The incidence of bone marrow failure and hematopoietic malignancies increases with aging, suggesting that the biological effects of aging promote the development of hematopoietic diseases in older adults. To examine the biology of aging in hematopoietic cells, many studies have investigated the functional effects of aging in murine model systems. Murine studies have found that hematopoietic stem cells (HSCs) from older mice have an increased proliferative capacity, disrupted cell cycle regulation, reduced repopulating potential and propensity toward myeloid skewing.1 In a search to discover novel genes causing age-associated functional changes, studies have compared the global expression changes between murine long-term repopulating HSCs (LTR-HSCs) from young and old mice, identifying a number of age-associated expression changes.2, 3 However, owing to logistic and scientific challenges, there have been fewer studies examining the effects of age on the human hematopoietic system, and there are no studies examining the age-associated expression changes in HSCs or even early hematopoietic progenitor cells in humans. Therefore, we examined the age-associated expression changes in human CD34+ cells and compared the age-associated expression changes in human CD34+ cells to murine LTR-HSCs.
Microarray studies were used to identify the age-associated expression changes in two populations of human hematopoietic progenitor cells: BMCD34+ cells (N=8, age range, 16–51 years) and PBCD34+/38− cells (N=4, age range, 22–53 years). Details about the methods and subjects are provided in Supplementary Methods and Supplementary Table 1, respectively. The microarray studies identified 161 and 917 genes with age-associated expression changes in BMCD34+ and PBCD34+38− cells, respectively (Supplementary Table 2). The slight majority of these genes (approximately 65%) displayed an increased expression with age in both populations. Ten genes had similar age-associated expression changes in both data sets (Figure 1a). Functional pathways affected by aging included gene expression, cellular proliferation, cancer and cellular development (Supplementary Table 2).
Although the human BMCD34+ and PBCD34+38− cells are enriched for HSCs, these CD34+ cells do not represent a pure population of LTR-HSCs. Recently, Rossi et al. and Chambers et al.2, 3 isolated near-homogeneous populations of murine LTR-HSCs and examined age-associated expression changes in these cells. Although murine studies have provided important insights into the age-associated expression changes in HSCs, interspecies differences have been reported.4 We wondered which age-associated expression changes in the human CD34+ cells also occurred in murine LTR-HSCs, believing that age-associated expression changes conserved across species would be of particular interest for future studies.
Therefore, we examined the expression profiles of murine HSCs from the Rossi and Chambers studies.2, 3 Analyses for each data set were performed independently of one another, as different murine array platforms were used for each study. We identified 494 and 3359 genes with age-associated expression changes from the Rossi and Chambers studies, respectively.2, 3 Eighty-four genes displayed age-associated expression changes in both murine data sets (Supplementary Table 3). Approximately 90 and 85% of the genes with age-associated expression changes in human BMCD34+ and PBSC34+38− cells, respectively, had at least one or more probe sets on the murine array platforms used by Chambers and Rossi (Supplementary Table 2).2, 3 Examining the overlap of age-associated expression changes between human CD34+ cells and murine LTR-HSCs, interferon regulatory factor 8 (IRF8), displayed an age-associated decrease in its expression in all four analyses (Figure 1).
To determine if similar age-associated expression changes occurred in more differentiated hematopoietic cells, we compared the expression profiles of expanded T-cells from younger (age ⩽30 years) and older (age >70 years) normal donors. Analyses identified 13 genes with age-associated expression changes (increased=4 and decreased=9, Supplementary Table 4). IRF8 was one of the 13 genes with age-associated expression changes in human T cells, and as in the previous microarray studies, IRF8 expression in T cells was significantly decreased in older adults.
Given the small numbers of samples, we sought to validate our findings using an independent sample set. Quantitative reverse transcription-PCR (Q RT-PCR) studies using Taqman assays found that IRF8 expression decreased with aging in BMCD34+ (N=6, R=0.77, P=0.07), PBCD34+38−, (N=2, P=N/A) and T cells (N=26, P=0.001) (Supplementary Figure 1), confirming the microarray data. To assess the relationship between mRNA and protein expression in non-immortalized human hematopoietic cells, we simultaneously examined mRNA and protein expression of IRF8 in a limited number of PBCD34+ samples that had both RNA and protein available. Quantitative reverse transcription-PCR and protein assays (Figure 2a) demonstrated a similar decrease in IRF8 expression with increasing age (mRNA, R=0.90, P=0.04 and protein, R=0.91, P=0.03, Figure 2b).
To our knowledge, this is the first report to identify IRF8 as a potential biomarker of aging. Similarly, it is the first study to compare the age-associated expression changes between human CD34+ cells and murine HSCs. Previous studies have established that IRF8, a transcription factor, plays a critical role in normal hematopoiesis, controlling the transcription of several pivotal regulatory genes,5 and inactivation of IRF8 causes a chronic myeloid leukemia-like syndrome in mice.6, 7 In addition, we and others have found that leukemic blasts frequently display decreased IRF8 expression, suggesting a possible link to malignant transformation in humans.8 It is intriguing to postulate that perhaps the loss of IRF8 expression in the aging hematopoietic cells may contribute to myeloid skewing and/or the development of hematopoietic diseases in older adults. However, additional studies are needed to better characterize the age-associated expression changes of IRF8 in other hematopoietic cells (for example, monocytes, B-cells, and so on) and to examine the functional consequences of its decreased expression with increasing age.
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This research was supported by the following NIH Grants: AG06781, CA92405, CA114563, CA114563, CA018029, HL66947, CA018029 and DK56465. DLS oversaw the design, analyses and writing of the paper. YEC, ELP and MRC performed most of the experiments. NES, FRA, JPR and SH helped with the design, analyses and interpretation of the studies as well as actively contributed to the writing of the paper. MY, EBL and BLW helped obtain the samples for the studies and actively contributed to the writing of the paper.
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Stirewalt, D., Choi, Y., Sharpless, N. et al. Decreased IRF8 expression found in aging hematopoietic progenitor/stem cells. Leukemia 23, 391–393 (2009) doi:10.1038/leu.2008.176
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