Human Side Population Keratinocytes Exhibit Long-Term Proliferative Potential and a Specific Gene Expression Profile and Can Form a Pluristratified Epidermis

Abstract

The aim of the present study was to characterize human side population (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 hematopoietic cells was adapted to keratinocytes. Three types of keratinocytes were studied: the 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 did unsorted and MP keratinocytes. In long-term cultures, SP cells exhibited an extensive expansion potential, performing a mean of 44 population doublings for up to 12 successive passages after cell sorting. Moreover, even in longterm cultures, SP keratinocytes were able to form a pluristratified epidermis when seeded on a dermal substrate. Unsorted and MP keratinocytes promoted a reduced expansion: mean values of 14 population doublings for five passages and 12 population doublings for four 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 (vs. MP) cells, with 37 upregulated genes and only four downregulated genes in SP cells. The majority of these genes were functionally related to the regulation of transcription and cell signaling. 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. STEM CELLS 2006;24:965-974

Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer

“…Similar to our observations, transcriptional profiling of SP cells from several non-malignant tissues showed that more genes were upregulated than downregulated and included genes associated with multi-drug resistance, regulation of transcription, cell signalling, Notch and Wnt pathways (Liadaki et al, 2005;Behbod et al, 2006;Larderet et al, 2006). Side population fraction cells examined in our studies have upregulated genes that are involved in pathways modulating stemness, including MYC, FGF1, OCT4, KLF4, NOTCH2 and WNT.…”

“…Overall, there were more upregulated stem cell genes in SP cells. These features are consistent with previous reports on enrichment in genes associated with growth and developmental pathways in SP cells from normal tissues (Behbod et al, 2006(Behbod et al, , 2006Larderet et al, 2006). In addition, several of the upregulated genes, including ABCG2, BMP1/2, FGF1, IGF1, MYC, SOX1/2, WNT1 and NOTCH2, have been recently associated with cancer stem cells and tumour-initiating cells in lung cancer and other tumour types, as well as with epithelial-tomesenchymal transition in tumours with poor prognosis (Fan et al, 2006;Moustakas and Heldin, 2007).…”

Niche regulation of corneal epithelial stem cells at the limbus

“…A similar finding was also noted in cultured epidermal keratinocytes [86]. These results suggest that clonal expansion of "quiescent" limbal SCs is less apt on 3T3 fibroblast feeder layers than "activated" limbal SCs, which generate sufficient numbers of TACs.…”

p63 induces key target genes required for epidermal morphogenesis

“…Initial reports suggested a role for p63 in epithelial stem cell maintenance (5,6). However, the finding that p63 is not enriched in epidermal stem cells challenges this notion (7)(8)(9)(10). In contrast, we previously demonstrated that TAp63 is required for the commitment to stratification during epidermal morphogenesis, a process that is mediated, in part, by induction of AP-2␥ (11,12).…”