Advances in Stem Cell and Cell-Based Gene Therapy Approaches for Experimental Acute Lung Injury: A Review of Preclinical Studies

Abstract

Given the failure of pharmacological interventions in acute respiratory distress syndrome (ARDS), researchers have been actively pursuing novel strategies to treat this devastating, life-threatening condition commonly seen in the intensive care unit. There has been considerable research on harnessing the reparative properties of stem and progenitor cells to develop more effective therapeutic approaches for respiratory diseases with limited treatment options, such as ARDS. This review discusses the preclinical literature on the use of stem and progenitor cell therapy and cell-based gene therapy for the treatment of preclinical animal models of acute lung injury (ALI). A variety of cell types that have been used in preclinical models of ALI, such as mesenchymal stem cells, endothelial progenitor cells, and induced pluripotent stem cells, were evaluated. At present, two phase I trials have been completed and one phase I/II clinical trial is well underway in order to translate the therapeutic benefit gleaned from preclinical studies in complex animal models of ALI to patients with ARDS, paving the way for what could potentially develop into transformative therapy for critically ill patients. As we await the results of these early cell therapy trials, future success of stem cell therapy for ARDS will depend on selection of the most appropriate cell type, route and timing of cell delivery, enhancing effectiveness of cells (i.e., potency), and potentially combining beneficial cells and genes (cell-based gene therapy) to maximize therapeutic efficacy. The experimental models and scientific methods exploited to date have provided researchers with invaluable knowledge that will be leveraged to engineer cells with enhanced therapeutic capabilities for use in the next generation of clinical trials.

Acute Lung Injury and Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a devastating complication of severe acute lung injury (ALI), and a significant cause of morbidity and mortality in critically ill patients.^1,2 ARDS is characterized by bilateral infiltrates on chest radiograph, no sign of left ventricular failure, and hypoxemia (defined by the ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen [PaO₂:FiO₂] ≤ 300 mmHg).³ Whereas some patients with ARDS may recover within 1 to 2 weeks, others suffer a lengthy clinical course with all the attendant complications of prolonged mechanical ventilation. Many survivors of ARDS are left with profound muscle weakness,⁴ and some develop persistent pulmonary limitation with compromised diffusion capacity⁵ in addition to reduced health-related quality of life.^6,7

ARDS can be caused by a direct insult to the lung epithelium, known as pulmonary ARDS, or by indirect lung injury caused by a systemic inflammatory process, known as extrapulmonary ARDS.⁸ Infectious etiologies, such as sepsis and pneumonia, are among the leading causes of ALI/ARDS.⁹ Most notably, sepsis, a condition characterized by a combination of infection and systemic inflammatory response, has the highest risk of progression to ARDS.¹⁰ ARDS is a relatively common and potentially lethal clinical syndrome, with an incidence as high as 80 cases per 100,000 population per year.¹¹ One report estimated that there are approximately 190,600 cases of ALI per year in the United States, with 74,500 reported deaths annually.² The mean intensive care unit (ICU) cost for a patient with ARDS was $97,810 in Canadian dollars (in 2002),¹² with similar costs reported for patients in Finland¹³ and the United States.¹⁴ Moreover, post-acute care of survivors is estimated to be as high as $3.5 million per one independently functioning survivor (alive with no functional dependency) at 1 year post-ICU discharge.⁷

Pathogenesis of Ali/ards

In general, ALI/ARDS is characterized by a severe acute inflammatory response in the lungs and neutrophilic alveolitis.¹ The physiological hallmark of ARDS is disruption of the alveolar–capillary barrier (i.e., pulmonary vascular leak), leading to the development of noncardiogenic pulmonary edema, in which a fibrin-rich proteinaceous exudate floods the alveolar spaces, impairs gas exchange, and precipitates respiratory failure.¹⁵ The physiological structure of the alveolar–capillary barrier is composed of both the alveolar epithelium and microvascular endothelium. Both alveolar epithelial cell and microvascular endothelial cell (EC) injury and/or death have been implicated in the pathogenesis of ALI/ARDS.¹ In addition, other features such as intraalveolar hemorrhage and fibrin deposition, cellular debris, and formation of hyaline membranes can be observed in ALI/ARDS after 3 to 7 days.

Animal Models Frequently Used to Study Ali/ards

Various experimental animal models have been used to investigate the mechanism and pathophysiology of ALI. Most rely on ways to simulate known clinical causes of ARDS, such as sepsis (or peritonitis), multiple transfusions, acid aspiration (aspiration of gastric contents), trauma, and ischemia–reperfusion of distal or pulmonary vasculature.¹⁶ Although none of these animal models can fully reproduce all the features of human ARDS, most exhibit some of the key physiological and pathological features of human ARDS, and are useful for the testing of hypotheses and novel therapeutic interventions.

