Animals C57BL/6 mice were purchased from Jackson Lab. Immuno- compromised mice (bg-nu/nu-xid) were purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN) and were used for in vivo transplantation experiments under the approved animal protocol of University of Southern California (USC #10874). Cell isolation and culture Keloid and matched peripheral normal skin tissues were obtained from the same patient donor and were used for isolation of stem cells. At least 5 sets of keloid derived progenitor cells (KPCs) with appropriately matched skin derived progenitor cells (SKPs) were derived from 5 unrelated patients [(3 African- Americans (2 females, 1 male), 2 Hispanics (1 female, 1 male)], and were used in all experiments described. SKPs were also derived from normal skin tissues from other donors. Tissues were treated aseptically, cut into 5–6 mm pieces, and incubated in sterile phosphate-buffered saline solution (PBS) containing 3 mg/mL dispase (Sigma) overnight at 4uC. The epidermis was manually stripped off; the dermal portion was minced into 1-mm3 pieces, digested in sterile PBS containing 4 mg/mL collagenase I (Worthington Biochemical Corporation, Lakewood, NJ) for 2 hours at 37uC [54,57], filtered through a 70 mm cell strainer (Falcon, Franklin Lakes, NJ), and the isolated cells were cultured under two different conditions. For sphere-forming culture [54,55,56], cells were seeded on uncoated dishes with growth medium, Dulbecco’s minimum essential medium with low glucose (DMEM-LG)/F12 (3:1) (Invitrogen, Carlsbad, CA) supplemented with 40 ng/ml fibroblast growth factor (FGF-2), 20 ng/ml epidermal growth factor (EGF) (Chemicon, Billerica, MA), B27, 1 mg/ml fungizone and 100 U/ml penicillin/100 mg/ml strepto- mycin (Invitrogen). After 2,4 weeks, spheres were formed and further sub-cultured in 60 mm ultra-low culture dishes (Corning Inc., Corning, NY). For cell attachment or MSC culture, cells were cultured as described previously with some modifications [53]. Briefly, resuspended cells were plated on non-treated 10-cm Petri dishes (VWR Scientific Products, Willard, OH) with minimum essential medium (MEM: Invitrogen) containing 10% fetal bovine serum (FBS: Clontech Laboratories, Inc., Mountain View, CA), 100 U/ml penicillin, 100 mg/ml streptomycin (Invi- trogen), 2 mM L-glutamine, 100 mM non-essential amino acid (NEAA), and 550 mM 2-mercaptoethanol (2-ME; Sigma-Aldrich), and cultured at 37uC in a humidified tissue-culture incubator with 5% CO2 and 95% O2. The confluent cells were passaged with 0.05% trypsin containing 1 mM EDTA, and continuously subcultured in the complete growth medium. Colony forming unit fibroblasts (CFU-F) assay The CFU-F assay was performed as previously described [58,59]. Cell aggregates containing more than 50 cells were counted as colonies using a dissecting microscope. The CFU-F assay was repeated in 5 independent experiments. Single cell cloning Precursor cells isolated from keloid tissues and matched peripheral normal skin samples of different donors were seeded in non-coated tissue culture 96-well plates (Falcon) at a concentration of 2 cells/ml (200 ml/well, at least 4 plates/donor). The plates were screened for presence of single cell colony while wells contained more than two colonies were excluded from further analysis. Wells containing a single cell were allowed to reach confluency, transferred to 24-well dishes, and further expanded in the growth medium [57]. Niche-Driven Overgrowth of KPC Population doubling assay Clonal dermal precursor cells at each passage (P2, P5, P10 and P20) were seeded at 1.06103 cells in 35-mm dishes in MSC medium for each period (0, 2, 4, 6, 8, 10 days). Cells were suspended with 0.05% trypsin-EDTA and cell number was determined by hemacytometer. Population doubling time (PDT) was calculated with the formula, PDT = (t-t0)*lg2/lg(N/N0) (N0 and N represent the cell numbers at time t0 and t, respectively). Meanwhile, the accumulated population doublings were deter- mined and calculated according to the standard 3T3 protocol as described previously [60]. Multipotent differentiation We tested the in vitro multi-differentiation capabilities of dermal precursor cells including osteogenesis, adipogenesis and neural cell differentiation as described in Methods S1. Stimulation of cells with IL-6 or IL-17 KPCs and SKPs at 70,80% confluence were serum-starved (medium containing 1% FBS) for 24 hours followed by exposure to different concentrations of IL-6 or IL-17 (R & D Systems). Under certain conditions, to clarify the role of IL-17/IL6 axis in the regulation of KPC functions, the serum-starved cells were pretreated with different concentrations of specific neutralizing antibodies for human IL-6 or IL-17 or an isotype-matched control IgG (R & D Systems) for 1 hour followed by stimulation with IL-6 or IL-17 for 24 hours. Then whole cell lysates or total RNA were extracted and subjected to Western blot and RT-PCR analysis, respectively. Cell proliferation assay The proliferation of dermal precursor cells was assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation using a BrdU staining kit (Invitrogen) as previously described [55]. To quantify cell proliferation capacity, ten representative images were used to enumerate BrdU-positive cells and quantitated as a percentage of BrdU-positive cells over total nucleated cells. Flow cytometric analysis Approximately 56105 cells were incubated with phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibodies specific for human CD34, CD45, CD105, CD146, CD29, CD73, CD90, and Stro-1, or incubated with purified mouse monoclonal antibodies specific for human IL- 6 receptor (IL-6R) and IL-17 receptor (IL-17R) followed by incubation with FITC-conjugated secondary antibodies (Table S1). The labeled cells were subjected to EpicsH XL-MCL Beckman Coulter flow cytometer, and analyzed using the Expo32 program (USC Clinical Pathology Reference Lab). Cell samples incubated in the absence of primary antibody or with an isotype- matched control IgGs (Southern Biotech, Birmingham, AL) were used as negative controls [58,59]. Reverse transcription–polymerase chain reaction (RT- PCR) and quantitative real-time PCR (qPCR) Total RNA was isolated from cultured cells using TRIZOLH Reagent (Invitrogen). RT-PCR was carried out using the One-step RT-PCR Kit (QIAGEN, Valencia, CA), whereby the number of amplification cycles and the amplifying condition for individual target genes were determined to be in the linear range. To confirm RT-PCR results in some experiments, qPCR was performed using the iScript one-step RT-PCR kit with SYBR Green (BioRad, Hercules, CA) according to the manufacturer’s instructions. As an PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7798