The role of pulmonary mesenchymal cells in airway epithelium regeneration during injury repair
- 165 Downloads
The airways of mammalian lung are lined with highly specialized cell types that are the target of airborne toxicants and injury. Several epithelial cell types and bone marrow-derived mesenchymal stem cells have been identified to serve as stem cells during injury repair. However, the contributions of endogenous mesenchymal cells to recruitment, expansion or differentiation of stem cells, and repair and reestablishment of the normal composition of airway epithelium following injury have not been addressed.
The role of mouse pulmonary mesenchymal cells was investigated by lineage tracing using Dermo1-Cre; ROSAmTmG mice. In experimental models of lung injury by lipopolysaccharide and naphthalene, GFP-labeled Dermo1+ mesenchymal cells were traced during injury repair. In vitro lung explant culture treated with or without lipopolysaccharide was also used to verify in vivo data.
During injury repair, a subgroup of GFP-labeled Dermo1+ mesenchymal cells were found to contribute to normal repair of the airway epithelium and differentiated into Club cells, ciliated cells, and goblet cells. In Club cell-specific naphthalene injury model, the process of Dermo1+ stem cell regenerating epithelial cells was dissected. The Dermo1+ stem cells was migrated into the airway epithelium layer sooner after injury, and sequentially differentiated transitionally to epithelial stem cells, such as neuroendocrine cells, and finally to newly differentiated Club cells, ciliated cells, and goblet cells in injury repair.
In this study, a population of Dermo1+ mesenchymal stem cell was identified to serve as stem cells in airway epithelial cell regeneration during injury repair. The Dermo1+ mesenchymal stem cell differentiated into epithelial stem cells before reestablishing various epithelial cells. These findings have implications for understanding the regulation of lung repair and the potential for usage of mesenchymal stem cells in therapeutic strategies for lung diseases.
KeywordsLung Dermo1 Mesenchymal stem cell Lipopolysaccharide Naphthalene Injury repair
Mesenchymal stem cell
Throughout life, multicellular organisms must regenerate cells to maintain the integrity and functions of their tissues after injury, but the capacity to repair the tissue damage may fail due to repeated injury and aging. The adult lung is one of the few organs that has a direct interface with the outside environment. The epithelial cells that line the airways are constantly exposed to potential toxic agents and pathogens. Therefore, it must be able to respond quickly and effectively to recover the cellular damage. The cellular hallmark of lung repair after injury is a rapid proliferative and differentiation response ultimately leading to restoration of the airway epithelium. Several origins of the stem cells that repair damaged airway epithelium have been identified [1, 2, 3, 4]. However, precise information regarding their emergence and diversification during injury repair is scant.
The conducting airways of the mammalian lung are composed of three major epithelial cell types, namely ciliated cells, non-ciliated Club cells, and neuroendocrine (NE) cells. These cells are arranged in an orderly manner to form branches and alveolar structures . The mesenchymal part is crucial in determining the shape and size of the lung, which can be subdivided into sub-mesothelial mesenchyme (marked by the expression of WNT2A and FGF10) and sub-epithelial mesenchyme (marked by the expression of NOGGIN) . Pulmonary mesenchyme contains a host of complex cell lineages including lymphatics, endothelial cells, smooth muscle cells, myofibroblasts, cartilage-forming cells, and mesothelial cells . The normal development of pulmonary mesenchyme is associated with the successful extension and branching of the airway [8, 9]. Coordination of pulmonary mesenchyme and epithelium is required to form a functional lung.
Various experimentally induced whole lung and airway injury models have been used, such as the toxicant naphthalene (NAPH) mediated abolishing of Club cells and the acute lung injury induced by lipopolysaccharide (LPS). Subsequently, the surviving cells are thought to serve as stem/progenitor cells to restore the epithelium. There are evidences that several cell types serve in this capacity. Fist, a subset of NAPH-resistant variant Club cells has been identified that seems to have the ability to self-renew as well as generate other cell types . Second, the pulmonary neuroendocrine (NE) cells are distinguished mainly by expression of PGP9.5. NE cells reside within a unique microenvironment known as neuroepithelial bodies and undergo expansion subsequent to NAPH injury . In bacterial LPS model, the lung parenchyma is damaged by the generation and release of proteases and reactive oxygen and nitrogen species produced by activated lung macrophages and transmigrated neutrophils in the interstitial and alveolar compartments. The end results are microvascular injury and diffuse alveolar damage with intrapulmonary hemorrhage, edema, and fibrin deposition . Mesenchymal stem cells (MSCs), commonly referred to as adult marrow stromal cells, show capacity to differentiate into a number of mature cell types, including fibroblasts, myofibroblasts, and epithelial cells . Treatment with MSCs protected LPS injured mice from death by decreasing the edema and reducing the inflammatory response . Recent study observed significant number of labeled MSCs had migrated from the bone marrow to the lung lesions and differentiated to macrophages, alveolar epithelial cells, and interstitial fibroblasts and myofibroblasts . As ongoing maintenance of the airways and repair after injury are key to normal respiratory function, precise knowledge of which cell type(s) are recruited to reestablish airway homeostasis and the precise mechanics of how repair is controlled is of significant interest.
