Abstract
COPD is a common, preventable and usually progressive disease associated with an enhanced chronic inflammatory response in the airways and lung, generally caused by exposure to noxious particles and gases. It is a treatable disease characterised by persistent respiratory symptoms and airflow limitation due to abnormalities in the airways and/or alveoli. COPD is currently the third leading cause of death worldwide, representing a serious public health problem and a high social and economic burden. Despite significant advances, effective clinical treatments have not yet been achieved. In this scenario, cell-based therapies have emerged as potentially promising therapeutic approaches. However, there are only a few published studies of cell-based therapies in human patients with COPD and a small number of ongoing clinical trials registered on clinicaltrials.gov. Despite the advances and interesting results, numerous doubts and questions remain about efficacy, mechanisms of action, culture conditions, doses, timing, route of administration and conditions related to homing and engraftment of the infused cells. This article presents the state of the art of cell-based therapy in COPD. Clinical trials that have already been completed and with published results are discussed in detail. We also discuss the questions that remain unanswered about cell-based regenerative and translational medicine for COPD.
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This review describes the current state of the art of cell-based, regenerative and translational medicine in COPD, presenting the clinical trials already completed and with published results. https://bit.ly/3P08Bmq
Introduction
According to the definition by the Global Initiative for Chronic Obstructive Lung Disease (GOLD), COPD is a heterogeneous lung condition characterised by chronic respiratory symptoms (dyspnoea, cough, sputum production, exacerbations) due to abnormalities of the airways (bronchitis, bronchiolitis) and/or alveoli (emphysema) that cause persistent, often progressive, airflow obstruction [1]. Chronic respiratory symptoms result from interactions among genetic, epigenetic and environmental factors, characterising a complex multifactorial disease [1–4]. Conversely, α1-antitrypsin deficiency is associated with an autosomal recessive monogenic inheritance pattern and is responsible for 1–3% of cases [2, 3]. The main environmental factors associated with COPD include tobacco smoke, pollution and occupational exposure to noxious gases and particles [1].
From an epidemiological perspective, COPD represents a serious public health problem worldwide and remains a therapeutic challenge for pulmonologists and general practitioners [5, 6]. COPD is currently the third leading cause of death worldwide and, according to the World Health Organization, ranks seventh among the top global causes of disability-adjusted life years [1, 7]. In this scenario, COPD is a major cause of chronic morbidity and mortality, with substantial economic and social burdens [1, 7–14]. The discovery regarding the origin of chronic respiratory disease in the first years of life modified the classic concept of COPD as solely a “smoker's disease” [15, 16]. A series of other relevant aetiologic factors, as well as the interactions between these factors, have been highlighted in the context of the pathophysiology and lung function trajectories over a life span that can impact and determine abnormal lung development and/or accelerated lung function decline.
Knowledge of the different trajectories and corresponding aetiology [17–19], as demonstrated in figure 1, as well as recent advances in the knowledge of COPD pathophysiology, have allowed expansions of the definition (a heterogeneous lung condition) and the understanding of COPD as a syndrome and not just a single disease, redefining the “range of conditions under the umbrella of COPD” and, as a consequence, opening up the possibility of individualising treatment according to the phenotypic characteristics of each patient [21]. As shown in figure 2, we attempted to synthesise the therapeutic potential resulting from different factors secreted by mesenchymal stromal/stem cells (MSCs) (secretome), as well as the physiological processes of these different factors that can act directly or indirectly as potential targets for cell-based therapy for COPD and other chronic diseases, through a paracrine mechanism or distant from the originating cell (MSC extracellular vesicles) or by cell-to-cell contact.
Different factors secreted (secretome) by mesenchymal stromal/stem cells (MSCs) and physiological processes that may act as potential targets for cell-based therapy. CAT: catalase; CCL: chemokine (C-C motif) ligand; CX3CL1: C-X3-C motif chemokine ligand 1; CXCL: chemokine (C-X-C motif) ligand; EGF: epidermal growth factor; FGF: fibroblast growth factor; FLT3: Fms related receptor tyrosine kinase 3; GM-CSF: granulocyte–macrophage colony-stimulating factor; Gpx: glutathione peroxidase; HGF: hepatocyte growth factor; IDO: indoleamine 2,3-dioxygenase; IGF-1: insulin-like growth factor 1; IL: interleukin; IP10: interferon-induced protein 10; KGF: keratinocyte growth factor; LAP: latency-associated peptide; NGF-3: nerve growth factor 3; PGE2: prostaglandin E2; PIGF: phosphatidylinositol glycan anchor biosynthesis class F; SOD: superoxide dismutase; TGF: transforming growth factor; TNF-α; tumour necrosis factor-α; VEGF: vascular endothelial growth factor.
This understanding of COPD in a broader context of pathogenesis and pathophysiology opens up new avenues of opportunity for early diagnosis, effective forms of prevention, the possibility of early intervention and new therapeutic approaches [22–33]. In this context, cell-based therapies have arisen as potentially promising new therapeutic approaches and different cell-based therapies have been proposed and tested in pre-clinical models of COPD/emphysema.
