Subretinal Transplantation of Embryonic Stem Cell–Derived Retinal Pigment Epithelium for the Treatment of Macular Degeneration: An Assessment at 4 Years

Abstract Purpose This study aimed to investigate whether the mouse embryonic stem cell (ESC) microenvironment improves the stem cell phenotype and proliferation properties of human retinal pigment epithelium (hRPE) cells by regulating the PI3K signaling pathway. Methods Primary hRPE cells were cocultured with either ESCs or human corneal epithelial cells (CECs) for 72 hours, after which their proliferation, apoptosis, cell cycle progression, and colony formation were assayed to evaluate changes in their biological characteristics. Gene expression was detected by real-time PCR and protein levels were determined by western blotting or immunofluorescence. LY294002, an antagonist of the PI3K signaling pathway, was used to further confirm the mechanism involved. Results In comparison to hRPE cells cultured alone, hRPE cells cocultured with ESCs had an increased proliferative capacity, reduced apoptotic rate, and higher colony-forming efficiency. The expression of the stem cell-associated marker KLF4 and the differentiation marker CRALBP increased and decreased, respectively, in hRPE cells isolated from the ESC coculture. Furthermore, PI3K pathway-related genes were significantly up-regulated in hRPE cells after exposure to ESCs. LY294002 reversed the pro-proliferative effect of ESCs on hRPE cells. In contrast, CECs did not share the ability of ESCs to influence the biological behavior and gene expression of hRPE cells. Conclusions Our findings indicate that the ESC microenvironment enhances stemness and proliferation of hRPE cells, partially via activation of the PI3K signaling pathway. This study may have a significant impact and clinical implication on cell therapy in regenerative medicine, specifically for age-related macular degeneration.

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Embryonic stem cell microenvironment enhances proliferation of human retinal pigment epithelium cells by activating the PI3K signaling pathway

10.21203/rs.3.rs-27573/v1 ◽

2020 ◽

Author(s):

Jiahui Liu ◽

Liu Yang ◽

Xiaoran Wang ◽

Shoubi Wang ◽

Zheqian Huang ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Retinal Pigment Epithelium ◽

Embryonic Stem Cell ◽

Signaling Pathway ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Embryonic Stem ◽

Pi3k Signaling ◽

Pi3k Signaling Pathway

Abstract Purpose. This study aimed to investigate whether the mouse embryonic stem cell (ESC) microenvironment improves the stem cell phenotype and proliferation properties of human retinal pigment epithelium (hRPE) cells by regulating the PI3K signaling pathway. Methods. Primary hRPE cells were cocultured with either ESCs or human corneal epithelial cells (CECs) for 72 hours, after which their proliferation, apoptosis, cell cycle progression, and colony formation were assayed to evaluate changes in their biological characteristics. Gene expression was detected by real-time PCR and protein levels were determined by western blotting or immunofluorescence. LY294002, an antagonist of the PI3K signaling pathway, was used to further confirm the mechanism involved. Results. In comparison to hRPE cells cultured alone, hRPE cells cocultured with ESCs had an increased proliferative capacity, reduced apoptotic rate, and higher colony-forming efficiency. The expression of the stem cell-associated marker KLF4 and the differentiation marker CRALBP increased and decreased, respectively, in hRPE cells isolated from the ESC coculture. Furthermore, PI3K pathway-related genes were significantly up-regulated in hRPE cells after exposure to ESCs. LY294002 reversed the pro-proliferative effect of ESCs on hRPE cells. In contrast, CECs did not share the ability of ESCs to influence the biological behavior and gene expression of hRPE cells. Conclusions. Our findings indicate that the ESC microenvironment enhances stemness and proliferation of hRPE cells, partially via activation of the PI3K signaling pathway. This study may have a significant impact and clinical implication on cell therapy in regenerative medicine, specifically for age-related macular degeneration.

Download Full-text