Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Abstract

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6-8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC) transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/ high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets. Introduction Human induced pluripotent stem cells (hiPSC) are selfrenewing and can differentiate in almost any adult cell type. These characteristics make hiPSC a useful tool for disease modeling in basic research and drug discovery 1. Human iPSC retains the donor genetic background which allows deriving disease-relevant cell types that are most

A multimodal 3D neuro-microphysiological system with neurite-trapping microelectrodes

“…5e) and high content imaging platforms open up the possibility to test hundreds of compounds each day while combining functional and structural data into a single platform. Automated screen was tested with small molecule–derived neural precursor cells (smNPCs) generated from commercial hiPSCs 35 , which are a powerful tool to model human neurodegenerative diseases 36 . We differentiated the smNPCs into dopaminergic neurons and cultured them in 3D following the same protocols as for primary hippocampal neurons.…”

“…The smNPCs were derived from iPS cells using the method described in Reinhardt et al . 53 , applying few adjustments 35 . The smNPCs were cultured on Matrigel (Corning, 354234) coated plates in the expansion medium (N2B27) supplemented with 3 µM CHIR (R&D System, 4423), 0.5 µM PMA (Cayman Chemical, 10009634) and 64mg/l Ascorbic acid (AA, Sigma-Aldrich, A8960) for up to 10 passages before use in any downstream experiments.…”

Integrated multi-omics analysis reveals common and distinct dysregulated pathways for genetic subtypes of Frontotemporal Dementia

“…The differentiation protocol from smNPC to neurons involves over-expression of Neurogenin-2 (NGN2) using a modified version of the NGN2 lentiviral inducible vector system (single vector pLV_TRET_hNgn2_UBC_BSD_T2A_rtTA3). The detailed description about protocol, reagents and media composition is available in Dhingra et al 53 .…”

Novel regulators of PrPCbiosynthesis revealed by genome-wide RNA interference

“…Counter with trypan blue (Gibco). Culturing of the cells for the smNPC-derived neurons was done using a published protocol (Dhingra et al, 2020).…”