Senior Scholar Award in Aging
Woodring E. Wright, M.D., Ph.D.
University of Texas Southwestern Medical Center

Functional Tests of Replicative Aging in Organotypic Skin Equivalents.

One of the major functions of cellular senescence is to serve as a brake against malignancy. At least 3-6 mutations are required to form a tumor. Each mutation probably requires 20-30 doublings for the initial mutation, elimination of the remaining wild-type allele (for recessive tumor suppressor genes) and expansion to a population size sufficient for the next mutation to occur. Cellular senescence would thus intervene after only one or two mutations and prevent the progression of premalignant cells. One of the critical steps in oncogenesis involves the upregulation or reactivation of telomerase in order to overcome this limit and provide the proliferative capacity required for the accumulation of additional mutations. Even if the major function of cellular senescence is to protect against tumor progression, the following logic suggests that replicative aging will have a significant impact on organismal aging. The number of permitted divisions should represent a balance between minimizing doublings to maximize its function as a brake against cancer and providing enough replication for cell turnover during maintenance and repair. Although the maximum human lifespan is thought to be unchanged over the past 100,000 years, with rare individuals living to 100 years, the functional lifespan in the wild has been estimated to be 35-40 years. There should thus be a selective pressure to have a sufficient number of cell divisions to provide for reasonable fitness until the time one was likely to have been killed (by age 40), but additional capacity beyond that point would come at the expense of a greater risk of cancer prior to age 40. The consequences of these opposing pressures should be that as we live beyond our historical lifespan, the limit on replicative capacity should have physiological consequences that contribute to the pathologies of aging. However, experimental proof of the contribution of replicative aging to organism aging remains lacking. The present studies represent a first step in moving beyond replicative aging in culture and establishing the model systems in which the relevance to organismal aging can be tested.

We propose to create model systems in which the contribution of replicative aging to the biology of aging skin can be investigated. Skin organotypic cultures can be created in vitro by first seeding human dermal fibroblasts into a collagen I matrix, allowing the matrix to contract, and then plating human epidermal keratinocytes on the upper surface. After the cells have attached overnight, the medium volume can be reduced so that the keratinocytes are at an air-liquid interface. They subsequently stratify and produce a basement membrane. This system allows one to create skin organotypic cultures using any combination of fibroblasts and keratinocytes of defined replicative age. We will use this system to explore the contributions of replicative age (“young” with long telomeres and 80% of their proliferative potential remaining, “old” with short telomeres and 10% of their proliferative potential remaining, and “near senescent” with only 1-2 doublings remaining) to various aspects of skin physiology.

Specific questions will include:

  1. Does replicative age influence epithelial migration? Lesions of defined depth can be produced using laser ablation, and the rates of epithelial migration to cover the wound can be measured over time.
  2. Does replicative age influence epithelial turnover? Living fibroblasts are required for good formation of skin organotypic cultures. We will determine whether, following stratification, the age of the fibroblast component influences the rate of turnover of the keratinocytes in the epidermal layer.
  3. Does replicative age influence dermal remodeling? Both young and near senescent fibroblasts are able to contract collagen matrices and support stratification of the keratinocytes. However, nothing is known about the long term remodeling of the matrix by the fibroblasts. We will determine whether over several months the thickness of the contracted matrix, the density of collagen or the cellular density changes using young versus old dermal components. This study will include potential remodeling of the dermis following laser wounding of the dermal equivalent.
  4. How are these questions influenced following transplantation of human skin organotypic cultures onto the backs of immuno-incompetent mice? Organotypic cultures lack a vasculature and the mobility of true skin. The most interesting results are likely to be observed in long-term changes in skin thickness or morphology following many months after vascularization and incorporation into the mouse skin.

Contact Dr. Wright.