Regenerative medicine is a branch that aims to develop methods and procedures for the growth, restoration or replacement of damaged and diseased cells, tissues and organs. Stem cell treatment is a branch of regenerative medicine (Caplan, 1991). The principle of stem cell therapy is to overcome the inability to regenerate damaged tissue after acute or chronic disease. It is gaining ground as a therapeutic option for many conditions, both in human and veterinary medicine. Of particular interest are chronic respiratory diseases, responsible for a lower quality of life, resulting from the remodelling of the lung tissue and subsequent loss of functional tissue (Hirota and Martin, 2013).
By definition, stem cells are undifferentiated cells capable of self-renewal and differentiation into various specialised cells (Morrison et al, 1997). Depending on the source of the tissue, they are classified as embryonic stem cells, adult stem cells or induced pluripotent stem cells (Evans and Kaufman, 1981; Takahashi and Yamanaka, 2006). Based on their stage of development and their ability to differentiate, they are further classified into pluripotent and multipotent cells (Wagers and Weissman, 2004). Two types of stem cells are naturally present in the adult organism: bone marrow and peripheral blood contain haematopoietic stem cells, and non-haematopoietic (or mesenchymal) stem cells are found in many other adult tissues (Dulak et al, 2015). These cells are multipotent and can differentiate into different types of body cells. Mesenchymal stem cells are activated in the body when needed to replace dead, damaged or diseased cells (Caplan, 1991) and can differentiate into cells of bone, cartilage, ligaments, tendons, fat, skin, muscle, neuroglia and connective tissue (Caplan, 1991; Morrison et al, 1997; Dulak et al, 2015). Compared to other types of stem cells, mesenchymal stem cells are ethically more acceptable, and the acquisition, isolation and culture of a large number of stem cells is relatively straightforward.
Depending on the source, mesenchymal stem cells can have different properties, which should be considered when choosing the optimal stem cell therapy procedure to rehabilitate a specific disease. The most commonly used mesenchymal stem cells are bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells. Adipose tissue in particular is a desirable source of mesenchymal stem cells because of the minimally invasive procedure required to obtain these cells (Kern et al, 2006; Fujita et al, 2018).
However, there are few standards for the isolation, cultivation and characterisation of animal mesenchymal stem cells, which excludes a consistent comparison of results between studies. Additionally, the diversity of tissue sources makes it difficult to establish criteria to define mesenchymal stem cells precisely (Dominici et al, 2006; Uder et al, 2018).
Therapeutic potential of mesenchymal stem cells
Depending on the relationship between the donor and the recipient, the stem cell source can be classified as autologous, allogeneic or xenogeneic (Food and Drug Administration, 2015). Isolation and propagation of autologous stem cells is time-consuming and expensive, while allogeneic cells represent the possibility of immediate access. Furthermore, the potential of autologous mesenchymal stem cells may be affected by the patient's age (Guercio et al, 2013; Sancak et al, 2016; Lee et al, 2017; Zajic et al, 2017; Taguchi et al, 2019) and existing clinical or subclinical disease (Marycz et al, 2016). A significant caveat to allogeneic stem cell therapy is the possibility that recipient CD8+ T cells recognise surface major histocompatibility complex molecules on allogeneic mesenchymal stem cells, leading to direct cytotoxicity or alloantibody production (Wieczorek et al, 2017).
The therapeutic effect of mesenchymal stem cells was first attributed to their migration to the damaged tissue area and their ability to differentiate into different cell lines. This was quickly rebutted by the finding that less than 1% of mesenchymal stem cells remain in the body within 1 week after systemic administration of these cells (Eggenhofer et al, 2012; Song et al, 2012; Fujita et al, 2018). Therefore, the immunoregulatory effect (which takes place with the help of paracrine signalling and interaction with local immune system cells) has been highlighted more in recent years (Voga et al, 2020). Mesenchymal stem cells inhibit the activation and proliferation of effector T cells, responsible for tissue damage in many diseases, and increase the proportion of regulatory T cells (T-reg), which inhibit the course of immune processes and prevent autoimmunity (Aggarwal and Pittenger, 2005; Duffy et al, 2011). Furthermore, they inhibit tissue damage and pro-inflammatory cytokine production through macrophage interaction (Tsyb et al, 2008; Németh et al, 2009). Mesenchymal stem cells negatively regulate the expression of chemotactic receptors and ligands of B cells, so their chemotactic properties are inhibited (Corcione et al, 2006; Luk et al, 2016).
