Takeshi Katsuda

Human adipose tissue-derived mesenchymal stem cells secrete functional neprilysin-bound exosomes

Alzheimers disease (AD) is characterized by the accumulation of β-amyloid peptide (Aβ) in the brain because of an imbalance between Aβ production and clearance. Neprilysin (NEP) is the most important Aβ-degrading enzyme in the brain. Thus, researchers have explored virus-mediated NEP gene delivery. However, such strategies may entail unexpected risks, and thus exploration of a new possibility for NEP delivery is also required. Here, we show that human adipose tissue-derived mesenchymal stem cells (ADSCs) secrete exosomes carrying enzymatically active NEP. The NEP-specific activity level of 1 μg protein from ADSC-derived exosomes was equivalent to that of ~ 0.3 ng of recombinant human NEP. Of note, ADSC-derived exosomes were transferred into N2a cells, and were suggested to decrease both secreted and intracellular Aβ levels in the N2a cells. Importantly, these characteristics were more pronounced in ADSCs than bone marrow-derived mesenchymal stem cells, suggesting the therapeutic relevance of ADSC-derived exosomes for AD.

The therapeutic potential of mesenchymal stem cell-derived extracellular vesicles.

Extracellular vesicles (EVs), membrane vesicles that are secreted by a variety of mammalian cell types, have been shown to play an important role in intercellular communication. The contents of EVs, including proteins, microRNAs, and mRNAs, vary according to the cell type that secreted them. Accordingly, researchers have demonstrated that EVs derived from various cell types play different roles in biological phenomena. Considering the ubiquitous presence of mesenchymal stem cells (MSCs) in the body, MSC‐derived EVs may take part in a wide range of events. In particular, MSCs have recently attracted much attention due to the therapeutic effects of their secretory factors. MSC‐derived EVs may therefore provide novel therapeutic approaches. In this review, we first summarize the wide range of functions of EVs released from different cell types, emphasizing that EVs echo the phenotype of their parent cell. Then, we describe the various therapeutic effects of MSCs and pay particular attention to the significance of their paracrine effect. We then survey recent reports on MSC‐derived EVs and consider the therapeutic potential of MSC‐derived EVs. Finally, we discuss remaining issues that must be addressed before realizing the practical application of MSC‐derived EVs, and we provide some suggestions for enhancing their therapeutic efficiency.

Trash or Treasure: extracellular microRNAs and cell-to-cell communication

Circulating RNAs in human body fluids are promising candidates for diagnostic purposes. However, the biological significance of circulating RNAs remains elusive. Recently, small non-coding RNAs, microRNAs (miRNAs), were isolated from multiple human body fluids, and these “circulating miRNAs” have been implicated as novel disease biomarkers. Concurrently, miRNAs were also identified in the extracellular space associated with extracellular vesicles (EVs), which are small membrane vesicles secreted from various types of cells. The function of these secreted miRNAs has been revealed in several papers. Circulating miRNAs have been experimentally found to be associated with EVs; however, other types of extracellular miRNAs were also described. This review discusses studies related to extracellular miRNAs, including circulating miRNAs and secreted miRNAs, to highlight the importance of studying not only secreted miRNAs, but also circulating miRNAs to determine the contribution of extracellular miRNAs especially in cancer development.

Comparative marker analysis of extracellular vesicles in different human cancer types

Several cell types, including tumour cells, secrete extracellular vesicles (EVs), and tumour-derived EVs play a role in cancer initiation and progression. These vesicles include both a common set of membrane and cytosolic proteins and origin-specific subsets of proteins that likely correlated to cell type–associated functions. To confirm the presence of EVs in the preparations, researchers have identified so-called EV marker proteins, including the tetraspanin family proteins and such cytosolic proteins as heat shock 70 kDa protein 4 (HSP70) and tumour susceptibility gene 101 (TSG101). However, studies have shown that some EV markers are not always present in all EVs, which not only complicates the identification of EVs but also precludes the quantitative evaluation of EV proteins. Thus, it is strongly required to explore well-conserved EV marker proteins that are present at similar levels, regardless of their tissue or cellular origin. In this study, we compared the presence of 11 well-known EV marker proteins by immunoblotting using EVs isolated from 4 human prostate cell lines and 5 human breast cell lines, including cancer cells with different phenotypes. We found that all the tested EVs were positive for CD9 and CD81, with similar abundance that was irrespective of the EV origin. In contrast, other EV marker proteins, such as TSG101, Rab-5b and CD63, were detected in an inconsistent manner, depending on the origin of the EVs. Thus, we propose that the detection of CD9 and/or CD81 should ensure the presence of EVs.

The roles of extracellular vesicles in cancer biology: Toward the development of novel cancer biomarkers

Recent important progress in cancer biology was the identification of the significant roles played by extracellular vesicles (EVs). EVs are secreted by a variety of mammalian cell types and have been revealed to play important roles in intercellular communications. EVs serve as unique communication vehicles in many ways. First, unlike cytokine signaling, EVs enable transportation not only of proteins, but also of nucleic acids, including mRNAs and microRNAs. Recent reports showing the functionality of these nucleic acids in the recipient cells have opened up a new avenue of cell‐to‐cell communication research. Second, EVs have been revealed to transport membrane components including receptors, such as epithelial growth factor receptor. These findings have provided significant insights into understanding the molecular mechanisms of cancer development. Third, EVs protect their contents from clearance by degrading enzymes present in the extracellular space, which allows for remote transportation of the contents, even between organs. This concept is highlighted by recent reports that suggest the deep involvement of cancer cell derived EVs in metastasis. From these points of view, we will summarize recent studies on the relevance of EVs in cancer biology. We will also highlight the possibility of novel diagnostic technologies using circulating EVs in body fluid.

