Retinal Pigment Epithelium Differentiation Research Articles

C-Jun-mediated microRNA-302d-3p induces RPE dedifferentiation by targeting p21Waf1/Cip1

Dedifferentiation of retinal pigment epithelium (RPE) cells and choroidal neovascularization (CNV) contributes to the pathogenesis of age-related macular degeneration (AMD). MicroRNAs (miRNAs) have crucial roles in AMD onset and progression. We thus aim to investigate the effects of miRNAs on RPE dedifferentiation and endothelium cell (EC) behavior, and analyze its downstream pathways. We have previously identified miR-302d-3p as the most downregulated miRNA signature along with RPE differentiation. Herein, in vitro study supported that miR-302d-3p induces RPE dedifferentiation typified by reduction of RPE characteristic markers, interrupts its phagocytosis, and promotes its migration, proliferation, and cell-cycle progression. c-Jun was identified as a potential upstream transcript factor for MIR302D, which might modulate RPE function by regulating miR-302d-3p expression. P21Waf1/Cip1, a cyclin-dependent kinase inhibitor encoded by the CDKN1A gene, was identified as a downstream target of miR-302d-3p. Our data suggested that p21Waf1/Cip1 could promote RPE differentiation, and inhibit its proliferation, migration, and cell-cycle progression. We also demonstrated that miR-302d-3p suppresses RPE differentiation through directly targeting p21Waf1/Cip1. In addition, the miR-302d-3p/CDKN1A axis was also involved in regulating tube formation of ECs, indicating its potential involvement in CNV formation. Taken together, our study implies that miR-302d-3p, regulated by c-Jun, contributes to the pathogenesis of both atrophic and exudative AMD. MiR-302d-3p promotes RPE dedifferentiation, migration, proliferation and cell-cycle progression, inhibits RPE phagocytosis, and induces abnormal EC behavior by targeting p21Waf1/Cip1. Pharmacological miR-302d-3p inhibitors are prospective therapeutic options for prevention and treatment of AMD.

LncRNA ZNF503-AS1 promotes RPE differentiation by downregulating ZNF503 expression

Long noncoding RNAs (lncRNAs) have important roles in various biological processes. Our previous work has revealed that dedifferentiation of retinal pigment epithelium (RPE) cells contributes to the pathology of age-related macular degeneration (AMD). Herein, we show roles of lncRNAs in RPE differentiation. We used microarray to identify lncRNA expression profiles in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived RPE cells. A total of 217 differentially expressed lncRNAs along with the differentiation were initially identified, among which 13 lncRNAs showed a consistent fold change of over 2. LncRNA ZNF503-AS1, located in the cytoplasm of RPE cells, was found consistently upregulated along with RPE differentiation, and downregulated in the RPE-choroid of AMD patients. In vitro study further suggested that ZNF503-AS1 insufficiency could inhibit RPE differentiation, and promote its proliferation and migration. As ZNF503-AS1 is transcribed from the antisense strand of the ZNF503 gene locus, we further revealed its regulatory role in ZNF503 expression. ZNF503-AS1 was reversely correlated with ZNF503 expression. Our results also suggested that ZNF503 could inhibit RPE differentiation, and promote its proliferation and migration. Thus, ZNF503-AS1 potentially promotes RPE differentiation through downregulation of ZNF503 expression. In addition, nuclear factor-κB was recognized as a potential upstream transcript factor for ZNF503-AS1, which might participate in promoting RPE differentiation by regulating the expression of ZNF503-AS1. Taken together, our study identifies a group of RPE differentiation relevant lncRNAs, and the potential role of ZNF503-AS1 in the pathology of atrophic AMD, which might help with the intervention of AMD patients.

Stem Cell Derived Retinal Pigment Epithelium: The Role of Pigmentation as Maturation Marker and Gene Expression Profile Comparison with Human Endogenous Retinal Pigment Epithelium.

