Fig. 5: Characterization of ganglioid cultures.

a Time points of ganglioid culture analysis. b At DIV4, cells derived from tdT⁺ EGCs (red) incorporate EdU (blue, arrowhead) and are labelled by pH3 (green/yellow, arrow). Scale bar: 100 μm. c–e Immunostaining of DIV20 ganglioids (arrows) with TuJ1 (green) and S100B (blue) (c), SYN1 (green, arrows) and NOS1 (blue, arrowheads) (d), CALB, NOS1, NPY and VIP (green, arrows) (e). Scale bars: 500 μm (c), 100 μm (d, e). f Immunostaining of retinoic acid-supplemented DIV20 ganglioid cultures for NOS1 (green, arrows, top) and VIP (green, arrows, bottom). Scale bars: 100 μm. All immunostainings (b–f) were performed at least 4 times. g Superimposed membrane voltage responses of representative action potential-generating DMSO (control; left) and retinoic acid-treated (right) neurons following 1 s current injections from −100 to +200 pA in 50 pA increments (scale bars: horizontal 200 ms, vertical 20 mV). Corresponding time derivative waveforms of action potentials are shown as inserts. Arrowhead points to the presence of a hump, which is a defining feature of AH-type enteric neurons (control 0/5 = 0%; RA 14/15 = 93.3%). h Bar plot showing proportion of cells firing action potentials in each treatment group: control (untreated and DMSO-ctrl cells) n = 29; RA treated: n = 26. i Mean (±standard deviation) scaled expression of GM16, 75 and 81 in bulk RNA-seq datasets (three biologically independent samples each DIV0, DIV4, DIV10, four biologically independent samples DIV20) generated from ganglioid cultures at the indicated time points. j Volcano plots showing mean log₂-transformed fold change (FC; x axis) and significance (−Log₁₀(adjusted P value)) of differentially expressed genes from the indicated GMs at DIV0 and DIV4 of ganglioid cultures. k Track plots showing Hmga2 and Igf2bp2 accessibility in bulk ATAC-seq data collected from DIV4. l UMAP representation of sequenced cells (27726) from ganglioid cultures colour-coded according to DIV. m Mean expression of developmental time-associated GMs on the UMAP shown in (l) (top) and expression of representative genes (bottom). n PCA representation of scRNA-seq data for ganglioid cultures coloured by Louvain cluster. Source data are provided as Source Data files. Cell images used in panel 5a were from BioRender.com.