Isolation of keratocytes from discarded corneal tissues and using keratocyte conditional medium for differentiation of stem cells to keratocytes
Mohsen ghiasi1 , Mehrdad Hashmi2 , Khosrow Jadidi3 , Ali Salimi4 , Hossein Aghamollaei5 *
- Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
- Department of Genetics, Faculty of Advanced Science and Technology, Islamic Azad University, Tehran Medical Sciences, Tehran, Iran
- Vision Health research Center, Semnan university of medical sciences, Semnan, Iran
- Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
- Chemical Injures Research Center. Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Abstract: Keratocytes are the major cellular components of the human corneal stroma. Cell therapy by keratocytes can be used in some corneal diseases such as keratoconus and corneal ectasia. Keratocytes are quiescent cells that cannot be easily cultured. Differentiation of stem cells into keratocytes is proposed as an alternative method. The aim of this study was to use keratocytes conditional medium for differentiation of mesenchymal stem cell to keratocyte.
Methods: Keratocyte cells were isolated from discarded DSEAK samples and identified by Real-time PCR by specific genes including keratocan, lumican, Aldehyde Dehydrogenase 3 Family Member A1 (ALDH3A1) and CD34. Keratocyte was cultured in an optimized culture media and this media was collected and stored as a conditional medium. Human adipose mesenchymal stem cells(hAMSCs) were cultured and exposed to the conditioned keratocyte medium for 21 days. Differentiation was evaluated by the expression of the main markers of keratocytes.
Results: The keratocytes were isolated and cultured by the method optimized in this study and confirmed by the expression of specialized markers. After exposing the hAMSCs with keratocytes conditional medium for 21 days, Real-time PCR analysis confirmed the significant increase expression of specific markers including keratocan, lumican, ALDH3A1, and CD34 compared to the control group (p<0.05).
Conclusion: Since the conditional medium is rich in specific keratocyte growth factors and microvesicles, the use of this medium leads to induction of differentiation in stem cells, which is a convenient and inexpensive method to supply keratocytes required in corneal tissue engineering
