Human retinal pigment epithelium secretes insulin-like growth factor 2 in vitro

Abstract

Purpose : Functional retinal pigment epithelium (RPE) cultures can be derived from human induced pluripotent stem cells (iPSCs) to investigate the molecular mechanisms underlying human disease via an in vitro model. The RPE secretes various molecules and growth factors which can play a critical role in eye development. Retrospective analysis from RNA-sequencing and gene expression data (https://eyeintegration.nei.nih.gov/) showed that insulin-like growth factor 2 (IGF2) is expressed in RPE at the transcript level. IGF2 is a peptide hormone known to regulate cell proliferation, growth, migration, differentiation, and survival (Bergman et al., 2013). We aimed to validate the data via protein studies and investigate whether IGF2 could be a growth factor secreted by the RPE.

Methods : Human iPSC lines were derived from unaffected individuals and differentiated toward RPE cells. The RPE cells were cultured on a 2-D trans-well system for 6-8 weeks to mature and form an epithelial monolayer. All experiments were performed after 6-8 weeks of post-selection maturation. Western blot was performed to study IGF2 protein levels in RPE. Immuno-fluorescence followed by confocal microscopy (Zeiss 880) was performed to study cellular localization of IGF2. ELISA was performed using cell-culture supernatant media to quantify secreted levels of IGF2 from the apical and basal sides of RPE monolayers.

Results : A 20 kDa band was consistently observed in RPE monolayer lysates from three different RPE sources when incubated with anti-IGF2 antibody (ab9574). Immuno-fluorescence staining of RPE monolayers was performed using the same anti-IGF2 antibody and counter stained with ZO1 and nuclear stain (Hoechest333142). ZO1 staining was observed on the apical cell borders, and IGF2 staining localized in the apical portion of the cells just basal to the ZO1 staining.
To confirm IGF2 protein is secreted from the RPE monolayer, we performed ELISA using the apical and basal media sides of the RPE monolayer 24 hours after media change. We consistently observed IGF2 in the range of 200-400 pg/mL in the supernatant media.

Conclusions : IGF2 is expressed at the transcript and protein levels and secreted from the human RPE monolayer in vitro. This novel finding is important as IGF2 could play a crucial role in the development of the neural retina. Ongoing work is testing this hypothesis through an in vivo mouse model.

This is a 2021 ARVO Annual Meeting abstract.