Human pluripotent stem cell derived midbrainPITX3^eGFP/w neurons: A versatile tool for pharmacological screening and neurodegenerative modeling

PITX3 expression is confined to adult midbrain dopaminergic (mDA) neurons. In this study we describe the generation and basic functional characteristics of mDA neurons derived from a human pluripotent stem cell (hPSC) line expressing eGFP under the control of the PITX3 promoter. Flow cytometry showed that eGFP was evident in 15% of the neuron population at day 12 of differentiation and this level was maintained until at least day 80. From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ~20% of PITX3^eGFP/w neurons exhibited spontaneous Ca²⁺ transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min⁻¹, respectively). These neurons also responded to any of ATP, glutamate, acetylcholine, or noradrenaline with elevations of intracellular calcium. As neuronal cultures matured more dopamine was released and single PITX3^eGFP/w neurons began to respond to more than one neurotransmitter. MPP⁺ and tumor necrosis factor (TNF), but not prostaglandin E₂, caused death of the ~50% of PITX3^eGFP/w neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time. This work now shows that these PITX3^eGFP/w neurons are amenable to flow cytometry, release dopamine and respond to multiple neurotransmitters with elevations of intracellular calcium, we believe that they represent a versatile system for neuropharmacological and neurotoxicological studies.