Int J Stem Cell Res Ther ISSN: 2469-570X

• Abstract
• Keywords
• Introduction
• Mesenchymal Stromal Cells (MSC)
• MSC for the Treatment of IPF: Preclinical Studies
• MSC for the Treatment of IPF: Human Clinical Data
• Conclusions and Considerations for Future
• Table 1
• Table 2
• References

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International Journal of Stem cell Research and Therapy

¹Institut de Investigació Sanitària de Palma, Fundaciód' Investigació Sanitària de les Illes Balears, Palma de Mallorca, Spain
²Servei de Pneumologia, Hospital Universitari Son Espases, Palma de Mallorca, Spain
³CIBER Enfermedades Respiratorias (CIBERES), Spain
⁴Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Abstract

Idiopathic pulmonary fibrosis (IPF) is a rare and very severe respiratory disease with high morbidity and mortality. For now, no drug-based therapy has been proven unambiguously to reverse the fibrotic process, and no lasting treatment is available other than lung transplantation.

Mesenchymal stromal cells (MSC) are adult multipotent cells capable of differentiating into a number of different cell lineages, which can be isolated from bone marrow, adipose tissue, umbilical cord, amniotic membrane and other tissues, expanded in culture and, subsequently, administered by systemic or local routes into injured animals or ill patients. As a result of their proliferative potential, multipotency, immunomodulatory effects, migratory ability and immunoprivileged state (MSC express very low levels of histocompatibility complex), interest has accelerated as to their imaginable therapeutic applications. Here, we review the properties of MSC, the preclinical studies in animal models of bleomycin-induced lung fibrosis followed by the inoculation of MSC from different origins, and the ongoing human clinical trials using these cells as a therapeutic alternative for IPF.

Keywords

Idiopatic pulmonary fibrosis, Adult stem cells, Mesenchymal stromal cells

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with high morbidity and mortality with a 5-year survival of approximately 20% [1-3]. It is the most common of the idiopathic interstitial pneumonias and one of the most prevalent interstitial lung diseases (ILD). Anatomically it is characterized by scarring of the lung, generating a parenchymal fibrosis, which diminishes lung volumes causing progressive functional loss [4,5] and is associated with the histopathologic and/or radiologic pattern of usual interstitial pneumonia (UIP) [5]. The diagnosis requires exclusion of other known causes of ILD, the presence of a UIP pattern on high-resolution computed tomography (HRCT) in patients not subjected to surgical lung biopsy, and specific combinations of HRCT and surgical lung biopsy pattern in patients subjected to surgical lung biopsy [5]. The natural course of IPF is unpredictable with periods of stability followed by episodes of worsening that can result in respiratory failure and death [6].

IPF is unlikely to occur in adults younger than 50, nonetheless it arises in approximately 0.2% of those older than 75 years [7,8]. In Europe and North America the estimated incidence is 3-9 cases per 100,000 per year, and is increasing worldwide [9]. It is likely that IPF accounts for much of the increased ILD-related mortality reported worldwide between 1990 and 2013 [10].

The manifestation of IPF is believed to be linked to one or more factors, depending on the patient: age, genetic predisposition, and repeated damage to the alveolar epithelium. Cigarette smoking, environmental/occupational pollutants, microbial agents, chronic micro-aspiration secondary to gastroesophageal reflux have been cited as some of the insults that could trigger the fibrotic process [11-13].

Inflammation is involved in the pathogenesis of IPF since several cytokines, such as IL-4, IL-5, IL-13, IL-18, and MIP-1α, are at higher concentration in cel¬lular cultures and bronchoalveolar lavage of patients with IPF, compared to normal individuals [14]. However, the leading point of view suggests that persistent micro-injuries to alveolar epithelial cells induce a fibrotic environment and that growth factors and other mediators secreted by the damaged epithelial cells, such as transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), connective-tissue growth factor, tumor necrosis factor (TNF)-α, fibroblast growth factor (FGF), insulin-like growth factor (IGF)-1 and Wnt-pathway components, among others, participate in the pathogenesis/progression of IPF, promoting fibroblast recruitment, proliferation, and differentiation to invasive myofibroblasts [15]. Consecutively, actively proliferating fibroblasts and myofibroblasts organize into fibroblastic foci and are responsible for the excessive collagen production that results in scarring of the lung and architectural deformation [15,16]. Additionally, matrix metalloproteinases (MMPs), such as MMP1, MMP2, MMP3, MMP7, MMP8, and MMP9 have also been implicated in IPF [17-19].

