Here we describe the isolation, characterisation and ex-vivo expansion of human epidermal neural crest stem cells (hEPI-NCSC) and we provide protocols for their directed differentiation into osteocytes and melanocytes. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. Multipotency and self-renewal were determined by in vitro clonal analyses. hEPI-NCSC generate all major neural crest derivatives, including bone/cartilage cells, neurons, Schwann cells, myofibroblasts and melanocytes. Furthermore, hEPI-NCSC express additional neural crest stem cell markers and global stem cell genes. To variable degrees and in a donor-dependent manner, hEPI-NCSC express the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28, OCT-4/POU5F1 and NANOG. hEPI-NCSC can be expanded ex vivo into millions of stem cells that remain mulitpotent and continue to express stem cell genes. The novelty of hEPI-NCSC lies in the combination of their highly desirable traits. hEPI-NCSC are embryonic remnants in a postnatal location, the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. hEPI-NCSC are multipotent somatic stem cells that can be isolated reproducibly and with high yield. By taking advantage of their migratory ability, hEPI-NCSC can be isolated as a highly pure population of stem cells. hEPI-NCSC can undergo robust ex vivo expansion and directed differentiation. As somatic stem cells, hEPI-NCSC are conducive to autologous transplantation, which avoids graft rejection. Together, these traits make hEPI-NCSC novel and attractive candidates for future cell-based therapies and regenerative medicine.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-011-9255-5) contains supplementary material, which is available to authorized users.

Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGFinduced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.

Individuals with germline mutations in the tumour suppressor gene CYLD are at high risk of developing disfiguring cutaneous appendageal tumours, the defining tumour being the highly organised cylindroma. Here, we analysed CYLD mutant tumour genomes by array comparative genomic hybridisation (aCGH) and gene expression microarray analysis. CYLD mutant tumours were characterised by an absence of copy number aberrations apart from loss-of-heterozygosity at chromosome 16q, the genomic location of the CYLD gene. Gene expression profiling of CYLD mutant tumours revealed dysregulated tropomyosin kinase (TRK) signalling with overexpression of TRKB and TRKC in tumours when compared to perilesional skin. Immunohistochemical analysis of a tumour microarray demonstrated strong membranous TRKB and TRKC staining in cylindromas, as well as elevated levels of ERK phosphorylation and BCL2 expression. Membranous TRKC overexpression was also observed in 70% of sporadic basal cell carcinomas. RNA interference mediated silencing of TRKB and TRKC, as well as treatment with the small molecule TRK inhibitor lestaurtinib, reduced colony formation and proliferation in three-dimensional primary cell cultures established from CYLD mutant tumours. These results suggest that TRK inhibition could be used as a strategy to treat tumours with loss of functional CYLD.

Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75NTR and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer’s disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild‐type and cleavage‐resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M‐proNGF and NGF bound to p75NTR with similar affinities, whilst M‐proNGF had a lower affinity than NGF for TrkA. M‐proNGF behaved neurotrophically, albeit less effectively than NGF. M‐proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M‐proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M‐proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M‐proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75NTR : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.