Purchase this article with an account.
Lea D Bennett, Bojana Radojevic; L-DOPA Stimulates Neurite Outgrowth during Early Retinal Organoid Development. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1694.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Human retinal organoids made from inducible pluripotent stem (iPS) cells mimic retinogenesis and have been crucial for uncovering diseases mechanisms that cannot be recapitulated in animal models. However, mature retinal organoids are disorganized and unlike fetal retina, do not express important cellular pathways (circadian entrainment and axon guidance). Therefore, the goal of this study was to evaluate a key regulator of circadian entrainment, dopamine (DA) in developing retinal organoids.
At day 30 (D30) and D60, human retinal organoids were dissociated and grown on laminin-coated coverslips for neurite outgrowth assay. At D30, l-DOPA with ascorbic acid was added to the media for 2-3 hours to stimulate DA synthesis. Cells were stained with neuron-specific marker TUJ1 (βIII tubulin) and MAP2 to visualize axon outgrowth. Axon-guiding signals (e.g. Irx4, Foxg1, Nfil3) and cell type-specific markers were quantified with RT-qPCR.
Exogenous l-DOPA with ascorbic acid induced expression of the penultimate enzyme, tyrosine hydroxylase (TH) and DA synthesis prior to the appearance of dopaminergic amacrine cells (DACs). Cells treated with l-DOPA and ascorbic acid at D30 modified neurite outgrowth in early retinogenesis and showed increased axon lengths as well as expression of axon guiding genes (RT-qPCR). Expression of TH was 3 fold higher in the cells treated with l-DOPA and ascorbic acid at D30 compared to cells that were not treated with l-DOPA.
This work aimed to determine how developing retinal neurons respond to DA to ultimately create functional retinal organoids that mimic the structure and physiology of human retina which will have broader potential for ophthalmic research.
This is a 2021 ARVO Annual Meeting abstract.
This PDF is available to Subscribers Only