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Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

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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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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Correspondence to Alice Pébay.

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The authors declare no potential conflicts of interest.

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Alex W. Hewitt, Kathryn C. Davidson and Alice Pébay are co-senior authors.

Electronic supplementary material

Supplementary Figure 1
figure4

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)

High resolution image (TIFF 1350 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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Keywords

  • Human induced pluripotent stem cells
  • Human embryonic stem cells
  • Retinal pigment epithelium
  • Differentiation
  • RNA sequencing
  • Electrophysiology