Polyester Nanoparticles and Polyurethane Nanocapsules Deliver Pirfenidone To Reduce Fibrosis and Scarring.

Pirfenidone has been shown to reduce fibrosis and modulate inflammation associated with conditions from pulmonary fibrosis to rheumatoid arthritis. It may also be useful for ocular diseases as well. However, for pirfenidone to be effective, it needs to be delivered to the tissue of interest, which, in the case of the eye, in particular, motivates the need for a system that permits local, long-term delivery to address the continuing pathology of the, condition. We investigated a set of delivery systems to determine the impact of encapsulation materials on the loading and delivery of pirfenidone. While the polyester system based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles exhibited higher loading than a polyurethane-based nanocapsule system, the delivery was short, with 85% of the drug being released in 24 h and no measurable drug after 7 days. Addition of different poloxamers impacted the loading but not the release of the drug. In contrast, the polyurethane nanocapsule system delivered 60% of the drug over the first 24 h and the remainder over the next 50 days. Furthermore, the polyurethane system permitted on-demand delivery via ultrasound. Being able to tune the amount of drug delivered via ultrasound has the potential to tailor the delivery of pirfenidone to modulate inflammation and fibrosis. We used a fibroblast scratch assay to confirm the bioactivity of the released drug. This work provides multiple platforms for the delivery of pirfenidone locally and over time in both passive and on-demand formulations with the potential to address a range of inflammatory and fibrotic conditions.

Evaluating Thera-101 as a Low-Volume Resuscitation Fluid in a Model of Polytrauma.

Traumatic brain injury (TBI) and hemorrhage remain challenging to treat in austere conditions. Developing a therapeutic to mitigate the associated pathophysiology is critical to meet this treatment gap, especially as these injuries and associated high mortality are possibly preventable. Here, Thera-101 (T-101) was evaluated as low-volume resuscitative fluid in a rat model of TBI and hemorrhage. The therapeutic, T-101, is uniquely situated as a TBI and hemorrhage intervention. It contains a cocktail of proteins and microvesicles from the secretome of adipose-derived mesenchymal stromal cells that can act on repair and regenerative mechanisms associated with poly-trauma. T-101 efficacy was determined at 4, 24, 48, and 72 h post-injury by evaluating blood chemistry, inflammatory chemo/cytokines, histology, and diffusion tensor imaging. Blood chemistry indicated that T-101 reduced the markers of liver damage to Sham levels while the levels remained elevated with the control (saline) resuscitative fluid. Histology supports the potential protective effects of T-101 on the kidneys. Diffusion tensor imaging showed that the injury caused the most damage to the corpus callosum and the fimbria. Immunohistochemistry suggests that T-101 may mitigate astrocyte activation at 72 h. Together, these data suggest that T-101 may serve as a potential field deployable low-volume resuscitation therapeutic.

Comparative evaluation of mesenchymal stromal cell growth and osteogenic differentiation on a shape memory polymer scaffold.

Trauma-induced, critical-size bone defects pose a clinical challenge to heal. Albeit autografts are the standard-of-care, they are limited by their inability to be shaped to various defect geometries and often incur donor site complications. Herein, the combination of a "self-fitting" shape memory polymer (SMP) scaffold and seeded mesenchymal stromal cells (MSCs) was investigated as an alternative. The porous SMP scaffold, prepared from poly(ε-caprolactone) diacrylate (PCL-DA) and coated with polydopamine, provided conformal shaping and cell adhesion. MSCs from five tissues, amniotic (AMSCs), chorionic tissue (CHSCs), umbilical cord (UCSCs), adipose (ADSCs), and bone marrow (BMSCs) were evaluated for viability, density, and osteogenic differentiation on the SMP scaffold. BMSCs exhibited the fastest increase in cell density by day 3, but after day 10, CHSCs, UCSCs, and ADSCs approached similar cell density. BMSCs also showed the greatest calcification among the cell types, followed closely by ADSCs, CHSCs and AMSCs. Alkaline phosphatase (ALP) activity peaked at day 7 for AMSCs, UCSCs, ADSCs and BMSCs, and at day 14 for CHSCs, which had the greatest overall ALP activity. Of all the cell types, only scaffolds cultured with ADSCs in osteogenic media had increased hardness and local modulus as compared to blank scaffolds after 21 days of cell culture and osteogenic differentiation. Overall, ADSCs performed most favorably on the SMP scaffold. The SMP scaffold was able to support key cellular behaviors of MSCs and could potentially be a viable, regenerative alternative to autograft.

