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Articles  |   April 2016
Mimicking Retinal Development and Disease With Human Pluripotent Stem Cells
Author Affiliations & Notes
  • Divya Sinha
    Waisman Center University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Jenny Phillips
    Waisman Center University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • M. Joseph Phillips
    Waisman Center University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • David M. Gamm
    Waisman Center University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Correspondence: David M. Gamm, T609 Waisman Center, 1500 Highland Avenue, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA;
Investigative Ophthalmology & Visual Science April 2016, Vol.57, ORSFf1-ORSFf9. doi:10.1167/iovs.15-18160
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      Divya Sinha, Jenny Phillips, M. Joseph Phillips, David M. Gamm; Mimicking Retinal Development and Disease With Human Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(5):ORSFf1-ORSFf9. doi: 10.1167/iovs.15-18160.

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      © ARVO (1962-2015); The Authors (2016-present)

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As applications of human pluripotent stem cells (hPSCs) continue to be refined and pursued, it is important to keep in mind that the strengths and weaknesses of this technology lie with its developmental origins. The remarkable capacity of differentiating hPSCs to recapitulate cell and tissue genesis has provided a model system to study stages of human development that were not previously amenable to investigation and experimentation. Furthermore, demonstration of developmentally appropriate, stepwise differentiation of hPSCs to specific cell types offers support for their authenticity and their suitability for use in disease modeling and cell replacement therapies. However, limitations to farming cells and tissues in an artificial culture environment, as well as the length of time required for most cells to mature, are some of the many issues to consider before using hPSCs to study or treat a particular disease. Given the overarching need to understand and modulate the dynamics of lineage-specific differentiation in stem cell cultures, this review will first examine the capacity of hPSCs to serve as models of retinal development. Thereafter, we will discuss efforts to model retinal disorders with hPSCs and present challenges that face investigators who aspire to use such systems to study disease pathophysiology and/or screen for therapeutics. We also refer readers to recent publications that provide additional insight and details on these rapidly evolving topics.

The process by which tissues and whole organisms develop from a single fertilized cell, or zygote, has intrigued biologists for centuries. In mammals, rapid cell division transforms the zygote to a blastocyst, which harbors a collection of pluripotent cells that together comprise the “inner cell mass.” This fleeting population of pluripotent stem cells is capable of producing every cell and tissue, each with its requisite specializations in composition, form, and function. 
Understanding cell differentiation and maturation not only is a core pursuit of developmental biologists, but also can provide critical insights into disease, thereby aiding the design of therapeutic strategies. Compared to many other species, the study of development and disease on a molecular level in humans is particularly challenging. Much of our knowledge is extrapolated from animal models, or from harvested prenatal or adult postmortem human organs since extraction of live cells often is too invasive or potentially harmful to the patient. Even when appropriate human cell samples can be obtained, quantities may be too limited for thorough investigation. Perhaps in no other human tissues are these limitations felt more strongly than in those belonging to the central nervous system, which includes the retina. However, the advancement of human pluripotent stem cell (hPSC) technology has circumvented several of these issues and provided a novel avenue to study retinogenesis and create in vitro models of retinal diseases. 
Two types of hPSCs currently exist: human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Human embryonic stem cells are derived directly from the inner cell mass and, when maintained under specific conditions, can be maintained in a pluripotent state indefinitely.1 Thus, the culture and differentiation of hESCs provide a means to study each step in the production of human cells and tissues, although the extent to which any in vitro system can truly mimic development is the subject of intense investigation. The subsequent discovery that differentiated human somatic cells can be reprogrammed to fully pluripotent hiPSCs2,3 brought another wave of potential applications for hPSCs, including the ability to probe disease mechanisms and develop personalized treatment strategies using patient-specific cell lines. 
In this review, we will examine the progress and future prospects of hPSC research as it relates to modeling retinal development and disease. We also will touch upon therapeutic aspects of hPSC technology, and emphasize how such applications are tied first and foremost to our understanding of the dynamics of lineage-specific differentiation and the cellular diversity inherent to these complex culture systems. The reader is also encouraged to consult other recent reviews that provide further information and opinions on this subject.49 
Modeling Retinal Development
The vast majority of our knowledge of human retinal development has come from limited and/or static analyses of human fetal retina,1022 or inferences from animal models ranging from flies to primates.2331 While animal studies have revealed numerous conserved developmental mechanisms across species, determining the extent to which they are used in human retinogenesis has remained elusive. With the availability of hPSCs,13 researchers now have an inexhaustible supply of source material to generate all major classes of human retinal cell types for research or clinical applications3252 and, equally as important, to study the steps and cues involved in retinal cell differentiation and maturation.34,4042,45 In actuality, these two pursuits are largely inseparable. Attention to the latter is necessary to appreciate the many “moving parts” within a differentiating hPSC culture system and the potential to introduce culture artifact, which could affect the characteristics and functionality of the end product. Indeed, given that no current model system (including hPSCs) can recapitulate every nuance of human retinogenesis, how confident can we be in the data obtained from hPSC models, or for that matter, in the authenticity of the cells they generate? A number of recent studies have sought to address this question, with the preponderance of evidence to date suggesting a remarkable capacity of hPSCs to yield bona fide retinal cell progeny at various stages of maturation. 
To date, researchers have shown that cell birth in hPSC-derived retinal cultures approximates the sequence and time frame of human retinogenesis.32,34,4042,45,46,52 Protocols exist to differentiate hPSCs sequentially from a pluripotent stem cell state to neuroectoderm, eye field, and optic vesicle stages.32,34,4043,45,46,52 From there, RPE and neural retinal progenitor cells (NRPCs) are generated, with hPSC-RPE often differentiating alongside hPSC-NRPCs.34,42 Thereafter, all major classes of neural retina (NR) cells are produced in a conserved birth order, with retinal ganglion cells (RGCs) born first, followed by horizontal cells (HCs), amacrine cells (ACs), and cones in an early cohort of cell types, followed by a late cohort that includes rods, bipolar cells (BCs), and Müller glia (MG).13,26,27,37,40,41,46 
Protocols for the differentiation of NR cell types can be divided into 2D36,37,44,50,51,53 and 3D methods.3843,46,47,52 Remarkably, 3D retinal cultures derived from hPSCs have shown the capacity to self-organize into laminated tissue reminiscent of the maturing retina in vivo.42,46,52 Photoreceptors derived from these 3D hPSC-retinae form synapses, possess characteristic electrophysiological signatures, elaborate primitive inner and outer segments, and can respond to light.40,42,46,52 Given these findings, which far exceed those from other renewable tissue culture systems, hPSCs currently represent the most promising cell or tissue source for modeling human retinogenesis. Furthermore, owing to their ability to mimic normal developmental processes, hPSCs also hold great potential to produce authentic human retinal cell types for use in retinal disease research or clinical applications. 
