Figure 3. scRNAseq analysis reveals that Nng2-induced iNs are sensory neurons.

Cell identity and heterogeneity were assessed using tSNE combined for all time points for ape (orange) and human (green) cells. (A) Filtered cell number for ape and human cells at different time points. (B) Identification of 8 different cell clusters by marker gene expression. NP, neural progenitor; SN, sensory neuron. (C) Marker gene expression (blue) for progenitor cells (SOX2), mature neurons (MAP2), and cortical cells (CUX1, BRN2, SATB2, and TRB1). Scale bars: uncorrected normalized expression. (D) Cell ratios of the three neuronal sub-classes of TAC1 (yellow), SSTR2 and GAL (green), and PIEZO and GAL (blue) expressing neurons for all cell lines and species. TAC1⁺, SSTR2⁺/GAL⁺, and PIEZO2⁺/GAL⁺ neurons on d5: 54%/36% (chimpanzees/humans), 16%/26%, and 31%/38; TAC1⁺, SSTR2⁺/GAL⁺, and PIEZO2⁺/GAL⁺ neurons on d14: 55%/57%, 9%/17%, and 36%/27%; TAC1⁺, SSTR2⁺/GAL⁺, and PIEZO2⁺/GAL⁺ neurons on d28: 71%/48%, 9%/20%, and 20%/32%; TAC1⁺, SSTR2⁺/GAL⁺, and PIEZO2⁺/GAL⁺ neurons on d35: 49%/31%, 12%/27%, and 40%/42%. (E) Heatmap showing scaled expression levels of top 10 significantly higher expressed genes in seven cell populations in iNs culture. The main populations we identified are fibroblasts (FB), neural progenitors, and neurons. Top annotation bars: ape (orange) and human (green). (F) The expression levels of sensory neurons markers are shown as a violin plot for neural progenitor’s clusters C1 and C2 and for neuron’s clusters C4, C5, C6, and C7. Note that the POU homeodomain transcription factors BRN3A (POU4F1) and Islet1 (ISL1) are highly expressed in a subset of early iNs. All iN clusters show high expression of growth factor FGF13 and voltage-gated sodium channel Nav1.7 (SCN9A) that regulate mechano-heat sensation in vivo.

Figure 3—figure supplement 1. Downregulation of stem cell markers in iNs.

(A,B) The violin plots show number of UMIs (A) and genes (B) per cell per cell line and time point of iN differentiation. (C) The violin plots show the expression in iPSCs and iNs at d5, d14, d28, and d35 of three stem cell marker genes (NANOG, OCT4, and SOX2) in apes (orange) and humans (green). All stem cell genes show a reduction in their expression at the transition from iPSCs to neurons. (D) Differences in gene expression between the different time points of iN differentiation and the stem cells. (E) Networks of enriched GO biological processes in the stem cells and iNs are shown in the top and bottom panels, respectively.

Figure 3—figure supplement 2. Expression of cortical excitatory neuron genes.

Heatmap of mRNA expression of all marker genes found in the single-cell quantitative RT-PCR analysis (Fluidigm) of Zhang et al. We could identify expression of almost all markers in all species and cell lines, with the absence of expression of few genes in our dataset including forebrain marker FOXG1 and GABA receptor GABRB2.

Figure 3—figure supplement 3. scRNAseq revealed that NGN2 induces cortical and sensory neuron fates.

(A–C) tSNE plots showing single iNs from each species (A) and cell line (B) at each time point (C). (D) scRNAseq analysis reveals that NGN2-induced iNs express both sensory neuron markers as well as cortical markers. Cell identity and heterogeneity was assessed using tSNE combined for all time points (d5, d14, d28 and d35) and ape and human cells together. Marker gene expression for single markers or a combination of sensory and cortical markers. Sensory neurons are shown in blue (PRPH and ISL1) and cortical markers are shown in red (BRN2 and CUX1). Quantifications show that around 66% of the neurons are PRPH positive: 22% of neurons are BRN2 positive and 13.7% of cells are PRPH and BRN2 positive; 31% of cells are CUX1 positive and 21.2% of cells are PRPH and CUX1 positive. Quantifications show that around 47% of neurons are ISL1 positive: 8.3% of cells are ISL1 and BRN2 positive; 15.9% of cells are ISL1 and CUX1 positive, suggesting that the system consists of sensory and cortical neurons and possibly of cortical sensory neurons.

Figure 3—figure supplement 4. NGN2 induces cortical sensory neuron fates.

The cell identity and the culture heterogeneity of the iNs was assessed by immunofluoresce and mRNA expression analyses for peripheral, cortical, and sensory markers (A) mRNA expression of the peripheral marker Peripherin (PRPH) in iNs. 70% of the iNs were found the express PRPH mRNA. (B) 12% of iNs were found to be immuno-positive for PRPH protein (magenta). (C) Marker genes for cortical layers (CUX1 for layers II–IV, BRN2 for layers II–II, and TBR1 for layers V–VI). (D) mRNA expression of the cortical markers TBR1, CUX1, and BRN2 in iNs. iNs were negative for TBR1, 27–45% of the iNs expressed CUX1 mRNA, and 18–35% BRN2 mRNA. (E) Immunofluorescence of the cortical marker proteins TBR1, CUX1, and BRN2 (magenta) in iNs. iNs were negative for TBR1, 90% of the iNs were immune positive for CUX1, and 50–60% were immune positive for BRN2. Scale bars are 10 µm. A Tukey's post hoc test revealed no significant pairwise differences between the species (see Supplementary file 6).

Figure 3—figure supplement 5. Sensory identity of iNs.

The sensory fate was assessed by immunostaining for the sensory neuron proteins ISLET1 and NAV1.7. (A) 60% of the iNs were positive for ISLET1 (green) at d21 and d35 of differentiation. A proportion of iNs co-expressed BRN2 (magenta) and ISLET1 (green) protein. (C) 60–65% of iNs were immune-positive for NAV1.7 protein at d21 of differentiation. A Tukey's post hoc test revealed no significant pairwise differences between the species (see Supplementary file 6).