Introduction

Transplantation of hematopoietic stem cells (HSCs) is curative for many patients with bone marrow failure and blood cancers, yet two-thirds of these patients have no suitable donor [1]. This unmet need has driven enterprising efforts to identify cues that dictate HSC fate such that alternative sources can be produced in a dish [2]. To date, HSCs remain intractable to de novo generation from pluripotent stem cells and other sources. We have previously shown that the force of blood flow in the embryonic arteries promotes HSC development [3, 4]. Indeed, mutant mouse embryos without a heartbeat (and no blood flow) do not produce HSCs. The present study was designed to understand how mechanotransduction regulates generation of the first HSCs in the embryo.

Methods

We leveraged various fluidics platforms and a cardiac-mutant mouse model to examine a continuum of hematopoietic specification in mouse embryos where effects of shear stress could be isolated. Using single-cell methodologies in RNA-seq, protein synthesis, and microscopy, we assessed how physical forces associated with arterial blood flow impact mitochondrial assembly, cristae formation, and energy-generating capacity as cells undergo an endothelial-to-hematopoietic transition. Hematopoietic function after selection for mitochondrial membrane potential was measured by transplantation assays in adult mice.

Results

Our research shows that force in the embryonic vasculature drives mitochondrial biogenesis that endows endothelial precursors with hematopoietic potential. Interestingly, embryos without a heartbeat produce hematopoietic precursors with immature mitochondria containing fewer cristae and reduced mitochondrial protein translation. Transmission electron microscopy of endothelial cells lining the aorta shows profound immaturity of cristae without blood flow in embryos with no heartbeat. We find in single-cell RNA-seq that blood flow regulates transcripts encoding PI3K-Akt and mitochondrial machinery in precursors just prior to expression of hematopoietic transcripts.

This process can be mimicked ex vivo in microfluidic cultures where shear stress of an intensity typical within the embryonic mouse aorta (5 dyne/cm2) increases mitochondrial activity of hematopoietic precursors with improved transplantation performance. Force triggers transcriptional activation of genes encoding mitochondrial ribosomes, mitochondrial protein import complexes, and electron transport chain proteins, but also induces anabolic processes that precede transcription. Shear stress directly stimulates PI3K-Akt signaling within 5 minutes of initiation to post-translationally modify the mTORC1 effector 4E-BP1, which is known to dictate translation initiation and elongation of peptides required for mitochondrial capacity.

We believe that shear stress acts via mTOR to control remodeling of the proteome which is necessary for building mitochondrial capacity in hemogenic endothelium. Importantly, we find that hemogenic endothelium with high mitochondrial membrane potential exhibits superior engraftment in adult recipient mice.

Discussion

We identify an overlooked role for force in maturation of mitochondrial machinery which appears to be essential for HSC emergence and population of the blood system. Adaptation in protein synthesis likely dictates this mitochondrial remodeling. Although in vitro specification of HSCs remains elusive, our study could provide clues to a flow-sensitive molecular mechanism that could aid generation of HSCs in culture.

References

  1. Besse et al, J Oncol Pract, 11:e120-130, 2015.

  2. Barcia Durán et al, FEBS Lett, 593:3253-3265, 2019.

  3. Adamo et al, Nature, 459:1131-1135, 2009.

  4. Diaz et al, J Exp Med, 212:665-680, 2015.

Acknowledgements

Grants to P.L.W. from the American Society of Hematology Scholar Award and National Institutes of Health (K01DK092365, R01DK111599) supported this work.

Disclosures

No relevant conflicts of interest to declare.

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2024
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