ES Cells from Primate Blastocysts Produced in vivo vs in vitro and Aging

Thus far, we have discussed aging in the sense of changes occurring in the fertility of an individual female primate as she becomes older. But in another sense, aging is a property of individual cells, and this not only may underlie age-related events in the individual but also account for functional changes in a cell as it reaches the end of its life. In this context, aging is a key aspect of cultured cells, including ES cells. We believe that a major focus on mitochondria is warranted in ES cell studies. A sufficient number of normal mitochondria is required in somatic cells to support normal functions. A high frequency of genetically abnormal mitochondria (muta-tional load) could reduce the number of functional mitochondria, leading to poor cell performance, manifested as slow cell divisions, karyotypic anomalies, and/or perturbation of mitochondrial distribution profiles. Moreover, the presence of mtDNA anomalies may be increased in cell lines derived from in vitro produced embryos compared with embryos produced in vivo.

Mitochondria play a key role in early development, though one that is far from being fully understood (Barnett and Bavister, 1996; Van Blerkom et al., 2000; Bavister and Squirrell, 2000; Barnett et al., 1996; Ludwig et al., 2001). By analogy with their fundamental metabolic roles in somatic cells (Yaffe, 1999), we can expect that mitochondria have similar importance in the inner cell mass (ICM) of blastocysts and in the ES cells derived from them, which are primordial somatic cells. Culture conditions that affect mitochondrial activity, such as providing oxidizable substrates, during oocyte maturation or early cleavage stages can profoundly alter embryo development in several species including nonhuman primates (McKiernan et al., 1991; Rose-Hellekant et al., 1998; Krisher and Bavister, 1999; Zheng et al., 2001,

2002). Mitochondria sequester cytoplasmic calcium, and ''uncoupling'' them caused apoptotic cell death in embryos (Liu et al., 2001). Moreover, mitochondrial structure and activity change strikingly during pre-implantation development, suggesting an increasingly important role once differentiation (blastocele formation) occurs. Some somatic cells such as fibroblasts and neurons also exhibit a pronounced clustering of active mitochondria (Yaffe, 1999; Mattson and Partin, 1999). In some cells, the distribution of mitochondria is under genetic control (Yaffe, 1999). In fibroblasts, mitochondria cluster around the nucleus while peripheral cytoplasm is devoid of mitochondria, the same profile as in fertilizing oocytes and early preimplantation embryos (Yaffe, 1999; Barnett et al., 1996; Squirrell et al., 2003). In neurons, mitochon-drial clustering is involved in establishing cell polarity and disrupting their distribution results in loss of polarity (Mattson and Partin, 1999).

In view of these observations on preimplantation embryos and on somatic cells, it seems very likely that clustering (nonrandom distribution) of mitochondria occurs in primate ES cells and thus may be important for their normal functions. If so, the distribution profile(s) could become a simple marker of ES cell competence, as well as an indicator of impending differentiation. We should examine primate (human and monkey) ES cells to determine this. Next, we should ascertain if mitochon-drial localization patterns are different in ES cells from IVP vs. in vivo produced blastocysts, because most of the IVP embryos are nonviable, while we expect most blastocysts produced in vivo to be viable. This difference should be reflected in ES cell quality and functional competence. However, because of the complete lack of human embryos produced in vivo, this comparative study can only be done in a suitable nonhuman primate model, such as the rhesus monkey, in which protocols both for IVP and for flushing uteri of mated animals have been established (Schramm and Bavister, 1996; Wolfgang et al., 2001). Once this study has been completed, we can move on to examination of effects of aging, in both senses of the term: (i) effects of age of the individual on mito-chondrial localization (and other aspects such as mtDNA errors as described above) in ES cells from IVP and in vivo produced embryos, and (ii) effects of aging of the ES cells in culture (i.e., increasing passage numbers) on these parameters.

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