Despite significant advances in clinical therapeutic approaches, the continuous incorporation of new drugs into the therapeutic arsenal against COPD and new pulmonary rehabilitation techniques [32, 34–36], curative clinical treatments have not yet been achieved. Currently employed treatments are palliative because in the COPD spectrum such approaches target only the potentially reversible components of the disease (such as inflammation, hypersecretion and smooth muscle contraction) but not its irreversible components (such as parenchymal destruction, emphysema, lung vasculature and airways remodelling).
In this narrative review, we present and discuss the current state of the art of cell-based therapies and regenerative medicine in patients with COPD. We include clinical trials that are registered at clinicaltrials.gov (National Institutes of Health, USA) and have already been completed and published. We also discuss the questions that remain unanswered about the efficacy, reproducibility and standardisation of cell-based therapies for COPD. To prepare this review, the following databases were consulted: Cochrane Library, PubMed, Medline, Scopus and Web of Science. Some articles referring to the theme of this review previously mentioned in the references of the publications consulted during the preparation of the article are also discussed. The literature review covered a period of 28 years, from 1995 to 2022.
Regenerative and translational medicine: from bench to bedside
Pre-clinical studies: from the bench
Experimental evidence from animal models of the migration and homing of mesenchymal precursor cells from bone marrow to the lung was first reported by Pereira et al. [37]. Several authors have subsequently shown the chimerism and migration of bone marrow mononuclear cells (BMMCs) to the lung in experimental models and in bone marrow transplantation in human patients [26]. These results strongly indicate the direction of tests for migration analysis and cell-based therapies in animal models.
Consistent results in experimental cell-based therapy in mice (C57BL/6) with lung injury and subsequent infusion of BMMCs were first reported in 2004 by Yamada et al. [38]. The authors reported the protection of lung parenchyma in animals treated with BMMCs after lipopolysaccharide-induced lung injury. In 2004, Ishizawa et al. [39] reported alveolar regeneration in animals with lipopolysaccharide-induced emphysema that were treated with retinoic acid and granulocyte colony stimulating factor to promote lung regeneration and an increase in BMMC numbers in alveoli; a concomitant treatment with both factors resulted in an additive effect. Rojas et al. [40] showed that infusion of bone marrow-derived mesenchymal stem cells (BM-MSCs) played a role in the repair of bleomycin-injured lungs in mice.
The first study of cell-based therapy with MSCs in an animal model of COPD was conducted in 2006 by Shigemura et al. [41]. Rats underwent emphysema induction by the intratracheal instillation of porcine pancreatic elastase. After 14 days, the treated group received an intravenous infusion of 5×107 MSCs per animal. Alveolar and vascular regeneration, improvements in gas exchange and greater tolerance to exercise were observed when compared with the control group. Since then, various pre-clinical and clinical studies of cell-based therapies for COPD have been published, with several comprehensive and consistent reviews [1, 32, 42–61].
A significant proportion of pre-clinical animal studies of COPD have reported consistent results in terms of the feasibility and efficacy of cell-based therapies with BMMCs and MSCs [1, 47, 56, 62, 63]. Despite these promising findings, animal models have limitations in terms of pathophysiological aspects and the therapeutic translation to human disease. Nonetheless, the consistent results in cell-based therapy in animal models supported the proposition and provided the basis for translational studies and the implementation of cell-based clinical trials in human patients.
Clinical trials: to bedside
The first clinical trial of a cellular therapy in patients with advanced-stage pulmonary emphysema was conducted in Brazil in 2011 by Ribeiro-Paes et al. [64]. In this phase I clinical trial (clinicaltrials.gov identifier: NCT01110252; approved by the Brazilian National Committee of Ethics in Research – CONEP, Report 233/2009), autologous freshly isolated BMMCs were infused in four patients with very severe COPD (GOLD stage IV) out of a total of 10 patients requested in the original project sent to CONEP. All patients underwent stimulation with granulocyte colony stimulating factor for three consecutive days before bone marrow harvest. A total of 1×108 BMMCs were infused per patient in the medial brachial vein. The results showed a slight improvement in forced expiratory volume in 1 s (FEV1) and forced expiratory volume (FVC) in all patients 30 days post-infusion. After this period, pulmonary function parameters showed a progressive downward trend, returning to pre-procedure baseline values or below. Importantly, during the follow-up period, no serious adverse events were observed that could be directly associated with the BMMC infusion. Complementing this study, Stessuk et al. [5] performed a follow-up of the same patients for to 3 years. Encouraging results were observed in one of these patients (patient IMC 4). During the follow-up period, after 23 months, this patient maintained a stable FEV1 value and a significant increase in FVC relative to pre-procedure values. During this long follow-up period of patient IMC 4, no adverse events that could be associated with the BMMC infusion procedure were detected, thus corroborating the proposition that cellular therapy with BMMC is safe.
