Phenotypic and genotypic profile of human tympanic membrane derived cultured cells

Abstract

The human tympanic membrane (hTM), known more commonly as the eardrum, is a thin, multi-layered membrane that is unique in the body as it is suspended in air. When perforated, the hTM's primary function of sound-pressure transmission is compromised. For the purposes of TM reconstruction, we investigated the phenotype and genotype of cultured primary cells derived from hTM tissue explants, compared to epithelial (HaCaT cells) and mesenchymal (human dermal fibroblasts (HDF)) reference cells. Epithelium-specific ets-1 (ESE-1), E-cadherin, keratinocyte growth factor-1 (KGF-1/FGF-7), keratinocyte growth factor-2 (KGF-2/FGF10), fibroblast growth factor receptor 1 (FGFR1), variants of fibroblast growth factor receptor 2 (FGFR2), fibroblast surface protein (FSP), and vimentin proteins were used to assess the phenotypes of all cultured cells. Wholemount and paraffin-embedded hTM tissues were stained with ESE-1 and E-cadherin proteins to establish normal epithelial-specific expression patterns within the epithelial layers. Immunofluorescent (IF) cell staining of hTM epithelial cells (hTMk) demonstrated co-expression of both epithelial- and mesenchymal-specific proteins. Flow cytometry (FCM) analysis further demonstrated co-expression of these epithelial and mesenchymal-specific proteins, indicating the subcultured hTMk cells possessed a transitional phenotype. Gene transcript analysis of hTMk cells by reverse transcriptase polymerase chain reaction (RT-PCR) revealed a down regulation of ESE-1, E-cadherin, FGFR2, variant 1 and variant 2 (FGFR2v1 and FGFR2v2) between low and high passages, and up-regulation of KGF-1, KGF-2, and FGFR1. All results indicate a gradual shift in cell phenotype of hTMk-derived cells from epithelial to mesenchymal.