Mesenchymal Stem Cells for the Treatment of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is an inexorably progressive lung disease of unknown origin. Prognosis is poor, with limited treatment options available, and the median survival remains just 3–5 years. Despite the use of pirfenidone and nintedanib for the treatment of IPF, curative therapies remain elusive and mortality remains high. Regenerative medicine and the use of cell-based therapies has recently emerged as a potential option for various diseases. Promising results of preclinical studies using mesenchymal stem cells (MSCs) suggest that they may represent a potential therapeutic option for the treatment of chronic lung diseases including IPF. Encouraging results of Phase 1 studies of MSCs various have reduced safety concerns. Nonetheless, there is still a pressing need for exploratory biomarkers and interval end-points in the context of MSCs investigation. This review intends to summarize the current state of knowledge for stem cells in the experimental and clinical setting of IPF, present important safety and efficacy issues, highlight future challenges and address the need for large, multicenter clinical trials coupled with realistic end-points, including biomarkers, to assess treatment efficacy.

Current evidence suggests that the areas of fibrosis seen in lungs of patients with IPF share many features with normal aging lung, such as genomic instability, telomere attrition, mitochondrial dysfunction, cellular senescence, and immune dysregulation (10,15,16). Due to the inefficacy of immunomodulatory and immunosuppressive agents in the past, the role of the immune system in the pathogenesis of IPF remains poorly understood (17-22). However, highly activated and proliferative CD4+ cells and functional impairment of T regulatory cells (Tregs) in patients with IPF, suggest a link between immunity and pulmonary fibrosis (10,23,24).
There are two approved compounds for the treatment of IPF: pirfenidone and nintedanib. Pirfenidone is an antifibrotic compound with an unclear mechanism of action targeting several molecules including transforming growth factor-β (TGFβ), tumor necrosis factor-α (TNF-α), and interleukin 6 (25). Nintedanib is a tyrosine-kinase inhibitor, targeting vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and platelet derived growth factor receptor (PDGFR) (22).While the use of pirfenidone and nintedanib have been shown to slow the progression of IPF (26-28), both compounds have significant side effects and neither is curative (28-30). Morbidity and mortality from IPF remains high and thus there is a pressing need for alternative therapeutic options for this complex disease (7,(31)(32)(33). The US National Institutes of Health database lists 493 complete or ongoing clinical trials of MSCs (34). Toward this end, regeneration and cell therapies such as the use of mesenchymal stem cells (MSCs) have emerged as a potential option.
MSCs are easily harvested from many tissues (peripheral blood, adipose tissue, bone marrow, and umbilical cord) and may be expanded in vitro with minimal modifications. MSCs represent the most extensively studied stem cell population (54). Research supports the immunomodulatory, anti-inflammatory, and potentially anti-fibrotic properties of MSCs (49, 55, 56). Importantly, MSCs are "immune privileged, " lacking expression of class II major histocompatibility complex (MHC-II). Therefore, allogeneic use of MSCs is possible (57).

PRECLINICAL STUDIES
Recent studies on the pathophysiology of IPF suggest that early alveolar injury activates abnormal alveolar epithelial cells and stimulates the release of mediators including matrix metalloproteinases and TGF beta-1 (2, 58-61). These mediators activate cytokines and chemokines including IL-1 and IL-13 leading to the phenotype of abnormal wound healing (62-65). Therefore, IPF is considered a complex and multifactorial disease characterized by alveolar epithelial injury and alveolar collapse, fewer alveolar epithelial type II cells, alveolar stem cell exhaustion, and myofibroblast deregulation due to living on a fibrotic matrix (66,67).
Several experimental studies have been conducted in order to investigate the effect of MSCs from various organs, mainly from bone marrow with a dosage ranging between 0.1 × 10 6 and 4 × 10 6 cells, in pathways associated with lung injury and pulmonary fibrosis and several end-points had been set (68, 69) ( Table 1).
The majority of studies investigating the effect of donor MSCs on BLM-induced pulmonary fibrosis have used young male mouse models (90,91). Young mice, however, undergo spontaneous resolution of BLM-induced pulmonary fibrosis in some studies (69,90,91). Although IPF is primarily a disease of individuals over the age of 50, most studies have also utilized young female mice to evaluate the molecular patterns and potential therapeutic targets for patients with IPF (92)(93)(94). Interestingly, in one study of bleomycin induced fibrosis in mice, MSCs were found to improve survival when compared with pirfenidone (64). This study also reported downregulation of IL-2, IL-1b, TNF-α, and TGFβ leading to a reduction in inflammation (64). In addition, downregulation of MMPs was noted with a reduction in collagen deposition and fibrosis (64).
Collectively, MSCs seem to exert pleiotropic effects in the site of lung injury including anti-inflammatory, immunomodulatory, antifibrotic effects (65), engagement in paracrine signaling (95), activation of resident stem cells, and differentiation into local cell types (56, 65,74,77,79,83,85,[96][97][98]. Preclinical studies have shown MSCs to be efficacious in the treatment and prevention of lung fibrosis (65,69). Nonetheless, concerns remain regarding the activity of MSCs within a pro-fibrotic microenvironment (99)(100)(101)(102). While some preclinical studies suggest that MSCs might promote fibrosis, to date, no human studies have found a similar pro-fibrotic effect (37, 42, 91, 100, 101, 103-112).   (113). Furthermore, studies of intravenously administered placental derived MSCs (105,109) found that administration of up to 2 × 10 6 cells per kilogram was safe in subjects with moderately severe IPF (109). Importantly, the authors reported only minor and transient alterations in peri-infusion hemodynamics and gas exchange, reducing the concerns for embolization of stem cells to an already compromised pulmonary vasculature. Subjects were followed for six months with no observed decline in forced vital capacity (FVC), diffusing lung capacity for carbon monoxide (DLCO), six-minute walk test (6MWT), or CT fibrosis score (90). The AETHER trial also showed favorable safety outcomes for the intravenous delivery of a single dose of allogeneic MSCs in patients with IPF up to 2 × 10 8 cells (105).
Although this was an underpowered study for the detection of significant changes in functional indices, the mean decline in % predicted FVC and DLCO were below the thresholds for disease progression (1,115). ReCell, an FDA approved phase 1b multidose, randomized, double-blind trial of 10 × 10 6 cells delivered intravenously to patients with IPF, has not yet begun enrollment.

