Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates (‘organoids’) or 3D structures with less physiological relevance (‘spheroids’). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.

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It is well established that Schwann cells (SCs) promote and enhance axon guidance and nerve regeneration by providing multiple cues, including extracellular matrix, cell surface molecules, neurotrophic factors and cellular topography. Which of the elements of the complex environment associated with SCs provides the essential information for directed nerve growth is unclear, because, until now, it has been impossible to investigate their contributions individually. Our development of biomimetic materials that replicate the micro- and nanoscale topography of SCs has allowed us to investigate for the first time the role of cellular topography in directing nerve growth. Dorsal root ganglion (DRG) neurons were cultured on flat poly(dimethyl siloxane) (PDMS) and on PDMS replicas with protruding SC topography. Image analysis showed that more neurons adhered to the replicas than to the flat substrates, and that neurite growth on the replicas followed the underlying SC pattern. Neuronal alignment was dependent on cell density. Live SCs derived from the DRG also grew along the replica SC pattern. These results suggest that the combination of micro- and nanoscale topographical cues provided by SCs can influence nerve growth and point toward design parameters for future nerve guidance channels.