Retinal Stem Cells

Retinal Stem Cells
Author: Tahani Baakdhah
Publisher:
Total Pages:
Release: 2021
Genre:
ISBN:

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Growing retinal stem cell (RSC) clonal spheres in traditional static culture is associated with low expansion rate and poor survival attributed to loss of cells during the enzymatic dissociation steps. Another problematic issue is the difficulty to mimic in vivo stem cell niche conditions. Using our traditional lab protocol, only 0.2% of the pigmented ciliary epithelium cells (CE) give rise to floating RSC clonal spheres in serum free conditions. These issues have hindered our ability to study the signaling pathways governing RSC proliferation and expansion in vitro. To overcome these limitations, I used microcarriers (MCs) in a suspension stirring bioreactor (SSB) to help achieve sufficient numbers suitable for differentiation and transplantation. Using this new protocol, I achieved a significant (10-fold) enrichment of RSC yield compared to conventional static culture techniques using a combination of FACTIII MCs and relative hypoxia (5%) inside the bioreactor. My work showed that hypoxia (5% O2) was associated with better RSC expansion across all platforms which was attributed to hypoxia-induced boosting survival and/or symmetric division of stem cells. RSC spheres were thought to be unvaried and were randomly picked and placed on laminin extracellular matrix (ECM) in static plates. In my work, I found noticeable variance in the pigment distribution between RSC spheres which led me to categorize them into three different groups according to their pigmentation, I also noted that this variance in pigment level and distribution was associated with contrasting differentiation potentials. RSCs were classified into three morphological groups: heavily pigmented (HP), lightly pigmented (LP) and centrally pigmented (CP) spheres. Unlike the other two sphere types, CP spheres were capable of producing highly proliferative progenitors (producing large number of cobblestone-like cell lawns) in adherent culture that differentiate into retinal pigment epithelium (RPE) cells. I found that the individual stem cells that (clonally) formed the three sphere types appear homogeneous, but it is their downstream progenitors that are different. I showed that CP spheres contain a population of early RPE progenitors that respond to proliferative signals from the surrounding non-pigmented neural retinal cells while The HP and LP spheres do not respond to these signals.

Differentiation and Characterization of Cell Types Associated with Retinal Degenerative Diseases Using Human Induced Pluripotent Stem Cells

Differentiation and Characterization of Cell Types Associated with Retinal Degenerative Diseases Using Human Induced Pluripotent Stem Cells
Author: Manav Gupta
Publisher:
Total Pages: 194
Release: 2013
Genre: Astrocytes
ISBN:

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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.

Ocular Neuroprotection

Ocular Neuroprotection
Author: Leonard A. Levin
Publisher: CRC Press
Total Pages: 343
Release: 2003-07-15
Genre: Medical
ISBN: 0203911946

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Many retinal and optic nerve disorders have no effective therapy, or are treated incompletely and with considerable side effects. Recent advances have suggested the significant benefits associated with neuroprotection - that is, when treatment strategies are directed to photoreceptors, retinal ganglion cells, or other neural targets. Enter Ocular N

Stem Cell Proliferation and Differentiation

Stem Cell Proliferation and Differentiation
Author:
Publisher: Academic Press
Total Pages: 258
Release: 2020-03-24
Genre: Science
ISBN: 0128128917

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Stem Cell Proliferation and Differentiation, Volume 138, the latest release in the Current Topics in Developmental Biology series, highlights new advances in the field, with this new volume presenting interesting chapters. Each chapter is written by an international board of authors. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in the Current Topics in Developmental Biology series Includes the latest information on stem cell proliferation and differentiation

Characterisation of Neural Progenitors from the Adult Retina and Ciliary Epithelium

Characterisation of Neural Progenitors from the Adult Retina and Ciliary Epithelium
Author: I. Kokkinopoulos
Publisher:
Total Pages:
Release: 2009
Genre:
ISBN:

