Depolymerizing F-actin accelerates the exit from pluripotency to enhance stem cell-derived islet differentiation

In this study, we demonstrate that cytoskeletal state at the onset of directed differentiation impacts the exit of human pluripotent stem cells (hPSCs) from pluripotency and downstream lineage specification. In particular, depolymerizing F-actin with latrunculin A (latA) during the first 24 h of definitive endoderm formation facilitates efficient loss of pluripotency and alters Activin/Nodal, BMP, c-Jun, and WNT signaling dynamics. These signaling changes influence downstream patterning of the gut tube, leading to improved pancreatic progenitor identity and decreased expression of markers associated with other endodermal lineages. Continued differentiation generates islets containing a higher percentage of β cells that exhibit improved maturation, insulin secretion, and ability to reverse hyperglycemia. Furthermore, this latA treatment reduces enterochromaffin cells in the final cell population and corrects differentiations from hPSC lines that otherwise fail to consistently produce pancreatic islets, highlighting the importance of cytoskeletal signaling at the onset of directed differentiation. This study demonstrates that cytoskeletal state at the onset of directed differentiation impacts the exit of stem cells from pluripotency and downstream lineage specification. These findings are utilized to improve stem cell-derived islet generation.