BPD is a multifactorial disease of premature infants resulting from mechanical injury, oxygen toxicity, and infection, subsequently leading to pulmonary inflammation and damage. The disease mainly affects preterm infants as their lungs are more vulnerable to injury due to poorly developed airway supporting structures, surfactant deficiency, decreased compliance, and underdeveloped antioxidant mechanisms. In the current era, with the increased survival of extremely low gestational age infants and the advent of surfactant treatment and gentler modes of ventilation, the pathology of BPD has evolved into a new pattern of injury characterized by impaired alveolarization and dysmorphic vasculogenesis. The prominent pathologic findings include disruption of lung development with decreased septation and alveolar hypoplasia leading to fewer and larger alveoli, thickened alveolar septa, inflammation, bronchial smooth muscle hypertrophy, and interstitial edema (23
). Vascular changes characteristic of pulmonary hypertension may also be evident with pulmonary arteriolar muscularization and right ventricular hypertrophy (24
). Data are lacking on the exact timing and relative roles of vasculogenesis, angiogenesis, and remodeling during lung development. Interplay between diverse signaling pathways, including transcription factors, growth factors, extracellular matrix, and mechanical forces leading to the precise development of the lung and its circulation is largely unknown. In recent years, a vascular hypothesis for the pathogenesis of BPD has been proposed with accumulating data to suggest that early disruption of vascular growth from endothelial damage leads to impaired growth of the distal airspaces, resulting in reduced alveolarization (25
). Disruption of lung vascular growth also sets the stage for late pulmonary hypertension. Lung injury results in increased elastic tissue formation and thickening of the interstitium, which in turn compromises septation and capillary development. At present, all the available interventions for the prevention and/or treatment of BPD lack efficacy and have undesirable side effects. Therefore, the search for an effective preventive and/or treatment modality is of paramount importance.
Research on stem cell and progenitor cell–based therapies on animal models of disease has produced promising results on the ability of BMSCs to repair tissue injury (7
). BMSCs belong to the select group of adult stem cells that have traditionally shown differentiation potential toward mesenchymal tissues such as bone (5
), cartilage (26
), and fat cells (27
), but recent work has demonstrated a greater plasticity beyond mesenchymal cell fate and includes the differentiation into endothelial and neuronal lineages (28
). In addition to their multilineage differentiating capability, BMSCs produce immunosuppressive cytokines and growth factors that may help in the reparative process (31
). The potent immunomodulatory and antiinflammatory properties of BMSCs in a clinical study involved treatment of graft-versus-host disease (32
) and cell-based treatments are currently the focus of intensive studies in graft enhancement, tissue protection, and regenerative medicine. We hypothesized that exogenous administration of BMSCs may protect the lung architecture in a neonatal mouse model of bronchopulmonary dysplasia. The saccular and alveolar stages of lung development in the mouse occur after birth, making it a suitable model to study lung injury at similar stages of lung development to those observed in the human preterm neonate between 24 and 28 weeks' gestation, a period most vulnerable to BPD from ventilator- and oxygen-induced injury. Pulmonary hypertension contributes significantly to the morbidity and mortality of patients with BPD (3
). Changes such as vascular remodeling and right ventricular hypertrophy due to chronically elevated pulmonary artery pressures have been successfully reproduced in rodent models of the pathology.
In our studies, neonatal mice exposed to 75% O2 showed the histological findings of BPD, including simplification of alveoli, thickened alveolar septa, inflammatory cell infiltration, and higher Fulton's index (right ventricular hypertrophy) and vascular remodeling. Using this well-characterized mouse model of BPD, we demonstrated that systemically administered BMSCs partially protect the lung architecture against hyperoxia-induced injury, whereas a single bolus injection of BMSC-CM is able to confer full protection, preventing alveolar simplification, preserving normal alveolar number, and ameliorating the lung vascular remodeling associated with the disease.
