br Experimental Procedures br Author Contributions br Acknow

Experimental Procedures
Author Contributions
Acknowledgments The authors thank Matthew Deater and the Grompe laboratory for supplying C57BL/6 Fancd2+/− mice and antibodies, and Michael Garbati for the advice on LY2228820. We are indebted to members of the Flow Cytometry Core Laboratory at OHSU and Nofar Movshovich for assistance with imaging analyses. K.J.S. was supported by the Medical Research Foundation of Oregon Early Clinical Investigator Grant, and A.N.K. was supported by the T32 Molecular Hematology Training Grant. The Endocrine Technologies Support Core (ETSC) at the Oregon National Primate Research Center (ONPRC) is supported by NIH Grant P51 OD011092 awarded to ONPRC. Aspects of this work were presented in part at the American Society of Hematology 55th Annual Meeting in New Orleans, LA, American Society of Hematology 57th Annual Meeting in Orlando, FL, and the American Society of Pediatric Hematology Oncology 27th Annual Meeting in Chicago, IL.
Introduction Differentiation of functional hematopoietic stem and progenitor orexin (HS/PCs) from human pluripotent stem cells (hPSCs) provides a unique source of therapeutic cells for blood diseases and thus generates wide research interests in the field (Daley and Lux, 2014; Liu et al., 2015; Singbrant et al., 2015). Indeed, significant progress have been made on how to drive hPSC differentiation toward different blood lineages (Doulatov et al., 2013; Kennedy et al., 2012; Vodyanik et al., 2005; Wang et al., 2012; Woods et al., 2011). However, HS/PCs derived from hPSCs through current differentiation protocols showed very limited engraftment and hematopoietic reconstitution in vivo (Doulatov et al., 2013; Wang et al., 2005; Woods et al., 2011). These findings indicate that the in vitro conditions for driving blood differentiation do not fully recapitulate the mechanisms of hematopoiesis in vivo. During development, numerous studies using different models such as zebrafish and mouse embryos have shown that hematopoietic stem cells (HSCs) emerge directly from a unique endothelial population, the hemogenic endothelial cells (HECs), through a special process called endothelial to hematopoietic transition (EHT) (Bertrand et al., 2010; Boisset et al., 2010; Tavian et al., 2010). During EHT, cells with endothelium phenotype gradually acquire hematopoietic morphology and characteristics. The EHT process has also been detected during the in vitro blood differentiation of human PSCs (Eilken et al., 2009; Rafii et al., 2013). Therefore, systematic analysis and comparison of the EHT process in vivo and in vitro at the molecular level might aid the generation of functional HS/PCs from hPSCs. To date, a number of key transcription factors (TFs) and signaling pathways that control EHT have been identified in mouse and zebrafish (Chanda et al., 2013; Clements and Traver, 2013; Kissa and Herbomel, 2010; Wang et al., 2013; Wei et al., 2014). For instance, in mouse embryo, Runx1 is highly expressed in both HECs and HSCs and plays essential roles in EHT (Chen et al., 2009). GATA2 is another factor that is known to be critical for hematopoiesis (Rodrigues et al., 2012; Vicente et al., 2012). Mouse embryo lacking Gata2 died at an early stage due to the severe anemia (Gao et al., 2013; Lim et al., 2012; Ling et al., 2004; Tsai et al., 1994). Notably, mouse HECs without Gata2 failed to produce long-term repopulating HSCs due to an impaired EHT (de Pater et al., 2013). We have also demonstrated that human embryonic stem cells (hESCs) with GATA2 deficiency exhibited a reduced EHT during blood differentiation (Huang et al., 2015). These reports suggest that the critical role of GATA2 in regulating EHT is conserved in different species and systems. In addition to the EHT process, TFs also play essential roles in determining the normal function of HS/PCs. For example, overexpression of Hoxb4 could enhance the engraftment of hematopoietic progenitor cells (HPCs) derived from mouse ESCs (Kyba et al., 2002). However, HOXB4 did not show a similar function in hESC-derived HPCs (Wang et al., 2005), indicating that different TFs need be identified for human cells. Indeed, many other factors such as HOXA9 ERG, RORA, SOX4, and MYB have been tested for promoting engraftment of HS/PCs generated in vitro. However, none of these factors were able to mediate long-term engraftment of these in vitro generated human HS/PCs (Doulatov et al., 2013; Ramos-Mejia et al., 2014; Vanhee et al., 2015). Another approach to generate HS/PCs in vitro is through direct specification of functional HECs into HS/PCs. Indeed, it has been shown that endothelial cells isolated from the aorta gonad mesonephros (AGM) region at embryonic day 10.5 (E10.5) to E11.5 mouse embryos efficiently generated HPCs in vitro (Li et al., 2013). However, how to precisely discriminate the functional HECs from non-hemogenic ECs remains challenging. The inaccessibility of HECs largely hampers the further understanding of their molecular determinants during hematopoiesis.