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Updated: Aug 7, 2026
In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors
Published on: July 19, 2014
Deletion of RFX6 impairs iPSC-derived islet organoid development and survival, with no impact on PDX1+/NKX6.1+
Noura Aldous1,2,3, Ahmed K Elsayed1,2,4, Bushra Memon3
1College of Health and Life Sciences, Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), Education City, Doha, Qatar.
Insights
Regulatory factor X6 (RFX6) loss impairs pancreatic islet development by increasing apoptosis and downregulating key endocrine genes, not by affecting pancreatic progenitors. This clarifies RFX6
Area of Science:
- Endocrinology
- Developmental Biology
- Genetics
Background:
- Mutations in Regulatory Factor X6 (RFX6) are linked to neonatal diabetes and MODY, indicating its crucial role in pancreatic islet development.
- While RFX6's function in islet development is known, the precise mechanisms by which its loss-of-function mutations lead to diabetes pathogenesis remain unclear.
Purpose of the Study:
- To elucidate the mechanisms underlying impaired pancreatic islet development and hypoplasia caused by RFX6 loss-of-function mutations.
- To investigate the specific role of RFX6 in the differentiation and function of pancreatic endocrine cells.
Main Methods:
- Examined RFX6 expression during human embryonic stem cell (hESC) differentiation into pancreatic islets.
- Utilized CRISPR/Cas9 to generate induced pluripotent stem cell (iPSC) lines lacking RFX6.
- Performed RNA-sequencing (RNA-seq) on pancreatic progenitors (PPs) and endocrine progenitors (EPs) to analyze transcriptional changes.
- Assessed islet organoid formation, cellular apoptosis, and gene expression changes in RFX6-deficient cells.
Main Results:
- RFX6 expression was identified in endocrine progenitor (EP) cells, co-localizing with neurogenin 3 and islet hormones, but not in PDX1+/NKX6.1+ pancreatic progenitor (PP) cells.
- RFX6 deficiency in iPSC-derived pancreatic islets led to decreased PDX1 expression at the posterior foregut (PF) stage but did not affect PDX1+/NKX6.1+ PPs.
- RNA-seq revealed downregulation of genes critical for pancreatic endocrine differentiation, insulin secretion, and ion transport in RFX6-deficient cells.
- RFX6 loss resulted in smaller islet organoids, increased cellular apoptosis, and reduced catalase expression, suggesting a protective role for RFX6.
- Overexpression of RFX6 rescued the defective phenotypes observed in RFX6-knockout PPs, EPs, and islets.
Conclusions:
- Pancreatic hypoplasia and reduced islet cell formation in RFX6 mutation carriers are primarily due to increased cellular apoptosis and the downregulation of essential pancreatic endocrine genes.
- The findings indicate that RFX6's critical role lies in promoting EP survival and function rather than in the initial specification of PDX1+/NKX6.1+ pancreatic progenitors.
- RFX6 deficiency impacts later stages of islet development, contributing to diabetes pathogenesis through impaired endocrine cell function and survival.
Aims/Hypothesis:
Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown RFX6 variants to be linked with type 2 diabetes. Despite RFX6's known function in islet development, its specific role in diabetes pathogenesis remains unclear. Here, we aimed to understand the mechanisms underlying the impairment of pancreatic islet development and subsequent hypoplasia due to loss-of-function mutations in RFX6.
Methods:
We examined regulatory factor X6 (RFX6) expression during human embryonic stem cell (hESC) differentiation into pancreatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell populations during islet development. Furthermore, induced pluripotent stem cell (iPSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed to explore the consequences of RFX6 loss across different developmental stages. Subsequently, we evaluated transcriptional changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrine progenitors (EPs).
Results:
RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the PPs, RFX6 did not co-localise with pancreatic and duodenal homeobox 1 (PDX1) or NK homeobox 1 (NKX6.1) but instead co-localised with neurogenin 3, NK2 homeobox 2 and islet hormones in the EPs and islets. Single-cell analysis revealed high RFX6 expression levels 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 this did not affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis 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 expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in RFX6-knockout PPs, EPs and islets.
Conclusions/Interpretation:
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.
Data Availability:
RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891 ).
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