title: Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions.
abstract: 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.
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Sacha
authors.initials: S
authors.lastname: Reichman
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Amélie
authors.initials: A
authors.lastname: Slembrouck
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Gagliardi
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Chaffiol
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Angélique
authors.initials: A
authors.lastname: Terray
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Nanteau
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Anais
authors.initials: A
authors.lastname: Potey
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Morgane
authors.initials: M
authors.lastname: Belle
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Oriane
authors.initials: O
authors.lastname: Rabesandratana
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Jens
authors.initials: J
authors.lastname: Duebel
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Gael
authors.initials: G
authors.lastname: Orieux
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Emeline F
authors.initials: EF
authors.lastname: Nandrot
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.|Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 1423, Paris, France.
authors.forename: José-Alain
authors.initials: JA
authors.lastname: Sahel
authors.affiliation: Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Goureau
mesh_terms: D002448:Cell Adhesion; D002454:Cell Differentiation; D002460:Cell Line; D015925:Cryopreservation; D061252:Feeder Cells / Q000166:cytology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D009940:Organoids / Q000166:cytology* / Q000648:ultrastructure; D010786:Photoreceptor Cells / Q000166:cytology; D011309:Preservation, Biological; D055213:Retinal Pigment Epithelium / Q000166:cytology*
keywords: Cellular therapy; Induced pluripotent stem cells; Retinal photoreceptors; Retinal pigmented epithelium