A toolkit for mapping cell identities in relation to neighbors reveals conserved patterning of neuromesodermal progenitor populations
Fig 6
Comparing local patterning between embryos, gastruloids and monolayer cell culture.
A: Protocol for generating hNMPs from hESC and representative confocal images of the culture fixed at 72 h and stained for SOX2, TBXT, TBX6, and LAMINB1 (further characterization S7 Fig). B: Protocol for generating gastruloids [51] with a representative confocal image of a gastruloid fixed at 120 h for SOX2, TBXT, TBX6, and LAMINB1. Red box indicates typical field of view for high resolution image acquisition and subsequent neighbor analysis. C: Density plots comparing each nucleus’s TF normalized fluorescence intensity (NFI) value to its neighbors shows strong linear correlations for TBXT and SOX2, but not for TBX6 with many ‘high’ TBX6 cells surrounded by ‘low’ TBX6 cells. Consistent in the E8 epiblast, gastruloids, and in vitro human NMP like cell monolayers. Black line indicates the median and the dashed line indicates x = y line. D: Violin superplots of TF local heterogeneity (coefficient of variation [CV]) for respective TF+ cells within NMP-like cells isolated in gastruloids (process outlined in S8 Fig) and hNMP monolayers compared to in vivo WT E8 bi-fated region. NMPLC populations within in vitro models are more heterogeneous than comparative in vivo NMP populations. All scale bars indicate 50 µm. Points represent replicate means. Error bars represent 5%–95% confidence intervals. Gastruloids n = 3 with 3 technical replicates. Data for Fig 6 (C–D): Data file 1 and Data file 2, https://doi.org/10.5281/zenodo.15802710.