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

Abstract

PurposeNonexudative (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.MethodsH9 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.ResultsPharmacological 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.ConclusionsROCK inhibition promotes attachment, proliferation, and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing.Translational RelevanceModulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies.

Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage

“…The complex RPE-derived wound healing process in AMD is weakly understood. In a recent study the healing of scratch wounded H9 hESC-RPE cells has been shown to have partial wound closure within 30 days [ 12 ]. Ca 2+ has an important role in the wound healing of epithelial cells but less is known about cellular Ca 2+ dynamics implicated in the response to fresh wounds and wound healing of RPE.…”

“…The functionality of RPE after wounding has been assessed in various animal models, for example, rabbit [ 5 , 6 ], chick [ 7 ], porcine [ 8 ], bovine RPE [ 9 ] and in donor human RPE cells, [ 10 , 11 ] and human ESC-RPE cells [ 12 ]. These studies demonstrated that RPE cells on the wound edge undergo dedifferentiation, changing their phenotype towards the phenotype of migrating cells: the cells evolve cytoskeletal reorganization and partially or completely lose their pigmentation [ 5 , 6 , 8 , 13 ].…”

“…In wounded monolayers, the cells located in the vicinity of the denuded area are important for triggering a wound healing process [ 17 , 18 ]. Furthermore, in the healed wounds, the RPE cells transiently change their morphology towards a less mature phenotype [ 5 , 12 , 13 ]. Thus, we hypothesize that these changes are reflected in Ca 2+ dynamics of cells located around the wounded area and inside of healed wounds.…”

ROCK Inhibitor Increases Proacinar Cells in Adult Salivary Gland Organoids

“…A well-known function of Y27632 in cells cultured ex vivo is to promote proliferation as well as to ablate apoptosis in dissociated cells (Watanabe et al, 2007;Chapman et al, 2014;Sun et al, 2015;Croze et al, 2016;Zhang et al, 2011). ROCK inhibition by Y27632 has been also shown to delay senescence in the salivary gland epithelium .…”

Trockene AMD – zelluläre und gentherapeutische Behandlungsansätze

“…Weiterführende Arbeiten hinsichtlich der Verwendung von hESC-RPE-Transplantaten beschäftigen sich u. a. mit der Etablierung verbesserter Kultivierungsmethoden. Beispiele hierfür sind xenofreie, definierte Medien, die essenziell für die klinische Anwendung und eine reduzierte Immunogenität sind [32] oder die Kultivierung in Anwesenheit von ROCK-Inhibitoren (ROCK: RhoA Coiled-Coil Kinase), welche die Anhaftung, Proliferation und Wundheilung der Zellen begünstigen [33]. Modifikationen hinsichtlich der Transplantationstechnik werden in vivo im Kaninchenein Tiermodell mit einer zum Menschen vergleichbaren Augengrößeausgetestet.…”