Characterization of Human ESC-Derived RPE

Human ESC-RPE authentication was performed as previously described [16 (link)]. Briefly, transepithelial electrical resistance (TEER) was triplicate measured with Millicell volt-ohm meter (Merck Millipore) [17 (link)]. The key RPE protein expression and localization were verified with indirect immunofluorescence labeling for zonula occludens-1 (ZO-1), claudin-3, claudin-19, sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase), bestrophin, and MER Proto-Oncogene, tyrosine Kinase (MERTK). Enzyme-linked immunoassay (ELISA) for pigment epithelium-derived factor (PEDF) was carried out from apical and basal media collected after overnight incubation and analyzed with the Human PEDF ELISA kit (BioVendor) following the manufacturer’s instructions. Phagocytosis assay was conducted with porcine photoreceptor outer segments (POS) by 4 h apical incubation at 37 °C in the presence of 10% fetal bovine serum (Thermo Fisher Scientific), followed by labeling with anti-rhodopsin antibody and tetramethylrhodamine (TRITC). The nuclei were counterstained with DAPI included in ProLong Gold mounting medium (Thermo Fisher Scientific). Images were acquired with an LSM 700-800 Confocal microscope (Carl Zeiss) and processed with the Zen 2.3 SP1 Black software (Carl Zeiss). All primary and secondary antibody details appear in Table S1 (Additional file 1).

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Liu Z., Ilmarinen T., Tan G.S., Hongisto H., Wong E.Y., Tsai A.S., Al-Nawaiseh S., Holder G.E., Su X., Barathi V.A., Skottman H, & Stanzel B.V. (2021). Submacular integration of hESC-RPE monolayer xenografts in a surgical non-human primate model. Stem Cell Research & Therapy, 12, 423.

Publication 2021

Millicell volt-ohm meter Human PEDF ELISA kit fetal bovine serum ProLong Gold mounting medium LSM 700-800 Confocal microscope Zen 2.3 SP1 Black software

Anti antibody Antibody Assay Bestrophin Claudin Claudin 3 Confocal microscope Dapi Electrical resistance Enzyme immunoassay Fetal bovine serum Gold Human Indirect immunofluorescence Mertk Na k atpase Nuclei Phagocytosis Photoreceptor Pigment epithelium derived factor Porcine Protein Proto oncogene Rhodopsin Tetramethylrhodamine Tritc Tyrosine kinase Zonula occludens

Corresponding Organization :

Other organizations : National University of Singapore, Tampere University, Singapore Eye Research Institute, Knappschaftsklinikum Saar, Agency for Science, Technology and Research, Institute of Molecular and Cell Biology

Top 5 similar protocols

Variable analysis

independent variables

Transepithelial electrical resistance (TEER) measurement method (Millicell volt-ohm meter)
Indirect immunofluorescence labeling for RPE protein expression and localization (ZO-1, claudin-3, claudin-19, Na+/K+-ATPase, bestrophin, MERTK)
Enzyme-linked immunoassay (ELISA) for pigment epithelium-derived factor (PEDF) secretion
Phagocytosis assay with porcine photoreceptor outer segments (POS)

dependent variables

Transepithelial electrical resistance (TEER) values
RPE protein expression and localization (ZO-1, claudin-3, claudin-19, Na+/K+-ATPase, bestrophin, MERTK)
PEDF secretion levels in apical and basal media
Phagocytosis of porcine photoreceptor outer segments (POS) by RPE cells

control variables

Overnight incubation duration for PEDF ELISA
Incubation temperature (37 °C) for phagocytosis assay
Presence of 10% fetal bovine serum for phagocytosis assay

controls

Positive control: None specified
Negative control: None specified

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