Clinical applications of mesenchymal stromal cell-based therapies for pulmonary diseases: An Update and Concise Review

Lung disorders are a leading cause of morbidity and death worldwide. For many disease conditions, no effective and curative treatment options are available. Mesenchymal stromal cell (MSC)-based therapy is one of the cutting-edge topics in medical research today. It offers a novel and promising therapeutic option for various acute and chronic lung diseases due to its potent and broad-ranging immunomodulatory activities, bacterial clearance, tissue regeneration, and proangiogenic and antifibrotic properties, which rely on both cell-to-cell contact and paracrine mechanisms. This review covers the sources and therapeutic potential of MSCs. In particular, a total of 110 MSC-based clinical applications, either completed clinical trials with safety and early efficacy results reported or ongoing worldwide clinical trials of pulmonary diseases, are systematically summarized following preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines, including acute/viral pulmonary disease, community-acquired pneumonia (CAP), chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia (BPD), interstitial lung diseases (ILD), chronic pulmonary fibrosis, bronchiolitis obliterans syndrome (BOS) and lung cancer. The results of recent clinical studies suggest that MSCs are a promising therapeutic approach for the treatment of lung diseases. Nevertheless, large-scale clinical trials and evaluation of long-term effects are necessary in further studies.


Introduction
Lung diseases across all ages have become one of the major public health issues worldwide with increased human activities, environmental changes, indoor and outdoor air pollution, long-term smoking, occupational exposures, and various pathogens [1]. Acute and chronic lung diseases have high morbidity and mortality [2,3]. While these diverse conditions require different specific therapeutic approaches (e.g., antimicrobial medications, inhaled corticosteroids, anti-inflammatory drugs, antifibrotic drugs, specific cytokine inhibitors, bronchodilators, respiratory support, mechanical ventilation, and restricted fluid input), persistent alleviation of clinical symptoms cannot be offered to most patients affected to date. Lung transplantation has evolved to represent the last option for many patients with end-stage lung diseases. However, there is a severe shortage of suitable donor lungs, and transplantation itself is associated with the potential for serious risks due to the need for lifelong immunosuppression, resulting in a high posttransplant mortality rate [4]. Thus, a new therapeutic strategy is desperately needed.
Mesenchymal stromal cell (MSC)-based therapy is one of the most cutting-edge and popular directions in medical research today [5]. Autologous or allogeneic-derived mesenchymal stem cells (MSCs) Ivyspring International Publisher are easier to obtain from multiple biological tissues, including bone marrow (BM), neonatal tissues, and adipose tissues. MSCs can be induced into proinflammatory MSC type 1 (MSC1) or anti-inflammatory MSC type 2 (MSC2) responding to different immune environments [6]. These cells have multiple potential advantages, including superior proliferation ability, lower immunogenicity, multidifferentiation potential, large-scale supply, and minimal ethical issues [7]. Upon administration by the intravenous route, the cells travel directly to the lungs, where the majority are sequestered, a great benefit for the treatment of pulmonary disease. These findings have paved the way for the development of clinical protocols and thereby provide off-the-shelf therapy.
To globally analyze clinical trials for MSC-based therapy of pulmonary diseases, a comprehensive search of the ClinicalTrials.gov database from 1990 to January 19, 2021, was conducted according to PRISMA guidelines. We systematically summarized completed and ongoing clinical trials worldwide of pulmonary diseases, including acute/viral pulmonary disease, community-acquired pneumonia (CAP), chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia (BPD), interstitial lung diseases (ILD), chronic pulmonary fibrosis, bronchiolitis obliterans syndrome (BOS) and lung cancer. Moreover, the sources and therapeutic potential of MSCs are also summarized. The results of current clinical studies support MSCs as a promising therapeutic approach for the treatment of lung diseases. Nevertheless, large-scale clinical trials and evaluation of long-term effects are necessary in further studies.

Adult and Neonatal Tissue Source of MSCs
MSCs are nonhematopoietic stem cells with multilineage potential and can be readily isolated and expanded from multiple biological tissues, including BM, neonatal tissues, and adipose tissues. MSCs are an attractive stem cell source for the regeneration of damaged tissues in clinical applications because these cells are characterized as undifferentiated cells, are able to self-renew with a high proliferative capacity, and possess mesodermal differentiation potential [8]. MSCs can not only modulate immune responses in different inflammatory microenvironments but also relieve cell death and tissue injury in pathological and physiological states [9]. MSCs can be extracted from both healthy donors and patients and are easily expanded in vitro to a therapeutic volume used as an "off-the shelf" therapeutic agent or can be stored for repetitive therapeutic usage [10]. BM is the most characterized and documented source of MSCs. BM-derived MSCs (BMSCs) have become the most common source of multipotent cells for transplantation in preclinical and clinical trials since they were first isolated in 1970 by Friedenstein et al. [11]. However, the harvest of MSCs from BM is a painful, invasive procedure, and there is a risk of viral exposure. In addition, the number, differentiation potential, and maximal life span of MSCs from BM decline with increasing age [12]. MSCs in the umbilical cord (UC) can be obtained from Wharton jelly, veins, arteries, the umbilical cord lining, and the subamnion and perivascular regions. UC-derived MSCs (UCMSCs) can be obtained through a painless collection method and have fewer associated ethical issues. They also renew faster than BMSCs [13]. Adipose tissues are another popular source and have significant advantages over MSCs derived from other sources, mainly because a large number of MSCs can be obtained through minimally invasive lipoaspiration methods and can easily be extracted [14]. The MSC concentration in adipose tissue is greater than that in all other tissues in the body, and the potency is maintained with the age of the donor, unlike BMSCs. In addition, adipose-derived MSCs (ADSCs) possess stronger immunomodulatory capability than BM-MSCs [15]. Taken together, these findings show that ADSCs have advantages in both autologous use and allogeneic use. Since the mid-2000s, thousands of clinical trials have used MSCs to test therapeutic interventions for numerous severe diseases, alone or in combination with other drugs. Notably, as a paracrine mediator, exosome-based therapy is now recognized as an emerging novel approach that contributes to the healing of injured or diseased tissues and organs [16]. Exosomes (50-150 nm in diameter) derived from MSCs may contain growth factors, cytokines, DNA, lipids, mRNAs, miRNAs, and mtRNAs, which function as intercellular mediators between MSCs and target cells, including MSCs. MSC-derived exosomes possess therapeutic properties, including stimulation of cell migration and extracellular matrix synthesis, antiapoptotic effects, immunomodulation and anti-inflammatory effects [17].

