Abstract
We generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells. After development of the optic vesicle from the induced stem cell embryoid body in three-dimensional culture, conversion to two-dimensional culture, achieved by supplementation with BDNF, resulted in differentiation of RGCs at a rate of nearly 90% as indicated by a marginal subregion of an extruded clump of cells, suggesting the formation of an optic vesicle. Axons extended radially from the margin of the clump. Induced RGCs expressed specific markers, such as Brn3b and Math5, as assessed using by quantitative PCR and immunohistochemistry. The long, prominent axons contained neurofilaments and tau and exhibited anterograde axonal transport and sodium-dependent action potentials. The ability to generate RGCs with functional axons uniformly and at a high rate may contribute to both basic and clinical science, including embryology, neurology, pathognomy, and treatment of various optic nerve diseases that threaten vision.
Highlights
We generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells
For purposes of generating RGCs, we modified a previously established17 3D retinal regeneration protocol (Fig. 1). This method called for addition of both foetal bovine serum (FBS) and Matrigel to the culture medium; these components were not required by other protocols[20,21], which produced retinal cells efficiently
We severed the optic vesicle (OV) from the embryoid bodies (EBs), which is predicted to lead to differentiation into neural retinal cells, as demonstrated previously[17]
Summary
We generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells. In subsequent experiments in 3D culture systems, the entire retinal layer, including the retinal ganglion cell layer and functional photoreceptors, was established from human iPSCs mainly using intrinsic cues[21] and addition of small amounts of exogenous factors, as reported in previous studies[22,23,24,25,26] In this experiment, an OV-like structure was directly differentiated into a retina without the process of optic cup formation. There is a critical difference between these two major www.nature.com/scientificreports methods; in the former, the OV-like structures were extruded from the embryoid body, while in the latter, aggregates of human iPSCs or ESCs were directly induced into OV-like structures In both methods[17,21], the entire retinal layer is regenerated, containing thick outer layers with well-differentiated photoreceptors and thin, immature inner layers comprised of RGCs. the formation of RGC axons has not been realised using either 3D retinogenesis method
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