The physiological relevance of Matrigel as a cell-culture substrate and in angiogenesis assays is often called into question. Here, we describe an array-based method for the identification of synthetic hydrogels that promote the formation of robust in vitro vascular networks for the detection of putative vascular disruptors, and that support human embryonic stem cell expansion and pluripotency. We identified hydrogel substrates that promoted endothelial-network formation by primary human umbilical vein endothelial cells and by endothelial cells derived from human induced pluripotent stem cells, and used the hydrogels with endothelial networks to identify angiogenesis inhibitors. The synthetic hydrogels show superior sensitivity and reproducibility over Matrigel when evaluating known inhibitors, as well as in a blinded screen of a subset of 38 chemicals, selected according to predicted vascular disruption potential, from the Toxicity ForeCaster library of the US Environmental Protection Agency. The identified synthetic hydrogels should be suitable alternatives to Matrigel for common cell-culture applications.

Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis.

PurposeThe role of β–adrenergic receptor (AR) signaling in neovascular ocular diseases has recently emerged. We have previously reported that intraperitoneal propranolol inhibits choroidal neovascularization (CNV) in vivo and β2-AR blockade reduces vascular endothelial growth factor (VEGF) expression in mouse retinal pigment epithelium and choroidal endothelial cells in culture. Here we tested the hypothesis that the β2-AR regulates CNV through modulation of VEGF and inflammatory cytokine expression.MethodsMice were subjected to laser burns, inducing CNV, and were treated with an intravitreal β2-AR antagonist. After 3 and 5 days, total eye interleukin-6 (IL-6) and VEGF protein levels were measured, respectively. After 14 days, CNV was measured on choroidal–scleral flatmounts. The effects of β-AR signaling on VEGF and IL-6 expression were investigated in various mouse retinal and human RPE cells by using specific β-AR agonists and antagonists.Resultsβ2–Adrenergic receptor signaling increased Vegf mRNA expression by approximately 3- to 4-fold in mouse retinal microglia and pericytes in culture. β2–Adrenergic receptor signaling upregulated IL-6 mRNA expression between 10- and 60-fold in mouse retinal microglia, pericytes, RPE, and choroidal endothelial cells in culture. Intravitreal injection of β2-AR antagonist ICI 118,551 reduced CNV by 35% and decreased IL-6 protein levels by approximately 50%. In primary human RPE cells, β2-AR activation also stimulated VEGF and IL-6 mRNA expression by 2- and 10-fold, respectively.ConclusionsAnti-VEGF therapy for CNV is highly effective; however, some patients are resistant to therapy while others undergo repeated, frequent treatments. β2–Adrenergic receptor signaling is a potential therapeutic target because of its angiogenic and inflammatory properties.

Induced pluripotent stem cells (iPSC)‐based therapies have been hailed as the future of regenerative medicine because of their potential to provide treatment options for most degenerative diseases. A key promise of iPSC‐based therapies is the possibility of an autologous transplant that may engraft better in the longer‐term due to its compatibility with the patient's immune system. Despite over a decade of research, clinical translation of autologous iPSC‐based therapies has been slow—partly due to a lacking pre‐defined regulatory path. Here, we outline regulatory considerations for developing an autologous iPSC‐based product and challenges associated with the clinical manufacturing of autologous iPSCs and their derivatives. These challenges include donor tissue source, reprogramming methods, heterogeneity of differentiated cells, controls for the manufacturing process, and preclinical considerations. A robust manufacturing process with appropriate quality controls and well‐informed, prospectively designed preclinical studies provide a path toward successful approval of autologous iPSC‐based therapies.