In age-related macular degeneration (AMD) the retinal pigment epithelium (RPE) deteriorates, leading to photoreceptor decay and severe vision loss. New therapeutic strategies aim at RPE replacement by transplantation of pluripotent stem cell (PSC)-derived RPE. Several protocols to generate RPE have been developed where appearance of pigmentation is commonly used as indicator of RPE differentiation and maturation. It is, however, unclear how different pigmentation stages reflect developmental stages and functionality of PSC-derived RPE cells. We generated human embryonic stem cell-derived RPE (hESC-RPE) cells and investigated their gene expression profiles at early pigmentation (EP) and late pigmentation (LP) stages. In addition, we compared the hESC-RPE samples with human endogenous RPE. We used a common reference design microarray (44 K). Our analysis showed that maturing hESC-RPE, upon acquiring pigmentation, expresses markers specific for human RPE. Interestingly, our analysis revealed that EP and LP hESC-RPE do not differ much in gene expression. Our data further showed that pigmented hESC-RPE has a significant lower expression than human endogenous RPE in the visual cycle and oxidative stress pathways. In contrast, we observed a significantly higher expression of pathways related to the process adhesion-to-polarity model that is typical of developing epithelial cells. We conclude that, in vitro, the first appearance of pigmentation hallmarks differentiated RPE. However, further increase in pigmentation does not result in much significant gene expression changes and does not add important RPE functionalities. Consequently, our results suggest that the time span for obtaining differentiated hESC-RPE cells, that are suitable for transplantation, may be greatly reduced.

Restoration of Mesenchymal RPE by Transcription Factor-Mediated Reprogramming.

Transforming growth factor β-mediated epithelial-to-mesenchymal transition (EMT) is a major component of the wound healing response and a negative determinant of retinal pigment epithelium (RPE) differentiation. We have shown previously that inhibition of TGFβ signaling restored the capacity of mesenchymal RPE to differentiate; however, the potential lessens with extensive passaging. We investigated TGFβ-independent mechanisms that regulate RPE differentiation following repetitive passaging. Retinal pigment epithelium-EMT was induced by repetitive passaging of fetal RPE at subconfluence. Suppression of EMT was achieved by the addition of A-83-01, a TGFβ receptor kinase inhibitor. Transcriptomic analysis was used to identify potential TGFβ independent processes that prevent differentiation after extensive passage. Downregulated transcription factors were identified and transduced into highly passaged RPE to restore cell differentiation. Restoration was evaluated according to morphology, expression of RPE/mesenchymal markers, transcriptomic analysis, cell doubling time, and senescence-associated β-galactosidase (SA-β-gal) activity. A-83-01-treated RPE failed to differentiate after 7 passages (P7). This failure was concomitant with downregulation of RPE genes, misregulation of cell cycle genes, a decline in proliferative potential, and cell senescence. Exogenous expression of MYCN and OTX2 in conjunction with A-83-01 restored P7-RPE differentiation to a status similar to minimally passaged RPE. Moreover, the treatment allowed cells to maintain their differentiation capacity after extended passaging. Retinal pigment epithelium subjected to chronic wound stimulus undergoes TGFβ-mediated EMT, loss of expression of signature RPE genes, and senescence. Targeting these aspects with a TGFβ receptor kinase inhibitor, a RPE transcription factor, and a cell cycle regulator restores the capacity of highly passaged RPE cells to regenerate and differentiate.

Inhibition by miR-410 facilitates direct retinal pigment epithelium differentiation of umbilical cord blood-derived mesenchymal stem cells.

Retinal pigment epithelium (RPE) is a major component of the eye. This highly specialized cell type facilitates maintenance of the visual system. Because RPE loss induces an irreversible visual impairment, RPE generation techniques have recently been investigated as a potential therapeutic approach to RPE degeneration. The microRNA-based technique is a new strategy for producing RPE cells from adult stem cell sources. Previously, we identified that antisense microRNA-410 (anti-miR-410) induces RPE differentiation from amniotic epithelial stem cells. In this study, we investigated RPE differentiation from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via anti-miR-410 treatment. We identified miR-410 as a RPE-relevant microRNA in UCB-MSCs from among 21 putative human RPE-depleted microRNAs. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including MITF, LRAT, RPE65, Bestrophin, and EMMPRIN. The RPE-induced cells were able to phagocytize microbeads. Results of our microRNA-based strategy demonstrated proof-of-principle for RPE differentiation in UCB-MSCs by using anti-miR-410 treatment without the use of additional factors or exogenous transduction.

MicroRNA-184 promotes differentiation of the retinal pigment epithelium by targeting the AKT2/mTOR signaling pathway.

