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 is critical for the specification of human pluripotent stem cells (hPSCs) to all three germ layers. In particular, a polymerized actin cytoskeleton facilitates directed ectoderm differentiation, while depolymerizing F-actin promotes mesendoderm lineages. Applying this concept to a stem cell-derived islet (SC-islet) differentiation protocol, we show that depolymerizing F-actin with latrunculin A (latA) during the first 24 hours of definitive endoderm formation facilitates rapid exit from pluripotency and alters Activin/Nodal, BMP, JNK-JUN, and WNT pathway 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.