Keywords

Intercellular Communication

Homeostasis and Intercellular Communication

Intercellular communication is a biological process necessary for the maintenance of homeostasis in the organism that relies on coordination between cells to complete their functions. It is crucial in the development, differentiation, and adaptation of cells to their environment (Armingol et al. 2021; Mittelbrunn and Sánchez-Madrid 2012; Song et al. 2019). Alterations in cell–cell communication are implicated in the development of pathophysiological processes in the organism such as tumorigenesis, metastasis, autoimmunity, senescence, and also in physiological processes such as ageing (Yang et al. 2021); concepts that we will develop in the chapter.

Intercellular communication generally requires the exchange of biochemical substances (ions, metabolites, hormones, cytokines, etc.) between them. This type of communication is called biochemical communication. There are two types of biochemical communication: direct and indirect (Fig. 11.1).

  • Direct, contact-dependent, or juxtacrine communication occurs between neighboring cells near each other in which their membrane structures facilitate the exchange of molecules without these being secreted into the extracellular space. In turn, there are three direct biochemical communication pathways:

    • Communication mediated by cell adhesion molecules (selectins, integrins, cadherins, and immunoglobulins).

    • Communication mediated by gap junctions.

    • Communication mediated by tunneling nanotubes, which are thin projections of the plasma membrane.

  • Indirect communication is characterized by the secretion of signaling molecules that are transported by flow or diffusion. These molecules are secreted freely and can either diffuse passively across the membrane (hydrophobic small molecules) or be transported into the cytoplasm of the target cell by endocytosis (hydrophilic molecules). However, there is another type of indirect communication in which the secretory cell releases extracellular vesicles containing the signaling molecules. Depending on the distance between the secretory cell and the target cell, indirect communication can be classified as follows:

    • Paracrine, in which communication only affects cells in the immediate neighborhood of the secretory cell.

    • Autocrine, in which the signals are directed to the cells that secrete them.

    • Endocrine, long-distance communication in which signaling molecules travel through the systemic circulation to reach the target cells (Armingol et al. 2021; Yang et al. 2021).

Fig. 11.1
An illustration depicts the direct cellular communication mediated by cell adhesion molecules, gap junctions and tunneling nanotubes, indirect cellular communication with paracrine, autocrine and endocrine.

The two types of biochemical communication. Direct cell communication is mediated by cell adhesion molecules, gap junctions, or by tunneling nanotubes. Indirect cellular communication includes paracrine, autocrine, and endocrine signaling. Effector cells (yellow) transmit signaling molecules to target cells (green). Created with BioRender.com

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In addition to biochemical communication, there is also mechanical communication. Here, cells communicate with their surroundings by exerting mechanical forces on the extracellular matrix. These signals are involved in various processes such as cytoskeleton reorganization and cell migration (Van Helvert et al. 2018), matrix remodeling (Swartz et al. 2001), epigenetic modifications (Yang et al. 2021), or recruitment of immune cells to the tissue (Pakshir et al. 2019) among others.

Intercellular Communication and Ageing

Ageing involves changes in cell–cell communication, mainly due to increased cellular senescence. Senescence is implicated not only in ageing but also in age-related diseases such as cancer, arthritis, atherosclerosis, and Alzheimer's disease. The main characteristic of senescence is the stable arrest of the cell cycle; however, its influence on tissue homeostasis and the development of age-related pathologies is due to the Senescence-associated secretory phenotype (SASP). SASP can be considered a type of intercellular communication specific to senescent cells that are characterized by abundant secretion of molecules of diverse nature such as cytokines, growth factors, chemokines, and matrix metalloproteinases. Recently, new components of the SASP such as extracellular vesicles, metabolites, and ions have been discovered (Cuollo et al. 2020; Fafián-Labora and O'Loghlen 2020; López-Otín et al. 2013).

It should be noted that the profile of secreted molecules of SAPS is diverse and depends on the tissue context. Sometimes SASP plays a beneficial role in tissue physiology as it is involved in the removal of senescent cells from tissue and is thus related to the maintenance of homeostasis and tissue regeneration. In addition, SASP is involved in the recruitment of innate and adaptive immune cells in the vicinity of tumor cells and premalignant lesions (Cuollo et al. 2020; Fafián-Labora and O'Loghlen 2020; Olivieri et al. 2018). SASP components involved in these functions are regulated by p53 and include nerve growth factor inducible (VGF), or insulin-like growth factor-binding protein (IGFB) (Lopes-Paciencia et al. 2019).

Despite this, ageing increases the number of senescent cells and alters the type of molecules secreted from the SASP and thus intercellular communication. In this situation, SASP plays a detrimental role in tissues and is associated with the development of age-related diseases (Cuollo et al. 2020; Olivieri et al. 2018). The components of SASP in ageing can be classified as pro-inflammatory, pro-oncogenic, paracrine senescent, and autocrine senescent.

SASP and Its Pro-inflammatory Components: Age-Related Inflammation

The C/EBP family of transcription factors and mainly the NF-κB factor regulate the secretion of cytokines and pro-inflammatory factors such as Interleukin-1β (IL-1β), IL-6, IL-7, IL-8, chemokine “C-X-C motif” ligand 1 (CXCL1), or transforming growth factor β1 (TGFβ1) among others, which contribute to the development of a chronic and systemic inflammation process known as “inflammageing” (Franceschi and Campisi 2014). This inflammageing situation contributes to the development of age-related diseases such as obesity, type 2 diabetes, atherosclerosis, and Alzheimer's disease. In addition, inflammageing and stress-mediated inflammatory responses are associated with activation of the NF-κB pathway in the hypothalamus and induce a signaling pathway that results in reduced production of gonadotropin-releasing hormone (GnRH). Decreased GnRH has been shown to contribute to age-related physiological alterations including bone fragility, muscle weakness, skin atrophy, and reduced neurogenesis (Zhang et al. 2013).

Other studies, such as those of Pont and colleagues, link inflammation to ageing through the AUF1 factor (Pont et al. 2012). This factor is responsible for the degradation of the messenger RNA of pro-inflammatory cytokines, including tumor necrosis factor α (TNFα) and IL-1β (Lu et al. 2006), and is therefore involved in halting the inflammatory response. In addition, AUF1 has also been found to be involved in the activation of the expression of the catalytic subunit of telomerase, which means that it contributes to maintaining telomere length. A reduction in the expression of this factor, therefore, contributes to inflammageing, increased cellular senescence, and premature ageing.

At the same time as the age-related chronic inflammation situation, the function of the immune system is increasingly impaired in ageing. This makes it more difficult to effectively eliminate pathogens and dysfunctional cells from tissues such as senescent cells. This leads to an accumulation of senescent cells in tissues resulting in an increase in pro-inflammatory cytokines and thus chronic inflammation.

SASP and Pro-oncogenic Components

The SASP in ageing is also formed by enzymes involved in the reorganization of the extracellular matrix such as the matrix metalloproteinases (MMPs). These enzymes, in addition to reorganizing the extracellular matrix, are responsible for processing chemokines in tissues. Therefore, the secretion of these proteases reduces the recruitment of macrophages that are responsible for eliminating senescent cells, thus increasing senescence in tissues (Fig. 11.2). In addition, this results in reduced immune surveillance which, together with the immunosenescence characteristic of ageing, results in the body's inability to detect cells on the verge of malignant transformation (Lopes-Paciencia et al. 2019; López-Otín et al. 2013). IL-6 and IL-8 have also been found to have protumorigenic effects on cells in the vicinity of the senescent cell. That is, paracrine protumorigenic activity (Kuilman et al. 2008; Pietras et al. 2013). Consequently, SASP can also lead, in addition to chronic inflammation, to the development of tumors.

Fig. 11.2
An illustration depicts the paracrine pre-oncogenic, senescence, pro inflammatory, and autocrine senescence.

