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.2021 Feb 10:20:688-702.
doi: 10.1016/j.omtm.2021.02.006. eCollection 2021 Mar 12.

Cell therapy with hiPSC-derived RPE cells and RPCs prevents visual function loss in a rat model of retinal degeneration

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Cell therapy with hiPSC-derived RPE cells and RPCs prevents visual function loss in a rat model of retinal degeneration

Anna Salas et al. Mol Ther Methods Clin Dev..

Abstract

Photoreceptor loss is the principal cause of blindness in retinal degenerative diseases (RDDs). Whereas some therapies exist for early stages of RDDs, no effective treatment is currently available for later stages, and once photoreceptors are lost, the only option to rescue vision is cell transplantation. With the use of the Royal College of Surgeons (RCS) rat model of retinal degeneration, we sought to determine whether combined transplantation of human-induced pluripotent stem cell (hiPSC)-derived retinal precursor cells (RPCs) and retinal pigment epithelial (RPE) cells was superior to RPE or RPC transplantation alone in preserving retinal from degeneration. hiPSC-derived RPCs and RPE cells expressing (GFP) were transplanted into the subretinal space of rats.In vivo monitoring showed that grafted cells survived 12 weeks in the subretinal space, and rats treated with RPE + RPC therapy exhibited better conservation of the outer nuclear layer (ONL) and visual response than RPE-treated or RPC-treated rats. Transplanted RPE cells integrated in the host RPE layer, whereas RPC mostly remained in the subretinal space, although a limited number of cells integrated in the ONL. In conclusion, the combined transplantation of hiPSC-derived RPE and RPCs is a potentially superior therapeutic approach to protect retina from degeneration in RDDs.

Keywords: RCS rat; RPE; cell therapy; human-induced pluripotent stem cell; iPSC; photoreceptors; regenerative medicine; retina degeneration; retinal pigment epithelium; subretinal injection.

