Ventral midbrain dopaminergic (mDA) Neuron Differentiation Protocol

Generating authentic functional ventral midbrain dopaminergic neurons, those most vulnerable in Parkinson's disease, from human induced pluripotent stem cells (iPSCs) has enabled cell replacement therapy clinical trials and increasingly sophisticated in vitro disease models. This protocol features further optimizations on a recently updated developmental patterning approach (Kim et al., 2021), focusing on optimizing key stages of t including cell maintenance, induction, cryopreservation, reassembly, and maturation. This comprehensive protocol details the conditions required for the development of midbrain cell types including caudal ventral midbrain dopaminergic neurons and other resident neural cell types, including morphogens, media, substrates, timing, and enables selective enrichment of DA neurons as well as other regional cell types, and integrations with organoid and assembloid cultures. Importantly, variability between different cell line genetic backgrounds, and isogenic pairs, is reduced by by reducing the impact of non-disease-related varitation in iPSC line-intrisic phenotypic variablkes like plating and repassaging efifciency at multiple bottlenecks,and leverages strategies for further inter-line normalization.