Therapeutic Exosome Preparations: Relative Bioactivities of Intra‐ and Extra‐Vesicular Components

Abstract

Adult progenitor cells from the heart known as cardiosphere derived cells (CDCs) are therapeutic candidates in active clinical development for conditions including heart failure, pulmonary hypertension and Duchenne muscular dystrophy. The mechanism of action of CDCs is paracrine, mediated principally by the secretion of extracellular vesicles (EVs) known as exosomes. Progenitor cell EVs represent an exciting therapeutic opportunity, but the ideal culture conditions and isolation methods are still under investigation. The objective of this study was to examine the effect of various conventional EV isolation methods upon signaling, uptake, and viability in human vein endothelial cells (HUVECs), cardiac fibroblasts, and neonatal rat cardiomyocytes (NRVMs). Specifically, we sought to distinguish effects attributable to the EVs themselves from those of external factors co‐isolated with, or adherent to, the EVs.EVs were concentrated from CDC‐conditioned medium by ultrafiltration using molecular weight cutoffs (MWCO) ranging from 3 kD to 1000 kD. We also concentrated the flow‐through from each preparation using 3 kD filters. HUVECs and NRVMs were exposed to concentrated conditioned medium (CCM) containing 5×108 EVs/mL, and ERK1/2 and Akt were assessed by Western blot. The degree of activation of ERK1/2 and Akt increased with increasing protein levels as MWCO decreased. The flow‐through from the 1000 kD and 100 kD preparations was also highly bioactive, indicating that freely‐diffusible growth factors contribute to ERK/Akt signaling. Preservation of viability in HUVECs subjected to growth factor withdrawal followed a similar pattern; however, the flow‐through did not enhance viability as effectively as it activated kinase signaling pathways. To assess the contribution of exosomal surface proteins or lipid mediators to activation of signaling, enhancement of viability, and uptake of EVs, we performed (1) thorough buffer exchange in 100 kD UFC filters, and (2) proteinase K treatment of the CCM. We found that “clean” 100 kD EVs did not activate ERK1/2 or Akt in any of the cell types tested, nor did they enhance viability upon growth factor withdrawal. Nevertheless, these EVs were taken up by HUVECs, and they were equally effective to 3 kD CCM/EVs (retaining all growth factors) in reducing the expression of leukocyte adhesion molecules after TNF stimulation. Proteinase K treated EVs did not induce ERK/Akt signaling or enhance viability.We conclude that soluble paracrine factors produced by CDCs activate pro‐survival and proliferative signaling pathways in cardiac cell types which enhance the effectiveness of EVs. While certain in vitro effects are attributable to EVs themselves, free growth factors contribute to bioactivity in conventional preparations of extracellular vesicles.Support or Funding InformationDepartment of Defense Award W81XWH‐16‐1‐0592This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.