Müller glial cell-dependent regeneration of the neural retina: An overview across vertebrate model systems.

Retinal dystrophies are a major cause of blindness for which there are currently no curative treatments. Transplantation of stem cell-derived neuronal progenitors to replace lost cells has been widely investigated as a therapeutic option. Another promising strategy would be to trigger self-repair mechanisms in patients, through the recruitment of endogenous cells with stemness properties. Accumulating evidence in the past 15 year0s has revealed that several retinal cell types possess neurogenic potential, thus opening new avenues for regenerative medicine. Among them, Müller glial cells have been shown to be able to undergo a reprogramming process to re-acquire a stem/progenitor state, allowing them to proliferate and generate new neurons for repair following retinal damages. Although Müller cell-dependent spontaneous regeneration is remarkable in some species such as the fish, it is extremely limited and ineffective in mammals. Understanding the cellular events and molecular mechanisms underlying Müller cell activities in species endowed with regenerative capacities could provide knowledge to unlock the restricted potential of their mammalian counterparts. In this context, the present review provides an overview of Müller cell responses to injury across vertebrate model systems and summarizes recent advances in this rapidly evolving field. Developmental Dynamics 245:727-738, 2016. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc.

CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in Müller glial cells.

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective agent in multiple retinal degeneration animal models. Recently, CNTF has been evaluated in clinical trials for the inherited degenerative disease retinitis pigmentosa (RP) and for dry age-related macular degeneration (AMD). Despite its potential as a broad-spectrum therapeutic treatment for blinding diseases, the target cells of exogenous CNTF and its mechanism of action remain poorly understood. We have shown previously that constitutive expression of CNTF prevents photoreceptor death but alters the retinal transcriptome and suppresses visual function. Here, we use a lentivirus to deliver the same secreted human CNTF used in clinical trials to a mouse model of RP. We found that low levels of CNTF halt photoreceptor death, improve photoreceptor morphology, and correct opsin mislocalization. However, we did not detect corresponding improvement of retinal function as measured by the electroretinogram. Disruption of the cytokine receptor gp130 gene in Müller glia reduces CNTF-dependent photoreceptor survival and prevents phosphorylation of STAT3 and ERK in Müller glia and the rest of the retina. Targeted deletion of gp130 in rods also demolishes neuroprotection by CNTF and prevents further activation of Müller glia. Moreover, CNTF elevates the expression of LIF and endothelin 2, thus positively promoting Müller and photoreceptor interactions. We propose that exogenous CNTF initially targets Müller glia, and subsequently induces cytokines acting through gp130 in photoreceptors to promote neuronal survival. These results elucidate a cellular mechanism for exogenous CNTF-triggered neuroprotection and provide insight into the complex cellular responses induced by CNTF in diseased retinas.

Time-delay signature of chaos in 1550 nm VCSELs with variable-polarization FBG feedback.

Based on the framework of spin-flip model (SFM), the output characteristics of a 1550 nm vertical-cavity surface-emitting laser (VCSEL) subject to variable-polarization fiber Bragg grating (FBG) feedback (VPFBGF) have been investigated. With the aid of the self-correlation function (SF) and the permutation entropy (PE) function, the time-delay signature (TDS) of chaos in the VPFBGF-VCSEL is evaluated, and then the influences of the operation parameters on the TDS of chaos are analyzed. The results show that the TDS of chaos can be suppressed efficiently through selecting suitable coupling coefficient and feedback rate of the FBG, and is weaker than that of chaos generated by traditional variable-polarization mirror feedback VCSELs (VPMF-VCSELs) or polarization-preserved FBG feedback VCSELs (PPFBGF-VCSELs).

PTEN regulates retinal interneuron morphogenesis and synaptic layer formation.

The lipid phosphatase PTEN is a critical negative regulator of extracellular signal-induced PI3K activities, yet the roles of PTEN in the neural retina remain poorly understood. Here, we investigate the function of PTEN during retinal development. Deletion of Pten at the onset of neurogenesis in retinal progenitors results in the reduction of retinal ganglion cells and rod photoreceptors, but increased Müller glial genesis. In addition, PTEN deficiency leads to elevated phosphorylation of Akt, especially in the developing inner plexiform layer, where high levels of PTEN are normally expressed. In Pten mutant retinas, various subtypes of amacrine cells show severe dendritic overgrowth, causing specific expansion of the inner plexiform layer. However, the outer plexiform layer remains relatively undisturbed in the Pten deficient retina. Physiological analysis detects reduced rod function and augmented oscillatory potentials originating from amacrine cells in Pten mutants. Furthermore, deleting Pten or elevating Akt activity in individual amacrine cells is sufficient to disrupt dendritic arborization, indicating that Pten activity is required cell autonomously to control neuronal morphology. Moreover, inhibiting endogenous Akt activity attenuates inner plexiform layer formation in vitro. Together, these findings demonstrate that suppression of PI3K/Akt signaling by PTEN is crucial for proper neuronal differentiation and normal retinal network formation.

