Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

Abstract

The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-alpha(6) integrin and anti-CD71 antibodies. RA cells express high levels of alpha(6) integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed alpha(6) integrin and beta(1) integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.

Localization of bromodeoxyuridine-incorporating, p63- and p75NGFR- expressing cells in the human gingival epithelium

“…Recently, it was reported that p63 was involved in maintenance of epidermal stem cells and stratification of the epidermis (21). The fact that cells rapidly adhering to collagen type IV successfully perform epidermal reconstruction (22) proved that epidermal stem cells were located in the basal layer. The p63-expression in a wide area covering the basal to spinous layers observed in the present study may explain the two functional roles of this protein-sustaining stem cells in the basal layer and stratification of the epidermis in the spinous layer.…”

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].…”

Human oral cancer cells with increasing tumorigenic abilities exhibit higher effective membrane capacitance

“…cell-like properties we have chosen here a functional method based on the cellular adherence to collagen IV. This method has been reported to successfully enrich for cells with stem cell-like properties in various epithelial tissues, such as normal human skin 13,30 . This method has been shown to robustly sort cell populations with different self-renewing capacities 13 and different potential for regenerating normal skin 30 and OSCC 5a .…”

“…This method has been reported to successfully enrich for cells with stem cell-like properties in various epithelial tissues, such as normal human skin 13,30 . This method has been shown to robustly sort cell populations with different self-renewing capacities 13 and different potential for regenerating normal skin 30 and OSCC 5a . We have chosen to use this method for CSC enrichment since it avoids possible cell surface modifications that might occur due to antibody binding, and thus prevents any alterations of their original electrophysiological properties that would occur after marker-based cell sorting.…”