Mutant Best1 Expression and Impaired Phagocytosis in an iPSC Model of Autosomal Recessive Bestrophinopathy

Abstract

Autosomal recessive bestrophinopathy (ARB) is caused by mutations in the gene BEST1 which encodes bestrophin 1 (Best1), an anion channel expressed in retinal pigment epithelial (RPE) cells. It has been hypothesized that ARB represents the human null phenotype for BEST1 and that this occurs due to nonsense mediated decay (NMD). To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with ARB and her parents. After differentiation to retinal pigment epithelial (iPSC-RPE) cells, both BEST1 mRNA and Best1 protein expression were compared to controls. BEST1 mRNA expression levels, determined by quantitative PCR, were similar in ARB iPSC-RPE, parental cells, and genetically unrelated controls. Western blotting revealed that CRALBP and RPE65 were expressed within the range delineated by unrelated controls in iPSC-RPE from the ARB donor and her parents. Best1 protein was detected in different clones of ARB iPSC-RPE, but at reduced levels compared to all controls. When tested for the ability to phagocytose photoreceptor outer segments, ARB iPSC-RPE exhibited impaired internalization. These data suggest that impaired phagocytosis is a trait common to the bestrophinopathies. Furthermore, ARB is not universally the result of NMD and ARB, in this patient, is not due to the absence of Best1.

Inhibition of Ca 2+ channel surface expression by mutant bestrophin‐1 in RPE cells

“…This result is in accordance with the localization of all mutations at the N‐terminal half of bestrophin‐1 whereas domains for β‐subunit binding are on the C‐terminus. Comparable to other expression systems, in CHO‐K1, mutant bestrophin‐1 showed reduced surface expression. The Ca V 1.3 interaction reduces Ca V 1.3 plasma membrane localization as well.…”

“…Studies of mutant bestrophin‐1 in heterologous expression reported data representative for absence of WT‐bestrophin‐1 . Studies using iPSC‐generated RPE cells from patients with BVMD report slightly different results showing that the presence of WT‐bestrophin‐1 influences the outcome . The patients` iPSC model, however, does not permit differential detection of mutant versus WT‐bestrophin‐1 and bears the disadvantage that different cell lines from the same iPSC show strong variances.…”

“…As the light‐peak in the EOG results from activation of Cl channels in the RPE basolateral membrane, these observations deliver an explanation for the patients’ phenotype. More recent work indicated that BEST1 mutations impair phagocytic activity by the RPE, probably by a loss of volume control that leads to lipofuscin accumulation . However, the hypothesis that the loss of Cl − conductance alone might explain the pathology of disease BVMD is insufficient.…”

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions

“…The wound healing response has provided the basis, for example, for the use of platelet lysates in MSC cell culture . We too have observed that platelet lysate is an excellent replacement for serum in the growth of cells differentiated from iPSCs . For these reasons, the use of other components of the wound healing response such as fibrinogen/fibrin seems logical.…”

“…The previously described iPSC line 006‐BIOTR‐0001, obtained from fibroblasts and reprogrammed with Sendai virus kit (Thermo Fisher, Waltham, MA; clone 1, Mayo Clinic) was used for all experiments . Additional lines used to validate reproducibility of the fibrinogen coating included the previously described IMR‐90‐4, obtained from a immortalized human fibroblast cell line and reprogrammed using lentiviral vectors (clone 4; WiCell; Madison, WI), and a new line, 018‐BIOTR‐0089 (clone 18), produced by reprogramming of human peripheral blood lymphocytes using episomal DNA transfection. mTESR (Stem Cell Technologies, Vancouver, BC, Canada) was used for iPSC growth, and ReLeSR (Stem Cell Technologies) was used to dissociate cells for passage.…”