Abstract
Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of 3D in vitro models for neurological diseases. Neurons cultured in 3D aggregates might not only serve to facilitate the development of more precise human brain models for basic mechanistic studies but also represent better predictors of drug responses in vivo. To evaluate the capacity of alginate to support the differentiation of hiPSCs to midbrain-specific dopaminergic neurons for Parkinson’s disease (PD) modeling, we encapsulated iPSCs in alginate microcapsules (1% or 2%) with or without fibronectin (Fn). Cells grown in 1% alginate-Fn and 2% alginate-Fn present increased differentiation capacity towards neural lineages with respect to both alginate (1%-2%) alone and 2D conditions. Gene expression analysis suggests an increase in TH+ neurons and a higher maturity compared to the neurons differentiated in 2D. Immunofluorescence analysis further supports these results, showing expression of synaptic markers as well as specific DA neuronal markers. The 3D neurons can be maintained in culture for more than 200 days. We envision that this differentiation protocol might enable the generation of midbrain-specific dopaminergic neurons in a shorter time-frame as compared to 2D cultures and will allow successful phenotype assessment and advanced therapy development.