CELLULAR CHARACTERIZATION AND SUCCESSFUL TRANSFECTION OF SERIALLY SUBCULTURED NORMAL HUMAN ESOPHAGEAL KERATINOCYTES

Background: In vitro cell culture models can provide unique insights i nto squamous epithelial proliferation, differentiation, and neoplastic transformation. Cultures of human esophageal keratinocytes could be a dvantageous for the study of these processes, Methods: Normal human es ophageal keratinocytes were cultivated on 3T3 fibroblast feeder layers in vitro and expanded through Four serial subcultivations. Confluent tertiary cultures were analyzed by morphological and immunohistochemic al techniques to define their basic properties. The ability to transie ntly transfect cultured esophageal epithelium was tested using a Rous sarcoma virus-luciferase reporter gene by the calcium phosphate and li pofection methods. Results: Postconfluent cultures displayed a predomi nantly basal cell phenotype with limited stratification, widespread ex pression of keratins 5 and 14, and production of attachment specializa tion proteins such as alpha 6 beta 4 integrin and collagen VII. Termin al differentiation markers (involucrin and transglutaminase) were prem aturely expressed. The cells expressed growth factors important in pro liferation and differentiation, such as transforming growth factor-bet a and interleukin-1 beta. Tertiary cultures were successfully transien tly transfected with a Rous sarcoma virus-luciferase reporter gene con struct. Conclusion: Normal human esophageal cells can be serially pass aged through extended numbers of cell generations and transfected by s tandard methods. This in vitro system may be useful in the study of fu ndamental cellular processes governing proliferation and differentiati on in the esophageal epithelium. J. Cell. Physiol. 177:274-281, 1998. (C) 1998 Wiley-Liss, Inc.