Differential Susceptibility of Fetal Retinal Pigment Epithelial Cells, hiPSC- Retinal Stem Cells, and Retinal Organoids to Zika Virus Infection.

Zika virus (ZIKV) causes microcephaly and congenital eye disease. The cellular and molecular basis of congenital ZIKV infection are not well understood. Here, we utilized a biologically relevant cell-based system of human fetal retinal pigment epithelial cells (FRPEs), hiPSC-derived retinal stem cells (iRSCs), and retinal organoids to investigate ZIKV-mediated ocular cell injury processes. Our data show that FRPEs were highly susceptible to ZIKV infection exhibiting increased apoptosis, whereas iRSCs showed reduced susceptibility. Detailed transcriptomics and proteomics analyses of infected FRPEs were performed. Nucleoside analogue drug treatment inhibited ZIKV replication. Retinal organoids were susceptible to ZIKV infection. The Asian genotype ZIKV exhibited higher infectivity, induced profound inflammatory response, and dysregulated transcription factors involved in retinal organoid differentiation. Collectively, our study shows that ZIKV affects ocular cells at different developmental stages resulting in cellular injury and death, further providing molecular insight into the pathogenesis of congenital eye disease.

The Effect of Anti-Inflammatory Topical Ophthalmic Treatments on In Vitro Corneal Epithelial Cells.

Purpose: To compare the effect of three commonly prescribed anti-inflammatory eye drops on corneal epithelial cells in vitro. Methods: Three different lines of human corneal epithelial cells were tested: primary cells cultured from donor tissue, commercially available primary cells, and immortalized cells. Cells were seeded on 96-well plates and treated with the following eye drops: cyclosporine 0.05%, lifitegrast 5%, and tacrolimus 0.03% or 0.1%. Exposure times tested were 30 seconds, 1 minute, 2 minutes, 1 hour, 2 hours, 4 hours, and 24 hours. Brightfield images and viability assays were analyzed 48 to 72 hours after the initiation of treatments. At least five replicates were tested per drug and time exposure. Results: Commercially obtained primary cells showed reduced viability following 1 hour with tacrolimus 0.1% (8%; P = 0.043%) and 4 hours with tacrolimus 0.03% (17%; P = 0.042%). Lifitegrast exposure reduced primary cell viability after 4 hours (10%; P = 0.042). Cell viability in primary cells was not deleteriously affected following exposure to cyclosporine for up to 4 hours. A similar trend was observed in both primary cells cultured from donor tissue and immortalized human corneal epithelial cells, demonstrating greater decreases in cell viability in tacrolimus compared to lifitegrast and cyclosporine. Light microscopy imaging for analysis of cell morphology and confluence supported the results. Conclusions: Tacrolimus showed the highest impact on corneal epithelium survival in vitro, and cyclosporine proved the most protective. Translational Relevance: Comparing anti-inflammatory eye drops on corneal epithelial cells in vitro may inform eye drop selection and development for clinical purposes.

Genome-wide association study identifies three novel loci in Fuchs endothelial corneal dystrophy.

The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (P<5 × 10-8): KANK4 rs79742895, LAMC1 rs3768617 and LINC00970/ATP1B1 rs1200114. We also observe an overwhelming effect of the established TCF4 locus. Interestingly, we detect differential sex-specific association at LAMC1, with greater risk in women, and TCF4, with greater risk in men. Combining GWAS results with biological evidence we expand the knowledge of common FECD loci from one to four, and provide a deeper understanding of the underlying pathogenic basis of FECD.

Corneal Endothelial Cell Migration and Proliferation Enhanced by Rho Kinase (ROCK) Inhibitors in In Vitro and In Vivo Models.

Purpose: To explore the role of Rho-associated kinases (ROCK) in corneal physiology and regeneration, and the effects of suppressing its activity in stimulating corneal endothelial cell proliferation and migration in vitro and in vivo. Methods: Immunohistochemistry was performed to detect RhoA and ROCK-1 and ROCK-2 in human corneal tissue. Adult porcine corneal endothelial cells (CECs) were isolated, grown to confluence, and further characterized. Under the treatment of ROCK inhibitors, changes in the cellular distribution profile of ZO-1 and F-actin were examined by immunofluorescence staining. Corneal endothelial cells migration was evaluated by scratch assay and analyzed with Axiovision software. Cell proliferation was quantified using Click-iT EdU HCS Assay. In vivo, the corneal endothelia of rabbits were surgically injured and H-1152 was topically applied for 10 days. Progress of wound healing was evaluated daily by monitoring corneal edema, inflammation, and thickness using slit-lamp examination, photography, and pachymetry. Rabbits were euthanized and enucleated for further evaluation. Results: H-1152 exhibited significant stimulatory effect on CEC migration and proliferation in vitro compared with both untreated and Y-27632-treated cells. Furthermore, topical administration of H-1152 led to marked reduction in corneal edema and formation of multinucleate CECs in vivo suggestive of proliferation associated with healing. Conclusions: H-1152 exhibited a better stimulatory effect on CEC migration and proliferation in vitro than Y-27632. Our findings suggest that topical administration of H-1152 promotes healing of injured corneal endothelium in vivo. These results demonstrate the efficacy of ROCK inhibitors as a potential topical therapy for patients with corneal endothelial disease.

