Lysyl oxidases are novel regulators of definitive hematopoiesis

项目摘要/摘要 造血干细胞(HSCs)是一种自我更新、可移植、具有重建能力的细胞。 所有的血统都是终身的。因此，造血干细胞移植(HSCT)被用于治疗白血病和其他疾病。 血液紊乱。原则上，诱导多能干细胞(IPSC)来源的造血干细胞可以提供无限的 供移植、针对特定患者的体外研究、药物研究和作为基因输送载体的造血干细胞来源 心理治疗。然而，到目前为止，还不可能从高植入率的IPSC中产生真正的HSC 潜在的和多血统的重建。从IPSC产生HSC的一种方法是复制 体外培养的HSCs的正常胚胎规格。肝星状细胞起源于血管内皮细胞。 胚胎背主动脉(DA)。细胞外基质(ECM)为HSC提供了物理环境 前体细胞接收信号，诱导其对造血谱系的承诺。尽管很可能 对于ECM的重要性，我们还不清楚ECM如何或是否会影响HSC规范。这个 赖氨酰氧化酶(Lox)家族的细胞外基质修饰酶控制细胞外基质的硬度，生长因子信号的显示， 以及通过催化包括胶原和纤维连接蛋白在内的ECM蛋白的交联而获得的其他特性。vbl.使用 斑马鱼的造血发育模型，我已经证明了LOX活性是规范 最早的人类干细胞。我的初步研究表明，至少需要三个洛克斯家庭成员才能 明确的造血系统的规范和成熟。 在目标1中，我将系统地研究Lox家族对HSC规范的贡献。 在目标2中，我将确定HSC规范中LOX调节的机制，以区分 最可能的可能性：LOX诱导的ECM僵硬直接通过整合素-ECM调节HSCs 相互作用，间接通过调节现有的生长因子信号，或两者兼而有之。 在这份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一名独立科学家的事业。目标建立在我以前的经验基础上，而 同时提供HSC生物学领域的培训。我将获得基因组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，我还会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.