Abstract 1406 Force remodels mitochondria by tuning protein metabolism in blood development

Hematopoietic stem cells (HSCs) replenish the blood and immune systems throughout life. Transplantation of HSCs is standard-of-care for patients with bone marrow failure and hematological malignancy, yet two-thirds of all patients needing a stem cell transplant have no suitable donor. This substantial unmet need has driven enterprising efforts to understand and manipulate the cues that shape HSC specification and self-renewal such that alternative sources can be generated in a dish. The present study was designed to understand the contribution of biophysical cues in the environment where the first HSCs emerge during embryogenesis. Methods: We have used a combination of microfluidic devices and in vivo genetic mouse models to examine fluid shear stress-induced changes in the transcriptome by RNA-seq, signaling by immunoblotting, population identities and single cell protein synthesis by flow cytometry, and mitochondrial ultrastructure and activity by extracellular flux assays and electron and super-resolution microscopy. The impact of biomechanical force and intracellular signaling on hematopoietic activity was measured by hematopoietic transplantation assays. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) for the University of Texas Health Science Center at Houston. Results: We found that the physical forces associated with blood flow are critical for defining metabolic features necessary for specification of HSC fate in the embryonic arteries that supply the endothelial precursor cells of HSCs. Mutant embryos without a heartbeat do not produce HSCs and have hematopoietic precursors with immature mitochondria containing fewer folds of the inner mitochondrial membrane known as cristae, as determined by electron and super-resolution microscopy. Force generated by blood flow stimulates mitochondrial protein translation, cristae formation, mitochondrial membrane potential, and oxidative phosphorylation. This process can be mimicked ex vivo in microfluidic cultures to increase mitochondrial activity of hematopoietic precursors with improved transplantation performance. Single-cell transcriptome and protein analyses suggest that force-responsive PI3K-Akt signaling regulates the mTORC1 effectors S6K and 4E-BP1 to promote translation of mRNAs encoding mitochondrial ribosomes and electron transport chain proteins. Conclusions: Our data reveal an overlooked role for force in maturation of mitochondrial machinery essential for HSC emergence and population of the blood system. Although in vitro specification of HSCs remains elusive, our study could provide clues to a flow-sensitive molecular mechanism essential for this process. Support for this research was provided to P.L. Wenzel from the NIH and American Society for Hematology.