were treated with 400ng/ml of SHH from day 0–4 followed by VPA from day 5–7 (Fig 1H). Abundant neurite outgrowth was observed at 8 days (Fig 1I). Between days 8–18 precursor cells were supplemented with growth factors BDNF, GDNF, IGF1 and second messenger cAMP to mature the neuronal cultures at a very high density (Fig 1J). Around days 17–19 highly dense neuronal precursors were dissociated, frozen and/or plated at low density to differentiate into neuronal networks (Fig 1K). To support maturation into functional neurons in low density, neuronal precursors were co-cultured with primary rat astrocytes. Human neurons (density of 62.5k cells/ 3.8 cm2 well) and rat astrocytes (density of 25k/ 3.8 cm2 well) were grown in either direct contact or indirect contact co-culture mode [32] as depicted in Fig 1M and 1N respectively. After 49 days of differentiation (Fig 1L) direct contact neuronal cultures were processed for functional analysis. Day 19 neuronal precursors, when cultured in low-density and in absence of astrocytes failed to mature and showed increased neuronal degradation around 35–40 days of differentiation. In conclusion, partial induction of hPSC-derived hNES cells with SHH and VPA produces cultures with mature neuronal morphology.
