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。这个系统的中心是一群造血干细胞（hsc），它们具有自我更新的能力，也能分化成成熟的血液细胞和免疫细胞类型。最近的证据还表明，与正常的造血系统类似，血癌（白血病）以类似的分层方式组织，长寿的白血病干细胞（LSC）位于其顶端。尽管在正常和恶性干细胞的纯化和功能表征方面取得了进展，但我们对单个HSC/LSCs在体内的行为的理解仍然很差。深入了解造血干细胞的数量及其对血系形成的贡献，对于基因治疗和移植等治疗干预措施的设计具有重要意义。尽管通过经典的逆转录病毒标记研究获得了这一过程的一些知识，但所有这些研究都涉及造血干细胞的离体操作，并将其移植到致命辐射的受体中，显然是非生理条件。在这个探索性的提议中，我们寻求开发一种新的策略，使我们能够在原位独特地标记和跟踪hsc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，其中造血干细胞可以在骨髓中被标记，因此在完全正常的生理条件下遵循它们的行为。首先，在HSC水平上对转座子标记进行表征。我们将确定有多少HSC可以被唯一标记，以及每个HSC是否具有唯一的转位标签。其次，一旦确定了标记参数，我们将在原位研究造血干细胞克隆的动态行为。在诱导转位后，我们将确定在稳态造血中有多少HSC克隆对多系造血有贡献，以及这种贡献的机制是什么。在第三部分，我们将研究白血病模型中克隆贡献的波动。这项研究将使我们第一次能够确定造血干细胞在稳态造血中的行为。如果成功的话，这个新系统可能用于研究各种组织和肿瘤类型中的干细胞动力学。

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.