Inflammatory stimuli from microbial pathogens, such as endotoxin (lipopolysaccharide [LPS]), are well recognized to induce pulmonary inflammation. LPS is the main component of the gram-negative bacterial wall and binds to the CD14/TLR4/MD2 receptor complex, activating transcription of a number of inflammation- and apoptosis-related genes via the transcription factor NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells). Experimental administration of LPS, either systemically or intratracheally, has been used to induce pulmonary inflammation and neutrophil recruitment in animal models of ALI.^17,18 The intravenous injection of LPS mimics some aspects of the septic response including massive systemic release of proinflammatory cytokines, with mild recruitment of neutrophils into the airspace. In contrast, direct targeting of the airway by intratracheal instillation of LPS causes large increases in neutrophils in the airspace, in addition to increased levels of albumin and proinflammatory cytokines, and subsequent apoptosis of endothelial cells. Furthermore, LPS increases the expression of surface adhesion molecules by alveolar ECs, including intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, P-selectin, and E-selectin, resulting in endothelial activation.

The LPS model of ALI has been used extensively because of its simplicity and reproducibility. As a potent activator of innate immune response, LPS activates the Toll-like receptor 4 pathway and elicits a host inflammatory response, mimicking the response to bacterial infection. However, LPS instillation alone does not reproduce the severe epithelial and endothelial damage seen in human ARDS, and therefore is only a partial model of bacteria-induced ALI.¹⁶

Oleic acid, one of the most commonly found fatty acids in mammals, represents half of the total fatty acids present in lipid pulmonary emboli, a well-recognized cause of ALI in patients after long bone trauma.¹⁹ Studies showed that oleic acid is toxic to both alveolar endothelial and epithelial cells (i.e., type I cells), inducing swelling and necrosis.²⁰ Because of the insolubility of oleic acid in water, oleic acid is usually dissolved in ethanol or emulsified in blood, before being administered via a peripheral vein, a central vein, intratracheally, or directly into the right atrium or pulmonary artery.¹⁶ In oleic acid-induced ALI, animals exhibit signs of increased microvascular permeability, elevation of extravascular lung water, accumulation of protein-rich edema within the interstitium and airspaces, hypoxemia, early systemic hypotension, and pulmonary hypertension. This model is highly reproducible and exhibits some basic characteristics of ALI.¹⁶ However, debate remains on the extent to which oleic acid-induced ALI mimics ARDS occurring in humans, because long bone trauma or lipid injury is only a rare cause of ARDS.

Even though mechanical ventilation has been used as a clinical strategy to manage patients with ARDS, large trials have now demonstrated that ventilation at a traditional tidal volume of 12–15 ml/kg (of predicted body weight) is actually detrimental, rather than beneficial, to patients with ARDS.^21,22 Mechanical ventilation (MV) at high tidal volume has been used as a method to induce ALI in animals (i.e., ventilation-induced lung injury [VILI] model), because high tidal volume MV promotes lung injury by repeatedly overstretching alveolar epithelial cells.²³ Pathological examination of lungs ventilated with a high tidal volume reveals alveolar hemorrhage, hyaline membranes, and recruitment of inflammatory cells. Patients with preexisting lung injury are even more susceptible to further insult from mechanical ventilation.²¹

Because sepsis is known as one of the most prevalent risk factors for ARDS, animal models of sepsis have been used to study ALI, including systemic or local administration of live bacteria, or systemic and local administration of a bacteria product such as LPS.¹⁶ Local administration of bacteria can result in pulmonary infection, leading to the development of pneumonia, with epithelial injury and inflammation.²⁴

Current Therapies for Ards

Mechanical ventilation is one of the most important clinical strategies in the management of ARDS. A study conducted by the ARDS Network compared traditional high tidal volume (12 ml/kg) with low tidal volume (6 ml/kg) ventilation in a large randomized clinical trial. The results of this study demonstrated that a protective ventilation strategy reduced pulmonary and systemic inflammatory responses, and resulted in an improvement in 28-day mortality.²¹ A follow-up trial by the ARDS Network investigated the potential to further improve clinical outcome, by increasing positive end-expiratory pressure (PEEP) in the setting of low tidal volume (V _t) mechanical ventilation.²⁵ No additional improvement in mortality and ventilation-free days was observed with higher levels of PEEP. More recently, Amato and colleagues showed that because respiratory system compliance (C_RS) is strongly related to the volume of remaining aerated functional lung during disease (termed functional lung size), the driving pressure (ΔP = V _t/C_RS) is a critical variable in stratifying patients at risk for ventilator-induced lung injury.²⁶ Using this premise, the group reanalyzed data from more than 3,562 patients enrolled in various ARDS trials to demonstrate that reducing ΔP significantly improved survival. These data also explain why PEEP increments do not consistently result in improved survival—this happens only when increasing PEEP changes lung mechanics in such a way as to allow the same V _t to be delivered at lower driving pressures (ΔP). In addition to protective ventilation strategies, proning has emerged as a fundamental strategy for ventilator management of patients with ARDS.²⁷

