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: 5.71). 02/2012; 1(2):96-109. DOI: 10.5966/sctm.2011-0057
Source: PubMed


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.

Download full-text


Available from: Gary Siuzdak, Sep 29, 2015
49 Reads
  • Source
    • "Several protocols for RPE cell differentiation have been described (Ukrohne et al. 2012; Buchholz et al. 2013; Zhu et al. 2013; Rowland et al. 2013; Maruotti et al. 2013; Singh et al. 2013b). We have slightly modified an earlier protocol by Ukrohne et al. (2012) which proofed highly efficient. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12017-014-8308-8) contains supplementary material, which is available to authorized users.
    Neuromolecular medicine 05/2014; 16(3). DOI:10.1007/s12017-014-8308-8 · 3.68 Impact Factor
  • Source
    • "However, hESC-derived RPE protected a significantly larger area of ONL than fibroblasts, pointing to a superior effect of these cells. In light of recent, promising transplantation approaches for the replacement of dysfunctional or lost RPE in pre-clinical models [3], [9], [10], [12], [19] and human patients [11] with ES or iPS cell-derived RPE [40], [41], [42], this rapid protocol for directly differentiating pure, human RPE cell populations from an unlimited in vitro cell source has high medical relevance. Regarding the possibility of using such cells for human transplantation, further caution on the purity of transplanted RPE cells should be considered. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.
    PLoS ONE 01/2013; 8(1):e54552. DOI:10.1371/journal.pone.0054552 · 3.23 Impact Factor
  • Investigative ophthalmology & visual science 05/2012; 53(5):2511-4. DOI:10.1167/iovs.12-9483p · 3.40 Impact Factor
Show more