Maintenance and expansion of long-term hematopoietic stem cells

摘要 造血干细胞(HSCs)是再生医学的基石，两者都是研究的模型系统 干细胞移植在治疗骨髓衰竭中的生物学和治疗学， 髓系肿瘤和其他造血系统疾病。在不久的将来，HSCT也将是 基因组编辑在治疗镰状细胞病等遗传性血液疾病中的应用 地中海贫血。然而，肝星状细胞是一种罕见的细胞，它们会迅速失去自我更新的能力。 造血生态位，对造血干细胞的体外研究和体外扩增提出了重大挑战 治疗应用。脐带血捐献者的造血干细胞移植在儿童中被广泛使用，因为 降低了对HLA配型所需的严格程度，降低了移植物抗宿主病的风险，但UCB 由于造血干细胞数量较少，成人移植受到限制。同样，基因组的一个主要障碍 遗传性血液病的编辑是基因组编辑后的HSCs数量较少，自我丧失 在体外操作后更新细胞。因此，增加HSC的方法将发生实质性变化 造血干细胞移植的治疗前景。我们以前建立了一套长期维持的文化体系 HSCs体外培养。然后，我们对2200种生物活性化合物进行了高通量筛选，以获得更多的小分子 允许HSC扩张的分子，并确定了一种先导化合物，可以使HSCs扩张约10倍 培养4天内的HSCs，经有限稀释分析和在小鼠体内长期植入证实 异种移植物。该化合物还可以扩增成人供者的CRISPR修饰的表型造血干细胞。 我们已经从筛选中确定了其他化合物，使表型HSCs能够在体外扩增 但还没有通过异种移植试验来验证这些。这项建议的具体目的是：1) 严格检测脐带血和CRISPR修饰的HSCs体外扩增的体内功能；2)建立 我们的先导化合物，翻译起始因子eIF4E的抑制剂，在HSC同种血肿中的作用机制； 以及3)探索在屏幕上确定的其他化合物，包括开发一种新的方法来 加快小鼠异种移植试验中多种化合物的体内测试。扩展的能力 功能干细胞的体外培养将为造血干细胞移植提供关键的治疗进展 造血干细胞是有限的，包括脐带血和基因组编辑的造血干细胞。

SUMMARY Hematopoietic stem cells (HSCs) are a cornerstone of regenerative medicine, both as a model system to study stem cell biology and therapeutically for HSC transplants (HSCT) in the treatment of bone marrow failure, myeloid neoplasms, and other hematopoietic disorders. In the near future, HSCT will also be a critical step in the application of genome editing to treat inherited blood disorders such as sickle cell disease and thalassemia. However, HSCs are rare cells that rapidly lose their capacity for self-renewal outside of the hematopoietic niche, presenting a major challenge to ex vivo study of HSCs and to the expansion of HSCs for therapeutic applications. HSCT with umbilical cord blood (UCB) donors is widely used in children because of the reduced stringency required for HLA matching and the lower risk of graft versus host disease, but UCB transplants are limited in adults because of the low number of HSCs. Similarly, a major obstacle to genome editing for inherited blood diseases is the low number of HSCs after genome editing and the loss of self- renewing cells after ex vivo manipulation. Therefore, a method to increase HSCs would substantially change the therapeutic landscape in HSCT. We previously established a culture system that maintains long-term HSCs ex vivo. We then performed a high throughput screen of >2200 bioactive compounds for additional small molecules that allow expansion of HSCs and identified a lead compound that confers ~10-fold expansion of HSCs within 4 days of culture, as confirmed by limiting dilution analysis and long-term engraftment in mouse xenografts. This compound also confers expansion of CRISPR modified phenotypic HSCs from adult donors. We have identified additional compounds from the screen that confer ex vivo expansion of phenotypic HSCs but have not yet validated these by xenotransplantation assays. The specific aims of this proposal are to: 1) rigorously test the in vivo function of HSCs expanded from UCB and CRISPR-modified HSCs; 2) establish the mechanism of our lead compound, an inhibitor of the translation initiation factor eIF4E, in HSC homoestasis; and 3) explore additional compounds identified in the screen, including the development of a novel approach to accelerate the in vivo testing of multiple compounds in mouse xenografts assays. The ability to expand functional HSCs ex vivo will provide a critical therapeutic advance for HSC transplant when the number of HSCs is limiting, including umbilical cord blood and genome-edited HSCs.