Like for other somatic tissues, isolation of a pure population of stem cells has been a primary goal in epidermal biology. We isolated discrete populations of freshly obtained human neonatal keratinocytes (HNKs) using previously untested candidate stem cell markers aldehyde dehydrogenase (ALDH) and CD44 as well as the previously studied combination of integrin α6 and CD71. An in vivo transplantation assay combined with limiting dilution analysis was used to quantify enrichment for long-term repopulating cells in the isolated populations. The ALDH(+) CD44(+) population was enriched 12.6-fold for long-term repopulating epidermal stem cells (EpiSCs) and the integrin α6(hi) CD71(lo) population was enriched 5.6-fold, over unfractionated cells. In addition to long-term repopulation, CD44(+) ALDH(+) keratinocytes exhibited other stem cell properties. CD44(+) ALDH(+) keratinocytes had self-renewal ability, demonstrated by increased numbers of cells expressing nuclear Bmi-1, serial transplantation of CD44(+) ALDH(+) cells, and holoclone formation in vitro. CD44(+) ALDH(+) cells were multipotent, producing greater numbers of hair follicle-like structures than CD44(-) ALDH(-) cells. Furthermore, 58% ± 7% of CD44(+) ALDH(+) cells exhibited label-retention. In vitro, CD44(+) ALDH(+) cells showed enhanced colony formation, in both keratinocyte and embryonic stem cell growth media. In summary, the CD44(+) ALDH(+) population exhibits stem cell properties including long-term epidermal regeneration, multipotency, label retention, and holoclone formation. This study shows that it is possible to quantify the relative number of EpiSCs in human keratinocyte populations using long-term repopulation as a functional test of stem cell nature. Future studies will combine isolation strategies as dictated by the results of quantitative transplantation assays, in order to achieve a nearly pure population of EpiSCs.
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