Types of SASP components. SASP components that mediate autocrine senescence are interleukin-6 (IL-6) and -8 (IL-8). SASP components that promote paracrine senescence include interleukin-1β (IL-1β), transforming growth factor-β1 (TGF-β1), and damage-associated molecular patterns (DAMPs). SASP components can also spread pro-oncogenic effects through matrix metalloproteinases (MMPs), IL-6, and IL-8; as well as pro-inflammatory effects through IL-1β, IL-6, IL-7, IL-8, chemokine “C-X-C motif” ligand 1 (CXCL1), and TGF-β1. Created with BioRender.com

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SASP and Paracrine Senescence

Acosta et al. (2013) demonstrated that there is a paracrine transmission of senescence from senescent cells with SASP to neighboring normal. However, this can spread locally with limitations, as the authors obtained results indicating that controls may exist to prevent the uncontrolled spread of senescence through an organ or tissue. The main components of SASP capable of triggering paracrine senescence are IL-1β and TGF-β (Pont et al. 2012). Senescent cells can also secrete damage-associated molecular patterns (DAMPs) capable of inducing paracrine senescence (Lopes-Paciencia et al. 2019).

SASP and Autocrine Senescence

The factors IL-6 and IL-8, which are part of the SASP, are capable of forcing cell cycle arrest of the senescent cell that secretes them, accentuating the senescence of this cell (Lopes-Paciencia et al. 2019; Pietras et al. 2013).

In short, ageing is a process that not only affects cells but also their communication. It is worth noting that there are currently therapeutic strategies that act in this sense, senomorphics. These are drugs that neutralize the detrimental effects of cell communication in ageing, i.e., they reduce SASP without killing senescent cells as senolytics do. Senomorphics, therefore, offers an opportunity to modulate and mitigate the deleterious effects of ageing at the level of cell communication. Despite this, intercellular communication is very complex, highlighting the need for further research in this field to improve the prospects for patients suffering from these pathologies.

Stem Cells for Regenerative Medicine

Introduction to Stem Cells

A stem cell is presented as one that divides asymmetrically to produce a copy of itself and a second cell that is on its path to differentiate (Martinez-Agosto et al. 2007). The definition of a stem cell inevitably requires an assessment of its potential to give rise to several differentiated progenies. Therefore, according to their potency, stem cells can be divided into totipotent, pluripotent, multipotent, and unipotent. Stem cells can also be classified according to their origin into two groups: embryonic stem cells (ESCs) and adult stem cells (ASCs). ESCs are pluripotent cells that can give rise to cells from the three germ layers: endoderm, mesoderm, and ectoderm (Amit et al. 2000; Itskovitz-Eldor et al. 2000; Reubinoff et al. 2000; Schuldiner et al. 2000). Adult stem cells (ASCs) can give rise to mature cell types within the same germ layer: ectodermal cells will give rise to skin and neurons; mesodermal cells will generate cardiac, muscle, blood, and bone cells; and endodermal cells will produce visceral cells, such as pancreatic, lung, kidney, or thyroid cells.

In the adult organism, ASCs reside in niches. Several niches have been identified in almost all adult tissues: lungs, skin, adipose tissue, vasculature, heart, central nervous system, liver, pancreas, kidneys, gastrointestinal tract, endometrium, muscle, bone marrow, oral tissues, etc. (Mas-Bargues et al. 2019). All of them being potential sources of stem cells. Thus, ASCs can be easily obtained for regenerative therapies.

Several strategies have been employed to induce the conversion of differentiated cells into an embryonic state. All these approaches led to the generation of a new cell type, commonly referred to as “induced pluripotent stem cells” or iPSCs. The iPSCs are reprogrammed from differentiated somatic cells by going back to an undifferentiated state similar to ESCs. Both ESCs and iPSCs are defined as pluripotent and can differentiate into three germ layers (endoderm, mesoderm, and ectoderm) as well as self-renew. Thus, iPSCs can also be used for regenerative therapies.

The usage of ASCs and iPSCs in cell-based therapies has advantages and disadvantages. The ideal stem cell for transplantation should be obtained from an easy-access source, with high and fast expansion rates in vitro, long-term survival capacity, immunologically inert for a successful host integration, and able to differentiate in situ to replace the damaged cells (Kim and Park 2017). Although iPSCs have a higher replicative lifespan and differentiation potential, this feature has been associated with an increased formation of teratomas following iPSCs administration (Deng et al. 2018; Iida et al. 2017), whereas ASCs are non-tumorigenic when transplanted in vivo (Itakura et al. 2017). Another key point is the ability to attach to the target site, which has been directly related to their growth and differentiation (Lam and Longaker 2012). In this context, ASCs displayed better migration and engraftment than iPSCs. Indeed, ASCs are the preferred cells for cell-based therapies.

Importance of Stem Cell Culture Conditions for Clinical Applications

Any regenerative therapy using ASCs or iPSCs needs a very high number of cells which can only be achieved by a long-term in vitro culture. During this expansion stage, stem cells can enter a senescence state, where they cease to proliferate and lose the ability to differentiate (Alves-Paiva et al. 2022). For this reason, in vitro culture conditions must be considered to obtain good-quality stem cells that can be used in cell therapies. Currently, stem cell expansion is mostly achieved via the conventional platform and protocol: tissue culture plastic (TCP) and 10% fetal bovine serum (FBS) in 21% O2/5% CO2 incubators. This combination has been a gold standard in obtaining millions of cells, but clinical studies require a new protocol for SCs expansion. Recently, new culture systems, reduced oxygen tensions, and media preconditioning have proven to improve stem cell proliferation and differentiation potentials toward a better outcome for regenerative therapies.

Stem cell culture systems can be two-dimensional (2D) or three-dimensional (3D) (Abdollahi 2021). 2D systems include common polystyrene flasks, whereas 3D systems mainly consist of bioreactors, such as two-chamber or hollow-fiber bioreactors, in which stem cells can be grown in higher densities (Gobin et al. 2021). Furthermore, 3D systems can capture physiological aspects that are missing in conventional 2D systems, such as tissue architecture, extracellular matrix composition, and heterotypic cell–cell interactions (Ader and Tanaka 2014). Indeed, a study suggested a decellularized extracellular matrix as an improved cell expansion setting for ASCs derived from bone marrow aspirates (Van et al. 2019). Another recent study reported that a gravity-controlled environment could enhance the proliferation and differentiation of ASCs (Nakaji-Hirabayashi et al. 2022). Moreover, there are several bioengineering strategies to create 3D constructs from iPSCs (Varzideh et al. 2022).

Most conventional in vitro cell cultures are performed under ambient oxygen concentration (20–21% pO2), which is often referred to as “normoxia.” In contrast, in vivo ASCs are not exposed to such a high concentration of oxygen. ASCs are developed in environments with low oxygen tension, that ranges between 0 and 1% pO2 in the bone marrow (Chow et al. 2001; Harrison et al. 2002), to 13–18% pO2 in blood and lungs (Steurer et al. 1997). Keeley and Mann (2019) published a comprehensive review of the specific oxygen concentration for each tissue of the human body and concluded that an average range of 3–6% pO2 would be considered as the physiological normoxia in which cells should be cultured in vitro. Indeed, culturing ASCs derived from dental pulps at 3% pO2 proved to maintain their proliferation rate, differentiation capacity, and stemness potential compared to those cultured at 21% pO2 (El Alami et al. 2014; Mas-Bargues et al. 2017, 2019). Moreover, it has been reported that low oxygen tension improves ASCs adhesion, proliferation, and differentiation in 3D scaffolds (Vina-Almunia et al. 2017). It is interesting to note that cultivating ASCs under physiological normoxia before transplantation improves tissue regenerative potential (Chen et al. 2022; Rosova et al. 2008).

