Characterization of the secretion of mesenchymal stem cells and its relevance to cardioprotection

Ph.DDOCTOR OF PHILOSOPH

Iodine Radiolabeled Mesenchymal Stem Cell (MSC)-Exosomes and Their CD73 Enzymatic Activities

IODINE RADIOLABELLED MESENCHYMAL STEM CELL (MSC)-EXOSOMES AND THEIR CD73 ENZYMATIC ACTIVITIES Chang-Tong Yang a,b*,Ruenn Chai Laic, Sai Kiang Limc, David Chee Eng Ng a,b a Department of Nuclear Medicine and Molecular Imaging, Radiological Sciences Division, Singapore General Hospital, Outram Road, 169608 Singapore; b Duke-NUS Medical School, 8 College Road, 169857 Singapore; cInstitute of Molecular & Cell Biology, 8A Biomedical Grove #05-16 Immunos, 138648, Singapore; *[email protected] Introduction MSC-derived exosomes have shown therapeutic potential in the areas of cardiovascular, orthopaedic, ophthalmologic, immune, dermatologic diseases and radiation sickness. Efficient radioisotope-labelling of exosomes remains as a challenging process. We demonstrate iodine-131 radiolabeled exosomes using both chloramine-T and Pierce Iodination methods, and characterized I-labelled exosomes via their CD73 enzymatic activities. Experimental methodology and results Two classic radio-iodination methods have been used to label exosomes due to several advantages: relatively long half-life of I-131 (half-life 8 days) and I-124 (half-life 4.2 days, 25.6% positron emission) could enable a desired tracking kinetics of exosomes in vitro and in vivo; radiolabeling of iodine to peptides and antibodies is a well-established chemistry; the unlabeled free iodine after radio-labelling can be easily removed to reach high radiochemical purity. By using chloramine-T, the radiolabeling yield of 131I-labeled-exosomes achieved ~30-40% with a radiochemical purity > 90% after running through PD10 column purification. Using Pierce Iodination, the radiolabeling yield drops to ~15-20%, radiochemical purity achieved >90% after the same purification process. The integrity of I-labeled-exosomes is important in the reproducibility and development of exosome clinical therapeutics. No radioactive iodine was labelled to exosome for characterization of their integrity. The results showed that chloramine T radiolabeling affected the structures of I-labeled-exosomes as the CD73 enzymatic activity of I-labeled exosomes was destroyed, the particle size became much larger and caused broader exosome size distribution. While with Pierce iodination the CD73 activity drops by 50 % when compared to that of the unlabeled exosomes, and the particles kept the same size. Conclusions Using chloramine T method showed that the CD73 enzymatic activity of I-labelled exosomes was destroyed, suggesting the labeling process damaged the structure of exosomes. By comparison, using Pierce Iodination method preserved the CD73 enzymatic activity, indicating that exosomes can be radiolabeled using Pierce Iodination for in vitro and in vivo tracking and pharmacokinetic studies

Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs

10.1186/1479-5876-9-47Journal of Translational Medicine9

Membrane lipid binding molecules for the isolation of bona fide extracellular vesicle types and associated biomarkers in liquid biopsy

Cancer exacts a heavy socioeconomic cost. Earlier detection and treatment are likely to mitigate this cost. Unfortunately, conventional tissue biopsy, the gold standard in cancer diagnosis cannot fulfill the goal of earlier detection. While liquid biopsy is a promising alternative to tissue biopsy, it has its challenges and limitations. A major challenge is the isolation of bona fide lipid membrane vesicles from biological fluids. In this review, we presented a new perspective of isolating different types of extracellular vesicles (EVs) by their affinity for membrane lipid binding ligands for liquid biopsy. EVs are lipid membrane particles naturally released by almost all cells and are found in almost all biological fluids suitable for liquid biopsy. They carry materials from the secreting cells that could affect the biology of the recipient cells and could thus inform on the state and progress of the disease. However, isolating bona fide EVs is a technical challenge as biological fluids have a complex composition and contain particles or aggregates that are physically similar to EVs. Here we review the use of membrane lipid-binding ligands to isolate different bona fide EV subtypes, and to circumvent the problem of co-isolating physically similar non-EV complexes in current EV isolation protocols. We will discuss the advantages of this technique and its potential for accelerated biomarker discovery and validation through examples of pre-clinical studies. We propose that isolating EV subtypes is a technically viable and robust strategy to overcome the current bottleneck of isolating EVs for liquid biopsy