Therapeutic Potential of MSCs
During the last decade, rapidly developing regenerative medicine in the treatment of tissue and organ injury has led to more widespread use of MSC technology. In vitro, MSCs show self-renewal, extensive proliferation ability, and multipotency. The therapeutic potential of MSCs for lung disorders is supported by several factors (Figure 1). First, MSCs are characterized by low immunogenicity. Generally, MSCs positively express CD73, CD90, and CD105 surface markers, have low expression of major histocompatibility complex (MHC) class I, and do not express hematopoietic or endothelial surface markers (CD11b, CD14, CD19, CD34, CD45, CD79alpha), human leukocyte antigen (HLA)-DR, major histocompatibility complex (MHC) class II, costimulatory molecules (e.g., CD40/CD40L, B7/CD28, ICOS/ICOSL, 4-1BB/4-1BBL, OX40/ OX40L), or adhesion molecules (e.g., CD31, CD18, and CD56) [18]; additionally, transmembrane 4 L6 family member 1 (TM4SF1) has been indicated effective as an MSC-specific surface marker [19]. Therefore, this low-immunogenicity phenotype of MSCs permits the use of allogeneic cells for patients and strongly reduces the risk of allograft rejection. No immunosuppressive therapy is needed.
Second, MSCs modulate the host immune response. The role of MSCs is to adjust the balance between inflammation and tissue reconstruction to provide damaged tissue with a relatively stable environment, which is beneficial for tissue repair. MSCs from the microenvironment are considered to constitute a double-edged sword in exerting multiple modulatory effects on diverse aspects of the immune response. That is, MSCs are capable of polarized differentiation [20]. MSCs can differentiate into MSC1 cells, which can promote a proinflammatory state and preserve the immune response to microorganisms through specific Toll-like receptors (TLRs) when the immune system is underactivated [21]. On the other hand, when the immune system is overactivated (cytokine storm), MSCs may differentiate into anti-inflammatory MSC2 cells to limit the inflammatory cytokine cascade and host tissue injury, ultimately avoiding self-overattack ( Figure 2) [22,23]. More specifically, MSCs can be used as therapy to strike a balance in the immune cells of patients with COVID-19. It has been proposed that MSCs suppress cytokine storms by negatively regulating the immune response in the case of major inflammation (as with COVID-19) [24]. Third, MSCs enhance migration/homing and tissue repair after injury, which is mediated partly by paracrine and/or directed differentiation mechanisms that enhance the resolution of tissue injury. After sensing the injury signal released from damaged tissues, MSCs can be mobilized and migrate into injured tissues through peripheral circulation; this trafficking process is regulated by multiple mechanical factors (e.g., mechanical strain, shear stress, matrix stiffness, and microgravity) and chemical factors (including stromal derived factor-1/CXC chemokine receptor 4 axis, osteopontin, basic fibroblast growth factor, vascular endothelial growth factor-A, hepatocyte growth factor, insulin-like growth factor-1, platelet-derived growth factor, transforming growth factor-β1) [25]. Subsequently, MSCs reach the damaged tissue site and perform wound healing of damaged tissues in two key ways, i.e., paracrine (e.g., releasing bioactive factors: chemokines, cytokines, and growth factors) and/or directed differentiation to replace damaged cells (e.g., osteocytes, chondrocytes, cardiomyocytes, and endothelial cell differentiation) [26]. Over the recent decades of intensive studies, the bone morphogenic protein (BMP) signaling and wingless and int-1 (Wnt) signaling pathways have been demonstrated to regulate osteoblast and adipocyte differentiation of MSCs [27]. The sustained activation of ERK by 5-azacytidine contributed to the induction of the differentiation of MSCs into cardiomyocytes [28]. Growth differentiation factor 11 (GDF11) binds to the TGF-β receptor and subsequently activates the RAS-RAF-MEK-ERK/EIF4E pathway to induce the endothelial differentiation of MSCs [29]. A body of evidence indicates that following systemic injection, most MSCs are trapped in capillary beds of various tissues, especially the lungs. MSC infusion might benefit alveolar epithelial cells, injured airways and lung tissue repair given the ability of these cells to differentiate into targeted cells to counteract pulmonary fibrosis and improve lung dysfunction.
Fourth, MSCs enhance trophic effects. The trophic properties of MSCs are believed to be a mechanism underpinning the therapeutic impact in preclinical studies. MSCs can either promote their own survival and proliferation through autocrine effects or secrete trophic factors that will act on adjacent cells through a paracrine effect in a hostile microenvironment [30]. For example, prostaglandin E2 secreted by MSCs contributes to the maintenance of self-renewal capacity through the E-prostanoid 2 receptor [31].
Fifth, MSCs induce pro-angiogenic properties. Angiogenesis is a complex biological process involving interactions between vascular cells and the extracellular environment, and its dysregulation can contribute to serious disease. A growing body of evidence has shown that MSC-based proangiogenic therapies have been increasingly utilized in the treatment of ischemic diseases [32]. This effect was mainly attributed to the modulation of angiogenic factors produced by MSCs. Roura et al. reported that umbilical cord blood-derived MSCs showed angiogenic potential since they directly self-organize, forming new functional vasculature connected with the host circulatory system once implanted [33]. Recent experimental studies have demonstrated that MSC-derived exosomes could be considered for use in therapeutic angiogenesis, especially for ischemic diseases [34]. More interestingly, miR29a-loaded exosomes from engineered BMSCs (miR-29a-loaded BMSC-Exos) showed a robust ability to promote angiogenesis and osteogenesis in vivo [35].
Sixth, MSCs may enhance host antimicrobial capacity. MSCs have demonstrated bactericidal effects both in vitro and in vivo through direct and indirect mechanisms to induce microbial killing. Direct mechanisms of MSC-mediated bacterial killing include scavenger receptor-mediated phagocytosis (macrophage receptor with collagenous structure (MARCO) and SR-B1), antimicrobial peptide (AMP) production, and the indoleamine 2,3-dioxygenase (IDO) and inducible nitric oxide synthase (iNOS) pathways [36]. Recent evidence has suggested that MSCs have the potential to break down biofilms via cysteine protease secretion and present a strategy to increase the efficacy of conventional antibiotics via combination therapy between degradation of the biofilm layer by MSCs and increased antibiotic penetration [37,38]. Indirect mechanisms of action are through the recruitment and activation of host immune cells. MSC administration can result in enhanced alveolar macrophage phagocytosis involved in promoting effective antigen presentation, phagocytosis, and bacterial killing. MSC-derived extracellular vesicles (EVs) carrying mitochondria are responsible for these effects through the promotion of oxidative phosphorylation in macrophages [39,40]. In addition, in an in vitro virus infection experiment, MSCs demonstrated antiviral effects and could inhibit virus-specific CD8 (+) T-cell proliferation activation and proliferation via IDO-mediated mechanisms [41]. Literature reviews demonstrate that specific TLR stimulation affects the immunomodulatory potency of MSCs. Given that TLRs are immediately capable of detecting internal and external hazard signals and that their stimulation has an intense effect on the ability to proliferate, differentiate, migrate, and survive, it seems that stimulation of these receptors can have a primary effect on the interaction of MSCs and immune cells, improving the antiviral activity [42].
Seventh, genetic engineering strategies represent a promising and effective approach to enhance the therapeutic efficacy of MSCs and improve the outcomes of diseases. In addition to applications in tissue engineering, to enhance their therapeutic efficacy, developing a cellular therapy using MSCs as attractive delivery vectors is the ultimate goal of this area of research. Genetic engineering methods to modify MSCs can be classified as those using viral transduction, nonviral transfection, or genome editing tools and techniques to overexpress therapeutic proteins that complement their innate properties ( Figure 3) [43][44][45][46]. A growing body of evidence indicates that the paracrine, homing, immunomodulatory, anti-inflammatory, and tissue repair properties of MSCs can be strengthened through genetic modification [47]. As therapeutic agents and novel carriers, genetically modified MSCs target metastasis and efficiently provide a local high concentration of therapeutic agents that target a specific disease (Table 1). These strategies offer therapeutic dosages of MSCs and therapeutic agents at the target site, circumventing the problems with toxicities for repetitive systemic administration.

