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Original Research: CRITICAL CARE |

IV Delivery of Induced Pluripotent Stem Cells Attenuates Endotoxin-Induced Acute Lung Injury in MiceInduced Pluripotent Stem Cells and Lung Injury FREE TO VIEW

Kuang-Yao Yang, MD, PhD; Hsin-Chin Shih, MD, PhD; Chorng-Kuang How, MD; Cheng-Yu Chen, MD, PhD; Han-Shui Hsu, MD, PhD; Ching-Wen Yang, BS; Yu-Chin Lee, MD, FCCP; Reury-Perng Perng, MD, PhD, FCCP; Chi-Hsien Peng, MD; Hsin-Yang Li, MD, PhD; Chia-Ming Chang, MD; Chung-Yuan Mou, PhD; Shih-Hwa Chiou, MD, PhD
Author and Funding Information

From the Department of Chest Medicine (Drs K.-Y. Yang, Lee, and Perng), the Department of Emergency Medicine (Drs Shih and How), the Department of Surgery (Dr Hsu), the Department of Medical Research and Education (Ms C.-W. Yang and Dr Chiou), the Department of Obstetrics and Gynecology (Drs Li and Chang), Taipei Veterans General Hospital; Institute of Clinical Medicine (Drs K.-Y. Yang, Shih, How, Chen, Peng, Li, and Chiou), Institute of Emergency and Critical Care Meicine (Drs Shih and How), Institute of Oral Biology (Dr Chang), and Institute of Pharmacology (Dr Chiou), School of Medicine (Drs K.-Y. Yang, Chen, Hsu, Lee, Perng, Li, Chang, and Chiou), National Yang-Ming University; and the Department of Medical Research and Education (Dr Chen), National Yang-Ming University Hospital; Shin Kong Wu Ho-Su Memorial Hospital and Fu-Jen Catholic University (Dr Peng); and the Department of Chemistry, College of Science (Dr Mou), National Taiwan University, Taipei, Taiwan, China.

Correspondence to: Shih-Hwa Chiou, MD, PhD, Department of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Rd, Taipei, 11217, Taiwan; e-mail: kyyang@vghtpe.gov.tw


Drs K.-Y. Yang and Chiou contributed equally to this work.

Funding/Support: This work was supported, in part, by Taiwan National Science Council Research Grants [NSC 97-3111-B-075-001-MY3, NSC 97-2314-B-075-046, NSC 100-2325-B-010-010, NSC 100-2314-B-075-047-MY3], Department of Health [DOH100-TD-C-111-007], Taipei Veterans General Hospital [V97C1-143, V98C1-072, V99C1-167, V100C-159, V97B1-006, E1-008, and F-001], the Joint Projects of UTVGH [VGHUST 98-G6-6], Yen-Tjing-Ling Medical Foundation, and National Yang-Ming University (Ministry of Education, Aim for the Top University Plan).

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/site/misc/reprints.xhtml).


© 2011 American College of Chest Physicians


Chest. 2011;140(5):1243-1253. doi:10.1378/chest.11-0539
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Background:  Induced pluripotent stem (iPS) cells are novel stem cell populations, but the role of iPS cells in acute lung injury (ALI) is not currently known. We investigated the effect of iPS cells in modifying the pathophysiology of endotoxin-induced ALI.

Methods:  Male C57BL/6 8- to 12-week-old mice were enrolled in this study. Mouse iPS cells were delivered through the tail veins of mice 4 h after intratracheal instillation of endotoxin. Lung histopathologic findings, the pulmonary levels of cytokines, and functional parameters were analyzed after either 24 h or 48 h.

Results:  More iPS cells integrated into the lungs of mice with ALI than those of the control mice, as demonstrated by in vivo radionuclide imaging and in vitro Hoechst-labeled fluorescent staining. iPS cells significantly diminished the histopathologic changes of ALI and the lung injury score. There was also a significant reduction in the activity of nuclear factor-κB (NF-κB) and neutrophil accumulation in the lung, confirmed by immunostaining, electrophoretic mobility shift assays, and the decrease of myeloperoxidase activity, in the iPS-cell-treated mice with ALI. These protective effects were not replicated by the control cell therapy with fibroblasts. iPS cells mediated a downregulation of the proinflammatory response to endotoxin (reducing tumor necrosis factor-α, IL-6, and macrophage inflammatory peptide-2). In addition, iPS cells rescued the hypoxemia and pulmonary function of ALI. Treatment with a conditioned medium of iPS cells showed effects similar to those of iPS cells, which may suggest the therapeutic benefits of iPS mediated by paracrine factors.

Conclusions:  IV delivery of iPS cells provides a beneficial effect to attenuate the severity of endotoxin-induced ALI and improve physiologic impairment, which is partly mediated by a reduction in NF-κB activity and neutrophils accumulation. The conditioned medium of iPS cells demonstrated effects equal to those of iPS cells.

Figures in this Article

Acute lung injury (ALI) is characterized by neutrophil accumulation in the lungs, interstitial edema, disruption of epithelial integrity, and leakage of protein into the alveolar space.14 Infection, associated with endotoxemia, is a frequent predisposing factor in the development of ALI.1 Neutrophils play a central role in this acute pulmonary inflammatory process, because their elimination can prevent the development of ALI.5 The neutrophils present in the lungs during ALI produce inflammatory mediators, including cytokines such as IL-6 and macrophage inflammatory peptide-2 (MIP-2), and demonstrate increased activation of transcriptional regulatory factors, including nuclear factor-κB (NF-κB).58 Binding elements for NF-κB are present in the enhancer/promoter regions of cytokine genes, such as MIP-2 and tumor necrosis factor-α (TNF-α), as well as other important immunoregulatory molecules.9 Inhibition of NF-κB activation prevents endotoxin-induced increases in proinflammatory cytokine expression in the lungs.10

Induced pluripotent stem (iPS) cells are novel stem cell populations induced from mouse and human adult somatic cells through reprogramming by transduction of defined transcription factors.11,12 These studies further suggest that iPS cells are indistinguishable from embryonic stem cells (ESC) in morphology, proliferative abilities, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity.13,14 iPS cells share the same features as ESC and are capable of self-renewal and differentiation into three germ layers, offering the potential for clinical cell therapies.11 iPS cells can be derived from the patient’s somatic cells to avoid potential immune rejection. Therefore, iPS cells are regarded as candidates for cell therapy and are used for autologous transplantation without the risk of rejection. Notably, Tsuji et al15 provided the evidence that iPS-derived neural stem cells can produce functional neurons and promote locomotor function recovery in a mouse spinal cord injury model. Our recent study also showed that iPS cells present the capability of multilineage differentiation16 and further reduce the severity of cerebral ischemic injury.17 Although these findings may suggest that iPS cells can be a therapeutic resource of cell transplantation, the roles of iPS cells in the immunomodulation in the transplanted host organs are still undetermined.