Dedifferentiation of retinal pigment epithelium (RPE) cells is a crucial contributing factor to the pathology of retinal degenerative diseases, including age-related macular degeneration (AMD). Herein, we aim to reveal the roles of microRNAs (miRNAs) in RPE dedifferentiation and seek for potential therapeutic targets. Based on the microarray data, miR-184 was sorted out as the most up-regulated signature along with the differentiation from human induced pluripotent stem cells (hiPSC) to RPE cells, suggesting its potential promotive role in RPE differentiation. In vitro study indicated that miR-184 insufficiency suppressed RPE differentiation, typified by reduction of RPE markers, and promoted cell proliferation and migration. The role of miR-184 in maintaining regular RPE function was further proved in zebrafish studies. We also noticed that miR-184 expression was reduced in the macular RPE-choroid from a donor with RPE dysfunction compared to a healthy control. We next demonstrated that RAC-beta serine/threonine-protein kinase (AKT2) was a direct target for miR-184. MiR-184 promoted RPE differentiation via suppression of AKT2/mammalian target of rapamycin (mTOR) signaling pathway. We also found that AKT2 was up-regulated in macular RPE-choroid of the donor with RPE dysfunction and dry AMD patients. Taken together, our findings suggest that miR-184 insufficiency is involved in the pathogenesis of dry AMD. MiR-184 promotes RPE differentiation via inhibiting the AKT2/mTOR signaling pathway. MiR-184 based supplementary therapeutics and mTOR blocker, like rapamycin, are prospective options for AMD treatment.

Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool.

PurposeTo establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC).MethodsRetinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated.ResultsWe obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation.ConclusionWe successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.

Epithelial-mesenchymal transition of the retinal pigment epithelium causes choriocapillaris atrophy.

Epithelial-to-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is commonly observed at sites of choroidal neovascularization in patients suffering from age-related macular degeneration. To learn in an experimental model how RPE EMT affects the biology of the choroidal vasculature, we studied transgenic mice (βB1-TGF-β1) with ocular overexpression of transforming growth factor-β1 (TGF-β1). RPE EMT was detectable at postnatal day (P)1 and included marked structural and functional alterations such as loss of the outer blood-retina barrier and reduced mRNA expression of the RPE-characteristic molecules Rlbp1, Rpe65, Rbp1 and Vegfa. Moreover, vascular endothelial growth factor (VEGF) was not detectable by immunohistochemistry at the RPE/choroid interface, while RPE cells stained intensely for α-smooth muscle actin. The choriocapillaris, the characteristic choroidal capillary network adjacent to the RPE, developed normally and was not obviously changed in embryonic transgenic eyes but was absent at P1 indicating its atrophy. At around the same time, photoreceptors stopped to differentiate and photoreceptor apoptosis was abundant in the second week of life. Structural changes were also seen in the retinal vasculature of transgenic animals, which did not form intraretinal vessels, and the hyaloid vasculature, which did not regress. In addition, the amounts of retinal HIF-1α and its mRNA were markedly reduced. We conclude that high amounts of active TGF-β1 in the mouse eye cause transdifferentiation of the RPE to a mesenchymal phenotype. The loss of epithelial differentiation leads to the diminished synthesis of RPE-characteristic molecules including that of VEGF. Lack of RPE-derived VEGF causes atrophy of the choriocapillaris, a scenario that disrupts photoreceptor differentiation and finally results in photoreceptor apoptosis. Lack of retinal vessel formation and of hyaloid vessel regression might be caused by the decrease in the metabolic requirements of the neuroretina leading to low amounts of retinal HIF-1α. In summary, our data indicate that failure of RPE differentiation may well precede and cause atrophy of the choriocapillaris. In contrast, RPE EMT is not sufficient to cause choroidal neovascularization.

