Advanced age drives the progressive degeneration of mammalian hair follicles (HF) and termination of its regeneration cycle. The role of hair follicle dermal stem cell (hfDSC) in this age-associated HF dysfunction remains largely unexplored. Here, we first characterize a novel function of hfDSCs in regenerating the full dermal papilla (DP) after laser ablation. Next, we performed long-term fate mapping, age-dependant in vivo clonal analysis and in vitro proliferation assay on hfDSCs to describe the age-related defects in mesenchymal stem cell self-renewal and differentiation capabilities. Moreover, single-cell RNA-sequencing of the HF mesenchymal population mapped the intrinsic transcriptional changes that may drive their age-related dysfunction. We highlight the requirement of hfDSCs for normal HF regeneration. Our findings characterize a novel function of mesenchymal stem cells, and how this crucial cell becomes dysfunctional with age to drive age-related hair loss.
Overall design: We enriched for mesenchymal cells within the mammalian hair follicle for analysis. We first generated α-SMACreERT2:ROSAYFP transgenic mice, and treated the mice with tamoxifen at 3 and 4 days of age to permanently label the hair follicle dermal stem cells and their progeny. The mice were aged to either young (2mo and 12mo) aged (18mo) cohorts before isolation. To isolate cells of the hair follicle mesenchyme, we utilized Fluorescence activated cell sorting (FACS). We collected two populations of cells for all samples, to a total of 10,000 cells per age cohort (N=2 per cohort). First, we collected 5000 cells that were YFP+ and (Itga9+ or Cd200+), then we collected 5000 cells that were Cd26+ and (Itga9+ or Cd200+). The two populations were mixed to generate two replicates of young (2mo), one replicate of 12mo and one replicate for 18mo. Samples were processed according to 10X Genomics ChromiumTM Single Cell 3’ Reagent Guidelines v2 or v3 Chemistry as per the manufacturer’s protocol. Single cells were sorted into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed by Genome Quebec using Illumina HiSeq4000. We recovered 2707cells for sample 1, 1906 for sample 2, 6998 for sample 2 and 5415 for sample 3. Our estimated doublet rate was approximately ≈3% due to loading of 10,000 cells and we achieved ~50,0000-100,000 reads/cell.
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