Media preconditioning consists in activating cytoprotective pathways by either exposing cells to a sublethal environment (Hu et al. 2008), or by transfecting cells with survival genes (Zhang et al. 2008), or by conditioning cells with pharmacological molecules to activate specific cellular survival pathways. Among these pharmacological agents, oxytocin, celasterol, or melatonin have been commonly used (Noiseux et al. 2012; Touani et al. 2021; Zhao et al. 2015). As an example, in vitro studies have demonstrated that melatonin exerts antioxidant and antiapoptotic effects on MSCs, thereby improving the outcome of stem cell transplantation (Lee et al. 2019; Zhang et al. 2017a). Melatonin in transplanted mice promoted neural stem cell proliferation and differentiation into oligodendrocytes and astrocytes while reducing oxidative stress (Mendivil-Perez et al. 2017).

Stem Cells in Clinical Trials

Stem Cell Properties Supporting Their Clinical Application

Among ASCs, mesenchymal stem cells (MSCs) are the most used in clinical trials. The therapeutic potential of MSCs includes their ability to differentiate into various cell lineages, modulate the immune response, and migrate to the exact site of injury.

Several in vitro and in vivo studies have reported that MSCs can differentiate into bone, cartilage, fat, muscle, tendon, and bone marrow cells (Okolicsanyi et al. 2015; Zheng et al. 2013). MSCs’ fate is regulated by specific signals from their microenvironment, which includes biological molecules and biomechanical forces. These factors play a key role in determining MSCs differentiation and thus, their contribution to the repair process (Han et al. 2019). Indeed, MSCs-based therapies must ensure that the host microenvironment meets the requirements for the specific type of cell. It has been reported that the host environment led to the development of cell types other than those needed for the therapy (Ponticiello et al. 2000).

MSCs can modulate the immune system; MSCs can interact with both the innate and adaptive immune systems, leading to the modulation of several effector functions. Indeed, both in vitro and in vivo studies have reported that MSCs can interact directly with lymphocytes, dendritic cells, macrophages, and natural killer cells to prevent an excessive immune response (Han et al. 2012; Uccelli et al. 2008). This is of utmost importance regarding graft-versus-host-disease (GVHD), a syndrome that is characterized by systemic inflammation. For instance, donor-derived MSCs have been shown to induce long-term allograft acceptance in a rat model of heart transplantation (Popp et al. 2008). Although to prevent GVHD, the best solution is to use autologous stem cell transplants.

The homing ability of MSCs relies on their ability to migrate to the target tissue. The mechanism involves several signaling molecules and the corresponding receptors on the MSCs cell surface. Some of these signaling molecules have been identified: chemokines, adhesion molecules, and matrix metalloproteinases (MMPs) (Ries et al. 2007; Sohni and Verfaillie 2013). MSCs are known to express C-X-C chemokine receptor type 4 (CXCR4) and receptor tyrosine kinase (RTK) which are involved in MSC migration (Neuss et al. 2004; Shi et al. 2007). Notably, homing-related molecules can be upregulated by inflammatory cytokines such as tumor necrosis factor (TNF) and interleukins, such as IL-1 (Ren et al. 2010). Thus, this means that different inflammation statuses in different patients might lead to different MSCs engraftments and efficiency (Wei et al. 2013). If MSCs are intravenously administered, they must adhere to the vascular endothelial wall and cross the endothelial and muscular layers (Schmidt et al. 2006). This transendothelial migration of MSCs is a three-step process: adhesion, rolling, and crossing endothelial cells, which seems to be mediated by the very late antigen-4/vascular cell adhesion molecule-1 (VLA-4/VCAM-1) (Ruster et al. 2006).

Notably, tracking studies on stem cell delivery in mice have been performed using gene reporters and noninvasive techniques (PET, bioluminescence, or fluorescence). The reported data suggests that MSCs successfully reach different organs depending on the previously induced pathology (Belmar-Lopez et al. 2022).

Stem Cell-Based Therapies: Clinical Trials

Accumulating evidence supports the increasing use of stem cells in clinical trials, suggesting their translation from the laboratory bench to the patient’s bedside. According to the clinicaltrials.gov database, as of today (April 28th, 2022), there are over 9000 studies employing stem cells for regenerative medicine. Figure 11.3 highlights the use of MSCs for a wide diversity of pathological conditions.

Fig. 11.3
A pie chart depicts the clinical trials using stem cells. Nervous system diseases have the highest percent of 11.

Clinical trials using stem cells classified by disease condition

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Another important parameter to describe the use of stem cells in clinical trials is the phase of the investigation. As shown in Fig. 11.4, most studies are on phase 2, which consists of the analysis of the safety and effectiveness of the treatment. However, this phase is the most limiting since less than 25% of the studies progress to phase 3, and less than 10% reach phase 4, which are FDA approved drugs. Thus, this figure reflects that the therapeutic effectiveness of stem cell therapies needs to be carefully addressed and should also focus on long-term effects, which have been poorly addressed.

Fig. 11.4
A bar graph presents the clinical trials using stem cells by the phase of the investigation. Phase 2 has the highest value of 4000. Early phase 1 has the lowest percent of around 50.

Clinical trials using stem cells classified by the phase of the investigation

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Cardiovascular Diseases

Several MSC-based clinical trials have addressed cardiovascular diseases, ischemic heart diseases, and congestive heart failure. Myocardial infarction causes a loss of cardiomyocytes, and these dead cardiac cells will become replaced by fibroblasts to form scar tissue, leading to contractile dysfunction. MSC-based therapy aims at regenerating the damaged myocardium (Mathiasen et al. 2009).

In the study performed by Chen et al. (2004), myocardial infarction patients were divided into two groups. One group received an intracoronary injection of bone marrow (BM)-MSCs whereas the other group received saline. Six months after the beginning of the treatment, the group that received BM-MSCs displayed increased movement over the infarcted area, and left ventricular ejection was also improved in comparison with the control group.

The study of Katritsis et al. (2005) enrolled patients who had suffered an anteroseptal myocardial infarction, either recently or a long time ago. All patients had previously been subjected to angioplasty and stent implantation in the left anterior descending artery. BM-MSCs were administered through the balloon of the catheter. They observed improved end-diastolic and end-systolic diameter and volume, fraction shortening, and ejection fraction. However, these positive effects were only observable in patients who recently suffered a myocardial infarction.

Mathiasen and colleagues conducted a phase II trial assessing the intramyocardial delivery of autologous MSCs in patients with chronic ischemic heart failure. A total of 15 injections were administered to the ischemic region of the myocardium. After 12 months, the damaged myocardial tissue was regenerated improving the functional capacity of the injured hearts (Mathiasen et al. 2012) (ClinicalTrials.gov: NCT00644410).

Similar results were obtained by Friis et al., where the authors evaluated the effectiveness of the intramyocardial injection of autologous MSCs in patients suffering from stable coronary artery disease (CAD) and refractory angina. The patients that received the cell-based therapy displayed enhanced left ventricular function and exercise capacity (Friis et al. 2011) (ClinicalTrials.gov: NCT00260338).

Neurological Disorders

Most MSC-based clinical trials involving the treatment of neurological diseases targeted Parkinson’s disease (PD), Huntington’s disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and spinal cord injury (SCI).

Previous experimentation has reported that transplanting MSC into the brain is followed by the re-establishment of neural capacity (improved dopaminergic activity) through the mechanism of differentiation (Lindvall et al. 2012). Similarly, studies performed in humans have shown that transplanted intracerebral stem cells acquire local astroglial morphology and enhance the activity of local dopaminergic neurons (Niclis et al. 2017). Moreover, there is an ongoing clinical trial project named TRANSEURO investigating the benefits of grafting allogeneic dopaminergic neuroblasts derived from fetal ventral mesencephalic tissue into PD patients to recuperate the impaired activity dopaminergic neurons (Barker and Consortium 2019).

