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Cell Biology International (2012) 36, 589–594 (Printed in Great Britain)
Role of bone marrow-derived mesenchymal stem cells in the prevention of hyperoxia-induced lung injury in newborn mice
Xiaoying Zhang*12, Hui Wang†1, Yun Shi*, Wei Peng*, Sheng Zhang*, Wanqiao Zhang*, Jing Xu*, Yabo Mei* and Zhichun Feng*2
*Department of Pediatrics, BaYi Childrens Hospital of The General Military Hospital of Beijing PLA, Beijing 100700, Peoples Republic of China, and †Department of Science Division, The General Military Hospital of Beijing PLA, Beijing 100700, Peoples Republic of China


BPD (bronchopulmonary dysplasia) is predominantly characterized by persistent abnormalities in lung structure and arrested lung development, but therapy can be palliative. While promising, the use of BMSC (bone marrow-derived mesenchymal stem cell) in the treatment of lung diseases remains controversial. We have assessed the therapeutic effects of BMSC in vitro and in vivo. In vitro co-culturing with injured lung tissue increased the migration-potential of BMSC; and SP-C (surfactant protein-C), a specific marker of AEC2 (type II alveolar epithelial cells), was expressed. Following intraperitoneal injection of BMSC into experimental BPD mice on post-natal day 7, it was found that BMSC can home to the injured lung, express SP-C, improve pulmonary architecture, attenuate pulmonary fibrosis and increase the survival rate of BPD mice. This work supports the notion that BMSC are of therapeutic benefit through the production of soluble factors at bioactive levels that regulate the pathogenesis of inflammation and fibrosis following hyperoxia.


Key words: bronchopulmonary dysplasia (BPD), bone marrow-derived mesenchymal stem cells, fibrosis, hyperoxia, surfactant protein-C, type II alveolar epithelial cells

Abbreviations: AEC2, type II alveolar epithelial cells, BMSC, bone marrow-derived mesenchymal stem cell, BPD, bronchopulmonary dysplasia, CFSE, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, DMEM, Dulbecco's modified Eagle's medium, IL-1β, interleukin-1β, RAC, radial alveolar counts, SP-C, surfactant protein-C, TGFβ1, transforming growth factor β1, TIMP-1, tissue inhibitor of metalloproteinases 1, TNFα, tumour necrosis factor α

1These authors have contributed equally to this work.

2Correspondence may be addressed to either of the authors (email fengzhichun81@sina.com or xiaoyingzhang79@163.com).


1. Introduction

Babies born prematurely or experience respiratory problems shortly after birth are at risk of BPD (bronchopulmonary dysplasia), a common chronic lung disease with a multifactorial aetiology that manifested in preterm neonates (Zhang et al., 2011). Although the incidence and severity of respiratory distress syndrome has been reduced through antenatal corticosteroid therapy and post-natal replacement surfactant treatment, the overall incidence of BPD has not decreased, but is on the rise (Zhang et al., 2011). BPD is predominantly characterized by persistent abnormalities in lung structure and arrested lung development, but therapy can be palliative (van Haaften et al., 2009).

Increasing attention has been focused on the use of BMSC (bone marrow-derived mesenchymal stem cells) for regenerating damaged organs (van Haaften et al., 2009). However, the use of BMSC in the treatment of lung diseases remains controversial. BMSC have the capacity for self-renewal and the potential to differentiate into multiple lineages (Yu et al., 2011), which make them an attractive option for cell therapeutic agents (Secchiero et al., 2011). However, some studies have suggested that BMSC do not contribute to the alveolar epithelium, which questions the fundamental rationale of this type of therapeutic strategy (Chang et al., 2005).

We have therefore investigated the effect of BMSC administration in a well-established hyperoxia-induced model of BPD. Consistent with our hypothesis, BMSC significantly improved survival rates and pulmonary architecture, attenuated inflammation and inhibited lung fibrosis in BPD mice.

2. Materials and methods

2.1. Generation of BMSC

BMSC were obtained from femoral bone marrow from mice (Kunming mouse, 3–4 weeks old males, purchased from the Chinese Academy of Sciences), as previously described (Rochefort et al., 2006).