2 years later, the first placebo-controlled randomised trial using MSCs for cell therapy in COPD was published (clinicaltrials.gov identifier: NTC 00683722). This study was sponsored by Osiris Therapeutics Inc. (Columbia, MD, USA) and published by Weiss et al. [65]. In this study, the authors used a pool of nonhuman leukocyte antigen matched MSCs from bone marrow that was commercially registered as Prochymal™ (Osiris Therapeutics Inc.), a pre-manufactured formulation of human MSCs from different donors [66]. Patients enrolled in this study received four monthly infusions (0, 30, 60 and 90 days) of 1×108 MSCs (Prochymal™) per patient or vehicle control and were subsequently followed for 2 years after the first infusion. The results showed a decrease in C-reactive protein (CRP), indicating a possible improvement in the inflammatory process. However, no clinical or laboratory improvements in lung function or the patients’ quality of life were found. No adverse events that were related to the MSC therapy were observed.
In 2016, Stolk et al. [67] published the results of a phase I prospective open-label study for autologous MSC infusion in patients with severe emphysema (clinicaltrials.gov identifier: NCT01306513). Seven patients completed the protocol. They underwent two lung volume reduction surgery (LVRS) procedures and received two autologous BM-MSC infusions, 3 and 4 weeks after the second LVRS. A significant increase in FEV1 was recorded after 12 months. At the 12-month follow-up, no adverse events were noted. Similar to previous studies, the procedure was considered feasible and safe.
The next year, de Oliveira et al. [68] published a phase I, prospective, patient-blinded, randomised, placebo-controlled study of 10 patients with severe emphysema (clinicaltrials.gov identifier: NCT04018729). In this interesting and innovative study, a one-way endobronchial valve (EBV) insertion was associated with an infusion of allogenic BM-MSCs in patients with severe and very severe COPD (GOLD stages III and IV). Patients (n=5 per group) randomly received either allogeneic BM-MSCs (108 cells) or 0.9% saline solution bronchoscopically immediately before insertion of the one-way EBV. The results showed a systemic decrease in the inflammatory process (CRP) in patients who were treated with an EBV and BM-MSCs. The procedure proved to be safe, but no improvements in the patients’ respiratory parameters were found at the 90-day follow-up.
In 2017, an initial phase I clinical trial by Comella et al. [69] evaluated the early and delayed safety of a stromal vascular fraction (SVF) infusion. In this study, 12 patients were enrolled and received an intravenous infusion of 1.3 and 2.3×108 SVF cells per patient. The SVF approach was innovative and the results showed no adverse effects. It would be important to reassess this study regarding the safety of the infusion volume (1 L), which can lead to cardiorespiratory decompensation when considering the susceptibility of patients with COPD. This protocol was approved by Angeles Hospital (Tijuana, Mexico), but no record of this trial was found on clinicaltrials.gov.
In 2018, Armitage et al. [70] performed a phase I pilot study in a cohort (n=9) of mild-to-very severe stable COPD patients (Australian clinical trials registry no. 12614000731695). The authors evaluated MSC biodistribution and inflammatory and clinical end-points following systemic MSC infusions. The patients received two intravenous infusions of cultured allogeneic BM-MSCs (≈2×106 MSCs·kg−1). The first infusion was performed with radiolabelled cells (indium-111). The second infusion was performed 1 week later with unlabelled cells. The recovered signal was smaller in the emphysematous lung. There was no significant improvement in pulmonary parameters, such as FEV1 and FVC. In contrast to previous results, there was an increase in CRP levels. However, reductions of several other inflammatory factors were found. The authors hypothesised that these reductions could be attributable to the release of trophic factors, including extracellular vesicles (EVs). Despite no improvements in the spirometric results, the authors concluded that systemic MSC infusions may be useful for attenuating inflammation in COPD patients.
In 2020, Le Thi Bich et al. [71] performed a clinical trial with 20 patients with moderate-to-severe COPD (ISRCTN Register: ISRCTN70443938) [72]. The patients received expanded allogeneic umbilical cord-derived MSCs (UC-MSCs) (1×106 cells·kg−1 infusion) and were followed for 6 months. The authors evaluated different pulmonary function parameters and inflammation (CRP). The results showed a significant decrease in the dyspnoea modified Medical Research Council (mMRC) score and a decrease in the number of exacerbations during the 6-month follow-up. However, no significant improvements in FEV1, the 6-min walk test or CRP were observed relative to initial values before treatment. In this pioneering pilot clinical study with UC-MSCs, no severe adverse events that were directly related to UC-MSC administration were reported.
Another group from Vietnam published a phase I/II matched case-control trial to evaluate the safety and efficacy of allogeneic human UC-MSCs in patients with moderate-to-severe COPD (clinicaltrials.gov identifier: NCT04433104) [73]. In this study, 40 patients were enrolled and assigned to two age-matched, gender-matched and COPD condition-matched groups (UC-MSC-treated group and control group). This is a very well-structured clinical protocol. It is expected that after conclusion of the trial and publication of the pulmonary function results, this study may provide new data on the safety and therapeutic efficacy of UC-MSCs in COPD patients. Each of these clinical trial procedures are summarised in table 1 and figure 3.
