Generation of retinal pigment epithelial cells from small molecules and OCT4-reprogrammed human induced pluripotent stem cells.

Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
pISSN (Impact Factor: 3.6). 02/2012; 1(2):96-109. DOI: 10.5966/sctm.2011-0057
Source: PubMed

ABSTRACT Autologous retinal pigment epithelium (RPE) grafts derived from induced pluripotent stem cells (iPSCs) may be used to cure blinding diseases in which RPE dysfunction results in photoreceptor degeneration. Four, two, and one factor-derived iPS (4F-, 2F-, and 1F-iPSCs, respectively) were differentiated into fully functional cuboidal shaped pigmented cells in polarized monolayers that express RPE-specific markers. 1F-iPS-RPE strongly resemble primary human fetal RPE (hfRPE) based on proteomic and untargeted metabolomic analyses, and, utilizing novel in vivo imaging technology coupled with electroretinography, we demonstrate that 1F-iPS-RPE mediate anatomical and functional rescue of photoreceptors after transplantation in an animal model of RPE-mediated retinal degeneration. 1F-iPS-RPE cells were injected subretinally as a suspension and formed a monolayer dispersed between host RPE cells. Furthermore, 1F-iPS-RPE do not simply provide trophic support to rescue photoreceptors as previously speculated, but actually phagocytose photoreceptor outer segments in vivo and restore visual cycling (based on high-resolution mass spectrometry based detection of recycled photoreceptor protein and lipid end products and electron microscopic analysis). Thus, 1F-iPS-RPE grafts may be superior to conventional iPS-RPE for clinical use since 1F-iPS-RPE closely resemble hfRPE, mediate anatomical and functional photoreceptor rescue in vivo and are generated using a reduced number of potentially oncogenic reprogramming factors.

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    ABSTRACT: Age-related macular degeneration (AMD) is the leading cause of legal blindness in older people in the developed world. The disease involves damage to the part of the retina responsible for central vision. Degeneration of retinal pigment epithelial (RPE) cells, photoreceptors, and choriocapillaris may contribute to visual loss. Over the past decades, scientists and clinicians have tried to replace lost RPE cells in patients with AMD using cells from different sources. In recent years, advances in generating RPE cells from stem cells have been made and clinical trials are currently evaluating the safety and efficiency of replacing the degenerated RPE cell layer with stem cell– derived RPE cells. However, the therapeutic success of transplantation of stem cell– derived RPE cells may be limited unless the transplanted cells can adhere and survive in the long term in the diseased eye. One hallmark of AMD is the altered extracellular environment of Bruch's membrane to which the grafted cells have to adhere. Here, we discuss recent approaches to overcome the inhibitory environment of the diseased eye and to enhance the survival rate of transplanted RPE cells. Our aim is to highlight novel approaches that may have the potential to improve the efficacy of RPE transplantation for AMD in the future.
    07/2014; 3(4). DOI:10.1167/tvst.3.4.4
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    ABSTRACT: To better characterize human retinal pigment epithelial (RPE) cells, their maturation was studied by time-lapse observation and immunostaining of the tight junction protein ZO-1. During subconfluency with active migration, the cells had an elongated shape. During cell division to reach confluency, RPE cells became small and tight, exhibiting cobblestone-like morphology. In addition, RPE maturation at the peripheral region of the culture vessel was delayed when compared with the central region, demonstrating local heterogeneity during maturation. To correlate cellular migration and maturation, we compared frequencies of migration rate and number of ZO-1-positive cells at the central and peripheral regions. Cells having migration rates less than 5.0 μm/h in the central region were 1.4-fold higher than in the peripheral region at day 5. Regardless of locational differences in the culture vessel, the frequency of cells having migration rates less than 5.0 μm/h showed 90% agreement with the frequency of ZO-1-positive cells. To inhibit cell migration, RPE cells were exposed to medium containing 50 μg/ml Rac1 inhibitor at day 5. Frequencies of ZO-1-positive cells and cells having migration rates less than 5.0 μm/h at the peripheral region were similar to those at the central region. The results show that migration is an important factor affecting maturation, and demonstrate that location heterogeneity during maturation is caused by different migratory behaviors in the culture vessel.
    Journal of Bioscience and Bioengineering 06/2014; DOI:10.1016/j.jbiosc.2014.05.025 · 1.79 Impact Factor
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    ABSTRACT: Purpose: Age-related macular degeneration (AMD) is a major leading cause of visual impairment and blindness with no cure currently established. Cell replacement of RPE is discussed as a potential therapy for AMD. Previous studies were performed in animal models with severe limitations in recapitulating the disease progression. We describe in detail the effect of systemic injection of sodium iodate in the mouse retina. We further evaluate the usefulness of this animal model to analyze cell-specific effects following transplantation of human embryonic stem cell (hESC)-derived RPE cells. Methods: Morphological, functional and behavioral changes following sodium iodate injection were monitored by histology, gene expression analysis, electroretinography and optokinetic head tracking. hESC-derived RPE cells were transplanted one week after sodium iodate injection and experimental retinae were analyzed three weeks later. Results: Injection of sodium iodate caused complete RPE cell loss, photoreceptor degeneration and altered gene and protein expression in outer and inner nuclear layers. Retinal function was severely affected by day 3 and abolished from day 14. Following transplantation, donor hESC-derived RPE cells formed extensive monolayers that displayed wild type RPE cell morphology, organization and function, including phagocytosis of host photoreceptor outer segments. Conclusions: Systemic injection of sodium iodate has considerable effects on RPE, photoreceptors and inner nuclear layer neurons, and provides a model to assay reconstitution and maturation of RPE cell transplants. The availability of an RPE-free Bruch's membrane in this model likely allows the unprecedented formation of extensive polarized cell monolayers from donor hESC-derived RPE cell suspensions.
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