Identification of Candidate Regulators of Hematopoietic Stem Cell Aging Using a Combined QTL Mapping and Microarray Analysis Approach.

A combination of quantitative trait locus (QTL) mapping and microarray expression profiling was used to identify five novel candidate genes for regulating hematopoietic stem cell aging in two strains of inbred mice, C57BL/6 (B6) and DBA/2 (D2). Despite their ability to self-renew and maintain essentially normal levels of hematopoietic function throughout the lifetime of an organism, it has become increasingly evident that hematopoietic stem cells (HSCs) are subject to qualitative and quantitative changes over time, and that these changes may affect both the rate of aging and longevity of organisms. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting that genetic regulation plays a role in HSC aging. Our lab has identified a QTL on murine chromosome 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In B6 mice, the frequency of HSCs increases with age, whereas in D2 mice the population of HSCs decreases as the animals age. Using a congenic mouse model in which the region surrounding the QTL in D2 mice was introgressed onto a B6 background, genome wide gene expression analyses were performed using sorted lineage negative hematopoietic cells, which are enriched for primitive stem and progenitor hematopoietic cells. RNA isolated from lineage negative cells from chromosome 2 congenic and B6 background mice at young and old ages was reverse transcribed, labeled, and hybridized to Affymetrix Murine Genome 430 arrays. Using an overall p-value cut-off of ≤ 0.01, a total of 4,368 transcripts were differentially expressed between background and congenic mice, out of 39,000 transcripts present on the mouse genome chip. Following a ‘QTL to Gene’ strategy, we identified 123 differentially expressed transcripts located in the congenic interval on chromosome 2. We then assessed for hematopoietic tissue specific expression patterns, the presence of single nucleotide polymorphisms (SNPs) in the transcripts, and cis-regulation of transcript expression. Five genes were identified that met these criteria, and thus are considered candidate regulators of hematopoietic stem cell aging. We used real time PCR to validate differential expression of a subset of the identified genes. Interestingly, a majority of the genes are associated with cell cycle control and/or protein targeting and transport processes. Additionally, all five cis-regulated genes were found to be down-regulated in aged congenic mice compared to aged B6 mice, thus suggesting coordinated regulation by a common promoter region(s) located within the chromosome 2 QTL. These findings suggest that stem cell aging is associated with specifc intrinsic cellular events and provide insight into the genetic mechanisms underlying hematopoietic stem cell aging.