There is considerable interest in transplanting stem cells or progenitors into the injured nervous system and enhancing their differentiation into mature, integrated, functional neurons. Little is known, however, about what intrinsic or extrinsic signals control the integration of differentiated neurons, either during development or in the adult. Here we ask whether purified, postmitotic, differentiated retinal ganglion cells (RGCs) directly isolated from rat retina or derived from in vitro-differentiated retinal progenitor cells can survive, migrate, extend neurites, and form morphologic synapses in a host retina, in vivo and ex vivo. We found that acutely purified primary and in vitro-differentiated RGCs survive transplantation and migrate into deeper retinal layers, including into their normal environment, the ganglion cell layer (GCL). Transplanted RGCs from a wide range of developmental ages, but not from adults, were capable of extending lengthy neurites in the normal and injured adult rat retina ex vivo and to a lesser degree after transplantation in vivo. We have also demonstrated that RGCs may be differentiated and purified from retinal precursor cultures and that they share many of the same cell biological properties as primary RGCs. We have established that progenitor-derived RGCs have similar capacity for integration as developing primary RGCs but appear to form a lower number of presynaptic punctae. This work provides insight for further understanding of the integration of developing RGCs into their normal environment and following injury.

Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina. Using this scaffold we observed an increase in RGC survival and no significant change in their electrophysiological properties. When analyzed for alignment, 81% of RGCs were observed to project axons radially along the scaffold fibers, with no difference in alignment compared to the nerve fiber layer of retinal explants. When transplanted onto retinal explants, RGCs on ES scaffolds followed the radial pattern of the host retinal nerve fibers, whereas RGCs transplanted directly grew axons in a random pattern. Thus, the use of this scaffold as a cell delivery device represents a significant step towards the use of cell transplant therapies for the treatment of glaucoma and other retinal degenerative diseases.

What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice , and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor in regulating RGC fate, and further describe a newly identified, Sox4-regulated site for post-translational modification with small ubiquitin-related modifier (SUMOylation) in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies. Glaucoma is the most common cause of blindness worldwide and, along with other optic neuropathies, is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in ∼14% of all diagnosed patients. Differentiated and transplanted RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby to restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)-to-RGC and human stem cell-to-RGC fate specification, and take a significant step toward understanding neuronal and retinal development and ultimately cell-transplant therapy.

Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs) after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα) is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP) after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury.

The central nervous system (CNS) consists of complex groups of individual cells that receive electrical, chemical, and physical signals from their local environment. Standard in vitro cell culture methods rely on two-dimensional (2D) substrates that poorly simulate in vivo neural architecture. Neural cells grown in three-dimensional (3D) culture systems may provide an opportunity to study more accurate representations of the in vivo environment than 2D cultures. Furthermore, each specific type of neuron depends on discrete compositions and physical properties of their local environment. Previously, we developed a library of hydrogels composed of poly(ethylene glycol) (PEG) and poly(L-lysine) (PLL) which exhibit a wide range of mechanical properties. Here, we identified specific scaffolds from this library that readily support the survival, migration and neurite outgrowth of purified retinal ganglion cells (RGCs) and amacrine cells (ACs). These data address important biological questions about the interaction of neurons with the physical and chemical properties of their local environment and provide further insight for engineering neural tissue for cell-replacement therapies following injury.