Human Embryonic Stem Cell-Derived Cardiomyocytes Migrate in Response to Gradients of Fibronectin and Wnt5a: Implications for cardiac repair & congenital heart defects

Abstract

An improved understanding of the factors that regulate the migration of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) would provide new insights into human heart development and suggest novel strategies to improve their electromechanical integration following intra-cardiac transplantation. Until now, nothing has been reported as to the factors controlling hESC-CM migration. Here we hypothesized that hESC-CMs would migrate in response to extracellular matrix and soluble signaling molecules previously implicated in heart morphogenesis. To test this, we screened candidate factors by transwell assay for effects on hESC-CM motility, followed by validation via live cell imaging and/or gap-closure assays. Fibronectin (FN) elicited a haptotactic response from hESC-CMs, with cells seeded on a steep FN gradient showing nearly five-fold greater migratory activity than cells on uniform FN. Studies with neutralizing antibodies indicated that adhesion and migration on FN are mediated by integrins α-5 and α-V. Next, we screened 10 soluble candidates by transwell assay and found that the non-canonical Wnt, Wnt5a, elicited a ~two-fold increase in migration over controls. This effect was confirmed using the gap closure assay, in which Wnt5a-treated hESC-CMs showed ~two-fold greater closure than untreated cells. Studies with microfluidics-generated Wnt5a gradients showed that this factor was chemoattractive as well as chemokinetic, and Wnt5a-mediated responses were inhibited by the Frizzled-1/2 receptor antagonist, UM206. In summary, hESC-CMs show robust pro-migratory responses to FN and Wnt5a, findings that have implications for both cardiac development and cell based therapies.