From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

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.retinal ganglion cells | rods | cones I rreversible 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, ...

Induced Pluripotent Stem Cell Therapies for Degenerative Disease of the Outer Retina: Disease Modeling and Cell Replacement

“…30 To avoid the presence of animal derivatives (animal-derived sera, or in most cases Matrigel), a chemically defined 3D retinal differentiation medium was recently reported, which only contains nicotinamide and 20 ng/mL of basic fibroblast growth factor. 121 To date, efforts to speed up the differentiation protocols 31,122,123 using molecules known to promote retinal differentiation 31,118,123 or by passing the EB formation 122 have generally reduced the survival of differentiated cells, 31,123 and/or the stratification obtained. 122 Whether correct lamination of pluripotent stem cell-derived donor retinal tissue is required for generation of transplantationcompetent human photoreceptor cells remains to be resolved.…”

“…126 Another important step for clinical cell therapy is the purification of the cell population of interest from the differentiated stem cell cultures, which typically contain multiple cell types. 30,115,122 For RPE cell transplantation, it is possible to grow monolayers of RPE free from contaminating cells. For photoreceptor transplantation, photoreceptor cells suitable for transplantation might be best produced in synthetic 3D optic vesicles containing other retinal neurons and RPE.…”

“…The cell surface markers used to sort murine rod precursors show similar expression patterns in the developing human retina and so could help to enhance the photoreceptor yield from the iPSC retinal differentiations, as it did with the mESC. 128,129 Human iPSC-derived photoreceptors are reported to express CD73 at day 42 of differentiation, 122 and based on transcriptomic analysis, other potentially useful surface markers such as Cacna2d4, Kcnv2, and Cnga1 have been identified. 131 …”

Engineering Cardiovascular Regeneration

“…Pluripotency describes the ability of a cell to differentiate into all cell types of the three germ layers (endoderm, ectoderm, mesoderm) and their derivations but not extraembryonic trophoblast, including retina cells [33], neuronal cells, embryonic erythrocytes, or cardiomyocytes among others (review in [34]). …”

Production de rétinesin vitroà partir de cellules pluripotentes humaines

“…Ces cellules peuvent être facilement amplifiées tout en conservant leur phénotype correspondant à leur état in vivo. La création rapide de banques de cellules de l'EPR destinées au traitement futur de la DMLA et d'autres maladies liées à l'EPR est donc tout à fait envisaaugmente la proportion de précurseurs de photorécepteurs dans les rétines in vitro, en forçant les progéniteurs réti-niens à quitter précocement le cycle de division cellulaire [4]. Ces précurseurs produits à l'aide de notre protocole expriment spécifiquement l'antigène de surface CD73 ( Figure 1C).…”