Biology subjects

Aldous, N.

Publications and source records attributed to Aldous, N..

3 recordsLinked to original sources

bioRxiv2024

RFX3 is essential for the development and maturation of human pancreatic islets derived from pluripotent stem cells

RFX3 in human pancreatic islet development has not been explored. This study aims to investigate the function of RFX3 in human pancreatic islet development using human islet organoids derived from iPSCs, hypothesizing that RFX3 regulates human islet cell differentiation. We generated RFX3 knockout (RFX3 KO) iPSC lines using CRISPR/Cas9 and differentiated them into pancreatic islet organoids. Various techniques were employed to assess gene expression, cell markers, apoptosis, proliferation, and glucose-stimulated insulin secretion. Single-cell RNA sequencing (scRNA-seq) datasets from hESC-derived pancreatic islets were re-analyzed to investigate RFX3 expression in specific cell populations at various developmental stages. Furthermore, bulk RNA sequencing was conducted to further assess transcriptomic changes. RFX3 was found to be highly expressed in pancreatic endocrine cell populations within pancreatic progenitors (PPs), endocrine progenitors (EPs), and mature islet stages derived from iPSCs. scRNA-seq further confirmed RFX3 expression across different endocrine cell clusters during differentiation. RFX3 loss disrupted pancreatic endocrine gene regulation, reduced hormone-secreting islet cells, and impaired beta-cell function and insulin secretion. Despite a significant reduction in pancreatic islet hormones, the pan-endocrine marker CHGA remained unchanged at both EP and islet stages, likely due to an increase in enterochromaffin cells (ECs). This was supported by our findings of high EC marker expression in RFX3 KO EPs and islets. In addition, RFX3 loss led to smaller islet organoids, elevated TXNIP levels, and increased apoptosis in EPs and islets. These findings underscore the crucial role of RFX3 in regulating human islet cell differentiation and its role in suppressing enterochromaffin cell specification. These insights into RFX3 function have implications for understanding islet biology and potential diabetes susceptibility.

developmental biology↗

bioRxiv2024

Identifying miRNA Signatures Associated with Pancreatic Islet Dysfunction in a FOXA2-Deficient iPSC Model

The pathogenesis of diabetes involves complex changes in the expression profiles of mRNA and non-coding RNAs within pancreatic islet cells. Recent progress in induced pluripotent stem cell (iPSC) technology have allowed the modeling of diabetes-associated genes. Our recent study using FOXA2-deficient human iPSC models has highlighted an essential role for FOXA2 in the development of human pancreas. Here, we aimed to provide further insights on the role of microRNAs (miRNAs) by studying the miRNA-mRNA regulatory networks in iPSC-derived islets lacking the FOXA2 gene. Consistent with our previous findings, the absence of FOXA2 significantly downregulated the expression of islet hormones, INS, and GCG, alongside other key developmental genes in pancreatic islets. Concordantly, RNA-Seq analysis showed significant downregulation of genes related to pancreatic development and upregulation of genes associated with nervous system development and lipid metabolic pathways. Furthermore, the absence of FOXA2 in iPSC-derived pancreatic islets resulted in significant alterations in miRNA expression, with 61 miRNAs upregulated and 99 downregulated. The upregulated miRNAs targeted crucial genes involved in diabetes and pancreatic islet cell development. In contrary, the absence of FOXA2 in islets showed a network of downregulated miRNAs targeting genes related to nervous system development and lipid metabolism. These findings highlight the impact of FOXA2 absence on pancreatic islet development and suggesting intricate miRNA-mRNA regulatory networks affecting pancreatic islet cell development.

developmental biology↗

bioRxiv2024

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.

developmental biology↗