Differentiating Human iPSCs into Dopaminergic Neurons that Express Classical Biochemical Traits and Show Neuronal Transmission and Excitability

Abstract

Parkinson’s disease (PD) is an incurable neurodegenerative disorder. Current therapeutic approaches are aimed to lessen the symptoms without slowing or halting the disease progression (Hammond, Constance et al., 2007). Models that allow to study the cellular and molecular mechanisms behind the neurological dysfunction are critical in developing new therapies. In vitro neuronal models have been used to recapitulate patient pathophysiology. However, it is crucial to choose the correct cell population to study the disease of interest. iPSC cellular models have the advantage of recapitulating the patient's own cellular background and for this reason they are considered the best model for generating disease-relevant neurons. Here we optimized a protocol to differentiate iPSC in mid-dopaminergic neurons (mDA, the cells affected by PD) and performed different assays to test the level of differentiation and neuronal phenotype. We tested samples from the 5 differentiation stages in our protocol and observed expression of both early and late classical dopaminergic neuronal markers such as SHH, FGF8, FOXA2, LMX1A, NURR1, TUBB and TH. To further characterize the maturation of our neuronal network upon differentiation of cells from neurospheres, we measured neuronal activity by using multi electrode array and calcium influx assay. Our data showed that the mean firing rate of iPSC-derived mDA increased from 0.04 Hz at day 9, to 2.43 Hz by day 20. The number of spikes also increased from 38 on day 5, to 2196 spikes on day 20, demonstrating action potentials are spontaneously being fired by the plated neurons. Finally, we observed that dysregulation of calcium influx in mature mDA neurons upon treatment with receptor agonists leads to cell death, as seen by the decrease in TH levels. Taken together the results we report here show we can efficiently differentiate human iPSCs in mature and active dopaminergic neurons and this model may be potentially used for further investigation of therapies against PD.