Search strategy
A comprehensive search of the ClinicalTrials.gov database from 2000 to January 19, 2021, was conducted according to Preferred Reporting Items of Systematic Reviews and Meta-analyses (PRISMA) guidelines. The keywords used to search for MSC-based therapy for lung disease in ClinicalTrials.gov were as follows: 1) Condition or disease: "acute respiratory distress" OR "acute respiratory syndrome" OR ARDS, "2019 novel coronavirus" OR "2019-nCoV" OR "COVID19" OR "interstitial pneumonia" OR "viral pneumonia" OR "virus pneumonia", "bacterial pneumonia", "chronic pulmonary diseases" OR "chronic obstructive pulmonary disease" OR COPD OR "emphysema", "bronchopulmonary dysplasia" OR BPD, "idiopathic pulmonary fibrosis", "pulmonary arterial hypertension", "asthma", "lung transplant reject", "lung disease" and "pulmonary disease"; and 2) Other terms: "Mesenchymal stromal cells" OR MSC OR MSCs. This therapeutic review provides an evaluation of the use of MSCs in acute and chronic pulmonary disease treatment. A total of 170 clinical trials were initially found. After the exclusion of 38 duplicates and 22 trials of "unknown", "terminated" and "withdrawn", 110 trials focused on MSC therapy in pulmonary diseases were reviewed using Prisma Flow ( Figure 4).

