Intratracheal Transplantation Research Articles

DJ-1 governs airway progenitor cell/eosinophil interactions to promote allergic inflammation

DJ-1 is an antioxidant protein known to regulate mast cell-mediated allergic response, but its role in airway eosinophilic interactions and allergic inflammation is not known. The aim of this study was to investigate the role of DJ-1 in airway eosinophilic inflammation invitro and invivo. Ovalbumin-induced airway allergic inflammation was established in mice. ELISA was adopted to analyze DJ-1 and cytokine levels in mouse bronchoalveolar lavage fluid. Transcriptional profiling of mouse lung tissues was conducted by single-cell RNA-sequencing technology. The role of DJ-1 in the differentiation of airway progenitor cells into goblet cells was examined by organoid cultures, immunofluorescence staining, quantitative PCR, and cell transplantation in normal, DJ-1 knockout (KO), or conditional DJ-1 KO mice. This study observed that DJ-1 was increased in the lung tissues of ovalbumin-sensitized and challenged mice. DJ-1 KO mice exhibited reduced airway eosinophil infiltration and goblet cell differentiation. Mechanistically, we discovered that eosinophil-club cell interactions are reduced in the absence of DJ-1. Organoid cultures indicated that eosinophils impair the proliferative potential of club cells. Intratracheal transplantation of DJ-1-deficient eosinophils suppresses airway goblet cell differentiation. Loss of DJ-1 inhibits the metabolism of arachidonic acid into cysteinyl leukotrienes in eosinophils while these secreted metabolites promote airway goblet cell fate in organoid cultures and invivo. DJ-1-mediated interactions between airway epithelial progenitor cells and immune cells are essential in controlling airway goblet cell metaplasia and eosinophilia. Blockade of the DJ-1 pathway is protective against airway allergic inflammation.

Intratracheal Transplantation of Mesenchymal Stem Cells Attenuates Hyperoxia-Induced Microbial Dysbiosis in the Lungs, Brain, and Gut in Newborn Rats.

We attempted to determine whether intratracheal (IT) transplantation of mesenchymal stem cells (MSCs) could simultaneously attenuate hyperoxia-induced lung injuries and microbial dysbiosis of the lungs, brain, and gut in newborn rats. Newborn rats were exposed to hyperoxia (90% oxygen) for 14 days. Human umbilical cord blood-derived MSCs (5 × 105) were transplanted via the IT route on postnatal day (P) five. At P14, the lungs were harvested for histological, biochemical, and microbiome analyses. Bacterial 16S ribosomal RNA genes from the lungs, brain, and large intestine were amplified, pyrosequenced, and analyzed. IT transplantation of MSCs simultaneously attenuated hyperoxia-induced lung inflammation and the ensuing injuries, as well as the dysbiosis of the lungs, brain, and gut. In correlation analyses, lung interleukin-6 (IL-6) levels were significantly positively correlated with the abundance of Proteobacteria in the lungs, brain, and gut, and it was significantly inversely correlated with the abundance of Firmicutes in the gut and lungs and that of Bacteroidetes in the lungs. In conclusion, microbial dysbiosis in the lungs, brain, and gut does not cause but is caused by hyperoxic lung inflammation and ensuing injuries, and IT transplantation of MSCs attenuates dysbiosis in the lungs, brain, and gut, primarily by their anti-oxidative and anti-inflammatory effects.

Intratracheal transplantation of trophoblast stem cells attenuates acute lung injury in mice

