Cloning of differentially expressed genes in skin fibroblasts from centenarians

Normal human fibroblasts undergoing serial passaging have been extensively used to identify genes linked with aging. Most of the isolated genes relate to growth retardation signals and the failure of homeostasis that accompanies aging and senescence. In contrast, there is still limited knowledge regarding the nature of the genes that influence positively the rate of aging and longevity. Healthy centenarians represent the best example of successful aging and longevity. Studies using samples from these individuals have proved very valuable for identifying a variety of factors that contribute to successful aging. The aim of the current work was to take advantage of skin fibroblast cultures established from healthy donors including centenarians in order to clone differentially expressed genes in centenarians. First, we demonstrate that centenarian derived cultures follow the typical Hayflick curve and they enter senescence after serial passaging. Application of differential screening techniques in minimally passaged cultures of four control donors of different ages (18-80 years old) and four centenarians has resulted in the cloning of six differentially expressed genes in centenarians. Four of the cloned genes, namely adlican, KBL, EST 38 and EST 39, were over-expressed in centenarians, while VDUP1 and OCIF were down-regulated in the same samples. We have also compared the expression levels of two representative cloned genes in cultures of human embryonic and adult fibroblasts to establish potential links with replicative senescence. Interestingly, VDUP1 was found over-expressed in late passage cells, while EST 39 was down-regulated in the same cultures. Thus our work demonstrates that a combination of the use of both biopsies derived cells and classical in vitro cells passaging will facilitate the better understanding of the biology of aging and longevity. Normal human fibroblasts undergoing serial passaging have been extensively used to identify genes linked with aging. Most of the isolated genes relate to growth retardation signals and the failure of homeostasis that accompanies aging and senescence. In contrast, there is still limited knowledge regarding the nature of the genes that influence positively the rate of aging and longevity. Healthy centenarians represent the best example of successful aging and longevity. Studies using samples from these individuals have proved very valuable for identifying a variety of factors that contribute to successful aging. The aim of the current work was to take advantage of skin fibroblast cultures established from healthy donors including centenarians in order to clone differentially expressed genes in centenarians. First, we demonstrate that centenarian derived cultures follow the typical Hayflick curve and they enter senescence after serial passaging. Application of differential screening techniques in minimally passaged cultures of four control donors of different ages (18-80 years old) and four centenarians has resulted in the cloning of six differentially expressed genes in centenarians. Four of the cloned genes, namely adlican, KBL, EST 38 and EST 39, were over-expressed in centenarians, while VDUP1 and OCIF were down-regulated in the same samples. We have also compared the expression levels of two representative cloned genes in cultures of human embryonic and adult fibroblasts to establish potential links with replicative senescence. Interestingly, VDUP1 was found over-expressed in late passage cells, while EST 39 was down-regulated in the same cultures. Thus our work demonstrates that a combination of the use of both biopsies derived cells and classical in vitro cells passaging will facilitate the better understanding of the biology of aging and longevity.