Using Cell-Based Strategies to Break the Link between Bronchopulmonary Dysplasia and the Development of Chronic Lung Disease in Later Life

Bronchopulmonary dysplasia (BPD) is the chronic lung disease of prematurity that affects very preterm infants. Although advances in perinatal care have changed the course of lung injury and enabled the survival of infants born as early as 23-24 weeks of gestation, BPD still remains a common complication of extreme prematurity, and there is no specific treatment for it. Furthermore, children, adolescents, and adults who were born very preterm and developed BPD have an increased risk of persistent lung dysfunction, including early-onset emphysema. Therefore, it is possible that early-life pulmonary insults, such as extreme prematurity and BPD, may increase the risk of COPD later in life, especially if exposed to secondary challenges such as respiratory infections and/or smoking. Recent advances in our understanding of stem/progenitor cells and their potential to repair damaged organs offer the possibility of cell-based treatments for neonatal and adult lung injuries. This paper summarizes the long-term pulmonary outcomes of preterm birth and BPD and discusses the recent advances of cell-based therapies for lung diseases, with a particular focus on BPD and COPD.


Introduction
Intrauterine and early postnatal environments have been shown to play an in�uential role in the development and maturation of the lung [1]. Suboptimal conditions that interfere with normal development may result in altered lung structure and function and increase the risk for disease later in life. Alarmingly, the onset of adult lung disease following inadequate development and maturation is becoming apparent at an early age. Recently, Wong and colleagues [2] showed that survivors of moderate-severe bronchopulmonary dysplasia (BPD) presented with emphysema in early adulthood (17-33 years of age). Understanding how the fetus and developing lung responds to intrauterine alterations and adapts to the postnatal environment can teach us about basic biology and the implications for adult lung diseases [3,4].

Early Life Origins of BPD
Development of the lung throughout gestation is a vital process required for adequate fetal to neonatal transition at birth. As the fetal lung proceeds through its developmental stages in utero, it becomes progressively prepared for exposure to the external environment. Successful transition to ex utero life at birth is dependent upon the ability of the lungs to effectively function as an organ of gas exchange. Indeed, organs of the developing fetus and newborn infant are extremely plastic and are particularly vulnerable to intrauterine and early postnatal environments. erefore, being born preterm with structurally immature and surfactant-de�cient lungs usually results in exposure to many environmental factors that can impact later lung development and function. It is likely that the long-term pulmonary outcomes of preterm birth Pulmonary Medicine and BPD are a result of complex programming mechanisms in�uenced by both environmental and epigenetic changes that take place during the period of development [1,5]. Many fetal and postnatal factors associated with preterm birth modulate the pathogenesis of BPD, including severity or prematurity, oxidative stress from supplemental oxygen therapy, ventilator-induced lung injury, fetal and/or postnatal infection or in�ammation, and nutrition [6,7]; therefore, identi�cation of the injurious factors contributing to the development of BPD is oen hampered by its multifactorial etiology.

Preterm Birth and BPD
Over the past few decades, advances in perinatal care have improved the outcome for infants born extremely preterm (i.e., between 24 and 28 weeks of gestation), and there is currently a rising trend in the rate of survival. However, with the shi in the limit of viability toward a lower gestational age, the task of protecting the more immature lung from injury becomes increasingly challenging. Severity of prematurity is one of the major risk factors for the development BPD [8]. Due to their immature pulmonary surfactant system, immature alveoli, and underdeveloped surface area for adequate gas exchange, almost all extremely preterm infants require prolonged respiratory support to ensure survival [6]; however, this further increases their risk of developing BPD. Furthermore, infants who were born extremely preterm have a high incidence of rehospitalization during their �rst year of life (over 40% of prematurely born infants), with the most common cause for re-hospitalization being respiratory infection [9]. It has been demonstrated by many studies that prematurely born infants who develop BPD carry a life-long risk of poor pulmonary health; this is discussed in detail later.

