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. 2012 Dec 6;18(1):1312-9.
doi: 10.2119/molmed.2012.00242.

Long-term safety and efficacy of human-induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa

Yao Li  1 , Yi-Ting Tsai, Chun-Wei Hsu, Deniz Erol, Jin Yang, Wen-Hsuan Wu, Richard J Davis, Dieter Egli, Stephen H Tsang
Affiliations

Long-term safety and efficacy of human-induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa

Yao Li et al. Mol Med. .

Abstract

The U.S. Food and Drug Administration recently approved phase I/II clinical trials for embryonic stem (ES) cell-based retinal pigmented epithelium (RPE) transplantation, but this allograft transplantation requires lifelong immunosuppressive therapy. Autografts from patient-specific induced pluripotent stem (iPS) cells offer an alternative solution to this problem. However, more data are required to establish the safety and efficacy of iPS transplantation in animal models before moving iPS therapy into clinical trials. This study examines the efficacy of iPS transplantation in restoring functional vision in Rpe65(rd12)/Rpe65(rd12) mice, a clinically relevant model of retinitis pigmentosa (RP). Human iPS cells were differentiated into morphologically and functionally RPE-like tissue. Quantitative real-time polymerase chain reaction (RT-PCR) and immunoblots confirmed RPE fate. The iPS-derived RPE cells were injected into the subretinal space of Rpe65(rd12)/Rpe65(rd12) mice at 2 d postnatally. After transplantation, the long-term surviving iPS-derived RPE graft colocalized with the host native RPE cells and assimilated into the host retina without disruption. None of the mice receiving transplants developed tumors over their lifetimes. Furthermore, electroretinogram, a standard method for measuring efficacy in human trials, demonstrated improved visual function in recipients over the lifetime of this RP mouse model. Our study provides the first direct evidence of functional recovery in a clinically relevant model of retinal degeneration using iPS transplantation and supports the feasibility of autologous iPS cell transplantation for retinal and macular degenerations featuring significant RPE loss.

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Figures

Figure 1
Figure 1
Generation of human iPS cell lines from skin fibroblasts. (A) Fibroblast monolayer. (B, C) Fibroblasts after transduction with OCT4, SOX2, KLF4 and MYC. iPS colonies appeared between 14 and 21 d (4×). (D) iPS colony (10×). Scale bar, 100 μm.
Figure 2
Figure 2
Expression of pluripotency markers in human iPS cells at d 14. Left column: DAPI stained nuclei. Middle column: cells labeled with TRA-1-60, SSEA4, NANOG or SOX2 antibodies; markers of pluripotency. Right column: merged images. Scale bar, 50 μm.
Figure 3
Figure 3
Human iPS cells differentiated into cell types of all three germ layers. (A) Gastrointestinal epithelium (endoderm). (B) Muscle (mesoderm). (C) Neuroepithelium with pigment (ectoderm). (D) Cartilage (mesoderm) (scale bar, 100 μm).
Figure 4
Figure 4
RPE morphology in differentiating human iPS cells. (A) MEF control (left) and differentiated pigmented cells (right). Monolayer cell domes are shown (arrowheads), suggesting apical-basal fluid transportation, an essential function of RPE. (B) Pigmented colonies transferred to feeder plates showing RPE fate. Arrowheads indicate different stages: early (blue), intermediate (red) and mature (green). (C) Colony with mixture of pigmented and nonpigmented hexagonal cells. (D) Colony with most hexagonal cells being pigmented. (E, F) iPS-derived RPE with native RPE morphological characteristics, including prominent melanin granules and perinuclear location of melanin granules. (G, H) Human autopsy RPE. Scale bar, 200 μm.
Figure 5
Figure 5
Morphology (A–F), expression (G–H) and functional analyses (I) of our iPS-derived-RPE. Arrowheads indicate apical microvilli (AMv) in iPS-RPE (A, B) and in human autopsy donor RPE (D, E) at 15,000× and 60,000×. Li, lipofusin granule. (C, F) Arrowheads indicate adherens junction-like structure between RPE cells (3,000×). (G) Quantitative RT-PCR of RPE65, BEST1 and MFRP expression in autopsy RP, iPS-RPE, undifferentiated iPS and donor fibroblast. (H) Immunoblot analysis of RPE65 (65 kDa) and CRALBP (34 kDa) expression in autopsy RPE, iPS-RPE, undifferentiated iPS and donor fibroblast. Total protein (20 μg/lane). (I) Phagocytosis of POS in iPS-RPE. Bovine POS (green) and ZO1 (red). White arrowheads indicate POS inside RPE. Scale bar in A–C, 1 μm.
Figure 6
Figure 6
Survival of transplanted iPS-derived pigmented RPE in albino Rpe65rd12/Rpe65rd12 mice. (A) Fundus photograph shows a patch of black pigmented iPS-RPE (arrowhead) behind the albino retinal 5 months after transplantation. No gross disruption of the host retina occurred. (B,C) Histology of host retina 5 months after transplantation. White arrowheads indicate mouse RPE (B). Human pigmented cells, indicated with black arrowheads, are adjacent to the host RPE (C). CH, choroid; PR, photoreceptor.
Figure 7
Figure 7
Retinal function is restored in Rpe65rd12/Rpe65rd12 in SCID mice after human iPS cell–derived RPE transplantation. (A) Representative maximum ERG traces from treated (blue), control fellow eye (red) and wild-type mice (black). (B) Maximal b-wave peak analysis. Enhancement is calculated as the difference in maximum responses between treated and untreated control eyes. iPS-RPE–treated eyes show a 13.7-μV average difference (*p = 0.0246, n = 7). Mice receiving mitomycin-C–treated ES cells (mito-C ES) failed to show any statistically significant differences (p = 0.2853, n = 6). The value is represented as the mean ± standard error of the mean, and the p value is conducted by a ratio-paired t test.
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