Tiny Messengers with Big Promises: Extracellular Vesicles
Regenerative medicine is a multidisciplinary field focused on restoring damaged, dysfunctional, or missing tissues, ultimately reinstating their normal function and physiology. Within this field, various approaches are employed, and three of them are instrumental in tissue regeneration. The first involves cell-based therapies, where cells are introduced directly or indirectly to facilitate tissue restoration, relying on their paracrine functions. The second approach, classical tissue engineering, combines cells with biodegradable scaffolds to construct functional tissues. In recent years, significant progress has been made in the third strategy, namely material-based strategies, where biodegradable materials play a crucial role, often incorporating cellular functions [Figure 1] (de Jong et al., 2014).
Extracellular vesicles, integral constituents of cellular homeostasis, are central in mediating intercellular communication. These vesicular entities actively transport molecular cargo across cellular membranes, encompassing both efflux and influx processes while also orchestrating the degradation of cellular components. Furthermore, the evolving landscape of technological innovations has paved the way for harnessing synthetic vesicles as transformative tools in regenerative medicine, thereby presenting novel avenues for therapeutic intervention (Crum, 2022).
Regenerative Medicine Evolution
Historically, tissue replacement predominantly relied on organ, tissue, or cell transplantation. Notable advancements in transplantation techniques occurred throughout the last century, starting with early cornea transplants (Zirm, 1989) and culminating in the pioneering kidney transplantation in the 1950s (Merrill et al., 1956). Whilst regenerative medicine has faced substantial technical constraints, there has been a noteworthy shift in focus over time. The challenges have evolved from primarily technical hurdles to critical issues such as the scarcity of suitable donor organs and the inherent risk of immune rejection. These challenges continue to shape the landscape of regenerative medicine. Nevertheless, it is essential to acknowledge that the field faces a spectrum of issues, including achieving functional tissue integration and addressing long-term safety and regulatory considerations, among others.
Tissue engineering and regenerative medicine have emerged as translational biomedical disciplines, focusing on innovative strategies to restore damaged tissues (Yahya et al., 2021). These strategies leverage cells, scaffolds, and signalling molecules—alone or in combination—to elicit constructive tissue remodelling, integration, and regeneration when implanted into the host. The ultimate objective is the generation of new functional tissues, which can be achieved through the supply of exogenous allogeneic or autologous cells or by employing bioactive materials that attract endogenous stem cells to the injury site. Regardless of the cell source, appropriate guidance for cell proliferation, differentiation, and spatial organisation is imperative for successful tissue regeneration (Abraham et al., 2021). Tissue and organ loss, often precipitated by grave illnesses or catastrophic accidents, burden patients significantly. Regeneration and engineering in medicine are making remarkable strides, propelled by extensive research on stem cells, biomaterials, and related subjects.
Mesenchymal stem cells (MSCs) have assumed a central role in regenerative medicine due to their robust self-renewal capabilities and versatile differentiation potential [Figure 2]. Nonetheless, using these cells in regenerative medicine faces challenges, encompassing limitations in cell sourcing, ethical dilemmas, and the risk of tumorigenesis after transplantation (MacPherson & Kimmelman, 2019). Current research is turning its attention toward harnessing the paracrine actions of cells and extracellular vesicles as a promising avenue to address these limitations and advance the field of regenerative medicine.
Extracellular Vesicles: Overview
The history of extracellular vesicles (EVs) can be traced back to the mid-20th century. In 1940, during the early days of subcellular exploration, a novel subcellular component was discovered within cell-free plasma through high-speed centrifugation techniques. Subsequently, in the 1960s, advancements in electron microscopy revealed the true nature of this subcellular fraction as small vesicles. The term "exosomes" was officially introduced in 1987 when researchers successfully isolated these vesicles from cell supernatant (Pan & Johnstone, 1983; van der Pol et al., 2012). Initially, exosomes were misinterpreted as cellular debris or remnants associated with cell death (Bobrie et al., 2011). However, extensive research has since been conducted to unveil the intricate biology, functions, and potential clinical applications of exosomes. It is now well-established that exosomes play a crucial role in cell-to-cell communication, facilitating the exchange of genetic material and proteins between parent cells and neighbouring cells in the extracellular environment, highlighting their diverse and vital biological roles (Colombo et al., 2014).