The study performed by Bachoud-Levi et al. analyzed the use of intrastriatal grafts of human fetal striatal cells. Their results reported improved daily cognitive and motor functions in three of five total patients as demonstrated by increased metabolic activity at the graft site (Bachoud-Levi et al. 2000). However, another study showed that a decade after the first transplantation in HD patients, the grafts yielded no survival in the caudate region (Cicchetti et al. 2009).

Karussis and colleagues studied the long-term effect of autologous BM-MSC in patients with intractable MS or progressive ALS. BM-MSCs were injected either intrathecally or intravenously, and none of them revealed any side effects in the 6–25 months of follow-up. Magnetic resonance imaging displayed the dissemination of BM-MSCs from the injection site to the meninges and spinal cord parenchyma. Thus, this data suggested safety and clinical stabilization and even improvement in some patients (Karussis et al. 2006).

Mendonça et al. conducted a phase I trial involving patients with chronic traumatic SCI. BM-MSCs were locally injected at the injury site. The treatment was safe and feasible and all patients displayed improvements in tactile sensitivity and gained lower limb motor function (Mendonca et al. 2014) (ClinicalTrials.gov: NCT01325103).

Taken together, stem cell therapy seems to have a potential neuroprotective effect. However, neurological disorders are often related to an advanced age. Ageing could diminish their therapeutic effects on these diseases, potentially leading to graft failure and other risks (Nguyen et al. 2019).

Bone and Cartilage Diseases

The ability of MSCs to differentiate into osteocytes and chondrocytes both in vitro and in vivo makes them suitable candidates for the treatment of bone and cartilage diseases, such as osteogenesis imperfecta (OI) and osteoarthritis (OA) (Liu et al. 2014).

Horwitz et al. (2002) demonstrated the feasibility of combined allogenic BM-MSC transplantation for children with severe OI. The authors showed that injected BM-MSCs in bone marrow could migrate to the bone, and differentiate into osteoblasts to improve bone structure. The clinical trial performed by Le Blanc et al. (2005) assessed in utero MSC allogenic transplantation in a female fetus with severe OI. After birth, the infant showed no immunoreactivity against the donor and only three fractures appeared in the first two years of age with normal psychomotor development and correct growth tendencies.

Orozco et al. administered autologous BM-MSCs through intra-articular injection in patients with chronic knee pain and radiological evidence of OA. One year after the beginning of the treatment, patients showed a rapid and progressive improvement of functional movement, along with a decrease in poor cartilage area and an enhancement in cartilage quality (Orozco et al. 2013) (ClinicalTrials.gov: NCT01183728). Similar results were obtained by Wong et al. (2013) in patients undergoing high tibial osteotomy and microfractures in knees with various cartilage defects. Their results showed improvement in repaired cartilage tissue after two years of postoperative outcomes.

Despite these optimistic results, the clinical application of MSCs in OA and OI remains in its infancy, and its effects need to be further addressed.

Other Pathologies

Several new clinical observations reported the efficacy of infused MSC in ameliorating tissue damage and/or improving function after injury to other organs, such as lungs, kidneys, liver, and eyes.

The study performed by Weiss et al. (2021) evaluated the time course of lung function, exercise performance, and exacerbation frequency following four monthly infusions of allogeneic BM-MSCs versus placebo. Their results reported that COPD patients treated with allogeneic BM-MSCs demonstrated significant improvements from baseline in forced expiratory volume in one second, forced vital capacity, and six-minute walk distance at 120 days after the first infusion. These improvements persisted variably over the 2-year observational period (ClinicalTrials.gov: NCT00683722).

A randomized study enrolled patients with liver cirrhosis, divided into a control group (receiving conventional treatment) and an umbilical cord-MSC (UC-MSC)-treated group (receiving three injections in 4-week intervals). The reported results showed an increased overall survival rate in the UC-MSC-treated group than in the control group. Moreover, UC-MSC treatment improved liver function, as demonstrated by the levels of serum albumin, prothrombin activity, cholinesterase, and total bilirubin (Shi et al. 2021) (ClinicalTrials.gov: NCT01220492).

A study analyzed patients with chronic kidney disease (CKD) due to different etiologies such as hypertension, or nephrotic syndrome. These patients received an intravenous infusion of autologous cultured MSCs. Follow-up visits of all patients did not reveal any cell-related adverse events. However, changes in glomerular filtration rate and serum creatinine were not statistically significant. This study showed the safety and tolerability of a single-dose infusion of autologous MSCs in patients with CKD but without beneficial results (Makhlough et al. 2018) (ClinicalTrials.gov: NCT02195323).

A phase I clinical trial enrolled 14 participants who were treated with a single-dose intravitreal BM-MSC injection. During the 12-month follow-up period, they found significant improvements in the best-corrected visual acuity (BCVA) compared to the control group, although they returned to the baseline at 12 months. The visual field (VF) and central subfield thickness (CST) remain unaltered, indicating no remarkable disease progression. Importantly, the authors noticed mild and transient adverse events after one year of the beginning of the treatment and observed one severe but manageable adverse effect in one patient in year 3 (Tuekprakhon et al. 2021) (ClinicalTrials.gov: NCT01531348).

Taken together, MSC-based therapies seem to have beneficial effects, but long-term side effects have also been reported.

Paracrine Effect of Stem Cells

A paradigm shift emerged suggesting that the beneficial effects of stem cells might not be restricted to cell replenishment alone, but also to their paracrine effect (Baraniak and McDevitt 2010).

The paracrine effect exerted by MSCs was hypothesized to support the observation that the number of implanted MSCs detected in target tissue was too low to explain tissue recovery or wound healing (Wang et al. 2011).

Stem cells secrete different kinds of factors that modulate the molecular composition of the environment to modulate responses from the resident cells’ dynamics (Galderisi and Giordano 2014). Stem cells actively contribute to their environment by secreting cytokines, growth factors, and extracellular matrix (ECM) molecules that act either on themselves (autocrine actions) or on neighboring cells (paracrine actions).

All these factors promote cell protection, proliferation, and migration. Indeed, these cells secrete proangiogenic factors, antifibrotic factors, factors responsible for ECM homeostasis, as well as anti-inflammatory, antiapoptotic, antioxidant, and immunosuppressive factors. Some of the identified factors involved in immune system signaling are interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-b (TGF-b). Other factors have been characterized as ECM remodelers, among them fibronectin, periostin, collagen, decorin, and matrix metalloproteinases (MMPs). Also, growth factors have been described, including vascular endothelial growth factor (VEGF), colony-stimulating factor (CSF), bone morphogenetic protein 2 (BMP-2), basic fibroblast growth factor (bFGF), and insulin-like growth factors (Baraniak and McDevitt 2010; Gnecchi et al. 2008; Linero and Chaparro 2014; Ratajczak et al. 2012; Squillaro et al. 2016).

In addition to soluble factors, stem cells also release small vesicles, spherical membrane fragments shed from the cell surface or secreted from the endosomal compartment (Ratajczak et al. 2006b). These vesicles also have an important role in improving the function of damaged organs as discussed in the following sections.

Taken together, the characteristics of stem cells, including differentiation, immunomodulation, homing and paracrine effects, make them suitable for cell-based therapies (Fig. 11.5).

Fig. 11.5
An illustration depicts the in vitro culture with differentiation, immunomodulation, homing, and paracrine effects. The parts Cell cellular system, oxygen tension, and preconditioning are labeled.

