Mol. Cells 2016; 39(10): 728-733
Published online October 31, 2016
https://doi.org/10.14348/molcells.2016.0095
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: ymoh55@amc.seoul.kr
Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The
Keywords gene profile, human cord blood-derived mesenchymal stem cells, microarray, smoke-induced chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation and alveolar wall destruction (emphysema) that result from exposure to cigarette smoke (Rabe et al., 2007). The developmental mechanism of COPD has been suggested to involve cigarette smoke-induced airway inflammation, oxidative stress, and alveolar destruction from an imbalance between proteases and anti-proteases (MacNee and Tuder, 2009; Yoshida and Tuder, 2007). Although current treatments are available to inhibit airway inflammation and relax contracted airways, there is no therapy that regenerates destroyed alveoli (Aaron et al., 2007; Calverley et al., 2007). Thus, recent studies to improve COPD treatment have focused on the control of anti-oxidative and anti-protease activities.
Cell therapy using various stem cells has shown beneficial therapeutic effects in animal models of COPD (Huh et al., 2011; Katsha et al., 2011; Schweitzer et al., 2011). Our recent studies showed that MSCs from the bone marrow and cord blood successfully improved the emphysema component of COPD (Huh et al., 2011). Recently, we reported that most stem cells injected systemically disappeared within one day, when the stem cells were tracked by fluorescent labeling or with human-based
Previous studies have analyzed gene profiles in the presence and absence of cigarette exposure. However, no studies have examined the changes in gene profiles following MSC injection in a cigarette smoke-induced COPD model. Therefore, the goal of the current study was to analyze the changes in gene expression profiles in the cigarette smoke-induced COPD mouse lung following systemic injection of human cord blood-derived (hCB)-MSCs over time on days 1, 4, and 14. Using a mouse WG-6 expression bead array based on Illumina, we compared gene expression profiles following cigarette exposure and non-exposure. Next, we compared the gene expression profiles of two groups, an hCB-MSC-injected group and a non-injected group, both of which were induced to have COPD lungs by a 6-month exposure to cigarette smoke before systemic injection of hCB-MSCs.
C57BL/6J mice were purchased from OrientBio (Korea). Mice were bred in specific pathogen-free facilities at the Asan Medical Center. All live mouse experiments were approved by the Institutional Animal Care and Use Committee of the Asan Medical Center (Korea). Seven-week-old female C57BL/6J mice were exposed to commercial cigarette smoke (Eighty Eight Lights, KT&G, Daejeon, Korea) 5 days per week for 6 months as we described previously(Huh et al., 2011). Briefly, 30?40 mice were settled in an inhalation box (50 × 40 × 30 cm) connected to a pump, and exposed to 12 cigarettes. Control mice were bred in cages with clean room air. Human cord blood MSCs were provided by MEDIPOST Co., Ltd (Korea). hCB-MSCs were cultured with alpha-minimum essential medium (Gibco Life Technologies, USA) and subcultured using 0.25% trypsin-EDTA (Gibco Life Technologies). Mice exposed to 6 months of cigarette smoke were intravenously injected with 5 × 104 hCB-MSCs. Mice were then sacrificed and their lungs were collected on days 1, 4, and 14 following injection of hCB-MSCs. RNA later was used to maintain RNA stability during mouse lung collection.
Total mRNA from lung tissue was prepared using Trizol (Invitrogen Life Technologies, USA) and purified using RNeasy columns (Qiagen, Germany) according to the manufacturer’s protocol. The purity and quantity of the isolated RNA were evaluated using aND-1000 spectrophotometer (NanoDrop Technologies Inc., USA), and a quality check of the RNA was performed using a 2100 Bioanalyzer (Agilent Technologies, USA). Only RNA samples with an RNA integrity number >7.5 were used for the microarray experiment.
RNA was amplified and purified using the AmbionIllumina RNA amplification kit (Ambion, USA) according to the manufacturer’s instructions. Biotinylated cRNA (1.5 μg) was hybridized to each MouseWG-6 expression bead array (Illumina, Inc., USA), and the array signal was detected using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Biosciences, UK). Arrays were scanned with an Illumina bead array reader confocal scanner.
The lung was inflated with 0.5% low-melting agarose and fixed using 4% formalin, and then embedded in paraffin and cut into 4-μm-thick sections for immunohistochemistry. The sections were stained using an Immunohistochemistry accessory kit (Bethyl Laboratories, Inc., USA) with anti-Egr-1 antibody (1:250, Abcam, UK) and then observed by microscopy.
