1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 25 Beisihuanxilu, Beijing 100080, Peoples Republic of China and 2 Graduate School of the Chinese Academy of Sciences, 19 Yu-quan Road, Beijing 100039, Peoples Republic of China
3 To whom correspondence should be addressed. e-mail: duane{at}panda.ioz.ac.cn
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Abstract |
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Key words:
CFE/epidermal stem cell/FACS/integrin 1/p63
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Introduction |
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Isolation and identification of putative human epidermal stem cells has been done previously. In earlier studies, it has been found that in vitro three types of colony, holoclone, paraclone and meroclone, could be formed because of the different amplification potentials of human keratinocytes (Barrandon and Green, 1987). Holoclones are thought to be derived from epidermal stem cells, while the other two types of clones are from TA cells. Although this assay is very useful to investigate the proliferative potential in vitro, the comparison between these cultures is hard to make. Keratinocytes with characteristics of stem cells can be isolated on the basis of high surface expression of
1 integrins and rapid adhesion to extracellular matrix proteins (Jones and Watt, 1993
). Stem cells can be isolated to >90% purity by fluorescence-activated cell sorting (FACS) using
1 integrin as a molecular marker. However, human epidermal stem cells expressed
2-fold higher levels of
1 integrin than TA cells, which make it difficult to reliably purify stem cells using FACS. Recently, p63 transcription factor, a p53 homologue, has been shown to be a secific marker for keratinocyte stem cells. It helps distinguish human keratinocyte stem cells from their TA progeny (Mills et al., 1999
; Pellegrini et al., 2001
).
-Catenin is a major structural component of adherens junctions and a downstream effector of the Wingless (Drosophila)/Wnt (vertebrate) signalling pathway (Wiechaus and Riggleman, 1987
; Ben-Zeev and Geiger, 1998
). The phosphorylated
-catenin, reflecting Wnt signalling activities, appears to be expressed at higher levels in cultured epidermal stem cells than TA cells (Zhu and Watt, 1999
).
Previous studies have shown that in the process of serial cultivation of human keratinocytes, the plating efficiency and culture lifetime were inversely related to the age of the skin donor (Rheinwald and Howard, 1975). However, integrin
1 expression does not change in abundance or distribution during skin development (Hertle et al., 1991
). Therefore, we have hypothesized that the fetal epidermis may contain more stem cells than the adult epidermis. To validate our hypothesis, we have isolated and characterized epidermal stem cells from human fetuses.
In this study, we have examined the expression of p63 in the human fetal skin using immunofluorescent histochemistry. The epidermal stem cells have been isolated from human fetal epidermis of different ages by FACS based on expression of integrin 1. The purified epidermal stem cells have been cultured in vitro and further characterized for expression of molecular markers, colony formation efficiency (CFE) and differentiation potential.
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Materials and methods |
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Isolation and cultivation of human fetal keratinocytes
The basal keratinocytes were isolated after digestion with 0.25% trypsin and 0.02% EDTA for overnight at 4°C, then cultured by using a mitomycin C-treated NIH3T3 cell as the feeder using the method as previously described (Jones and Watt, 1993). The medium was made up of three parts of Dulbeccos modified Eagles medium plus one part of Hams F-12 medium containing 10% fetal calf serum (Hyclone), 20 ng/ml epidermal growth factor (Sigma), 5 µg/ml transferrin (Sigma), 1.8x104 mol/l adenine (Amresco), 5 µg/ml insulin (Sigma), 1010 mol/l cholera toxin (Sigma) and 0.4 µg/ml hydrocortisone (Sigma). The cultures were grown in a humidified 5% CO2 atmosphere at 37°C and the medium changed every 2 days. Keratinocytes were passaged at 80% confluence after removing the feeder cells with 0.02% EDTA.
Antibodies
The final concentration of FITC-labelled anti-integrin 1 mouse monoclonal antibody (sc-9970 FITC; Santa Cruz) and isotype-matched negative control antibody (sc-2855; Santa Cruz) were used at 1 µg/ml in the FACS. The anti-p63 antibody (sc-8431; Santa Cruz), anti-involucrin antibody (sc-15223; Santa Cruz), anti-nestin antibody (MAB353; Chemicon) and anti-active-
-catenin antibody (05-665; Upstate) were used in the immunofluorescent staining at dilutions suggested by the suppliers.
Cell preparation for FACS
Newly confluent cultures at first passage were used for staining. Cells were harvested by removing the feeders first with 0.02% EDTA and then treating the keratinocyte with trypsinEDTA. After washing with 1% bovine serum albumin (BSA), the cells were then incubated in 1% BSA with the FITC-labelled anti-integrin 1 antibody for 1 h at 37°C. After washing with 1% BSA three times, cells were filtered with a cell mesh (70 µm). Immediately before analysis, PI (1 µg/ml) was added for viability gating. Cells were kept on ice until the flow cytometric procedure.