Aims and outcomes
This review included registered clinical trials that evaluated the safety and/or efficacy of MSCs administered to patients with lung diseases from any cause, either complete or ongoing. The use of MSCs as monotherapy and/or combined therapy was included. Additionally, one unregistered study with results was identified on PubMed and discussed briefly here. The primary outcomes were the comprehensive safety and efficacy evaluation of MSC use in pulmonary disease therapy. Secondary outcomes were changes in pulmonary function and biomarkers. All results collected from the studies were reported with the same measurements retrieved from the papers.

Results
In general, new registrations of clinical trials with MSC-based therapy reached a peak in 2020, accompanied by one startling discovery of 58 registered MSC trials specifically targeting COVID-19 ( Figure 5). The first clinical trial involving the use of MSCs for pulmonary disease was conducted in 2008, and the results were published in 2013 [48]. These clinical studies involved acute/viral pulmonary disease, CAP, COPD/emphysema, BPD, ILD, chronic pulmonary fibrosis, CLAD, BOS and lung cancer. Additionally, clinical trials are underway for cystic fibrosis (CF), non-CF bronchiectasis, pulmonary arterial hypertension (PAH), and even poisoninduced lung injury ( Figure 6). These clinical trials are listed in Table 2 (completed and published trials) and Table 3 (ongoing trials). The majority of clinical trials are still in Phase I (safety studies), Phase II (proof of concept for efficacy in human patients), or a mixture of Phase I/II, as shown in Figure 7.

ARDS
ARDS is a devastating disorder characterized by acute and refractory hypoxia, noncardiogenic pulmonary edema, diffuse alveolar-capillary membrane damage, and reduced compliance [49]. ARDS and pneumonia are interrelated in critically ill patients [49]. Despite decades of research, there is still no effective pharmacotherapy for ARDS. Although some supportive care approaches have been established, ARDS remains devastating and life-threatening. ARDS constitutes a spectrum of increasingly severe acute respiratory failure with growing prevalence and high mortality and morbidity that increase with age [50,51].
To date, there have been 8 registered clinical trials using MSC-and MSC-derived exosomes for the treatment of ARDS (Table 2 and Table 3). In the first early-stage clinical trial, MSCs were utilized for the treatment of ARDS (NCT01902082) in Shaoxing Second Hospital of China between January and April 2013 [52]. The study population comprised 6 patients randomized to the MSC group and 6 patients randomized to the placebo group, in which the patients in the MSC group received a single intravenous dose of 1×10 6 ADSCs per kilogram of weight. The results showed no infusion toxicities or serious adverse events related to MSC administration. However, the two groups were similar in the length of hospital stay, ventilator-free days, and ICU-free days within 28 days after the treatment. Subsequently, Wilson et al. [53] reported the START trial (NCT01775774), a Phase I, multicenter, open label, dose escalation pilot study designed to test the safety of a single-dose systemic injection of allogeneic BMSCs in patients with moderate to severe ARDS. Nine patients received intravenous infusions of BMSCs at a low dose (n=3, 1×10 6 cells/kg), an intermediate dose (n=3, 5×10 6 cells/kg) or a high dose (n=3, 10×10 6 cells/kg). High dose BM-MSCs improved daily sequential organ failure assessment (SOFA) score compared to lower doses. However, no signifcant differences in inflammatory and endothelial injury markers were detected in any of the samples collected. The trial demonstrated that a single intravenous dose of MSCs of up to 1×10 6 BMSCs/kg was well tolerated. Another Phase I trial (NCT02804945) have completed in June 2019. The participants received a maximum dose of 3×10 6 cells per kilogram of weight intravenously. However, the result has not been posted yet. In addition, Chen et al. [54] reported that the transplantation of menstrual blood-derived MSCs could reduce mortality in patients with H7N9 virus-induced ARDS without adverse effects after a five-year follow-up period in China. Because H7N9 and COVID-19 share similar complications, MSC transplantation may be useful for treating COVID-19.

