A, Size and volume of KPC transplants (mean±SEM). Activated CD4⁺CD25⁻ T lymphocytes were infused at 1×10⁶/mouse through the tail vein two days before KPC transplants. KPCs were mixed with hydrogel with or without IL-6 and transplanted subcutaneously for 8 weeks. * P<0.05; ** P<0.01. B, Dual-color immunostaining for mouse CD4 (red) and IL-17 (green) using paraffin-embedded sections of KPC transplants. Scale bars, 50 µm. (C) Tissue lysates were prepared from the transplant tissues, and IL-17 expression was analyzed by ELISA (mean±SEM). * P<0.05, ** P<0.01. The results are representative of five independent experiments using KPCs and the matched SKPs from different patient donors.

A, IL-17 stimulated IL-6 secretion in SKPs and KPCs as determined by ELISA analysis (mean±SEM). ** P<0.01; ns, no significance. B, IL-17 stimulated the expression of hTERT and Oct-4 in SKPs and KPCs as determined by Western blot analysis. C, Blocking IL-6, but not IL-17 attenuated the basal expression of hTERT and Oct-4 in SKPs and KPCs. Cells were treated with 5 µg/mL of neutralizing antibody for either IL-6 (IL-6 Ab), or IL-17 (IL-17Ab), or both for 24 hours and expression of hTERT and Oct-4 was determined by Western blot analysis. D, IL-17-induced upregulation of hTERT and Oct-4 expression in SKPs and KPCs was significantly attenuated by treatment with neutralizing antibodies for either IL-17, or IL-6, or both. After serum-starved for 24 hours, cells were pretreated with neutralizing antibodies, followed by incubation with 20 ng/mL of IL-17. The expression of hTERT and Oct-4 was determined by Western blot analysis. E, IL-6-induced upregulation of hTERT and Oct-4 expression in SKPs and KPCs was attenuated only by IL-6Ab, not by IL-17Ab. Following serum-starvation for 24 hours, cells were pretreated with neutralizing antibodies and incubated with 20 ng/mL of IL-6. Expression of hTERT and Oct-4 was determined by Western blot analysis. An isotype-matched normal mouse IgG (mIgG) was used as negative controls. The results are representative of at least five independent experiments using KPCs and the matched SKPs from different patient donors (n = 5).

A, Size and volume of transplants generated from SKPs and KPCs using Gelfoam as a carrier for 8 weeks (mean±SEM). ** 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 µm. C, H & E histological stain of transplants. Scale bars, 50 µm. D, In vivo bone regeneration by SKPs or KPCs using hydroxyapatite/tricalcium phosphate (HA/TCP) as carrier. Scale bars, 50 µm. E, Serial transplantation of KPCs. KPCs (2×10⁶) 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 µm. The results are representative of five independent experiments.

A, Cytokine expression profiling of keloid and matched normal skin tissue lysates by human cytokine antibody array. B, Determination of IL-17 levels in keloid and normal skin tissue lysates by ELISA (mean±SEM). * P<0.05. C, Immunohistochemical studies of IL-6 and IL-17 expression in keloid and matched normal skin tissues. Scale bars, 50 µm. D, Confocal immunofluorescence studies show co-localization of IL-17 around CD3⁺ cells as determined by dual-color staining. Scale bars, 100 µm. E and F, Western analysis of IL-17 (E) and IL-6 expression (F) in keloid tissues and matched normal skins. G and H, Expression of IL-6 and IL-6 receptor (IL-6R) in precursor cells derived from different keloid samples (K1, K2) and matched normal skins (N1, N2) as determined by Western blot (G) and RT-PCR analyses (H). I, Expression of IL-17 receptor (IL-17R) in precursor cells derived from different keloid samples (K1, K2) and matched normal skins (N1, N2) as determined by western blot and RT-PCR analyses. Data are representative of at least five independent experiments using KPCs and matched SKPs from different patient donors (n = 5).