Despite the aforementioned accolades, hPSC technology for human retinal developmental studies has limitations that should be taken into account. Many traditional markers used to identify individual NR cell types can be found elsewhere in the central nervous system or in nonneural tissues. For example, eye field transcription factors, such as RAX, PAX6, LHX2, OTX2, SIX3, and SIX6, also are expressed in the developing human forebrain at various stages of development,5463 and markers for NR cell types, such as ATOH7 and POU4F2 (RGCs), CALB1 (HCs), PKCα (BPs), VIMENTIN, and GLUTAMINE SYNTHASE (MGs), and SLC6A9, GAD1, and GAD2 (ACs), are only specific when used to distinguish cells within the retina.32,6472 On the other hand, photoreceptors are a highly specialized cell type in the body and, therefore, have more specific markers available, although many photoreceptor genes also are expressed in pinealocytes.60,7375 Thus, expression of one or even multiple markers may not assure the identity of a NR cell type in a mixed lineage culture of differentiating hPSCs. However, a reasonable level of confidence is achievable if the target cell's ancestry can be traced back to an isolated population of multipotent retinal progenitors. Three-dimensional hPSC differentiation protocols are particularly well-suited for this task, as optic vesicle-like structures (OVs) can be readily separated from nonretinal tissues, including forebrain.40,43,45,46,52 Cells within hPSC-OVs are highly proliferative and express the homeodomain transcription factor Visual Systems Homeobox 2 (VSX2), an early and highly selective marker of NRPCs. Together, knowledge of the course of retinal differentiation combined with enrichment of hPSC-OVs provide a critical quality control step for in vitro retinal modeling studies. 
The extracellular environment also has a key role in cell and tissue development, and, therefore, represents another potential limitation of hPSC-based studies of retinogenesis. In vivo, the developing optic vesicle receives diffusible and perhaps mechanical signals from surrounding tissues, such as the surface ectoderm and periocular mesenchyme, and later from the developing lens.7680 These extraretinal influences are thought to be involved in retinal patterning, morphogenesis, and cellular differentiation. Despite lacking most of these cues, isolated 3D hPSC-OVs grown in minimal medium recapitulate many, but certainly not all, of the hallmarks of normal retinogenesis, suggesting that retinal differentiation can be driven largely through cell- or tissue-autonomous means. However, it is also possible for unwanted and/or undefined cell types to be present in 2D or 3D hPSC cultures, which could secrete factors or otherwise exert influences foreign to normal developing retinal tissue. As such, in depth characterization of hPSC-derived retinal culture systems is an essential aspect of all modeling studies. 
The relatively austere culture environment of differentiating hPSCs and hPSC-OVs also provides a convenient means of testing the function(s) of key developmental signaling molecules at defined developmental time points. An example comes from a recent study examining the role of BMP4 during early retinal differentiation in hESCs. Under conditions shown to generate FOXG1+ telencephalic neuroepithelium, BMP4 supplementation at early stages of differentiation redirected these cells to VSX2+ NRPCs, which in turn gave rise to NR progeny. However, treating these NRPC cultures with the WNT agonist CHIR99021 and the FGF receptor inhibitor SU5402 resulted in a relative conversion from NR to RPE.35 A similar study in chicks also showed that early exposure to BMP4 caused an overproduction of NR at the expense of telencephalic neuroepithelium,81 while conditional deletion of BMP4 signaling in mouse led to RPE production at the expense of NR.82 These studies highlight how hPSC cultures can be used to discover or confirm the roles and function of key extracellular signaling molecules in human retinal development. 
In addition to serving as in vitro models of normal retinal development, patient-specific or gene-edited hPSCs also can offer insights into mechanisms and consequences of developmental disorders. Human induced pluripotent stem cells made from patients with known mutations in critical retinal transcription factors are prime targets for these investigations. One recent study used hiPSCs created from a patient with severe microphthalmia (small, malformed eyes) caused by a rare, homozygous R200Q mutation in the homeodomain region of VSX2.45 Previous studies in the mouse showed that this mutation results in a functional null phenotype, although Vsx2 protein is expressed normally.83 Before the onset of VSX2 expression in differentiating hiPSC-NRPCs, (R200Q)VSX2 mutant neuroectodermal cultures developed identically to control cultures generated from an unaffected sibling. However, upon initiation of VSX2 expression, multiple phenotypic differences between (R200Q)VSX2 and control hiPSCs became evident. Unlike the latter, (R200Q)VSX2 hiPSC-OVs demonstrated a preference toward RPE production at the expense of NRPCs, reduced cell proliferation, decreased hiPSC-OV growth over time, absence of bipolar cells, and delayed photoreceptor maturation. Furthermore, these effects could be ameliorated partially via ectopic expression of wild type VSX2. These findings mimicked those obtained from the ocular retardation mouse (Vsx2 null)84,85 and/or the (R200Q)Vsx2 mouse mutant models,83 providing further evidence for the bona fide nature of hiPSC-derived retinal progeny, as well as for the conserved function of VSX2 in human retinogenesis. 
Human embryonic stem cells also can be used to study the roles of key developmental regulators through gene manipulation or ectopic expression strategies. Both shRNA knock-down and bacterial artificial chromosome (BAC)-mediated homologous recombination knockout approaches were used to examine the function of microphthalmia-associated transcription factor (MITF) in early human retinal differentiation.34 The resulting perturbation of MITF expression in hESC-retinal cultures caused reductions in eye field gene expression and early OV cell proliferation, along with disrupted RPE maturation and pigmentation. Taken together, evidence from these and other studies investigating normal and abnormal retinal development provide reason to be optimistic regarding the authenticity of hPSC-derived retinal progeny. However, further effort is needed to determine whether and to what extent the in vitro retinal differentiation program diverges from that followed in normal embryogenesis. That information then can be used to refine expectations for current and future applications of hPSC technology, such as cell replacement therapy and retinal disease modeling. 
