Single cell RNA seq of micropatterns derived from Human ESC Micropatterns

During mouse gastrulation, the formation of germ layers proceeds concurrently with the transition pluripotency state and the allocation of the multipotent epiblast cells to the germ layer derivatives. Lineage analysis and fate-mapping studies have revealed that groups of cells in different regions of the epiblast and the primitive streak of the gastrulating embryo are allocated to separate progenitors of the ectoderm, mesoderm and endoderm lineages three germ layers with descendants of each progenitor contributing to specific types of germ layer derivatives. Spatiotemporal transcriptomic analysis of the epiblast cell population has inferred that besides the allocation of the epiblast cells to progenitors of germ layers, a population of putative mesendoderm progenitors may be present in the gastrulating embryos. Overall design: Differentiation of the cells on micropatterned coverslips was a protocol adapted from Warmflash et al. 2014 (Warmflash, Sorre et al. 2014).? HES3 Mixl1:GFP cells were passaged as single cells with StemPro Accutase Cell Dissociation Reagent (ThermoFisher Scientific) and seeded on micropatterned coverslips at a density of 2.5x105 cells/cm2 with 10µM Y-27632 dihydrochloride Rock inhibitor (TOCRIS) in mTeSR Plus (StemCell Technologies) into a total volume of 0.5 mL per well of a 24-well plate. Cells were incubated at 37 °C with 5% CO2. After 24 hours, micropatterns were incubated with either 50 ng/mL recombinant human BMP-4 (R&D Systems), 6µM CHIR99021 (StemCell Technologies) alone or 3 µM CHIR99021 and 100 ng/ml recombinant h/m/rActivin A (R&D Systems) in mTeSR Plus and incubated for a further 48 hours at 37 °C with 5% CO2.? The micropatterned cells were lifted off the gelled coverslips by first washing with PBS to remove debris, followed by incubating in PBS at 37°C for 5 min and then by vigorous tapping of the plate. Whole micropatterned cells floating in PBS were visualized under a microscope and approximately 10 micropatterns for each treatment were picked up with a 200µl-capacity pipette tip. Cells were dissociated with StemPro Accutase Cell Dissociation Reagent (ThermoFisher Scientific) at 37°C for 5 min. Cells were assessed for viability and concentration, before loading into a 10X Chromium Chip G (10X Genomics) for an output of 10,000 cells using single cell droplet capture performed on the Chromium Controller (10X Genomics).