Browsing by Author "Gupta, Manav"

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    Differentiation and characterization of cell types associated with retinal degenerative diseases using human induced pluripotent stem cells
    (2014-07-31) Gupta, Manav; Meyer, Jason S.; Belecky-Adams, Teri; Randall, Stephen Karl, 1953-
    Human induced pluripotent stem (iPS) cells have the unique ability to differentiate into 200 or so somatic cell types that make up the adult human being. The use of human iPS cells to study development and disease is a highly exciting and interdependent field that holds great promise in understanding and elucidating mechanisms behind cellular differentiation with future applications in drug screening and cell replacement studies for complex and currently incurable cellular degenerative disorders. The recent advent of iPS cell technology allows for the generation of patient-specific cell lines that enable us to model the progression of a disease phenotype in a human in vitro model. Differentiation of iPS cells toward the affected cell type provides an unlimited source of diseased cells for examination, and to further study the developmental progression of the disease in vitro, also called the “disease-in-a-dish” model. In this study, efforts were undertaken to recapitulate the differentiation of distinct retinal cell affected in two highly prevalent retinal diseases, Usher syndrome and glaucoma. Using a line of Type III Usher Syndrome patient derived iPS cells efforts were undertaken to develop such an approach as an effective in vitro model for studies of Usher Syndrome, the most commonly inherited disorder affecting both vision and hearing. Using existing lines of iPS cells, studies were also aimed at differentiation and characterization of the more complex retinal cell types, retinal ganglion cells (RGCs) and astrocytes, the cell types affected in glaucoma, a severe neurodegenerative disease of the retina leading to eventual irreversible blindness. Using a previously described protocol, the iPS 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 process was monitored for a period of 70 days for the differentiation of retinal cell types and 150 days for astrocyte development. The different stages of differentiation and the individually derived somatic cell 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. The results of this study successfully demonstrated that Usher syndrome patient derived iPS cells differentiated to the affected photoreceptors of Usher syndrome along with other mature retinal cell types, chronologically analogous to the development of the cell types in a mature human retina. This study also established a robust method for the in vitro derivation of RGCs and astrocytes from human iPS cells and provided novel methodologies and evidence to characterize these individual somatic cell types. Overall, this study provides a unique insight into the application of human pluripotent stem cell biology by establishing a novel platform for future studies of in vitro disease modeling of the retinal degenerative diseases: Usher syndrome and glaucoma. In downstream applications of this study, the disease relevant cell types derived from human iPS cells can be used as tools to further study disease progression, drug screening and cell replacement strategies.
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    Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells
    (Nature Publishing Group, 2014-11) Prasain, Nutan; Lee, Man Ryul; Vemula, Sasidhar; Meador, Jonathan Luke; Yoshimoto, Momoko; Ferkowicz, Michael J.; Fett, Alexa; Gupta, Manav; Rapp, Brian M.; Saadatzadeh, Mohammad Reza; Ginsberg, Michael; Elemento, Olivier; Lee, Younghee; Voytik-Harbin, Sherry L.; Chung, Hyung Min; Hong, Ki Sung; Reid, Emma; O'Neill, Christina L.; Medina, Reinhold J.; Stitt, Alan W.; Murphy, Michael P.; Rafii, Shahin; Broxmeyer, Hal E.; Yoder, Mervin C.; Department of Pediatrics, IU School of Medicine
    The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.
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    Enhancing The Specification Of Retinal Neurons From Human Induced Pluripotent Stem Cells
    (Office of the Vice Chancellor for Research, 2013-04-05) Iglesias, Clara; Gupta, Manav; Meyer, Jason S.
    A 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.
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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.