Deletion of RFX6, a Diabetes-Associated Gene, Impairs iPSC-Derived Islet Organoid Development and Survival, With No Impact on the Generation of PDX1+/NKX6.1+ Progenitors

RFX6 is essential for pancreatic development and insulin secretion, while its role in diabetes pathogenesis is unclear. Here, RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the pancreatic progenitors (PPs), RFX6 did not co-localize with PDX1 and NKX6.1, but instead with NEUROG3, NKX2.2, and islet hormones in the endocrine progenitor (EPs) and islets. Single-cell analysis revealed high RFX6 expression in endocrine clusters across various hESC-derived pancreatic differentiation stages. Upon differentiating iPSCs lacking RFX6 into pancreatic islets, a significant decrease in PDX1 expression at the PF stage was observed, although it did not affect PPs co-expressing PDX1 and NKX6.1. RNA sequencing showed the downregulation of essential genes involved in pancreatic endocrine differentiation, insulin secretion, and ion transport due to RFX6 deficiency. Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced Catalase (CAT) expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in PPs and EPs. These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1+/NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes.