Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

Revisiting the Role of CD63 as Pro-Tumorigenic or Anti-Tumorigenic Tetraspanin in Cancers and its Theragnostic Implications.

Cluster of differentiation antigen 63 (CD63) belongs to a superfamily of proteins, usually defined as tetraspanins which are known to transverse the bilayer membranes four times. The expression of CD63 has been shown to get altered in several cancers, where it has been demonstrated to act as both a tumor promoter and tumor suppressor. The present review describes the mechanism of how CD63 promotes tumor formation in certain cancer types while inhibiting in some other specific cancers. Glycosylation, a post-translational process plays a significant role in regulating the expression and function of these membrane proteins. Being a crucial exosomal flag protein, CD63 has been found to get involved in endosomal cargo sorting as well as the production of extracellular vesicles. Increased expression of exosomal CD63 derived from advanced tumors has demonstrated its role in promoting metastasis. CD63 also regulates the characteristic and function of stem cells on which they get expressed. This particular tetraspanin has been discovered to participate in gene fusion to perform distinctive roles in certain specific cancer types like breast cancer and pigmented epithelioid melanocytoma. Furthermore, this review mentions twelve different microRNAs obtained from miRDB that might target CD63. A few theragnostic uses of this membrane protein are also discussed. Thereby, the review indicates that further studies on CD63 might prove it to be an effective therapeutic target in different cancers in the coming future.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

A potential target gene CD63 for different degrees of intervertebral disc degeneration.

Understanding molecular mechanisms of intervertebral disc degeneration (IDD) and providing a novel target for the treatment of IDD have important implications. We sought to explore a new promising gene target for the treatment of IDD. This study integrated 19,678 genes of 38 IDD patients from two gene datasets. Differentially Expressed Genes (DEGs) of annulus fibrosus were analyzed in groups with mild disc degeneration (MDD) and severe disc degeneration (SDD). We screened the hub gene through biological information technology (bioinformatic) methods. Then, we further validated the hub gene using annulus fibrosus and nucleus pulposus tissues from 12 patients with qRT-PCR. In addition, we explored its underlying molecular mechanism with GO, KEGG and GSEA. Through multiple screening bioinformatics methods, the hub gene CD63 was identified. The qRT-PCR explored that CD63 decreased significantly in SDD group compared to that in MDD group (P < 0.001). The GO, KEGG and GSEA of CD63 explored significant enrichment of the molecular features (P < 0.001), including the cellular component (Extracellular matrix, P < 0.001), the molecular function (collagen binding, P < 0.001), the biological processes (protein targeting, collagen fibril organization and platelet degranulation, P < 0.001) and the signaling pathways. Our research explored and validated a new regulatory gene, CD63 for different degrees of IDD. A new novel form of therapeutic target for IDD may be developed.

Nucleofection of Adipose Mesenchymal Stem/Stromal Cells: Improved Transfection Efficiency for GMP Grade Applications.

Nucleofection (NF) is a safe, non-viral transfection method, compatible with Good Manufacturing Practice guidelines. Such a technique is useful to improve therapeutic effectiveness of adipose tissue mesenchymal stem cells (ASC) in clinical settings, but improvement of NF efficiency is mandatory. Supernatant rich in growth factors (SRGF) is a clinical-grade medium additive for ASC expansion. We showed a dramatically increased NF efficiency and post-transfection viability in ASC expanded in presence of SRGF (vs. fetal bovine serum). SRGF expanded ASC were characterized by increased vesicle endocytosis but lower phagocytosis properties. SRGF increased n-6/n-3 ratio, reduced membrane lipid raft occurrence, and lowered intracellular actin content in ASC. A statistical correlation between NF efficiency and lipid raft availability on cell membranes was shown, even though a direct relationship could not be demonstrated: attempts to selectively modulate lipid rafts levels were, in fact, limited by technical constraints. In conclusion, we reported for the first time that tuning clinical-grade compatible cell culture conditions can significantly improve ASC transfection efficiency by a non-viral and safe approach. A deep mechanistic characterization is extremely complex, but we can hypothesize that integrated changes in membrane structure and intracellular actin content could contribute to explain SRGF impact on ASC NF efficiency.