Extracellular vesicles encompass a diverse group of lipid-bilayer enclosed vesicles released by various cell types, including immune cells, endothelial cells, epithelial cells, neuronal cells, cancer cells, Schwann cells, and mesenchymal stem cells (Tsiapalis & O’Driscoll, 2020). These microscopic structures play a pivotal role as messengers in intercellular communication, influencing various physiological and pathological processes. These processes include immune responses, the maintenance of homeostasis, inflammation regulation, promoting angiogenesis, and even the modulation of cancer progression through transmitting essential biological signals (Yáñez-Mó et al., 2015).
EVs have been classified into five categories based on their characteristics and origins [Figure 3]:
Exosomes, typically ranging in size from 30 to 150 nanometers, originate from the endosomal system when multivesicular bodies fuse with the cell membrane (Bebelman et al., 2018);
Microvesicles, also known as shedding vesicles or ectosomes, are larger vesicles, typically between 100 to 1,000 nanometers, formed through the outward budding and fission of the cell membrane (Zaborowski et al., 2015);
Apoptotic bodies, which can be relatively large, up to several micrometres, are released during programmed cell death (apoptosis) and contain cellular fragments (Borges et al., 2013);
Biomineralisation vesicles, although less commonly studied, are involved in the mineralisation of tissues (Cui et al., 2016);
Matrix-bound nanovesicles, associated with the extracellular matrix, play a role in tissue remodelling (Huleihel et al., 2016).
It is worth noting that there is still an ongoing effort to establish a complete agreement on the terminology and categorisation of vesicles within the scientific community (Gould & Raposo, 2013).
The significance of EVs in tissue repair and regenerative medicine is becoming increasingly apparent. Recent studies suggest that the therapeutic benefits of MSCs in tissue regeneration primarily stem from their paracrine action, characterised by the secretion of EVs rather than direct MSC engraftment and proliferation (Lai et al., 2015). EVs transmit vital signals and possess a unique ability to attract endogenous cells, thereby enriching the regenerative environment through the release of various chemokines. These chemokines significantly contribute to processes such as angiogenesis and tissue repair (Silva et al., 2017). EVs emerge as a promising tissue repair and regeneration tool. Their lipid-bounded vesicular structure, filled with functional cargo, including lipids, proteins, and nucleic acids, enables them to profoundly influence recipient cells, impacting cellular functions and phenotypes. With documented involvement in development, tissue homeostasis, wound healing, and implications in various disease contexts, EVs represent a dynamic and versatile frontier in biomedicine (Wang & Pan, 2023).
Mesenchymal stem cells exhibit remarkable differentiation capabilities, spanning multiple tissue types such as adipocytes, chondrocytes, osteocytes, hepatocytes, and neurocytes (Crapnell et al., 2013; Gang et al., 2004; Lin, 2012). Beyond their mechanical differentiation prowess, MSCs are prolific secretors of exosomes and a repertoire of essential biomolecules, encompassing cytokines, chemokines, and growth factors. Although early investigations suggested MSCs as pivotal players in tissue repair, their limited survival and engraftment in damaged regions have curtailed their effectiveness in tissue regeneration (Kucharzewski et al., 2019). However, subsequent studies have unveiled a transformative role for paracrine signalling in mediating the therapeutic benefits of MSC applications. This paracrine activity includes the secretion of EVs (Barreca et al., 2020). Research has showcased the potential of MSC-derived exosomes to supplant traditional MSC-based stem cell therapies across a spectrum of injury and disease models (Yin et al., 2019).