Generation of Human Corneal Endothelial Cells via In Vitro Ocular Lineage Restriction of Pluripotent Stem Cells.

Purpose: We generate a renewable supply of corneal endothelial cells (CEC) from human pluripotent stem cells (PSCs) under defined culture conditions. Methods: Corneal endothelial cell induction was driven by small molecules in a stepwise fashion of lineage specification. During the initial phase, PSC fate was restricted to the eye field-like state and became eye field stem cells (EFSCs). In the second phase, PSC-derived EFSCs were further directed toward either neural crest lineage or retinal lineage. The CECs were directly induced from ocular neural crest stem cells (NCSCs) by suppressing TGF-ß and ROCK signaling. Results: Under chemically defined conditions, PSCs were massively converted into EFSCs and subsequently NCSCs. Eye field cell identity was characterized by the expression of key fate restriction factors for early eye field cells, such as PAX6, LHX2, and VSX2. The induction of ocular NCSCs was initiated by promoting WNT signaling in EFSCs. Within 2 weeks of induction, the majority of cells expressed the typical neural crest markers p75NTR and HNK-1. Eye field stem cell-derived NCSCs can be propagated and cryopreserved. Subsequently, a CEC monolayer was induced from adherent NCSCs in the presence of small molecular inhibitors to suppress TGF-ß and ROCK signaling. The polygon-shaped CEC-like cells became visible after a week in culture. The NCSC-derived CECs expressed typical CEC markers, such as N-Cadherin and Na+/K+-ATPase. Conclusions: A novel small molecule-based approach was developed to derive human CECs from PSCs via ocular lineage specification. Moreover, EFSC-derived NCSCs could serve as an immediate source cell for rapid CEC induction in vitro.

Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP.

Uveal melanoma (UM) is the most common cancer in adult eyes. Approximately 80% of UMs harbor somatic activating mutations in GNAQ or GNA11 (encoding Gq or G11, respectively). Herein, we show in both cell culture and human tumors that cancer-associated Gq/11 mutants activate YAP, a major effector of the Hippo tumor suppressor pathway that is also regulated by G protein-coupled receptor signaling. YAP mediates the oncogenic activity of mutant Gq/11 in UM development, and the YAP inhibitor verteporfin blocks tumor growth of UM cells containing Gq/11 mutations. This study reveals an essential role of the Hippo-YAP pathway in Gq/11-induced tumorigenesis and suggests YAP as a potential drug target for UM patients carrying mutations in GNAQ or GNA11.

Neural Stem Cells Derived by Small Molecules Preserve Vision.

PURPOSE: The advances in stem cell biology hold a great potential to treat retinal degeneration. Importantly, specific cell types can be generated efficiently with small molecules and maintained stably over numerous passages. Here, we investigated whether neural stem cell (NSC) derived from human embryonic stem cells (hESC) by small molecules can preserve vision following grafting into the Royal College Surgeon (RCS) rats; a model for retinal degeneration. METHODS: A cell suspension containing 3 × 104 NSCs or NSCs labeled with green fluorescent protein (GFP) was injected into the subretinal space or the vitreous cavity of RCS rats at postnatal day (P) 22; animals injected with cell-carry medium and those left untreated were used as controls. The efficacy of treatment was evaluated by testing optokinetic response, recording luminance threshold, and examining retinal histology. RESULTS: NSCs offered significant preservation of both photoreceptors and visual function. The grafted NSCs survived for long term without evidence of tumor formation. Functionally, NSC treated eyes had significantly better visual acuity and lower luminance threshold than controls. Morphologically, photoreceptors and retinal connections were well preserved. There was an increase in expression of cillary neurotrophic factor (CNTF) in Müller cells in the graft-protected retina. CONCLUSIONS: This study reveals that NSCs derived from hESC by small molecules can survive and preserve vision for long term following subretinal transplantation in the RCS rats. These cells migrate extensively in the subretinal space and inner retina; there is no evidence of tumor formation or unwanted changes after grafting into the eyes. TRANSLATIONAL RELEVANCE: The NSCs derived from hESC by small molecules can be generated efficiently and provide an unlimited supply of cells for the treatment of some forms of human outer retinal degenerative diseases. The capacity of NSCs migrating into inner retina offers a potential as a vehicle to delivery drugs/factors to treat inner retinal disorders.

