efficient-derivation-retinal-pigment-epithelium-cells-from-stem

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

The Scripps Research Institute

Lowy Medical Research Institute

Benign familial neonatal convulsions (BFNC) is a rare autosomal dominant generalized epilepsy of the newborn infant. Seizures occur repeatedly in the first days of life and remit by approximately 4 months of age. Previously our laboratory cloned two novel potassium channel genes, KCNQ2 and KCNQ3, and showed that they are mutated in patients with BFNC. In this report, we characterize the breakpoints of a previously reported interstitial deletion in the KCNQ2 gene and show that only KCNQ2 is deleted. We identify 11 novel mutations in KCNQ2 and one novel mutation in the KCNQ3 potassium channel genes. In one family, the phenotype extends beyond neonatal seizures and includes rolandic seizures, and a subset of families has onset of seizures in infancy. In the Xenopus oocyte expression system, we characterize five KCNQ2 and one KCNQ3 disease-causing mutations. These mutations cause a variable loss of function, and selective effects on the biophysical properties of KCNQ2/KCNQ3 heteromultimeric channels. We report here the first dominant negative mutation in KCNQ2 that has a phenotype of neonatal seizures without permanent clinical CNS impairment.

KCNQ2 and KCNQ3 potassium channel genes in benign familial neonatal convulsions: expansion of the functional and mutation spectrum.

Long QT syndrome (LQTS) predisposes affected individuals to sudden death from cardiac arrhythmias. Although most LQTS individuals do not have cardiac events, significant phenotypic variability exists within families. Probands can be very symptomatic. The mechanism of this phenotypic variability is not understood.

Compound mutations: a common cause of severe long-QT syndrome.

KCNQ1 (Kv 7.1) alpha-subunits and KCNE1 beta-subunits co-assemble to form channels that conduct the slow delayed rectifier K+ current (IKs) in the heart. Mutations in either subunit cause long QT syndrome (LQTS), an inherited disorder of cardiac repolarization. Here, the functional consequences of the LQTS-associated missense mutation V310I and several nearby residues were determined. Val310 is located at the base of the pore helix of KCNQ1, two residues below the TIGYG signature sequence that defines the K+ selectivity filter. Channels were heterologously expressed in Xenopus laevis oocytes and currents were recorded using the two-microelectrode voltage-clamp technique. V310I KCNQ1 reduced IKs amplitude when co-expressed with wild-type KCNQ1 and KCNE1 subunits. Val310 was also mutated to Gly, Ala or Leu to explore the importance of amino acid side chain volume at this position. Like V310I, V310L KCNQ1 channels gated normally. Unexpectedly, V310G and V310A KCNQ1 channels inactivated strongly and did not close normally in response to membrane hyperpolarization. Based on a homology model of the KCNQ1 channel pore, we speculate that the side group of residue 310 can interact with specific residues in the S5 and S6 domains to alter channel gating. When volume of the side chain is small, the stability of the closed state is disrupted and the extent of channel inactivation is enhanced. We mutated putative interacting residues in S5 and S6 and found that mutant Leu273 and Phe340 channels also can disrupt close states and modify inactivation. Together these findings indicate the importance of a putative pore helix-S5-S6 interaction for normal KCNQ1 channel deactivation and confirm its role in KCNQ1 inactivation. Disturbance of these interactions might underly LQTS associated with KCNQ1 mutant channels.

Mutation of colocalized residues of the pore helix and transmembrane segments S5 and S6 disrupt deactivation and modify inactivation of KCNQ1 K+ channels.