One of the limitations of using BMSCs is the lack of unique cell surface markers to isolate and characterize them with several early studies reporting significant variability in isolation and culture methods. The BMSC isolation and characterization used in this study included both positive and negative selection methods using specific cell surface markers according to and exceeding the criteria set forth by the International Society for Cellular Therapy (21
) to achieve a homogeneous cell population. In addition, differentiation assays confirmed that these cells have the ability to differentiate into osteoblasts and adipocytes in vitro
. Sex-mismatch experiments with donor male cells and female recipient neonatal mice allowed us to quantify transplanted cells and show persistence of a small number of donor cells 2 weeks after injection. We were unable to further characterize whether the donor cells differentiated into lung cell types or to address cell fusion possibilities. We did observe that a higher number of donor BMSCs can be detected in the injured lung compared with the normal lung at 10 days post injection. Nevertheless, the overall donor cell retention is minimal, in the order of one BMSC per 10,000 lung cells, and we cannot formally exclude the possibility that the observed donor cells represent rare, nonmesenchymal contaminants in our cultures, despite the strict selection regimen we used. Based on these data, we cannot propose that donor BMSCs extensively replace injured lung cells to effectively improve lung architecture. The minimal BMSC engraftment after transplantation combined with the therapeutic efficacy of cell-free conditioned media point to paracrine effects of BMSC action rather than direct tissue repair.
Similar results of low engraftment, associated with a significant beneficial response, have been reported in other studies. Togel and colleagues observed a rapid clearance of BMSCs within 24 hours of intravenous delivery but demonstrated significant protection from ischemic acute renal failure in a rat model of disease (34
). A similar “early benefit” effect was observed in a cardiac injury model within 72 hours of BMSC injection that was attributed to the paracrine effects of growth factors released by the transplanted cells (35
). However, none of these studies tested the potential in vivo
cytoprotective actions of secreted factors derived from cultured BMSCs. More recently it has become apparent that many (but not all) of the beneficial effects of stem and progenitor cells in animal disease models are the result of immunomodulatory and trophic support properties delivered by the transplanted cells acting in a paracrine manner. Using a bleomycin-induced lung inflammation and fibrosis model, Ortiz and colleagues (36
) suggested that BMSC-secreted interleukin 1 receptor antagonist (IL1ra) represents a key candidate for the observed beneficial effects of BMSC treatment, and the antiapoptotic effect of BMSCs on neutrophils was shown to depend on IL-6 secretion but not on cell-to-cell contact (37
). Chen and colleagues have demonstrated a beneficial effect of BMSC-CM on wound healing compared with fibroblast conditioned media (38
). Parekkadan and colleagues have demonstrated a significant survival benefit in rats with fulminant hepatic failure via intravenous administration of sonicated BMSCs (39
). Similar paracrine effects of stem or progenitor cells have been observed in other studies (40
), and the assertion has been advanced that, in certain systems, it may not be just a singular factor but rather a specific milieu of secreted factors that confer the reparative and trophic action of stem or progenitor cells (43
). Although paracrine effects may explain certain of the observed cytoprotective properties of BMSCs, cell-to-cell interactions, resulting in reprogramming of immune cells by BMSCs, have been shown to be paramount for protection in an animal model of sepsis (44
None of the above studies identified the factor(s) responsible for BMSC-CM efficacy in vivo
. In our system, the beneficial response appears to be stem cell–specific, because we did not detect any effect with mouse lung fibroblast or PASMC injection. BMSCs were administered after the animals had been already exposed to high oxygen for 3 days and the observed protective effect suggests that BMSCs, or the milieu of proteins they secrete, could be used clinically as a prophylaxis tool to prevent further injury. BMSC-mediated release of growth factors and potential immunosuppressive effects may explain some of the observed physiological benefits. Our mass spectroscopic analysis on BMSC-CM identified protein classes associated with cell proliferation and apoptosis, cell–cell interactions and cell motility, immune modulation, and respiratory disease. A previous gene expression profiling study by Ohnishi and colleagues (45
) identified similar classes of molecules expressed in high abundance (> 100-fold) specifically by BMSCs compared with bone marrow–derived mononuclear cells. In our analysis, out of several proteins identified in BMSC-CM, two were of particular interest: Spp1 and Csf1. Spp1, also known as osteopontin (Opn), has been identified as a protein with a pivotal role in immune and vascular models of injury. Opn regulates cytokine production by macrophages, inhibits macrophage accumulation in vascular systems, mediates cell adhesion and migration, and can act as a survival factor (46
). Osteopontin can have an antiinflammatory or proinflammatory effect depending on the injury model and stage of injury. Opn is important for Th1-mediated immune and autoimmune disease modulation. Xanthou and colleagues have demonstrated effects of Opn on Th2-mediated allergic disease, observing a proinflammatory effect of Opn on primary systemic sensitization and an antiinflammatory effect during secondary pulmonary antigenic challenge (48
). Csf1 is a key differentiation, growth, and survival factor for monocytes/macrophages and its action on these cells results in enhanced cytotoxicity, superoxide production, phagocytosis, chemotaxis, and secondary cytokine production (49
). Exogenous administration of recombinant Csf1 was shown to have protective effects in human fungal infections (50
). The role of both Spp1 and Csf1 in this neonatal mouse model of disease is unknown and these molecules represent attractive candidates for further functional studies to determine whether in isolation, or in combination with other secreted proteins, they are responsible for the observed BMSC-CM efficacy. It is likely that a combination of these proteins rather than one single factor confers cytoprotective epithelial, vascular, and antiinflammatory effects on the developing neonatal lung.