Although transplant of bone marrow-derived mesenchymal stem cells (MSCs)1820 or ESC21 for the cell therapy of ALI has been attempted, the role of iPS cells in ALI has never been explored. In the present study, we first transplanted iPS cells into endotoxin-induced ALI mice to determine the homing potential of iPS cells to injured lungs. Next, we investigated their role in modifying the inflammatory process of ALI, based on the results of lung histopathologic findings, the expression of inflammatory cytokines, and functional parameters. This novel cellular therapy opened an era of cell-based transplantation in ALI.

Mice

Male C57BL/6 mice, 8 to 12 weeks of age, were purchased from the National Experimental Animal Center (Taipei, Taiwan). The mice were kept on a 12-h light/dark cycle, with free access to food and water. All experiments were conducted in accordance with institutional review board-approved protocols.

Mouse iPS Cells

Murine-iPS cells were generated from mouse embryonic fibroblasts (MEFs) derived from C57BL/6 mice. The iPS cells were reprogrammed by the transduction of retroviral vectors encoding four transcription factors, Oct-4, Sox2, c-Myc, and Klf4, as described previously.11,16,17 Details are provided in e-Appendix 1.

Experimental Design

The model of endotoxin-induced ALI was modified from a previous report20 and was as described next. After anesthesia, the mice received an intratracheal injection of endotoxin (Escherichia coli 0111:B4 endotoxin; Sigma; St. Louis, Missouri) at a dose of 4 mg/kg in 50-μL phosphate-buffered saline (PBS). A similar dose has been demonstrated previously to produce acute neutrophilic alveolitis, histologically consistent with ALI in mice.22 In addition, the control mice received an intratracheal injection of 50 μL PBS. In designated experiments, mice received either iPS cells (2 × 106 cells in 200 μL of PBS) (referred to as iPS-treated ALI mice) or PBS, 200 μL (referred to as PBS-treated ALI mice) via tail vein injection, 4 h after the induction of lung injury. These iPS cell loads have been used previously by our laboratory and have a rescue effect in brain injury.17 Further experiments were done in which non-reprogrammed MEFs, apoptotic iPS cells, and the conditioned medium from iPS cells (iPS-CM) were injected via tail vein 4 h after the induction of lung injury as additional control cell therapy (2 × 106 cells in 200 μL of PBS) and paracrine therapy (200 μL of iPS-CM). Apoptotic iPS cells were made following the protocol and achieving > 90% apoptosis (e-Appendix 1). At the end of either 24 or 48 h, samples were collected from each mouse for assessment of lung injury, histology, immunohistochemistry, electrophoretic mobility shift assays (EMSAs), cytokines and myeloperoxidase (MPO) analysis, and pulmonary physiology.

Histology and Immunohistochemistry

Lungs from each group were excised 24 and/or 48 h after ALI. Hematoxylin and eosin stain and immunohistochemistry stain were performed with the primary antibody Ly6C, total NF-κB p65 (Santa Cruz Biotechnology; Santa Cruz, California), and phospho-NF-κB p65 (Cell Signaling Technologies; Danvers, Massachusetts). Details are provided in e-Appendix 1.

Lung Injury Score

To quantify the severity of lung injury on histology, the lung injury score was assessed. Each hematoxylin-and-eosin-stained slide was evaluated by two separate investigators (K.-Y. Y. and S.-H. C.) in a blinded manner. To generate the lung injury score, a total of 300 alveoli were counted on each slide at × 400 magnification. Within each field, points were assigned according to the predetermined criteria used in a previous study.23

MPO Assay, Cytokine Enzyme-Linked Immunosorbent Assay, and EMSA

Excised lungs from mice from each treatment group were frozen in liquid nitrogen, weighed, and stored at −80°C. Lungs were homogenized in different buffer solutions for MPO, EMSA, and cytokine assay. MPO activity was assayed as reported previously24 and the supernatant was assayed for peroxidase activity corrected to lung weight. Protein content of the supernatants was determined using a bicinchoninic acid protein assay kit (Pierce; Rockford, Illinois). Immunoreactive TNF-α, IL-6, IL-10, and MIP-2 were quantitated using commercially available enzyme-linked immunosorbent assay kits (R&D Systems; Minneapolis, Minnesota). Nuclear extracts were prepared, and nuclear translocation of NF-κB was determined as described previously by our laboratory.5,25 The LightShift Chemiluminescent EMSA Kit (Pierce) was used to detect nuclear levels of NF-κB.

Coculture of Lipopolysaccharide-Stimulated Pulmonary Neutrophils and iPS Cells

To determine whether iPS cells were able to regulate production of inflammatory cytokines by stimulated neutrophils, by a cell-cell contact-dependent or independent mechanism, pulmonary neutrophils were cocultured with iPS cells in either a standard single well or a Transwell (Costar; Cambridge, Massachusetts) (Fig 1A) and then stimulated with lipopolysaccharide (LPS). Details are provided in e-Appendix 1.

Figure Jump LinkFigure 1. iPS cells moderate the production of MIP-2 by LPS-stimulated pulmonary neutrophils isolated from mice with acute lung injury (ALI) in a cell-to-cell contact-independent manner. A, MIP-2 was measured in the supernatant of the coculture system as described in the “Materials and Methods” section. B, Administration of iPS cells in the culture wells decreased the production of MIP-2 by LPS-stimulated pulmonary neutrophils, and this decrease was similar when a Transwell insert was placed to prevent direct cell-to-cell contact. Data are presented as mean ± SD. * and #P < .05 for comparison with the condition of neutrophils alone, n = 5 per condition. iPS = induced pluripotent stem; LPS = lipopolysaccharide; MIP-2 = macrophage inflammatory peptide-2.Grahic Jump Location
Pulmonary Function Measurements: Arterial Blood Gas and Plethysmography

Arterial blood gas was sampled from the left ventricle of the mice. Pulmonary function, including tidal volume and Penh (airway resistance), was evaluated using whole-body plethysmography at a preset time course after the procedure. The mice were placed in a whole-body plethysmography chamber (Buxco Electronics; Shorn, Connecticut) to analyze respiratory waveforms. The mechanism of determining Penh has been described by Hamelmann et al.26 Details are provided in e-Appendix 1.

