Extracellular Vesicles

Applications to Regenerative Medicine, Therapeutics and Diagnostics

Wojciech Chrzanowski, Chwee Teck Lim, Sally Yunsun Kim, Wojciech Chrzanowski, Chwee Teck Lim, Sally Yunsun Kim

• Publisher: Royal Society of Chemistry
• Year: 2021
• ISBN:
  9781839164569
• Edition: 1
• Topic: Tecnologia e ingegneria
• Subtopic: Scienza biomedica

Extracellular and biofluids vesicles (EVs) are highly specialised yet ubiquitous nanoscale messengers secreted by cells. With the development of stem cell engineering, EVs promise to deliver next generation tools in regenerative medicine and tissue engineering, as well as in diagnostics. A vibrant and promising field, this book provides the first resource to the field.

Covering basic cell biology, including EV production and intracellular communication, this book will provide material scientists and engineers with a foundation to the necessary biology. The reader will then learn about the isolation of extracellular vesicles their physicochemical characterisation and therapeutic application of EVs in regenerative medicine as well as their potential as biomarkers in medical diagnostic. This book will also discuss the regulatory landscape of EVs.

Bridging cell biology, biomaterials, biophysics and biomedical engineering the content of this book is written with a broad interdisciplinary audience in mind. Researchers, new and established will find this a must-have on their shelf.

CHAPTER 1

Therapeutic Potential of Mesenchymal Stromal Cell-derived Small Extracellular Vesicles

FABIOLA NARDI BAUER ^a AND BERND GIEBEL^*a

^a Institute for Transfusion Medicine, University Hospital Essen, Virchowstraβe 179, 45147 Essen, Germany,
^*E-mail: [email protected]

Small extracellular vesicles (sEVs) are biological nanoparticles with sizes below 200 nm that are released by virtually all cells and are detected in all body fluids. Assembled in a cell type specific manner, at least a proportion of the sEVs can transmit complex information from the sending to specifically selected target cells. Depending on their origin, signalling sEVs can modulate physiological and pathophysiological processes. sEVs released from mesenchymal stromal cells (MSCs), for example, mediate therapeutic activities in an increasing number of different diseases. Although their concrete mechanisms of action (MoA) as well as their site(s) of action (SoA) have not been sufficiently dissected yet, MSC-sEVs apparently act in a multimodal manner. Evidence has been provided that, as part of their therapeutic activities, MSC-sEV preparations can modulate immune (pro-inflammatory immune responses) into regulatory/tolerogenic immune responses, promote angiogenic and pro-regenerative processes and act in an anti-apoptotic manner as well as restoring energy. Here, we summarize the history behind the discovery of the therapeutic activities of MSC-sEVs, some of their therapeutic application fields, and challenges connected with their effective translation into clinics.

1.1 Concepts in Regenerative Medicine

Degenerative diseases are classically associated with irreversible tissue loss. In this context, it is widely assumed that the potential of endogenous stem and progenitor cells, which normally control tissue homeostasis, is insufficient to promote tissue regeneration. Consequently, stem and/or progenitor cells with developmental potentials comparable to endogenous stem cells have been applied to different preclinical models as well as to a variety of different patient cohorts suffering from degenerative diseases.

In the early 2000s, as interest in stem cell biology grew exponentially, a number of observations implicated plasticity in somatic stem cell compartments, i.e. the capability of somatic stem cells to generate differentiated cells far beyond their normal developmental routes. Observations implied, for example, that immature brain cells, under the right extrinsic conditions, could create blood cells and vice versa. ^1,2 Mesenchymal stem/stromal cells (MSCs) especially became an attractive research object. MSC-like fibroblasts were originally described by Friedenstein and colleagues in the late 1960s. ^3,4 These fibroblastoid cells, initially grown from adult bone marrow, can be easily expanded as plastic adherent cells. Within differentiation assays they can develop towards the osteogenic, adipogenic and chondrogenic lineage, all being mesodermal derivatives, ⁵ thus fulfilling the potential of mesenchymal stem cells, whose existence had been predicted before. ⁶ Quickly it became evident that MSCs can be raised from various tissues types including fat, umbilical cord, umbilical cord blood and placental tissue. In addition, some MSC types, including the mesodermal adult progenitor cells (MAPC) and the unrestricted somatic stem cells (USSCs) appeared as somatic stem cells with germ layer spanning developmental potentials comparable to the pluripotent embryonic stem (ES) cells, which also became popular at that time. ⁷ In contrast to the ES cells, however, MSC-like cells showed no teratogenic potentials and quickly were considered as therapeutic agents for degenerative diseases. ^8,9 It was proposed that the administration of MSCs, systematically applied to the body and guided by environmental factors, home to affected tissues and differentiate to replace lost cell types. Since MSCs also generated cells mimicking features of neurons or myocardial cells, respectively, ^10,11 they were quickly discussed as allogeneic off-the-shelf products for acute diseases like ischaemic stroke and myocardial infarction. Consequently, groups have started to study their interaction with allogenic immune cells. While it was initially assumed that MSCs would be rejected in principle, they were shown to modulate the activity of different types of immune cells. Specifically, without the need for direct intercellular contacts, they were found to reversibly inhibit proliferation of stimulated CD4 and CD8 T cells ^12,15 and to promote the generation, recruitment and regulation of regulatory T cells. ^16,17 MSCs also effectively inhibit the proliferation of B cells and the differentiation of monocyte-derived dendritic cells. ^18,19 Furthermore, MSCs inhibit the proliferation of IL-2 induced NK cells and prevent NK cell activation, downregulating NKp30 and NKG2D receptors. ^20,21 Thus, MSCs suppress various types of immune effector responses and promote regulatory immune cell functions. Coupled to these functions, they secrete a number of different soluble factors including cytokines known to modulate immune responses, such as transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), tryptophan degrading enzyme indoleamine 2, 3-dioxygenase (IDO), prostaglandin E2 (PGE₂) and human leukocyte antigen (HLA) class I molecule G (HLA-G). ^12,14,22,26 Consequently, in addition to their assumed cell replacement potential for the treatment of degenerative diseases, MSCs became increasingly discussed as therapeutic agents for inflammatory diseases, especially for acute Graft-versus-Host Disease (aGvHD).

