Karl J. Wahlin

Angiopoietin 2 expression in the retina: upregulation during physiologic and pathologic neovascularization

Vascular development in the embryo requires coordinated signaling through several endothelial cell‐specific receptors; however, it is not known whether this is also required later during retinal vascular development or as part of retinal neovascularization in adults. The Tie2 receptor has been implicated in stabilization and maturation of vessels through action of an agonist ligand, angiopoietin 1 (Ang1) and an antagonistic ligand, Ang2. In this study, we have demonstrated that ang2 mRNA levels are increased in the retina during development of the deep retinal capillaries by angiogenesis and during pathologic angiogenesis in a model of ischemic retinopathy. Mice with hemizygous disruption of the ang2 gene by insertion of a promoterless β‐galactosidase (βgal) gene behind the ang2 promoter, show constitutive βgal staining primarily in cells along the outer border of the inner nuclear layer identified as horizontal cells by colocalization of calbindin. During development of the deep capillary bed or retinal neovascularization, other cells in the inner nuclear layer and ganglion cell layer, in regions of neovascularization, stain for βgal. Thus, there is temporal and spatial correlation of Ang2 expression with developmental and pathologic angiogenesis in the retina, suggesting that it may play a role. J. Cell. Physiol. 184:275–284, 2000.

Inactivation of the microRNA-183/96/182 cluster results in syndromic retinal degeneration

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated “miR-183CGT/GT,” using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.

A Simple and Scalable Process for the Differentiation of Retinal Pigment Epithelium From Human Pluripotent Stem Cells

Age‐related macular degeneration (AMD), the leading cause of irreversible vision loss and blindness among the elderly in industrialized countries, is associated with the dysfunction and death of the retinal pigment epithelial (RPE) cells. As a result, there has been significant interest in developing RPE culture systems both to study AMD disease mechanisms and to provide substrate for possible cell‐based therapies. Because of their indefinite self‐renewal, human pluripotent stem cells (hPSCs) have the potential to provide an unlimited supply of RPE‐like cells. However, most protocols developed to date for deriving RPE cells from hPSCs involve time‐ and labor‐consuming manual steps, which hinder their use in biomedical applications requiring large amounts of differentiated cells. Here, we describe a simple and scalable protocol for the generation of RPE cells from hPSCs that is less labor‐intensive. After amplification by clonal propagation using a myosin inhibitor, differentiation was induced in monolayers of hPSCs, and the resulting RPE cells were purified by two rounds of whole‐dish single‐cell passage. This approach yields highly pure populations of functional hPSC‐derived RPE cells that display many characteristics of native RPE cells, including proper pigmentation and morphology, cell type‐specific marker expression, polarized membrane and vascular endothelial growth factor secretion, and phagocytic activity. This work represents a step toward mass production of RPE cells from hPSCs.

MICAL flavoprotein monooxygenases : Expression during neural development and following spinal cord injuries in the rat

MICALs comprise of a family of phylogenetically conserved, multidomain cytosolic flavoprotein monooxygenases. Drosophila (D-)MICAL binds the neuronal Sema1a receptor PlexA, and D-MICAL-PlexA interactions are required in vivo for Sema1a-induced axon repulsion. The biological functions of vertebrate MICAL proteins, however, remain unknown. Here, we describe three rodent MICAL genes and analyze their expression in the intact rat nervous system and in two models of spinal cord injury. MICAL-1, -2, and -3 expression patterns in the embryonic, postnatal, and adult nervous system support the idea that MICALs play roles in neural development and plasticity. In addition, MICAL expression is elevated in oligodendrocytes and in meningeal fibroblasts at sites of spinal cord injury but is unchanged in lesioned corticospinal tract neurons. Furthermore, we find that the selective monooxygenase inhibitor EGCG attenuates the repulsive effects of Sema3A and Sema3F in vitro, but not those of several other repulsive cues and substrates. These results implicate MICALs in neuronal regeneration and support the possibility of employing EGCG to attenuate Sema3-mediated axon repulsion in the injured spinal cord.

Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs

The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.

Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells

The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

Significance Cell-based approaches utilizing retinal pigment epithelial (RPE)-like cells derived from human pluripotent stem cells (hPSCs) are being developed for the treatment of retinal degeneration. In most research published to date, the choice of the factors used to induce RPE differentiation is based on data from developmental studies. Here, we developed an unbiased approach directed at identifying novel RPE differentiation-promoting factors using a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. We identified chetomin, a dimeric epidithiodiketopiperazine, as a strong inducer of RPE; combination with nicotinamide resulted in efficient RPE differentiation. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Molecular dynamics of photoreceptor synapse formation in the developing chick retina

The cellular and molecular mechanisms underlying photoreceptor synaptogenesis are poorly understood. Furthermore, a detailed picture of the molecular composition of photoreceptor synapses, or their subtypes, is not yet available, nor do we know what differences, if any, exist among those subtypes. To address these questions, we investigated temporal and spatial patterns of expression and assembly of photoreceptor presynaptic components during chick embryo retinal development and early posthatched life by using reverse transcriptase polymerase chain reaction (RT‐PCR), dissociated retinal cells, laser‐capture microdissection (LCM), immunocytochemistry and confocal microscopy. Immunocytochemistry in tissue sections and dissociated cells showed many similarities and few differences in the synaptic composition of rods and cone subtypes, which, however, were found to project to different strata within the outer plexiform layer. A striking finding was the precise timetable of expression of synaptic genes and proteins during synaptogenesis. Although mRNAs for some synaptic molecules appeared as early as embryonic day (ED) 5–8 (the time of inner retina synaptogenesis), others were undetectable before the time of onset of photoreceptor synaptogenesis on ED13, including CAST, rim2, synapsin‐2, syntaxin‐3, synaptotagmin, glutamate receptors ‐1, ‐4, and ‐5, homer‐1 and ‐2, and tenascin‐R. Most synaptic proteins in photoreceptors followed a similar sequence of expression: they were negative or weakly positive before ED13, appeared in inner segments between ED13 and ED15, became subsequently detectable in perinuclear and axonal regions, and by ED18 were assembled into synaptic terminals and became undetectable in the inner segments. The identity of the signals that regulate the coordinated expression of these synaptic components remains to be investigated. J. Comp. Neurol. 506:822–837, 2008.

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

Background Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. Methods A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. Results 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. Conclusion This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

Transcription Factor SOX9 Plays a Key Role in the Regulation of Visual Cycle Gene Expression in the Retinal Pigment Epithelium

Background: The visual cycle is an enzymatic cascade that regenerates the visual chromophore. Results: Visual cycle gene expression is regulated by SOX9 in combination with OTX2 or LHX2 and can be modulated by common microRNAs. Conclusion: A core transcriptional network involving SOX9 regulates visual cycle genes. Significance: Understanding visual cycle gene regulation may have implications for treating retinal degenerative diseases. The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.