Lysyl oxidases are novel regulators of definitive hematopoiesis

项目总结/摘要 造血干细胞（HSCs）是具有自我更新能力的可移植细胞， 所有的血统因此，HSC移植（HSCT）被用于治疗白血病和其他疾病。 血液疾病原则上，诱导多能干细胞（iPSC）衍生的HSC可以提供无限的造血干细胞。 用于移植、患者特异性体外研究、药物研究和作为基因递送载体的HSC来源 疗法然而，迄今为止，尚不可能从具有高植入率的iPSC产生真正的HSC。 潜力和多谱系重建。从iPSC产生HSC的一种方法是复制 HSC在体外的正常胚胎特化。HSC起源于造血内皮（HE）细胞， 胚胎背主动脉（DA）。细胞外基质（ECM）提供了HSC生长的物理环境。 前体接受诱导其定型为造血谱系的信号。尽管可能 虽然ECM的重要性，但我们还不了解ECM如何或是否影响HSC规格。的 ECM修饰酶的赖氨酰氧化酶（Lox）家族控制ECM硬度，生长因子信号的显示， 以及通过催化包括胶原和纤连蛋白在内的ECM蛋白的交联而具有其它性质。使用 在斑马鱼造血发育模型中，我已经证明Lox活性是造血发育特异化所必需的。 最早的HSC我的初步研究表明，至少有三个家庭成员需要适当的 最终造血系统的特化和成熟。 在目标1中，我将系统地研究Lox家族对HSC特化的贡献。 在目标2中，我将确定HSC特化的Lox调节机制，以区分 最可能的可能性：Lox诱导的ECM刚度直接通过整合素-ECM调节HSC 相互作用，间接通过调节现有的生长因子信号，或两者。 在F32申请中提出的研究和培训将在我的导师Dr。 威尔逊·克莱门茨和我在圣裘德儿童研究医院的导师团队。该提案描述了 研究，将提供特殊的培训机会，并产生原创和有意义的科学 这些发现开启了我作为独立科学家的职业生涯。这些目标建立在我以前的经验上， 同时提供HSC生物学领域的培训。我将获得基因编辑和切割方面的专业知识- 用于ECM可视化和分析的边缘技术，包括二次谐波产生（SHG）和 偏振光显微镜此外，我将利用众多的职业发展资源。

Project Summary/Abstract Hematopoietic stem cells (HSCs) are self-renewing, transplantable cells that have the capacity to reconstitute all blood lineages for life. Consequently, HSC transplantation (HSCT) is used in treatment of leukemia and other blood disorders. In principle, induced pluripotent stem cell (iPSC)-derived HSCs could provide an unlimited source of HSCs for transplant, patient-specific in vitro studies, drug studies and as a vector for delivery of gene therapy. However, to date, it has not been possible to generate bona fide HSCs from iPSCs with high engraftment potential and multilineage reconstitution. One means of generating HSCs from iPSCs would be to reproduce the normal embryonic specification of HSCs in vitro. HSCs arise from hemogenic endothelial (HE) cells in the embryonic dorsal aorta (DA). The extracellular matrix (ECM) provides the physical environment in which HSC precursors receive signals that induce their commitment to the hematopoietic lineage. Despite the likely importance of the ECM, we do not yet understand how or whether the ECM influences HSC specification. The lysyl oxidase (Lox) family of ECM-modifying enzymes controls ECM stiffness, display of growth factor signals, and other properties by catalyzing the cross-linking of ECM proteins including collagen and fibronectin. Using the zebrafish model of hematopoietic development, I have shown that Lox activity is required for specification of the earliest HSCs. My preliminary studies indicate that at least three Lox family members are required for proper specification and maturation of the definitive hematopoietic system. In Aim 1, I will systematically examine Lox family contribution to HSC specification. In Aim 2, I will determine the mechanism of Lox regulation of HSC specification to distinguish between the most likely possibilities: Lox-induced ECM stiffness regulates HSCs directly through integrin-ECM interactions, indirectly through modulating existing growth factor signals, or both. The research and training proposed in this F32 application will take place under the guidance of my mentor, Dr. Wilson Clements and my mentorship team at St. Jude Children’s Research Hospital. The proposal describes research that will provide exceptional training opportunities and generate original and meaningful scientific findings to launch my career as an independent scientist. The aims build on my prior experience while simultaneously providing training in the field of HSC biology. I will gain expertise in genome editing and cutting- edge techniques for visualization and analysis of ECM, including second harmonic generation (SHG) and polarized light microscopy. In addition, I will take advantage of numerous career development resources.