MSC−sEV Treatment Polarizes Pro−Fibrotic M2 Macrophages without Exacerbating Liver Fibrosis in NASH

Mesenchymal stem/stromal cell small extracellular vesicles (MSC−sEVs) have shown promise in treating a wide range of animal models of various human diseases, which has led to their consideration for clinical translation. However, the possibility of contraindication for MSC−sEV use is an important consideration. One concern is that MSC−sEVs have been shown to induce M2 macrophage polarization, which is known to be pro−fibrotic, potentially indicating contraindication in fibrotic diseases such as liver fibrosis. Despite this concern, previous studies have shown that MSC−sEVs alleviate high−fat diet (HFD)−induced non−alcoholic steatohepatitis (NASH). To assess whether the pro−fibrotic M2 macrophage polarization induced by MSC−sEVs could worsen liver fibrosis, we first verified that our MSC−sEV preparations could promote M2 polarization in vitro prior to their administration in a mouse model of NASH. Our results showed that treatment with MSC−sEVs reduced or had comparable NAFLD Activity Scores and liver fibrosis compared to vehicle− and Telmisartan−treated animals, respectively. Although CD163+ M2 macrophages were increased in the liver, and serum IL−6 levels were reduced in MSC−sEV treated animals, our data suggests that MSC−sEV treatment was efficacious in reducing liver fibrosis in a mouse model of NASH despite an increase in pro−fibrotic M2 macrophage polarization

Topical application of mesenchymal stem cell exosomes alleviates the imiquimod induced psoriasis-like inflammation

10.3390/ijms22020720International Journal of Molecular Sciences2221-1

Assessment of tumorigenic potential in mesenchymal-stem/stromal-cell-derived small extracellular vesicles (MSC-sEV)

10.3390/ph14040345Pharmaceuticals14434

Isolation and characterization of exosome from human embryonic stem cell-derived c-myc-immortalized mesenchymal stem cells

Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular between 2006 and 2012. However, defined mechanisms of action underpinning the therapeutic efficacy of MSCs are lacking, but they are increasingly attributed to MSC trophic secretion rather than their differentiation potential. A promising secreted therapeutic candidate is an extracellular vesicle (EV) known as the exosome. The use of exosomes instead of cells as a therapeutic agent provides several advantages. A critical advantage is the prospect of a conventional pharmaceutical manufacturing process that is highly scalable and amenable to the stringent manufacturing process. For example, MSCs used as producers of therapeutics, and not as therapeutics per se, could be immortalized to generate infinitely expansible clonal lines to enhance the reproducible production of therapeutic exosomes. In this chapter, we will describe the immortalization of MSCs, and the production, isolation, and characterization of exosomes from immortalized MSC

Therapeutic MSC exosomes are derived from lipid raft microdomains in the plasma membrane

Background: Mesenchymal stem cell (MSC) was previously shown to secrete lipid vesicles that when purified by high performance liquid chromatography as a population of homogenously sized particles with a hydrodynamic radius of 55–65 nm reduce infarct size in a mouse model of myocardial ischemia/reperfusion injury. As these vesicles exhibit many biophysical and biochemical properties of exosomes, they were identified as exosomes. Here we investigated if these lipid vesicles were indeed exosomes that have an endosomal biogenesis. Method: In most cells, endocytosis is thought to occur at specialized microdomains known as lipid rafts. To demonstrate an endosomal origin for MSC exosomes, MSCs were pulsed with ligands e.g. transferrin (Tfs) and Cholera Toxin B (CTB) that bind receptors in lipid rafts. The endocytosed ligands were then chased to determine if they were incorporated into the exosomes. Results: A fraction of exogenous Tfs was found to recycle into MSC exosomes. When MSCs were pulsed with labelled Tfs in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, Tf incorporation in CD81-immunoprecipitate was reduced during the chase. CTB which binds GM1 gangliosides that are enriched in lipid rafts extracted exosome-associated proteins, CD81, CD9, Alix and Tsg101 from MSC-conditioned medium. Exogenous CTBs were pulse-chased into secreted vesicles. Extraction of Tf- or CTB-binding vesicles in an exosome preparation mutually depleted each other. Inhibition of sphingomyelinases reduced CTB-binding vesicles. Conclusion: Together, our data demonstrated that MSC exosomes are derived from endocytosed lipid rafts and that their protein cargo includes exosome-associated proteins CD81, CD9, Alix and Tsg101