Abstract
Purpose :
Purpose: Müller glia (MG) cells have been identified as key cell types involved in several retinal fibrotic complications. MG cells can undergo hypertrophy, increased proliferation, and phenotypic change to become fibroblasts and myofibroblasts, which play an essential role in formation and contraction of epiretinal membranes and fibrotic scars. In this study, the combined effect of substrate stiffness and growth factor conditions on MG cell proliferation and phenotypic change to myofibroblasts were examined.
Methods :
MG cells were isolated from retina of freshly enucleated porcine eyes using a papain dissociation kit. Following 7-8 day culture in 10%FBS-DMEM, MG cells were trypsinized and plated on collagen-coated polyacrylamide gels of various stiffness in 96 wells. Four different culture conditions: 2.5% FBS, 2.5% FBS with 10ng/ml TGFbeta1, 2.5% FBS with 100ng/ml IGF-1, 2.5% FBS with TGFbeta1 and IGF-1, were used to culture cells for 2 days (for proliferation assay) or 6 days (to assay phenotypic change). Proliferation was assessed by EdU incorporation and cell density on day 2, and myofibroblast transdifferentiation was assessed by expression of alpha-smooth muscle actin (aSMA) expression on day 6.
Results :
MG cell proliferation was both stiffness and growth factor dependent. Higher stiffness supported proliferation, especially for IGF-1 treated cells, on day 2. Myofibroblast transdifferentiation, examined by expression of aSMA, was also enhanced on substrates with higher stiffness in the presence of growth factors.
Conclusions :
Substrate stiffness and growth factor conditions play key roles in fibrotic changes associated with fibrosis in MG cells. IGF-1 plays a prominent role in inducing proliferation on stiffer substrates, and stiffer substrates also support myofibroblast transdifferentiation of MG cells. Further study into the crosstalk of mechanosignaling and growth factor signaling is warranted to understand key events in regulating fibrotic transformation of MG cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.