2.2. BMSC immunophenotype

Phycoerythrin labelling for mouse monoclonal antibodies against CD34, CD45, CD105 and CD106 (AbD Serotec) was used according to the manufacturer's protocol. Cells were assessed at passage 2 by flow cytometry and CFlow® software (Accuri C6 Flow Cytometer).

2.3. BMSC differentiation

P2 BMSC differentiation potential was evaluated by adipogenic and osteogenic induction using Differentiation Medium (Catalogue Nos. GUXMX-9003 and GUXMX-90021, Cyagen) and by light microscopy imaging (Nikon, TE2000-S).

2.4. BMSC migration assay

BMSC were seeded in the medium without serum in the upper chamber of a Transwell Permeable Supports chamber (Corning) with an 8 μm mesh separating the upper and lower chambers. Cells were allowed to migrate for 6 h at 37°C into the lower chamber, which contained one of the following: DMEM (Dulbecco's modified Eagle's medium) (15% serum), DMEM (15% serum) plus normoxic lung, or DMEM (15% serum) plus hyperoxic lung.

2.5. Co-culture assay

BMSC seeded in the bottom chamber of a Transwell Permeable Supports chamber (Corning) were exposed to either hyperoxia-injured or normoxic lung in the upper chamber, with a 0.4 μm mesh separating the upper and lower chambers. Cells were stained for SP-C (surfactant protein-C) (antibody used at a 1:200 dilution; Santa Cruz), nuclear DNA (Hoechst 33258, 0.1%; Sigma) or microfilament (FITC-conjugated phalloidin, 500 ng/ml; Sigma) and imaged with a confocal laser scanning microscope (Nikon,C1 si).

2.6. Animal model

The use of animals was approved by Hospital of Beijing IACUC (Institutional Animal Care and Use Committee) and conformed to the guidelines of the National Institutes of Health concerning the care and use of laboratory animals. The experimental BPD mouse model was induced as we described elsewhere (Zhang et al., 2011). Newborn Kunming mice (Chinese Academy of Sciences) were placed in sealed plexiglas chambers and exposed to normoxia (room air) or hyperoxia (FiO2 =  60%, BPD model) conditions from birth until post-natal day (P) 45 (or P45).

2.7. In vivo experimental design

Newborn mouse pups were randomly assigned to one of three groups: (i) normoxia (room air, air control group), (ii) hyperoxia plus PBS and (iii) hyperoxia plus BMSC. Each group was composed of 20 mice. Cells were administered on post-natal day 7 via an intraperitoneal injection (105 cells per animal) and every following week. Prior to administration, BMSC were labelled with the intravital green fluorescent dye CFSE [5(6)-carboxyfluorescein diacetate N-succinimidyl ester; Sigma, 0.5μM]. Animals were killed on P45.

2.8. Necropsy, sample, histology, fibrosis and inflammation assessment

Female mice were anaesthetized with 50 mg/kg sodium pentobarbital. Blood samples were collected from the orbital sinus and plasma samples were snap-frozen in liquid nitrogen for later analysis. Mice were killed, and lungs were prepared for histology (Zhang et al., 2011). The severity of pulmonary fibrosis in the lung sections was assessed by the trained histopathologist after staining for collagen using Sirius red or Masson's trichrome stain (Y.M.) (Henderson et al., 2010). Immunostaining for SP-C (antibody used at 1:200 dilution; Santa Cruz) was as previously described (Zhang et al., 2009). RAC (radial alveolar counts) were performed also as previously described at ×100 magnification (Zhang et al., 2011).

2.9. Y chromosome in situ hybridization

In situ hybridization for mouse Y chromosome involved a Y-chromosome-specific SRY DNA labelling kit (HAO YANG).

2.10. ELISA

Plasma samples were tested for IL-1β (interleukin-1β) and TNFα (tumour necrosis factor α) activities using appropriate ELISA kits (NeoBioscience).

