During embryonic development, hemodynamic forces caused by blood flow support vascular remodeling, arterialization of luminal endothelium, and hematopoietic stem cell (HSC) emergence. Previously, we reported that fluid shear stress plays a key role in stimulating nitric oxide (NO) signaling in the aorta-gonad-mesonephros (AGM) and is essential for definitive hematopoiesis. We employed a Dynamic Flow System modified from a cone-and-plate assembly to precisely regulate in vitro exposure of AGM cells to a defined pattern of laminar shear stress. Here, we present the design of a microfluidic platform accessible to any research group that requires small cell numbers and allows for recirculation of paracrine signaling factors with minimal damage to nonadherent hematopoietic progenitors and stem cells. We detail the assembly of the microfluidic platform using commercially available components and provide specific guidance in the use of an emerging standard in the measurement of embryonic HSC potential, intravenous neonatal transplantation.
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This work was funded by grants from the American Society of Hematology, State of Texas Emerging Technology Fund, and National Institutes of Health to P.L.W.
Zovein AC, Hofmann JJ, Lynch M, French WJ, Turlo KA, Yang Y et al (2008) Fate tracing reveals the endothelial origin of hematopoietic stem cells. Cell Stem Cell 3:625–636CrossRefPubMedCentralPubMedGoogle Scholar
Eilken HM, Nishikawa S, Schroeder T (2009) Continuous single-cell imaging of blood generation from haemogenic endothelium. Nature 457:896–900CrossRefPubMedGoogle Scholar
Lancrin C, Sroczynska P, Stephenson C, Allen T, Kouskoff V, Lacaud G (2009). The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage. Nature 457:892–895CrossRefPubMedCentralPubMedGoogle Scholar
Bertrand JY, Chi NC, Santoso B, Teng S, Stainier DY, Traver D (2010) Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464:108–111CrossRefPubMedCentralPubMedGoogle Scholar
Boisset JC, van Cappellen W, Andrieu-Soler C, Galjart N, Dzierzak E, Robin C (2010) In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464:116–120CrossRefPubMedGoogle Scholar
Kissa K, Herbomel P (2010) Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464:112–115CrossRefPubMedGoogle Scholar
Lam EY, Hall CJ, Crosier PS, Crosier KE, Flores MV (2010) Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells. Blood 116:909–914CrossRefPubMedGoogle Scholar
Swiers G, Rode C, Azzoni E, de Bruijn MF (2013) A short history of hemogenic endothelium. Blood Cells Mol Dis 51:206–212CrossRefPubMedGoogle Scholar
Adamo L, Naveiras O, Wenzel PL, McKinney-Freeman S, Mack PJ, Gracia-Sancho J et al (2009) Biomechanical forces promote embryonic haematopoiesis. Nature 459:1131–1135CrossRefPubMedCentralPubMedGoogle Scholar