Niche-Driven Overgrowth of KPC Figure 2. In vivo transplantation of dermal stem cells. A, Size and volume of transplants generated from SKPs and KPCs using Gelfoam as a carrier for 8 weeks (mean6SEM). ** P,0.01. B, Immunohistological studies of KPC transplant tissues using a specific antibody for human mitochondria (purple color) and type I collagen (brown color), respectively. Scale bars, 50 mm. C, H & E histological stain of transplants. Scale bars, 50 mm. D, In vivo bone regeneration by SKPs or KPCs using hydroxyapatite/tricalcium phosphate (HA/TCP) as carrier. Scale bars, 50 mm. E, Serial transplantation of KPCs. KPCs (26106) with hydrogel were injected subcutaneously into nude mice. After 4 weeks, the transplanted were harvested and the recovered cells were expanded in vitro and re-transplanted into mice for another 4 weeks. The transplant tissues were harvested for H & E staining or immunohistochemical (IHC) staining with a specific antibody for human mitochondria (purple color). Scale bars, 50 mm. The results are representative of five independent experiments. doi:10.1371/journal.pone.0007798.g002 Elevated IL-17 and IL-6 coordinately contribute to the unique inflammatory niche of KPCs To further explore the concept of niche regulated keloid fibroproliferative growth we next attempted to define the distinct ‘‘pathological’’ niche of keloid by screening a panel of candidate inflammatory cytokines utilizing fresh keloid tissues from different donors. Using a focused human cytokine array we found that the levels of several mediators, including growth-regulated IL-6, oncogene alpha (GROa), IL-1b, MIP-1d, RANTES, stem cell factor (SCF), TGF-b1, TNF-a, angiogenin II, vascular endothelial growth factor (VEGF) and platelet-derived growth factor beta homodimer (PDGF-BB), were increased by more than 2-folds in keloid tissues as compared to matched normal skin tissues (Figure 3A; Table S3). Particularly, an elevated level of IL-6, a critical cytokine of the TH2 cells, and its receptor IL-6R, were detected in all keloid tissues screened and further confirmed by immunohistochemical studies (Figure 3C; Figure S4A) and Western blot analysis (Figure 3F; Figure S4B). Both IL-6 and IL-6R were expressed in SKPs and KPCs, with a relatively higher level in KPCs as compared to the matched SKPs, as determined by Western blot, RT-PCR and flow cytometry (Figures 3G and 3H; Figure S4C). Another important inflammatory cytokine of the TH17 cells [38–41], IL-17, was abundantly expressed in keloid tissues as demonstrated by ELISA (Figure 3B) and further confirmed by immunohistochemical staining (Figure 3C) and Western blot analysis (Figure 3E). The increased expression of IL-17 in keloid tissues was discernible around T-cell receptor CD3-positive cells as determined by dual-color immunofluorescence staining (Figure 3D). IL-17 has been reported to be produced almost exclusively by activated T cells, but its receptor is widely expressed in various cell types [42]. Herein, we observed an elevated expression of IL-17R in KPCs as compared to SKPs (Figure 3I; Figure S4C). However, the expression of IL-17 was undetectable in both KPCs and SKPs (data not shown). Taken together, these findings suggest that IL-6 and IL-17 are abundant components of the unique inflammatory niche of keloid and may contribute to the regulation of self-renewal and differentiation of keloid stem cells. IL-17 is involved in the regulation of hTERT expression in KPCs via coordinating IL-6 paracrine loop IL-6 plays an important role in regulating self-renewal and differentiation of stem cells [21,22,25], as well as tumor growth [25–27]. Our data showed that IL-6 stimulated BrdU incorpora- tion in both KPCs and SKPs (Figure 4A). Exposure of KPCs and SKPs to exogenous IL-6 resulted in a dose-dependent increase in Oct-4 expression at both mRNA and protein levels (Figures 4B and 4C). In parallel studies, we found that incubation with IL-6 enhanced telomerase (hTERT) expression and enzyme activities in both KPCs and SKPs (Figures 4D, 4E and 4F). The basal level of hTERT was always higher in KPCs as compared to SKPs. Since KPCs constitutively expressed a higher level of IL-6 and IL-6R than SKPs (Figures 3G and 3H; Figure S4C), we next tested whether the intrinsically secreted IL-6 might contribute to the elevated telomerase expression and activity in KPCs. Our data showed that treatment with IL-6 neutralizing antibodies sup- pressed hTERT expression in both KPCs and SKPs (Figures 4G and 4H) in a dose-dependent manner. However, IL-6 showed no obvious effects on SSEA-4 expression in both SKPs and KPCs as determined by flow cytometry (Figure S5A). Taken together, these data suggest that IL-6 plays a critical role in the regulation of self- renewal and proliferation of KPCs by regulating essential functional genes such as hTERT and Oct-4. PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7798