Osakada, F. et al. In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. J. Cell Sci. 122, 3169-3179

Laboratory for Retinal Regeneration, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe, Japan.
Journal of Cell Science (Impact Factor: 5.43). 09/2009; 122(Pt 17):3169-79. DOI: 10.1242/jcs.050393
Source: PubMed


The use of stem-cell therapy to treat retinal degeneration holds great promise. However, definitive methods of retinal differentiation that do not depend on recombinant proteins produced in animal or Escherichia coli cells have not been devised. Here, we report a defined culture method using low-molecular-mass compounds that induce differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into retinal progenitors, retinal pigment epithelium cells and photoreceptors. The casein kinase I inhibitor CKI-7, the ALK4 inhibitor SB-431542 and the Rho-associated kinase inhibitor Y-27632 in serum-free and feeder-free floating aggregate culture induce retinal progenitors positive for RX, MITF, PAX6 and CHX10. The treatment induces hexagonal pigmented cells that express RPE65 and CRALBP, form ZO1-positive tight junctions and exhibit phagocytic functions. Subsequent treatment with retinoic acid and taurine induces photoreceptors that express recoverin, rhodopsin and genes involved in phototransduction. Both three-factor (OCT3/4, SOX2 and KLF4) and four-factor (OCT3/4, SOX2, KLF4 and MYC) human iPS cells could be successfully differentiated into retinal cells by small-molecule induction. This method provides a solution to the problem of cross-species antigenic contamination in cell-replacement therapy, and is also useful for in vitro modeling of development, disease and drug screening.

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    • "Accordingly, 30% of ESC/iPSC differentiate into retinal progenitors(Ikeda et al., 2005), a number that increases to 80% for both ESC (Lamba et al., 2006) and iPSC(Tucker et al., 2011) by incorporating BMP/Wnt inhibiton with IGF and FGF treatments. These ESC/iPSC-derived retinal progenitors successfully mature into photoreceptors as well as RPE cells and integrate into retinal explants after co-culture with adult retina/retinal neurons(Osakada et al., 2008) or after the addition of a cocktail of small molecules(Osakada et al., 2009b). Comparisons of the gene expression profiles of ESC-derived retinal cells with primary developing foetal retinal cells using microarray analysis shows them to be highly conserved between the two cell sources throughout development(Lamba and Reh, 2011). "
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    ABSTRACT: Stem cell therapies are being explored extensively as treatments for degenerative eye disease, either for replacing lost neurons, restoring neural circuits or, based on more recent evidence, as paracrine-mediated therapies in which stem cell-derived trophic factors protect compromised endogenous retinal neurons from death and induce the growth of new connections. Retinal progenitor phenotypes induced from embryonic stem cells/induced pluripotent stem cells (ESCs/iPSCs) and endogenous retinal stem cells may replace lost photoreceptors and retinal pigment epithelial (RPE) cells and restore vision in the diseased eye, whereas treatment of injured retinal ganglion cells (RGCs) has so far been reliant on mesenchymal stem cells (MSC). Here, we review the properties of non-retinal-derived adult stem cells, in particular neural stem cells (NSCs), MSC derived from bone marrow (BMSC), adipose tissues (ADSC) and dental pulp (DPSC), together with ESC/iPSC and discuss and compare their potential advantages as therapies designed to provide trophic support, repair and replacement of retinal neurons, RPE and glia in degenerative retinal diseases. We conclude that ESCs/iPSCs have the potential to replace lost retinal cells, whereas MSC may be a useful source of paracrine factors that protect RGC and stimulate regeneration of their axons in the optic nerve in degenerate eye disease. NSC may have potential as both a source of replacement cells and also as mediators of paracrine treatment. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Feb 2015 · Stem Cell Research
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    • "The iPSCs were differentiated to RPE according to the protocol established by Dr. Osakada (Osakada et al., 2009). The iPSCs clumps were first incubated in human ES cell culture medium supplemented with 10 μM Y-27632 (WAKO), 5 μM SB431542 (Sigma–Aldrich) and 3 μM CKI-7 (Sigma– Aldrich) for 1 day. "
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    ABSTRACT: Age-related macular degeneration (AMD) is one retinal aging process that may lead to irreversible vision loss in the elderly. Its pathogenesis remains unclear, but oxidative stress inducing retinal pigment epithelial (RPE) cells damage is perhaps responsible for the aging sequence of retina and may play an important role in macular degeneration. In this study, we have reprogrammed T cells from patients with dry type AMD into induced pluripotent stem cells (iPSCs) via integration-free episomal vectors and differentiated them into RPE cells that were used as an expandable platform for investigating pathogenesis of the AMD and in-vitro drug screening. These patient-derived RPEs with the AMD-associated background (AMD-RPEs) exhibited reduced antioxidant ability, compared with normal RPE cells. Among several screened candidate drugs, curcumin caused most significant reduction of ROS in AMD-RPEs. Pre-treatment of curcumin protected these AMD-RPEs from H2O2-induced cell death and also increased the cytoprotective effect against the oxidative stress of H2O2 through the reduction of ROS levels. In addition, curcumin with its versatile activities modulated the expression of many oxidative stress-regulating genes such as PDGF, VEGF, IGFBP-2, HO1, SOD2, and GPX1. Our findings indicated that the RPE cells derived from AMD patients have decreased antioxidative defense, making RPE cells more susceptible to oxidative damage and thereby leading to AMD formation. Curcumin represented an ideal drug that can effectively restore the neuronal functions in AMD patient-derived RPE cells, rendering this drug an effective option for macular degeneration therapy and an agent against aging-associated oxidative stress.
    Full-text · Article · Aug 2014 · Frontiers in Aging Neuroscience
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    • "Furthermore, the RPE layer is situated adjacent to the vestigial embryonic retinal ventricular space, and the relative ease of generating a transient retinal detachment at this site has been routinely exploited in the subretinal delivery of donor graft materials and other therapeutics. The RPE carries out a number of complex functions that are critical for maintenance of photoreceptor health and activity (Strauss, 2005) and, as mentioned previously, many of these functions have been recapitulated in vitro by RPE derived from hESCs and hiPSCs (Kokkinaki et al., 2011; Meyer et al., 2011; Osakada et al., 2009; Singh et al., 2013b). "
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    ABSTRACT: Human pluripotent stem cells have made a remarkable impact on science, technology and medicine by providing a potentially unlimited source of human cells for basic research and clinical applications. In recent years, knowledge gained from the study of human embryonic stem cells and mammalian somatic cell reprogramming has led to the routine production of human induced pluripotent stem cells (hiPSCs) in laboratories worldwide. hiPSCs show promise for use in transplantation, high throughput drug screening, "disease-in-a-dish" modeling, disease gene discovery, and gene therapy testing. This review will focus on the first application, beginning with a discussion of methods for producing retinal lineage cells that are lost in inherited and acquired forms of retinal degenerative disease. The selection of appropriate hiPSC-derived donor cell type(s) for transplantation will be discussed, as will the caveats and prerequisite steps to formulating a clinical Good Manufacturing Practice (cGMP) product for clinical trials.
    Full-text · Article · Jun 2014 · Experimental Eye Research
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