Abstract
Purpose:
To evaluate cellular expression profiles of retinal pigment epithelium-65 (RPE65) and zonular occludens-1 (ZO-1) markers during spontaneous differentiation human embryonic stem cell into retinal pigment epithelial cells (hESC-RPE)
Methods:
Human embryonic stem cells (hESC), from the WA09-DL-11 feeder dependent line, were removed from liquid nitrogen and grown on a confluent layer of inactivated mouse embryonic fibroblast. Differentiated pigmented embryoid body (EB) clusters were dissected from undifferentiated hESC colonies. After dissection, EBs were isolated onto 6-well gelatin coated plates for further monolayer expansion into hESC-RPE cells. Confluent monolayers were passaged 2 - 3 weeks following EB isolation and plated onto gelatin-coated slides. Each slide was placed into a 10 mm petri dish filled with RPE maintenance media and allowed to grow to either 7, 21, or 35 days post passage. Slides were extracted on designated time points and prepared for immunohistocytochemistry with antibodies targeted to RPE65 and ZO-1. Immunofluorescence was performed under 10x magnification on an inverted Olympus IX51-IX2-SL microscope with Olympus U-RFL-T fluorescence lamp.
Results:
Day seven following hESC-RPE precursor cell plating showed no evidence of RPE65 or ZO-1 expression. Two weeks later on day 21, maturation of hESC-RPE precursor cells demonstrated an enhanced fluorescence of RPE65 and a slight fluorescence increase in ZO-1 expression. Four weeks after precursor cell plating, fluorescence intensity for both RPE65 and ZO-1 was more intense than the prior two time points.
Conclusions:
This study shows enhanced in vitro cellular expression patterns of RPE65 and ZO-1 during differentiation of hESC-RPE precursor cells into a more mature state. Based on the above findings, precursor hESC-RPE cells demonstrate RPE marker patterns between one to three weeks after plating. This information can assist with therapeutic implementation especially when attempting to identify points of hESC-RPE precursor maturation.
Keywords: 701 retinal pigment epithelium •
721 stem cells •
554 immunohistochemistry