Establishing induced pluripotent stem cell lines from two dominant optic atrophy patients with distinct OPA1 mutations and clinical pathologies.

Dominant optic atrophy (DOA) is an inherited disease that leads to the loss of retinal ganglion cells (RGCs), the projection neurons that relay visual information from the retina to the brain through the optic nerve. The majority of DOA cases can be attributed to mutations in optic atrophy 1 (OPA1), a nuclear gene encoding a mitochondrial-targeted protein that plays important roles in maintaining mitochondrial structure, dynamics, and bioenergetics. Although OPA1 is ubiquitously expressed in all human tissues, RGCs appear to be the primary cell type affected by OPA1 mutations. DOA has not been extensively studied in human RGCs due to the general unavailability of retinal tissues. However, recent advances in stem cell biology have made it possible to produce human RGCs from pluripotent stem cells (PSCs). To aid in establishing DOA disease models based on human PSC-derived RGCs, we have generated iPSC lines from two DOA patients who carry distinct OPA1 mutations and present very different disease symptoms. Studies using these OPA1 mutant RGCs can be correlated with clinical features in the patients to provide insights into DOA disease mechanisms.

Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells.

Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.

Ciliary neurotrophic factor-mediated neuroprotection involves enhanced glycolysis and anabolism in degenerating mouse retinas.

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective cytokine in multiple models of retinal degeneration. To understand mechanisms underlying its broad neuroprotective effects, we have investigated the influence of CNTF on metabolism in a mouse model of photoreceptor degeneration. CNTF treatment improves the morphology of photoreceptor mitochondria, but also leads to reduced oxygen consumption and suppressed respiratory chain activities. Molecular analyses show elevated glycolytic pathway gene transcripts and active enzymes. Metabolomics analyses detect significantly higher levels of ATP and the energy currency phosphocreatine, elevated glycolytic pathway metabolites, increased TCA cycle metabolites, lipid biosynthetic pathway intermediates, nucleotides, and amino acids. Moreover, CNTF treatment restores the key antioxidant glutathione to the wild type level. Therefore, CNTF significantly impacts the metabolic status of degenerating retinas by promoting aerobic glycolysis and augmenting anabolic activities. These findings reveal cellular mechanisms underlying enhanced neuronal viability and suggest potential therapies for treating retinal degeneration.

Generation of a homozygous LRPAP1 knockout human embryonic stem cell line (FDCHDPe009-B) by CRISPR/Cas9 system.

The homozygous autosomal recessive truncating mutations of LDL receptor related protein associated protein 1 (LRPAP1) is a possible reason for Nonsyndromic Extreme Myopia, patients with which show typical chorioretinal degeneration. We generated an LRPAP1 knockout FDCHDPe009-B embryonic stem cell line to study mechanisms of retinal degeneration underlying LRPAP1 deficiency with the help of the CRISPR/Cas9 system. Two distinct biallelic deletions in the cell line have been confirmed, which causing a frameshift and premature stop codons thus influence the translation of LRPAP1. FDCHDPe009-B has maintained normal stem cell morphology, pluripotent gene expression, parental karyotype, and ability to differentiate into three germ layers.

Generation of a homozygous LRP2 knockout human embryonic stem cell line (FDCHDPe010-A-56) by CRISPR/Cas9 system.

LRP2 is mainly expressed in the cell membrane of epithelia, maintaining normal endocytosis of nutrients from the extracellular microenvironment and mediating growth factor signals. The deficiency of LRP2 can result in abnormal lysosomal and mitochondrial function as well as insufficient resistance to oxidative stress. LRP2-KO animals show enlarged eyes and malfunction of the retinal pigment epithelium (RPE). We were able to generate an LRP2-KO human embryonic stem (ES) cell line using CRISPR/Cas9 gene editing and differentiate the mutant ES cells into RPE cells. Thus, this LRP2-KO human ES line will facilitate studying cellular mechanisms of eye disease due to LRP2 deficiency.

Single Cell Transcriptomic Analyses Reveal the Impact of bHLH Factors on Human Retinal Organoid Development.

The developing retina expresses multiple bHLH transcription factors. Their precise functions and interactions in uncommitted retinal progenitors remain to be fully elucidated. Here, we investigate the roles of bHLH factors ATOH7 and Neurog2 in human ES cell-derived retinal organoids. Single cell transcriptome analyses identify three states of proliferating retinal progenitors: pre-neurogenic, neurogenic, and cell cycle-exiting progenitors. Each shows different expression profile of bHLH factors. The cell cycle-exiting progenitors feed into a postmitotic heterozygous neuroblast pool that gives rise to early born neuronal lineages. Elevating ATOH7 or Neurog2 expression accelerates the transition from the pre-neurogenic to the neurogenic state, and expands the exiting progenitor and neuroblast populations. In addition, ATOH7 and Neurog2 significantly, yet differentially, enhance retinal ganglion cell and cone photoreceptor production. Moreover, single cell transcriptome analyses reveal that ATOH7 and Neurog2 each assert positive autoregulation, and both suppress key bHLH factors associated with the pre-neurogenic and states and elevate bHLH factors expressed by exiting progenitors and differentiating neuroblasts. This study thus provides novel insight regarding how ATOH7 and Neurog2 impact human retinal progenitor behaviors and neuroblast fate choices.

Distinct effects of Hedgehog signaling on neuronal fate specification and cell cycle progression in the embryonic mouse retina.

Cell-extrinsic signals can profoundly influence the production of various neurons from common progenitors. Yet mechanisms by which extrinsic signals coordinate progenitor cell proliferation, cell cycle exit, and cell fate choices are not well understood. Here, we address whether Hedgehog (Hh) signals independently regulate progenitor proliferation and neuronal fate decisions in the embryonic mouse retina. Conditional ablation of the essential Hh signaling component Smoothened (Smo) in proliferating progenitors, rather than in nascent postmitotic neurons, leads to a dramatic increase of retinal ganglion cells (RGCs) and a mild increase of cone photoreceptor precursors without significantly affecting other early-born neuronal cell types. In addition, Smo-deficient progenitors exhibit aberrant expression of cell cycle regulators and delayed G(1)/S transition, especially during the late embryonic stages, resulting in a reduced progenitor pool by birth. Deficiency in Smo function also causes reduced expression of the basic helix-loop-helix transcription repressor Hes1 and preferential elevation of the proneural gene Math5. In Smo and Math5 double knock-out mutants, the enhanced RGC production observed in Smo-deficient retinas is abolished, whereas defects in the G(1)/S transition persist, suggesting that Math5 mediates the Hh effect on neuronal fate specification but not on cell proliferation. These findings demonstrate that Hh signals regulate progenitor pool expansion primarily by promoting cell cycle progression and influence cell cycle exit and neuronal fates by controlling specific proneural genes. Together, these distinct cellular effects of Hh signaling in neural progenitor cells coordinate a balanced production of diverse neuronal cell types.

Function and mechanism of CNTF/LIF signaling in retinogenesis.

Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) exhibit multiple biological effects in the developing vertebrate retina. CNTF/LIF inhibits rod photoreceptor, and promotes bipolar cells and Muller glia differentiation. In addition, CNTF/LIF has been shown to have proliferative and apoptotic effects. Moreover, LIF also inhibits retinal vascular development. CNTF/LIF signaling components CNTFRalpha, LIFRbeta, gp130, and a number of STAT proteins are expressed in the retina. CNTF/LIF activates Jak-STAT, ERK, and Notch pathways during retinal development. Perturbation of CNTF induced signal transduction reveals that different combinations of CNTF/LIF signaling pathways regulate differentiation of retinal neurons and glia. Gene expression studies show that CNTF/LIF affects retinogenesis by regulating various genes involved in transcription, signal transduction, protein modification, apoptosis, protein localization, and cell ion homeostasis. Most past studies have deployed ectopic expression or addition of exogenous CNTF/LIF, thus further ana-lysis of mice with conditional mutations in CNTF/LIF signaling components will allow better understanding of in-vivo functions of CNTF/LIF associated signaling events in retinogenesis.

