Influence of the isolation method on characteristics and functional activity of mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND AIMS: Extracellular vesicle (EV) isolation methods are based on different physicochemical properties and may result in the purification of distinct EV populations. We compared two different isolation methods suitable for producing clinical-grade mesenchymal stromal cell-derived EVs (MSC-EVs)-ion exchange chromatography (IEX) and ultrafiltration (UF)-and evaluated their impact on the composition and functional properties of EVs. METHODS: EVs were purified from conditioned culture medium using an anion exchange resin (IEX) or Amicon filters with a 100-kDa cutoff (UF) (MilliporeSigma, Burlington, MA, USA). We assessed nanoparticle size and distribution by nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) and morphology by transmission electron microscopy. We also measured protein, lipid and total RNA concentration and immunophenotyped both EV populations by flow cytometry (MACSPlex assay; Miltenyi Biotec, Bergisch Gladbach, Germany). Moreover, immunomodulatory activity was tested using a standardized macrophage polarization assay and T-cell stimulation assay. Finally, proteomic analysis and cytokine quantification were carried out to better characterize both EV populations. RESULTS: We found by both TRPS and NTA that IEX and UF yielded a comparable amount of total particles with similar size and distribution. In addition, a similar quantity of lipids was obtained with the two procedures. However, IEX yielded 10-fold higher RNA quantity and a larger amount of proteins than UF. MSC-EVs isolated from IEX and UF were positive for the exosome markers CD9, CD63 and CD81 and showed a comparable surface marker expression pattern. Both populations demonstrated immunomodulatory activity in vitro, as they prevented acquisition of the M1 phenotype in lipopolysaccharide-stimulated macrophages and inhibited acquisition of the activation markers CD69 and CD25 on T cells, but the IEX-EVs exerted a significantly greater immunomodulatory effect on both macrophages and T cells compared with UF-EVs. Proteomic analysis and gene ontology enrichment analysis revealed no major differences between the preparations. Finally, cytokine quantification revealed that IEX-EVs were more enriched in some crucial anti-inflammatory and immunomodulatory cytokines (e.g., IL-2, IL-10, transforming growth factor beta and vascular endothelial growth factor) compared with UF-EVs. CONCLUSIONS: MSC-EVs isolated by IEX and UF displayed similar physicochemical, phenotypic and functional characteristics. In our conditions, both EV populations demonstrated important anti-inflammatory activity in macrophages and T cells. However, IEX-EVs were more potent than UF-EVs, which may indicate the superiority of this method for the production of clinical-grade EVs.

Exosome engineering for efficient and targeted drug delivery: Current status and future perspective.

Exosomes are membrane-bound vesicles that are released by most cells. They carry nucleic acids, cytokines, growth factors, proteins, lipids, and metabolites. They are responsible for inter- and intracellular communications and their role in drug delivery is well defined. Exosomes have great potential for therapeutic applications, but the clinical use is restricted because of limitations in standardized procedures for isolation, purification, and drug delivery. Bioengineering of exosomes could be one approach to achieve standardization and reproducible isolation for clinical use. Exosomes are important transporters for targeted drug delivery because of their small size, stable structure, non-immunogenicity, and non-toxic nature, as well as their ability to carry a wide variety of compounds. These features of exosomes can be enhanced further by bioengineering. In this review, possible exosome bioengineering approaches, their biomedical applications, and targeted drug delivery are discussed.

Klotho Deficiency Causes Heart Aging via Impairing the Nrf2-GR Pathway.

RATIONALE: Cardiac aging is an important contributing factor for heart failure, which affects a large population but remains poorly understood. OBJECTIVE: The purpose of this study is to investigate whether Klotho plays a role in cardiac aging. METHODS AND RESULTS: Heart function declined in old mice (24 months), as evidenced by decreases in fractional shortening, ejection fraction, and cardiac output. Heart size and weight, cardiomyocyte size, and cardiac fibrosis were increased in old mice, indicating that aging causes cardiac hypertrophy and remodeling. Circulating Klotho levels were dramatically decreased in old mice, which prompted us to investigate whether the Klotho decline may cause heart aging. We found that Klotho gene mutation (KL-/-) largely decreased serum klotho levels and impaired heart function. Interestingly, supplement of exogenous secreted Klotho prevented heart failure, hypertrophy, and remodeling in both old mice and KL (-/-) mice. Secreted Klotho treatment inhibited excessive cardiac oxidative stress, senescence and apoptosis in old mice and KL (-/-) mice. Serum phosphate levels in KL (-/-) mice were kept in the normal range, suggesting that Klotho deficiency-induced heart aging is independent of phosphate metabolism. Mechanistically, Klotho deficiency suppressed GR (glutathione reductase) expression and activity in the heart via inhibition of transcription factor Nrf2 (nuclear factor-erythroid 2 p45-related factor 2). Furthermore, cardiac-specific overexpression of GR prevented excessive oxidative stress, apoptosis, and heart failure in both old and KL (-/-) mice. CONCLUSIONS: Klotho deficiency causes cardiac aging via impairing the Nrf2-GR pathway. Supplement of exogenous secreted Klotho represents a promising therapeutic strategy for aging-associated cardiomyopathy and heart failure.