Short-and Long-Term Pulmonary Outcomes of Preterm Birth and BPD
Recent evidence suggests that BPD has long-term respiratory complications that reach beyond childhood into adulthood.
Numerous follow-up studies show an increased risk of impaired lung function in infancy, childhood, adolescence, and early adulthood. Few studies have investigated the longterm pulmonary outcome in adults exceeding their early 20's. is is particularly important because it is only now that large populations of very preterm subjects are reaching middleage adulthood (i.e., 35-45 years of age) and may be at risk of persistent respiratory morbidity.

Pulmonary Outcomes during Infancy.
Several studies have documented abnormal pulmonary function during infancy (up to approximately 2 years of age) following preterm birth and development of BPD [10][11][12][13][14][15]. ese studies indicate that prematurely born infants (both with and without BPD) experience reduced lung function in the �rst few months of life. Compared to term-born infants, preterm infants with BPD are more likely to be symptomatic with recurrent wheezing [14] and require re-hospitalization during the �rst 2 years of life due to acute respiratory distress and respiratory tract illness [13,15]. Functional tests show signi�cantly decreased airway function in prematurely born infants compared to term born controls, including decreased forced expiratory volume in 0.5 seconds (FEV 0.5 ) [12,14], reduced forced expiratory �ow rates (FEF 50% , FEF 75% , and FEF 25-75% ) [10,12,14], reduced functional residual capacity (FRC) [11,15], and increased lung residual volume (RV) [14]. Reduced pulmonary diffusing capacity has also been demonstrated in clinically stable infants and toddlers with chronic lung disease of prematurity, suggesting impairment of alveolar development, albeit alveolar volume appeared normal [16]. Furthermore, respiration variables of prematurely born infants have been shown to be signi�cantly altered during the �rst 2 years of life, indicated by a faster breathing frequency [11,15], greater tidal volume ( ) [11], increased amount of dead space [11], and greater minute ventilation ( ) [11]. ere are also considerable differences in compliance and resistance of the respiratory system, with preterm infants presenting with lower compliance and higher resistance compared to term-born controls [11].
Some of the childhood studies described earlier were conducted in children who were born in the presurfactant and preantenatal steroid era [13,23,30]. However, the remaining studies discussed earlier were performed in the postsurfactant and postantenatal steroid era, which indicates that even with the introduction of pre and postnatal interventions to reduce the severity of BPD, poor pulmonary outcomes are still evident in childhood, perhaps as a consequence of earlier interference with normal lung development.

Pulmonary Outcomes during Adolescence and Young
Adulthood. Poor pulmonary outcomes observed in prematurely born children throughout childhood remain prevalent into adolescence and young adulthood [17]. In comparison to the numerous extensive studies performed in ex-preterm children, there are few studies that have investigated expreterm adults older than 20 years. Recent studies of adolescents and adults have assessed pulmonary outcomes from approximately 14 to 22 years of age [32][33][34][35]. Respiratory symptoms still persist to late adolescence and early adulthood, with signi�cantly more ex-preterm subjects reporting the occurrence of cough, wheeze, and asthma compared to term-born controls [32][33][34]. Prematurely born adolescents also had a signi�cantly greater risk of being hospitalized Pulmonary Medicine 5 for respiratory problems [32]. As with pulmonary function outcomes in childhood, FEV 1 , FEF 25%−75% , and FVC all remained signi�cantly lower in late adolescence and young adulthood following preterm birth compared to control subjects [25,29,35,36]. Additionally, exercise capacity remained lower than that of controls, with subjects exhibiting signi�cantly lower maximal heart rate, faster breathing frequency, reduced O 2 max , reduced max , and shorter exercise distance in response to exercise tests [25,35]. Of particular interest is the recent evidence showing higher exhaled breath condensate 8-isoprostane levels in ex-preterm nonBPD and BPD adolescents, indicative of signi�cantly increased oxidative stress in their airways; this suggests the presence of an ongoing airway disease [36]. More alarmingly, studies are surfacing of impaired alveolar development and emphysema at adult age in former preterm infants with BPD [2,37]. Data from such follow-up studies suggest that very preterm birth, or associated factors, can permanently affect the small conducting airways and gas exchanging region and may contribute to impaired lung function observed further into adulthood.
It is important to note that the adolescent and young adulthood studies described earlier were undertaken with individuals born during the presurfactant era (birth years of the subjects ranged from 1977 to 1985), with the exception of the studies by Kotecha et al., 2012 [29], and Filippone et al., 2012 [36]. Although similar pulmonary outcomes are still observed in the postsurfactant era during childhood, further longitudinal follow-up studies are required from the present survivors of preterm birth who were treated with surfactant to gain a thorough understanding of the long-term pulmonary outcomes. To date, there are limited functional studies that have been undertaken in adolescent and young adult subjects from the postsurfactant era (i.e., infants born aer 1990). It is anticipated that in the forthcoming years, further data will become available, since preterm individuals that were the �rst to be treated with synthetic surfactant are now entering this age range.