Some substances, which mesenchymal stem cells otherwise secrete through a paracrine action, are also secreted by extracellular vesicles and therefore allow the transfer of effectors over longer distances throughout the body (Hyvärinen et al, 2018; Crain et al, 2019; Park et al, 2019; An et al, 2020). Extracellular vesicles are membrane-protected carriers of micro- and messenger RNA, proteins and mitochondria. They also represent the potential to exploit the effects of mesenchymal stem cells in a cell-free manner, which can reduce the potential side effects of cell therapy (Jung et al, 2013; Mäkelä et al, 2015). However, intercellular contact is probably essential for some of the immunomodulatory properties of mesenchymal stem cells (Fu et al, 2012; Luk et al, 2016; 2017; Gao et al, 2017). The main limitation in the search for applications for the therapeutic use of extracellular vesicles is the lack of stand-ardised techniques for the isolation and purification of these cells. Ligands and cargo differ between types of extracellular vesicles, leading to the assumption that each type of extracellular vesicles has a different function (Tan et al, 2013; Reiner et al, 2017; Toh et al, 2018). Confirmation of the functionality of extracellular vesicle therapy requires that the therapeutic effect occurs without inter-cellular contact and that this is not achieved by soluble paracrine factors without extracellular vesicles (Iso et al, 2007).
The immune response after the application of mesenchymal stem cells not only depends on its active immunomodulatory activity, but also indirectly results from the reaction of other cells, which is triggered by the presence of mesenchymal stem cells (Luk et al, 2016). Mesenchymal stem cells undergo apoptosis after administration in the body; the apoptotic cells are then phagocytosed by macrophages, which ultimately achieve immunosuppressive activity (Galleu et al, 2017). de Witte et al (2018) further demonstrated that mesenchymal stem cells phagocytosis after intravenous application in the lung results in the expression of a specific monocyte phenotype at the local and systemic level, further regulating the acquired immune response by inducing T-reg cells.
The transfer of mitochondria is also one of the mechanisms of action of mesenchymal stem cells, as they are capable of intercellular transfer of organelles via tunnelling nanotubes. This activity allows for activating or upgrading aerobic metabolism in somatic cells of mammals with poorly functional or non-functional mitochondria (Spees et al, 2006). In a murine pneumonia model, human bone marrow-derived mesenchymal stem cells could transfer their mitochondria through tunnelling nanotubes to alveolar macrophages, leading to enhanced phagocytosis and an antimicrobial effect (Jackson et al, 2016).
In addition to the complex mechanisms of immunomodulation, the ability of mesenchymal stem cells to populate or migrate to the area of damaged tissue is also important. The migration of mesenchymal stem cells is closely related to chemical factors such as chemokines, cytokines and growth factors. Mechanical factors such as mechanical stress in the tissues, stiffness of the matrix and microgravity are also crucial for cell guidance (Fu et al, 2019).
To ensure a high local number of cells in a relatively short time, the most efficient method of administering mesenchymal stem cells is local transplantation. However, intraparenchymal injection of mesenchymal stem cells is sometimes impossible (Nowakowski et al, 2016). Therefore, systemic applications are simpler, among which the intravenous route is the least invasive but greatly limits the number of mesenchymal stem cells in the target tissue (Gao et al, 2001; Iso et al, 2007; Lee et al, 2009; Fujita et al, 2018). Migration of mesenchymal stem cells is limited by their exit from the systemic circulation (Ullah et al, 2019). Because of their size, the capture of mesenchymal stem cells in the pulmonary microcirculation is also very evident (Gao et al, 2001; Eggenhofer et al, 2012). An additional reason for the accumulation of mesenchymal stem cells in the lungs is their molecular interactions (which happen as a result of their overexpression and activation of integrins) with the pulmonary endothelium (Wang et al, 2015).