Statistical Analysis

To limit variability for each experimental condition, the entire group of mice was prepared and studied at the same time. Separate groups of mice were used for the lung injury score, immunostaining, MPO assay, EMSA, enzyme-linked immunosorbent assay, arterial blood gas, and plethysmography data. Data are presented as mean ± SEM or mean ± SD for each experimental group. One-way analysis of variance and the Tukey-Kramer Multiple Comparisons test (for multiple groups) or Student t test (for comparisons between two groups) were used. P < .05 was considered significant.

Characterization of MEF-Derived iPS Cells

In previous studies,16,17 we successfully established mouse-iPS cells from MEF. See e-Appendix 2 and e-Figure 1 for details.

IV Delivery of iPS Cells Had Homing Potential for Injured Lung Tissue

To investigate the homing potential, 131I-IdUrd-labeled iPS cells transplanted via tail vein injection were located using a planar γ camera (in vivo radionuclide imaging) and were obtained from anesthetized animals at 4 and 8 h after iPS cell delivery. More 131I-IdUrd-labeled iPS cells accumulated over the lung area in mice with endotoxin-induced ALI (LPS ± iPS) than in control mice (CON ± iPS) (Fig 2A). In addition, to explore the microscopic fate of transplanted cells, we labeled iPS cells with Hoechst and located Hoechst-positive cells in the mice with ALI or control mice. We observed Hoechst-labeled iPS cells located around the pulmonary vessels in both the mice with ALI and the control mice (data not shown). Of interest, after 24 h of iPS cell delivery, we found that Hoechst-labeled cells were more scattered in the injured lung sections of mice with ALI (LPS ± iPS) than in the lung sections of control mice (CON + iPS) (Fig 2B). To quantify the scattered density of the incorporated iPS cells in the lungs, a total of 10 lung sections of each sample were counted on each slide at ×100 magnification; the number of incorporated iPS cells was higher in the LPS-injured lungs (from mice with ALI) than in the control lungs (from control mice) at 24 h after iPS cell delivery, (cell count per lung section field in mean ± SD, 94.6 ± 10.6 vs 20.3 ± 5.1, P = .006) (Fig 2C).

Figure Jump LinkFigure 2. More transplanted iPS cells were trafficking in the injured lung in mice with ALI. A, Transplanted 131I-IdUrd-labeled iPS cells were located using in vivo radionuclide imaging (autoradiography) at 4 and 8 h after IV iPS administration in intratracheal LPS (ALI) or intratracheal phosphate-buffered saline (CON) mice. Selective uptake of 131I-IdUrd was observed in the lung area of the anesthetized mice, and there was more radio uptake accumulation in the lung area in the endotoxin-induced lung-injury mice (LPS + iPS) than in the control mice (CON + iPS). B, Transplanted Hoechst-labeled iPS cells were located using fluorescent microscopy (original magnification ×100) at 24 h after IV iPS administration in the mice with ALI or control mice. Isolated Hoechst-labeled iPS cells delivered by IV were noted to be more scattered in the injured lung area of mice with ALI (LPS + iPS) than in the lung area of control mice (CON + iPS). Bright light-blue staining represents the Hoechst-labeled cells. C, To quantify the scattered density of the incorporated iPS cells in the lungs at 24 h, the number of labeled iPS cells in the injured lungs (from mice with ALI) and control lungs (from control mice) was determined by counting Hoechst-labeled iPS cells in 10 lung sections of each sample; there were more transplanted iPS cells in the injured lungs (LPS + iPS) than in the control lungs (CON + iPS). Data are presented as mean ± SD. *P = .006, n = 6 per group. CON = control. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Effects of iPS Cells in the Histopathology of Endotoxin-Induced ALI

As shown in Figure 3A, endotoxin intratracheal injection resulted in significant ALI. All of these endotoxin-induced events included lung edema, coupled with neutrophil infiltration and elevations in inflammatory cytokine production (Fig 4), which is indicative of the development of ALI. To explore the role of iPS cells in ALI, we compared lung sections from ALI mice that underwent treatment with iPS cells, control cells (MEFs or apoptotic iPS cells), iPS-CM, or PBS via the tail vein. Histologic evaluations of iPS-cell-recipient lungs at 24 and 48 h after LPS-induced ALI showed that the numbers of infiltrative neutrophils and injury areas were dramatically reduced by iPS cell treatment compared with the PBS treatment. (Fig 3A). Furthermore, mice treated with iPS cells had a significantly lower lung injury score than did mice treated with PBS (Fig 3B). In contrast, mice that received the apoptotic iPS cells showed minimal effect, but MEFs had no effect on the severity of ALI compared with iPS-cell-treated mice at 24 h. However, mice treated with iPS-CM after ALI had a similar result to those treated with iPS cells (Fig 3).

Figure Jump LinkFigure 3. iPS cells improved lung injury as assessed by histologic methods. A, Hematoxylin and eosin staining of lung sections demonstrated attenuated lung injury in the mice receiving iPS cells at both 24 and 48 h after endotoxin-induced ALI. B, Histopathologic lung injury score showed a significant reduction in the mice with ALI receiving iPS cells compared with those receiving phosphate-buffered saline (PBS) treatment (LPS 24 h vs LPS + iPS 24 h, P = .039 and LPS 48 h vs LPS + iPS 48 h, P = .037). Minimal histopathologic abnormalities were also present in mice that were injected with intratracheal PBS (control group). In contrast, mice that received the MEF (LPS + MEF) or aiPS cells (LPS + aiPS) showed no improvement in the severity of acute lung injury induced by LPS compared with iPS-cell-treated mice at 24 h. However, mice treated with iPS-CM after ALI (LPS + iPS-CM) had a similar result to that with iPS cells (LPS + iPS). The score represents the average of the findings of two independent investigators who each read the hematoxylin-and-eosin-stained slide in a blinded manner. The categories used to generate the score were alveolar septal congestion, alveolar hemorrhage, intraalveolar fibrin deposition, and intraalveolar infiltrates. Data are presented as mean ± SD. *P< .05; n = 6 per group. aiPS = apoptotic induced pluripotent stem; iPS-CM = conditioned medium from induced pluripotent stem cell; MEF = mouse embryonic fibroblast. See Figure 1 and 2 legends for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 4. A-C, Levels of proinflammatory cytokines, TNF-α, IL-6, and MIP-2 were reduced in mice receiving iPS cells at both 24 and 48 h after endotoxin-induced ALI. MIP-2 (A), IL-6 (B), and TNF-α (C) were significantly reduced in the lungs of mice treated with iPS cells at both 24 and 48 h after ALI. Furthermore, mice treated with iPS-CM at 24 h after ALI had similar results to those with iPS cells. In contrast, mice that received the MEF (LPS + MEF) had increases in the levels of proinflammatory cytokines compared with iPS-cell-treated mice at 24 h after ALI. Data are presented as mean ± SD. *P < .05, n = 5 per group. TNF-α = tumor necrosis factor-α. See Figure 1-3 legends for expansion of other abbreviations.Grahic Jump Location
Transplanted iPS Cells Diminished Inflammatory Cell Accumulation and Decreased MPO Activity in the Lung