Impacts of ciliary neurotrophic factor on the retinal transcriptome in a mouse model of photoreceptor degeneration.

Ciliary neurotrophic factor (CNTF) has been tested in clinical trials for human retinal degeneration due to its potent neuroprotective effects in various animal models. To decipher CNTF-triggered molecular events in the degenerating retina, we performed high-throughput RNA sequencing analyses using the Rds/Prph2 (P216L) transgenic mouse as a preclinical model for retinitis pigmentosa. In the absence of CNTF treatment, transcriptome alterations were detected at the onset of rod degeneration compared with wild type mice, including reduction of key photoreceptor transcription factors Crx, Nrl, and rod phototransduction genes. Short-term CNTF treatments caused further declines of photoreceptor transcription factors accompanied by marked decreases of both rod- and cone-specific gene expression. In addition, CNTF triggered acute elevation of transcripts in the innate immune system and growth factor signaling. These immune responses were sustained after long-term CNTF exposures that also affected neuronal transmission and metabolism. Comparisons of transcriptomes also uncovered common pathways shared with other retinal degeneration models. Cross referencing bulk RNA-seq with single-cell RNA-seq data revealed the CNTF responsive cell types, including Müller glia, rod and cone photoreceptors, and bipolar cells. Together, these results demonstrate the influence of exogenous CNTF on the retinal transcriptome landscape and illuminate likely CNTF impacts in degenerating human retinas.

Generation of an integration-free induced pluripotent stem cell line (FDEENTi003-A) from a patient with pathological myopia.

Pathological myopia (PM) is a major cause of irreversible vision impairment worldwide. We have successfully reprogrammed the peripheral blood mononuclear cells (PBMCs) from a PM patient to induced pluripotent stem cells and characterized their pluripotency and genetic stability, as well as the potential to differentiate to retinal pigment epithelium (RPE). This line may serve as a useful tool to explore the pathogenesis of PM.

Establishment of an induced pluripotent stem cell line (FDEENTi002-A) from a patient with Best's disease carrying c.888C > A mutation in BEST1 gene.

Best's disease (BD) is an inherited retinal degenerative disease caused by mutations in BEST1 gene. A human induced pluripotent stem cell (iPSC) line has been generated with integration-free Sendai virus method from peripheral blood mononuclear cells (PBMCs) of a BD patient carrying c.888C > A mutation in BEST1 gene. This cell line may serve as a model for the study of pathogenesis of BD.

Single-Cell RNA Sequencing of hESC-Derived 3D Retinal Organoids Reveals Novel Genes Regulating RPC Commitment in Early Human Retinogenesis.

The development of the mammalian retina is a complicated process involving the generation of distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell-fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA sequencing of cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinctive subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found that genes related to the Notch and Wnt signaling pathways, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified that CCND1, a G1-phase cell-cycle regulator, was coexpressed with ASCL1 in a cell-cycle-independent manner. Temporally controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in early retinogenesis of humans.

Elevated expression of human bHLH factor ATOH7 accelerates cell cycle progression of progenitors and enhances production of avian retinal ganglion cells.

The production of vertebrate retinal projection neurons, retinal ganglion cells (RGCs), is regulated by cell-intrinsic determinants and cell-to-cell signaling events. The basic-helix-loop-helix (bHLH) protein Atoh7 is a key neurogenic transcription factor required for RGC development. Here, we investigate whether manipulating human ATOH7 expression among uncommitted progenitors can promote RGC fate specification and thus be used as a strategy to enhance RGC genesis. Using the chicken retina as a model, we show that cell autonomous expression of ATOH7 is sufficient to induce precocious RGC formation and expansion of the neurogenic territory. ATOH7 overexpression among neurogenic progenitors significantly enhances RGC production at the expense of reducing the progenitor pool. Furthermore, forced expression of ATOH7 leads to a minor increase of cone photoreceptors. We provide evidence that elevating ATOH7 levels accelerates cell cycle progression from S to M phase and promotes cell cycle exit. We also show that ATOH7-induced ectopic RGCs often exhibit aberrant axonal projection patterns and are correlated with increased cell death during the period of retinotectal connections. These results demonstrate the high potency of human ATOH7 in promoting early retinogenesis and specifying the RGC differentiation program, thus providing insight for manipulating RGC production from stem cell-derived retinal organoids.

Molecular and cellular alterations induced by sustained expression of ciliary neurotrophic factor in a mouse model of retinitis pigmentosa.

