Mesenchymal stromal cells and autoimmunity.

Abstract Mesenchymal stromal cells (MSCs) are committed progenitors of mesodermal origin that are found virtually in every organ and exhibit multilineage differentiation into osteocytes, adipocytes and chondrocytes. MSCs also mediate a wide spectrum of immunoregulatory activities that usually dampen innate and adaptive immune responses. These features have attracted interest in the perspective of developing novel cell therapies for autoimmune disease. However, depending on the microenvironmental conditions, MSCs may show a plastic behavior and switch to an immunostimulatory phenotype. After thorough characterization of the effects of MSCs on the immune system, MSC cell therapy has been tested in animal models of autoimmunity using different cell sources, protocols of in vitro expansion and routes and schedules of administration. The pre-clinical results have been encouraging in some models [e.g. Crohn's disease (CD), multiple sclerosis] and heterogeneous in others (e.g. graft-versus-host disease, systemic lupus erythematosus, rheumatoid arthritis). Clinical trials have been carried out and many are ongoing. As discussed, the results obtained are too preliminary to draw any conclusion, with the only exception of topical administration of MSCs in CD that has proven efficacious. The mechanism of action of infused MSCs is still under investigation, but the apparent paradox of a therapeutic effect achieved in spite of the very low number of cells reaching the target organ has been solved by the finding that MSC-derived extracellular vesicles (EVs) closely mimic the therapeutic activity of MSCs in pre-clinical models. These issues are critically discussed in view of the potential clinical use of MSC-derived EVs

