Integrative Epigenetic Analysis and Ex Vivo Generation of Megakaryocytes from Umbilical Cord Blood

在美国和欧洲，每年有数百万个单位的捐献者血小板被输入，用于治疗接受放射治疗、化疗或器官移植手术的患者。尽管输注供者来源的血小板是治疗血小板减少患者的一种有效方法，但由于需求的增加、5天的保质期和污染的可能性，许多人提出了担忧。血小板是由骨髓中的巨核细胞(MK)产生的，来源于造血干细胞和祖细胞(HSPC)。这项提议的长期目标是从HSPC浓缩的脐带血中体外产生血小板，这将使我们能够满足巨大的公众需求。最近的研究表明，组蛋白脱乙酰酶抑制剂(HDACi)的加入可以极大地影响祖细胞的扩增，但会降低MK的分化效率。我的中心假设是，表观遗传和转录改变启动或减慢HSPC对MK分化线索的敏感性。为了研究表观遗传信号在HSPC向MK转变过程中的作用，我建议进行高通量芯片和RNA测序，以发现扩增的(低潜能)和新鲜的脐带血HSPC之间的差异活性调节元件。这项高度跨学科和协作的提案利用计算生物学和机器学习来识别不同活跃的调控元件，并将遗传特征与MK命运联系起来。为了验证我的发现，并建立某些区域的组蛋白修饰与MK潜力之间的因果关系，我将产生一种dCas9-融合蛋白，它可以直接在目标位置操纵表观遗传标记。我的发现将建立染色质修饰剂如HDACi的作用机制，以及表观基因组和MK分化之间的因果关系。我与米勒、巴格里和马哈茂德实验室的高度合作提案将大大增强我们对造血分化为制造血小板的理解。

Several million units of donor platelets are transfused annually in the US and Europe to treat patients undergoing radiation treatment, chemotherapy, or organ transplant surgery. Although transfusions of donor- derived platelets are an effective way to treat thrombocytopenic patients, numerous concerns have been raised due to the increase in demand, the five-day shelf-life, and the possibilities for contamination. Platelets are produced by megakaryocytes (MKs) in the bone marrow and are derived from hematopoietic stem and progenitor cells (HSPCs). The long-term objective of this proposal is to generate platelets ex vivo from HSPC- enriched umbilical cord blood that will allow us to address a vast public need. Recent studies have shown that the addition of histone deacetylase inhibitors (HDACi) can greatly affect expansion of progenitor cells, but can reduce MK differentiation efficiency. My central hypothesis is that epigenetic and transcriptional changes prime or desensitize HSPC to MK differentiation cues. To investigate the role of epigenetic signals during HSPC-to- MK transition, I propose to perform high-throughput ChIP and RNA-sequencing to discover differentially active regulatory elements between expanded (low potential) and fresh cord blood HSPCs. This highly interdisciplinary and collaborative proposal utilizes computational biology and machine-learning to identify differentially active regulatory elements, and associate genetic features with MK fate. To validate my findings and establish a causal relationship between histone modifications of certain regions and MK potential, I will generate a dCas9-fusion protein that can directly manipulate the epigenetic marker at a targeted site. My findings will establish the mechanism of action of chromatin-modifying agents such as HDACi, and the causal relationship between the epigenome and MK differentiation. My highly collaborative proposal with the Miller, Bagheri, and Mahmud lab will substantially enhance our understanding of hematopoietic differentiation toward manufacturing platelets.