Neurotrophic Factors Secreted by Induced Pluripotent Stem Cell-Derived Retinal Progenitors Promote Retinal Survival and Preservation in an Adult Porcine Neuroretina Model.

Purpose: Paracrine factors released by pluripotent stem cells have shown great potential as therapeutic agents in regenerative medicine. The purpose of this study was to characterize trophic factor secretion of retinal progenitor cells (RPCs) derived from human induced pluripotent stem cells (iPSCs) and to assess its impact on retinal survival ex vivo. Methods: RPCs were generated from human 3D1 iPSCs following previously established protocols with modifications. Conditioned medium (CM) was harvested from iPSC-derived retinal progenitors and analyzed for trophic factor composition through multiplex enzyme-linked immunosorbent assay. Retina-preserving capability of the collected CM was examined using a degenerative porcine neuroretina model. Viability of the CM-treated retina explants was evaluated using the resazurin-based PrestoBlue reagent, whereas the lactate dehydrogenase (LDH) assay was used to assess retinal cytotoxicity. Retina explants were also analyzed morphologically through immunohistochemistry for glial cell activation and apoptosis. Results: We have successfully generated and characterized iPSC-derived RPCs that secreted an array of neuroprotective factors, including osteopontin, hepatocyte growth factor, stromal cell-derived factor 1, and insulin-like growth factor-1. Retina explants cultured in CM derived from iPSC-RPCs (iPSC-RPC-CM) showed better preservation of the retinal microarchitecture and fewer terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)+ nuclei, and reduced reactive gliosis. Furthermore, we saw a reduction in extracellular LDH levels in CM-treated retina explants, which also exhibited higher metabolic activity than the untreated controls. Conclusions: iPSC-derived RPCs secrete many trophic factors that have been shown to promote neuroprotection, tissue repair, and regeneration in the retina. Overall, we have demonstrated the neuroprotective effects of iPSC-RPC-CM through a degenerative neuroretina model ex vivo.

Repeat low-level blast exposure increases transient receptor potential vanilloid 1 (TRPV1) and endothelin-1 (ET-1) expression in the trigeminal ganglion.

Blast-associated sensory and cognitive trauma sustained by military service members is an area of extensively studied research. Recent studies in our laboratory have revealed that low-level blast exposure increased expression of transient receptor potential vanilloid 1 (TRPV1) and endothelin-1 (ET-1), proteins well characterized for their role in mediating pain transmission, in the cornea. Determining the functional consequences of these alterations in protein expression is critical to understanding blast-related sensory trauma. Thus, the purpose of this study was to examine TRPV1 and ET-1 expression in ocular associated sensory tissues following primary and tertiary blast. A rodent model of blast injury was used in which anesthetized animals, unrestrained or restrained, received a single or repeat blast (73.8 ± 5.5 kPa) from a compressed air shock tube once or daily for five consecutive days, respectively. Behavioral and functional analyses were conducted to assess blast effects on nocifensive behavior and TRPV1 activity. Immunohistochemistry and Western Blot were also performed with trigeminal ganglia (TG) to determine TRPV1, ET-1 and glial fibrillary associated protein (GFAP) expression following blast. Increased TRPV1, ET-1 and GFAP were detected in the TG of animals exposed to repeat blast. Increased nocifensive responses were also observed in animals exposed to repeat, tertiary blast as compared to single blast and control. Moreover, decreased TRPV1 desensitization was observed in TG neurons exposed to repeat blast. Repeat, tertiary blast resulted in increased TRPV1, ET-1 and GFAP expression in the TG, enhanced nociception and decreased TRPV1 desensitization.

Polarized Secretion of Matrix Metalloproteinases and Their Inhibitors by Retinal Pigment Epithelium Derived from Induced Pluripotent Stem Cells During Wound Healing.

PURPOSE: To characterize the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) by induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. We hypothesize that iPS-RPE secretes mediators of tissue remodeling such as MMPs and TIMPs to promote migration and proliferation of cells during wound healing. METHODS: iPS-RPE was grown on transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Conditioned media were collected from the apical and basolateral sides of the transwells every 72 h for 12 days. The media were analyzed by multiplex ELISA assays to detect secreted MMPs and TIMPs. Activity assays were performed to detect the active form of MMP-2 in conditioned media. RESULTS: MMP-2 and TIMP-1, -2, -3, and -4 were detected in conditioned media from iPS-RPE. The proteins were found to be secreted in a polarized manner. The apical secretion and activation of MMP-2 was elevated from days 3 to 12 after wounding. TIMP-1, -2, -3, and -4 were detected in conditioned media from both the apical and basolateral sides of wounded cells. Apical secretion of all 4 TIMPs increased within 3 days after wounding. CONCLUSIONS: These results indicate that iPS-RPE secretes MMP-2 and all 4 TIMPs in a polarized manner. After wounding, apical secretion of MMP-2 was higher compared to control. Apical secretion of all 4 TIMPs increased compared to control, while only TIMP-1 showed increased basolateral secretion compared to control.

Secretion Profile of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium During Wound Healing.

PURPOSE: The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing. METHODS: iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67, ß-catenin, e-cadherin, n-cadherin, and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction, cell proliferation, and migration were measured. RESULTS: iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of ß-catenin to the nucleus, cadherin switch, and expression of S100A4. GRO, GM-CSF, MCP-1, IL-6, and IL-8 were secreted by both the control and the wounded cell cultures. VEGF, FGF-2, and TGFß expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction, as well as proliferation and migration of ARPE 19 cells. CONCLUSIONS: These results indicate that after the monolayer is wounded, iPS-RPE secretes proteins into the culture medium that promote increased proliferation, contraction, and migration.

Trichostatin A Inhibits Retinal Pigmented Epithelium Activation in an In Vitro Model of Proliferative Vitreoretinopathy.

PURPOSE: Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however, the mechanisms leading to enhanced RPE proliferation, migration, and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease. METHODS: ARPE-19 cells, primary cultures of porcine RPE, and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGFß2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor, trichostatin A (TSA; 0.1 µM), were assessed for contraction and migration through collagen contraction and scratch assays, respectively. Western blotting and immunofluorescence analysis were performed to assess α-smooth muscle actin (α-SMA) and ß-catenin expression after TGFß2 treatment alone or in combination with TSA. RESULTS: TGFß2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 µM). In agreement with these data, immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased α-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGFß2-mediated iPS-RPE cell migration. CONCLUSIONS: Our findings indicate a role of acetylation in RPE activation. Specifically, the HDAC inhibitor TSA decreased RPE cell proliferation and TGFß2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR.