Journal: bioRxiv
Article Title: GluN2A-mediated currents and calcium signal in human iPSC-derived neurons
doi: 10.1101/2025.09.26.678211
Figure Lengend Snippet: A) Schema of the iPSCs neurodifferentiation using either BPM or NMM. B) Brightfield microscope images showing iPSC-derived neural cultures using either BPM or NMM at different time points of differentiation after NPCs platting. C) Relative mRNA levels of MAP2 and GFAP respect to GAPDH at different time points during the process of neurodifferentiation. For MAP2 , “*” indicates statistical differences between time points for BPM and NMM conditions; for GFAP , “*” indicates statistical differences between time points for BPM only, and “+” indicates statistical differences between BPM and NMM. Two-tailed Student’s t test * p < 0.05, ++ p < 0.01. D) Dispersion plots from FACS experiment of cultures in either BPM or NMM conditions showing cell singlets; neurons are immunolabeled with NeuN-488 and astrocytes with GFAP-594. To ensure high-confidence data, only singlets with strong signals from both fluorophores were included in the statistical analysis (indicated within the black boxes). E, F) Immunofluorescence images of neural cultures in either BPM or NMM conditions at Day 30 (E) and Day 60 (F), stained for endogenous MAP2 (neuronal marker) and GFAP (astrocyte marker). The white arrowhead shows a canonical star-shaped GFAP+ astrocyte (immunopositive for GFAP); white arrows show PAX6+/GFAP-cells; empty arrowhead indicates PAX6+/GFAP+ cells (only present in cells cultured in NMM). At Day 60 many astrocytes were GFAP+/S100β+ in both conditions. G) Volcano plot showing the distribution of differentially expressed genes between neural cultures (BPM vs NMM) at Day 60 of neurodifferentiation. H, I) Heatmap extracted from the RNA-seq data showing genes associated with the GO cluster “Neuronal differentiation” (H) and “Astrocytic differentiation” (I). Thresholds of differentially expressed genes: p -value < 0.05, Average Log2 Fold-Change of 1.5 in the Partek Flow platform. Expression values are mean ± SD of n= 3-6 RNA samples, each of which consists of 2 pooled 12-well plates of iPSC-derived neurons.
Article Snippet: Primary antibodies used were S100β (rabbit, 1:500, Proteintech 15146-1-AP), GFAP (mouse, 1:500, Thermo Fischer MA5-12023), TUJ1 (beta-3-tubulin)(mouse, 1:500, abcam ab14545), MAP2 (chicken, 1:500, Thermo Fischer PA1-10005), Synaptophysin (rabbit, 1:250, Thermo Fischer MA5-14532), PAX6 (rabbit, 1:500, Thermo Fischer 42-6600), Nanog (mouse, 1:500, BioLegend 674002), OCT4 (rabbit, 1:500, Thermo Fischer MA5-14845), GluN1 N-terminal (rabbit, 1:500, Alomone AGP-046).
Techniques: Microscopy, Derivative Assay, Two Tailed Test, Dispersion, Immunolabeling, Immunofluorescence, Staining, Marker, Cell Culture, RNA Sequencing, Expressing
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