Identification of cellular and noncellular components of mature intact human peripheral nerve.

BACKGROUND AND AIMS: The goal of this study was to define basic constituents of the adult peripheral nervous system (PNS) using intact human nerve tissues. METHODS: We combined fluorescent and chromogenic immunostaining methods, myelin-selective fluorophores, and routine histological stains to identify common cellular and noncellular elements in aldehyde-fixed nerve tissue sections. We employed Schwann cell (SC)-specific markers, such as S100ß, NGFR, Sox10, and myelin protein zero (MPZ), together with axonal, extracellular matrix (collagen IV, laminin, fibronectin), and fibroblast markers to assess the SC's relationship to myelin sheaths, axons, other cell types, and the acellular environment. RESULTS: Whereas S100ß and Sox10 revealed mature SCs in the absence of other stains, discrimination between myelinating and non-myelinating (Remak) SCs required immunodetection of NGFR along with axonal and/or myelin markers. Surprisingly, our analysis of NGFR+ profiles uncovered the existence of at least 3 different novel populations of NGFR+/S100ß- cells, herein referred to as nonglial cells, residing in the stroma and perivascular areas of all nerve compartments. An important proportion of the nerve's cellular content, including circa 30% of endoneurial cells, consisted of heterogenous S100ß negative cells that were not associated with axons. Useful markers to identify the localization and diversity of nonglial cell types across different compartments were Thy1, CD34, SMA, and Glut1, a perineurial cell marker. INTERPRETATION: Our optimized methods revealed additional detailed information to update our understanding of the complexity and spatial orientation of PNS-resident cell types in humans.

Vitamin C regulates Schwann cell myelination by promoting DNA demethylation of pro-myelinating genes.

Ascorbic acid (vitamin C) is critical for Schwann cells to myelinate peripheral nerve axons during development and remyelination after injury. However, its exact mechanism remains elusive. Vitamin C is a dietary nutrient that was recently discovered to promote active DNA demethylation. Schwann cell myelination is characterized by global DNA demethylation in vivo and may therefore be regulated by vitamin C. We found that vitamin C induces a massive transcriptomic shift (n = 3,848 genes) in primary cultured Schwann cells while simultaneously producing a global increase in genomic 5-hydroxymethylcytosine (5hmC), a DNA demethylation intermediate which regulates transcription. Vitamin C up-regulates 10 pro-myelinating genes which exhibit elevated 5hmC content in both the promoter and gene body regions of these loci following treatment. Using a mouse model of human vitamin C metabolism, we found that maternal dietary vitamin C deficiency causes peripheral nerve hypomyelination throughout early development in resulting offspring. Additionally, dietary vitamin C intake regulates the expression of myelin-related proteins such as periaxin (PRX) and myelin basic protein (MBP) during development and remyelination after injury in mice. Taken together, these results suggest that vitamin C cooperatively promotes myelination through 1) increased DNA demethylation and transcription of pro-myelinating genes, and 2) its known role in stabilizing collagen helices to form the basal lamina that is necessary for myelination.

Heregulin Activity Assays for Residual Testing of Cell Therapy Products.

BACKGROUND: Heregulin is a ligand for the protooncogene product ErbB/HER that acts as  a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a ß1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. RESULTS: Western blotting was used to measure the activity of three different ß1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of ß1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect ß1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive ß1-heregulin molecules prior to transplantation. CONCLUSIONS: These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than ß1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.

The properties of human Schwann cells: Lessons from in vitro culture and transplantation studies.

Human Schwann cells (hSCs) can be isolated directly from peripheral nerve and cultured using methods similar to those used for SCs from other species. Yet, important interspecies differences are revealed when the primary or expanded hSCs are compared to their nonhuman counterparts. This review addresses the special properties of nerve-derived hSCs that have resulted to date from both in vitro studies and in vivo research on cell transplantation in animal models and human subjects. A consensus has yet to emerge about the essential attributes of cultured normal hSCs. Thus, an emphasis is placed on the importance of validating hSC cultures by means of purity, identity, and biological activity to reliably use them as in vitro models of the SC phenotype and cell therapy products for injury repair. Combining traditional immunological methods, high-resolution omics approaches, and assorted cell-based assays is so far the best approach to unequivocally identify hSC populations obtained by direct isolation or derivation from stem cells. Special considerations are required to understand and manage the variability and heterogeneity proper of donor batches, as well as to evaluate risk factors. This is particularly important if the intended use of the hSCs is implantation in the human body, diagnosis of disease, or drug testing aimed at targeting any aspect of SC function in human patients. To conclude, in view of their unique properties, new concepts and methods are needed to better understand the biology of hSCs and exploit their full potential in basic science and regenerative medicine.

