Figure 3.

Schematic representation of ex vivo gene therapy and clonal analysis. (A) Scheme of combined cell and gene therapy for LAMB3-dependent junctional epidermolysis bullosa (JEB), as reported in Hirsch et al. (2017). Clonogenic keratinocytes, consisting of stem cells (rhodamine red) and transient amplyfing cells (light pink), were cultivated from a skin biopsy, transduced with γRVs, containing LAMB3 and used to prepare transgenic epidermal sheets, which were then transplanted on surgically prepared skin lesions. Of note, clonal tracing performed on cultures initiated from the restored skin (Hirsch et al. 2017) has shown that long-term skin regeneration (right part of the panel) is sustained only by long-lived, self-renewing stem cells detected as holoclone-forming cells (see panel B), as indicated by the rhodamine red color. (B) Clonal analysis. Keratinocytes are inoculated (0.5 cells per well) onto 96-multiwell plates containing irradiated 3T3-J2 cells. After 7 days of cultivation, single clones are transferred to two dishes and cultivated. One dish (one-quarter of the clone) is stained 12 days later for the classification of clonal type, which is determined by the percentage of aborted colonies formed by the progeny of the founding cell. The clone is scored as a holoclone when 0%–5% of colonies are terminal. When 95%–100% of colonies are terminal (or when no colonies formed), the clone is classified as a paraclone. When the number of terminal colonies is between 5% and 95%, the clone is classified as a meroclone. The second dish (three-quarters of the clone) is used for further analyses.