Studies that evaluated the use of stem cell therapies in COPD with published results
Outline of cell-based therapy methodologies for COPD treatment in nine published clinical trials. Studies by a) Ribeiro-Paes et al. [64] and Stessuk et al. [5], b) Weiss et al. [65], c) Stolk et al. [67], d) de Oliveira et al. [68], e) Armitage et al. [70], f) Le Thi Bich et al. [71], g) Hoang et al. [73] and h) Squassoni et al. [74]. 6MWT: 6-min walk test; AD-MSC: adipose tissue-derived mesenchymal stem cell; BM-MSC: bone marrow-derived mesenchymal stem cell; BMMC: bone marrow mononuclear cell; CBC: complete blood count; CSE: clinical simulation; CT: computed tomography; EBV: endobronchial valve; LVRS: lung volume reduction surgery; PFT: pulmonary function test; UC-MSC: umbilical cord-derived mesenchymal stem cell.
Co-infusion of BMMCs and MSCs: additive or synergistic therapeutic effects
The results with cell-based therapy in COPD reporting morphological regeneration of the parenchyma in pre-clinical animal models and the improvement of pulmonary function parameters in human patients has been attributed to local paracrine effects resulting from the modulation of the inflammatory response and the release of cytokines and growth factors that can stimulate tissue regeneration. Several research groups have proposed that BMMCs are related to neoangiogenesis and tissue neovascularisation, thereby promoting the regeneration of damaged tissue [48, 62, 75–79]. In parallel, there are some consistent results from pre-clinical animal studies that show that MSC from different sources (e.g. bone marrow and adipose tissue) play a modulatory role in the immune response and can attenuate and modulate the inflammatory response by inhibiting the expression of pro-inflammatory cytokines and chemokines, such as interleukin (IL)-1β, IL-6 and tumour necrosis factor-α, and promoting anti-inflammatory cytokines, such as IL-4 and IL-10 [50, 58, 59, 80–87]. Infusions of MSCs may also increase the release of various growth factors, such as vascular endothelial growth factor [77, 78, 81, 83–85, 88–90], fibroblast growth factor 2, hepatocyte growth factor and insulin-like growth factor 1 [58, 59, 83, 84, 86, 88, 91, 92].
Another important effect in the context of COPD is related to a decrease in the synthesis of metalloproteinases (MMP), mainly MMP1, MMP9 and MMP12, in alveolar macrophages that act in the degradation of alveolar walls and thus play a fundamental role in the pathophysiology of pulmonary emphysema [59, 83, 90, 93].
Considering the properties and mechanisms of action of BMMCs and MSCs, we hypothesise that concomitant infusions of BMMCs and MSCs may result in additive or synergistic effects in stimulating neoangiogenesis, decreasing the inflammatory process and delaying disease progression. Starting from this premise, we proposed a new randomised, open and controlled phase I clinical trial. The first end-point was to assess the procedure's safety. As a secondary end-point, we evaluated the efficacy of concomitant infusions of BMMCs and autologous adipose tissue-derived mesenchymal stromal cells (ADSCs) in patients with advanced COPD [74]. The experimental design of this clinical trial is shown in figure 4. This clinical protocol was approved by the National Commission of Ethics in Research (CONEP) in Brazil (Report 926.672) and registered at clinicaltrials.gov (NTC02412332).
In this study, 20 patients with moderate-to-severe COPD who met the inclusion criteria were enrolled. The patients were randomly distributed into four groups (n=5 per group), as follows: the control group (patients who were maintained on conventional clinical treatment for COPD), the BMMC group (patients who were treated with autologous BM-MSCs), the ADSC group (patients who were treated with ADSCs) and the co-infusion group (patients who received concomitant infusions of ADSCs and BMMCs). The infusions of BMMCs, ADSCs and ADSCs+BMMCs were performed through the middle brachial vein. The total number of cells per infusion was 1×108 cells per patient. Patients who received ADSCs+BMMCs were infused with a total of 5×107 of each cell type. Patients were evaluated relative to the pre-treatment baseline with regard to their biochemical profile, pulmonary function, cardiopulmonary test, radiological examinations, 6-min walk test result and quality of life. The control and treated groups were periodically evaluated for 12 months. No adverse events were detected that were directly related to the BMMC, ADSC and ADSC+BMMC infusion procedures, thus corroborating previous results from our group and other research groups demonstrating that BMMC and ADSC infusions are safe. Regarding efficacy, the best results were obtained for some pulmonary function parameters in the BMMC group (i.e. decrease in partial pressure of CO2) and the ADSC+BMMC group (i.e. lung capacity, including oxygen uptake and diffusing capacity of the lung for CO). The small number of patients in each group likely impacted the results and did not allow validation of the hypothesis that co-infusions of BMMCs and ADSCs could result in additive or synergistic therapeutic effects in COPD [74]. Additional studies are needed with more patients to allow more valid and confirmatory assessments of possible additive or synergistic therapeutic effects of concomitant BMMC and ADSC infusions. Despite a number of limitations, this study represents a starting point for the evaluation and review of the influence on the biological and clinical response as a function of the source of MSCs employed.
