulge cells are derived from the bulge region of the hair follicle and are characterized by low + high alpha 6 integrin surface levels and are positive for CD34 but negative for Sca-1.

2009-11-16 | E-MEXP-928 | biostudies-arrayexpress

Transcription profiling of human stem cells enriched for hair follicle bulge cells

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.

2007-11-08 | E-GEOD-3419 | biostudies-arrayexpress

Akt signalling leads to stem cell activation and promotes tumour development in epidermis.

Project description:A permantly active form of the oncogene Akt was expressed in the keratinocytes of the basal proliferative layer of the epidermis. Stem cells of the hair follicle expressing the cell surface marker CD34 were isolated. RNA form the CD34(+) and CD34(-) keratinocytes was extracted and and hybridized to Mouse Genome 430 2.0 Affymetrix arrays. Gene expression was compared between CD34(+) hair follicle stem cells and CD34(-) cells isolates from the back skin of K5-myrAkt transgenic mice.

2014-01-26 | E-GEOD-48522 | biostudies-arrayexpress

Bald scalp retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells

Project description: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. This SuperSeries is composed of the SubSeries listed below.

2011-01-12 | GSE21569 | GEO

Transcriptional profiling of bulge stem cells

Project description:In adult skin, each hair follicle contains a reservoir of stem cells (the bulge), which can be mobilized to regenerate the new follicle with each hair cycle and to reepithelialize epidermis during wound repair. Here we report new methods that permit their clonal analyses and engraftment and demonstrate the two defining features of stem cells, namely self-renewal and multi-potency. We also show that, within the bulge, there are two distinct populations, one of which maintains basal lamina contact and temporally precedes the other, which is suprabasal and arises only after the start of the first postnatal hair cycle. This spatial distinction endows them with discrete transcriptional programs, but surprisingly, both populations are growth inhibited in the niche but can self-renew in vitro and make epidermis and hair when grafted. These findings suggest that the niche microenvironment imposes intrinsic “stemness” features without restricting the establishment of epithelial polarity and changes in gene expression. FACS was used to isolate pure populations of living total K14-GFP-actin expressing cells and K14-GFP-actin ?6high CD34high cells from postnatal d49 mouse back skins for RNA extraction and hybridization on Affymetrix microarrays. By comparing the transcriptional profile of FACS-isolated GFP ?6high CD34high expressing cells with FACS-isolated total GFP cells, we determined the genes differentially regulated in basal bulge stem cells in telogen stage comparing to total epidermis

2012-10-19 | E-GEOD-41704 | biostudies-arrayexpress

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