Our studies indicate that mechanisms of immunological protection may be the major effectors of BMSC treatment. In agreement, several reports have demonstrated immunological protection with BMSC administration, including down-regulation of T cell proliferation and dendritic cell maturation (51
), and antiproliferative effects on B lymphocytes (54
) and natural killer cells (55
). We speculate that the low retention of BMSCs into the lung may limit their ability to secrete factor(s) in sufficient amounts to achieve complete tissue recovery in response to hyperoxic or potentially other lung injury. The concentrated administration of active immunomodulators produced by BMSCs in culture may achieve significant in vivo
levels to trigger signaling pathways of repair and immunological protection that can have long-lasting effects. Inflammation is considered the key mediator of alveolar and vascular injury in BPD. It likely results from an imbalance between pro- and antiinflammatory mediators within the lung. In their review of BPD, Thompson and colleagues have found higher levels of proinflammatory cytokines (IL1β, IL-6, and IL-16) and lower levels of antiinflammatory cytokines (IL-10 and IL-13) in premature infants with BPD (56
). In a multicenter study of extremely low birth weight infants with BPD, Ambalavanan and colleagues demonstrated a correlation of higher IL1β and lower IL-17 serum concentrations with BPD (57
). In our study, hyperoxia exposure resulted in significant inflammation within the lungs of animals treated with MLFs or PASMCs, as shown by significant neutrophil and macrophage levels in the BALF. Treatment of hyperoxic animals with BMSCs or BMSC-CM prevented the development of inflammation resulting in a significantly reduced number of neutrophils and macrophages within the BALF. To characterize the inflammation further we measured the cytokine and chemokine profile within the BALF via the Luminex 200 system. Our analysis identified higher levels of proinflammatory cytokines in the BALF of hyperoxic animals treated with PASMC-CM (IL-17, IL-5, and TNF-α) compared with the BMSC-CM. The rest of the proinflammatory cytokine levels, although higher in the hyperoxic animals treated with PASMC-CM compared with BMSC-CM, did not reach statistical significance. One likely explanation is the presence of low cytokine levels in the BALF compared with the levels in serum or whole lung. Also the peak timing of cytokine elevation is unknown and it is possible that some of these cytokines are secreted in higher amounts in the early or late phase of inflammation. We have measured these cytokine levels at 14 days post hyperoxia. Further studies using early and late time frames, as well as measurement of cytokine profile within the lungs in addition to BALF, will determine the exact cytokine and chemokine alterations in BPD.
In conclusion, this report demonstrates a potential beneficial effect of BMSC treatment on the pathophysiology of oxygen-induced lung injury. Further in vivo and in vitro studies are required to optimize dose, timing, and duration of both stem cell and cell-free media treatment and to delineate the mechanisms underlying BMSC protection in our model of BPD. The findings of this study point to the beneficial use of stem cells and/or a pool of factors they secrete in culture as a therapeutic approach to protect the newborn injured lung.