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Summer Research Fellowship Programme of India's Science Academies

Understanding the molecular basis of endothelium to mesenchymal transition in case of human corneal endothelium cells, cultured in vitro

Debdyuti Bhadra

Student, Zoology Department, Ramakrishna Mission Vidyamandira, Belur Math, Howrah, West Bengal 711202

Dr. Charanya Ramachandran

Scientist, Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034

Abstract

Endothelium is the posterior-most layer of the cornea, which supplies nutrients to the avascular cornea and maintaining the swelling pressure of stroma. The adult endothelial cells do not divide in vivo and rather compensates by cell enlargement. If there is a critical loss of these cells due to endothelial disease or any trauma, it leads to corneal edema and visions loss. Currently, only surgical interventions are available, where clinicians transplant a healthy Descemet membrane to replace the dysfunctional endothelium. But the scarcity of healthy tissues obliged the researchers to find some alternatives like in vitro culture of human corneal endothelial (hCE) cells and tissue engineering. Numerous culture techniques have been used to standardize a protocol for culturing hCE cells in vitro but still there are some major limitations. Cultured adult hCE have been observed to be become senescent or undergo endothelial to mesenchymal transition (EnMT) to a fibroblastic phenotype after one or two population doublings in vitro. The exact molecular pathway behind this transition is not yet understood properly. This project aims to map the onset of EnMT and determine the molecular mechanism underlying this transition by performing a systematic study on some hCE specific markers such as, Zona occludens 1 (ZO-1), N-cadherin and Na+, K+-ATPase. Immunofluorescence staining was used to check the expressional status of these markers in the freshly peeled hCE cells and in vitro cultured cell. RNA was isolated for real-time PCR to check the expressional status of these genes at the transcriptome level. We did not find any difference in expression patterns of markers within freshly peeled tissue and P1 cells except in case of Na+, K+-ATPase. This study suggested that hCEC can be expanded in vitro and the cells can sustain their characters at P1. Further continuation is needed to fulfil the objectives of this project.

 Keywords: cell culture, endothelium-mesenchymal transition, corneal regeneration

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