Retinal ganglion cell polarization using immobilized guidance cues on a tissue-engineered scaffold

Abstract

Cell transplantation therapies to treat diseases related to dysfunction of retinal ganglion cells (RGCs) are limited in part by an inability to navigate to the optic nerve head within the retina. During development, RGCs are guided by a series of neurotrophic factors and guidance cues; however, these factors and their receptors on the RGCs are developmentally regulated and often not expressed during adulthood. Netrin-1 is a guidance factor capable of guiding RGCs in culture and relevant to guiding RGC axons toward the optic nerve head in vivo. Here we immobilized Netrin-1 using UV-initiated crosslinking to form a gradient capable of guiding the axonal growth of RGCs on a radial electrospun scaffold. Netrin-gradient scaffolds promoted both the percentage of RGCs polarized with a single axon, and also the percentage of cells polarized toward the scaffold center, from 31% to 52%. Thus, an immobilized protein gradient on a radial electrospun scaffold increases RGC axon growth in a direction consistent with developmental optic nerve head guidance, and may prove beneficial for use in cell transplant therapies for the treatment of glaucoma and other optic neuropathies.

Towards the development of a human glaucoma disease-in-a-dish model using stem cells

“…However, once grown on the scaffold, not only did neurites grow in the same orientation, but were also seen to bundle together in areas of higher density, a phenomenon of axonal organization seen in vivo. More recently, the same group published their latest advancement, an implantable scaffold containing a chemical gradient of Netrin-1, a chemotropic factor expressed in the developing eye that polarizes axonal growth towards the center, not just in a radial orientation [86]. For an in-depth review on the various forms of ocular scaffolds being used in stem cell research, we direct the reader to Kador and Goldberg's 'Scaffolds and stem cells: delivery of cell transplants for retinal degenerations' [87].…”

“…For example, there are many obstacles in successfully co-culturing RGCs with superior colliculi explants in a 2D system, while attempting to study axonal growth guided by 3D signaling gradients and mechanical boundaries. Although techniques have been designed to bridge the gap between the artificial arrangement of co-cultured tissues and their in vivo counterparts, such as NASA's HRB, electrospun biodegradable scaffolding and ex vivo perfused human outflow pathway models, the field is lacking reliable substrates/ techniques for accurate modeling of the proposed co-culture systems [46,[58][59][60][61]85,86,88]. Even the complex 3D retina or optic nerve models that we described and proposed for future experiments are not physiologically representative of an in vivo system due to various reasons.…”

The Potential of Human Stem Cells for the Study and Treatment of Glaucoma

“…Additionally, it may be possible to engineer artificial scaffolds to guide RGC axons to their proper brain targets. 69,70 Through combinatorial approaches involving genetic engineering of pluripotent stem cells, differentiation of such cells into RGCs, and the incorporation of bioengineering guidance scaffolds, it may be possible to direct stem cell–derived RGCs to integrate into the retina, repopulate the optic nerve with new axons, and then reach and synapse with brain targets to make visual recovery possible. Although there have not yet been any reports of stem cell–derived RGC axon growth into the optic nerve, a parallel experiment has been reported describing mouse embryo–derived retinal precursor cell transplantation into the adult mouse, demonstrating a limited but clear donor RGC contribution to the optic nerve.…”

Control of Retinal Ganglion Cell Positioning and Neurite Growth: Combining 3D Printing with Radial Electrospun Scaffolds

“…7 The addition of guidance factor gradients on the surface of tissueengineered scaffolds enhanced the polarization of axon growth toward the optic nerve head, mimicking the physiology of axon guidance found during early retinal devel-opment. 8 These methods, however, have not been able to recreate the more complex organization of the ganglion cell layer (GCL) and retinal nerve fiber layer (RNFL) around the fovea, thus limiting their clinical potential.…”