Exogenously infused MSCs modulate tissue injury and repair. These properties have led to novel therapeutic strategies involving exogenous administration of MSCs in various injury and disease settings. Despite the broad therapeutic potential of this cell type, the in vivo role of endogenous MSCs remains undefined due to the absence of specific markers. In the current study, we demonstrate that a subgroup of Dermo1+ mesenchymal cells serve as MSCs to regenerate airway epithelial cells during LPS and NAPH-induced injury repair in mouse lung. These endogenous MSCs sequentially differentiated transitionally to epithelial stem cells, such as neuroendocrine cells, and finally to newly differentiated Club cells, ciliated cells and goblet cells. Moreover, the Dermo1+ MSCs are not HH/Gli1 signaling regulated mesenchymal cells.
Materials and methods
Dermo1-Cre, Gli1-CreERT2, and Gt (ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (referred as ROSAmTmG) mice were gifts from Parviz Minoo (University of Southern California, USA). Dermo1-Cre; ROSAmTmG and Gli1-CreERT2; ROSAmTmG mice were generated by crossing Dermo1-Cre and Gli1-CreERT2 with ROSAmTmG mice, respectively. All animals were maintained on a 12-h light/dark cycle with ad libitum access to water and feed in individually ventilated units in the specific-pathogen-free facility. During the experiment, all procedures, care, and handling of animals were in accordance with the guidelines developed by Beijing Association on Laboratory Animal Care and were approved by China Agricultural University (SKLAB-2015-10).
Tamoxifen (Sigma, USA) was dissolved in corn oil (Sigma, USA) at a concentration of 20 mg/mL. For lineage-tracing studies, Gli1-CreERT2; ROSAmTmG mice received five continuous doses of 75 mg/kg bodyweight tamoxifen via intraperitoneal injection to induce CRE-mediated GFP expression. Injury was induced after 10 days of chasing.
Adult mice (8–12 weeks) were selected for injury with no gender distinction. For LPS injury, 20 mL/kg bodyweight avertin (Sigma, USA, 20 mg/mL) was intraperitoneally injected to anesthetize the mice. Five milligrams per kilogram bodyweight LPS (Sigma, USA, 1 mg/mL, PBS for control mice) dissolved in PBS (phosphate-buffered saline, pH 7.4) was intratracheally instilled via a 24-gauge venous indwelling needle and a 1-mL syringe. An extra of 0.8 mL of gas was supplied to flush the liquid uniformly into the more distal bronchioles. Mice woke up naturally and sacrificed at 1, 3, 5, 7, or 14 days post injury (DPI). For naphathalene injury, 300 mg/kg bodyweight NAPH (Sigma, USA, 30 mg/mL, corn oil for control mice) dissolved in corn oil was intraperitoneally injected. Mice were sacrificed at 1, 3, 5, or 7 DPI. Three to 5 mice were analyzed per injury stage. Each injury process was repeated over three times.
RNA isolation and real-time quantitative polymerase chain reaction (qPCR)
Tissue RNAs were extracted by Qiagen RNeasy Mini Kit (QIAGEN, Germany) according to the handbook. One microgram of total RNAs was applied to synthesize the first-strand cDNAs by promega M-MLV Reverse Transcriptase (Promega, USA). Primers used for qPCR were designed via Primer3 software. Melting curve and amplification analyses were used to validate the primers. Quantification of targeted genes was performed on Roche LightCycler480 instrument with LightCycler 480 SYBR Green-based real-time qPCR kit reagents (Roche, Switzerland). PCR was conducted using the default thermal cycling parameter setting. Sample expression level was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression.
Tissue collection, fixation, and HE staining
The lung tissues were collected from mice around 2 months old and fixed in 4% PFA for 16 h in 4 °C. For paraffin sections, tissues were dehydrated by gradient ethanol solutions, embedded in paraffin. For frozen sections, tissues were dehydrated by gradient sucrose solutions, embedded in OCT. Tissue sections in 5 μm were prepared for hematoxylin-eosin (HE) staining and immunohistochemistry analysis.