Stem cell characteristics and culture conditions for clinical trials. After mesenchymal stem cell (MSC) obtention, an in vitro culture phase is modulated by the cell culture system, the oxygen tension used, and the media preconditioning. During the in vitro phase, the main characteristics of MSC (differentiation, immunomodulation, homing, and paracrine effects) can be checked before their use in clinical trials

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Extracellular Vesicles as Tools for Intercellular Communication

EVs: Definition and Classification

Extracellular vesicles (EVs) are key effectors of intercellular communication. They can be defined as particles naturally released from a cell to the extracellular space, which are bound by a lipid bilayer and lacks a functional nucleus, hence cannot replicate (Théry et al. 2018). EVs serve as vehicles for intercellular transfer of cytosolic proteins, lipids, and nucleic acids, allowing for higher biostability and targeting capacity than other non-capsulated secretome components (Zaborowski et al. 2015). Although they are of utmost importance to maintain homeostasis in multicellular organisms, unicellular organisms such as bacteria are also capable of secreting EVs, named outer membrane vesicles (Yanez-Mo et al. 2015).

Traditionally, EVs have been classified according to their biogenetic pathway (Raposo and Stoorvogel 2013). Due to the difficulty in confirming their origin, a designation based on physical characteristics, such as size, is currently preferred. EVs size can range from 10 to 20 nm in diameter, although most research focuses on EVs of 100 nm and above (Margolis and Sadovsky 2019). According to the International Society for Intercellular Vesicles, EVs under 200 nm in diameter should be considered small EVs, while those over 200 nm are medium or large EVs. Alternative classifications may be based on properties such as density, biochemical composition or type, and culture condition of the cells of origin (Théry et al. 2018).

A Brief History of EVs

The first observations of EVs were reported by Chargaff and West (1946), who described them as procoagulant platelet-derived particles that were sedimented after plasma centrifugation. They were referred to as “platelet dust” in 1967 by Peter Wolf, who published the first electron microscopy images of these particles (Wolf 1967). Crawford termed those “microparticles” and showed that their cargo included lipids, RNA, and contractile proteins (Crawford 1971). Throughout the decade, EVs were isolated from a variety of sources, ranging from human tumor cell cultures to other body fluids (Yanez-Mo et al. 2015). In the early 1980s, parallel works from Johnstone and Stahl named these particles “exosomes” and proposed they served as a major route for the externalization of damaged proteins (Johnstone et al. 1991). A decade later, Raposo et al. (1996) found that these exosomes, which they isolated from Epstein Barr virus-infected B cells, were able to present antigens and induce a T-cell response. Their work suggested that exosomes’ biological functions may go far beyond cellular waste disposal (Couch et al. 2021). This hypothesis was confirmed in 2006 by Ratzjack et al., who evidenced that EVs mediate horizontal RNA transfer capable of reprogramming gene expression of recipient cells (Ratajczak et al. 2006a). Ever since EVs have acquired renewed interest as vehicles of cell-to-cell communication.

EVs Biogenesis

There are currently three known EV biogenetic pathways, giving rise to (i) exosomes, (ii) microvesicles, and (iii) apoptotic bodies (Fig. 11.6). Exosomes, which range between 40 and 120 nm in diameter, are produced by endocytic budding of the cell membrane and the formation of an intra-luminal vesicle (ILV) inside the cell. The ILV is loaded with a particular cargo in an endosomal complex required for transport (ECRT)-dependent or independent pathway and matures to form a multivesicular body (MVB). The MVB is transferred alongside microtubules to fuse with the plasma membrane and exosomes are secreted into the extracellular space. Microvesicles, also called ectosomes, measure between 100 nm and 1 μm and are formed by outward budding and fission of the plasma membrane. Apoptotic bodies, from 50 nm to 2 μm in diameter, are released by blebbing of cells undergoing apoptosis (Doyle and Wang 2019; Yanez-Mo et al. 2015; Zaborowski et al. 2015).

Fig. 11.6
An illustration depicts the apoptotic bodies, exosomes, and micro vesicles between the donor and recipient cells.

Extracellular vesicles biogenesis. Extracellular vesicles (EVs) mainly include exosomes, microvesicles, and apoptotic bodies. Exosomes are derived from multivesicular bodies (MVB) inside the cells that fuse with the plasma membrane. Microvesicles are formed by outward budding and fission of the plasma membrane. Apoptotic bodies are released by blebbing of cells undergoing apoptosis. Recipient cells can incorporate EV cargo through endocytosis or phagocytosis. Created with BioRender.com

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EV Composition

EVs’ cargo can include proteins, lipids, genetic materials, and small-molecules metabolites (amino acids, ATP, amides, sugars, cytokines, etc.) (Hade et al. 2021).

EV biogenesis can be used to understand their proteome (Doyle and Wang 2019). EVs’ formation and transportation are regulated by ESCRT proteins, thus, these proteins and their accessory proteins (Alix, TSG101, HSC70, and HSP90) are expected to be found in EVs (Morita et al. 2007). A wide range of proteins have been confirmed to be integrated into or attached to the membranes of EVs, or are present in their intra-luminal space. CD63, CD9, and CD81 are transmembrane proteins of the tetraspanin family and are commonly found in EV membranes (van der Koog et al. 2022). On the other hand, heat shock proteins (Hsp70 and Hsp90), lysosomal-associated membrane proteins (Lamp2a and Lamp2b), cytoskeletal proteins (actin, tubulin, and cofilin), integrins, and proteoglycans are found within EVs (Elsharkasy et al. 2020). Also, functional proteins might be characteristic of EVs isolated from specific cell sources. For instance, EVs derived from antigen-presenting cells (APCs), such as B-lymphocytes, dendritic cells, microglia, and macrophages, are enriched with major histocompatibility complex (MHC) proteins (Raposo et al. 1996).

EVs have a lipid bilayer and lipidomic analyses have shown that EVs contain lipid species that are present in the plasma membrane, although EVs are enriched in glycosphingolipids, sphingomyelins, phosphatidylserines, phosphatidylcholines, and cholesterol compared to the cellular plasmatic membrane (van der Koog et al. 2022). EVs are rounded vesicles, thus the lipid bilayer has a highly curved structure. This is reflected in the presence of lipid species that allow the curvature in the outer membrane, such as lipids with one fatty acid chain (Haraszti et al. 2016). EVs mainly contain monounsaturated fatty acids, polyunsaturated fatty acids, and saturated fatty acids, though the lipid composition depends on the parent cell (Skotland et al. 2019, 2020). Interestingly, EVs can transport several bioactive lipids and lipid metabolism-related enzymes, such as arachidonic acid, leukotrienes prostaglandins, phosphatidic acid, docosahexaenoic acid, and lysophosphatidylcholine (Subra et al. 2007).

The genetic cargo of EVs includes both DNA and RNA. RNAs have been extensively studied and several analyses have reported that EVs include various biotypes that represent a selected portion of the RNA content of the parent cell (O'Brien et al. 2020). Among them, small and long noncoding RNAs, including small nuclear RNAs, small nucleolar RNAs, ribosomal RNAs, PIWI-interacting RNAs, transfer RNAs, mitochondrial RNAs, Y RNAs, and vault RNAs (Chakrabortty et al. 2015; Lener et al. 2015; Xiang et al. 2013). Also, mRNA, small interfering RNAs, microRNAs, antisense oligonucleotides, guide RNAs, self-amplifying RNAs, and circular RNAs have been described (Fanale et al. 2018; Geng et al. 2020; Li et al. 2014).

The RNA within EVs reflects the type and the physiological/pathological state of the parent cell. However, EVs’ RNA content differs from the cellular RNA content, in terms of both RNA types and their relative concentrations, thus suggesting that some RNA species are selectively incorporated into extracellular vesicles (Baglio et al. 2015; Guduric-Fuchs et al. 2012; Mittelbrunn et al. 2011).

EV Internalization and Function

EVs can enter a recipient cell through many routes, including macropinocytosis, lipid-raft-mediated uptake, phagocytosis, and membrane fusion (Mathieu et al. 2019; Mulcahy et al. 2014), as well as tunneling nanotubes mediating transfer (Rustom et al. 2004). The main route of EV uptake is likely via clathrin/caveolin-mediated endocytosis (Mulcahy et al. 2014).