A, KPCs and SKPs were cultured in 1% FBS for 24 hours followed by exposure to different concentrations of IL-6, and BrdU incorporation in KPCs and SKPs was determined (mean±SEM). ** P<0.01. B, Expression of Oct-4 mRNA and protein were determined by Western blotting (WB) and RT-PCR, respectively. C, Immunofluorescence studies of Oct-4 expression in SKP and KPC after incubated with 20 ng/ml IL-6 for 24 hours. Scale bars, 20 µm. D, Expression of hTERT mRNA and protein were determined by Western blotting (WB) and RT-PCR, respectively. E, Immunofluorescence studies of hTERT expression in SKP and KPC following incubation with 20 ng/ml IL-6 for 24 hours. Scale bars, 20 µm. F, Telomerase enzyme activity of SKPs and KPCs in response to IL-6 as determined by TeloTAGGG Telomerase PCR ELISA. G and H, Treatment with neutralizing antibody for IL-6 (IL-6Ab) decreased the basal level of hTERT as determined by Western blotting (G) and TeloTAGGG Telomerase PCR ELISA (H). An isotype-matched normal mice IgG (mIgG) was used as negative control (mean±SEM). * P<0.05; ** P<0.01; *** P<0.001; ns, no significance. The results are representative of at least five independent experiments using KPCs and the matched SKPs from different patient donors (n = 5).

A, Frozen sections of keloid tissues and their matched peripheral normal skins were immunostained with specific antibodies for human Oct-4 and SSEA-4. Scale bars, 50 µm. B, Expression of Oct-4 and SSEA-4 in keloids (K1, K2) and matched normal skins (N1, N2) were determined by Western blot analysis and scanning densitometer. C, Colony formation analysis of cells derived from keloids and normal skin (mean±SEM). * P<0.05; ** P<0.01. D, Cell population doubling numbers were determined in single cell colony-derived stem cells from keloids (KPCs) and matched peripheral normal skins (SKPs) by standard 3T3 cell culture protocol (mean±SEM). E, Flow cytometric analysis of cell surface markers of KPCs and SKPs. F, RT-PCR (upper panel) and qPCR (lower panel) analysis of stem cell genes of KPCs (K1, K2) and SKPs (N1, N2). G and H, Analysis of telomerase activity using TeloTAGGG telomerase PCR ELISA kit (mean±SEM). * P<0.05; ** P<0.01; *** P<0.001. Data are representative of at least five independent experiments using specimens obtained from different patient donors with matched normal controls (n = 5).

A, KPCs or SKPs (2×10⁶) were mixed with hydrogel incorporated with or without IL-6 and subcutaneously injected into immunocompromised mice for 8 weeks. Size and volume of transplants generated from KPCs and SKPs were measured (mean±SEM). Scale bars, 1 mm. ** P<0.01; *** P<0.001. B, Histological analysis of KPC and SKP transplants by hematoxylin and eosin (H & E) staining. Scale bars, 50 µm. C, Immunohistochemical studies (IHC) of transplants of KPCs and SKPs using antibody specific for human mitochondria (purple color) and type I collagen (brown color) or an isotype-matched control IgG (IgG isotype), and then counterstained with hematoxylin (nuclei, blue). Scale bars, 50 µm. D, Increased synthesis and secretion of collagen by IL-6 in KPCs versus SKPs generated transplants as demonstrated by electron microscopy (EM). N, nucleus; C, cytoplasm; IC, intracellular collagen; EC, extracellular collagen. Scale bars, 500 nm. E, Expression of Oct-4 and hTERT in KPC and SKP transplants with or without IL-6 as determined by Western blot analysis. F, Expression of Oct-4 and hTERT in KPC and SKP transplants with or without IL-6 as determined by immunohistochemical studies. Scale bars, 50 µm. G, Telomerase enzyme activity of SKPs and KPCs transplants with or without IL-6 as determined by TeloTAGGG Telomerase PCR ELISA (mean±SEM). * P<0.05; ** P<0.01. H, Increased expression of proliferating cell nuclear antigen (PCNA) in transplants of SKPs and KPCs in the presence of IL-6 as determined by immunohistochemical studies. Scale bars, 50 µm. I and J, Treatment with neutralizing antibodies against IL-6 inhibited in vivo formation and growth maintenance of KPC transplants. KPCs were mixed with hydrogel with or without IL-6 neutralizing antibody (IL-6Ab, 10 µg/ml) and injected subcutaneously into immunocompromised mice. KPC transplants were evaluated after 8 weeks (I). To test the inhibitory effect of IL-6 neutralizing antibody on growth of KPC-derived transplant, IL-6 neutralizing antibody was locally injected into KPC-transplants twice a week (5 µg/time) for another 4 weeks (J). An isotype-matched normal mice IgG (IgG isotype) was used as negative controls. The results are representative of five independent experiments using KPCs and the matched SKPs from different patient donors (mean±SEM). ** P<0.01; ns, no significance.