All data analysis of differentially expressed genes was conducted using R 2.15.1. Raw expression data was log2-transformed and normalized by a quantile method. Statistical significance of the expression data was determined using a
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A
Figure 4 shows the genes specifically altered by injection of hCB-MSCs at each time point and in common between the different time points using a Venn diagram. Seven genes and 21 genes were continuously up- and down-regulated, respectively, following injection of hCB-MSCs. The common up- and down-regulated genes are shown in Table 2. The number of up-regulated genes was the lowest at 1 day after injection of hCB-MSCs compared to the other time points and increased in a time-dependent manner. In contrast, the largest number of down-regulated genes showing differential expression was observed on day 1 after injection of hCB-MSCs. Figure 5 was shows the immunohistochemistry results for Egr-1 in the lungs. The Egr-1 gene was down-regulated in both groups following hCB-MSC injection. The results revealed only a minimal correlation between mRNA expression and protein expression.
The DEGs between the hCB-MSCs-injected and non-injected groups were categorized based on time and direction of expression. We also performed GO analysis using DAVID. The top 20 up-regulated genes at each time point are listed in Table 3. We also represent the fold-change following cigarette exposure in Table 3. The expression of oxygen transport- and anti-oxidant function-related genes such as
In the present study, we analyzed the gene expression profiles following cigarette exposure or non-exposure as well as hCBMSC-injected or non-injected mice. We focused on the changes in gene profiles between hCB-MSCs-injected vs. non-injected mouse lungs following the induction of COPD by chronic exposure to cigarette smoke and found that gene expression profiles varied with after injection of hCB-MSCs. Genes involved in oxidative stress, xenobiotic metabolic processes, and anti-protease functions were detected most frequently. In addition, GO term analysis showed that genes related to immune responses, blood vessel development, and metabolic processes were differentially expressed on days 1 and 4 after hCB-MSC injection compared to controls. DEGs on day 14 after injection of hCB-MSCs were related to cell growth regulation. Our results suggest that immune responses, oxidative stress, and transcription were regulated at a nearly stage after hCB-MSC injection (on days 1 and 4),and that blood vessel development and cell growth regulation predominated at a later stage after hCB-MSC injection (on day 14).
In previous reports, MSCs showed regenerative properties in animal models with smoke- and elastase-induced COPD (Huh et al., 2011; Katsha et al., 2011; Schweitzer et al., 2011). Recently, we reported that systemically injected MSCs were observed for only 1 day after cell injection in an elastase-induced emphysema model (Kim et al., 2014). Other reports failed to detect injected MSCs; therefore, stem cell biologists have attempted to explain the therapeutic effect of mesenchymal stem cells in these models in the context of the regulation of host cells or host microenvironment (Henning et al., 2014; Liang et al., 2014; Suga et al., 2014). To understand the detailed mechanisms of stem cell therapy, understanding the changes in host genes following MSC injection is required. Thus, we focused on the host changes based on the profiles of DEGs in this study.
Previous studies analyzed the gene expression profiles in the lungs of smoke-exposed mice (Halappanavar et al., 2009; Rangasamy et al., 2009).Inflammatory signaling was up-regulated following cigarette exposure. We also detected up-regulation of inflammatory-related genes such as
Therefore, MSCs may have regenerative roles that reestablish a balance between elastase and anti-elastase by up-regulating
There were some limitations to this study. One limitation is that the differential change in gene expression profiles did not involve exposure to normal air and exposure to normal air plus hCB-MSCs. Therefore, further studies of mice exposed to normal air and the use of injected cell controls are needed to correctly identify the detailed regeneration mechanisms of stem cells. Another limitation is that our microarray experiment used whole lungs. Although we determined organ-level changes in molecular signals following the injection of hCB-MSCs, it remains unclear how the gene profiles change in individual cells in the lung such as bronchial and alveolar epithelial cells, fibroblasts, and endothelial cells. More advanced techniques are needed to analyze the gene expression profiles using sections of the lung tissues or specific lung cell types.
This is the first study to determine the time-dependent molecular changes induced by treatment with hCB-MSCs in a cigarette smoke-induced COPD model. MSCs systemically injected into an animal disappear within several days; it is thought that these cells rarely differentiate into tissue resident cells in the lungs, such as epithelial cells. Therefore, studies focused on molecular changes in host tissues may reveal the regenerative mechanisms of MSCs. Our present report improves the understanding of the regenerative mechanisms induced by injection of hCB-MSCs based on the gene expression profile changes in a cigarette smoke-induced COPD animal model.
Mol. Cells 2016; 39(10): 728-733
Published online October 31, 2016 https://doi.org/10.14348/molcells.2016.0095
Copyright © The Korean Society for Molecular and Cellular Biology.
