Cellular Aging: Basic Phenomena

What has generally been accepted as the strongest evidence supporting the usefulness of the cell senescence model of aging has been reports from several laboratories of a negative correlation between donor age and proliferative life span in vitro. On the basis of these observations, it follows that the physiological effects of aging in vivo are reflected by the life span of cells maintained in vitro. Other laboratories report that the colony-forming capacity of individual cells also declines as a function of donor age. These observations, and the relationship between growth potential and maximal species life span is generally regarded as clear evidence of a direct relationship between aging in vivo and in vitro.

In spite of the fact that the relationship between donor age and proliferative lifespan became widely accepted, the correlations reported were always quite low. Furthermore, the studies were never standardized for biopsy site or culture conditions, which further added to individual variation and further decreased the correlation. Probably the most important problem with human studies was that so few of them actually assessed the health status of the donors from whom the cell lines were established. Many of the cell lines used were established from cadavers. It is known that diseases such as diabetes exert a profound affect on the proliferative potential of cell cultures. It is probable that other diseases can also affect in vitro proliferative capacity. The failure to screen for donor health probably skewed the results of many studies. These factors were seldom discussed when considering the correlations between donor age and proliferative lifespan. Instead, the relatively low correlations were seen as the result of individual variations in a highly outbred and otherwise uncontrolled population, and it was generally accepted that stronger correlations would be found in a longitudinal study where cell lines were established from a small group of individuals throughout their life. Due to the relatively large numbers of lines examined, the correlations reported found general acceptance, even though they were weak. Nevertheless, it remained difficult to assess whether the reported correlations between donor age and replicative life span indicated any compromise of physiology or proliferative homeostasis in vivo.

In order to address some of the problems with standardization and health status, 124 cell lines, established from 116 donors who participated in the Baltimore Longitudinal Study of Aging (BLSA), and 8 samples of fetal skin were examined by Cristofalo and coworkers (Cristofalo, et al., 1998). In this study, all of the donors were medically evaluated and determined to be healthy (by the criteria of the BLSA). None of the donors had diabetes when the cell lines were established. The results of this study showed that in healthy donors there was no relationship between donor age and proliferative life span. A longitudinal study was also performed to evaluate the effects of age on in vitro life span within individuals. Multiple cell lines established from the same individuals at time intervals spanning as long as fifteen years were compared. Surprisingly, this longitudinal study also failed to reveal any relationship between donor age and proliferative capacity in the five individuals studied. In fact, the proliferative potentials of cultures established when donors were older was found to be greater than that in lines established at younger donor ages in four of the five donors.

In spite of these findings, the fact that the colony-forming capacity of individual cells had also been reported to decline with donor age still seemed to support a relationship between donor age and proliferative potential in culture. However, the colony-forming assay is strongly influenced by variations in growth rates. Both the initial growth rates and the rate of thymidine incorporation into cells is dramatically higher in fetal fibroblast lines than in adult lines, which indicates that cell lines established from fetal skin initially divided more frequently, even though the replicative life span of these cell lines do not exceed that observed in the postnatal cell lines. It would seem equally relevant that the rate of thymidine incorporation and initial growth rates of large groups of lines established from adults do not vary significantly with respect to age. In view of differences in initial growth rates and intraclonal variations in the proliferative potential of single cells, it seems probable that the clone-size distribution method of estimating proliferative life span and an actual determination of replicative life span measure different things.

It should be noted that studies supporting the inverse relationship between donor age and proliferative life span are not limited to human cells. Studies of rodent skin fibroblasts also appeared to support the existence of a small, though significant, inverse correlation between donor age and replicative life span. Furthermore, it was observed that treatment of hamster skin fibroblasts with growth promoters could extend the proliferative life of cultures established from young donors but had negligible effects on cultures established from older donors. However, even in rodents, the relationship between donor age and proliferative potential is not entirely clear. For example, an examination of hamster skin fibroblast cultures established from the same donors at different ages reveals no age-associated changes in proliferative potential in animals older than twelve months.

In vitro studies on aging have used two kinds of cell cultures. The predominant one has been fetal- or neonatal-derived cultures that show aging changes when subcultured at regular intervals; some of these alterations parallel aging changes in vivo. The other related paradigm is that of cells derived from donors of different ages and studied after only one, or a few, subcultivations. In either case, attempts to relate changes in these individual cells to changes occurring in organisms as they age forms one basis of the use of the in vitro model for studies of in vivo aging.