Bikash R. Pattnaik

Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three‐dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)‐like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease‐specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV‐like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine. STEM CELLS 2011;29:1206‐1218

iPS cell modeling of Best disease: Insights into the pathophysiology of an inherited macular degeneration

Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.

Terpenoids from Zingiber officinale (Ginger) Induce Apoptosis in Endometrial Cancer Cells through the Activation of p53

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC50 of 1.25 µg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC50 10 µM (2.3 µg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer.

A Novel KCNJ13 Nonsense Mutation and Loss of Kir7.1 Channel Function Causes Leber Congenital Amaurosis (LCA16)

Mutations in the KCNJ13 gene that encodes the inwardly rectifying potassium channel Kir7.1 cause snowflake vitreoretinal degeneration (SVD) and leber congenital amaurosis (LCA). Kir7.1 controls the microenvironment between the photoreceptors and the retinal pigment epithelium (RPE) and also contributes to the function of other organs such as uterus and brain. Heterologous expressions of the mutant channel have suggested a dominant‐negative loss of Kir7.1 function in SVD, but parallel studies in LCA16 have been lacking. Herein, we report the identification of a novel nonsense mutation in the second exon of the KCNJ13 gene that leads to a premature stop codon in association with LCA16. We have determined that the mutation results in a severe truncation of the Kir7.1 C‐terminus, alters protein localization, and disrupts potassium currents. Coexpression of the mutant and wild‐type channel has no negative influence on the wild‐type channel function, consistent with the normal clinical phenotype of carrier individuals. By suppressing Kir7.1 function in mice, we were able to reproduce the severe LCA electroretinogram phenotype. Thus, we have extended the observation that Kir7.1 mutations are associated with vision disorders to include novel insights into the molecular mechanism of disease pathobiology in LCA16.

Snowflake Vitreoretinal Degeneration (SVD) Mutation R162W Provides New Insights into Kir7.1 Ion Channel Structure and Function

Snowflake Vitreoretinal Degeneration (SVD) is associated with the R162W mutation of the Kir7.1 inwardly-rectifying potassium channel. Kir7.1 is found at the apical membrane of Retinal Pigment Epithelial (RPE) cells, adjacent to the photoreceptor neurons. The SVD phenotype ranges from RPE degeneration to an abnormal b-wave to a liquid vitreous. We sought to determine how this mutation alters the structure and function of the human Kir7.1 channel. In this study, we expressed a Kir7.1 construct with the R162W mutation in CHO cells to evaluate function of the ion channel. Compared to the wild-type protein, the mutant protein exhibited a non-functional Kir channel that resulted in depolarization of the resting membrane potential. Upon co-expression with wild-type Kir7.1, R162W mutant showed a reduction of IKir7.1 and positive shift in ‘0’ current potential. Homology modeling based on the structure of a bacterial Kir channel protein suggested that the effect of R162W mutation is a result of loss of hydrogen bonding by the regulatory lipid binding domain of the cytoplasmic structure.

Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies

While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases. DOI: http://dx.doi.org/10.7554/eLife.19264.001

Oxytocin expression and function in the posterior retina: a novel signaling pathway.

PURPOSE Oxytocin (OXT) is recognized as an ubiquitously acting nonapeptide hormone that is involved in processes ranging from parturition to neural development. Its effects are mediated by cell signaling that occurs as a result of oxytocin receptor (OXTR) activation. We sought to determine whether the OXT-OXTR signaling pathway is also expressed within the retina. METHODS Immunohistochemistry using cell-specific markers was used to localize OXT within the rhesus retina. Reverse transcriptase PCR and immunohistochemistry were used to assess the expression of OXTR in both human and rhesus retina. Single-cell RT-PCR and Western blot analyses were used to determine the expression of OXTR in cultured human fetal RPE (hfRPE) cells. Human fetal RPE cells loaded with FURA-2 AM were studied by ratiometric Ca(2+) imaging to assess transient mobilization of intracellular Ca(2+) ([Ca(2+)]i). RESULTS Oxytocin was expressed in the cone photoreceptor extracellular matrix of the rhesus retina. Oxytocin mRNA and protein were expressed in the human and rhesus RPE. Oxytocin mRNA and protein expression were observed in cultured hfRPE cells, and exposure of these cells to 100 nM OXT induced a transient 79 ± 1.5 nM increase of [Ca(2+)]i. CONCLUSIONS Oxytocin and OXTR are present in the posterior retina, and OXT induces an increase in hfRPE [Ca(2+)]i. These results suggest that the OXT-OXTR signaling pathway is active in the retina. We propose that OXT activation of the OXTR occurs in the posterior retina and that this may serve as a paracrine signaling pathway that contributes to communication between the cone photoreceptor and the RPE.

A Novel Approach to Single Cell RNA‐Sequence Analysis Facilitates In Silico Gene Reporting of Human Pluripotent Stem Cell‐Derived Retinal Cell Types

Cell type‐specific investigations commonly use gene reporters or single‐cell analytical techniques. However, reporter line development is arduous and generally limited to a single gene of interest, while single‐cell RNA (scRNA)‐sequencing (seq) frequently yields equivocal results that preclude definitive cell identification. To examine gene expression profiles of multiple retinal cell types derived from human pluripotent stem cells (hPSCs), we performed scRNA‐seq on optic vesicle (OV)‐like structures cultured under cGMP‐compatible conditions. However, efforts to apply traditional scRNA‐seq analytical methods based on unbiased algorithms were unrevealing. Therefore, we developed a simple, versatile, and universally applicable approach that generates gene expression data akin to those obtained from reporter lines. This method ranks single cells by expression level of a bait gene and searches the transcriptome for genes whose cell‐to‐cell rank order expression most closely matches that of the bait. Moreover, multiple bait genes can be combined to refine datasets. Using this approach, we provide further evidence for the authenticity of hPSC‐derived retinal cell types. Stem Cells 2018;36:313–324

High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression

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.

Oxytocin (OXT)-stimulated inhibition of Kir7.1 activity is through PIP 2 -dependent Ca 2 + response of the oxytocin receptor in the retinal pigment epithelium in vitro

Oxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G-protein coupled receptor. Our localization of OXTR to the retinal pigment epithelium (RPE), in close proximity to OXT in the adjacent photoreceptor neurons, leads us to propose that OXT plays an important role in RPE-retinal communication. An increase of RPE [Ca2+]i in response to OXT stimulation implies that the RPE may utilize oxytocinergic signaling as a mechanism by which it accomplishes some of its many roles. In this study, we used an established human RPE cell line, a HEK293 heterologous OXTR expression system, and pharmacological inhibitors of Ca2+ signaling to demonstrate that OXTR utilizes capacitative Ca2+ entry (CCE) mechanisms to sustain an increase in cytoplasmic Ca2+. These findings demonstrate how multiple functional outcomes of OXT-OXTR signaling could be integrated via a single pathway. In addition, the activated OXTR was able to inhibit the Kir7.1 channel, an important mediator of sub retinal waste transport and K+ homeostasis.