Replicative lifespan of fibroblasts in ageing studies

A recent paper by Maier and Westendorp (2009) focused on the replicative capacity of fibroblasts from patients with accelerated ageing syndromes, patients with age-related diseases and donors of varying chronological age. Their findings were as follows:

(1) Fibroblasts from patients with accelerated ageing syndromes are lower when compared with strains from age-matched controls.

(2) No difference in replicative capacity was found in fibroblasts from patients with age-related diseases when compared to age-matched controls.

(3) No relationship between replicative capacity of fibroblasts and donor age.

It is probably not surprising that there is a lower replicative capacity in skin fibroblasts taken from patients with Werner- and Hutchinson-Gilford syndrome patients as the mechanisms underlying these syndromes are probably universally found throughout all the somatic cells in these patients. For example, Werner syndrome is caused by a mutation in the WRN gene and is associated with short telomeres and accelerated cellular senescence (Cox and Faragher, 2007). This mutation is going to be present in all cell types, therefore it does not matter which cell type is investigated, the result of a reduced replicative capacity is likely to be the same. However, the same result is unlikely to be true when investigating the replicative capacity of skin fibroblasts in subjects suffering from diseases associated with a completly different cell type.

Maier and Westendorp investigated the replicative capacity of skin fibroblasts in patients with age-related disease. However, some of the diseases classed as age related in this instance are not. These include cystic fibrosis and familial Alzheimer’s disease. This is not the main point in question. It is not surprising that there is no relationship between the replicative capacity of skin fibroblasts in patients suffering from say cardiovascular disease or diabetes because this cell type has no involvement in the development or progression of those particular diseases. If they looked at cell types related to a particular disease such as vascular endothelial cells in cardiovascular disease (Minamino et al, 2002), microglial cells in Alzheimer’s (Streit et al 2007) or pancreatic beta cells in diabetes (Sone and Kagawa, 2005) they would most likely see a decline in replicative capacity compared to age-matched controls. This was the case for lung fibroblasts in lung emphysema, demonstrated in this investigation.

Different cell types have different replicative capacities, have different functions, are maintained within different environments and thus undergo varying degrees of stresses. In addition to this, there are risk factors such as sun exposure, smoking and diet which have the potential to accelerate cellular ageing. As such, different tissues age at different rates. Therefore, the presence of disease in one tissue is not necessarily going to reflect the biological condition of another. The replicative capacity of skin fibroblasts is not necessarily going to be influenced by the presence of disease in other tissues.

A theoretical scenario where a particular disease may impact on the replicative capacity of skin fibroblasts, is if the presence of disease uses up the stem cell/progenitor cell reserve needed for cellular repair and replacement, or somehow impacts on the functioning of stem cell/progenitor cells. In this instance, damaged or lost skin cells can no longer be replaced by the stem cell/progenitor cell reserve, causing local cells to divide and replace instead. This in turn reduces the replicative capacity of those cells. This may occur in advanced stages of a disease where constant cell replacement has been undertaken. This may explain results of studies investigated in this paper which demonstrated that the replicative capacity of fibroblasts in patients with severe diabetes was diminished when compared with controls, but was insignificantly decreased in patients with mild to moderate diabetes. Also, Kuki et al (2006) has demonstrated that endothelial progenitor cells (EPCs) cultured under high glucose levels (associated with diabetes) undergo accelerated senescence. The presence of elevated oxidised low density lipoproteins (ox-LDL) observed in diabetics has also been shown to reduce the number and impair function of circulating EPCs. In addition to this, it is known that stem cells lose the capacity for self renewal when removed from the stem cell niche, suggesting that the local environment plays a crucial role in determining stem cell behaviour (Boyle et al, 2007). Therefore, the presence of diseases in advanced stages, especially those associated with inflammation, may alter the environment of stem cell niches and thus impacting on their ability to function. In this scenario, the presence of disease has the potential to impact other tissues by impairing the function of stem/progenitor cells needed for repair and maintenance.

It has often been shown that a decline in the replicative capacity of fibroblasts is correlated with an increase in chronological age of a donor. However, if the health state of donors is taken into consideration and only “healthy” subjects are investigated in this regard, there appears to be no correlation (Cristofalo et al, 1998). This suggests that the replicative capacity of a tissue only reflects biological age and not chronological age. Of course it is true, that a longer a person lives, the increased likelihood that cells become damaged, lost and replaced and this in turn would reduce the replicative capacity of those cells. However, if factors which result in cellular damage/loss such as the presence of disease (not necessarily age-related), infection or environmental factors such as smoking and sun exposure are reduced, then damage/loss of cells is reduced and the replicative capacity of those cells remains high.