It is generally accepted that functional effects on recipient cells, besides direct signaling from the plasma membrane, might require EV internalization followed by cargo release into the recipient cell. Overall, so far little is known about the intracellular processing of EVs’ cargo and how delivered instructions are interpreted by the recipient cell (van Niel et al. 2022).

Indeed, the evidence supports the conclusion that uptake does not equate to functionality. When assessing the functional delivery of cargo, it became evident that some cargo was retained in the endosomal compartment. The authors showed that some of the EVs content remained within the cell in a “non-functional” manner. Almost 17% of the internalized signal was retained in the recipient cell for 24 h after removing the external source of the signal (O'Brien et al. 2022). Similarly, other studies suggest that EVs, or at least the dyes or labels that are used to visualize them, eventually end up in lysosomes. These results suggest a possible mechanism of retaining cargo in endosomes of the recipient cell before degradation or possible re-release.

At the same time, there is overwhelming evidence that EVs transfer their cargo into the cytosol of recipient cells and induce epigenetic modifications, which indicates that EVs are capable of avoiding the endolysosomal pathway. It is still unknown whether the fate of EVs internalized cargo depends on intrinsic differences between EV subtypes or the fate is dictated by the characteristics of the recipient cell (van Niel et al. 2022).

Most therapeutic uses for EVs require the uptake of encapsulated content into the cytoplasm (Gurunathan et al. 2019). Although the functional delivery of EV content is often the desired outcome, it should be taken into account that endosomal uptake often results in lysosomal degradation with the purpose of recycling or eliminating molecules from the recipient cell (Tian et al. 2010).

Isolation Methods

EVs can be isolated from the supernatant of cultured cells and biological fluids. There are a plethora of different methods to isolate EVs, however, these have proven to produce different EV populations and purities. Moreover, some isolation and purification methods undergo cellular stress that affects EV function (Mas-Bargues and Borrás 2021).

EVs can be isolated based on their density, size, immunoaffinity, and solubility or aggregation properties. The most used method is based on the particles’ density to separate them by ultracentrifugation at 100,000 g for 1–2 h (Crescitelli et al. 2013; Gudbergsson et al. 2016). Several protocols isolate EVs according to their size, such as ultrafiltration and size exclusion chromatography (SEC) (Varderidou-Minasian and Lorenowicz 2020; Xu et al. 2016). Both of them are a time and cost-effective alternative to the gold standard ultracentrifugation. Immunoaffinity-based techniques rely on the use of an antibody (such as tetraspanins CD9, CD63, or CD81) to interact with the molecules on the EVs’ outer surface. These antibodies are attached to well plates, chromatography columns, magnetic beads, or microfluidic platforms, thereby allowing EV isolation (Doyle and Wang 2019; Li et al. 2017; Sidhom et al. 2020). Polymer-induced precipitation is another used method for EV isolation based on solubility or aggregation properties. Highly hydrophilic polymers (such as polyethylene glycol (PEG)) interact with water molecules surrounding the EVs to create a hydrophobic microenvironment, resulting in EV precipitation (Weng et al. 2016). This technique is inexpensive and allows an EV recovery of 90% and a high yield, but entails sample contamination with protein aggregates and nucleic acids.

Extracellular Vesicles in Regenerative Medicine

Regenerative Medicine: From a Cell-Based to a Cell-free Approach

Although stem cell-based regenerative medicine has yielded encouraging results, we are currently witnessing a transition to a cell-free approach, and notably to an EV-based approach (Gomzikova and Rizvanov 2017; Moghadasi et al. 2021). The reason for this is twofold: EVs might mirror most of the beneficial effects of cell therapy while facing considerably fewer safety concerns.

On the one hand, it has recently been questioned whether the benefits of cell therapy are explained by their structural role; that is, by the capacity of stem cells (SCs) to engraft in damaged tissues and repair them through differentiation. Contrarily, the paracrine hypothesis postulates that these regenerative effects might also, and perhaps more importantly, be attributed to the factors that SCs secrete to the microenvironment, including EVs. This last idea has received extensive support to date. First, some studies found that injected SCs have a limited engrafting capacity in vivo, which is aggravated by their short half-life. Indeed, when injected intravenously, most of them are trapped and damaged in the narrow lung capillaries (Eggenhofer et al. 2012; Kurtz 2008). Second, the beneficial effects of SC transplantation have been shown to extend to tissues where SCs do not settle, as was shown by hematopoietic SC transplantation in renal damage (Duffield et al. 2005) and myocardial infarction (Jackson et al. 2001; Takahashi et al. 2006), or muscular SCs on a murine progeria model (Lavasani et al. 2012). As shown by this last study, young healthy SCs are capable of rescuing proliferation defects in aged SCs; this suggests that tissue repair processes could be carried out by the recipient’s SCs after reactivation by donor SCs, in line with the paracrine hypothesis (Lavasani et al. 2012).

On the other hand, EV-based therapies offer remarkable benefits over cell-based therapies (Prockop et al. 2010). The most relevant risks of cell-based approaches are oncogenic transformation potential and undesired differentiation (Breitbach et al. 2007; Miura et al. 2006; Røsland et al. 2009), which do not occur with EVs since they lack a functional nucleus. Other safety issues include emboli formation with intravascular delivery (Furlani et al. 2009), pulmonary first-pass effect, and immune rejection (Moghadasi et al. 2021). Being nano-sized, biocompatible, and barely immunogenic, EVs overcome these drawbacks. Finally, their stability to freezing cycles, as well as their targeting capability and organotropism, make EVs an excellent candidate for both, cells and non-capsulated secretome components (Moghadasi et al. 2021).

EVs in Regenerative Medicine: Sources and Delivery Routes

Several sources have been described for EVs’ isolation in regenerative medicine, both natural and artificial. Natural sources include SCs and plasma from young individuals or even centenarians (Yin et al. 2021). Among all types of SCs, mesenchymal SCs (MSCs) are the most used due to their many advantages, such as their presence in a wide range of tissues, their effective isolation methods, and their capacity to grow in adherent cultures. EVs are frequently obtained from bone marrow MSCs (BM-MSCs), adipose-derived MSCs (AD-MSCs), dental pulp MSCs (DP-MSCs), and umbilical cord MSCs (uc-MSCs), among others (Álvarez-Viejo 2020). Artificial sources include induced pluripotent SCs (iPSCs), as well as EVs from natural SCs subject to media preconditioning and coated EVs (Yin et al. 2021). While media preconditioning can alter EVs’ content, coating only affects their surface, thus improving EVs’ features such as skin absorption (Zhang et al. 2020). Regarding delivery routes, EVs are commonly delivered through direct intravenous or intraperitoneal injection, although the topical application is also being tested. Alternative routes are hydrogel injection, which allows for slower absorption, or coating of bioactive scaffolds made of electrospun fibers (de Jong et al. 2014).

Role of EVs in the Ageing Process

As fundamental vehicles of intercellular communication, EVs are a double-edged sword throughout the ageing process. EVs from young organisms, healthy tissues, and SCs may work as beneficial signaling molecules and stimulate cellular repair. On the contrary, EVs from old or injured tissues, such as SASP-related EVs, could spread cell senescence and promote chronic inflammation. In vivo, the circulating EV pool is likely to represent a continuum from “healthy” to “unhealthy” EVs, reflecting the status of the different microenvironments. Over time, the balance could tip in favor of ageing promoting EVs, which positively reinforce organ damage and lead to age-related diseases in the long term (Lananna and Imai 2021). Hence, two possible gerotherapeutic strategies emerge. First, target cells that secrete “unhealthy” EVs; this is the endpoint of senolytic and senomorphic drugs (Childs et al. 2017). Second, to replace those with “healthy” EVs, as is the purpose of EV-based regenerative medicine. The latter strategy is the subject of this chapter.