Azithromycin Protects Retinal Glia Against Oxidative Stress-Induced Morphological Changes, Inflammation, and Cell Death.

The reactivity of retinal glia in response to oxidative stress has a significant effect on retinal pathobiology. The reactive glia change their morphology and secret cytokines and neurotoxic factors in response to oxidative stress associated with retinal neurovascular degeneration. Therefore, pharmacological intervention to protect glial health against oxidative stress is crucial for maintaining homeostasis and the normal function of the retina. In this study, we explored the effect of azithromycin, a macrolide antibiotic with antioxidant, immunomodulatory, anti-inflammatory, and neuroprotective properties against oxidative stress-induced morphological changes, inflammation, and cell death in retinal microglia and Müller glia. Oxidative stress was induced by H2O2, and the intracellular oxidative stress was measured by DCFDA and DHE staining. The change in morphological characteristics such as the surface area, perimeter, and circularity was calculated using ImageJ software. Inflammation was measured by enzyme-linked immunosorbent assays for TNF-α, IL-1ß, and IL-6. Reactive gliosis was characterized by anti-GFAP immunostaining. Cell death was measured by MTT assay, acridine orange/propidium iodide, and trypan blue staining. Pretreatment of azithromycin inhibits H2O2-induced oxidative stress in microglial (BV-2) and Müller glial (MIO-M1) cells. We observed that azithromycin inhibits oxidative stress-induced morphological changes, including the cell surface area, circularity, and perimeter in BV-2 and MIO-M1 cells. It also inhibits inflammation and cell death in both the glial cells. Azithromycin could be used as a pharmacological intervention on maintaining retinal glial health during oxidative stress.

Analysis of Nonbattle Deaths Among U.S. Service Members in the Deployed Environment.

OBJECTIVE: Describe etiologies and trends in non-battle deaths (NBD) among deployed U.S. service members to identify areas for prevention. BACKGROUND: Injuries in combat are categorized as battle (result of hostile action) or nonbattle related. Previous work found that one-third of injured US military personnel in Iraq and Afghanistan had nonbattle injuries and emphasized prevention. NBD have not yet been characterized. METHODS: U.S. military casualty data for Iraq and Afghanistan from 2001 to 2014 were obtained from the Defense Casualty Analysis System (DCAS) and the Department of Defense Trauma Registry (DoDTR). Two databases were used because DoDTR does not capture prehospital deaths, while DCAS does not contain clinical details. Nonbattle injuries and NBD were identified, etiologies classified, and NBD trends were assessed using a weighted moving average and time-series analysis with autoregressive integrated moving average. Future NBD rates were forecast. RESULTS: DCAS recorded 59,799 casualties; 21.0% (n = 1431) of all deaths (n = 6745) were NBD. DoDTR recorded 29,958 casualties; 11.5% (n = 206) of all deaths (n = 1788) were NBD. After early fluctuations, NBD rates for both Iraq and Afghanistan stabilized at approximately 21%. Leading causes of NBD were gunshot wounds and vehicle accidents, accounting for 66%. Approximately 25% was self-inflicted. A 24% NBD rate was forecasted from 2015 through 2025. CONCLUSIONS: Approximately 1 in 5 deaths were NBD. The majority were potentially preventable, including a significant proportion of self-inflicted injuries. A single comprehensive data repository would facilitate future mortality monitoring and performance improvement. These data may assist military leaders with implementing targeted safety strategies.

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.

Topical delivery of a small molecule RUNX1 transcription factor inhibitor for the treatment of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment surgery failure. Despite significant advances in vitreoretinal surgery, it still remains without an effective prophylactic or therapeutic medical treatment. After ocular injury or retinal detachment, misplaced retinal cells undergo epithelial to mesenchymal transition (EMT) to form contractile membranes within the eye. We identified Runt-related transcription factor 1 (RUNX1) as a gene highly expressed in surgically-removed human PVR specimens. RUNX1 upregulation was a hallmark of EMT in primary cultures derived from human PVR membranes (C-PVR). The inhibition of RUNX1 reduced proliferation of human C-PVR cells in vitro, and curbed growth of freshly isolated human PVR membranes in an explant assay. We formulated Ro5-3335, a lipophilic small molecule RUNX1 inhibitor, into a nanoemulsion that when administered topically curbed the progression of disease in a novel rabbit model of mild PVR developed using C-PVR cells. Mass spectrometry analysis detected 2.67 ng/mL of Ro5-3335 within the vitreous cavity after treatment. This work shows a critical role for RUNX1 in PVR and supports the feasibility of targeting RUNX1 within the eye for the treatment of an EMT-mediated condition using a topical ophthalmic agent.

