Therapeutic potential of mesenchymal stromal cells in the treatment of ARDS (2018)

Acute respiratory distress syndrome (ARDS) is a syndrome of acute respiratory failure caused by noncardiogenic pulmonary edema. The most common clinical disorders associated with the development of ARDS are bacterial and viral pneumonia, aspiration of gastric contents, or less commonly, pancreatitis and drug reactions.1 In addition, a recent study showed that 30% of trauma patients develop ARDS,2 resulting in a threefold increase in mortality in these patients.3 Criteria for the diagnosis of ARDS have evolved over time, but the current definition includes impaired oxygenation (arterial hypoxemia with PaO2/FiO2 ratio < 300 mm Hg) and bilateral infiltrates on chest imaging in the absence of left atrial hypertension as the dominant cause of pulmonary edema.4 Advances in supportive care have markedly improved survival for patients with ARDS, but the mortality of ARDS remains high, at approximately 25% to 40%.5, 6 Despite five decades of research, there is still no effective goal-directed pharmacotherapy for this syndrome, and novel treatment options are needed to improve the morbidity and mortality associated with this condition. A growing body of evidence suggests that cell-based therapy with mesenchymal stromal cells (MSCs) holds substantial therapeutic potential for a variety of inflammatory conditions, including ARDS. MSCs are nonhematopoietic stromal cells that express characteristic cell surface markers and can differentiate in vitro into chondrocytes, osteoblasts, and adipocytes, although they do not have true stem cell properties in vivo.7 MSCs can be isolated from most types of mesenchymal tissue including bone marrow, umbilical cord, placenta, and adipose tissue. There are several properties of MSCs that make them attractive therapeutic candidates. First, they have low constitutive expression of major histocompatibility complex (MHC) type I and lack both MHC type II and T-cell costimulatory molecules, so they seem to be nonimmunogenic, theoretically allowing for allogeneic transplantation without immunosuppression or human leukocyte antigen matching.8 Second, MSCs can be rapidly expanded ex vivo allowing short time to administration. Third, MSCs are thought to have a short lifespan in vivo and low tumorigenicity, unlike embryonic stem cells, making them ideal for the treatment of acute disease with a favorable safety profile.9 The therapeutic potential of MSCs have been studied in a number of medical and surgical conditions including sepsis,10, 11 diabetes,12 myocardial infarction,13 hepatic failure,14 acute renal failure,15, 16 chronic obstructive pulmonary disease,17 neurologic injuries,18 graft-versus-host disease,19 and trauma.20 Therefore, it was hypothesized that MSCs might also be beneficial in the treatment of ARDS. To test this hypothesis, several groups studied whether MSCs can reduce the severity of lung injury in preclinical models, and preliminary data is quite promising. In this review, we first summarize preclinical data that support the use of MSCs in ARDS treatment. Second, we review our current understanding of the mechanisms that explain the therapeutic benefit of MSCs in ARDS treatment. Finally, we discuss ongoing challenges and future directions in this field. Given the therapeutic benefit of MSCs in a variety of clinical applications,10-20 it was hypothesized that MSCs may also be effective for the treatment of ARDS. To test this hypothesis, in 2007, Gupta et al.21 studied the effects of mouse bone marrow–derived MSCs in a murine model of acute lung injury caused by Escherichia coli endotoxin. The authors found that MSC-treated mice had a reduction in the quantity of pulmonary edema by both gravimetric and histologic criteria, a reduction in proinflammatory cytokines (tumor necrosis factor-α [TNF-α] and macrophage inflammatory protein-2 [MIP-2]), an increase in anti-inflammatory cytokines (interleukin-1 receptor agonist [IL1-ra], IL-13), and overall improved mortality compared to control mice. Subsequently, Lee et al.22 used a similar model of E. coli endotoxin-induced lung injury in a human ex vivo perfused lung model to test the therapeutic efficacy of allogeneic human bone marrow-derived MSCs. The investigators found that administration of MSCs or even MSC-conditioned media reduced lung endothelial injury and restored alveolar fluid clearance to normal