A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain.

The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.

CD63 is regulated by iron via the IRE-IRP system and is important for ferritin secretion by extracellular vesicles.

Extracellular vesicles (EVs) transfer functional molecules between cells. CD63 is a widely recognized EV marker that contributes to EV secretion from cells. However, the regulation of its expression remains largely unknown. Ferritin is a cellular iron storage protein that can also be secreted by the exosome pathway, and serum ferritin levels classically reflect body iron stores. Iron metabolism-associated proteins such as ferritin are intricately regulated by cellular iron levels via the iron responsive element-iron regulatory protein (IRE-IRP) system. Herein, we present a novel mechanism demonstrating that the expression of the EV-associated protein CD63 is under the regulation of the IRE-IRP system. We discovered a canonical IRE in the 5' untranslated region of CD63 messenger RNA that is responsible for regulating its expression in response to increased iron. Cellular iron loading caused a marked increase in CD63 expression and the secretion of CD63+ EVs from cells, which were shown to contain ferritin-H and ferritin-L. Our results demonstrate that under iron loading, intracellular ferritin is transferred via nuclear receptor coactivator 4 (NCOA4) to CD63+ EVs that are then secreted. Such iron-regulated secretion of the major iron storage protein ferritin via CD63+ EVs, is significant for understanding the local cell-to-cell exchange of ferritin and iron.

Expression profiling, immune functions, and molecular characteristics of the tetraspanin molecule CD63 from Amphiprion clarkii.

CD63, a member of the tetraspanin family, is involved in the activation of immune cells, antiviral immunity, and signal transduction. The economically important anemonefishes Amphiprion sp. often face disease outbreaks, and the present study aimed to characterize CD63 in Amphiprion clarkii (denoted AcCD63) to enable better disease management. The in-silico analysis revealed that the AcCD63 transcript is 723 bp long and encodes 240 amino acids. The 26.2 kDa protein has a theoretical isoelectric point of 5.51. Similar to other tetraspanins, AcCD63 consists of four domains: short N-/C-terminal domains and small/large extracellular loops. Pairwise sequence alignment revealed that AcCD63 has the highest identity (100%) and similarity (99.2%) with CD63 from Amphiprion ocellaris. Multiple sequence alignment identified a conserved tetraspanin CCG motif, PXSCC motif, and C-terminal lysosome-targeting GYEVM motif. The quantitative polymerase chain reaction analysis showed that AcCD63 was highly expressed in the spleen and head kidney tissue, with low levels of expression in the liver. Temporal expression patterns of AcCD63 were measured in the head kidney and blood tissue after injection of polyinosinic:polycytidylic acid (poly (I:C)), lipolysacharides (LPS), or Vibrio harveyi (V. harveyi). AcCD63 was upregulated at 12 h post-injection with poly (I:C) or V. harveyi, and at 24 h post-injection with all stimulants in the head kidney. At 24 h post-injection, poly (I:C) and LPS upregulated, whereas V. harveyi downregulated AcCD63 expression in the blood. All viral hemorrhagic septicemia virus transcripts (M, G, N, RdRp, P, and NV) were downregulated in response to AcCD63 overexpression, and removal of viral particles occurred via the involvement of AcCD63. The expression of antiviral genes MX dynamin-like GTPase 1, interferon regulatory factor 3, interferon-stimulated gene 15, interferon-gamma, and viperin in CD63-overexpressing fathead minnow cells was downregulated. Collectively, our findings suggest that AcCD63 is an immunologically important gene involved in the A. clarkii pathogen stress response.

Upregulation of miR-96-5p by bone marrow mesenchymal stem cells and their exosomes alleviate non-alcoholic steatohepatitis: Emphasis on caspase-2 signaling inhibition.