2.11. Reverse transcription reaction and quantitative real-time PCR

Total RNAs were purified with the mirVana™ PARIS™ Kit (Ambion). A one-step SYBR PrimeScript RT–PCR (reverse transcription–PCR) kit (TakaRa) was used to quantify the copy number of cDNA targets (Zhang et al., 2009). Primers are listed in the Supplementary Table S1 (available at http://www.cellbiolint.org/cbi/036/cbi0360589add.htm).

2.12. Statistical analysis

Data are presented as the means±S.D. of the samples. One-way ANOVA analyses and LSD (least significant difference) test was used to compare different groups. Survival curves were derived using the Kaplan–Meier method and differences were compared by log-rank tests. Difference was deemed statistically significant with P<0.05.

3. Results

3.1. Morphology, immunophenotype and differentiation analysis of BMSC

BMSC formed a homogeneous population of cells at passage 2 (Supplementary Figure S1A available at http://www.cellbiolint.org/cbi/036/cbi0360589add.htm). Analysis of cell surface immunophenotype showed that the BMSC population was positive for CD105 and CD106, but negative for CD34 and CD45 (Supplementary Figure S1B). We also verified that these BMSC could differentiate into specific mesenchymal lineages (adipocytes or osteocytes) when grown in appropriate induction media (Supplementary Figure S1C).

3.2. Injured lung promotes BMSC differentiation and migration in vitro

To assess whether hyperoxia-damaged lung contributes to differentiation of BMSC into AEC2 (type II alveolar epithelial cells) in vitro were studied in culture. BMSC co-cultured with hyperoxia-damaged lung (Figure 1A) expressed SP-C mRNA (Figure 1B) and proteins (Figure 2C), specific markers for AEC2 identity. Conversely, BMSC did not express SP-C mRNA, or the proteins, when co-cultured with normoxic lung or medium alone (Figures 1B and 1C). Importantly, confocal microscopy showed a significant microfilament rearrangement within BMSC co-cultured with hyperoxia-damaged lung compared with normoxic lung or medium alone (Figure 1D). The results suggest that injured lung can promote BMSC migration in vitro. To confirm the results, migration experiments showed that cell migration was significantly increased in BMSC co-cultured with hyperoxia-damaged lung compared with normoxic lung or medium alone (Figure 1E).

3.3. Intraperitoneal delivery of BMSC homes to injured lung and contributes to engraftment as AEC2

Y-chromosome in situ hybridization carried out on female BPD mice indicated the presence of brown Y-chromosome stained cells in the corners of alveoli and around the airway of the BMSC-transplanted group, but not in PBS controls (Figure 2A). The data suggest that BMSC delivered intraperitoneally can home on injured lungs. Immunofluorescent staining to determine whether the homed BMSC can adopt the phenotype of AEC2 indicated that SP-C expression co-localized with CFSE-labelled BMSC (Figure 2B). Importantly, BPD mice treated with BMSC increased expression levels of SP-C mRNA compared with PBS controls (Figure 2C), also suggesting that BMSC contribute to engraftment as AEC2.

3.4. Intraperitoneal delivery of BMSC improves survival and prevents hyperoxia-induced pulmonary fibrosis of BPD mice

Survival in the BMSC-treated group was significantly higher than in the PBS control group (Figure 3A). Intraperitoneal delivery of BMSC also improved alveolarization, as seen by histological analysis and quantified using RAC. PBS treatment had no protective effect on lung architecture (Figure 3B).

BMSC administration significantly reduced the severity of pulmonary fibrosis around the airways and alveoli (Figures 4A and 4B) compared with PBS controls, as assessed by Sirius Red and Masson's trichrome staining for collagen. Real-time PCR and ELISA also showed that BMSC-treated group had decreased pulmonary expression of a number of genes associated with extracellular matrix remodelling and fibrosis, such as TGFβ1 (transforming growth factor β1), collagen 1α and TIMP-1 (tissue inhibitor of metalloproteinases 1) (Figure 4C). Plasma expression levels of IL-1 and TNFα were also lower in the experimental group compared with the PBS-treated group (Figure 4D).

