internal control, levels of b-actin were determined in parallel with the target genes. All primers (Table S2) were synthesized at the Core Facility at Norris Comprehensive Cancer Center, USC. Western blotting We performed electrophoresis of protein extracts prepared from cultured cells or tissue lysates followed by immunoblotting as previously described [57]. Blots were incubated with primary antibodies at a dilution of 1:1000,1:2000 followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (PIERCE, Rockford, IL), and visualized by the enhanced chemiluminescence light method (PIERCE). For standardization, blots were re-probed with mouse antibody to human b-actin. Cytokine antibody array To analyze the cytokine expression profile of tissue lysates we utilized the RayBio Human Cytokine Antibody Array 3 (RayBiotech, Inc., Norcross, GA), which allows the detection of 42 cytokines, chemokines and growth factors in one experiment following manufacturer’s protocol. Telomerase activity and telomeric length assay The telomerase activity was measured using the telomere repeat amplification protocol (TRAP) TeloTAGGG telomerase PCR ELISA kit (Roche Diagnostics, Indianapolis, IN). The telomerase- mediated elongated product was detected by hybridization to digoxigenin-labeled probes and enzyme activity quantified by photometric enzyme immunoassay. Enzyme-linked immunoassay (ELISA) The level of IL-6 and IL-17 in tissue and cell lysates was detected using human IL-6 ELISA Ready-SET-Go (eBioscience, San Diego, CA) and human or mouse IL-17 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN), respectively, following the manufacturers’ instructions. In vivo transplantation Transplantation studies were carried out using KPCs and matched SKPs derived from five different donors, and each transplantation was done in triplicates (n = 3). Approximately 2.06106 stem cells mixed with 40 mg of hydroxyapatite/ tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Inc., Warsaw, IN), or Gelfoam (3 mm63 mm62 mm, Pharmacia, Piscataway, NJ), were subcutaneously transplanted into the dorsal surface of 8–10-week-old female immunocompromised mice as previously described [55,56]. Serial transplantation was described in Methods S. For the hydrogel carrier transplant, we mixed 2.06106 cells with HeprasilTM (CMHA-S, thiol-modified hya- luronic acid with thiol-modified heparin) and Gelin-STM (GTN- DTPH, thiol-modified gelatin) (1:1) with or without IL-6, and then combined with ExtralinkTM (PEGDA, polyethylene glycol diacry- late) (4:1) (Glycosan Biosystems, Salt Lake City, UT). The immobilized heparin in HeprasilTM mimics the heparan sulfate proteoglycans of the extracellular matrix with increased avidity for growth factors by forming non-covalent binding to the polymer network, prevents proteolysis, and maintains their bioactive state with a slow release over several weeks (Glycosan Biosystems) [61,62]. The mixtures were immediately injected subcutaneously into the dorsal surface of 8–10-week-old female immunocompro- mised mice. To further investigate the role of IL-6 in the initiation of KPC- generated transplants, KPCs mixed with hydrogel with or without human IL-6 neutralizing antibody (10 mg/ml) were injected Niche-Driven Overgrowth of KPC subcutaneously into immunocompromised mice for 8 weeks. On the other hand, to investigate the role of IL-6 in the maintenance of KPC-generated transplants, KPCs mixed with hydrogel were injected subcutaneously into immunocompromised mice for 4 weeks, followed by intra-lesional injection of IL-6 neutralizing antibody twice a week (5 mg/time) for another 5 weeks. In all cases, an isotype-matched normal mice IgG was used as negative control. Adaptation of activated CD4+CD252 T-lymphocytes in immunocompromised mice Spleens were harvested from C57BL/6 mice for isolation of CD4+CD252 T-lymphocytes using MidiMACS separator (Milte- nyi Biotec) and mouse CD4+CD25+ T lymphocyte isolation kit (Miltenyi Biotec) following manufacturer’s instruction. The purity of the CD4+CD252 T cells was .95%. The cells were seeded at 16106/well on 24-well multi-plates and cultured in RPMI-1640 medium containing 10% FBS, 50 mM 2ME, 10 mM HEPES, 1 mM sodium pyruvate, 1% NEAA, 2 mM L-glutamine, 100 U/ ml penicillin and 100 mg/ml streptomycin followed by stimulation with 5 mg/ml plate-bounded anti-CD3 antibody (BD Biosciences) and 2 mg/ml soluble anti-CD28 (BD Biosciences). After three days, activated cells were collected and washed with normal saline [63]. 16106/mouse of the activated CD4+CD252 T lymphocytes were injected into immunocompromised mice through tail vein two days before KPCs mixed with hydrogel with or without IL-6 were transplanted subcutaneously. In parallel, transplants in mice without infusion of T cells were used as controls. Histology and immunohistochemistry Keloid and matched peripheral normal skin tissues, normal skin and normal scar tissues from both African-American and Caucasian patient donors, and transplant samples were fixed with 10% formalin in PBS or embedded in O.C.T. compound (Sakura, Tokyo, Japan) and snap frozen in liquid nitrogen. Procedures for immunohistochemical studies and electron microscopy were described in Methods S1. Immunofluorescence studies 4% paraformaldehyde-fixed cultured cells and sections of normal and keloid tissue samples were immunolabeled with specific primary antibodies followed by FITC- and/or PE- conjugated secondary antibodies (BD Biosciences). After the nuclei were stained with 49, 6-diamidino-2-phenylindole (DAPI) the samples were observed under a confocal microscope. Isotype- matched control antibodies (Invitrogen) were used as negative controls. Positive signals in at least 5 random fields were visualized and counted. Statistical analysis All data are expressed as mean6SEM from at least five independent experiments. Differences between experimental and control groups were analyzed by two-tailed unpaired Student’s t-test using SPSS. P-values less than 0.05 were considered statistically significant. Results Keloid contains benign tumor-like stem cells Aside from the characteristic histological features of abundant ECM stroma, specifically large collagen bundles (Figure S1B), hyper-cellular and inflammatory states, the keloid tissue harbors an elevated level of Octamer-4 (Oct-4) and stage specific PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7798