Regenerative medicine in COPD: perspectives and challenges
Clinical trials registered at clinicaltrials.gov
Approximately 5100 clinical trials for stem cell/cell therapy are registered at clinicaltrials.gov (accessed: 9 September 2022). Despite notable advances, as well as many potentially promising results and well-conducted studies with regard to the safety of stem cell-based therapies, there is still no broad consolidation of clinical efficacy [60]. In the literature and the clinicaltrials.gov database, there is a marked predominance of studies that have as an end-point or outcome measure the safety of clinical protocols (phase I). For COPD, there are 22 studies that are registered in clinicaltrials.gov, as shown in table 2. Most of these studies used MSCs from different sources (adipose tissue and umbilical cord), with a predominance of phase I or phase I/II studies.
Clinical trials for COPD registered at clinicaltrials.gov (accessed 9 September 2022)
Therapeutic potential of cell-based therapy: issues to be better understood
A consistent result in all the previously published clinical trials of cell therapy in COPD patients refers to safety of the procedure. No notable adverse effects were described during the different follow-up periods in any of the previously published protocols, which ranged from 3 to 30 months [64, 65, 67, 68, 70–72, 74]. However, several questions remain unanswered about the cell-based therapies’ effectiveness and mechanisms of action, especially for MSCs from different sources [60, 94, 95].
Among the numerous issues that need to be better understood regarding the therapeutic potential of cell-based regenerative medicine for COPD are the tissue of origin, intrinsic factors of the donor/recipient, culture conditions, the number of passages during the cell proliferation process, dose and timing of administration (acute or chronic phase of the disease), the route of administration (intratracheal or intravenous), and conditions related to the homing and engraftment of infused cells [33, 74, 95]. Some of the answers to these questions may come from new pre-clinical study models, as well as from new clinical trials that can objectively and unquestionably prove clinical efficacy.
Standardisation of cell culture protocols
One important aspect that requires better evaluation and standardisation among different research groups is linked to the cultivation conditions of protocols of cell therapy for COPD, particularly the number of passages of MSCs during the cell proliferation phase. In cell therapy procedures, many cells are necessary to achieve the desired therapeutic effect. Ex vivo cell proliferation represents an artificial and inhospitable condition for the cell, which can result in genetic instability and a risk of genotoxic effects [57, 96].
Dose and timing of infusion
The dose and infusion timing that have been adopted in clinical protocols for COPD/emphysema have been extrapolated from results of experimental models, especially in rats and mice, and previous studies of cell-based therapies in other human pathologies, such as myocardial infarction [97, 98]. Kim et al. [99] performed a comparative analysis between different doses in an experimental model of elastase-induced emphysema in C57BL/6J mice. Doses of the order of 5×104 MSCs exerted a significant effect on the emphysematous mouse lung. Kennelly et al. [100] proposed that the most effective doses in an animal model would be between 5×104 and 5×105 cells per mouse. In clinical trials of human patients that have been published to date, doses of 1×108 BMMCs per patient were used [5, 64]. The same dose of MSCs was used in the study with Prochymal™ [65]. In the clinical trial by Stolk et al. [67], 1–2×106 BM-MSCs·kg−1 was used. Equivalent doses were used in the nine published protocols with human patients. However, controversies remain about dose-response effects. Kennelly et al. [100] showed that doses above 105 cells per mouse are less effective. In Crohn's disease, for example, high doses were shown to not improve therapeutic efficacy [101]. The issue of dosage is one aspect that needs to be reviewed in human therapy, considering that the number of cells that are used in clinical trials is much lower than in mouse models. Considering the standard mouse weight of 20 g, the range of effective doses in mice that was proposed by Kim et al. [99] and Kennelly et al. [100] would correspond to patients who weigh 70 kg. By extrapolation, the human dose would be approximately 1.8–18 times higher than in clinical studies of COPD [64, 65, 67, 68, 70, 71, 74].
With regard to timing of the infusion, there is no consensus in the literature about the disease stage that best responds to BMMC or MSC infusions. Considering the anti-inflammatory effect of MSCs and considering the chronicity of inflammation in COPD, two approaches can be proposed. Therapeutic efficacy could be maximised if treatment is initiated in the acute phase, thereby limiting progression of the disease. In the case of diagnosis in more advanced or severe phases of the disease with repeated exacerbations (which are more common in COPD), chronic therapy with repeated infusions could be adopted. In animal models, Richardson et al. [97] compared the immediate and late administration of two doses of MSCs in a rat model of myocardial infarction. The authors used allogeneic MSCs (1×106 “low” dose or 2×106 “high” dose) by trans-epicardial injection immediately after myocardial infarction (“early-low” and “early-high”) or 1 week later (“late-low” and “late-high”). The results showed that the therapeutic effects of MSC infusions that occurred after acute myocardial infarction are strongly influenced by the timing of cell delivery, with dose-dependent effects that are most evident with early intervention. Similar results were reported by Kennelly et al. [100], indicating that the effectiveness of treatment with MSCs is time dependent and more effective in the acute phase, but lung function may improve when therapy is introduced in the chronic phase; in this case, however, it has lower efficacy.
