Bulk RNA sequencing of pancreatic progenitor spheroids treated with CHIR99021 and control (DMSO) conditions
H9 human embryonic stem cells with one allele of the PDX1 gene genetically modified into a reporter PDX1-P2A-H2B-GFP and one allele of NEUROG3 gene genetically modified into NEUROG3-P2A-tagRFP-T-NLS were differentiated into the pancreatic lineage until Stage 4 Day 3 of the in vitro differentiation protocol (Rezania et al., 2014, as modified in Petersen et al., 2017) and expanded as pancreatic progenitor (PP) organoids as described in Gonçalves et al 2021. PP organoids were seeded on day 0 and treated with 3 µM CHIR 99021 in the expansion medium from day 3 to day 10, with DMSO as negative control. Organoids from treated and control conditions were harvested on day 4 and day 10 for gene expression analysis by bulk RNA sequencing. d10_CHIR_7days samples were treated with CHIR from day 3 to day 10, whereas d10_CHIR_4days samples were treated with CHIR from day 3 to day 7, followed by DMSO from day 7 to day 10.