Exosomes: Formation and Release
While this paragraph concentrates on exosomes, it is essential to note that these processes share commonalities with the formation and release of other extracellular vesicles. Exosome biogenesis is a complex cellular process that plays a pivotal role in intercellular communication [Figure 6]. It centres around the formation of intraluminal vesicles (ILVs) within late endosomes, referred to as multivesicular bodies (MVBs). These endosomes act like cellular sorting hubs, processing and directing various molecules to their proper destinations, including recycling cell surface receptors and breaking down waste materials. In the context of exosome formation, endosomes play an important role in packaging and releasing these specialised vesicles loaded with specific molecules for cell-to-cell communication. (F. T. Borges et al., 2013). Exosomes, which are the end product of this process, are secreted by a variety of cell types and have been isolated from diverse physiological fluids, including sperm (Sullivan et al., 2005), urine (Pisitkun et al., 2004), plasma (Caby et al., 2005), and bronchial lavage fluid (Admyre et al., 2003). These nanoscale vesicles typically exhibit a diameter ranging from 40 to 100 nanometers and display a consistent spherical shape (Familtseva et al., 2019). Exosomes share a common set of proteins, including tetraspanins, Alix, and Tsg101, which are evolutionarily conserved and integral to their structure and function. Additionally, exosomes carry specific proteins that mirror their cellular origin. These remarkable vesicles originate from MVBs, intracellular endosomal organelles distinguished by multiple intraluminal vesicles enclosed within a single outer membrane. MVBs are derived from early endosomes, which are necessary for the degradative endosomal pathway for internalised proteins (Hessvik & Llorente, 2018).
Exosome biogenesis involves early endosomes, which interact with both the Golgi apparatus and the endoplasmic reticulum. Exosomes are formed through the endocytosis of the early endosome membrane, denoted by its unique orientation of the involuted cytoplasmic side (Crum, Capella-Monsonís, et al., 2022). The generation of MVBs, as well as the secretion of exosomes, is mediated through the concerted action of endosomal complexes required for transport (ESCRT complexes). This complex process is mediated by the concerted action of endosomal complexes required for transport (ESCRT complexes), consisting of approximately 20 proteins organised into four distinct complexes (ESCRT-0, -I, -II, and -III), and associated proteins like VPS4, VTA1, and Alix. These ESCRT complexes are remarkably conserved across species (Henne et al., 2011). Exosome formation starts with the budding of a nascent vesicle toward the interior of an MVB, connected to the endosomal limiting membrane by a membranous stalk. The ESCRT complex subsequently severs this nascent ILV from the limiting membrane. The precise triggers that designate a nascent bud to become an MVB remain elusive (van Niel et al., 2011). The origin of exosomes suggests that their production is influenced by changes in the cellular microenvironment. Studies have shown an increase in the formation of early endosomes and MVBs in response to signalling via growth factor receptors, highlighting the cell's capacity to adjust exosome production according to its specific need (Borges et al., 2013).
As mentioned, extracellular vesicle membranes originate from the parent cell's plasma membrane. Notably, lipid rafts are believed to undergo invagination within the cell, forming early endosomes. ILVs are subsequently generated through the inward budding of these early endosomal membranes. Of particular significance, lipid rafts are specialised membranes characterised by a rich presence of integral membrane proteins, including signalling receptors, ceramides, and cholesterol. Exosomes, thus, carry these membrane proteins on their surface. Specific membrane proteins have been identified on exosomes and are recognised as crucial mediators of signal transfer to recipient cells. Remarkably, these membrane proteins facilitate cell-to-cell communication over long distances, akin to cell-cell junctions (de Jong et al., 2014) [Figure 7].
Applications of EVs
One area where EV-based therapies have shown substantial potential is the cardiovascular system, particularly in addressing the limited regenerative capacity of the heart. Notable successes have been observed in animal models of cardiac injury, including myocardial infarction and ischemia-reperfusion injury, where exosomes derived from MSCs have demonstrated the ability to mitigate disease progression (Sahoo & Losordo, 2014). These exosomes have been shown to reduce cardiac fibrosis, improve blood flow to the site of injury, and contribute to overall cardiac tissue regeneration. Furthermore, placental MSC-derived exosomes have exhibited promise in promoting angiogenesis, which holds significance in treating conditions like cerebral vascular accidents. In tissue transplantation, the local delivery of microvesicles containing IL-33 has emerged as a potential strategy for prophylaxis against chronic graft rejection by modulating pro-inflammatory macrophages (Li et al., 2020).
Exploration of EV-based therapies has extended to the respiratory system, where tissue healing often involves pathways associated with excessive fibrosis and pulmonary hypertension. Exosomes derived from MSCs have demonstrated their capacity to counteract pulmonary hypertension while suppressing overactive proliferation and fibrosis pathways (Lee et al., 2012). Additionally, lung-derived microvesicles have proven valuable for reprogramming marrow cells into pulmonary epithelial phenotypes, offering potential sources for developing engineered lung tissues (Aliotta et al., 2007).
While EV research in the renal system has predominantly centred on diagnostic applications, recent findings indicate that microvesicles originating from endothelial progenitor cells and MSCs may provide protective effects for kidney epithelial cells in models of acute kidney injury and ischemia-reperfusion injury, suggesting their regenerative potential (Liu et al., 2020).
EV-based therapies have exhibited promise in promoting neural regeneration in the central and peripheral nervous systems, characterised by limited innate regenerative abilities. Exosomes derived from MSCs have demonstrated the capacity to promote neurite outgrowth and remyelination of central nervous system nerve fibres after injury (Xin et al., 2013). In the peripheral nervous system, Schwann cell-derived exosomes have facilitated axon growth and regeneration in models of peripheral nerve injury (Lopez-Verrilli et al., 2013; Lu et al., 2019).
In contrast to other organs, the liver possesses a remarkable innate regenerative capacity. Nevertheless, liver diseases and failure continue to pose significant health challenges globally. There is hope to utilise EVs as a complementary strategy. Hepatocyte-derived EVs have been shown to enhance hepatocyte proliferation, viability, and regeneration by activating stellate cells in the liver (Rodríguez-Suárez et al., 2014). Furthermore, microvesicles from human liver stem cells have accelerated liver regeneration in hepatectomised rats through EV-mediated RNA transfer to target hepatocytes (Herrera et al., 2010).
In the context of skin and wound healing, a plethora of products and approaches have been developed, with EVs emerging as a promising supplement. EVs derived from various cell sources have consistently demonstrated their ability to promote re-epithelialisation, angiogenesis, extracellular matrix remodelling, and migration of crucial skin cell types, offering potential advantages in wound healing (Narauskaitė et al., 2021).
In bone and cartilage regeneration, EVs - particularly those from MSC sources - have displayed the potential to enhance tissue repair. MSC-derived exosomes have been associated with increased type II collagen production and the inhibition of cartilage degradation in preclinical studies (Vonk et al., 2018). In bone regeneration, EVs derived from osteoblasts, periodontal-ligament stem cells, and biomineralisation vesicles have all demonstrated the potential to promote bone formation and facilitate osteoclast activation (Man et al., 2022).
Many research groups have investigated the therapeutic potential of extracellular vesicles within the axial skeleton, with a primary focus on addressing intervertebral disc degeneration. Notably, exosomes derived from various sources of MSCs, including bone marrow (Liao et al., 2019), human urine (Guo et al., 2021), adipose tissue (Xing et al., 2021), human placenta (Yuan et al., 2020), and the human umbilical cord (Yuan et al., 2021), have demonstrated significant promise in alleviating inflammation and damage associated with intervertebral disc degeneration. Furthermore, exosomes from nucleus pulposus cells, endplate chondrocytes, annulus fibrosus cells (Sun et al., 2021), platelet-rich plasma (Xu et al., 2021), and notochordal cells (Sun et al., 2020) have exhibited similar early potential in treating this condition.
In addition to their role in bone and cartilage regeneration, EVs have been explored as therapeutic agents for joint-related pathologies, such as rheumatoid arthritis (RA). Researchers have employed various strategies, including genetic modification of bone marrow-derived dendritic cells to produce exosomes enriched with anti-inflammatory molecules like IL-10 (Ruffner et al., 2009) and IL-4 (Kim et al., 2007), pro-apoptotic molecules like FasL (Kim et al., 2006), and immune checkpoint inhibitors like CTLA4-Ig (Ruffner et al., 2009). These innovative approaches have demonstrated preclinical efficacy in modulating innate and adaptive immune cell populations in RA. Furthermore, matrix-bound nanovesicle-based therapies have shown promise in mitigating RA by modulating locally and systemically macrophage phenotypes in rodent models. These therapies provide an alternative to fluid-phase exosome therapies characterised by minimal processing and manipulation, making them a noteworthy addition to the therapeutic arsenal for these conditions. (Crum et al., 2022).
