Characterization of Stargardt iPSC-derived RPE cells

Abstract

Purpose :
Stargardt disease (STGD) is a blinding disease of children and young adults, caused predominantly by mutations in the ABCA4 gene. The underlying STGD pathogenesis resides mainly within the supportive cells, the retinal pigment epithelium (RPE). We generated induced pluripotent stem cell (iPSC) derived RPE cells from three STGD patients with single pathogenic variants in the ABCA4 gene for whom other STGD genes have been excluded. In the current study, morphological and molecular features were evaluated in iPSC-derived RPE cells from one STGD patient with a heterozygous ABCA4 variant (R1129L / C.3386G>T).

Methods :
Fibroblasts from skin biopsies of STGD patients were reprogrammed to generate three independent iPSC lines, using the non-integrating Simplicon^TM RNA Reprogramming method. One of these STGD patient-derived lines and a normal control iPSC line (Source: UCLA BSCRC iPSC Core) were differentiated into RPE cells that were analyzed at passage 2, after 2 and 5 months (mo) in culture. Transcription levels of ABCA4 and markers of cell pluripotency, development, melanogenesis, and RPE-specificity were determined by qRT-PCR. Protein levels were evaluated by immunohistochemistry and immunoblotting. Morphological features of the iPS-derived RPE cells were analyzed by light and electron microscopy (EM). Transepithelial resistance (TER) was measured and formation of tight junctions was evidenced by immunofluorescence.

Results :
Normalized RPE-specific marker (RPE65, LRAT, BEST1) mRNA levels were several-fold lower in STGD iPSC-derived RPE cells compared to control in both 2 and 5 mo cultures. ABCA4 mRNA expression was reduced at 2 mo in culture compared with control cells and normalized by 5 mo. By EM analysis, STGD iPSC-derived RPE cells were highly polarized demonstrating basal nuclei, basal infoldings, apical microvilli and intercellular junctions, similar to the controls. By immunohistochemistry, ZO-1 localized on the lateral border of the cells with a heterogeneous distribution in the STGD-derived RPE. TER was significantly reduced in STGD iPSC-derived RPE cells compared to control.

Conclusions :
Differentiation of the STGD patient iPSCs into RPE cells was similar to the control iPSCs. Both STGD and control iPSC-derived RPE cells express robust levels of ABCA4. This will allow further molecular and biological analyses, including vitamin A metabolism and complement reactivity.

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