BackgroundAcute lung injury (ALI) is a common lung disorder that affects millions of people every year. The infiltration of inflammatory cells into the lungs and death of the alveolar epithelial cells are key factors to trigger a pathological cascade. Trophoblast stem cells (TSCs) are immune privileged, and demonstrate the capability of self-renewal and multipotency with differentiation into three germ layers. We hypothesized that intratracheal transplantation of TSCs may alleviate ALI.MethodsALI was induced by intratracheal delivery of bleomycin (BLM) in mice. After exposure to BLM, pre-labeled TSCs or fibroblasts (FBs) were intratracheally administered into the lungs. Analyses of the lungs were performed for inflammatory infiltrates, cell apoptosis, and engraftment of TSCs. Pro-inflammatory cytokines/chemokines of lung tissue and in bronchoalveolar lavage fluid (BALF) were also assessed.ResultsThe lungs displayed a reduction in cellularity, with decreased CD45+ cells, and less thickening of the alveolar walls in ALI mice that received TSCs compared with ALI mice receiving PBS or FBs. TSCs decreased infiltration of neutrophils and macrophages, and the expression of interleukin (IL) 6, monocyte chemoattractant protein-1 (MCP-1) and keratinocyte-derived chemokine (KC) in the injured lungs. The levels of inflammatory cytokines in BALF, particularly IL-6, were decreased in ALI mice receiving TSCs, compared to ALI mice that received PBS or FBs. TSCs also significantly reduced BLM-induced apoptosis of alveolar epithelial cells in vitro and in vivo. Transplanted TSCs integrated into the alveolar walls and expressed aquaporin 5 and prosurfactant protein C, markers for alveolar epithelial type I and II cells, respectively.ConclusionIntratracheal transplantation of TSCs into the lungs of mice after acute exposure to BLM reduced pulmonary inflammation and cell death. Furthermore, TSCs engrafted into the alveolar walls to form alveolar epithelial type I and II cells. These data support the use of TSCs for the treatment of ALI.

Type 2 Alveolar Epithelial Cells Differentiated from Human Umbilical Cord Mesenchymal Stem Cells Alleviate Mouse Pulmonary Fibrosis Through β-Catenin-Regulated Cell Apoptosis.

Pulmonary fibrosis (PF) is a chronic, progressive, and lethal disease with little response to available therapies. One of the major mechanisms of PF is the repeated injury and inadequate regeneration of the alveolar epithelium. In this study, we induced human umbilical cord mesenchymal stem cells (hUC-MSCs) to differentiate into type 2 alveolar epithelial cells (AEC2s), and we provided evidence that intratracheal transplantation of hUC-MSC-derived AEC2s (MSC-AEC2s) could improve mortality and alleviate fibrosis in bleomycin-induced PF mice. Transplantation of MSC-AEC2s could increase the AEC2 cell count in these mice, and the results of the cell tracing experiment exhibited that the increased AEC2s originated from the self-renewal of mouse alveolar epithelium. The AEC2 survival was controlled by the apoptosis of AEC2s via the expression of β-catenin in PF mice. In in vitro experiments, MSC-AEC2s could alleviate the apoptosis of MLE-12 cells induced by transforming growth factor beta (TGF-β1), which could be eliminated by using PRI-724, a β-catenin inhibitor, suggesting β-catenin signaling involved in the protection against apoptosis provided by MSC-AEC2s. Our study demonstrated that MSC-AEC2s could protect PF mice through regulating apoptosis mediated by β-catenin, which provided a viable strategy for the treatment of PF.

Induced pluripotent stem cell-derived lung alveolar epithelial type II cells reduce damage in bleomycin-induced lung fibrosis

BackgroundIdiopathic pulmonary fibrosis is a chronic, progressive, and severe disease with a limited response to currently available therapies. Epithelial cell injury and failure of appropriate healing or regeneration are central to the pathogenesis of idiopathic pulmonary fibrosis. The purpose of this study is to investigate whether intratracheal transplantation of alveolar type II-like cells differentiated from induced pluripotent stem cells can stop and reverse the fibrotic process in an experimental model of bleomycin-induced lung fibrosis in rats.MethodsHuman induced pluripotent stem cells were differentiated to alveolar type II-like cells and characterized. Lung fibrosis was induced in rats by a single intratracheal instillation of bleomycin. Animals were transplanted with human induced pluripotent stem cells differentiated to alveolar type II-like cells at a dose of 3 × 106 cells/animal 15 days after endotracheal bleomycin instillation when the animal lungs were already fibrotic. Animals were sacrificed 21 days after the induction of lung fibrosis. Lung fibrosis was assessed by hydroxiprolin content, histologic studies, and the expression of transforming growth factor-β and α-smooth muscle actin.ResultsCell transplantation of alveolar type II-like cells differentiated from induced pluripotent stem cells can significantly reduce pulmonary fibrosis and improve lung alveolar structure, once fibrosis has already formed. This is associated with the inhibition of transforming growth factor-β and α-smooth muscle actin in the damaged rat lung tissue.ConclusionTo our knowledge, this is the first data to demonstrate that at the fibrotic stage of the disease, intratracheal transplantation of human induced pluripotent differentiated to alveolar type II-like cells halts and reverses fibrosis.