In contrast to MV strategies, pharmacological approaches to treat ALI/ARDS have largely focused on suppressing the endogenous inflammatory response either globally or via inhibition of specific mediators. Despite advances in our understanding of the biology of ALI/ARDS and the promising results of preclinical studies, the translation of basic science into a clinically useful and widely applicable strategy for therapeutic intervention has not yet occurred. The largely disappointing results from clinical trials can be explained in part by our incomplete understanding of the molecular responses to injury, the development of immunosuppressive complications, limitations of delivery systems to the lung, the role of timing and combination therapy, and the superimposed effects of MV.^{11,21,28
–30} ARDS continues to be an important contributor to prolonged mechanical ventilation in the ICU, and ARDS-associated mortality remains high at 30–50%.^1,29

Experimental Cell Therapy for Acute Lung Injury

Mesenchymal stem cells for the treatment of experimental acute lung injury

Mesenchymal stem cells (MSCs), more appropriately called mesenchymal stromal cells or marrow stromal cells, were originally described as bone marrow-derived cells with the ability to self-renew and differentiate into various tissues of mesenchymal lineage, such as bone, muscle, fat, and even vascular tissue.^31

–34 Their relative ease of isolation, ability to be expanded to large numbers ex vivo, and genetic stability make them a robust cell type that can be employed in a variety of applications, including in the treatment of cardiovascular diseases, pulmonary fibrosis, spinal cord injury, bone repair, and cartilage repair.³⁵ MSCs play a role in vascular repair by stimulating angiogenesis. Historically, MSCs were thought to arise from the bone marrow and to home to sites of injury, to differentiate into cells of the residing tissue, and to engraft into these tissues to restore function.^31,32 However, more recently it has been appreciated that MSCs are present in all tissues of the body, in particular adipose tissue, umbilical cord, placenta, lung, heart, and many others.³⁶ It has been suggested that MSCs may represent a subpopulation of vascular pericytes that play key roles in vascular homeostasis and repair.³⁷ Moreover, MSCs lack class II major histocompatibility complex (MHC) antigens and express few or no costimulatory molecules, which allow them to be used in allogeneic transplantation with a low risk of immune rejection.^38
–40 Therefore, it is no surprise that MSCs are one of the most widely studied stem and progenitor cell types for cell therapy to date^41,42 (Table 1).

Table 1.

Mesenchymal stem and progenitor cell therapy for acute lung injury in animal models

Cell type, source	Cell dose, route of administration, timing	Animal species	Reference	Model
MSC, mouse BM	2.5 × 10⁵, IV, 1/2 hr post	Mouse	Mei et al (2007)⁴⁸	LPS (IT)
MSC, mouse BM	5 × 10⁵, IV, 1 hr post	Mouse	Xu et al (2007)⁴⁹	LPS (IP)
MSC, mouse BM	7.5 × 10⁵, IT, 4 hr post	Mouse	Gupta et al (2007)⁴⁷	LPS (IT)
MSC,^a mouse BM	Unknown, IV, 2 hr post	Mouse	Xu et al (2008)⁹¹	LPS (NB)
MSC, human BM	10⁶, IT, 4 hr post	Mouse	Krasnodembskaya et al (2010)⁵⁴	E. coli (IT)
MSC, rat BM	10⁶, IV, 2 hr post	Rat	Liang et al (2011)⁷⁰	LPS (IV)
MSC, human UC	10⁶, IT, 4 hr post	Mouse	Sun et al (2011)⁵⁹	LPS (IT)
MSC, human UCB	10⁵, IT, 3 hr post	Mouse	Kim et al (2011)⁶¹	E. coli (IT)
MSC, human BM	5 × 10⁵, OA, 4 hr post	Mouse	Danchuk et al (2011)⁶⁸	LPS (OA)
MSC, human UC	5 × 10⁵, IV, 4 hr post	Rat	Li et al (2012)⁶⁰	LPS (IP)
MSC, mouse BM	7.5 × 10⁵, IT, 4 hr post	Mouse	Gupta et al (2012)⁵⁵	E. coli (IT)
MSC, rat BM	10⁶, intrapleural, immediately	Rat	Qin et al (2011)⁷¹	LPS (IT)
MSC, human OF	3 × 10⁵, IV, 1/3 hr post	Mouse	Chien et al (2012)⁶²	LPS (IT)
MSC, rat BM	2 × 10⁶, IV, 1/6 hr post	Rat	Xu et al (2012)⁵⁷	Oleic acid (IV)
MSC, mouse BM	5 × 10⁶, IV, 1 hr post	Mouse	Tai et al (2012)⁷²	LPS (IT)
MSC, mouse BM	2.5 × 10⁵, IT, 4 hr post	Mouse	Ionescu et al (2012)⁶⁹	LPS (IT)
MSC, mouse BM	10⁵, IV, 24 hr post	Mouse	Maron-Gutierrez et al (2013)⁹⁸	LPS (IT or IP)
MSC, rat BM	4 × 10⁶, IT or IV, 2.5–3 hr post	Rat	Curley et al (2013)⁵⁸	Ventilation
MSC, human adipose	10⁶, IV, 6 hr post	Mouse	Martínez-González et al (2013)⁶³	LPS (IT)
MSC, mouse BM	5 × 10⁵, IV, 2 hr post	Mouse	Chen et al (2013)⁹⁴	LPS (IT)
MSC, mouse BM	5 × 10⁵, IV, 24 hr pre or 12 hr post	Mouse	Shalaby et al (2014)⁵⁶	E. coli (IT)
MSC, mouse BM	5 × 10⁵, IV, 1 hr post	Mouse	Zhao et al (2014)⁹⁵	LPS (IP)
MSC, human BM	5 or 10 × 10⁶/kg, IV, 1 hr post	Sheep	Asmussen et al (2014)⁶⁷	Cotton smoke + P. aeruginosa (IT)
MSC, mouse BM	5 × 10⁵, IV, 4 hr post	Mouse	He et al (2015)⁹³	LPS (IT)
MSC, mouse BM	5 × 10⁶, IV, 1 hr post	Mouse	Zhao et al (2015)⁹⁹	LPS (IP)

