Julien Maruotti

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.

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.

Nuclear Transfer‐Derived Epiblast Stem Cells Are Transcriptionally and Epigenetically Distinguishable from Their Fertilized‐Derived Counterparts

Mouse embryonic pluripotent stem cells can be obtained from the inner cell mass at the blastocyst stage (embryonic stem cells, ESCs) or from the late epiblast of postimplantation embryos (epiblast stem cells, EpiSCs). During normal development, the transition between these two stages is marked by major epigenetic and transcriptional changes including DNA de novo methylation. These modifications represent an epigenetic mark conserved in ESCs and EpiSCs. Pluripotent ESCs derived from blastocysts generated by nuclear transfer (NT) have been shown to be correctly reprogrammed. However, NT embryos frequently undergo abnormal development. In the present study, we have examined whether pluripotent cells could be derived from the epiblast of postimplantation NT embryos and whether the reprogramming process would affect the epigenetic changes occurring at this stage, which could explain abnormal development of NT embryos. We showed that EpiSCs could be derived with the same efficiency from NT embryos and from their fertilized counterparts. However, gene expression profile analyses showed divergence between fertilized‐ and nuclear transfer‐EpiSCs with a surprising bias in the distribution of the differentially expressed genes, 30% of them being localized on chromosome 11. A majority of these genes were downregulated in NT‐EpiSCs and imprinted genes represented a significant fraction of them. Notably, analysis of the epigenetic status of a downregulated imprinted gene in NT‐EpiSCs revealed complete methylation of the two alleles. Therefore, EpiSCs derived from NT embryos appear to be incorrectly reprogrammed, indicating that abnormal epigenetic marks are imposed on cells in NT embryos during the transition from early to late epiblast. STEM CELLS 2010;28:743–75228:743–752

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.

Modeling Retinal Dystrophies Using Patient-Derived Induced Pluripotent Stem Cells

Retinal degenerative disease involving photoreceptor (PR) cell loss results in permanent vision loss and often blindness. Generation of induced pluripotent stem cell (iPSC)-derived retinal cells and tissues from individuals with retinal dystrophies is a relatively new and promising method for studying retinal degeneration mechanisms in vitro. Recent advancements in strategies to differentiate human iPSCs (hiPSCs) into 3D retinal eyecups with a strong resemblance to the mature retina raise the possibility that this system could offer a reliable model for translational drug studies. However, despite the potential benefits, there are challenges that remain to be overcome before stem-cell-derived retinal eyecups can be routinely used to model human retinal diseases. This chapter will discuss both the potential of these 3D eyecup approaches and the nature of some of the challenges that remain.

Axon Guidance Signaling Modulates Epithelial to Mesenchymal Transition in Stem Cell-Derived Retinal Pigment Epithelium

The critical role of epithelial to mesenchymal transition (EMT) in embryonic development, malignant transformation, and tumor progression has been well studied in normal and cancerous tissues and cells. Interestingly, EMT has also been reported to play a key role in the early progression of several retinal degenerative diseases, including scarring associated proliferative vitro-retinopathy (PVR), choroidal neo-vascularization induced “wet” age-related macular degeneration (AMD) and diabetic retinopathy (DR). Despite these studies, many questions remain unexplored regarding EMT-associated retinal pigment epithelium (RPE) degeneration and dysfunction. We hypothesize that RPE cells undergo EMT prior to cell death during the progression of atrophic “dry” AMD. Utilizing human stem cell-derived RPE (hRPE) as a model to study RPE EMT, we optimized two independent but complementary RPE EMT induction systems: 1) enzymatic dissociation of hRPE monolayer cultures and 2) co-treatment of hRPE monolayer cultures with transforming growth factor beta (TGF-β) and the inflammatory cytokine, tumor necrosis factor alpha (TNF-α). To further understand the molecular mechanisms of RPE EMT regulation, we performed an RNA-Sequencing (RNA-Seq) time course examination across 48 hours beginning with EMT induction. Our transcriptome profiling provides a comprehensive quantification of dynamic signaling events and associated biological pathways underlying RPE EMT and reveals an intriguing significance for widespread dysregulation of multiple axon guidance molecules in this process.

Collagen vitrigels with low-fibril density enhance human embryonic stem cell-derived retinal pigment epithelial cell maturation

Structural and biochemical cues of extracellular matrix can substantially influence the differentiation and maturation of cultured retinal pigment epithelial (RPE) cells. In this study, thin collagen vitrigels were engineered to create collagen nanofibrillar structures of different fibril densities in an effort to evaluate the maturation of human embryonic stem cell–derived retinal pigment epithelial (hESC‐RPE) cells. The ultrastructure of the different collagen vitrigels was characterized by transmission electron microscopy, and the mechanical properties were evaluated by tensile testing. The pigmentation and polarization of cells, in addition to key RPE marker gene and protein expression levels, were analyzed to determine the differentiation of hESCs on the gels. The hESC‐RPE differentiation was most significant in collagen vitrigels with low fibril density with mature collagen fibrils with diameter of around 60 nm and Youngs modulus of 2.41 ± 0.13 MPa. This study provides insight into the influence of collagen nanofibrillar structures on hESC‐RPE maturation and presents a potential bioengineered substratum for hESC‐RPE for future preclinical and clinical applications.

Reprogramming and Pluripotency of Epiblast Stem Cells

Pluripotent stem cells established in vitro from mammalian embryos are essential tools to understand pluripotency. From the mouse embryo, two kinds of stem cells can be derived, both being pluripotent: embryonic stem cells (ESCs) are derived from the pluripotent cells in the inner cell mass of the blastocyst, whereas epiblast stem cells (EpiSCs) are derived from the post-implantation late epiblast, at the time of gastrulation. These two types of stem cells share some common properties but each of them also display specific features that clearly define two states of pluripotency, now referred as naive for ESCs and primed for EpiSCs. Although being derived from the inner cell mass at the blastocyst stage, human ESCs are in fact distinct from mouse ESCs and closer to the primed state. In this chapter we describe how EpiSCs are obtained in the mouse and what their molecular and functional characteristics are in comparison with mouse ESCs. We present the two states of pluripotency and their in vivo equivalence. In other species where pluripotent stem cells derived from the embryo are available, we show that these cells are indeed in the primed state. Then, we present a state of art of experiments exploring pathways allowing conversion of EpiSCs into ESCs or vice-versa and means to stabilize cells in one or the other state. From the current knowledge, it seems that the primed state not only represents a more advanced epiblast developmental stage, but may also be a more easily stabilized state, compared to the naive one. At last we expose reprogramming experiments in the mouse, where somatic cells are reprogrammed into the primed state, through induction using exogenous factors or by nuclear transfer into enucleated oocytes.