Conclusions
EV-based therapies hold immense promise in regenerative medicine, offering innovative solutions to address tissue repair and regeneration challenges across various organ systems. These tiny vesicles have demonstrated success in mitigating cardiac injury, countering respiratory fibrosis, and aiding renal recovery. Moreover, EVs have shown promise in promoting neural regeneration, enhancing liver regeneration, and accelerating skin and wound healing. In bone and cartilage repair and joint-related conditions like rheumatoid arthritis, EVs have displayed substantial potential. These findings underscore the transformative potential of EV-based treatments in regenerative medicine. While challenges and complexities persist in translating these promising therapies to clinical applications, the growing body of evidence suggests that extracellular vesicles represent a versatile and effective tool for addressing tissue damage and promoting regeneration in diverse organ systems. Continued research and development in this field promise to improve the lives of patients suffering from a wide range of debilitating conditions.
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Cover Image: [Illustration showing extracellular vesicles.] (n.d., 2023). Nature. https://www.nature.com/collections/hjjfdgedbg
Figure 1: Fields of regenerative medicine. Fontanazza (2016) [Illustration]. MedTechIntelligence. https://medtechintelligence.com/news_article/the-steady-progress-of-regenerative-medicine/
Figure 2: [Examples of potential applications of MSC-EVs in tissue engineering and regenerative medicine.] Tsiapalis, D., & O’Driscoll, L. (2020). [Illustration]. Mesenchymal Stem Cell Derived Extracellular Vesicles for Tissue Engineering and Regenerative Medicine Applications. Cells, 9(4), 991. Retrieved from http://dx.doi.org/10.3390/cells9040991
Figure 3: Major subclasses of extracellular vesicles. Crum, R. J., Capella-Monsonís, H., Badylak, S. F., & Hussey, G. S. (2022). [Illustration]. Extracellular vesicles for regenerative medicine applications. Applied Sciences (Switzerland), 12(15). https://doi.org/10.3390/app12157472
Figure 4: [Overview - using EVs for regeneration.] Nagelkerke, A., Ojansivu, M., van der Koog, L., Whittaker, T. E., Cunnane, E. M., Silva, A. M., Dekker, N., & Stevens, M. M. (2021). [Illustration]. Extracellular vesicles for tissue repair and regeneration: Evidence, challenges and opportunities. In Advanced Drug Delivery Reviews, 175, 113775. https://doi.org/10.1016/j.addr.2021.04.013
Figure 5: Typical exosomes. Hade, M. D., Suire, C. N., & Suo, Z. (2021). [Illustration]. Mesenchymal stem cell-derived exosomes: Applications in regenerative medicine. Cells, 10(8), 1–48. https://doi.org/10.3390/cells10081959
Figure 6: Exosome biogenesis pathways. Kita, S., & Shimomura, I. (2022). [Illustration]. Extracellular Vesicles as an Endocrine Mechanism Connecting Distant Cells. Molecules and Cells 45(1), 771–780. https://doi.org/10.14348/molcells.2022.0110
Figure 7: [EV-mediated cell cross-talk, clearance mechanisms and immune responses.] Herrmann, I. K., Wood, M. J. A., & Fuhrmann, G. (2021). [Illustration]. Extracellular vesicles as a next-generation drug delivery platform. Nature Nanotechnology, 16(7), 748–759. https://doi.org/10.1038/s41565-021-00931-2
Figure 8: [Schematic representation of tissue engineering approaches in exosome- based cardiac repair therapies.] Hade, M. D., Suire, C. N., & Suo, Z. (2021). [Illustration]. Mesenchymal stem cell-derived exosomes: Applications in regenerative medicine. Cells, 10(8), 1–48. https://doi.org/10.3390/cells10081959
Figure 9: [Schematic representation of functions of exosomes in neural cell communication.] Hade, M. D., Suire, C. N., & Suo, Z. (2021). [Illustration]. Mesenchymal stem cell-derived exosomes: Applications in regenerative medicine. Cells, 10(8), 1–48. https://doi.org/10.3390/cells10081959
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