We administered the iPS cells after endotoxin-induced ALI and then determined the severity of inflammatory cell accumulation and the activity of MPO. As shown in Figure 5A, intratracheal LSP resulted in a significant increase in the accumulation of inflammatory cells in the lungs, which was demonstrated visually by Ly6C staining (positive for neutrophils and monocytes). In contrast, the addition of iPS cells transplanted through tail veins was associated with a significant decrease in this phenomenon. Quantification of Ly6C staining confirmed that iPS cells reduced the degree of inflammatory cell accumulation in the lung (Fig 5B). Moreover, iPS cells decreasing neutrophils burden was demonstrated by the reduction of MPO activity in the lungs (Fig 5C). In contrast, MEF administration did not diminish LPS-induced elevations in the MPO activity in the lung, and apoptotic iPS cells had only a small effect on it.

Figure Jump LinkFigure 5. iPS cell administration prevented neutrophil accumulation and decreased MPO activity after endotoxin-induced ALI. A, Neutrophil accumulation in the lung was significantly increased after endotoxin exposure. As was similarly concluded visually by Ly6C staining, transplant of iPS cells at 24 h after endotoxin-challenge significantly reduced neutrophil accumulation in the alveolus (original magnification × 400). B, Quantification of Ly6C staining showed a significant reduction in the degree of neutrophil accumulation in the ALI mice receiving iPS cells (LPS 24 h vs LPS + iPS 24 h, P = .013). C, iPS cells significantly decreased the activity of MPO after endotoxin-induced ALI, as determined by MPO assay at 24 and 48 h after injury. In contrast, MEFs, or a-iPS cell administration did not significantly diminish LPS-induced elevations in the MPO activity. Data are presented as mean ± SD. *P < .05, n = 5 per group. MPO = myeloperoxidase. See Figure 1-3 legends for expansion of other abbreviations.Grahic Jump Location
Transplanted iPS Cells Reduced the Activity of NF-κB in Endotoxin-Induced ALI

The effect of stem cells on the NF-κB activity of ALI has never been examined. The activity levels of NF-κB demonstrated by immunostaining of NF-κB p65 in the lung were significantly enhanced at 24 h after endotoxin-induced ALI, but iPS cell administration significantly reduced this phenomenon (Figs 6A, 6C). Similarly, the levels of phospho-NF-κB p65 in the lung were significantly lower in the iPS-cell-treated ALI mice compared with the mice with PBS-treated ALI (Fig 6B, 6D). In contrast, mice that received the MEF did not moderate the activation of NF-κB induced by ALI compared with iPS-cell-treated mice, but apoptotic iPS cells did partially diminish the activity of NF-κB p65. Furthermore, we performed an EMSA test to reconfirm nuclear translocation of NF-κB (Fig 7). NF-κB levels were significantly increased in nuclear extracts from lung tissues of ALI mice compared with those in control mice. In iPS-treated ALI mice, nuclear levels of NF-κB were significantly decreased as compared with ALI mice or MEF-treated mice. ALI mice that received iPS-CM had nuclear levels of NF-κB similar to those treated with iPS cells.

Figure Jump LinkFigure 6. iPS cell administration reduced the levels of NF-κB activity after endotoxin-induced ALI. A, Activity levels of NF-κB demonstrated by immunostaining of NF-κB p65 in the lung were significantly enhanced after endotoxin-induced ALI, but iPS cell administration significantly diminished this effect to levels near those of uninjured lungs. C, Quantification of NF-κB p65 staining showed a significant reduction in the activity levels of NF-κB p65 in the mice receiving iPS cells at 24 h after endotoxin-induced ALI (LPS 24 h vs LPS + iPS 24 h, P = .04). B and D, Activity levels of phospho-NF-κB (p-NF-κB) p65 demonstrated by immunostaining in the lung were significantly lower in the iPS-treated ALI mice compared with the PBS-treated ALI mice at 24 h (LPS 24 h vs LPS + iPS 24 h, P = .04). In contrast, mice that received MEF (LPS + MEF) showed no moderation of the activation of NF-κB p65 (total or phospho-) induced by LPS compared with iPS-cell-treated mice, but apoptotic iPS cells (LPS + aiPS) partially reduced the activity of NF-κB p65. Data are presented as mean ± SD *P < .05, n = 5 per group. IHC = immunohistochemistry; NF-κB = nuclear factor-κB. See Figure 1-3 legends for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 7. iPS cells reduced nuclear translocation of NF-κB (using electrophoretic mobility shift assay [EMSA]) compared with PBS-treated ALI. NF-κB levels were significantly increased in nuclear extracts from lung tissues of ALI mice compared with those found in control mice. In iPS-treated ALI mice, nuclear levels of NF-κB were significantly decreased as compared with ALI mice or MEF-treated ALI mice. ALI mice that received iPS-CM had levels of nuclear NF-κB similar to those with iPS cells. Representative EMSA gel is presented. Densitometry data are shown as mean ± SD. *P < .05, n = 5 per group. See Figure 1-3 and 6 legends for expansion of other abbreviations.Grahic Jump Location
iPS Cells Decreased the Production of Inflammatory Cytokines in Endotoxin-Induced ALI

An intratracheal injection of LPS resulted in increased production of the inflammatory cytokines TNF-α and IL-6 and the chemokine MIP-2 in mice lung (Fig 4A-C). In contrast, the addition of iPS cells significantly decreased LPS-induced elevations in the protein release of TNF-α, IL-6, and MIP-2 at both 24 and 48 h after ALI. Moreover, mice treated with iPS-CM at 24 h after ALI had similar results as those with iPS cells. On the contrary, mice that received MEF had increases in the levels of proinflammatory cytokines compared with iPS-cell-treated mice at 24 h after ALI. However, the protein release of the antiinflammatory cytokine IL-10 in the lung was similar in the iPS-cell- and PBS-treated mice at any time point (e-Fig 2).

iPS Cells Through a Cell-Cell Contact-Independent Mechanism Reduced the Cytokine Levels of Neutrophils Stimulation by LPS

