Hematopoietic Stem Cells (HSCs) as Juvenile Protective Factors that Alter Aging

描述(由申请人提供)：衰老的有害影响可能部分是由于包括干细胞在内的青少年保护因素的丧失。小鼠造血是研究干细胞的理想方法，因为可以非常精确地测量骨髓和血液前体的谱系。在BALB/cByJ(BALB)小鼠中，每个造血干细胞(HSC)的再繁殖能力随着年龄的增长而下降20-50倍，而在C57BL/6J(B6)小鼠中，没有这种下降。目的1-3在6周和6、12、18和24个月龄的胚胎肝脏和骨髓中HSC对细胞应激的敏感性的测试菌株差异，以确定B6-BALB差异是否存在于青少年中，或者它们是否随着年龄的增长而缓慢出现。长期(LT)-造血干细胞、短期(ST)-造血干细胞和普通髓系前体(CMPS)将通过针对这些菌株的衰老研究而优化的流式细胞仪标记来鉴定。目的4检测内源性和外源性幼年保护因子移植到老龄小鼠体内是否能改善HSC功能，延长寿命。了解干细胞老化的因素和机制将有助于识别青少年中预测老年功能缺陷的表型。使用这些方法，我们将检验以下假设：目标1：B6细胞比BALB细胞更好地保持基因组稳定性。来自B6和BALB小鼠的LT-HSCs、ST-HSCs和CMPS将暴露于导致基因组不稳定的治疗：伽玛辐射、DNA聚合酶抑制和百草枯。基因组不稳定性将被量化为a)染色体结构损伤(光谱核型)，b)节段性拷贝数异常(阵列比较基因组杂交)，以及c)DNA双链断裂(组蛋白H-2AX磷酸化)。HSC功能与活性氧(ROS)和基因组不稳定性的相关性将被测试。目的2：B6 HSCs衰老较慢，因为其增殖速度慢于BALB HSCs。HSC亚群将通过a)暴露于5-FU(5-FU)后的细胞损失率和b)在饮用水中1、3和10天后对溴脱氧尿苷(BrdU)的摄取和稀释来评估。目的3：B6细胞的凋亡率低于BALB细胞。HSC亚群将通过比较Annexin V和丙二醇碘双重标记鉴定的凋亡细胞比例来评估。目的4：年轻或胎儿的HSC移植将使老年BALB受者受益。在未经治疗的老年BALB受者中，年轻的HSC可能会竞争有缺陷的HSC的再生功能。如果有必要，老年受者的造血干细胞将被生长因子动员起来，为年轻的造血干细胞打开壁龛，或者通过放射或化疗杀死它们，使其开放壁龛并移除转化的细胞。潜在的外源性缺陷将通过将胎儿脾(带有基因标记的细胞)移植到受体肾胶囊中进行测试。如果移植脾的微环境随着年龄的增长而受损，与年轻受者相比，老年受者移植脾的年轻微环境中会出现更多的内源性HSC更新和分化细胞的产生。为了公众健康，该计划的目标是：1)确定防止C57BL/6J小鼠的造血干细胞功能随年龄丧失的机制和因素，而不是BALB/cByJ小鼠；2)测试将年轻的造血干细胞和微环境移植到老年受者体内的益处。潜在的长期临床益处包括：造血干细胞延迟耗尽；改善长期健康；增加涉及老年捐赠者或受者的造血干细胞移植的长期成功率；以及改善由干细胞或前体细胞功能丧失引起的老年病的治疗。

DESCRIPTION (provided by applicant): Detrimental effects of aging might be due in part to loss of juvenile protective factors, including stem cells. Murine hematopoiesis is ideal for investigating stem cells, because lineages of marrow and blood precursors can be measured with great precision. In BALB/cByJ (BALB) mice, the repopulating ability per hematopoietic stem cell (HSC) declines 20- to 50-fold with age, while in C57BL/6J (B6) mice, there is no such decline. Aims 1-3 test strain differences in HSC susceptibility to cellular stress in fetal liver and in marrow at 6 weeks plus 6, 12, 18 and 24 months of age, to determine if the B6-BALB differences are present in juveniles or if they slowly emerge with age. Long term (LT)-HSCs, short term (ST)-HSCs and common myeloid precursors (CMPs) will be identified by flow cytometry markers optimized for aging studies in these strains. Aim 4 tests whether transplants of intrinsic or extrinsic juvenile protective factors into aged mice will improve HSC function and increase lifespan. Understanding the factors and mechanisms of stem cell aging will help identify phenotypes in juveniles that predict functional defects in old age. Using these approaches, we will test the following hypotheses: Aim 1: That B6 cells maintain genomic stability better than BALB cells. LT-HSCs, ST-HSCs and CMPs from B6 and BALB mice will be exposed to treatments that cause genomic instability: gamma radiation, DNA polymerase inhibition, and paraquat. Genomic instability will be quantified as a) chromosome structural lesions (spectral karyotyping), b) segmental copy number aberrations (array comparative genomic hybridization), and c) DNA double strand breaks (histone H-2AX phosphorylation). Correlations of HSC functions with reactive oxygen species (ROS) and genomic instabilities will be tested. Aim 2: That B6 HSCs age less because they proliferate more slowly than BALB HSCs. HSC subpopulations will be assessed by a) rates of cell loss after exposure to 5-fluorouricil (5-FU) and b) uptake and dilution of bromodeoxyuridine (BrdU) after 1, 3, and 10 days in drinking water. Aim 3: That the rate of apoptosis is slower in B6 than in BALB. HSC subpopulations will be assessed by comparing proportions of apoptotic cells identified using dual labeling of Annexin V and propidium iodide. Aim 4: That young or fetal HSC grafts will benefit old BALB recipients. Young HSCs may out compete the repopulating functions of the defective HSCs in untreated old BALB recipients. If necessary, HSCs in old recipients will be mobilized by growth factors to open niches for young HSCs, or killed by irradiation or chemotherapy to both open niches and remove transformed cells. Potential extrinsic defects will be tested by transplanting fetal spleens (with genetically marked cells) into recipient kidney capsules. If microenvironments are damaged with age, more endogenous HSC renewal and production of differentiated cells will occur in the youthful microenvironment of the grafted spleens in old, compared to young, recipients. TO PUBLIC HEALTH The objectives of this program are to 1) identify mechanisms and factors that prevent the loss of hematopoietic stem cell (HSC) function with age in C57BL/6J mice but not in BALB/cByJ mice, and 2) test the benefits of transplanting youthful HSCs and microenvironments into old recipients. Potential long-term clinical benefits include the following: delayed exhaustion of HSCs; improving long-term health; increased long-term success of HSC transplants involving elderly donors or recipients; and improved treatments for diseases of aging that are mediated by the loss of stem cell or precursor cell function.