Magnetic separation of peripheral nerve-resident cells underscores key molecular features of human Schwann cells and fibroblasts: an immunochemical and transcriptomics approach

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.

Heregulin Activity Assays for Residual Testing of Cell Therapy Products

“…We should emphasize that these assays are flexible and can be adapted accordingly to assess the presence of ligands/factors other than heregulin, and signaling pathways other than ErbB, ERK and Akt. In particular, the phosphorylation of the latter kinases can serve as a readout for undefined serum factors or autocrine molecules released by the cultured cells themselves, since hSCs and fibroblasts express the transcripts of a wide variety of ligands for membrane receptors that use ERK and/or Akt as signal transducers [ 25 ]. Though the heregulin activity in the hSC medium is expected to be derived from the recombinant heregulin peptide used as an additive during culture, the contribution of endogenous activity cannot be ruled out, as the hSC cultures themselves can synthesize NRG-related molecules.…”

“…Though the heregulin activity in the hSC medium is expected to be derived from the recombinant heregulin peptide used as an additive during culture, the contribution of endogenous activity cannot be ruled out, as the hSC cultures themselves can synthesize NRG-related molecules. It has been established that fibroblasts in hSC populations express the transcript for NRG1 [ 25 ], thus raising the possibility that some of the heregulin activity detected in hSC-conditioned medium originates from fibroblasts which are highly proliferative and usually populate these cultures in various proportions. It should be noted that our assays cannot discriminate the effect of experimentally added heregulin and heregulin secreted by cells in culture, and/or other factors in the conditioned medium.…”

Using a transection paradigm to enhance the repair mechanisms of an investigational human cell therapy

“…The copyright holder for this preprint this version posted February 23, 2022. ; https://doi.org/10.1101/2022.02.22.22271022 doi: medRxiv preprint associated genes (TWIST1 35 , PDGFRA 35 , and ADAM12) and endoneurial mesenchymal cells (Fig. 2C, D).…”

Human Schwann Cell Transplantation for Spinal Cord Injury: Prospects and Challenges in Translational Medicine

“…For instance, the presence of nerve-derived fibroblasts in hSC cultures has resulted in the deposition of large amounts of collagen and extensive axonal degeneration when transplanted in areas of acute demyelination in the spinal cord of adult rats (Brierley et al, 2001). Whether human fibroblasts are seemingly deleterious in traumatic SCI is unknown, but we suspect the fibroblast populations to be supportive of nerve repair because of their immaturity and their strong proregenerative transcriptional signature (Peng et al, 2020). It should be mentioned that SCs alone are usually ineffective to favorably modify the glial scar.…”