The results of Longhini-dos-Santos et al. [85] found time-dependent morphological improvements in the lung parenchyma (mean alveolar diameter) in mice with induced emphysema that were treated with BMMCs 7, 14 and 21 days after intranasal porcine elastase administration, but the response was directly proportional to the infusion time, which was contrary to the results that are reported above. The best and most statistically significant response was obtained with BMMC infusion after 21 days. Wang et al. [98] published a systematic review and meta-analysis of the ideal time and dose for MSC infusion to obtain the best end-points in patients with acute myocardial infarction. The authors concluded that infusion of no more than 107 MSCs within 1 week after a percutaneous coronary intervention might improve left ventricular systolic function. In human patients, however, further controlled studies of the most appropriate infusion timing are needed. Additionally, a particular problem is related to the fact that COPD is usually diagnosed in symptomatic and later stages of the disease.
Route and multiple infusions
Another aspect that is still scarcely explored in clinical cell therapy protocols for COPD is the possibility of adopting cell therapy with multiple infusions when considering its chronic inflammatory characteristics and repeated exacerbations during the clinical course of the disease. Poggio et al. [102] performed a comparative study of the effects of one or two infusions of MSCs that were administered 1 week apart in a mouse model of elastase-induced emphysema. The results showed that both one and two doses of MSCs improved lung function, but two doses resulted in improvements in ventilatory mechanics, histological recovery of the lung parenchyma and the recovery of cardiac function in animals with elastase-induced emphysema. A clinical trial by Weiss et al. [65] repeatedly infused Prochymal™ and found no improvements in any of the analysed clinical parameters, with the exception of a drop in CRP levels in treated patients compared with controls.
Hoang et al. [73] published a very well-designed phase I/II clinical trial of the safety and efficacy of UC-MSC infusions in 20 COPD patients. In this clinical trial, patients received two intravenous infusions of 1×106 cells·kg−1 at a 3-month interval between the first and the second dose. It is hoped that consistent data on the use of UC-MSCs for COPD will be added to the literature with the publication of these results. In a study that was published by our group that assessed the safety (phase I) of cell therapy, improvements in FEV1 and FVC were observed 30 days after intravenous BMMC infusion in the brachial vein. After this period, there was a progressive decrease in pulmonary function parameters, which returned to pre-treatment values in the four patients who underwent the procedure [64]. These findings suggest the possibility of maintaining pulmonary function parameters through repeated infusions, but the low number of patients who were enrolled in this phase I study did not allow more thorough analyses of the results. This hypothesis needs to be tested with a greater number of patients to allow more robust statistical analyses.
With regard to the most appropriate cell infusion route, results from animal models of the intra-tracheal instillation of BM-MSCs or ADSCs show that it is safe and effective. Liu et al. [47] suggested that intra-tracheal or intra-bronchial infusion is a preferred and safer route of MSC administration. Our research group adopted the intravenous route for ADSCs [64, 74]. The rationale for using intravenous infusion is based on the principle that the lung acts as a primary cell retention filter in the microvasculature of the lung. This transient trap and homing triggers a paracrine action on the lung parenchyma, stimulating chemotaxis and the release of growth factors and anti-inflammatory cytokines. Additionally, in our view, infusion into a peripheral vein (e.g. brachial) in human patients has fewer risks when considering that patients with COPD are in a borderline unstable pathological condition and intratracheal interventions or intrabronchial infusions can trigger disease exacerbation or serious adverse effects.
Target tissue microenviroment, homing and engraftment of the transplanted cells
Another important aspect that is still debatable refers to possible pathophysiological conditions of donor patients (i.e. heterologous infusions), recipient patients as a whole and target tissue, which may be an inhospitable microenvironment that can compromise the homing, engraftment, viability and survival of transplanted cells [103–108]. Considering that survival and viability of MSCs in target tissue are key factors for the success of cell-based therapies, the biochemical and molecular regulatory mechanisms that are involved in homing and engraftment processes need to be precisely defined to improve the therapeutic efficacy of these cells [103–109]. In addition to these aspects, in the case of COPD, the possible influence of pharmacological therapies, such as bronchodilators and corticosteroids, needs to be considered.
