Human Hematopoietic Progenitor Cells Exhibit Increased Microsatellite Instability Associated with Advanced Age.

Abstract

The etiology of the aging process in human hematopoietic progenitor cells (HPCs) and how this relates to diseases associated with aging, such as leukemia myelodysplasia, anemia, bone marrow failure, and altered immune function, remains unclear. We hypothesize that DNA mismatch repair (MMR) defects, specifically in HPCs, might contribute to genomic instability and in turn hematopoietic diseases of the elderly. An increase in microsatellite instability (MSI), a characteristic of MMR defects, has been shown in the peripheral blood (Neri, et al. J. Gerontol., 2005) as well as in T-cells of healthy individuals (Kirchevsky, et al. Exp. Gerontol., 2004) as a function of age but has not been examined in clonal hematopoiesis of normal individuals. Mice who receive bone marrow transplants from MMR deficient mice show increased incidence of MSI, as well as mortality due to bone marrow stem cell failure (Reese, et al. Blood, 2003). Loss of MMR pathway components have also been reported in non-polyposis colorectal cancer, myelomas, and T-cell leukemia. To study MMR deficiency in HPCs during the normal aging process, we have tested DNA of 24 CFU grown in human methylcellulose from the mononuclear fraction of three human umbilical cord blood samples (at birth), 3 human bone marrow aspirates ages 18–49 yr (middle age), and 3 human bone core samples ages 50–85 yr (older) from total hip or knee replacement patients. Each CFU was tested for MSI at five loci (BAT25, BAT26, D2S123, D5S346, and D17S250) and given a ranking: stable (MSS), no MSI at any of the 5 loci; low frequency MSI (MSI-L), MSI seen at only one locus; or high frequency MSI (MSI-H) MSI seen at two or more loci. All five of the MSI loci were informative in at least one sample. 5′ fluorescently labeled forward primers for the MSI loci were used with unlabeled reverse primers to amplify DNA prepared from CFU. These fragments were analyzed by denaturing polyacrylamide gel and Typhoon 9200 phosphoimager. A positive MSI was scored for a CFU at a particular locus if significant differences were seen between the fragment lengths of other CFU from the same patient sample at the same locus. The average frequency of MSS CFU for the, “at birth” samples was: 69% ± 40%; for the, “middle age” samples: 47% ± 6%; for the, “older” samples: 54% ± 17%. The average frequency of MSI-L CFU for the “at birth” samples was: 28% ± 34%; for the, “middle age” samples: 51% ± 6%; for the, “older” samples: 40% ± 10%. Finally, the average frequency of MSI-H CFU for the “at birth” samples was 1% ± 2%; for the “middle age” samples was 1% ± 2%; for the “older” samples was 13% ± 8%. Our preliminary results show a nearly ten fold increase in frequency of MSI-H CFU seen in the older donors over younger age donors. This suggests that there may be a dramatic decline in MMR function associated with advanced age. This is the first data showing MMR deficiency associated with age in clones derived from normal human hematopoietic progenitor cells. If confirmed MMR may be an important target for both risk assessment and intervention to prevent marrow associated disorders.