Rancourt, DerrickHeale, Kali2022-03-072022-03-072022-02Heale, K. (2022). A biophysical and molecular characterization of human embryonic stem cell-derived exosomes (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/114449Exosomes are endocytic nanovesicles that facilitate intercellular communication via the transfer of biomolecules. There are currently several exciting applications for exosomes being developed in therapeutics and diagnostics, reflected in their increased appearance in the academic and commercial spheres. Despite the prospective uses of exosomes, their utility in research is complicated by their often inadequate characterization. This investigation aimed to characterize the biophysical and molecular properties of exosomes harvested from human embryonic stem cells (hESCs) following the guidelines of the International Society for Extracellular Vesicles. Aim 1 addressed a basic characterization of hESC-derived exosomal properties. This included an examination of exosome morphology, size, and protein marker presence via Transmission Electron Microscopy (TEM) and western blot. In Aim 2, the miRNA content of hESC-derived exosomes was investigated via RNA-Sequencing (RNA-Seq). This report begins with a look at the current and prospective uses of exosomes from stem cells in the academic and patent literature. This provides a robust list of potential applications which can be improved by successful characterizations. In Aim 1, while the data is unable to provide definitive evidence towards the isolation of hESC-derived exosomes, this report provides insight into improving practices for exosomal immunogold labelling and western blotting. Additionally, high-resolution imaging of samples isolated from a common exosome isolation procedure revealed the presence of vesicle-like structures with the morphology and size of exosomes. Finally, in Aim 2, sequencing data provided novel preliminary information on the miRNA contents of hESC-derived exosomes. Over 400 miRNAs were detected from the samples, with several of these being involved in mediating pluripotency and cellular reprogramming.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.ExosomeExtracellular VesicleBiologyBiology--CellBiology--MolecularBiochemistrymaster thesis10.11575/PRISM/39623