Qin Wan

A Multiplex High-Throughput Gene Expression Assay to Simultaneously Detect Disease and Functional Markers in Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

There is continuing interest in the development of lineage‐specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into “adult‐like” cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage‐specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained “adult‐like” phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high‐throughput format. Here, we use a multiplex high‐throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell‐derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell‐derived RPE that was then grown in 96‐ and 384‐well plates. As a proof of principle, we demonstrate differential expression of eight genes in iPS cells, iPS cell‐derived RPE at two different differentiation stages, and primary human RPE using this multiplex assay. The data obtained from the multiplex gene expression assay are significantly correlated with standard quantitative reverse transcription‐polymerase chain reaction‐based measurements, confirming the ability of this high‐throughput assay to measure relevant gene expression changes. This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases.

IFNγ regulates retinal pigment epithelial fluid transport

The present experiments show that IFNgamma receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNgamma applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNgamma in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.

In Pursuit of Authenticity: Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for Clinical Applications

Induced pluripotent stem cells (iPSCs) can be efficiently differentiated into retinal pigment epithelium (RPE), offering the possibility of autologous cell replacement therapy for retinal degeneration stemming from RPE loss. The generation and maintenance of epithelial apical‐basolateral polarity is fundamental for iPSC‐derived RPE (iPSC‐RPE) to recapitulate native RPE structure and function. Presently, no criteria have been established to determine clonal or donor based heterogeneity in the polarization and maturation state of iPSC‐RPE. We provide an unbiased structural, molecular, and physiological evaluation of 15 iPSC‐RPE that have been derived from distinct tissues from several different donors. We assessed the intact RPE monolayer in terms of an ATP‐dependent signaling pathway that drives critical aspects of RPE function, including calcium and electrophysiological responses, as well as steady‐state fluid transport. These responses have key in vivo counterparts that together help determine the homeostasis of the distal retina. We characterized the donor and clonal variation and found that iPSC‐RPE function was more significantly affected by the genetic differences between different donors than the epigenetic differences associated with different starting tissues. This study provides a reference dataset to authenticate genetically diverse iPSC‐RPE derived for clinical applications.

Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue.

PURPOSE We tested what native features have been preserved with a new culture protocol for adult human RPE. METHODS We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used. RESULTS Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 μM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 μL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01). CONCLUSIONS The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells

SUMMARY Primary cilia are sensory organelles that protrude from the cell membrane. Defects in the primary cilium cause ciliopathy disorders, with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation and that RPE maturation defects in ciliopathies precede photoreceptor degeneration. Pharmacologically enhanced ciliogenesis in wild-type induced pluripotent stem cells (iPSC)-RPE leads to fully mature and functional cells. In contrast, ciliopathy patient-derived iPSC-RPE and iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Proteins of the primary cilium regulate RPE maturation by simultaneously suppressing canonical WNT and activating PKCδ pathways. A similar cilium-dependent maturation pathway exists in lung epithelium. Our results provide insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC epithelial cells for clinical applications.

Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes

Surface proteins localized on the apical and basal plasma membranes are required for a cell to sense its environment and relay changes in ionic, cytokine, chemokine, and hormone levels to the inside of the cell. In a polarized cell, surface proteins are differentially localized on the apical or the basolateral sides of the cell. The retinal pigment epithelium (RPE) is an example of a polarized cell that performs a variety of functions that are dependent on its polarized state including trafficking of ions, fluid, and metabolites across the RPE monolayer. These functions are absolutely crucial for maintaining the health and integrity of adjacent photoreceptors, the photosensitive cells of the retina. Here we present a series of approaches to identify and validate the polarization state of cultured primary human RPE cells using immunostaining for RPE apical/basolateral markers, polarized cytokine secretion, electrophysiology, fluid transport, phagocytosis, and identification of plasma membrane proteins through cell surface capturing technology. These approaches are currently being used to validate the polarized state and the epithelial phenotype of human induced pluripotent stem (iPS) cell derived RPE cells. This work provides the basis for developing an autologous cell therapy for age-related macular degeneration using patient specific iPS cell derived RPE.