Polarized Expression of Kir7.1 Channels in a 3D Organoid Culture Model

Abstract

BackgroundLeber Congenital Amaurosis (LCA) is an autosomal recessive genetic condition of the retina that leads to blindness in infants and young children. Previous data indicate that mutations in Kir7.1, an inwardly rectifying potassium channel, located in the retinal pigmented epithelium apical membrane contributes to a LCA blindness subtype. The specific localization of the Kir7.1 in the polarized RPE cell is critical as it contributes to retina function. Using standard cell culture models it is impossible to reproduce Kir7.1 distribution, molecular interaction and hence function. Culturing cells in 3D‐organoids will likely provide an appropriate in vitro system to study Kir7.1 disease mechanism and develop therapeutics.HypothesisWe hypothesize that Kir7.1 protein will have a polarized distribution in differentiated Mardin Darby Canine Kidney (MDCK) 3D Cell Culture Model that allows us to study channel biology.MethodsWe isolated mRNA from cultured MDCK cells that were either transfected with Kir7.1 or non‐transfected. We then utilized PCR and ran the PCR products on an agarose gel. We have optimized a 3D cell culture model of MDCK cells. About 3×104 cells/ml were grown in a collagen cocktail (24 mM glutamine, 2.8 mM NaHCO3, 1X MEM alpha, 20 mM HEPES, 2 mg/ml Collagen I) for 5 days in a compartmentalized chamber coverslip. On Day 5 or when organoids were noticed, we transduced with GFP Kir7.1 through lentiviral particles (viral titer 3.54×109) 500 MOI. Approximately one week after transduction, we stained the organoids with Hoescht and WGA‐Alexa 594 for nucleus and membrane staining respectively. Using Nikon C2‐confocal microscope, z‐stack images were acquired and subjected to off‐line analysis.ResultsOur PCR results indicate that MDCK cells do not natively express Kir7.1 as it was only expressed in the transfected cells. After 5 days in culture, we began to observe well developed spherical organization of mature MDCK cells. A single layer of Hoescht positive cells could be visualized. Kir7.1 expression was mostly detected by GFP positive signal in the membrane lining the inner surface of the sphere. WGA‐Alexa 594, however, lined the outer membrane of the organoids.ConclusionThe development of the MDCK 3D cell culture model allows for the study of polarized epithelium. Kir7.1 localization to the inner membrane of the organoid confirms that the basolateral membrane localization in MDCK cells corresponds with apical distribution in the RPE cells. Our data also indicates that MDCK cells are a good model for studying Kir7.1 because they do not natively express the protein. The experimental set up is thus robust and time efficient to study Kir7.1 channel biology in its most native setting.Support or Funding InformationNIH T32HD041921 NIH EY024995This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.