Modeling Human Retinal Diseases
It is implicitly understood that diseases—with all of their direct and indirect manifestations within an organism—cannot be wholly modeled in a dish. However, one can endeavor to recapitulate cell-based (or perhaps tissue-specific) pathophysiological mechanisms that contribute to one or more of the outward features of a particular disease. Moreover, efforts to examine human cellular functions for the purpose of understanding retinal disease and/or testing drugs or toxins are by no means the sole purview of hPSCs. Immortalized cell lines and primary cultures from cadaver tissue have been used for decades as sources of human retinal cells to study diseases. Alternatively, heterologous expression systems can be used to test the function of normal and mutant retinal proteins in nonretinal cell types.86,87 However, while each of the aforementioned in vitro systems is informative, all have disadvantages. Immortalization produces cells with cancer-like properties, while primary adult NR cultures, when available, cannot be expanded unless they undergo spontaneous immortalization. A notable exception is adult human RPE, which possesses a stem cell population that can be cultured and propagated post mortem,88 although obtaining donor eyes with specific inherited retinal diseases (IRDs) poses challenges. Prenatal NR and RPE are known to undergo limited expansion in vitro,8992 but face the same hurdles as adult cultures with regard to disease modeling and also evoke ethical concerns. Lastly, heterologous expression systems do not mimic endogenous gene expression levels or ratios, nor do they necessarily provide the proper cellular environment to assess protein function. Human pluripotent stem cell technology circumvents or minimizes many of these limitations by providing customizable or patient-specific lines that can be perpetuated indefinitely and produce every major retinal cell class with high fidelity. Producing a desired cell type or tissue, however, is only one of the important steps needed to model disease mechanisms in a dish. Numerous other considerations also must be taken into account when evaluating the suitability of hPSCs to study a particular retinal disorder, as outlined below. 
While hESCs and hiPSCs can potentially be used for disease modeling studies, patient-specific hiPSCs are intrinsically well-suited for this application. Human induced pluripotent stem cell lines from patients with IRDs not only possess the precise gene mutation(s) of interest, but also harbor known and unknown genetic modifications that might contribute to that particular individual's disease course and phenotype. Given the tremendous genetic diversity of IRDs and the inter- and intrafamilial phenotypic heterogeneity they commonly exhibit, having an hiPSC option is becoming increasingly important. Indeed, the slow pace, unpredictable success, and/or questionable relevance of traditional approaches to modeling human IRDs has, over the last three decades, created a wide gap between our knowledge of the genetics of these disorders compared to their biological underpinnings. Human induced pluripotent stem cells, while neither inexpensive nor rapid to produce and validate, do provide a reasonably efficient means to generate relevant, IRD gene-expressing cell types for study and testing. However, when consent for hiPSC derivation, access to affected patients, or funds to derive new lines is not forthcoming, normal hiPSCs and/or hESCs can be modified through various gene editing strategies9399 to produce hPSC lines with desired gene mutations. This approach benefits from built-in isogenic controls, but does not offer direct clinical correlation or account for genetic modifications associated with the disease. Thus, we contend that the generation of patient-specific hiPSCs, along with unaffected sibling and/or gene-corrected control lines, currently is the optimal approach for IRD modeling. 
Even under ideal conditions there is no guarantee that hPSC-based IRD models will yield meaningful and reliable data. However, the chances of a successful outcome are greater when the limitations of these model systems are factored into the study design and interpretation. Potential issues include batch-to-batch variability of media components, the acquisition of genomic abnormalities after extensive passaging,48 spurious effects of off-target cell types within mixed cultures, and/or the absence of extraretinal tissue influences that might normally impact the course of retinal disease in vivo. As mentioned earlier in this review, it also is important to keep in mind that differentiating hPSC cultures follow a human developmental timeline. Thus, the length of time it takes for human retinal cell types to adopt mature characteristics in vitro is on the order of months, compared to the days or weeks it takes for retinal progeny from mouse PSCs to develop. In addition to these limitations, it is worth noting that all diseases are not equally amenable to hPSC-based modeling. The following are important questions to pose when choosing a disease to model (see Fig.): (1) Can adequate amounts of highly enriched, disease-relevant retinal cell type(s) be produced? (2) Do the cell types in question attain a sufficient level of structural and functional maturity to demonstrate a reproducible and measurable phenotype? (3) Are assays available to monitor cultures reliably for anticipated and perhaps unanticipated outcomes? (4) To what extent can reliable controls be built into the system? These questions provide a context in which to evaluate the increasing number of hPSC-based retinal disease models being published, examples of which are discussed in further detail below. 
Assessing the potential to model diseases with hiPSCs. (A) Venn diagram showing four major categories to consider when planning an hiPSC modeling study. Few diseases are optimal in all categories, although that does not preclude more complex diseases from being candidates for modeling. (B) Features within each category from (A) that contribute to the challenges of modeling a particular disease with hiPSCs.
Assessing the potential to model diseases with hiPSCs. (A) Venn diagram showing four major categories to consider when planning an hiPSC modeling study. Few diseases are optimal in all categories, although that does not preclude more complex diseases from being candidates for modeling. (B) Features within each category from (A) that contribute to the challenges of modeling a particular disease with hiPSCs.
Modeling Monogenetic Diseases Using hiPSC-RPE
Of all degenerative diseases, those targeting the RPE may offer the most straightforward hiPSC modeling opportunities. To begin with, RPE often is generated readily by hiPSC cultures under either directed100,101 or spontaneous differentiation conditions.102104 Furthermore, RPE grows as a monolayer structure that can be identified in culture by its distinctive pigmentation and cellular morphology,105 which greatly facilitates its subsequent isolation and passaging48 on various substrates.106 Given their substantial ability to proliferate, hiPSC-RPE are capable of providing large quantities of essentially pure cells for experimentation. Perhaps most important, hiPSC-RPE mature in culture to the point where they can perform many native functions at measurable levels, such as tight junction formation, photoreceptor outer segment (POS) phagocytosis and degradation, ion and fluid transport, and polarized growth factor secretion, among others.49,102 
Given the advantages of modeling RPE-based IRDs, it is not surprising that diseases falling within this category were among the first to be modeled with hiPSCs. One of the earliest of these studies generated hiPSCs from a patient with gyrate atrophy40,107 (GA), an IRD with a purely ocular phenotype that results in progressive loss of vision and eventual blindness due to primary RPE damage and secondary photoreceptor death. Gyrate atrophy is caused by mutations in the OAT gene, which encodes the vitamin B6–dependent mitochondrial matrix enzyme ornithine-δ–aminotransferase. There is no available treatment for the majority of GA patients, although high dose vitamin B6 supplementation can slow disease progression in some affected individuals. Since RPE cannot be biopsied in living patients, testing for vitamin B6 responsiveness is done traditionally using blood or fibroblast samples, and treatment is discontinued if a negative response is recorded due to possible long-term side effects of the supplement. In the aforementioned report, GA hiPSC-RPE showed significantly decreased OAT activity compared to normal and gene-corrected control cultures.107 However, GA hiPSC-RPE OAT activity increased dramatically upon exposure to therapeutic vitamin B6 levels, even though this patient had been deemed previously unresponsive based on surrogate in vitro tests. 
Human induced pluripotent stem cell–RPE models also can be used to probe disease pathophysiology and test novel treatments. Best disease (BD), an autosomal dominant macular degenerative disease caused by mutations in the RPE gene BEST1, is one such disorder where the mechanism leading to RPE dysfunction and photoreceptor death remains unclear. Human induced pluripotent stem cell–RPE generated from patients with BD and their unaffected siblings were subjected to a series of assays performed under baseline culture conditions and following exposure to physiological stimuli, which revealed reduced fluid flux and increased accumulation of autofluorescent material after long-term POS feeding in the mutant cultures.49 Additional testing showed delayed degradation of POS proteins, altered calcium transients, and elevated oxidative stress levels in BD versus control hiPSC-RPE. Given that calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, the authors surmised that these disruptions in cellular function could contribute to the clinical picture of BD.48 More recently, Lukovic et al.108 produced a patient-specific hiPSC model of retinitis pigmentosa (RP) caused by a mutation in the RPE-specific gene MERTK. The hiPSC-RPE cultures harboring the MERTK mutation demonstrated defective POS phagocytosis, which is consistent with the retinal pathophysiology observed in animal models109 and human patients110 with MERTK deficiency. The existence of hiPSC-RPE models of BD and MERTK-associated RP sets the stage for focused screening of drugs that target pathways involved in the cellular phenotype of these diseases. Indeed, Singh et al.111 recently showed that two compounds, valproic acid and rapamycin, may mitigate the delay in POS degradation observed in BD hiPSC-RPE cultures. 
In addition to screening for pharmacologic compounds, hiPSC-RPE may prove to be a convenient and reproducible platform on which to test gene therapy strategies. Li et al.112 developed an hiPSC-RPE model of autosomal recessive RP resulting from mutations in the gene encoding membrane frizzled-related protein (MFRP). The authors first confirmed that hiPSC-RPE expressing mutant MFRP or altered levels of its dicistronic partner CTRP5 exhibited actin disorganization and abnormal apical microvilli. Thereafter, they showed that gene therapy using an AAV vector expressing human MFRP could restore hiPSC-RPE actin organization and cell morphology. These promising results subsequently were confirmed in a relevant mouse model. Thus, in this case hiPSC-RPE served an adjunctive role in efficacy testing by providing a supply of human cells affected by the disease. 
Ideally, preclinical efficacy data from disease-specific hiPSCs and relevant animal models would be available for analysis, but in some circumstances hiPSCs may be the only viable option for therapeutic testing. The autosomal recessive IRD choroideremia, a progressive blinding disease caused by mutations in the gene encoding Rab escort protein 1 (REP1), is an example of such a scenario. Cereso et al.113 used an AAV2/5 vector expressing REP1 to overcome the biochemical phenotype of hiPSC-RPE cultures derived from a patient with choroideremia. However, unlike MRFP-deficient RP, no animal models are available for this disease. As a result, emphasis was placed on hiPSC-generated efficacy data when researchers sought (and ultimately received) approval for an AAV-mediated gene therapy trial for choroideremia.114 
In addition to testing AAV-mediated gene therapy, hiPSC-RPE model systems can be used to develop and test methods to bypass gene defects, as demonstrated by Schwarz et al.115 using translational read-through drugs and hiPSCs derived from a patient with a premature stop mutation in the RP2 gene. Together, these reports illustrate how knowledge gained from custom, disease-specific hiPSC-RPE models can shed light on disease mechanisms, alter patient management, and/or lead to new therapeutic alternatives for patients with blinding disorders, particularly in situations where no suitable animal model exists. 
Modeling Photoreceptor-Based IRDs
Inherited retinal diseases that primarily affect photoreceptors also have been the target of a number of hiPSC modeling efforts despite the additional challenges that such studies face relative to hiPSC-RPE models. Whereas hiPSC-RPE is comparatively straightforward to identify, enrich, and expand, no equivalent method to purify human photoreceptors and culture them in isolation currently exists. In addition, producing functional photoreceptors with mature features is expensive, time-consuming, and rather inefficient at present, taking upwards of 200 days to see rudimentary outer segments in a minority of cells.52 Also, multiple subtypes of photoreceptors (S, L, and M cones and rods) develop in culture over prolonged and nonsynchronous timelines. As a result, at any given time point the photoreceptor population in hiPSC-derived retinal cultures consists of a mixture of subtypes at varying stages of differentiation, all of which exist within an even more heterogeneous population of NR cell types, as well as RPE in many circumstances. In 2D cultures that do not include an OV enrichment step, substantial contamination from nonretinal lineages also is a concern. That being said, significant progress has been made in recent years producing and differentiating hiPSC-photoreceptors in 3D OV cultures to an extent where they display higher order structure and respond to physiological stimuli.40,52 In addition, for selected IRDs (e.g., those affecting gene splicing or expression, development, metabolism, and other basic functions), mature photoreceptor structure and function may not be necessary to at least partially probe disease mechanism. Thus, despite the aforementioned limitations, hiPSC models have the capacity to expand our knowledge of photoreceptor-based disorders and advance therapeutic development. 
The first published report describing hiPSC models of inherited photoreceptor degenerative diseases was provided by Jin et al.,116 who created hiPSCs from individual RP patients with mutations in the RP1, RP9, PRPH2, or RHODOPSIN genes. Comparison of cell survival in RP versus normal hiPSC 2D cultures showed higher rates of rod death in the RP lines. Three different pharmacologic interventions then were tested, one of which (α-tocopherol) yielded a positive effect on rod survival in a single RP line. A subsequent study using an hiPSC line containing a RHODOPSIN missense mutation examined disease pathophysiology in greater detail, identifying endoplasmic reticulum stress as a likely factor in the death of mutant hiPSC-rods.117 In addition to pharmacologic testing, hiPSC-based retinal disease models offer a convenient and informative platform to test gene therapy strategies. Recently, Burnight et al.118 generated patient-specific hiPSCs and photoreceptor precursor cells to evaluate lentivirus-mediated delivery of full length CEP290, an IRD-associated gene involved in POS production and maintenance. Results from this study revealed a dose-dependent toxicity associated with ectopic CEP290 gene expression, which will be used to refine the gene delivery vector and improve patient safety. 
