Abstract
Purpose: :
To study the effect of plating density of primary cultures of human corneal endothelial cells (HCECs) and of a feeder layer on the level of expression of Sp1, a positive transcription factor involved in cell cycle and proliferation of many cell systems, in HCECs.
Methods: :
Corneal endothelial cells from a 68 year old donor were seeded at the seventh passage at low, mid and high density (2x103, 8x103, and 1.6x104 cells/cm2, respectively) with irradiated 3T3 murine fibroblasts (plated at either 1x104 or 2x104 cells/cm2). As controls, murine fibroblasts and endothelial cells were seeded alone. Electrophoretic mobility shift assays (EMSAs) were conducted by incubating a synthetic double–stranded oligonucleotide bearing the target sequence recognized by Sp1 with crude nuclear extracts prepared from these cultures. A densitometric analysis of the Sp1 DNA–protein complexes was conducted using the Image software and the data expressed relative to the level observed on seeding endothelial cells without the presence of 3T3 (and considered as 100%).
Results: :
Co–culturing HCECs with 1–day confluent, irradiated murine 3T3 fibroblasts (which express no detectable Sp1 protein) as a feeder layer considerably increased the level of Sp1 expression by approximately 3.5–fold when cells had been plated at mid–density. This positive influence of irradiated 3T3 cells on Sp1 expression is cell–density dependent as maximal Sp1 expression is observed at mid–plating density. In addition, doubling the concentration of co–cultured, irradiated 3T3 from 1x104 to 2x104 cells/cm2 also potentiated the positive influence of this feeder layer by 181% and 180% at mid– and high plating density, respectively. The ability of irradiated 3T3 to improve Sp1 expression in HCECs was considerably reduced when endothelial cells, co–cultured with 3T3 cells, had been maintained at post–confluence for 7 days (from a 3.5–fold increase with subconfluent HCECs to 1.9–fold with 7–day post–confluent HCECs).
Conclusions: :
Expression of Sp1 in corneal endothelial cells is considerably increased, in a cell–density dependent manner, by the presence of a feeder layer (for instance, irradiated 3T3 cells). Higher levels of Sp1 might therefore contribute to preserve the morphological characteristics of HCECs and delay their progression toward terminal differentiation in vitro.
Keywords: cornea: endothelium • transcription factors • cornea: basic science