1356 Characterization of human epidermal stem cells in situ using multiplex immunofluorescent analysis identifies a quiescent KRT5+/MECP2+ subpopulation

Abstract

The nature of human epidermal stem cells is still poorly defined. Several markers are have been used to identify populations that include the stem cells such as ITGB1, p63, or KRT14. However, these and other markers are relatively broadly expressed within the epidermis. Better methods to define the human epidermal stem cells in situ within the tissue are required to better understand the many processes that rely on proper stem cell function including normal tissue homeostasis, wound healing, inflammation and cancer. To overcome the limitations of individual markers to identify epidermal stem cells, we are using an approach that allows us to interrogate several stem cell markers simultaneously in situ on tissue sections. This allows us to detect relationships between cells in the basal cell layer of the epidermis that have been previously unnoticed and can evaluate stem cell marker expression in relationship e.g. to proliferation. This multiplex approach allows us to stain for up to six markers sequentially, image and compare all six markers and define their relationship at high resolution. For example, a comparison of KRT15 and ITGB1, two markers associated with epidermal stem cells, shows that KRT15 demarcates a quiescent cell population while ITGB1+ cell populations also contain proliferating basal cells. Furthermore, novel stem cell markers such as MECP2 and XPC are co-expressed in these quiescent KRT15 positive cells indicating a protected, quiescent stem cell population within the basal cell layer that shows similarities to the stem cell layer of the oral mucosa or esophagus. The stem cells of human oral mucosa and the human epidermis are MECP2+/XPC+/KRT15+ basal keratinocytes and in a state of deep quiescence indicated by the absence of any proliferation marker including key cell cycle markers such as RB1. Therefore, populations sorted using ITGB1 or KRT14 as markers represent heterogeneous populations of which only a subpopulation represent cells in a deep quiescent state. Our data suggest that a careful description of the co-expression pattern of stem cell markers can allow novel insights into epidermal stem cell biology that cannot be defined in mouse models or sorted cells.