Regulation of Stem Cell Aging by Retinoblastoma Like-1 (p107)

It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. We have previously mapped a quantitative trait locus (QTL) to murine chromosome 2 that is associated with the variation in frequency of HSCs between aged C57BL/6 (B6) and DBA/2 (D2) mice. In B6 mice the HSC population steadily increases with age, whereas in D2 mice, this population declines. A QTL regulating the natural variation in lifespan between the two strains was mapped to the same location on mouse Chr 2, thus leading to the hypothesis that stem cell function affects longevity. B6 alleles of this locus, associated with expansion of the stem cell pool, are also associated with a ~50% increase in lifespan. In the present study, we characterize a congenic mouse model which was generated by introgressing D2 alleles in the QTL onto a B6 background. Using a surrogate assay to mimic aging, we analyzed the cell cycle, apoptotic and self-renewal capabilities of congenic and B6 HSCs and show that D2 alleles in the QTL affect the apoptotic and self-renewal capabilities of HSCs. Next, we used oligonucleotide arrays to compare the differential expression of B6 and congenic cells using a population enriched for primitive stem and progenitor cells. Three variables were examined using Affymetrix M430 arrays:

• the effect of strain—congenic versus background;

• the effect of age—2 months versus 22 months; and

• the effects of 2 Gy of irradiation because previous studies indicated that congenic animals were highly sensitive to the effects of mild radiation compared to B6 background animals.

Extensive analysis of the expression arrays pointed to a strong candidate, the gene encoding Retinoblastoma like protein 1, otherwise known as p107. The B6 allele is associated with increased p107 expression in old HSCs therefore p107 in this context is a positive regulator of stem cell number in aged mice. Real-time PCR was used to validate the differential expression of p107 in lineage negative and lineage negative Sca-1+, c-kit+ (LSK) cells. Detailed sequence analysis of the gene revealed the presence of 4 non-synonymous, coding region single nucleotide polymorphisms (SNPs) between B6 and D2 mice, which may contribute to the differential expression of the gene and function of the protein. Perhaps most importantly, we show that overexpression of p107 in congenic HSCs increases day 21, day 28, and day 35 CAFC numbers in vivo by 2- to 4-fold, therefore confirming its role as a positive regulator of primitive progenitor populations including HSCs. These studies uncover a novel role for p107 and provide additional clues in the complex regulation of stem cell aging.