Diabetes is characterized by an absolute or relative deficiency of insulin‐producing β‐cells. Strategies capable of restoring endogenous β‐cell mass therefore represent a major therapeutic objective. Human pancreatic α‐cells exhibit intrinsic plasticity and constitute a promising source for β‐cell replacement approaches. However, current reprogramming strategies primarily rely on viral overexpression of the β‐cell transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and MAF bZIP transcription factor A (Mafa) (ectopically Pdx1‐ and Mafa‐expressing α‐cell (αPM) pseudoislets) and generate hybrid cells that retain substantial α‐cell identity. This thesis aimed to better understand and potentially promote α‐to‐β‐like reprogramming efficiency using complementary model, pharmacological, and genetic approaches.
The evaluation of the αTC1.6 mouse cell line to study α‐cell conversion revealed limited plasticity compared with human α‐cells, emphasizing the importance of human models for translational studies.
Pharmacological modulation of αPM pseudoislets demonstrated that efficient reprogramming requires a permissive signaling in addition to transcription factor expression. Coordinated activity of histone deacetylase (HDAC), forkhead box O (FOXO1), protein kinase C (PKC), and WNT pathways was required for sustained insulin (INS) expression. In addition, jagged 1 (JAG1), a NOTCH pathway activator, tended to enhance INS expression. These findings indicate that lineage conversion depends on integrated transcriptional and signaling regulation.
The effects of ARX loss, a key regulator of α‐cell identity, were investigated using clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR‐Cas9)‐mediated genome editing and two complementary approaches. In purified primary α‐cells isolated by fluorescence‐activated cell sorting (FACS) transcriptional changes were assessed by bulk RNA sequencing. In parallel, ARX deletion in whole islets was examined by single‐nucleus multiome profiling to characterize transcriptional heterogeneity and associated alterations in chromatin accessibility. Deletion of ARX induced a transcriptomic shift characterized by downregulation of α‐cell identity genes and upregulation of β‐ and δ‐cell associated genes, indicating partial activation of alternative endocrine programs. Sn‐multiome analysis further revealed altered chromatin accessibility at regulatory regions associated with β‐ and δ‐cell lineages, supporting a model in which α‐cell identity is actively maintained by lineage‐restricting transcriptional and epigenetic programs. These results identify ARX as a key gatekeeper of human primary α‐cell fate and demonstrate that its disruption promotes broader endocrine plasticity.
Overall, this work demonstrates that adult human α‐cells retain significant reprogramming potential and can be redirected toward β‐cell trait acquisition through coordinated modulation of transcriptional, epigenetic, and signaling networks.
