HSCIntrinsic Mechanisms of Functional Decline Lead to Changes in Stem Cell Number with

We have cited several studies in which knockout (KO) mice were used to demonstrate a role for certain molecules in the regulation of the hematopoietic system. Unfortunately, embryonic lethality and the unpredictable and nonphysiological downstream effects of gene KO studies prohibit the use of KO or conditional KO strategies to investigate many genes that may have unique roles in adult HSCs. Inbred strains of laboratory mice provide excellent models to study HSC properties because strain-specific phenotypic differences can be directly attributed to underlying genetic differences in the stem cells of mice that are otherwise healthy and viable. For example, the genetic regulation of the age-related decline in stem cell function has been demonstrated extensively by comparing divergent phenotypes among various mouse strains. Stem cells in the relatively long-lived C57BL/6 (B6) mouse strain increase in number during ageing, whereas stem cells in DBA/2 (D2) and BALB/cBy (BALB) strains decrease over time and display overt signs of ageing (Morrison et al. 1996, Chen et al. 1999, de Haan 1999, Kamminga 2005, Yuan et al. 2005). When equal numbers of bone marrow cells from old and young B6 mice are mixed with heterogeneous competitor cells and injected into lethally irradiated recipients, the old cells have a competitive advantage compared to young cells (Harrison et al. 1989, Morrison et al. 1996). In stark contrast to these results, old stem cell populations in the relatively short-lived D2 and BALB mice display a significant competitive disadvantage compared to young cells (Harrison et al. 1989, Van Zant et al. 1990, Chen et al. 2000). Strain-dependent differences in B6 and D2 stem cell ageing were demonstrated in a series of experiments in allo-phenic (cellular chimeras) mice. Eight-cell embryos from B6 and D2 mice were combined ex vivo to form chimeric embryos that were implanted into the uteri of pseudopregnant females (Van Zant et al. 1990). All tissues in the resulting chi-meric mice consisted of a mixture of B6 and D2 cells. During ageing, D2 HSC function progressively declined, evidenced by a shift to the B6 genotype in all blood cell lineages by two years of age. Importantly, the chimeric mice also showed complete tolerance to B6 and D2 cells, thus ruling out all possibility that the D2 genotype is selectively eliminated during ageing (Mintz and Silvers 1967). The chimeras allow simultaneous assessment of two stem cell populations in a common environment, demonstrating the cell intrinsic nature of stem cell ageing properties in DBA and B6 mice. D2 hematopoiesis in the chimeras ceased around the time of the strain's natural life span, which supports the existence of a link between viability of HSC populations and organismal longevity.

Following this important observation in B6/DBA allophenic mice, several other studies followed in which hematopoiesis was characterized more thoroughly in both B6 and DBA strains (de Haan et al. 1997, de Haan and Van Zant 1997, de Haan and Van Zant 1999, Geiger et al. 2001). In B6 mice, the frequency of cells that bear cell surface markers associated with long-term self-renewal increase three- to fivefold as the animals age. It is not surprising that, in whole bone marrow transplant studies, old B6 cells perform better than young ones. Marrow cells derived from old B6 mice simply have a higher proportion of primitive stem cells compared to young mice. However, despite the fact that old B6 cells have a competitive advantage in transplant settings, they possess several qualitative defects. (Nonetheless, in most physiological situations their numerical advantage clearly compensates for qualitative defects.) A true assessment of a stem cell's regenerative capacity is its ability to be serially transplanted through multiple mouse recipients. Serial bone marrow transfer imparts severe proliferative demands on the HSC compartment and leads to stem cell exhaustion. In this setting, stem cell exhaustion is thought to lead to replicative senescence of the transplanted cells, resembling physiological HSC ageing (Bell and Van Zant 2004). Like most inbred strains, old B6 cells cannot be serially transplanted indefinitely and undergo a finite number of population doublings (Siminovitch et al. 1964, Ogden and Mickliem 1976m Harrison 1979, Harrison and Astle 1982, Ross et al. 1982, Kamminga 2005).

Recently, Kamminga et al. (2005) demonstrated that the maximum number of population doublings in primitive cells from B6 mice is approximately 30, and only 20 in D2 Although the maximum expansion potential was greater in B6 mice, serial transplantation resulted in severe functional impairment in both strains, evidenced by decreased clonogenic activity, reduced competitive repopulating activity, and impaired peripheral blood cell recovery. This study again emphasizes that intrinsic genetic regulation influences stem cell function in B6 and DBA mice during ageing.

Similar to the patterns of change in stem cell number in DBA/2 mice with age, the number of primitive hematopoietic cells (CD34+) that can be obtained from human bone marrow aspirates decreases with age. Regression analysis on data collected from almost 50 human marrow samples showed that 24% of the variation in CD34+ cell numbers can be attributed to age (Fig. 6.2). We suspect that comparing genetic and phenotypic differences between the HSCs of B6 and DBA mice will reveal loci involved in regulation of stem cell number in humans. Furthermore, it is conceivable that these loci will prove to be targets of therapeutic intervention that can be manipulated to limit malignancy or stimulate self-renewal or proliferation in bone marrow failure syndromes.

Age in Years

Fig. 6.2 Whole bone marrow aspirates were obtained from 43 adult humans. Mononucleated cells were collected by Ficoll-gradient centrifugation and stained for the presence of CD34. Flow cytometric analysis was used to identify the percentage of CD34+ cells in each sample. Linear regression analysis revealed that approximately 24% of the variation in the number of CD34+ cells in human bone marrow can be attributed to age (See Color Plate)

Age in Years

Fig. 6.2 Whole bone marrow aspirates were obtained from 43 adult humans. Mononucleated cells were collected by Ficoll-gradient centrifugation and stained for the presence of CD34. Flow cytometric analysis was used to identify the percentage of CD34+ cells in each sample. Linear regression analysis revealed that approximately 24% of the variation in the number of CD34+ cells in human bone marrow can be attributed to age (See Color Plate)

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