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，既是研究的模型系统干细胞生物学和治疗HSC移植（HSCT）在治疗骨髓衰竭时髓样肿瘤和其他造血疾病。在不久的将来，HSCT也将是关键的一步基因组编辑在治疗遗传疾病（例如镰状细胞疾病和地中海贫血。但是，HSC是罕见的细胞，它们迅速失去了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）供体的HSCT因儿童而广泛使用 HLA匹配所需的严格程度降低，移植物与宿主疾病的风险较低，但UCB 由于HSC数量较少，因此成人的移植受到限制。同样，基因组的主要障碍基因组编辑后的HSC数量较少，自我丧失是遗传性疾病的编辑离体操纵后更新细胞。因此，增加HSC的方法将大大改变 HSCT中的治疗景观。我们以前建立了一种维持长期的文化体系 HSCS ex Vivo。然后，我们进行了> 2200个生物活性化合物的高吞吐量屏幕允许HSC膨胀并鉴定出铅化合物的分子，该化合物赋予了约10倍膨胀的铅化合物 HSC在培养后4天内，通过限制稀释分析和长期植入小鼠证实异种移植物。这种化合物还赋予了成人捐助者的CRISPR修改表型HSC的扩展。我们已经从屏幕上确定了其他化合物，以赋予表型HSC的体内扩展但尚未通过异种移植测定来验证这些。该提议的具体目的是：1）严格测试从UCB和CRISPR修饰的HSC扩展的HSC的体内功能； 2）建立 HSC同觉中的铅化合物的机理，这是翻译起始因子EIF4E的抑制剂； 3）探索屏幕上确定的其他化合物，包括开发一种新方法加速小鼠异种移植分析中多种化合物的体内测试。扩展的能力当数量的数量 HSC是有限的，包括脐带血和基因组编辑的HSC。