EVs as Gerotherapeutics: Lifespan and Healthspan

Several studies have sought to understand the role of circulating EVs in lifespan and healthspan in mammals, and whether they could be extended through an EV-based approach. The first evidence was provided by Zhang et al., who proved that ageing speed is largely controlled by the hypothalamus through EV-contained miRNAs (Zhang et al. 2017b). In their work, they showed that hypothalamic neural SCs (NSCs) were lost with age in a murine model, and implantation of young NSCs extended mice longevity. Interestingly, treatment with NSC-EVs achieved similar improvements in healthspan, measured through motor coordination, endurance, sociality, and memory tests. Subsequent studies have supported this hypothesis, achieving a healthspan extension by reducing hypothalamic NSCs senescence (Xiao et al. 2020).

Recently, Yoshida et al. identified another EV component involved in ageing and longevity regulation in mammals: extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a nicotinamide adenine dinucleotide (NAD+) biosynthetic enzyme (Yoshida et al. 2019). It has been established that NAD+ bioavailability declines with age, causing a variety of age-related changes (Verdin 2015). Some studies show that eNAMPT secreted by adipose tissue—enhanced by certain stimuli, like fasting, through a SIRT1-mediated pathway—reaches the hypothalamus and locally increases NAD+, improving physical performance in mice (Yoon et al. 2015). Recent works prove that EV-contained eNAMPT decreases over time, therefore treatment with eNAMPT-containing EVs isolated from plasma of young mice can increase the lifespan and healthspan of old mice (Yoshida et al. 2019).

EVs as Gerotherapeutics: Hallmarks of Ageing

Many studies have questioned whether EVs from different sources can ameliorate different hallmarks of ageing, with promising results. Of all the hallmarks, the most frequently studied concerning EVs is cellular senescence. Recent work from Fafián-Labora et al. found that small EVs from young fibroblasts could ameliorate senescence-related biomarkers in fibroblasts from old or Hutchinson-Gilford progeria syndrome donors, as well as mice. They propose glutathione-S-transferase mu 2 (GSTM2), an antioxidant EV-contained enzyme whose activity declines with age, is responsible for the observed changes, in line with the decreased reactive oxygen species (ROS) levels and lipid peroxidation after treatment (Fafián-Labora et al. 2020). Another work with an iPSC-EVs treatment on senescent MSCs was able to decrease senescent phenotype by delivery of different antioxidant enzymes, peroxiredoxins 1 and 2 (Liu et al. 2019). Further studies in UVB-radiated fibroblasts support the senotherapeutic potential of EVs, although the underlying mechanism has not been elucidated yet (Deng et al. 2020; Oh et al. 2018). Recently, Dorronsoro et al., injected old mice intraperitoneally with MSC-EVs from young mice and measured senescence markers in different organs. They observed a decrease in most markers, including senescence-associated ß-galactosidase, p16, p21, and pro-inflammatory cytokines IL-1ß and IL-6. Consequently, they proposed MSC-EVs might work as senomorphics; that is, they might downregulate the expression of SASP-related factors, hence impairing the spread of this senescent state to adjacent cells (Dorronsoro et al. 2021).

Regarding other hallmarks, evidence is less abundant but still optimistic. MSC-EVs may be able to attenuate stem cell exhaustion, as shown by in vitro studies on BM-MSC-EVs and DP-MSC-EVs (Dorronsoro et al. 2021; Mas-Bargues et al. 2020). In this last study, treated cells recovered their stemness as evidenced by pluripotency factor (OCT4, SOX2, KLF4, cMYC) overexpression and the shift toward a highly glycolytic and low oxidative metabolism (Mas-Bargues et al. 2020). In some recent studies, treatment with EVs from young serum and MSCs has increased the expression of telomere-related genes, which could attenuate telomere shortening (Lee et al. 2018; Sonoda et al. 2020). The first study also found that mTOR and IGF-1 levels were generally lower after treatment, therefore, they might play a role in alleviating deregulated nutrient sensing (Lee et al. 2018). Furthermore, research on cardiovascular and neurodegenerative diseases shows that the therapeutic potential of MSC-EVs point to an increase in autophagy as the underlying mechanism; for this reason, it has been suggested that MSC-EVs may also help preserve proteostasis (Chen et al. 2020; He et al. 2020; Liu et al. 2017).

EVs and Age-related Diseases

Nowadays, more than 20% of the total burden of disease is due to people aged 60 years and over, with special emphasis on cardiovascular disease (30%), cancer (15%), and pulmonary, musculoskeletal, and neurodegenerative diseases (Prince et al. 2015). Given the promising results of EVs-based therapy to slow or even reverse some hallmarks of physiological ageing, it is not surprising that they are currently under study as next-generation therapies for age-related diseases (Fig. 11.7).

Fig. 11.7
An illustration depicts the extracellular vesicles with young plasma, M S C s between aging with lifespan and hallmarks of aging, and age-related disease with metabolic and others.

Extracellular vesicles in ageing and age-related diseases. EVs can be isolated from plasma or MSC obtained from dental pulp, adipose tissue, umbilical cord, or bone marrow. EVs can modulate ageing and age-related diseases. Regarding the former, EVs’ content (miRNAs and extracellular nicotinamide phosphoribosyltransferase (eNAMPT)) could be involved in longevity regulation, and EVs from young donors could ameliorate some hallmarks of ageing such as cellular senescence and stem cell exhaustion. Regarding the latter, EVs-based therapies are being used for cardiovascular, musculoskeletal, neurodegenerative, and metabolic diseases. Created with BioRender.com

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Cardiovascular Diseases

The cardioprotective properties of MSC-EVs have been investigated in a variety of age-related diseases, such as myocardial infarction (MI), ischemic stroke, and atherosclerosis. Recovery of cardiac function after MI has been in the spotlight since the origins of stem cell therapy (Jackson et al. 2001), and this interest has continued to rise with cell-free approaches, given its long-term morbidity as a cause of heart failure. Several studies have shown that MSC-EVs notably contribute to myocardial repair after MI, reducing infarct size and ultimately improving functional parameters such as stroke volume and cardiac output (Bian et al. 2014; Teng et al. 2015). Not only MSCs but different cell types, such as cardiomyocyte progenitor cells, hematopoietic stem cells (HSCs), and iPSCs have been used for EV isolation with similar results (Rezaie et al. 2019). Nevertheless, the underlying mechanism of these effects remains elusive. Since one of the main processes driving the recovery of ischemic myocardium is angiogenesis, it has been proposed that MSC-EVs could induce capillary development through the delivery of miR-210 (Bian et al. 2014; Teng et al. 2015; Wang et al. 2017a). Another mechanism under study is the modulation of the pro-inflammatory microenvironment (Teng et al. 2015), which could be achieved by activating the S1P/SK1/S1PR1 pathway and M2 macrophage polarization, in close relation to EV-contained miR-182 (Deng et al. 2019). Lastly, MSC-EVs could prevent myocardiocyte apoptosis and cardiac fibrosis through miR-221, which downregulates PUMA (p53 upregulated modulator of apoptosis). Ex vivo assays, where cardiac stem cells were pretreated with MSC-EVs, have also shown promising results (Zhang et al. 2016). Regarding ischemic stroke, evidence suggests that both neural plasticity and revascularization promoted by MSC-EVs may accelerate functional recovery in animal models (Xin et al. 2013a, b).

Since atherosclerosis is the disease underlying the aforementioned events, it has become an emerging therapeutic target. MSC-EVs’ contribution to the stabilization of atherosclerotic plaques is two-sided. First, they can inhibit endothelial cell (EC) apoptosis and promote proliferation by activating Nrf2, which protects ECs against oxidative stress (Chen et al. 2021). Secondly, the let-7c miRNA family carried by EVs downregulates the NF-κB inflammation pathway. This anti-inflammatory effect is enhanced through the shifting of macrophage phenotype from M1 to M2 (Li et al. 2019; Ma et al. 2021).