�. �eonatal Lung �n�ur� and the �n�uence of Aging on Lung Structure and Function
It is well known that aging is associated with progressive decline in lung function, a result of many age-induced structural alterations to the lung [38,39]. Increases in the total alveolar airspace volume with enlargement of the alveolar ducts are common consequences of aging and are thought to arise from increases in the size and frequency of interalveolar pores [40]. A reduction in the supporting parenchymal tissue within the lung can result in collapse of the small conducting airways during normal breathing, especially during expiration, potentially causing gas trapping and hyperin�ation, more commonly known as "senile emphysema. " e increase in airspace size resulting from loss of supporting tissue and a decrease in elastic recoil are likely the cause of reported increases in lung compliance with aging [38,39,41]. Furthermore, aging is thought to be associated with a decline in pulmonary immune function [40], and increases in the number of immune cells in bronchoalveolar lavage �uid have been reported in the aging human population [42]. is evidence of persistent low-grade in�ammation in the respiratory tract is thought to cause oxidant-mediated injury to the lung matrix, resulting in a loss of alveoli and impaired gas exchange [42]. e recent reports of increased oxidative stress in the airways of ex-preterm nonBPD and BPD adolescents [36] in conjunction with reports of early-onset emphysema in expreterm BPD adults [2,37] suggest that preterm birth and the subsequent development of BPD may accelerate and/or exacerbate the normal age-induced pulmonary changes. erefore, it is reasonable to consider that early life pulmonary insults, such as preterm birth per se and BPD, may increase the risk of chronic obstructive pulmonary disease (COPD) later in life [43], especially if the lungs of ex-preterm BPD adults are exposed to secondary challenges such as respiratory infections and/or smoking.
Progress toward decreasing the incidence/severity of BPD over the next few years using currently available techniques and strategies is likely (i.e., optimization of antenatal management combined with surfactant and early noninvasive ventilatory support targeting lower oxygen saturations) [44]. However, currently, there is a lack of treatment for both BPD and the chronic lung disease that consequently ensues later in life. erefore, a further understanding of the mechanisms involved in lung development, injury, and repair are necessary in order to advance toward preventing lung injury and/or promoting lung development/regeneration in extremely and very prematurely born infants. Exciting discoveries in stem cell biology over recent years may offer new insight into the pathogenesis of BPD and, more importantly, open new therapeutic avenues. Consequently, as we advance in the quest to provide therapeutic stem/progenitor cell-based strategies for the prevention and repair of neonatal lung injury, our focus may need to extend to potential therapies for the ex-preterm BPD adult lung exhibiting early-onset emphysema/COPD. Indeed, BPD and COPD exhibit common therapeutic targets [3].

Therapeutic Potential of Stem/Progenitor Cells for Lung Repair/Regeneration
Recent animal and human studies suggest that damage or depletion of stem/progenitor cells in the developing lung likely contributes to the pathogenesis of both BPD and COPD (including emphysema), thus highlighting the potential of stem/progenitor cell supplementation for the prevention or repair of lung injury. e use of stem/progenitor cells is being increasingly examined in experimental animal models and provides compelling evidence for the bene�cial effects of stem cell therapy approaches for a wide variety of lung diseases [45][46][47]. Given the perturbations of the resident lung stem/progenitor cells in both BPD and COPD, the ideal therapeutic approach involves replenishing the damaged lung with healthy multipotent stem/progenitor cells that could repopulate, repair, and regenerate the injured lung. Indeed, several recent studies have demonstrated promising 6 Pulmonary Medicine outcomes using different types of stem/progenitor cell types in animal models of BPD (Table 1) and COPD (Table 2).

