An In Vivo Competitive Repopulation Assay System to Evaluate Human Keratinocyte Stem Cells  Atsushi Terunuma, Melissa B. Shaya, Mark C. Udey, Jonathan C. Vogel  Journal of Investigative Dermatology  Volume 123, Issue 5, Pages 993-995 (November 2004) DOI: 10.1111/j.0022-202X.2004.23456.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The in vivo assay system to compare the relative abundance of repopulating cells in two populations of epidermal cells. Human epidermal cells were prepared from six pieces each of HLA-A2-positive and -negative neonatal foreskins by enzymatic treatments (Pfutzner et al, 1999). The equivalent numbers of A2-positive and -negative cells were mixed, and 600,000 cells were seeded onto a dermal equivalent prepared by established methods to make human skin equivalents (HSE;Garlick and Taichman, 1994; a). HSE showed histology of close resemblance to the normal human skin (b; E: epidermis; D: dermis). HSE were grafted on the backs of immunocompromised mice 3 d after seeding epidermal cells. The grafted HSE (pigmented patch on panel c) were chased for 5–26 wk and harvested by wide excision containing the surrounding mouse skin. An epidermal cell suspension was prepared by the enzymatic treatments and stained with anti-HLA-A, B, C (FITC-conjugate; BD Biosciences Pharmingen, San Diego, California), anti-HLA-A2 (clone BB7.2; BD Biosciences Pharmingen) and 7-AAD (BD Biosciences Pharmingen) to identify living cells. Goat anti-mouse IgG2b conjugated with AlexaFluor 647 (Molecular Probes, Eugene, Oregon) was used as a secondary antibody for BB7.2. Cells were analyzed using FACSCalibur and CellQuest software (BD Biosciences Immunocytometry Systems, San Jose, CA). Living human cells gated with a blue box (d) were evaluated for the percentages of HLA-A2-positive and -negative cells (e). Journal of Investigative Dermatology 2004 123, 993-995DOI: (10.1111/j.0022-202X.2004.23456.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The system stably maintained the 1:1 ratio between two populations of cells that should contain the equivalent numbers of repopulating cells. The percentages of HLA-A2-positive cells in the HSE, started with 47%, remained constant on the backs of immunocompromised mice (Swiss nude and scid-beige: Taconic, Germantown, NY; NOD-scid: The Jackson Laboratory, Bar Harbor, Maine) for 5–26 wk (n=2–3 at each time point in each strain). Averages (±SEM) in one of two independent sets of experiments with similar results are shown. Journal of Investigative Dermatology 2004 123, 993-995DOI: (10.1111/j.0022-202X.2004.23456.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions