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COMMENTARY |
1 The Lankenau Institute for Medical Research, Wynnewood, Pennsylvania.
2 The Coriell Institute for Medical Research, Camden, New Jersey.
3 The Sally Balin Medical Center, Media, Pennsylvania.
THE limited replicative life span of fibroblasts derived from various human tissues is commonly studied as a model of biological aging (1,2). There is little doubt that organismic failures in aging have a cellular basis. Replicative senescence in culture fits the description and definition of cell senescence; with subcultivation, there is a gradual loss of proliferative capacity in the population until the culture can no longer be subcultivated. In vivo, there is a gradual attenuation of proliferation rates of some cell types with age, suggesting parallel changes in proliferative regulation in vivo and in vitro (3). Thus the study of replicative senescence in vitro as a model for cellular changes in vivo is attractive since it brings the experimental advantage of cells that exhibit some features of senescence that are maintained in an environment that is under the control of the investigator.
Despite numerous published studies on replicative senescence over the last 40 years (e.g., Ref. 3), the relevance of in vitro studies to aging in vivo has been controversial. The question of major interest is whether the changes we observe in replicative senescence duplicate the pathways and mechanisms of cell senescence in situ. One of the major sources of support for the direct relationship of replicative senescence to cell senescence in situ has been the putative decline in the replicative life span of skin fibroblasts (and other cell types) in culture as a function of donor age (4,5). However, failure to consistently show this inverse relationship of donor age to proliferative life span has been problematic for defining the relevance of the cell culture model to organismic aging.
In fact, several studies (6–8) using healthy donors of different ages have not demonstrated a relationship between donor age and replicative life span. In the most recent of these studies that address this putative relationship, Smith and colleagues (6) make four major points: (a) colony size distribution (CSD) analysis estimates the total replicative life span of fibroblasts; (b) in cultures using CSD to estimate replicative life span, the authors report no significant decline of replicative life span as a function of donor age. However, when comparing cultures derived from females only, they detected a small but significant decline in estimated replicative life span; (c) for both male and female donors, the estimated replicative life span was greater for the pool of donors under 30 years of age compared with those over 30 years of age; and (d) when cultures established from sequential biopsies from the same individuals at different ages were evaluated for estimated replicative life span, there was a nonsignificant trend toward shorter life spans as the donors grew older.
Although finding (b) above essentially confirms and extends our findings (7) and those of others (8), namely, that there is large variability and no statistically significant age association with replicative life span for human fibroblast cultures (7,8), there are some differences in the data and interpretation between Smith (6) and other authors (7,8) that we think require clarification. This commentary is written in part to clarify these differences and also to comment on the implications of these findings to the role of cell culture studies for research on aging.
Existing evidence indicates that the replicative life span of a culture reflects the maximum replicative capacity of the longest-lived clone in the population (9). Thus, the procedure of comparing donor age to replicative life span is confounded by selection pressures against slower-growing cells that take place from the first outgrowth, through serial subcultivation, to senescence. On the other hand, the CSD assay as used by Smith and colleagues (6) evaluates the capacity of cell populations to form colonies of a certain minimal size within a defined period of time ("usually before population doubling [PDL] 10") (6). While populations examined by the CSD method are not free of selection effects, the cells have experienced "less selection" than populations subcultivated multiple times over long periods in culture. Therefore, it might seem safe to conclude that CSD more accurately reflects the proliferative potential of cells in the tissue than the serial cultivation method. However, for several reasons (see below), we suggest that CSD is not useful as a method for estimating total replicative life span. Furthermore, regardless of how the total replicative life span of fibroblasts in culture is determined, it is evident that it is not an accurate reflection of donor age.
Although Smith and colleagues (6) present compelling data to support the existence of a correlation between life span estimated by CSD and replicative life span determined in mass culture, several of the results that they obtained with CSD cannot be confirmed using standard published methods for direct proliferative life span determination (7). For example, a large number of the same Baltimore Longitudinal Study on Aging-derived cultures have been compared for replicative life span versus CSD at the Coriell Institute for Medical Research in Camden, New Jersey. The results of this study (Figure 1) do not confirm the observations of Smith and colleagues (6) that CSD is correlated with actual cumulative PDL.
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In previous studies, we (7) have shown that the growth rates of early PDL skin fibroblast cultures derived from young donors were significantly higher than that for cultures derived from older donors. This is evident both in differences between fetal- and adult-derived cultures as well as differences between cultures derived from donors aged <30 versus >30 years (7). This may be one basis for the discrepancy of the estimated life spans with those measured directly. Since the CSD method counts the number of colonies that have reached a particular size within a fixed time period, the assay should predict longer life spans for fetal-derived cell lines that grow more rapidly. It is informative that, using the CSD method, the replicative life span of some fetal lines was overestimated by a factor of 2 (7, and Coriell Aging Cell Bank Catalog).
