Ex vivo and in vitro analysis of epidermal progenitors using flow cytometry

Summary: In this study, we use flow cytometric analysis to characterize the expression patterns of CD90, a proposed marker of epidermal stem cells. We find that CD90+ cells in human epidermal samples are evenly split between keratinocyte and melanocyte lineages, suggesting this is a marker for both types of progenitors. Similarly, both melanocytes and keratinocytes dividing in culture also express CD90. While keratinocytes can maintain a stem-like phenotype <i>in vitro</i>, we find that all melanocytes begin to express the differentiation marker CD117 after the first passage. Growing melanocytes on collagen, in the presence of melanogenesis inhibitors, or co-cultured with keratinocytes, can all increase CD90 expression, yet none of these conditions are able to prevent the universal differentiation that occurs. Further, we find that all pre-passaged melanocytes have intermediate levels of both CD117 and melanosomes, but after the first passage a second population emerges with high expression of both. These CD117-high cells are significantly less likely to co-express CD90, highlighting loss of proliferative capacity. Though cells double positive for both CD90 and CD117 appear to drive population growth <i>in vitro</i>, we find these cells to be extremely rare <i>ex vivo</i>, suggesting they more likely represent a transitional state in the skin, rather than a proliferative pool as seen in culture. Lastly, we find that aging increases the percentage of CD90 positive epidermal cells, though we suspect this to be due to age-related thinning of the upper epidermis, as other basal cell markers are also overrepresented with age. Brook H. Abegaze^{1, 2}, T R. Parenteau³, Merisa Piper⁴, Ruby Ghadially^{1, 2}, Theodora Mauro^{1, 2} 1. Dermatology, University of California San Francisco, San Francisco, CA, United States. 2. Dermatology, San Francisco VA Health Care System, San Francisco, CA, United States. 3. Geriatrics, University of California San Francisco, San Francisco, CA, United States. 4. Plastic Surgery, University of California San Francisco, San Francisco, CA, United States. Pigmentation, Melanoma, and Melanoma Immune Surveillance