Cytidine deamination-induced perpetual immunity to SAR-CoV-2 infection is a potential new therapeutic target.

As the world is racing to develop perpetual immunity to the SARS-CoV-2 virus. The emergence of new viral strains, together with vaccination and reinfections, are all contributing to a long-term immunity against the deadly virus that has taken over the world since its introduction to humans in late December 2019. The discovery that more than 95 percent of people who recovered from COVID-19 had long-lasting immunity and that asymptomatic people have a different immune response to SARS-CoV-2 than symptomatic people has shifted attention to how our immune system initiates such diverse responses. These findings have provided reason to believe that SARS-CoV-2 days are numbered. Hundreds of research papers have been published on the causes of long-lasting immune responses and variations in the numbers of different immune cell types in COVID 19 survivors, but the main reason of these differences has still not been adequately identified. In this article, we focus on the activation-induced cytidine deaminase (AID), which initiates molecular processes that allow our immune system to generate antibodies against SARS-CoV-2. To establish lasting immunity to SARS-CoV-2, we suggest that AID could be the key to unlocking it.

Controlled Nutrient Delivery to Pancreatic Islets Using Polydopamine-Coated Mesoporous Silica Nanoparticles.

In the present study, we created a nanoscale platform that can deliver nutrients to pancreatic islets in a controlled manner. Our platform consists of a mesoporous silica nanoparticle (MSNP), which can be loaded with glutamine (G: an essential amino acid required for islet survival and function). To control the release of G, MSNPs were coated with a polydopamine (PD) layer. With the optimal parameters (0.5 mg/mL and 0.5 h), MSNPs were coated with a layer of PD, which resulted in a delay of G release from MSNPs over 14 d (57.4 ± 4.7% release). Following syngeneic renal subcapsule islet transplantation in diabetic mice, PDG-MSNPs improved the engraftment of islets (i.e., enhanced revascularization and reduced inflammation) as well as their function, resulting in re-establishment of glycemic control. Collectively, our data show that PDG-MSNPs can support transplanted islets by providing them with a controlled and sustained supply of nutrients.

SAR-CoV-2 infection, emerging new variants and the role of activation induced cytidine deaminase (AID) in lasting immunity.

As the world faces a fourth COVID-19 spike, scientists are learning a lot more about the new SARS-CoV-2 strains that were previously unknown. Currently, the Delta versions of SARS-CoV-2 have become the prevalent strains in much of the world since it first appeared in India in late 2020. Researchers believe they have discovered why Delta has been so successful: those infected with it create significantly more virus than those infected with the original strain of SARS-CoV-2, making it extremely contagious. This has redirected the focus to how our immune system defends us from these various pathogens and initiates such varied responses. Hundreds of research papers have been published on the origins of long-lasting immune responses and disparities in the numbers of different immune cell types in COVID 19 survivors, but the primary architect of these discrepancies has yet to be discovered. In this essay, we will concentrate on the primary architect protein, activation induced cytidine deaminase (AID), which triggers molecular processes that allow our immune system to produce powerful antibodies and SARS-CoV-2 specific B cells, allowing us to outwit the virus. We believe that if we ever achieve permanent immunity to SARS-CoV-2 infection, AID will be the key to releasing it.

HSP70-Mediated NLRP3 Inflammasome Suppression Underlies Reversal of Acute Kidney Injury Following Extracellular Vesicle and Focused Ultrasound Combination Therapy.

Acute kidney injury (AKI) is the abrupt loss of renal function, for which only supportive therapies exist. Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have been shown to be therapeutically effective in treating AKI by spurring endogenous cell proliferation and survival while suppressing inflammation. Pre-treating kidneys with pulsed focused ultrasound (pFUS) has also been shown to enhance MSC therapy for AKI, but its role in MSC-derived EV therapy remains unexplored. Using a mouse model of cisplatin-induced AKI, we show that combination therapy with pFUS and EVs restores physiological and molecular markers of kidney function, more so than either alone. Both pFUS and EVs downregulate heat shock protein 70 (HSP70), the NLRP3 inflammasome, and its downstream pro-inflammatory cytokines IL-1ß and IL-18, all of which are highly upregulated in AKI. In vitro knockdown studies suggest that HSP70 is a positive regulator of the NLRP3 inflammasome. Our study therefore demonstrates the ability of pFUS to enhance EV therapy for AKI and provides further mechanistic understanding of their anti-inflammatory and regenerative effects.

COVID-19 pandemic: Mechanistic approaches and gender vulnerabilities.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly pathogenic virus that causes coronavirus-19 disease (COVID-19), a severe respiratory damaging syndrome with serious health complications worldwide. SARS-CoV-2 was unfamilar before the epidemic started in Wuhan, China, in December 2019. COVID-19 is currently a pandemic influencing several countries worldwide. One of the mysteries of the new coronavirus is that it is deadlier for men than women with the male mortality rate is twice as high as that of females.

Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs).

Patients with breast cancer often develop malignant regrowth of residual drug-resistant dormant tumor cells years after primary treatment, a process defined as cancer relapse. Deciphering the causal basis of tumor dormancy therefore has obvious therapeutic significance. Because cancer cell behavior is strongly influenced by stromal cells, particularly the mesenchymal stem/stromal cells (MSCs) that are actively recruited into tumor-associated stroma, we assessed the impact of MSCs on breast cancer cell (BCC) dormancy. Using 3D cocultures to mimic the cellular interactions of an emerging tumor niche, we observed that MSCs sequentially surrounded the BCCs, promoted formation of cancer spheroids, and then were internalized/degraded through a process resembling the well-documented yet ill-defined clinical phenomenon of cancer cell cannibalism. This suspected feeding behavior was less appreciable in the presence of a rho kinase inhibitor and in 2D monolayer cocultures. Notably, cannibalism of MSCs enhanced survival of BCCs deprived of nutrients but suppressed their tumorigenicity, together suggesting the cancer cells entered dormancy. Transcriptome profiles revealed that the resulting BCCs acquired a unique molecular signature enriched in prosurvival factors and tumor suppressors, as well as inflammatory mediators that demarcate the secretome of senescent cells, also referred to as the senescence-associated secretory phenotype. Overall, our results provide intriguing evidence that cancer cells under duress enter dormancy after cannibalizing MSCs. Importantly, our practical 3D coculture model could provide a valuable tool to understand the antitumor activity of MSCs and cell cannibalism further, and therefore open new therapeutic avenues for the prevention of cancer recurrence.

Rapid analysis of cell surface N-glycosylation from living cells using mass spectrometry.

Cell surfaces are covered with a dense carbohydrate layer referred to as the glycocalyx. Because different cell types express different glycan signatures, it is of paramount importance to have robust methods to analyze the glycome of living cells. To achieve this, a common procedure involves cell lysis and extraction of membrane (glyco)proteins and yields a major proportion of high-mannose N-glycans that most likely stem from intracellular proteins derived from the ER. Using HEK 293 cells as a model system, we developed a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells. We directly released glycopeptides from cell surfaces through tryptic digestion of freshly harvested and vital cells, thereby improving the detection and quantification of complex-type N-glycans by increasing their relative amount from 14 to 85%. It was also possible to detect 25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1, and 51 in Hep G2 cells. The additional signals provided deeper insight into cell-type-specific N-glycan features such as antennarity, fucosylation, and sialylation. Thus, this protocol, which can potentially be applied to any cells, will be useful in the fields of glycobiotechnology and biomarker discovery.

Transdifferentiation of mesenchymal stem cells-derived adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and have a correlation with cell cycle arresting and driving genes.

It is generally accepted that after differentiation bone marrow mesenchymal stem cells (MSC) become lineage restricted and unipotent in an irreversible manner. However, current results imply that even terminally differentiated cells transdifferentiate across lineage boundaries and therefore act as a progenitor cells for other lineages. This leads to the questions that whether transdifferentiation occurs via direct cell-to-cell conversion or dedifferentiation to a progenitor cells and subsequent differentiation, and whether MSC potency decreases or increases during differentiation. To address these questions, MSC were differentiated into adipogenic lineage cells, followed by dedifferentiation. The process of dedifferentiation was also confirmed by single cell clonal analysis. Finally the dedifferentiated cells were used for adipogenesis, osteogenesis and chondrogenesis. Histology, FACS, qPCR and GeneChip analyses of undifferentiated MSC, adipogenic-differentiated and dedifferentiated cells were performed. Interestingly, gene profiling and bioinformatics demonstrated that upregulation (DHCR24, G0S2, MAP2K6, SESN3) and downregulation (DST, KAT2, MLL5, RB1, SMAD3, ZAK) of distinct genes have an association with cell cycle arrest in adipogenic-differentiated cells and perhaps narrow down the lineage potency. However, the upregulation (CCND1, CHEK, HGF, HMGA2, SMAD3) and downregulation (CCPG1, RASSF4, RGS2) of these genes have an association with cell cycle progression and maybe motivate dedifferentiation of adipogenic-differentiated cells. We found that dedifferentiated cells have a multilineage potency comparable to MSC, and also observed the associative role of proliferation genes with cell cycle arrest and progression. Concluded, our results indicate that transdifferentiation of adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and correlates with cell cycle arresting and deriving genes. Regarding clinical use, the knowledge of potency and underlying mechanisms are prerequisites.

From seaweed to healing: the potential of fucoidan in wound therapy.

This bibliometric review examines the current state of research on fucoidan, a sulphated polysaccharide found in brown seaweed species, and its potential for wound healing. The review included 58 studies that investigated fucoidan's effects on wound healing, revealing that it possesses anti-inflammatory and antioxidant properties that could aid in the healing process. Fucoidan was also found to promote cell proliferation, migration, and angiogenesis, essential for wound healing. However, the optimal dosage, treatment duration, safety, and efficacy of fucoidan in various wound types and patient populations still require further investigation. Additionally, advanced wound dressings like hydrogels have garnered significant attention for their potential in wound healing. While this review indicates promise for fucoidan as a natural wound healing compound, it underscores the need for additional clinical trials to determine its optimal use as a commercial therapeutic agent in wound healing.

The current trend of exosome in epithelial ovarian cancer studies: A bibliometric review.

Background: Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer. About 90% of ovary tumors are epithelial. The current treatment for EOC involves surgical debulking of the tumors followed by a combination of chemotherapy. While most patients achieve complete remission, many EOCs will recur and develop chemoresistance. The cancer cells can adapt to several stress stimuli, becoming resistant. Therefore, new ways to fight resistant cells during the disease are being studied. Recently, exosomes, which reflect cell behavior in normal and pathological conditions such as epithelial ovarian cancer, are of academic interest as new biomarkers for diagnosis and therapy. Consequently, the current study aimed to investigate the research output of exosomes in EOC. Method: A bibliometric method was used for analyzing publications on exosome and epithelial ovarian cancer from the beginning to 15 October 2022 by searching keywords in Scopus, PubMed and Google scholar. Annual scientific publications, authors, citations, journals, co-authorships, and keywords co-occurrence were analyzed and plotted using Microsoft Office Excel and VOS viewer. 39 original journal articles and 3 reviews have been published since 2015 up to 15 October 2022. Results: The findings showed that China is the top country in research output, international collaborations, organization, author, and sponsorship. The top journals were the Journal of Ovarian Research, Oncotarget, and Tumor Biology, all in the United States. The top institution was Shanghai Jiao Tong University in China. The top author was Xipeng Wang. Co-occurrence analysis showed that academics' interest is toward:1) 1) Exosomes as prognostic biomarkers of EOC as well as their role in the proliferation and migration of cells. 2) The role of exosomes in metastasis through different mechanisms; 3) The role of exosomes in epithelial-mesenchymal transition of ovarian cancer cells; 4) The diagnostic role of EVs in EOC; and 5) Conferring chemoresistance in EOC through the exosomal transfer of miRNAs. Conclusion: Research on the exosome and EOC has an increasing trend, and China is much more involved than other countries in research, financial support, and international cooperation. These findings could aid researcher in understanding novel ideas and subjects interested by sponsors in this field.

Role of CD9 Sensing, AI, and Exosomes in Cellular Communication of Cancer.

