Clonal Analysis of Progenitor Activities During Native and Transplant Hematopoiesis

 描述(申请人提供)：血液是一个高度动态的系统，每天都有数十亿成熟细胞被补充。传统上认为，细胞再生的持续过程完全依赖于造血干细胞；而祖细胞是造血干细胞的丰富后代，通常被认为是瞬时扩增阶段，在此阶段，稀有的原始细胞在最终产生功能血细胞之前被戏剧性地扩增。尽管在实验和治疗移植中HSC在血液再生中的重要作用已被反复证明，但最近使用新的原位谱系追踪技术的研究表明，在自然、未受干扰的条件下，HSC对稳定状态下的血细胞生成的贡献非常有限。相比之下，祖细胞群体虽然不能移植，但在自然造血过程中具有更持久的造血能力。这些发现共同证明了在祖细胞阶段对血液动态平衡的调节达到了前所未有的水平。他们还强调了祖细胞在骨髓移植过程中用于强健的血淋巴再生的潜在用途。这一建议旨在进一步表征祖细胞维持其体内长期造血活动的细胞和分子机制。目标1将试图阐明不同亚群的祖细胞在正在进行的血细胞生产中的贡献。这将通过比较不同血统之间的克隆重叠，以及在转座子标记诱导后的多个时间点直接测量祖细胞亚群的克隆输出来实现。在目标2中，将在小鼠寿命的不同阶段检查祖细胞的克隆复杂性和克隆大小。这一分析的结果将有助于确定祖细胞的自我更新是在单细胞水平上实现的，还是通过种群动态完成的。细胞休眠在保存祖细胞活性中的作用，以及不同克隆行为的分子基础也将在这方面进行探索。目的3将重点介绍骨髓移植对祖细胞功能特性的影响。将使用H2B-GFP标记保留模型来测试移植对祖细胞增殖和存活的全球影响。转座子技术将有助于比较原始和移植造血过程中祖细胞在自我更新和分化过程中的克隆行为。总而言之，这些拟议的研究将为血液再生的细胞机制的旧问题带来新的见解。拟议工作的结果也可能为设计更好的策略来保护移植期间的祖细胞活性提供线索。

 DESCRIPTION (provided by applicant): Blood is a highly dynamic system, in which billions of mature cells are replenished every day. It is traditionally thought that this ongoing process of cel regeneration exclusively depends on hematopoietic stem cells (HSC); whereas progenitors, the abundant progeny of HSCs, are generally thought to represent transient amplifying stages, where rare primitive cells are dramatically expanded before the final production of functional blood cells. While the essential role of HSC in blood regeneration has been repeatedly demonstrated in both experimental and therapeutic transplantation, recent studies using novel in situ lineage tracing techniques have revealed very limited contribution of HSC to blood cell generation at steady state under native, unperturbed conditions. In contrast, the progenitor populations, although non-transplantable, have a much more persistent ability for blood generation during native hematopoiesis. These findings collectively demonstrate a previously unappreciated level of regulation of blood homeostasis at the stage of progenitor cells. They also highlight the potential use of progenitors for robust hematolymphoid regeneration during bone marrow transplantation. This proposal aims to further characterize the cellular and molecular mechanisms by which the progenitors sustain their long-term in vivo hematopoietic activities. Aim 1 will attempt to elucidate the contribution by the different subsets of the progenitors in ongoing blood cell production. This will be achieved by comparing clonal overlapping between different blood lineages, and also by directly measuring the clonal output of the progenitor subsets at multiple time points following induction of transposon labeling. In Aim 2, the clonal complexity and clone size of progenitors will be examined at different stages of the mouse lifespan. Results of this analysis will help to determine if progenitor self-renewal is achieved at the level of single cells, or is accomplished through population dynamics. The role of cellular dormancy in preserving progenitor activity, and molecular basis of distinct clonal behaviors will be explored as well in this aspect of the study. Aim 3 will focus on the impact of bone marrow transplantation on functional properties of progenitors. The H2B-GFP label-retaining model will be used to test the global impact of transplantation on progenitor proliferation and survival. And the transposon technology will help compare the clonal behaviors of progenitors in self-renewal and differentiation during native and transplant hematopoiesis. In the aggregate, these proposed studies will bring new insights to the old questions of cellular mechanisms of blood regeneration. Outcomes of the proposed work may also provide clues for the design of better strategies for preserving progenitor activity during transplantation.