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The mammalian central neural retina (CNR) lacks the capability to regenerate, a phenomenon retained by lower vertebrates. However, retinal stem cells have been isolated from the ciliary epithelium of the mammalian retina. Chx10 is a paired-like homeobox transcription factor gene expressed in the presumptive neural retina of the invaginating optic vesicle. The Chx10 gene is expressed in the proliferating retinal progenitor cell population throughout retinal development hence is one of the earliest characterised RPC-specific markers. Mutations in the Chx10 homeobox gene cause reduced proliferation of retinal progenitor cells during development, leading to microphthalmia. Recently, it was showed that in the ocular retardation mouse model lacking Chx10 (Chx10orJ/orJ), dividing cells persist in the adult CNR, suggesting the existence of a dormant stem/progenitor population. The neurosphere-forming assay is a tool which has allowed scientists to study the behaviour of neural stem/progenitor cells in vitro. Here, I show that cells deriving from the CNR of the adult microphthalmic retina are proliferative and give rise to neurospheres in vitro, a characteristic of neural stem cells. However, these adult-derived CNR progenitors differ from those of the wildtype CE, leading to de-pigmented, larger and more numerous neurospheres expressing Müller glial cell markers. My results suggest that lack of Chx10 leads to maintenance of a dormant neural progenitor population in the adult CNR possible deriving from the abnormal appearance of GFAPpos Müller glia in late embryonic stages of the Chx10orJ/orJ retina. Furthermore, Chx10 is not required for in vitro proliferation of these progenitors. One of the cardinal features of stem cells is their differentiation potential and multipotency. My experiments illustrate that Chx10orJ/orJ CNR-derived neurospheres are able to differentiate in a similar fashion to wildtype CE-derived neurospheres. Furthermore, when neurospheres lacking Chx10 are placed in conditions that promote differentiation, they significantly up-regulate the expression of photoreceptor genes in comparison to wildtype. Hitherto, the developmental origin of CE-derived neurosphere-forming retinal stem cell is unclear. The ciliary body, where the CE is located in adult mammals, includes cells of mesodermal, neural crest and neural ectodermal origin. Here, data collected from lineage tracing analysis and in vivo BrdU-tagging experiments suggest that neurospheres are formed from BrdUpos cells observed in vivo, and that these cells originate from the embryonic anterior forebrain. The comparative analysis of the microphthalmic CNR retinal progenitors and CE-derived progenitors provides valuable information on cell properties relevant for potential cell-based replacement therapies, as well for retinal regeneration potential in mammals.

Retinal Differentiation of Pluripotent Stem Cells

Retinal Differentiation of Pluripotent Stem Cells
Author: Debarchana Sarkar
Publisher:
Total Pages:
Release: 2017-01-26
Genre:
ISBN: 9781360998589

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This dissertation, "Retinal Differentiation of Pluripotent Stem Cells" by Debarchana, Sarkar, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: The retina is an internal photosensitive neural tunic which absorbs light and prevents it from reflecting back. The light receptors and neurons of the retina are initial processor of visual information. Various anomalies of the retina such as retinitis pigmentosa, cone-rod dystrophy to retinal degenerative diseases cause severe loss of vision since they affect photoreceptors directly or indirectly. Conventional therapies have never been fully successful in restoring vision in such diseases. However current research in stem cell therapies has shown remarkable potential. In this project, induced pluripotent stem cells from mouse were coxed into photoreceptor fate in presence and absence of Dorsomorphin using specific media in a stepwise differentiation process. Dorsomorphin is an inhibitor of Bone Morphogenic Protein (BMP) whose suppression may influence neural differentiation. Studies were done using conventional inverted microscopy and fluorescent microscopy on mouse induced pluripotent stem cells (miPS cells). Immunolabelling techniques involving Pax6, Crx, RPE65, Rhodopsin and Opsin were used to evaluate the advantage of these as markers for stem cells differentiation. Reverse Transcriptase PCR was done to confirm the gene expression on the differentiated cells. Human iPS derived Mesenchymal stem cells were cultured and the effect of different concentrations of Retinoic Acid such as 0mM, 0.1mM and 0.5mM on cell proliferation was tested in both presence and absence of Dorsomorphin. The results revealed both control and Dorsomorphin treated miPS cells successfully differentiated into photoreceptors-like cells as detected by positive staining of Rhodopsin and Opsin. The cells were however negative for Pax6, and very weak staining for RPE and Crx. The presence or absence of Dorsomorphin did not make any difference on miPS differentiation. The same observation was made on differentiating human iPS-MSC where Dorsomorphin did not reveal much effect. However highest cell count of proliferating cells was observed in the subgroups containing 0.1mM Retinoic Acid on Day7 groups as control had an average of 590 317.23 and treatment 1206 .33 114.99 cells, with statistical significance of PIn conclusion, the study reveals that iPS can be another potential stem cell source for therapies of retinal diseases involving photoreceptors where the question of ethical issue is not a problem unlike embryonic stem cells. Also it reveals the concentration of Retinoic Acid most suited for human iPS-MSC cell proliferation. Dorsomorphin did not seem to have much effect on either type of stem cells in terms of promoting photoreceptor differentiation. DOI: 10.5353/th_b5071270 Subjects: Retina - Differentiation Stem cells

Proliferation and Potential of Neural and Retinal Stem Cells

Proliferation and Potential of Neural and Retinal Stem Cells
Author: Laura Donaldson
Publisher:
Total Pages: 610
Release: 2011
Genre:
ISBN: 9780494781692

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The term "stem cell" is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

Retinal Degenerative Diseases

Retinal Degenerative Diseases
Author: Matthew M. LaVail
Publisher: Springer Science & Business Media
Total Pages: 867
Release: 2011-12-21
Genre: Medical
ISBN: 1461406315

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This book will contain the proceedings of the XIV International Symposium on Retinal Degeneration (RD2010), held July 13-17, 2010, in Mont-Tremblant, Quebec, Canada. The volume will present representative state-of-the-art research in almost all areas of retinal degenerations, ranging from cytopathologic, physiologic, diagnostic and clinical aspects; animal models; mechanisms of cell death; candidate genes, cloning, mapping and other aspects of molecular genetics; and developing potential therapeutic measures such as gene therapy and neuroprotective agents for potential pharmaceutical therapy.