Comparative single cell RNA seq of gastruloids from mESCs grown in 3 distinct media conditions facilitating distinct pluripotent states at 3 different developmental time points
Comparative single cell transcriptomic study (using 10x Genomics v3.1 kits) of gastruloids, aggregates of mESCs, at different developmental timepoints. mESCs, corresponding to the 0h timepoint, were also sequenced. The source mESCs were grown in 2 distinct media conditions: DMEM-based Serum-LIF (SL), SL + 0.3uM PD03 (SL + 1i) and SL + 1.5uM CGP77 + 3uM CHIR99 (SL + a2i). The following sets of timepoints were compared: 0hpa (hours post-aggregation) SL, 0hpa SL + 1i, 0hpa SL + a2i (corresponding to the mESC timepoint; 48hpa SL, 48hpa SL + 1i, 72hpa SL + a2i (corresponding to the pre-CHIR99 timepoint); 72hpa SL, 72hpa SL + 1i, 96hpa SL + a2i (corresponding to the post-CHIR99 timepoint); 120hpa SL, 120hpa SL + 1i, 144hpa SL + a2i (corresponding to the maximum elongation timepoint). The aim of this study was to delineate whether mESC pluripotent state influences cell and tissue fate acquisition dynamics. Gastruloids used in this study were generated from a transgenic G4 mESC line (George et al., PNAS 2007) modified via CRISPR-Cas9 mediated heterozygous knock-in of a Bra/T reporter allele (p2a-H2B-eGFP) as well as a ubiquitous nuclear marker allele (CAG::H2B-mKO2).