Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)–derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD.
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Atala, A. Human embryonic stem cells: early hints on safety and efficacy. Lancet 379, 689–690 (2012).
Carr, A.J. et al. Development of human embryonic stem cell therapies for age-related macular degeneration. Trends Neurosci. 36, 385–395 (2013).
Nazari, H. et al. Stem cell based therapies for age-related macular degeneration: The promises and the challenges. Prog. Retin. Eye Res. 48, 1–39 (2015).
Bharti, K. et al. Developing cellular therapies for retinal degenerative diseases. Invest. Ophthalmol. Vis. Sci. 55, 1191–1202 (2014).
Bhutto, I. & Lutty, G. Understanding age-related macular degeneration (AMD): relationships between the photoreceptor/retinal pigment epithelium/Bruch's membrane/choriocapillaris complex. Mol. Aspects Med. 33, 295–317 (2012).
Rosenfeld, P.J. et al. Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 355, 1419–1431 (2006).
Muthiah, M.N. et al. Adaptive optics imaging shows rescue of macula cone photoreceptors. Ophthalmology 121, 430–431.e3 (2014).
Schwartz, S.D. et al. 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, 509–516 (2015).
Mandai, M. et al. Autologous induced stem-cell-derived retinal cells for macular degeneration. N. Engl. J. Med. 376, 1038–1046 (2017).
Vugler, A. et al. Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation. Exp. Neurol. 214, 347–361 (2008).
Haruta, M. et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells. Invest. Ophthalmol. Vis. Sci. 45, 1020–1025 (2004).
International Stem Cell Initiative. et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat. Biotechnol. 25, 803–816 (2007).
Klimanskaya, I. et al. Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells 6, 217–245 (2004).
Idelson, M. et al. Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 5, 396–408 (2009).
Skottman, H., Dilber, M.S. & Hovatta, O. The derivation of clinical-grade human embryonic stem cell lines. FEBS Lett. 580, 2875–2878 (2006).
Allegrucci, C. et al. Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome. Hum. Mol. Genet. 16, 1253–1268 (2007).
Lund, R.D. et al. Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats. Cloning Stem Cells 8, 189–199 (2006).
Sparrow, J.R. & Duncker, T. Fundus autofluorescence and RPE lipofuscin in age-related macular degeneration. J. Clin. Med. 3, 1302–1321 (2014).
Holz, F.G., Schmitz-Valckenberg, S., Spaide, R.F. & Bird, A.C. (eds.) The Atlas of Fundus Autofluorescence Imaging (Springer, 2007).
Bressler, N.M. et al. Submacular Surgery Trials (SST) Research Group. Surgery for hemorrhagic choroidal neovascular lesions of age-related macular degeneration: ophthalmic findings: SST report no. 13. Ophthalmology 111, 1993–2006 (2004).
Amer, M.H., White, L.J. & Shakesheff, K.M. The effect of injection using narrow-bore needles on mammalian cells: administration and formulation considerations for cell therapies. J. Pharm. Pharmacol. 67, 640–650 (2015).
Tezel, T.H., Del Priore, L.V. & Kaplan, H.J. Reengineering of aged Bruch's membrane to enhance retinal pigment epithelium repopulation. Invest. Ophthalmol. Vis. Sci. 45, 3337–3348 (2004).
Diniz, B. et al. Subretinal implantation of retinal pigment epithelial cells derived from human embryonic stem cells: improved survival when implanted as a monolayer. Invest. Ophthalmol. Vis. Sci. 54, 5087–5096 (2013).
Stanga, P.E. et al. Retinal pigment epithelium translocation after choroidal neovascular membrane removal in age-related macular degeneration. Ophthalmology 109, 1492–1498 (2002).
van Zeeburg, E.J., Maaijwee, K.J., Missotten, T.O., Heimann, H. & van Meurs, J.C. A free retinal pigment epithelium-choroid graft in patients with exudative age-related macular degeneration: results up to 7 years. Am. J. Ophthalmol. 153, 120–7 e2 (2012).
