The emergence of several cell therapy candidates in the clinic is an encouraging sign for human diseases/disorders that currently have no effective treatment; however, scalable production of these cell therapies has become a bottleneck. To overcome this barrier, three-dimensional (3D) cell culture strategies have been considered for enhanced cell production. Here, we demonstrate a high-throughput 3D culture platform used to systematically screen 1200 culture conditions with varying doses, durations, dynamics, and combinations of signaling cues to derive oligodendrocyte progenitor cells and midbrain dopaminergic neurons from human pluripotent stem cells (hPSCs). Statistical models of the robust dataset reveal previously unidentified patterns about cell competence to Wnt, retinoic acid, and sonic hedgehog signals, and their interactions, which may offer insights into the combinatorial roles these signals play in human central nervous system development. These insights can be harnessed to optimize production of hPSC-derived cell replacement therapies for a range of neurological indications.

Advancing our knowledge of how neural stem cell (NSC) behavior in the adult hippocampus is regulated has implications for elucidating basic mechanisms of learning and memory as well as for neurodegenerative disease therapy. To date, numerous biochemical cues from the endogenous hippocampal NSC niche have been identified as modulators of NSC quiescence, proliferation, and differentiation; however, the complex repertoire of signaling factors within stem cell niches raises the question of how cues act in combination with one another to influence NSC physiology. To help overcome experimental bottlenecks in studying this question, we adapted a high-throughput microculture system, with over 500 distinct microenvironments, to conduct a systematic combinatorial screen of key signaling cues and collect high-content phenotype data on endpoint NSC populations. This novel application of the platform consumed only 0.2% of reagent volumes used in conventional 96-well plates, and resulted in the discovery of numerous statistically significant interactions among key endogenous signals. Antagonistic relationships between fibroblast growth factor 2, transforming growth factor β (TGF-β), and Wnt-3a were found to impact NSC proliferation and differentiation, whereas a synergistic relationship between Wnt-3a and Ephrin-B2 on neuronal differentiation and maturation was found. Furthermore, TGF-β and bone morphogenetic protein 4 combined with Wnt-3a and Ephrin-B2 resulted in a coordinated effect on neuronal differentiation and maturation. Overall, this study offers candidates for further elucidation of significant mechanisms guiding NSC fate choice and contributes strategies for enhancing control over stem cell-based therapies for neurodegenerative diseases.