The retinal pigment epithelium (RPE) consists of a monolayer of cuboidal, pigmented cells that is located between the retina and the choroid. The RPE is vital for growth and function of the vertebrate eye and improper development results in congenital defects, such as microphthalmia or anophthalmia, or a change of cell fate into neural retina called transdifferentiation. The transcription factors microphthalmia-associated transcription factor (Mitf) and orthodenticle homolog 2 (Otx2) are crucial for RPE development and function; however, very little is known about their regulation. Here, by using a Wnt-responsive reporter, we show that the Wnt/beta-catenin pathway is activated in the differentiating mouse RPE. Cre-mediated, RPE-specific disruption of beta-catenin after the onset of RPE specification causes severe defects, resulting in microphthalmia with coloboma, disturbed lamination, and mislocalization of adherens junction proteins. Upon beta-catenin deletion, the RPE transforms into a multilayered tissue in which the expression of Mitf and Otx2 is downregulated, while retina-specific gene expression is induced, which results in the transdifferentiation of RPE into retina. Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that beta-catenin binds near to and activates potential TCF/LEF sites in the Mitf and Otx2 enhancers. We conclude that Wnt/beta-catenin signaling is required for differentiation of the RPE by directly regulating the expression of Mitf and Otx2. Our study is the first to show that an extracellular signaling pathway directly regulates the expression of RPE-specific genes such as Mitf and Otx2, and elucidates a new role for the Wnt/beta-catenin pathway in organ formation and development.

Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression.

Development of the retinal pigment epithelium (RPE) is controlled by intrinsic and extrinsic regulators including orthodenticle homeobox 2 (Otx2) and the Wnt/β-catenin pathway, respectively. Otx2 and β-catenin are necessary for the expression of the RPE key regulator microphthalmia-associated transcription factor (Mitf); however, neither factor is sufficient to promote Mitf expression in vivo. The study was conducted to determine whether Otx2 and β-catenin act in a combinatorial manner and tested whether co-expression in the presumptive chick retina induces ectopic Mitf expression.

Ectopic Mitf in the embryonic chick retina by co-transfection of β-catenin and Otx2.

Successful transition from embryonic to adult circulation is critical for survival of mammalian organisms. This shift occurs in the central cardiovascular circulation and in the eye as oxygen tension increases. However, its regulation is not well understood. We have used combinatorial gene deletion and overexpression assays to assess the effect of astrocyte-targeted deletion of von Hippel-Lindau tumor suppressor (Vhl), hypoxia-inducible factor-αs (Hif-αs), and Vegf on the normal regression of the hyaloidal vessels, the fetal ocular circulation system. Astrocytic Vhl deletion induced accelerated hyaloidal regression and subsequent massive secondary outgrowth. Combinatorial gene deletion involving Vhl, Hif-αs, and Vegf genes revealed that HIF-2α/vascular endothelial growth factor signaling induces secondary outgrowth in Vhl mutants. Conversely, HIF-1α regulated macrophage migration inhibitory factor and promoted macrophage infiltration that accelerates hyaloidal vessel regression. The phenotype observed in Vhl mutants strongly resembles human persistent hyperplastic primary vitreous cases and may provide insights into vascular remodeling mechanisms in other systems.

Astrocyte pVHL and HIF-α isoforms are required for embryonic-to-adult vascular transition in the eye.

Autologous retinal pigment epithelium (RPE) grafts derived from induced pluripotent stem cells (iPSCs) may be used to cure blinding diseases in which RPE dysfunction results in photoreceptor degeneration. Four-, two-, and one-factor-derived iPSCs (4F-, 2F-, and 1F-iPSCs, respectively) were differentiated into fully functional cuboidal pigmented cells in polarized monolayers that express RPE-specific markers. 1F-iPSCs-RPE (1F-iPS-RPE) strongly resembles primary human fetal RPE (hfRPE) based on proteomic and untargeted metabolomic analyses, and using novel in vivo imaging technology coupled with electroretinography, we demonstrated that 1F-iPS-RPE mediate anatomical and functional rescue of photoreceptors after transplantation in an animal model of RPE-mediated retinal degeneration. 1F-iPS0RPE cells were injected subretinally as a suspension and formed a monolayer dispersed between host RPE cells. Furthermore, 1F-iPS-RPE do not simply provide trophic support to rescue photoreceptors as previously speculated but actually phagocytose photoreceptor outer segments in vivo and maintain visual cycling. Thus, 1f-iPS-RPE grafts may be superior to conventional iPS-RPE for clinical use because 1F-IPS-RPE closely resemble hfRPE, mediate anatomical and functional photoreceptor rescue in vivo, and are generated using a reduced number of potentially oncogenic reprogramming factors.

