Abstract
We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40–60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.
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References
Davidson, K. C., et al. (2014). Human pluripotent stem cell strategies for age-related macular degeneration. Optometry and Vision Science, 91(8), 887–893.
Kamao, H., et al. (2014). Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Reports, 2(2), 205–218.
Reardon, S., & Cyranoski, D. (2014). Japan stem-cell trial stirs envy. Nature, 513(7518), 287–288.
Schwartz, S. D., et al. (2012). Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet, 379(9817), 713–720.
Schwartz, S. D., et al. (2015). Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet, 385(9967), 509–516.
Leach, L. L., & Clegg, D. O. (2015). Concise review: making stem cells retinal: methods for deriving retinal pigment epithelium and implications for patients with ocular disease. Stem Cells, 33(8), 2363–2373.
Klimanskaya, I., et al. (2004). Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning and Stem Cells, 6(3), 217–245.
Buchholz, D. E., et al. (2013). Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Translational Medecine, 2(5), 384–393.
Buchholz, D. E., et al. (2009). Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells, 27(10), 2427–2434.
Vaajasaari, H., et al. (2011). Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells. Molecular Vision, 17, 558–575.
Zahabi, A., et al. (2012). A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. Stem Cells and Development, 21(12), 2262–2272.
Zhu, D., et al. (2011). Polarized secretion of PEDF from human embryonic stem cell-derived RPE promotes retinal progenitor cell survival. Investigative Ophthalmology & Visual Science, 52(3), 1573–1585.
Idelson, M., et al. (2009). Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell, 5(4), 396–408.
Osakada, F., et al. (2009). In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. Journal of Cell Science, 122(17), 3169–3179.
Reichman, S., et al. (2014). From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium. Proceedings of the National Academy of Sciences, 111(23), 8518–8523.
Kuwahara, A., et al. (2015). Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue. Nature Communications, 6.
Leach, L. L., et al. (2015). Canonical/beta-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells. Investigative Ophthalmology & Visual Science, 56(2), 1002–1013.
Zhu, Y., et al. (2013). Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium. PloS One, 8(1), e54552.
Nakano, T., et al. (2012). Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell, 10(6), 771–785.
Cho, M. S., et al. (2012). Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses. Stem Cell Research, 9(2), 101–109.
Lamba, D. A., et al. (2006). Efficient generation of retinal progenitor cells from human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 103(34), 12769–12774.
Lamba, D. A., et al. (2010). Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells. PloS One, 5(1), e8763.
Maminishkis, A., et al. (2006). Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Investigative Ophthalmology & Visual Science, 47(8), 3612–3624.
Carr, A. J., et al. (2009). Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PloS One, 4(12), e8152.
Yu, J., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318(5858), 1917–1920.
Thomson, J. A., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282(5391), 1145–1147.
Molday, R. S., Hicks, D., & Molday, L. (1987). Peripherin. A rim-specific membrane protein of rod outer segment discs. Investigative Ophthalmology & Visual Science, 28(1), 50–61.
Singh, R., et al. (2013). Functional analysis of serially expanded human iPS cell-derived RPE cultures. Investigative Ophthalmology & Visual Science, 54(10), 6767–6778.
Stone, A. B. (1974). A simplified method for preparing sucrose gradients. The Biochemical Journal, 137(1), 117–118.
Crombie, D. E., et al. (2015). Characterization of the retinal pigment epithelium in friedreich ataxia. Biochemistry and Biophysics Reports, 4, 141–147.
Blankenberg, D., et al. (2010). Manipulation of FASTQ data with galaxy. Bioinformatics, 26(14), 1783–1785.
Trapnell, C., et al. (2012). Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and cufflinks. Nature Protocols, 7(3), 562–578.
Love, M. I., Huber, W., & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15(12), 550.
Amirpour, N., et al. (2013). Comparing three methods of Co-culture of retinal pigment epithelium with progenitor cells derived human embryonic stem cells. International Journal of Preventive Medecine, 4(11), 1243–1250.
Gabrielian, K., et al. (1992). In vitro stimulation of retinal pigment epithelium proliferation by taurine. Current Eye Research, 11(6), 481–487.