Lastly, hiPSC technology has been used to help identify previously unrecognized genetic mutations leading to photoreceptor degeneration. In an innovative study by Tucker et al.,119 exome sequencing of a patient with sporadic RP identified an Alu insertion at exon 9 of the male germ cell-associated kinase (MAK) locus. The pathogenic nature of this mutation then was confirmed using hiPSCs, which showed loss of a tissue-specific splice variant of MAK in retinal lineage cells. Another study from the same group identified a novel mutation in an intron of the USH2A that caused protein misfolding and increased endoplasmic reticulum stress.51 Of course, hiPSC-RPE models also are useful in the search for causative IRD gene mutations,120,121 and can be employed alongside hiPSC-derived NR cultures when the primary cell target of the disease is not known. 
The Future of hiPSC-Based Retinal Disease Modeling
In addition to RPE- and photoreceptor-based IRDs, disorders affecting RGCs, the output neurons of the NR, are now being targeted for hiPSC modeling studies. Such investigations are aided by the fact that RGCs are early born NR cells that are identifiable in live, plated cultures by virtue of their long neuronal processes, and have the potential to be purified via immunopanning.122,123 However, markers attributed to RGCs, including cell surface markers, also are found in nonretinal neurons within the central nervous system. Therefore, it is advisable that hiPSC models of RGC disorders incorporate an early OV enrichment step to minimize confounding data from other neuronal lineages.43 Furthermore, it is not clear which of the numerous subtypes of RGCs are produced by hiPSCs or whether recapitulating this diversity is important for modeling inner retinal diseases. 
As the production and analysis of hiPSC models of Mendelian, cell-autonomous IRDs continue to be refined, investigators are beginning to apply this technology to more complex retinal disorders. Many of these diseases, including age-related macular degeneration (AMD), are human-specific and do not translate well to other species, and, thus, pose an area of need for model system development. However, with increased complexity come additional questions and challenges as researchers attempt to recreate them in a dish (see Fig.). Taking AMD as an example, issues pertaining to genetic and environmental risk factors, cellular age, multicellular and multiorgan involvement, and regional differences in tissue anatomy must be considered, as discussed below. 
A host of risk factor gene variants are implicated in AMD that are associated with several cellular pathways spanning complement activation, lipid and retinoid metabolism, oxidative stress, iron metabolism, angiogenesis, and extracellular matrix and basement membrane integrity. Several nongenetic risk factors like obesity, nutrition, and smoking also have a role, as does perhaps the most difficult variable to control—time. Since AMD generally is not seen before the sixth decade of life, the impact of genetics and environment is likely subtle, requiring many years for deleterious effects to accumulate and derail cellular physiology. Furthermore, although AMD is considered largely a primary RPE disease, it only affects the macula and its pathogenesis involves multiple retinal layers, including the choriocapillaris and Bruch's membrane. While it is conceptually feasible to build ocular structures that incorporate these layers (or their facsimiles), we currently lack the fundamental knowledge and cellular signatures needed to produce a macula in vitro. 
While modeling AMD in totality remains a daunting proposition, valuable information about this disease can be gleaned potentially by asking focused questions using relevant cell-based systems. In this “divide and conquer” approach, individual molecules and pathways suspected of contributing to AMD pathophysiology are examined in relative isolation, with or without manipulation of culture genetics and environment to simulate the effects of aging and/or chronic cellular stress. Yang et al.124 fed N-retinylidene-N-retinylethanolamine (A2E), a toxic compound believed to have a role in AMD progression, to hiPSC-RPE derived from individuals with protective and high risk haplotypes of the ARMS2/HTRA1 alleles. A2E-treated hiPSC-RPE accumulated autofluorescent material akin to what is observed in aged patients with AMD. Interestingly, mass spectrometry protein profiling and activity assays showed defective mitochondrial superoxide dismutase 2 responses in hiPSC-RPE expressing the high risk haplotypes. Of note, a similar strategy was discussed earlier using BD hiPSC-RPE, which employed physiological stimuli to uncover subtle, disease-specific alterations in cell functions. 
Primary open-angle glaucoma (POAG) is another complex disease for which a good model system is lacking, yet the potential exists for hiPSCs to provide useful information regarding the pathologic roles of individual genes and pathways. Tucker et al.125 used hiPSCs derived from a patient with a rare inherited form of normal tension glaucoma caused by a TBK1 gene duplication to examine its effect on autophagy in RGC-like cells. While it is unclear whether similar differences are found in POAG, studies like this provide a platform to test hypotheses and build further complexity into the system. However, while adding more elements to a culture system may lead to a closer approximation of a disease process, it also can add confounding variables and complicate interpretations, even with appropriate experimental controls. 
As protocols and tools for producing specific cell types and 3D tissues continue to improve, almost any retinal disease may become the focus of an hPSC model. However, choosing an appropriate disease to model in a dish requires an appreciation of the inherent advantages and disadvantages of hPSC technology, many of which are rooted in developmental biology. When these issues are taken into consideration, hPSCs have the potential to close the gap between our knowledge of the genetics and biology of retinal disease, and to facilitate therapeutic development and testing at a faster pace than previously possible. 
Supported by the Retina Research Foundation Emmett A. Humble Distinguished Directorship of the McPherson Eye Research Institute, the Sandra Lemke Trout Chair in Eye Research, and the Muskingum County Community Foundation (DMG). 
Disclosure: D. Sinha, None; J. Phillips, None; M.J. Phillips, None; D.M. Gamm, None 
Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282: 1145–1147.
Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131: 861–872.
Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318: 1917–1920.
Forest DL, Johnson LV, Clegg DO. Cellular models and therapies for age-related macular degeneration. Dis Model Mech. 2015; 8: 421–427.
Jayakody SA, Gonzalez-Cordero A, Ali RR, Pearson RA. Cellular strategies for retinal repair by photoreceptor replacement. Prog Retin Eye Res. 2015; 46: 31–66.
Yvon C, Ramsden CM, Lane A, et al. Using stem cells to model diseases of the outer retina. Comput Struct Biotechnol J. 2015; 13: 382–389.
Wiley LA, Burnight ER, Songstad AE, et al. Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases. Prog Retin Eye Res. 2015; 44: 15–35.
Nguyen HV, Li Y, Tsang SH. Patient- specific iPSC- derived RPE for modeling of retinal diseases. J Clin Med. 2015; 4: 567–578.
Wahlin KJ, Maruotti J, Zack DJ. Modeling retinal dystrophies using patient-derived induced pluripotent stem cells. Adv Exp Med Biol. 2014; 801: 157–164.
Chu Y, Hughes S, Chan-Ling T. Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma. FASEB J. 2001; 15: 2013–2015.