No improvement was seen.

Note: Except where indicated, improvements were seen in acute lung injury. Only studies conducted with a therapeutic scenario examined in animal disease models have been included here.

BM, bone marrow; IP, intraperitoneal; IT, intratracheal; IV, intravenous; LPS, lipopolysaccharide; MSC, mesenchymal stem cell; NB, nebulization; OA, oropharyngeal aspiration; OF, orbital fat; UC, umbilical cord; UCB, umbilical cord blood.

It has become increasingly apparent that MSCs also have an ability to modulate the immune response in the host, and this may be an important mechanism for their efficacy in a variety of inflammatory conditions.⁴³ There is an extensive body of literature suggesting that MSCs can interact with various types of immune cells, including but not limited to T cells, macrophages, B cells, dendritic cells, and nature killer cells.⁴⁴ Proliferation of T cells can be suppressed by MSCs, which have been activated by proinflammatory cytokines such as interferon (IFN)-γ and/or tumor necrosis factor (TNF)-α. The suppressive activity is mediated by the generation of more nitric oxide (NO) via upregulation of inducible nitric oxide synthase in mouse MSCs, or more indoleamine-2,3-dioxygenase (IDO) in the case of human MSCs. Both in vitro and in vivo studies have demonstrated that MSCs can increase the number of T regulatory cells, which are a subset of T cells that maintain tolerance to self-antigen and prevent autoimmunity, mainly through cell–cell interaction or with factors such as transforming growth factor (TGF)-β or prostaglandin E₂ (PGE₂). These immune-modulatory properties, specifically the ability of MSCs to inhibit T cell proliferation or to induce IDO activities, have since been used widely by researchers as a strategy to assess MSC potency in vitro. For further details on how MSCs may interact with various immune cells to modulate the host immune response the reader is referred to excellent review articles by Gebler and colleagues⁴⁵ and Shi and colleagues.⁴⁶

In 2007, three research groups independently reported therapeutic benefit of bone marrow-derived MSCs in experimental models of acute lung injury^47
–49 (Table 1). Mei and colleagues showed that bone marrow-derived MSCs reduced ALI when administered into the pulmonary circulation (cells delivered via jugular vein) in an LPS-injured mouse lung, possibly by reducing LPS-induced cellular and humoral inflammation in the host. Using a systemic LPS-induced ALI model (by injection of LPS intraperitoneally), Xu and colleagues reported that MSCs prevented LPS-induced edema and neutrophil recruitment in the airways (although MSCs had no effect on cytokine level measured in bronchoalveolar lavage fluid). They isolated MSCs by culturing bone marrow, followed by immunodepletion of macrophages (CD11b⁺ cells) and hematopoietic cells (CD45⁺ cells) from the plastic-adherent culture.⁴⁹ MSCs were infused intravenously to each mouse one hour after the LPS injury. In a report by Gupta and colleagues,⁴⁷ MSCs were administered directly into the airspaces 4 hr after intratracheal instillation of LPS. Administration of MSCs increased survival, decreased pulmonary edema and systemic/pulmonary inflammation, and increased antiinflammatory cytokine IL-10 levels compared with untreated mice. This study found that MSCs inhibited the production of TNF-α by LPS-stimulated alveolar macrophages in a contact-independent manner.