Maier and Westendorp suggest an alternative explanation for the lack of relationship between donor age and replicative lifespan of skin fibroblasts: “The overall replicative capacity might decline with age but rare fibroblasts clones with extended replicative potential continue to be present at old age but do not nessesarily reflect the properties of the overall population. Therefore, the replicative capacity in vitro reflects only the expansive propagation of the longest surviving clone, which seems to have comparable in vitro characteristics when obtained from young and old individuals.”

Data on the replicative capacity of cells in regard to ageing and age-related disease is only important because the shorter the replicative capacity of a tissue, the increased likelihood that senescent cells will appear or are present. The presence of senescent cells in tissues is thought to play a role in ageing and age-related disease. Thus, it is more important to investigate the distribution and frequency of senescent cells in tissues associated with accelerated ageing syndromes, age-related diseases and chronological age.

Relationship between replicative capacity and organismal ageing

Leonard Hayflick was the first to propose that the senescence of normal cells may contribute to the organismal ageing. Investigations into this proposal started by comparing the replicative potential of cells, usually fibroblasts, extracted from individuals at various ages.

The first of these studies showed an inverse relationship between donor age and the number of population doublings achieved in vitro (Martin et al, 1970). This study looked at the replicative lifespan of fibroblasts taken from 100 subjects with an age range from foetal to 90 years. These cells were cultured and the number of population doublings before entering senescence was recorded. The results showed that the replicative potential decreased as donor age increased. A later study showed similar results (Schneider, 1979). This study looked at the ability of fibroblasts taken from young (20-35 years) and old (65+ years) to proliferate in culture. It was reported that cell cultures from old human donors have a reduction in their proliferative capacity. A more recent study looked at the replicative capacity of human adrenocortical cells to proliferate as a function of donor age (Yang et al, 2001). Again, it was found that younger cells have a higher proliferative capability than the old. In this instance, population doubling fell from 50 for foetal cells to almost a total lack of division in culture from older cells.

To investigate the possible link between replicative lifespan and organismal ageing, a few studies compared replicative capacity with longevity in animals. One such study investigated the relationship between longevity of eight mammalian species (mouse, rat, rat-kangaroo, mink, rabbit, bat, horse and human) and the lifespan of normal fibroblasts in vitro (Röhme, 1981). It was reported that there was a direct relationship found between the longevity of the eight mammalian species and the replicative capacity of their cultured fibroblasts. A much later, but similar study, compared animal life spans and in vitro replicative capacity of skin fibroblasts in groupings of small, middle, large, and very large breeds of dogs of specific ages (Li et al, 1996). It was found that the life spans were inversely correlated to the frame sizes of the breeds. It was shown that all the small breeds studied have a longer life span than that of the large breeds. The replicative capacity of fibroblasts from the large dogs (Great Dane and Irish Wolfhound) was significantly decreased compared with that of the small dogs. The reasoning behind these observations may again be due to varying degrees of cell turnover between the species. Large dogs consist of more cells than small dogs and as a result more cell turnover was initially required in their development compared to small dogs. This increase in cell turnover would subsequently lead to a decrease in replicative potential and an increase in the rate of senescent cell formation.

Interestingly, a recent study looked at the replicative capacity of 124 skin fibroblast cell lines from donors of different ages which were medically examined and declared “healthy” (Cristofalo et al, 1998). Healthy people were used specifically as previous studies, discussed later, have shown that disease states may accelerate the reduction in replicative capacity. Results indicated that there was no significant correlation between the replicative capacity of the cell lines and donor age. In the same study, a comparison of multiple cell lines established from the same donors of different ages also failed to show any significant differences. It was concluded that the replicative capacity of fibroblasts in vitro does not correlate with donor age. However, differences in replicative capacity with age may only be observed as a result of increased cell turnover in response to disease and cellular injury. Therefore, a healthy old person who has had little or no cellular injuries or disease would have had little cell turnover and therefore have cells which may have a replicative capacity similar to someone much younger. Thus, this study supports the notion that replicative capacity is an indicator of biological age.