치료 효능이 증가된 중간엽 줄기세포 유래 나노베지클

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 화학생물공학부, 2020. 8. 김병수.중간엽 줄기세포 이식의 안전 관련 쟁점과 낮은 이식 생존율로 인해, 중간엽 줄기세포 유래 엑소좀은 심근경색과 척수 손상을 포함한 다양한 질병의 대체 치료제로서 각광받고 있다. 중간엽 줄기세포 유래 엑소좀이 중간엽 줄기세포 치료에 비해 갖는 장점에도 불구하고, 낮은 생산성과 낮은 표적 효능으로 인해 임상 적용에 여러 가지의 제한이 있는 상황이다. 이러한 점들을 극복하기 위해, 엑소좀-모방 나노베지클이 기존 엑소좀의 대체제로서 연구되고 있다. 그러나, 나노베지클 또한 개질 없이 표적 장기에 충분한 양이 축적되지 못한다. 본 연구에서는, 가공을 통해 치료 효능과 표적 물질이 향상된 중간엽 줄기세포로부터 얻은, 치료 효능이 증가된 나노베지클을 개발하였다. 먼저, 우리는 철나노입자가 함유된 중간엽 줄기세포 유래 나노베지클(철나노입자-나노베지클)을 개발하였다. 철나노입자-나노베지클은 경색이 온 심장에서의 잔류량은 자성 유도로 인해 극적으로 증가하였다. 게다가, 철나노입자는 철나노입자를 함유한 중간엽 줄기세포와 철나노입자-나노베지클의 치료성 RNA와 단백질을 증가시켰고, 이는 엑소좀의 낮은 생산성에 대한 우려를 감소시켰다. 철나노입자-나노베지클의 심장으로의 주사와 자성 유도는 염증 단계에서 치료 단계로의 이른 전환과 세포 사멸 및 섬유화 감소, 그리고 혈관 생성과 심기능 회복 효과를 향상시켰다. 이 접근법은 중간엽 줄기세포 유래 나노베지클 치료법의 치료 효능을 높이고, 심근 경색에 대한 임상적용의 길을 열어줄 것이다. 두 번째로, 우리는 대식 세포 세포막이 융합된 중간엽 줄기세포 (대식세포막 융합-중간엽 줄기세포)로부터 대식 세포 세포막이 융합된 엑소좀-모방 나노베지클(대식세포막 융합-나노베지클)을 개발하였고, 이의 치료 효능을 쥐의 척수손상 모델에서 확인하였다. 대식세포막 융합-나노베지클은 일반 중간엽 줄기세포 유래 나노베지클에 비해 허혈성 부위 표적 효능물질을 많이 포함하였다. 대식세포막으로부터 유래된 대식세포막 융합-나노베지클에 포함된 표적 효능물질은 대식세포막 융합-나노베지클을 전신 주사하였을 때 척수로의 축적을 증가시켰다. 대식세포막 융합-나노베지클의 증가된 축적은 세포 사멸과 염증을 감소시켰고, 축삭 손실을 예방, 혈관 형성을 촉진, 섬유화를 감소하였고, 이어서 척수 기능 향상시켰다. 이 결과들은 대식세포막 융합-나노베지클의 임상적용 가능성을 제시한다. 종합하여, 우리는 철나노입자-중간엽줄기세포와 대식세포막 융합-중간엽줄기세포로부터 얻은 나노베지클에서 경색이 온 심장과 다친 척수로의 축적을 증가시켜주는 치료 효능물질 또는 표적 효능물질이 증가한 것을 보여주었다. 개질된 중간엽 줄기세포로부터 유래된 나노베지클의 치료 효능을 높이는 우리의 새로운 접근법은 기존의 중간여 ㅂ줄기세포 또는 중간엽 줄기세포 유래 엑소좀 치료법의 좋은 대안이 될 것이다. 우리의 기술은 허혈성/염증성 질환에 대한 치료법으로 미래에 임상적용이 가능할 것이다.Due to the safety issues and poor engraftment of mesenchymal stem cell (MSC) implantation, MSC-derived exosomes have been spotlighted as an alternative therapy for wide variety of diseases including myocardial infarction (MI) and spinal cord injury (SCI). Despite of the advantages of exosomes over MSC therapies, there are several limitations for clinical application due to the very small productivity and poor targeting ability to diseased organs after administration. To overcome these obstacles, exosome-mimetic extracellular nanovesicles (NVs) have been developed as an alternative to conventional exosomes. However, NVs also poorly accumulate in target organs without modification. Here, we developed therapeutic potential-enhanced NVs derived from MSCs that were engineered to possess increased therapeutic and targeting molecules. First, we developed NVs derived from iron oxide nanoparticles (IONPs)-incorporated MSCs (IONP-MSCs). The retention of injected IONP-MSC-derived NVs (IONP-NVs) within the infarcted heart was dramatically augmented by magnetic guidance. Furthermore, IONPs significantly increased the levels of therapeutic RNAs and proteins in IONP-MSCs as well as IONP-NVs, which can reduce the concern of low exosome-productivity. The injection of IONP-NVs into the infarcted heart and magnetic guidance induced an early shift from the inflammation phase to the reparative phase, reduced apoptosis and fibrosis, and enhanced angiogenesis and cardiac function recovery. This approach can enhance the therapeutic potency of an MSC-derived NV therapy and may pave the way for the clinical application of MI. Second, we fabricated macrophage membrane-fused-exosome-mimetic nanovesicles (MF-NVs) from macrophage membraned-fused MSCs (MF-MSCs) and confirmed their therapeutic potential in a clinically relevant mouse SCI model (controlled mechanical compression injury model). MF-NVs contained larger quantity of ischemic region-targeting molecules compared to normal MSC-derived nanovesicles. The targeting molecules in MF-NVs, which were derived from macrophage membranes, increased the accumulation of MF-NVs in the injured spinal cord after the in vivo systemic injection. Increased accumulation of MF-NVs attenuated apoptosis and inflammation, prevented axonal loss, enhanced blood vessel formation, decreased fibrosis, and consequently improved spinal cord function. These results