Neurodegenerative Diseases

In the central nervous system (CNS), EVs are studied with particular interest because they solve a difficulty of conventional drugs: the crossing of the blood–brain barrier (BBB). Being small-sized and highly lipophilic, they reach deep into the CNS, where they exert regenerative and immunomodulatory effects (Guy and Offen 2020). Recent studies on Alzheimer’s Disease (AD) mice models show that MSC-EVs improved cognition and alleviated memory deficits, which was consistent with the observed decrease in Amyloid-β (Aβ) plaques in the brain. MSC-EVs decreased astrocyte and microglia activation by tipping the balance from pro-inflammatory IL-1 and TNF-a to anti-inflammatory cytokines like IL-10. This led to the discovery of miR-21, an EV-contained miRNA capable of downregulating the STAT3–NF-κB axis responsible for the amplification of neural damage in AD (Cui et al. 2018). Similar results have been obtained with intraventricular injection of MSCs-EVs, where a marked increase of synaptogenesis in the hippocampus was attributed to miR-146a (Nakano et al. 2020). In another study, neural recovery from Aβ-induced damage and ROS decrease was achieved by MSC-EVs through the transfer of enzymatically active catalase (Bodart-Santos et al. 2019). Likewise, blood marrow and umbilical cord MSC-EVs have shown therapeutic effects on Parkinson’s disease rat models by stimulating neuronal differentiation in the substantia nigra (Mendes-Pinheiro et al. 2019), as well as inducing autophagy and decreasing apoptosis, thereby upregulating dopamine levels in the striatum and improving motor function (Chen et al. 2020).

Musculoskeletal Diseases

Current studies have unveiled the potential of MSC-EVs in musculoskeletal disorders, which are responsible for the exponential increase in frailty among the elderly. Osteoarthritis (OA) stands as the most prevalent disease in this group, several strategies have been pursued to restore cartilage and bone homeostasis. On the one side, MSC-EVs from adipose tissue were able to promote chondrocyte survival, decrease apoptosis, and improve autophagy; these effects were due to EV-contained miR-100-5p, which downregulates the mTOR pathway (Wu et al. 2019). Moreover, ADSC-EVs effectively tackled senescence features in OA osteoblasts (Tofiño-Vian et al. 2017). On the other side, MSC-EVs promoted repair of extracellular matrix (ECM) by increasing collagen type II synthesis and downregulating ECM-degrading enzymes, such as ADAMTS5 and metalloproteinases MMP1 and MMP13 (Cosenza et al. 2017; Jin et al. 2020; Wang et al. 2017b). The functional translation of these effects was remarkable, with recovery from gait abnormalities in murine models (Cosenza et al. 2017).

Positive OA osteoblasts results have driven interest in MSC-EVs to another major disease, osteoporosis. In vitro experiments showed that MSC-EVs obtained from human iPSC could increase protein expression of osteoblast-related genes and proliferation rates (Qi et al. 2016). In vivo models of osteoporosis in ovariectomized rats support these osteogenic and angiogenic effects of EVs, administered by two different routes: direct intraosseous injection or implantation of an EVs-releasing scaffold (Qi et al. 2016). MSC-EVs may as well have regenerative effects on the intervertebral disc via antioxidant and anti-inflammatory properties (Xia et al. 2019), thus constituting an attractive option for herniated disc prevention.

Sarcopenia is a condition that affects mostly elderly people to a greater or lesser extent and is particularly aggravated by episodes of illness or hospital admission. Although little has been published on age-related sarcopenia, there is growing evidence that MSC-EVs could be of value in preventing drug-induced muscle wasting (Cho et al. 2021; Li et al. 2021). A recent study found increased myotube diameter both in vitro and in vivo after treatment and an increase in muscle weight and strength, which may be explained by EV-contained miR-486-5p and its inhibitory effect on FoxO1 (Li et al. 2021). Similar work with tonsil MSC-EVs identified miR-145-5p as the responsible component for increased myogenic differentiation and total muscle mass in mice (Cho et al. 2021).

Other Diseases

One of the most important causes of morbimortality among the elderly is metabolic diseases, in particular, type 2 diabetes mellitus (T2DM), which is intimately related to obesity, non-fatty liver disease, and metabolic syndrome. T2DM is caused by increased peripheral resistance to insulin action, which drives an increased hepatic glucose output and, ultimately, impaired insulin secretion. In one study, uc-MSC-EVs proved useful in promoting hepatic glycogen storage and reducing gluconeogenesis, via AMPK signaling and autophagy induction, both in hepatocyte culture and in a T2DM rat model (He et al. 2020). Likewise, they reduced glucose levels and improved insulin sensitivity, promoting the expression of GLUT-4 receptors in muscle (Sun et al. 2018). Even though pancreatic damage does not play a role until the late stages of the disease, results in this regard are also encouraging and leave the door open for future studies in T1DM (Sun et al. 2018) (ClinicalTrials.gov: NCT02138331).

Chronic kidney disease (CKD) is a prevalent disease that greatly compromises the quality of life in the elderly, because the only available treatments, dialysis, and renal transplant, are potentially risky and have high costs. MSC-EVs have successfully been used to increase renal function parameters, such as glomerular filtration rate or urea and creatinine levels, in advanced stages of CKD in a recent clinical trial (Nassar et al. 2016). Interestingly, stimulation of MSCs with melatonin, a proposed renoprotective hormone, may enhance EVs’ regenerative potential in a murine model of CKD.

Cancer accounts for one of the highest burdens in the elderly (Prince et al. 2015). MSC-EVs have not received so much attention in cancer therapy, given that many of the biological processes they stimulate, such as cell proliferation, angiogenesis, and immunosuppression, could favor tumor growth and extension (Xunian and Kalluri 2020). Nonetheless, a clinical trials are currently in progress using modified MSC-EVs as drug carriers for cancer therapy, such as colon cancer (ClinicalTrials.gov NCT01294072) and pancreatic cancer (ClinicalTrials.gov: NCT03608631).

In conclusion, preclinical data provide evidence of the efficacy and safety of MSC-EVs-based therapies against a variety of age-related diseases. A number of these studies have already reached the stage of clinical trials, as is the case in ischemic stroke (ClinicalTrials.gov: NCT03384433), AD (ClinicalTrials.gov: NCT04388982), OA (ClinicalTrials.gov: NCT04223622), CKD (Nassar et al. 2016), and cancer. While EV studies are an evolving field in preclinical ageing research, some factors still hinder their translation to the clinical setting. Among them, we could underscore the lack of established cell culture conditions, the absence of scalable EV isolation protocols, more thorough investigations on the optimal therapeutic dose, administration routes and schedule, and reliable assays to evaluate the efficacy of EV-based therapies.

Concluding Remarks

Stem cells play a key role in maintaining tissue homeostasis, both after injury and during ageing. Stem cells have unique characteristics, such as differentiation, homing, and immunomodulation which make them suitable candidates for regenerative medicine. Stem cell-based therapies are currently aiming to improve damaged tissue functionality with promising results. However, long-term issues have been associated with stem cell therapies, such as poor engraftment at the injury site. This led to the hypothesis that stem cell-derived effects were due to their ability to modulate the microenvironment. Indeed, paracrine effects proved to be as effective as stem cell transplants. The study of the released molecules evolved into non-cell-based therapies; the golden star being mediated by extracellular vesicles. EVs’ content includes bioactive molecules, such as lipids, proteins, and genetic material that modulate the fate of the target cell. EVs-based therapy could slow down or even reverse some hallmarks of physiological ageing. Moreover, EVs are being used in many clinical trials for several age-related diseases, both as biomarkers and treatments.

Overall, these studies need to be developed, and more research should be performed to address several questions, such as EVs’ in vivo biodistribution and fate, the different processing of EVs cargo by target cells, or the relative potency of EVs in directing cell–cell communication compared to other secretome components. These and many other questions are key questions and challenges that remain to be addressed.