Intranasal versus Intraperitoneal Delivery of Human Umbilical Cord Tissue–Derived Cultured Mesenchymal Stromal Cells in a Murine Model of Neonatal Lung Injury
2014; Elsevier BV; Volume: 184; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2014.08.010
ISSN1525-2191
AutoresLiansheng Liu, Quanfu Mao, Sharon Chu, Marwan Mounayar, Reza Abdi, William L. Fodor, James F. Padbury, Monique E. De Paepe,
Tópico(s)Mesenchymal stem cell research
ResumoClinical trials investigating mesenchymal stromal cell (MSC) therapy for bronchopulmonary dysplasia have been initiated; however, the optimal delivery route and functional effects of MSC therapy in newborns remain incompletely established. We studied the morphologic and functional effects of intranasal versus i.p. MSC administration in a rodent model of neonatal lung injury. Cultured human cord tissue MSCs (0.1, 0.5, or 1 × 106 cell per pup) were given intranasally or i.p. to newborn severe combined immunodeficiency–beige mice exposed to 90% O2 from birth; sham controls received an equal volume of phosphate-buffered saline. Lung mechanics, engraftment, lung growth, and alveolarization were evaluated 8 weeks after transplantation. High-dose i.p. MSC administration to newborn mice exposed to 90% O2 resulted in the restoration of normal lung compliance, elastance, and pressure-volume loops (tissue recoil). Histologically, high-dose i.p. MSC administration was associated with alveolar septal widening, suggestive of interstitial matrix modification. Intranasal MSC or lower-dose i.p. administration had no significant effects on lung function or alveolar remodeling. Pulmonary engraftment was rare in all the groups. These findings suggest that high-dose systemic administration of human cultured MSCs can restore normal compliance in neonatally injured lungs, possibly by paracrine modulation of the interstitial matrix. Intranasal delivery had no obvious pulmonary effects. Clinical trials investigating mesenchymal stromal cell (MSC) therapy for bronchopulmonary dysplasia have been initiated; however, the optimal delivery route and functional effects of MSC therapy in newborns remain incompletely established. We studied the morphologic and functional effects of intranasal versus i.p. MSC administration in a rodent model of neonatal lung injury. Cultured human cord tissue MSCs (0.1, 0.5, or 1 × 106 cell per pup) were given intranasally or i.p. to newborn severe combined immunodeficiency–beige mice exposed to 90% O2 from birth; sham controls received an equal volume of phosphate-buffered saline. Lung mechanics, engraftment, lung growth, and alveolarization were evaluated 8 weeks after transplantation. High-dose i.p. MSC administration to newborn mice exposed to 90% O2 resulted in the restoration of normal lung compliance, elastance, and pressure-volume loops (tissue recoil). Histologically, high-dose i.p. MSC administration was associated with alveolar septal widening, suggestive of interstitial matrix modification. Intranasal MSC or lower-dose i.p. administration had no significant effects on lung function or alveolar remodeling. Pulmonary engraftment was rare in all the groups. These findings suggest that high-dose systemic administration of human cultured MSCs can restore normal compliance in neonatally injured lungs, possibly by paracrine modulation of the interstitial matrix. Intranasal delivery had no obvious pulmonary effects. The incidence of premature delivery in the United States is currently 11% to 12% (National Center for Health Statistics, final natality data; retrieved from March of Dimes, http://www.marchofdimes.com/peristats, last accessed May 5, 2014). Although programmatic efforts at reducing late preterm births have shown regional and national success, the incidence of extremely low-birth-weight infants has remained unchanged. Improved survival has resulted in an increased number of infants at risk for complications of prematurity. 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Mesenchymal stem cells transplantation protects against rat pulmonary emphysema.Front Biosci. 