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Poster by Yasmin Abu Aqel (Hamad Bin Khalifa University), Racha Chouaib (Hamad Bin Khalifa University), Aldana I. Alnesf (Hamad Bin Khalifa University), Idil Ahmed (Hamad Bin Khalifa University), Gowher Ali (Hamad Bin Khalifa University), Ahmed K. Elsayed (Hamad Bin Khalifa University), Zeyaul Islam (Hamad Bin Khalifa University), Sara Al Khawaga (Hamad Bin Khalifa University), Prasanna R. Kolatkar (Hamad Bin Khalifa University), Paul J. Thornalley (Hamad Bin Khalifa University), Khalid Hussain (Sidra Medicine), and Essam M. Abdelalim (Hamad Bin Khalifa University) Background: Glucokinase (GCK, hexokinase IV) phosphorylates glucose to glucose-6-phosphate during glycolysis process in pancreatic β-cells and hepatocytes. This is the rate limiting step in glucose metabolism and enables those cells to respond appropriately to blood glucose level. Mutations in the GCK gene can either result in hyperglycemia or hypoglycemia. Heterozygous loss of function mutation causes maturity‐onset diabetes of the young 2 (MODY2), while homozygous inactivating mutation leads to permanent neonatal diabetes mellitus (PNDM). The mechanism of diabetes development due to GCK mutations is not fully understood due to the lack of human models Objective: This study aims to understand the effect of GCK mutations on both pancreatic β-cells and mature hepatocytes to identify the molecular mechanisms underlying the defects associated with GCK mutations by using human iPSCs models. Methods: We generated induced pluripotent stem cells (iPSCs) from blood cells of two patients diagnosed with heterozygous and homozygous mutations in the GCK gene. The mutations were confirmed in patient’s samples using whole exome sequencing (WES) and Sanger sequencing. The iPSC lines were extensively characterized using different approaches. Several experiments were done to understand the effect of this mutation. CRISPR/Cas9 knock-in approach was used to generate isogenic controls by correcting the GCK mutation in the generated iPSCs. Mutated and corrected iPSC lines were differentiated into pancreatic and hepatic lineages to understand the effect of GCK mutations in the development of MODY2 and PNDM. in silico study was done to capture the protein’s 3D structure of GCK and predict the effect of this GCK mutation on protein level. Results: Our findings showed that the generated iPSC lines displayed pluripotency characteristics, were able to differentiate into the three germ layers spontaneously and showed normal karyotypes. Our enzymatic preliminary results showed that the mutated GCK protein is less stable compared to the wild type (WT) controls although they are both well folded. Furthermore, it showed higher binding ability and affinity to glucose. Moreover, in silico analysis suggested that this GCK mutation may affect the binding affinity of GCK with glucokinase regulatory protein (GKRP). All generated iPSCs and their isogenic controls were successfully differentiated into pancreatic β-cells and mature hepatocytes. Additional functional studies were performed on both pancreatic β-cells and mature hepatocytes to understand the molecular mechanisms underlying the defects associated with GCK mutations. Conclusion: In conclusion, these human iPSC models can provide valuable insights into the underlying mechanisms of monogenic diabetes, which will pave the way towards personalized treatment.

Poster by Ahmed K. Elsayed, Nehad M. Alajez, and Essam M. Abdelalim (Hamad Bin Khalifa University) Background: Our recent studies showed that FOXA2 plays a key role in the development of endocrine and exocrine pancreas. Downregulation of FOXA2 expression during differentiation of induced pluripotent stem cells (iPSCs) into pancreatic islets leads to a significant reduction in α-and β-cell masses. However, whether these changes are associated with alterations in the expression profile of the long non-coding RNAs (lncRNAs) remains unclear. Objective: the objective of this study is to investigate the alterations in the lncRNA profile and its relationship with the dysregulated mRNAs at the pancreatic progenitors (PPs) and pancreatic islet stages lacking FOXA2. Methods: Therefore, here, we used our recently established iPSCs lacking FOXA2 (FOXA2-/- iPSCs). we screened the dysregulated lncRNAs in the pancreatic progenitors (PPs) and β-cell stage derived from FOXA2-/- iPSCs in comparison to its control. Results: PPs and islet cells lacking FOXA2 showed several dysregulated lncRNAs in comparison to controls. Differential expression analysis identified 442 downregulated and 114 upregulated lncRNAs in PPs and 177 downregulated and 59 upregulated in the β-cell stage. Correlation analysis revealed 12 DE-lncRNAs, which were strongly correlated to key downregulated pancreatic genes in both pancreatic progenitors and beta cell stages (MEG3, H19, ZNF667-AS1, LINC00543, LINC00261, AC097639.1, AL035661.1, SLC25A25-AS1, U73166.1, ZNF790-AS1, MNX1-AS2, and AC091563.1). Conclusion: Our results showed a strong correlation of lncRNAs to several key pancreatic genes and TFs during pancreatic differentiation. Furthermore, these data suggest that the FOXA2-related defective pancreatic islet development is associated with significant alterations in the expression profile of lncRNAs.

Additional file 1: Table S1. List of primers used for PCR and qPCR.

FOXA2 has been identified as an essential factor for pancreas development and emerging evidence supports an association between FOXA2 and diabetes. Although the role of FOXA2 during pancreatic development is well-studied in animal models, its role during human islet cell development remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from a patient with FOXA2 haploinsufficiency (FOXA2+/− iPSCs) followed by beta-cell differentiation to understand the role of FOXA2 during pancreatic beta-cell development. Our results showed that FOXA2 haploinsufficiency resulted in aberrant expression of genes essential for the differentiation and proper functioning of beta cells. At pancreatic progenitor (PP2) and endocrine progenitor (EPs) stages, transcriptome analysis showed downregulation in genes associated with pancreatic development and diabetes and upregulation in genes associated with nervous system development and WNT signaling pathway. Knockout of FOXA2 in control iPSCs (FOXA2−/− iPSCs) led to severe phenotypes in EPs and beta-cell stages. The expression of NGN3 and its downstream targets at EPs as well as INSUILIN and GLUCAGON at the beta-cell stage, were almost absent in the cells derived from FOXA2−/− iPSCs. These findings indicate that FOXA2 is crucial for human pancreatic endocrine development and its defect may lead to diabetes based on FOXA2 dosage.Other Information Published in: Cell Death & Disease License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1038/s41419-021-03390-8

Additional file 2: Table S2. List of antibodies used for immunostaining, flow cytometry and Western blotting.

Additional file 3: Table S3. FPKM values for select genes for IR samples compared to IS samples derived from RNA-Seq.