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

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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.