COVID-19/severe influenza
The cure of COVID-19 is essentially dependent on the patients' own immune system. When the immune system is over activated in an attempt to kill the virus, this can lead to the production of a large number of inflammatory factors, resulting in severe cytokine storm. The cytokine storm may induce organ damage followed by the edema, dysfunction of air exchange, ARDS, acute cardiac injury, and secondary infection, which may lead to death [55]. Thus, preventing the severe acute respiratory infection and cytokine storm form of COVID-19 as the most dangerous phase of this disease can be helpful for the treatment and reduction of the death rate [56]. In this regard, MSC-based immunomodulation treatment has been proposed as a suitable therapeutic approach, and several clinical trials have begun. More recently, a growing number of clinical investigations of cell-based therapies, primarily involving MSCs but also involving MSC-derived exosomes, have been initiated worldwide for COVID-19.   MSCs were utilized for the first time for the therapeutic application of COVID-19 pneumonia in Beijing YouAn Hospital, China, from Jan 23, 2020 to Feb 16, 2020 [57]. In this clinical study, seven confirmed COVID-19 patients received single dose of clinical grade MSCs (1×10 6 cells per kilogram of weight). The pulmonary function and symptoms of these seven patients were significantly improved 2 days after MSC transplantation. Analysis of immune cells revealed that there was an increment of blood lymphocyte concentrations, Tregs and DCs with decreased NK cells. Meanwhile, the plasma level of C-reactive protein (CRP) and TNF-α was significantly decreased, while IL-10 and vascular endothelialderived growth factor (VEGF), which correlated with pulmonary regeneration, increased in the MSC treatment group compared to the placebo control group. The satisfactory results of the MSCs therapy gave hope for more critically ill COVID-19 patient. Another clinical study is a case report of a 65-year-old woman diagnosed with critically ill-type COVID-19 along with acute respiratory failure and acute diarrhea on January 31, 2020 [58]. During the treatment, three doses of 5×10 7 /administration UCMSCs were used, 3 days apart. Stem cell therapy was used with conventional therapy to which the patient did not respond. After the third infusion, the patient was negative for SARS-CoV-2 and discharged with no side effects. Additionally, a case report study also described the therapeutic efficacy of the human umbilical cord Wharton's jelly-derived MSCs (hWJCs) (1×10 6 cells per kilogram of weight) on a patient with COVID-19 pneumonia [59]. This report suggested that the adoptive transfer therapy of hWJCs might be an ideal choice to be used for COVID-19 treatment.
While basic studies using MSC-derived exosomes have not been sufficiently performed for COVID-19, clinical studies using exosomes are in the planning stage or have recently been initiated. Recently, a pilot study using allogenic ADSC-derived exosomes for treating severe COVID-19 was completed in China (NCT04276987) [60]. This trial is a Phase I, randomized, single-group assignment study whose primary objective is to explore the safety and efficiency of exosomes in the treatment of severe COVID-19 patients (Table 2). Moreover, there was a similar clinical trial had been registered in Russia. The COVID-19EXO trial (NCT04491240), a Phase I/II, randomized, open-label, parallel-group study, was completed. This trial enrolled 30 patients, and all eligible study subjects were randomized, doubleblinded, to either one of the two treatment groups or placebo group. The patients in the treatment groups received inhalation of 3 ml of special solution containing 0.5-2×10 10 exosomes twice a day for 10 days in combination with standard therapy. The primary outcome measure was the number of patients with nonserious and serious adverse events during the trial. Inspiringly, according to the results posted on ClinicalTrial.gov, no adverse events were registered [61]. Up to January 19, 2021, there were 58 registered clinical trials of MSC (n=56) and MSCs-dervied exosomes (n=2), of which 33 are active and recruiting patients and six have completed their trials (Table 2 and Table 3). The sources of MSCs are umbilical cord (n=21), Wharton's jelly (n=3), placental tissue (n=1), bone marrow (n=9), adipose tissue (n=6), dental pulp (n=1), olfactory mucosa (n=1), and unmentioned origin (n=14) ( Table 2 and Table 3). The first trial was registered on Feb 5, 2020 by Beijing 302 Hospital. This phase І clinical trial (NCT04252118) was done to inspect the safety of UCMSCs therapy for pneumonia patients infected with SARS-CoV-2 [62]. The second trial (NCT04269525) was registered on Feb 13, 2020 by Zhongnan Hospital. This phase Ⅱ trial was being conducted to assess the role of UCMSCs (100×10 6 cells/time at D1, D3, D5, D7) in treating COVID-19 pneumonia [63].
The present preliminary clinical data reveal that MSCs succeed in managing severe and critically severe COVID-19 patient, and have a benefit in reducing inflammation, improving pulmonary function, and reducing death in COVID-19 patients. The factors considered to be vital for effective treatment include the route, timing, dose, volume, source, and duration of the MSC administration. Adequately powered clinical trials are urgently needed to test clinical outcomes in patients with COVID-19.

Community-acquired bacterial pneumonia
Community-acquired bacterial pneumonia (CABP), as an acute lung infection, can lead to sepsis and is associated with high mortality rates in patients presenting with shock and/or respiratory failure who require mechanical ventilation and admission to intensive care units, thus reflecting the limited effectiveness of current therapy [64,65]. Very recently, Laterre et al. [66] first reported an ongoing Phase I/II, randomized, double-blind, multicenter trial (NCT03158727) to assess the safety and efficacy of expanded allogeneic ADSCs for the treatment of patients with severe CABP (sCABP) admitted to the ICU. The study was initiated in January 2017 and is expected to be completed by December 2021 (Table 3).

Tuberculosis
Tuberculosis (TB) remains an important cause of CAP. Mycobacterium tuberculosis has developed the ability to continually resist antitubercular agents. Multidrug-resistant TB (MDR-TB), defined by resistance to isoniazid and rifampicin, the two front-line antimicrobial drugs used to treat TB, presents one of the most urgent and difficult challenges facing global TB control [67]. The first open-label Phase I clinical trial of 30 MDR-TB and extensively drug-resistant TB patients who received single-dose autologous bone marrow-derived MSCs (1×10 6 cells per kilogram of weight) was conducted in 2010 by a specialist center in Minsk, Belarus, and the results were published in 2014 [68]. There were no serious adverse events reported. Subsequently, in a small cohort study comprising 36 patients with MDR TB, intravenous infusions of autologous BM-MSCs were administered 4 weeks after starting TB treatment [69]. The results showed that autologous transplantation of MSCs could vastly improve outcomes for 81% of MDR-TB patients. This result could revolutionize therapy options and have strong implications for future directions of MDR-TB therapy research.

