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High resolution single cell transcriptomics reveals heterogeneity of self-renewing hair follicle stem cells

ABSTRACT: Purpose: To dissect the transcriptomic profiles and unravel population-specific transcriptional heterogeneity of self renewing hair follicle stem cells in vivo Methods: We performed 10x genomics single-cell RNA sequencing (scRNA-seq) of FACS sorted CD34+/K14-H2BGFP+ hair follicle stem cells from mouse skin at mid-anagen. FACS purified CD34+/K14-H2BGFP+ single-cell suspension was processed for the barcoded single-cell 3′ cDNA libraries generation using Chromium Single Cell 3′ gel bead and library Kit v3. The final libraries were quantified using Agilent Bioanalyzer high sensitivity DNA chip and sequenced using an Illumina NextSeq-500. The raw data files were demultiplexed to generate the sample-specific FASTQ files, which were aligned to the mouse reference genome (mm10-3.0.0) using the 10x Genomics Cell Ranger pipeline (v3.1.0). The raw scRNA-seq data was processed using Cell Ranger from the 10x platform to generate an expression matrix that was further analyzed in R using the Seurat package version 3.1. Only high-quality cells that had between 200 and 5000 genes expressed and had under 10% of the UMIs mapped to mitochondrial genes were retained. Results: We obtained a total of 6736 high quality cells from two datasets for further analysis by Seurat workflow Conclusions: Obtained high quality single cell transcriptomic data to dissect molecular heterogeneity of hair follicle stem cells

ORGANISM(S): Mus musculus

PROVIDER: GSE162333 | GEO | 2021/04/14

REPOSITORIES: GEO

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Project description:Increasing evidence suggests that microRNAs may play important roles in regulating self-renewal and differentiation in mammalian stem cells (SCs). Here, we explore this issue in skin. We first characterize microRNA expression profiles of skin SCs versus their committed proliferative progenies and identify a microRNA subset associating with “stemness”. Of these, miR-125b is dramatically downregulated in early SC-progeny. We engineer an inducible mice system and show that when miR-125b is sustained in SC-progenies, tissue balance is reversibly skewed towards stemness at the expense of epidermal, oil-gland and HF differentiation. Using gain-and-loss of function in vitro, we further implicate miR-125b as a repressor of SC differentiation. In vivo, transcripts repressed upon miR-125b induction are enriched >700% for predicted miR-125b targets normally downregulated upon SC-lineage commitment. We verify some of these miR-125b targets, and show that Blimp1 and VDR in particular can account for many tissue imbalances we see when miR-125b is deregulated. We used microarrays to compare the global miRNA expression profile of P4 stage hair follicle ORS cells from DTG (K14-rtTA,TRE-miR-125b) and control littermates. Hair follicle cells were isolated from P4 Backskin (Dox since P3 for 24hrs) of DTG (K14-rtTA/TRE-miR-125b/K14-H2BGFP), TRE (TRE-miR-125b/K14-H2BGFP), KrtA (K14-rtTA/K14-H2BGFP) as following: interfollicular epidermis sheet was pealed from hair follicle & dermis after dispase treatment.The hair follicle & dermis were first digested by collagenase (Sigma). Intact hair follicles were separated from dermal cells by low speed spinning (20g). The hair follicles were then digested by Trypsin and filtered by 40 µm cell strainers. The isolated hair follicle cells were FACS sorted. During FACS, cells were first gated against CD34 (endothelial cells), CD45 (immune cells), CD114 (melanocytes) and DAPI (dead cells). ORS cells were sorted from the remaining cells as α6HiGFPHi.

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High resolution single cell transcriptomics reveals heterogeneity of self-renewing hair follicle stem cells

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