Extracellular Vesicles Derived From Stem Cells in Intervertebral Disc Degeneration

Intervertebral disc degeneration (IVDD) is the leading cause of low back pain related to degradation of cartilaginous tissues, mainly resulting from oxidative stress, cell apoptosis, and extracellular matrix degradation. Extracellular vesicles (EVs) exist in all bodily fluids and can be produced by all types of cells. Stem cell-derived EVs (SC-EVs), which are the main paracrine components of stem cells, have gained significant attention in the field of regenerative medicine. Over the past years, accumulating evidence indicates the therapeutic and diagnostic potentials of EVs in IVDD. The main mechanisms involve the induction of regenerative phenotypes, apoptosis alleviation, and immune modulation. In addition, the efficiency of SC-EVs can be enhanced by choosing appropriate donor cells and cell phenotypes, optimizing cell culture conditions, or engineering EVs to deliver drugs and targeting molecules. Given the importance and novelty of SC-EVs, we give an overview of SC-EVs and discuss the roles of SC-EVs in IVDD.

Sonic Hedgehog acts as a macrophage chemoattractant during regeneration of the gastric epithelium

Sonic Hedgehog (Shh), secreted from gastric parietal cells, contributes to the regeneration of the epithelium. The recruitment of macrophages plays a central role in the regenerative process. The mechanism that regulates macrophage recruitment in response to gastric injury is largely unknown. Here we tested the hypothesis that Shh stimulates macrophage chemotaxis to the injured epithelium and contributes to gastric regeneration. A mouse model expressing a myeloid cell-specific deletion of Smoothened (LysMcre/+;Smof/f) was generated using transgenic mice bearing loxP sites flanking the Smo gene (Smo loxP) and mice expressing a Cre recombinase transgene from the Lysozyme M locus (LysMCre). Acetic acid injury was induced in the stomachs of both control and LysMcre/+;Smof/f (SmoKO) mice and gastric epithelial regeneration and macrophage recruitment analyzed over a period of 7 days post-injury. Bone marrow-derived macrophages (BM-Mø) were collected from control and SmoKO mice. Human-derived gastric organoid/macrophage co-cultures were established, and macrophage chemotaxis measured. Compared to control mice, SmoKO animals exhibited inhibition of ulcer repair and normal epithelial regeneration, which correlated with decreased macrophage infiltration at the site of injury. Bone marrow chimera experiments using SmoKO donor cells showed that control chimera mice transplanted with SmoKO bone marrow donor cells exhibited a loss of ulcer repair, and transplantation of control bone marrow donor cells to SmoKO mice rescued epithelial cell regeneration. Histamine-stimulated Shh secretion in human organoid/macrophage co-cultures resulted in macrophage migration toward the gastric epithelium, a response that was blocked with Smo inhibitor Vismodegib. Shh-induced macrophage migration was mediated by AKT signaling. In conclusion, Shh signaling acts as a macrophage chemoattractant via a Smo-dependent mechanism during gastric epithelial regeneration in response to injury.

Promising Roles of Exosomal microRNAs in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the loss of immune tolerance. Lupus nephritis (LN) is still a major cause of the morbidity and mortality of SLE. In clinical practice, diagnosis, and therapy of SLE is complicated and challenging due to lack of ideal biomarkers. Exosomes could be detected from numerous kinds of biological fluids and their specific contents are considered as hallmarks of autoimmune diseases. The exosomal miRNA profiles of SLE/LN patients significantly differ from those of the healthy controls making them as attractive biomarkers for renal injury. Exosomes are considered as optimal delivery vehicles owing to their higher stable, minimal toxicity, lower immunogenicity features and specific target effects. Endogenous miRNAs can be functionally transferred by exosomes from donor cells to recipient cells, displaying their immunomodulatory effects. In addition, it has been confirmed that exosomal miRNAs could directly interact with Toll-like receptors (TLRs) signaling pathways to regulate NF-κB activation and the secretion of inflammatory cytokines. The present Review mainly focuses on the immunomodulatory effects of exosomal-miRNAs, the complex interplay between exosomes, miRNAs and TLR signaling pathways, and how the exosomal-miRNAs can become non-invasive diagnostic molecules and potential therapeutic strategies for the management of SLE.