You-Sun Kim1,2, Nurdan Kokturk3, Ji-Young Kim2, Sei Won Lee1,4, Jaeyun Lim1, Soo Jin Choi5, Wonil Oh5, and Yeon-Mok Oh1,4,*
1University of Ulsan College of Medicine, Seoul 05505, Korea, 2Asan Institute for Life Sciences, Seoul 05505, Korea, 3Department of Pulmonology, Gazi University, Ankara, Turkey, 4Department of Pulmonary and Critical Care Medicine, Asan Medical Center, Seoul 05505, Korea, 5Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, Korea
Correspondence to:*Correspondence: ymoh55@amc.seoul.kr
Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The
Keywords: gene profile, human cord blood-derived mesenchymal stem cells, microarray, smoke-induced chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation and alveolar wall destruction (emphysema) that result from exposure to cigarette smoke (Rabe et al., 2007). The developmental mechanism of COPD has been suggested to involve cigarette smoke-induced airway inflammation, oxidative stress, and alveolar destruction from an imbalance between proteases and anti-proteases (MacNee and Tuder, 2009; Yoshida and Tuder, 2007). Although current treatments are available to inhibit airway inflammation and relax contracted airways, there is no therapy that regenerates destroyed alveoli (Aaron et al., 2007; Calverley et al., 2007). Thus, recent studies to improve COPD treatment have focused on the control of anti-oxidative and anti-protease activities.
Cell therapy using various stem cells has shown beneficial therapeutic effects in animal models of COPD (Huh et al., 2011; Katsha et al., 2011; Schweitzer et al., 2011). Our recent studies showed that MSCs from the bone marrow and cord blood successfully improved the emphysema component of COPD (Huh et al., 2011). Recently, we reported that most stem cells injected systemically disappeared within one day, when the stem cells were tracked by fluorescent labeling or with human-based
Previous studies have analyzed gene profiles in the presence and absence of cigarette exposure. However, no studies have examined the changes in gene profiles following MSC injection in a cigarette smoke-induced COPD model. Therefore, the goal of the current study was to analyze the changes in gene expression profiles in the cigarette smoke-induced COPD mouse lung following systemic injection of human cord blood-derived (hCB)-MSCs over time on days 1, 4, and 14. Using a mouse WG-6 expression bead array based on Illumina, we compared gene expression profiles following cigarette exposure and non-exposure. Next, we compared the gene expression profiles of two groups, an hCB-MSC-injected group and a non-injected group, both of which were induced to have COPD lungs by a 6-month exposure to cigarette smoke before systemic injection of hCB-MSCs.
C57BL/6J mice were purchased from OrientBio (Korea). Mice were bred in specific pathogen-free facilities at the Asan Medical Center. All live mouse experiments were approved by the Institutional Animal Care and Use Committee of the Asan Medical Center (Korea). Seven-week-old female C57BL/6J mice were exposed to commercial cigarette smoke (Eighty Eight Lights, KT&G, Daejeon, Korea) 5 days per week for 6 months as we described previously(Huh et al., 2011). Briefly, 30?40 mice were settled in an inhalation box (50 × 40 × 30 cm) connected to a pump, and exposed to 12 cigarettes. Control mice were bred in cages with clean room air. Human cord blood MSCs were provided by MEDIPOST Co., Ltd (Korea). hCB-MSCs were cultured with alpha-minimum essential medium (Gibco Life Technologies, USA) and subcultured using 0.25% trypsin-EDTA (Gibco Life Technologies). Mice exposed to 6 months of cigarette smoke were intravenously injected with 5 × 104 hCB-MSCs. Mice were then sacrificed and their lungs were collected on days 1, 4, and 14 following injection of hCB-MSCs. RNA later was used to maintain RNA stability during mouse lung collection.
Total mRNA from lung tissue was prepared using Trizol (Invitrogen Life Technologies, USA) and purified using RNeasy columns (Qiagen, Germany) according to the manufacturer’s protocol. The purity and quantity of the isolated RNA were evaluated using aND-1000 spectrophotometer (NanoDrop Technologies Inc., USA), and a quality check of the RNA was performed using a 2100 Bioanalyzer (Agilent Technologies, USA). Only RNA samples with an RNA integrity number >7.5 were used for the microarray experiment.
RNA was amplified and purified using the AmbionIllumina RNA amplification kit (Ambion, USA) according to the manufacturer’s instructions. Biotinylated cRNA (1.5 μg) was hybridized to each MouseWG-6 expression bead array (Illumina, Inc., USA), and the array signal was detected using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Biosciences, UK). Arrays were scanned with an Illumina bead array reader confocal scanner.
The lung was inflated with 0.5% low-melting agarose and fixed using 4% formalin, and then embedded in paraffin and cut into 4-μm-thick sections for immunohistochemistry. The sections were stained using an Immunohistochemistry accessory kit (Bethyl Laboratories, Inc., USA) with anti-Egr-1 antibody (1:250, Abcam, UK) and then observed by microscopy.
