Abstract
A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina in vitro using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-differentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.
Highlights
A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy
By chance, we added feeder cells together with dissociated Ff-hiPSCs on day 0, we found that the added mouse embryonic fibroblasts (MEFs) improved aggregate growth in 3D-differentiation culture (Fig. S1a,b)
We reasoned that the initial state of Ff-hiPSCs may be different from that of human pluripotent stem cells (hPSCs) on feeder cells and hypothesized that addition of certain signaling molecules to the culture medium around day 0 may bias the initial state of Ff-hiPSCs toward a state suitable for 3D-differentiation culture
Summary
A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. The self-organizing stem cell culture technique developed in these studies, named SFEBq (serum-free floating culture of embryoid body-like aggregates with quick aggregation), is used to generate 3D-organoids as an attractive model for recapitulating mouse and human organogenesis in vitro[24,25,26]. Human 3D-retina, generated from hESCs using the BMP method and the induction-reversal culture method, differentiated into rod and cone photoreceptors that developed a structured outer nuclear layer in a primate model of severe retinal degeneration[38]. We recently published another paper about functional maturation of human 3D-retina transplantation in the end-stage retinal degeneration model mice[39] These studies demonstrate the potential of hPSC-derived 3D-retina as a graft source for retinal transplantation therapy
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