COPD/Emphysema
COPD is an umbrella term used to describe chronic lung diseases, such as emphysema and chronic bronchitis, which cause limitations in airflow [70]. The disease burden from COPD, in contrast to that of TB, appears to be growing, despite the development of new therapeutics such as long-acting antimuscarinic agents, long-acting β-agonists, inhaled corticosteroids, and phosphodiesterase inhibitors [71]. Interest in using MSCs for the treatment of COPD or emphysema has translated into clinical trials. The first Phase II clinical trial (NCT00683722) involved the use of allogeneic BMSCs for the treatment of moderate-to-severe COPD from May 20, 2008, to August 24, 2010 [48]. Thirty patients received four monthly infusions (100×10 6 cells/infusion) and completed the 2-year follow-up. This trial demonstrated that systemic administration of multiple doses of MSCs appears to be safe and may decrease inflammation in an older, comorbid population of patients with compromised lung function due to moderate to severe COPD.
In addition, Stolk et al. [72] reported another Phase I clinical trial (NCT01306513) that aimed to study the safety and feasibility of intravenous administration of autologous BMSCs to patients with severe emphysema. Seven patients received bone marrow aspiration for BMSC collection, while the first underwent lung volume reduction surgery (LVRS) on one lung. The second LVRS on the contralateral lung was preceded by two intravenous infusions of autologous BMSCs (1-2×10 6 cells/kg). After LVRS and MSC infusions, alveolar septa showed a 3-fold increased expression of the endothelial marker CD31. One year after the second LVRS, all patients presented increased forced expiratory volume in 1 second (FEV 1 ) and body weight and changes in lung densitometry compared to their own values before the first LVRS. The results showed that autologous MSC administration in patients with severe emphysema is feasible and safe. However, a main limitation of the study was the lack of a placebo group. At present, there are 10 registered clinical trials using MSCs for the treatment of COPD or emphysema ( Table 2 and  Table 3). Moreover, with respect to cellular sources, only controlled trials with a strict comparison between different tissues might determine the suitability and efficacy of specific cell types to treat COPD or emphysema.

BPD
BPD is the most prevalent respiratory disorder among infants born extremely preterm and is characterized by the arrest of alveolarization, fibroblast activation, and inflammation [73]. It is one of the leading causes of chronic lung disease in children [74]. The pathogenesis of BPD involves multiple prenatal and postnatal mechanisms affecting the development of very immature lungs. Their combined effects alter the lung's morphogenesis, disrupt capillary gas exchange in the alveoli, and lead to the pathological and clinical features of BPD [75].
Chang et al. [76] reported the first Phase I dose-escalation clinical trial (NCT01297205) in 2014 to evaluate the safety and efficacy of intratracheal transplantation of human UCMSCs in preterm infants at high risk for BPD. This trial demonstrated that the treatment was well tolerated, without serious adverse effects or dose-limiting toxicity: all 9 infants who underwent MSC transplantation survived, and only 3 of these infants developed moderate BPD. A two-year follow-up (NCT01632475) by the same researchers indicated that one of 9 infants in the MSC group died of Enterobacter cloacae sepsis at 6 months, and 8 infants survived without any transplantation-related adverse outcomes [77]. Intratracheal transplantation of allogeneic UCMSCs in preterm infants is safe and feasible. The next Phase II clinical trial (NCT03392467) and follow-up (NCT04003857) for intratracheal instillation of UCMSCs to preterm infants with BPD are ongoing [78,79]. Recently, Wu et al. [80] reported the first randomized, single-center, open-label, dose-escalation, Phase II trial (NCT03601416) using MSCs intravenously administered in children with severe BPD. In this study, the safety and efficacy of treatment with low-(n=24, 2.5×10 6 cells/kg) and high-dose (n=24, 5×10 6 cells/kg) intravenous infusions of allogeneic UCMSCs were compared with those of traditional supportive treatments for BPD. These results will provide new evidence of MSC-based therapy for severe BPD.

IPF
IPF is the most lethal ILD, characterized by fibrosis following failed epithelial repair and chronic progressive scarring of the lungs [81]. Although the precise etiology is unknown, a number of risk factors may contribute to disease development, including smoking, drug exposure, infectious agents, and genetic predisposition [82]. Currently, its associated mortality remains high, and no effective pharmacotherapy or artificial ventilation and transplantation exists. The administration of MSCs is investigated as a new therapeutic method for IPF [83].
The first pilot IPF clinical trial (NCT01385644) with placenta-derived MSC therapy was conducted in 2010 in Australia, and the results were published in 2014 [84]. In this single-center, nonrandomized, dose escalation Phase Ib study, four out of the 8 patients participating in the trial received intravenous infusion of placenta-derived MSCs at 1×10 6 cells/kg, and another 4 patients received 2×10 6 cells/kg by the same delivery. Both dose schedules were well tolerated, with only minor and transient acute adverse effects. At 6 months postinfusion, most adverse events of this trial were mild and selflimiting, and lung function and computed tomography (CT) fibrosis scores were all unchanged from baseline, with no evidence of worsening fibrosis [84]. These results demonstrated that intravenous MSCs for patients with moderately severe IPF are feasible and have a good short-term safety profile. Subsequently, in a Phase I/II clinical trial (NCT02594839), twenty patients with a rapid progressive course of severe to moderate IPF were randomized into two groups: one group received two intravenous doses of allogeneic BMSCs (2×10 8 cells) every 3 months (total amount: 1.6×10 9 cells). After the study was completed, no significant adverse effects were found in the MSC-administrated group, and they were observed having a better outcome for the 6-min walk test distance, for DLCO in 26 weeks, and for forced ventilation capacity in 39 weeks compared with the placebo group [85]. Therapy with high doses of BMSCs is a promising method for reducing rapid pulmonary function decline in patients with IPF.
Another trial (NCT02013700) also supports the safety of a single infusion of BMSCs in patients with mild-moderate IPF [86]. Moreover, the authors nicely discuss the limitations of the study, which include the small sample size (nine patients), the lack of randomization, and the absence of a placebo control arm for comparison. These trials demonstrate that therapy with high doses of allogeneic MSCs is a safe and promising method for reducing disease progression in patients with IPF. Ultimately, we need a large number of Phase II/III clinical trials of MSCs for IPF to evaluate their efficacy.

