Maintenance and expansion of long-term hematopoietic stem cells

长期造血干细胞的维持和扩增

基本信息

批准号：
9767274
负责人：
PETER S KLEIN
金额：
$ 54.03万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-20 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9767274
关键词：
Adult Biological Assay Biological Models Blood donor Cells Chemicals Child Clustered Regularly Interspaced Short Palindromic Repeats Development Differentiation and Growth Disease Engraftment Extracellular Signal Regulated Kinases Future Genetic Goals Hematological Disease Hematopoietic Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Homeostasis Human Inherited Laboratories Lead Libraries MEKs Maintenance Mediating Methods Mus Myelodysplastic/Myeloproliferative Disease Myeloproliferative disease Pancytopenia Pathway interactions Peptide Initiation Factors Phenotype Play Recovery Regenerative Medicine Risk Role Sickle Cell Anemia Signal Transduction System Testing Thalassemia Therapeutic Toxic effect Translation Initiation Translations Transplantation Umbilical Cord Blood Umbilical Cord Blood Transplantation Validation Work Xenograft procedure base genome editing graft vs host disease hematopoietic stem cell expansion hematopoietic stem cell self-renewal high throughput screening improved in vivo in vivo evaluation inhibitor/antagonist knock-down leukemia/lymphoma next generation sequencing novel novel strategies peripheral blood regenerative ribosome profiling self renewing cell self-renewal small molecule stem cell biology stem cell therapy

项目摘要

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.

概括造血干细胞（HSC）是再生医学的基石，既可作为研究的模型系统干细胞生物学和造血干细胞移植（HSCT）治疗骨髓衰竭，骨髓肿瘤和其他造血系统疾病。在不久的将来，HSCT也将是迈出的关键一步。应用基因组编辑治疗遗传性血液疾病，例如镰状细胞病和地中海贫血。然而，HSC 是一种罕见的细胞，在体外会迅速丧失自我更新的能力。造血生态位，对 HSC 的离体研究和 HSC 的扩展提出了重大挑战治疗应用。脐带血 (UCB) 捐献者的 HSCT 广泛应用于儿童，因为 HLA 匹配所需的严格性降低，移植物抗宿主病的风险降低，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造血干细胞数量减少以及自我修复能力的丧失。离体操作后更新细胞。因此，增加 HSC 的方法将大大改变 HSCT 的治疗前景。我们之前建立了一个长期保持的文化体系 HSC 离体。然后，我们对 >2200 种生物活性化合物进行了高通量筛选，以寻找其他小分子化合物。允许 HSC 扩增的分子，并鉴定出一种先导化合物，可将 HSC 扩增约 10 倍通过有限稀释分析和小鼠长期植入证实 HSC 培养 4 天内异种移植物。该化合物还可以扩增来自成年供体的 CRISPR 修饰的表型 HSC。我们从筛选中鉴定出其他化合物，可实现表型 HSC 的离体扩增但尚未通过异种移植试验验证这些。该提案的具体目标是：1) 严格测试从 UCB 扩增的 HSC 和 CRISPR 修饰的 HSC 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 稳态中的作用机制； 3) 探索筛选中发现的其他化合物，包括开发一种新方法加速小鼠异种移植试验中多种化合物的体内测试。扩展能力当离体功能性 HSC 的数量增加时，将为 HSC 移植提供关键的治疗进展。 HSC 是有限的，包括脐带血和基因组编辑的 HSC。