Generation of retinal pigment epithelial cells from small molecules and OCT4 reprogrammed human induced pluripotent stem cells.

Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS).

Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells.

Current therapies directed at controlling vascular abnormalities in cancers and neovascular eye diseases target VEGF and can slow the progression of these diseases. While the critical role of VEGF in development has been well described, the function of locally synthesized VEGF in the adult eye is incompletely understood. Here, we show that conditionally knocking out Vegfa in adult mouse retinal pigmented epithelial (RPE) cells, which regulate retinal homeostasis, rapidly leads to vision loss and ablation of the choriocapillaris, the major blood supply for the outer retina and photoreceptor cells. This deletion also caused rapid dysfunction of cone photoreceptors, the cells responsible for fine visual acuity and color vision. Furthermore, Vegfa deletion showed significant downregulation of multiple angiogenic genes in both physiological and pathological states, whereas the deletion of the upstream regulatory transcriptional factors HIFs did not affect the physiological expressions of angiogenic genes. These results suggest that endogenous VEGF provides critical trophic support necessary for retinal function. Targeting factors upstream of VEGF, such as HIFs, may be therapeutically advantageous compared with more potent and selective VEGF antagonists, which may have more off-target inhibitory trophic effects.

Targeted deletion of Vegfa in adult mice induces vision loss.

Vascular networks develop from a growing vascular front that responds to VEGF and other guidance cues. Angiogenesis is required for normal tissue function, but, under conditions of stress, inappropriate vascularization can lead to disease. Therefore, inhibition of angiogenic sprouting may prevent neovascularization in patients with blinding neovascular eye diseases, including macular degeneration. VEGF antagonists have therapeutic benefits but also can elicit off-target effects. Here, we found that the Ras pathway, which functions downstream of a wide range of cytokines including VEGF, is active in the growing vascular front of developing and pathological vascular networks. The endogenous Ras inhibitor p120RasGAP was expressed predominately in quiescent VEGF-insensitive endothelial cells and was ectopically downregulated in multiple neovascular models. MicroRNA-132 negatively regulated p120RasGAP expression. Experimental delivery of α-miR-132 to developing mouse eyes disrupted tip cell Ras activity and prevented angiogenic sprouting. This strategy prevented ocular neovascularization in multiple rodent models even more potently than the VEGF antagonist, VEGF-trap. Targeting microRNA-132 as a therapeutic strategy may prove useful for treating multiple neovascular diseases of the eye and for preventing vision loss regardless of the neovascular stimulus.

Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting.

Over a span of two decades, it has become increasingly clear that vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of retinal diseases including age-related macular degeneration (AMD) and diabetic retinopathy (DR). Based on these observations, anti-VEGF therapies are being developed and approved for clinical use in the treatment of neovascular eye diseases. Hypoxia-inducible factors (HIFs) are transcriptional factors that are stabilized and activated under hypoxic conditions and induce expression of gene products, including VEGF, that are required for cell survival under hypoxia. Here we discuss recent findings from our lab and others that define roles of the HIF-VEGF axis in the retina.

Hypoxia-inducible factor (HIF)/vascular endothelial growth factor (VEGF) signaling in the retina.

Degeneration or dysfunction of the retinal pigment epithelium (RPE) can induce secondary photoreceptor atrophy and catastrophic vision loss in patients with age-related macular degeneration (AMD). AMD is the leading cause of vision loss in the elderly in industrialized countries and no cure exists for the "dry" or atrophic form to date. However, recent pre-clinical data from several groups suggests that embryonic stem cell-derived RPE cell transplantation may prevent photoreceptor degeneration in animal models of RPE degeneration. Another approach may be to derive RPE cells from autologous induced pluripotent stem cells (iPSCs) reprogrammed from dermal tissue. However, the safety of this approach has been questioned on several levels. In this chapter we will summarize work reported by several groups, including our own, that clearly demonstrate that transplanted RPE cells can provide anatomical and functional photoreceptor rescue in animal models of retinal degeneration. We will also discuss some of the prevailing concerns and challenges associated with this technique.