PURPOSE: To characterize molecular and cellular changes induced by sustained expression of ciliary neurotrophic factor (CNTF) in the rds mutant mouse retina. METHODS: Recombinant adeno-associated virus (rAAV) expressing CNTF was injected subretinally, for transduction of peripherin/rds(+/)(-) transgenic mice that carry the P216L mutation found in human retinitis pigmentosa. Characterization of retinal neurons and glia was performed by immunocytochemistry with cell-type-specific markers. Activation of signaling molecules was examined by Western blot and immunostaining. Alterations of gene transcription profiles were studied by microarray analyses. RESULTS: CNTF viral transduction maintained rhodopsin expression in surviving rod photoreceptors, but greatly reduced both S- and M-opsin normally expressed in cones. In addition, CNTF treatment resulted in increased numbers and dispersion of Müller glia and Chx10-positive bipolar cells within the inner nuclear layer. Persistent CNTF signaling also caused enhanced phosphorylation of STAT1, STAT3, and p42/44 ERK, as well as their levels of expression. Moreover, altered transcription profiles were detected for a large number of genes. Among these, Crx and Nrl involved in photoreceptor differentiation and several genes involved in phototransduction were suppressed. CONCLUSIONS: Despite the rescue from cell death, continuous exposure to CNTF changed photoreceptor cell profiles, especially resulting in the loss of cone immunoreactivity. In addition, the Müller glia and bipolar cells became disorganized, and the number of cells expressing Müller and bipolar cell markers increased. Constitutive CNTF production resulted in sustained activation of cytokine signal transduction and altered the expression of a large number of genes. Therefore, stringent regulation of CNTF may be necessary for its therapeutic application in preventing retinal degeneration.

Cytokine-induced activation of signal transducer and activator of transcription in photoreceptor precursors regulates rod differentiation in the developing mouse retina.

Ciliary neurotrophic factor (CNTF) exhibits multiple biological effects during vertebrate retinogenesis, including regulation of photoreceptor cell differentiation. In the early postnatal mouse retina, CNTF induces rapid and transient phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3 and the extracellular signal-regulated kinase (ERK). Although both proliferating progenitor cells and postmitotic neurons respond directly to cytokine signals, CNTF elicits distinct phosphorylation patterns of STAT3 and ERK. CNTF stimulation induces low levels of STAT3 phosphorylation in progenitors and differentiated neurons but a robust STAT3 activation among postmitotic photoreceptor precursors expressing the cone-rod homeobox gene Crx and newly differentiated rod photoreceptors. In contrast, CNTF causes preferential phosphorylation of ERK in progenitor cells and photoreceptor precursors. Inhibition of the cytokine receptor gp130 using neutralizing antibodies reveals that gp130 is required for both CNTF-induced STAT3 and ERK phosphorylation. Perturbation of STAT signaling by a STAT inhibitor peptide or a dominant-negative STAT3 mutant causes enhanced production of rod photoreceptors in the absence of exogenous cytokines, whereas inhibiting ERK activation by a MEK (mitogen-activated protein kinase kinase)-specific inhibitor has no effect on rod photoreceptor differentiation in vitro. Furthermore, disrupting the function of epidermal growth factor (EGF) receptors, which modulate rod development in vivo, indicates that the EGF family of ligands does not mediate the inhibitory effect of cytokine on rod differentiation. These results demonstrate that cytokine signal transduction is dynamic and heterogeneous in the developing retina, and that endogenous ligand-induced STAT activation in retinal progenitor and/or photoreceptor precursor cells plays an important role in regulating photoreceptor development.

Expression of the Flk1 receptor and its ligand VEGF in the developing chick central nervous system.

The receptor tyrosine kinase Flk1 is known to mediate signals of vascular endothelial growth factor (VEGF) during vasculogenesis and hematopoiesis. We demonstrate by in situ hybridization that in addition to endothelial cells, chick Flk1 mRNA is also expressed in the notochord and in the neural epithelial cells of the ventral diencephalon, hindbrain, and spinal cord. During the development of the avascular chick retina, Flk1 mRNA is detected in the proliferative zone of the neural epithelium, whereas the VEGF ligand is expressed by differentiated retinal ganglion cells. Moreover, expression patterns of Flk1 in the retina are conserved among chick, quail and mouse, thus suggesting a distinct role of Flk1 and VEGF in the development of the vertebrate central nervous system.