ROCK Inhibition Promotes Attachment, Proliferation, and Wound Closure in Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium

DOI: 10.1167/tvst.5.6.7 Article ROCK Inhibition Promotes Attachment, Proliferation, and Wound Closure in Human Embryonic Stem Cell–Derived Retinal Pigmented Epithelium Roxanne H. Croze, William J. Thi, and Dennis O. Clegg Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, Department of Molecular, Cellular & Developmental Biology, University of California, Santa Barbara, CA, USA Correspondence: Dennis O. Clegg, University of California, Santa Bar- bara, Neuroresearch Institute, Build- ing 571, Room 6131, Santa Barbara, CA 93106, USA. e-mail: dennis. clegg@lifesci.ucsb.edu Received: 4 June 2016 Accepted: 10 October 2016 Published: 22 November 2016 Keywords: age related macular degeneration; retinal pigment ep- ithelium; ROCK inhibition Citation: Croze RH, Thi WJ, Clegg DO. ROCK inhibition promotes at- tachment, proliferation, and wound closure in human embryonic stem cell–derived retinal pigmented epi- thelium. Trans Vis Sci Tech. 2016; 5(6):7, doi:10.1167/tvst.5.6.7 Purpose: Nonexudative (dry) age-related macular degeneration (AMD), a leading cause of blindness in the elderly, is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy, which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However, the factors regulating RPE responses to AMD-associated lesions are not well understood. Here, we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell–derived RPE (hESC-RPE) attachment, proliferation, and wound closure. Methods: H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment, and proliferation and cell size within an in vitro scratch assay were examined. Results: Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation, and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain, suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. Conclusions: ROCK inhibition promotes attachment, proliferation, and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. Translational Relevance: Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies. Introduction Age-related macular degeneration (AMD) is a progressive disease that is the leading cause of blindness in the aged population of the developed world. 1 While the mechanisms underlying AMD are still under investigation, most evidence suggests that death or dysfunction of the retinal pigmented epithe- lium (RPE) is responsible for onset of the disease. The RPE cells are critical for maintaining the function and viability of the photoreceptors, the main light sensing neuron. Therefore, when the RPE cells degenerate, the photoreceptors in turn begin to die and vision is lost. 2,3 There are two forms of the disease, wet or exudative, and dry or atrophic AMD. Wet AMD is characterized by neovascularization, which disrupts the RPE monolayer. This form, although rapidly progressive, can be treated with intraocular injections of anti–vascular epithelial growth factor (VEGF) antibodies to suppress the overactive blood vessel formation. 1 The dry form, affecting 80% to 90% of patients diagnosed with AMD, can only be treated with a vitamin cocktail and antioxidant supplements, which merely slows disease progression in 25% of patients. 4,5 Geographic atrophy (GA), areas devoid of RPE cells and photoreceptors, often occurs in late stage AMD patients, leading to large deficits in the central visual field. Unfortunately, RPE have limited proliferative abilities to fill in areas affected by GA prior to photoreceptor death. 6,7 TVST j 2016 j Vol. 5 j No. 6 j Article 7 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.