Abstract
Purpose :
The retinal pigment epithelium (RPE) is a polarized tissue that has important functions like supporting photoreceptors of the eye, transporting nutrients and secreting growth factors and cytokines. Our lab studied the surface proteome of RPE by cell surface capturing technology which revealed the presence of ABCA4 on the surface of RPE. We focused on the role of the ABCA4 protein as mutations in ABCA4 gene have been associated with various retinal degenerative diseases including the Stargardt disease. To specifically understand the function of ABCA4 protein in RPE, we developed an in-vitro Stargardt disease model using ABCA4-/- induced pluripotent stem cells (iPSC)-derived RPE and Stargardt patient iRPE.
Methods :
We used CRISPR/Cas9 technology to generate iPSCs with a knockout of the ABCA4 gene targeting exon 1 of the ABCA4 gene. Resultant targeted clones were screened and two independent clones (C1 and C2) that resulted in out-of-frame deletions on both alleles were selected. For the Stargardt patient, fibroblasts were isolated from skin biopsy which were reprogrammed to form iPSCs. ABCA4-/- iPSCs and Stargardt patient iPSCs were differentiated to form Stargardt iRPE. ABCA4 knockout was confirmed by qRT-PCR, dd PCR, immunostaining and Sanger sequencing. Cells were evaluated for RPE-specific morphology by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) and functions such as electrophysiological response.
Results :
Stargardt iRPE and the corresponding isogenic control iPSCs displayed normal karyotype and expressed similar levels of developmental and mature RPE markers. Immunostaining of iRPE monolayers for maturity markers RPE65 and EZRIN further confirmed qRT-PCR observation. TEM of iRPE monolayers showed normal polarized RPE features like abundant apical processes and tight junctions between neighboring cells in Stargardt iRPE. SEM also suggested the formation of normal and fully confluent apical processes in Stargardt iRPE compared to isogenic Control iRPE. In addition, Stargardt-iRPE showed similar electrophysiological response compared to the Control iRPE.
Conclusions :
We generated an in vitro Stargardt disease model using ABCA4-/- and Stargardt patient iRPE. We found that the genetic profile, cell morphology and structural features of patient and ABCA4-/- iRPE behaved like Control iRPE suggesting there are no developmental defects in RPE due to ABCA4 loss of function.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.