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Original Article |
1 Service de Génomique Fonctionnelle, CEA, Evry; Université d'Evry Val d'Essonne (EA 2541-Université/CEA, Evry
2 L'OREAL, Life Sciences Advanced Research, Centre C. Zviak, Clichy, France
3 UMR 217 CEA/CNRS, Fontenay aux Roses
4 Service de Génomique Fonctionnelle, CEA, Evry
* To whom correspondence should be addressed. E-mail: michele.martin{at}cea.fr.
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Abstract |
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The aim of the present study was to characterize human SP epidermal keratinocytes isolated from primary cell cultures. For that purpose, keratinocytes were isolated from normal adult breast skin samples and the Hoechst 33342 exclusion assay described for haematopoietic cells was adapted to keratinocytes. Three types of keratinocytes were studied: the side population (SP), the main population (MP) and the unsorted initial population. SP keratinocytes represented 0.16% of the total population. In short-term cultures, they exhibited an increased colony forming efficiency and produced more actively growing colonies than unsorted and MP keratinocytes. In long-term cultures, SP cells exhibited an extensive expansion potential, performing a mean of 44 population doublings over up to 12 successive passages after cell sorting. Moreover, SP keratinocytes were able in long-term cultures to form a pluristratified epidermis when seeded on a dermal substrate. Unsorted and MP keratinocytes promoted a reduced expansion: mean of 14 population doublings over 5 passages and 12 population doublings over 4 successive passages, respectively. To further characterize SP cells, cDNA microarrays were used to identify their molecular signature. Transcriptome profiling showed that 41 genes were differentially expressed in SP versus MP cells, with 37 up-regulated genes and only 4 down-regulated genes in SP cells. The majority of these genes were functionally related to the regulation of transcription and cell signalling. In conclusion, SP human keratinocytes isolated from primary cultures exhibited both short and long-term high proliferative potential, formed a pluristratified epidermis and were characterized by a specific gene expression profile.
Key Words. Side population, human keratinocytes, tissue culture, proliferative potential, organotypic 3D culture, microarray gene profiling
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