ILD associated with autoimmune disorders
ILD can manifest as a pulmonary complication of an underlying autoimmune and connective tissue disease (CTD-ILD), such as systemic sclerosis (SSc-ILD). ILD associated with SSc, together with pulmonary hypertension, represents the most common cause of death [87]. The most common agents currently utilized for the treatment of CTD-ILD include corticosteroids, azathioprine (AZA), mycophenolate mofetil (MMF) and cyclophosphamide (CYC) [88,89]. In recent years, researchers have attempted to determine more about the safety of MSC treatment or CTD-ILD, especially as MSCs can counteract the three main pathogenic axes of the disease: fibrosis, angiogenic defects, and autoimmunity [90]. The first Phase I trial (NCT03929120) designed to evaluate the safety of MSCs for patients with CTD-ILD is ongoing [91]. Another clinical trial is ongoing (NCT04432545) in Colombia, which aims to evaluate the therapeutic effects of allogeneic MSC infusion as a treatment in patients with SSc-ILD refractory to conventional therapy [92] (Table 3).

Pneumoconiosis
Pneumoconiosis is a kind of lung disease caused by inhalation of dust, such as silica (commonly named siliconosis), coal and rock dust and is characterized by inflammation, coughing, and fibrosis [93]. Early pneumoconiosis may be asymptomatic, but advanced stages of pneumoconiosis result in airflow limitation, hypoxia, pulmonary hypertension, respiratory or heart failure, and premature death, even without further exposure to the dust [94]. Currently, there is no effective drug treatment. The first Phase I clinical trial (NCT02668068) using UCMSCs for pneumoconiosis was registered in January 2016 [95]. This study was completed in China and observed and evaluated the safety and efficacy of combined large volume WLL with MSC transplantation for the treatment of pneumoconiosis. However, no results have been reported yet.

Radiation-induced pulmonary fibrosis
The lung is a radiosensitive organ, and pulmonary damage after high-dose radiation can cause radiation pneumonitis in the early stages and pulmonary fibrosis later on [96]. Effective treatments for improving patient prognosis are lacking. A Phase I, open, single-center, nonrandomized clinical study (NCT02277145) on radiation-induced pulmonary fibrosis treated with umbilical cord-derived MSCs was completed in December 2018 [97]. Patients received 1×10 6 cells per kilogram of weight of clinical grade UCMSCs injected via fiberoptic bronchoscopy after full lavage of the localized lesions. However, no results of this trial have been reported.

BOS
BOS, characterized by persistent airflow obstruction, is a devastating complication after lung transplantation [98] and allogeneic hematopoietic stem cell transplantation (allo-HSCT) [99]. The key clinical feature of BOS is the development of airway obstruction with a reduction in FEV 1 that does not respond to bronchodilators. The first clinical trial (NCT01175655) for patients with BOS after lung transplantation treated with allogeneic MSCs was published in July 2017 [100]. In this trial, a total of ten lung transplant recipients diagnosed with BOS received MSC infusions at a dose of 2×10 6 cells per kilogram of weight for each infusion twice weekly for 2 weeks. Study data confirmed the feasibility and safety of such intravenous delivery of allogeneic MSCs in patients with advanced BOS. Another multicenter, open-label, Phase I/II, prospective cohort study (NCT02543073) evaluated the safety and efficacy of allogeneic BMSCs for allo-HSCT associated BOS recipients [101]. In the MSC group, MSCs were intravenously given at a median dose of 1×10 6 cells per kilogram of weight once weekly for 4 consecutive weeks as a cycle. If tolerated, a second cycle was given at a 2-week interval. The outcome of the study revealed that MSCs may be a safe and effective therapy for BOS patients after allo-HSCT.

Lung cancer
As genetically modified vectors, combining the tumor-homing capacity of MSCs and genetic engineering of the cells to express tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) will enable the specific targeting of cancer stem cells (CSCs), which would be an attractive cytotherapeutic option for cancer [102]. A Phase I clinical trial (NCT03298763) of MSC-TRAIL for lung cancer is ongoing in the UK, which aims to establish the recommended MSC-TRAIL dose when given in combination with cisplatin/pemetrexed chemotherapy in metastatic non-small cell lung cancer (NSCLC) patients [103]. The study was initiated in March 2019 and is expected to be completed by September 2025.

