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Isfahan University of Medical Sciences
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| Document Type
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Latin Dissertation
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| Language of Document
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English
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| Record Number
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102660
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| Doc. No
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T10975
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| Call number
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WL,300,A992e,2007
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| Main Entry
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Azari, Hassan
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| Title & Author
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Establishment Of a Flow Cytometry- Based Method For the Generation Of Highly Enriched Immature Neuros From Neural Stem Cells/حسن آذری
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| College
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Schools, Medical
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| Date
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, 2007
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| Degree
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Anatomy, Ph.D
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| Page No
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165 p.: ill( som col ), diag, tab
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| Note
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Orginal Works
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| Abstract
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Introduction: Transplanting foetal tissues, embryonic and adult stem cells has brought some hope for treatment of neurological diseases, but still there are a lot of problems like moral and ethical concerns of using foetal tissues and it's shortage, tumor formation of embryonic stem cells, poor neuronal differentiation of neural stem cells, immune rejection of transplanted cells from non-autologous sources that needs to be resolved before one can successfully apply basic scientific findings into clinic. In this study our attempt was to establish a flow cytometry based method to generate a highly enriched immature neuronal population from neural stem cells. Materials and Methods: Neural stem cells were isolated and propagated using the neurosphere assay. Differentiating neural stem cell cultures were fixed for microscopic examination or dissociated into a single cells suspension to analyse and sort by flow cytometry. The cellular composition of each population was determined by immunocytochemistry. The frequency of neural stem / progenitors and bona fide neural stem cells were determined using neurosphere assay and neural colony forming cell assay, respectively. 04 positive and 04/ A2B5 positive were excluded to further purify immature neuronal cells. PSA-NCAM antibody was applied to directly purify immature neuronal cells. Different factors including foetal calf serum, BDNF, CNTF, GH, Noggin and BMP-4 were screened for their survival effects on sorted immature neurons. The phenotype and functional maturation were investigated using immunocytochemistry and patch clamp electrophysiology, respectively., Sorted immature neurons were also transplanted into adult mice intact striatum.Results: The neuroblast assay could significantly generate more 13-111 tubulin IR cells and less GFAP IR cells than the conventional method. FACS analysis showed two distinct cell populations; low FCS, low SSC (P1) and high FCS, high SSC (P2). Immunocytochemistry showed that the 131 population were mainly immature neuronal cells whereas the majority of the P2 population were astrocytes. It has been shown that the majority of the neurosphere forming cells and colony forming cells could be separated from neuronal population using this sorting strategy. FACS exclusion of 04 IR cells and 04/A2B5 IR cells increased the purity of sorted immature neurons from 75.2 ▒3.3 to 84.1 ▒2.2 and 91.6 +0.93, respectively. Growth factor screening showed that only BMP4 could increase survival of sorted immature neurons relative to the control condition. It has also been shown that sorted immature neurons can differentiate in vitro and express mature neuronal markers of which the majority were GAD 65/67 IR nd DARPP-32 IR and show basic criteria of functionally mature neuronal cells. Transplantation results revealed that sorted immature neurons can survive if supplemented with appropriate trophic factor like BMP4. Conclusion: A highly enriched immature neuronal population from differentiating neural stem cell progeny can be isolated based on their physical properties that can be used for drug screening, electrophysiology, and transplantation. Key words: Neural stem cell, Flow cytometry, Screening, Transplantation, and Neurosphere assay..
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| Descriptor
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1.Stem Cells.- Descripors: Central Nervous System
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Cell Culture
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Cytolological Techniques
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Flow Cytometry
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Stem Cell Transplantion
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| Added Entry
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Esfandiary, Ebrahim, Supervisor
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Mardani, Mohammad, Supervisor
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A.Rynolds, Brent, Supervisor
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| Translated Title Supplied by Cataloguer
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راهاندازی یک روش فلوسیتومتری برای نورونهای نابالغ از سلولهای بنیادی عصبی
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http://elib.mui.ac.ir/site/catalogue/102660
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