Lung Progenitor/Stem Cells in Health and Disease.
Observations from our lab in an oxygen-challenged neonatal rat model of BPD have shown signi�cantly reduced numbers of circulating and resident mesenchymal stem cells (MSCs) in the lung [48]. It has also been shown that the umbilical cord blood (UCB) of preterm infants yields a higher amount of endothelial colony-forming cells (ECFCs; a speci�c subset of endothelial progenitor cells, EPCs) than that of termborn infants; however, those preterm ECFCs exhibited an increased susceptibility to in vitro oxygen exposure [49].
Of particular interest is the reduced number of ECFCs in preterm infants who subsequently developed BPD compared to nonBPD preterm infants [50]. Similarly, resident lung stem/progenitor cells in the presence of COPD exhibit comparable perturbed characteristics. A recent study by Liu and Xie [51] found that the number of early outgrowth EPCs (a subset of EPCs that exhibit a spindlelike morphology in-vitro culture at 4-7 days and express the hematopoietic lineage cell markers CD34, CD133, and VEGFR-2) [52] isolated from patients with COPD was sig-ni�cantly less than that of control subjects. Furthermore, the EPCs of COPD patients exhibited reduced cluster-forming numbers and impaired cell migratory capacity, suggesting that the capacity of EPCs to repair dysfunctional endothelium is compromised in COPD [51]. ese dysfunctional characteristics of COPD EPCs were also con�rmed in a study by Kim and colleagues [53], where they observed signi�cantly lower colony-forming units and lower migratory capacity of circulating EPCs isolated from patients with emphysema. ese �ndings from BPD and COPD studies highlight the potential of stem/progenitor cell supplementation for repair/regeneration of the lung.

erapeutic Potential of Mesenchymal Stem Cells in BPD and COPD.
Of the many different types of stem/progenitor cell therapies that have been used in experimental models, MSCs appear to be the most extensively examined cell type. MSCs possess the ability to differentiate and form various mesenchymal cell types, including bone, cartilage, and adipose cells, and can be sourced from the bone marrow, UCB, umbilical cord Wharton's jelly, the placenta, and adipose tissue.
Low engrament and differentiation of these MSCs into the injured neonatal lung suggest that the potential mechanisms through which MSCs exert their actions are paracrine mediated. ese speculations are supported by in vitro and in vivo studies demonstrating that administration of conditioned media (CdM) from MSCs has the ability to protect alveolar epithelial and lung microvascular endothelial cells from oxidative stress, prevent oxygen-induced alveolar growth arrest, and stimulate a subset of stem/progenitor cells, known as bronchoalveolar stem cells (BASCs), to aid in lung repair [48,54,55,57,58]. Furthermore, the therapeutic bene�ts of MSC-CdM may surpass those of MSCs, with invivo �ndings indicating a more profound therapeutic effect of MSC-CdM in preventing/repairing lung injury than that of MSCs [54].
Compared to the various available MSC sources, UCB represents a very appealing source of MSCs for therapeutic use in the newborn due to its clinically relevant, easily accessible, ethically viable, and readily available source of stem/progenitor cells. Chang and colleagues [59,60] demonstrated that MSCs obtained from human UCB prevent hyperoxia-induced alveolar growth arrest and alleviate �brotic changes in the neonatal rat lung. Interestingly, Chang et al. also show that the route of administration may alter the outcome, with intratracheal transplantation resulting in a more prominent attenuation of hyperoxiainduced lung injury than intraperitoneal transplantation [60]. Furthermore, Chang et al. recently demonstrated the dose-dependent effects of human UCB-derived MSCs in the oxygen-challenged neonatal rat lung [59]. is study indicated that intratracheal delivery of a minimum of 5 × 10 4 cells is required to exhibit e�cient antiin�ammatory, anti�brotic, and antioxidative effects following hyperoxiainduced lung injury in neonatal rats [59]. In light of these �ndings, further studies determining the optimal dose of MSCs for potential clinical bene�t in human neonates are anticipated. More recently, data from our own lab has demonstrated the bene�cial long-term effects of UCB-derived MSCs in an oxygen-challenged rat model of BPD [58]. Long-term assessment of 6-month-old rats showed no adverse effects of either MSC or MSC-CdM therapy and also demonstrated persistent improvements in adult exercise capacity and lung structure [58].

