The secretome of Mesenchymal stem cells protects both primary trabecular meshwork cells and retina ganglion cells

Abstract

Purpose: To investigate the effect of Mesenchymal Stem Cells (MSCs) secretome (MSC-CM) on primary human trabecular meshwork cells and primary retinal ganglion cells

Methods: Primary human trabecular meshwork (hTM) cells (ScienceCell) from passage 1 to 8 were cultured in 6-well plates till 90% confluence before exposure to MSC-CM, TGF-β₂ (20µg/ml) or both for 3 or 72 hours. We investigated the hTM-myofibroblast phenotype transition induced by TGF-β₂+/-MSC-CM, and α-SMA, col3 and col4 mRNA level expression after 72h. Next, hTM exposure to TGF-β₂+/-MSC-CM for 3h, we looked for the Akt- and myosin (MLC) phosporylation using Western-Blot assays. Purified primary RGCs was obtained from rat retina using immunopanning methods. Cells were seeded in 96-well plates at 12 000cells/well and maintained for 6 days in neurobasal+glutamine (NBAg), MSC-CM or NBAg+B27 medium. Cell viability was evaluated by calcein green fluorescence using live-dead test kit and a cell counting software.

Results: MSC-CM displayed favorable effects on viability, contractibility and phenotype conservation of hTM cells especially by impeding the deleterious effect of TGF-β₂. MSC-CM clearly triggered the phosphorylation of the antiapoptotic Akt pathway resulting in an increased survival as proved in an in vitro BAK cytotoxic model. MSC-CM significantly inhibited the TGF-β₂-dependent MLC phosphorylation on hTM and α-SMA, col3 and col4 mRNA increase. Furthermore, MSC-CM promoted cell viability and growth in a purified primary culture of rat RGC in vitro.

Conclusions: Through these in vitro approaches, we highlighted the beneficial effects of MSC-CM on primary cells directly involved in the physiopathology features of glaucoma. These results support the promising concept of MSCs-based therapy to treat glaucoma disease.