Identifying and maintaining the epidermal stem cell compartment

Epidermal regeneration obtained with autologous cultured keratinocytes can be life saving for patients suffering from massive full-thickness burns. However, the widespread use of cultured epidermal autografts has been hampered by inconsistent clinical results reported by several leading burn centers world wide. An important cause for poor results is depletion of epidermal stem cells during the culture period.
Therefore, our ultimate goal is to transplant composite skin grafts containing a functional stem cell compartment. To achieve this we are currently focusing on:
1) the reliable identification of epidermal stem cells
2) the enrichment and/or maintenance of an appropriate number of vital epidermal stem cells throughout the culture period and beyond.

The identification of the so called side population (SP) has already been accomplished in our laboratory using a dye exclusion technique based on the function of membrane pumps of the ABC transporter family (also known as BCRP or MXR). These pumps were shown to be specifically expressed in stem cells of various organs. We have been able to highly enrich the keratinocyte SP by FACS sorting. In addition, we have developed a bioassay to determine the “stemness” of the isolated SP.

Despite these critical achievements we still have to answer the question whether the typical characteristics of epidermal stem cells can be maintained during a culture period of about 3 weeks. It is becoming increasingly clear that the properties required to ensure sustained stem cell function are vested in neighboring differentiated cells. Through specific signals (e.g. induced by Wnt growth factors), these cells control the behavior of stem cells. Thus, location rather than specialized patterns of gene expression appear to characterize stem cells. These specific locations are generally referred to as ‘niches’. A niche is considered to be a subset of cells and extracellular substrates that can house stem cells and control both self-renewal and progeny production in vivo.
Starting off from a highly enriched keratinocyte SP fraction and utilizing appropriate extracellular scaffolds and combinations of growth factors, we are aiming at creating a dermo-epidermal environment that will allow the maintenance of stem cells in vitro.