present their possibility of clinical application for SCI. Together, we showed that NVs derived from IONP-MSCs or MF-MSCs contain greater therapeutic molecules or targeting molecules that can facilitate enhanced accumulation in infarcted myocardium or injured spinal cord. Our novel approach to augment therapeutic efficacy of NVs derived from modified MSCs can be a good replacement of conventional MSC or MSC-derived exosome therapies. Our technology can be applied the clinical applications in the future as therapies for acute ischemic/inflammatory diseases.Chapter 1. Research backgrounds and objective 1 1.1. Conventional MSc-based therapies and limitations 3 1.2. Extracellular vesicles: potential therapeutic agent 4 1.2.1 Extracellular vesicles 4 1.2.2. Extracellular vesicles as an alternative to cell-based therapies 6 1.3. Exosome-mimetic extracellular nanovesicles 9 1.4. Modification of extracellular vesicles 11 1.5. Research objective of thesis 12 Chapter 2. Experimental methods 14 2.1. Fabrication and characterization of NVs 15 2.1.1. Preparation of IONP 15 2.1.2. Cell culture 16 2.1.3. Isolation of macrophage membranes and fabrication of MF-MSCs 17 2.1.4. Characterization of IONP-MSCs and MF-MSCs 18 2.1.5. Preparation of NVs 19 2.1.6. Characterization of NVs 20 2.2. In vitro assays 21 2.2.1. Quantitative PCR and analysis of RNA 21 2.2.2. Evaluation of cellular viability 21 2.2.3. Western blot analysis 22 2.2.4. Flow cytometry 23 2.2.5. Immunocytochemistry 23 2.2.6. In vitro cell assay in hypoxic conditions and macrophage polarization by the NV treatment 24 2.2.7. Capillary tube formation 24 2.2.8. Cell migration assay 25 2.2.9. In vitro NV binding assay 25 2.3. In vivo assays 26 2.3.1. MI model and treatment 26 2.3.2. SCI model and treatment 27 2.3.3. Ex vivo biodistribution of NVs 27 2.3.4. MRI 28 2.3.5. Immunohistological assessment in vivo 29 2.3.6. Apoptosis assessment in vivo 30 2.3.7. Capillary density determination in heart 30 2.3.8. Determination of fibrosis in heart 31 2.3.9. Evaluation of cardiac functions 31 2.3.10. Behavior evaluation 32 2.4. Statistical analysis 32 Chapter 3. Nanovesicles derived from iron oxide nanoparticles-incorporated mesenchymal stem cells for cardiac repair 33 3.1. Introduction 34 3.2. Results and discussion 38 3.2.1. IONPs internalization into MSCs 38 3.2.2. Cellular modification of MSCs by IONPs 40 3.2.3. Production of IONP-NVs from IONP-MSCs 42 3.2.4. Characterization of IONP-NVs 45 3.2.5. Cellular uptake of IONP-NVs in vitro 49 3.2.6. Cardioprotective effects of IONP-NVs in vitro 52 3.2.7. Antifibrotic effects of IONP-NVs in vitro 58 3.2.8. Anti-inflammatory effects of IONP-NVs in vitro 60 3.2.9. Proangiogenic effects of IONP-NVs in vitro 62 3.2.10. Increased retention of IONPs by magnetic guidance in the infarcted myocardium 65 3.2.11. Cytotoxicity of IONP-NVs with magnetic guidance in vivo 68 3.2.12. Attenuation of cell apoptosis and inflammation IONP-NVs with magnetic guidance in vivo 72 3.2.13. Increased blood vessel density and improved left ventricular remodeling by IONP-NVs with magnetic guidance in vivo 76 3.2.14. Enhanced cardiac function recovery by IONP-NVs with magnetic guidance in vivo 79 Chapter 4. Targeted delivery of mesenchymal stem cell-derived nanovesicles for spinal cord injury treatment 82 4.1. Introduction 83 4.2. Results and discussion 87 4.2.1. Production and characterization of MF-MSCs 87 4.2.2. Production and characterization of MF-NVs 89 4.2.3. Neuroprotective effects of MF-NVs in vitro 92 4.2.4. Anti-inflammatory effects of MF-NVs in vitro 94 4.2.5. Proangiogenic effects of MF-NVs in vitro 96 4.2.6. Enhanced targeting efficiency of MF-NVs in vitro and in vivo 98 4.2.7. Reduced glial scar formation and improved function recovery by MF-NVs in vivo 100 4.2.8. Enhanced neuroprotection, anti-inflammation, and pro-angiogenesis by MF-NVs in vivo 104 Chapter 5. Conclusions 109 References 112 요약 (국문초록) 134Docto

