In Vivo Transplantation Assay at Limiting Dilution to Identify the Intrinsic Tissue Reconstitutive C

This protocol describes an in vivo grafting approach to investigate the intrinsic long-term tissue reconstitutive capabilities of interfollicular keratinocyte stem cells and their committed progeny—the committed progenitors or transit amplifying and early differentiating cells. This approach utilizes the previously described skin reconstitution rat trachea assay, which has been adapted to investigate differences between stem cells and their more committed progeny. Limiting dilutions of each cell fraction reveal that both stem cells and their progeny are capable of skin tissue reconstitution, but at a limiting dilution of 100 cells per rat trachea only the keratinocyte stem cells maintain the reconstituted skin for as long as 10 weeks. The thorough analysis of reconstituted tissues using skin specific proliferation and stage-specific differentiation markers is also described because it provides qualitative distinction of the epithelial sheets reconstituted by stem cells and their progeny.