Abstract
Purpose :
Schlemm’s canal (SC) cells are important for controlling conventional outflow facility and thus regulating intraocular pressure. However, isolation of SC cells from donor tissue is difficult. Since SC cells exhibit both vascular and lymphatic markers, it may be possible to differentiate them from stem cells. Adipose-derived stem cells (ADSCs) are capable of differentiating into both vascular and lymphatic endothelial cells, via VEGF-A and -C. Shear stress also plays a critical role in maintaining SC integrity, function, and PROX1 expression. We hypothesize that subjecting ADSCs to the optimal cocktail of VEGF, shear stress and co-culture with human trabecular meshwork (HTM) cells provides biochemical and mechanical cues necessary for differentiation.
Methods :
Human ADSCs were cultured in Endothelial Growth Media with VEGF-A for 14 days, followed by 7 days of VEGF-C treatment. On day 3, samples (N=16) were placed onto an orbital shaker to generate ~14 dynes/cm2 of shear stress. Changes in cell morphology, gene expression of PROX1, CD31 and VEGFR2 were evaluated using SEM, qPCR and immunocytochemistry. Cell alignment was examined using Image J. To further optimize differentiation, undifferentiated ADSCs were co-cultured with HTM cells (N=32) on microfabricated, porous SU8 scaffolds for 14 days. Expression of characteristic SC markers were examined, followed by perfusion studies and treatment with dexamethasone. One-way ANOVA was used for statistical analysis.
Results :
ADSCs were highly sensitive to the combination of VEGF-A and shear stress, forming cord-like structures throughout the culture, indicative of endothelial differentiation. VEGF-C treatment significantly increased PROX1 expression at both protein and mRNA level (2.8-fold; p<0.0001). SEM images showed 62.7% increase in cell alignment. Co-culture samples showed full coverage of differentiated ADSCs and HTM cells on each side of the SU8 scaffold. Perfusion of the co-cultured constructs showed higher flow resistance than HTM control, and dexamethasone treatment decreased the outflow facility.
Conclusions :
VEGF treatments, shear stress and co-culture were effective in differentiating ADSC into PROX1 expressing cells that respond to dexamethasone treatment. Further phenotyping and evaluation of drug responses are needed to confirm SC differentiation. This study makes in vitro high-throughput, pharmacological screening of glaucoma drugs possible.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.