Isolating a pure population of epidermal stem cells for use in tissue engineering

Continuously renewing tissues, such as the epidermis, are maintained by ste m cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Ho echst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and nonproliferative bas al cells. More than 90% of freshly isolated stem cells showed a G(0)/G(1) c ell cycle profile, while greater than 20% of the TA cells were actively div iding. Both stern and TA cells retained proliferative capacity, but the ste m cells formed larger, more expandable colonies in culture. Both population s could be transduced with a retroviral vector and used to bioengineer an e pidermis. However, only the epidermis from the stem cell population continu ed to grow and express the reporter gene for 6 months in organotypic cultur e. The epidermis from the transient amplifying cell fraction completely dif ferentiated by 2 months. This novel sorting method yields;pure viable epith elial stem cells that can be used to bioengineer a tissue and to test perma nent recombinant gene expression.