Divergent transcriptional responses to pancreatic transcription factors are dictated by the cellular context.

Objective. The outcome of directed differentiation for cell-based therapies is profoundly influenced by the starting cell's intrinsic state, or cellular context. How pluripotent versus lineage-primed cells interpret the same developmental cues remains unclear. To address this, we performed a comparative transcriptomic analysis using two mouse cell lines engineered for episomal expression: a pluripotent embryonic stem cell line (PLT-ES) and a multipotent pancreatic stem cell line (PLT-PPPD). Methods. We introduced a core set of pancreatic transcription factors - PMN: Pdx1 (pancreatic and duodenal homeobox 1), MafA (MAF BZIP transcription factor A), and NeuroD1 (neurogenic differentiation 1) - into both cell types and analyzed global gene expression and insulin promoter activity. Results. The two cell types exhibited strikingly divergent fates. In lineage-primed PLT-PPPD cells, PMN expression induced a mixed pancreatic/hepatic phenotype. In stark contrast, PMN expression in pluripotent PLT-ES cells failed to initiate an endodermal program, instead robustly driving differentiation towards mesodermal lineages. Intriguingly, reporter assays revealed that the PMN factors could activate the insulin promoter in both PLT-ES and PLT-PPPD cells, regardless of the overall differentiation outcome. Conclusions. Our findings demonstrate that cellular context is a paramount determinant of cell fate. The activation of a single promoter is insufficient to orchestrate a complete differentiation program. While pancreatic stem cells exhibit plasticity within the endodermal lineage, pluripotent cells are misdirected towards entirely different germ layers if not appropriately primed. This study underscores the critical importance of using lineage-committed cells for predictable therapeutic outcomes.