levels. Aside from endotoxin models of lung injury, MSCs have also been studied in live bacterial models of lung injury. In 2010, Krasnodembskaya et al.23 reported that human bone marrow–derived MSCs or MSC-conditioned media caused marked inhibition of bacterial growth in both gram-negative (E. coli and Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) organisms. The authors also found that intratracheal administration of human bone marrow–derived MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the broncheoaveolar lavage fluid in a mouse model of E. coli pneumonia. Similarly, intratracheal delivery of mouse bone marrow–derived MSCs enhanced survival and improved bacterial clearance in E. coli infected mice.24 Not only were MSCs effective for acute lung injury caused by bacterial infections, but preclinical data also suggested that MSCs have therapeutic benefit in acute lung injury caused by trauma. In 2011, Pati et al.25 demonstrated that the administration of intravenous human bone marrow–derived MSCs reduced the severity of lung injury in rat models of hemorrhagic shock. Based on preclinical data suggesting the efficacy of MSCs in the treatment of ARDS, there are current efforts to study the safety and efficacy of MSCs in patients with ARDS. A randomized trial of 12 patients in China with ARDS was the first to show that a single dose of 1 million allogeneic adipose-derived MSCs/kg can be safely administered in humans.26 Subsequently, a phase 1 clinical trial with nine patients in the United States showed no safety issues with a standard dose escalation of 1, 5, or 10 million allogeneic bone marrow–derived MSC/kg ideal body weight in patients with moderate to severe ARDS (NCT 01775774).27 With this phase 1 experience, a 60-patient phase 2a safety trial of MSCs for moderate to severe ARDS was recently completed, and the results are being analyzed. In this trial, subjects were randomized 2:1 to human bone marrow–derived MSCs at 10 million MSC/kg ideal body weight or placebo (NCT 02097641). The primary endpoint for this trial was safety, and thus it was not powered for efficacy endpoints, so future larger phase 2b trials will be needed to study respiratory endpoints. Given the therapeutic potential of MSCs, there has been much interest in understanding the mechanisms by which MSCs promote tissue repair, and significant advances have been made in this field over the past decade. Initially, it was thought that MSCs engrafted at the site of tissue injury and provided direct structural benefit.28 However, with more detailed cell identification methods, engraftment is now thought to be a rare event of unclear physiologic significance.29 Instead, preclinical models of MSCs suggest that the therapeutic benefit of MSCs does not require direct cell contact. For example, in a murine model of bleomycin-induced lung injury, the administration of mouse bone marrow–derived MSCs normalized proinflammatory cytokines despite the fact that only a small number of the MSCs localized to the injured lung.30 Moreover, multiple studies have also demonstrated that administration of MSC-conditioned media is sufficient for these beneficial effects. Curley et al.31 found that both rat bone marrow–derived MSCs and MSC-conditioned media reduced inflammation and decreased mortality in a rat model of ventilator-induced lung injury, suggesting that a secreted factor in the conditioned media was sufficient for the beneficial therapeutic effect.32 Together, these studies suggest that extracellular factors may be responsible for the beneficial effects of MSCs. To date, most work has focused on the hypothesis that paracrine factors are responsible for this effect, but more recently it has been proposed that the transfer of mitochondria and/or extracellular vesicles may also be important in this process. We will discuss each of these mechanisms separately in this section. Given the hypothesis that MSCs secrete paracrine factors that are important for tissue repair, there has been interest in identifying key factors involved in this process. Multiple paracrine factors have been implicated to date that modulate the resolution of ARDS through four mechanisms: 1) anti-inflammatory effects on host cells, 2) reduction of lung alveolar epithelium permeability, 3) increased rate of alveolar fluid clearance, and 4) enhancement of host mononuclear cell phagocytic activity. Here, we will illustrate examples of paracrine