Cornish EE, Hendrickson AE, Provis JM. Distribution of short-wavelength-sensitive cones in human fetal and postnatal retina: early development of spatial order and density profiles. Vision Res. 2004; 44: 2019–2026.
Cornish EE, Xiao M, Yang Z, Provis JM, Hendrickson AE. The role of opsin expression and apoptosis in determination of cone types in human retina. Exp Eye Res. 2004; 78: 1143–1154.
Diaz-Araya C, Provis JM. Evidence of photoreceptor migration during early foveal development: a quantitative analysis of human fetal retinae. Vis Neurosci. 1992; 8: 505–514.
Hendrickson A, Bumsted-O'Brien K, Natoli R, Ramamurthy V, Possin D, Provis J. Rod photoreceptor differentiation in fetal and infant human retina. Exp Eye Res. 2008; 87: 415–426.
Hollenberg MJ, Spira AW. Human retinal development: ultrastructure of the outer retina. Am J Anat. 1973; 137: 357–385.
Hollenberg MJ, Spira AW. Early development of the human retina. Can J Ophthalmol. 1972; 7: 472–491.
Katusic A, Juric-Lekic G, Jovanov-Milosevic N, et al. Development of the fetal neural retina in vitro and in ectopic transplants in vivo. Coll Antropol. 2008; 32: 201–207.
Kozulin P, Natoli R, O'Brien KM, Madigan MC, Provis JM. Differential expression of anti-angiogenic factors and guidance genes in the developing macula. Mol Vis. 2009; 15: 45–59.
Provis JM, Leech J, Diaz CM, Penfold PL, Stone J, Keshet E. Development of the human retinal vasculature: cellular relations and VEGF expression. Exp Eye Res. 1997; 65: 555–568.
Provis JM, van Driel D. Retinal development in humans: the roles of differential growth rates, cell migration and naturally occurring cell death. Aust N Z J Ophthalmol. 1985; 13: 125–133.
Spira AW, Hollenberg MJ. Human retinal development: ultrastructure of the inner retinal layers. Dev Biol. 1973; 31: 1–21.
van Driel D, Provis JM, Billson FA. Early differentiation of ganglion amacrine, bipolar, and Muller cells in the developing fovea of human retina. J Comp Neurol. 1990; 291: 203–219.
Bras-Pereira C, Bessa J, Casares F. Odd-skipped genes specify the signaling center that triggers retinogenesis in Drosophila. Development. 2006; 133: 4145–4149.
De Robertis E. Morphogenesis of the retinal rods; an electron microscope study. J Biophys Biochem Cytol. 1956; 2: 209–218.
Hoar RM. Embryology of the eye. Environ Health Perspect. 1982; 44: 31–34.
Liu W, Khare SL, Liang X, et al. All Brn3 genes can promote retinal ganglion cell differentiation in the chick. Development. 2000; 127: 3237–3247.
Passini MA, Levine EM, Canger AK, Raymond PA, Schechter N. Vsx-1 and Vsx-2: differential expression of two paired-like homeobox genes during zebrafish and goldfish retinogenesis. J Comp Neurol. 1997; 388: 495–505.
Turner DL, Cepko CL. A common progenitor for neurons and glia persists in rat retina late in development. Nature. 1987; 328: 131–136.
Turner DL, Snyder EY, Cepko CL. Lineage-independent determination of cell type in the embryonic mouse retina. Neuron. 1990; 4: 833–845.
Wetts R, Fraser SE. Multipotent precursors can give rise to all major cell types of the frog retina. Science. 1988; 239: 1142–1145.
Wong LL, Rapaport DH. Defining retinal progenitor cell competence in Xenopus laevis by clonal analysis. Development. 2009; 136: 1707–1715.
Andrabi M, Kuraku S, Takata N, Sasai Y, Love NR. Comparative transcriptome analysis of self-organizing optic tissues. Sci Data. 2015; 2: 150030.
Boucherie C, Mukherjee S, Henckaerts E, Thrasher AJ, Sowden JC, Ali RR. Brief report: self-organizing neuroepithelium from human pluripotent stem cells facilitates derivation of photoreceptors. Stem Cells. 2013; 31: 408–414.
Capowski EE, Simonett JM, Clark EM, et al. Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation. Hum Mol Genet. 2014; 23: 6332–6344.
Kuwahara A, Ozone C, Nakano T, Saito K, Eiraku M, Sasai Y. Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue. Nat Commun. 2015; 6: 6286.
Lamba DA, Gust J, Reh TA. Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice. Cell Stem Cell. 2009; 4: 73–79.
Lamba DA, McUsic A, Hirata RK, Wang PR, Russell D, Reh TA. Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells. PLoS One. 2010; 5: e8763.
Maekawa Y, Onishi A, Matsushita K, et al. Optimized culture system to induce neurite outgrowth from retinal ganglion cells in three-dimensional retinal aggregates differentiated from mouse and human embryonic stem cells [published online ahead of print August 19, 2015]. Curr Eye Res. doi:10.3109/02713683.2015.1038359.
Mellough CB, Collin J, Khazim M, et al. IGF-1 Signaling plays an important role in the formation of three-dimensional laminated neural retina and other ocular structures from human embryonic stem cells. Stem Cells. 2015 ;33:2416–2430.
Meyer JS, Howden SE, Wallace KA, et al. Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment. Stem Cells. 2011; 29: 1206–1218.
Meyer JS, Shearer RL, Capowski EE, et al. Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2009; 106: 16698–16703.
Nakano T, Ando S, Takata N, et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell. 2012; 10: 771–785.
Ohlemacher SK, Iglesias CL, Sridhar A, Gamm DM, Meyer JS. Generation of highly enriched populations of optic vesicle-like retinal cells from human pluripotent stem cells. Curr Protoc Stem Cell Biol. 2015; 32:1H.8.1–1H.8.20.
Osakada F, Ikeda H, Mandai M, et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol. 2008; 26: 215–224.
Phillips MJ, Perez ET, Martin JM, et al. Modeling human retinal development with patient-specific induced pluripotent stem cells reveals multiple roles for visual system homeobox 2. Stem Cells. 2014; 32: 1480–1492.
Phillips MJ, Wallace KA, Dickerson SJ, et al. Blood-derived human iPS cells generate optic vesicle-like structures with the capacity to form retinal laminae and develop synapses. Invest Ophthalmol Vis Sci. 2012; 53: 2007–2019.
Reichman S, Terray A, Slembrouck A, et al. From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium. Proc Natl Acad Sci U S A. 2014; 111: 8518–8523.
