Purification and characterisation of extracellular vesicles released from pluripotent stem cells

Extracellular vesicles (EVs) are nano-sized particles constitutively released from cells into biological fluids such as blood, plasma, saliva and urine. Interestingly, these vesicles contain genetic cargoes including proteins, RNA and bioactive lipids that can be functionally delivered and affect recipient cells. Hence, EVs are postulated to be an alternative source of cell-cell communication in both normal physiological systems and pathological situations. Recently, EVs from mesenchymal stem cells have been shown to promote regeneration of injured cells. Induced pluripotent stem cells (iPSCs) are a relative newer type of stem cells that is emerging as useful tools for re-modeling diseases. However, knowledge about EVs from iPSCs is relatively sparse. In initial experiments, we successfully purified EVs from mouse iPSCs using the differential ultracentrifugation (UC) method. However, we noticed a discrepancy between total particle counts and expression of EV markers, across different EV batches. One crucial prerequisite for EV research is the availability of a standardized workflow for collection and purification of EVs from biological sources. Increasing evidence from recent studies has suggested that the original UC method is limited by several shortcomings such as low EV yield, purity and altered biophysical properties. Hence, this has led to a new wave in the development of alternative EV purification strategies. In this thesis, we start with a systematic comparison study between UC and an alternative purification protocol, size-exclusion liquid chromatography (LC) of EVs from serum-free conditions. We found that LC is better than UC in terms of overall EV yields, purity and vesicle integrity. Subsequently, we demonstrate that LC allowed for the derivation of pure EVs from complex media sources used for growing stem cells like iPSCs, which was previously impossible with UC. Lastly, we describe novel data on the characterization of EVs from pluripotent stem cell sources and discuss the possible roles of these EVs in stem cell biology.