Recent clinical trials have been testing new molecules with antifibrotic properties (e.g., pirfenidone and nintedanib) instead of just using anti-inflammatory and immunosuppressant drugs [20]. Nintedanib [21,22], an intracellular inhibitor of several tyrosine kinases that targets multiple growth factor receptors and pirfenidone [23-26], reduced the rate of forced vital capacity (FVC) decline, and are recommended in the treatment of patients with IPF [20]. However, no drug-based therapy has been proven unequivocally to reverse the fibrotic process, and no lasting option for therapy is available other than transplantation merely in a limited number of patients.

Stem cells are considered to represent one of the best hopes for regenerating permanently damaged tissue, and because mesenchymal stem (stromal) cells (MSC) are known to home to sites of injury, inhibit inflammation, and contribute to epithelial tissue repair, their use has been suggested as a potential therapy for the treatment of IPF.

Mesenchymal Stromal Cells (MSC)

Stem cells are undifferentiated cells that can continuously self-renew and also differentiate into multiple cell types [27]. Adult stem cells, which are derived from postnatal fully developed tissue, have a limited lifespan and a restricted differentiation potential [28].

MSC are adult multipotent cells capable of differentiating into a number of different cell lines that can be isolated from bone marrow (BM), adipose tissue (AT), umbilical cord (UC), amniotic membrane(AM) or other sources and expanded in culture and subsequently administered by systemic or local routes into injured animals. Because of their combination of proliferative potential, multipotency, immunomodulatory effects [29-31], migratory ability and immunoprivileged state (MSC do not express major histocompatibility factor II) [32], interest has grown regarding their potential therapeutic applications [33].

A growing body of preclinical literature supports the efficacy of MSC administration in a range of experimentally induced lung pathologies, including those used as models of acute lung injury, lung infections, asthma, bronchopulmonary dysplasia, bronchiolitis obliterans, chronic obstructive pulmonary disease, pulmonary ischemia-reperfusion injury, pulmonary hypertension [34], elastase-induced emphysema [35], asbestos-induced lung injury [36] and radiation-induced injury [37]. These studies show that MSC administration improves part or all of the model-specific disease endpoints.

In the lung, at sites of injury, MSC contribute to tissue regeneration and repair [38-42]. Further, using Y chromosome fluorescence in situ hybridization, transplanted Y chromosome-positive MSC from male donors can be found at sites of lung injury in recipient female mice [39,42]. These male MSC appear to adopt epithelial cell morphology, suggesting that they can contribute to tissue regeneration, either by fusion with resident epithelial cells or by mesenchymal-to-epithelial transition [42]. MSC cultured in airway growth media differentially express lung-specific epithelial markers, including club cell (Clara cell) secretory protein, surfactant protein-C, and thyroid transcription factor-1 [43,44].

Also, MSC secrete multiple trophic factors that suppress the immune system [45], inhibit apoptosis [46], stimulate mitosis [47] and differentiation [29-31], and enhance angiogenesis [46,48]. The benefits of MSC trophic factors secretion have been confirmed using co-cultures with endothelial cell populations [49], as well as conditioned media [50] from MSC. MSC are hypothesized to reduce inflammation primarily by release of soluble anti-inflammatory mediators and microvesicles without the need for engraftment or recapitulation of lung morphogenesis. Other mechanisms may be involved, and although significant deficiencies persist regarding our understanding of the disease-specific molecular mechanisms by which MSC mediate the regenerating outcomes, these studies provide a rational basis for the clinical application of MSC in human lung diseases.