Stem cell-derived exosomes in the therapy of inflammatory diseases

Τα εξωσώματα, ένας υπότυπος εξωκυτταρικών μικροσωματιδίων με διάμετρο 30-150 nm, είναι νανοσωματίδια που εκκρίνονται από όλους τους τύπους κυττάρων, τόσο ευκαρυωτικά όσο και προκαρυωτικά. Για πολλά χρόνια, οι επιστήμονες πίστευαν ότι τα εξωσώματα ήταν ένας μηχανισμός απομάκρυνσης των ανεπιθύμητων ουσιών του κυττάρου, ωστόσο, τις τελευταίες δεκαετίες, τα εξωσώματα έλαβαν πρωτοφανή επιστημονική προσοχή όταν ανακαλύφθηκε ότι φέρουν γενετικό υλικό και ενεργούν ως μεσολαβητές της διακυτταρικής επικοινωνίας. Το εξωσωμικό φορτίο περιλαμβάνει πρωτεΐνες, ριβονουκλεϊκά οξέα, όπως mRNAs και microRNAs, και λιπίδια. Μεταφέροντας βιοδραστικά μόρια, τα εξωσώματα μπορούν να ρυθμίσουν τη φλεγμονή, την ανοσοαπόκριση, τη μετάσταση των όγκων, την αναγέννηση των ιστών και πολλές άλλες βιολογικές διεργασίες. Η έρευνα σχετικά με τον πιθανό ρόλο τους στη διάγνωση, την εξέλιξη και τη θεραπεία πολλών ασθενειών αυξάνεται εκθετικά τα τελευταία χρόνια. Τα εξωσώματα που εκκρίνονται από βλαστικά κύτταρα είναι πολλά υποσχόμενα στη θεραπεία διαφόρων ασθενειών λόγω των μοναδικών ιδιοτήτων που προέρχονται από τα γονικά τους κύτταρα. Παρέχουν νέες προοπτικές για την ανάπτυξη μιας θεραπευτικής προσέγγισης χωρίς κύτταρα, η οποία αποτελεί σημαντική επιδίωξη λόγω των αναφερθεισών παρενεργειών της θεραπείας με βλαστικά κύτταρα, όπως η απόρριψη από το ανοσοποιητικό σύστημα, η λανθασμένη διαφοροποίηση, ο σχηματισμός όγκων και η παγίδευση στους πνεύμονες. Τα εξωσώματα που προέρχονται από βλαστοκύτταρα έχουν την ικανότητα να επιδιορθώνουν κατεστραμμένους ιστούς, να προάγουν την αγγειογένεση, να ρυθμίζουν τις ανοσολογικές αποκρίσεις και άλλες φυσιολογικές ή παθολογικές διεργασίες και, έτσι, είναι ιδανικοί υποψήφιοι για τη θεραπεία χρόνιων φλεγμονωδών και αυτοάνοσων καταστάσεων. Η φλεγμονή εμφανίζεται ως ένας σημαντικός προδιαθετικός παράγοντας στην παθογένεση μιας πληθώρας ασθενειών. Συμπερασματικά, η θεραπευτική δυναμική των εξωσωμάτων που προέρχονται από βλαστικά κύτταρα, καθώς και ο μηχανισμός που τη διέπει, διερευνάται διεξοδικά μέσω in vitro και in vivo μελετών σε ζώα. Τα εξωσώματα που προέρχονται από βλαστοκύτταρα εμφανίζουν πολλά υποσχόμενα αποτελέσματα στη θεραπεία χρόνιων φλεγμονωδών παθήσεων της γαστρεντερικής οδού, όπως της φλεγμονώδους νόσου του εντέρου και της περιοδοντίτιδας, σε νευροεκφυλιστικές διαταραχές, όπως η νόσος του Alzheimer και η σκλήρυνση κατά πλάκας και σε διάφορες αυτοάνοσες ασθένειες, όπως η αρθρίτιδα και ο διαβήτης. Αυτή η εργασία περιγράφει την πρόοδο σχετικά με τις επιδράσεις των εξωσωμάτων των βλαστικών κυττάρων στη θεραπεία πολλών φλεγμονωδών ασθενειών και τον μηχανισμό δράσης τους.Exosomes, a subtype of extracellular microvesicles with a diameter of 30–150 nm, are nanovesicles secreted by all types of cells, both eukaryotic and prokaryotic. For many years, scientists believed that exosomes were a disposal mechanism of the cell, however, over the last decades, exosomes received unprecedented scientific attention when it was discovered that they carry genetic material and they act as mediators of cellular communication. The exosomal cargo includes proteins, ribonucleic acids, such as mRNAs and microRNAs, and lipids. By transferring bioactive molecules, exosomes can modulate inflammation, immune response, tumor invasion and metastasis, tissue regeneration and several other biological processes. The research regarding their potential role in the diagnosis, progression and therapy of many diseases is growing exponentially over the last years. Exosomes secreted by stem cells hold great potential in the treatment of various diseases due to the unique properties that are derived from their parental cells. They provide new perspectives for the development of a cell-free therapeutic approach, which is an important pursue due to the reported side effects of stem cell-based therapy, such as immune rejection, mal-differentiation, tumor formation and lung entrapment. Stem cell-derived exosomes have the capacity to repair damaged tissues, promote angiogenesis, regulate immune responses and other physiological or pathological processes, and thus, they are ideal candidates for the treatment of chronic inflammatory and autoimmune conditions. Inflammation is emerging as a major predisposal factor in the pathogenesis of a plethora of diseases. Hence, the therapeutic potential of stem cell-derived exosomes, as well as the mechanism underlying it, is being thoroughly investigated through in vitro and in vivo animal studies. Stem-cell derived exosomes exhibit promising results in the treatment of chronic inflammatory diseases of the gastrointestinal tract, like inflammatory bowel disease and periodontitis, neurodegenerative disorders, such as Alzheimer’s disease and multiple sclerosis, and various autoimmune diseases, such as arthritis and diabetes. This report describes the progress regarding the effects of stem cell-derived exosomes in the treatment of many inflammatory diseases and the mechanism behind their action

