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归因于青少年保护因子的丧失，包括干细胞。小鼠造血是研究干细胞的理想方法，因为骨髓和血液前体的谱系可以非常精确地测量。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C57BL/6J （B6）小鼠中则没有这种下降。目的1-3测试6周加上6、12、18和24个月大的胎肝和骨髓中HSC对细胞应激的易感性差异，以确定B6-BALB差异是否存在于幼崽中，还是随着年龄的增长而缓慢出现。长期(LT)-造血干细胞，短期(ST)-造血干细胞和共同髓系前体（CMPs）将通过优化的流式细胞术标记物在这些菌株中进行衰老研究。目的4检验内源性或外源性少年保护因子移植到老年小鼠体内是否能改善HSC功能并延长寿命。了解干细胞老化的因素和机制将有助于确定预测老年功能缺陷的青少年表型。利用这些方法，我们将验证以下假设：目的1:B6细胞比BALB细胞更能保持基因组稳定性。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的处理：伽马辐射、DNA聚合酶抑制和百草枯。基因组不稳定性将被量化为a)染色体结构病变（谱核型），b)片段拷贝数畸变（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）。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比BALB hsc衰老更少，因为它们比BALB hsc增殖更慢。将通过以下方法评估HSC亚群：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天后溴脱氧尿嘧啶（BrdU）的摄取和稀释。目的3:B6细胞的凋亡速度比BALB细胞慢。HSC亚群将通过比较使用膜联蛋白V和碘化丙啶双重标记鉴定的凋亡细胞的比例来评估。目的4：年轻或胎儿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的再填充功能。如有必要，老年受体中的造血干细胞将被生长因子动员，为年轻造血干细胞打开壁龛，或通过照射或化疗杀死，以打开壁龛并去除转化细胞。潜在的外源性缺陷将通过将胎儿脾脏（带有遗传标记细胞）移植到受体肾胶囊中来检测。如果微环境随着年龄的增长而受损，与年轻受体相比，老年移植脾脏的年轻微环境中会发生更多的内源性HSC更新和分化细胞的产生。该项目的目标是：1)确定在C57BL/6J小鼠中，而在BALB/cByJ小鼠中，防止造血干细胞（HSC）功能随年龄增长而丧失的机制和因素；2)测试将年轻造血干细胞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：延缓造血干细胞耗竭；改善长期健康；老年供体或受体造血干细胞移植的长期成功率增加；改进了由干细胞或前体细胞功能丧失介导的衰老疾病的治疗方法。

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.