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

Abstract

SUMMARYPrimary 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.

Functional Voltage-Gated Calcium Channels Are Present in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium

“…D, Supporting Information Fig. S2D), characteristic to mature RPE . This data suggest that with increasing maturation, hESC‐RPE started to gain the homogeneous apical localization of Ca V 1.3 detected in mouse RPE (Fig.…”

“…We addressed this considering the localization of the primary Ca V channel subtype, Ca V 1.3, during hESC‐RPE maturation. We immunolabeled Ca V 1.3 together with pericentrin (PCNT), a protein localized in the centrosomes at the base of the primary cilia, that have been recently shown to be important for RPE maturation . Figure shows how the localization of these proteins changed remarkably during maturation.…”

Gene augmentation and read-through rescue channelopathy in an iPSC-RPE model of congenital blindness

“…We thus confirmed maturity of these cultured hiPSC-RPE cells unlike the ones generated from ciliopathy patients. 28 Matured LCA16 hiPSC-RPE cells did not show Kir7.1 function, their membrane potential was depolarized and they were unable to phagocytose photoreceptor outer segments as compared to control hiPSC-RPE cells. A defect in the phagocytosis of shed photoreceptor outer segments by LCA16 hiPSC-RPE is thus secondary and consistent with the slow progression toward blindness observed in LCA16 patients, as documented through clinical manifestations, including electroretinogram abnormalities and retinal pigmentation.…”

Primary Cilia as Signaling Hubs in Health and Disease

“…For example, transcriptome analysis uncovered that TGFβ signaling that is activated at the ciliary pocket, the proximal part of the primary cilium residing in the cytoplasm within an invagination of the plasma membrane, is associated with differentiation into cardiomyocytes. [26,214] The molecular mechanisms underlying the regulation of cell proliferation by growth factors and morphogens through primary cilia remain to be completely elucidated. [213] Inducible pluripotent stem cells and animal models, such as mice and zebrafish, combined with genome-editing technology, such as CRISPR/ Cas9, can also be used to examine the complex phenotypes induced by the impairment of target genes in primary cilia.…”