Isolation And Evaluation Of Exosomes Originating From Human Adipose Tissue-Derived Mesenchymal Stem Cells On The Expression Of Some Angiogenesis Related Genes

Abstract

In regenerative medicine, human adipose tissue-derived mesenchymal stem cells (hadMSCs) have exhibited tremendous promise in providing an efficient approach for chronic cardiovascular diseases. Noble effects of mesenchymal stem cells (MSCs) have been acknowledged to attribute not only to cellular engraftment and response to the site of injury but paracrine action via the release of extracellular vesicles (EVs), notably, exosomes. This study aims to investigate more about the roles of exosomes derived from hadMSCs (hadMSC-Exo) in the angiogenesis effect. Firstly, hadMSCs were characterized and cultured at a density of 500,000 cells in a T75 flask for 48 hours. The conditioned medium was ultracentrifuged for exosome isolation. Exosomes were confirmed for CD9, CD63, and CD81 markers by flow cytometry and imaged by TEM assessment. Human umbilical vein endothelial cells (HUVECs) culture was established for the angiogenesis model under treatment of hadMSC-Exo. The expression of some specific angiogenesis-related genes including growth and adheren factors were identified by RTqPCR. The result of the Bradford assay shown that the concentration of total protein in isolated exosomes was 0.51 ± 0.04 µg/ml. Exosomes were successfully isolated and characterized by their morphology and immunophenotype. The results showed that exosomes significantly escalated the mRNA expression of TGF by 11.56 ± 2.03, Flk-1 by 4.04 ± 0.01, VE-Cadherin by 7.25 ± 2.30, and CD31 by 3.02 ± 1.13 (P < 0.05). The study suggested that exosomes promoted angiogenesis of endothelial cells in vitro. Summary: The study aims to investigate more about the roles of exosomes derived from hadMSCs (hadMSC-Exo) on the expression of some specific angiogenesis-related genes including growth and adheren factors in vitro. Briefly, exosomes were ultracentrifuged from the conditioned medium of hadMSCs, and further characterized by their morphology and immunophenotype. The exosomes were used to treat human umbilical vein endothelial cells for 24hrs to isolate RNA and conduct RT-qPCR subsequently. The results showed that exosomes significantly escalated the mRNA expression of TGF, Flk-1, VE-Cadherin, and CD31 (P<0.05).