MSCs express a range of damage- and pattern-associated molecular pathogen receptors, such as the Toll-like receptors, a property of MSCs that may play an important role in their immunomodulatory actions. MSCs are postulated to sample the ambient environment and react to inflammatory stimuli with release of a spectrum of antiinflammatory molecules including antiinflammatory peptides, antibacterial peptides, extracellular vesicles (including exosomes and microvesicle [MV] particles; for extensive review on how extracellular vesicles derived from MSCs may contribute to benefit seen in cell therapy studies, the reader is referred to an excellent review article by Rani et al.⁵⁰) containing inhibitory microRNAs, lipid mediators (such as lipoxins⁵¹), and other agents.⁵² One report even suggested that part of the ability of MSCs to repair injured airway epithelium could be contributed by the transferral of MSC mitochondria, which resulted in increased ATP levels in host alveolar cells.⁵³ Overall, most MSC therapy reports used ALI models induced by LPS administration (either direct ALI with intratracheal or nebulization delivery of LPS, or indirect ALI with intraperitoneal injection of LPS). However, similar results were also documented using other models of lung injury including ALI induced by live bacteria (Escherichia coli),^54
–56 oleic acid,⁵⁷ or injurious mechanical ventilation.⁵⁸

Although bone marrow-derived MSCs have been used in the majority of reported studies (Table 1), promising results have also been reported using MSCs derived from alternative sources, such as umbilical cord,^59,60 umbilical cord blood,⁶¹ orbital fat,⁶² or adipose tissue.⁶³ It has been suggested that MSCs represent a subset of pericytes, mural cells that surround small blood vessels in the bone marrow as well as many other organs,^64,65 and contribute to vascular homeostasis and tissue repair after injury. Interestingly, preclinical studies using xenotransplantation of human MSCs in experimental ALI in immune-competent animal models still demonstrated therapeutic benefit.^{54,59

–63,66
–68} This finding suggests that MSCs may enjoy a remarkable degree of immune privilege, and avoid triggering acute immune responses even across species. In addition, different delivery routes to administer MSCs have also been used in studies listed in Table 1, including intravenous delivery via jugular or tail vein, intraperitoneal injection, intratracheal instillation, and nebulization.

Because almost all published studies of preclinical cell therapy have reported low levels of MSC persistence in the treated animals, it is widely believed MSCs most likely exert their therapeutic benefit via their secreted factors through paracrine, immune-modulatory mechanism(s). To examine this hypothesis, Ionescu and colleagues conducted an elegant study, in which they used not only MSCs but also conditioned medium generated from MSCs to treat LPS-induced ALI in mice.⁶⁹ The authors were able to show that conditioned medium alone (concentrated from an equivalent cell dose) was almost as effective as using the MSCs in rescuing ALI. Insulin-like growth factor (IGF)-I was identified from the MSC-conditioned medium, and implicated as partly responsible for promoting the switching of the host alveolar macrophages to an M2 antiinflammatory phenotype, which promoted healing and attenuation of lung inflammation in animals suffering from ALI. By using techniques such as RNA interference to knock down expression of the putative factor expressed by MSCs, Danchuk and colleagues identified TNF-α-induced protein 6 (TSG-6) as one of the released factors that is partially responsible for the observed therapeutic benefit of MSC treatment.⁶⁸ The antiinflammatory cytokine IL-10 has also been reported by several studies as one of the main contributors to the antiinflammatory properties of MSCs.^{47,57,63,66,70
–72} Using a live E. coli-induced pneumonia model, Krasnodembskaya and colleagues showed that MSCs delivered into the airway can reduce bacterial growth in the lungs (demonstrated via bacteria colony assay using lung homogenates and bronchoalveolar lavage fluid).⁵⁴ These authors further identified an antimicrobial peptide, LL-37, that is secreted by MSCs and responsible for the observed antimicrobial activity by MSCs in their study. This result is consistent with another study that showed a similar antimicrobial concept of MSC action with a different animal model of bacterial infection: through the cecal ligation and puncture (CLP)-induced model of sepsis, a study by Mei and colleagues demonstrated that MSCs possess the ability to modulate the host response to fight bacterial infection,⁷³ potentially through upregulation of genes responsible for promoting phagocytosis and killing of bacteria.^73,74

Endothelial progenitor cells for the treatment of experimental acute lung injury

First reported by Asahara and colleagues in 1997, endothelial progenitor cells (EPCs), representing precursor cells of hematopoietic and vascular systems, were described as a population of circulating bone marrow-derived cells that contribute to in vivo postnatal vasculogenesis.⁷⁵ Transplanted EPCs have been shown to localize within small blood vessels and to improve circulation to sites of ischemic injury in animal models of hind-limb ischemia. Current literature suggests that there are two main phenotypes of EPCs. The first are called early outgrowth EPCs (or circulating angiogenic cells), which have a spindle-shaped morphology in culture and exhibit potent proangiogenic activity, but neither proliferate nor survive past a few weeks.⁷⁶ The second are late outgrowth EPCs, which emerge as a highly proliferative subpopulation of cells from prolonged culture of mononuclear cells, show a typical endothelial cobblestone appearance, and express mature endothelial surface markers.^77,78

Given the putative role of circulating EPCs in repairing endothelium after vascular injury, several studies examined the benefit of using EPCs derived from various sources (peripheral blood, bone marrow, or umbilical cord blood) or various species (rabbit, rat, or human) in ALI models (Table 2). The cells used in these studies were mostly early outgrowth EPCs^79
–81 (except for one study by Yin and colleagues⁸²) and had all been characterized for their ability to incorporate acetyl-low density lipoprotein (acetyl-LDL)^79

–82 and binding of lectin.^79,80,82 All showed that the administration of EPCs after the induction of lung injury (by LPS or oleic acid) was effective in reducing lung water and lung injury scores, potentially via the ability of EPCs to differentiate into endothelial cells and/or to repair function and integrity of the alveolar–capillary barrier. Furthermore, some even showed improvement of PaO₂ (arterial blood gas)^79,82 and survival⁸¹ with EPC treatment, suggesting therapy targeting repair of damaged endothelium can lead to positive physiological outcomes.

