Small Molecules Restore Bestrophin 1 Expression and Function of Both Dominant and Recessive Bestrophinopathies in Patient-Derived Retinal Pigment Epithelium.

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy. Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp. Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1. Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.

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

Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three-dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two-dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1-60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin-based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications. Stem Cells Translational Medicine 2019;8:512-521.

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

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.

Fibrin hydrogels as a xenofree and rapidly degradable support for transplantation of retinal pigment epithelium monolayers.

Recent phase 1 trials of embryonic stem cell and induced pluripotent stem cell (iPSCs) derived RPE transplants for the treatment of macular degeneration have demonstrated the relative safety of this process. However, there is concern over clumping, thickening, folding, and wrinkling of the transplanted RPE. To deliver a flat RPE monolayer, current phase 1 trials are testing synthetic substrates for RPE transplantation. These substrates, however, cause localized inflammation and fibrosis in animal models due to long degradation times. Here we describe the use of thin fibrin hydrogels as a support material for the transplantation of RPE. Fibrin was formed into a mechanically rigid support that allow for easy manipulation with standard surgical instruments. Using fibrinolytic enzymes, fibrin hydrogels were degraded on the scale of hours. The rate of degradation could be controlled by varying the fibrinolytic enzyme concentration used. RPE cells degraded fibrin spontaneously. To preserve the fibrin support during differentiation of iPSCs to RPE, media was supplemented with the protease inhibitor aprotinin. iPSC-RPE on fibrin gels remained viable, generated monolayers with characteristic cobblestone appearance and dark pigmentation, and expressed mRNA and protein markers characteristic of RPE in the eye. Following differentiation of the cells, addition of fibrinolytic enzymes fully and rapidly degraded the fibrin support leaving behind an intact, viable iPSC-RPE monolayer. In conclusion, human fibrin hydrogels provide a xeno-free support on which iPSCs can be differentiated to RPE cells for transplant which can be rapidly degraded under controlled conditions using fibrinolytic enzymes without adverse effects to the cells. STATEMENT OF SIGNIFICANCE: Stem cell-derived retinal pigment epithelial (RPE) cell transplantation is currently in phase 1 clinical trials for macular degeneration (MD). A major obstacle in these studies is delivering the RPE as a living, flat sheets without leaving behind foreign materials in the retina. Here we investigate the suitability of using hydrogels made from human blood-derived proteins for RPE transplant. Our data shows that these fibrin hydrogels are rigid enough for use in surgery, support growth of stem cell-derived RPE, and are easily degraded within hours without damage to the RPE sheet. These fibrin hydrogels offer a promising solution to transplant RPE for patients with MD.