| Abstract: | Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.Irreversible blindness caused by retinal diseases, such as inherited retinopathies, age-related macular degeneration (AMD), or glaucoma, is mainly due to the impairment or loss of function of photoreceptor cells, supporting retinal pigmented epithelium (RPE) or retinal ganglion cells (RGCs). Rescuing the degenerated retina is a major challenge for which specific cell replacement is one of the most promising approaches (1, 2). Pluripotent stem cells, like human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs), have the ability to be expanded indefinitely in culture and could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases (3, 4). Several publications have indicated that hESCs and hiPSCs can be differentiated into RPE cells spontaneously after fibroblast growth factor (FGF) 2 removal (5–7) or by different floating aggregate methods (8–11). Concerning neural retinal cells, a growing body of convergent data has demonstrated the ability of hESCs or hiPSCs to be committed into the neural retinal lineage and further differentiated into cells expressing photoreceptor markers (12–15). Recent innovative approaches using 3D cultures from embryoid bodies (EBs) of hESCs or hiPSCs allowed the self-formation of optic cup (OC) structures (16) or the generation of optic vesicle (OV)-like structures (17), depending on the addition of exogenous molecules and different substrates used. These protocols require multiple steps and trained handling, which are not always compatible with the manufacturing process for therapeutic approach or drug screening that need a large-scale production of cells of interest. Therefore, very simple and reliable approaches minimizing the use of exogenous molecules should be developed to generate hESCs or hiPSC-derived retinal cells.In the present study, we report a new retinal differentiation process using confluent hiPSCs, without cell clumps or EB formation and in the absence of Matrigel or serum. We demonstrate that integration-free hiPSCs derived from adult human dermal fibroblasts (AHDFs) cultured in proneural medium can simultaneously generate RPE cells and self-forming neural retinal (NR)-like structures within 2 wk and that, when switched to floating cultures, structures containing retinal progenitor cells (RPCs) can differentiate into all retinal cell types, including RGCs and precursors of photoreceptors, needed for therapeutic applications. |