Intratracheal Instillation of Stem Cells in Term Neonatal Rats.

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26-28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Intratracheal Transplantation of Amnion-Derived Mesenchymal Stem Cells Ameliorates Hyperoxia-Induced Neonatal Hyperoxic Lung Injury via Aminoacyl-Peptide Hydrolase.

Background and ObjectivesBronchopulmonary dysplasia (BPD) has major effects in premature infants. Although previous literature has indicated that mesenchymal stem cells (MSCs) can alleviate lung pathology in BPD newborns and improve the survival rate, few research have been done investigating significantly differentially expressed genes in the lungs before and after MSCs therapy. The aim of this study is to identify differentially expressed genes in lung tissues before and after hAD-MSC treatment.Methods and ResultsHuman amnion-derived MSCs (hAD-MSCs) were cultured and met the MSCs criteria for cell phenotype and multidirectional differentiation. Then we confirmed the size of hAD-MSCs-EXOs and their expressed markers. An intratracheal drip of living cells showed the strongest effect on NHLI compared to cellular secretions or exosomes, both in terms of ameliorating pulmonary edema and reducing inflammatory cell infiltration. Through gene chip hybridization, PCR, and western blotting, acylaminoacyl-peptide hydrolase (APEH) expression was found to be significantly decreased under hyperoxia, and significantly increased after hAD-MSC treatment.ConclusionsThe intratracheal transplantation of hAD-MSCs ameliorated NHLI in neonatal rats through APEH.

M2 macrophages promote pulmonary endothelial cells regeneration in sepsis-induced acute lung injury.

Macrophages can polarize to M2 phenotype to decrease inflammation and encourage tissue repair. Nonetheless, its role in sepsis-induced acute lung injury and its effect on endothelial cells (ECs) regeneration remains unknown. The aim of the current study was to explore the impact of M2 macrophages on pulmonary ECs proliferation in sepsis-induced acute lung injury. We co-cultured mouse lung microvascular endothelial cells (MLMVECs) with M2 macrophages following LPS challenge. M2 macrophages were intratracheally transplanted into mice subjected to cecal ligation and puncture (CLP). We further performed cytokine array for the supernatant from M2 macrophages and serum from mice subjected with CLP. We found both co-culture with M2 macrophages and treating with supernatant from M2 macrophages increased ECs viability following LPS challenge. Intratracheal transplantation of M2 macrophages markedly promoted pulmonary ECs proliferation, manifesting as attenuation of lung microvascular permeability and lung tissue edema, as well as improvement of survival rate. We further found that CXCL12, IL-1ra, TIMP-1, IL-4, and CXCL1 were increased in the supernatant of M2 macrophages in vitro. G-CSF and Complement Component 5a (C5/C5a) were increased in the serum of the M2-transplanted mice. The present study suggested M2 macrophages could promote ECs proliferation in sepsis-induced ALI through secretion of anti-inflammatory cytokines and growth factors.

Intratracheal transplantation of mesenchymal stem cells attenuates hyperoxia-induced lung injury by down-regulating, but not direct inhibiting formyl peptide receptor 1 in the newborn mice.

Formyl peptide receptor 1 (FPR1) has been shown to be a key regulator of inflammation. However, its role in bronchopulmonary dysplasia (BPD) has not been delineated yet. We investigated whether FPR1 plays a pivotal role in regulating lung inflammation and injuries, and whether intratracheally transplanted mesenchymal stem cells (MSCs) attenuate hyperoxic lung inflammation and injuries by down-regulating FPR1. Newborn wild type (WT) or FPR1 knockout (FPR1-/-) C57/BL6 mice were randomly exposed to 80% oxygen or room air for 14 days. At postnatal day (P) 5, 2×105 MSCs were intratracheally transplanted. At P14, mice were sacrificed for histopathological and morphometric analyses. Hyperoxia significantly increased lung neutrophils, macrophages, and TUNEL-positive cells, while impairing alveolarization and angiogenesis, along with a significant increase in FPR1 mRNA levels in WT mice. The hyperoxia-induced lung inflammation and lung injuries were significantly attenuated, with the reduced mRNA level of FPR1, in WT mice with MSC transplantation and in FPR1-/- mice, irrespective of MSCs transplantation. However, only MSC transplantation, but not the FPR1 knockout, significantly attenuated the hyperoxia-induced increase in TUNEL-positive cells. Our findings indicate that FPR1 play a critical role in regulating lung inflammation and injuries in BPD, and MSCs attenuate hyperoxic lung inflammation and injuries, but not apoptosis, with down regulating, but not direct inhibiting FPR1.

