Associate Director, Pluripotent Stem Cell Biology STEMCELL Technologies Inc. Vancouver, British Columbia, Canada
Abstract: Differentiation of human pluripotent stem cells (hPSCs) into neuronal cell types has become a cornerstone of neurological research by providing unique cellular models of human diseases. Lentiviral delivery of the transcription factor neurogenin-2 (NGN2) has been widely adopted over the past decade to rapidly convert hPSCs into neurons. However, this method faces challenges such as genomic integration risks, variability, labor-intensive steps and viral safety requirements. To address these limitations we developed the STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit, an integration-free system that simplifies the generation of highly-pure hPSC-derived neurons using mRNA-lipid nanoparticle (LNP) delivery of NGN2. hPSCs from four independent cell lines (H9, H1, WLS-1C, and SCTi003-A) were seeded at a density of 7.5 x 10^4 cells/cm2 into 24-well plates. Synthetic NGN2 mRNA encapsulated in LNPs was then added to the culture for three consecutive days. By day 6, we observed efficient neuron conversion, with 96% ± 3% of cells expressing neuronal marker TUJ1, and a yield of 7.6 × 10^5 ± 3 × 10^3 (mean ± SD; n = 8, 4 cell lines) neurons per 24-well plate. After two weeks of maturation using BrainPhys™ hPSC Neuron Kit, 94% ± 2.6% of neurons expressed MAP2, and 90% ± 4.9% expressed the synaptic vesicle marker synapsin I (n = 8, 4 cell lines). Transcriptomic profiling revealed a mostly forebrain identity, with high expression of FOXG1, TBR1 and SLC17A7. To characterize the electrophysiological properties of these neurons, hPSCs were seeded into 96-well plates, and their electrical activity was assessed using a multiwell microelectrode array over several weeks. Spontaneous electrical firings were detectable on day 8 (mean firing rate [MFR] = 0.11 Hz ± 0.03 Hz), peaked on day 21 (MFR = 1.5 Hz ± 0.5 Hz) and persisted for > 48 days in culture (n = 8, 4 cell lines), demonstrating sustained functional activity. In summary, STEMdiff™-TF Forebrain Induced Neuron Differentiation Kit provides a user-friendly, efficient, and integration-free method for generating functional forebrain neurons from hPSCs using NGN2. This system enables rapid acquisition of electrical activity, providing a platform for studying human neurons and advancing neurobiological research.
