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  • CB-5083 br Methods and materials br Results

    2018-11-06


    Methods and materials
    Results
    Discussion In vitro differentiation of ESCs closely mimics early embryonic development and can produce large quantity of hematopoietic cells. However, segregation of the EryPs from surrounding CB-5083 differentiated from mESCs has not been reported. Here, we demonstrated that CD71high could be utilized as a marker to purify EryPs from differentiated ESCs. Although overlapped with markers for hematopoietic progenitors, CD41 and c-kit, the CD71high population was exclusive for myeloid-lymphoid cells which were positive for CD45. In addition, CD71high cells sorted from day 5 EBs appeared to be homogenous, highly enriched for erythroblasts that gave rise about 800 red colonies per 10,000 cells. These data clearly demonstrate that EryPs in the CD71high population are the dominant blood lineage. As previously reported, EryPs differ significantly from EryDs in size, globin expression, and transcriptional regulation (Baron et al., 2012; Fraser, 2013; Palis et al., 2010). The molecular mechanisms underlying these differences are however only partially described because significant amounts of EryPs are required for such mechanistic investigation. Efficient production and segregation of CD71high EryPs from differentiated ESCs will therefore allow us to explore these fundamental questions during erythropoiesis. Similarly to EryPs from YS around 8.5dpc, βH1 was the predominant type of embryonic goblin in CD71high cells. These putative CD71high EryPs were able to go through “maturational globin switching” and exhibited similar expression pattern with YS-derived EryPs. More importantly, we further demonstrated that these CD71high cells matured into Ter119+ enucleated red blood cells when co-culturing with macrophages in vitro. These data thus validate our claim that CD71high population represents an in vitro counterpart of YS-derived EryPs that can mature and enucleate. Notably, enucleation of erythrocytes in vitro has been a challenge in the field of hematopoietic research. It will be of great interest to explore the specific contribution of macrophages may have during this process in the future study. According to current model, bi-potential megakaryocyte-erythroid precursors (MEPs) exist during primitive hematopoiesis (Klimchenko et al., 2009; Tober et al., 2007). However, it remains unclear how the fate specification of EryPs and megakaryocytes is regulated. We found that Scl promoted the formation of all blood lineages from mESCs as a master regulator of hematopoiesis. In contrast, megakaryopoietic differentiation depends more stringently on Ets family members, such as Erg and Fli1. At the expense of megakaryocytic development, induction of Klf1 favored the commitment of erythrocytes by upregulating CD71high population and transcript levels of βH1 and εy globins. Similarly, Eaf1 depletion blocked the development of erythrocytes as demonstrated by reduced percentage of CD71high cells, thus indicating a critical requirement of Eaf1 in the formation of EryPs. Therefore, our approach to utilize CD71high EryPs from ESCs coupled with genetic modification may represent a valuable model to examine cell-intrinsic regulation of primitive erythropoiesis during early embryonic development. The following are the supplementary materials related to this article.
    Acknowledgment The Scl−/− ESC line is a kind gift from Dr. Stuart Orkin\'s lab at Harvard Medical School. We would like to thank Dr. Bing Du at East China Normal University for the help in macrophage derivation from BM. This work was supported by grants from the Ministry of Science and Technology of China (2010CB945403 and 2014CB964800), the National Science Foundation of China (31271589 and 30971522), and the Science and Technology Commission of Shanghai Municipality (11DZ2260300 and 13JC1406402).
    Introduction Cell differentiation in adults involves flexible but precise control of the expression of genes specific for each cell fate or pluripotency. Crucial changes in their expression are most likely controlled by specific epigenetic rearrangements (Vincent & Van Seuningen, 2009). Extensive studies conducted to characterise embryonic stem cells (ESC) at the epigenetic level have shown that ESC harbour a permissive chromatin with hypomethylation of their genome compared to differentiated cells (Bibikova et al., 2006) and bivalent histone marks where large regions containing trimethylated K27H3 (gene silencing) colocalise with smaller regions containing methylated K4H3 (gene activation) (Bernstein et al., 2006). This allows rapid transcriptional activation of genes specific for a cell fate. One can hypothesise that the plasticity of adult SC involves similar epigenetic mechanisms. However, only a few studies mostly conducted in vitro have addressed this concept.