Therapeutic potential of mesenchymal stem cell treatment on chronic respiratory diseases
Although many respiratory disorders and their treatment with conservative and surgical methods are well defined, vets remain limited to symptomatic treatment for some respiratory conditions. Insufficient immune response and/or medical treatment can lead to permanent pathological tissue remodelling and a permanent loss of lung function.
The immunomodulatory effects of mesenchymal stem cells represent the possibility of cure, or at least mitigation, of some persistent chronic diseases that current therapeutic strategies cannot cure or significantly alleviate. Several studies based on mesenchymal stem cell therapy have been published in respiratory medicine. The results of these studies are variable when defining the impact of mesenchymal stem cells on the pathogenesis of inflammation, especially on the expression and translation of inflammatory mediators. However, a significant reduction in inflammation and a noticeable effect on lung tissue remodelling and/or clinical improvement are consistently reported after mesenchymal stem cell application (Harrell et al, 2019). Mesenchyman stem cells can even differentiate into alveolar epithelial cells under in vitro conditions (Ma et al, 2011).
A growing body of research points to the potential therapeutic effects of mesenchymal stem cells in the treatment of asthma. Positive results of cellular therapy for asthma have been reported in both experimental studies in mouse models (Bonfield et al, 2010; Lee et al, 2011; Cho et al, 2014; Mariñas-Pardo et al, 2014; Cruz et al, 2015; Mohammadian et al, 2016; Abreu et al, 2017; de Castro et al, 2017; Dai et al, 2017; 2018), naturally asthmatic cats (Trzil et al, 2016) and horses (Barussi et al, 2016; Adamič et al, 2022). The effect of mesenchymal stem cells found in a preclinical trial for asthma treatment is mainly related to the regulation of cytokines derived from the T-helper (Th)1, Th2, and Th17 types of immune response (Cho et al, 2014; Mariñas-Pardo et al, 2014; Cruz et al, 2015; de Castro et al, 2017; Zhang and He, 2019).
In a mouse model of asthma, Cho et al (2014) reported a decrease in clinical symptoms and inflammation after intravenous administration of adipose-derived mesenchymal stem cells. These positive effects coincided with reduced cytokine expression in the Th2 type of immune response and increased cytokine expression of cytokines involved in the Th1 type of immune response in bronchoalveolar lavage fluid. Mariñas-Pardo et al (2014) reported similar conclusions. They found rapid relief from airway inflammation related to Th1 cytokine expression and accumulation of cells in the lungs, excluding the integration of cells into the lung parenchyma and, therefore, their potential promodular effect. However, it should be emphasised that the effect of mesenchymal stem cell treatment decreased with continuous antigenic challenge (Mariñas-Pardo et al, 2014). Administration of bone marrow-derived mesenchymal stem cells significantly reduced the experimentally elevated inflammatory index and the number of eosinophil and neutrophil granulocytes in the bronchoalveolar lavage fluid of rats to the levels of healthy control animals without asthma (Lee et al, 2011). In the group that received bone marrow-derived mesenchymal stem cells, the number of goblet cells and the amount of collagen decreased significantly. Similar beneficial effects of mesenchymal stem cell treatment of experimentally induced asthma have been reported in mice (Cho et al, 2014; Mohammadian et al, 2016; Dai et al, 2017; 2018) and cats (Trzil et al, 2016). Trzil et al (2016) reported a reduction in inflammatory cells, airway hyper-responsiveness and airway remodelling detected 9 months after treating cats with adipose-derived mesenchymal stem cells.
Current evidence suggests that the use of mesenchymal stem cells to treat lung disease is considered safe (Fujita et al, 2018). A study found a significant decrease in C-reactive protein in patients with chronic obstructive lung disease after mesenchymal stem cell treatment (Weiss et al, 2013). Two studies reported that chest computed tomography 6 months and 1 year after mesenchymal stem cell treatment did not detect fibrotic or tumorigenic changes (Chambers et al, 2014; Stolk et al, 2016). Furthermore, Stolk et al (2016) analysed differences in lung histology and expression of specific genes in patients with chronic obstructive lung disease after treatment with bone marrow-derived mesenchymal stem cells. Treatment did not increase the expression of profibrotic genes or proliferation markers in lung tissue, nor was any change in histological structure detected (Stolk et al, 2016).
