Haematopoietic stem cells derive directly from aortic endothelium during development

Abstract

A major goal of regenerative medicine is to instruct formation of multipotent, tissue-specific stem cells from induced pluripotent stem cells (iPSCs) for cell replacement therapies. Generation of haematopoietic stem cells (HSCs) from iPSCs or embryonic stem cells (ESCs) is not currently possible, however, necessitating a better understanding of how HSCs normally arise during embryonic development. We previously showed that haematopoiesis occurs through four distinct waves during zebrafish development, with HSCs arising in the final wave in close association with the dorsal aorta. Recent reports have suggested that murine HSCs derive from haemogenic endothelial cells (ECs) lining the aortic floor. Additional in vitro studies have similarly indicated that the haematopoietic progeny of ESCs arise through intermediates with endothelial potential. Here we have used the unique strengths of the zebrafish embryo to image directly the generation of HSCs from the ventral wall of the dorsal aorta. Using combinations of fluorescent reporter transgenes, confocal time-lapse microscopy and flow cytometry, we have identified and isolated the stepwise intermediates as aortic haemogenic endothelium transitions to nascent HSCs. Finally, using a permanent lineage tracing strategy, we demonstrate that the HSCs generated from haemogenic endothelium are the lineal founders of the adult haematopoietic system.

Figure 1. Direct imaging of HSC emergence from the embryonic aortic floor

a–d, Time-lapse imaging of a double transgenic cmyb:eGFP, kdrl:memCherry embryo between 30–38 hpf. Four sequences from Supplementary video 1 are presented, documenting the stepwise emergence of HSCs from hemogenic endothelium in denoted region (blue box, upper panel). For each time point, the GFP, memCherry and merged images are shown. memCherry; GFP double positive cells are denoted by white arrowheads (A, aorta; V, vein).

Figure 2. Prospective isolation of aortic hemogenic endothelium and nascent HSCs

a–b, Double transgenic cmyb:eGFP; kdrl:RFP embryos were dissected to separate anterior from posterior, AGM containing tissues at 36 hpf. Throughout the figure, the cellular fraction including hemogenic endothelium is denoted by red boxes or bars, nascent HSCs by orange boxes or bars, maturing HSCs by yellow boxes or bars, and mature HSCs by green boxes or bars. c, Correlation of FACS expression profiles to stepwise HSC emergence in kdrl:memCherry; cmyb:eGFP embryos (i, hemogenic endothelium; ii, nascent HSC; iii, maturing HSC). Images captured from Supplementary video 1. d, Quantitative PCR expression for endothelial (top panel) and hematopoietic (bottom panel) genes in purified kdrl⁺cmyb⁻ (red), kdrl⁺cmyb^lo (orange), kdrl^locmyb⁺ (yellow) and kdr⁻cmyb⁺ cells (green). Units on Y-axis represent fold changes from the kdrl⁺cmyb⁻ reference standard, which is set at 1.0. We note that the kdr⁻cmyb⁺ population contains some neuronal cells, effectively diluting the vascular and hematopoietic signals. Error bars, standard deviation.

Figure 3. Long-term lineage tracing of embryonic endothelial cells

a, Flow cytometric analysis of WKM from a transgenic βactin:switch adult animal. b, Bar graphs show the percentage of switched cells (DsRed^hi shaded black; DsRed^lo grey) in WKM of double transgenic kdrl:Cre; βactin:switch adult animals (n=32). DsRed⁻ cells are represented in white. c, Histogram plots show percentages of switched hematopoietic lineages at 6 months of age in WKM (average ± standard deviation, n=5). d, Quantitative PCR expression of switched (DsRed^hi in black; DsRed^lo in grey) and non-switched cells (white bars) at 3 months, for B lymphoid (pax5), myeloid (pu.1) and erythroid (gata1) genes. Units on Y-axis represent fold changes from WKM, the reference standard set at 1.0. Error bars, standard deviation.