Chen, F.K. et al. Long-term visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age-related macular degeneration. Clin. Experiment. Ophthalmol. 37, 275–285 (2009).
van Romunde, S.H.M., Polito, A., Peroglio Deiro, A., Guerriero, M. & Pertile, G. Retinal pigment epithelium-choroid graft with a peripheral retinotomy for exudative age-related macular degeneration: long-term outcome. Retina doi: 10.1097/IAE.0000000000001945 (2017).
Cereda, M.G., Parolini, B., Bellesini, E. & Pertile, G. Surgery for CNV and autologous choroidal RPE patch transplantation: exposing the submacular space. Graefes Arch. Clin. Exp. Ophthalmol. 248, 37–47 (2010).
Uppal, G. et al. New algorithm for assessing patient suitability for macular translocation surgery. Clin. Experiment. Ophthalmol. 35, 448–457 (2007).
Algvere, P.V., Gouras, P. & Dafgard Kopp, E. Long-term outcome of RPE allografts in non-immunosuppressed patients with AMD. Eur. J. Ophthalmol. 9, 217–30 (1999).
Wang, S. et al. Morphological and functional rescue in RCS rats after RPE cell line transplantation at a later stage of degeneration. Invest. Ophthalmol. Vis. Sci. 49, 416–421 (2008).
Lu, B. et al. Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells 27, 2126–2135 (2009).
Pertile, G. & Claes, C. Macular translocation with 360 degree retinotomy for management of age-related macular degeneration with subfoveal choroidal neovascularization. Am. J. Ophthalmol. 134, 560–565 (2002).
Chen, F.K. et al. A comparison of macular translocation with patch graft in neovascular age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 50, 1848–1855 (2009).
Hewitt, Z.A., Amps, K.J. & Moore, H.D. Derivation of GMP raw materials for use in regenerative medicine: hESC-based therapies, progress toward clinical application. Clin. Pharmacol. Ther. 82, 448–452 (2007).
Carr, A.J. et al. Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay. Mol. Vis. 15, 283–295 (2009).
Mao, Y. & Finnemann, S.C. Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture. Methods Mol. Biol. 935, 285–295 (2013).
Ramsden, C.M. et al. Rescue of the MERTK phagocytic defect in a human iPSC disease model using translational read-through inducing drugs. Sci. Rep. 7, 51 (2017).
We acknowledge H. Moore, Stem Cell Derivation Facility, Centre for Stem Cell Biology (CSCB), University of Sheffield for derivation of the original SHEF-1 hESC line and P. Keane and M. Cheetham for comments on the paper. We thank R. McKernan for support and input throughout the project. L.d.C. and P.J.C. received the following grants and donations and would like to acknowledge that they were used to fund the studies reported in this article: Anonymous Donor, USA, Establishment of The London Project to Cure Blindness - Donation. Lincy Foundation, USA, The London Project To Cure Blindness: Funding Towards The Production Of A Cell Based Therapy For Late Stage Age-Related Macular Degeneration - P12761. Macular Disease Society Studentship – Donation. MRC, Stem Cell Based Treatment Strategy For Age-Related Macular Degeneration (AMD) - G1000730. CIRM (California Institute of Regenerative Medicine) LA1_C2-02086. Pfizer Inc, The Development Plan For A Phase I/IIa Clinical Trial Implanting HESC Derived RPE for AMD - PF-05406388. Moorfields Biomedical Research Centre, National Institute for Health Research (NIHR) - BRC2_011. The Michael Uren Foundation R170010A.
J.K., M.B., M.F., J.S., T.H., G.F., M.W., P.T.L., and P.W. were all employees of Pfizer during the period of this clinical trial. This study was sponsored by Pfizer Inc. L.d.C. and P.J.C. are named on two patents lodged by University College London Business. They are Patent Application No. PCT/GB2009/000917 (for the patch) and International Patent Application No. PCT/GB2011/051262 (for the surgical tool).
