Cell-Surface Marker Signature for Enrichment of Ventricular Cardiomyocytes Derived from Human Embryonic Stem Cells.

To facilitate understanding of human cardiomyocyte (CM) subtype specification, and the study of ventricular CM biology in particular, we developed a broadly applicable strategy for enrichment of ventricular cardiomyocytes (VCMs) derived from human embryonic stem cells (hESCs). A bacterial artificial chromosome transgenic H9 hESC line in which GFP expression was driven by the human ventricular-specific myosin light chain 2 (MYL2) promoter was generated, and screened to identify cell-surface markers specific for MYL2-GFP-expressing VCMs. A CD77+/CD200- cell-surface signature facilitated isolation of >97% cardiac troponin I-positive cells from H9 hESC differentiation cultures, with 65% expressing MYL2-GFP. This study provides a tool for VCM enrichment when using some, but not all, human pluripotent stem cell lines. Tools generated in this study can be utilized toward understanding CM subtype specification, and enriching for VCMs for therapeutic applications.

Polo-like kinase 2 regulates angiogenic sprouting and blood vessel development.

Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates.

The atypical Rho GTPase, RhoU, regulates cell-adhesion molecules during cardiac morphogenesis.

The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.

UBIAD1-mediated vitamin K2 synthesis is required for vascular endothelial cell survival and development.

Multi-organ animals, such as vertebrates, require the development of a closed vascular system to ensure the delivery of nutrients to, and the transport of waste from, their organs. As a result, an organized vascular network that is optimal for tissue perfusion is created through not only the generation of new blood vessels but also the remodeling and maintenance of endothelial cells via apoptotic and cell survival pathways. Here, we show that UBIAD1, a vitamin K2/menaquinone-4 biosynthetic enzyme, functions cell-autonomously to regulate endothelial cell survival and maintain vascular homeostasis. From a recent vascular transgene-assisted zebrafish forward genetic screen, we have identified a ubiad1 mutant, reddish/reh, which exhibits cardiac edema as well as cranial hemorrhages and vascular degeneration owing to defects in endothelial cell survival. These findings are further bolstered by the expression of UBIAD1 in human umbilical vein endothelial cells and human heart tissue, as well as the rescue of the reh cardiac and vascular phenotypes with either zebrafish or human UBIAD1. Furthermore, we have discovered that vitamin K2, which is synthesized by UBIAD1, can also rescue the reh vascular phenotype but not the reh cardiac phenotype. Additionally, warfarin-treated zebrafish, which have decreased active vitamin K, display similar vascular degeneration as reh mutants, but exhibit normal cardiac function. Overall, these findings reveal an essential role for UBIAD1-generated vitamin K2 to maintain endothelial cell survival and overall vascular homeostasis; however, an alternative UBIAD1/vitamin K-independent pathway may regulate cardiac function.