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      Accelerated and Improved Differentiation of Retinal Organoids from Pluripotent Stem Cells in Rotating-Wall Vessel Bioreactors

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          Summary

          Pluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV) bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25) reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.

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          Highlights

          • Optimization of improved retinal organogenesis using bioreactors

          • Better growth and differentiation of retinal neurons in bioreactor organoid culture

          • Recapitulation of in vivo retina development in bioreactor organoids

          • Requirement of additional factors for functional maturation of the retina in vitro

          Abstract

          Swaroop and colleagues demonstrate that rotating-wall vessel bioreactor culture provides a favorable environment for improved growth and differentiation of retinal organoids, closely recapitulating early stages of development in vivo.

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          Most cited references43

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          Stem cells and niches: mechanisms that promote stem cell maintenance throughout life.

          Niches are local tissue microenvironments that maintain and regulate stem cells. Long-predicted from mammalian studies, these structures have recently been characterized within several invertebrate tissues using methods that reliably identify individual stem cells and their functional requirements. Although similar single-cell resolution has usually not been achieved in mammalian tissues, principles likely to govern the behavior of niches in diverse organisms are emerging. Considerable progress has been made in elucidating how the microenvironment promotes stem cell maintenance. Mechanisms of stem cell maintenance are key to the regulation of homeostasis and likely contribute to aging and tumorigenesis when altered during adulthood.
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            Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.

            The potential to generate virtually any differentiated cell type from embryonic stem cells (ESCs) offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. To realize this potential, it is essential to be able to control ESC differentiation and to direct the development of these cells along specific pathways. Embryology has offered important insights into key pathways regulating ESC differentiation, resulting in advances in modeling gastrulation in culture and in the efficient induction of endoderm, mesoderm, and ectoderm and many of their downstream derivatives. This has led to the identification of new multipotential progenitors for the hematopoietic, neural, and cardiovascular lineages and to the development of protocols for the efficient generation of a broad spectrum of cell types including hematopoietic cells, cardiomyocytes, oligodendrocytes, dopamine neurons, and immature pancreatic beta cells. The next challenge will be to demonstrate the functional utility of these cells, both in vitro and in preclinical models of human disease.
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              Feeder-free growth of undifferentiated human embryonic stem cells.

              Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.
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                Author and article information

                Contributors
                Journal
                Stem Cell Reports
                Stem Cell Reports
                Stem Cell Reports
                Elsevier
                2213-6711
                07 December 2017
                09 January 2018
                07 December 2017
                : 10
                : 1
                : 300-313
                Affiliations
                [1 ]Neurobiology, Neurodegeneration, and Repair Laboratory (N-NRL), National Eye Institute (NEI), National Institutes of Health, Bldg 6/338, 6 Center Drive, Bethesda, MD 20814, USA
                [2 ]Trans-NIH Shared Resources on Biomedical Engineering and Physical Sciences (BEPS), National Institutes of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health, Bldg 13/3N18B, 13 South Drive, Bethesda, MD 20814, USA
                [3 ]Signal Processing and Instrumentation Section, Center for Information Technology (CIT), National Institutes of Health, Bldg 12A/2021, 12 South Drive, Bethesda, MD 20814, USA
                Author notes
                []Corresponding author swaroopa@ 123456nei.nih.gov
                [4]

                Co-first author

                Article
                S2213-6711(17)30487-3
                10.1016/j.stemcr.2017.11.001
                5768666
                29233554
                d5800308-d631-4330-b8e5-c10f2a3bc873

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 27 July 2017
                : 2 November 2017
                : 3 November 2017
                Categories
                Resource

                ipsc,embryonic stem cell,in vitro organogenesis,retina development,3-d organoid culture,retinal disease,transcriptome,rna-seq,bioreactor

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