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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 unknown1. Indeed, whether blood cells arise from mesodermal cells, mesenchymal progenitors, bipotent endothelial–haematopoietic precursors or haemogenic endothelial cells remains controversial2,3,4,5,6,7,8,9. 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 technology10 it has been impossible to observe blood cell emergence continuously at the single-cell level, and the postulated existence of haemogenic endothelial cells remains disputed1. 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.

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Figure 1: ESC-derived mesodermal cells give rise to haemogenic endothelial colonies.
Figure 2: Continuous observation of endothelial and haematopoietic molecular markers.
Figure 3: Nascent blood cells generated from ECs express CD41 before CD45.
Figure 4: Mesodermal cells derived from mouse embryos give rise to haemogenic endothelial colonies.

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Acknowledgements

We are grateful to S. Nishikawa, M. A. Rieger, A. Hermann and M. Yoder for technical advice and discussions, M. Ogawa and H. Niwa for the VEVC2 and EB3 ESC lines, respectively, B. Schauberger for programming contributions, and A. Roth and C. Raithel for technical support. We thank M. Goetz, A. Hermann, M. A. Rieger and A. IJpenberg for critical reading of the manuscript. Part of this study was financed by the Deutsche Forschungsgemeinschaft to T.S. and by the Leading Project for the Realization of Regenerative Medicine to S.-I.N.

Author Contributions H.M.E. planned and performed experiments; S.-I.N. discussed results and commented on the manuscript; T.S. designed the study and experiments, developed the time-lapse imaging and cell-tracking technology, performed initial experiments, analysed data with H.M.E. and wrote the paper with H.M.E.

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Correspondence to Timm Schroeder.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8 with Legends, Supplementary Tables 1-3 and Supplementary Movies Legends 1-8 (PDF 2443 kb)

Supplementary Video 1

This time-lapse movie, covering a period of about 4.5 days, shows that cells with endothelial morphology can give rise to blood cells (see file s1 for full legend). (MOV 9773 kb)

Supplementary Video 2

This time-lapse movie, covering a period of about 1 day, proofs the cell identity of endothelial and blood cells by molecular and functional markers (see file s1 for full legend). (MOV 3788 kb)

Supplementary Video 3

This time-lapse movie, covering a period of about 2.5 days, shows that nascent blood cells generated by endothelial cells express the surface marker CD41 prior to CD45 expression (see file s1 for full legend). (MOV 4070 kb)

Supplementary Video 4

This time-lapse movie, covering a period of about 1.5 days, shows that mesodermal cells purified from 7.5 dpc mouse embryos can generate hemogenic endothelial cells (see file s1 for full legend). (MOV 3892 kb)

Supplementary Video 5

This time-lapse movie, covering a period of about 6.5 days, shows that nascent blood cells generated by endothelial cells have colony forming potential with high proliferative potential and multilineage (including megakaryocytic) potential. (MOV 8815 kb)

Supplementary Video 6

This time-lapse movie, covering a period of about 5 hours, demonstrates the temporal and optical resolution of the primary image sequences used for cell tracking (see file s1 for full legend). (MOV 2331 kb)

Supplementary Video 7

This time-lapse movie, covering a period of about 8.5 hours, demonstrates the temporal and optical resolution of the primary image sequences used for cell tracking (see file s1 for full legend). (MOV 6633 kb)

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Eilken, H., Nishikawa, SI. & Schroeder, T. Continuous single-cell imaging of blood generation from haemogenic endothelium. Nature 457, 896–900 (2009). https://doi.org/10.1038/nature07760

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