Abstract
ABSTRACTThe genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17−CD34+CD43− endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17−CD34+CD43+ blood cells and SOX17+CD34+CD43− endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.
Highlights
Blood cells develop from an endothelial intermediate at multiple stages during embryonic development in vertebrates (Dzierzak and Bigas, 2018; Ivanovs et al, 2017)
To facilitate the dissection of early human haematopoiesis, we used a dual reporter human pluripotent stem cell (hPSC) line, SOX17mCHERRY/wRUNX1CGFP/w (Ng et al, 2016), in which GFP targeted to RUNX1C marks hematopoietic progenitor cells (Corada et al, 2013; Sroczynska et al, 2009), and mCHERRY targeted to SOX17 marks vascular endothelium (Burtscher et al, 2012; Challen and Goodell, 2010; Clarke et al, 2013)
We have modelled extra-embryonic human haematopoiesis and dissected the role of the transcription factor RUNX1 in analyses facilitated by the use of a reporter line in which GFP reported cells expressing the haematopoietic specific, C isoform, of RUNX1 and mCHERRY, expressed from the SOX17 locus, marked endothelium
Summary
Blood cells develop from an endothelial intermediate at multiple stages during embryonic development in vertebrates (Dzierzak and Bigas, 2018; Ivanovs et al, 2017). The methylcellulose-based blast colony-forming cell (BL-CFC) assay has been used to interrogate formation of the earliest human blood cells that correspond to the first products of yolk sac haematopoiesis (Kennedy et al, 2007). In this assay, the BL-CFC proliferates and differentiates to form a core structure (D’Souza et al, 2005) that includes haemogenic endothelial cells, which in turn generate blood cells via an endothelial-to-haematopoietic transition (EHT) (Lancrin et al, 2009). EMPs from the mouse yolk sac, and HSCs and preHSCs emerging from the mouse AGM all require Runx, whereas the earliest wave of yolk sac erythrocytes still appears in Runx1-null mouse embryos (de Bruijn and Dzierzak, 2017; Frame et al, 2016; Okuda et al, 1996; Wang et al, 1996)
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