Clinical relevance and therapeutic significance of microRNA-133a expression profiles and functions in malignant osteosarcoma-initiating cells.

Novel strategies against treatment‐resistant tumor cells remain a challenging but promising therapeutic approach. Despite accumulated evidence suggesting the presence of highly malignant cell populations within tumors, the unsolved issues such as in vivo targeting and clinical relevance remain. Here, we report a preclinical trial based on the identified molecular mechanisms underlying osteosarcoma‐initiating cells and their clinical relevance. We identified key microRNAs (miRNAs) that were deregulated in a highly malignant CD133high population and found that miR‐133a regulated the cell invasion that characterizes a lethal tumor phenotype. Silencing of miR‐133a with locked nucleic acid (LNA) reduced cell invasion of this cell population, and systemic administration of LNA along with chemotherapy suppressed lung metastasis and prolonged the survival of osteosarcoma‐bearing mice. Furthermore, in a clinical study, high expression levels of CD133 and miR‐133a were significantly correlated with poor prognosis, whereas high expression levels of the four miR‐133a target genes were correlated with good prognosis. Overall, silencing of miR‐133a with concurrent chemotherapy would represent a novel strategy that targets multiple regulatory pathways associated with metastasis of the malignant cell population within osteosarcoma. Stem Cells 2014;32:959–973

Dark side of the exosome: the role of the exosome in cancer metastasis and targeting the exosome as a strategy for cancer therapy

Cell-cell communication is essential for the regulation of various biological phenomena in multicellular organisms, including development and homeostasis. Deregulation of these interactions leads to inappropriate cell-cell communication, resulting in disease development. Cancer cells communicate closely with the cells in their microenvironment, and this communication promotes malignancy via abnormal growth, invasion, drug resistance and metastasis. Understanding cell-cell interactions in cancer is essential for the development of novel anticancer agents. As a result, discovering the communication tools used by cancer cells is important to understanding these interactions. In this review, we summarize the recent findings regarding exosome-mediated cancer development. In addition, we propose that targeting the exosome represents a novel strategy for cancer therapy.

Transplantation of a fetal liver cell-loaded hyaluronic acid sponge onto the mesentery recovers a Wilson's disease model rat

An auxiliary liver represents a promising alternative for liver transplantation. The use of a large amount of mature hepatocytes, however, despite their high function, is limited in a clinical setting. Here, we propose a novel transplantation system that dramatically improved a diseased animal by incorporating fetal liver cells (FLCs) as a cell source, the mesentery as a transplantation site and a hyaluronic acid (HA) sponge as a cell scaffold. We transplanted wild-type Long Evans Agouti rat FLCs embedded in HA sponges onto the mesentery of Long Evans Cinnamon (LEC) rats, an animal model for Wilsons disease. The FLC-loaded HA sponges successfully grafted and consequently prevented jaundice. Accordingly, the treated animals showed a significant reduction in blood copper concentration, which consequently led to significant decreases in serum total bilirubin and direct bilirubin, and to a significant increase in albumin productivity. Furthermore, haematoxylin and eosin staining of the host livers demonstrated that fibrosis at the periportal area was moderated in the treated animals. In conclusion, we transplanted FLC-loaded HA sponges onto the mesenteric blood vessels, leading to thick, liver-like tissue possessing blood vessels, and the liver tissue engineered thus exhibited a remarkable therapeutic effect on the copper metabolism deficiency of LEC rats.

Commitment of Annexin A2 in recruitment of microRNAs into extracellular vesicles.

Extracellular vesicles (EVs) contain microRNAs (miRNAs). However, the exact molecular mechanisms of the recruitment of miRNAs in EVs are not well characterized. Based on proteomic analysis, we identified that silencing of Annexin A2 (ANXA2) significantly decreased the amount of miRNAs in EVs. In addition, microarray analysis revealed that ANXA2 regulated the loading of miRNAs into EVs in a sequence independent manner. Lastly, immunoprecipitation analysis confirmed that ANXA2 could bind miRNAs in EVs in the presence of Ca2+. These observations demonstrate that ANXA2 plays an important role in the packaging process of miRNAs into EVs.

Molecular signatures of mesenchymal stem cell-derived extracellular vesicle-mediated tissue repair

Extracellular vesicles (EVs) play important roles in intercellular communications via their content molecules, and mimic, at least in part, the roles that are played by their originating cells. Consistent with this notion, an increasing number of reports have suggested that EVs derived from mesenchymal stem cells (MSCs), which are therapeutically beneficial to a wide range of diseases, can serve as drugs to treat multiple diseases. EVs contain a variety of molecules, including proteins, microRNAs, and mRNAs, and are associated with biological processes in a content molecule-dependent manner. In this article, we review the latest reports regarding the therapeutic potential of MSC-EVs by focusing on the underlying molecular mechanisms of their effects. Specifically, we feature the effects of MSC-EVs in terms of their content molecules and of the tissue recovery processes endowed by these molecules.