MPZL2 is a novel gene associated with autosomal recessive nonsyndromic moderate hearing loss.

While recent studies have revealed a substantial portion of the genes underlying human hearing loss, the extensive genetic landscape has not been completely explored. Here, we report a loss-of-function variant (c.72delA) in MPZL2 in three unrelated multiplex families from Turkey and Iran with autosomal recessive nonsyndromic hearing loss. The variant co-segregates with moderate sensorineural hearing loss in all three families. We show a shared haplotype flanking the variant in our families implicating a single founder. While rare in other populations, the allele frequency of the variant is ~ 0.004 in Ashkenazi Jews, suggesting that it may be an important cause of moderate hearing loss in that population. We show that Mpzl2 is expressed in mouse inner ear, and the protein localizes in the auditory inner and outer hair cells, with an asymmetric subcellular localization. We thus present MPZL2 as a novel gene associated with sensorineural hearing loss.

EGFP transgene: a useful tool to track transplanted bone marrow mononuclear cell contribution to peripheral remyelination.

Bone marrow mononuclear cells (BMMC) constitute a heterogeneous population with potential to promote tissue regeneration. For this reason, this cell fraction has recently become a therapeutic alternative to mesenchymal stem cells, as culture is not required and phenotypic transformations can be hence avoided. In this work, and in order to attain long-lasting cell labeling and study longer survival times, we used BMMC isolated from adult transgenic rats expressing GFP to reproduce our wild type model and evaluate their remyelination ability in a reversible model of Wallerian degeneration. RT-PCR and flow cytometry analysis confirmed that cells isolated from the transgenic strain exhibited similar expression levels of markers specific to multipotent progenitors (CD34, CD90 and CD105) and Schwann cells (MPZ, MBP, S100ß and p75NTR) compared to wild type BMMC. BMMC expressing GFP retained their migration capacity, arriving exclusively at the injured nerve. Most importantly, and as detected through long-lasting cell tracking, some of these BMMC settled in the demyelinated area, mingled with endogenous cells, underwent phenotypic changes and colocalized with Schwann cell markers MBP and S100ß. Also worth highlighting, transgenic BMMC replicated wild type BMMC effects in terms of MBP organization and levels. On the basis of these findings, BMMC isolated from transgenic animals constitute a useful tool to evaluate their role in peripheral nervous system demyelination-remyelination and the underlying mechanisms.

Axon contact-driven Schwann cell dedifferentiation.

Mature Schwann cells (SCs) retain dedifferentiation potential throughout adulthood. Still, how dedifferentiation occurs remains uncertain. Results from a variety of cell-based assays using in vitro cultured cAMP-differentiated and myelinating SCs revealed the existence of a novel dedifferentiating activity expressed on the surface of dorsal root ganglion (DRG) axons. This activity had the capacity to prevent SC differentiation and elicit dedifferentiation through direct SC-axon contact. Evidence is provided showing that a rapid loss of myelinating SC markers concomitant to proliferation occurred even in the presence of elevated cAMP, a signal that is required to drive and maintain a differentiated state. The dedifferentiating activity was a membrane-bound protein found exclusively in DRG neurons, as judged by its subcellular partitioning, sensitivity to proteolytic degradation and cell-type specificity, and remained active even after disruption of cellular organization. It differed from the membrane-anchored neuregulin-1 isoforms that are responsible for axon contact-induced SC proliferation and exerted its action independently of mitogenic signaling emanating from receptor tyrosine kinases and mitogen-activated protein kinases such as ERK and JNK. Interestingly, dedifferentiation occurred without concomitant changes in the expression of Krox-20, a transcriptional enhancer of myelination, and c-Jun, an inhibitor of myelination. In sum, our data indicated the existence of cell surface axon-derived signals that override pro-differentiating cues, drive dedifferentiation and allow SCs to proliferate in response to axonal mitogens. This axonal signal may negatively regulate myelination at the onset or reversal of the differentiated state. GLIA 2017;65:851-863.

Human Schwann Cells in vitro III. Analytical Methods and a Practical Approach for Quality Control.

This paper introduces simple analytical methods and bioassays to promptly assess the identity and function of in vitro cultured human Schwann cells (hSCs). A systematic approach is proposed to unequivocally discriminate hSCs from other glial cells, non-glial cells, and non-human SCs (authentication), identify hSCs at different stages of differentiation, and determine whether individual hSCs are proliferative or senescent. Examples of how to use distinct cell-based approaches for quality control and routine troubleshooting are provided to confirm the constitution (identity, purity, and heterogeneity) and potency (bioactivity) of hSC cultures from multiple sources. The bioassays are valuable for rapidly gauging the responses of hSCs to mitogenic and differentiating factors and ascertaining the cells' basic properties before performing co-culture or cell grafting studies. The assays are image based and use adherent hSCs established in monoculture to simplify the experimental setup and interpretation of results. Finally, all sections contain thorough background information, notes, and references to facilitate decision making, data interpretation, and ad hoc method development for diverse applications.

Human Schwann Cells in vitro II. Passaging, Purification, Banking, and Labeling of Established Cultures.

This manuscript describes step-by-step procedures to establish and manage fresh and cryopreserved cultures of nerve-derived human Schwann cells (hSCs) at the desired scale. Adaptable protocols are provided to propagate hSC cultures through serial passaging and perform routine manipulations such as enzymatic dissociation, purification, cryogenic preservation, live-cell labeling, and gene delivery. Expanded hSCs cultures are metabolically active, proliferative, and phenotypically stable for at least three consecutive passages. Cell yields are expected to be variable as determined by the rate of growth of individual batches and the rounds of subculture. The purity, however, can be maintained high at >95% hSC regardless of passage. The cells obtained in this manner are suitable for various applications, including small drug screens, in vitro modeling of neurodevelopmental processes, and cell transplantation. One caveat of this protocol is that continued expansion of same-batch hSC populations is eventually restricted due to senescence-linked growth arrest.

Human Schwann Cells in vitro I. Nerve Tissue Processing, Pre-degeneration, Isolation, and Culturing of Primary Cells.

This paper presents versatile protocols to prepare primary human Schwann cell (hSC) cultures from mature peripheral nervous system tissues, including fascicles from long spinal nerves, nerve roots, and ganglia. This protocol starts with a description of nerve tissue procurement, handling, and dissection to obtain tissue sections suitable for hSC isolation and culturing. A description follows on how to disintegrate the nerve tissue by delayed enzymatic dissociation, plate the initial cell suspensions on a two-dimensional substrate, and culture the primary hSCs. Each section contains detailed procedures, technical notes, and background information to aid investigators in understanding and managing all steps. Some general recommendations are made to optimize the recovery, growth, and purity of the hSC cultures irrespective of the tissue source. These recommendations include: (1) pre-culturing epineurium- and perineurium-free nerve fascicles under conditions of adherence or suspension depending on the size of the explants to facilitate the release of proliferative, in vitro-activated hSCs; (2) plating the initial cell suspensions as individual droplets on a laminin-coated substrate to expedite cell adhesion and thereby increase the recovery of viable cells; and (3) culturing the fascicles (pre-degeneration step) and the cells derived therefrom in mitogen- and serum-supplemented medium to accelerate hSC dedifferentiation and promote mitogenesis before and after tissue dissociation, respectively. The hSC cultures obtained as suggested in this protocol are suitable for assorted basic and translational research applications. With the appropriate adaptations, donor-relevant hSC cultures can be prepared using fresh or postmortem tissue biospecimens of a wide range of types and sizes.

Endogenous Glycoprotein GPM6a Is Involved in Neurite Outgrowth in Rat Dorsal Root Ganglion Neurons.

The peripheral nervous system (PNS) has a unique ability for self-repair. Dorsal root ganglion (DRG) neurons regulate the expression of different molecules, such as neurotrophins and their receptors, to promote axon regeneration after injury. However, the molecular players driving axonal regrowth need to be better defined. The membrane glycoprotein GPM6a has been described to contribute to neuronal development and structural plasticity in central-nervous-system neurons. Recent evidence indicates that GPM6a interacts with molecules from the PNS, although its role in DRG neurons remains unknown. Here, we characterized the expression of GPM6a in embryonic and adult DRGs by combining analysis of public RNA-seq datasets with immunochemical approaches utilizing cultures of rat DRG explants and dissociated neuronal cells. M6a was detected on the cell surfaces of DRG neurons throughout development. Moreover, GPM6a was required for DRG neurite elongation in vitro. In summary, we provide evidence on GPM6a being present in DRG neurons for the first time. Data from our functional experiments support the idea that GPM6a could contribute to axon regeneration in the PNS.

Single Fraction and Hypofractionated Radiation Cause Cochlear Damage, Hearing Loss, and Reduced Viability of Merlin-Deficient Schwann Cells.

