Abstract Background Retinal ganglion cell (RGCs) loss underlies several retinal degenerative diseases, including Glaucoma and NF1-OPG. RGCs are essential cells in the visual pathway. These cells transmit the signal gathered from the retinal photoreceptors to the brain via the optic nerve and loss of these cells leads to permanent loss of vision. Presently there is no treatment, however, in the last decade cell replacement has shown potential to restore vision in these conditions. Materials and Methods In this study, we have established protocol with a dose dependent response differentiating porcine retinal progenitors (pRPCs) into retinal ganglion cells (RGC) with DAPT and Forskolin treatments. Cells were initially treated with a known concentration of DAPT and Forskolin that has been widely used to culture RGCs. Results We identify the optimal concentration and time course that yields the highest expression of RGCs, pRPCs were cultured for 4 weeks in 25 different concentrations of small molecules. At week 2 and 3 approximately 40% of the pRPCs population expressed the neuronal marker TUJ1 and RGCs markers such as RBPMS and Thy1.1. Differentiated cells showed the lowest proliferative index as indicated by Ki-67 expression. Conclusion This data demonstrates that specific concentrations of DAPT and Forskolin can induce RPCs to undergo differentiation into RGCs linage.

Engineering matrices for cell therapy requires design criteria that include the ability of these materials to support, protect and enhance cellular behavior in vivo. The chemical and mechanical formulation of the biomaterials can influence not only target cell phenotype but also cellular differentiation. In this study, we have demonstrated the effect of a gelatin (Gtn)—hyaluronic acid (HA) hydrogel on human retinal progenitor cells (hRPCs) and show that by altering the mechanical properties of the materials, cellular behavior is altered as well. We have created an interpenetrating network polymer capable of encapsulating hRPCs. By manipulating the stiffness of the hydrogel, the differentiation potential of the hRPCs was controlled. Interpenetrating network 75 (IPN 75; 75% HA) allowed higher expression of rod photoreceptor markers, whereas cone photoreceptor marker expression was found to be higher in IPN 50. In vivo testing of these living matrices performed in Long–Evans rats showed higher levels of rod photoreceptor marker expression when IPN 75 was injected versus IPN 50. These biomaterials mimic biological cues that are required to simulate the dynamic complexity of natural retinal ECM. These hydrogels can be used as a vehicle for cell delivery in vivo as well as for expansion and differentiation in an in vitro 3D system in a highly reproducible manner.

Despite notable efforts and significant therapeutical advances, age-related macular degeneration remains the single most common reason for vision loss. Retinal progenitor cells (RPCs) are considered promising candidates for cellular treatments that repair and restore vision. In this allogenic study, the phenotypic profile of pig and human RPCs derived using similar manufacturing processes is compared. The long-term (12-week) survival of green fluorescent protein-pig retinal progenitor cells GFP-pRPC after subretinal transplantation into normal miniature pig (mini-pig) retina is investigated. Human eyes are both anatomically and physiologically mimicked by pig eyes, so the pig is an ideal model to show an equivalent way of delivering cells, immunological response and dosage. The phenotypic equivalency of porcine and clinically intended human RPCs was established. Thirty-nine mini-pigs are used in this study, and vehicle-injected eyes and non-injected eyes serve as controls. Six groups are given different dosages of pRPCs, and the cells are found to survive well in all groups. At 12 weeks, strong evidence of integration is indicated by the location of the grafted cells within the neuro-retina, extension of processes to the plexiform layers and expression of key retinal markers such as recoverin, rhodopsin and synaptophysin. No immunosuppression is used, and no immune response is found in any of the groups. No pRPC-related histopathology findings are reported in the major organs investigated. An initial dose of 250 k cells in 100 µl of buffer is established as an appropriate initial dose for future human clinical trials.

Zwitterionic surfaces are increasingly explored as antifouling coatings due to their propensity to resist protein, bacterial, and cell adhesion and are typically applied as polymeric systems. Here, the self-assembly of strongly interacting small molecule amphiphiles is reported to produce nanoribbons for antifouling applications. Synthesized amphiphiles spontaneously form micrometers-long nanoribbons with nanometer-scale cross-sections and intrinsically display a dense coating of zwitterionic moieties on their surfaces. Substrates coated with nanoribbons demonstrate concentration-dependent thicknesses and near superhydrophilicity. These surface coatings are then probed for antifouling properties and substantial reductions are demonstrated in protein adsorption, bacterial biofilm formation, and cell adhesion relative to uncoated controls. Harnessing cohesive small molecule self-assembling nanomaterials for surface coatings offers a facile route to effective antifouling surfaces.

Biomaterial-based cell replacement approaches to regenerative medicine are emerging as promising treatments for a wide array of profound clinical problems. Here we report an interpenetrating polymer network (IPN) composed of gelatin-hydroxyphenyl propionic acid and hyaluronic acid tyramine that is able to enhance intravitreal retinal cell therapy. By tuning our bioinspired hydrogel to mimic the vitreous chemical composition and mechanical characteristics we were able to improve in vitro and in vivo viability of human retinal ganglion cells (hRGC) incorporated into the IPN. In vivo vitreal injections of cell-bearing IPN in rats showed extensive attachment to the inner limiting membrane of the retina, improving with hydrogels stiffness. Engrafted hRGC displayed signs of regenerating processes along the optic nerve. Of note was the decrease in the immune cell response to hRGC delivered in the gel. The findings compel further translation of the gelatin-hyaluronic acid IPN for intravitreal cell therapy.

