Transient immortalisation of human cells using human corneal endothelium (posterior corneal epithelium) as a model

• Applicant: Dr. Monika Valtink
• Subject Area: Ophthalmology ; Nuclear Medicine, Radiotherapy, Radiobiology
• Term: from 2013 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 232772305

Terminally differentiated cells are characterised by leaving the cell cycle, an actively maintained process that may be reversible upon manipulation. Human corneal endothelial cells (HCEC) are such post-mitotic cells, which show almost no proliferative activity in vivo and only limited proliferation in vitro. Age, degeneration, diseases or surgical interventions can cause a loss of corneal endothelial cells and by this the need for a corneal transplant, but the number of qualitatively sufficient donor corneas is limited. To develop a method for in vitro and in situ multiplication of HCEC without loss of their typical morphological features in order to improve the quality of insufficient donor corneas and also to analyse these cells in more detail would greatly expedite corneal research. In this project, cellular growth factors and viral/cellular oncogenes shall be identified, which allow simple and reproducible immortalisation of primary HCEC after stable expression using retroviral gene transfer. Subsequently, such identified factors will be analysed regarding their ability to expand primary cultures of HCEC by transient transgene expression as a result of temporary promotion of proliferation following two strategies: 1) transient transgene expression after non-integrating retroviral gene transfer, and 2) supplementation of the growth medium with recombinant chimeric proteins that are taken up by HCEC through protein transduction domains and develop their proliferation promoting activity intracellularly.

• DFG Programme: Research Grants