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bioRxiv · 10.1101/2025.06.20.660764

Extended NGN2 Expression in iPSCs Dramatically Enhances Purity of Neuronal Cultures

Munoz-Estrada, J.·Mostafania, A.·Halwatura, L.·Haghani, A.·Jiang, Y.·Meyer, J. G.
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Abstract

iPSC-derived neuronal cultures can provide valuable insights into the pathogenesis of neurological disease. However, single-cell iPSC clones expressing NGN2 and mCherry exhibit spontaneous loss of mCherry fluorescence, raising questions about the homogeneity of neurons derived from what appear to be heterogeneous iPSCs. We find that mCherry silencing does not influence iNeurons with two lines of evidence. First, using single-cell proteomics, we found that spontaneous mCherry silencing does not drive heterogeneity in iPSCs. Second, bulk proteomics and immunofluorescence analysis indicated that iNeurons from iPSCs expressing or lacking mCherry both resemble cortical glutamatergic neurons. The primary confounding factor in iNeuron generation was that suboptimal neuronal conversion led to cell aggregates comprised of actively proliferating neuronal progenitor cells and astrocytes as the culture developed. Our results indicate that extended NGN2 dosage substantially improves neuron purity. HighlightsO_LIEF1-mCherry undergoes spontaneous silencing at the AAVS1 locus C_LIO_LISingle-cell proteomics reveals heterogeneity in edited iPSCs independent of mCherry silencing C_LIO_LImCherry silencing has minimal impact on NGN2-mediated neuronal differentiation. C_LIO_LIExtended NGN2 induction produces more homogenous neuronal cultures C_LI eTOC blurbExpression of a mCherry reporter under the EF1 promoter and integrated into the AAVS1 locus is silenced in undifferentiated iPSCs, whereas CAG-driven rtTA3G remains active for NGN2-induced neuronal differentiation. Optimizing NGN2 induction in iPSC cultures is crucial for generating homogeneous neuronal cultures, as suboptimal conditions result in heterogeneous populations enriched with neural progenitor cells (NPCs) and astrocytes, and a disrupted neuronal organization.

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Munoz-Estrada, J., Mostafania, A., Halwatura, L., Haghani, A., Jiang, Y., Meyer, J. G.. 2025-06-21. Extended NGN2 Expression in iPSCs Dramatically Enhances Purity of Neuronal Cultures. https://doi.org/10.1101/2025.06.20.660764

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