Progresses of cell-based therapy and tissue engineering in the treatment of age-related macular degeneration

Age-related macular degeneration (AMD) has become one of the leading causes of irreversible blindness worldwide. With the advancement of stem cell technology, tissue engineering and biomaterials, cell-based therapy has been inspiring for many degenerative diseases. For its unique advantages, AMD has become one of the most promising fields for cell-based therapy, which involve retinal pigment epithelium(RPE)cells, induced differentiation of neural retina cells and related cytokine regulations. RPE cells can be derived from human embryonic stem cells (hESC) or Induced pluripotent stem cells (iPS). Recently hESC-derived RPE cells have been applied to patients with dry AMD with initial success in clinical trials. In terms of tissue engineering, studies are focused on factors affecting the long-term survival of transplanted cells, including tissue scaffolds, soluble hybrid materials and scaffold anchoring. This article briefly reviews the RPE differentiation, neural retina differentiation and related cytokines of cell-based therapy and scaffolds, materials, and cell-scaffolds interactions of tissue engineering in AMD treatment. Key words: Macular degeneration/therapy; Tissue therapy; Tissue engineering; Review

Transcriptional Reactivation of OTX2, RX1 and SIX3 during Reprogramming Contributes to the Generation of RPE Cells from Human iPSCs.

Directed differentiation of human induced pluripotent stem cells (iPSCs) into retinal pigmented epithelium (RPE) holds great promise in cell replacement therapy for patients suffering from degenerative eye diseases, including age-related macular degeneration (AMD). In this study, we generated iPSCs from human dermal fibroblasts (HDFs) by electroporation with episomal plasmid vectors encoding OCT4, SOX2, KLF4, L-MYC together with p53 suppression. Intriguingly, cell reprogramming resulted in a metastable transcriptional activation and selective demethylation of neural and retinal specification-associated genes, such as OTX2, RX1 and SIX3. In contrast, RPE progenitor genes were transcriptionally silent in HDFs and descendant iPSCs. Overexpression of OCT4 and SOX2 directly stimulated the expression of OTX2, RX1 and SIX3 in HDFs and iPSCs. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified an OCT4- and two SOX2-binding sites located in the proximal promoter of OTX2. Histone acetylation and methylation on the local promoter also participated in the reactivation of OTX2. The transcriptional conversion of RX1 and SIX3 genes partially attributed to DNA demethylation. Subsequently, iPSCs were induced into the RPE cells displaying the characteristics of polygonal shapes and pigments, and expressing typical RPE cell markers. Taken together, our results establish readily efficient and safe protocols to produce iPSCs and iPSC-derived RPE cells, and underline that the reactivation of anterior neural transcription factor OTX2, eye field transcription factor RX1 and SIX3 in iPSCs is a feature of pluripotency acquisition and predetermines the potential of RPE differentiation.

Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model.

SummaryHuman embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

Yap and Taz regulate retinal pigment epithelial cell fate

The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Tead-responsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap-dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosage-dependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson's chorioretinal atrophy and congenital retinal coloboma.

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

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.

MicroRNAs are essential for differentiation of the retinal pigmented epithelium and maturation of adjacent photoreceptors

Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPE and are important for its development and maintenance, virtually nothing is known about the in vivo roles of non-coding transcripts. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and a few were implicated to play role in RPE based on studies in cell lines. Here, through RPE-specific conditional mutagenesis of Dicer1 or Dgcr8 in mice, the importance of miRNAs for RPE differentiation was uncovered. miRNAs were found to be dispensable for maintaining RPE fate and survival, and yet they are essential for the acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly, miRNAs of the RPE are required for maturation of adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The alterations in the miRNA and mRNA profiles in the Dicer1-deficient RPE point to a key role of miR-204 in regulation of the RPE differentiation program in vivo and uncover the importance of additional novel RPE miRNAs. This study reveals the combined regulatory activity of miRNAs that is required for RPE differentiation and for the development of the adjacent neuroretina.

Concise Review: Making Stem Cells Retinal: Methods for Deriving Retinal Pigment Epithelium and Implications for Patients With Ocular Disease.

Stem cells provide a potentially unlimited source of cells for treating a plethora of human diseases. Regenerative therapies for retinal degenerative diseases are at the forefront of translation to the clinic, with stem cell-derived retinal pigment epithelium (RPE)-based treatments for age-related macular degeneration (AMD) already showing promise in human patients. Despite our expanding knowledge of stem cell biology, methods for deriving cells, including RPE have remained inefficient. Thus, there has been a push in recent years to develop more directed approaches to deriving cells for therapy. In this concise review, we summarize recent efforts that have been successful in improving RPE derivation efficiency by directing differentiation from human pluripotent stem cells using developmental cues important for normal RPE specification and maturation in vivo. In addition, potential obstacles for clinical translation are discussed. Finally, we review how derivation of RPE from human induced pluripotent stem cells (hiPSCs) provides in vitro models for studying mechanisms of retinal disease and discovering new avenues for treatment.

