Human epidermal neural crest stem cells as a source of Schwann cells

Published by The Company of Biologists Ltd | Development Human epidermal neural crest stem cells as a source of Schwann cells Motoharu Sakaue 0 1 Maya Sieber-Blum () 0 0 Institute of Genetic Medicine, Newcastle University, Centre for Life , Newcastle upon Tyne NE1 3BZ , UK 1 Present address: Department of Veterinary Medicine, Laboratory of Anatomy II, School of Veterinary Medicine, Azabu University , 1-17-71 Fuchinobe, Chuo-ku, Sagamihara 252-5201 , Japan We show that highly pure populations of human Schwann cells can be derived rapidly and in a straightforward way, without the need for genetic manipulation, from human epidermal neural crest stem cells [hEPI-NCSC(s)] present in the bulge of hair follicles. These human Schwann cells promise to be a useful tool for cell-based therapies, disease modelling and drug discovery. Schwann cells are glia that support axons of peripheral nerves and are direct descendants of the embryonic neural crest. Peripheral nerves are damaged in various conditions, including through trauma or tumour-related surgery, and Schwann cells are required for their repair and regeneration. Schwann cells also promise to be useful for treating spinal cord injuries. Ex vivo expansion of hEPI-NCSC isolated from hair bulge explants, manipulating the WNT, sonic hedgehog and TGFβ signalling pathways, and exposure of the cells to pertinent growth factors led to the expression of the Schwann cell markers SOX10, KROX20 (EGR2), p75NTR (NGFR), MBP and S100B by day 4 in virtually all cells, and maturation was completed by 2 weeks of differentiation. Gene expression profiling demonstrated expression of transcripts for neurotrophic and angiogenic factors, as well as JUN, all of which are essential for nerve regeneration. Co-culture of hEPINCSC-derived human Schwann cells with rodent dorsal root ganglia showed interaction of the Schwann cells with axons, providing evidence of Schwann cell functionality. We conclude that hEPINCSCs are a biologically relevant source for generating large and highly pure populations of human Schwann cells. hEPI-NCSC; Neural crest; Schwann cell; Peripheral nerve; SOX10; KROX20; Protein zero; EPI-NCSCs - INTRODUCTION Schwann cells are neural crest-derived glia that support axons of peripheral nerves. Injuries to peripheral nerves are common; they can be due to neuropathies, traumatic injury, tumour-related surgery or repetitive compression (Pfister et al., 2011; Griffin et al., 2014; Evans, 2001). Overall, peripheral nerve injury carries a high cost to healthcare systems (Rosberg et al., 2005; Noble et al., 1998). After peripheral nerve injury, the proximal stump of the nerve is capable of regeneration and reinnervation. Surgical repair for minor nerve reconstruction involves direct end-to-end anastomosis (Griffin et al., 2014). In significant nerve injuries, nerve repair requires bridging the gap and the introduction of Schwann cells, together with Schwann cell-derived growth factors and an extracellular matrix for guiding axonal extension and nerve regeneration (Shirosaki et al., 2014). Axons regenerate along aligned Schwann cells called bands of Büngner, which form a permissive environment (Allodi et al., 2012). Scaffolds and tubes are used as guidance materials for nerve grafts. Currently, autografts from sensory nerves are typically used to repair large peripheral nerve damage. Autografts have several disadvantages, however, including damage to the donor nerve caused by the biopsy. Schwann cells have also proven useful to treat spinal cord injuries (e.g. Wiliams and Bunge, 2012; Bunge, 2008). The availability of large numbers of human Schwann cells for autologous use and for disease modelling and drug discovery is highly desirable. Here, we provide a methodology for using hEPI-NCSC to generate large and highly pure populations of human Schwann cells using a straightforward strategy that avoids genetic modification and includes manipulation of pertinent signalling pathways with small molecules. hEPI-NCSC are multipotent adult stem cells that derive from the neural crest, a transient tissue in vertebrate embryos that originates from the dorsal aspect of the developing neural tube, the future spinal cord. Neural crest cells become migratory and translocate away from the forming neural tube to various locations within the embryo, where they give rise to a wide array of cell types and tissues, including the Schwann cells of the peripheral nervous system (Le Douarin and Kalcheim, 1999). A subset of multipotent embryonic neural crest cells invade the ectoderm early in development (Richardson and SieberBlum, 1993; Narytnyk et al., 2014a), some of which become located in a stem cell niche of the hair follicle, the ‘bulge’, where they persist postnatally and into adulthood (Sieber-Blum et al., 2004; Sieber-Blum and Grim, 2004; Hu et al., 2006; Clewes et al., 2011; Sieber-Blum, 2014). Another type of neural crest-derived skin progenitor cell is a Schwann cell precursor that a (...truncated)