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First published online 2 September 2003
doi: 10.1242/jcs.00701

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Desmosomal proteins, including desmoglein 3, serve as novel negative markers for epidermal stem cell-containing population of keratinocytes

¹ Department of Cell and Molecular Pathology, St John's Institute of Dermatology
² Richard Dimbleby Department of Cancer Research/Cancer Research UK Laboratory, Guy's, King's and St Thomas' School of Medicine, St Thomas' Hospital, London, UK
³ FACS Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, UK

View larger version (90K): [in a new window]   Fig. 1. Diversity of Dp expression in human palm keratinocytes both in vivo and in vitro. (A) Confocal microscopic image of human palm skin labelled for Dp (green) and Dsgs (red). Both proteins show a low level of expression at the tips of the epidermal ridge (arrow head) but abundant immunoreactivity at the side of the ridge or above the dermal papillae (arrows), as well as in the suprabasal layers. (B) In situ hybridisation showing Dp mRNA expression in palm skin, and (C) the negative control with the randomer. Note clusters of small rounded cells located at the tips of the palm ridge express little or low level of Dp mRNA compared with the suprabasal cells. (D) Immunofluorescent staining for Dp in cultured palm keratinocytes. Diversity of Dp expression is seen and a few small rounded cells show a very low level of Dp expression, which is largely restricted to the cell boundary (arrow). Larger cells show Dp staining throughout the cells. (E) In situ hybridisation showing Dp mRNA expression in cultured palm keratinocytes. Note heterogeneous expression of Dp mRNA in vitro and little or no expression in clusters of small cells (arrows). Scale bar: (A-D) 20 µm, (E) 50 µm.

View larger version (40K): [in a new window]   Fig. 2. Human adult palm keratinocyte populations, separated by rapid and slow adhesion to type IV collagen, show different desmosomal protein expression. Two cell populations, separated by rapid (<20 minutes, lane 1) or slow (>20 minutes-24 hours, lane 2) adhesion to type IV collagen, were analysed by western blotting for the desmosomal proteins Dp, Dsgs, Dscs, Pg and Pkp1. Non-separated cells were used as the control (lane 3). The graph shows densitometric analysis of eight blots from five experiments on Dp expression (mean±s.e.m.). Note Dp expression in the slowly adhering cell population is about fourfold that of the rapidly adhering cell population. Control cells show an intermediate level of expression as expected. Similar expression profiles were observed for the other desmosomal proteins. ß-actin demonstrates equal loading of the protein samples.

View larger version (54K): [in a new window]   Fig. 3. The Dsg3dim population of adult palm keratinocytes shows a higher CFE than the Dsg3bri population. (A) Dot plot showing the FSC and SSC of a suspension of cultured keratinocytes (a). Each dot represents one cell, and both FSC and SSC are measured in arbitrary units on a linear scale. Cells were sorted into two groups: fluorescence dim (Dsg3dim: 20% of the cells with the least Dsg3) and fluorescence bright (Dsg3bri: 40% of the cells with the highest Dsg3) populations (b). Alexa Fluor 488-conjugated IgG was used as the negative control (unfilled). (B) The sorted cells were plated onto the J2 feeder cells at 400 cells per well, and grown for 11 days in culture. Colonies were stained for keratins and visualised with anti-mouse HRP. More colonies grew in the Dsg3dim population (a) than in the Dsg3bri population (b). (C) Colony density analysis, as indicated by total colony area per dish (i.e. percentage of total area of dish) between Dsg3dim and Dsg3bri populations (mean±s.e.m. of triplicate samples). There is a higher total colony area in the Dsg3dim, than in the Dsg3bri, population and by 11 days the total colony area per dish in the Dsg3dim population is significantly larger (*P<0.05), nearly twofold that of Dsg3bri. (D) CFE (ratio of colony number to plated cell number) at 11 days (mean±s.e.m. of triplicate). A slightly higher CFE is seen in Dsg3dim than in the Dsg3bri population. (E) Mean colony area (size) of Dsg3dim and Dsg3bri populations at three time points. The colony area increases with time and by 11 days the mean colony area in both Dsg3dim and Dsg3bri is significantly larger than that at 7 days or 9 days (*P<0.05). In particular that of Dsg3dim is twofold that of Dsg3bri by 11 days (average 1.4 mm2 vs 0.6 mm2, **P<0.05).

View larger version (62K): [in a new window]   Fig. 4. Dsg3dim population of palm keratinocytes survived better than Dsg3bri population in long-term culture. (A) Phase-contrast images (a,b; arrows delineate margin of colony) of different clone types in Dsg3dim and Dsg3bri at passage 2. Large colonies are observed in the Dsg3dim (a) but not the Dsg3bri population, in which almost all the colonies are abortive (b). (B) Assessment of the frequency of abortive colonies in Dsg3dim and Dsg3bri at passage 2. Of 96 colonies in Dsg3dim only 59 are abortive colonies, however, 32 out of 33 are abortive colonies in Dsg3bri (P<0.05). Scale bar: (in A) 50 µm.

View larger version (53K): [in a new window]   Fig. 5. Sorting keratinocytes for a ß1bri/Dsg3dim population further enhanced the enrichment of colony forming cells. (A) Keratinocytes dual labelled for ß1 integrin and Dsg3 were sorted into two groups: ß1bri/Dsg3dim (11.5% of the cells, with the highest ß1 and least Dsg3) and ß1bri/Dsg3bri (5.39% of the cells, with both the highest ß1 and Dsg3) populations (a). (b,c) FSC and SSC plot of ß1bri/Dsg3dim and ß1bri/Dsg3bri populations, respectively. Note that 85% of cells in ß1bri/Dsg3dim (b) but only 40% of cells in ß1bri/Dsg3bri (c) have low SSC characteristics. Relatively more cells in c show high FSC and SSC characteristics. (B) Sorted sole (thick skin; a) and breast (thin skin; b) keratinocytes (Kc) were plated onto the feeders at 1000 or 500 cells per well, respectively, and grown for 11 days. Colonies were stained for keratins and visualised using anti-mouse HRP. There are significantly more colonies in ß1bri/Dsg3dim than ß1bri/Dsg3bri wells of both cell types. (C) Quantitative colony analyses include colony density (total colony area per dish) and CFE (ratio of colony number to plated cell number) (left graph), colony size (mean colony area) and colony number (mean colony number per dish) (right graph). There is a 4- or 5-fold increase in colony density, and a 7- or 3-fold increase in CFE in ß1bri/Dsg3dim compared with ß1bri/Dsg3bri in either thick or thin skin keratinocytes, respectively (*P<0.05). Significantly more colonies are seen in ß1bri/Dsg3dim rather than ß1bri/Dsg3bri population (*P<0.05) and larger colonies show in thin skin Kc (*P<0.05).

View larger version (13K): [in a new window]   Fig. 6. Keratinocytes in Dsg3dim orß1bri/Dsg3dim populations have a greater overall proliferative potential than cells in the Dsg3bri or ß1bri/Dsg3bri populations. (A) Passage number and (B) duration of cell culture of different keratinocyte fractions in six independent experiments. The age of the skin donor was between 15 and 50 years old. Cells used for FACS were all from passage 1 following initial cultivation from the tiny biopsies. At each passage, cells were plated at the same density for each fraction. Note both the passage number and culture duration show significant differences (*P<0.05), and cells in Dsg3dim or ß1bri/Dsg3dim populations are capable of expanding more passages and surviving for a longer periods in culture than cells in the Dsg3bri or ß1bri/Dsg3bri populations.

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