An insight into Exosomal Cargo Isolated from three tissue specific Mesenchymal Stem Cells: Implication in Translational Research
Adult Mesenchymal stem cells (MSCs) derived exosomes have recently gained importance as a cell-free therapy for several chronic and inflammatory diseases. It is believed that these exosomes contain several biologically active molecules (i.e. miRNAs, proteins) which help to cure the diseases. The majority of published reports to date that have investigated the secretome of MSCs have done so using canonical expansion of cell culture conditions. However, the microenvironment experience by MSC post administration into animal models and patients is strikingly different. Thus instead of using secretome, Exosomes purified from adipose tissue (AD), Wharton Jelly (WJ) and Bone marrow BM) are widely studied. However, the content of the exosomes may differ based on their sources from where they are originated. It remains unclear, however, which types of proteins are packaged into exosomes compared with the cells from which they are derived. To explore these exosomes as a cell-free therapy for specific diseases, it is therefore, important to get comprehensive knowledge of the bioactive molecules present in the exosomes. In this study, we have purified the exosomes from three different sources (AD, WJ, and BM) and using RNA-Seq  approach, we have categorized the common and different microRNAs present in the exosomes. Overall design: Mesenchymal Stem Cells (MSCs) were isolated from three different sources i.e. Bone Marrow, Adipose tissue and Wharton's Jelly. These MSCs were propagated in DMEM (LG) media (Life Technologies, USA) containing 10% FBS (HyClone, USA), 2mM L-glutamine,100 U/ml of penicillin and 100 U/ml of streptomycin (Life Technologies, USA). These cells were sub-cultured at 70% confluence. The MSCs were stained for tri-lineage differentiation (adipocytes, osteocytes and chondrocytes) and cell specific surface markers by flow-cytometry which included CD 105, CD 73, CD 29 and CD 90, HLA-I, CD 34/45 and HLA Class II, as per the International Society for Cellular Therapy (ISCT) guidelines. For enrichment of exosomes, the MSCs were cultured in serum free media -STEMPRO® MSC SFM CTS (Thermo Fisher Scientific, USA) for 48 hours. The conditioned media from MSCs cultured for 48 h was pooled together for exosome isolation by ultracentrifugation and characterization for their size, morphology and surface markers by Nanoparticle Tracking Analysis (NanoSight LM20 (Malvern Instruments Company, Nanosight, Malvern, United Kingdom)), Electron Microscopy (Tecnai, FEI, USA) and Western Blotting respectively. After this, Exosomes were processed for total RNA isolation using miRCURY RNA Exosome Isolation kit (Exiqon, Denmark, Europe) following manufacturer's recommendations. These were then processed further for RNA sequencing.