Integrated supplementary information
The device for introducing the therapeutic patch into the sub-retinal space. The device consists of a Handle containing a mechanism driven by the Wheel which advances a flexible Rod through the Shaft that in turn pushes the therapeutic patch out of the Tip of the device. The surgeon rolls the Wheel forward to advance the Rod.
Supplementary Figure 2 TRA-1-60 flow cytometry on SHEF1.3 hESC, P0 and P1 RPE (Fluorophore - TO-PRO®-3)
A: Forward scatter (FSC) / side scatter (SSC) plots of hESC, P0 and P1 RPE. hESC and RPE lie in slightly different positions on this plot, with RPE being smaller (FSC) and more granular (SSC) than hESC.B: Overlays of IgM (black) and TRA-1-60 (red) staining for hESC, P0 RPE and P1 RPE. Positive staining is clearly visible for hESC, whereas the isotype control (IgM, negative) and TRA-1-60 samples clearly overlay for both P0 and P1 RPE. Percentages indicate the percentage of positive events occurring within the gate shown (M12, M18 or M16 in hESC, P0 and P1 RPE plots respectively).C: Percentage positive events recorded for IgM isotype control and TRA-1-60 in hESC, P0 RPE and P1 RPE. Average values are hESC 0.08% IgM, 93.5% TRA-1-60; P0 RPE 0.10% IgM, 0.30% TRA-1-60 and P1 RPE 0.09% IgM and 0.32% TRA-1-60. All values except for the TRA-1-60 stained hESC are below the lower limit of detection (1.03%).D: An example of the 0.3% of TRA-1-60 events that show slightly higher staining than the isotype control in P1 RPE (even though these are within the background limits of the assay) displayed on a FSC/SSC plot. (For example taking all TRA-1-60 events in gate M16 in figure 2B and displaying them as FSC/SSC). This is compared to a representative hESC FSC/SSC plot to show that the majority of these events are separated from hESC based on SSC ie they are more granular and very unlikely to be hESC cells.
Supplementary Figure 3 Propidium iodide (PI) staining of SHEF1.3 hESC dissociated and seeded into RPE culture conditions (Fluorophore - TO-PRO®-3)
A & B: On day 0 over 95% of hESC cells seeded into DMEM/CellStart (RPE conditions) or mTeSR1/Matrigel were viable as they excluded PI (A). By 2 days post seeding on average 96%±1% (n=5) of cells in DMEM conditions, including both adherent cells and those in the media (as very few actually adhered) were dead or dying, as judged by flow cytometry for PI. The same was observed on day 4 (97%±0.6% dead, n=3). By contrast, around 40-50% of cells in mTeSR conditions were dead at 2 days post seeding, demonstrating that under these conditions around half of SHEF1.3 hESC survive.C: Virtually all SHEF1.3 hESC seeded into DMEM/CellStart (RPE culture conditions) appear rounded and dead as well as staining positively for PI (red). By contrast, though dead cells are apparent, many of the cells seeded into mTeSR/Matrigel (MGL) conditions appear morphologically viable and do not stain with PI.D and E: Blue = HOECHST (nuclei) and green = TRA-1-60 (D) or Ki67 (E). By 6 weeks post seeding, a small number of live cells were occasionally visible in DMEM/CellStart conditions (4 out of 9 experiments). These did not appear morphologically like SHEF1.3 hESC cells (compare the large, elongated cells in D and E with single cell hESC on Matrigel (MGL) in C. These did not stain positively for TRA-1-60 (compare D with hESC on MGL in figure 4C) or KI67 (compare CellStart and MGL seeded cells in E).
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da Cruz, L., Fynes, K., Georgiadis, O. et al. Phase 1 clinical study of an embryonic stem cell–derived retinal pigment epithelium patch in age-related macular degeneration. Nat Biotechnol 36, 328–337 (2018). https://doi.org/10.1038/nbt.4114
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