Cell isolation and culture
The experiments using keloid patient–derived cells were approved by the Medical and Ethics Committee of Nippon Medical School Hospital (B-2021-368). All patients signed informed consent prior to enrolling in this study. All patients in this study were Japanese. Keloid tissue was collected during keloid surgical treatment from 27 patients who were confirmed to have clinical evidence of keloids (Supplementary Table 1). No patients received chemotherapy, radiotherapy, or intralesional steroid treatment before surgery. Normal skin and scar tissue were obtained from four patients who underwent elective scar excision surgery. Keloids and normal skin and scars were diagnosed on the basis of their clinical appearance and pathology. Primary fibroblast cultures were established as previously described69. Briefly, the surgically resected keloid tissue and the normal skin tissue used only the dermis layer by removing the epidermis and fat. The dermal tissue was washed with PBS, cut into pieces approximately 1 mm in size, and cultured in Dulbecco’s modified eagle medium (DMEM) containing 4.5 mg/ml glucose (DMEM hi-glucose: FUJIFILM Wako Pure Chemicals, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS: ThermoFisher Scientific [Gibco]: Waltham, MA USA) and penicillin-streptomycin mixture (Nacalai Tesque, Kyoto, Japan). Keloid fibroblasts and normal fibroblasts were cultured in the same condition. When fibroblasts reached approximately 80% confluence, cells were passed at 1/3. Only low-passage cultures (passages 1 or 2) were used in this study. For keloid-derived sphere culture, cells were seeded in 6-well ultra-low attachment plates in DMEM/F-12 (3:1) (Gibco) supplemented with B-27 supplement (Gibco), 40 ng/ml basic FGF (bFGF, FUJIFILM Wako Pure Chemicals), and 20 ng/ml EGF (R&D Systems, Minneapolis, MN USA). Lenti-X 293T cells were purchased from Takara Bio (Kusatsu, Japan) and cultured in DMEM hi-glucose supplemented with 10% FBS.
shRNA, recombinant lentivirus production and infection, and stable cell lines
shRNAs, which generate siRNAs for target genes, were inserted into the pLKO.1 vector (Addgene, Watertown, MA USA). The target sequences are listed in Supplementary Table 2. Recombinant lentiviruses were produced in Lenti-X293T packaging cells by transient transfection with pLKO.1 plasmids and Lentiviral High Titer Packaging Mix (Takara Bio) using TransIT-Lenti transfection reagent (Mirus Bio, Madison, WI USA). Twenty-four hours after transfection, the medium was changed, and cells were incubated for an additional 24 h. Medium containing lentivirus was collected and debris was removed using a syringe filter (pore size 0.45 mm; CORNING, Corning, NY USA). The filtered culture media was supplemented with 8 µg/ml polybrene and used to infect cells. Stable shRNA-expressing cells were selected by puromycin (2.5 mg/ml: Sigma-Aldrich, Saint Louis, MO).
RNA isolation and qPCR
Total RNA was isolated using Nucleospin RNA (Takara Bio). RNA concentration and purity were assessed by a NanoDrop spectrophotometer (Cytiva, Marlborough, MA USA). An equal amount of total RNA was reverse transcribed by the PrimeScript RT reagent kit: Perfect Real Time (Takara Bio). cDNA was subjected to qPCR using LUNA real-time PCR Mastermix (New England Biolabs, Ipswich, MA USA) and qPCR probes (ThermoFisher Scientific) with the StepOnePlus Real-Time PCR System (ThermoFisher Scientific). The qPCR probes are listed in Supplementary Table 3. Each amplification reaction was performed in triplicate. The mean and S.D. of three threshold cycles were used to calculate the amount of transcript in the sample (StepOne software v2.2.3; ThermoFisher Scientific). Quantification of mRNA was represented in arbitrary units as the ratio of the sample quantity to the calibrator or to the mean values of control samples. All values were normalized to mRNA levels of human b-actin, the endogenous control. The Taqman probes used in this study are listed in Supplementary Table 4.