Anti-inflammatory Role of Mesenchymal Stem Cells in an Acute Lung Injury Mouse Model.

Background Mesenchymal stem cells (MSCs) attenuate injury in various lung injury models through paracrine effects. We hypothesized that intratracheal transplantation of allogenic MSCs could attenuate lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice, mediated by anti-inflammatory responses.Methods Six-week-old male mice were randomized to either the control or the ALI group. ALI was induced by intratracheal LPS instillation. Four hours after LPS instillation, MSCs or phosphate-buffered saline was randomly intratracheally administered. Neutrophil count and protein concentration in bronchoalveolar lavage fluid (BALF); lung histology; levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein-2; and the expression of proliferation cell nuclear antigen (PCNA), caspase-3, and caspase-9 were evaluated at 48 hours after injury.Results Treatment with MSCs attenuated lung injury in ALI mice by decreasing protein level and neutrophil recruitment into the BALF and improving the histologic change. MSCs also decreased the protein levels of proinflammatory cytokines including IL-1β, IL-6, and TNF-α, but had little effect on the protein expression of PCNA, caspase-3, and caspase-9.Conclusions Intratracheal injection of bone marrow-derived allogenic MSCs attenuates LPS-induced ALI via immunomodulatory effects.

Therapeutic Effects of Human Umbilical Cord–Derived Mesenchymal Stem Cells on Canine Radiation-Induced Lung Injury

To investigate the effect of human umbilical cord-derived mesenchymal stem cell (MSC) transplantation on canine radiation-induced lung injury. Beagle dogs received localized 15-Gy x-ray radiation to the right lower lung to establish the model of radiation-induced lung injury. After 180 days, dogs were divided into 2 groups (4 per group). The MSC group received intratracheal MSC transplantation, and the saline group received the same volume of normal saline by lavage. The effect of MSC transplantation on lung injury was then evaluated 180 days after transplantation. At 180 days after 15-Gy radiation, canine arterial blood oxygen partial pressure was significantly decreased, and the levels of hydroxyproline and transforming growth factor (TGF)-β in peripheral blood were significantly increased, whereas that of TGF-α was significantly decreased. Computed tomography evaluation revealed visible honeycomb shadows in the right middle and lower pulmonary pleurae. Blood oxygen partial pressure of the MSC group gradually increased over time, whereas the levels of hydroxyproline and TGF-β in the peripheral blood showed a decreasing trend; TGF-α levels gradually increased, which differed significantly from the results observed in the saline group. In addition, computed tomography and pathologic examination showed that the degree of lung injury in the MSC group was milder. The MSC group also showed significantly increased pulmonary superoxide dismutase levels and significantly decreased tumor necrosis factor-α, Interleukein-1, and hyaluronic acid levels. Further study confirmed that MSC transplantation inhibited the activation of TGF-β-Smad2/3 in lung tissues, and invitro experiments showed that medium conditioned with MSCs effectively inhibited the increase in Smad2 and 3 levels induced by TGF-β1. Canine radiation-induced lung injury could be observed at 180 days afterradiation at 15 Gy. MSC transplantation can reduce oxidative stress, inflammatory reactions, and TGF-β-Smad2/3 pathway activation, thereby reducing lung injury.

Human mesenchymal stem cells ameliorate experimental pulmonary hypertension induced by maternal inflammation and neonatal hyperoxia in rats.

