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    • Reliable cell surface markers in defining hematopoietic cell

    2018-10-24

    Reliable cell-surface markers in defining hematopoietic ryanodine with different potential are valuable in assessing well-defined populations for further investigation. Cell-surface markers are of particular significance for hPSC differentiation, as the in vitro system lacks anatomical and morphological information. To date, a few surface markers have been identified in defining blood cells at different stages of hESC/iPSC differentiation (Choi et al., 2012; Vodyanik et al., 2006). For example, CD43 has been identified as a marker for hPSC-derived HPCs with the potential to form CFUs, thus providing an easy way to isolate HPCs in vitro (Vodyanik et al., 2006). However, defining functional HECs has been complex and challenging due to the limitation of reliable surface markers. Our finding that the HECs are almost exclusively GATA2/eGFP+ allows us to identify reliable surface markers for functional HECs. Indeed, we identified a panel of cell-surface markers that differentially expressed between GATA2/eGFP+ HECs and GATA2/eGFP− ECs. The lists include some previously identified hematopoietic markers such as CD62P and CD41, and many other unidentified ones. Among them, CD61 is of particular significance as it almost exclusively marked the small portion of HECs capable of producing HPCs in hPSC differentiation. Interestingly, almost all HPCs generated from hPSC-derived HECs are also CD61+. In addition, the CFU-Mix (also referred as GEMM) potential cells were restricted in the CD61low cells in YS of E10 mouse embryo (Figure S5A), indicating that CD61low also labels HPCs in mouse early embryo. In another report, Boisset et al. (2013) also showed that the HSCs with transplantation potential are restricted in the CD61low fraction in E11 AGM and the E11 placenta, while at a later stage, as in the E12 YS and E14 fetal liver, transplantable HSCs were found in both CD61− and CD61low fractions, indicating that CD61 marks HSCs at the specific stage of mouse embryo. Another integrin, CD41, was also increased in GATA2/GFP+ HECs. CD41 has been known as a marker to identify HPCs from mouse embryo (Ferkowicz, 2003). However, mouse HECs were defined as CD41− (CD31+CD41−CD45−Ter119−). Interestingly, CD61 is usually co-expressed with CD41 in many different cell types. However, our findings show that at an early stage of hematopoiesis both in vitro and in vivo, CD61 is expressed independently of CD41 in HECs. Indeed, hematopoietic cells isolated from E8 or E10 mouse embryo exhibited significant differential expression of CD61 and CD41 (Figures S5B and S5C). Similarly, the expressions of CD41 and CD61 are not consistent in hematopoietic cells differentiated from hPSCs in vitro (Figures S4B and S4C). These data indicate that CD61 or CD41 might act independently in certain cell types or stages of hematopoiesis, but their exact roles and timing need to be investigated further. Nonetheless, our finding that CD61 is conservative in defining HECs both in vitro for hPSC differentiation and in vivo for mouse embryo provides valuable information on how to define and access the bipotent HECs. It is noteworthy that the percentage of HECs labeled by CD61 from either YS or AGM is very low, varying between 2% and 10% of previously recognized ECs (Figure 5A). Therefore, the identification of CD61 would allow dozen-fold enrichment for HECs and would greatly facilitate future research in understanding the mechanisms of HE determination and HSC generation both in vitro and in vivo.
    Experimental Procedures
    Author Contributions
    Introduction Erythroblasts are one of the first blood cells to be generated during embryonic hematopoiesis (Palis and Yoder, 2001). Investigating the early erythropoiesis is helpful in comprehending the occurrence and regulation of early embryonic hematopoiesis. Research in early erythropoiesis is largely performed on murine models (Keller et al., 1999; Palis et al., 1999). The lack of proper in vitro and in vivo systems hampers the observation of this process during human embryonic/fetal development. The isolation of human embryonic stem cells (hESCs) (Thomson et al., 1998) and the establishment of human induced pluripotent stem cells (hiPSCs) (Takahashi et al., 2007) have provided excellent tools to investigate early events in human hematopoiesis. Large-scale production of hPSC-derived erythroblasts can be achieved in vitro by mimicking in vivo erythropoiesis (Lu et al., 2008; Ma et al., 2008). Previously, we found that erythroblasts were derived from hESC/mouse fetal liver stromal cell (mFLSC) co-culture and matured continuously (Ma et al., 2008). In this study, we have modified this method by co-culturing hPSCs with aorta-gonad-mesonephros (AGM)-S3 cells (Xu et al., 1998), which were obtained from the earliest definitive hematopoiesis-supporting region (Matsuoka et al., 2001), to study human early erythropoiesis.