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, ...

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self-forming neuroretinal-like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation-compatible CD73 photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC-derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73 photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP-compliant retinal cell manufacturing protocol allowing large-scale production and banking of hiPSC-derived retinal cells and tissues. Stem Cells 2017;35:1176-1188.

SummaryPhotoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.

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Parkinsonian symptoms are currently thought to be related to hyperactivity of the subthalamic nucleus (STN). Because the STN is known to receive many inputs including glutamatergic cortical afferent fibers, we sought to determine whether the activity of this pathway is altered after dopaminergic denervation to estimate its contribution to the impairment of STN activity. A precise mapping of the origin of the corticosubthalamic projection was first performed using retrograde and anterograde tracing methods. Cortical neurons projecting to the STN were found to originate in layer V of the motor, anterior cingulate, and dorsal insular cortices, and the most anterior tip of the frontal lobe, leading to different functional corticosubthalamic inputs. The metabolic activity of the neurons projecting to the STN, first identified by retrograde tracing, was then evaluated by in situ hybridization of the first subunit of cytochrome oxidase (COI), a marker of metabolic activity, in unilateral 6-hydroxydopamine-lesioned rats. Measurements of COI mRNA expression showed a 38 and 41.5% decrease after dopaminergic denervation in the neurons projecting to the STN located in the motor and dorsal insular areas, respectively, whereas neuronal activity was mildly changed in neurons of the anterior cingulate cortex. The modified activity of STN neurons in parkinsonism may thus result in part from complex interactions between glutamatergic hyperactive fibers originating in the thalamus and the pedunculopontine nucleus and hypoactive fibers originating in the cerebral cortex.

The reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) has broad applications in regenerative medicine. The generation of self-organized retinal structures from these iPSCs offers the opportunity to study retinal development and model-specific retinal disease with patient-specific iPSCs and provides the basis for cell replacement strategies. In this study, we demonstrated that the major type of glial cells of the human retina, Müller cells, can be reprogrammed into iPSCs that acquire classical signature of pluripotent stem cells. These Müller glial cell-derived iPSCs were able to differentiate toward retinal fate and generate concomitantly retinal pigmented epithelial cells and self-forming retinal organoid structures containing retinal progenitor cells. Retinal organoids recapitulated retinal neurogenesis with differentiation of retinal progenitor cells into all retinal cell types in a sequential overlapping order. With a modified retinal maturation protocol characterized by the presence of serum and high glucose levels, our study revealed that the retinal organoids contained pseudolaminated neural retina with important features reminiscent of mature photoreceptors, both rod and cone subtypes. This advanced maturation of photoreceptors not only supports the possibility to use 3D retinal organoids for studying photoreceptor development but also offers a novel opportunity for disease modeling, particularly for inherited retinal diseases.