Development and characterization of patient-specific iPSC-derived retinal pigmentary epithelia (RPE)-like cells as a model of LCHAD-associated retinopathy.

Tiffany DeVine, PhD1 Garen Gaston, MS1, Dietrich Matern, MD, PhD2, Cary O. Harding, MD1, Melanie B. Gillingham, PhD1

1Department of Molecular & Medical Genetics at Oregon Health & Science University, Portland, Oregon, 2Mayo Clinic Biochemical Genetics Laboratory, Rochester, MN, USA

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) is one of three enzymatic domains found within the trifunctional protein (TFP) complex that mediates long chain fatty acid oxidation (FAO) in mitochondria. Unlike other FAO disorders, patients with LCHAD deficiency develop vision loss from progressive chorioretinopathy. Currently, there is no animal model available to study LCHAD associated chorioretinopathy.  While the mechanism of retinal pathogenesis in LCHAD deficient patients remains poorly understood, there is evidence to suggest that the initial physiologic perturbations begin in retinal pigment epithelial (RPE) cells and progress to other retinal cell layers. We sought to develop an in-vitro RPE cell model as a tool to uncover pathogenic mechanisms caused by loss of LCHAD. Additionally, this model can be used to explore novel therapies for the treatment of LCHAD associated retinopathy. In this study we reprogrammed patient fibroblasts harboring the HADHA (G1528C) mutation into induced pluripotent stem cells (iPSC).  These cells, along with wild type control iPSCs were subsequently differentiated into retinal pigment epithelium (RPE) through a directed method. Using immunofluorescence and RNA expression analysis, we have confirmed that iPSC derived RPE are histologically similar to primary human RPE despite remaining homozygous for the G1528C mutation. We have also confirmed that several fatty acid oxidation proteins (HADHA, HADHB, VLCAD, CPT1) are expressed in both control and HADHA mutant RPE. Cellular energetics were evaluated in both wildtype and mutant RPE cells using the Seahorse Biosciences XFe analyzer. Our results indicate that wild type RPE exposed to 200uM BSA-palmitate + carnitine in glucose limited media show a steady increase in the oxygen consumption rate (OCR), a 1.5-fold increase over 45 minutes, when compared to cells treated with BSA alone.

In contrast, LCHAD deficient cells showed no change in OCR when exposed to palmitate, suggesting that their ability to utilize long-chain fatty acids as an energy source is impaired. LCHAD deficient RPE that were fed BSA-palmitate (200uM) + carnitine showed a dramatic increase in C16-OH acylcarnitine in the media after 48 hours (.31uM) compared to LCHAD deficient RPE cells that were exposed to BSA alone (.02uM).  Media collected from wildtype RPE under the same conditions showed no change either in the presence of palmitate (.002uM) or in the BSA control (.002uM). These results demonstrate that LCHAD deficient RPE derived from patient iPSCs may be an effective model to study the pathophysiology of LCHAD-associated retinopathy and for the evaluation of potential novel therapies to treat this severely disabling disease.

Funded by the Scully/Peterson, Myers, and Marcello Miracle Family Foundations

Share this post