Subretinal Transplantation of Photoreceptor Precursors and Retinal Pigment Epithelium Derived from Human Embryonic Stem Cells in Retinal Degeneration Rats

Abstract

Introduction : Degeneration and loss of photoreceptor or retinal pigment epithelium (RPE) is the major pathologic change in retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. The transplantation of RPE or photoreceptors derived from stem cells, has been attempted as a possible therapeutic method to regenerate retina and restore lost vision. Successful differentiation of RPE or photoreceptor precursors from human embryonic stem cells or induced pluripotent stem cells has been reported. However, obtaining a sufficient amount of cells still requires a great amount of time, and moreover, the efficacy of differentiation is still low. This study demonstrates a defined, effective method to differentiate photoreceptor precursors and RPEs from human embryonic stem cells with a relatively high efficiency and short incubation time. Furthermore, to determine a therapeutic potential of the derived cells, photoreceptor precursors and RPEs were characterized and the anatomical and functional changes were evaluated after a subretinal transplantation. Methods : Photoreceptor precursors and RPEs were differentiated from human embryonic stem cells (hESC) via the formation of cell clumps with neural structures, spherical neural masses (SNMs). It took four weeks to form SNM from hESC and additional two weeks to differentiate into photoreceptor precursors, or a week to differentiate into RPEs. The differentiated photoreceptor precursors and RPEs were characterized with immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The microstructure on electomicrography and the phagocytic function of RPEs were also evaluated. After characterization of these hESCs-derived retinal cells, the differentiated photoreceptor precursors or RPEs were transplanted into subretinal space of the retinal degeneration rats, Royal College Surgeon (RCS) rats. The RCS rats were divided into three groups, 1) as photoreceptor group of which the eyes were transplanted with photoreceptor precursors (n=25)

2) an RPE group of which the eyes were transplanted with RPEs (n=25)

and 3) a control group that was injected only with culture media (n=26). The integration of transplanted cells and occurrences of the tumor were observed for up to 24 weeks after the transplantation. An electroretinogram (ERG) was recorded at 4, 12, and 24 weeks after transplantation and the amplitude of the b-wave was analyzed. A histologic examination was performed at 4 and 24 weeks after transplantation to analyze the thickness of the whole retina, inner nuclear layer, and outer nuclear layer. Results : The differentiated photoreceptor precursors expressed photoreceptor-specific markers such as rhodopsin, recoverin and opsin as well as neural markers, βIII-tubulin and nestin in immunocytochemistry and RT-PCR. The efficiency of differentiation of the photoreceptor precursors was highest at two weeks after culture of SNM, showing that about 80% of the cells expressed these photoreceptor specific markers. The differentiated RPE cells showed the typical morphology of RPE, such as a polygonal shape and pigmentation. Transmission electron microscopy revealed apical microvilli, pigmented granules in the cytoplasm and tight junctions between cells. The RPEs also expressed molecular markers of RPE, including MITF, ZO-1, RPE65, and bestrophin. Over 90% of the cells expressed markers for RPE after a week of differentiation from SNM. They also showed phagocytosis of the bovine photoreceptor outer segment. After subretinal transplantation of photoreceptor precursors or RPEs, the transplanted cells were integrated into the retina and there was no evidence of severe inflammation or tumor formation until 24 weeks of observation. The amplitude of b-wave in the photoreceptor (74.51 ± 27.78 uV) and RPE groups (37.48 ± 13.75 uV) were higher compared with the control group (9.28 ± 1.56 uV) after 4 weeks of transplantation (p=0.018 and 0.043, respectively). There was a tendency that the amplitude of b-wave in the photoreceptor group was higher, compared with the RPE group, although there was no statistical significance. After 12 weeks of transplantation, the amplitude of the b-wave on ERG was slightly decreased in all three groups, but the photoreceptor group and RPE group showed higher amplitude compared to the control group (56.47 ± 20.14 uV, 23±8.07 uV and 9.07 ± 2.10 uV, respectively). At 24 weeks after transplantation, the b-wave amplitude in the photoreceptor group was still higher, compared with the control group (50.33 ± 15.81 uV vs 5.63 ± 1.45 uV, p=0.0058). However, there was no statistical significant difference between the RPE group (22.80 ± 10.16 uV) and control group with the aspect to the b-wave amplitude (p=0.095). The thickness of outer nuclear layer was also higher in the photoreceptor (14.03 ± 1.48 um, p=0.011) and the RPE groups (12.49 ± 1.20 um, p=0.048) compared with the control group (9.38 ± 0.92 um) at 4 weeks after transplantation. At 24 weeks after transplantation, the thickness of the outer nuclear layer was still higher in the photoreceptor (12. 01 ± 1.48 um) or RPE group (9.85 ±1.23 um), compared with the control group (6.95 ± 0.68 um). The thickness of whole retina and inner nuclear layer were not different among groups at 4 weeks and 24 weeks after transplantation. Conclusions: Photoreceptor precursors and RPE cells can be derived effectively in short time via SNMs from hESCs. Subretinal transplantation of photoreceptor precursors or RPE was well tolerated and delayed retinal degeneration, implying possibilities for cell therapy for retinal degenerative diseases.