Controlled clustering enhances PDX1 and NKX6.1 expression in pancreatic endoderm cells derived from pluripotent stem cells.

Pluripotent stem cell (PSC)-derived insulin-producing cells are a promising cell source for diabetes cellular therapy. However, the efficiency of the multi-step process required to differentiate PSCs towards pancreatic beta cells is variable between cell lines, batches and even within cultures. In adherent pancreatic differentiation protocols, we observed spontaneous local clustering of cells expressing elevated nuclear expression of pancreatic endocrine transcription factors, PDX1 and NKX6.1. Since aggregation has previously been shown to promote downstream differentiation, this local clustering may contribute to the variability in differentiation efficiencies observed within and between cultures. We therefore hypothesized that controlling and directing the spontaneous clustering process would lead to more efficient and consistent induction of pancreatic endocrine fate. Micropatterning cells in adherent microwells prompted clustering, local cell density increases, and increased nuclear accumulation of PDX1 and NKX6.1. Improved differentiation profiles were associated with distinct filamentous actin architectures, suggesting a previously overlooked role for cell-driven morphogenetic changes in supporting pancreatic differentiation. This work demonstrates that confined differentiation in cell-adhesive micropatterns may provide a facile, scalable, and more reproducible manufacturing route to drive morphogenesis and produce well-differentiated pancreatic cell clusters.