Culture of Highly Differentiated Human Retinal Pigment Epithelium for Analysis of the Polarized Uptake, Processing, and Secretion of Retinoids

The retinal pigment epithelium (RPE) occupies a strategic position within the eye, given its location between the neurosensoryretina and the vascular bed (choroid) that nourishes the photoreceptor cells (rods and cones). Among the many attributes ofthis versatile monolayer of cells is its unique ability to convert vitamin A (retinol) into the prosthetic group (11-
cis
-retinal) for the rod and cone opsins, the photopigments essential for vision. It does so by absorbing retinol via a receptor-mediatedprocess that involves the interaction of a carrier protein secreted by the liver, retinol-binding protein (RBP), and a receptor/channelthat is the gene product of STRA6 (stimulated by retinoic acid 6). Following its uptake through the basolateral plasma membraneof the RPE, retinol encounters a brigade of binding proteins, membrane-bound receptors, and enzymes that mediate its multi-stepconversion to 11-
cis
-retinal and the transport of this visual chromophore to the light-sensitive photoreceptor cell outer segment, the portionof the cell that houses the phototransduction cascade. This process is iterative, repeating itself via the retinoid visualcycle. Most of the human genes that code for this cohort of proteins carry disease-causing mutations in humans. The consequencesof these mutations range in severity from relatively mild dysfunction such as congenital stationary night blindness to totalblindness. The RPE, although post-mitotic in situ, is capable of proliferation when removed from its native milieu. This offersone the opportunity to study the retinoid visual cycle in modular form, providing insights into this intriguing process inhealth and disease. This chapter describes a cell culture method whereby the entire visual cycle can be created in vitro.