Fig. 4. Stem cell-derived motor neurons undergo maturation-relevant gene expression changes.

a Image of embryoid body differentiated from Chat-Cre; Sun1-GFP stem cell line showing co-expression of SUN1-GFP (green) with motor neuron markers ISL1/2 (red) and HB9 (blue). Boxed region on the top left is expanded in the remaining three panels. b Schematic of in vitro maturation experiments: motor neurons are co-cultured with astrocyte-enriched glial cells. c SUN1-GFP expressing motor neurons stained with motor neuron marker ISL1/2 (magenta), and Neurofilament heavy chain (NFH in white) at DIV7 and DIV28 of co-culture. Arrows point to SUN1-GFP+ ISL1/2+ motor neurons. d Plots showing differential gene expression between consecutive in vitro timepoints. Each dot represents one gene, colored dots are genes that are upregulated (green) or downregulated (blue) at least 2-fold with a p value <0.001, as determined by EdgeR. The percent of expressed genes that are dynamic is also reported. e Principal component analysis on expression of in vitro differential genes (>2-fold change between DIV0 and DIV28) at all in vivo and in vitro timepoints. Each dot is the average expression of all biological replicates. f Principal component analysis on expression data from in vivo dynamic, in vitro stable genes (2168 + 2254 genes from g), labels same as e. g Correlation between changes in gene expression in vitro and in vivo. Each dot represents a gene, and its placement on the plot shows how its expression changes between DIV0 and DIV28 in vitro (x-axis) and between E10.5 and P21 in vivo (y-axis). Blue genes have shared regulation while pink genes have opposite regulation in vitro and in vivo. The total number of genes in each category is also reported. h, i Expression of shared up- and downregulated genes. Center line of box plots is the median, the interquartile range is 25–75th percentile, and outliers are eliminated; n.s. p value >0.05 as determined by two-tailed t-tests; n = 1623 up and 1173 down genes. j, k Images and scoring of Thy1-P2A-SUN1-GFP reporter and SPP1 in cultured motor neurons, as detected by immunostaining, and heatmap showing expression of Thy1 and Spp1 in vivo. Error bars show SEM; n = 3 independent biological replicates shown as red dots. All scale bars are 50 μm. Source data are provided as a Source Data file.