RNA-sequencing of sorted epidermal cells from murine dorsal skin
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE149065 | GEO | 2022/04/21
REPOSITORIES: GEO
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Project description:The skin epidermis is a highly compartmentalised tissue consisting of a cornifying epithelium called the interfollicular epidermis (IFE) and associated hair follicles (HFs). Several stem cell populations have been described that mark specific sub compartments in the skin but none of them is IFE-specific. Here we identify Troy as a marker of IFE and HF infundibulum basal layer cells in embryonic and adult human and mouse epidermis. Genetic lineage-tracing experiments demonstrate that Troy-expressing basal cells contribute to long-term renewal of all layers of the cornifying epithelium. Single-cell transcriptomics and organoid assays of Troy-expressing cells as well as their progeny confirmed stem cell identity as well as the ability to generate differentiating daughter cells. In conclusion, we define Troy as a marker of epidermal basal cells that govern interfollicular epidermal renewal and cornification.
2021-08-05 | GSE165379 | GEO
Project description:There are multiple stem cells in adult mammalian epidermis, but the mechanisms controlling lineage specification are poorly understood. To identify gene expression signatures of the three major epidermal differentiation compartments we micro-dissected individual SG, IFE and HF from adult epidermis. The RNA was isolated from age and sex matched wild-type mice and performed transcriptome analysis with Affymetrix Exon microarrays We micro-dissected individual interfollicular epidermis (IFE), hair follicle (HF) and sebaceous gland (SG) from adult tail epidermis. The RNA was isolated from age and sex matched wild-type mice. Three biological replicates for each epidermal differentiation compartment were analyzed.
2017-05-16 | E-GEOD-62604 | biostudies-arrayexpress
Project description:The epidermis and associated appendages ensure a number of critical functions necessary for survival and social interactions. Perturbations of epidermal stem cell homeostasis lead to a variety of skin diseases affecting humans and dogs. Therefore, the establishment of an in vitro system to investigate canine epidermal stem cells, representing a model for human diseases, is essential. Here we report the establishment and characterization of organoids derived from microdissected canine hair follicles (HFs) and interfollicular epidermal (IFE). Gene and protein expression analysis revealed high mRNA and protein levels of keratin 5 and 14, IFE differentiation markers and intercellular molecules (e.g., keratin 10 and desmoglein), indicating a strong basal cell signature as well as differentiation towards mature epidermis. Key markers of HF stem cells were lacking. This suggests that, independently of the tissue of origin, both organoid lines develop into the same cell type (basal IFE-like keratinocytes). Signaling pathways members important for regulation of HF growth and cycling (such as Wnt, Hh, BMP and Notch) were present in both HF and IFE organoids at low levels. Withdrawal of growth factors (Noggin, R-spondin and FGFs) resulted in upregulation of markers such as KRT16, IVL, KRT17 and SOX9, showing the potential of the organoids to develop towards more differentiated tissue. However, for induction of HF signatures or hair growth, addition of different growth factors or dermal papilla co-culture might be required. Taken together, our in vitro culture system can provide the basis to address hair growth and explore epidermal function/regeneration, allowing us to further investigate pathomechanisms of cutaneous disorders in dogs and potentially human patients.
2018-07-02 | GSE104622 | GEO
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
Project description:There are multiple stem cells in adult mammalian epidermis, but the mechanisms controlling lineage specification are poorly understood. To identify gene expression signatures of the three major epidermal differentiation compartments we micro-dissected individual SG, IFE and HF from adult epidermis. The RNA was isolated from age and sex matched wild-type mice and performed transcriptome analysis with Affymetrix Exon microarrays
2017-05-16 | GSE62604 | GEO
Project description:Using lineage-tracing in a well-established psoriasis-like mouse model with inducible epidermal deletion of c-Jun and JunB, we found that mutant HF-SCs survive and express a broad group of pro-inflammatory cytokines, whereas mutant inter-follicular epidermal cells (IFE) disappear over time. Mutant HF-SCs initiate epidermal hyperplasia and skin inflammation by priming neighboring non-mutant epidermal cells to acquire a psoriasis-like phenotype. To explore the molecular mechanisms that govern the behavior of these distinct mutant and non-mutant HF-SCs and IFE cell populations during psoriasis-like disease, RNA sequencing analyses of sorted GFP+, Tomato+ HF-SCs and b-KCs from DKO*-mT/mG mice.