Flow cytometry
Cells were analysed on a BectonDickinson FACScan and sorted using a Cytomation Plus (Dako). For cell sorting, gates were set using forward light scatter (FSC) and side light scatter (SSC). A total f 50 000 events was acquired in list mode for each sample. The integrin fluorescence was detected on the FL1 channel (520560 nm band pass filter). PI was excited with 100 mW of UV radiation (351364 nm). Debris and cells positive for PI were gated out.
Electron microscopy
To observe the morphology of stem cells, the sorted cells were fixed with glutararaldehyde. Then the samples were prepared and treated according to standard procedures. Samples were observed under a TEM (H-7500; Hitachi) and results recorded.
Cultivation of sorted epidermal stem cells
Sorted epidermal stem cells have been cultured on a collagen type IV-coated (100 µg/ml) plate. After sorting, the epidermal stem cells were seeded into a 24-well plate at a density of 105 cells per well. The medium was as for those mentioned above and was changed every 2 days.
Determination of CFE
To determine the CFE of sorted populations, the sorted cells were seeded in 6-well plate at a density of 1000 cells/well. The plate was seeded with mitomycin C-treated feeders 24 h before use and the medium changed every 2 days. After culturing for 2 weeks, these cells were fixed with 4% formaldehyde, washed in PBS, and stained with 1% Rhodamine B (Sigma) (James and Howard, 1975). Colonies were then counted under a microscope. Only colonies >32 cells were scored. The CFE was designated as the number of colonies divided by the total number of seeded cells.
Immunocytochemistry
For the immunocytochemistry analysis of sorted cells, cells were air-dried onto glass slides, fixed with a mixture of methanol and acetone (1:1) for 10 min at room temperature, rinsed in PBS for three times, and incubated with an primary antibody at 4°C for overnight. The other steps were the same as described above for immunohistochemistry.
Suspension-induced terminal differentiation
Suspension-induced terminal differentiation was performed as previously described (Green, 1975). In brief, the sorted cells were placed in a medium containing 1.6% methyl cellulose (Sigma). After 24 h at 37°C, the cells were harvested and used for immunocytochemistry analysis.
Statistics
Results were presented as the average ± SD of at least three independent experiments. Statistical differences were evaluated by analysis with t-test; values of P < 0.05 were accepted as significant.
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Results |
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Discussion |
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There are at least two factors important for isolating epidermal stem cells. One is choosing specific surface molecular markers, and the other is separating basal keratinocyte population containing epidermal stem cells from the rest of the epidermis. In addition to integrin 1, integrin
6 has also been suggested to be a better stem cell marker (Li et al., 1998
). In fact, lack of specific molecular markers has been a major obstacle for stem cell research (Lavker and Sun, 2000
; Tani et al., 2000
). In the mouse, side population (SP) cells have been isolated as genuine epidermal stem cells using FACS and Hoechst staining (Dunwald et al., 2001
; Liang and Bickenbach, 2002
). But in humans, our study suggested there were no SP cells in the epidermis (Zhou JX, Jia LW, Liu WM, Liu S, Cao YJ, Duan EK unpublished data). This study has shown that integrin
1 can function as a reliable marker for isolating human epidermal stem cells.
We show that the sorted putative epidermal stem cells express integrin 1, p63 and phosphorylated
-catenin, but not nestin. Identification of human epidermal stem cells mainly depended on two parameters, molecular marker expression pattern and CFE determination (Lavker and Sun, 2000
; Watt, 2001
). In addition to integrin
1, p63 has served as an important marker for epidermal stem cells (Mills et al., 1999
; Pellegrini et al., 2001
). Active
-catenin has been shown to contribute to the high proliferative potential of keratinocytes with the characteristics of epidermal stem cells (Zhu and Watt, 1999
). Our sorted putative epidermal stem cells have been further verified by p63 and active
-catenin expression and high CFE potential. We also tried to investigate whether nestin, a widely distributed marker of adult stem cells (Lendahl et al., 1990
; Hunziker and Stein, 2000
), appeared in the sorted epidermal stem cells. The results suggested that it did not appear in our sorted epidermal stem cells. The sorted epidermal stem cells could proliferate in vitro although perpaps they are slow to enter a proliferative phase. However, it appears that the CFE of our sorted putative stem cells was lower than that previously described (Jones and Watt, 1993
; Janes et al., 2002
). This may imply that our sorted stem cells were less pure. It may also be due to sorting efficiency and/or difference in integrin
1 expression between fetal and adult epidermal cells. Finally, the cultured stem cells can be induced to generate differentiated cells, which show the expression of involucrin.
In conclusion, epidermal stem cells have been successfully isolated from human fetal epidermis using FACS and expanded in vitro. The isolated epidermal stem cell population will be extremely useful in the future for studying stem cell biology and the potential of generating functional differentiated cells other than epidermal cells.
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Acknowledgements |
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References |
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Submitted on November 24, 2003; accepted on January 8, 2004.
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