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Title: Isolation and characterisation of muscle satellite cells from differentiating human embryonic stem cells.
Authors: Parris, James Joseph
Issue Date: 2011
Publisher: Newcastle University
Abstract: Muscular dystrophies are a category of diseases in which the muscle fibres degrade over time. At present there is no known cure, however a great deal of promise exists in cell replacement therapy, which has been successful in alleviating animal models of muscular dystrophy. Unfortunately, attempts to use stem cell therapy to cure or treat muscular dystrophies in humans have been unsuccessful, despite many different approaches to isolating and transplanting potentially myogenic cells. While skeletal muscle differentiation of embryonic stem cells has previously been reported, a simple and efficient method for the isolation of myogenic precursors from human ES cells has not been established. Recently, advances in induced pluripotent stem cell technology have brought the possibility of patient-specific pluripotent cell lines within reach, though a great deal of work needs to be done to understand the reprogramming process and the differentiation potential of these cells. This technology provides another avenue for cell therapy treatment of muscular dystrophies. Aims: The primary goals of the work described in this thesis were to develop a novel method of differentiating human embryonic stem cells to muscle satellite cells or comparable myogenic precursors and to isolate them using fluorescence activated cell sorting based on the expression of satellite cell-specific genes or surface proteins. Results: Myoblast conditioned medium was used as the primary means of driving myogenic differentiation of hES cells, measured by flow cytometry analysis of surface marker expression and quantitative PCR analysis of myogenic gene expression. During ES cell differentiation, isolation of a pure, differentiated population of cells can be difficult. A variety of satellite cell surface markers were examined in human adult and foetal myoblast lines to test potential targets for FACS isolation. In addition, a reporter construct was developed with the intent of having the PAX7 promoter drive expression of GFP and a line of hES cells containing this construct was established. The differentiation strategy developed for hES cells was also tested on a line of iPS cells and a new line of iPS cells were generated from a patient with Duchenne muscular dystrophy. Conclusions: Several viable candidates for surface marker selection of satellite cells were identified including CD56, CD106, and M-cadherin. However, despite trying a number of different approaches of differentiating hES cells, none resulted in a highly efficient method for generating myogenic precursors. The small number of myogenic cells produced was confirmed by flow cytometry, qPCR, and immunostaining analysis.
Description: PhD Thesis
Appears in Collections:Institute of Human Genetics

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