Visualization and Lineage Tracing of Hematopoietic Stem Cell Heterogeneity

描述（由申请人提供）：骨髓中的造血干细胞（HSC）在整个生命过程中不断产生各种类型的血细胞。老年人HSC的活性表现出异常，如淋巴细胞发育减少，髓细胞发育增强，易患骨髓增生性疾病。最近的证据表明，HSC包括两个不同的亚群：自我更新能力有限的“淋巴细胞偏倚”HSC和具有优越长期重建能力的“髓细胞偏倚”HSC。后一亚群随着年龄的增长而变得主要，限制了淋巴细胞的生成并易发生骨髓增生。虽然“HSC异质性”模型为理解HSC动力学和衰老提供了必要的框架，但由于我们目前无法前瞻性地识别两个HSC亚群或在完整生物体中追踪它们的后代，它受到了限制。我们的初步研究表明，转基因菌株在部分HSC中表达绿色荧光蛋白或Cre重组酶，与“骨髓偏向”HSC一致。我们建议这些报告菌株可用于两个HSC亚群的鉴定和谱系追踪。在Aim 1中，表达分析和过继转移将用于验证荧光和Cre报告者对功能性HSC亚群的标记。在Aim 2中，这些报告基因将用于动员和免疫介导应激后的HSC的量化和谱系追踪。此外，将描述HSC亚群对髓性白血病发展的贡献。这些实验将建立一个新的遗传系统来分析HSC异质性，并表征未过继转移的完整动物的HSC动力学。一个成功的结果将为研究白血病发生过程中的HSC异质性提供有用的工具和初步数据。了解HSC活性的细胞基础，将有助于为年龄或应激损伤的造血系统“返老还童”和造血恶性肿瘤的治疗提供新方法。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSC) in the bone marrow continuously give rise to all blood cell types throughout the entire life. The activity of HSC in aged individuals shows abnormalities such as reduced lymphoid development, enhanced myeloid development, and predisposition to myeloproliferative diseases. Recent evidence suggests that the HSC comprise two distinct subsets: the "lymphoid-biased" HSC with limited self-renewal capacity, and the "myeloid-biased" HSC with superior long-term reconstitution capacity. The latter subset becomes predominant with age, limiting lymphopoiesis and predisposing to myeloproliferation. While the "HSC heterogeneity" model provides essential framework for understanding HSC dynamics and aging, it is limited by our current inability to prospectively identify the two HSC subsets or to trace their progeny in intact organisms. Our preliminary studies characterized transgenic strains expressing green fluorescent protein or Cre recombinase in a fraction of HSC consistent with "myeloid-biased" HSC. We propose that these reporter strains can be used for the identification and lineage tracing of the two HSC subsets. In Aim 1, expression analysis and adoptive transfers will be used to verify the marking of functional HSC subsets by the fluorescent and Cre reporters. In Aim 2, these reporters will be used for the quantification and lineage tracing of the HSC after mobilization and immune-mediated stress. In addition, the contribution of HSC subsets to the development of myeloid leukemia will be characterized. These experiments would establish a novel genetic system to analyze HSC heterogeneity, and characterize HSC dynamics in intact animals without adoptive transfer. A successful outcome would provide useful tools and initial data for the study of HSC heterogeneity during leukemogenesis. Understanding the cellular basis of HSC activity should facilitate novel approaches to the "rejuvenation" of age- or stress-impaired hematopoiesis and to the treatment of hematopoietic malignancies. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cells (HSCs) in the bone marrow continuously give rise to all blood cell types throughout the entire life. We will characterize novl genetic systems to identify and trace the functional subsets of HSCs in naive animals and during leukemia development. Understanding the cellular basis of HSCs activity should facilitate novel approaches to the "rejuvenation" of age- or stress- impaired blood development and to the treatment of blood cancers.