Journal: Nature

Article Title: Morphodynamics of human early brain organoid development

doi: 10.1038/s41586-025-09151-3

Figure Lengend Snippet: a ) Photographs of the sample holding chamber with four separated sub-chambers each containing four microwells, for growing and imaging organoids with sixteen different organoids arranged one per microwell n = 16 organoids. b ) Schematic overview of tiled image acquisition and example images showing two timepoints, before and after tiled acquisition and image fusion. Scale Bar is 100 micrometers. Time is in hours. n = 16 organoids. c ) Cross section images (day 7) from 16 simultaneously imaged organoids, generated with cells lines labeled with nuclear membrane (lamin, RFP, magenta), plasma membrane (CAAX, RFP, magenta), actin (GFP, green), tubulin (RFP, magenta), and nuclei (histone, GFP, green) and unlabeled WTC-11. d ) Images showing cross section (z-plane) and orthogonal views (y-plane, x-plane) of one organoid from a timecourse lightsheet imaging experiment shown in c. Scale Bar is 100 micrometers. Time is in hours. e ) Maximum intensity projections from a 2-week imaging acquisition of a mosaic organoid (histone, GFP, green; CAAX, RFP, magenta), unlabeled WTC-11). Scale Bar is 500 micrometers. Time is in hours. n = 16 organoids. f ) Images from a 3-week continuous imaging experiment using a NKX2-1:GFP reporter line. The organoids were given SHH morphogen treatment to induce ventral telencephalic patterning of the organoids. Images are false-colored with the green-fire-blue LUT. Scale Bar is 100 micrometers. Time is in hours. n = 4 organoids.

Article Snippet: The organoids were treated with the average morphogen concentration of SHH (140 ng/ml, Miltenyi, 130-095-727) from day 3–14 together with purmorphamine (0.21 μM, Miltenyi, 130-104-465).

Techniques: Imaging, Generated, Labeling, Membrane, Clinical Proteomics

Journal: bioRxiv

Article Title: Mapping the temporal and functional landscape of Sonic Hedgehog signaling reveals new insights into early human forebrain development

doi: 10.1101/2025.05.20.654466

Figure Lengend Snippet: (A) Schematic illustration of the differentiation protocol for generating dorsal (dAN) and ventral (vAN) anterior neuroectoderm from hiPSCs over a 12-day period. Neural induction and ventralization are initiated by a combination of small molecules: LDN193189 (500 nM), SB431542 (10 µM), from day 0 to day 12, and XAV939 (5 µM) from day 0 to day 5. Additional exposure to human recombinant Sonic Hedgehog (SHH, 500 ng/ml) from day 0 to day 6 selectively drives ventral patterning in vAN cultures. (B) Representative Immunofluorescent images at day 12 show distinct marker expression in dAN and vAN cells. Markers analyzed include OTX2 (anterior neuroectoderm marker), PAX6 and EMX2 (dorsal forebrain markers), EOMES (early neural cortical marker), MEIS2 (forebrain marker), NKX2.1 (ventral forebrain marker), FOXA2 (floor plate marker), and SHH (ventral marker). Expression levels are quantified as the percentage of positively stained cells relative to DAPI (mean values from 5 independent images). Scale bar: 40 µm. (C) Principal Component Analysis (PCA) of day 12 dAN and vAN samples (LON and WTC lines) from 5 replicates revealed clear segregation by identity and lineage. PC1 (52% total variance) strongly distinguished dAN versus vAN conditions (one-way ANOVA p.value of 8.9e-12), while PC2 (30% total variance) separates samples by cell lineage origin (one-way ANOVA p.value of 3.1e-12). (D) Heatmap displaying normalized RNA-seq expression profiles of day 12 dAN and vAN samples. Samples were manually ordered by type (vAN/dAN) and cell line (LON/WTC). Marker genes sets specific for hiPSCs, forebrain, ventral/dorsal Forebrain, Midbrain and Hindbrain identity were curated from literature. (E) Heatmap of scaled normalized RNA-seq data showing differentially expressed genes (DEGs) between day 12 dAN and vAN samples (|log2FC| ≥ 1, FDR < 0.01), across 2 hiPSC lines (LON, WTC). Both genes and samples were hierarchical clustered (cluster vAN and cluster dAN), two cluster were identified and then delimited using cutree R function with k=2. (F) Brain development-related Gene Ontology terms among the top 20 most enriched (in term of gene ratio) biological process for both DEGs clusters (dAN and vAN). Color scale indicates gene sets enrichment statistical significance.

Article Snippet: To induce ventralisation and generate ventral anterior neuroectoderm 500ng/ml of human recombinant SHH (C24II, #78065, STEMCELL) was added to media mix from day 0 to day 6.

Techniques: Recombinant, Marker, Expressing, Staining, RNA Sequencing