The coculture of iPS cells and pulmonary neutrophils stimulated by LPS had lower levels of MIP-2 in the cell supernatant compared with pulmonary neutrophils alone and those stimulated by LPS (Fig 1). The degree of reduction in MIP-2 was similar in the presence of a Transwell that prohibits cell contact between the iPS cells and neutrophils. iPS cells alone did not produce MIP-2 in response to LPS stimulation.

iPS Cells Rescued the Hypoxemia and Physiologic Impairment of Endotoxin-Induced ALI

As demonstrated by the experimental results shown in Figure 3, exposure of the lungs of mice to LPS caused extensive alveolar damage. In contrast, most of the alveolar damage was reduced if iPS cells were administered to these ALI mice. Therefore, it was hypothesized that normal lung function could be restored to the ALI mice if iPS cells were transplanted. To test this hypothesis, the pulmonary function parameters of Penh, a measurement of airway resistance, lung tidal volumes, and Pao2, were measured in mice that were treated with and without iPS cells following endotoxin-induced lung injury. As illustrated in Figure 8A, Penh was increased significantly in endotoxin-induced ALI mice when compared with the control mice; however, iPS cell administration significantly attenuated Penh levels at 6 h (0.6485 ± 0.0665 vs 1.3499 ± 0.1853, P = .048) and 24 h (0.4529 ± 0.0597 vs 0.8088 ± 0.0845, P = .047) after endotoxin-induced ALI. Similar to Penh, lung tidal volume during spontaneous respiration was significantly decreased at 6 h (0.291 ± 0.022 mL vs 0.182 ± 0.011 mL, P = .033) and 24 h (0.287 ± 0.039 mL vs 0.214 ± 0.013 mL, P = .043) after endotoxin-induced ALI; in contrast, the lung tidal volume in the ALI mice that were treated with iPS cells recovered to near normal at 24 h after ALI (Fig 8B). Moreover, Pao2 levels in the ALI mice declined significantly at 24 h after endotoxin-induced ALI (Fig 8C). Impressively, the LPS-injured mice that underwent a transplant with iPS cells exhibited improved Pao2 levels at 24 h after the LPS challenge (38.33 ± 3.28 Torr vs 73.33 ± 9.87 Torr, P = .022). IV iPS-CM treatment after ALI showed similar effects as iPS cells. In contrast, mice that received control cells (MEF) had no improvement in the severity of hypoxemia induced by LPS.

Figure Jump LinkFigure 8. iPS cells improved pulmonary function as assessed by plethysmography and arterial blood gas measurement. A, Penh was determined during spontaneous breathing using a mouse pulmonary plethysmograph. The Penh measured 3 to 48 h after endotoxin-induced ALI was significantly increased in the ALI mice compared with the control mice at the 6- and 24-h time points. In contrast, when iPS cells were transplanted into the endotoxin-challenged mice after endotoxin-induced ALI, the airway resistance of the mice was reduced at 6 and 24 h after endotoxin-challenge. B, Lung tidal volumes were significantly decreased in the endotoxin-induced ALI mice compared with the control mice at the 6- and 24-h time points. On the other hand, when endotoxin-induced ALI mice were transplanted with iPS cells, their lung tidal volumes returned to normal by 6 and 24 h after the endotoxin-challenge. C, Pao2 levels in the ALI mice declined significantly at 24 h after the endotoxin challenge. Moreover, the ALI mice that had been transplanted with iPS cells exhibited improved Pao2 levels at 24 h after the endotoxin challenge. Mice treated with iPS-CM after ALI (LPS + iPS-CM) had a result similar to those treated with iPS cells (LPS + iPS). In contrast, mice that received the MEF (LPS + MEF) had no effect on the severity of hypoxemia induced by LPS compared with iPS-cell-treated mice at 24 h. Data are presented as mean ± SD. *P < .05, n = 8 per group. Penh = airway resistance. See Figure 1-3 legends for expansion of other abbreviations.Grahic Jump Location

Previous studies using MSC and ESC cell-based therapy in lung injury have been reported1821; however, iPS cells have never been investigated in this area. This is the first study to demonstrate the therapeutic potential and immunomodulatory effect of iPS cells in ALI. There are four major discoveries in our study: (1) iPS cells via IV injection migrated and accumulated in the area of lung injury; (2) iPS cell administration diminished the pathologic severity of endotoxin-induced ALI; (3) iPS cells improved the pulmonary physiologic functions of ALI; and (4) the advantageous effects with iPS cells were partly mediated by reducing the activity of NF-κB and paracrine factors.

The ultimate goal of cell therapy is to use functional cell types relevant to a specific disease, such as cardiac infarction or ALI.1821 However, avoiding immunorejection is an important concern in current transplantation medicine. The potential advantage of iPS cells over ESC is that iPS cells can be derived from a patient’s own somatic cells, thereby avoiding immune rejection after transplant and the ethical concerns raised by ESC.13,14,2730 In contrast to ESC and iPS cells, MSC have a limited life span and become senescent when cultured in vitro.31 This study demonstrated that our MEF-derived iPS cells expressed stem cell marker genes and possessed a pluripotent capacity to differentiate into different cell types of all three germ layers (e-Fig 1). Although pluripotent differentiation in iPS cells has been demonstrated recently,14,27,28 the therapeutic potential and usefulness of iPS cells in improving the severity of ALI has never been investigated. In the present study, we demonstrated the homing effect of iPS cells in the injured lung as well as their immunomodulatory effect in ALI. Furthermore, this study provides the first evidence that the transplant of iPS cells could effectively reduce severity and promote functional recovery in ALI. However, the benefits with iPS cell therapy were not replicated with the administration of MEF or apoptotic iPS cells, which suggests that true, undifferentiated, viable iPS cells have a functional potential to moderate the inflammatory process of endotoxin-induced ALI. In addition, iPS cells possess immunomodulatory properties in other inflammatory diseases, such as ischemic brain injury, which was confirmed in our recent study.17

Neutrophil recruitment into the lung plays a central role in the acute pulmonary inflammatory process.5 However, we demonstrated that iPS cell instillation not only reduced the levels of MIP-2 in the lung, but also diminished neutrophil accumulation in ALI; these findings were confirmed by the reduction of MPO activity. Additionally, we used in vitro coculture experiments with endotoxin-stimulated pulmonary neutrophils and iPS cells to demonstrate the interactive mechanism between both cells. Using a Transwell system to physically separate the neutrophils and iPS cells and inhibit cell-cell contact, we showed that the iPS cells suppressed the activity of neutrophils to secrete a chemokine by endotoxin stimulation in a cell-contact independent style. Thus, iPS cells may produce some paracrine mediator to regulate the neutrophil activity in response to endotoxin stimulation and reduce the inflammatory cascade in endotoxin-induced ALI in vivo. A recent study about conditioned medium from MSC has suggested that IV conditioned medium from MSC therapy reduces myocardial infarct size and preserves cardiac function in pigs with acute myocardial infarction.32 Likewise, our study also demonstrated that iPS-CM therapy in ALI mice had effects similar to those of the iPS cell treatment. The real mechanism of iPS-CM deserves further investigation.