Singh R, Phillips MJ, Kuai D, et al. Functional analysis of serially expanded human iPS cell-derived RPE cultures. Invest Ophthalmol Vis Sci. 2013; 54: 6767–6778.
Singh R, Shen W, Kuai D, et al. iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degeneration. Hum Mol Genet. 2013; 22: 593–607.
Tucker BA, Anfinson KR, Mullins RF, Stone EM, Young MJ. Use of a synthetic xeno-free culture substrate for induced pluripotent stem cell induction and retinal differentiation. Stem Cells Transl Med. 2013; 2: 16–24.
Tucker BA, Mullins RF, Streb LM, et al. Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. Elife. 2013; 2: e00824.
Zhong X, Gutierrez C, Xue T, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat Commun. 2014; 5: 4047.
Osakada F, Ikeda H, Sasai Y, Takahashi M. Stepwise differentiation of pluripotent stem cells into retinal cells. Nat Protoc. 2009; 4: 811–824.
Conte I, Morcillo J, Bovolenta P. Comparative analysis of Six 3 and Six 6 distribution in the developing and adult mouse brain. Dev Dyn. 2005; 234: 718–725.
Del Bene F, Tessmar-Raible K, Wittbrodt J. Direct interaction of geminin and Six3 in eye development. Nature. 2004; 427: 745–749.
Jean D, Bernier G, Gruss P. Six6 (Optx2) is a novel murine Six3-related homeobox gene that demarcates the presumptive pituitary/hypothalamic axis and the ventral optic stalk. Mech Dev. 1999; 84: 31–40.
Jones L, Lopez-Bendito G, Gruss P, Stoykova A, Molnar Z. Pax6 is required for the normal development of the forebrain axonal connections. Development. 2002; 129: 5041–5052.
Kobayashi M, Nishikawa K, Suzuki T, Yamamoto M. The homeobox protein Six3 interacts with the Groucho corepressor and acts as a transcriptional repressor in eye and forebrain formation. Dev Biol. 2001; 232: 315–326.
Muranishi Y, Terada K, Furukawa T. An essential role for Rax in retina and neuroendocrine system development. Dev Growth Differ. 2012; 54: 341–348.
Nishida A, Furukawa A, Koike C, et al. Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development. Nat Neurosci. 2003; 6: 1255–1263.
Pichaud F, Desplan C. Pax genes and eye organogenesis. Curr Opin Genet Dev. 2002; 12: 430–434.
Porter FD, Drago J, Xu Y, et al. Lhx2, a LIM homeobox gene, is required for eye, forebrain, and definitive erythrocyte development. Development. 1997; 124: 2935–2944.
Simeone A. Otx1 and Otx2 in the development and evolution of the mammalian brain. EMBO J. 1998; 17: 6790–6798.
Haverkamp S, Ghosh KK, Hirano AA, Wassle H. Immunocytochemical description of five bipolar cell types of the mouse retina. J Comp Neurol. 2003; 455: 463–476.
Mitchell CK, Rowe-Rendleman CL, Ashraf S, Redburn DA. Calbindin immunoreactivity of horizontal cells in the developing rabbit retina. Exp Eye Res. 1995; 61: 691–698.
Mu X, Fu X, Beremand PD, Thomas TL, Klein WH. Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2. Proc Natl Acad Sci U S A. 2008; 105: 6942–6947.
Schnitzer J. Immunocytochemical studies on the development of astrocytes Müller (glial) cells, and oligodendrocytes in the rabbit retina. Brain Res Dev Brain Res. 1988; 44: 59–72.
Sinn R, Peravali R, Heermann S, Wittbrodt J. Differential responsiveness of distinct retinal domains to Atoh7. Mech Dev. 2014; 133: 218–229.
Vardimon L, Fox LE, Moscona AA. Developmental regulation of glutamine synthetase and carbonic anhydrase II in neural retina. Proc Natl Acad Sci U S A. 1986; 83: 9060–9064.
Cherry TJ, Trimarchi JM, Stadler MB, Cepko CL. Development and diversification of retinal amacrine interneurons at single cell resolution. Proc Natl Acad Sci U S A. 2009; 106: 9495–9500.
Cubelos B, Leite C, Gimenez C, Zafra F. Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles. Neurochem Int. 2014; 73: 204–210.
Trifonov S, Yamashita Y, Kase M, Maruyama M, Sugimoto T. Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization expression and quantification in the mouse brain. BMC Neurosci. 2014; 15: 114.
Magnoli D, Zichichi R, Laura R, et al. Rhodopsin expression in the zebrafish pineal gland from larval to adult stage. Brain Res. 2012; 1442: 9–14.
Rath MF, Rohde K, Klein DC, Moller M. Homeobox genes in the rodent pineal gland: roles in development and phenotype maintenance. Neurochem Res. 2013; 38: 1100–1112.
Venkataraman V, Nagele R, Duda T, Sharma RK. Rod outer segment membrane guanylate cyclase type 1-linked stimulatory and inhibitory calcium signaling systems in the pineal gland: biochemical, molecular, and immunohistochemical evidence. Biochemistry. 2000; 39: 6042–6052.
Pittack C, Grunwald GB, Reh TA. Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos. Development. 1997; 124: 805–816.
Fuhrmann S, Levine EM, Reh TA. Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development. 2000; 127: 4599–4609.
Zhao S, Hung FC, Colvin JS, et al. Patterning the optic neuroepithelium by FGF signaling and Ras activation. Development. 2001; 128: 5051–5060.
Fuhrmann S. Eye morphogenesis and patterning of the optic vesicle. Curr Top Dev Biol. 2010; 93: 61–84.
Graw J. Eye development. Curr Top Dev Biol. 2010; 90: 343–386.
Pandit T, Jidigam VK, Patthey C, Gunhaga L. Neural retina identity is specified by lens-derived BMP signals. Development. 2015; 142: 1850–1859.
Huang J, Liu Y, Oltean A, Beebe DC. Bmp4 from the optic vesicle specifies murine retina formation. Dev Biol. 2015; 402: 119–126.
Zou C, Levine EM. Vsx2 controls eye organogenesis and retinal progenitor identity via homeodomain and non-homeodomain residues required for high affinity DNA binding. PLoS Genet. 2012; 8: e1002924.
Bone-Larson C, Basu S, Radel JD, et al. Partial rescue of the ocular retardation phenotype by genetic modifiers. J Neurobiol. 2000; 42: 232–247.
Burmeister M, Novak J, Liang MY, et al. Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. Nat Genet. 1996; 12: 376–384.