4. Discussion

Previous studies have questioned the therapeutic potential of BMSC in treating lung diseases, pointing to the low survival rate of transplanted BMSC in recipient mice and the failure of bone marrow to reconstitute lung epithelium (Chang et al., 2005). However, we find that BMSC home to injured lung, as detected by Y-chromosome in situ hybridization and confocal microscope (Figure 2A and 2B). Additionally, our in vitro and in vivo experiments demonstrate that injured lung can promote the migration potential of BMSC (Figures 1D and 1E) and induce BMSC to express SP-C, a specific marker of AEC2 identity (Figures 1B, 1C, 2B and 2C). The data support the potential use of BMSC in therapeutic strategy for lung injury, similar to previous reports (van Haaften et al., 2009).

However, engrafted BMSC exhibited their therapeutic benefit via a significant decrease in pulmonary fibrosis. At least one previous study found evidence of BMSC conferring anti-fibrotic effects in vitro (Ohnishi et al., 2007), whereas, Salazar et al. (2009) reported conflicting results. The latter authors hypothesized that BMSC mediated the proliferation and increased expression of procollagen by lung fibroblasts via Wnt isoforms and TGFβ1. Our results independently confirm the importance of BMSC in the prevention of pulmonary fibrosis induced by hyperoxia (Figure 4). We have also shown that BMSC home to injured lung (Figure 2A), decreases collagen deposition (Figures 4A and 4B), improves BPD mice survival, improves pulmonary architecture (Figure 3) and reduces pulmonary expression of a number of genes (such as TGFβ1, collagen 1α and TIMP-1) involved in extracellular matrix remodelling and fibrosis (Figure 4C). Numerous reports have documented the fibrotic and inflammatory effects of TGFβ1 expression in pathogenesis of lung fibrosis (Kang et al., 2007; Salazar et al., 2009; Henderson et al., 2010), and that overexpression of TGFβ1 in the lungs of newborn animals causes pathological features consistent with BPD (Nakanishi et al., 2007). Nakanishi et al. demonstrated that the neutralization of the abnormal TGF-activity can improve somatic growth in hyperoxic mouse pups and decrease pulmonary inflammation (Nakanishi et al., 2007). It is also important to note that TIMP-1 is stimulated by TGFβ1 via Bax- and Bid-dependent mechanisms, and plays an important role in TGFβ1-stimulated pulmonary fibrosis (Kang et al., 2007). Thus, the decrease in TGFβ1 expression levels observed in BPD mice treated with BMSC further illustrates the potential therapeutic benefits for BMSC in treating BPD. The role that BMSC plays in the prevention of lung fibrosis through modulation of TGFβ1 remains to be elucidated.

We have shown that expression levels of IL-1 and TNFα in the plasma of BMSC-treated group are significant decreased compared with PBS control group. There is evidence that BMSC play a key role in anti-inflammatory and anti-fibrotic effect by mediating the IL-1RN (interleukin-1 receptor antagonist), which blocks IL-1 and TNFα in the injured lung (Ortiz et al., 2007).

These results demonstrate the therapeutic efficacy of BMSC in preventing pulmonary fibrosis in hyperoxia-induced BPD, and further support the notion that BMSC exerts their therapeutic benefit through the production of bioactive levels of soluble factors.

Author contribution

Xiaoying Zhang, Hui Wang and Zhichun Feng designed all the experiments and wrote the paper; Xiaoying Zhang performed the animal model, treatment, necropsy, sample, histology and statistical analysis; Hui Wang performed stem cell migration assay and real-time PCR assays; Yun Shi performed generation of BMSC and BMSC immunophenotype; Wei Peng performed BMSC differentiation and Y-chromosome in situ hybridization assays; Sheng Zhang performed co-culture assay; Wanqiao Zhang performed ELISA; Jing Xu performed animal necropsy, sample, histology; Yabo Mei performed fibrosis and inflammation assessment; Xiaoying Zhang and Zhichun Feng provided funding.

Acknowledgements

We thank our technical staff for technical assistance and also thank Professor Xiyu He, Professor Chunzhi Huang and Dr Shaodong Hua for fruitful discussion of the results.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 30772036, 81070524 and 30973210].