TCF7L2 variation and proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) is the most severe vision-threatening complication of diabetes. For investigation of genetic association between TCF7L2 and PDR in Caucasian type 2 diabetes mellitus (T2DM) and its functional consequences, 383 T2DM patients with PDR (T2DM-PDR) and 756 T2DM patients without diabetic retinopathy (T2DM-no DR) were genotyped with rs7903146 in TCF7L2. We found that risk allele (T) frequency of rs7903146 was significantly higher in T2DM-PDR patients (allelic P = 2.52E-04). In lymphoblastoid cells induced to undergo endoplasmic reticulum (ER) stress by treatment of tunicamycin, higher fold change of TCF7L2 and VEGFA mRNA levels were observed in rs7903146-TT cells than in rs7903146-CC cells (P = 0.02 for TCF7L2; P = 0.004 for VEGFA), suggesting that ER stress plays a role in PDR pathogenesis. Silencing TCF7L2 resulted in decreased mRNA levels of both TCF7L2 and VEGFA (P < 0.001). Retinas of oxygen-induced retinopathy mice (a model for PDR) had higher TCF7L2 and VEGFA mRNA levels than those of controls (P = 2.9E-04 for TCF7L2; P = 1.9E-07 for VEGFA). Together, data from our study show that TCF7L2-rs7903146 is associated with PDR in Caucasian T2DM and suggest that TCF7L2 promotes pathological retinal neovascularization via ER stress-dependent upregulation of VEGFA.

Integration and long distance axonal regeneration in the central nervous system from transplanted primitive neural stem cells.

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.

Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.

The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.

[Maturation of Cordyceps sinensis associates with alterations of fungal expressions of multiple Ophiocordyceps sinensis mutants in stroma of Cordyceps sinensis].

OBJECTIVE: To examine maturational changes in expressions of Ophiocordyceps sinensis (O.sinensis) transition and transversion mutation genotypes in Cordyceps sinensis (C.sinensis) stroma. METHODS: MassARRAY single nucleotide polymorphism (SNP) matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum genotyping was used, and 8 SNP extension primers were designed based on the scattered, multiple point mutations of known sequences for the O.sinensis mutants within their internal transcribed spacer (ITS) segments. Of the extension primers, 5 (not capable of distinguishing between the 2 AT-biased genotypes) located in rDNA ITS1 and ITS2 regions: 067721-211, 067721-240, 067721-477, 067721-531 and 067721-581. The other 3 extension primers located in 5.8S rDNA region: 067740-324, 067740-328 and 067740-360, to distinguish between the 2 AT-biased genotypes. RESULTS: MS chromatograms at the 8 SNP sites showed dynamic alterations of mutant alleles in C.sinensis stroma. The allele for the AT-biased genotypes at 067721-211 site showed higher peak height than its GC-biased counterpart in the premature C.sinensis stroma, but disappeared with C.sinensis maturation. Chromatograms displayed not only the transition mutation alleles, but also transversion mutants. Some of the transversion mutation alleles displayed higher peak heights than those for GC- and AT-biased alleles, but their peak heights and detection rates tended to be decreased with C.sinensis maturation. When distinguishing between the 2 AT-biases, AB067744 and AB067740 genotype alleles co-existed in the premature C.sinensis stroma. The allele peak height for AB067744 genotype was greatly decreased with C.sinensis maturation, while that for AB067740 genotype increased. CONCLUSION: Co-existence of at least 5 transition and transversion mutant genotypes of O.sinensis and the dynamic changes in their expressions in C.sinensis stroma along with C.sinensis maturation may be of extreme importance in C.sinensis stroma germination and maturation, enabling C.sinensis to complete its life cycle.

The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation.

Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. YAP is phosphorylated and inhibited by the Lats kinase, a key component of the Hippo tumor suppressor pathway. Elevated YAP protein levels and gene amplification have been implicated in human cancer. In this study, we report that YAP is inactivated during embryonic stem (ES) cell differentiation, as indicated by decreased protein levels and increased phosphorylation. Consistently, YAP is elevated during induced pluripotent stem (iPS) cell reprogramming. YAP knockdown leads to a loss of ES cell pluripotency, while ectopic expression of YAP prevents ES cell differentiation in vitro and maintains stem cell phenotypes even under differentiation conditions. Moreover, YAP binds directly to promoters of a large number of genes known to be important for stem cells and stimulates their expression. Our observations establish a critical role of YAP in maintaining stem cell pluripotency.