NGN2 induces diverse neuron types from human pluripotency.

Diverse subpopulations emerge during NGN2-directed neuron differentiation into iNs from human iPSCs(A) Schematic of scRNA-seq time course experiment and representative images from human iPSCs differentiating into NGN2-iNs. Cells were analyzed with scRNA-seq (10× Genomics) at multiple time points during differentiation. Immunohistochemical staining of NGN2-iNs at d28 with MAP2 (green) and DAPI (blue). Scale bars, 10 μm.(B) SPRING embedding shows the developmental relationships of 409B2-derived NGN2-iNs with cells colored by time points (left) or cluster (right).(C) Expression feature plots stem cell, mesenchymal, and neural marker genes.(D) Expression feature plots of NGN2-iN clusters.(E) Proportion of PRPH+ and PRPH− cells quantified using scRNA-seq (10×) or immunofluorescence.See also Figures S1A and S1B.

NGN2-iN neuron diversity is recapitulated in multiple iPSC lines(A–C) (A) scRNA-seq was performed on d35 NGN2-iNs from polyclonal 409B2, monoclonal 409B2, and polyclonal Sc102a1 iPSCs. (B and C) UMAP embedding of Seurat 3.0 integrated scRNA-seq data, with cells colored by cell source (B) or cluster annotated by marker genes (C).(D) Feature plots showing the expression of marker genes.(E) Stacked bar plot showing proportions of clusters in each sample.(F) Dot plot of marker gene expression patterns and detection rates across clusters.

Molecular signatures of NGN2-iN compared with primary neuronal cell types mouse reference atlases(A) NGN2-iN subpopulation signatures were compared with diverse reference atlases.(B) Spatial expression patterns of selected markers as maximum intensity projections across sagittal sections in the embryonic day 13.5 mouse brain from the Allen Developing Mouse Brain Atlas.(C) Transcriptomic similarities between NGN2-iNs, other iPSC-derived neurons, and primary neurons represented as Pearson correlations between expression profiles. SC, spinal cord.(D) Average expression of various marker genes of primary neuron subtypes in NGN2-iN clusters and primary mouse PNS and CNS neuron subtypes.(E) Proportions of the estimated CNS component in NGN2-iNs, cerebral organoid neurons, and human/mouse primary mature PNS/CNS neurons.(F) Expression of exogenous (left) and endogenous (right) NGN2 in different NGN2-iN clusters. The boxes show the lower and upper quartiles of the distributions. the bars extend to the min/max or 1.5x interquartile range.

Neuronal fate specification is sensitive to NGN2 dosage(A) Schematic for the doxycycline (Dox) treatment duration experiment.(B) Detection rates for cells expressing exogenous NGN2 (>5 UMI) and endogenous NGN2 (>0 UMI) from each sample.(C) UMAP embedding of NGN2-iN cells from each sample. scRNA-seq data were integrated using cluster similarity spectrum-based integration (He et al., 2020).(D) UMAP plots colored by marker gene expression. DCX marks the neural lineages. LHX4 marks the identity of the off-target cluster.(E) Proportion of cells per Dox treatment time point in each of the three neural clusters.(F) Expression level of exogenous NGN2 from the time course experiment presented in Figure S1A. The boxes show the lower and upper quartiles of the distributions. the bars extend to the min/max or 1.5x interquartile range.(G) Numbers of exogenous NGN2 transcripts in the three neural clusters from different time points of NGN2-iN development. The boxes show the lower and upper quartiles of the distributions. the bars extend to the min/max or 1.5x interquartile range.(H) Proportion of cells from different time points of NGN2-iN development.