Transplantation of retinal organoids in vivo and long-term survival of photoreceptor cells
10.3760/cma.j.cn.115989-20200331-00227
- VernacularTitle:视网膜类器官在体移植及感光细胞长期存活实验研究
- Author:
Xiao SUN
1
;
Haohuan XIE
;
Mei ZHANG
;
Tian XUE
Author Information
1. 中国科学技术大学生命科学与医学部眼科中心 中国科学技术大学生命科学学院 中国科学院脑功能与疾病重点实验室，合肥 230026
- From:
Chinese Journal of Experimental Ophthalmology
2020;38(10):829-836
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the differentiation process of human-induced pluripotent stem cells (hiPSCs) into retinal organoids (ROs) in vitro and its potential application in cell transplantation, and to provide a fundamental study for stem-cell therapy. Methods:BC1-eGFP hiPSCs were differentiated into neurospheres in directional differentiation medium via suspension culture.On day 7, hiPSCs-induced-neurospheres were seeded onto Geltrex-coated dishes to induce neural retinal (NR) domains.On day 28, the NR domains were manually detached and collected.These NRs were cultured until the maturation of ROs.The quantitative real-time PCR (at week 0, week 7, week 15, week 21 and week 30 group) and immunohistochemistry (at day 8, day 15, week 15, week 21 and week 30 group) were used to characterize the differentiation process of hiPSCs.For ROs transplantation, the ROs were digested, and the cell suspension was injected into the subretinal cavity of Gnat1-/- mice with the outer limiting membrane damaged in retina.Immunohistochemistry was also used to detect the survival and integration ability of the implanted cells 5 months after transplantation. Results:The morphology and immunofluorescence staining illustrated that the cells induced from hiPSCs highly expressed the neural-retinal-epithelial specific markers PAX6 and SOX1 in the early stage, then the cells expressed the retinal-progenitor-cell specific marker LHX2 and a transparent and horseshoe-shaped NR domain was formed at the outer region of the colony.ROs was obtained by manual isolation and suspension culture.The diameter of ROs was about 1 millimeter.The retinal-like tissue gradually became thicker, even formed retinal pigment epithelial cells.Quantitative real-time PCR results showed that the expression of retinal-progenitor marker VSX2 peaked at week 7 and maintained its high expression thereafter ( F=168.30, P<0.01); the expression of retinal-precursor marker RCVRN also appeared at week 7 and increased thereafter ( F=271.60, P<0.01); the expressions of RHO was detected at week 15 ( F=95.02, P<0.01), and the expression of OPN1LW/MW was detected at week 21 ( F=40.57, P<0.01). Moreover, the expression of photoreceptor protein RHO maintained in a relatively high expression state at week 30.Three weeks after the transplantation of RO cells, cells with green fluorescence were successfully moved into the outer nuclear layer of the host retina.Four to six months after transplantation, the implanted cells expressed the functional light signal transduction protein GNAT1. Conclusions:Transplantation of retinal organoids in vivo can recapitulate the development of human retina.Transplanted RO cells can effectively move into the outer nuclear layer, differentiate into photoreceptors and survive in the recipient mice's retina over several months.