2019-08-23 | GSE119762 | GEO
Project description:The skin interfollicular epidermis (IFE) is the first barrier against the external environment and its maintenance is critical for survival. Two seemingly opposite theories have been proposed to explain IFE homeostasis. One posits that IFE is maintained by a long-lived slow-cycling stem cell (SC) population that give rise to short-lived transit-amplifying (TA) cell progeny, while the other suggests that homeostasis is achieved by a single committed progenitor (CP) that balances stochastic fate. Here, we probed the cellular heterogeneity within the IFE using two different inducible CREER targeting IFE progenitors. Quantitative analysis of clonal fate data and proliferation dynamics demonstrate the existence of two distinct proliferative cell compartments composed of slow-cycling SC and CP, both of which undergo population asymmetric self-renewal. However, following wounding, only SCs contribute substantially to the repair and long-term regeneration of the tissue, while CP cells make a minimal and transient contribution. Transcriptional profile of InvCREER/RosaYFP and K14CREER/RosaYFP targeted FACS-isolated alpha6 integrin-high CD34-neg basal cells from mouse tail epidermis. Skin epidermis was removed from tail bone and incubated overnight in HBSS (Gibco) 0.25% trypsin (Gibco) at 4M-BM-0C. Epidermis was separated from the dermis and incubated on a rocking plate (100 rpm) at room temperature for 5 min. Basal cells were mechanically separated from the epidermis by flushing 10 times under the epidermis. Tissues were then cut in pieces of 1 mm2 with scalpel, and trypsin was neutralized by adding DMEM medium (Gibco) supplemented with 2% Chelex Fetal Calf Serum (FCS). Samples were filtrated on 70 and 40M-BM-5m filter (Falcon). Immunostaining was performed using biotin-conjugated anti-CD34 (clone RAM34; BD Biosciences) PE-conjugated anti-M-NM-16-integrin (clone GoH3; BD biosciences). Primary antibodies were washed with 2% FCS/PBS and cells were incubated for 30 min in APC conjugated streptavidin (BD Biosciences) secondary antibodies, on ice, with shaking every 10 min. Living K14- and involucrin-expressing epidermal cells were gated by forward scatter, side scatter, negative staining for Hoechst dye and by following the YFP signal. Basal cells from the interfollicular epidermis were targeted using CD34 negative alpha 6 high gating. Fluorescence-activated cell sorting analysis was performed using FACSAria I at high pressure (70 psi) and FACSDiva software (BD Biosciences). Sorted cells were harvested directly in the lysis buffer provided by the RNeasy microkit (QIAGEN) supplemented with 1M-BM-5l of beta-mercaptoethanol for every 100M-BM-5l of lysis buffer. RNA extraction was performed on freshly sorted cells according to the manufacturerM-bM-^@M-^Ys protocol.
2012-09-07 | E-GEOD-36688 | biostudies-arrayexpress
Project description:Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker-positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD34- and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD34- cells (p = 0.03), whereas mRNA of the mesenchymal marker Pdgfrα (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p = 0.04). FACS analysis confirmed the existence of a Pdgfrα positive subpopulation within Epcam+/Sca-1+/CD34- epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD34- cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,- cells in the biology of the murine lung.
2014-01-01 | GSE52215 | GEO
Project description:Our study examined a population of radial glial-like cells (RGLCs) in the dorsal spinal cord midline that we showed provide long-distance growth support for longitudinal rapidly adapting (RA) mechanoreceptor axons during development of spinal cord dorsal column. To evaluate potential molecular markers of these cells, we isolated the RGLCs using hematoxylin and eosin staining to visualize the cell bodies in the dorsal column midline from E14.5 mouse embryos, and used laser capture microdissection for each sample ("LCM"). To compare transcript expression to the adjacent RA mechanoreceptive axons, we performed FACS of dorsal root ganglion of E14.5 Ret-Tdtomato+ RA mechanoreceptors. Our analyses revealed a high enrichment of radial glial-specific markers in the LCM replicates compared to Ret-Tdt samples. In contrast, neuronal-specific markers were more highly enriched in the Ret-Tdt samples, as expected. These data suggest the midline RGLCs are of radial glial identity. Others may find these data helpful in determining potential RGLC-mechanoreceptor molecular interactions in subsequent studies.
2018-05-30 | GSE114193 | GEO
Project description:Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker-positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD34- and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD34- cells (p = 0.03), whereas mRNA of the mesenchymal marker PdgfrM-NM-1 (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p = 0.04). FACS analysis confirmed the existence of a PdgfrM-NM-1 positive subpopulation within Epcam+/Sca-1+/CD34- epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD34- cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,- cells in the biology of the murine lung. Single cells of 10 Sca-1+/CD34+/CD31-/CD45-, 7 Sca-1+/CD34-/CD31-/CD45-, and 12 Sca-1-/CD34-/CD31-/CD45- were analyzed. Although the raw data are two channel only Cy5 signal of each file as analyzed. The Cy5 channel for each gene is normalized to the average Cy5 intensity of the gene across all samples.
2014-01-01 | E-GEOD-52215 | biostudies-arrayexpress