1. ¹Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, Centro de Investigación Biomédica en Red de Enfermedades, Raras, Madrid, Spain
2. ²Laboratory of Tissue Engineering and Cutaneous Physiopathology, IDI-IRCCS, Rome, Italy
3. ³Laboratory of Molecular and Cell Biology, IDI-IRCCS, Rome, Italy
4. ⁴Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy

Correspondence: Fernando Larcher, Head, Cutaneous Disease Modeling Unit, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, Avenida Complutense 22, Edificio 7, 28040 Madrid, Spain. E-mail: fernando.larcher@ciemat.es

^*These two authors contributed equally to this work.

Received 13 March 2007; Accepted 21 May 2007; Published online 19 June 2007.

Abstract

Predicting the risks of permanent gene therapy approaches involving the use of integrative gene-targeting vectors has become a critical issue after the unfortunate episode of a clinical trial in children with X-linked severe combined immunodeficiency (X-SCID). Safety pre-assessment of single isolated gene-targeted stem cells or their derivative clones able to regenerate their tissue of origin would be a major asset in addressing untoward gene therapy effects in advance. Human epidermal stem cells, which have extensive proliferative potential in vitro, theoretically offer such a possibility as a method of assessment. By means of optimized organotypic culture and grafting methods, we demonstrate the long-term in vivo regenerative capacity of single gene-targeted human epidermal stem cell clones (holoclones). Both histopathological analysis of holoclone-derived grafts in immunodeficient mice and retroviral insertion site mapping performed in the holoclone in vitro and after grafting provide proof of the feasibility of pre-assessing genotoxicity risks in isolated stem cells before transplantation into patients. Our results provide an experimental basis for previously untested assumptions about the in vivo behavior of epidermal stem cells prospectively isolated in vitro and pave the way for a safer approach to cutaneous gene therapy.