Transcriptional Regulation of Retinal Fate Determination from Human Induced Pluripotent Stem Cells

poster abstractVertebrate eye development is a complex process that is dependent upon the activity of numerous transcription factors. However, the process by which a retinal fate is specified from a primitive anterior neural progenitor cell remains largely elusive. Human induced pluripotent stem cells (hiPSCs) allows for the unique ability to recapitulate events during human development at stages that would otherwise be inaccessible to investigation. Building upon our previous studies, we sought to establish the role of key transcription factors during the establishment of a retinal fate. hiPSCs were directed to differentiate toward a retinal lineage using a targeted, stepwise differentiation process that mimics human retinogenesis. Experiments were designed to assess the developmental stages at which retinal cell fate determination was established from a primitive anterior neural population. Samples were collected every two days over the first twenty days of differentiation and gene expression analysis was performed via qPCR and immunocytochemistry. From a primitive anterior neural population derived from hiPSCs, populations of retinal and forebrain progenitor cells could be readily identified within the first 20 days of differentiation. During this timecourse, retinal populations were characterized by the expression of key transcription factors which were absent from other non-retinal cell types. The effects of these candidate genes were determined via qPCR and immunocytochemistry analyses to establish their ability to specify an early retinal fate. The work presented in this study helps to elucidate the mechanisms by which a retinal fate is specified from a more primitive population. The results of this study will assist in the establishment of efficient methods to generate retinal cells from hiPSCs and help establish these cells as a unique in vitro model system for studies of human development

TRANSCRIPTIONAL ANALYSIS OF RETINAL AND FOREBRAIN PROGENITOR CELLS DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

poster abstractEye development has been extensively studied in traditional model sys-tems but studies related to humans have been limited. The recent develop-ment of induced pluripotent stem cells (iPSCs) enabled the study of human development in culture at stages that would otherwise be inaccessible to in-vestigation. By definition, Pluripotent stem cells are cells that have the ca-pacity to generate any adult cell type, such as the muscle cell or the blood cell. A defined set of genes, known as eye field transcription factors (EFTFs) have proven to play an important role in eye development. Utilizing iPSCs as our model system, we sought to identify EFTFs that might play an essential role in the specification of the retina of the human eye. iPSCs were directed to develop into retinal cells as previously estab-lished. Since these events occur early in the developmental process, sam-ples were collected every two days over the first twenty days of differentia-tion. The development of retinal cells was determined by the characteriza-tion of gene expression patterns of six EFTFs over this timecourse in order to highlight important trends in retinal development. Retinal populations were identified by the expression of numerous EFTFs which were absent from other non-retinal cell types. Our preliminary data utilizing iPSCs highlights similar trends in the expression of these EFTFs as anticipated. However, the expression patterns of two key EFTFs varied from the others in a manner which implicated them to be critical for retinal devel-opment from an unspecified stem cell source. Thus, these candidate EFTFs were investigated further to establish their specific roles in retinal develop-ment using a combination of genetic and molecular biology approaches. The work presented in this study helps to elucidate the mechanisms by which retinal cells are specified and help establish iPSCs as a unique model system for studies of human development. 1also Indiana University Center for Regenerative Biology and Medicine, Indiana University Department of Medical and Molecular Genetics, and Stark Neurosciences Research Institute, Indianapolis IN 46202 This work was supported by a grant from the Indiana University Collaborative Research Grant fund of the Office of the Vice president for Research as well as startup funds from the School of Science at IUPUI

Human Pluripotent Stem Cells Serve as an Effective In Vitro Model for Studies of Early Stages of Retinogenesis

poster abstractSpecification of the neural retina is one of the first events in human development and thus, efforts to study the initial stages of retinal specification have been largely limited. This is particularly true for the earliest event in retinogenesis, the establishment of a definitive retinal fate from a more primitive neural progenitor source. With the advent of human pluripotent stem cells (hPSCs), the complex interplay of transcription factors involved in early events of retinal development from an unspecified pluripotent population can be studied in an in vitro model. To examine this potential, hPSCs were directed to differentiate to a neuroretinal lineage in which a subpopulation of cells adopts a definitive retinal fate whereas others develop toward a forebrain lineage. Samples were collected over the first month of differentiation, starting from the undifferentiated state through when cells acquired either retinal or nonretinal forebrain identities and gene expression patterns were characterized using immunocytochemistry and quantitative RT-PCR. Results demonstrated that while neural transcription factors such as PAX6, OTX2, and LHX2 were expressed early in development, definite retinal transcription factors such as CHX10 were expressed later in differentiation. Furthermore, the expression of CHX10 was found to be uniquely associated with retinal populations and remained absent from the other neural populations, thereby illustrating the ability of this protocol to uniquely identify and isolate retinal populations and further study them in vitro. Overall, these studies will serve to further understand the specification of a retinal fate from a pluripotent population. Such information will assist in the establishment of more efficient methods to generate retinal cells from hiPSCs for translational purposes, as well as establish hiPSCs as a unique in vitro model system for studies of the earliest stages of human development. Mentor: Jason S. Meyer1-

Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration

Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies

Transcriptional regulation of retinal progenitor cells derived from human induced pluripotent stem cells

In order to develop effective cures for diseases and decipher disease pathology, the need exists to cultivate a better understanding of human development. Existing studies employ the use of animal models to study and model human development and disease phenotypes but the evolutionary differences between humans and other species slightly limit the applicability of such animal models to effectively recapitulate human development. With the development of human pluripotent stem cells (hPSCs), including Human induced Pluripotent stem cells (hiPSCs) and Human Embryonic Stem cells (hESCs), human development can now be mirrored and recapitulated in vitro. These stem cells are pluripotent, that is, they possess the potential to generate any cell type of the body including muscle cells, nerve cells or blood cells. One of the major focuses of this study is to use hiPSCs to better understand and model human retinogenesis. The retina develops within the first three months of human development, hence rendering it inaccessible to investigation via traditional methods. However, with the advent of hiPSCs, retinal cells can be generated in a culture dish and the mechanisms underlying the specification of a retinal fate can be determined. Additionally, in order to use hiPSCs for successful cell replacement therapy, non-xenogeneic conditions need to be employed to allow for fruitful transplantation tests. Hence, another emphasis of this study has been to direct hiPSCs to generate retinal cells under non-xenogeneic conditions to facilitate their use for future translation purposes

Build Your Own Retina: Modeling Retinogenesis and Disease Using Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) allow for the unprecedented ability to recapitulate early stages of human development, which are otherwise inaccessible to investigation. This is especially true for one of the earliest events in human development, the establishment of a neuroretinal fate from an unspecified pluripotent population. To test the ability of hPSCs to serve in this capacity, hPSCs were generated using mRNA-reprogamming methods and maintained in xenogeneic-free differentiation conditions. These cells were directed to differentiate using a three-dimensional approach to analyze their ability to successfully recapitulate early events in human development in a temporal and developmentally-appropriate fashion. To do so, hPSCs were first directed to an anterior neural phenotype, which was confirmed by analysis of stage-specific neural transcription factors via immunocytochemistry and quantitative RT-PCR. Next, the cells were directed to an optic vesicle-like stage, where the presumptive retinal cells were identified by the expression of specific transcription factors. Finally, three-dimensional optic vesicle-like retinal organoids were identified, isolated, and further analyzed for the expression of markers associated with some of the differentiated cell types of the neural retina. Upon establishment of hPSC-derived retinal organoids, this system was further utilized to study the neurodegeneration in glaucoma and provide insights into the disease mechanisms. Overall, the results of this study help to demonstrate the suitability of hPSC-differentiation approaches as an effective tool to model retinal development and disease

Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

The ability and efficiency of mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs) to yield retinal cell types in a directed, stepwise manner was tested. hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes. Such methods represent a promising new approach for retinal stem cell research, especially translational applications

Astrocytes Regulate the Development and Maturation of Retinal Ganglion Cells Derived from Human Pluripotent Stem Cells

Summary: Retinal ganglion cells (RGCs) form the connection between the eye and the brain, with this connectivity disrupted in numerous blinding disorders. Previous studies have demonstrated the ability to derive RGCs from human pluripotent stem cells (hPSCs); however, these cells exhibited some characteristics that indicated a limited state of maturation. Among the many factors known to influence RGC development in the retina, astrocytes are known to play a significant role in their functional maturation. Thus, efforts of the current study examined the functional maturation of hPSC-derived RGCs, including the ability of astrocytes to modulate this developmental timeline. Morphological and functional properties of RGCs were found to increase over time, with astrocytes significantly accelerating the functional maturation of hPSC-derived RGCs. The results of this study clearly demonstrate the functional and morphological maturation of RGCs in vitro, including the effects of astrocytes on the maturation of hPSC-derived RGCs. : In this article, VanderWall and colleagues demonstrate the morphological and functional maturation of hPSC-derived RGCs over time, including the role of astrocytes in their functional maturation. Results indicated that hPSC-derived RGCs were capable of exhibiting robust neurite outgrowth and functional properties, with the direct contact with astrocytes significantly enhancing this maturation. As astrocytes are a major component of the nerve fiber layer and optic nerve, the results of these studies constitute a model for studying these cellular interactions in vitro. Keywords: stem cell, retina, retinal ganglion cell, astrocyte, development, differentiation, pluripotent stem cel

Three-Dimensional Retinal Organoids Facilitate the Investigation of Retinal Ganglion Cell Development, Organization and Neurite Outgrowth from Human Pluripotent Stem Cells

Abstract Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs