Continuous single-cell imaging of blood generation from haemogenic endothelium

Abstract

Despite decades of research, the identity of the cells generating the first haematopoietic cells in mammalian embryos is unknown. Indeed, whether blood cells arise from mesodermal cells, mesenchymal progenitors, bipotent endothelial-haematopoietic precursors or haemogenic endothelial cells remains controversial. Proximity of endothelial and blood cells at sites of embryonic haematopoiesis, as well as their similar gene expression, led to the hypothesis of the endothelium generating blood. However, owing to lacking technology it has been impossible to observe blood cell emergence continuously at the single-cell level, and the postulated existence of haemogenic endothelial cells remains disputed. Here, using new imaging and cell-tracking methods, we show that embryonic endothelial cells can be haemogenic. By continuous long-term single-cell observation of mouse mesodermal cells generating endothelial cell and blood colonies, it was possible to detect haemogenic endothelial cells giving rise to blood cells. Living endothelial and haematopoietic cells were identified by simultaneous detection of morphology and multiple molecular and functional markers. Detachment of nascent blood cells from endothelium is not directly linked to asymmetric cell division, and haemogenic endothelial cells are specified from cells already expressing endothelial markers. These results improve our understanding of the developmental origin of mammalian blood and the potential generation of haematopoietic stem cells from embryonic stem cells.

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis

“…19 Again, standard LeGO vectors should be particularly useful for analyzing hematopoiesis. Indeed, unambiguous genetic marking may become very important in the context of single-cell imaging studies 20 and for the analysis of novel ex vivo models of definitive hematopoiesis. 21 The set of already available LeGO vectors, together with the possibility to rapidly include additional markers (based on their modular structure) make them ideal tools for simultaneous marking of different cell populations.…”

Definitive human and mouse hematopoiesis originates from the embryonic endothelium: a new class of HSCs based on VE-cadherin expression

“…Another possibility is that hemogenic precursors contained within EL and fetal BM CD34 + CD31 + CD45 -cell populations could strictly correspond to hematopoietic precursors, which is only possible if we admit that commitment to HCs arise before CD45 expression by these cells. Indeed, previous studies in human and murine ES models demonstrated that during hematopoietic development CD41 is expressed before CD45 (Eilken et al, 2009;Lancrin et al, 2009;Mikkola et al, 2003;Vodyanik et al, 2006). Then, despite the fact that they are CD45 -, the CD34 + CD31 + CD45 -cells that we assimilated as ECs could correspond to already committed HCs.…”

A short history of hemogenic endothelium

“…More recently, lineage-tracing studies in which Cre recombinase was expressed under the regulatory control of the endothelial marker VE-Cadherin (VECadh) lent further support to this model [27,28]. Arguably, the strongest support for an endothelial origin of hematopoietic cells came from the recent time-lapse imaging studies that visualized the transition of endothelium into blood in real-time, both in vitro [29] and in/ex vivo [30][31][32][33]. The process of endothelial cells taking on a hematopoietic fate has been termed the endothelial-to-hematopoietic transition (EHT), and involves the bending out and rounding up of an endothelial cell and its subsequent detachment from the vascular wall and formation of a free-moving hematopoietic cell [30].…”