RESUMO
Oxidative stress is an important cause for several retinal aging diseases. Cell therapy using a decellularized human amniotic membrane (dHAM) as a tissue scaffold for retinal pigment epithelial cells has a potential therapeutic role under such pathological conditions. This is attributed by the anti-inflammatory, antimicrobial, low-immunogenicity aspects of dHAM, apart from harboring a drug reservoir potential. The underlying mechanisms for maintaining the physiological properties of transplanted cells and their survival in a diseased milieu using dHAM has remained unexplored/unanswered. Hence, we investigated the potential role of dHAM in preserving the cellular functions of retinal pigment epithelium in an oxidative stress environment. Adult human retinal pigment epithelial (ARPE-19) cells were cultured on dHAM or tissue culture dishes under hyperoxia. Gene expression, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA), and scanning electron microscopy (SEM) were performed to assess the levels of reactive oxygen species, proliferation, apoptosis, epithelial-mesenchymal transition, phagocytosis, and secretion of vascular endothelial factors. These results indicate reduced epithelial-mesenchymal transition, generation of reactive oxygen species (p ≤ 0.0001), and apoptosis (p ≤ 0.05) in cells cultured on dHAM, compared to those on tissue culture dishes under oxidative stress conditions. Concomitantly, the secretion of the vascular endothelial growth factor was significantly reduced (p ≤ 0.01) on dHAM. Phagocytic activity was significantly higher (p ≤ 0.001) in cells cultured on dHAM and were comparable to those cells cultured on tissue culture dishes. SEM images showed a clustered growth pattern on dHAM compared to an elongated morphology when cultured on tissue culture dishes under oxidative stress conditions. These findings demonstrate the utility of dHAM as a scaffold for growing retinal epithelial cells and to maintain their physiological properties in an oxidative stress condition with a potential to develop regenerative medicine strategies to treat degenerative eye diseases.
RESUMO
Differentiation is a major histological parameter determining tumor aggressiveness and prognosis of the patient; cancer stem cells with their slow dividing and undifferentiated nature might be one of the factors determining the same. This study aims to correlate cancer stem cell markers (CD44 and CD147) with tumor differentiation and evaluate their subsequent effect on prognosis. Immunohistochemical analysis in treatment naïve oral cancer patients (n = 53) indicated that the expression of CD147 was associated with poorly differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma (p < 0.01). Furthermore, co-expression analysis showed that 45% each of moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma patients were CD44high/CD147high as compared to only 10% of patients with well-differentiated squamous cell carcinoma. A three-way analysis indicated that differentiation correlated with recurrence and survival (p < 0.05) in only the patients with CD44high/CD147high cohort. Subsequently, relevance of these cancer stem cell markers in patterning the differentiation characteristics was evaluated in oral squamous cell carcinoma cell lines originating from different grades of oral cancer. Flowcytometry-based analysis indicated an increase in CD44+/CD147+ cells in cell lines of poorly differentiated squamous cell carcinoma (94.35 ± 1.14%, p < 0.001) and moderately differentiated squamous cell carcinoma origin (93.49 ± 0.47%, p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin (23.12% ± 0.49%). Expression profiling indicated higher expression of cancer stem cell and epithelial-mesenchymal transition markers in SCC029B (poorly differentiated squamous cell carcinoma originated; p ≤ 0.001), which was further translated into increased spheroid formation, migration, and invasion (p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin. This study suggests that CD44 and CD147 together improve the prognostic efficacy of tumor differentiation; in vitro results further point out that these markers might be determinant of differentiation characteristics, imparting properties of increased self-renewal, migration, and invasion.