Non-alcoholic steatohepatitis (NASH) has evolved as the most common and devastating chronic liver disease. This study aimed to explore the underlined mechanism for the therapeutic potentials of bone marrow mesenchymal stem cells (BM-MSCs) and their derived exosomes (BM-MSCs-Exo) in an experimental model of high fat diet (HFD) induced NASH. Rats were fed with HFD for 12 weeks. At the seventh week, BM-MSCs were given at a dose of 1x106 cell i.v., per rat. A total of three doses of BM-MSCs were given per each rat in six weeks. BM-MSCs-Exo were given at a dose of 15, 30 and 120 µg/kg i.v., twice per week for six weeks. Perfect homing to the liver was detected. Beneficial effects were reported to BM-MSCs or BM-MSCs-Exo cotreatment; where the highest anti-steatotic effects were attributed to BM-MSCs-Exo (120 µg/kg) showing significant downregulation of fatty acid synthesis (SREB1, 2, ACC), downregulation in lipid uptake (CD36); accompanied by significant upregulation in fatty acid oxidation (PPARα, CPT1). These events were associated with abrogation of hepatic steatosis and ballooning in HFD-induced NASH. BM-MSCs or BM-MSCs-Exo cotreatment exerted significant anti-apoptotic effects mediated by significant decrease in Bax/Bcl2 ratio. Besides, significant increase in mitochondrial mitophagy genes (Parkin, PINK1, ULK1, BNIP3L, ATG5, ATG7, ATG12) were detected in BM-MSCs or BM-MSCs-Exo cotreated groups. These findings are thought to be modulated through upregulation of miRNA-96-5p which leads to downregulation of its downstream target caspase-2. Being a critical player in NASH development, caspase-2 targeting by miRNA-96-5p could be a promising therapeutic modality to treat NASH.

Molecular cloning and characterization of CD63 in common carp infected with koi herpesvirus.

CD63 is a member of the four-transmembrane-domain protein superfamily and is the first characterized tetraspanin protein. In the present study, we cloned the common carp (Cyprinus Carpio) CD63 (ccCD63) sequence and found that the ccCD63 ORF contained 711 bp and encoded a protein of 236 amino acids. Homology analysis revealed that the complete ccCD63 sequence had 84.08% amino acid similarity to CD63 of Sinocyclocheilus anshuiensis. Subcellular localization analysis revealed that ccCD63 was localized in the cytoplasm. Quantitative real-time PCR (qRT-PCR) analysis indicated that ccCD63 was expressed in the gill, intestine, liver, spleen, brain and kidney, with higher expression in spleen and brain tissues than in the other examined tissues. After koi herpesvirus (KHV) infection, these tissues exhibited various expression levels of ccCD63. The expression level was the lowest in the liver and highest in the brain; the expression level in the brain was 8.7-fold higher than that in the liver. Furthermore, knockdown of ccCD63 promoted KHV infection. Moreover, ccCD63 was correlated with the regulation of RIG-I/MAVS/TRAF3/TBK1/IRF3 and may be involved in the antiviral response through the RIG-I viral recognition signalling pathway in a TRAF3/TBK1-dependent manner. Taken together, our results suggested that ccCD63 upregulated the interaction of KHV with the host immune system and suppressed the dissemination of KHV.

Epstein-Barr Virus LMP1 Modulates the CD63 Interactome.

Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains; it is associated with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in the cellular trafficking of different proteins, EV cargo sorting, and vesicle formation. We have previously shown that CD63 is important in LMP1 trafficking to EVs, and this also affects LMP1-mediated intracellular signaling including MAPK/ERK, NF-κB, and mTOR activation. Using the BioID method combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as a network of proximal interacting proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism, and transportation. The CD63-only interactome was enriched in Rab GTPases, SNARE proteins, and sorting nexins, while adding LMP1 into the interactome increased the presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of protein enrichment from protein localization and vesicle-mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTOR, Nedd4 L, and PP2A, indicating the formation of a multiprotein complex with CD63, thereby potentially regulating LMP1-dependent mTOR signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into the network of partners required for endocytic trafficking and extracellular vesicle cargo sorting, formation, and secretion.