Additionally, various populations of lung resident progenitor cells have been identified. Adult pulmonary tissue resident MSC demonstrates a phenotype and function similar to BM-MSC and have been identified in the side population of cells from both murine and human lung tissue [51,52] as well as bronchoalveolar lavage fluid from human lung allografts [53]. Depending on their microenvironment, the lung MSC demonstrate properties similar to other tissue MSC including multilineage differentiation, paracrine antiinflammatory properties, suppression of T cell proliferation as well as the ability to differentiate to myofibroblasts [52,54]. Lung MSC exhibit high telomerase activity which indicates the capacity for self-renewal [51,52,54]. These properties allow a small number of cells to contribute substantially to both tissue regeneration and to proliferative diseases [54-62]. However, in addition to their reparative properties, several studies indicate that MSC may be a critical factor in the development of dysfunctional lung remodeling in some diseases [63-67].

MSC for the Treatment of IPF: Preclinical Studies

MSC may support the restoration of the alveolar epithelium and reduce fibrosis through their anti-apoptotic and anti-scarring effects even in the absence of a substantial and sustained structural engraftment. The MSC effects on lung fibrosis have been investigated with syngeneic, allogeneic, or xenogeneic MSC administration in mouse or rat models of bleomycin-induced lung fibrosis, suggesting that MSC may be efficacious in the treatment of IPF (Table 1).

Of all bleomycin-induced lung fibrosis, improvement in fibrosis was reported in several studies [54,68-72], and a decrease in inflammation was reported in some others [39,40,42,72]. In addition, 3 studies reported near normalization of histopathological change [42,69,73] and four more showed an improvement in alveolar injury [68-70,72]. Moreover, one study reported less improvement with MSC alone than with MSC transfected with ACE2 [74].

On lung collagen, Moodley et al. [40] noted that the effect of MSC was significant 14 and 28 days after bleomycin injury (13 and 27 days after cell administration), but not after 7 days. Ortiz et al. [42] showed that the effect was meaningful when cells were given immediately, but not 7 days after bleomycin administration. In Moodley et al. [75], a significant decrease was seen when human amniotic membrane MSC (AM-MSC) were used, but not with human BM-MSC. Lung collagen content was not decreased by repeated AT-MSC administration at 8, 10, 12, and 14 weeks in a chronic injury model in which bleomycin was given every 2 weeks for 16 weeks [72]. Lung collagen assessed at 14 days was also not decreased by MSC administration twice, at 8 hours and 3 days after bleomycin injury [76].

MSC therapy decreased lung TGF-β levels [39,40,69,72,73,75], including the lung TGF-β protein [39,40,72,73,75] and lung TGF-β mRNA level [40,69]. Lung TNF-α protein level was decreased by amnion MSC but not by BM-MSC. Lung TNF-α mRNA level was also decreased by UC-MSC in [40]. In another study, MSC therapy increased TNF-α protein level when measured 13 days after MSC administration but not at earlier time points [74]. There was no change in TNF-α protein level in [77]. There was no significant change in lung TNF-α mRNA level with MSC therapy in [78]. By contrast, IL-1 levels were significantly decreased by stem cell therapy at all time points, up to 28 days [39]. In a study of Moodley et al. the effect was significant when using amnion MSC but not with BM-MSC [75]. There was no significant effect on lung IL-1 level in [77]. In another study [40], lung IL-1 mRNA level was assessed and was not affected by UC-MSC. Lung IL-6 protein level was decreased by MSC therapy in [75], but lung IL-6 mRNA level was not decreased in [40]. There was no significant change in lung IL-6 protein level with MSC therapy in [77] whereas BAL IL-6 was decreased in [39].