Exosomes are small membrane-bound vesicles that are released by various types of cells, including cancer cells, and play a role in intercellular communication. CD9 is a protein that is involved in cell signaling and adhesion. It is found on the surface of various cells, including cancer cells, and has been implicated in the communication between cancer cells and their microenvironment. Exosomes are small membrane-bound vesicles that are released by cells and contain various bioactive molecules, such as proteins, lipids, and nucleic acids. Exosomes have been shown to play a role in intercellular communication, and they have been implicated in the progression of cancer. There is evidence to suggest that CD9 is involved in the packaging and release of exosomes by cancer cells. CD9 has been shown to be important for the formation of tetraspanin-enriched microdomains (TEMs) on the surface of exosomes. These TEMs are thought to be important for the sorting and packaging of specific molecules into exosomes. In summary, CD9 appears to play an important role in the communication between cancer cells and their microenvironment via exosomes. The precise mechanisms by which CD9 mediates this communication are still being investigated, but the involvement of CD9 in exosome packaging and uptake suggests that it may be a promising target for the development of novel cancer therapies. Furthermore, CD9 has been shown to be involved in the uptake of exosomes by recipient cells. For example, studies have shown that CD9-positive exosomes released by cancer cells can be taken up by other cancer cells, leading to the transfer of oncogenic molecules and the promotion of cancer progression.

Different Sourced Extracellular Vesicles and Their Potential Applications in Clinical Treatments.

Extracellular vesicles (EVs) include a heterogeneous group of natural cell-derived nanostructures that are increasingly regarded as promising biotherapeutic agents and drug delivery vehicles in human medicine. Desirable intrinsic properties of EVs including the ability to bypass natural membranous barriers and to deliver their unique biomolecular cargo to specific cell populations position them as fiercely competitive alternatives for currently available cell therapies and artificial drug delivery platforms. EVs with distinct characteristics can be released from various cell types into the extracellular environment as a means of transmitting bioactive components and altering the status of the target cell. Despite the existence of a large number of preclinical studies confirming the therapeutic efficacy of different originated EVs for treating several pathological conditions, in this review, we first provide a brief overview of EV biophysical properties with an emphasis on their intrinsic therapeutic benefits over cell-based therapies and synthetic delivery systems. Next, we describe in detail different EVs derived from distinct cell sources, compare their advantages and disadvantages, and recapitulate their therapeutic effects on various human disorders to highlight the progress made in harnessing EVs for clinical applications. Finally, knowledge gaps and concrete hurdles that currently hinder the clinical translation of EV therapies are debated with a futuristic perspective.

Methodologies to Isolate and Purify Clinical Grade Extracellular Vesicles for Medical Applications.

The use of extracellular vesicles (EV) in nano drug delivery has been demonstrated in many previous studies. In this study, we discuss the sources of extracellular vesicles, including plant, salivary and urinary sources which are easily available but less sought after compared with blood and tissue. Extensive research in the past decade has established that the breadth of EV applications is wide. However, the efforts on standardizing the isolation and purification methods have not brought us to a point that can match the potential of extracellular vesicles for clinical use. The standardization can open doors for many researchers and clinicians alike to experiment with the proposed clinical uses with lesser concerns regarding untraceable side effects. It can make it easier to identify the mechanism of therapeutic benefits and to track the mechanism of any unforeseen effects observed.

Diagnostic and Therapeutic Potential of Extracellular Vesicles.

Extracellular vesicles (EVs) are naturally phospholipid enclosed nanovesicles released by many cells in the body. They are stable in circulation, have low immunogenicity, and act as carriers for functionally active biological molecules. They interact with target organs and bind to the receptors. Their target specificity is important to use EVs as noninvasive diagnostic and prognostic tools. EVs play a vital role in normal physiology and cellular communication. They are known to protect their cargo from degradation, which makes them important drug carriers for targeted drug delivery. Using EVs with markers and tracking their path in systemic circulation can be revolutionary in using them as diagnostic tools. We will discuss the scope of this in this paper. Although there are limitations in EVs isolation and storage, their high biocompatibility will fuel more innovations to overcome these challenges.

Artificial intelligence and guidance of medicine in the bubble.

Microbubbles are nanosized gas-filled bubbles. They are used in clinical diagnostics, in medical imaging, as contrast agents in ultrasound imaging, and as transporters for targeted drug delivery. They can also be used to treat thrombosis, neoplastic diseases, open arteries and vascular plaques and for localized transport of chemotherapies in cancer patients. Microbubbles can be filled with any type of therapeutics, cure agents, growth factors, extracellular vesicles, exosomes, miRNAs, and drugs. Microbubbles protect their cargo from immune attack because of their specialized encapsulated shell composed of lipid and protein. Filled with curative medicine, they could effectively circulate through the whole body safely and efficiently to reach the target area. The advanced bubble-based drug-delivery system, integrated with artificial intelligence for guidance, holds great promise for the targeted delivery of drugs and medicines.