Many progenitor and stem cell-based therapies in pre-clinical development and in clinical trial have failed to achieve desired outcomes, in part, due to: low cell viability; dispersion of cells from the injection site; and/or failure of the cells to differentiate and engraft into host tissue. These problems may be exacerbated by the elevated shear stress applied to the cells as they pass through a small-bore needle. Our supposition is that these issues can be mitigated by replacing the phosphate buffered saline (PBS), which is likely employed as the cell carrier, with an injectable gelatin-based hydrogel incorporating an appropriate regulatory molecule (human epidermal growth factor). The first objectives of the study were to evaluate in vitro: the effects of injection through a 31-gauge needle on viability, proliferation, and phenotypic expression of human retinal progenitor cells (hRPCs), when employing PBS as the carrier and compare it to a gelatin-based gel as a carrier. The second objective of the work, conducted in vivo, in a rat model, was to: determine the degree of engraftment of injected cells; and evaluate markers of an immune response to the xenogeneic cells.Hydroxyphenyl propionic acid was conjugated to gelatin (Gtn-HPA) to enable in situ, enzyme-mediated covalent cross-linking, with independent control over the gelation time and degree of cross-linking. After 1 and 6 culture days, hRPC exposed to shear stress showed 50% or more reduction in viability of cells in PBS compared to cells in medium and in gel. Shear stress decreased proliferation and increased apoptosis in the PBS group by 50% or more, while cells in the gel group were protected from shear-induced apoptosis and their proliferation remained 2-fold higher than the PBS group. Also, of note was the preservation of or increase in stemness markers in the gel with hEGF group compared to cells in PBS and the gel alone, with or without shear stress. In vivo studies, performed 3 days after subretinal injection into non-immunosuppressed rats revealed: a) a greater number of cells at the injection site in the gel group, with signs of engraftment of cells to the retina; and b) a significantly lower immune response in the gel group, compared to cells injected in PBS.This work demonstrates that the shear stress experienced by hRPCs in PBS as a result of injection through a small-bore needle adversely affects cell viability, proliferation, apoptosis, phenotype, in vivo retention of cells at the injection site, migration, engraftment and immune reaction. These findings suggest the use of a biomaterial hydrogel as a replacement for PBS for retinal cell therapy.

Using stem and progenitor cells to treat retinal disorders holds great promise. Using defined culture conditions to maintain the desires phenotype is of utmost clinical importance. We cultured human retinal progenitor cells (hRPCs) in different conditions: such as normoxia (20% oxygen), and hypoxia (5% oxygen) with and without knock-out serum replacement (KOSR) to evaluate its effect on these cells. KOSR is known nutrient supplement often used to replace bovine serum for culturing embryonic or pluripotent stem cells, especially those destined for clinical applications. The purpose of this study was to identify the impact of different environmental and chemical cues to determine if this alters the fate of these cells. Our results indicate that cells cultured with or without KOSR do not show significant differences in viability, but that the oxygen tension can significantly change their viability (higher in hypoxia than normoxia). However, cells with KOSR in hypoxia condition expressed significantly higher stemness markers such as C-myc and Oct4 (31.20% and 13.44% respectively) in comparison to hRPCs cultured in KOSR at normoxia (12.07% and 4.05%). Furthermore, levels of markers for retinal commitment such as rhodopsin were significantly lower in the KOSR supplemented cells in hypoxia culture compared to normoxia. KOSR is known to improve proliferation and maintain stemness of embryonic cells and our experiments suggest that hRPCs maintain their proliferation and stemness characteristics in hypoxia with KOSR supplement. Normoxia, however, results in mature cell marker expression, suggesting a profound effect of oxygen tension on these cells.

The optic pathway glioma (OPG) is a slow-growing brain tumor that arises along the optic nerve or its downstream connections and causing vision to gradually worsen with time. This tumor forms in children with a genetic condition called neurofibromatosis type 1 (NF1), causing tumors to grow on nerves. In normal conditions, glial cells are there to support and protect nerve cells but, in NF1-OPG, glial cells have a genetic defect and grow out of control forming a tumor called a glioma. There are no rat models of NF1-OPG that can be used to explore various treatment options, and mouse models make interventional studies difficult due to their small eye size. We have created a model in which to study the progression of tumor growth in the optic nerve and establish the anatomical and functional consequences of the model and determine its suitability to serve as a surrogate for human disease. C6 rat glioma cells were injected into the optic nerve of Long-Evans rats and allowed to proliferate for 2 weeks. The eye clearly showed proptosis and lens opacity was observed, likely due to increased intraocular pressure caused by growing tumors. Hematoxylin–eosin staining showed marked cellularity, with hyperchromatism and pleomorphism. There was prominent area of necrosis with neoplastic cells palisading around the penumbra. Immunostaining with markers such as S100, β-tubulin III, Foxp3, CD45, Vimentin, and Ki67 confirmed low-grade tumor formation, with a mild immune response. Our results show the utility of a surgically induced rat model of OPG that may be used for exploring various treatment options for NF1 ocular tumors.