© 2021 The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

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Graphical abstract
Figure 1
Figure 1
Characterization of human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells (A) RPE-like cells exhibited typical pigmented hexagonal cell morphology under bright-field and expressed green fluorescent protein (GFP). Representative immunostaining images show expression of RPE markers bestrophin-1 (BEST1), ZO-1 (zonula occludens 1), MITF (microphthalmia-associated transcription factor), and a human antigen (Ku80). Nuclei are stained with DAPI. Scale bars, 75 μm in left panels and 50 μm in middle and right panels. (B) Gene-expression levels in RPE-like cells by quantitative real-time PCR. Values are normalized toGAPDH and relative to undifferentiated hiPSC, expressed as 2−ΔΔCt (log scale). Data presented as mean ± standard deviation (SD; n = 3), five independent biological replicates. (C) Quantitative analysis by flow cytometry of RPE65, MITF, and BEST1 in RPE cell culture. Histograms show both the undifferentiated hiPSC (blue) and RPE cells (red). Results are representative of two independent experiments performed with cells from different differentiations. (D) Representative transmission electron microscopy (TEM; a−c) and scanning electron microscopy (SEM; d−f) images of RPE cells in culture. RPE monolayers show polarization with apical microvilli, melanosomes, mitochondria, tight junction (red arrow), and adherent junction (red arrowhead) at the apical border and basal nuclei (N). Scale bars, 2 μm (a and b) and 0.2 μm (c) for TME images. Scale bars, 10 μm (d), 5 μm (e), and 0.5 μm (f) for SEM images. (E)In vitro phagocytosis of tetramethylrhodamine (TRITC)-labeled photoreceptor (PR) outer segments (POS; red) by RPE-like cells (GFP in green). Nuclei are stained with DAPI. Scale bar, 25 μm.
Figure 2
Figure 2
Generation and characterization of retinal precursor cells (RPCs) from hiPSCs (A) Schematic diagram of the three-step protocol at different stages of differentiation. Bright-field images of cell morphology of undifferentiated hiPSC colonies at day −5, embryoid bodies at day 1, early retinal rosettes containing RPCs at day 10, and optic cup-like structures at day 60. Scale bars, 500 μm and 100 μm. (B) At day 21, confocal images of retinal progenitor cells forming neural rosettes and expressing human antigen Ku80; eye-field primordial and neural retina markers SOX1, CHX10, OTX2, and PAX6; and early PR markers CRX and NRL. Scale bars, 50 μm. (C) Quantitative real-time PCR of gene expression at day 21 relative to undifferentiated hiPSC shows downregulation of pluripotent genesOCT4 andNANOG and upregulation of neural (PAX6,OTX2, andCHX10) and photoreceptor (CRX,RAX, andrecoverin [Recov]) genes. Values are normalized toGAPDH and relative to undifferentiated hiPSC, expressed as 2−ΔΔCt (log scale). Data presented as mean ± SD (n = 3), three independent biological replicates. (D) Representative bright-field and immunocytochemistry images of RPC cultures transduced with the lentivirus SparQ-GFP at day 45 (before transplantation). The expression of GFP, neural retina marker (CHX10, PAX6, and RAX), and PR progenitor markers (NRL, RECOV, and CRX) is shown. Magnification of Recov + cells with axonal projections (white arrows). Scale bars, 75 μm (bright field); 50 μm and 10 μm in the magnifications. Nuclei are stained with DAPI. (E) uantitative real-time PCR of gene expression in RPCs at day 45 relative to undifferentiated hiPSC shows no expression of pluripotent genes (NANOG andOCT4) but upregulation of retinal markers, including mature PR markersopsin andrhodopsin. Values are normalized toGAPDH and relative to undifferentiated hiPSC, expressed as 2−ΔΔCt (log scale). Data presented as mean ± SD (n = 3), three independent biological replicates. (F) Quantitative flow cytometry analysis of RPC at day 45 with surface marker Tra-1-60 and intracellular markers RECOV, RHO, RG-OPSIN, and PAX6 (specific for RPC), MITF (for RPE cells), and Ki67 (proliferation) and the appropriate controls (undifferentiated hiPSC, retinal progenitors at day 21, and secondary antibody are shown in Figure S1). The numbers in the corner show the percentage of stained cells in this gate. Secondary antibody was used as control. Results are representative of two biological replicates.
Figure 3
Figure 3
In vivo time course of GFP-fluorescent cell survival in the subretinal space of the RCS rat (A) Images from the same eye injected with RPE cells alone at 1, 2, 4, 8, and 12 weeks postinjection (PI). (B) Images from the same eye injected with RPC alone. (C) Images from the same eye injected with the combination of RPE + RPC alone. Images show fundus retinographies (a, c, e, g, i, and k) and fluorescent retinographies under blue filter (b, d, f, h, j, and l) in the same area, where grafted GFP+ cells are observed as green spots.
