Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.

We demonstrate for the first time that human blood-derived iPSCs can generate retinal cell types, providing a highly convenient donor cell source for iPSC-based retinal studies. We also show that cultured TiPSC-OVs have the capacity to self-assemble into rudimentary neuroretinal structures and express markers indicative of chemical and electrical synapses.

Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.

Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions hiPSCs form optic vesicle-like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC-derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, Visual System Homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC-OVs displayed a significant growth deficit compared to control hiPSC-OVs, as well as increased production of retinal pigmented epithelium (RPE) at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC-OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC-OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wildtype VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC-OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2-mediated gene regulation. Our results establish hiPSC-OVs as versatile model systems to study retinal development at stages not previously accessible in humans, and support the bona fide nature of hiPSC-OV-derived retinal progeny.