BACKGROUND: Vestibular schwannomas (VS) are benign intracranial tumors caused by loss of function of the merlin tumor suppressor. We tested three hypotheses related to radiation, hearing loss (HL), and VS cell survival: (1) radiation causes HL by injuring auditory hair cells (AHC), (2) fractionation reduces radiation-induced HL, and (3) single fraction and equivalent appropriately dosed multi-fractions are equally effective at controlling VS growth. We investigated the effects of single fraction and hypofractionated radiation on hearing thresholds in rats, cell death pathways in rat cochleae, and viability of human merlin-deficient Schwann cells (MD-SC). METHODS: Adult rats received cochlear irradiation with single fraction (0 to 18 Gray [Gy]) or hypofractionated radiation. Auditory brainstem response (ABR) testing was performed for 24 weeks. AHC viabilities were determined using immunohistochemistry. Neonatal rat cochleae were harvested after irradiation, and gene- and cell-based assays were conducted. MD-SCs were irradiated, and viability assays and immunofluorescence for DNA damage and cell cycle markers were performed. RESULTS: Radiation caused dose-dependent and progressive HL in rats and AHC losses by promoting expression of apoptosis-associated genes and proteins. When compared to 12 Gy single fraction, hypofractionation caused smaller ABR threshold and pure tone average shifts and was more effective at reducing MD-SC viability. CONCLUSIONS: Investigations into the mechanisms of radiation ototoxicity and VS radiobiology will help determine optimal radiation regimens and identify potential therapies to mitigate radiation-induced HL and improve VS tumor control.

Using a Transection Paradigm to Enhance the Repair Mechanisms of an Investigational Human Cell Therapy.

One promising strategy in cell therapies for Parkinson's disease (PD) is to harness a patient's own cells to provide neuroprotection in areas of the brain affected by neurodegeneration. No treatment exists to replace cells in the brain. Thus, our goal has been to support sick neurons and slow neurodegeneration by transplanting living repair tissue from the peripheral nervous system into the substantia nigra of those with PD. Our group has pioneered the transplantation of transection-activated sural nerve fascicles into the brain of human subjects with PD. Our experience in sural nerve transplantation has supported the safety and feasibility of this approach. As part of a paradigm to assess the reparative properties of human sural nerve following a transection injury, we collected nerve tissue approximately 2 weeks after sural nerve transection for immunoassays from 15 participants, and collected samples from two additional participants for single nuclei RNA sequencing. We quantified the expression of key neuroprotective and select anti-apoptotic genes along with their corresponding protein levels using immunoassays. The single nuclei data clustered into 10 distinctive groups defined on the basis of previously published cell type-specific genes. Transection-induced reparative peripheral nerve tissue showed RNA expression of neuroprotective factors and anti-apoptotic factors across multiple cell types after nerve injury induction. Key proteins of interest (BDNF, GDNF, beta-NGF, PDGFB, and VEGF) were upregulated in reparative tissue. These results provide insight on this repair tissue's utility as a neuroprotective cell therapy.

Merlin-Deficient Schwann Cells Are More Susceptible to Radiation Injury than Normal Schwann Cells In Vitro.

Objectives Vestibular schwannomas (VS) are intracranial tumors, which are caused by NF2 gene mutations that lead to loss of merlin protein. A treatment for VS is stereotactic radiosurgery, a form of radiation. To better understand the radiobiology of VS and radiation toxicity to adjacent structures, our main objectives were (1) investigate effects of single fraction (SF) radiation on viability, cytotoxicity, and apoptosis in normal Schwann cells (SCs) and merlin-deficient Schwann cells (MD-SCs) in vitro, and (2) analyze expression of double strand DNA breaks (γ-H2AX) and DNA repair protein Rad51 following irradiation. Study Design This is a basic science study. Setting This study is conducted in a research laboratory. Participants Patients did not participate in this study. Main Outcome Measures In irradiated normal SCs and MD-SCs (0-18 Gy), we measured (1) viability, cytotoxicity, and apoptosis using cell-based assays, and (2) percentage of cells with γ-H2AX and Rad51 on immunofluorescence. Results A high percentage of irradiated MD-SCs expressed γ-H2AX, which may explain the dose-dependent losses in viability in rodent and human cell lines. In comparison, the viabilities of normal SCs were only compromised at higher doses of radiation (>12 Gy, human SCs), which may be related to less Rad51 repair. There were no further reductions in viability in human MD-SCs beyond 9 Gy, suggesting that <9 Gy may be insufficient to initiate maximal tumor control. Conclusion The MD-SCs are more susceptible to radiation than normal SCs, in part through differential expression of γ-H2AX and Rad51. Understanding the radiobiology of MD-SCs and normal SCs is important for optimizing radiation protocols to maximize tumor control while limiting radiation toxicity in VS patients.

