High-efficiency differentiation into functional dopaminergic neurons from Parkinson’s patients-derived induced pluripotent stem cells

The objective of the study was to compare two neuronal differentiation protocols, Pluripotent Stem Cell (PSC) Dopaminergic (DA) Neuron Differentiation Kit (Prototype, Thermo Fisher, catalog Number A30416SA) and our dopaminergic differentiation protocol (Mak et al. 2012) for length of protocol, efficiency of DA neuron generation, and electrophysiological properties. Parkinson disease (PD) patient specific induced PSCs (iPSC) differentiated into DA neurons lay the foundation for exploring disease mechanism and drug discovery. However, there remain critical challenges that must be addressed to reduce variability. It is important to develop standardized and validated iPS cell laboratory practices to generate and characterize cells. Critical factors for successful neuronal differentiation are yield of desired, functional target tissue type, reproducibility of the protocol, duration of differentiation, and cost. We tested two different protocols to achieve high yield of DA neurons using iPSCs from PD patients. Two approaches were 1. embryoid body (EB)/rosette derived neural stem cells induced with dual SMAD inhibition and NCAM sorting that are further differentiated into mature neurons (via GDNF/BDNF) (Mak et al. 2012) in ~60 days and 2. direct differentiation of iPSCs through floorplate progenitor into mature DA neurons via PSC Dopaminergic Neuron Differentiation Kit in 35 days. Higher amount of DA neuron specific enzyme, tyrosine hydroxylase, were obtained after 35 days of differentiation with the PSC Dopaminergic Neuron Differentiation Kit compared to cells derived with EB/dual SMAD inhibition protocol after 60 days. The PSC Dopaminergic Neuron Differentiation Kit showed a sharp increase in floorplate/mesencephalic markers such as FOXA2, Corin, LMX1A, and EN-1 at Day 10 (expression range of 10-10,000 fold increase in comparison to day 0). Neuronal cultures showed spontaneous activity on multielectrode arrays (MEA, Axion Biosystems) of about 3000 spikes and an average amplitude of 22µV(range of 17µV to 40µV). In summary, dopaminergic differentiation with the ThermoFisher PSC Dopaminergic Neuron Differentiation Kit provided reproducible culture conditions, shorter differentiation time, and a high-yield of functional dopaminergic neurons.