Acute GvHD is triggered by allogeneic immune cells being transplanted as part of allogeneic haematopoietic stem cell (alloSC) transplants to myeloablative patients. The co-transplantation of immune cells including T cells is required to eliminate residual tumour (leukaemic) cells that have not been eradicated by the myeloablative treatment regimen before alloSC transplantation. Thus, alloSC transplantation provides a combined regenerative and immunotherapeutic approach. Despite the fact that the therapy can be curative, up to 50% of patients receiving alloSC transplants develop mild to severe forms of aGvHD; severe forms are associated with high mortality rates. ²⁷ First-line aGvHD patients are treated with steroids which are lympholytic and can successfully suppress disease inducing T cell functions in approximately half of the aGvHD patients. For the remaining, the steroid-refractory aGvHD patients until now, no second-line treatment has been approved. MSCs have been and are still considered as potential second-line therapeutic agents for steroid refractory aGvHD patients. Indeed, in 2004, Katarina Le Blanc and her team successfully treated a 9 year-old steroid-refractory aGvHD patient with allogenic MSCs. ²⁸

Since then, more than 1200 clinical MSC trials have been registered at the American National Institute of Health (NIH), most of them either intending to apply MSCs as regenerative or immunotherapeutic agents to a diversity of different patient cohorts (clinicaltrials.gov). From 2006 to 2009, the first phase 3 clinical trial (NCT00366145; [Remestemcel-l]) was conducted to assess the efficacy of human MSCs as second-line therapy for steroid-refractory aGvHD. Despite the reported safety of Remestemcel-l, the overall response rate beyond day 28 was comparable within the Remestemcel-l group and the placebo group. ²⁹ However, clinical studies on paediatric aGvHD patients or on patients with high aGvHD risks revealed better outcomes in MSC-treated than in placebo-treated groups. ³⁰ As a consequence, a phase 3 clinical study had been performed (NCT02336230), single-armed and prospective, to treat steroid-refractory paediatric patients with Remestemcel-l. The MSC product was dosed 2 × 10⁶ cells kg⁻¹ bodyweight and applied over a 4 week period twice per week. Within the MSC treated group, 74% of the patients showed improvements beyond day 28. In contrast, in the control group the aGvHD symptoms of only 45% of the patients were improved beyond day 28. The overall response improvement was sustained in a huge proportion of the patients with confirmed improvement beyond day 180. ^31,32 Still, with the exception of Japan, which has licenced a commercial MSC product (TEMCELL) for aGVHD treatment, and a conditional clinical approval for paediatric aGvHD treatment with Prochymal, another commercial MSC product, in Canada and New Zealand, ^33,34 we are not aware of any other licensed MSC product at the moment.

1.2 MSCs Act in a Paracrine Rather than Cellular Manner

Starting with the observation that MSCs can mediate inhibition of T cell proliferation in the absence of physical intercellular contacts ¹² and coupled with the fact that they secrete various cytokines, it became evident, over the years, that MSCs exert proportions of their therapeutic functions via their secretome, both in pro-regenerative and in immunotherapeutic approaches. ³⁵

Initially, studies that investigated whether cells need to engraft into affected tissues to achieve their therapeutic effects showed that, in most cases, systemically administered MSCs end up in the lungs of treated animals and were rarely recovered in high numbers in their assumed target tissues. ^36,38 The group of Darwin Prockop, for example, demonstrated that intravenously applied human MSCs improved the heart function following myocardial infarction in mice. Specifically, applied MSCs decreased inflammatory responses, reduced infarct sizes and improved cardiac functions. Upon exploring the bio-distribution of the administered human MSCs by screening the animals, human DNA was almost exclusively detected in the animals' lungs and only marginally in other tissues. Furthermore, MSCs trapped in the lung were shown to secrete TNF-α-induced protein 6 (TNAIP6 or TSG-6), an anti-inflammatory cytokine. Subsequent experiments in which recombinant TSG-6 was administered to infarcted mice confirmed that TSG-6 administration decreases myocardial infarct-induced inflammatory responses and reduced infarction sizes. Supporting the important role of TSG-6, human MSCs following siRNA mediated knockdown of the TSG-6 expression failed to improve myocardial infarction sizes. ³⁶ Comparably, MSC administration was found to i...