Microarray assay and pathway analysis
RNA isolation from normal fibroblasts, normal fibroblast–derived stem cells, keloid fibroblasts, and keloid fibroblast–derived stem cells was described above. Total RNA was isolated from three keloid patients and two donors. Microarray assay was outsourced to a contracted analysis service from Takara Bio. Briefly, RNA integrity was measured in the Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA USA), and samples with an RNA integrity number ≥ 1.6 were subjected to microarray analysis. Total RNA (100 ng) was used to generate ss-cDNA, which was hybridized on the Human Clariom S Assay gene chip (Affymetrix). To investigate pathways associated with differentially expressed genes, upregulated or downregulated genes with a fold change of >1.3 (p < 0.2) in KNS, KMS, or KCS compared with NS were analyzed by Ingenuity Pathway Analysis software (QIAGEN, Venlo, The Netherlands.).
In vitro drug treatment and quantification of viable cells
To evaluate the influence of SMO inhibitor on fibroblasts, 3×103 cells were seeded in each well of 96-well plates. When cells reached 100% confluence, the growth medium was changed to DMEM hi-glucose supplemented with 0.2% FBS, and vismodegib (Selleck, Houston, TX USA) was treated for 72 h. To evaluate the effect of vismodegib on fibroblast-derived stem cells, 1×104 cells were seeded in each well of 96-well ultra-low attachment plates, and vismodegib was treated for 7 days. The quantification of viable cells was performed using a CCK-8 staining kit (Dojindo, Kumamoto Japan) following the manufacturer’s instructions.
Immunohistochemistry
Tissues were fixed in 10% formaldehyde for 48 h and embedded in paraffin. Samples were sectioned (4 µm) and mounted onto glass slides. For visualizing collagen bundles, tissue slides were deparaffinized and subjected to hematoxylin and eosin (H&E) staining as described elsewhere70. For immunohistochemical analysis, deparaffinized tissue slides were subjected to antigen retrieval by the antibody manufacturers’ recommended method. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 30 min. Nonspecific binding sites in tissues were blocked with 1% bovine serum albumin (BSA). The tissue samples were incubated with the appropriate dilution of a primary antibody overnight at 4°C. The primary antibodies used in this study are listed in Supplementary Table 3. Biotin-conjugated secondary antibodies were incubated for 30 min at room temperature, and interaction of the antigen and the primary antibody was detected with the streptavidin-biotin-peroxidase staining kit (Histofine: Nichirei Biosciences, Japan) and chromogen 3,3′-diaminobenzidine (DAB). Nuclei were stained by hematoxylin. Negative controls included the omission of primary antibody and the substitution with nonimmune sera. Antibodies used in the immunohistochemical analysis are listed in Supplementary Table 3. Adobe Photoshop 2022 (Adobe, San Jose, CA USA) was used for image analysis.
Immunofluorescent staining
Keloid fibroblasts seeded on cover glass slips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were incubated with 1% BSA for 15 min at room temperature, followed by incubation with primary antibody for 30 min at room temperature. Texas red–conjugated anti-mouse IgG antibody (ThermoFisher Scientific [Invitrogen]) was incubated for 20 min at room temperature in the absence of light. Nuclei were stained with Hoechst33342 (Invitrogen), and the coverslip was mounted onto a ProLong glass slide (Invitrogen). Cells were visualized by BioZERO fluorescence microscopy (KEYENCE, Osaka Japan).
Keloid tissues were also subjected to immunofluorescence staining. Slices of frozen sections (4 mm-thick) were mounted on a slide glass and dried for 30 min at room temperature. The tissue was fixed in 4% paraformaldehyde for 10 min at room temperature and incubated with 1% BSA for 30 min at room temperature. The tissue was then incubated with anti-GLI1 and anti-OCT4 or anti-NANOG antibodies overnight at 4℃. The sections were incubated with Alexa 488–conjugated anti-mouse (for GLI1) and Alexa 546–conjugated anti-rabbit (for OCT and NANOG) secondary antibodies for 30 min at room temperature, avoiding light. Nuclei were stained with VECTASHILD® Antifade Mounting Medium with 4′,6′-diamidino-2-phenylindole (DAPI: Vector Laboratories, Burlingame, CA USA), and the tissue was covered with cover glass. Immunofluorescent images were visualized by XA70 fluorescence microscopy (EVIDENT [Olympus], Tokyo Japan). Antibodies used in the immunofluorescent staining are listed in Supplementary Table 3.