MSCs in Preclinical Studies for COPD.
Repair of lung injury in experimental models of COPD/emphysema has also been demonstrated by many studies utilizing MSCs, derived from bone marrow, adipose tissue, and umbilical cord Wharton's jelly [61][62][63][64][65][66][67][68][69][70]. Huh and colleagues [61] used a cigarette smoke-induced experimental model of COPD/emphysema and showed that bone marrow-derived MSCs and their CdM were capable of restoring the alveolar structure and increased pulmonary vascularity. Zhen and colleagues [62] also used bone marrow-derived MSCs, but in a papaininduced model of COPD/emphysema, and showed alveolar structure improvements. e bene�cial effects of MSCs have also been demonstrated in an elastase-induced experimental model of COPD/emphysema, where Katsha and colleagues [65] also showed preservation of the alveolar structure, reduced in�ammation, and upregulation of growth factors. e bleomycin-induced adult lung injury model has also been implemented in many studies that demonstrate the bene�cial effects of MSCs, as evident by reduced lung �brosis and modulation of pulmonary in�ammation [66][67][68][69][70]. Similar to the use of MSCs in experimental BPD animal models, the proposed mechanism of action is through a paracrine and immunomodulatory effect rather than cell engrament [71].
Apart from MSCs, other reparative cells of interest include EPCs, amnion epithelial cells (AECs), and perivascular cells (PCs). e therapeutic potential of EPCs in neonatal lung injury has been effectively demonstrated in an oxygeninduced BPD mouse model [72]. Treatment of neonatal mice exposed to hyperoxia with intravenously administered bone marrow-derived angiogenic cells (a population of bone marrow myeloid-like precursor cells) showed restoration of the alveolar structure and vessel density to that of control (room air-exposed) levels [72]. Recently, the therapeutic potential of human AECs has been investigated in a sheep model of neonatal lung injury, induced by LPS administration in fetal sheep [73]. Since human AECs are sourced from placentae, which are normally discarded aer birth, they present an easily accessible and ethically viable cell therapy candidate. Administration of AECs to fetal sheep exposed to LPS attenuated in�ammation-induced changes in lung function and structure and reduced pulmonary in�ammation [73]. Of particular interest is the ability of AECs to signi�cantly increase the expression levels of SP-A and -C. e potential therapeutic effects of human AECs have also been examined in bleomycin-induced adult lung injury models [74,75]. Administration of AECs to adult mice exposed to bleomycin attenuated lung �brosis, reduced collagen content, and decreased pulmonary in�ammation [74,75]; partial restoration of lung function was also observed following AEC administration [75]. Recently, umbilical cordderived PCs have been shown to exhibit similar reparative potential to UCB-derived MSCs in a rat model of neonatal lung injury [58]. Umbilical cord-derived PCs, as a whole cell therapy or growth factor producers (i.e., CdM), rescued oxygen-induced arrested alveolar growth and improved longterm lung function [58]. e low engrament into the lungs indicates that these cells act via immune modulation, rather than cell engrament and differentiation. More detailed assessment of the therapeutic potential of these cells in other models of neonatal and adult lung injury will be of interest.

Summary
e long-term pulmonary outcomes reported in ex-preterm BPD children, adolescents, and adults highlight the potential link between altered early-life development and environment and chronic lung disease in adulthood. Findings from several exciting studies indicate that a variety of stem/progenitor cells can prevent and/or regenerate neonatal and adult lung injury in various experimental models. In terms of neonatal lung injury, additional studies in different animal models of BPD are necessary to broaden the current knowledge and understanding of the therapeutic potential of stem/progenitor cells. In doing so, further evidence for creating a strong rationale for translating this potential breakthrough into the clinic can be generated. Furthermore, by preventing or repairing neonatal lung injury in prematurely born infants, it is possible that the overall risk of COPD/emphysema development in later life may be reduced in this subset of the population.