The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation.
This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy.
The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/26510 |
| Date | 16 March 2011 |
| Creators | Manceur, Aziza |
| Contributors | Audet, Julie |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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