GSE125422
Comparison of human skin- and nerve-derived Schwann cells reveals many similarities and subtle genomic and functional differences
Skin is an easily accessible tissue and a rich source of Schwann cells (SCs). Toward potential clinical application of autologous SC therapies, we aim to improve the reliability and specificity of our protocol to obtain SCs from small skin samples. As well, to explore potential functional distinctions between skin-derived SCs (Sk-SCs) and nerve-derived SCs (N-SCs), we used single-cell RNA-sequencing and a series of in vitro and in vivo assays. Our results showed that Sk-SCs expressed typical SC markers. Single-cell sequencing of Sk- and N-SCs revealed an overwhelming overlap in gene expression with the exception of HLA genes which were preferentially up-regulated in Sk-SCs. In vitro, both cell types exhibited similar levels of proliferation, migration, uptake of myelin debris and readily associated with neurites when co-cultured with human iPSC-induced motor neurons. Both exhibited ensheathment of multiple neurites and early phase of myelination, especially in N-SCs. Interestingly, dorsal root ganglion (DRG) neurite outgrowth assay showed substantially more complexed neurite outgrowth in DRGs exposed to Sk-SC conditioned media compared to those from N-SCs. Multiplex ELISA array revealed shared growth factor profiles, but Sk-SCs expressed a higher level of VEGF. Transplantation of Sk- and N-SCs into injured peripheral nerve in nude rats and NOD-SCID mice showed close association of both SCs to regenerating axons. Myelination of rodent axons was observed infrequently by N-SCs, but absent in Sk-SC xenografts. Overall, our results showed that Sk-SCs share near-identical properties to N-SCs but with subtle differences that could potentially enhance their therapeutic utility. Overall design: Human skin and nerve samples were dissected and minced to liberate single cells. Schwann cells were selected by immunopanning based on p75 expression. Schwann cells enriched for p75 were subsequently expanded in vitro for 1 month. All Schwann cell cultures were fed every fourth day and treated identically. Human skin-derived fibroblasts were selected by immnopanning based on Thy-1 expression. Fibroblast cultures were fed every fourth day. Approximately 10,000 single cells from each culture were loaded onto the micro-fluidic platform for barcoding. All samples were processed according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v2 Chemistry as per the manufacturer's protocol. In brief, cultured Schwann cells and fibroblasts were 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. 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. D11 primers, D12 and C12  primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using DTape station 1000. Sequencing was performed first using Illumina MiSeq SR50 to approximate the number of recovered cells in each sample. We recovered 7123 skin-derived Schwann cells , 7137 nerve-derived Schwann cells and 9585 skin-derived fibroblasts. Based on this, we determined lane distributions for sequencing using Illumina HiSeq 4000 PE (75 bp paired-end reads) with a targeted sequencing depth of ~40,000 reads/cell.