Biomechanical Determinants of Hematopoietic Stem Cell Potential

项目摘要 造血干细胞（HSC）的供体匹配来源的可用性继续限制了获得和移植造血干细胞的机会。 异基因HSC移植后的结果。对改进HSC来源的未满足需求促使全球 在供体招募和匹配方面的改进，以及开发患者源性或 普遍兼容的造血细胞目前，培养皿中HSC的特化效率低， 大多数使用共培养和造血基因表达的方法仅产生具有有限的 谱系和移植潜力。 我们的研究表明，由血液流过血管系统引起的生物力学力是一个关键的调节器 并可促进具有长期造血重建潜力的细胞的植入。在 在我们之前的资助期间，我们发现血流的启动是能量代谢的关键决定因素， HSC前体（称为生血内皮）中的线粒体动力学。 我们目前研究的目的是确定促进确定性造血的代谢适应， 长期目标是利用生物物理线索，如剪切应力，在定向分化和扩增， 用于治疗性移植和血液病建模的定制HSC。具体来说，我们的目标是定义 线粒体成熟对造血内皮细胞发育的贡献，造血内皮细胞产生HSC。 我们将采用一种组合的方法，提供代谢活动，线粒体， 超微结构和造血功能。我们的第一个目标是解决中断的影响， 线粒体成熟对生血内皮前体细胞向造血细胞转化的影响 药理学靶向、体外平台仿生建模和心脏突变小鼠模型。在我们 第二个目标，我们利用来自补充数据集的试点数据，支持线粒体的作用， 血管内皮细胞分化中的通透性转换孔（mPTP）。我们讨论了双相调节 在内皮细胞向造血细胞转化（EHT）的连续过程中，mPTP的开放决定了 造血命运在EHT期间，将通过以下方式定义mPTP活性中断或增强的后果： 评估代谢和造血能力的指标。拟议的研究将解决一个主要缺陷， 在我们对线粒体成熟如何促进内皮细胞向造血细胞命运转变的理解中， 并有望激发通过代谢重编程在体外产生HSC的新方法。

PROJECT SUMMARY The availability of donor-matched sources of hematopoietic stem cells (HSCs) continues to limit access to and outcomes following allogeneic HSC transplant. Unmet need for improved HSC sources has motivated global improvements in donor recruitment and matching, as well as enterprising attempts to develop patient-derived or universally compatible hematopoietic cells. At present, specification of HSCs in a dish has been inefficient, and most methods using co-culture and expression of hematopoietic genes only produce progenitors with limited lineage and engraftment potential. Our studies show that biomechanical force caused by flow of blood through the vasculature is a critical regulator of hematopoiesis and can promote engraftment of cells with long-term hematopoietic reconstitution potential. In our prior funding period, we found that initiation of blood flow is a critical determinant of energy metabolism and mitochondrial dynamics in the HSC precursor known as the hemogenic endothelium. The objective of our current research is to define metabolic adaptations that promote definitive hematopoiesis, with the long-term goal of exploiting biophysical cues such as shear stress in directed differentiation and expansion of customized HSCs for therapeutic transplant and blood disease modeling. Specifically, we aim to define the contribution of mitochondrial maturation to development of the hemogenic endothelium that gives rise to HSCs. We will employ a combination of methods that provide single-cell resolution of metabolic activity, mitochondrial ultrastructure, and hematopoietic function. Our first aim is designed to address the effects of interrupting mitochondrial maturation on commitment of hemogenic endothelial precursors to the hematopoietic fate via pharmacological targeting, biomimetic modeling with in vitro platforms, and cardiac mutant mouse models. In our second aim, we leverage pilot data from complementary datasets that support a role for the mitochondrial permeability transition pore (mPTP) in differentiation of arterial endothelium. We address how biphasic regulation of mPTP opening over the continuum of the endothelial-to-hematopoietic transition (EHT) dictates acquisition of hematopoietic fate. Consequences of disrupted or enhanced mPTP activity will be defined during EHT by assessing indicators of metabolic and hematopoietic capacity. The proposed study will address a major deficiency in our understanding of how mitochondrial maturation contributes to transition of endothelial to hematopoietic fate and promises to inspire novel methods for generation of HSCs in vitro by metabolic reprogramming.