REFERENCES

Chang, JC, Summer, R, Sun, X, Fitzsimmons, K and Fine, A (2005) Evidence that bone marrow cells do not contribute to the alveolar epithelium. Am J Respir Cell Mol Biol 33, 335-42
Crossref   Medline   1st Citation   2nd  

Henderson, WR, Chi, EY, Ye, X, Nguyen, C, Tien, YT and Zhou, B (2010) Inhibition of Wnt/beta-catenin/CREB binding protein (CBP) signaling reverses pulmonary fibrosis. Proc Natl Acad Sci USA 107, 14309-14
Crossref   Medline   1st Citation   2nd  

Kang, HR, Cho, SJ, Lee, CG, Homer, RJ and Elias, JA (2007) Transforming growth factor (TGF)-beta1 stimulates pulmonary fibrosis and inflammation via a Bax-dependent, bid-activated pathway that involves matrix metalloproteinase-12. J Biol Chem 282, 7723-32
Medline   1st Citation   2nd  

Nakanishi, H, Sugiura, T, Streisand, JB, Lonning, SM and Roberts, JD (2007) TGF-beta-neutralizing antibodies improve pulmonary alveologenesis and vasculogenesis in the injured newborn lung. Am J Physiol Lung Cell Mol Physiol 293, L151-61
Crossref   Medline   1st Citation   2nd  

Ohnishi, S, Sumiyoshi, H, Kitamura, S and Nagaya, N (2007) Mesenchymal stem cells attenuate cardiac fibroblast proliferation and collagen synthesis through paracrine actions. FEBS Lett 581, 3961-6
Crossref   Medline   1st Citation  

Ortiz, LA, Dutreil, M, Fattman, C, Pandey, AC, Torres, G and Go, K (2007) Interleukin 1 receptor antagonist mediates the anti-inflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci USA 104, 11002-7
Crossref   Medline   1st Citation  

Rochefort, GY, Delorme, B, Lopez, A, Herault, O, Bonnet, P and Charbord, P (2006) Multipotential mesenchymal stem cells are mobilized into peripheral blood by hypoxia. Stem Cells 24, 2202-8
Crossref   Medline   1st Citation  

Salazar, KD, Lankford, S and Brody, AR (2009) Mesenchymal stem cells produce Wnt isoforms and TGF-beta1 that mediate proliferation and procollagen expression by lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 297, L1002-11
Crossref   Medline   1st Citation   2nd  

Secchiero, P, Zorzet, S, Tripodo, C, Corallini, F, Melloni, E and Caruso, L (2011) Human bone marrow mesenchymal stem cells display anti-cancer activity in SCID mice bearing disseminated non-Hodgkins lymphoma xenografts. PLoS ONE 5, e11140
1st Citation  

van Haaften, T, Byrne, R, Bonnet, S, Rochefort, GY, Akabutu, J and Bouchentouf, M (2009) Airway delivery of mesenchymal stem cells prevents arrested alveolar growth in neonatal lung injury in rats. Am J Respir Crit Care Med 180, 1131-42
Crossref   Medline   1st Citation   2nd   3rd  

Yu, JM, Wu, X, Gimble, JM, Guan, X, Freitas, M and Bunnell, BA (2011) Age-related changes in mesenchymal stem cells derived from rhesus macaque bone marrow. Aging Cell 10, 66-79
Crossref   Medline   1st Citation  

Zhang, X, Liu, S, Hu, T, Liu, SP, He, Y and Sun, S (2009) Up-regulated microRNA-143 transcribed by nuclear factor kappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression. Hepatology 50, 490-9
Crossref   Medline   1st Citation   2nd  

Zhang, X, Peng, W, Zhang, S, Wang, C, He, X and Zhang, Z (2011) MicroRNA expression profile in hyperoxia-exposed newborn mice during the development of bronchopulmonary dysplasia. Respir Care 56, 1009-15
Crossref   Medline   1st Citation   2nd   3rd   4th   5th  


Received 11 September 2011/18 January 2012; accepted 20 February 2012

Published as Cell Biology International Immediate Publication 20 February 2012, doi:10.1042/CBI20110447


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)