OUTSTANDING CHALLENGES
While it now appears that it is safe to use MSCs in patients with IPF, many questions and challenges remain. In the preclinical realm, there is a need for animal models more representative of chronic IPF (91,116) for the continued study of how MSCs exert their effects. Bleomycin induced pulmonary fibrosis is still considered the best available animal model for preclinical testing (91,117). However, there is increasing criticism that potential therapies usually administered the first 7 days following bleomycin exposure may act mainly through prevention of the inflammatory cascade rather than reversal of fibrosis, thus limiting their applicability to human IPF (69). First, improved animal models will enable the identification of biomarkers that may be useful as measures of disease activity and/or treatment effect. Second, the timing of treatment for best effect needs to be better elucidated. Furthermore, the most efficacious source of MSCs and the role of age need to be more fully explored. One report suggesting that adipose-derived MSCs from young, but not old, mice prevent bleomycin induced lung fibrosis in an aged mouse model (118) highlights the need for further research in this area. Several challenges in the clinical setting also remain to be addressed. The optimal source of MSCs, the best route of administration, the number and timing of administrations, and the appropriate dosing interval. Thus, allogeneic human bone marrow-derived and autologous adipose derived MSCs have been the most studied in the context of IPF. There are limited studies on endogenous stem cells from the lungs of patients with IPF and concerns remain surrounding the risk of biopsy and the potential for intervention-induced IPF exacerbation and the possibility of detrimental effects on lung function from biopsy. Lung tissue obtained at the time of lung transplant remains the best tool for study.
It is also critical to characterize appropriate endpoints to assess treatment effectiveness in these patients (100,(119)(120)(121)(122)(123)(124)(125)(126)(127)(128)(129). Molecular biomarkers would be the optimal choice for the assessment of cell based therapies. Finally, well-designed and meticulously conducted multicenter randomized clinical trials of MSCs for the treatment of IPF are needed to assess efficacy.

MOVING FORWARD
While preclinical trials suggest that MSCs may be effective in the treatment of IPF, and early clinical trials suggest that they are likely to be safe in the population, insufficient data exists at this time to definitely state that the use of MSCs for the treatment of IPF is either safe or efficacious. Despite this lack of evidence, cell based therapies are being aggressively marketed to this vulnerable patient population. A recent study found that as of August of 2016, there were at least 351 stem cell related businesses registered in the United States. These sites offer unproven, experimental treatments for a wide variety of conditions (130,131). In the case of IPF, desperate patients and their physicians continue to succumb to an onslaught of marketing and branding of as yet unproven "stem cell" treatments. Unfortunately, these businesses are also almost wholly unregulated (132). Publication of case reports of harm arising from the misuse of unproven treatments support increased government oversight in the interest of patient safety.

CONCLUSION
Idiopathic Pulmonary Fibrosis (IPF) is a debilitating lung disease characterized by a progressive decline in lung function ultimately resulting in death. The lack of curative treatments for this disease has created an urgency for other potential therapeutic options. Preclinical studies suggest that because MSCs have immunomodulatory, anti-inflammatory, and potentially antifibrotic properties, they may be efficacious in the treatment of IPF. Early clinical trials have shown that MSCs may be safely administered to patients with IPF, but large multicenter randomized trials still need to be performed.

AUTHOR CONTRIBUTIONS
AT, RT and TK wrote the initial manuscript. The manuscript was supervised and significantly modified by MG, AT, DB. KM, IN, VA offered significant intellectual contribution. All authors approved the final form of the manuscript.