Sun H, Tsunenari T, Yau KW, Nathans J. The vitelliform macular dystrophy protein defines a new family of chloride channels. Proc Natl Acad Sci U S A. 2002; 99: 4008–4013.
Johnson AA, Lee YS, Chadburn AJ, et al. Disease-causing mutations associated with four bestrophinopathies exhibit disparate effects on the localization, but not the oligomerization, of Bestrophin-1. Exp Eye Res. 2014; 121: 74–85.
Salero E, Blenkinsop TA, Corneo B, et al. Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives. Cell Stem Cell. 2012; 10: 88–95.
Gamm DM, Wright LS, Capowski EE, et al. Regulation of prenatal human retinal neurosphere growth and cell fate potential by retinal pigment epithelium and Mash1. Stem Cells. 2008; 26: 3182–3193.
Kelley MW, Turner JK, Reh TA. Regulation of proliferation and photoreceptor differentiation in fetal human retinal cell cultures. Invest Ophthalmol Vis Sci. 1995; 36: 1280–1289.
Wright LS, Pinilla I, Saha J, et al. VSX2 and ASCL1 are indicators of neurogenic competence in human retinal progenitor cultures. PLoS One. 2015; 10: e0135830.
Yang P, Seiler MJ, Aramant RB, Whittemore SR. In vitro isolation and expansion of human retinal progenitor cells. Exp Neurol. 2002; 177: 326–331.
Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science. 2003; 300: 764.
Christian M, Cermak T, Doyle EL, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010; 186: 757–761.
Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339: 819–823.
Hockemeyer D, Wang H, Kiani S, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011; 29: 731–734.
Mali P, Aach J, Stranges PB, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013; 31: 833–838.
Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science. 2003; 300: 763.
Ran FA, Hsu PD, Lin CY, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013; 154: 1380–1389.
Buchholz DE, Pennington BO, Croze RH, Hinman CR, Coffey PJ, Clegg DO. Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl Med. 2013; 2: 384–393.
Leach LL, Buchholz DE, Nadar VP, Lowenstein SE, Clegg DO. Canonical/beta-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells. Invest Ophthalmol Vis Sci. 2015; 56: 1002–1013.
Buchholz DE, Hikita ST, Rowland TJ, et al. Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells. 2009; 27: 2427–2434.
Croze RH, Clegg DO. Differentiation of pluripotent stem cells into retinal pigmented epithelium. Dev Ophthalmol. 2014; 53: 81–96.
Carr AJ, Vugler AA, Hikita ST, et al. Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PLoS One. 2009; 4: e8152.
Maminishkis A, Chen S, Jalickee S, et al. Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci. 2006; 47: 3612–3624.
Rowland TJ, Blaschke AJ, Buchholz DE, Hikita ST, Johnson LV, Clegg DO. Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins. J Tissue Eng Regen Med. 2013; 7: 642–653.
Howden SE, Gore A, Li Z, et al. Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci U S A. 2011; 108: 6537–6542.
Lukovic D, Artero Castro A, Delgado AB, et al. Human iPSC derived disease model of MERTK-associated retinitis pigmentosa. Sci Rep. 2015; 5: 12910.
D'Cruz PM, Yasumura D, Weir J, et al. Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Hum Mol Genet. 2000; 9: 645–651.
Gal A, Li Y, Thompson DA, et al. Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat Genet. 2000; 26: 270–271.
Singh R, Kuai D, Guziewicz KE, et al. Pharmacological modulation of photoreceptor outer segment degradation in a human iPS cell model of inherited macular degeneration [published online ahead of print August 24, 2014]. Mol Ther. doi:10.1038/mt.2015.141.
Li Y, Wu WH, Hsu CW, et al. Gene therapy in patient-specific stem cell lines and a preclinical model of retinitis pigmentosa with membrane frizzled-related protein defects. Mol Ther. 2014; 22: 1688–1697.
Cereso N, Pequignot MO, Robert L, et al. Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient. Mol Ther Methods Clin Dev. 2014; 1: 14011.
Vasireddy V, Mills JA, Gaddameedi R, et al. AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models. PLoS One. 2013; 8: e61396.
Schwarz N, Carr AJ, Lane A, et al. Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells. Hum Mol Genet. 2015; 24: 972–986.
Jin ZB, Okamoto S, Osakada F, et al. Modeling retinal degeneration using patient-specific induced pluripotent stem cells. PLoS One. 2011; 6: e17084.
Jin ZB, Okamoto S, Xiang P, Takahashi M. Integration-free induced pluripotent stem cells derived from retinitis pigmentosa patient for disease modeling. Stem Cells Transl Med. 2012; 1: 503–509.
Burnight ER, Wiley LA, Drack AV, et al. CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype. Gene Ther. 2014; 21: 662–672.
Tucker BA, Scheetz TE, Mullins RF, et al. Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa. Proc Natl Acad Sci U S A. 2011; 108: E569 –E.
Lustremant C, Habeler W, Plancheron A, et al. Human induced pluripotent stem cells as a tool to model a form of Leber congenital amaurosis. Cell Reprogram. 2013; 15: 233–246.
Zahabi A, Shahbazi E, Ahmadieh H, et al. A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. Stem Cells Dev. 2012; 21: 2262–2272.
Hong S, Iizuka Y, Kim CY, Seong GJ. Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation. Mol Vis. 2012; 18: 2922–2930.
Zhang XM, Li Liu DT, Chiang SW, et al. Immunopanning purification and long-term culture of human retinal ganglion cells. Mol Vis. 2010; 16: 2867–2872.
Yang J, Li Y, Chan L, et al. Validation of genome-wide association study (GWAS)-identified disease risk alleles with patient-specific stem cell lines. Hum Mol Genet. 2014; 23: 3445–3455.
Tucker BA, Solivan-Timpe F, Roos BR, et al. Duplication of TBK1 stimulates autophagy in iPSC-derived retinal cells from a patient with normal tension glaucoma. J Stem Cell Res Ther. 2014; 3: 161.
Assessing the potential to model diseases with hiPSCs. (A) Venn diagram showing four major categories to consider when planning an hiPSC modeling study. Few diseases are optimal in all categories, although that does not preclude more complex diseases from being candidates for modeling. (B) Features within each category from (A) that contribute to the challenges of modeling a particular disease with hiPSCs.
Assessing the potential to model diseases with hiPSCs. (A) Venn diagram showing four major categories to consider when planning an hiPSC modeling study. Few diseases are optimal in all categories, although that does not preclude more complex diseases from being candidates for modeling. (B) Features within each category from (A) that contribute to the challenges of modeling a particular disease with hiPSCs.

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