2008; 13: 3415-3422Crossref PubMed Scopus (102) Google Scholar These properties are thought to promote an optimal milieu for repair and regeneration of the injured tissue. Exogenously administered MSCs are believed to exert their effects by cell contact–dependent and paracrine mechanisms involving secretion of specific mediators and transfer of cellular materials, such as proteins, nucleic acids, and cellular organelles (including mitochondria), to host cells via microvesicles30Islam M.N. Das S.R. Emin M.T. Wei M. Sun L. Westphalen K. Rowlands D.J. Quadri S.K. Bhattacharya S. Bhattacharya J. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury.Nat Med. 2012; 18: 759-765Crossref PubMed Scopus (943) Google Scholar, 31Zhu Y.G. Feng X.M. Abbott J. Fang X.H. Hao Q. Monsel A. Qu J.M. Matthay M.A. Lee J.W. 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Short-term, long-term and paracrine effect of human umbilical cord-derived stem cells in lung injury prevention and repair in experimental bronchopulmonary dysplasia.Thorax. 2013; 68: 475-484Crossref PubMed Scopus (189) Google Scholar, 36Ionescu L. Byrne R.N. van Haaften T. Vadivel A. Alphonse R.S. Rey-Parra G.J. Weissmann G. Hall A. Eaton F. Thebaud B. Stem cell conditioned medium improves acute lung injury in mice: in vivo evidence for stem cell paracrine action.Am J Physiol Lung Cell Mol Physiol. 2012; 303: L967-L977Crossref PubMed Scopus (251) Google Scholar Umbilical cord blood or tissue (including Wharton substance) may represent an attractive tissue source of MSCs for pulmonary regenerative therapy in newborns. Human umbilical cord (blood or tissue) stem cells can be collected at no risk to the donor, have low immunoreactivity, and have low inherent pathogen transmission. Cord stem cells are especially attractive for the treatment of neonatal diseases because, ideally, the infant's own cord-derived stem cells may be used as an autologous transplant, thus limiting the risk of infection or rejection. The demonstrated efficiency of human umbilical cord–derived MSCs in preclinical models of neonatal lung injury/BPD has culminated in recent phase 1 clinical trials (reviewed by Antunes et al37Antunes M.A. Laffey J.G. Pelosi P. Rocco P.R. Mesenchymal stem cell trials for pulmonary diseases.J Cell Biochem. 2014; 115: 1023-1032Crossref PubMed Scopus (64) Google Scholar). Currently, six clinical trials of MSC therapy for BPD have been registered with ClinicalTrials.gov (NCT01297205, NCT01632475, NCT01828957, NCT02023788, NCT01897987, and NCT01207869, http://clinicaltrials.gov, last accessed September 12, 2014). Five trials based in South Korea are using or have used Pneumostem MSCs (Medipost, Seoul, Republic of Korea), a human umbilical cord blood MSC preparation developed commercially for the purpose of cell therapy in premature infants with BPD. The results of the first completed study from the Republic of Korea, which was an open-label, single-center, phase 1 clinical study to evaluate the safety and efficacy of Pneumostem MSCs for BPD treatment, were recently published.38Chang Y.S. Ahn S.Y. Yoo H.S. Sung S.I. Choi S.J. Oh W.I. Park W.S. Mesenchymal stem cells for bronchopulmonary dysplasia: phase 1 dose-escalation clinical trial.J Pediatr. 2014; 164: 966-972.e6Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar In this study, intratracheal MSC transplantation (10 or 20 × 106 cells/kg) was reported to be safe and associated with a significant reduction in various cytokines, including IL-6, IL-8, and transforming growth factor β1, in tracheal aspirates on posttransplantation day 7.38Chang Y.S. Ahn S.Y. Yoo H.S. Sung S.I. Choi S.J. Oh W.I. Park W.S. Mesenchymal stem cells for bronchopulmonary dysplasia: phase 1 dose-escalation clinical trial.J Pediatr. 2014; 164: 966-972.e6Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar One clinical trial from Taiwan describing the use of umbilical cord MSCs for severe BPD is registered with ClinicalTrials.gov (NCT01207869, http://clinicaltrials.gov/show/NCT01207869, last accessed September 12, 2014). The status of this latter study is unknown. All six clinical trials registered involve the delivery of MSCs via intratracheal administration at doses ranging from 3 to 20 × 106/kg body weight. The recent expansion of MSC therapy for neonatal lung diseases from preclinical studies to the clinical arena has introduced a sense of urgency to gain a better understanding of the characteristics, limitations, and benefits of this approach. Several important unresolved issues and knowledge gaps remain, including the optimal delivery route (intrapulmonary versus i.v./systemic), the importance of dose effects, the exact mechanisms of action, the importance of timing of administration, and the long-term effects.19Borghesi A. Cova C. Gazzolo D. Stronati M. Stem cell therapy for neonatal diseases associated with preterm birth.J Clin Neonatol. 