Analysis of Endothelial-to-Haematopoietic Transition at the Single Cell Level identifies Cell Cycle Regulation as a Driver of Differentiation

Abstract

Haematopoietic stem cells (HSC) first arise during development in the aorta-gonad-mesonephros (AGM) region of the embryo from a population of haemogenic endothelial cells which undergo endothelial-to-haematopoietic transition (EHT). Despite the progress achieved in recent years, the molecular mechanisms driving EHT are still poorly understood, especially in human where the AGM region is not easily accessible. In this study, we took advantage of a human pluripotent stem cell (hPSC) differentiation system and single-cell transcriptomics to recapitulate EHT in vitro and uncover mechanisms by which the haemogenic endothelium generates early haematopoietic cells. We show that most of the endothelial cells reside in a quiescent state and progress to the haematopoietic fate within a defined time window, within which they need to re-enter into the cell cycle. If cell cycle is blocked, haemogenic endothelial cells lose their EHT potential and adopt a non-haemogenic identity. Furthermore, we demonstrated that CDK4/6 and CDK1 play a key role not only in the transition but also in allowing haematopoietic progenitors to establish their full differentiation potential. Therefore, we propose a direct link between the molecular machineries that control cell cycle progression and EHT.

VEGFC/FLT4-induced cell-cycle arrest mediates sprouting and differentiation of venous and lymphatic endothelial cells

“…By mating these fish with Tg(kdrl:tagBFP) (Matsuoka et al, 2016) animals, which feature non-nuclear, blue fluorescence in all ECs, we can distinguish ECs not labeled by either FUCCI fluorophore, by virtue of cytoplasmically expressed tagBFP (Figures 1B and 1D). Based on previous reports using FUCCI (Canu et al, 2020;Pauklin and Vallier, 2013), such cells (i.e., tagBFP+;FUCCIÀ) are expected to be in early G1. Confocal imaging of Tg(fli1a:Gal4FF;UAS:FUCCI;kdrl:tagBFP) embryos revealed significant changes in the cell cycle phase distribution of PCV-ECs, between 26 and 42 hours post-fertilization (hpf).…”

First blood: the endothelial origins of hematopoietic progenitors

“…Although pro- and pre-HSCs have thus far only been defined in the mouse, the human AGM also produces blood clonogenic cells without the capability of long-term engraftment prior to bona fide HSC emergence [ 40 , 41 ]. Moreover, in vitro differentiation of human pluripotent stem cells yields hematopoietic progenitors with multilineage differentiation potential, but no stem cell activity [ 42 , 43 ], and the transcriptional signature of these in vitro-derived hematopoietic progenitors is similar to that of hematopoietic cells in the human AGM in vivo [ 44 ]. Altogether, these findings suggest that hematopoietic precursors similar to mouse pro-HSCs and pre-HSCs exist in human before mature HSCs are produced.…”

“…In this context, the in vitro differentiation of human pluripotent stem cells (hPSCs) provides a valuable tool to recapitulate the earliest stages of human hematopoietic development. This approach has allowed, for example, to elucidate signalling pathways required for human primitive vs. definitive hematopoiesis [ 14 , 15 ] and the importance of cell cycle entry as a necessary condition for EHT [ 44 ]. Moreover, hPSC systems allow hematopoietic disease modeling and provide a useful resource for regenerative medicine.…”

Large‐scale generation of megakaryocytes from human embryonic stem cells using transgene‐free and stepwise defined suspension culture conditions

“…We also applied an immunoselection technique to harvest CD34 + cells, which acted as the seed cells for hematopoietic and MK differentiation process at the end of stage 2 induction. Many researchers have used this method to improve the differentiation efficiency of hematopoietic cells from pluripotent stem cells 35‐37 . Both Q‐CTS‐hESC‐2 and H9 hESCs showed high differentiation efficiency of CD41a + MKs.…”