Browsing by Subject "induced pluripotent stem cells (iPSCs)"

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    Toward the Establishment of an In-Vitro Model of Glaucoma Using Human Induced Pluripotent Stem Cells
    (Office of the Vice Chancellor for Research, 2013-04-05) Gupta, Manav; Meyer, Jason S.
    Glaucoma 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.
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    TRANSCRIPTIONAL ANALYSIS OF RETINAL AND FOREBRAIN PROGENITOR CELLS DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS
    (Office of the Vice Chancellor for Research, 2012-04-13) Sridhar, Akshayalakshmi; Steward, Melissa M.; Gupta, Manav; Meyer, Jason S.
    Eye 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.