Engineered extracellular vesicles as versatile ribonucleoprotein delivery vehicles for efficient and safe CRISPR genome editing.

Transient delivery of CRISPR-based genome editing effectors is important to reduce off-target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer-binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com-binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three-component complex including CD63-Com fusion protein, com-modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV-delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety.

Resistance Training Diminishes the Expression of Exosome CD63 Protein without Modification of Plasma miR-146a-5p and cfDNA in the Elderly.

Aging-associated inflammation is characterized by senescent cell-mediated secretion of high levels of inflammatory mediators, such as microRNA (miR)-146a. Moreover, a rise of circulating cell-free DNA (cfDNA) is also related to systemic inflammation and frailty in the elderly. Exosome-mediated cell-to-cell communication is fundamental in cellular senescence and aging. The plasma changes in exercise-promoted miR-146a-5p, cfDNA, and exosome release could be the key to facilitate intercellular communication and systemic adaptations to exercise in aging. Thirty-eight elderly subjects (28 trained and 10 controls) volunteered in an 8-week resistance training protocol. The levels of plasma miR-146a-5p, cfDNA, and exosome markers (CD9, CD14, CD63, CD81, Flotillin [Flot]-1, and VDAC1) were measured prior to and following training. Results showed no changes in plasma miR-146a-5p and cfDNA levels with training. The levels of exosome markers (Flot-1, CD9, and CD81) as well as exosome-carried proteins (CD14 and VDAC1) remained unchanged, whereas an attenuated CD63 response was found in the trained group compared to the controls. These findings might partially support the anti-inflammatory effect of resistance training in the elderly as evidenced by the diminishment of exosome CD63 protein expression, without modification of plasma miR-146a-5p and cfDNA.

Environmental pH stress influences cellular secretion and uptake of extracellular vesicles.

Exosomes (extracellular vesicles/EVs) participate in cell-cell communication and contain bioactive molecules, such as microRNAs. However, the detailed characteristics of secreted EVs produced by cells grown under low pH conditions are still unknown. Here, we report that low pH in the cell culture medium significantly affected the secretion of EVs with increased protein content and zeta potential. The intracellular expression level and location of stably expressed GFP-fused CD63 (an EV tetraspanin) in HeLa cells were also significantly affected by environmental pH. In addition, increased cellular uptake of EVs was observed. Moreover, the uptake rate was influenced by the presence of serum in the cell culture medium. Our findings contribute to our understanding of the effect of environmental conditions on EV-based cell-cell communication.

Generation of cd63-deficient zebrafish to analyze the role of cd63 in viral infection.

The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys-Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.

A fusion of CD63-BCAR4 identified in lung adenocarcinoma promotes tumorigenicity and metastasis.

BACKGROUND: Recently, fusion variants of the breast cancer anti-oestrogen-resistance 4 (BCAR4) gene were recurrently discovered in lung adenocarcinoma from the genome-wide studies. However, the functional characterisation of BCAR4 fusion has not been investigated. METHODS: Based on the analysis of RNA-sequencing data, we identified a fusion transcript of CD63-BCAR4 in a Korean patient with lung adenocarcinoma who did not harbour any known activating mutations in EGFR and KRAS genes. To investigate the oncogenic effect of CD63-BCAR4, in vitro and in vivo animal experiments were performed. RESULTS: In vitro experiments showed strongly enhanced cell migration and proliferation by the exogenous expression of CD63-BCAR4 protein in bronchial epithelial cells. Cell migration was notably reduced after knockdown of BCAR4 fusion by small-interfering RNA. The tumorigenic and metastatic capability of the CD63-BCAR4 fusion was confirmed by using the mouse xenograft model. Fusion-overexpressed cells result in metastasis to the liver and lung as well as the primary tumours after subcutaneous injection into mice. Cyclin D1, MMP1, Slug and mesenchymal markers were significantly increased after CD63-BCAR4 overexpression in the in vitro and in vivo experiments. CONCLUSIONS: Taken together, our results suggest a newly identified fusion gene, CD63-BCAR4 as a potential novel oncogene in lung adenocarcinoma.

