Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

Abstract

The enrichment and identification of human epidermal stem cells (EpSCs) are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rapidly adherent keratinocytes into three populations that range in size from 5-7 μm (population A), to 7-9 μm (population B), to ≥9 μm (population C) in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of β1-integrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency (CFE). When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to β1-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

Isolation, culture and identification of epidermal stem cells from newborn mouse skin

“…The isolation, identification, and enrichment of human EPS cells are of paramount importance. Isolation and identification of putative stem cells from human skin has been done previously in 1993 (Germain et al 1993;Jones and Watt 1993), 1996 (Jones 1996), 1998(Li et al 1998(Papini et al 2003), 2004Park et al 2004;Zhou et al 2004a(Dong et al 2005Toma et al 2005;Yano et al 2005), 2006(Spichkina et al 2006, and 2008 Li et al 2008). The cultured mouse putative EPS cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy (Fusenig and Worst 1975;Mackenzie et al 1989;Tani et al 2000;Hakkinen et al 2001;Zhou et al 2004b;Yano et al 2005).…”

In vitro keratinocyte expansion for cell transplantation therapy is associated with differentiation and loss of basal layer derived progenitor population

“…Changes in keratin composition and cell size increase occur also during the physiological in vivo differentiation process and maturation of stratified epithelia (Izumi et al, 2007;Li et al, 2008;Barrandon and Green, 1985). The increase in cell size is described as an indicator of the differentiation process (Barrandon and Green, 1985), especially the change between the nucleus and cytoplasm ratio (Li et al, 2008;Izumi et al, 2007).…”

“…Location Description Literature α6 integrin (α6int) Stratum basale Transmembrane protein that leads cell-cell and cell-ECM connexions Kim et al (2004), Li et al (2008Li et al ( , 1998, Redvers et al (2006), Ruetze et al (2010), Kloepper et al (2008), and Fortunel et al (2010).…”

p63 and its isoforms as markers of rat oral mucosa epidermal stem cells in vitro

“…After 24 h, the primary medium was changed to epidermal stem cell medium, a mixture of supernatant of NIH3T3 cells and keratinocyte-serum-free medium (K-SFM) (Gibco, CA, USA) supplemented with 5 ng ml À1 epidermal growth factor (EGF) and 50 mg ml À1 bovine pituitary extract (BPE). Once the cell clones emerged, they were removed via ethylenediamine tetraacetic acid (EDTA) digestion and cultured in collagen IV coated six well plates at 48C overnight at a density of 1000 cells per cm 2 . Cells not attached after 10 min were discarded to separate the rapidly attaching stem cells from the slower adhering keratinocytes.…”

“…However, enrichment should be based either on cell size 2 or require identifying the appropriate cell surface markers. It has been reported that K14, K15, K19, CD90, integrin-b1, and integrin-a6 are epidermal stem cell markers.…”