Niche-Driven Overgrowth of KPC Figure 6. In vivo effect of IL-6-linked hydrogel on growth of transplanted KPCs and SKPs. A, KPCs or SKPs (26106) 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 (mean6SEM). 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 mm. 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 mm. 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 mm. G, Telomerase enzyme activity of SKPs and KPCs transplants with or without IL-6 as determined by TeloTAGGG Telomerase PCR ELISA (mean6SEM). * 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 mm. 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 mg/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 mg/ 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 (mean6SEM). ** P,0.01; ns, no significance. doi:10.1371/journal.pone.0007798.g006 inflammatory niche of keloid, plays a critical role in the regulation of self-renewal and differentiation of keloid stem cells. To explore whether IL-6 played a role in the initiation of KPC- generated transplants, KPCs mixed with hydrogel with or without human IL-6 neutralizing antibody were injected subcutaneously into immunocompromised mice whereas isotype-matched anti- body (mouse IgG) was used as negative control. Our results indicated that treatment with IL-6 neutralizing antibody signifi- cantly suppressed the in vivo growth of KPC-generated transplants (Figure 6I). We next examined whether IL-6 was capable of maintaining the growth of KPC-generated transplants. KPCs mixed with hydrogel were injected subcutaneously into immuno- PLoS ONE | www.plosone.org 11 November 2009 | Volume 4 | Issue 11 | e7798