Lung tissue sections were stained with hematoxylin and eosin for HE analysis. α-SMA (A5228, Sigma, US); CC10 (07-623, Meck Millipore, US); CD44 (ab25340), CD90 (ab3105), GFP (ab13970), MUC5AC (ab3649), and SMMHC (ab53219) were from Abcam; DESMIN (sc-271677), FOXJ1 (sc-53139), and PDGFA (sc-128) were from Santa Cruz; KI67 (Rm-9106-S0, Thermos Fisher); PGP9.5 (53772, Anaspec); SOX2 (3579) and SOX9 (82630) were from Cell Signaling Technology.
Lung explant culture
Whole mouse lungs were dissected into small pieces and embedded into growth factor reduced BD matrigel matrix (BD Biosciences, USA), diluted 1:1 in DMEM/Ham’s F12 medium. After polymerization of the matrigel at 37 °C in a humidified incubator, lung explants were covered with culture medium (DMEM/Ham’s F12, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% FBS) with or without 10 μg/mL LPS and cultured at 37 °C in a humidified incubator under 5% CO2 for 6 h.
Cell counting and image analysis
Images were captured by an Olympus BX53 microscope under × 20 objective or Nikon A1 laser scanning confocal microscope under × 100 objective after immunostaining (Additional file 2). By ImageJ software, more than 10 random fields per section under × 20 objective were analyzed for cell quantification.
The experimental data are presented as the mean ± SD and were analyzed by paired Student’s t test using SPSS21.0 software to compare the difference between samples. P value < 0.05 was considered significant.
Dermo1+ mesenchymal cells modulated regeneration of airway epithelium after LPS injury
Dermo1 is a basic helix-loop-helix transcription factor that is highly expressed in mesodermal tissues in mice . To investigate whether lung endogenous mesenchymal progenitor/stem cells contribute to regeneration of the airway epithelium in injury repair, we have generated Dermo1-Cre; ROSAmTmG mice by crossing Dermo1-Cre with ROSAmTmG mice (Fig. 1e) . In Dermo1-Cre; ROSAmTmG mice, the Dermo1 positive (Dermo1+) mesenchymal cells and all of their progeny express green fluorescent protein (GFP) (Fig. 1f). After LPS injection, Dermo1+ cells were observed on the epithelial layer in both proximal and distal airways on day 1. The number of Dermo1+ epithelial cells was increased on day 3 and decreased once the injury was repaired (Fig. 1g). The above results suggested that the Dermo1+ mesenchymal cells acted as stem cells to regenerate airway epithelium in LPS injury repair.
Dermo1+ stem cells proliferated in airway epithelial cell regeneration after LPS injury
Dermo1+ stem cells differentiated into various airway epithelial cell types in LPS injury repair
Dermo1+ stem cells differentiated into airway epithelial cells after LPS treatment in vitro
Dermo1+ stem cells regenerated Club cells through NE cells in NAPH-induced airway injury
Dermo1+ stem cells were not Gli1 signaling related
Dermo1+ stem cells were endogenous mesenchymal stem cells
To address whether the Dermo1+ epithelial cells occurred in injury repair maintained characteristic of mesenchymal cells, the expression of pulmonary mesenchymal cell markers, PDGFA, DESMIN, αSMA, and SMMHC, were analyzed in Dermo1+ epithelial cells. In multiple experiments, we detected no expression of any of these genes in Dermo1+ epithelial cells (Fig. 7b).
The purpose of the current study was to determine the precise role of endogenous mesenchymal stem cells in lung injury repair. The choice of Dermo1 was based on its specific expression in the pulmonary mesenchyme. In Dermo1-Cre; ROSAmTmG mice, Dermo1+ cells were lineage traced by GFP labeling. Examination of airway epithelial regeneration in the LPS- and NAPH-induced injury/repair model revealed a process of Dermo1+ cell population dynamics characterized by expression of epithelial cell markers that lead to regeneration of Club cells, ciliated cells, and goblet cells. During this well-orchestrated process, Dermo1+ mesenchymal stem cells (MSCs) sequentially transdifferentiate into epithelial stem cells and terminal differentiated epithelial cells. These studies provide novel evidence that a subgroup of Dermo1+ cells serves as endogenous pulmonary MSCs in the re-establishment of a functional airway epithelium by regulating the step-wise transition of putative stem cells to transitional epithelial stem cell intermediates and, finally, to newly differentiated epithelial cells.