factors that act via these distinct mechanisms (also see Fig. 1). First, ARDS is usually associated with increased inflammation, so it was hypothesized that MSCs may secrete proteins that have anti-inflammatory properties. Several anti-inflammatory paracrine factors have been identified to date. In 2007, Ortiz et al.33 found that a subpopulation of mouse bone marrow–derived MSCs secrete IL1-ra. This protein inhibits cytokine stimulation of helper T lymphocytes and suppresses the production of TNF-α to reduce inflammation, so the authors proposed that IL1-ra mediates the anti-inflammatory effect of MSCs. Another anti-inflammatory paracrine factor secreted by MSCs is TNF-alpha stimulated tumor necrosis factor inducible gene 6 (TSG-6). In 2011, Danchuk et al.34 found that human bone marrow–derived MSCs significantly reduced the expression of proinflammatory cytokines, neutrophil counts, and total protein in bronchoalveolar lavage samples in a mouse model of lung injury, but showed that this anti-inflammatory effect was not observed when TSG-6 expression was knocked down in MSCs. Subsequent work corroborated this finding by demonstrating that intravenous administration of recombinant TSG-6 alone reduced inflammatory markers and improved survival of mice with bleomycin-induced lung injury.35 In addition, insulin-like growth factor-1 (IGF-1) may be another important anti-inflammatory mediator secreted by MSCs. In 2012, Ionescu et al.36 reported that the addition of mouse bone marrow-derived MSC-conditioned media improved the histological appearance of murine LPS-induced lung injury. The authors found abundant quantities of IGF-1 in the conditioned media and showed that addition of recombinant IGF-1 alone partially reproduced the tissue repair effect in vitro and in vivo. Most recently, lipoxin A4, a pro–resolving lipid mediator, has been identified as a possible anti-inflammatory paracrine factor secreted by MSCs. A 2015 study by Fang et al.37 found that mouse bone marrow–derived MSCs release lipoxin A4, and they showed that blockade of the lipoxin A4 receptor attenuated the beneficial effects of MSCs in endotoxin-induced lung injury in mice. Aside from the anti-inflammatory properties of MSCs, there is evidence to suggest that MSCs affect lung endothelial and alveolar epithelial permeability via a paracrine mechanism. In 2010, Fang et al.38 cocultured human bone marrow–derived MSCs with human alveolar type II cells and exposed them to a cytokine mixture to induce cellular injury. In this study, addition of MSCs restored type II cell epithelial permeability to protein control levels, but direct cell contact was not needed, suggesting a paracrine mechanism of action. The investigators then used small double-stranded interfering RNA knockdown of potential paracrine soluble factors and found that angiopoietin-1 was responsible for this beneficial effect. More recently, Goolaerts et al.39 demonstrated that coculture of human bone marrow–derived MSCs with rat alveolar epithelial cells exposed to inflammatory and hypoxic stimuli in vitro caused normalization of epithelial cell protein permeability and suggested a role for IL1-ra and prostaglandin E2 (PGE2) in this process. Third, removal of alveolar edema fluid via vectoral transport across alveolar epithelial cells is critical for recovery from acute lung injury, and recent data suggest that MSCs may secrete paracrine factors that improve the rate of alveolar fluid clearance. A growing body of evidence suggests that fibroblast growth factor 7 (FGF-7) may be particularly important in this process. In 2014, Goolaerts et al.39 used an in vitro model of acute lung injury in rat alveolar epithelial cells and found that the addition of human bone marrow–derived MSCs increased transepithelial sodium transport by preventing the decrease in apical expression of the epithelial sodium ion channel that usually occurs during ARDS. The investigators demonstrated that this beneficial effect was not seen with MSC-conditioned media when FGF-7 was depleted. Studies in ex vivo human perfused lungs confirmed and extended these findings. In 2009, Lee et al.40 demonstrated that the administration of human bone marrow–derived MSCs to an ex vivo perfused human lung injured with endotoxin normalized alveolar fluid clearance and reduced lung water in an FGF-7–dependent manner. Specifically, FGF-7–depeleted media had minimal effect on improving alveolar