Abstract
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.
Highlights
Type 1 diabetes is caused by the autoimmune destruction of the insulin-producing beta cells found in the islets of Langerhans in the pancreas
NKX6.1 represses the formation of multihormonal endocrine cells[25] and higher NKX6.1 expression correlates with accelerated maturation of human embryonic stem cells (hESCs)-derived pancreatic endoderm (PE) cells into insulin-expressing cells after engraftment in diabetic mice[26]
Commitment from pluripotent states towards pancreatic lineages was confirmed by the downregulation of pluripotency gene, OCT3/4, compared to induced PSC (iPSC), as well as upregulation of definitive endoderm genes, SOX17 and FOXA2, relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Summary
Type 1 diabetes is caused by the autoimmune destruction of the insulin-producing beta cells found in the islets of Langerhans in the pancreas. These aggregates are typically heterogenous which may explain batch variability observed in insulin-producing cell yield, maturity, and purity More advanced techniques such as microfluidic methods[28] or cell-repellent microwells can result in homogenous structures, but these are challenging to scale up, can require complex equipment and/or multiple manual operation steps which leads to significant loss of valuable cell material. Cell-adhesive microwells induced higher levels of PDX1 and NKX6.1 nuclear transcription factor accumulation in the overall cell population, and this increase was associated with the clustering phenotype in which multilayer tissues are formed Overall, this system maintains the simplicity and ease of handling possible with simple adherent 2D culture systems, while enhancing differentiation efficiency and may provide a scalable route towards cell therapy manufacturing
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