Mechanisms and consequences of sickle cell disease-induced cycling in hematopoietic stem cells

项目摘要 镰状细胞病(SCD)是一种痛苦的终生衰弱疾病，由编码基因突变引起。 血红蛋白β亚基，引起异常的血红蛋白聚合导致溶血，反复发作 血管闭塞和慢性全身炎症导致相当大的全球发病率和早期死亡率。 SCD的治疗依赖于造血干细胞(HSC)移植，但其破坏性作用 SCD对HSCs的病理生理学研究仍未定性化，我们试图填补当前知识的这一空白。我们的 初步研究表明，在转基因SCD小鼠模型中，随着年龄的增长，HSCs表现出更多的周期， SCD患者来源的HSCs的体外分化偏重于髓系。作为一名博士后研究员 麦金尼-弗里曼实验室，我将研究其功能后果和分子机制 在小鼠模型中SCD介导的HSC循环，并将这些发现翻译到人HSC中 SCD。在目标1中，我将使用集落形成试验，限制稀释一次和二次HSC移植， 和连续暴露于化疗以评估SCD诱导的循环对HSC的有害影响 频率和功能。在目标2中，我将探索分子和表观遗传失调背后的增加 SCD期间的HSC循环。从SCD小鼠和对照小鼠分离的HSCs将受到Bulk RNA-seq和 ATAC-SEQ用于定义与基因启动子可及性变化相关的转录失调 冥想增加了骑车的次数。最后，我将通过询问细胞周期将我们的发现转化为人类 从SCD患者分离的HSCs的调节失调(目标3)。SCD患者衍生的骨髓抽吸物将被 用流式细胞仪和体外EDU分析表型HSCs的频率和细胞周期状态的变化 成立为法团。将通过集落形成来分析SCD HSCs的谱系潜能和造血量 体外单个HSC分化实验。最后，SCD HSC的再填充潜力和自我更新将是 限制稀释一次和二次移植到表达免疫的人细胞因子中 有缺陷的小鼠。总之，拟议的研究有助于加深我们对以前未曾探索过的 通过检测SCD的影响来研究HSC生物学方面的问题。对SCD介导的机制有更深入的了解 随着基因编辑后的自体HSC移植，HSC损伤的发生变得格外重要 或者，SCD的基因治疗在频率上有所改善和增加。这些目标借鉴了我以前在癌症方面的培训 恶性血液病的生物学研究，也为人类干细胞领域提供了大量新的培训机会 生物学和职业生涯发展。麦金尼-弗里曼实验室和圣裘德儿童研究 医院是接受HSC生物学研究的理想环境，结合最先进的技术 机构资源、职业发展资源和优秀的导师，唯一的目标是 晋升为独立的学术首席调查员职位。

Project Summary Sickle cell disease (SCD) is a painful debilitating life-long condition resulting from mutations in the gene encoding hemoglobin β subunit, causing abnormal hemoglobin polymerization leading to hemolysis, repeated vasooclusion, and chronic systemic inflammation resulting in substantial global morbidity and early mortality. Curative therapy for SCD relies on hematopoietic stem cell (HSC) transplantation, however the damaging effects of SCD pathophysiology on HSCs remain uncharacterized and we seek to fill this gap in current knowledge. Our preliminary studies show that HSCs display increased cycling in a transgenic SCD mouse model upon aging, and myeloid lineage biased in vitro differentiation of SCD patient derived HSCs. As a postdoctoral fellow in the McKinney-Freeman laboratory, I will investigate the functional consequences and molecular mechanisms underlying SCD mediated HSC cycling in a murine model and translate these findings to human HSC during SCD. In Aim 1, I will use colony formation assays, limiting dilution primary and secondary HSC transplantation, and serial exposure to chemotherapy to assess the detrimental impact of SCD-induced cycling on HSC frequency and function. In Aim 2, I will probe the molecular and epigenetic dysregulation underlying increased HSC cycling during SCD. HSCs isolated from SCD and control mice will be subjected to bulk RNA-seq and ATAC-seq to define transcriptional dysregulation correlated with changes in gene promoter accessibility meditating increased cycling. Finally, I will translate our findings to humans by interrogating cell cycle dysregulation in HSCs isolated from SCD patients (Aim 3). SCD patient-derived bone marrow aspirates will be profiled for frequency of phenotypic HSCs and alterations in cell cycle status by flow cytometry and ex vivo EdU incorporation. Lineage potential and hematopoietic output of SCD HSCs will be analyzed by colony formation and in vitro single HSC differentiation assays. Lastly, SCD HSC repopulating potential and self-renewal will be examined by limiting dilution primary and secondary transplantation into human cytokine expressing immune deficient mice. Together the proposed studies serve to deepen our understanding of a previously unexplored aspect of HSC biology by examining the impact of SCD. Greater understanding of SCD mediated mechanisms of HSC impairment become exceptionally important as autologous HSC transplantation following gene editing or gene therapy for SCD improve and increase in frequency. These aims draw on my prior training in cancer biology of hematologic malignancies but also provide abundant novel training opportunities in the field of HSC biology and for professional career development. The McKinney-Freeman lab and St. Jude Children’s Research Hospital are ideal environments in which to receive training in the study HSC biology, combining state-of-the-art institutional resources, career development resources, and excellent mentorship with the singular goal of advancing to an independent academic primary investigator position.