Abstract
Purpose :
Corneal endothelial disorders, such as Fuchs endothelial corneal dystrophy, are a leading cause for corneal transplantation. Due to a global shortage of donor corneal tissue, it Is essential for alternative therapeutic strategies to restore vision to be developed. Current research in regenerative medicine is investigating the development of cell therapies for corneal endothelial repair. The umbilical cord is a rich source of stem and endothelial progenitor cells, including human umbilical vein endothelial cells (HUVECs). We hypothesize that the optimisation of HUVECs for corneal endothelial cell (CEC) replacement will provide a therapeutic pathway for the treatment of corneal endothelial disorders.
Methods :
HUVECs were isolated from human umbilical cords (n=10) and characterised by polymerase chain reaction (PCR) and immunocytochemistry (ICC). HUVECs were differentiated into CEC-like cells using a CEC-conditioned medium collected from an immortalised CEC line. Differentiation was measured by changes in morphology, ICC, PCR, and flow cytometry. The conditioned medium was fractionated using protein concentrators of different molecular weights (3, 5, 10, 30, 50, and 100 kDa). HUVECs were then differentiated using the concentrated medium samples, to identify the molecular weights of the proteins of interest. Subsequently, mass spectrometry was used to identify the target proteins which may be driving CEC differentiation.
Results :
Isolated HUVECs showed expression of HUVEC markers, CD31 and CD146. Following differentiation by conditioned medium, a clear change in cell morphology was observed with the cells displaying a hexagonal morphology. This finding was supported by an increase in cell circularity and the formation of tight junctions between cells, as seen with ZO1 labelling. PCR analysis showed the upregulation of CEC markers ATP1A1 and ZO1. Fractionation of the conditioned medium determined that proteins driving differentiation have a molecular weight >100 kDa, after which mass spectrometry analysis identified target proteins in the conditioned medium which may be responsible for CEC differentiation.
Conclusions :
These results show that HUVECs can be differentiated into CEC-like cells. Proteins responsible for CEC differentiation can be identified, which will allow for the development of targeted CEC replacement therapies in which differentiated HUVECs are used to re-populate the corneal endothelium.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.