A NOVEL SYSTEM FOR THE STUDY OF IN VIVO CLONAL HEMATOPOIETIC STEM CELL DYNAMICS

描述(申请人提供)：造血是一个复杂的细胞分化程序。在这个系统的中心是一群具有自我更新和分化为成熟血液和免疫细胞类型的造血干细胞(HSCs)。最近的证据还表明，与正常的造血系统类似，血癌(白血病)是以类似的分级方式组织的，长期存活的白血病干细胞(LSC)位于其顶端。尽管在正常和恶性干细胞的纯化和功能鉴定方面取得了进展，但我们对单个HSC/LSCs在体内的行为仍然了解很少。深入了解造血干细胞的数量和对血系形成的贡献，对于设计基因治疗和移植等治疗干预措施具有重要意义。虽然通过经典的逆转录病毒标记研究已经获得了一些关于这一过程的知识，但所有这些研究都涉及对造血干细胞的体外操作，并将其移植到受到致命性照射的受者体内，显然是在非生理条件下。在这个探索性的建议中，我们试图开发一种新的策略，使我们能够在原位唯一地标记和跟踪HSCs和LSCs的后代。利用四环素诱导和睡美人(SB)转座酶系统的组合，我们将创建一个小鼠模型，在该模型中，HSCs可以在仍在骨髓中进行标记，从而在完全正常的生理条件下跟踪它们的行为。首先，提出了在HSC水平上表征转座子标记的方法。我们将确定有多少个HSC可以被唯一标记，以及每个单独的HSC是否有唯一的转座标签。其次，一旦标记参数确定，我们将在原位研究造血干细胞克隆的动态行为。在转位诱导后，我们将确定有多少HSC克隆在稳态造血中对多系造血做出了贡献，以及这种贡献的机制是什么。在第三部分中，我们将研究白血病模型中克隆贡献的涨落。这项研究将使我们第一次确定HSCs在稳态造血中的行为。如果成功，这个新的系统可以用来研究潜在的每一种组织和类型的肿瘤中的干细胞动力学。 项目简介：这项提议将使我们能够了解造血干细胞如何在生物体中发挥作用。因此，我们的结果将使我们能够了解干细胞功能在临床条件下是如何受到影响的，例如骨髓衰竭综合征和血癌。

DESCRIPTION (provided by applicant): Hematopoiesis is a complex program of cellular differentiation. At the center of this system is a population of hematopoietic stem cells (HSCs) endowed with the ability to self-renew as well as to differentiate into mature blood and immune cell types. Recent evidence also suggests that akin to the normal hematopoietic system, blood cancers (leukemias) are organized in a similar hierarchical fashion with long-lived leukemic stem cells (LSC) at their apex. Despite advances in the purification and functional characterization of normal and malignant stem cells, our understanding of how individual HSC/LSCs behave in vivo, is still very poor. Insight into the number and the contribution of HSCs to the formation of blood lineages would have great significance for the design of therapeutic interventions such as gene therapy and transplantation. Although some knowledge of this process has been gained through classic retroviral marking studies, all of those studies involved ex-vivo manipulation of HSCs and their transplantation into lethally-irradiated recipients, clearly non-physiological conditions. In this exploratory proposal, we seek to develop a novel strategy which will allow us to uniquely tag and follow the progeny of HSCs and LSCs in situ. Utilizing a combination of the tetracycline-inducible and the sleeping beauty (SB) transposase systems, we will create a murine model in which HSCs can be marked while still in the bone marrow and therefore follow their behavior under completely normal physiological conditions. It is proposed, firstly, to characterize transposon marking at the level of the HSC. We will determine how many HSCs can be uniquely marked and whether each individual HSC has a unique transposition tag. Secondly, once the marking parameters are defined, we will study the dynamic behavior of hematopoietic stem cell clones in situ. After induction of transposition we will determine how many HSC clones contribute to multi-lineage hematopoiesis in steady state hematopoiesis, and what the mechanisms of this contribution are. In the third part, we will study the fluctuation of clonal contribution in leukemia model. This study will allow us for the first time to define the behavior of HSCs in steady state hematopoiesis. If successful, this novel system could be used to study stem cell dynamics in potentially every tissue and type of tumor. PROJECT NARRATIVE: This proposal will allow us to understand how blood-forming stem cells function in an organism. Therefore our results will allow us to understand how stem cell function is affected in clinical conditions such as bone marrow failure syndromes and cancers of the blood.