2013; 2: 1-7Crossref Google Scholar The aim of this study was to perform a systematic, comparative analysis of the dose-dependent effects of intranasal (i.n.)/intrapulmonary versus (i.p.)/systemic MSC delivery. We carefully examined the functional effects of stem cells on long-term lung mechanics, histologic features, and structure. As a source of MSCs, we used human umbilical cord tissue–derived cultured MSCs. Hyperoxic exposure of newborn mice was used to induce early neonatal lung injury. As described by us and others,39Mao Q. Gundavarapu S. Patel C. Tsai A. Luks F.I. De Paepe M.E. The Fas system confers protection against alveolar disruption in hyperoxia-exposed newborn mice.Am J Respir Cell Mol Biol. 2008; 39: 717-729Crossref PubMed Scopus (15) Google Scholar, 40Fritzell Jr., J.A. Mao Q. Gundavarapu S. Pasquariello T. Aliotta J.M. Ayala A. Padbury J.F. De Paepe M.E. Fate and effects of adult bone marrow cells in lungs of normoxic and hyperoxic newborn mice.Am J Respir Cell Mol Biol. 2009; 40: 575-587Crossref PubMed Scopus (29) Google Scholar, 41Crapo J.D. Barry B.E. Foscue H.A. Shelburne J. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen.Am Rev Respir Dis. 1980; 122: 123-143PubMed Google Scholar moderate to severe hyperoxia during the neonatal period provides a faithful replication of the early acute injury and subsequent alveolar simplification typical of preterm infants with BPD. Deeper insight into the characteristics of MSC therapy in neonatal lung diseases may contribute to evidence-based translation of preclinical experience to a much-needed clinical application. Human cultured umbilical cord tissue–derived MSCs (tissue cord MSCs, further described as MSCs) were used in all the experiments. Umbilical cord tissue was procured from uncomplicated full-term deliveries at The Christ Hospital (Cincinnati, OH) according to protocols approved by the hospital's Institutional Review Board and were sent to the Viacord Processing Lab (Cincinnati, OH). On receipt, the cord was cleaned with a chlorhexadine wipe and then was placed into a sterile cup with 10 mL of antibiotic solution (25 μg/mL gentamicin, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B; all from Lonza Group AG, Basel, Switzerland). After rinses with sterile phosphate-buffered saline (PBS), the cord tissue underwent overnight digestion in collagenase (Collagenase NB 6, GMP grade, 0.75 mg/mL; SERVA Electrophoresis GmbH, Heidelberg, Germany) with antibiotics in a calcium chloride–buffered digestion solution (37°C). The homogenate was centrifuged to pellet the cell suspension, washed several times, and resuspended in dimethyl sulfoxide freezing media. Frozen cell aliquots were thawed at 37°C and resuspended in culture media (Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum (both from STEMCELL Technologies Inc., Vancouver, BC, Canada), 1% penicillin/streptomycin, and 1% l-glutamine (Lonza Group AG). Cells were cultured on collagen-coated plates (37°C, 5% CO2); medium was replaced every 3 to 4 days. On reaching 70% to 80% confluence, MSCs were trypsinized (0.25% Trypsin-EDTA; Life Technologies, Carlsbad, CA) to a new passage. Cultured MSCs at passages 4 to 10 were used in all the experiments. The cells were surface stained using a panel of flow cytometry anti-human antibodies against CD73, CD90, CD105, CD34, CD45, CD14, HLA-ABC, CD49c, CD49e, HLA-DR (BD Biosciences-BD Pharmingen, San Jose, CA), CD49d, and CD49f (eBioscience Inc., San Diego, CA) and were analyzed by flow cytometry. The MSC line selected for this study expressed the mesenchymal stem cell markers CD73, CD90, and CD105. In addition, the cells also expressed HLA class I and various cell adhesion markers (CD49c, CD49d, CD49e, and CD49f). The cells were negative for hematopoietic cell surface antigens CD34, CD45, CD14, CD19, and HLA-DR (HLA class II, not shown). These molecular characteristics conform to the consensus criteria for defining (human) MSCs established by the International Society for Cellular Therapy.42Dominici M. Le Blanc K. Mueller I. Slaper-Cortenbach I. Marini F. Krause D. Deans R. Keating A. Prockop D. Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement.Cytotherapy. 2006; 8: 315-317Abstract Full Text Full Text PDF PubMed Scopus (12394) Google Scholar Six-week-old timed pregnant severe combined immunodeficiency–beige mice (Fox Chase SCID beige, T- and B-cell deficient, natural killer cell impaired; Charles River Laboratories, Wilmington, MA) were maintained under pathogen-free conditions. Newborn mice were exposed to room air or hyperoxia (90% O2) from birth until postnatal day 7 (day of birth is postnatal day 1). For hyperoxia exposure, mice were placed in an airtight Plexiglas chamber. Oxygen concentrations were continuously monitored and controlled with a ProOx 110 in-line oxygen analyzer and controller system (BioSpherix, Redfield, NY). Nursing dams were rotated daily between air- and oxygen-exposed litters to minimize maternal oxygen toxicity. On postnatal day 5, corresponding to a time point of intense acute lung injury and active tissue remodeling, the pups were randomly assigned to MSC administration by the i.n. or i.p. route. For i.n. inoculation, 20 μL of cell suspension containing 0.1, 0.5, or 1 × 106 cells was placed over the nasal orifices, as previously described,40Fritzell Jr., J.A. Mao Q. Gundavarapu S. Pasquariello T. Aliotta J.M. Ayala A. Padbury J.F. De Paepe M.E. Fate and effects of adult bone marrow cells in lungs of normoxic and hyperoxic newborn mice.Am J Respir Cell Mol Biol. 2009; 40: 575-587Crossref PubMed Scopus (29) Google Scholar thus ensuring aspiration of stem cells into the lungs. For i.p. delivery, a 25-μL Hamilton syringe (Hamilton Co., Reno, NV) with a 26-gauge needle was used for injection of the cell suspension (0.1, 0.5, or 1 × 106 cells in 20 μL of PBS) into the left lower quadrant. The injection was preceded by aspiration to ensure proper localization of the needle. Hyperoxia-exposed sham controls received equal volumes of vehicle buffer (PBS). The i.n. and i.p. deliveries were well tolerated by the normoxic and hyperoxia-exposed pups. The animals were sacrificed 48 hours or 8 weeks after transplantation. All the animal experiments were approved by the institutional animal care and use committee at Lifespan Health System's Rhode Island Hospital and were conducted in accordance with institutional guidelines for the care and use of laboratory animals. Invasive lung function testing was performed 8 weeks after transplantation by the forced oscillation technique in anesthetized, nonparalyzed, tracheotomized animals with intact chest wall.43Vanoirbeek J.A. Rinaldi M. De Vooght V. Haenen S. Bobic S. Gayan-Ramirez G. Hoet P.H. Verbeken E. Decramer M. Nemery B. Janssens W. Noninvasive and invasive pulmonary function in mouse models of obstructive and restrictive respiratory diseases.Am J Respir Cell Mol Biol. 2010; 42: 96-104Crossref PubMed Scopus (237) Google Scholar Mice were deeply anesthetized with an i.p. injection of 140 mg/kg of ketamine and 14 mg/kg of xylazine to eliminate all spontaneous breathing under anesthesia. Body weights were recorded at the start of the procedure. The tracheal cannula was connected to a flexiVent computer-controlled small animal ventilator (SCIREQ Scientific Respiratory Equipment Inc., Montreal, QC, Canada). The mice were ventilated with a tidal volume of 10 mL/kg at an average breathing frequency of 150 breaths/min and a positive end-expiratory pressure of 3 cm H2O to prevent alveolar collapse. Lung function parameters were calculated by fitting pressure and volume data to the single-compartment and constant-phase models.44Bates J. Lung Mechanics: An Inverse Modeling Approach. Cambridge University Press, New York2009Crossref Scopus (265) Google Scholar We measured resistance, compliance, and elastance of the en
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