Human mesenchymal stem cells differentiate into corneal tissue of mouse

Abstract

Purpose: The dry eye syndrome or keratoconjunctivitis sicca represents one of the most commons ocular affections causes a very severe hyposecretor dry eye to the conventional treatment and that requires new therapeutic approaches. The mesenchymal stem cells (MSCs) are beginning to look as a possible alternative due to their multipotentiality, to their immunosuppression properties and to their ability of differentiation towards cells from damaged tissues, in addition to secreting factors that contributes to the tissue regeneration. The aim of this work is to analyze the therapeutic potential of the MSCs in the cornea and the ocular surface.

Methods: The cells were isolated from bone marrow cells of 5 healthy volunteer donors (3 males/2females) between 28 and 52 years. This hMSCs were expanded in T75 culture flasks and cultured as previously described. Then, hMSCs were transduced with a lentiviral vector containing the green fluorescent protein (GFP). Female BALB/c (H2d) mice (n=20) were anesthetized with isoflurane in air and received a 20 µl subconjuntival injection containing 2 x 105 hMSCs at 1-2 mm of the right eye esclerocorneal limbo. Left eyes were used as control. The eyes were enucleated and fixed in 4% paraformaldehyde. Then, were cryoprotected in 50% sucrose and cut in 12 µm sections on a cryostat. The fate of the hMSCs was analysed by immunohistochemistry with an anti-GFP antibody. We also used anti-MIF protein and anti-human Mitochondria antibodies in order to analyse the possibility of cell fusion between the human and mouse cells. Specificity of all antibodies was tested by western blot analysis.

Results: Even after 30 days, we found hMSCs across the whole mouse cornea. In epithelium, MSC appeared at different layers, with morphologies varying according with the epithelial cells at their position. Furthermore, they showed ultrastructural features typical of epithelial cells, such as bundles of intermediate filaments and interdigitations and desmosomes with other cells. In the stroma, we observed GFP+ cells with a keratocyte-like morphology, lying between bundles of collagen fibrils. We found GFP labeled cells in the endothelium too. These cells were Mitochondria+/MIF- what discarded any fusion process.

Conclusions: The successful integration of hMSCs in mouse cornea without any negative consequences is a good starting point for the use of these cells in the dry eye syndrome.

Keywords: 482 cornea: epithelium • 555 immunomodulation/immunoregulation • 479 cornea: clinical science