Inducing Corneal Keratocyte Differentiation in Human Wharton's Jelly-derived Mesenchymal Stem Cells via Keratocyte-derived Exosomes

Document Type : Research Article

Authors

1 Department of Cellular and Molecular Biology, Islamic Azad University, Tehran, Iran

2 Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

3 Institute of Converging sciences and Technologies, SR.C., Islamic Azad University, Tehran, Iran.

Abstract

One of the primary and most important components of the corneal stroma is keratocyte cells. These cells are of great interest in cell therapy for corneal diseases such as keratoconus and corneal ectasia due to their unique characteristics, including quiescence and difficulty in cultivation. Therefore, differentiation of mesenchymal stem cells (MSCs) into keratocytes has long been considered as an alternative approach. The present study was designed to explore potential of directing human Wharton's jelly-derived mesenchymal stem cells (hWJMSCs) toward corneal keratocyte-like cells using keratocyte-derived exosomes. First, keratocyte cells were isolated and cultured from discarded corneal tissue. Their culture medium was collected, and keratocyte-derived exosomes were extracted. In the next step, the optimal dose of extracted exosomes was determined using an MTT assay. Differentiation induction in hWJMSCs was then using the optimal dose of keratocyte exosomes (KExo), and the differentiation outcomes were evaluated by two techniques: real-time PCR and immunocytochemistry (ICC) at two time points (7 and 14 days). Exosomes were isolated from the conditioned medium of keratocyte cells, and SEM imaging confirmed their presence. PKH26 staining verified the successful uptake of exosomes by MSCs. Expression of the keratocan, lumican, ALDH3A1, and CD34 genes showed a significant increase (p< 0.05) after differentiation induction at both 7 and 14 days compared to the control group. Furthermore, keratocan protein expression significantly increased in cells treated with keratocyte-derived exosomes. Since the conditioned medium of keratocytes is rich in keratocyte growth factors, including exosomes, isolation and application of these exosomes effectively induce differentiation of hWJMSCs into keratocyte-like cells. Therefore, the present method offers as a suitable and cost-effective approach to generate keratocytes needed for corneal tissue engineering.

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References

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Journal of Genetic Resources
Volume 12, Issue 2
In Progress
2026
Pages 195-205

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