Direct Differentiation of Human Pancreatic Islet-Derived Mesenchymal Stromal Cells into Glucose-Responsive Insulin-Producing Cells

Background Type 1 diabetes mellitus affects millions of people worldwide, with β-cell replacement representing a final therapeutic goal. Direct lineage reprogramming offers a promising alternative to pluripotent stem cell–based approaches, bypassing the need for complex stepwise differentiation. Mesenchymal stromal cells derived from human pancreatic islets (hPD-MSCs) retain a pancreatic niche–related transcriptional profile and stellate cell–associated identity, suggesting an intrinsic predisposition toward endocrine differentiation. In this study, we aimed to develop a rapid and efficient protocol for direct reprogramming of hPD-MSCs into glucose-responsive insulin-producing cells. Methods hPD-MSCs were reprogrammed using a sequential viral delivery strategy. Cells were first transduced with lentiviral vectors encoding PAX6, ISL1, and shRNA targeting REST1/2, followed 24 hours later by adenoviral delivery of PDX1, NGN3, and MAFA (PMN factors). Reprogrammed cells were cultured in differentiation medium for 7 days and evaluated by RT-qPCR, C-peptide ELISA, intracellular calcium imaging, and bulk RNA sequencing. Results Adenoviral-mediated PMN overexpression induced significant upregulation of INS and NKX2.2, accompanied by measurable C-peptide secretion and a partial calcium response to glucose stimulation. The addition of ISP factors substantially enhanced reprogramming efficiency across all measured parameters, resulting in significantly higher insulin mRNA expression, increased basal and glucose-stimulated C-peptide secretion, and improved intracellular calcium dynamics upon high-glucose challenge. Transcriptomic analysis revealed induction of endocrine- and neuroendocrine-associated gene programs, including genes involved in secretory granule function, exocytosis, and stimulus–secretion coupling. Conclusions This study demonstrates that hPD-MSCs can be rapidly directed toward an insulin-producing β-cell-like phenotype using a combined ISP–PMN transcription-factor approach. The resulting cells acquire key molecular and functional features of normal β-cells, including glucose-responsive C-peptide secretion, while retaining a partially intermediate transcriptional state. These findings establish hPD-MSCs as a promising cell source for endocrine reprogramming and provide a basis for future development of cell-based diabetes therapies.