Niche-Driven Overgrowth of KPC embryonic antigen-4 (SSEA-4) positive signals randomly dispersed in the reticular dermis as compared to matched normal skin of the same donor (Figure 1A; Figure S1C). The relatively increased expression of two major markers for embryonic and mesenchymal stem cells [54,64,65], Oct-4 and SSEA-4, in keloid tissues as compared to matched normal skin was further confirmed by Western blot (Figure 1B), suggesting that keloid may harbor more postnatal stem cells than normal skin. Next, we isolated a population of adult stem cells from keloid dermis, named keloid derived precursor cells (KPCs), from 5 independent donors and assess their colony formation and proliferation. Normal skin-derived precursor cells, termed SKPs [54], were derived from matched normal skin of the same donor. Colony formation was observed on day 3 (Figure S1D) in 6,8% of KPCs whereas only 2,4% in normal skin precursor cells (SKPs) (Figure 1C), suggesting higher colony-forming capabilities of the tumor-like stem cells in keloids. Single colony formation was further confirmed using diluting seeding density at 2 cells/ml (200 ml per well) (Figure S1E). At early passages (less than passage 5), both KPCs and SKPs exhibited similar doubling time and cumulative population doublings; however, at a later passage, KPCs showed a relatively increased growth rate compared to Figure 1. Identification of dermal derived precursor cells from keloid tissues. A, Frozen sections of keloid tissues and their matched peripheral normal skins were immunostained with specific antibodies for human Oct-4 and SSEA-4. Scale bars, 50 mm. B, Expression of Oct-4 and SSEA-4 in keloids (K1, K2) and matched normal skins (N1, N2) were determined by Western blot analysis and scanning densitometer. C, Colony formation analysis of cells derived from keloids and normal skin (mean6SEM). * P,0.05; ** P,0.01. D, Cell population doubling numbers were determined in single cell colony-derived stem cells from keloids (KPCs) and matched peripheral normal skins (SKPs) by standard 3T3 cell culture protocol (mean6SEM). E, Flow cytometric analysis of cell surface markers of KPCs and SKPs. F, RT-PCR (upper panel) and qPCR (lower panel) analysis of stem cell genes of KPCs (K1, K2) and SKPs (N1, N2). G and H, Analysis of telomerase activity using TeloTAGGG telomerase PCR ELISA kit (mean6SEM). * P,0.05; ** P,0.01; *** P,0.001. Data are representative of at least five independent experiments using specimens obtained from different patient donors with matched normal controls (n = 5). doi:10.1371/journal.pone.0007798.g001 PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7798