Table 2.

Endothelial progenitor cell therapy for acute lung injury in animal models

Cell type, source	Cell dose, route of administration, timing	Animal species	Reference	Model
EPC, rabbit PB	10⁵, IV, 1/2 hr post	Rabbit	Lam et al (2008)⁸⁰	Oleic acid (IV)
EPC, rat BM	5 × 10⁶, IV, 1/2 hr post	Rat	Mao et al (2010)⁸¹	LPS (IV)
EPC, rabbit PB	10⁶, IV, 4 hr post	Rabbit	Cao et al (2012)⁷⁹	LPS (IV)
EPC, rat BM	2 × 10⁶, IV, 1/2 hr post	Rat	Yin et al (2015)⁸²	LPS (IT)

Only studies conducted with a therapeutic scenario examined in disease animal disease models have been included here.

BM, bone marrow; EPC, endothelial progenitor cell; IT, intratracheal; IV, intravenous; LPS, lipopolysaccharide; PB, peripheral blood.

Other stemlike or progenitor cells for the treatment of experimental acute lung injury

Other types of stemlike or progenitor cells have also been studied in preclinical ALI models (Table 3). In a study by Araújo and colleagues, mononuclear cells (MNCs) were isolated from mouse bone marrow via Ficoll density separation and injected directly, without culture, into two different models of ALI mice (a “pulmonary” ALI model induced by direct intratracheal LPS, or an “extrapulmonary” ALI model induced by indirect intraperitoneal LPS).⁸³ The study was designed to investigate the potential of using a simple-to-isolate and ready-to-use cell product for the treatment of patients with ARDS, who typically require immediate intervention after being admitted to a hospital ICU. Encouragingly, results showed that MNCs can reduce lung static elastance (lung mechanics), collagen fiber content, lung inflammation, and ultimately ALI-induced mortality, with more improvement seen in the extrapulmonary ALI model. In a large-animal model of ALI (intravenous infusion of LPS in pigs), Rojas and colleagues showed that BM-derived MNCs, either as an unselected cell population or a CD45^– BM cell population (no Ficoll density separation, with or without being further expanded in culture), reduced LPS-induced acute pulmonary hypertension, edema, hypoxemia, and systemic inflammation (levels of IL-1β and TNF-α).⁸⁴ However, the same group showed that an MNC product isolated from peripheral blood by Ficoll density centrifugation, or the CD45⁺ BM cell fraction, were both ineffective in the ALI model. In a separate study, Huang and colleagues demonstrated the therapeutic and survival benefit of CD34⁺ cells (freshly isolated from human umbilical cord blood using immunomagnetic beads conjugated to an antibody against CD34), providing evidence of the potential of cord blood progenitor cells for vascular repair in diseases such as ARDS.⁸⁵ Finally, other stemlike cells such as those isolated from orbital fat⁶⁶ and even induced pluripotent stem cells (iPSCs)⁸⁶ have also been investigated in preclinical models of ALI, further suggesting there may be alternative sources of cells with the potential to treat ARDS in patients.

Table 3.

Other stemlike and progenitor cell therapy for acute lung injury in animal models

Cell type, source	Cell dose, route of administration, timing	Animal species	Reference	Model
BMPC, mouse BM	3 × 10⁵, IV, 6 hr post	Mouse	Zhao et al (2009)¹⁰⁰	LPS (IP)
MNC, mouse BM	2 × 10⁶, IT, 6 hr post	Mouse	Araújo et al (2010)⁸³	LPS (IT or IP)
iPSC, mouse FB	2 × 10⁶, IV, 4 hr post	Mouse	Yang et al (2011)⁸⁶	LPS (IT)
ASC, human or mouse adipose	7.5 × 10⁵, OA, 4 hr post	Mouse	Zhang et al (2013)⁶⁶	LPS (IT)
MNC, pig BM	100 × 10⁶, IV, 1/4 hr post	Pig	Rojas et al (2013)⁸⁴	LPS (IV)
CD34⁺ cells, human UCB	2 × 10⁵, RO, 3 hr post	Mouse	Huang et al (2014)⁸⁵	LPS (IP)

Only studies conducted with a therapeutic scenario examined in animal disease models have been included in this table.

ASC, adipose-derived stem cells; BM, bone marrow; BMPC, bone marrow progenitor cell; IP, intraperitoneal; iPSC, induced pluripotent stem cell; IT, intratracheal; IV, intravenous; LPS, lipopolysaccharide; OA, oropharyngeal aspiration; RO, retro-orbital; UCB, umbilical cord blood.

