Mesenchymal stem cell derived-exosomes: a modern approach in translational medicine
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Mesenchymal stem cell derived-exosomes: a modern approach in translational medicine
Mesenchymal stem cells (MSCs) have captured great attention in regenerative treatment for more than decades based on their differentiation capacity, strong immunomodulator properties, and their ability to be kululiped and manipulated. The recent investigation implies that the MSCS pleiotropic effect is not related to their differentiation capabilities, but is more mediated by the secretion of soluble paracrine factors. Exosom, nano extracellular vesicles, is one of these paracrine mediators.
Exosom functional cargo transfers such as MIRNA and MRNA molecules, peptides, protein, cytokines, and lipids from MSc to the recipient’s cell. Exocomic participates in communication events between cells and contributes to healing tissues and injured organs. Studies report that exosom alone is responsible for the therapeutic effect of MSC in various experimental models. Therefore, the exosom derived MSC can be manipulated and applied to establish a new cell-free therapeutic approach to the treatment of various diseases including the heart, kidney, liver, immune, and neurological disease, and healing of skin wounds. Compared to their donor cells, the exosom derived MSC offers more stable entities and reduced risk of safety regarding living cell administration, eg. Microvascular occlusion risk.
This review discusses the exosome insulation method found and utilized in clinical settings so far and present the latest information summary of the MSC exosomes in the treatment of translation. Exosom is an extracellular type of vesicle (EVS) issued by several types of mammal cells and involved in communication between cells. A number of studies have explored the potential diagnostic and exosomal therapeutic. The main challenge is the lack of standard and standard techniques for insulation and analysis of downstream nanovesicles. Conventional insulation method, such as ultracentrification, precipitation, filtration, chromatography, and an immune affinity-based approach, depends on certain physical properties or in surface biomarkers. However, one of the existing methods has limitations. Various parameters, such as efficacy, specificity, labor input, cost and scalability, and standardization options, must be considered for the appropriate approach.
The communication of extracellular vesicles between mesenchymal stroma cells and immune effect cells
Mesenchymal Stem / Stromal Cell (MSCS) is a multiphotic cell that is on the body’s stroma network and is able to promote network repair and weaken the inflammatory process through its immunomodulatory properties. Preclinical and clinical observations reveal that it is not only inter-cell communication that directly mediates MSC property; In fact, an important role is also played by the release of soluble and bioactive factors, such as cytokines, growth factors and extracellular vesicles (EVs). EVS is a membrane plated vesicel that contains various kinds of bioactive molecules, including lipids, proteins, and nucleic acids, such as RNA.
EVS let go of their content into target cells, thus affecting the fate of cells through intracellular process control. In addition, EV which is revealed MSC can mediate the effect of modulation towards different effector cells belonging to innate and adaptive immunity. In this review, we will discuss the literature data on EVS obtained by MSC, including the current standard method for their isolation and characterization, the mechanism that supports its immunoregulator properties, and their potential clinical applications as an alternative MSc-based therapy for inflammatory reactions, such as grafting disease Versus-hosted (GVHD).
Mesenchymal stem cell derived-exosomes: a modern approach in translational medicine
Large extracellular vesicles can be characterized by multiplex labeling using imaging flow cytometry
The extracellular vesicles (EVS) are heterogeneous (30 Nm-10 μm), content (lipid, RNA, DNA, protein) and potential function (s). Many isolation techniques systematically throw large EVS in the early stages of the small or exosomatic isolation protocols. We describe here a standardized method for isolating large eV from medulloblastoma cells and examine the expression of the EV marker and the diameter using the imaging flow cytometry. Our approach allows the characterization of each large EVS as an individual event, decorated with several fluorescent conjugal markers with the additional advantage of visualizing each event to ensure an application of robust gage strategies.
Methods: We describe the isolation of the step and characterization of a subset of large EVS from the UW228-2 medulloblastoma cell line evaluated by fluorescent light microscopy, transmission electron microscopy (TEM) and Tunable resistance detection (TRP). The viability of parental cells was evaluated by the Annexine V exposure by flow cytometry. Imagery flow cytometry (ImageReam Mark II) identified EVS by direct fluorescent membrane marking with orange cell mask (CMO) in conjunction with the EV markers. A rigorous guarantee algorithm based on the intensity of lateral diffusion and fluorescence has been applied and the expression of the EV markers CD63, CD9 and the lamp 1 evaluated.
Results: UW228-2 cells extend prolifically EVS upwards up to 6 μm. We show that the Imaging Flow Cytometer II allows a robust and reproducible analysis of the large EVS, including the evaluation of the diameter. We also demonstrate a correlation between the increase in the size of the EV and the co-expression of the filtered markers.
Description: The Viral DNA/RNA Isolation Kit is intended for rapid co-extraction of viral DNA and RNA from a variety of biofluid samples, such as, plasma, serum, milk and swap samples. The proprietary microspherical paramagnetic beads used in the kit have a large binding surfaces and a high affinity towards nucleic acids. Going through sample lysis/binding, washing, and elution steps, the whole process can be completed under 35 minutes, and yields highly pure nucleic acids elute. The recovered nucleic acids can be used in a wide range of applications, such as PCR, RT-PCR, Sanger Sequencing, NGS, and gene chips.
Description: Saliva DNA Isolation Kit provides a fast and simple spin column procedure for isolating high quality DNA from saliva samples collected and preserved using AcceGen's Saliva DNA Collection and Preservation Devices, as well as fresh saliva samples.Saliva DNA purified using AcceGen's kit is of the excellent quality, and is compatible with a number of downstream research applications including PCR, qPCR, Southern Blot analysis, sequencing and microarray analysis.
Description: EcoSpin Plasmid Isolation Kit is designed as a simple, convenient, and cost-effective purification of high quality plasmid DNA from recombinant E. coli cultures. The standard protocol lasts less than 25 minutes and yields up to 20 µg of plasmid DNA. The kit can be effectively used for purification of any size plasmids and cosmids. The relative plasmid yield and optimal culture size depend on the plasmid copy number and medium used for the bacterial culture.
EASYspin RNA Isolation Kit (For bone tissues)(need not DNase I digestion)
Conclusions: We have developed a labeling and gating strategy strictly capable of exploring the eV marker expression (CD63, CD9 and LAMP1) on individual EV in a largely heterogeneous population. Taken together, the data presented here strongly support the value of the exploration of large EVS in clinical samples for potential biomarkers, useful in the monitoring of diagnosis and disease surveillance.
