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Extended NGN2 Expression in iPSCs Dramatically Enhances Purity of Neuronal Cultures

ABSTRACT: iPSC-derived neuronal cultures can provide valuable insights into the pathogenesis of neurological disease. However, single cell iPSC clones with NGN2 and mCherry show spontaneous loss of mCherry fluorescence, leading to questions about the homogeneity of neurons derived from apparently 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

INSTRUMENT(S): timsTOF Pro, Orbitrap Astral

ORGANISM(S): Ipscs Homo Sapiens (ncbitaxon:9606)

SUBMITTER: jesse meyer

PROVIDER: MSV000098268 | MassIVE | Thu Jun 19 17:14:00 BST 2025

REPOSITORIES: MassIVE

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