fluid clearance and blocking FGF-7 expression, preventing the therapeutic effect in vivo, whereas the addition of recombinant FGF-7 restored this activity. Similarly, a 2014 study using human perfused lungs rejected for transplant showed that treatment with human bone marrow–derived MSCs normalized alveolar fluid clearance, but pretreatment with an FGF-7–blocking antibody reduced this effect.41 Finally, apoptosis of both immune and structural cells occurs during ARDS, so it has been suggested that paracrine factors secreted by MSCs may have antiapoptotic effects. In 2008, Raffaghello et al.42 found that both human bone marrow-derived MSCs and MSC conditioned media had anti-apoptotic effects on human neutrophils in vitro, which was at least partially mediated by MSC production of the anti-apoptotic cytokine IL-6. In 2013, Lee et al.43 found that keratinocyte growth factor, a soluble factor produced by MSCs, decreased apoptosis of human monocytes through AKT phosphorylation, an effect that increased bacterial clearance and promoted lung recovery. The authors demonstrated that inhibition of keratinocyte growth factor by a neutralizing antibody reduced the antimicrobial effects of MSCs in the ex vivo perfused human lung and in vitro monocyte cultures that were injured with E. coli. While there is robust evidence supporting the role of paracrine factors in mediating MSC interactions with host tissue and most mechanistic research has focused on these pathways, newer data suggest alternative mechanisms of MSC action. One such hypothesis is that MSCs may be able to affect tissue repair through direct transfer of mitochondrial DNA to host cells. In 2012, Islam et al.44 showed that mouse bone marrow–derived MSCs transfer mitochondrial DNA to host alveolar cells in a mouse model of lung injury and found that this transfer normalized alveolar epithelial cell adenosine triphosphate concentration, restored surfactant secretion, and reduced mortality. Subsequently, Phinney et al.45 reported that mitochondrial transfer from human bone marrow–derived MSCs to macrophages by extracellular vesicles resulted in enhanced macrophage bioenergetics in human cells. New data suggests a possible mechanism of mitochondrial transfer from MSCs to host cells. In 2016, Jackson et al.46 used fluorescent imaging and flow cytometry to demonstrate extensive mitochondrial transfer from human bone marrow–derived MSCs to human macrophages in vitro, which occurred through the formation of intercellular cytoplasmic bridges, termed tunneling nanotubes. Of note, the presence of tunneling nanotubes suggests close cellular contact between MSCs and host cells, so this mechanism would not explain why MSC conditioned media alone has beneficial therapeutic effects. Thus, it is unlikely that mitochondrial transfer is the sole mechanism of MSC action, and further studies are needed to clarify the specific role that mitochondrial transfer plays in mediating MSC effect. Alternatively, it has been proposed that extracellular vesicles that bud off from MSC cells are responsible for the beneficial therapeutic effects of MSCs. Extracellular vesicles include exosomes (20–100 nm) and microvesicles (10–1000 nm) that are formed by the budding of multivesicular bodies from the cell membrane. Exosomes carry RNA, micro-RNA, proteins, and lipids that are delivered to host cells when they fuse with host cell membranes, but mitochondria are too large to fit into these structures. Previous studies have shown that extracellular vesicles derived from MSCs have protective effects in both in vitro and in vivo models of kidney injury,47, 48 so it was hypothesized that MSC-derived microvesicles may also be important for reducing acute lung injury. To address this hypothesis, in 2014, Zhu et al.49 utilized a murine model of E. coli endotoxin-induced lung injury and found that intratracheal and intravenous delivery of microvesicles isolated from human bone marrow–derived MSCs reduced inflammation and alveolar edema. In 2017, Morrison et al.50 found that extracellular vesicles from human bone marrow–derived MSC-conditioned media alter the function of macrophages and reduce the impact of the endotoxin-induced acute lung injury in a mouse model. Most recently, Park et al. demonstrated that MSC microvesicle administration increased alveolar fluid clearance and reduced bacterial load in a well-established ex vivo human perfused lung model of bacterial