Experimental Cell-Based Gene Therapy for Acute Lung Injury

There are many advantages to using combined gene and cell therapies versus using either gene or cell therapy alone. Given the physical size of cells themselves, transfected or transduced cells carrying a therapeutic gene can be effectively filtered by the small blood vessels in the lungs, and therefore directly target the pulmonary circulation to maximize therapeutic efficiency. In addition, as discussed previously, stem or progenitor cells such as MSCs or EPCs have shown, on their own, considerable efficacy in ALI models, thereby providing the possibility of synergistic effects of cell and gene therapy. In cell-based gene therapy, autologous or allogeneic cells can be first isolated and even further expanded in vitro, before being genetically modified to express a potentially therapeutic gene and subsequently injected back into the animals or patients. Genetically modified MSCs and EPCs have been used in a number of preclinical models of pulmonary diseases, such as pulmonary arterial hypertension,^87,88 acute lung injury (Table 4), lung tumor metastasis,⁸⁹ and cystic fibrosis,⁹⁰ and even early-phase clinical trials in patients (Pulmonary Hypertension and Angiogenic Cell Therapy [PHACeT]).⁸⁸ Given that MSCs are one of the most commonly used sources of cells in preclinical ALI studies, it is not surprising that these cells are also one of the most commonly used vehicles for cell-based gene therapy (Table 4).

Table 4.

Cell-based gene therapy strategies for acute lung injury in animal models

Cell type, source	Cell dose, timing, animal species	Gene	Reference	Model
MSC, mouse BM	2.5 × 10⁵, IV, 1/2 hr post, mouse	Angiopoietin-1 (ANGPT1)	Mei et al (2007)⁴⁸	LPS (IT)
MSC, mouse BM	Unknown, IV, 2 hr post, mouse	Angiopoietin-1 (ANGPT1)	Xu et al (2008)⁹¹	LPS (NB)
MSC, human adipose	10⁶, IV, 6 hr post, mouse	Soluble IL-1 receptor-like-1 (sST2)	Martínez-González et al (2013)⁶³	LPS (IT)
MSC, mouse BM	5 × 10⁵, IV, 2 hr post, mouse	Keratinocyte growth factor (KGF)	Chen et al (2013)⁹⁴	LPS (IT)
MSC, mouse BM	5 × 10⁵, IV, 1 hr post, mouse	Developmental endothelial locus-1 (Del-1)	Zhao et al (2014)⁹⁵	LPS (IP)
MSC, mouse BM	5 × 10⁵, IV, 4 hr post, mouse	Angiotensin-converting enzyme 2 (ACE-2)	He et al (2015)⁹³	LPS (IT)
MSC, mouse BM	5 × 10⁶, IV, 1 hr post, mouse	Basic fibroblast growth factor (FGF2)	Zhao et al (2015)⁹⁹	LPS (IP)
EPC, rat BM	2 × 10⁶, IV, 1/2 hr post, rat	Bone morphogenetic protein 2 (BMP2)	Yin et al (2015)⁸²	LPS (IT)

Only studies conducted with a therapeutic scenario examined in animal disease models have been included here.

IP, intraperitoneal; IT, intratracheal; IV, intravenous; LPS, lipopolysaccharide; MSC, mesenchymal stem cell; NB, nebulization.

The earliest cell and gene therapy studies for ALI used angiopoietin-1 (ANGPT1) as a therapeutic transgene.^48,91 ANGPT1 is a glycoprotein ligand for the TIE2 receptor tyrosine kinase. In addition to its roles in angiogenesis, ANGPT1 has well-known homeostatic functions, maintaining the normal quiescent phenotype of vascular ECs, protecting against vascular inflammation, reducing permeability, and promoting EC survival.⁹² Using a plasmid encoding ANGPT1, Mei and colleagues examined the potential therapeutic role of MSCs alone or together with ANGPT1 in mice with LPS-induced ALI.⁴⁸ They demonstrated that the administration of ANGPT1-transfected MSCs enhanced the immunomodulatory action of MSCs alone and provided additional antipermeability effects, resulting in nearly complete abrogation of the ALI phenotype. Similarly, Xu and colleagues studied the effect of MSCs alone, or an ANGPT1 lentiviral vector alone, compared with MSCs transduced with lentiviral ANGPT1 in the LPS–nebulization mouse model.⁹¹ Although no significant effect was observed with either lentiviral ANGPT1 or MSCs alone, a significant, albeit modest, benefit was seen with MSCs transduced with lentiviral ANGPT1.