Lung mesenchyme is a critical determinant of the shape and size of the lung, the extent and patterning of epithelial branching, and the formation of the pulmonary vasculature and interstitial mesenchymal components of the adult lung . Compared with epithelium, the composition of the pulmonary mesenchyme is more complicated. Crosstalk between pulmonary mesenchyme and epithelium has been demonstrated during lung development and injury repair. Different mesenchymal progenitor/stem cells have been identified to provide the niches for epithelia to maintain their stem cell capacity. For example, Axin2+Pdgfrα+ mesenchymal cells serve as alveolar stem cell niche supporting alveolar cell growth and regeneration, and Axin2+Pdgfrα− myofibrogenic progenitors contribute to pathologically deleterious myofibroblasts . Lgr6+ mesenchymal cells are also found to promote epithelial progenitors differentiate into airway cells via Wnt-Fgf10 cooperation, whereas Lgr5+ cells promote epithelial progenitors differentiate into alveolar cells through Wnt pathway . Meanwhile, whether MSCs can differentiate into epithelial cells in injury repair is wildly studied. BMMSCs have shown the capability to ameliorate lung injury and generated a significant amount of interests as a potential therapy for acute or chronic lung diseases [24, 25]. MSCs work through multiple mechanisms, including cell engraftment, immunomodulation, alveolar fluid clearance, lung protein permeability, and antibacterial properties . However, a number of issues central to whether or not using stem cells in therapeutic approaches will be successful. Problems with interpreting the results of these studies include having limited controls, failure of the stem cells to engraft, and the generation of inflammatory events by the treatment itself. In addition, the processing procedures, like isolation, in vitro amplification, immunological analysis , and uncontrollable change of cell morphology , will greatly limit their wide clinical applications. Thus, more and more tissue-resident endogenous mesenchymal stem cells are studied in a variety of tissues or organs, such as fat , placenta and blood , and skeletal muscle .
The crosstalk between pulmonary epithelium and mesenchyme was widely reported. Among which, Shh signaling pathway played an important role. Gli1+ cells traced by Shh signaling pathway reporter mice acted as progenitor cells and promoted repair of the damaged epithelial cells through paracrine actions . Gli1+ cells were peri-bronchial mesenchymal cells anatomically closer to the epithelial cell layer, which were also a subgroup of Dermo1+ cells. By tracing Gli1+ mesenchymal cells, surprisingly, Gli1+ cells did not contribute to the regeneration of airway epithelial after LPS injury. We realized that the current data could not distinguish which signaling pathways activated the Dermo1+ MSCs. The detailed mechanisms and the factors involved in required further investigation.
Acute lung injury (ALI) is an important problem in humans; however, its pathogenesis is poorly understood. To investigate the molecular mechanisms of ALI, various experimental models have been used, the most common being the endotoxin (bacterial LPS) model. In our study, intratracheal instillation of high dose of LPS caused severe lung injury as the inflammatory reaction was still intense at 7 days after injury. Various epithelial and mesenchymal derived cells were extensively damaged. Interestingly, the emergence of Dermo1+ MSCs in the airway epithelium was found in 1 day after injury. The quick response of Dermo1+ MSCs also occurred in the NAPH-induced injury model, which was even earlier than the presence of epithelial stem cells. Based on our observations, activation of endogenous mesenchymal stem cells, such as Dermo1+ MSCs, maybe a more preferable therapeutic approach than engrafting of bone marrow-derived MSCs.
In sum, the results of this study suggest that, during mouse lung LPS and naphthalene injury repair, a population of Dermo1+ mesenchymal cells serve as a reservoir for epithelial cell regeneration and re-establishment of the normal airway epithelium. The Dermo1+ mesenchymal stem cell differentiated into epithelial stem cells before reestablishing various epithelial cells. These findings have implications for understanding the regulation of lung repair and the potential for usage of mesenchymal stem cells in therapeutic strategies for lung diseases.
YX contributed to the study concept and design, analysis and interpretation of data, drafting of the manuscript. SF and SZ contributed acquisition of data, analysis and interpretation of data. HD contributed to the acquisition of data. TT contributed to the technical and material support. XH provided the animals. CL contributed to the research design, analysis and interpretation of data. All authors read and approved the final manuscript.
This work is supported by National Basic Research Program (2016YFA0100202), the Project for Extramural Scientists of Beijing Advanced Innovation Center for Food Nutrition and Human Health, the Project for Extramural Scientists of State Key Laboratory of Agrobiotechnology (2019SKLAB6-22) and China Postdoctoral Science Foundation Funded Project (2018M641550 & 2019T120161).
Ethics approval and consent to participate
All experimental procedures for handling and care of mice were following the instructions of the Institutional Animal Care Committee and approved by the Ethics Committee of the Agriculture University of China (Permission Number:
CAU20161010-1, valid period: 10,10,2016-9,22,2019).
Consent for publication
The authors declare that they have no competing interests.
- 25.Sun J, Han ZB, Liao W, Yang SG, Yang Z, Yu J, et al. Intrapulmonary delivery of human umbilical cord mesenchymal stem cells attenuates acute lung injury by expanding CD4+CD25+ Forkhead Boxp3 (FOXP3)+ regulatory T cells and balancing anti- and pro-inflammatory factors. Cell Physiol Biochem. 2011;27:587–96.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.