Mesenchymal stem cell based therapy for restoring corneal graft transparency

Abstract

Purpose: Mesenchymal stem cells (MSC) are being extensively tested in the field of organ transplantation due to their potent anti-inflammatory and tissue regenerative properties. In the case of corneal transplantation, the additional anti-angiogenic properties of MSC make them an attractive candidate for maintaining corneal clarity. This project aims to investigate the effect of allogeneic vs syngeneic MSC in preventing loss of and/or restoring corneal graft clarity in mouse corneal allograft model

Methods: Recipient-derived (syngeneic-Balb/c) and donor-derived (allogeneic-B6) bone marrow MSC were isolated, depleted of CD45+ cells and characterized as lineage negative. The effect of MSC on in vitro T cell proliferation was assessed by CFSE labelling. MSC(1x10⁶ cells) were administered in vivo either intravenously (IV) or subconjuctivally (SC) 7 days before (1^st dose) and on the day (2^nd dose) of corneal allotransplantation (B6 to Balb/c). Corneal graft opacity was evaluated for 2 months post grafting. Calcein/CFSE labelled MSC were administered IV to determine where MSC trafficked after inoculation. In addition, in vivo induction of T regulatory cells (Tregs) in secondary lymphoid organs was assessed by flow cytometry

Results: Both allo and syn-MSC had strong inhibitory effects on T cell proliferation in vitro. Only IV administered allo-MSC significantly reduced corneal opacification (p<0.0001) compared to syn-MSC and untreated (PBS) controls at 2 months post-transplant. Calcein/CFSE labelled splenocytes were tracked to the spleen and the eye-draining lymph nodes 16 hrs after inoculation by both IV and SC routes. However, labelled MSC could not be detected when administrated IV. Flow cytometric analysis at day 4 after IV showed syn-MSC but not allo-MSC induce significantly higher frequency of splenic Tregs (CD4+ Foxp3+) compared to PBS treated controls (n=4/group)

Conclusions: Our data show that IV but not SC administration of allo-MSC significantly improved corneal allograft survival. Syn-MSC were not effective. This data suggest that allo-MSC induce immunological tolerance to corneal allograft. However the effect did not correlate with induction of Tregs. In addition, the inability to track MSC after IV inoculation suggests that they are rapidly removed from the system presumably by phagocytic engulfment. Engulfment of apoptotic allo-MSC may be a possible mechanism for inducing allospecific tolerance in this model