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Pooja Teotia, Negar Firoozi, Iqbal Ahmad; Modeling human optic nerve regeneration in microfluidic devices: screening of genes and signaling pathways. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2501.
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© ARVO (1962-2015); The Authors (2016-present)
The degeneration of retinal ganglion cells (RGCs) is the unifying theme in glaucoma, a complex group of disorders. However, their susceptibility in terms of optic nerve degeneration and their ability to regenerate remains largely unknown. Though significant progress has been made in identifying genes and pathways involved in optic nerve de- and re-generation, the clinical relevance of these studies remains poorly established because they have been carried out in rodents. Modeling optic nerve regeneration using human induced pluripotent stem cell (hiPSC)-derived RGCs may identify molecular targets for therapeutic optic nerve regeneration.
hiPSCs were differentiated along the hRGC lineage using our chemically defined protocol in a microfluidic device, which allows directional orientation of hRGCs axons in microgrooves from the soma to axonal chamber. Axotomy was performed between 10-12 DIV by first removing the medium and adding 0.5% saponin for 3 minutes in the axonal chamber, after which the axonal chamber was re-coated with matrigel and medium restored for regeneration. The following conditions were created to examine the effect of specific pathways/genes: mTOR signaling: activation and inhibition by shRNA-mediated silencing of tuberous sclerosis complex 2 (TSC2) and rapamycin, respectively; JAK-STAT3 pathway: activation and inhibition by hyper IL-6 and AG490, respectively; Lin28: increasing and decreasing expression of Lin28.
The axons began to regenerate within 48 hours and their quantification carried out 5 days post axotomy revealed that the proportion of axons in the axonal chamber was significantly higher in mTOR/JAK-STAT pathway group versus controls (75.33±1.76%/71.00±2.43% vs 45.40±2.70%/49.87±6.23%). Regeneration was inhibited in the presence of rapamycin/AG490, demonstrating the specificity of the mTOR/JAK-STAT pathway on axon regeneration. Lin28, which mediated its effects on neurogenesis via IGF-1 recapitulated for the regenerative response.
Our results show that the role of specific pathways in optic nerve regeneration is evolutionarily conserved, suggesting their clinical implications in glaucomatous degeneration. We demonstrate a functional in vitro model of human optic nerve de- and re-generation for studying the development and pathology of axons in controlled conditions and identifying molecular targets for therapeutic regeneration.
This is a 2020 ARVO Annual Meeting abstract.
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