Enhanced differentiation and delivery of mouse retinal progenitor cells using a micropatterned biodegradable thin-film polycaprolactone scaffold.

The deterioration of retinal tissue in advanced stages of retinitis pigmentosa and age-related macular degeneration and the lack of signaling cues for laminar regeneration are significant challenges highlighting the need for a tissue engineering approach to retinal repair. In this study, we fabricated a biodegradable thin-film polycaprolactone (PCL) scaffold with varying surface topographies using microfabrication techniques. Mouse retinal progenitor cells (mRPCs) cultured on PCL scaffolds exhibited enhanced potential to differentiate toward a photoreceptor fate in comparison to mRPCs cultured on control substrates, suggesting that PCL scaffolds are promising as substrates to guide differentiation of mRPCs toward a photoreceptor fate in vitro before transplantation. When cocultured with the retinal explants of rhodopsin null mice, mRPC/PCL constructs showed increased mRPC integration rates compared to directly applied dissociated mRPCs. Moreover, these mRPC/PCL constructs could be delivered into the subretinal space of rhodopsin null mice with minimal disturbance of the host retina. Whether cocultured with retinal explants or transplanted into the subretinal space, newly integrated mRPCs localized to the outer nuclear layer and expressed appropriate markers of photoreceptor fate. Thus, the PCL scaffold provides a platform to guide differentiation and organized delivery of mRPCs as a practical strategy to repair damaged retina.

Low-oxygen culture conditions extend the multipotent properties of human retinal progenitor cells.

PURPOSE: Development of an effective cell-based therapy is highly dependent upon having a reproducible cell source suitable for transplantation. One potential source, isolated from the developing fetal neural retina, is the human retinal progenitor cell (hRPC). One limiting factor for the use of hRPCs is their in vitro expansion limit. As such, the aim of this study was to determine whether culturing hRPCs under 3% O2 would support their proliferative capacity while maintaining multipotency. METHODS: To determine the effect of low oxygen on the ability of hRPCs to self-renew, rates of proliferation and apoptosis, telomerase activity, and expression of proliferative, stemness, and differentiation markers were assessed for hRPCs cultured in 3% and 20% oxygen conditions. RESULTS: Culture under 3% oxygen increases the proliferation rate and shifts the proliferation limit of hRPCs to greater 40 divisions. This increased capacity for proliferation is correlated with an upregulation of Ki67, CyclinD1, and telomerase activity and a decrease in p53 expression and apoptosis. Increased expression of cMyc, Klf4, Oct4, and Sox2 in 3% O2 is correlated with stabilization of both HIF1α and HIF2α. The eye field development markers Pax6, Sox2, and Otx2 are present in hRPCs up to passage 16 in 3% O2 . Following in vitro differentiation hRPCs expanded in the 3% O2 were able to generate specialized retinal cells, including rods and cones. CONCLUSIONS: Low-oxygen culture conditions act to maintain both multipotency and self-renewal properties of hRPCs in vitro. The extended expansion limits permit the development of a clinical-grade reagent for transplantation.