Schwann cell dedifferentiation is independent of mitogenic signaling and uncoupled to proliferation: role of cAMP and JNK in the maintenance of the differentiated state.

Myelinating Schwann cells (SCs) are highly plastic cells that are able to dedifferentiate and re-enter the cell cycle. However, the molecular signals controlling dedifferentiation are not completely understood. Because a connection between mitogenic signaling and myelin loss has been suggested, we investigated the role of cAMP, a strong inducer of the myelinating phenotype, and mitogenic factors activating receptor tyrosine kinases (RTKs) on SC dedifferentiation. We herein provide evidence indicating that cAMP was required to not only initiate but also maintain a state of differentiation because SCs rapidly dedifferentiated and became competent to resume proliferation upon the removal of cAMP stimulation. Surprisingly, isolated SCs could undergo multiple cycles of differentiation and dedifferentiation upon cAMP addition and removal, respectively, in the absence of mitogenic factors and without entering the cell cycle. Conversely, the activation of RTKs and the ERK cascade by a variety of growth factors, including neuregulin, was not sufficient to initiate dedifferentiation in the presence of cAMP. Importantly, a reduction of cAMP triggered dedifferentiation through a mechanism that required JNK, rather than ERK, activity and an induction of the expression of c-Jun, a transcriptional inhibitor of myelination. In summary, the reversible transition from an undifferentiated to a myelinating state was dependent on cAMP but independent of RTK signaling and cell cycle progression, further indicating that dedifferentiation and proliferation are uncoupled and differentially regulated events in SCs.

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

The benefits of transplanting cultured Schwann cells (SCs) for the treatment of spinal cord injury (SCI) have been systematically investigated in experimental animals since the early 1990s. Importantly, human SC (hSC) transplantation for SCI has advanced to clinical testing and safety has been established via clinical trials conducted in the USA and abroad. However, multiple barriers must be overcome to enable accessible and effective treatments for SCI patients. This review presents available information on hSC transplantation for SCI with the intention to uncover gaps in our knowledge and discuss areas for future development. To this end, we introduce the historical progression of the work that supports existing and prospective clinical initiatives and explain the reasons for the choice of hSCs while also addressing their limitations as cell therapy products. A search of the relevant literature revealed that rat SCs have served as a preclinical model of reference since the onset of investigations, and that hSC transplants are relatively understudied, possibly due to the sophisticated resources and expertise needed for the traditional processing of hSC cultures from human nerves. In turn, we reason that additional experimentation and a reexamination of the available data are needed to understand the therapeutic value of hSC transplants taking into consideration that the manufacturing of the hSCs themselves may require further development for extended uses in basic research and clinical settings.

Characterization of calcium oxalate biominerals in some (non-Cactaceae) succulent plant species.

The water-accumulating leaves of crassulacean acid metabolism plants belonging to five different families were investigated for the presence of biominerals by infrared spectroscopic and microscopic analyses. Spectroscopic results revealed that the mineral present in succulent species of Agavaceae, Aizoaceae, and Asphodelaceae was calcium oxalate monohydrate (whewellite, CaC2O4 x H2O). Crystals were predominantly found as raphides or solitary crystals of various morphologies. However, representative Crassulaceae members and a succulent species of Asteraceae did not show the presence of biominerals. Overall, these results suggest no correlation between calcium oxalate generation and crassulacean acid metabolism in succulent plants.

Schwann Cell Cultures: Biology, Technology and Therapeutics.

Schwann cell (SC) cultures from experimental animals and human donors can be prepared using nearly any type of nerve at any stage of maturation to render stage- and patient-specific populations. Methods to isolate, purify, expand in number, and differentiate SCs from adult, postnatal and embryonic sources are efficient and reproducible as these have resulted from accumulated refinements introduced over many decades of work. Albeit some exceptions, SCs can be passaged extensively while maintaining their normal proliferation and differentiation controls. Due to their lineage commitment and strong resistance to tumorigenic transformation, SCs are safe for use in therapeutic approaches in the peripheral and central nervous systems. This review summarizes the evolution of work that led to the robust technologies used today in SC culturing along with the main features of the primary and expanded SCs that make them irreplaceable models to understand SC biology in health and disease. Traditional and emerging approaches in SC culture are discussed in light of their prospective applications. Lastly, some basic assumptions in vitro SC models are identified in an attempt to uncover the combined value of old and new trends in culture protocols and the cellular products that are derived.

Oscillatory cAMP signaling rapidly alters H3K4 methylation.

Epigenetic variation reflects the impact of a dynamic environment on chromatin. However, it remains elusive how environmental factors influence epigenetic events. Here, we show that G protein-coupled receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target-Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H+-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II).

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

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.