Source and conditions of the donor/receptor cells
Regarding the source of cells for cell therapy (e.g. conditions of donor tissue), Varghese et al. [110] performed a systematic review of the effects of different donor-related factors on ADSC functionality. These authors analysed 41 scientific papers and found that decreases in the proliferation and differentiation potential of ADSCs are directly related to age, a high body mass index, diabetes mellitus, radiation exposure and tamoxifen use. Advanced patient age has also been related to lower angiogenesis capacity [111] and changes in morphological patterns and cellular senescence factors, such as telomere shortening and genetic instability [52, 112–119]. Therefore, these factors may play a key role in therapeutic efficacy. The use of cells from neonatal tissues, such as the placenta and umbilical cord, which have greater differentiation potential and a higher proliferation rate [71, 73, 120], represent an attractive and potentially promising therapeutic alternative for the treatment of COPD. Additionally, the secretome may vary, depending on the origin tissue of MSC donor and recipient patients [58, 59]. Another important aspect that needs to be explored, as pointed out by Glassberg et al. [60], is to identify potential markers and patient phenotypes that correlate with the clinical response of patients who undergo cell-therapy procedures. Hoang et al. [95], in a recently published consistent and comprehensive review, emphasised the impact of MSC sources as a current challenge for MSC-based therapies. A better understanding of the molecular, functional mechanism and phenotypic determinants of the regenerative process of MSCs should broaden perspectives regarding the use and efficacy of cell-based therapy [32, 33, 60, 121].
Lung structure and cell-based regenerative medicine
Lung tissue regeneration is impacted by its complex tissue structure, which is composed of many ramifications and cell types. Furthermore, COPD and emphysema are diseases with a multifactorial aetiopathogenesis. The clinical course of these diseases is impacted by interactions among environmental, epigenetic and genetic factors that are patient specific [1, 4, 74, 122]. The lung represents a particular challenge in the context of tissue engineering and regenerative and translational medicine. As proposed by Squassoni et al. [74], new studies and well-designed randomised trials with large numbers of patients that allow statistically significant results and consistently support the initiation of randomised phase III, placebo-controlled clinical trials and large national and international multicentre collaborative studies of cell therapy for COPD are needed [32].
Due to the complex factors described above, advances in our knowledge of the cell therapies for COPD have occurred slowly. However, significant advances in terms of both safety and therapeutic efficacy have been made for other inflammatory conditions, such as graft-versus-host disease [123, 124], perianal fistulas and Crohn's disease [101, 125–127]. These and other promising advances in our understanding and management of chronic inflammatory conditions open up future promising perspectives in regenerative medicine and new clinical trials of cell-based therapies for COPD.
New perspectives to be explored: acellular or cell-free therapy
Translational studies of COPD/emphysema are still scarce in the literature. There are very few clinical reports of cell-based therapies for COPD in human patients to support more consistent and comprehensive conclusions, in contrast to findings from animal models, especially with regard to efficacy. Overall, clinical findings are still insufficient to confirm the efficacy of cell-based therapy for COPD [60]. In this context, it can be proposed, in summary, that there are two major types of challenges with regard to cell-based regenerative and translational medicine in COPD, namely assessing the biological response and unequivocally demonstrating therapeutic (clinical) efficacy.
Despite a series of gaps in the knowledge of the different parameters to be better defined and the limited number of clinical trials on cell-based therapy for COPD, the results to date represent an initial body of evidence and encouraging perspectives for new and standardised prospective studies [128]. In the study by Stessuk et al. [5], one of the patients maintained a stable FEV1 after a 23-month follow-up. Le Thi Bich et al. [71] reported a clinical trial of allogenic UC-MSCs that significantly reduced the mMRC score and number of exacerbations in patients with moderate-to-severe COPD. The results of the clinical protocol by Stolk et al. [67] showed a three-fold increase in the expression of CD31+ after an LVRS+ BM-MSC infusion. The higher expression of CD31, an endothelial marker, could indicate the occurrence of angiogenesis that results in a protective effect or repair of the lung parenchyma.
A new methodological approach that is potentially very promising in regenerative medicine is the use of MSC EVs a new acellular or cell-free therapy. EVs are membrane-linked structures that are released by cells. According to their diameter and function, they can be divided into microvesicles, exosomes and apoptotic bodies. Their release is involved in the process of cellular apoptosis. However, with the exception of apoptotic bodies, EVs are also released by healthy cells. This fact has opened up new perspectives and been studied as an alternative to cell therapies. EVs can be isolated from MSCs and other sources, such as dendritic cells, lymphocytes, adipocytes, neurons, epithelial cells and endothelial cells [129, 130].
The use of EVs derived from the MSC secretome may represent a new and promising therapeutic alternative, either alone or combined with other cell-based therapies, which could represent a paradigm shift [131]. The application of MSC EVs for therapeutic purposes has shown promising results, similar to the direct application of MSCs in injured tissues, regulating some functions through the transfer and release of proteins, DNA, RNA and growth factors into the extracellular medium, possibly even promoting mitochondrial transfer [61, 131, 132]. Furthermore, EVs can participate in cell-to-cell communication between distant and local cells and as paracrine mediators, in addition to regulating the release of chemokines that regulate the cellular microenvironment and interrupt apoptotic processes, their systemic circulation can also be used as a diagnostic indicator for various diseases [32, 131, 133].
