Enhancement of Constructing Ultra-thin Corneal Stromal Membranes-Based Bioengineered Retinal Pigment Epithelial Sheet in iPS - Conditioned Medium

Abstract

Purpose : Developing a bioengineered retinal pigment epithelium (RPE) sheet is difficult and fundamentally determines the success of RPE cell transplantation,which requires high dynamic RPE cells and a suitable scaffold.Here,we test the hypothesis that a ultra-thin corneal stromal membrane (CSM) can be used as a natural scaffold to construct a befitting bioengineered RPE sheet after cultured with induced pluripotent stem cells conditioned medium (iPS-CM) in vitro.

Methods : Primary hRPE cells were isolated from human eye balls and cultured. The optimum concentration of iPS-CM for the viability of RPE was screened by CCK-8 assays. Then cell morphology and proliferation were compared between iPS-CM group (iPS-CM supplemented DMEM-F12 medium and 10% serum) and control (DMEM-F12 medium and 10% serum) in hRPE cells by q-PCR, Edu and flow cytometry analysis. RPE cells were seeded on CSM. And they were assayed by immunofluorescence, qPCR and SEM.

Results : Primary hRPE cells positively expressed RPE cell markers such as RPE-65, Mitf, EMMPRIN. The optimum concentration of iPS-CM (iPS supernatant ratio DMEM-F12) is 1:2. hRPE cells at P1-P5 in iPS-CM displayed a polygonal and compact pattern, whereas hRPE cells at P1-P5 in control displayed an irregular and fusiform pattern. iPS-CM promoted proliferation and inhibited apoptosis of hRPE cells compared to controls (p=0.01). iPS-CM also decreased the expression of epithelial-mesenchymal transition (EMT)-related genes and proteins, such as fibronenctin, β-catenin and α-SMA in cultured hRPE. hRPE cells grown on CSM in iPS-CM showed more mottled pigmentation (Fig.1). They exhibited a correctly orientated monolayer sheet with a polygonal cell shape (Fig.2) and abundant microvilli on their apical surfaces.

Conclusions : iPS-CM can significantly promote the proliferation and inhibit EMT and apoptosis in hRPE cells. iPS-CM can also enhance the construction of CSM based bioengineered RPE sheet. A ultra-thin corneal stromal membrane may provide a promising vehicle for further RPE implantation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Fig.1. hRPE cells at P1 presented a fusiform pattern in control and a uniform epithelioid morphology in iPS-CM. They showed more mottled pigmentation after cultured on corneal stromal membrane (CSM).

 

Fig.2. The tissue sections of CSM based bioengineered RPE sheet showed monolayer RPE cells with the expressions of actin and DAPI.