Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype

Abstract

Despite the central role of human epidermal stem cells in tissue homeostasis, wound repair, and neoplasia, remarkably little is known about these cells, largely due to the absence of molecular markers that distinguish them from other proliferative cells within the germinative͞basal layer. Epidermal stem cells can be distinguished from other cells in the basal layer by their quiescent nature in vivo and their greater overall proliferative capacity. In this study, we demonstrate enrichment and isolation of a subpopulation of basal epidermal cells from neonatal human foreskin based on cell surface phenotype, which satisfy these criteria. These putative stem cells are distinguished from other basal cells by their characteristic expression of high levels of the adhesion molecule ␣ 6 , a member of the integrin family (␣ 6 bri ), and low levels of a proliferation-associated cell surface marker recognized by recently described mAb 10G7 (10G7 dim ). We conclude that cells with the phenotype ␣ 6 bri 10G7 dim represent the epidermal stem cell population based on the demonstration that these cells (i) exhibit the greatest regenerative capacity of any basal cells, (ii) represent a minor subpopulation (Ϸ10%) of immature epidermal cells, which (iii) are quiescent at the time of isolation from the epidermis, as determined by cell cycle analysis.

Stem/Progenitor Cell-Like Properties of Desmoglein 3dim Cells in Primary and Immortalized Keratinocyte Lines

“…Cells with low levels of Dsg3 expression (Dsg3 dim ) exhibited increased CFE and proliferative potential relative to cells with a high level of Dsg3 expression (Dsg3 bri ) [30], a result consistent with the possibility that low Dsg3 levels may serve as a marker of the "stem/progenitor cell-like" phenotype. In this study, we demonstrate that (a) Dsg3 dim keratinocytes are comparable with ␣ 6 bri /CD71 dim stem cells [23,24] in their ability to exhibit high CFE; (b) they are capable of generating well-ordered hierarchical skin structure in vitro, including all the layers that exist in the native skin; and (c) these cells are small and more proliferative in our culture conditions and express enhanced levels of p63 at both the transcript and protein levels. We also have explored the stem/progenitor cell-like properties of two well-established immortalized human keratinocyte cell lines: HaCaT, a line that frequently is used as a paradigm of skin development in vitro [31][32][33], and NTERT, human keratinocytes expressing hTERT [34].…”

“…Putative stem cells isolated by such an approach have been shown to be multipotent and enriched for LRC [21,22]. A range of other cell surface phenotypes, such as high levels of ␣ 6 integrin and low levels of CD71 (transferrin receptor) [23,24] also have been used, in conjunction with CD34 expression, to isolate the bulge keratinocyte stem cells [25,26]. Apart from these cell surface markers, the p63 transcription factor, a p53 homolog essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their transit-amplifying progeny [27].…”

Ovol2 Suppresses Cell Cycling and Terminal Differentiation of Keratinocytes by Directly Repressing c-Myc and Notch1

“…These results suggest that both decreased cell cycle time and compromised proliferation potential of progenitor cells are critical for the observed growth phenotype of Ovol2-deficient culture. Indeed, we observed a remarkable decrease in the expression of ␣6 integrin, which when highly expressed, marks keratinocytes that possess long term proliferation potential and increased clonogenecity (43,44), in Ovol2 knockdown cells (Fig. 3D).…”

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

“…2I-L). It has been noted that stem cells isolated from other tissues such as the skin and hair follicle [43][44][45] have smaller cell sizes than their more differentiated progeny. The SP cell frequency in rat corneal epithelium (4.6%) was also much higher than in the limbus, as well as results reported for several tissues and organs [13,16,20,23], and the presence of corneal epithelial SP cells in rat eyes were contrary to our previous results for both human [23] and rabbit [24].…”