Differentiation of human iPSCs into dopaminergic neurons: comparative analysis of 2D and 3D protocols for disease modeling and pharmacology

This pragmatic review addresses those protocols containing innovative steps for differentiating human inducible Pluripotent Stem Cells (iPSCs) into mature midbrain dopaminergic (mDA) neurons, leading to successful implementation in other articles of in vitro models for neurodegenerative or psychiatric disorders and for pharmacological tests, in the period 2004-2025. Article were selected according to the presence of protocols that allowed the iPSC differentiation into mDA neurons as defined by the expression of DA neural lineage markers and Thyrosine Hydroxylase, by the evidence of neuronal morphology, electrophysiology, neurochemistry, biomarkers and intracellular depositions that recapitulate various stages of differentiation and maturation of the mammalian brain mDA neuron. The critical innovative aspects that allowed the article selection was the presence of a significative methodological step in increasing the protocol performance and the useful replicability in term the number of peer-reviewed articles implementing this procedure. Out of 108 articles of protocols based on stem cells-derived mDA neurons, we identified 12 articles regarding 2-dimensional (2D) cultures and 11 articles regarding 3-dimensional (3D)/organoid cultures. For 2D cultures, the most effective protocol would include a floor plate induction with dual-SMAD inhibition improved by a biphasic WNT activation process and by a pre-differentiation step of neural precursor expansion. Regarding the more recent protocols for 3D midbrain-like organoids, less disease model articles were generated, requiring more time for assessing of the most performing protocols. 3D/organoids were found superior to 2D cultures since they better represent the original midbrain neuronal circuitry, allowing better personalized disease modelling.