TitleEngineering embryonic stem cells for myelin cell therapy
NameSadowski, Dorota (author), McKinnon, Randall (chair), Shi, Yufang (internal member), Pan, Zui (internal member), Rutgers University, Graduate School - New Brunswick,
SubjectPhysiology and Integrative Biology,
DescriptionCell replenishment using stem cell-derived transplants holds great promise for therapy in human disease. However, clinical therapeutics in the central nervous system (CNS) requires targeted migration for lesion repair. For CNS grafts, homing the graft population to the appropriate site for tissue repair is non trivial since surgical access to wound is not risk free. Our objective is to modify stem cells to generate lineage specified CNS glia that 'home' into lesions, focusing on myelin repair for multiple sclerosis and spinal cord injury. Myelin protects neuronal axons, and in pre-clinical models ES-derived OL progenitor cell (OPC) grafts can regenerate myelin forming oligodendrocytes (OLs) and rescue the phenotype of myelin-defective mice. Our studies focus on engineering ES cells to expresss PDGFRα, the trans-membrane receptor for Platelet-Derived Growth Factor, which directs OPC migration during brain development. Prior work identified a 'rheostat' function for PDGFRα, with low levels of PDGF activating PI3K to in initiate migration and high levels activating PLCγ to inhibit migration. This predicts that PLCγ-uncoupled receptors would direct ES-derived OPCs toward PDGF infused brain lesions. We addressed the rheostat model by engineering mouse ES cells to express wild-type and signal transduction mutant versions of PDGFRα. First we generated a medium cocktail named '2i5S' that allows the the long-term expansion of mouse ES cell lines, which is optimal for our engineering experiments. Next we introduced PDGFRα transgenes into mES cells in vitro. A very low number of stable ES transformants were obtained after transfection, and this directed us towards using a set of vectors (TRE.PDGFRα) with tetracycline inducible promoter for control of transgene expression. Lastly, we used myelin mutant shiverer mice to evaluate the potential of ES-derived OPCs to generate myelin in vivo. Our results showed substantial improvements in both motor function and longevity in animals with 7% of ESwtderived cells present in the pup. Therefore this suggests that 7% ES-derived stem cell engraftment is the minimal level that is required for functional cell replacement therapy.
NoteIncludes bibliographical references (p. 39-42)
Noteby Dorota Sadowski
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
RightsThe author owns the copyright to this work.