Extracellular Vesicles as Innovative Tools for AssessingAdverse Effects of Immunosuppressant Drugs

Background: Extracellular vesicles (EVs) are a heterogeneous family of small vesicles released by donor cells and absorbed by recipient cells, which represent important mediators with fundamental roles in both physiological and pathological conditions. EVs are present in a large variety of biological fluids and have a great diagnostic and prognostic value. They have gained the interest of the scientific community due to their extreme versatility. In fact, they allow us to hypothesize new therapeutic strategies since, in addition to being cell signal mediators, they play an important role as biomarkers, drug vehicles, and potential new therapeutic agents. They are also involved in immunoregulation, have the ability to transmit resistance to a drug from one cell to a more sensitive one, and can act as drug delivery systems. Objective: The main reciprocal interactions between EVs and immunosuppressive drugs will be presented. Results: The known interactions between EVs and immunosuppressive drugs, in particular, cyclosporin, glucocorticoids, rapamycin, methotrexate, cyclophosphamide, eculizumab, infliximab, certolizumab, etanercept, glatiramer acetate, and fingolimod are presented. Conclusion: This review provides relevant information on the links between EVs and immunosuppressive drugs with a focus on EVs' role as tools to assess effects of immunosuppressants, suggesting innovative properties and new possible therapeutic uses.

Mitochondrial Transfer in Saccharomyces cerevisiae

<p>This thesis investigated mitochondrial transfer in Saccharomyces cerevisiae, between respiratory compromised B18p⁰ recipient and respiratory competent donor cells. The respiratory compromised strain had three red fluorescent proteins tagged to the membrane, nucleus and cytoplasm (triple RFP-B18p⁰) and is referred to as the B18p⁰ strain. B18p⁰ cells did not contain mitochondrial DNA, causing it to be respiratory compromised and required a fermentable carbon source, such as glucose/dextrose, for proliferation. The respiratory competent strain used had a green fluorescent protein tagged to the Tom70 mitochondrial protein (Tom70-GFP) and is referred to as the Tom70 strain. The Tom70 cells contained the nuclear encoded URA3 cassette, allowing for negative selectivity of this strain using 5-FOA. S. cerevisiae strains were co-cultured together in media containing only non-fermentable carbon sources (YPGE), plated on YPGE plates containing 5-FOA and colonies grown were distinguished post-co-culture based on their distinct phenotypic and genotypic characteristics. Fluorescent analysis of co-culture colonies revealed the presence of 5-FOA resistant Tom70 cells and some red B18p⁰ cells that had acquired the ability to grow on non-fermentable carbon sources. Genotypic analysis revealed that the majority of these red colonies had acquired mtDNA as well as the nuclear encoded, Tom70 specific URA3 cassette. Several permutations of co-cultures were performed, using different ratios of recipient and donor cells and single-gene deletion donor cells. Purified mitochondria from Tom70 cells were tried to be transferred into B18p⁰ cells using centrifugation forces to induce a higher occurrence frequency of mitochondrial transfer. Metabolic support experiments were conducted to investigate if the Tom70 strain could provide metabolic support to the B18p⁰ strain without mitochondrial transfer. Results indicate that no permutation induced potential mitochondrial transfer at a higher rate than others. However, results indicate that mitochondrial transfer did occur at low frequencies, potentially through the fusion of respiratory competent and respiratory compromised cells. Forced transfer did not increase the occurrence frequency of B18p⁰ cells to take up mitochondria and Tom70 cells did not provide metabolic support to B18p⁰ cells.</p>

Mitochondrial Transfer in Saccharomyces cerevisiae

<p>This thesis investigated mitochondrial transfer in Saccharomyces cerevisiae, between respiratory compromised B18p⁰ recipient and respiratory competent donor cells. The respiratory compromised strain had three red fluorescent proteins tagged to the membrane, nucleus and cytoplasm (triple RFP-B18p⁰) and is referred to as the B18p⁰ strain. B18p⁰ cells did not contain mitochondrial DNA, causing it to be respiratory compromised and required a fermentable carbon source, such as glucose/dextrose, for proliferation. The respiratory competent strain used had a green fluorescent protein tagged to the Tom70 mitochondrial protein (Tom70-GFP) and is referred to as the Tom70 strain. The Tom70 cells contained the nuclear encoded URA3 cassette, allowing for negative selectivity of this strain using 5-FOA. S. cerevisiae strains were co-cultured together in media containing only non-fermentable carbon sources (YPGE), plated on YPGE plates containing 5-FOA and colonies grown were distinguished post-co-culture based on their distinct phenotypic and genotypic characteristics. Fluorescent analysis of co-culture colonies revealed the presence of 5-FOA resistant Tom70 cells and some red B18p⁰ cells that had acquired the ability to grow on non-fermentable carbon sources. Genotypic analysis revealed that the majority of these red colonies had acquired mtDNA as well as the nuclear encoded, Tom70 specific URA3 cassette. Several permutations of co-cultures were performed, using different ratios of recipient and donor cells and single-gene deletion donor cells. Purified mitochondria from Tom70 cells were tried to be transferred into B18p⁰ cells using centrifugation forces to induce a higher occurrence frequency of mitochondrial transfer. Metabolic support experiments were conducted to investigate if the Tom70 strain could provide metabolic support to the B18p⁰ strain without mitochondrial transfer. Results indicate that no permutation induced potential mitochondrial transfer at a higher rate than others. However, results indicate that mitochondrial transfer did occur at low frequencies, potentially through the fusion of respiratory competent and respiratory compromised cells. Forced transfer did not increase the occurrence frequency of B18p⁰ cells to take up mitochondria and Tom70 cells did not provide metabolic support to B18p⁰ cells.</p>

