Regenerative medicine refers to innovative therapies aimed at the permanent restoration of diseased tissues and organs. Regeneration of self-renewing tissues requires specific adult stem cells, which need to be genetically modified to correct inherited genetic diseases. Cultures of epithelial stem cells permanently restore severe skin and mucosal defects, and genetically corrected epidermal stem cells regenerate a normal epidermis in patients carrying junctional epidermolysis bullosa. The keratinocyte stem cell is therefore the only cultured stem cell used both in cell therapy and gene therapy clinical protocols. Epithelial stem cell identification, fate and molecular phenotype have been extensively reviewed, but not in relation to tissue regeneration. In this paper we focus on the localization and molecular characterization of human limbal stem cells in relation to corneal regeneration, and the gene therapy of genetic skin diseases by means of genetically modified epidermal stem cells.