Maintenance and expansion of long-term hematopoietic stem cells

长期造血干细胞的维持和扩增

• 批准号: 10162647
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-20 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10162647
• 关键词: Adult; Bar Codes; 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; Human; Inherited; Laboratories; Lead; Libraries; Maintenance; Mediating; Methods; Mus; Myelodysplastic/Myeloproliferative Disease; Myeloproliferative disease; Pancytopenia; Pathway interactions; Peptide Initiation Factors; Phenotype; Play; Recovery; Regenerative Medicine; Regenerative capacity; 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; ribosome profiling; self renewing cell; self-renewal; small molecule; stem cell biology; stem cell growth; stem cell homeostasis; stem cell therapy

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.

总结 造血干细胞（HSCs）是再生医学的基石，既作为模型系统进行研究 干细胞生物学和造血干细胞移植（HSCT）治疗骨髓衰竭， 骨髓肿瘤和其它造血系统疾病。在不久的将来，HSCT也将是 基因组编辑在治疗遗传性血液疾病如镰状细胞病和 地中海贫血然而，HSC是一种罕见的细胞，在细胞外迅速失去自我更新的能力。 造血生态位，提出了一个主要的挑战，离体研究的HSC和扩增的HSC， 治疗应用。脐带血（UCB）供体的HSCT广泛用于儿童，因为 HLA配型所需的严格性降低，移植物抗宿主病的风险降低，但UCB 由于HSC数量少，移植在成人中受到限制。同样，基因组的主要障碍 遗传性血液病的基因编辑是基因组编辑后HSC数量少， 在离体操作后更新细胞。因此，增加HSC的方法将大大改变 HSCT的治疗前景我们以前建立了一个文化体系， 离体HSC。然后，我们对>2200种生物活性化合物进行了高通量筛选，以获得额外的小分子化合物。 分子，允许HSC的扩增，并确定了一种先导化合物，赋予~10倍的扩增， 通过有限稀释分析和小鼠长期植入证实，培养4天内的HSC 异种移植该化合物还赋予来自成年供体的CRISPR修饰的表型HSC的扩增。 我们已经从筛选中鉴定出额外的化合物，其赋予表型HSC的离体扩增 但还没有通过异种移植试验验证这些。这项建议的具体目标是：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eIF 4 E的抑制剂，在HSC内稳态中的机制; 和3）探索在筛选中鉴定的其他化合物，包括开发一种新的方法， 在小鼠异种移植物试验中加速多种化合物的体内测试。能够实施拓展 当体外功能性HSC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。

项目成果

Lithium and Therapeutic Targeting of GSK-3.

• DOI: 10.3390/cells10020255
• 发表时间: 2021-01-28
• 期刊: Cells
• 影响因子: 6
• 作者: Snitow ME;Bhansali RS;Klein PS
• 通讯作者: Klein PS

Non-acylated Wnts Can Promote Signaling.

非酰化 Wnt 可以促进信号传导。

• DOI: 10.1016/j.celrep.2018.12.104
• 发表时间: 2019
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Speer,KelseyF;Sommer,Anselm;Tajer,Benjamin;Mullins,MaryC;Klein,PeterS;Lemmon,MarkA
• 通讯作者: Lemmon,MarkA