Regulating the production and biological function of small extracellular vesicles: current strategies, applications and prospects

Numerous studies have confirmed the great application potentials of small extracellular vesicles (sEVs) in biological medical field, especially in tissue repair and regeneration. However, the production capability of sEVs by noncancerous cells is very limited, while their dosage requirements in disease treatments are usually very high. Meanwhile, as cell aging, the sEV production capability of cells decreases and the biological function of sEVs changes accordingly. In addition, for special applications, sEVs carrying desired bioactive substances should be designed to perform their expected biological function. Therefore, improving the production of sEVs and precisely regulating their biological function are of great significance for promoting the clinical applications of sEVs. In this review, some of the current classic strategies in affecting the cellular behaviors of donor cells and subsequently regulating the production and biological function of their sEVs are summarized, including gene engineering methods, stress-inducing conditions, chemical regulators, physical methods, and biomaterial stimulations. Through applying these strategies, increased yield of sEVs with required biological function can be obtained for disease treatment and tissue repair, such as bone regeneration, wound healing, nerve function recovery and cancer treatment, which could not only reduce the harvest cost of sEV but promote the practical applications of sEVs in clinic. Graphical Abstract

Strategies for Engineering Exosomes and Their Applications in Drug Delivery

Exosomes are representative of a promising vehicle for delivery of biomolecules. Despite their discovery nearly 40 years, knowledge of exosomes and extracellular vesicles (EVs) and the role they play in etiology of disease and normal cellular physiology remains in its infancy. EVs are produced in almost all cells, containing nucleic acids, lipids, and proteins delivered from donor cells to recipient cells. Consequently, they act as mediators of intercellular communication and molecular transfer. Recent studies have shown that, exosomes are associated with numerous physiological and pathological processes as a small subset of EVs, and they play a significant role in disease progression and treatment. In this review, we discuss several key questions: what are exosomes, why do they matter, and how do we repurpose them in their strategies and applications in drug delivery systems. In addition, opportunities and challenges of exosome-based theranostics are also described and directions for future research are presented.

Harmful behaviour through plasmid transfer: a successful evolutionary strategy of bacteria harbouring conjugative plasmids

Conjugative plasmids are extrachromosomal mobile genetic elements pervasive among bacteria. Plasmids' acquisition often lowers cells' growth rate, so their ubiquity has been a matter of debate. Chromosomes occasionally mutate, rendering plasmids cost-free. However, these compensatory mutations typically take hundreds of generations to appear after plasmid arrival. By then, it could be too late to compete with fast-growing plasmid-free cells successfully. Moreover, arriving plasmids would have to wait hundreds of generations for compensatory mutations to appear in the chromosome of their new host. We hypothesize that plasmid-donor cells may use the plasmid as a ‘weapon’ to compete with plasmid-free cells, particularly in structured environments. Cells already adapted to plasmids may increase their inclusive fitness through plasmid transfer to impose a cost to nearby plasmid-free cells and increase the replication opportunities of nearby relatives. A mathematical model suggests conditions under which the proposed hypothesis works, and computer simulations tested the long-term plasmid maintenance. Our hypothesis explains the maintenance of conjugative plasmids not coding for beneficial genes. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Systemic Supplementation of Collagen VI by Neonatal Transplantation of iPSC-Derived MSCs Improves Histological Phenotype and Function of Col6-Deficient Model Mice

Collagen VI is distributed in the interstitium and is secreted mainly by mesenchymal stromal cells (MSCs) in skeletal muscle. Mutations in COL6A1-3 genes cause a spectrum of COL6-related myopathies. In this study, we performed a systemic transplantation study of human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) into neonatal immunodeficient COL6-related myopathy model (Col6a1KO/NSG) mice to validate the therapeutic potential. Engraftment of the donor cells and the resulting rescued collagen VI were observed at the quadriceps and diaphragm after intraperitoneal iMSC transplantation. Transplanted mice showed improvement in pathophysiological characteristics compared with untreated Col6a1KO/NSG mice. In detail, higher muscle regeneration in the transplanted mice resulted in increased muscle weight and enlarged myofibers. Eight-week-old mice showed increased muscle force and performed better in the grip and rotarod tests. Overall, these findings support the concept that systemic iMSC transplantation can be a therapeutic option for COL6-related myopathies.