pneumonia.51 Thus, there is a growing body of evidence that MSCs may produce extracellular vesicles that transfer biologically active material to host cells, including monocytes and macrophages. More information is needed to understand what cellular contents are carried by the MSC-derived extracellular vesicles, sometimes called the MSC-exosome transcriptome, and how these components modulate host cells. Over the past 30 years, substantial advances have been made in our understanding of MSCs, their mechanism of action, and their therapeutic potential. Preclinical studies have suggested the efficacy of these cells in in vitro and in vivo animal models of acute lung injury, and human trials are currently underway to test the safety and efficacy of MSCs for patients with moderate to severe ARDS. Current data suggest that paracrine factors are at least partially responsible for the therapeutic potential of MSCs in ARDS. If paracrine factors are indeed able to enhance lung recovery, the isolation and use of MSC-derived paracrine factors or exosomes may allow for a cell-free therapeutic for ARDS. A cell-free therapeutic would have some potential benefits in terms of immune tolerance and ease of administration, but it is also possible that intact MSCs may be more effective. For example, in one preclinical study, rat bone marrow–derived MSCs were more effective than MSC-conditioned media in recovery from ventilator-induced lung injury in a rat model.52 Further studies are needed to address whether any isolated paracrine factor may have therapeutic benefit alone, or instead whether multiple mechanisms are at play so intact MSCs are more effective at reducing lung injury. Besides paracrine factors, more recent work has suggested that mitochondrial transfer and/or extracellular vesicle transfer may be important mechanisms for MSC therapeutic effect in ARDS. However, it can be challenging to dissect the effect of each of these mechanisms individually when intact MSCs are administered. Therefore, future studies will need to clarify the individual roles and therapeutic effects of each of these mechanisms. It is essential to ensure that preparations of MSCs are of standardized quality for the clinical application of these cells. MSCs can be cultured from multiple sites, such as bone marrow, muscle, umbilical cord, and adipose tissue, and it is not fully understood whether MSCs derived from different sites possess different biological properties. Moreover, environmental factors such as temperature and culture density affect MSC phenotype. Therefore, it is important to take these factors into consideration when trying to generalize the safety profile and therapeutic effects of MSC preparations. More studies are needed to evaluate the ideal dose, efficacy, and mode of delivery of MSCs, and standardized protocols for MSC collection and expansion should be used. It is also important to determine which patients with ARDS will be the best candidates for MSCs or MSC-derived therapies. Recent research indicates that there is considerable heterogeneity in ARDS phenotypes, as identified by both clinical and biological markers,53 so it will be critical to determine which patients with ARDS will be optimal candidates for cell-based therapies. Overall, the therapeutic promise of MSCs remains high. For ARDS in particular, given that there are few effective therapeutics at this time, this cell-based therapy could be useful in clinical management. Future studies are needed to better understand the mechanisms by which MSCs exert therapeutic benefit, to clarify the safest and most effective method of therapeutic administration, and to determine the optimal ARDS patient population for cell-based therapy. In this review, we have summarized preclinical data indicating that MSCs promote the resolution of alveolar edema fluid and tissue repair in the injured lung and thus have therapeutic potential for patients with ARDS. Progress has been made in understanding the mechanisms that mediate this therapeutic effect, with evidence for the role of paracrine factors, mitochondrial transfer, and the exchange of extracellular vesicles. Further studies are needed to fully characterize the mechanism of action of MSCs and study their potential therapeutic use in patients with ARDS. Drs. Matthay and Liu were supported in part by and The authors for the for this was in the absence of any or that could be as a potential of