Proliferative Vitreoretinopathy After Combat Ocular Trauma in Operation Iraqi Freedom and Operation Enduring Freedom: 2001-2011.

BACKGROUND AND OBJECTIVE: To determine the risk factors associated with developing proliferative vitreoretinopathy (PVR) from combat ocular injuries in U.S. service members. PATIENTS AND METHODS: Retrospective review of associated risk factors and outcomes of PVR within the Walter Reed Ocular Trauma Database. Ocular injuries in U.S. service members wounded during Operation Enduring Freedom and Operation Iraqi Freedom from 2001 to 2011 were assessed, and of these all cases of PVR were studied. Principal outcome measures were the risk factors associated with PVR development and rate of final visual acuity (VA) less than 20/200. RESULTS: Eight hundred ninety eyes of 651 U.S. service members were evaluated. A total of 76 eyes (8.5%) of 66 patients developed PVR. Five patients had bilateral PVR. Nineteen patients had bilateral eye injuries. Sixty-one eyes (80.2%) had a final VA less than 20/200. PVR was found to be a significant risk factor for a poor final VA (P < .001). Retinal detachment (RD) was found in 52 eyes (68.4%) of patients. In patients with a RD, intraocular foreign bodies (IOFBs) (P < .001), unsuccessful repair (P = .002), and macular hemorrhage (P = .04) were significant risk factors for the development of PVR. Time to initial retina surgery was not found to be a risk factor for PVR development (P = .5). Time to initial retina surgery was available in 41 patients and the time to surgery on average was 22.56 days (range: 3 to 87 days). CONCLUSIONS: PVR occurs frequently in combat trauma and is a significant cause of poor final VA. In patients with PVR and RD, injuries caused by an IOFB, macular hemorrhage, or unsuccessful repair were significant risk factors for the development of PVR. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:556-563.].

Multidrug-Resistant Organisms from Ophthalmic Cultures: Antibiotic Resistance and Visual Acuity.

INTRODUCTION: There is a growing trend of multidrug-resistant organisms (MDRO). The goal of this study was to characterize MDRO at a single center from ophthalmic cultures to better understand how treatments were tailored and to assess effect on visual acuity. MATERIALS AND METHODS: The MDRO data were collected by the Multidrug-Resistant Organism Repository and Surveillance Network from the Brooke Army Medical Center clinical laboratory. Both patient- and isolate-specific data were collected and qualitatively analyzed. Primary outcome measures were organism and type of resistance, anatomic location of isolate, initial and final antibiotic choice, and visual acuity. RESULTS: Thirty-one bacterial culture samples were analyzed from 29 patients. Twenty-two (72%) were Gram-positive and all were methicillin-resistant Staphylococcus aureus (MRSA). Nine (29%) were Gram-negative and of these five were Pseudomonas spp. Fourteen (45%) isolates were cultured from the cornea, nine (29%) from the lid, four (13%) from the conjunctiva, and four (13%) from other locations. The majority (66.6%) required adjustment of initial antibiotics following ocular culture results. Sixteen adult patients had recorded initial and final visual acuities. Fifteen of those 16 patients had stable or improved visual acuities following treatment of the infection, but five patients had a final visual acuity less than 20/200. CONCLUSION: This study demonstrated a high frequency of corneal MDRO infections and specifically MRSA and Pseudomonas spp. isolates. Antibiotic treatments frequently required adjustment. Further prospective study of visual outcomes from ophthalmic MDRO cultures is needed.

Detection of Retinal Fibrosis in a Rabbit Model of Penetrating Eye Injury.

INTRODUCTION: To establish a rabbit model of posterior penetrating eye injury as a platform to test potential therapeutics. MATERIALS AND METHODS: Anesthetized rabbits received posterior penetrating eye injury in one eye, whereas contralateral eyes were maintained as uninjured controls. Rabbits were randomized into two experimental groups. Group A was euthanized on Day 14 postinjury to determine retinal fibrosis at an early phase of disease progression. Group B was euthanized on Day 28 postinjury to examine retinal fibrosis at a late phase of disease progression. We examined animals on postinjury Days 7, 14, 21, and 28 with indirect ophthalmoscope and fundus photography. After euthanasia, eyes were processed for histology and immunofluorescence labeling of fibrotic proteins α-smooth muscle actin and collagen I. RESULTS: Early fibrosis was detected by Day 14, as indicated by indirect ophthalmoscopy and fundus imaging. Fibrotic membranes were visible at sites of injury. Immunofluorescence analysis detected α-smooth muscle actin and collagen I within the fibrotic membranes. CONCLUSIONS: These data show that ocular fibrosis can be detected within 14 days after initial injury, with more severe fibrosis detected at 28 days postinjury. These results will be used to determine the optimal time points for later studies designed to test treatment strategies.

