Alexandre Taoum (Heidelberg, DE), Annika Jeschke (Heidelberg, DE), Lea Adams (Heidelberg, DE), Andrea Frank (Heidelberg, DE), Prof. Dr. Matthias Fuest (Aachen, DE), Prof. Dr. Daniela Duarte Campos (Heidelberg, DE)
Abstract text (incl. figure legends and references)
Introduction: Corneal transplantation is the gold standard procedure to cure corneal diseases that can lead to blindness. Fabrication of corneal tissues is a promising solution to overcome the shortage of human donors. Our future goal is to utilize a bioprinter mounted to a robotic arm to print corneal tissue directly onto the eye of the patient. One interesting cell type for in situ and in vivo corneal bioprinting is the use of bone marrow-derived mesenchymal stromal cells (MSCs) in their differentiated state toward the keratocyte lineage. Corneal stromal keratocytes are the main cell type present in the native tissue, and for this reason in this work we investigated the keratocyte differentiation potential of MSCs.
Objective: The main goal of this work was to investigate the ability of MSCs to differentiate towards keratocytes by analyzing and comparing the expression of specific markers of these differentiated cells and primary corneal stromal keratocytes (CSKs).
Materials & Methods: MSCs were expanded according to previously established protocols1,2. Keratocyte differentiation was initiated by subculturing the MSCs in a differentiation medium supplemented with 10 ng/ml FGF-b and 1 ng/ml TGF-β3 for 14 days. The expression of the keratocyte specific markers keratocan, lumican, and ALDH3A1 were investigated by immunofluorescence staining, Western Blot and qPCR.
Results: Keratocyte differentiated cells showed positive immunofluorescent staining of the keratocyte markers keratocan, lumican and ALDH3A1 and the success of differentiation was confirmed by qPCR and Western Blot.
Conclusion: We showed successful keratocyte differentiation of MSCs in 2D cultures. After validating our differentiation protocol in 2D, we begin to investigate if the keratocyte differentiation potential is also observed in 3D hydrogel cultures compatible with bioprinting.
Hin-Fai Yam G et al. J Cell Mol Med.2018;doi:10.1111/jcmm.13589 Dos Santos et al. IOVS 2019;doi:10.1167/iovs.19-27008