In vivo keloid fibroblast transplantation and Gene Set Enrichment Analysis (GSEA)
The animal experiment committee at the Nippon Medical School approved the animal experiments in this study (approval number: 29-045), and animal care was conducted by institutional guidelines. In vivo transplantation was performed as described elsewhere35. Briefly, 2×106 keloid fibroblasts from the marginal area (KMF) from three patients or normal fibroblasts from two donors were mixed with hydrogel including synthesized extracellular matrix (HyStem®-HP Cell Culture Scaffold Kit, Sigma-Ardrich) supplemented with recombinant human IL-6 (50 ng/ml: PeproTech, Cranbury, NJ USA). The mixtures were immediately injected subcutaneously into the dorsal surface of 6–8-week-old male nude mice (Japan CREA, Tokyo, Japan). KMF-transplanted mice were treated with vehicle or vismodegib by intraperitoneal injection once a day. Mice were killed three months after implantation, and the harvested transplant volume was evaluated. Transplant volume (mm3) was measured with calipers and was calculated as (length x width2)/2. RNA was isolated from the transplant for qPCR analysis and microarray assay. For GSEA of microarray assay data, G5:GO gene sets (The Molecular Signature Database [www.broadinstitute.org/gsea/msigdb]) were used for the enrichment of keloid pathogenesis using GSEA software V3.0 (Broad Institute, MIT).
Ex vivo keloid tissue culture
Ex vivo keloid tissue culture was performed as described elsewhere53. Keloid tissue culture media was composed of William’s E (WE) medium (Merck) supplemented with 10 mg/ml of recombinant human insulin (FUJIFILM Wako Pure Chemicals), 10 ng/ml of hydrocortisone (FUJIFILM Wako Pure Chemicals), 2 mM of L-glutamine (FUJIFILM Wako Pure Chemicals), and antibiotics. The keloid tissue biopsies were taken using a 4 mm diameter punch biopsy size and embedded in keloid tissue culture media supplemented with calf collagen matrix (3 mg/ml: KOKEN, Tokyo, Japan) in 24-well plates. Keloid tissue culture media was added to the culture plate to expose the surface of the epidermis to the air. For tissue maintenance, the media was changed every three days and supplemented with fresh media.
Statistics and reproducibility
The statistical significance of the difference between mean values was tested using a two-tailed Student’s t-test with unequal variance in Microsoft Excel. The significance threshold was set at p ≤ 0.05, except for microarray analysis. P-values are indicated in each figure. The representative immunofluorescent images in Figures 2d, f, Supplementary Figures 3b and c are from five independent experiments from five keloid patients (K8, K9, K10, K11, K12) and three independent experiments from three donors with normal dermis (N3, N4, N5). The representative immunohistochemical images in Figures 3a and 4f are from five independent experiments from five keloid patients (K1, K2, K3, K4, K9). The representative H&E staining image in Supplementary Firgure 7 of keloid fibroblasts transplants is from three keloid patient–derived fibroblasts (K12, K13, K18) and two donors with normal dermis–derived fibroblasts (N3, N4). The representative ex vivo histology images in Figure 7 are from four independent experiments from four keloid patients (K19, K20, K21, K22) per condition. The representative ex vivo histology images in Supplementary Figure 8 are from two independent experiments from keloid patients (K23, K24) per condition. The representative image in Supplementary Figure 2a is from five (POU5F1 [K2, K3, K4, K5 and K6] and NANOG [[K2, K3, K4, K5 and K6]] or six (SOX2 [K1, K2, K3, K4, K5 and K6] independent experiments. The representative image in Supplementary Figure 5b is from four (K1, K3, K7, K10) independent experiments. The representative image in Supplementary Figure 9c is from two (K22 and K23) independent experiments.