Reprogramming of Adult Retinal Müller Glial Cells into Human-Induced Pluripotent Stem Cells as an Efficient Source of Retinal Cells

Hindawi Stem Cells International Volume 2019, Article ID 7858796, 13 pages https://doi.org/10.1155/2019/7858796 Research Article Reprogramming of Adult Retinal Müller Glial Cells into Human-Induced Pluripotent Stem Cells as an Efficient Source of Retinal Cells Amélie Slembrouck-Brec,1 Amélie Rodrigues,1 Oriane Rabesandratana,1 Giuliana Gagliardi,1 Céline Nanteau,1 Stéphane Fouquet,1 Gilles Thuret,2 Sacha Reichman,1 Gael Orieux,1 and Olivier Goureau 1 1 2 Sorbonne Université, INSERM, CNRS, Institut de la Vision, F-75012 Paris, France Biologie, Ingénierie et Imagerie de la Greffe de Cornée, EA2521, Faculté de Médecine, Université Jean Monnet, Saint-Etienne, France Correspondence should be addressed to Olivier Goureau; Received 4 April 2019; Accepted 9 June 2019; Published 15 July 2019 Academic Editor: Holm Zaehres Copyright © 2019 Amélie Slembrouck-Brec et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. 1. Introduction Human pluripotent stem cells (PSCs) represent a valuable tool to study human neuronal development and neurodegenerative diseases and to develop future stem cell-based therapies [1]. As concerns the retina, both human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been shown to be able to produce retinal cells [2–7], including retinal ganglion cells, photoreceptors, and retinal pigmented epithelial (RPE) cells, corresponding to the major cell types affected in the most common retinal degenerative diseases. The differentiation of human PSCs toward the retinal lineage has evolved considerably in the last few years, and several innovative protocols allowing the self-formation of 3D retinal organoids have been reported [8–16]. Many types of somatic cells, such as skin fibroblasts, blood cells, keratinocytes, or urine-derived cells, have been successfully used for reprogramming and the production of human iPSCs [1, 17]. Due to their high availability through noninvasive and routine sampling in clinical settings, blood and urine-derived cells have been considered as a preferred source for reprogramming. Nevertheless, independently of the initial somatic identity of reprogrammed cells, human iPSCs can be guided to differentiate into retinal organoids with relatively similar efficiency using different retinal differentiation protocols [11, 18–21]. Recently, Wang et al. reprogrammed four 2 different neuronal cell types (rod photoreceptors, cone photoreceptors, bipolar cells, and amacrine/horizontal cells) from early and late postnatal mouse retina into iPSCs and further differentiated them into retinal organoids [22]. Mouse Müller glial cells (MGCs), which originate from the same pool of retinal progenitors as retinal neurons, can also be reprogrammed to pluripotency with high efficiency and can be guided to differentiate into retinal organoids [22]. Even though no data regarding the reprogramming of human retinal cells has been reported, human glial cells such as astrocytes isolated from cerebellar tissue can be efficiently reprogrammed into iPSCs and further differentiated into different neural cell types [23, 24]. Here, we report the generation of iPSCs from human MGCs. By applying our multistep retinal differentiation protocol [19], we differentiated human MGC-derived iPSCs into pseudolaminated retinal organoids that contained all major retinal cell types. 2. Materials and Methods 2.1. Human Postmortem Tissue and Müller Glial Cell Cultures. Postmortem eye tissues were collected within 24 h after death from bodies donated to science (Laboratory of Anatomy, Faculty of Medicine of St-Etienne, France) in accordance with the French bioethics law. Handling of donor tissues adhered to the tenets of the Declaration of Helsinki of 1975 and its 1983 revision in protecting donor confidentiality. The retina was dissected from globes by circumferential hemisection behind the ora serrata to gently remove the anterior segment including lens. The retina was carefully separated from the vitreous by transection of the papillary head and around the peripheral regions and transferred into a CO2-independent medium (Thermo Fisher Scientific). After removing the major blood vessels, the retina was chopped into small fragments (<2 mm2) and cells dissociated as previously described [25, 26] with some modifications. After 3 washes in Ringer’s solution (NaCl 155 mM; KCl 5 mM; CaCl2 2 mM; MgCl2 1 mM; NaH2PO4 2 mM; HEPES 10 mM; and glucose 10 mM), retinal fragments were dissociated with preactivated papain at 31.8 U/mg (Worthington) in Ringer’s solution during 30 min at 37°C. Digestion was arrested by the addition of 1 ml of DMEM (Thermo Fisher Scientific) containing 10% of fetal bovine serum (FBS) (Thermo Fisher Scientific) and 25 μg/ml of DNase1 (Sigma-Aldrich). The cells were homogeneously suspended with gentle up and down pipetting in prewarmed DMEM with 10% FBS and 10 μg/ml gentamycin (MGC-medium) and seeded in 6 cm dishes previously coated with Poly-D-Lysin at 2 μg/cm2 and Laminin at 1 μg/cm2 (Sigma-Aldrich). Dissociated cells were incubated at 37°C in a standard 5% CO2/95% air incubator, and medium was left unchanged for 3 to 5 days and then renewed every 2 to 3 days. By the end of the first week in culture, large flattened glial cells were observed with few neuron-like cells scattered on the glial surface. Mitotic glial cells became confluent within (...truncated)