A combined immunochemical and transcriptomics approach to reveal the purity and identity of human peripheral nerve Schwann cells and fibroblasts separated by magnetic-activated cell sorting

PAULA MONJE; BLANCHE KUO; DAVID SANT; AISHA KHAN; KRISTINE RAVELO; GAOFENG WANG; RISSET SILVERA; NATALIA ANDERSEN

Buenos Aires

Simposio; The Role of Glia in Health and Disease of the Nervous System: Clinical and Basic Science walking together.; 2017

IQUIFIB

This study was undertaken to develop magnetic-activated cell sorting (MACS) methods suitable for fibroblast elimination in primary Schwann cell (SC) cultures from adult human peripheral nerve tissue. First, we performed an exhaustive immunological characterization of the composition of heterogeneous adherent human SC cultures to justify a purification strategy based on cell surface immuno-labeling with human-specific p75NGFR antibodies. Second, we adjusted experimental conditions to efficiently label, sort and detect the MACS-purified cells that result from using mixed cultures containing various proportions of SCs and contaminating cells. Third, we optimized and validated the use of immunofluorescence microscopy analysis, flow cytometry analysis and next generation RNA sequencing to unambiguously determine the identity and purity of the post-MACS cellular products. Characterization of the MACS-purified cells from different human donors indicated purities >95% SCs and fibroblasts in the retained and eluted fractions, respectively, irrespective of the magnitude of the contaminating population. Transcriptome analysis not only confirmed the presence of known SC- and fibroblast-specific RNAs in the expected fraction but also highlighted a battery of differentially expressed genes that have not been previously appreciated. Overall, our results clearly showed the many advantages of using MACS to efficiently deprive SC cultures from contaminating cells while recovering highly viable fibroblasts for follow up experimentation. Furthermore, the combination of traditional immunochemical methods and state-of-the-art transcriptomics analysis seems ideal to exhaustively characterize the phenotype, purity and gene expression profiles of both SCs and fibroblasts of human origin.