In vivo tracking of the hematopoietic stem cell clonal dynamics using a novel multi-fluorescent transgenic mouse model

 描述（由申请人提供）：血癌是美国的主要死亡原因之一。NCI估计2014年有123，180例新病例和44，080例白血病和淋巴瘤死亡。造血干细胞（HSC）移植仍然是治疗这些血液恶性肿瘤的唯一方法。但即使在移植后的治疗目的，高达50%的患者死亡的前3年内。细胞治疗的成功取决于许多因素，其中选择正确的细胞来源是至关重要的。然而，研究HSC异质性是一个困难的挑战，因为使用细胞表面标记的抗体识别的干细胞的常规分离只能产生不纯的群体。已经尝试使用单细胞移植或使用条形码DNA的病毒整合追踪个体HSC来研究HSC的克隆行为。然而，单细胞移植不能研究克隆竞争，并且DNA的病毒标记需要离体操作细胞，这可能由于人工细胞培养条件而改变细胞特性。我们建立了一种新的多荧光转基因小鼠模型，命名为“HUe”小鼠，以研究体内造血细胞的克隆动力学。该小鼠允许在条件表达的荧光团随机重排后通过一系列荧光标签标记细胞。其优点是可以容易地进行流式细胞定量，并且可以分离细胞用于分子谱分析以及功能评估。该系统允许在体内单细胞水平上跟踪大量克隆，避免了细胞的离体操作，并使我们能够以前所未有的方式研究HSC克隆行为。我们建议使用这个新工具来实现以下目标，其长期目标是了解基本的HSC行为并改善治疗性干细胞选择/操作：目标（1）定义赋予干细胞抗应激/损伤能力的表观遗传景观，目标（2）确定控制干细胞特定谱系命运的表观遗传特征。这些研究的完成将极大地推进我们对干细胞亚型、它们的功能差异以及细胞行为独特的表观遗传调控的理解。这些研究是第一个定义特定干细胞功能的分子特征和干细胞亚型的前瞻性分离。这些研究将为使用患者定制的干细胞亚型提高HSC移植治疗的成功率提供见解。因此，该提案与NHLBI的使命结盟，以推进干细胞校正和改善血液病移植患者的生存率。

 DESCRIPTION (provided by applicant): Blood cancer is one of the leading causes of death in the US. The NCI estimates 123,180 new cases and 44,080 deaths from leukemia and lymphomas in 2014. Hematopoietic stem cell (HSC) transplantation remains the only cure for these hematological malignancies. But even after transplantation with a curative intent, up to 50% of patients die within the first 3 years. Success of cell therapy lies upon many factors among which, choosing the right cell source, is paramount. However, studying HSC heterogeneity is a difficult challenge because conventional isolation of stem cells using antibody recognition of cell surface markers can only yield an impure population. Attempts have been made to investigate the clonal behavior of HSCs using single cell transplantation or tracking individual HSCs using viral integration of barcoded DNAs. However, single cell transplantation cannot study clonal competition, and viral labeling of DNA requires ex vivo manipulation of cells, which may alter cell properties due to the artificial cell culture conditions. We developed a new multi-fluorescent transgenic mouse model, named `HUe' mouse, to study the clonal dynamics of hematopoietic cells in vivo. This mouse permits cell labeling by a range of fluorescent tags after stochastic rearrangement of conditionally expressed fluorophores. The advantage is that flow cytometric quantification could be readily performed and cells could be isolated for molecular profiling as well as functional assessment. This system allows tracking of a large number of clones at the single cell level in vivo, avoiding ex vivo manipulation of cells and enables us to study HSC clonal behavior in an unprecedented manner. We propose to use this new tool to address the following aims with the long-term goal of understanding fundamental HSC behavior and improving therapeutic stem cell selection/manipulation: aim (1) defining the epigenetic landscape that confer stem cell resistance to stress/injury, and aim (2) determining the epigenetic signatures that control specific lineage fate of stem cell. Completion of these studies will greatly advance our understanding of stem cell subtypes, their functional differences, and unique epigenetic regulation of cell behavior. These studies are the first to define the molecular features that defines a specific stem cell function and for prospective isolation of stem cell subtypes. These studies will provide insights to enhance the success of HSC transplant therapies using patient-tailored stem cell subtypes. Therefore, this proposal is in alliance with the mission of NHLBI in advancing stem cell correction and improving the survival of transplant patients inflicted with hematological diseases.