In addition to angiopoietin-1, other genes targeting permeability and inflammation have also been shown to enhance the existing benefit of stem or progenitor cells to treat ALI. Martínez-González and colleagues studied the benefit of adipose tissue-derived MSCs (hASCs) overexpressing soluble IL-1 receptor-like-1 (sST2, a decoy receptor for cytokine IL-33) in a mouse LPS-induced ALI model, because the interaction of IL-33 and its natural receptor ST2 had been previously linked to the induction and amplification of helper T cell type 1 (Th1) and Th2 immune responses in lungs.⁶³ The renin–angiotensin system (RAS) has also been implicated in the pathogenesis of ALI, and increased level of angiotensin (Ang) II can initiate lung inflammatory responses and damage the pulmonary endothelial barrier. Thus, He and colleagues used angiotensin-converting enzyme 2 (ACE2), which is known to convert the proinflammatory Ang II into the antiinflammatory Ang 1–7 peptide, to lessen the detrimental effects of Ang II in ALI models.⁹³ Yin and colleagues overexpressed bone morphogenetic protein 2 (BMP2), which is a member of the transforming growth factor-β superfamily and is involved in endothelial cell survival and barrier integrity.⁸² Similarly, Chen and colleagues overexpressed keratinocyte growth factor in MSCs (MSCs-KGF) to protect against pulmonary epithelial cell damage during ALI.⁹⁴ KGF is a potent factor that stimulates the proliferation of alveolar type II (ATII) cells, which can differentiate into alveolar type I (ATI) cells as a compensatory response to the loss of ATI cells during lung injury. To reduce inflammation, Zhao and colleagues overexpressed a factor called developmental endothelial locus-1 (Del-1), which plays a role in inflammatory cell migration and infiltration by inhibiting the receptor called major leukocyte adhesion receptor (LFA-1), and therefore mitigating leukocyte–endothelium adhesion.⁹⁵ MSCs transfected with Del-1 reduced LPS-induced ALI, specifically by decreasing neutrophil recruitment to the airway and lung injury scores. Overall, these studies demonstrated that combining a lung injury-reparative gene with stem/progenitor cells could provide synergistic benefit to treating ALI. However, whether the enhanced therapeutic benefit seen was directly contributed by the transfected gene, indirectly by the change in the secretion profile of soluble factors of the transfected cells, or both, would require further investigation.

Clinical Translation and Future Directions

On the basis of encouraging preclinical results of MSC therapy for ALI, two research teams have proceeded to early-phase clinical testing of stem cell therapy for patients with ARDS. In 2014, Zheng and colleagues reported the results of the first clinical trial that assessed adipose-derived MSCs in ARDS (NCT01902082).⁹⁶ This was a single-center, randomized, double-blind, and placebo-controlled phase I trial. The primary goal was to evaluate the safety and feasibility of intravenously infused, fresh allogeneic MSCs. Cryopreserved cells were thawed and cultured for 24–48 hours in the patient's own serum before infusion, and delivered at a dose of 1 × 10⁶ cells/kg body weight (cells were suspended in 100 ml of normal saline before infusion). Although no significant benefit of MSC therapy was seen, there was no infusion-associated toxicity or serious adverse events reported related to MSC administration. The STem cells for ARDS Treatment (START) trial, a multicenter, open-label, and dose-escalation phase I clinical trial (NCT01775774),⁹⁷ used freshly thawed, allogeneic bone marrow-derived MSCs, at a dose of 1 × 10⁶, 5 × 10⁶, or 10 × 10⁶ cells/kg predicted body weight (PBW). In contrast to the trial conducted by Zheng and colleagues, in which freshly cultured MSCs were used, the freshly thawed MSCs used in the START trial were further washed to remove dimethyl sulfoxide (DMSO) before being resuspended in 100 ml (regardless of cell dose) of Plasmalyte-A. MSCs were infused intravenously in nine patients with modest to severe ARDS, and although no MSC infusion-associated adverse events were observed, three patients experienced serious adverse events during the subsequent weeks after the cell infusion. However, all these events were deemed unrelated to the MSC therapy by a scientific review committee and data safety monitoring board. Some favorable physiological changes were also observed in patients who had received the highest dose panel of MSCs (10 × 10⁶ cells/kg PBW), including improvements in lung injury score and sequential organ failure assessment score, although none were statistically significant (compared with patients who had received lower doses of MSCs). With the favorable safety profile of MSC infusion seen in this phase I trial, a randomized, double-blind, placebo-controlled phase II START trial has since been initiated, in which a target of 60 patients with moderate to severe ARDS will receive 10 × 10⁶ MSCs/kg PBW.

In conclusion, there is great enthusiasm in the field to continue the journey to further explore the therapeutic potential of stem and progenitor cell therapy in ARDS, which continues to be a significant health burden not only to the patients themselves but to society as a whole. There are still a number of challenges before cell-based therapies can be translated into successful therapeutics in acute care settings. These include determining the best cell type, dosing, route, and timing of treatment administration; optimizing cell potency with preconditioning strategies and/or genetic modification; and improving methods to efficiently recruit cells to the lungs to maximize efficacy. Ultimately, these critical issues will need to be addressed in order to promote protection and optimal repair of injury in ARDS.

Footnotes

Acknowledgment

The authors thank Adrienne Szalamin for proofreading the manuscript.

Author Disclosure

D.J.S. owns a patent through Northern Therapeutics and stock options in United Therapeutics. S.H.J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.C.d.S. has no competing financial interests to disclose.

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