Fig. 1: Defined maturity stage neuronal cultures from induced pluripotent stem cells.

A Schematic representation of the differentiation protocol used for generation of human forebrain neurons from iPSCs. Brightfield images exemplify the different stages throughout differentiation. Scale bars, 100 µm. B Representative fluorescence microscopy images of immature neuronal cultures at day 5 and more mature neuronal cultures at day 35 of differentiation. Scale bars, 50 µm. Upper bar graph shows the quantification of the percentage of NeuN+ nuclei in d5 and d35 cultures. Graph shows mean with S.D., P < 0.0001, two-tailed t test. Lower bar graph shows the quantification of the degree of neurite expansion in d5 and d35 neuronal cultures. Bar graph shows mean with S.E.M. P = 0.0031, two-tailed t test. Every data point represents the result obtained for a single microscopy image. Three images were analyzed per differentiation and three independent differentiations were assessed. C Characterization of electrophysiological properties of individual neurons at d4-5 (n = 20 cells) and d25-28 (n = 24 cells) of differentiation. Shown are representative AP firing patterns of a neuron at d5 and d26 during 300 ms of depolarization. Box plot shows the number of action potentials evoked during 300 ms of depolarization (P < 0.0001, two-tailed Mann-Whitney U test). Each data point represents a single neuron. D Sample distance matrix with hierarchical clustering based on rlog normalized counts (upper panel). The degree of sample similarity is expressed by Euclidean distance. With extended time in culture, bulk transcriptomes become more similar. PCA biplot showing PC1 and PC2 based on the 500 most variable features (lower panel). Data shows biological replicates grouping together and sample separation based on time point of differentiation. Samples are color-coded by time point. Grey arrow indicates timeline of differentiation. E Heat map (z-scaled normalized counts) showing time point-dependent expression of canonical NPC, early neuron and more mature neuron marker genes resolved for the individual biological replicates. NPCs express the proliferation marker MKI67 and progenitor-associated genes such as SOX2, HES1, HES5, NES and PAX6. Immature d5 neuronal cultures prominently expressed early neuron markers like TUBB3, STMN1, FAT3 and DCX. From d25 onward, neuronal cultures expressed a wide range of neurotransmitter receptors including NMDA (GRIN1/2B), AMPA (GRIA1/2), GABA (GABRA3, GABRAB2) receptors and synaptic components like SNAP25, PSD95 (DLG4), SYP, SYN1 and SYT1 and the two major structural building blocks of the axon initial segment (AIS) ankyrin-G (ANK3) and βIV spectrin (SPTBN4) as well as the AIS-associated voltage-gated sodium channel Nav1.2 (SCN2A). F Dimensional reduction plot in UMAP dimensions. Seurat clustering of single cell transcriptomes reveals six distinct cell populations. Data from RNAseq was used for guided dimensional reduction of scRNAseq data (upper panel). Monocle3 pseudotime analysis based on dimensional reduction from upper panel. Grey arrow indicates progression of pseudotime with yellow data points marking cells that are most advanced along the trajectory (lower panel). G Heat map of marker gene expression in single cells ordered by pseudotime (z-scaled). H Scoring maturity of bulk neuronal cultures using the neuMatIdx R package described by He and Yu [8] The discriminating neuron maturity index (dNMI) relies on transcriptional modules best explaining the differences between immature and mature neurons. The neuron functionality index (NFI) only incorporates modules specifically enriched in mature neurons. Bar graphs show mean with S.D., one-way ANOVA with Bonferroni correction. dNMI and NFI score for the neuronal clusters identified in scRNAseq analysis showing advancing transcriptomic maturity from cluster 2 to 5.