Other lung diseases
CF CF is a common autosomal recessive disease that primarily affects the lungs and digestive system and is characterized by obstruction of airways, microbial infection, digestive disorders, and other complications due to mutations in CF transmembrane conductance regulator (CFTR) [104]. MSCs could be used to restore abnormal CFTR function. Moreover, the ability of MSCs to secrete the antimicrobial peptide LL-37, which is associated with the capacity to slow bacterial growth [105], will be a promising treatment for MSCs in patients with CF. The CEASE-CF trial (NCT02866721), a Phase І, single-center, open label, dose escalation study, was completed in April 2020, and the results have not yet been reported [106].

Non-CF bronchiectasis
Non-CF bronchiectasis is a syndrome of chronic inflammation leading to dilatation of airways and structural lung damage, which imposes a significant burden on patients. The observed cause of death is due primarily to bronchiectasis or related respiratory failure [107]. To demonstrate the safety of BMSCs in patients with non-CF bronchiectasis receiving standard of care therapy and to explore treatment efficacy, a Phase I investigation (NCT02625246) was completed in May 2019; however, the results are not available [108].

PAH
PAH is a rare, progressive disorder characterized by increased blood pressure in the arteries of the lungs. Although PAH is manageable, there is no effective therapy able to reduce mortality [109]. One trial (NCT04055415) evaluating the safety and initial impact of a single intravenous dose of a cell-based product made from allogeneic ADSCs (1×10 6 cells per kilogram of weight) to treat PAH is ongoing [110].

Poison-induced lung injury
Sulfur mustard (SM) is a potent alkylating toxic chemical compound that targets several organs, especially the lungs. Acute lung injury due to SM inhalation causes the formation of airway fibrin casts that obstruct airways at multiple levels, which is associated with chronic obstructive pulmonary deficiency, leading to acute respiratory failure and death [111]. Currently, effective medical countermeasures for SM are lacking. Ghazanfari et al. [112] showed that short-term SM exposure led to a decline in circulating MSC count after more than two decades. The lower number of peripheral MSCs in SM-exposed patients was not affected by taking corticosteroids or antibiotics, but comorbidities are probably involved in MSC frequency. In 2017, Nejad-Moghaddam et al. [113] reported a clinical trial (NCT02749448) using multiple doses of ADSC therapy for a male patient with SM-exposed lung injury at the Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. The patient received 100×10 6 cells every 20 days for a total of 4 injections within a 2-month period, and precise evaluations were performed. The results indicated that systemic ADSC administration appears to be safe and shows promising results with improvement of the patient's physical activity and 6MWT, FEV 1 and COPD assessment test (CAT) scores.

Discussion
Accumulating evidence supports MSC-based therapy as a promising therapeutic strategy in clinical trials of refractory and unmanageable pulmonary illnesses for targeting viral infection, fibrotic processes, and excessive inflammatory response, as well as combating organ failure [114]. Systemically infused MSCs have been found to migrate directly to the lungs, where they can ameliorate cytokine release syndrome, protect alveolar epithelial cells, repair injured airways, aid in alveolar fluid clearance, promote epithelial and endothelial recovery, resist pulmonary fibrosis, reduce the risk of allograft rejection, and improve lung function by secreting many kinds of factors and modulating multiple biological processes of the immune response, which are great benefits for treating severe pulmonary disease [115][116][117].
To date, several clinical trials have evaluated the safety, tolerance, and severe adverse events of MSC administration, and many clinical trials are still ongoing. Published phase I/II clinical trials seem to reasonably prove the safety and clinical improvement of MSC administration, with no significant adverse events, in acute and chronic lung diseases. Given that most clinical trials are in the early phase, undoubtedly, placebo-controlled, multicenter, more randomized large-scale phase II/III trials are needed to reach more convincing conclusions regarding the safety, effect sustainability and adverse effects of MSC therapies [118]. Additionally, evaluations of long-term safety or efficacy and the duration of local or systemic MSC transplantation are required.
Currently, the optimum therapeutic dosage of MSCs for treating lung diseases is unknown. In these published clinical trials, a wide dosage array of 1×10 6 cells~10×10 6 cells per kg of weight was used. Wilson et al. [53] reported that the application of three doses was administered in three cohorts (1×10 6 cells/kg, 5×10 6 cells/kg, and 10×10 6 cells/kg) in patients with ARDS, resulting in a corresponding reduction in the lung injury score of 30%, 36%, and 45%, respectively, and that the maximal dosage was well tolerated by patients. Recent studies have shown that MSC-based therapy significantly dampens cytokine storms in critically ill COVID-19 patients by negatively regulating the immune response. Accordingly, an optimal dosage of transplanted MSCs should be clearly defined, with the aim of finding the right balance between their beneficial and undesired effects, which could occur due to excessive immunosuppression [119]. In addition, the therapeutic effects of MSC administration should be carefully monitored since the differentiation potential, capacity for migration, immunomodulation and maximal life span of transplanted MSCs decline with increasing age.

Conclusion
MSC-based therapy approaches for lung diseases and critical illness continue to evolve at a rapid pace and offer hope for treating these devastating and currently incurable diseases. Further studies are expected to improve the standardization of MSC treatment protocols in terms of the donor source (autologous vs. allogeneic), sources of MSCs, MSCs culture status (fresh vs. cryopreserved/ thawed), manufacturing protocols, quality control provisions, routes of delivery (systemic vs. local), and cell dosing. Additionally, strict patient inclusion/ exclusion criteria should be defined, well-designed and controlled clinical trials should be performed, and rigorous ethical considerations must ensure patient safety before MSCs can be used in large-scale and long-term clinical applications for cell therapy.