Stem cell activity of human side population (SP) and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay

¹ Dermatology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
² Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

^* To whom correspondence should be addressed. E-mail: jonvogel{at}mail.nih.gov.

	Abstract

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP-KC) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow-derived side population (SP) hematopoietic stem cells, have been identified in human skin but their potential to function as keratinocyte stem cells (KSC) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of 1 and 6 integrins, and are distinct from SP-KC which express low levels of integrins, may be enriched for KSC based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP-KC and integrin-bright keratinocytes, we could not document their superior long-term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP-KC and integrin-bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different MHC Class I antigens is grafted onto immunocompromised mice, and the intrinsic MHC Class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP-KC showed little competitive expansion in vivo and were not enriched for KSC. In contrast, keratinocytes expressing elevated levels of 6 integrin and low levels of CD71 (6-bright/CD71-dim) expanded over 200-fold during the 33-week in vivo study. These results definitively demonstrate that human 6-bright/CD71-dim keratinocytes are enriched with KSC whereas SP-KC are not.

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