Developmental stage-dependent cellular plasticity governs β-cell reprogramming efficiency in human fibroblasts

Direct reprogramming of somatic cells into alternative lineages, bypassing a pluripotent intermediate state, represents a promising strategy to reduce the risks of tumorigenesis and genomic instability associated with induced pluripotent stem cell-based approaches. Although β-cell-like cells have been efficiently generated from developmentally related endodermal sources such as acinar cells, ductal cells and α-cells, their derivation from phylogenetically distant cell types, such as fibroblasts, remains challenging. In this study, we employed lentiviral-mediated reprogramming using a polycistronic construct encoding the pancreatic transcription factors Neurog3, Pdx1 and MafA to enable β-cell-like formation from human fibroblasts. To assess the influence of developmental stage on reprogramming efficiency, fibroblasts derived from neonatal, juvenile and adult stages were analyzed comparatively. Transgene delivery, expression and functional effects were validated by reporter gene analysis, microscopy, flow cytometry, RT-qPCR and immunoblotting. Reprogrammed cells derived from neonatal fibroblasts exhibited the highest reprogramming efficiency, as demonstrated by robust induction of INS and MAFA expression, along with concurrent downregulation of fibroblast marker PRRX1. In contrast, reprogrammed cells derived from adult fibroblasts displayed markedly reduced responsiveness. Flow cytometry analysis revealed that approximately 38% of reprogrammed cells derived from neonatal fibroblasts acquired an INS-positive phenotype. Notably, reprogrammed cells derived from juvenile fibroblasts exhibited a higher proportion of GCG-expressing cells compared to those derived from neonatal fibroblasts, indicating a more pronounced heterogeneous endocrine-like state characteristic of intermediate stages of pancreatic differentiation in the former compared to the latter. Collectively, these findings demonstrate that the developmental stage significantly influences reprogramming competence, with neonatal fibroblasts representing a more permissive cell source for β-cell-like conversion. These results highlight developmental stage-dependent cellular plasticity as a key determinant of reprogramming efficiency and reveal the optimal cell sources for β-cell generation.