Principal Investigator/ Associate Professor Sidra Medicine Doha, Qatar
Abstract: We recently identified the role of Regulatory Factor X 6 (RFX6) in human islet development, revealing how biallelic mutations in RFX6 contribute to monogenic diabetes and hypoplastic pancreas formation. However, the role of RFX3 in pancreatic islet development remains unexplored. In this study, we investigated RFX3 function using iPSC-derived islet organoids. RFX3 knockout (KO) iPSCs were generated via CRISPR/Cas9 and differentiated into pancreatic islet organoids. We evaluated gene expression, cell markers, apoptosis, proliferation, and function. Single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing were performed to examine RFX3 expression and transcriptomic changes, with RFX3 overexpression used to reverse dysregulated gene expression. RFX3 was highly expressed in pancreatic endocrine cell populations at various stages, including pancreatic progenitors (PPs), endocrine progenitors (EPs), and mature islets derived from hESCs and iPSCs. scRNA-seq further confirmed RFX3 expression across different endocrine cell clusters during differentiation. Loss of RFX3 disrupted pancreatic endocrine gene regulation, reduced islet cells, and impaired beta-cell function and insulin secretion. Despite a significant reduction in all pancreatic islet hormones, the pan-endocrine marker CHGA remained unchanged, due to an increase in enterochromaffin cells (ECs), which are also known to express CHGA. This was further supported by elevated expression of EC markers, including SLC18A1, FEV, CDX2, and LMX1A, in RFX3 KO EPs and islets, suggesting a shift in cell lineage commitment. Furthermore, RFX3 loss resulted in smaller islet organoids, and increased apoptosis, associated with elevated levels of the pro-apoptotic gene TXNIP in EPs and islets. Restoration of RFX3 expression in KO pancreatic cells rescued the dysregulated expression of endocrine genes and corrected the defective phenotypes, underscoring its critical role in pancreatic islet development. Our study highlights RFX3 as a key regulator of human pancreatic islet cell differentiation and a suppressor of EC lineage specification, which is linked to beta cell immaturity. These findings highlight RFX3's role in islet biology and diabetes, with potential to improve islet cell differentiation for diabetes therapies.
Funding Source: This work was funded by grants from QBRI (QBRI-HSCI Project 1) and from Sidra Medicine (SDR400217).
