Endothelial-Derived Exteracellular Vesicles Promote In Vitro Human Embryonic Stem Cell-Derived Retinal Pig-ment Epithelium Proliferation

Background: Human embryonic stem cell-derived retinal pig-mented epithelium (hESC-RPE) transplantation inspires some hopes for the treatment of retinal degenerative diseases. There are some studies that confirmed the powerful role of endothelial cells conditioned medium (ECs-CM) on RPE cells expansion. A current study aimed to investigate the effect of ECs extracel-lular vesicles (EVs), as an important part of paracrine factors on hESC-RPE Proliferation.Materials and Methods: The EVs were isolated from HU-VECs conditioned, then characterized by BCA protein assay, Dynamic light scattering, and western blot. 24 h post 50,000 cells/cm2 hESC-RPE cells seeding, the 75 µg/ml EVs were add-ed every two each day. At 9th days, both groups including PBS and EVs were considered for further experiments. To evaluate the effect of EVs on hESC-RPE proliferation, we conducted Ki-67 immunostaining and evaluate the monolayer organization by ZO1 staining.Results: The isolated EVs passed all criteria, the mean parti-cle diameter was 88.6 nm, and the expression of EVs marker was approved by CD81 western blot. At the 9th day, Ki-67 posi-tive cells in EVs treated hESC-RPE were 143.41 ± 9.72 which is significantly higher compared to the control group 41.00 ± 10.39 (P<0.0001). The result of ZO1 staining also indicates that EVs could accelerate the hESC-RPE monolayer formation.Conclusion: These results confirmed that the EVs secreted by HUVECs promoted expansion and monolayer formation that could be beneficial for obtaining higher numbers of hESC-RPE cells. Also, it is plausible that EVs could potentially stimulate RPE proliferation and regeneration at the early stages of retinal degenerative diseases. Further investigations on the effects of HUVECs- EVs are needed.

Keywords:

hESC-RPE , HUVECs , Extracellular Vesicles , Prolif-eration