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

In this study, we performed single-cell RNA sequencing and single-nuclei multiomic sequencing (RNA + ATAC) at various stages during the differentiation of human pluripotent stem cells to stem cell-derived islets after treatment with the actin depolymerizing compound latrunculin A (latA) during the first 24 hours of differentiation. We show that depolymerizing F-actin with latA during the first 24 hours of definitive endoderm formation (stage 1) 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.