Although endotoxemia has been demonstrated to modulate the activation of transcriptional factors such as NF-κB, as well as the expression of proinflammatory cytokines by neutrophils and other pulmonary cell populations,24,33 our previous study suggested that early alterations in neutrophil activation patterns, particularly involving the ability to accumulate NF-κB at the nucleus after endotoxin stimuli, contributed to the subsequent clinical course in ALI.34 However, the roles of stem cells in affecting transcriptional factors of ALI have not been examined. Based on recent reports on the immunomodulatory properties of MSC,20,3537 we hypothesized that the benefit of iPS cell therapy was mediated through a downregulation of NF-κB and the downstream proinflammatory cytokine response to endotoxin stimulation. Analysis of lung samples from the iPS-cell-treated and control mice confirmed this hypothesis, demonstrating lower levels of NF-κB activity in the iPS-cell-treated mice. Because the production of proinflammatory cytokines, including the chemokine MIP-2, is regulated by the activity of NF-κB,38 iPS cells regulating NF-κB activity reduced the neutrophil influx into the alveolus and ameliorated ALI, which was also validated by reducing the levels of TNF-α and IL-6. All of this suggests that iPS cells have a specific immunomodulatory effect.

iPS cell administration improved the impairment of pulmonary function in endotoxin-induced ALI. Hypoxemia is the major symptom and sign of ALI, in both the mice model and human cases. The effect of iPS cell treatment was to rescue the hypoxemia using a therapeutic agent in an animal model of lung injury, similar to the results of other studies.21,39 Our study showed that the IV injection of iPS cells led to recovery of the impairment of both airway resistance and lung tidal volume induced by endotoxin. In a previous mice model of early ALI, most changes in BAL suggestive of acute pulmonary irritation were compatible with the changes in pulmonary function, such as airway resistance (Penh) and tidal volume.40 Thus, iPS cell therapy not only abolished the pathologic changes in ALI mice, but also improved the changes in pulmonary physiologic function.

Some of the enthusiasm for using human ESC to regenerate damaged tissue has been tempered by the observation that direct application of human ESC in vivo may cause teratoma formation, resulting in a possible lethal outcome.41,42 In this study, we modified the route of delivery with IV administration via tail veins instead of direct intratracheal instillation into the lung. It is worthwhile to note that all of the iPS-cell-challenged mice remained alive and healthy and without teratoma formation for the observational period of this study (90 days, e-Figure 3). However, the risk of tumor formation is still a concern in stem cell therapy, and the long-term effect of iPS cell therapy may deserve to be followed up.

The limitation of this study is the lack of an in vitro investigation into the interaction between iPS cells and alveolar or endothelial cells or even other inflammatory cells. Because the process of ALI is complex, and different types of cells are involved in different stages and diverse parts of ALI, exploring the interaction between only neutrophils and iPS cells does not represent the entire inflammatory reaction of iPS cell therapy in ALI. Further study focusing on the detailed molecular mechanism of iPS cells in ALI would be worthwhile. In addition, one of the practical potential limitations of iPS cell therapy for patients with ALI is that each patient would have to have their own cells harvested, which would take several days using present technical skills. Harvesting iPS cells (autologous) from a patient with ALI and giving them to him or her within a reasonable time period could pose a practical challenge, and, therefore, autologous therapy might be difficult for the treatment of ALI in patients.

This study demonstrated that IV delivery of iPS cells provides a beneficial effect to attenuate the severity of histopathology and physiologic impairment in endotoxin-induced ALI. The effect is partly mediated by a reduction in NF-κB activity, as well as by decreasing neutrophil accumulation (eg, MPO and MIP-2 levels), and may be not cell contact dependent. These discoveries provide important evidence that iPS-cell-based therapy has a specific antiinflammatory effect in a mouse model of ALI, and support the use of iPS cells as an another applicable source of stem cells for the future management of ALI.

Author contributions: Drs K.-Y. Yang and Chiou take responsibility for the integrity of the manuscript and the accuracy of the data analysis.

Dr K.-Y. Yang: contributed to study conception and design, data collection, analysis, and interpretation; drafting of the manuscript; and critical revision of the article for important content.

Dr Shih: contributed to data analysis and interpretation and review of the manuscript.

Dr How: contributed to data collection, analysis, and interpretation and critical revision of the manuscript.

Dr Chen: contributed to data collection, analysis, and interpretation and critical revision of the manuscript.

Dr Hsu: contributed to data analysis and interpretation and review of the manuscript.

Ms C.-W. Yang: contributed to data collection, analysis, and interpretation and revision of the manuscript.

Dr Lee: contributed to data analysis and interpretation and revision of the manuscript.

Dr Perng: contributed to data analysis and interpretation and revision of the manuscript.

Dr Peng: contributed to data analysis and interpretation and review of the manuscript.

Dr Li: contributed to data analysis and interpretation and review of the manuscript.

Dr Chang: contributed to data analysis and interpretation and review of the manuscript.

Dr Mou: contributed to data analysis and interpretation and review of the manuscript.

Dr Chiou: contributed to study conception and design, iPS cell production, data analysis and interpretation, and editing of the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.

Additional information: The e-Appendixes and e-Figures can be found in the Online Supplement at http://chestjournal.chestpubs.org/content/140/5/1243/suppl/DC1.

