Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions.

Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.

Mutant Best1 Expression and Impaired Phagocytosis in an iPSC Model of Autosomal Recessive Bestrophinopathy.

Autosomal recessive bestrophinopathy (ARB) is caused by mutations in the gene BEST1 which encodes bestrophin 1 (Best1), an anion channel expressed in retinal pigment epithelial (RPE) cells. It has been hypothesized that ARB represents the human null phenotype for BEST1 and that this occurs due to nonsense mediated decay (NMD). To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with ARB and her parents.

Fibrin hydrogels as a xenofree and rapidly degradable support for transplantation of retinal pigment epithelium monolayers.

Unlabelled: Recent phase 1 trials of embryonic stem cell and induced pluripotent stem cell (iPSCs) derived RPE transplants for the treatment of macular degeneration have demonstrated the relative safety of this process. However, there is concern over clumping, thickening, folding, and wrinkling of the transplanted RPE. To deliver a flat RPE monolayer, current phase 1 trials are testing synthetic substrates for RPE transplantation.

Control of Maintenance and Regeneration of Planarian Eyes by ovo.

Purpose: Following decapitation, the planarian Schmidtea mediterranea regenerates its head and eyes. The gene ovo is required for eye maintenance and regeneration in response to wounding. In this study, we investigated whether eye regeneration in S.

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a Novel BEST1 Mutation.

Purpose: Mutations in BEST1, encoding bestrophin-1 (Best1), cause autosomal recessive bestrophinopathy (ARB). Encoding bestrophin-1 is a pentameric anion channel localized to the basolateral plasma membrane of the RPE. Here, we characterize the effects of the mutations R141H (CGC > CAC) and I366fsX18 (c.

Objective assessment of the corneal endothelium in Fuchs' endothelial dystrophy.

Purpose: To develop a standardized method of endothelial cell density (ECD) assessment in Fuchs' endothelial dystrophy that maximizes the sample area and uses the clearest endothelial cells in confocal images.

Methods: The corneal endothelium of 51 eyes from 30 patients, with varying degrees of Fuchs' endothelial dystrophy, was examined using confocal microscopy. In two or three distinct images of the central endothelium, local contiguous cell density was determined using a variable frame method.

Anterior keratocyte depletion in fuchs endothelial dystrophy.

Objective: To determine if keratocyte populations are different in corneas with Fuchs dystrophy compared with control corneas.

Methods: Eleven corneas excised during penetrating keratoplasty for Fuchs dystrophy and 5 control corneas of eyes enucleated for choroidal melanoma were examined using light microscopy. Twenty control corneas age-matched to the corneas with Fuchs dystrophy were examined using confocal microscopy in vivo.

Comparison of flex-center, center, and corner methods of corneal endothelial cell analysis.

Purpose: The center method of corneal endothelial cell analysis is rapid but excludes the outermost digitized cells of a contiguous group from analysis; the flex-center method (Konan, Inc) is a modification that includes analysis of the outermost cells, which is advantageous in images with few cells. In this study, we examined agreement among the flex-center, center, and corner (standard) methods of endothelial analysis.

Methods: Identical cells in endothelial images of 10 normal corneas and 10 corneas after penetrating keratoplasty (PK) were analyzed by each method.

Human corneal endothelial cell transplantation in a human ex vivo model.

Purpose: To determine the effects of incorporating superparamagnetic microspheres (SPMs) into cultured human corneal endothelial cells (HCECs) and to describe preliminary experiments of HCEC transplantation, facilitated by SPMs and an external magnetic field, in a human anterior segment ex vivo model.

Methods: HCECs were cultured as monolayers and incorporated with magnetite oxide SPMs (900, 300, and 100 nm) at different concentrations. Cell viability, migration toward a magnetic field, and light transmittance were measured after incorporation of the SPMs.

Dendritic cells facilitate accumulation of IL-17 T cells in the kidney following acute renal obstruction.

Acute urinary obstruction causes interstitial inflammation with leukocyte accumulation and the secretion of soluble mediators. Here we show that unilateral ureteral ligation caused a progressive increase in renal F4/80(+) and F4/80(-) dendritic cells, monocytes, neutrophils and T-cells 24-72 h following obstruction. Depletion of dendritic cells by clodronate pretreatment showed these cells to be the most potent source of tumor necrosis factor and other pro-inflammatory mediators in the obstructed kidney.

Regulation of relB in dendritic cells by means of modulated association of vitamin D receptor and histone deacetylase 3 with the promoter.

The NF-kappaB component RelB is essential for dendritic cell (DC) differentiation and maturation. The vitamin D receptor (VDR) is a nuclear receptor that mediates inhibition of DC maturation and transcriptional repression of relB after engagement of its ligand, 1alpha,25-dihydroxyvitamin D(3), or related analogs (D(3) analogs). Ligand-dependent relB suppression was abolished by a histone deacetylase (HDAC) inhibitor.

Antigen presentation by dendritic cells in renal lymph nodes is linked to systemic and local injury to the kidney.

Background: Dendritic cells (DCs) uniquely serve as conduits between innate and cognate arms of the immune system. The normal kidney contains an extensive population of interstitial DCs but their role in the pathogenesis of acute renal injury is not known.

Methods: Renal DCs were studied by flow cytometric analysis of collagenase-digested mouse kidneys, by immunohistochemistry, and by immunofluorescence microscopy.

Direct transcriptional regulation of RelB by 1alpha,25-dihydroxyvitamin D3 and its analogs: physiologic and therapeutic implications for dendritic cell function.

The nuclear factor-kappaB (NF-kappaB) protein RelB plays a unique role in dendritic cell (DC) function and, as such, is an important regulator of antigen presentation and immune regulation. In this study, inhibition of RelB expression in DCs exposed to an analog of the active form of vitamin D3 (1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3)) was observed and shown to be mediated by the vitamin D receptor (VDR). Potential vitamin D response elements were identified within promoter regions of human and mouse relB genes.

Generation of antigen-specific, interleukin-10-producing T-cells using dendritic cell stimulation and steroid hormone conditioning.

The mechanisms by which regulatory T-cell populations are generated in vivo are poorly understood. Nonetheless, the possibility of generating T-cells with regulatory capacity ex vivo using pharmacologic agents or modified antigen presenting cells has been raised by a number of recent studies. In this study, the effect of combined glucocorticoid and 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) agonists on dendritic cell (DC)-stimulated, antigen-specific CD4(+ve) T-cells was investigated.

Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

Interactions between porcine antigen presenting cells (pAPCs) and host lymphocytes may be important in cellular and humoral rejection of porcine organ xenografts. To investigate the role of pAPCs in the activation of xenogeneic lymphocytes, porcine bone marrow cells were stimulated using porcine GM-CSF with or without porcine IL-4 to generate populations of pAPCs that had phenotypic characteristics of myeloid dendritic cells. These bone marrow-derived pAPCs were weak stimulators of xenogeneic (mouse and human) T cells in vitro but induced primary B-cell proliferation and augmented CD40-induced B-cell proliferation.

Distinctive dendritic cell modulation by vitamin D(3) and glucocorticoid pathways.

Dendritic cell (DC) maturation plays a central role in regulating immunity. We show that glucocorticoid and 1alpha,25(OH)(2)D(3) agonists modulate DCs via distinct and additive signaling pathways. Phenotypic and functional indices were examined in DCs treated with dexamethasone (DEX) and/or a 1alpha,25(OH)(2)D(3) analog (D(3) analog).