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

描述（由申请人提供）：老化的有害影响可能部分是由于幼年保护因子（包括干细胞）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）的再生能力随着年龄的增长而下降20- 50倍，而在C57 BL/6 J（B6）小鼠中，没有这种下降。目的1-3测试6周龄加6、12、18和24月龄时胎肝和骨髓中HSC对细胞应激敏感性的品系差异，以确定B6-BALB差异是否存在于青少年中或是否随年龄缓慢出现。长期（LT）-HSC，短期（ST）-HSC和常见的骨髓前体细胞（CMP）将通过流式细胞术标记物进行鉴定，这些标记物针对这些菌株的老化研究进行了优化。目的4：探讨将内源性或外源性保幼因子移植到老年小鼠体内是否能改善HSC功能，延长寿命。了解干细胞衰老的因素和机制将有助于确定青少年的表型，预测老年时的功能缺陷。使用这些方法，我们将测试以下假设：目的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-HSC、ST-HSC和CMP将暴露于导致基因组不稳定性的处理：γ辐射、DNA聚合酶抑制和百草枯。基因组不稳定性将量化为a）染色体结构病变（光谱核型分析），B）节段性拷贝数畸变（阵列比较基因组杂交）和c）DNA双链断裂（组蛋白H-2AX磷酸化）。将测试HSC功能与活性氧（ROS）和基因组不稳定性的相关性。目的2：B6 HSC的老化程度较低，因为它们的增殖速度比BALB HSC慢。HSC亚群将通过a）暴露于5-氟尿嘧啶（5-FU）后的细胞损失率和B）在饮用水中1、3和10天后溴脱氧尿苷（BrdU）的摄取和稀释进行评估。目的3：B6细胞凋亡率低于BALB。将通过比较使用膜联蛋白V和碘化丙啶的双重标记鉴定的凋亡细胞的比例来评估HSC亚群。目的4：年轻或胎龄HSC移植对老年BALB受体有良好的保护作用。年轻的造血干细胞可能会在未经治疗的老年BALB受体中竞争有缺陷的造血干细胞的重建功能。如有必要，老年受者中的HSC将通过生长因子动员以为年轻HSC打开龛，或通过照射或化疗杀死以打开龛并去除转化细胞。将通过将胎脾（含遗传标记细胞）移植到受体肾囊中来检测潜在的外源性缺陷。如果微环境随着年龄的增长而受损，与年轻的受体相比，更多的内源性HSC更新和分化细胞的产生将发生在老年人移植脾的年轻微环境中。该项目的目标是：1）确定防止C57 BL/6 J小鼠（而不是BALB/cByJ小鼠）造血干细胞（HSC）功能随年龄增长而丧失的机制和因素，2）测试将年轻HSC和微环境移植到老年受体中的益处。潜在的长期临床益处包括：HSC的延迟耗尽;改善长期健康;增加涉及老年供体或受体的HSC移植的长期成功率;以及改善对由干细胞或前体细胞功能丧失介导的衰老疾病的治疗。

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.