DNA methylation profiles of human embryonic stem cells (HUES8) differentiated to pancreatic duct-like organoids.

To investigate Keratin 19 (KRT19) expression dynamics in pancreatic development, we established a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in various genetic backgrounds to monitor KRT19 expression from ist endogenous gene locus. A genuine differentiation protocol to generate mature pancreatic duct-like organoids (PDLO) was applied. While KRT19/mCherry expression became evident at early endoderm stage, mCherry signal was present in nearly all cells at pancreatic endoderm (PE) and pancreatic progenitor (PP) stage. Interestingly, despite homogenous KRT19 expression, the mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates with site specific DNA methylation patterns occuring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. DNA methylation profiles of the human embryonic stem cell line HUES8. HUES8 cells were differentiated into pancreatic progenitors (PP) and pancreatic duct like organoids (PDLOs). A stage-resolved genome-wide DNA methylation analysis was performed using the Infinium MethylationEPIC BeadChip array (n = 20). One sample was treated with 5-Aza-2’-deoxycytidine (5-Aza).