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    • Human pluripotent stem cells hPSCs including

    2018-10-26

    Human pluripotent stem over here (hPSCs), including human embryonic stem cells (Thomson et al., 1998) and induced pluripotent stem cells (iPSCs) (Takahashi et al., 2007), provide an ideal in vitro model to recapitulate human hematopoietic development. We have shown that hPSC-derived HSC-like cells possess lymphoid and myeloid differentiation ability, a key feature of HSCs (Ronn et al., 2015). Recent studies have functionally demonstrated an endothelial precursor to blood (hemogenic endothelium) from hPSC differentiation cultures (Ditadi et al., 2015; Slukvin, 2013), further establishing hPSCs as a suitable model to study human hematopoietic cell development. However, the signals regulating hemogenic endothelium and newly emergent HSCs in the human developmental context remain undefined. In addition, for functional transplantable HSCs it is vital to reduce reactive oxygen species (ROS) and oxidative stress, as reduced ROS is crucial for HSC functionality (Ito et al., 2006; Jang and Sharkis, 2007; Yahata et al., 2011).
    Results
    Discussion Using an hPSC differentiation system to model human hematopoietic cell emergence and development, our findings suggest that cAMP and signaling through its Epac axis is an important factor for the generation of hemogenic endothelium, from which the first hematopoietic cells arise. Thus cAMP is a crucial second messenger molecule regulating the in vitro hPSC-derived HSC-like cells. Interestingly, cAMP induction upregulated the frequency of HSC-like cells in the differentiation cultures and also mitigated oxidative stress, created redox-state balance, and enhanced CXCR4 expression in hPSC-derived hematopoietic cells, thus suggesting multiple independent functions of cAMP signaling in endothelial and hematopoietic cells. Because of the importance of low ROS levels in maintaining repopulating human HSCs (Jang and Sharkis, 2007; Yahata et al., 2011), the identification of cAMP-mediated ROS reduction in hPSC differentiation systems provides a mechanism for how ROS can be regulated in in vitro systems to better mimic in vivo HSC development. We speculate that the presence of mature immune cells elevates ROS activity in our differentiation system. The reduction in mature cells and decreased macrophage numbers following cAMP induction agrees with cAMP-mediated redox-state balance, leading to decreased ROS levels. ROS reduction was in concert with the increase of anti-oxidant gene response, decrease in the p38 stress pathway components, and reduced prevalence of mature hematopoietic cells. In terms of cAMP-mediated ROS reduction and reduction of mature progenitors, the reduced abundance of differentiated cells could in part result in decreased ROS, or, alternatively, low ROS levels could be playing a role to reduce differentiation, either way both benefiting the system for increasing the prevalence of HSC-like cells. Thus, benefits of cAMP, in terms of decreased ROS/p38 and maintenance of redox balance together, created a better environment for the HSC-like cells, presumably helping their survival, preventing premature senescence, and maintaining their functionality, as has been shown for adult HSCs. cAMP induction reduced p38 signaling, which is beneficial for the maintenance of human hematopoietic stem and progenitor cells (Baudet et al., 2012; Zou et al., 2012). The ability of cAMP to regulate inflammation and ROS has been shown previously, either by reducing pro-inflammation cytokines and enhancing the T helper 2 responses in inflamed human blood over here cells (Harris et al., 2002; Snijdewint et al., 1993; Yoshimura et al., 1997) or via suppressing inflammation/ROS through the cAMP-Epac axis (Remans et al., 2004; Xu et al., 2008). In our assay, cAMP-mediated ROS/p38 reduction, increased anti-oxidant response, and reduced differentiation could in part be attributed to these inflammation-regulating actions of cAMP. We speculate that ROS reduction has an important, but not exclusive, regulatory role in the cAMP-mediated maintenance of HSC-like cells. Moreover, cAMP-mediated ROS/p38 reduction and increased CXCR4 receptor expression provide a correlational benefit to HSC-like cells, as low ROS levels specifically increased the CXCR4high HSC-like cells. These findings suggest that cAMP induction imparts important functional properties to the derived hematopoietic cells, as low oxidative stress (Ito et al., 2006; Jang and Sharkis, 2007; Yahata et al., 2011) and high CXCR4 expression (Nie et al., 2008; Peled et al., 1999) are properties of HSCs with long-term transplantation potential.