Development of a protocol utilizing single-cell analysis for the differentiation of human iPSCs into SOX6+ midbrain dopaminergic neurons

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of midbrain dopaminergic (mDA) neurons in the substantia nigra, leading to motor symptoms. Current pharmacological treatments are insufficient in halting disease progression, making cell therapy a promising alternative for replacing lost neurons. Advances in stem cell research have enabled the generation of mDA progenitors from human pluripotent stem cells (hPSCs), demonstrating success in preclinical models and clinical trials. However, challenges such as dyskinesias from non-mDA contaminations, graft rejection, and the need for more specific cell types persist. This study focuses on utilizing single-cell multiomic and transcriptomic data to design mDA progenitor differentiation protocols, aiming to generate more specific subtypes of mDA neurons, particularly those vulnerable to degeneration in PD. By refining the protocol with enhanced ventralization and prolonged Wnt activation, we achieved improved midbrain patterning, cell viability, and the accelerated generation of SOX6-expressing mDA neurons in vitro with proven therapeutical properties in grafted hemiparkinsonian mice models. This approach aims to present a protocol designed using single-cell analysis to enrich the most PD-affected mDA neuron populations, offering potential for better cell modelization, and more targeted cell replacement therapies.