Comparative proteomic profiling of Small Extracellular vesicles derived from iPSCs and tissue specific mesenchymal stem cells.

BACKGROUND: Small Extracellular vesicles (EV) are emerging as crucial intercellular messengers that contribute to the physiological processes. EVs contain numerous functional proteins and nucleic acids derived from their parent cells and have different roles depending on their origin. Functionally, EVs transfer these biological materials from the parent cell to the recipient and thus exhibits a novel therapeutic platform for delivering therapeutics molecules to the target tissue. In this regard, EVs derived from stem cells such as Mesenchymal Stem Cells and iPSCs have demonstrated a higher ability to benefit regenerative medicine. Even though these stem cells share some common properties, due to the differences in their origin (cell sources, the hierarchy of potency, etc) the EVs cargo profiling and functionality may vary. METHOD: We used iTRAQ-based proteomic analysis to conduct a comprehensive and quantitative evaluation of EVs derived from iPSCs and various tissue-specific MSCs in this study. Additionally, the data was analyzed using a variety of bioinformatic tools, including ProteinPilot for peptide and protein identification and quantification; Funrich, GO, Reactome, and KEGG (Kyoto Encyclopedia of Genes and Genomes) for pathway enrichment; the STRING database, and the inBio Discover tool for identifying known and predicted Protein-Protein networks. RESULTS: Bioinformatics analysis revealed 223 differentially expressed proteins in these EVs; however, Wharton's jelly MSC-EV contained more exclusive proteins with higher protein expression levels. Additionally, 113 proteins were abundant in MSC-EVs, while others were shared between MSC-EVs and iPSC-EVs. Further, based on an in-depth examination of the proteins, their associated pathways, and their interactions with other proteins, it was determined that these proteins are involved in bone regeneration (9.3%), wound healing (4.4%), immune regulation (8.9%), cardiac regeneration (6.6%), neuro regeneration (8.9%), and hepatic regeneration (3.5%). CONCLUSION: Overall, the results of our proteomic analysis indicate that EVs derived from MSCs have a more robust profile of proteins with higher expression levels than iPSCs. This is a significant finding, as it demonstrates the critical therapeutic role of EVs in a variety of diseases, as demonstrated by enrichment analysis, their versatility, and broad application potential.

Hypoxia preconditioning elicit differential response in tissue-specific MSCs via immunomodulation and exosomal secretion.

Mesenchymal stromal cells (MSCs) are emerging as an ideal candidate for regenerative medicine. It is known that the culture conditions impact the cellular properties of MSCs and their therapeutic behavior. Moreover, maintenance of MSCs in low oxygen tension for a short duration has shown to be beneficial for MSCs as it is similar to that of their physiological niche. However, the precise mechanism through which hypoxia pre-conditioning affects MSCs is not clear yet. Thus, in this study, we have investigated the effect of hypoxia exposure (1% O2) on tissue-specific MSCs over a period of time under serum-free culture conditions and evaluated the changes in expression of immuno-modulatory molecules and exosome biogenesis and secretion markers. It was observed that all MSCs responded differentially towards hypoxia exposure as indicated by the expression of HIF-1α. Moreover, this short-term exposure did not induce any changes in MSCs cellular morphology, proliferation rate, and surface marker profiling. In addition, we observed an enhancement in the expression of immunomodulatory factors (HLA-G, PGE-2, and IDO) after hypoxia exposure of 12 to 24 h in all tissue-specific MSCs. Interestingly, we have also observed the upregulation in exosome secretion that was further corelated to the upregulation of expression of exosome biogenesis and secretion markers (ALIX, TSG101, RAB27a, RAB27b). Though there was a differential response of MSCs where WJ-MSCs and BM-MSCs showed upregulation of these markers at 6-12 h of hypoxia pre-conditioning, while AD-MSCs showed similar changes beyond 24 h of hypoxia exposure.

MicroRNA-Enriched Exosomes from Different Sources of Mesenchymal Stem Cells Can Differentially Modulate Functions of Immune Cells and Neurogenesis.

Adult Mesenchymal stem cells-derived exosomes carry several biologically active molecules that play prominent roles in controlling disease manifestations. The content of these exosomes, their functions, and effect on the immune cells may differ depending on their tissue sources. Therefore, in this study, we purified the exosomes from three different sources and, using the RNA-Seq approach, highly abundant microRNAs were identified and compared between exosomes and parental cells. The effects of exosomes on different immune cells were studied in vitro by incubating exosomes with PBMC and neutrophils and assessing their functions. The expression levels of several miRNAs varied within the different MSCs and exosomes. Additionally, the expression profile of most of the miRNAs was not similar to that of their respective sources. Exosomes isolated from different sources had different abilities to induce the process of neurogenesis and angiogenesis. Moreover, these exosomes demonstrated their varying effect on PBMC proliferation, neutrophil survival, and NET formation, highlighting their versatility and broad interaction with immune cells. The knowledge gained from this study will improve our understanding of the miRNA landscape of exosomes from hMSCs and provide a resource for further improving our understanding of exosome cargo and their interaction with immune cells.

Mesenchymal Stem Cell Derived Exosomes: a Nano Platform for Therapeutics and Drug Delivery in Combating COVID-19.

The recent pandemic situation transpired due to coronavirus novel strain SARS-CoV-2 has become a global concern. This human coronavirus (HCov-19) has put the world on high alert as the numbers of confirmed cases are continuously increasing. The world is now fighting against this deadly virus and is leaving no stone unturned to find effective treatments through testing of various available drugs, including those effective against flu, malaria, etc. With an urgent need for the development of potential strategies, two recent studies from China using Mesenchymal Stem Cells (MSCs) to treat COVID-19 pneumonia have shed some light on a potential cure for the COVID-19 infected patients. However, MSCs, despite being used in various other clinical trials have always been questioned for their tendency to aggregate or form clumps in the injured or disease microenvironment. It has also been reported in various studies that exosomes secreted by these MSCs, contribute towards the cell's biological and therapeutic efficacy. There have been reports evaluating the safety and feasibility of these exosomes in various lung diseases, thereby proposing them as a cell-free therapeutic agent. Also, attractive features like cell targeting, low-immunogenicity, safety, and high biocompatibility distinguish these exosomes from other synthetic nano-vesicles and thus potentiate their role as a drug delivery nano-platform. Building upon these observations, herein, efforts are made to give an overview of stem cell-derived exosomes as an appealing therapeutic agent and drug delivery nano-carrier. In this review, we briefly recapitulate the recent evidence and developments in understanding exosomes as a promising candidate for novel nano-intervention in the current pandemic scenario. Furthermore, this review will highlight and discuss mechanistic role of exosomes to combat severe lung pathological conditions. We have also attempted to dwell into the nano-formulation of exosomes for its better applicability, storage, and stability thereby conferring them as off the shelf therapeutic.