Pulmonary hypertension is a critical problem in infants with bronchopulmonary dysplasia. This study determined the therapeutic effects of human mesenchymal stem cells (MSCs) on pulmonary hypertension in an animal model. Pregnant Sprague–Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS, 0.5 mg/kg/day) on gestational days 20 and 21. The pups were randomly assigned to two treatment conditions: room air (RA) or an O2-enriched atmosphere. On postnatal day 5, they were intratracheally transplanted with human MSCs (3 × 105 and 1 × 106 cells) in 0.03 mL of normal saline (NS). Five study groups were examined: normal, LPS+RA+NS, LPS+O2+NS, LPS+O2+MSCs (3 × 105 cells), and LPS+O2+MSCs (1 × 106 cells). On postnatal day 14, the pup lungs and hearts were collected for histological examinations. The LPS+RA+NS and LPS+O2+NS groups exhibited a significantly higher right ventricle (RV):left ventricle (LV) thickness ratio and medial wall thickness (MWT) and higher β-myosin heavy chain (β-MHC) and toll-like receptor (TLR) 4 expression than did the normal group. Human MSC transplantation in LPS- and O2-treated rats reduced the MWT, RV:LV thickness ratio, and β-MHC and TLR4 expression to normal levels. Thus, intratracheal human MSC transplantation ameliorates pulmonary hypertension, probably by suppressing TLR4 expression in newborn rats.

Two-Year Follow-Up Outcomes of Premature Infants Enrolled in the Phase I Trial of Mesenchymal Stem Cells Transplantation for Bronchopulmonary Dysplasia

To determine the long-term safety and outcomes of mesenchymal stem cells (MSCs) for bronchopulmonary dysplasia in premature infants enrolled in a previous phase I clinical trial up to 2 years of corrected age (CA). We assessed serious adverse events, somatic growth, and respiratory and neurodevelopmental outcomes at visit 1 (4-6 months of CA), visit 2 (8-12 months of CA), and visit 3 (18-24 months of CA) in a prospective longitudinal follow-up study up to 2 years' CA of infants who received MSCs (MSC group). We compared these data with those from a historical case-matched comparison group. One of 9 infants in the MSC group died of Enterobacter cloacae sepsis at 6 months of CA, the remaining 8 infants survived without any transplantation-related adverse outcomes, including tumorigenicity. No infant in the MSC group was discharged with home supplemental oxygen compared with 22% in the comparison group. The average rehospitalization rate in the MSC group was 1.4/patient because of respiratory infections during 2 years of follow-up. The mean body weight of the MSC group at visit 3 was significantly higher compared with that of the comparison group. No infant in the MSC group was diagnosed with cerebral palsy, blindness, or developmental delay; in the comparison group, 1 infant was diagnosed with cerebral palsy and 1 with developmental delay. Intratracheal transplantation of MSCs in preterm infants appears to be safe, with no adverse respiratory, growth, and neurodevelopmental effects at 2 years' CA. ClinicalTrials.gov: NCT01632475.

Intratracheal transplantation of endothelial progenitor cells attenuates smoking-induced COPD in mice.

BackgroundEndothelial progenitor cells (EPCs) might play a protective role in COPD. The aim of this study was to investigate whether intratracheal allogeneic transplantation of bone-marrow-derived EPCs would attenuate the development of smoking-induced COPD in mice.MethodsIsolated mononuclear cells from the bone marrow of C57BL/6J mice were cultured in endothelial cell growth medium-2 for 10 days, yielding EPCs. A murine model of COPD was established by passive 90-day exposure of cigarette smoke. On day 30, EPCs or phosphate-buffered saline alone was administered into the trachea. On day 90, EPCs or 30 μL phosphate-buffered saline alone was administered into the trachea, and on day 120, inflammatory cells, antioxidant activity, apoptosis, matrix metalloproteinase (MMP)-2, and MMP-9 were measured.ResultsAfter EPC treatment, the lung function of the mice had improved compared with the untreated mice. Mean linear intercept and destructive index were reduced in the EPCs-treated group compared with the untreated group. In addition, the EPCs-treated mice exhibited less antioxidant activity in bronchoalveolar lavage fluid compared with the untreated mice. Moreover, decreased activities of MMP-2, MMP-9, and TUNEL-positive cells in lung tissues were detected in EPCs-treated mice.ConclusionIntratracheal transplantation of EPCs attenuated the development of pulmonary emphysema and lung function disorder probably by alleviating inflammatory infiltration, decelerating apoptosis, inhibiting proteolytic enzyme activity, and improving antioxidant activity.