Intratracheal application of biological therapeutic agents for clinical use has some advantages over systemic administration. The need for the number of cells administered and the possibility of developing unwanted systemic side effects are reduced. Intratracheal application also allows direct access to the local pulmonary immune system and faster interaction with it (Fujita et al, 2018). Considering the currently known effects of mesenchymal stem cell treatment, its therapeutic use in respiratory diseases (and other diseases) has a high potential for relief, healing and reducing suffering in both humans and animals.
Mesenchymal stem cells and severe equine asthma
Because of the recognised ability to change the course of inflammation, mesenchymal stem cell therapy is of particular interest in treating equine inflammatory respiratory diseases, primarily severe equine asthma. The essential features of the disease are increased hypersensitivity and responsiveness of the airways to antigens in inhaled air and the consequent development of chronic neutrophilic inflammation of the airways, bronchoconstriction and mucus accumulation. As a result of the changes mentioned above, lung function weakens and, in the long term, the airway tissue structures are transformed (Robinson et al, 2001; Lavoie, 2007). Although it is possible to achieve a certain period of clinical remission with consistent environmental management and symptomatic treatment of affected horses, no definitive cure can be achieved with currently available therapeutic strategies as they do not have a significant effect on the pathological remodelling of the airway walls (Robinson, 2001; Lavoie, 2007). Therefore, regenerative forms of treatment, mainly stem cell treatment, represent an attractive option. To date, two studies have been published that examined the impact of cellular therapy on severe equine asthma. Barussi et al (2016) studied the influence of intratracheal application of bone marrow mononuclear cells on the course of respiratory inflammation in horses with severe equine asthma. A comparison of treatment with a single intratracheal administration of autologous cells and treatment with oral administration of dexamethasone showed that bone marrow-derived mononuclear cells improved clinical signs and the inflammatory response in asthmatic horses. Anti-inflammatory interleukin-10 levels increased after cell treatment and were significantly higher than in the dexamethasone-treated control group (Barussi et al, 2016). Another group of researchers studied the safety and therapeutic efficacy of intrabronchial administration of adipose-derived mesenchymal stem cells for the treatment of severe equine asthma. A decrease in the expression of some pro-inflammatory cytokines in the broncheolar lavage fluid was found after treatment with adipose-derived mesenchymal stem cells. A long-term positive effect on the clinical signs of severe equine asthma was also detected. Based on the findings of this research, it can be concluded that intrabronchial application of autologous adipose-derived mesenchymal stem cells had a limited short-term anti-inflammatory effect and a positive long-term effect on the stability of severe equine asthma symptoms (Adamič et al, 2022).
Conclusions
Because of the recognised influence of mesenchymal stem cells on the course of local inflammation and remodelling of lung tissue in chronic diseases, they represent hope for more successful therapies that could improve the lives of individuals in the future. Research on the impact of mesenchymal stem cells on the development and immunological background of severe equine asthma is currently in its infancy but presents solid groundwork for future research. Further studies are needed to define the exact mechanism of action of mesenchymal stem cells and to establish guidelines for the most appropriate acquisition, dosage and application method of mesenchymal stem cells for asthmatic horses.
KEY POINTS
- Stem cells are non-differentiated cells that can self-renew and differentiate into various specialised cells.
- Mesenchymal stem cells may have different properties which should be taken into account when choosing the best methods of stem cell therapy to rehabilitate a specific disease.
- The immunomodulatory effects of mesenchymal stem cells are recognised as their primary therapeutic effect.
- A noticeable amelioration of airway inflammation and clinical improvement after mesenchymal stem cell application is consistently reported in scientific literature. It therefore presents a novel interest to investigate the option of treating severe equine asthma in horses.