Cellular characterization and successful transfection of serially subcultured normal human esophageal keratinocytes

Abstract

Background: In vitro cell culture models can provide unique insights into squamous epithelial proliferation, differentiation, and neoplastic transformation. Cultures of human esophageal keratinocytes could be advantageous for the study of these processes.

Methods: Normal human esophageal keratinocytes were cultivated on 3T3 fibroblast feeder layers in vitro and expanded through four serial subcultivations. Confluent tertiary cultures were analyzed by morphological and immunohistochemical techniques to define their basic properties. The ability to transiently transfect cultured esophageal epithelium was tested using a Rous sarcoma virus-luciferase reporter gene by the calcium phosphate and lipofection methods.

Results: Postconfluent cultures displayed a predominantly basal cell phenotype with limited stratification, widespread expression of keratins 5 and 14, and production of attachment specialization proteins such as alpha6beta4 integrin and collagen VII. Terminal differentiation markers (involucrin and transglutaminase) were prematurely expressed. The cells expressed growth factors important in proliferation and differentiation, such as transforming growth factor-beta and interleukin-1beta. Tertiary cultures were successfully transiently transfected with a Rous sarcoma virus-luciferase reporter gene construct.

Conclusion: Normal human esophageal cells can be serially passaged through extended numbers of cell generations and transfected by standard methods. This in vitro system may be useful in the study of fundamental cellular processes governing proliferation and differentiation in the esophageal epithelium.