Using our established keloid-like animal models we will be able to further investigate the interplay between stem cells and their immediate niche. In summary, we have successfully identified multipotent precursor cells from keloid scars and provided evidence supporting IL-17/IL-6 axis as an essential component of the unique keloid niche that provides extra proliferation-promoting signals to resident stem cells. Based on our findings, we postulate that the inflammatory stimuli, i.e., inflammatory infiltrates and enriched cytokines, induced by wounding, surgical injury, or infection, may produce a persistent state of chronic inflammation at the wound site. The increased IL-6, a major component of the inflammatory niche, in conjunction with TGFb1, may trigger the differentiation of na¨ıve T cells into TH17 cells, which further stimulate IL-6 secretion via IL-17, thus creating an enriched pro-inflammatory cytokine milieu. The functionally altered niche can promote the resident stem cells to acquire a benign tumor-like stem cell phenotype characterized by increased cell proliferation and differentiation, therefore directly drives the benign tumor growth as seen in keloids (Figure S6). Findings from this study have not only substantiated our current knowledge regarding the potential role of adult stem cells in pathologic diseases, specifically skin fibrosis, and other fibro-proliferative disorders, but most impor- tantly, have provided a promising niche-related disease model, to further explore the intricate interactions between stem cells and their functional niche components, and ultimately lead to the development of an animal model for keloid fibrosis. Finally, this study should open a new avenue for stem cell research on benign tumor and lead to the rational design and development of innovative methods for prevention and treatment of niche-related diseases by specifically modulating or targeting the unique niche microenvironment of adult stem cells. Supporting Information Methods S1 Found at: doi:10.1371/journal.pone.0007798.s001 (0.05 DOC) Table S1 Antibodies for immunostaining and neutralization Found at: doi:10.1371/journal.pone.0007798.s002 (0.09 MB PDF) Table S2 RT-PCR primer sequences Found at: doi:10.1371/journal.pone.0007798.s003 (0.06 PDF) Table S3 Quantification of cytokine antibody array Found at: doi:10.1371/journal.pone.0007798.s004 (0.05 PDF) MB MB MB Table S4 Transplantation with a range of number of dermal stem cells Found at: doi:10.1371/journal.pone.0007798.s005 (0.01 MB PDF) Figure S1 Isolation of precursor cells from keloid tissues. (A) Keloid scar displays benign tumor phenotype in terms of growth and recurrence. (B) H&E histological stain of keloid tissues and the matched peripheral normal skins. PD: papillary dermis; RD: reticular dermis. Scale bars, 50 mm. (C) Semi-quantification of immunohistochemical staining of Oct-4 and SSEA-4 in keloid and the matched normal skin (Fig. 1A) as described in Materials & Methods. **P,0.01. (D) Colony formation of stem cells derived from keloid (KPCs) and normal skin (SKPs). (E) Subcloning and culture of mesenchymal stem cells from keloids (KPCs) in a-MEM medium supplemented with 10% FBS, 1 x NEAA (non-essential Niche-Driven Overgrowth of KPC amino acid) and antibiotics. Scale bars, 100 mm. (F) Determination of doubling time of KPCs and SKPs as described in Materials and Methods (mean6SEM). (G) Karyotyping of the SKP and KPC clones at passage 10. The results are representative of at least five independent experiments. Found at: doi:10.1371/journal.pone.0007798.s006 (7.14 MB TIF) Figure S2 Multipotent differentiation of keloid derived precur- sor cells (KPCs). (A–C) Adipogenic differentiation of SKP or KPC as determined by Oil Red O staining (A and B) and RT-PCR analysis of specific adipocyte genes (C). (D–F) Osteogenic differentiation as determined by Alizarin Red S staining (D and E) and RT-PCR analysis of osteocalcin gene (F). Human bone marrow mesenchymal stem cells (hBMMSCs) were used as positive controls whereas KPCs culturing under normal growth medium were served as non-induction control. Scale bars, 50 mm. Data are representative of at least five independent experiments using KPCs and the matched SKPs from 5 different patient donors (mean6SEM). * P,0.05; ns, no significance. Found at: doi:10.1371/journal.pone.0007798.s007 (5.20 MB TIF) Figure S3 Sphere-colony formation of KPCs. (A) Subcloning and expansion of sphere-colonies derived from keloids in DMEM- LG/F12 (3:1) supplemented with 40 ng/mL FGF-2, 20 ng/mL EGF, B27 and antibiotics. Scale bars, 100 mm. (B and C) Expression of stem cell markers and BrdU incorporation by keloid-derived sphere colonies (K1,K3) as determined by immunofluorescence staining (B) and RT-PCR analysis (C). Scale bars, 50 mm. (D) Multipotent differentiation of keloid-derived sphere colonies into different lineages of neural cells, adipocytes, and osteocytes as determined by immunofluorescence staining with specific neural cell markers, Oil Red O and von Kossa staining. Scale bars, 50 mm. The results are representative of at lease five independent experiments. Found at: doi:10.1371/journal.pone.0007798.s008 (4.37 MB TIF) Figure S4 Expression of IL-6 receptor (IL-6R) in keloids (K1, K2) and matched normal skins (N1, N2). (A) Paraffin-embedded sections of keloid and the matched normal skin were immuno- stained with a specific antibody for human IL-6R or an isotype- matched control IgG. Scale bars, 50 mm. (B) Western blot analysis of IL-6R in tissue lysates. (C) Flow cytometric analysis of IL-6R and IL-17R expression in cultured SKPs or KPCs. The results are representative of five independent experiments. Found at: doi:10.1371/journal.pone.0007798.s009 (1.72 MB TIF) Figure S5 Effect of IL-6 on the expression of SSEA-4 and BrdU incorporation in SKPs and KPCs. (A) Cells were stimulated with 20 ng/ml IL-6 for 24 h followed by immunostained with antibodies for SSEA-4 and FITC-conjugated secondary antibody and analyzed by flow cytometry. (B and C) Effects of IL-6 and IL- 17 neutralizing antibodies on cell viability and proliferation in SKPs and KPCs. Cells were treated for 24 h with 5 mg/ml of neutralizing antibodies for human IL-6, or IL-17, or both in the absence of IL-6 and IL-17, whereby an isotype-matched normal mice antibody (mIgG) was used as negative controls. Cell viability and proliferation were determined by MTT (B) and BrdU incorporation assay (C), respectively. The results are representative of at least five independent experiments (mean6SEM). #, no significant difference; * P,0.05, as compared with non-treatment control. Found at: doi:10.1371/journal.pone.0007798.s010 (0.45 MB DOC) Figure S6 Schemed inflammatory niche-driven benign tumor growth model. Under the chronic inflammatory microenviron- ment, keloid-derived precursor cells (KPCs) are persistently PLoS ONE | www.plosone.org 14 November 2009 | Volume 4 | Issue 11 | e7798