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Establishment of 3-D Neurosphere Cultures from Human iPSC-derived Neural Progenitor Cells

Green neural progenitor stem cells.

SOT 60th Annual Meeting and ToxExpo

Virtual Event

March 12, 2021


Human induced pluripotent stem cells (iPSCs)–derived neurospheres provide an advanced in vitro system for modeling the human brain. This system can be used to investigate areas of research focused on brain development, disease pathology, drug screening, and the toxic effects of environmental chemicals. Here, we use ATCC iPSC-derived neural progenitor cells (NPCs) to generate 3-D neurospheres in vitro. We describe a straightforward method of generating neurospheres and further support their utilization for experimental applications such as dopaminergic differentiation and drug toxicity assays. We found that NPC-derived neurospheres grew exponentially and maintained their progenitor state for up to two weeks in culture. Further we found that neurospheres were able to successfully differentiate to multiple brain lineage cells including dopaminergic neurons in 3-D and displayed higher tyrosine hydroxylase (TH)-positive cells compared to NPCs in 2D cultures. Neurospheres from normal donor cells successfully differentiated and expressed the TH marker uniformly however, neurospheres from Parkinson’s disease donor cells displayed different patterning post differentiation than the neurospheres from normal donor cells. Neurospheres treated with various chemotherapeutic agents in multiple doses gave differential responses between healthy and disease cells. Viability of healthy neurospheres were significantly affected by paclitaxel and vincristine at all three dosages as compared to the control whereas no significant differences were seen for neurospheres from disease donor cells. Similar difference in sensitivity to paclitaxel and vincristine was observed in the two different NPCs. Interestingly, NPCs from healthy donors demonstrated greater sensitivity to paclitaxel and vincristine as compared to the neurospheres derived from the same cells. And this trend was also seen for NPCs and neurospheres from the diseased donor. Responses to amiodarone and chlorhexidine were found to be similar between both neurospheres from healthy and diseased. However, NPCs were slightly more sensitive to amiodarone and chlorhexidine as compared to their neurosphere counterparts. These data demonstrate that iPSCs-derived neurospheres are a powerful tool for developmental studies, drug screening, and toxicity testing compared to 2D NPC cultures.

Download the poster to explore the development and application of iPSC-derived neurospheres