Figure 4
Figure 4
Structural analysis of transplanted retinas by optical coherence tomography (OCT) Eyes injected with medium (sham; A, G, and M; n = 6 eyes), RPE cells (B, H, and N; n = 8 eyes), RPC (C, I, and O; n = 6 eyes), or the combination of RPE and RPC (RPE + RPCs; D, J, and P; n = 10 eyes) were analyzed at 4, 8, and 12 weeks PI by OCT, obtaining retina micrographs of the grafted area and a contralateral area of the same eye (not shown). Cross-sections of the retinas were analyzed, quantifying total retina thickness (TOTAL RETINA; area between blue and orange lines) and PR layer thickness (PR LAYER; area between green and orange lines). The preservation of the thickness of total retina and PR layer was compared among groups at 4 (E and F), 8 (K and L), and 12 weeks PI (Q and R), calculating the difference between the grafted area and the contralateral area of the same eye. Data presented as mean ± SD. Statistical significance was calculated by one-way ANOVA, followed by Tukey’s multiple comparison tests. ∗p < 0.05; ∗∗p < 0.005 between RPE + RPC and sham groups; #p < 0.05; ##p < 0.005 between RPE + RPC and RPE groups.
Figure 5
Figure 5
Visual function analysis by electroretinogram (A−C) Representative electroretinogram recordings at 4, 8, and 12 weeks PI. Scotopic electroretinogram responses were recorded from eyes injected with medium (sham; blue lines, n = 7 eyes), RPE-differentiated cells (RPE; red lines, n = 8 eyes), RPC-differentiated cells (RPC; green lines, n = 8 eyes), or the combination of RPE and RPCs (RPE + RPC; black lines, n = 9 eyes). Measurements were recorded under a light stimulus of 1.9 log cd · s · m−2 and 1 ms of duration. (D−G) Electroretinographic waves were quantified and compared among treatment groups at the three time points by the measurement of the b-wave amplitude (D) and its implicit time (F) and the a-wave amplitude (E) and its implicit time (G). Data presented as mean ± SD. Statistical significance was calculated by two-way ANOVA, followed by Tukey’s multiple comparison tests comparing sham with RPE, RPC, and RPE + RPC data (∗), comparing RPE with RPE + RPC data (#), or comparing RPC with RPE + RPC data (+). ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; ∗∗∗∗p < 0.0001; #p < 0.05; ##p < 0.005; ###p < 0.0005; +p < 0.05; ++p < 0.005; +++p < 0.0005.
Figure 6
Figure 6
Postmortem analysis of RPE and RPC integration in the RCS rat retina (A) Hematoxylin and eosin of rat eye cryosections injected with sham (control), RPE cells, RPC, and RPE + RPC at 8 weeks (a) and 12 weeks (b) PI (n = 3 eyes/group). Scale bar, 50 μm. (B) Immunofluorescence analysis of Rho, blue opsin, RPE65, and Recov in sections of RCS rat retinas at 8 weeks PI with sham (control), RPE cells, RPC, and RPE + RPC cell suspensions. Scale bars, 25 μm. (C) Immunofluorescence staining with Rho, blue opsin, RPE65, and Recov in sections of RCS rat retinas at 12 weeks PI with sham (control), RPE, RPC, and RPE + RPC cell suspensions. Brackets indicate outer nuclear layer (ONL). Scale bars, 25 μm. Nuclei were stained with DAPI. (D) Quantification of ONL nuclei rows (top graph) and ONL thickness (bottom graph) in retinal paraffin sections (n = 3−5 eyes/group). Data are expressed as mean ± SD. ∗p < 0.01; ∗∗∗p < 0.0005; ∗∗p < 0.001 versus 8 weeks; #p < 0.01; ##p < 0.001 versus control at 8 weeks; †p < 0.05; ††p < 0.00001 versus control at 12 weeks, calculated using Student’s t test. GCL, ganglion cell layer; INL, inner nuclear layer.
Figure 7
Figure 7
Identification of RPE and RPC engrafted in the RCS rat retina (A) Immunostaining of retinal sections at 8 weeks PI with RPE showing engraftment of GFP+ RPE cells in the host RPE layer (n = 4 eyes). (a) Bright-field image corresponding to Figure 6B showing pigmented GFP+ RPE cells. (b) GFP+ RPE cells expressed RPE65. (c and c’) Costaining with Rho and opsin showing interaction of engrafted GFP+ RPE with endogenous photoreceptors. (B) Immunostaining of retinal sections at 8 weeks PI with RPC showing integration of RPC into the ONL (n = 4 eyes). (d and e) Staining with Recov. White dashed square in (d) indicates the area enlarged in (d’). (f) Magnification of Figure 6B showing that GFP+ RPC expressed Rho. (C) Immunostaining of retinal sections at 8 weeks PI with PRE + RPC (n = 5 eyes). (g−j) Staining with Recov, RPE65, and human antigen Ku80. White arrowheads indicate GFP+ RPC cells integrated to ONL in (d), (e), (f), (g), and (h). GFP+ RPE cells integrate into the host RPE layer as a discrete monolayer (i) or as clusters (g) and (j). Scale bars, 25 μm. GFP in green. Nuclei were stained with DAPI.
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