Multiple factors through binding to GPCRs

Multiple factors, through binding to GPCRs, orchestrate the regulation of cAMP levels in a cell- and context-dependent manner, an effect that is most likely to be preserved during embryonic hematopoietic emergence and development (Diaz et al., 2015; Goessling et al., 2011; Hoggatt et al., 2009; Jing et al., 2015; Kim et al., 2015; Li et al., 2014; North et al., 2007). Thus, the regulatory networks involved in cAMP-mediated regulation of hematopoietic development are likely to be multifactorial and interconnected. Detailed dissection of such signals is therefore required to elucidate the signaling networks that trigger cAMP-mediated benefits to hematopoietic cells. Our findings on the pro-hematopoietic effects of cAMP on hPSC-derived hematopoietic cell specification are in agreement with reports describing the pivotal role of cAMP signaling in promoting and enhancing mouse and zebrafish hematopoietic development (Diaz et al., 2015; Jing et al., 2015; Kim et al., 2015), as well as human CD34+ cord blood/mobilized peripheral blood survival and engraftment (Goessling et al., 2011; Li et al., 2014). These studies demonstrated that cAMP activation via fluid shear stress in the murine system (Diaz et al., 2015; Kim et al., 2015), adenosine signaling in the zebrafish hematopoietic system (Jing et al., 2015), and PGE2 in human CD34+ 4-ap (Goessling et al., 2011; Li et al., 2014), instructs hematopoietic specification, mediated through the cAMP-PKA axis. In our in vitro model of human hematopoietic development, using an hPSC differentiation system, we show that by inhibiting the downstream effectors of cAMP signaling, PKA and Epac, only Epac signaling is required for hematopoietic development, notwithstanding that apart from affecting the underlying endothelium, Epac inhibition appears to affect the newly formed HSC-like cells. As experimental modulation of cAMP signaling to instruct HSC fate from hPSCs has not yet been achieved (Traver, 2015), our finding describing the pro-hematopoietic benefits of cAMP on hPSC-derived human hematopoietic cells is a key step toward refining modalities of human HSC generation using cAMP. Toward understanding the in vivo emergence and development of human HSCs, our findings on human PSC-derived hematopoietic cell generation necessitate further evaluation of cAMP and its signaling components during definitive human hematopoiesis, i.e., either directly in human fetuses or by developing culture systems to propagate human AGM region in vitro, notwithstanding the ethical and technical challenges related to this pursuit. cAMP-mediated Epac upregulation has been reported to reorganize cortical actin, enhance vascular endothelial cadherin-mediated cell adhesion, and induce integrin-mediated cell adhesion, leading to decreased endothelial cell permeability and enhanced endothelial barrier function (Cullere et al., 2005; Fukuhara et al., 2005; Rangarajan et al., 2003). Developmentally, in the early embryo hematopoietic cells (including cells with the potential to form HSCs) emerge from hemogenic endothelium in the AGM region through EHT (Chen et al., 2009; Zovein et al., 2008). From our Epac inhibition experiments, we demonstrate the critical role of Epac signaling in modulating hPSC-derived hemogenic endothelium and HSC-like cells. Epac being one of the downstream effectors of cAMP that regulates endothelial cell-cell adhesion, permeability, and barrier functions may suggest a link between endothelial cell mechanobiology and hemogenic endothelial cell function. However, due to the inability to currently evaluate pure populations of human endothelial cells with hematopoietic potential, confirmation of Epac\'s role, whether specifying hematopoietic function at the hemogenic endothelium (HE) cell stage, or acting indirectly on HE via an upstream cell intermediate, is still necessary.
Experimental Procedures
Author Contributions
Acknowledgments We are grateful for the support from ES/iPSC core facility, Lund University. This work was funded by grants from: The Swedish Research Council, Swedish Cancer Society, Swedish Children\'s Cancer Society, AFA Insurance (Sweden), Lund University Medical Faculty, The HematoLinné Program Grant, and Stem Therapy Program Grant.