Utilizing stem cell-derived RPE cells as a therapeutic intervention for age-related macular degeneration.

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor-A (VEGF) biologics, termed VEGF "Sticky-traps," with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky-traps could locally inhibit angiogenesis to at least the same extent as the original VEGF-trap that also gains whole-body access. Sticky-traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky-trap remained localized to various regions of the eye, such as the inner-limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky-trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre-maturity.

Local acting Sticky-trap inhibits vascular endothelial growth factor dependent pathological angiogenesis in the eye.

XCMS Online (xcmsonline.scripps.edu) is a cloud-based informatic platform designed to process and visualize mass-spectrometry-based, untargeted metabolomic data. Initially, the platform was developed for two-group comparisons to match the independent, "control" versus "disease" experimental design. Here, we introduce an enhanced XCMS Online interface that enables users to perform dependent (paired) two-group comparisons, meta-analysis, and multigroup comparisons, with comprehensive statistical output and interactive visualization tools. Newly incorporated statistical tests cover a wide array of univariate analyses. Multigroup comparison allows for the identification of differentially expressed metabolite features across multiple classes of data while higher order meta-analysis facilitates the identification of shared metabolic patterns across multiple two-group comparisons. Given the complexity of these data sets, we have developed an interactive platform where users can monitor the statistical output of univariate (cloud plots) and multivariate (PCA plots) data analysis in real time by adjusting the threshold and range of various parameters. On the interactive cloud plot, metabolite features can be filtered out by their significance level (p-value), fold change, mass-to-charge ratio, retention time, and intensity. The variation pattern of each feature can be visualized on both extracted-ion chromatograms and box plots. The interactive principal component analysis includes scores, loadings, and scree plots that can be adjusted depending on scaling criteria. The utility of XCMS functionalities is demonstrated through the metabolomic analysis of bacterial stress response and the comparison of lymphoblastic leukemia cell lines.

Article	Year
KCNQ2 and KCNQ3 potassium channel genes in benign familial neonatal convulsions: expansion of the functional and mutation spectrum. Brain : a journal of neurology\| PubMed ID: 14534157	2003
Compound mutations: a common cause of severe long-QT syndrome. Circulation\| PubMed ID: 15051636	2004
Mutation of colocalized residues of the pore helix and transmembrane segments S5 and S6 disrupt deactivation and modify inactivation of KCNQ1 K+ channels. The Journal of physiology\| PubMed ID: 15649981	2005
Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression. Development (Cambridge, England)\| PubMed ID: 19553286	2009
Ectopic Mitf in the embryonic chick retina by co-transfection of β-catenin and Otx2. Investigative ophthalmology & visual science\| PubMed ID: 20463321	2010
Astrocyte pVHL and HIF-α isoforms are required for embryonic-to-adult vascular transition in the eye. The Journal of cell biology\| PubMed ID: 22084310	2011
Generation of retinal pigment epithelial cells from small molecules and OCT4 reprogrammed human induced pluripotent stem cells. Stem cells translational medicine\| PubMed ID: 22532929	2012
Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells. Investigative ophthalmology & visual science\| PubMed ID: 22871841	2012
Targeted deletion of Vegfa in adult mice induces vision loss. The Journal of clinical investigation\| PubMed ID: 23093773	2012
Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting. The Journal of clinical investigation\| PubMed ID: 24084735	2013
Hypoxia-inducible factor (HIF)/vascular endothelial growth factor (VEGF) signaling in the retina. Advances in experimental medicine and biology\| PubMed ID: 24664708	2014
Utilizing stem cell-derived RPE cells as a therapeutic intervention for age-related macular degeneration. Advances in experimental medicine and biology\| PubMed ID: 24664714	2014
Local acting Sticky-trap inhibits vascular endothelial growth factor dependent pathological angiogenesis in the eye. EMBO molecular medicine\| PubMed ID: 24705878	2014
Interactive XCMS Online: simplifying advanced metabolomic data processing and subsequent statistical analyses. Analytical chemistry\| PubMed ID: 24934772	2014

Peter Westenskow

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