Differentiation of nestin-negative human hair follicle outer root sheath cells into neurons in vitro

A specialized quiescent population of hair follicle stem cells, residing in the hair follicle outer root sheath cells (ORSCs), has previously demonstrated pluripotency for differentiation into neural stem cells (NSCs). A previous study indicated that nestin-positive hair follicle ORSCs are able to differentiate into neurons. However, little has been reported on the isolation of nestin-negative human ORSCs and whether they can successfully differentiate into neurons in vitro. In the present study, nestin-positive ORSCs were significantly reduced with a prolonged incubation time in vitro. Following 9 days of primary culture, nestin-expressing ORSCs disappeared entirely, and ORSCs remained nestin-negative following 5 days of subculture. Notably, nestin was identified in ORSCs following a three-step process of neuro-induction. In addition, neruodevelopmental markers were detected in the ORSC-derived nestin-positive spherical cell mass, including the induction of the neuronal specific markers growth associated protein-43, neurotensin receptor-3 and p75 neurotrophin receptor, and also the gliocyte markers, glial fibrillary acidic protein and S100. These sphere-forming cells did not express the mature neuron-associated markers neurofilament medium, neuronal nuclei and neuron-specific enolase, which suggested that sphere-forming cells may preferentially differentiate into neural stem cell-like cells as opposed to mature neurons or neurogliocyte. In conclusion, ORSC-driven neural differentiation may be a suitable treatment strategy for neurodegenerative diseases and may possess an important value in regenerative medicine.