The use of EVs has been tested in several trials and pre-clinical studies. Nonetheless, there are important challenges in the clinical use of EVs. One important limitation is related to in vivo isolation because they are difficult to identify when considering the absence of specific markers. Therefore, the methodological standardisation, characterisation and isolation of EVs are necessary. Additionally, the optimal therapeutic dose needs to be defined to obtain greater efficacy and reproducibility [130, 131]. Promising results have been reported for the treatment of several chronic degenerative renal, cardiovascular, hepatic, immunological and neurological disorders [130, 134–136].
Kaspi et al. [137] verified the efficacy of MSC EVs in mice with acute respiratory distress syndrome. These results indicate the possibility of applying MSC EVs in coronavirus disease 2019 (COVID-19) cases. In the specific case of COPD, a study by Maremanda et al. [138] found protective effects of combined MSCs and MSC EVs in cigarette smoke-induced COPD in rats. The authors attributed these protective effects to anti-inflammatory actions [137, 138] and the ability of mitochondrial transfer by EVs [129, 138].
Sengupta et al. [139] conducted the first clinical trial of the autologous application of BM-MSC EVs in 24 COVID-19 patients. They found a reversal of hypoxia, immune reconstitution and the downregulation of proinflammatory cytokines. A pilot clinical study by Zhu et al. [135] showed the efficacy of exosomes isolated from AD-MSCs in severe pneumonia that was caused by COVID-19. Allogenic exosomes were administered by inhalation for five consecutive days in seven patients with severe COVID-19. There was resolution of lesions and no evidence of adverse effects during or after inhalation with the nebuliser [128, 135].
In the specific case of regenerative medicine in pre-clinical models of lung diseases, the intratracheal use of MSC-EVs showed therapeutic efficacy in acute injury caused by endotoxins in the lungs of mice. There was a decrease in the release of proinflammatory cytokines by neutrophils and macrophages and a delay in lesion progression [134, 135]. The intravenous application of MSC-EVs 4 h after the inoculation of bacteria in mice improved survival and attenuated acute lesions [134]. The anti-inflammatory activity of MSC-EVs has also been demonstrated in porcine models, in addition to anti-influenza effects [136].
As noted by Wang et al. [140], previous results provide promising evidence of the use of EVs to treat several infectious and chronic degenerative diseases [135]. EVs represent a promising treatment alternative for diseases that have inflammatory aspects, such as COPD [139], specifically when considering the specific characteristics of these vesicles, such as their role in immunomodulation and tissue regeneration [134, 140, 141].
Overall, EVs are a promising therapeutic alternative, either alone or combined with other cellular or acellular therapies, but many more studies are needed to overcome their limitations and more accurately elucidate their mechanism of action.
Points for clinical practice and questions for future research
Despite significant advances in therapeutic approaches, an effective clinical treatment has not yet been achieved for COPD.
In this scenario, cell-based therapies have arisen as potentially promising new therapeutic approaches for COPD.
The clinical results available are still insufficient to confirm the efficacy of cell-based therapy for COPD.
The biggest problems in cell-based regenerative medicine in COPD involve methodological aspects and protocol standardisation.
It is crucial that new studies and well-designed randomised trials are developed with large numbers of patients and large national and international multicentre collaborative studies.
Despite notable advances, a large number of potentially promising results and well-conducted studies with regard to the safety of MSC-based therapies, there is still no broad consolidation of clinical efficacy.
Conclusion
As extensively discussed in this review, there are still many unanswered questions and unresolved issues, many of which will likely be addressed by current and future clinical trials. Many issues with cell therapies for COPD involve, among other factors, methodological aspects, protocol standardisation, donor/host factors, as well as a better understanding of the mechanism of action underlying the therapeutic effect and unequivocal demonstration of clinical efficacy. There is still a long way to go before cell-based regenerative medicine can be incorporated as a well-established routine therapeutic approach for COPD. In this context, as proposed by Tzouvelkis et al [142], we need to “separate the hope from the hype” when we inform patients about the real state of the art of cell-based therapy for COPD, such that we do not give patients false hope for a cure while not depriving them of the hope of improving their quality of life.
Footnotes
Provenance: Submitted article, peer reviewed.
Author contributions: J.T. Ribeiro-Paes conceived and designed the study. L.P. Guarnier, L.G. Moro, F.A.d.R. Livero and J.T. Ribeiro-Paes undertook formal analysis, review and editing the text. E.J. Sekiya, C.A. de Faria; M.F. Azevedo; B.P. Roma; M.J. Malagutti-Ferreira; E.R. Albuquerque; A.G.D. Rodrigues and A.A. da Silva undertook literature review, editing and critical analysis of the text. All authors contributed to the final version of the manuscript.
Conflict of interest: The authors declare no conflicts of interest.
Support statement: L.P. Guarnier was financially supported by the Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES – Brazil) and L.G. Moro was financially supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Brazil).
- Received November 14, 2022.
- Accepted May 23, 2023.
- Copyright ©The authors 2023
This version is distributed under the terms of the Creative Commons Attribution Non-Commercial Licence 4.0. For commercial reproduction rights and permissions contact permissions{at}ersnet.org
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