Functional Integrin Regulation Through Interactions with Tetraspanin CD9.

Integrins are adhesion receptors that mediate many intercellular and cell-extracellular matrix interactions with relevance in physiology and pathology. Unlike other cellular receptors, integrins critically require activation for ligand binding. Through interaction in cis with other molecules and the formation of tetraspanin-enriched membrane microdomains (TEMs), the tetraspanin CD9 regulates integrin activity and avidity. Here we present three techniques used to study CD9-integrin interactions and integrin activation.

Identification and characterization of dynamically regulated hepatitis-related genes in a concanavalin A-induced liver injury model.

BACKGROUND: Concanavalin A (ConA)-induced liver damage of mice is a well-established murine model mimicking the human autoimmune hepatitis (AIH). However, the pathogenic genes of the liver injury remain to be revealed. METHODS: Using time-series liver transcriptome, top dynamic genes were inferred from a set of segmented regression models, and cross-checked by weighted correlation network analysis (WGCNA). AIH murine models created by ConA were used to verify the in vivo effect of these genes. RESULTS: We identified 115 top dynamic genes, of which most were overlapped with the hub genes determined by WGCNA. The expression of several top dynamic genes including Cd63, Saa3, Slc10a1, Nrxn1, Ugt2a3, were verified in vivo. Further, Cluster determinant 63 (Cd63) knockdown in mice treated with ConA showed significantly less liver pathology and inflammation as well as higher survival rates than the corresponding controls. CONCLUSION: We have identified the top dynamic genes related to the process of acute liver injury, and highlighted a targeted strategy for Cd63 might have utility for the protection of hepatocellular damage.

Anti-Tumor Effects of Exosomes Derived from Drug-Incubated Permanently Growing Human MSC.

Similar to growth-limited human primary cultures of mesenchymal stroma/stem-like cells (MSC), the continuously proliferating human MSC544 cell line produced extracellular vesicles as characterized by expression of the tetraspanin molecules CD9, CD63, and CD81. Release of these particles was predominantly detectable during continuous cell growth of MSC544 in contrast to confluency-mediated transient growth arrest. For therapeutic use, these particles were isolated from proliferating MSC544 after taxol treatment and applied to different cancer cell cultures. A pronounced cytotoxicity of lung, ovarian, and breast cancer cells was observed primarily with taxol-loaded exosomes, similar to the effects displayed by application of taxol substance. While these findings suggested pronounced cancer cell targeting of MSC544 exosomes, a tumor therapeutic approach was performed using a mouse in vivo breast cancer model. Thus, intravenous injection of taxol-loaded MSC544 exosomes displayed superior tumor-reducing capabilities as compared to application of taxol exosomes by oral gavage. To broaden this therapeutic spectrum, epirubicin was applied to MSC544, and the derived exosomes likewise exhibited significant cytotoxic effects in different cancer cell cultures. These findings suggest an unlimited source for large-scale exosome production with reproducible quality to enable variable drug targeting of tumors or other diseases.

Visualization of extracellular vesicles in the regenerating caudal fin blastema of zebrafish using in vivo electroporation.

Zebrafish have high regenerative ability in several organs including the fin. Although various mechanisms underlying fin regeneration have been revealed, some mechanisms remain to be elucidated. Recently, extracellular vesicles (EVs) have been the focus of research with regard to their role in cell-to-cell communication. It has been suggested that cells in regenerating tissues communicate using EVs. In this study, we examined the involvement of EVs in the caudal fin regeneration of zebrafish using an in vivo electroporation method. The process of regeneration appeared normal after in vivo electroporation, and the transferred plasmid showed mosaic expression in the blastema. We took advantage of this mosaic expression to observe the distribution of exosomal markers in the blastema. We transferred exosomal markers by in vivo electroporation and identified EVs in the regenerating caudal fin. The results suggest that blastemal cells communicate with other cells via EVs during caudal fin regeneration.