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

使用新型多荧光转基因小鼠模型体内跟踪造血干细胞克隆动态

• 批准号: 9134179
• 负责人: David T Scadden
• 金额: $ 23.95万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9134179
• 关键词: Address; Antibodies; Behavior; Binding; Biological Assay; Blood; Bone Marrow Diseases; Cause of Death; Cell Culture Techniques; Cell Therapy; Cell Transplantation; Cell physiology; Cell surface; Cells; Cessation of life; Characteristics; Clonal Hematopoietic Stem Cell; DNA; Data; Epigenetic Process; Fluorescence; Future; Goals; Health; Hematologic Neoplasms; Hematological Disease; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heterogeneity; Individual; Injury; Label; Methods; Mission; Modeling; Modification; Molecular; Molecular Profiling; Mus; Names; National Heart, Lung, and Blood Institute; Natural regeneration; Output; Patients; Physiological; Population; Property; Regenerative Medicine; Resistance; Siblings; Source; Stem cells; Stress; System; Tissues; Transgenic Mice; Transplant Recipients; Transplantation; Viral; Virus Integration; base; biological adaptation to stress; cell behavior; epigenetic regulation; fluorophore; improved; in vivo; insight; interest; leukemia/lymphoma; mouse model; novel; prospective; stem cell fate; stem cell therapy; success; tool

 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.

 描述（由适用提供）：血液癌是美国死亡的主要原因之一。 NCI估计2014年白血病和淋巴瘤死亡123,180例新病例和44,080例死亡。造血干细胞（HSC）移植仍然是这些血液学恶性肿瘤的唯一治愈方法。但是，即使在治愈意图移植后，最多50％的患者在头三年内死亡。细胞疗法的成功在于许多因素，其中选择正确的细胞来源至关重要。但是，研究HSC异质性是一个困难的挑战，因为使用抗体识别细胞表面标记的常规分离干细胞只能产生不纯的种群。已经尝试使用单细胞移植或使用条形码DNA的病毒整合来研究HSC的克隆行为。然而，单细胞移植不能研究克隆竞争，而DNA的病毒标记需要对细胞进行离体操纵，这可能会因人工细胞培养条件而改变细胞性质。我们开发了一种新的多荧光转基因小鼠模型，称为“色调”小鼠，以研究体内造血细胞的克隆动力学。该小鼠允许在有条件表达的荧光团随机重排后通过一系列荧光标签进行细胞标记。优势在于，可以很容易地进行流式细胞仪定量，并且可以分离细胞进行分子分析和功能评估。该系统允许在体内在单细胞水平上跟踪大量克隆，避免了对细胞的离体操纵，并使我们能够以前所未有的方式研究HSC克隆的行为。我们建议使用这种新工具来解决以下目标，其长期目标是了解基本的HSC行为并改善治疗。干细胞的选择/操纵：目标（1）定义了会议干细胞对应激/损伤的抗性表观遗传景观，而目标（2）确定控制干细胞特定谱系命运的表观遗传特征。这些研究的完成将大大提高我们对干细胞亚型的理解，其功能差异以及对细胞行为的独特表观遗传调节。这些研究是第一个定义定义特定干细胞功能和前瞻性分离干细胞亚型的分子特征的研究。这些研究将提供洞察力，以使用患者量的干细胞亚型增强HSC移植疗法的成功。因此，该提案与NHLBI的使命结盟，以推进干细胞矫正和改善血液学疾病造成的移植患者的存活率。