All data analysis of differentially expressed genes was conducted using R 2.15.1. Raw expression data was log2-transformed and normalized by a quantile method. Statistical significance of the expression data was determined using a
A
A
Figure 4 shows the genes specifically altered by injection of hCB-MSCs at each time point and in common between the different time points using a Venn diagram. Seven genes and 21 genes were continuously up- and down-regulated, respectively, following injection of hCB-MSCs. The common up- and down-regulated genes are shown in Table 2. The number of up-regulated genes was the lowest at 1 day after injection of hCB-MSCs compared to the other time points and increased in a time-dependent manner. In contrast, the largest number of down-regulated genes showing differential expression was observed on day 1 after injection of hCB-MSCs. Figure 5 was shows the immunohistochemistry results for Egr-1 in the lungs. The Egr-1 gene was down-regulated in both groups following hCB-MSC injection. The results revealed only a minimal correlation between mRNA expression and protein expression.
The DEGs between the hCB-MSCs-injected and non-injected groups were categorized based on time and direction of expression. We also performed GO analysis using DAVID. The top 20 up-regulated genes at each time point are listed in Table 3. We also represent the fold-change following cigarette exposure in Table 3. The expression of oxygen transport- and anti-oxidant function-related genes such as
In the present study, we analyzed the gene expression profiles following cigarette exposure or non-exposure as well as hCBMSC-injected or non-injected mice. We focused on the changes in gene profiles between hCB-MSCs-injected vs. non-injected mouse lungs following the induction of COPD by chronic exposure to cigarette smoke and found that gene expression profiles varied with after injection of hCB-MSCs. Genes involved in oxidative stress, xenobiotic metabolic processes, and anti-protease functions were detected most frequently. In addition, GO term analysis showed that genes related to immune responses, blood vessel development, and metabolic processes were differentially expressed on days 1 and 4 after hCB-MSC injection compared to controls. DEGs on day 14 after injection of hCB-MSCs were related to cell growth regulation. Our results suggest that immune responses, oxidative stress, and transcription were regulated at a nearly stage after hCB-MSC injection (on days 1 and 4),and that blood vessel development and cell growth regulation predominated at a later stage after hCB-MSC injection (on day 14).
In previous reports, MSCs showed regenerative properties in animal models with smoke- and elastase-induced COPD (Huh et al., 2011; Katsha et al., 2011; Schweitzer et al., 2011). Recently, we reported that systemically injected MSCs were observed for only 1 day after cell injection in an elastase-induced emphysema model (Kim et al., 2014). Other reports failed to detect injected MSCs; therefore, stem cell biologists have attempted to explain the therapeutic effect of mesenchymal stem cells in these models in the context of the regulation of host cells or host microenvironment (Henning et al., 2014; Liang et al., 2014; Suga et al., 2014). To understand the detailed mechanisms of stem cell therapy, understanding the changes in host genes following MSC injection is required. Thus, we focused on the host changes based on the profiles of DEGs in this study.
Previous studies analyzed the gene expression profiles in the lungs of smoke-exposed mice (Halappanavar et al., 2009; Rangasamy et al., 2009).Inflammatory signaling was up-regulated following cigarette exposure. We also detected up-regulation of inflammatory-related genes such as
Therefore, MSCs may have regenerative roles that reestablish a balance between elastase and anti-elastase by up-regulating
There were some limitations to this study. One limitation is that the differential change in gene expression profiles did not involve exposure to normal air and exposure to normal air plus hCB-MSCs. Therefore, further studies of mice exposed to normal air and the use of injected cell controls are needed to correctly identify the detailed regeneration mechanisms of stem cells. Another limitation is that our microarray experiment used whole lungs. Although we determined organ-level changes in molecular signals following the injection of hCB-MSCs, it remains unclear how the gene profiles change in individual cells in the lung such as bronchial and alveolar epithelial cells, fibroblasts, and endothelial cells. More advanced techniques are needed to analyze the gene expression profiles using sections of the lung tissues or specific lung cell types.
This is the first study to determine the time-dependent molecular changes induced by treatment with hCB-MSCs in a cigarette smoke-induced COPD model. MSCs systemically injected into an animal disappear within several days; it is thought that these cells rarely differentiate into tissue resident cells in the lungs, such as epithelial cells. Therefore, studies focused on molecular changes in host tissues may reveal the regenerative mechanisms of MSCs. Our present report improves the understanding of the regenerative mechanisms induced by injection of hCB-MSCs based on the gene expression profile changes in a cigarette smoke-induced COPD animal model.
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