Effects of extracellular osteoanabolic agents on the endogenous response of osteoblastic cells

The complex multidimensional skeletal organization can adapt its structure in accordance with external contexts, demonstrating excellent self-renewal capacity. Thus, optimal extracellular environmental properties are critical for bone regeneration and inextricably linked to the mechanical and biological states of bone. It is interesting to note that the microstructure of bone depends not only on genetic determinants (which control the bone remodeling loop through autocrine and paracrine signals) but also, more importantly, on the continuous response of cells to external mechanical cues. In particular, bone cells sense mechanical signals such as shear, tensile, loading and vibration, and once activated, they react by regulating bone anabolism. Although several specific surrounding conditions needed for osteoblast cells to specifically augment bone formation have been empirically discovered, most of the underlying biomechanical cellular processes underneath remain largely unknown. Nevertheless, exogenous stimuli of endogenous osteogenesis can be applied to promote the mineral apposition rate, bone formation, bone mass and bone strength, as well as expediting fracture repair and bone regeneration. The following review summarizes the latest studies related to the proliferation and differentiation of osteoblastic cells, enhanced by mechanical forces or supplemental signaling factors (such as trace metals, nutraceuticals, vitamins and exosomes), providing a thorough overview of the exogenous osteogenic agents which can be exploited to modulate and influence the mechanically induced anabolism of bone. Furthermore, this review aims to discuss the emerging role of extracellular stimuli in skeletal metabolism as well as their potential roles and provide new perspectives for the treatment of bone disorders

