Toward the Establishment of an In-Vitro Model of Glaucoma Using Human Induced Pluripotent Stem Cells

poster abstractGlaucoma is a severe neurodegenerative disease of the retina, leading to eventual irreversible blindness. A crucial element in the pathophysiology of all forms of glaucoma is the death of retinal ganglion cells (RGCs), a population of CNS neurons with their soma in the inner retina and axons fasciculating together to form the optic nerve. Retinal astrocytes have also been associated with glaucomatous neurodegeneration, although the direct or indirect role for these cells in the disease process remains unclear. Human induced pluripotent stem cells (iPSCs) provide a promising approach to develop cellular models to study such neurodegenerative diseases in vitro. Directed differentiation of several somatic cell types from human iPSCs have been successfully achieved with great implications for disease modeling and cell replacement strategies. Using existing lines of iPSCs, efforts were undertaken to successfully differentiate and characterize RGCs and astrocytes, the affected cell types in glaucoma. Using a previously described protocol, these cells were directed to differentiate toward a retinal fate through a step-wise process that proceeds through all of the major stages of neuroretinal development. The differentiation of RGCs was observed within the first forty days of differentiation whereas astrocytes were observed only after at least 70 days of differentiation. Using techniques including immunocytochemistry and RT-PCR, the individually derived somatic cells types were characterized by the expression of developmentally associated transcription factors specific to each cell type. Further approaches were undertaken to characterize the morphological differences between RGCs and other neuroretinal cell types derived in the process. Overall, this study demonstrates a robust method to derive the complex cell types associated with glaucoma, with prospects for further investigations into the developmental progression of the disease

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

Enhancing The Specification Of Retinal Neurons From Human Induced Pluripotent Stem Cells

poster abstractA variety of retinal degenerative diseases, including retinitis pigmentosa and age-related macular degeneration, result in the loss of retinal neurons leading to a gradual loss of vision. An in vitro model to study the development of human retinal cells would provide a better understanding of the structure and functionality of the retina, eventually leading to new therapeutic approaches to blinding disorders that could involve replacing cells that had been lost to disease. Following previously established protocols, two types of populations of cells are observed early in the differentiation process, those that lead to retinal cells and those that lead to other anterior phenotypes of the central nervous system. These cells arise from a common progenitor population derived from induced pluripotent stem cells, yet the mechanism underlying the differentiation of these two different types of cells remains elusive. To further study the specification of retinal cells from this common progenitor population, a more efficient method to produce these cells needs to be developed. The purpose of this experiment is to test several candidate growth factors and observe their effect on the production of retinal cells. This study tests five different growth conditions using insulin-like growth factor-1, fibroblast growth factor-2, the sonic hedgehog agonist purmorphamine, retinoic acid and an untreated control. Treatment was carried out from Day 7 until Day 20, a period during which previous studies have demonstrated an ability to influence the decision of these cells to become retinal non-retinal. Immunocytochemistry (ICC) and RT-PCR analysis was used to monitor the expression of proteins characteristic of retinal and non-retinal cells. These results can be used to devise a more efficient protocol for retinal specification from human induced pluripotent stem cells and in turn, will further our understanding of the development of the retina

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

Differentiation and Three-dimensional Organization of Retinal Ganglion Cells using Human Induced Pluripotent Stem Cells

poster abstractRetinal Ganglion Cells (RGCs) are a type of neuron which function to relay visual messages between the retina and brain, and are characterized by their long axons which form part of the optic nerve. Dysfunction in this communication pathway is highly implicated in degenerative blinding disorders such as glaucoma. Unique applications using human induced pluripotent stem cells (hiPSCs) offer the ability to model human diseases, and potentially develop novel therapeutic approaches to rescue or replace damaged cells. In order to better understand the progression of degenerative eye diseases, a remaining challenge is to precisely identify the sequence of events which contribute to the diseased state, and how their features differ from non-diseased cells. Efforts were therefore undertaken to visually document the maturation of RGCs by analyzing their morphology and three-dimension organization at varying stages of development. Induced retinal cells were harvested at six different stages of development and fixed in 4% paraformaldehyde (PFA) solution to arrest their development. Cells were then cryoprotected in combinations of sucrose and Optimal Cutting Temperature (OCT) solutions, and frozen using powered dry ice. Following cryostat sectioning, samples were subject to immunocytochemistry staining to visualize for retinal-like organization of cells. Preliminary results have indicated the presence of the RGC marker Brn3, as well as markers for other retinal cell types. Future tests intend to characterize these retinal cell types according to their morphology and three-dimensional organization

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-

Spatial stochasticity and non-continuum effects in gas flows

We investigate the relationship between spatial stochasticity and non-continuum effects in gas flows. A kinetic model for a dilute gas is developed using strictly a stochastic molecular model reasoning, without primarily referring to either the Liouville or the Boltzmann equations for dilute gases. The kinetic equation, a stochastic version of the well-known deterministic Boltzmann equation for dilute gas, is then associated with a set of macroscopic equations for the case of a monatomic gas. Tests based on a heat conduction configuration and sound wave dispersion show that spatial stochasticity can explain some non-continuum effects seen in gases