Functional Characterization of Quiescent Keratinocyte Stem Cells and Their Progeny Reveals a Hierarchical Organization in Human Skin Epidermis

Abstract

Although homeostatic renewal of human skin epidermis is achieved by the combined activity of quiescent stem cells (SCs) and their actively cycling progeny, whether these two populations are equipotent in their capacity to regenerate tissue has not been determined in biological assays that mimic lifelong renewal. Using fluorescence activated cell separation strategy validated previously by us, human epidermis was fractionated into three distinct subsets: that is, α 6briCD71dim, α 6briCD71bri, and α 6dim with characteristics of keratinocyte stem, transient amplifying, and early differentiating cells, respectively. The global gene expression profile of these fractions was determined by microarray, confirming that the α 6briCD71dim subset was quiescent, the α 6briCD71bri was actively cycling, and the α 6dim subset expressed markers of differentiation. More importantly, functional evaluation of these populations in an in vivo model for tissue reconstitution at limiting cell dilutions revealed that the quiescent α 6briCD71dim fraction was the most potent proliferative and tissue regenerative population of the epidermis, capable of long‐term (LT) epidermal renewal from as little as 100 cells for up to 10 weeks. In contrast, the cycling α 6briCD71bri fraction was the first to initiate tissue reconstitution, although this was not sustained in the LT, while differentiating α 6dim cells possessed the lowest demonstrable tissue regenerative capacity. Our data suggest that in human skin, the epidermal proliferative compartment is not composed of equipotent cells, but rather is organized in a functionally hierarchical manner with the most potent quiescent SCs at its apex (i.e., α 6briCD71dim) followed by cycling progenitors (i.e., α 6briCD71bri) and finally early differentiating keratinocytes (i.e., α 6dim). STEM CELLS 2011;29:1256–1268

The effect of isolation and culture methods on epithelial stem cell populations and their progeny—toward an improved cell expansion protocol for clinical application

“…Retaining populations of stem cells and their actively cycling progeny in culture is beneficial for rapid population expansion and is most compatible with tissue regeneration on re-introduction into the wound. Importantly, our results confirmed a distinct lack of differentiating keratinocytes, which make the smallest contribution to tissue regeneration on implantation [17]. This result was confirmed by use of qRT-PCR.…”

“…Schluter et al defined stem cells as being at the apex of cell proliferation and tissue regeneration, followed by their transiently amplifying daughter cells [17]. In contrast, differentiating keratinocytes exhibit the lowest tissue regenerative potential [17].…”

“…The optimal length of time required for incubation was initially investigated according to the size of the SC population released (Figure 1). Flow cytometric analysis of keratinocytes labeled for integrin a6 (Ia6) and CD71 was used to identify stem cells as Ia 6 bri CD71 dim , whereas TA keratinocytes were Ia 6 bri CD71 bri [17,35] (Figure 1A,B). Shorter incubation times in trypsin (5e10 min) yielded significantly greater numbers of SCs than incubation times of 20 and 30 min ( Figure 1C).…”

“…Whether transplanted in suspension or on a carrier, keratinocytes used clinically must maintain good re-implantation potential-that is, engraft permanently, produce progeny and construct an effective epidermis [16]. It is thought that keratinocyte stem cells (SCs) and their actively cycling transiently amplifying (TA) progeny harbor significant long-term tissue regenerative capacities, whereas differentiating cells do not contribute to the regeneration of a functional epidermis [16,17]. Thus, retaining the SC and TA phenotypes during keratinocyte culture is most likely to result in the successful contribution of implanted cells to the development of a robust epidermis.…”

Cell motion predicts human epidermal stemness

“…S1 A). The level of ATP-binding cassette subfamily G member 2, Delta1, melanoma chondroitin sulfate proteoglycan, and leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1) can also be used to identify human keratinocyte stem cells (Jones and Watt, 1993;Jones et al, 1995;Li et al, 1998;Lowell et al, 2000;Legg et al, 2003;Terunuma et al, 2003;Jensen and Watt, 2006;Schlüter et al, 2011). However, none of these cell surface proteins is stem cell specific, and the number of clonogenic keratinocytes in populations enriched by cell sorting based on these cell surface markers is always smaller than that of stem cells simply selected based on size with a Pasteur pipet (up to 28%; Barrandon and Green, 1985).…”