Antonetti, D. A., et al. (2002). Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin. Journal of Neurochemistry, 80(4), 667–677.
Marsh-Armstrong, N., et al. (1999). Asymmetric growth and development of the Xenopus laevis retina during metamorphosis is controlled by type III deiodinase. Neuron, 24(4), 871–878.
Rosenthal, R., & Strauss, O. (2003). Investigations of RPE cells of choriodal neovascular membranes from patients with age-related macula degeneration. Advances in Experimental Medicine and Biology, 533, 107–113.
Wimmers, S., Karl, M. O., & Strauss, O. (2007). Ion channels in the RPE. Progress in Retinal and Eye Research, 26(3), 263–301.
Kawasaki, H., et al. (2002). Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proceedings of the National Academy of Sciences of the United States of America, 99(3), 1580–1585.
Meyer, J. S., et al. (2011). Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment. Stem Cells, 29(8), 1206–1218.
Pera, E. M., et al. (2001). Neural and head induction by insulin-like growth factor signals. Developmental Cell, 1(5), 655–665.
Streit, A., et al. (2000). Initiation of neural induction by FGF signalling before gastrulation. Nature, 406(6791), 74–78.
Zhang, K., et al. (2014). Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors. Protein & Cell, 5(1), 48–58.
Acknowledgments
We thank Prof James Thomson (University of Wisconsin) for providing the iPSC line iPS (Foreskin) 4 clone 2, the Lions Eye Donation Service (Melbourne) for providing human donor eyes and the Melbourne Brain Centre’s flow Cytometry core facility. We thank Dr. Mirella Dottori (University of Melbourne) for providing access to essential equipment for the dissection of POS.
This work was supported by grants from the NHMRC (1059369), the National Stem Cell Foundation of Australia, the Ophthalmic Research Institute of Australia and the Stafford Fox Medical Foundation. Further support was provided by Australian Postgraduate Award Scholarships (GL, KPG), a NHMRC Career Development Award Fellowship (AP), a NHMRC-CSL Gustav Nossal Postgraduate Research Scholarship (DEC), a NHMRC Early Career Fellowship (AWH), an Australian Research Council (ARC) Future Fellowship (AP, FT140100047), a Cranbourne Fellowship (RCBW), a Gerard Crock Fellowship (KCD), the University of Melbourne and Operational Infrastructure Support from the Victorian Government.
Author Contributions
K.P.G., R.A., S.Y.L., D.H., A.C., D.E.C., H.S.W., R.C.B.W., H.H.L., J.K., A.W.H.: collection and/or assembly of data, data analysis and interpretation, final approval of manuscript. G.L., K.D., A.P.: concept and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.
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Alex W. Hewitt, Kathryn C. Davidson and Alice Pébay are co-senior authors.
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Supplementary Figure 1
GFC is more robust than basal conditions to induce RPE gene expression. (A) Representative image of cells obtained in RPEM only (Basal, top) or with GFC (bottom) at day 60 (P0). (B, C) qRT-PCR analysis of SIX3, RAX, PAX6, MITF, PMEL, CRALBP and RPE65 for (B) H9- and (C) ES4CL2-derived cells at P0, cultivated with RPEM in the absence or presence of GFC. All data is normalized to GAPDH housekeeping and undifferentiated hPSCs at day 0. Data expressed are mean ± SEM of at least three independent experiments, significance established by two-way ANOVA followed by Sidak multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001. Relative quantification: relative expression level of each gene in comparison to its expression level on Day 0 (undifferentiated). (GIF 74 kb)
Supplementary Figure 2
Heatmap displaying the top 200 differentially expressed genes between undifferentiated H9 and H9-derived-RPE cells. The corresponding genes in human native adult RPE cells are also displayed. Note the tight cluster of hPSC-derived RPE cells and primary human adult RPE cells. (PDF 257 kb)
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Lidgerwood, G.E., Lim, S.Y., Crombie, D.E. et al. Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium. Stem Cell Rev and Rep 12, 179–188 (2016). https://doi.org/10.1007/s12015-015-9636-2
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DOI: https://doi.org/10.1007/s12015-015-9636-2