Abstract
The basal layer of human interfollicular epidermis is thought to contain a minor compartment of quiescent or slowly cycling epithelial stem cells. These primitive keratinocytes give rise to the progenitors, which are the proliferating keratinocytes and which can be defined as early to late progenitors, according to their differentiation status. Because of the intrinsic heterogeneity of the basal layer, the development of new methods suitable for functional analysis of basal keratinocytes directly isolated from skin samples is greatly needed. We describe here a new method that allows a rapid and multiparallel deposition of single keratinocytes into 96-well plates, using flow cytometry. The first step of the process allows the clonal analysis of the growth potential of freshly isolated epithelial cells in primary cultures. In a second step, various techniques of functional characterization can be performed on the progeny of the cloned cell, including the generation of reconstructed epidermis, colony assays, and secondary cloning. In a third step, a long-term characterization of the progeny of the cloned keratinocytes can be performed, either by successive subclonings or mass expansion cultures.
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Kaur P. (2006). Interfollicular epidermal stem cells: identification, challenges, potential. J Invest Dermatol 126, 1450–1458.
Blanpain C., Horsley V., and Fuchs E. (2007). Epithelial stem cells: turning over new leaves. Cell 128, 445–458.
Watt F.M., Lo Celso C., and Silva-Vargas V. (2006). Epidermal stem cells: an update. Curr Opin Genet Dev 16, 518–524.
Barrandon Y. and Green H. (1987). Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84, 2302–2306.
Jones P.H., Harper S., and Watt F.M. (1995). Stem cell patterning and fate in human epidermis. Cell 80, 83–93.
Li A., Simmons P.J., and Kaur P. (1998). Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 95, 3902–3907.
Fortunel N.O., Hatzfeld J.A., Rosemary P.A., Ferraris C., Monier M.N., Haydont V., Longuet J., Brethon B., Lim B., Castiel I., Schmidt R., and Hatzfeld A. (2003). Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-beta1 concentrations. J Cell Sci 116, 4043–4052.
Larderet G., Fortunel N.O., Vaigot P., Cegalerba M., Maltère P., Zobiri O., Gidrol X., Waksman G., and Martin M.T. (2006). Human side population keratinocytes exhibit long-term proliferative potential and a specific gene expression profile and can form a pluristratified epidermis. Stem Cells 24, 965–974.
Rochat A., Kobayashi K., and Barrandon Y. (1994). Location of stem cells of human hair follicles by clonal analysis. Cell 76, 1063–1073.
Di Iorio, E., Barbaro V., Ruzza A., Ponzin D., Pellegrini G., De Luca M. (2005). Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration. Proc Natl Acad Sci USA 102, 9523–9528.
Ronfard V., Rives J.M., Neveux Y., Carsin H. and Barrandon Y. (2000). Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix. Transplantation 70, 1588–1598.
Mavilio, F., Pellegrini G., Ferrari S., Di Nunzio F., Di Iorio E., Recchia A., Maruggi G., Ferrari G., Provasi E., Bonini C., Capurro S., Conti A., Magnoni C., Giannetti A., and De Luca M. (2006). Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 12, 1397–1402.
Regnier M., Schweizer J., Michel S., Bailly C., and Prunieras M. (1986). Expression of high molecular weight (67 K) keratin in human keratinocytes cultured on dead de-epidermized dermis. Exp Cell Res 165, 63–72.
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Fortunel, N.O., Vaigot, P., Cadio, E., Martin, M.T. (2010). Functional Investigations of Keratinocyte Stem Cells and Progenitors at a Single-Cell Level Using Multiparallel Clonal Microcultures. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 585. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-380-0_2
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DOI: https://doi.org/10.1007/978-1-60761-380-0_2
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-379-4
Online ISBN: 978-1-60761-380-0
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