Abstract
BackgroundTransplantation of human pluripotent stem cell-derived retinal pigment epithelium (RPE) is an urgently needed treatment for the cure of degenerative diseases of the retina. The transplanted cells must tolerate cellular stress caused by various sources such as retinal inflammation and regain their functions rapidly after the transplantation. We have previously shown the maturation level of the cultured human embryonic stem cell-derived RPE (hESC-RPE) cells to influence for example their calcium (Ca2+) signaling properties. Yet, no comparison of the ability of hESC-RPE at different maturity levels to tolerate cellular stress has been reported.MethodsHere, we analyzed the ability of the hESC-RPE populations with early (3 weeks) and late (12 weeks) maturation status to tolerate cellular stress caused by chemical cell stressors protease inhibitor (MG132) or hydrogen peroxide (H2O2). After the treatments, the functionality of the RPE cells was studied by transepithelial resistance, immunostainings of key RPE proteins, phagocytosis, mitochondrial membrane potential, Ca2+ signaling, and cytokine secretion.ResultsThe hESC-RPE population with late maturation status consistently showed improved tolerance to cellular stress in comparison to the population with early maturity. After the treatments, the early maturation status of hESC-RPE monolayer showed impaired barrier properties. The hESC-RPE with early maturity status also exhibited reduced phagocytic and Ca2+ signaling properties, especially after MG132 treatment.ConclusionsOur results suggest that due to better tolerance to cellular stress, the late maturation status of hESC-RPE population is superior compared to monolayers with early maturation status in the transplantation therapy settings.
Highlights
Transplantation of human pluripotent stem cell-derived retinal pigment epithelium (RPE) is an urgently needed treatment for the cure of degenerative diseases of the retina
Our results indicate that the late maturity status of human embryonic stem cell (hESC)-RPE cells can tolerate cellular stress more effectively than the population with early maturation status, with potential implications to hPSC-RPE cell therapy
Mitochondria remained active after treatments with MG132 and Hydrogen peroxide (H2O2) To assess functional consequences of the stressors rather than cell death, hESC-RPE cells cultured for 3 weeks or 12 weeks were treated with sublethal concentrations of MG132 or H2O2, as previously determined for mature hESC-RPE [23, 24]
Summary
Transplantation of human pluripotent stem cell-derived retinal pigment epithelium (RPE) is an urgently needed treatment for the cure of degenerative diseases of the retina. We have previously shown the maturation level of the cultured human embryonic stem cell-derived RPE (hESC-RPE) cells to influence for example their calcium (Ca2+) signaling properties. The developmental stage of non-polarized cadaveric adult human RPE stem cells has been shown to affect transplantation efficacy when suspension transplantation was used, with intermediate differentiation times (4 weeks) producing the most consistent vision rescue in a rat model [10]. Polarized human embryonic stem cell (hESC)-derived RPE cells cultured for 4 weeks, on the other hand, have been shown to decrease their sensitivity to oxidative stress compared to non-polarized cells, suggesting potential advantages of sheet transplantation over the suspension approach [11]. Despite potentially highly impacting therapy efficacy, the development of tolerance to cellular stress during further in vitro maturation of hESC-RPE has not been examined. We evaluated the impact of culture time on the ability of the hESC-RPE to endure treatments with chemical stressors
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