Improved Human Endothelial Cell Proliferation in the Presence of a Feeder Layer: Effect on the Activation of the Transcription Factors SP1 and NF1

Abstract

Abstract: : Purpose: To evaluate the influence of media conditioned by various cell types on the proliferation of human corneal endothelium. To examine whether or not the transcription factors SP1 and NF1 are modified by co–culture of endothelial cells with a feeder cell layer. Methods: Nonconfluent cultures of human corneal endothelium were exposed to one of the following media: conditioned by 1) human corneal fibroblasts (FC), 2) murine irradiated fibroblasts (3T3), 3) bovine corneal endothelium (EB), 4) Optimem control medium with serum (T+), or 5) without serum (T–). Proliferation was evaluated by BrdU incorporation in cells exposed to media during 24 hours, and flow cytometric analysis (FACS). Electrophoretic mobility shift assay (EMSA) with labeled oligonucleotides bearing the target sequence recognized by Sp1/Sp3 or NF1 transcription factors were incubated with nuclear extracts from control monocultures of irradiated 1) 3T3 or 2) bovine corneal endothelial cells, or from human endothelial cells grown 3) in the absence or presence of a feeder layer of irradiated 4) murine fibroblasts or 5) bovine corneal endothelial cells. Endogenous expression of Sp1 and NF1 was also monitored by western blot (WB). Results: According to FACS analysis, the proportion of cells synthesizing DNA was 22, 24, 28, 37 and 61% for T–, EB, 3T3, FC and T+, respectively. EMSA and WB revealed that expression of Sp1/SP3 and NF1, practically undetectable when the human endothelium was cultured alone or in the presence of irradiated bovine corneal endothelial cells, was dramatically increased when endothelial cells were cultured with a 3T3 feeder layer. Conclusions: The culture of human corneal endothelia seems activated by the presence of a feeder cell layer containing human corneal or murine fibroblasts. Improvement of the proliferative properties of endothelial cells was correlated with an increased expression of Sp1/SP3. The present results could lead to better conditions for culturing human corneal endothelia, a population of cells very difficult to preserve in vitro.

Keywords: cornea: endothelium • cornea: basic science • transcription factors