Fig. 5. Differentiation of AHNPs into neuronal cell types. (A) Proliferating cells (30 PDs) assume a compacted morphology immediately after removal of mitogens and addition of dibutyl cAMP, IBMX and NGF. (B) Three days after induction of differentiation, intermediate cells displaying a developmentally intermediate phenotype are appreciated. (C) Five days after induction of differentiation, maturing cells concurrently lose GFAP and continue to strongly express ß-III-tubulin. (D) Seven days after induction of differentiation, newly generated neurons in vitro frequently co-express immature neuron markers, and assume typical bipolar morphologies. (E) Current and voltage clamp analysis of 7-day-old neurons. New neurons exhibit prominent Na⁺ and K⁺ channels, and were able to fire elicited action potentials when polarized to -60 mV. (F) ß-III-tubulin neurons generated in the presence of thymidine analog universally incorporate BrdU. Cells generated in this manner display additional type-specific neuronal markers, including PSA-NCAM (G) and neurofilament M (NF-M, H). Scale bars: 75 µm in A; 25 µm in B,H; 100 µm in C,G. Cells counterstained with DAPI.