Utility of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for an In Vitro Model of Proliferative Vitreoretinopathy.

The advent of stem cell technology, including the technology to induce pluripotency in somatic cells, and direct differentiation of stem cells into specific somatic cell types, has created an exciting new field of scientific research. Much of the work with pluripotent stem (PS) cells has been focused on the exploration and exploitation of their potential as cells/tissue replacement therapies for personalized medicine. However, PS and stem cell-derived somatic cells are also proving to be valuable tools to study disease pathology and tissue-specific responses to injury. High-throughput drug screening assays using tissue-specific injury models have the potential to identify specific and effective treatments that will promote wound healing. Retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE) are well characterized cells that exhibit the phenotype and functions of in vivo RPE. In addition to their role as a source of cells to replace damaged or diseased RPE, iPS-RPE provide a robust platform for in vitro drug screening to identify novel therapeutics to promote healing and repair of ocular tissues after injury. Proliferative vitreoretinopathy (PVR) is an abnormal wound healing process that occurs after retinal tears or detachments. In this chapter, the role of iPS-RPE in the development of an in vitro model of PVR is described. Comprehensive analyses of the iPS-RPE response to injury suggests that these cells provide a physiologically relevant tool to investigate the cellular mechanisms of the three phases of PVR pathology: migration, proliferation, and contraction. This in vitro model will provide valuable information regarding cellular wound healing responses specific to RPE and enable the identification of effective therapeutics.

CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells.

Dysregulation in the extracellular matrix (ECM) microenvironment surrounding the retinal pigment epithelium (RPE) has been implicated in the etiology of proliferative vitreoretinopathy and age-related macular degeneration. The regulation of ECM remodeling by RPE cells is not well understood. We show that membrane-type matrix metalloproteinase 14 (MMP14) is central to ECM degradation at the focal adhesions in human ARPE19 cells. The matrix degradative activity, but not the assembly, of the focal adhesion is regulated by chloride intracellular channel 4 (CLIC4). CLIC4 is co-localized with MMP14 in the late endosome. CLIC4 regulates the proper sorting of MMP14 into the lumen of the late endosome and its proteolytic activation in lipid rafts. CLIC4 has the newly-identified "late domain" motif that binds to MMP14 and to Tsg101, a component of the endosomal sorting complex required for transport (ESCRT) complex. Unlike the late domain mutant CLIC4, wild-type CLIC4 can rescue the late endosomal sorting defect of MMP14. Finally, CLIC4 knockdown inhibits the apical secretion of MMP2 in polarized human RPE monolayers. These results, taken together, demonstrate that CLIC4 is a novel matrix microenvironment modulator and a novel regulator for late endosomal cargo sorting. Moreover, the late endosomal sorting of MMP14 actively regulates its surface activation in RPE cells.

Quantitative Assessment of Retina Explant Viability in a Porcine Ex Vivo Neuroretina Model.

PURPOSE: Given that porcine and human retinas have similar structures and characteristics, ex vivo culture of porcine neuroretina provides an attractive model for studying mechanisms of human retinal injury and degenerative disease. Here, we describe the method that was used to establish and characterize an adult porcine retina culture system as a rapid screening tool for retinal survival in real time. METHODS: Neuroretina explants 8 mm in diameter were harvested from adult swine and cultured on porous cell culture inserts with adjustable heights. Retina explant viability was evaluated at 1, 4, 7, 11, and 14 days of culture using a resazurin-based metabolic assay. The explants were analyzed morphologically through immunohistochemistry for glial activation and apoptosis. Morphometric analysis was also performed on hematoxylin and eosin-stained retina sections from each time point. RESULTS: The viability of retina explants gradually decreased over time in culture. The laminar structure of the neuroretina was well preserved during the first 7 days. However, by day 14, most explants showed significant loss of cells in each laminar layer and obvious thinning. Overall, the progressive loss of retinal lamination and thickness, and increase in apoptotic nuclei with activated hypertrophic Müller cells were well correlated with the metabolic activity of the ex vivo neuroretina explants. CONCLUSIONS: This study was the first report to describe the use of a high-throughput and quantitative method for monitoring retina explant viability in real time. Ex vivo neuroretina cultures closely mimic the functional dynamics of the organ, and can be used efficiently to screen novel therapeutics for retinal neurodegenerative disease.

Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems.

Retinal degenerative diseases such as macular degeneration, glaucoma, and diabetic retinopathy constitute the leading cause of blindness in the industrialized world. There is a continuous demand in investigative ophthalmic research for the development of new treatment modalities for retinal therapy. Unfortunately, efforts to identify novel neuroprotective and neuroregenerative agents have often been hindered by an experimental model gap that exists between high-throughput methods via dissociated cells and preclinical animal models. Even though dissociated cell culture is rapid and high-throughput, it is limited in its ability to reproduce the in vivo conditions. In contrast, preclinical animal models may offer greater fidelity, albeit they lack efficiency and experimental control. Retina explant cultures provide an ideal bridge to close this gap and have been used to study an array of biological processes such as retinal development and neurodegeneration. However, it is often difficult to interpret findings from these studies due to the wide variety of experimental species and culture methods used. This review provides a comprehensive overview of current ex vivo neuroretina culture methods and assessments, with a focus on their suitability, advantages, and disadvantages, along with novel insights and perspectives on the organotypic culture model as a high-throughput platform for screening promising molecules for retinal regeneration.

Sealing of Corneal Lacerations Using Photoactivated Rose Bengal Dye and Amniotic Membrane.

PURPOSE: Watertight closure of perforating corneoscleral lacerations is necessary to prevent epithelial ingrowth, infection, and potential loss of the eye. Complex lacerations can be difficult to treat, and repair with sutures alone is often inadequate. In this study, we evaluated a potentially sutureless technology for sealing complex corneal and scleral lacerations that bonds the amniotic membrane (AM) to the wound using only green light and rose bengal dye. METHODS: The AM was impregnated with rose bengal and then sealed over lacerations using green light to bond the AM to the deepithelialized corneal surface. This process was compared with suture repair of 3 laceration configurations in New Zealand White rabbits in 3 arms of the study. A fourth study arm assessed the side effect profile including viability of cells in the iris, damage to the blood-retinal barrier, retinal photoreceptors, retinal pigment epithelium, and choriocapillaris in Dutch Belted rabbits. RESULTS: Analyses of the first 3 arms revealed a clinically insignificant increase in polymorphonuclear inflammation. In the fourth arm, iris cells appeared unaffected and no evidence of breakdown of the blood-retinal barrier was detected. The retina from green light laser-treated eyes showed normal retinal pigment epithelium, intact outer segments, and normal outer nuclear layer thickness. CONCLUSIONS: The results of these studies established that a light-activated method to cross-link AM to the cornea can be used for sealing complex penetrating wounds in the cornea and sclera with minimal inflammation or secondary effects.

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.

Recombinant Xeno-Free Vitronectin Supports Self-Renewal and Pluripotency in Protein-Induced Pluripotent Stem Cells.

Patient safety is a major concern in the application of induced pluripotent stem cells (iPSCs) in cell-based therapy. Efforts are being made to reprogram, maintain, and differentiate iPSCs in defined conditions to provide a safe source of stem cells for regenerative medicine. Recently, human fibroblasts were successfully reprogrammed into pluripotent stem cells using four recombinant proteins (OCT4, c-Myc, KLF4, and SOX2) fused with a cell-penetrating peptide (9R). These protein-induced pluripotent stem cells (piPSCs) are maintained and propagated on a feeder layer of mouse embryonic fibroblasts. Use of animal-derived products in maintenance and differentiation of iPSCs poses risks of zoonotic disease transmission and immune rejection when transplanted into humans. To avoid potential incorporation of xenogenic products, we cultured piPSCs on recombinant human matrix proteins. We then tested whether recombinant human matrix proteins can support self-renewal and pluripotency of piPSCs. After long-term culture on recombinant human vitronectin in xeno-free conditions, piPSCs retained the expression of pluripotent markers. The pluripotency of these cells was further evaluated by differentiating toward ectoderm, mesoderm, and endoderm lineages in vitro. In conclusion, recombinant human vitronectin can support the long-term culture and maintain the stemness of piPSCs in defined nonxenogenic conditions.

Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies.

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.