Biomechanical Determinants of Hematopoietic Stem Cell Potential

项目总结 供者匹配的造血干细胞(HSCs)来源的可获得性继续限制获得和 异基因造血干细胞移植后的结果。对改善HSC来源的未得到满足的需求推动了全球 改进捐赠者招募和匹配，以及积极尝试开发患者来源的或 普遍相容的造血细胞。目前，在一道菜中指定HSCs的效率很低，并且 大多数使用共培养和表达造血基因的方法只能产生有限的祖细胞 血统和嫁接潜力。 我们的研究表明，血液流经血管系统所产生的生物机械力是一个关键的调节因素。 能促进具有长期造血重建潜力的细胞的植入。在……里面 在我们之前的资助期，我们发现血流的启动是能量代谢和 HSC前体细胞中的线粒体动力学称为血源性内皮细胞。 我们目前研究的目标是定义促进最终造血的代谢适应， 在定向分化和扩展中利用生物物理线索(如剪切力)的长期目标 用于治疗性移植和血液病建模的定制HSCs。具体而言，我们的目标是定义 线粒体成熟对血液内皮细胞发育的贡献，从而导致造血干细胞的产生。 我们将使用提供代谢活动的单细胞分辨的方法组合，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 线粒体成熟对血管内皮细胞前体参与造血命运的影响 药物靶向、体外平台仿生建模和心脏突变小鼠模型。在我们的 第二个目标是，我们利用来自互补数据集的试点数据，这些数据支持线粒体的作用 通透性转换孔(MPTP)在动脉内皮细胞分化中的作用我们解决了两相监管如何 MPTP在内皮向造血细胞转化(EHT)的连续体上开放指示获得 造血命。中断或增强的MPTP活动的后果将在EHT期间定义为 评估代谢和造血能力的指标。拟议的研究将解决一个主要不足之处 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 并有望通过代谢重编程启发体外生成HSCs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.