Exosomes from Reprogrammed Human Hepatic Stem Cells for the Treatment of Liver Diseases: A Confirmation and Mechanism Analysis of Therapeutic Effects

Abstract

Selective isolation of low volume nanobioparticles from complicated biological samples containing a highly abundant content such as proteins, exosomes, and other cellular debris is crucial for bioanalysis. To promote the bioanalytical applications of separated nanobioparticles, factors like isolation methods and their therapeutic potential must be compared and investigated. Previously proteins from biofluids have been separated using the most widely used physical methods including ultracentrifugation (UC) and syringe-based extensive equipment which are both time-consuming, laborious, and expensive. Several methods including but not limited to UC, size exclusion column-based, and chemical-based separations have been used to isolate nanobioparticles from cells and other microorganisms to perform downstream applications including disease therapy. However, there are several limitations and challenges in the isolation methods and biological utilization of nanobioparticles in disease therapy due to a limited understanding of nanobioparticles’ properties and purification of intact and functional exosomes with sufficient yields in existing methodologies, especially for stem cell exosomes. Therefore, it is important to put major research efforts to develop cost-effective devices and opt for gentle particle isolation methods which could allow easy manipulation and maximum utilization of potential of biological samples in disease therapy. In this dissertation, we discussed the isolation and importance of exosomes from reprogrammed stem cell. Exosomes from chemically reprogrammed cells were isolated and investigated for their therapeutic properties in liver normal, disease and cancer cells. It was observed that reprogrammed stem cells can release nanobioparticles with physical properties of exosomes that can exert anti-proliferative effects and lipid suppresive properties by regulating related process-associated genes. Nanobioparticles with properties of exosomes expressing CD81 and CD63 with the size distribution peak observed at 155 nm were obtianed. We analyzed that as compared to non trreated control cells, exosomes have reduced cell proliferation of Hep G2 by 93.6% after 72 h. Furthermore, it was also identified that B-cell lymphoma 2 (BCL2) has been reduced by 89.6% and activation of caspase-3 (CAS-3) has occurred in treated conditions via mitochondrial pathway in cancer cells. Further, the effects of exosomes on liver disease models were studied . Their treatment reduced lipogenesis of nonalcoholic fatty liver disease (NAFLD) by 77.1% and alcoholic fatty liver disease (AFLD) by 63.2%. It was identified that AMPK pathway was regulated by exosomal miRNA-34 which resulted the regulation of lipogenesis. Finally, we discussed the comparative analysis based on yield, concentration and biological effectivity of exosome isolation method, Flow Amplification Separation Technology (FAST) with other existing methods. The utilization of gentle and effective isolation method can impact the functional activity of isolated nanobioparticles. Therefore, our results may highlight the potential of isolated nanobioparticles from saliva , reprogrammed stem cells and other microorganisms to play a vital role in the detection and treating fatal liver diseases and cancers.