Lung MMP-2 and MMP-9 levels were also assessed [40,42,75,77]. In the study of Min et al. [77], MMP-2 was increased while MMP-9 was reduced. In one of the Moodley et al. studies [40] but not in the other [75], MMP-2 was increased. However, MMP-9 was increased by AM-MSC but not by BM-MSC in [75], but was not significantly affected by MSC therapy in [40]. Lung MMP-2 and MMP-9 mRNA level was decreased by immediate administration of MSC in [42]. Lung MMP-13 was not significantly affected in [40,75], but was significantly decreased by MSC in [42].

Regarding the timing of stem cell administration and chronicity of lung injury, most studies administered MSC within hours to a few days after bleomycin injury. Few studies administered MSC after 7 [68,79] or 10 [75] days, and 1 study, Lee et al. examined chronic lung injury with bleomycin injection twice weekly for 16 weeks and MSC administration at weeks 8, 10, 12, and 14 [72]. In this latter study MSC administration had no effect on lung collagen content, but interestingly it improved the Ashcroft score, BAL total cell count and neutrophil count, and lung TGF-β levels. In the study of Huang et al., immediate administration of MSC resulted in significantly better effects on hydroxyproline content and alveolitis and fibrosis scores [68].

Considering the origin of the cells, Ortiz et al. [42,78] used allogeneic BALB/c mice BM-MSC in C57BL/6 mice. In [42], allogeneic cell administration reduced the extent of inflammation and lung collagen deposition when administered immediately after lung injury, but not after 7 days. However, MSC were unable to decrease totalBAL cell count or lung TNF-αmRNA levels [42]. There were no studies comparing allogeneic and syngeneic cells. Xenogeneic cells, human cells in mice or rats, were used in vivo in many studies [40,41,70,72-75,77], and were able to improve lung collagen content in some of them [40,70,73,75,77]. In summary, improvement in severity and extent of fibrosis 14 days after bleomycin injury and MSC administration was observed with allogeneic cells, whereas treatment with xenogeneic cells resulted in improvement in severity but not extent of fibrosis.

Finally, although the precise mechanism of action of MSC remains elusive [80], in several successful clinical trials [81,82], paracrine factors secretion has been proposed as a mechanism of action. Several paracrine factors released by MSC may alter the microenvironment and may contribute to a beneficial effect in IPF [78,81]. In addition to paracrine signaling, MSC may initiate an effect via cell fusion, cell to cell interactions [83], differentiation, and promotion of neovascularization. Each of these mechanisms alone or in combination may contribute to beneficial effects [84].

MSC for the Treatment of IPF: Human Clinical Data

As of March 2016, there are 5 recruiting, active or completed clinical trials registered on https://clinicaltrials.gov/ (U.S. National Institutes of Health) trying to evaluate the safety and efficacy of MSC therapy in IPF (Table 2). For the time being, the only completed is a small open-label, single center, non-randomized study with 8 patients with moderately severe IPF (FVC ≥ 50% and DLCO ≥ 25%) [85]. These IPF patients received either 1 × 10⁶ (n = 4) or 2 × 10⁶ (n = 4) unrelated-donor, placenta-derived MSC/kg via a large peripheral vein and were followed for 6 months with lung function (FVC and DLCO), 6-min walk distance (6MWD) and CT scan of the chest. Eight patients with median FVC 60% and DLCO 34.5% predicted, were treated. Both dose schedules were well tolerated with only minor and transient acute adverse effects. MSC infusion was associated with a transient (1% (0-2%)) fall in SaO2 after 15 min. At 6 months FVC, DLCO, 6MWD and CT fibrosis score were unchanged compared with baseline, supporting a good safety profile after 6 months follow-up [85].