The authors also report that cultures from young women exhibit a greater estimated proliferative life span than women aged >30 years, and that a negative trend is observed when CSD-predicted life spans of cell lines from women are plotted. This result does not agree with either the two studies of healthy donors (7,8), which failed to identify any donor age effect, or with the studies of Martin and colleagues (4), who failed to observe a sex effect or an age effect that was dependent on individuals under 30 years of age. Additionally, the data of Smith and colleagues (6) from longitudinal studies of estimated replicative life spans determined for cell lines established sequentially from the same individuals at different in vivo ages appeared to suggest an age-related decline that was not seen when replicative life span was determined directly by subcultivation to exhaustion (7).
Finally, it is surprising that Smith and colleagues (6) failed to observe a reduced proliferative potential for cells derived from diabetics while others found that the total number of cell divisions possible in fibroblast cultures established from individuals with either type I or II diabetes tended to be lower than was observed in age-matched, nondiabetic controls; the effect is greater in type II diabetes (8,10–12). Furthermore, the deleterious effects of diabetes on proliferative potential are progressive and become more pronounced with increasing donor age (8,12). Type I diabetes decreases plating efficiency as well (13), which would be expected to interfere with the CSD assay.
Taken together, these results suggest that CSD and serial cultivation methods to determine replicative life span may measure two different, but perhaps overlapping, biological parameters. CSD is an estimate of initial proliferation rate and cloning efficiency, soon after establishing the culture. In contrast to CSD, replicative life span determines the actual number of doublings it takes to exhaust the proliferative capacity of the initial population emerging from the biopsy.
Balin and colleagues (14) have shown that no matter how soon cells are examined after their initial outgrowth from biopsy pieces, there may still be artifacts that complicate the interpretation of results. They found that the way tissue cultures are typically established (for PDL or CSD), the initial outgrowth population does not accurately reflect the total population of viable cells in a tissue biopsy. Thus, with regard to human aging, methods that determine either total cumulative life span or CSD may be misleading when interpreted to reflect the proliferative capacity of the tissue from which the culture was derived.
An additional problem in evaluating the relevance of replicative senescence arises from the fact that several authors have shown that modulation of the expression of certain genes (15–17) as well as some changes in the culture environment (18–20) can produce a phenotype that is apparently indistinguishable from replicative senescence. Thus, the senescent phenotype can be achieved irrespective of proliferation. The fact that many different stimuli can produce the so-called senescent phenotype suggests that this phenotype may be a final, common pathway for replicating cells in which signaling or metabolic imbalances occur. When placed in culture, cells may be unable to achieve their "true" differentiated fate (identical to the one in vivo) either because of signals missing from the culture medium or failure to process these signals. The fact that the senescent phenotype has never been confirmed in healthy tissues in vivo is consistent with this view (18,21).
Another confounding issue relates to the "telomere hypothesis of aging," which proposes that replicative aging may be regulated by telomere shortening. One study that examined this correlation directly (22) claimed that both telomere length and proliferative life span were marginally correlated to donor age. Yet, a much stronger correlation was observed between the proliferative capacity of clones and telomere length than between donor age and telomere length. In fact, the large number of variations and exceptions to the original formulation of the Telomere Hypothesis, as well as the evidence for selection in serial subcultivations, have made this original interpretation untenable. For example, telomere length is highly variable in multiple clones established from a single individual (23). Thus, as in the case of proliferative life span, the data fail to clearly support a relationship between donor age and average telomere length.
In summary, no method of determining or estimating the replicative life span of fibroblasts in culture supports the relationship between replicative capacity in vitro and donor age. Thus, the generally accepted relationship between replicative life span in vitro and donor age as evidence for the direct applicability of studies in fibroblast culture to organismic aging is fundamentally flawed. In addition, the estimation of total replicative life span by CSD may introduce additional confounding factors in interpretation of in vivo/in vitro relationships.
The fact that this relationship cannot be demonstrated reproducibly will undoubtedly lead some to conclude that the cell culture senescence model is not relevant for studies on aging. However, while the putative inverse relationship between donor age and proliferative life span has been widely used to support the utility of the cell culture model, it is not a requirement for using cell culture as a model of aging. In fact, the original speculation by Hayflick and Moorhead of cell culture senescence as a model of aging was proposed before any relationship between donor age and proliferative capacity had been published or suggested.
Human fibroblasts in culture express human genetic, metabolic, and regulatory behavior. The use of cell cultures permits study of the mechanisms of changes that occur while the cells undergo a predictable and reproducible deterioration in a constant environment. Fibroblast cultures are valuable for examining a variety of questions and hypotheses relevant to the biology of aging. In fact, the human fibroblast model has already been valuable for explaining the cellular basis of some mechanisms underlying cellular aging changes observed in situ; also for testing hypotheses that address what may be common mechanisms underlying cell deterioration and loss of integrative function such as the effects of reactive oxygen species, overexpression and underexpression of signaling molecules, and other modulations particularly important to the mechanisms underlying aging.
Acknowledgments
Supported by grants AG00378 and AG20955 from the National Institute on Aging (NIA) and contract number N01-AG-0-2101 also from the NIA.
Address correspondence to Vincent J. Cristofalo, PhD, The Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096. E-mail: cristofalov{at}mlhs.org
References
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