To further investigate t http www apexbt com media

To further investigate the molecular program involved in HEC determination during human hematopoiesis, we generated a GATA2/eGFP reporter in H1 hESCs through gene targeting, referred as GATA2 hESCs. Based on an hPSC blood differentiation protocol in co-culturing with OP9 (Vodyanik et al., 2005), we show that GATA2/eGFP expression almost exclusively marks the functional HECs with the potential to produce CD34+CD43+ HPCs. We then separated HECs from non-hemogenic ECs in hESC differentiation by cell sorting based on GATA2/eGFP expression. Through further comparative analysis of whole-transcriptome data on GATA2/eGFP+ HECs and GATA2/eGFP− non-hemogenic ECs, we constructed a regulatory network positive or negative for hemogenic endothelial (HE) determination. Moreover, we identified a list of differentially expressed cell-surface markers between GATA2/eGFP+ HECs and GATA2/eGFP− ECs. Among them, CD61 precisely labeled functional HECs not only in hESC differentiation but also in yolk sac (YS) or AGM region at E10.0 in mouse embryos. The identification of CD61 provides a reliable marker for accessing and enriching HECs, which might greatly facilitate the understanding of HEC determination both in vivo and in vitro.
Results
Discussion Hematopoiesis is a highly regulated process controlled by the coordination of TFs and diverse signaling pathways. Knowledge of the mechanisms that drive HSC development is critical for generation of functional HSCs in vitro using hPSC differentiation. To date, significant progress has been made in understanding the regulation of HSC development, as well as identification of cell-surface markers defining hematopoietic b catenin inhibitor at different developmental stages and regions in animal models (Choi et al., 2012; Kennedy et al., 2012; Wang et al., 2013). However, information on human hematopoiesis remains quite limited due to the inaccessibility of human materials. Hematopoietic differentiation of hESCs in vitro provides a valuable model for understanding human hematopoiesis. It has been known that the in vitro hematopoietic differentiation of hESCs follows the basic principle of the in vivo embryonic hematopoiesis in mouse (Chanda et al., 2013). For example, EHT has been observed during differentiation of hESCs for the generation of HPCs (Rafii et al., 2013). Our previous work also showed ethylene the role of GATA2, a critical factor for mouse hematopoiesis, is conserved in a hESC model, as GATA2−/− hESCs exhibited a significant defect in EHT and then HPC generation (Huang et al., 2015). In the current study, we extended our previous work to generate and analyze the blood differentiation of a hESC GATA2 reporter cell line in vitro. We show that both HECs capable of producing HPCs and the generated HPCs are almost exclusively GATA2/eGFP+ cells. These data further highlight the critical role of GATA2 in the regulation of hematopoiesis. It is worth noting that a recent report showed that some HPCs in mice are independent of GATA2, although the HSCs were proved to be exclusively GATA2 expressing (Kaimakis et al., 2016). Our previous work also showed that GATA2−/− hPSC-derived HPCs can produce a certain number of CFUs in vitro (Huang et al., 2015). More detailed analysis of GATA2+ HSCs and GATA2− HPCs is needed in future research to enable full understanding of the role of GATA2 during hematopoiesis. Nevertheless, given the advantage of GATA2/eGFP as a reporter, we were able to discriminate HECs from non-hemogenic ECs in hESC hematopoietic differentiation. To investigate the molecular determinants for HE, we analyzed and compared the transcriptome of GATA2/eGFP+ HECs and GATA2/eGFP− ECs derived from hESCs. A panel of TFs that are positive and negative for HECs or ECs were identified in a human model (Figure S3A). Many important TFs for hematopoiesis are relatively conserved between human and mouse. For example, the well-known TFs identified in mouse hematopoietic development, such as GFl1, RUNX1, MYB, and SPI1, are more highly expressed in GATA2/eGFP+ HECs than in GATA2/eGFP− ECs (Figure S3B). Future work might need to investigate in detail the individual role of each previously unidentified TF in hematopoiesis using both human and mouse models.