AUTHOR=Watmuff Bradley , Hartley Brigham J. , Hunt Cameron P. J. , Fabb Stewart A. , Pouton Colin W. , Haynes John M. 

TITLE=Human pluripotent stem cell derived midbrain PITX3eGFP/w neurons: a versatile tool for pharmacological screening and neurodegenerative modeling

JOURNAL=Frontiers in Cellular Neuroscience

VOLUME=Volume 9 - 2015

YEAR=2015

URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2015.00104

DOI=10.3389/fncel.2015.00104

ISSN=1662-5102

ABSTRACT=Abstract
PITX3 expression is confined to adult midbrain dopaminergic neurons. In this study we describe the generation and basic functional characteristics of midbrain dopaminergic neurons derived from a human pluripotent stem cell line expressing eGFP under the control of the PITX3 promoter. Flow cytometry shows that eGFP is evident in 15% of the neuron population at day 12 of differentiation and this level is maintained until at least day 80.  From day 20-80 of differentiation intracellular chloride decreases and throughout this period around ~20% of PITX3eGFP/w neurons exhibit spontaneous Ca2+ transients (from 3.3+/-0.3 to 5.0+/-0.1 min-1, respectively).  These neurons also respond to any of ATP, glutamate, acetylcholine or noradrenaline with elevations of intracellular calcium. As neuronal cultures mature more dopamine is released and single PITX3eGFP/w neurons begin to respond to more than one neurotransmitter.  MPP+ and tumor necrosis factor(TNF), but not prostaglandin E2, caused death of the ~50% of PITX3eGFP/w neurons (day 80).  Tracking eGFP using time lapse confocal microscopy over 24 hours demonstrated significant TNF-mediated neurite retraction over time.  These PITX3eGFP/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.