Advanced Therapy Medicinal Products for Eye Diseases: Goals and Challenges

The concept of advanced therapy medicinal products (ATMPs) encompasses novel kinds of medicines for human use that are based on genes, cells or tissues. These intend to offer not only regeneration, but complete functional recovery of diseased tissues and organs using different strategies. Gene therapy, cell therapy and tissue engineering are the main areas in which promising advanced therapies are emerging. The eye is a very complex organ whose main structures, the cornea and the retina, play a pivotal role in maintaining normal vision, as severe alterations in these tissues can lead to blindness. Ocular tissues are starting to benefit from ATMPs by fighting against the enormous complexity and devastating potential of many ocular diseases. However, developments arising from this field of work face important challenges related to vectors to deliver drugs and genetic material to target tissues, suitable biomaterials to prepare cell scaffolds and cell stemness, among others—not to mention the complicated legislation around ATMPs, the complexity in production and quality control and the absence of standardized protocols.The purpose of this Special Issue is to serve as an overview of the current progress in the application of cell and gene therapies, as well as tissue engineering to restore functionality in diseased ocular structures, and the challenges linked to reaching patients

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

C

Mesenchymal stem cell exosomes: a two-edged sword in cancer therapy

Faezeh Vakhshiteh,1 Fatemeh Atyabi,1,2 Seyed Nasser Ostad3 1Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 2Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 3Department of Toxicology & Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Abstract: Mesenchymal stem cells (MSCs) are multipotent stromal cells present in various adult tissues. Several studies suggest that MSCs secrete exosomes that perform as mediators in the tumor niche and play several roles in tumorigenesis, angiogenesis, and metastasis. In contrast, there are other studies supporting the tumor-suppressing effects of MSC-derived exosomes. Therefore, the exact association of MSC exosomes and tumor cells remains open to debate. This review aimed to demonstrate the present knowledge of MSC-derived exosomes in cancer research and to illustrate current approaches to make use of modified exosomes as a platform in therapeutic strategies in cancer. Keywords: mesenchymal stem cells, exosome, drug delivery, exosome engineering, cancer therap

Exosomes Derived from Human Primed Mesenchymal Stem Cells Induce Mitosis and Potentiate Growth Factor Secretion.

Mesenchymal stem cells (MSCs) facilitate functional recovery in numerous animal models of inflammatory and ischemic tissue-related diseases with a growing body of research suggesting that exosomes mediate many of these therapeutic effects. It remains unclear, however, which types of proteins are packaged into exosomes compared with the cells from which they are derived. In this study, using comprehensive proteomic analysis, we demonstrated that human primed MSCs secrete exosomes (pMEX) that are packaged with markedly higher fractions of specific protein subclasses compared with their cells of origin, indicating regulation of their contents. Notably, we found that pMEX are also packaged with substantially elevated levels of extracellular-associated proteins. Fibronectin was the most abundant protein detected, and data established that fibronectin mediates the mitogenic properties of pMEX. In addition, treatment of SHSY5Y cells with pMEX induced the secretion of growth factors known to possess mitogenic and neurotrophic properties. Taken together, our comprehensive analysis indicates that pMEX are packaged with specific protein subtypes, which may provide a molecular basis for their distinct functional properties

Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation

<div><p>Human mesenchymal stem cell (hMSC)-derived exosomes have shown regenerative effects, but their role in osteogenesis and the underlying mechanism are yet to be determined. In this study, we examined the time-course secretion of exosomes by hMSCs during the entire process of osteogenic differentiation. Exosomes derived from hMSCs in various stages of osteogenic differentiation committed homotypic cells to differentiate towards osteogenic lineage, but only exosomes from late stages of osteogenic differentiation induced extracellular matrix mineralisation. Exosomes from expansion and early and late stages of osteogenic differentiation were internalised by a subpopulation of hMSCs. MicroRNA profiling revealed a set of differentially expressed exosomal microRNAs from the late stage of osteogenic differentiation, which were osteogenesis related. Target prediction demonstrated that these microRNAs enriched pathways involved in regulation of osteogenic differentiation and general mechanisms how exosomes exert their functions, such as “Wnt signalling pathway” and “endocytosis”. Taken together, the results show that MSCs secrete exosomes with different biological properties depending on differentiation stage of their parent cells. The exosomal cargo transferred from MSCs in the late stage of differentiation induces osteogenic differentiation and mineralisation. Moreover, it is suggested that the regulatory effect on osteogenesis by exosomes is at least partly exerted by exosomal microRNA.</p></div