Human mesenchymal stem cells attenuate pulmonary hypertension induced by prenatal lipopolysaccharide treatment in rats.

Intra-amniotic injection of lipopolysaccharide (LPS) induces pulmonary hypertension in newborn rats. This study was designed to test whether human mesenchymal stem cells (MSCs) reduce pulmonary hypertension and alleviate cardiac hypertrophy in prenatal LPS-treated rats. Pregnant Sprague-Dawley rats were injected intraperitoneally with LPS (0.5mg/kg per day) or untreated on gestational days 20 and 21. Human MSCs (3×10(5) cells and 1×10(6) cells) in 0.03mL of normal saline (NS) were transplanted intratracheally on postnatal day 5. Four study groups were considered: normal, LPS+NS, LPS+MSCs (3×10(5) cells), and LPS+MSCs (1×10(6) cells). On postnatal day 14, lung and heart tissues were collected for measuring the arterial medial wall thickness (MWT) and β-myosin heavy chain (β-MHC) level as markers of pulmonary hypertension and cardiac hypertrophy, respectively. The LPS+NS group exhibited a significantly higher right ventricle (RV)/[left ventricle (LV)+ interventricular septum (IVS)] thickness ratio and MWT, a greater cardiomyocyte width, a greater number of cardiomyocyte nuclei per squared millimeter, and higher β-MHC expression than those observed in the normal group. Human MSC transplantation (3×10(5) cells and 1×10(6) cells) in LPS-treated rats reduced MWT and the RV/(LV+IVS) thickness ratio to normal levels. This improvement in right ventricular hypertrophy was accompanied by a decrease in toll-like receptor 4 (TLR4), nuclear factor-κB, and tumor necrosis factor-α expression in the heart. Intratracheal human MSCs transplantation can attenuate pulmonary hypertension and right ventricular hypertrophy in prenatal LPS-treated rats; this attenuation may be associated with suppression of TLR4 expression via paracrine pathways.

Intratracheal transplantation of mesenchymal stem cells simultaneously attenuates both lung and brain injuries in hyperoxic newborn rats.

Bronchopulmonary dysplasia is an independent risk factor for adverse neurodevelopmental outcomes in premature infants. We investigated whether attenuation of hyperoxic lung injury with intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells (MSCs) could simultaneously mitigate brain damage in neonatal rats. Newborn Sprague-Dawley rats were exposed to hyperoxia or normoxia conditions for 14 d. MSCs (5 × 10(5) cells) were transplanted intratracheally at postnatal day (P) 5. At P14, lungs and brains were harvested for histological and biochemical analyses. Hyperoxic lung injuries, such as impaired alveolarization evident from increased mean linear intercept (MLI) and elevated inflammatory cytokine levels were significantly alleviated with MSC transplantation. Hyperoxia decreased brain weight, increased brain cell death, and induced hypomyelination. MSC transplantation significantly ameliorated hyperoxia-induced increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the dentate gyrus and reduced myelin basic protein. In correlation analyses, brain weight and myelin basic protein (MBP) were significantly inversely correlated with lung MLI and inflammatory cytokines, while TUNEL-positive brain cell number showed a significant positive correlation with lung MLI. Despite no significant improvement in short-term neurofunctional outcome, intratracheal transplantation of MSCs simultaneously attenuated hyperoxic lung and brain injuries in neonatal rats, with the extent of such attenuation being closely linked in the two tissues.

Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease withlimited response to currently available therapies. Alveolar type II (ATII) cells act as progenitor cells in the adult lung, contributing to alveolar repair during pulmonary injury. However, in IPF, ATII cells die and are replaced by fibroblasts and myofibroblasts. In previous preclinical studies, we demonstrated that ATII-cell intratracheal transplantation was able to reduce pulmonary fibrosis. The main objective of this study was to investigate the safety and tolerability of ATII-cell intratracheal transplantation in patients with IPF. We enrolled 16 patients with moderate and progressive IPF who underwent ATII-cell intratracheal transplantation through fiberoptic bronchoscopy. We evaluated the safety and tolerability of ATII-cell transplantation by assessing the emergent adverse sideeffects that appeared within 12months. Moreover, pulmonary function, respiratory symptoms, and disease extent during 12months of follow-up were evaluated. No significant adverse events were associated with the ATII-cell intratracheal transplantation. After 12months of follow-up, there was no deterioration in pulmonary function, respiratory symptoms, or disease extent. Our results support the hypothesis that ATII-cell intratracheal transplantation is safe and well tolerated in patients with IPF. This study opens the door to designing a clinical trial to elucidate the potential beneficial effects of ATII-cell therapy in IPF.

Antibacterial effect of mesenchymal stem cells against Escherichia coli is mediated by secretion of beta- defensin- 2 via toll- like receptor 4 signalling.

SummaryRecently, we demonstrated that intratracheal transplantation of human umbilical cord blood‐ derived mesenchymal stem cells (MSCs) attenuates Escherichia (E) coli‐ induced acute lung injury primarily by down‐ modulating inflammation and enhancing bacterial clearance iQn mice. This study was performed to elucidate the mechanism underlying the antibacterial effects of MSCs. The growth of E. coli in vitro was significantly inhibited only by MSCs or their conditioned medium with bacterial preconditioning, but not by fibroblasts or their conditioned medium. Microarray analysis identified significant up‐ regulation of toll‐ like receptors (TLR)‐ 2 and TLR‐ 4, and β‐ defensin 2 (BD2) in MSCs compared with fibroblasts after E. coli exposure. The increased BD2 level and the in vitro antibacterial effects of MSCs were abolished by specific antagonist or by siRNA‐ mediated knockdown of TLR‐ 4, but not TLR‐ 2, and restored by BD2 supplementation. The in vivo down‐ modulation of the inflammatory response and enhanced bacterial clearance, increased BD2 secretion and the resultant protection against E. coli‐ induced pneumonia observed only with MSCs, but not fibroblasts, transplantation in mice, were abolished by knockdown of TLR‐ 4 with siRNA transfection. Our data indicate that BD2 secreted by the MSCs via the TLR‐ 4 signalling pathway is one of the critical paracrine factors mediating their microbicidal effects against E. coli, both in vitro and in vivo. Furthermore, TLR‐ 4 from the transplanted MSCs plays a seminal role in attenuating in vivo E. coli‐ induced pneumonia and the ensuing acute lung injury through both its anti‐ inflammatory and antibacterial effects.

Efficient intratracheal delivery of airway epithelial cells in mice and pigs.

Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases.

Critical Role of Vascular Endothelial Growth Factor Secreted by Mesenchymal Stem Cells in Hyperoxic Lung Injury

Intratracheal transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) protects against neonatal hyperoxic lung injury by a paracrine rather than a regenerative mechanism. However, the role of paracrine factors produced by the MSCs, such as vascular endothelial growth factor (VEGF), has not been delineated. This study examined whether VEGF secreted by MSCs plays a pivotal role in protecting against neonatal hyperoxic lung injury. VEGF was knocked down in human UCB-derived MSCs by transfection with small interfering RNA specific for human VEGF. The in vitro effects of MSCs with or without VEGF knockdown or neutralizing antibody were evaluated in a rat lung epithelial (L2) cell line challenged with H2O2. To confirm these results in vivo, newborn Sprague-Dawley rats were exposed to hyperoxia (90% O2) for 14 days. MSCs (1 × 10(5) cells) with or without VEGF knockdown were administered intratracheally at postnatal Day 5. Lungs were serially harvested for biochemical and histologic analyses. VEGF knockdown and antibody abolished the in vitro benefits of MSCs on H2O2-induced cell death and the up-regulation of inflammatory cytokines in L2 cells. VEGF knockdown also abolished the in vivo protective effects of MSCs in hyperoxic lung injury, such as the attenuation of impaired alveolarization and angiogenesis, reduction in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive and ED-1-positive cells, and down-regulation of proinflammatory cytokine levels. Our data indicate that VEGF secreted by transplanted MSCs is one of the critical paracrine factors that play seminal roles in attenuating hyperoxic lung injuries in neonatal rats.