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 multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Genetic interaction between Pax6 and β-catenin in the developing retinal pigment epithelium.

Wnt/β-catenin signaling plays an essential role in the retinal pigment epithelium (RPE) determination. Since activity of Pax6 (together with Pax2) is also required for the RPE determination, we investigated a possible genetic interaction between Pax6 and Wnt/β-catenin signaling pathway by analyzing Pax6, β-catenin, and Pax6/β-catenin conditional knockout mice. Although Pax6 inactivation alone had no impact on initial specification determined by the expression of Mitf and Otx2, melanin pigmentation was reduced in the RPE. This suggests that along with Mitf and Otx2, Pax6 is required for the full differentiation of RPE. Reporter gene assays in vitro suggest that hypopigmentation is at least in part due to the direct regulation of genes encoding enzymes involved in melanin synthesis by Pax6, Mitf, and β-catenin. The RPE of a β-catenin/Pax6 double mutant was differentiated into the neural retina; however, the tissue was thinner than that of the conditional β-catenin mutant due to reduced proliferation. Together, our data demonstrate that Pax6 is required for the RPE differentiation by regulating pigmentation and accountable for hyperproliferation in the transdifferentiated RPE. In this context, Pax6 appears to function as a pleiotropic regulator, directing development of ocular tissues in concert with the signaling pathway and, at the same time, regulating expression of structural component of the eye, such as shielding pigment.

Canonical/ -Catenin Wnt Pathway Activation Improves Retinal Pigmented Epithelium Derivation From Human Embryonic Stem Cells

The purpose of this study was to better understand the role canonical/β-catenin Wnt signaling plays in the differentiation of human embryonic stem cells (hESCs) into retinal pigmented epithelium (RPE), with the goal of improving methods for derivation. Fluorescent reporters were generated to monitor RPE differentiating from hESCs by using a previously described 14-day derivation protocol. Reporters were used to test the effects of the canonical/β-catenin Wnt pathway agonist CHIR99021 on differentiating RPE. Cells derived from differentiation studies were characterized by lineage-specific transcription factor expression, morphology, pigmentation, and function. The RPE derivation efficiency was determined from percentage positive PMEL17 expression. Fluorescent reporters mimicked expression of endogenous genes during 14-day differentiation to RPE. Analysis of Wnt pathway gene expression showed that the pathway components are expressed in differentiating RPE cells. Addition of CHIR99021 improved RPE derivation based on morphology, expression of RPE-specific lineage markers, and genes involved in melanogenesis. Additionally, expression of the neural retina marker CHX10 was suppressed during differentiation with CHIR99021. Addition of soluble WNT3A, but not WNT5A, had the same result. The CHIR99021-modified protocol yielded cell populations that were 97.77% ± 0.1% positive for the RPE marker PMEL17 at day 14. After cells were expanded to passage 3, they were shown to express RPE markers, carry out phagocytosis of rod outer segments, and secrete pigment epithelium-derived factor apically and vascular endothelial growth factor basally. Our findings demonstrated the importance of canonical/β-catenin Wnt signaling in RPE differentiation and showed that manipulating the pathway significantly improves RPE derivation from hESC.

MicroRNAs are essential for differentiation of the retinal pigmented epithelium and maturation of adjacent photoreceptors.

Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPE and are important for its development and maintenance, virtually nothing is known about the in vivo roles of non-coding transcripts. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and a few were implicated to play role in RPE based on studies in cell lines. Here, through RPE-specific conditional mutagenesis of Dicer1 or Dgcr8 in mice, the importance of miRNAs for RPE differentiation was uncovered. miRNAs were found to be dispensable for maintaining RPE fate and survival, and yet they are essential for the acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly, miRNAs of the RPE are required for maturation of adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The alterations in the miRNA and mRNA profiles in the Dicer1-deficient RPE point to a key role of miR-204 in regulation of the RPE differentiation program in vivo and uncover the importance of additional novel RPE miRNAs. This study reveals the combined regulatory activity of miRNAs that is required for RPE differentiation and for the development of the adjacent neuroretina.

Yap and Taz regulate retinal pigment epithelial cell fate.

The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Tead-responsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap-dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosage-dependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson's chorioretinal atrophy and congenital retinal coloboma.