Endogenous Nodal switches Wnt interpretation from posteriorization to germ layer differentiation in geometrically constrained human pluripotent cells

We conducted bulk RNA sequencing on cells sorted by fluorescence from micropatterned colonies treated with FGF8 and different concentrations of WNT3a, using a SOX2::mCitrine cell line to distinguish between SOX2 positive and negative cells. The sequencing revealed that SOX2 negative cells exhibited a definitive endoderm identity, while SOX2 positive cells showed epiblast identity. SOX2::mCitrine cells were seeded into 4 separate micropattern glass coverslips CYTOO: (1) Control consisting of colonies in N2B27 medium only, (2) Colonies treated with FGF8 (200 ng/ml) with WNT3a (100 ng/ml), (3) Colonies treated with FGF8 (200 ng/ml) with WNT3a (300 ng/ml), and (4) Colonies treated with FGF8 (200 ng/ml) with WNT3a (1000 ng/ml); all for 3 days. Colonies were dissociated into single-cell suspensions using Accutase at 37ºC for 5 minutes. Cells were collected, centrifuged at 1000 rpm for 4 minutes, and re-suspended in PBS-EDTA (10 mM). Cells were then filtered through a 5ml Polystyrene Round-Bottom tube with cell-strainer mesh 40um cap (FALCON). A Sony SH800S Cell Sorter with a 488 nm laser was used to separate the SOX2 positive cells from the negative ones. RNA was collected with the Invitrogen RNAqueous-Micro Total RNA Isolation Kit. Sequencing was performed by Novogene Co. using the Illumina paired-end 150 platform (Novaseq 6000). Another biological repeat of all conditions except (3) was performed using the same protocol.