ALI

acute lung injury

EMSA

electrophoretic mobility shift assay

ESC

embryonic stem cell

iPS

induced pluripotent stem

iPS-CM

conditioned medium from induced pluripotent stem cell

LPS

lipopolysaccharide

MEF

mouse embryonic fibroblast

MIP-2

macrophage inflammatory peptide-2

MPO

myeloperoxidase

MSC

mesenchymal stem cell

NF-κB

nuclear factor-κB

PBS

phosphate-buffered saline

TNF-α

tumor necrosis factor-α

Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000;34218:1334-1349 [CrossRef] [PubMed]
 
Chollet-Martin S, Jourdain B, Gibert C, Elbim C, Chastre J, Gougerot-Pocidalo MA. Interactions between neutrophils and cytokines in blood and alveolar spaces during ARDS. Am J Respir Crit Care Med. 1996;1543 pt 1:594-601 [PubMed]
 
Goodman RB, Strieter RM, Martin DP, et al. Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am J Respir Crit Care Med. 1996;1543 pt 1:602-611 [PubMed]
 
Suter PM, Suter S, Girardin E, Roux-Lombard P, Grau GE, Dayer JM. High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am Rev Respir Dis. 1992;1455:1016-1022 [CrossRef] [PubMed]
 
Abraham E, Carmody A, Shenkar R, Arcaroli J. Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2000;2796:L1137-L1145 [PubMed]
 
Parsey MV, Tuder RM, Abraham E. Neutrophils are major contributors to intraparenchymal lung IL-1 beta expression after hemorrhage and endotoxemia. J Immunol. 1998;1602:1007-1013 [PubMed]
 
Shenkar R, Abraham E. Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia: roles of reactive oxygen intermediates, NF-kappa B, and cyclic AMP response element binding protein. J Immunol. 1999;1632:954-962 [PubMed]
 
Xing Z, Jordana M, Kirpalani H, Driscoll KE, Schall TJ, Gauldie J. Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor-alpha, macrophage inflammatory protein-2, interleukin-1 beta, and interleukin-6 but not RANTES or transforming growth factor-beta 1 mRNA expression in acute lung inflammation. Am J Respir Cell Mol Biol. 1994;102:148-153 [PubMed]
 
Foo SY, Nolan GP. NF-kappa B to the rescue: RELs, apoptosis and cellular transformation. Trends Genet. 1999;156:229-235 [CrossRef] [PubMed]
 
Liu SF, Ye X, Malik AB. In vivo inhibition of nuclear factor-kappa B activation prevents inducible nitric oxide synthase expression and systemic hypotension in a rat model of septic shock. J Immunol. 1997;1598:3976-3983 [PubMed]
 
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;1264:663-676 [CrossRef] [PubMed]
 
Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007;4487151:313-317 [CrossRef] [PubMed]
 
Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;1315:861-872 [CrossRef] [PubMed]
 
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Figures

Figure Jump LinkFigure 1. iPS cells moderate the production of MIP-2 by LPS-stimulated pulmonary neutrophils isolated from mice with acute lung injury (ALI) in a cell-to-cell contact-independent manner. A, MIP-2 was measured in the supernatant of the coculture system as described in the “Materials and Methods” section. B, Administration of iPS cells in the culture wells decreased the production of MIP-2 by LPS-stimulated pulmonary neutrophils, and this decrease was similar when a Transwell insert was placed to prevent direct cell-to-cell contact. Data are presented as mean ± SD. * and #P < .05 for comparison with the condition of neutrophils alone, n = 5 per condition. iPS = induced pluripotent stem; LPS = lipopolysaccharide; MIP-2 = macrophage inflammatory peptide-2.Grahic Jump Location
Figure Jump LinkFigure 2. More transplanted iPS cells were trafficking in the injured lung in mice with ALI. A, Transplanted 131I-IdUrd-labeled iPS cells were located using in vivo radionuclide imaging (autoradiography) at 4 and 8 h after IV iPS administration in intratracheal LPS (ALI) or intratracheal phosphate-buffered saline (CON) mice. Selective uptake of 131I-IdUrd was observed in the lung area of the anesthetized mice, and there was more radio uptake accumulation in the lung area in the endotoxin-induced lung-injury mice (LPS + iPS) than in the control mice (CON + iPS). B, Transplanted Hoechst-labeled iPS cells were located using fluorescent microscopy (original magnification ×100) at 24 h after IV iPS administration in the mice with ALI or control mice. Isolated Hoechst-labeled iPS cells delivered by IV were noted to be more scattered in the injured lung area of mice with ALI (LPS + iPS) than in the lung area of control mice (CON + iPS). Bright light-blue staining represents the Hoechst-labeled cells. C, To quantify the scattered density of the incorporated iPS cells in the lungs at 24 h, the number of labeled iPS cells in the injured lungs (from mice with ALI) and control lungs (from control mice) was determined by counting Hoechst-labeled iPS cells in 10 lung sections of each sample; there were more transplanted iPS cells in the injured lungs (LPS + iPS) than in the control lungs (CON + iPS). Data are presented as mean ± SD. *P = .006, n = 6 per group. CON = control. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 3. iPS cells improved lung injury as assessed by histologic methods. A, Hematoxylin and eosin staining of lung sections demonstrated attenuated lung injury in the mice receiving iPS cells at both 24 and 48 h after endotoxin-induced ALI. B, Histopathologic lung injury score showed a significant reduction in the mice with ALI receiving iPS cells compared with those receiving phosphate-buffered saline (PBS) treatment (LPS 24 h vs LPS + iPS 24 h, P = .039 and LPS 48 h vs LPS + iPS 48 h, P = .037). Minimal histopathologic abnormalities were also present in mice that were injected with intratracheal PBS (control group). In contrast, mice that received the MEF (LPS + MEF) or aiPS cells (LPS + aiPS) showed no improvement in the severity of acute lung injury induced by LPS compared with iPS-cell-treated mice at 24 h. However, mice treated with iPS-CM after ALI (LPS + iPS-CM) had a similar result to that with iPS cells (LPS + iPS). The score represents the average of the findings of two independent investigators who each read the hematoxylin-and-eosin-stained slide in a blinded manner. The categories used to generate the score were alveolar septal congestion, alveolar hemorrhage, intraalveolar fibrin deposition, and intraalveolar infiltrates. Data are presented as mean ± SD. *P< .05; n = 6 per group. aiPS = apoptotic induced pluripotent stem; iPS-CM = conditioned medium from induced pluripotent stem cell; MEF = mouse embryonic fibroblast. See Figure 1 and 2 legends for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 4. A-C, Levels of proinflammatory cytokines, TNF-α, IL-6, and MIP-2 were reduced in mice receiving iPS cells at both 24 and 48 h after endotoxin-induced ALI. MIP-2 (A), IL-6 (B), and TNF-α (C) were significantly reduced in the lungs of mice treated with iPS cells at both 24 and 48 h after ALI. Furthermore, mice treated with iPS-CM at 24 h after ALI had similar results to those with iPS cells. In contrast, mice that received the MEF (LPS + MEF) had increases in the levels of proinflammatory cytokines compared with iPS-cell-treated mice at 24 h after ALI. Data are presented as mean ± SD. *P < .05, n = 5 per group. TNF-α = tumor necrosis factor-α. See Figure 1-3 legends for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 5. iPS cell administration prevented neutrophil accumulation and decreased MPO activity after endotoxin-induced ALI. A, Neutrophil accumulation in the lung was significantly increased after endotoxin exposure. As was similarly concluded visually by Ly6C staining, transplant of iPS cells at 24 h after endotoxin-challenge significantly reduced neutrophil accumulation in the alveolus (original magnification × 400). B, Quantification of Ly6C staining showed a significant reduction in the degree of neutrophil accumulation in the ALI mice receiving iPS cells (LPS 24 h vs LPS + iPS 24 h, P = .013). C, iPS cells significantly decreased the activity of MPO after endotoxin-induced ALI, as determined by MPO assay at 24 and 48 h after injury. In contrast, MEFs, or a-iPS cell administration did not significantly diminish LPS-induced elevations in the MPO activity. Data are presented as mean ± SD. *P < .05, n = 5 per group. MPO = myeloperoxidase. See Figure 1-3 legends for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 6. iPS cell administration reduced the levels of NF-κB activity after endotoxin-induced ALI. A, Activity levels of NF-κB demonstrated by immunostaining of NF-κB p65 in the lung were significantly enhanced after endotoxin-induced ALI, but iPS cell administration significantly diminished this effect to levels near those of uninjured lungs. C, Quantification of NF-κB p65 staining showed a significant reduction in the activity levels of NF-κB p65 in the mice receiving iPS cells at 24 h after endotoxin-induced ALI (LPS 24 h vs LPS + iPS 24 h, P = .04). B and D, Activity levels of phospho-NF-κB (p-NF-κB) p65 demonstrated by immunostaining in the lung were significantly lower in the iPS-treated ALI mice compared with the PBS-treated ALI mice at 24 h (LPS 24 h vs LPS + iPS 24 h, P = .04). In contrast, mice that received MEF (LPS + MEF) showed no moderation of the activation of NF-κB p65 (total or phospho-) induced by LPS compared with iPS-cell-treated mice, but apoptotic iPS cells (LPS + aiPS) partially reduced the activity of NF-κB p65. Data are presented as mean ± SD *P < .05, n = 5 per group. IHC = immunohistochemistry; NF-κB = nuclear factor-κB. See Figure 1-3 legends for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 7. iPS cells reduced nuclear translocation of NF-κB (using electrophoretic mobility shift assay [EMSA]) compared with PBS-treated ALI. NF-κB levels were significantly increased in nuclear extracts from lung tissues of ALI mice compared with those found in control mice. In iPS-treated ALI mice, nuclear levels of NF-κB were significantly decreased as compared with ALI mice or MEF-treated ALI mice. ALI mice that received iPS-CM had levels of nuclear NF-κB similar to those with iPS cells. Representative EMSA gel is presented. Densitometry data are shown as mean ± SD. *P < .05, n = 5 per group. See Figure 1-3 and 6 legends for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 8. iPS cells improved pulmonary function as assessed by plethysmography and arterial blood gas measurement. A, Penh was determined during spontaneous breathing using a mouse pulmonary plethysmograph. The Penh measured 3 to 48 h after endotoxin-induced ALI was significantly increased in the ALI mice compared with the control mice at the 6- and 24-h time points. In contrast, when iPS cells were transplanted into the endotoxin-challenged mice after endotoxin-induced ALI, the airway resistance of the mice was reduced at 6 and 24 h after endotoxin-challenge. B, Lung tidal volumes were significantly decreased in the endotoxin-induced ALI mice compared with the control mice at the 6- and 24-h time points. On the other hand, when endotoxin-induced ALI mice were transplanted with iPS cells, their lung tidal volumes returned to normal by 6 and 24 h after the endotoxin-challenge. C, Pao2 levels in the ALI mice declined significantly at 24 h after the endotoxin challenge. Moreover, the ALI mice that had been transplanted with iPS cells exhibited improved Pao2 levels at 24 h after the endotoxin challenge. Mice treated with iPS-CM after ALI (LPS + iPS-CM) had a result similar to those treated with iPS cells (LPS + iPS). In contrast, mice that received the MEF (LPS + MEF) had no effect on the severity of hypoxemia induced by LPS compared with iPS-cell-treated mice at 24 h. Data are presented as mean ± SD. *P < .05, n = 8 per group. Penh = airway resistance. See Figure 1-3 legends for expansion of other abbreviations.Grahic Jump Location

