Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells

Abstract

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-β (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity. Glaucoma encompasses a heterogenous group of optic neuropathies, united by their exhibition of permanent damage to the optic nerve 1. This degeneration is due to the death of retinal ganglion cells (RGCs), with subsequent visual field loss 2. Primary open-angle glaucoma (POAG), the most common form of glaucoma, is characterized by chronic and progressive optic nerve degeneration and corresponding visual field deficits in the presence of an open and normal iridocorneal chamber angle 3. This disease is the leading cause of irreversible blindness worldwide 4 , with an estimated 11.1 million expected to become blind from POAG by 2020 5. Despite the prevalence of POAG, its pathogenesis remains poorly understood. The complexity of glaucoma certainly makes it possible, if not probable, that RGCs become inherently susceptible to this disease process. RGCs are found in the ganglion cell layer of the retina and serve as the projection neurons of the retina, utilizing long axons to effectively connect the eye to the brain. They transmit both image-forming and non-imageforming visual information, processed by retinal cells such as photoreceptors and bipolar cells, to higher visual centers in the lateral geniculate body through the optic nerve 6. Currently, there are many treatments that slow the disease, but no precision treatment exists for glaucoma or RGC degeneration. Although shown to be effective in animal models of glaucoma, neuroprotective approaches have not proven practical in human settings 7,8 .

Generation of a Transplantable Population of Human iPSC-Derived Retinal Ganglion Cells

“…In order to promote RGC survival and functional maturation, retinal organoids have been dissociated at D56, a stage of development at which many RGCs are present, and retinal cells were replated onto an adherent substrate. In agreement with previous studies, the adherent culture of retinal cells allowed RGCs to grow extensions showing βIII-tubulin or THY1 immunoreactivity (Ohlemacher et al, 2016;Li et al, 2017;Fligor et al, 2018;Langer et al, 2018;Chavali et al, 2020). The capacity of neurons such as RGCs to trigger an action potential depends on different hallmarks including the expression of specific voltage-gated channels and, importantly, a resting membrane potential (V rest ) allowing their activation (Catterall, 2012).…”

“…This crucial point is illustrated by a recent study showing that only hiPSC-derived RGCs displaying an adequately negative V rest were able to spike (Teotia et al, 2019). In the present study, the V rest was approximatively −43 mV, a slightly more depolarized value than that reported by Riazifar et al (2014) or Chavali et al (2020) in hiPSC-derived RGC-like cells, but lower than that observed by Teotia et al (2019). Consistent with previous studies (Teotia et al, 2019;VanderWall et al, 2019), we demonstrated that 3 weeks after plating, the mature RGCs, in contrast to photoreceptors, displayed voltage-gated ionic currents enabling these RGCs to evoke spikes after stimulation and to spontaneously fire.…”

“…Based on initial protocols developed with mouse and human ESCs (Eiraku et al, 2011;Nakano et al, 2012), different groups including ours developed three-dimensional (3D) culture systems recapitulating key steps of retinal development and allowing the generation of self-organizing retinal organoids containing RGCs (Reichman et al, 2014;Zhong et al, 2014;Maekawa et al, 2015;Ohlemacher et al, 2016;Fligor et al, 2018). Very recently, RGCs were differentiated from human induced pluripotent stem cells (hiPSCs) using a chemically defined medium resulting in dual SMAD and Wnt inhibition bypassing retinal organoid formation (Chavali et al, 2020). Patient-specific iPSCs can be useful to better characterize the pathogenesis and molecular mechanisms of different inherited optic neuropathies (Chen et al, 2016;Ohlemacher et al, 2016;Wu et al, 2018;VanderWall et al, 2020).…”

Hereditary Optic Neuropathies: Induced Pluripotent Stem Cell-Based 2D/3D Approaches

“…They generated over 80% of the pure RGC cultures in about 40 days and purified the Thy1+ cells thanks to the unique expression of the surface glycoprotein Thy1 in the RGCs. Employing magnetic-activated cell sorting (MACS) and CD90.2 microbeads, they obtained 95% of the cells positive for BRN3A [ 89 ]. In all these differentiation protocols, elongated axons were observed.…”

Molecular Genetics and Functional Analysis Implicate CDKN2BAS1-CDKN2B Involvement in POAG Pathogenesis

“…Cells were washed in 1X PBS and mounted with prolong gold mounting medium with DAPI for nuclei staining. Images were captured by Olympus FV1000 Confocal microscope with the use of appropriate filters and lasers [35].…”