Transplantation of rat embryonic stem cell-derived retinal progenitor cells preserves the retinal structure and function in rat retinal degeneration

Abstract

Introduction: Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model.

Methods: The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined.

Results: We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats.

Conclusions: We have successfully differentiated rESCs to glia enriched RPCs and retinal neuron enriched RPCs in vitro. The retinal neuron enriched rESC-RPC2 protected the structure and function of retina in rats with genetic retinal degeneration and could be a candidate cell source for treating some degenerative retinal diseases in human trials.

Fig. 1

In vitro differentiation of rat ESCs into RPCs. a Schematic diagram illustrating the strategy for differentiating rESCs into RPCs in this study. b Images of morphological changes of differentiating rESCs from day 1 to the formation of neuroectoderm-like structure at day 8. c Whole mount immunofluorescence staining, using antibodies against Sox2 and Otx2 (red), and bright images of rESC-derived neuroectoderm-like structure at day 8. DAPI (blue) was used to highlight the nuclei. Scale bar: 50 μm. d Whole mount immunofluorescence staining using antibody against Rax (red) and bright images of Rax::EGFP rESC-derived neuroectoderm-like structure at day 8. DAPI (blue) was used to highlight the nuclei. Scale bar: 50 μm. e Immunofluorescence images of cryosection of the neuroectoderm-like structure derived from Rax::EGFP rESCs at day 8. Antibody against EGFP (green) and antibodies against Pax6, N-cadherin/Ncad, Nestin/Nes, E-cadherin/Ecad and Otx2 (red) were used. DAPI (blue) was used to highlight the nuclei. Scale bar: 50 μm. f A representative result of RT-PCR analyses for marker expression during the differentiation process (rESC-RPC2). rESCs rat embryonic stem cells, RPCs retinal progenitor cells, DAPI 4′,6-diamidino-2-phenylindole, EGFP enhanced green fluorescent protein, RT-PCR reverse transcription polymerase chain reaction

Fig. 2

Characterization of rESC-RPCs at D16. a Comparisons of various marker expressions among rESCs, rESC-RPCs and other controls, including samples of differentiating rESCs at day 10 and P-RPCs. The relative mRNA levels of marker genes were determined by RT-qPCRs. Error bars represent the mean ± SD, n = 3. b Representative confocal immunofluorescence images of rESC-RPCs for NPC markers (Sox2, Nestin/Nes), anterior neural marker (Otx2), proliferation markers (pH3, PCNA and BrdU), retinal progenitor markers (Rax and Pax6), neuronal marker (Tuj1) and astrocyte and radial glia cell marker (GFAP). Scale bar: 50 μm. c Representative FCM profiles of rESC-RPCs for subpopulations expressing Sox2, Nestin, CD133, GFAP, Ki67, CD73 and CD24. Corresponding IgG was applied as the negative isotype control. rESC rat embryonic stem cell, RPCs retinal progenitor cells, RT-qPCR reverse transcription-quantitative polymerase chain reaction, NPC neural progenitor cell

Fig. 3

Microarray analysis of gene expression of rESC-RPC1, rESC-RPC2 and P-RPCs. a Heat map analysis of differentially expressed genes among rESC-RPC1, rESC-RPC2 and P-RPC. b GO analysis of highly expressed genes in P-PRC. Bar graph showing significance of enrichment terms for sets of genes in the blue box in a. c GO analysis of highly expressed genes in rESC-RPC1. Bar graph showing significance of enrichment terms for sets of genes in the yellow box in a. d GO analysis of highly expressed genes in rESC-RPC2. Bar graph showing significance of enrichment terms for sets of genes in the magenta box in a. e Heat map analysis of various genes expressed by RPC, retinal neurons and non-retinal tissues as listed in Additional file 5: Table S2. Log2 expression levels of the genes are shown in a blue-black-yellow gradient. rESC-RPCs rat embryonic stem cell-derived retinal progenitor cells, P-RPCs primary retinal progenitor cells, GO gene ontology

Fig. 4

Visual function evaluation of the RCS rats after the transplantation of rESC-RPCs and P-RPCs. a ERG b-wave of the RCS rats at different time points after different treatments. Error bars represent the mean ± SEM, ANOVA, **P < 0.01 (rESC-RPC2 vs rESC-RPC1), n = 7 for two weeks and four weeks, n = 6 for six weeks. b Typical ERG waves of the RCS rats at four-week time point after the transplantation. c ERG b-wave of the RCS rats four weeks after rESC-RPCs and P-RPC transplantation at different light intensity stimuli. Error bars represent the mean ± SEM, n = 7 for each group. RCS Royal College of Surgeons, rESC-RPCs rat embryonic stem cell-derived retinal progenitor cells, P-RPCs primary retinal progenitor cells, ERG electroretinogram, W/O without treatment

Fig. 5

Subretinal transplantation of EGFP labeled rESC-RPCs in the RCS model. a Generation of rESC-RPC2 from EF1α::EGFP lentivirus labeled rESCs. Scale bars: 50 μm. b Immunofluorescence staining of EGFP-rESC-RPC2 at D16 before transplantation. Antibody against EGFP (green) and antibodies against Nestin/Nes and Tuj1 (red) were used. DAPI (blue) was used to highlight the nuclei. Scale bars: 50 μm. c Flow cytometric analysis of EGFP-rESC-RPC2 in comparison with unlabeled rESC-RPC2. d Grafted rESC-RPC2 could be detected in the retina at different times after transplantation. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Scale bar: 50 μm. EGFP enhanced green fluorescent protein, rESC rat embryonic stem cell, RPC retinal progenitor cell, RCS Royal College of Surgeons, DAPI 4′,6-diamidino-2-phenylindole

Fig. 6

Differentiated EGFP-rESC-RPC2 integrate with host retina and preserve the retinal structure 4w after transplantation. a-d Differentiation of rESC-RPC2 into various retinal cells. Antibody against EGFP (green) and antibodies against retinal cell markers (red) were used for immunofluorescence staining. DAPI (blue) was used to highlight the nuclei. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Scale bars: 50 μm. e and f Integration of EGFP-rESC-RPC2 with retinal neurons. Colocalization of presynaptic markers Bassoon and Synaptophysin with EGFP expressed by donor cells in OPL was confirmed with immunostaining. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Scale bars: 50 μm. e' and f'. The magnified images of the rectangles in e and f. Arrow heads indicate the colocalization of EGFP and Bassoon or Synaptophysin. GCL ganglion cell layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer. g Preservation of the retinal structure by EGFP-rESC-RPC2 transplantation. The thickness of ONL of rESC-RPC2 treated eye was compared with that of vehicle (PBS) injected retina. Data are shown as mean ± SEM, t test, *P < 0.05 and **P < 0.01, n = 6 (rats) for each group. EGFP enhanced green fluorescent protein, rESC rat embryonic stem cell, RPC retinal progenitor cell, DAPI 4′,6-diamidino-2-phenylindole