There are other studies supposedly not registered in this database, like the one published by Tzouvelekis et al. [86], a phase Ib, prospective, non-randomized clinical trial, designed to study the safety of 3 endobronchial infusions, at monthly intervals, of 0.5 million autologous adipose derived stromal cells-stromal vascular fraction (ADSC-SVF) per kg/body weight per infusion (40 million cells per infusion) in 14 IPF patients of mild to moderate disease severity (FVC > 50% predicted value and DLCO > 35% of predicted value). The incidence of treatment emergent adverse events within 12 months was the primary end-point and alterations of functional, exercise capacity and quality of life parameters at serial time points (baseline, 6 and 12 months after first infusion) were exploratory secondary end-points. No cases of serious or clinically meaningful adverse events including short-term infusion toxicities as well as long-term ectopic tissue formation were recorded in all patients. Also, detailed safety monitoring through several time-points indicated that cell-treated patients did not deteriorate in both functional parameters and indicators of quality of life [86].

For the time being, these studies demonstrate a precious safety profile, but nevertheless lack the requirements of a double-blind prospective randomized controlled trial to scientifically and clinically assess the efficacy of a treatment.

Conclusions and Considerations for Future

In conclusion, MSC are a promising alternative for the therapy of IPF, particularly for their suitability to easily be isolated, expanded to big numbers in culture and their immunomodulatory and regenerative properties. However, a number of questions have still no answers: what is the most efficacious source of MSC? Are allogeneic cells as safe and efficacious as autologous MSC? Do MSC retain efficacy after passage? Are MSC most effective in the lung when administered intra-tracheally, intravenously or by some other method? [87].

In this sense, some authors argue that the somehow contradictory results found in the preclinical studies are probably dependent on the native tissue from which these cells are derived. Tissue-specific MSC may retain characteristics of their original tissue source in terms of their differentiation capability and specific cytokine gene expression profile [88]. According to Li et al. [89], AD-MSC as compared to BM-MSC have biological benefits in the proliferative capacity, secreted proteins (FGF, interferon-γ, and IGF-1) and immunomodulatory effects. On the other hand BM-MSC seem to have advantages in osteogenic and chondrogenic differentiation potential and other secreted proteins (stem cell-derived factor-1 and hepatocyte growth factor [HGF]) [89].

Profiles of paracrine factor secretion and gene expression of BM-MSC and MSC derived from perinatal tissues (UC-MSC and AM-MSC), have revealed that UC and AM are noticeably different from BM. Although MSC from all sources were found to express similar surface markers, UC-MSC and AM-MSC revealed higher potential of immunomodulatory capacity than BM-MSC (higher levels of HGF, MCP-1, and M-CSF in supernatants). Cytokine IL-1ra was the only factor associated with anti-inflammatory and anti-fibrotic effects of MSC which was secreted significantly higher by BM-MSC compared to perinatal MSC [90]. Perinatal MSC may also be of meaningful value in view of the fact that the treatment of aged patients, as it the case of IPF, may require an alternative source of stem cells, other than their own autologous MSC [91].

Regarding the administration route, in an animal model of neonatal hyperoxic lung injury that induces up-regulation of genes associated with the inflammatory and fibrosis response and cell death, the local intra-tracheal transplantation of human UC-MSC was more effective than the systemic intravenous infusion, with respect to delivery efficiency and therapeutic efficacy [92]. In any case, both of these inoculation routes seem to be better than the intraperitoneal route [93].

There are many alternatives when talking about the "timing" of MSC administration in animal models. Most of the bleomycin preclinicial protocols discussed above are actually testing the prophylactic or preventive effect of these cells, since they are administered at the same time as, or soon after, the damage is caused. In contrast, IPF in humans is most of the times diagnosed when the disease is already in the advanced stage, consequently the genuine regenerative potential of these cells could be at stake unless we are very stringent during the patient selection process, mostly defining very well the magnitude or grade of the disease in order to be counted in the clinical study.

Hence, biological variations between MSC from different pedigrees (BM-MSC, AD-MSC, UC-MSC, AM-MSC, etc.), inoculation routes (intravenous, intra-tracheal, etc.) and extent of the disease ("timing") should be thoroughly considered when choosing a specific clinical application for these cells. And, either way, is still early to draw conclusions on the best source of MSC and the best way to administer, although, possibly, intra-tracheal instillation of MSC obtained from perinatal tissues may have some advantages.

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