A Bioreactor Simulating Pulse-Pressure Mediated Circumferential Stretch Stimulates Hematopoietic Stem Cell Formation

During fetal development, heartbeat precedes blood circulation and definitive hematopoietic stem cell (HSC) formation in the aorta-gonad-mesonephros (AGM) region. However, how the function and growth of heart and blood vessels are linked to the development of blood is largely unknown. Blood flow-mediated shear stress on the endothelial lining of the AGM stimulates the endothelial emergence of HSCs. A zebrafish mutant for cadherin 5, which has normal blood formation despite an early circulation arrest, enabled us to analyze the timing and intertwined roles of heartbeat in blood formation. Here we show that heartbeat and stretching of the blood vessel stimulate the HSC formation. We used three-dimensional (3D) digital Doppler ultrasound, microangiography, and time-lapse confocal microscopy to demonstrate that a beating heart, in cdh5-silenced zebrafish embryos, produces pulsation in blood vessels despite the lack of cardiac output and active blood circulation. Our time-lapse confocal imaging of cd41:eGFP+ HSCs emerging from flk1:mCherry+ endothelium from the cdh5-silenced embryos, followed by machine learning analysis of pulsating blood vessels, further establishing that pulse-pressure on the arterial endothelial lining of AGM regulates the endothelial emergence of HSCs. To understand the relationship between pulse pressure, biomechanical stretching of endothelial lining, and HSC formation, we recreated pulsating blood vessel-like conditions in a bioreactor by applying cyclic strain on mouse E11.5 AGM-derived hemogenic endothelial cells. Cyclic strain-mediated Trpv4 activation in hemogenic endothelial cells both stimulated HSC development and rescued hematopoiesis in the silent heart (tnnt2)-silenced embryos, which lacked blood flow and heartbeat. Therefore, heartbeat-mediated biomechanical stretching of hemogenic endothelial cells in the AGM stimulates trpv4 channels during the endothelial-to-HSC transition. Our findings advance our fundamental understanding of the developmental cues and 3D microenvironmental mechanisms regulating HSC formation. Our model could establish hemogenic endothelial cells as a potential source of bone marrow-independent HSCs.