Abstract
Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Fibroblasts are an ill-defined population consisting of highly proliferative cells that, contrary to human SCs, do not undergo senescence in culture. We initiated this study by performing an exhaustive immunological and functional characterization of adult nerve-derived human SCs and fibroblasts to reveal their properties and optimize a protocol of magnetic-activated cell sorting (MACS) to separate them effectively both as viable and biologically competent cells. We next used immunofluorescence microscopy imaging, flow cytometry analysis and next generation RNA sequencing (RNA-seq) to unambiguously characterize the post-MACS cell products. High resolution transcriptome profiling revealed the identity of key lineage-specific transcripts and the clearly distinct neural crest and mesenchymal origin of human SCs and fibroblasts, respectively. Our analysis underscored a progenitor- or stem cell-like molecular phenotype in SCs and fibroblasts and the heterogeneity of the fibroblast populations. In addition, pathway analysis of RNA-seq data highlighted putative bidirectional networks of fibroblast-to-SC signaling that predict a complementary, yet seemingly independent contribution of SCs and fibroblasts to nerve regeneration. In sum, combining MACS with immunochemical and transcriptomics approaches provides an ideal workflow to exhaustively assess the identity, the stage of differentiation and functional features of highly purified cells from human peripheral nerve tissues.
Highlights
Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth
Fibroblasts are a major component of the contaminating populations (Fig. 2a) but other cell types such as immune cells, stem cells and pericytes can prevail in cultures of human origin
The non-glial cells can effectively adhere to uncoated, plastic dishes and this differential attribute can be exploited to reduce the impact of fibroblast contamination in SC cultures[22]
Summary
Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Our primary goal was to perform a phenotypic and functional analysis of peripheral nerve-derived human SC cultures to reveal the prevalent cell types in the populations and ascertain the most effective strategy for magnetic cell labeling, separation by MACS and characterization of the post-MACS cell products. The RNA-seq profiles of human SCs and fibroblasts revealed their non-overlapping molecular signatures underlying their respective neural crest and mesenchymal origins. Both SCs and fibroblasts displayed gene expression profiles consistent with progenitor cells capable to promote nerve tissue regeneration though independent mechanisms involving cell–cell signaling via direct membrane contact, secreted soluble factors, and interactions with the ECM. Our workflow can suit the needs of assorted basic and translational projects in which understanding human SC function, or using the cells themselves as a cell product, has relevance to investigations on peripheral nerve development, therapy, or neurodegenerative disease
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