Tables

References

Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000;34218:1334-1349 [CrossRef] [PubMed]
 
Chollet-Martin S, Jourdain B, Gibert C, Elbim C, Chastre J, Gougerot-Pocidalo MA. Interactions between neutrophils and cytokines in blood and alveolar spaces during ARDS. Am J Respir Crit Care Med. 1996;1543 pt 1:594-601 [PubMed]
 
Goodman RB, Strieter RM, Martin DP, et al. Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am J Respir Crit Care Med. 1996;1543 pt 1:602-611 [PubMed]
 
Suter PM, Suter S, Girardin E, Roux-Lombard P, Grau GE, Dayer JM. High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am Rev Respir Dis. 1992;1455:1016-1022 [CrossRef] [PubMed]
 
Abraham E, Carmody A, Shenkar R, Arcaroli J. Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2000;2796:L1137-L1145 [PubMed]
 
Parsey MV, Tuder RM, Abraham E. Neutrophils are major contributors to intraparenchymal lung IL-1 beta expression after hemorrhage and endotoxemia. J Immunol. 1998;1602:1007-1013 [PubMed]
 
Shenkar R, Abraham E. Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia: roles of reactive oxygen intermediates, NF-kappa B, and cyclic AMP response element binding protein. J Immunol. 1999;1632:954-962 [PubMed]
 
Xing Z, Jordana M, Kirpalani H, Driscoll KE, Schall TJ, Gauldie J. Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor-alpha, macrophage inflammatory protein-2, interleukin-1 beta, and interleukin-6 but not RANTES or transforming growth factor-beta 1 mRNA expression in acute lung inflammation. Am J Respir Cell Mol Biol. 1994;102:148-153 [PubMed]
 
Foo SY, Nolan GP. NF-kappa B to the rescue: RELs, apoptosis and cellular transformation. Trends Genet. 1999;156:229-235 [CrossRef] [PubMed]
 
Liu SF, Ye X, Malik AB. In vivo inhibition of nuclear factor-kappa B activation prevents inducible nitric oxide synthase expression and systemic hypotension in a rat model of septic shock. J Immunol. 1997;1598:3976-3983 [PubMed]
 
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;1264:663-676 [CrossRef] [PubMed]
 
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