Growth factors regulate the survival and fate of cells derived from human neurospheres

Cells isolated from the embryonic, neonatal, acid adult rodent central nervous system divide in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2), while retaining the ability to differentiate into neurons and glia(1,2). These cultures can be grown in aggregates termed neurospheres, which contain a heterogeneous mix of both multipotent stem cells and more restricted progenitor populations(3,4). Neurospheres can also be generated from the embryonic human brain(5-7) and in some cases have been expanded for extended periods of time in culture(8-10). However, the mechanisms controlling the number of neurons generated from human neurospheres are poorly understood. Here we show that maintaining cell-cell contact during the differentiation stage, in combination with growth factor administration, can increase the number of neurons generated under serum-free conditions from 8% to > 60%. Neurotrophic factors 3 and 4 (NT3, NT4) and platelet-derived growth factor (PDGF) were the most potent, and acted by increasing neuronal survival rather than inducing neuronal phenotype. Following differentiation, the neurons could survive dissociation and either replating or transplantation into the adult rat brain. This experimental system provides a practically limitless supply of enriched, non-genetically transformed neurons. These should be useful for both neuroactive drug screening in vitro and possibly cell therapy for neurodegenerative diseases.