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Murthy S, Seabold DA, Gautam LK, Caceres AM, Sease R, Calvert BA, Busch SM, Neely A, Marconett CN, Ryan AL. Culture conditions differentially regulate the inflammatory niche and cellular phenotype of tracheobronchial basal stem cells. Am J Physiol Lung Cell Mol Physiol 2025; 328:L538-L553. [PMID: 39982813 DOI: 10.1152/ajplung.00293.2024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2024] [Revised: 10/08/2024] [Accepted: 02/07/2025] [Indexed: 02/23/2025] Open
Abstract
Bronchial epithelial cells derived from the tracheobronchial regions of human airways (HBECs) provide a valuable in vitro model for studying pathological mechanisms and evaluating therapeutics. This cell population comprises a mixed population of basal cells (BCs), the predominant stem cell in airways capable of both self-renewal and functional differentiation. Despite their potential for regenerative medicine, BCs exhibit significant phenotypic variability in culture. To investigate how culture conditions influence BC phenotype and function, we expanded three independent BC isolates in three media: airway epithelial cell growth medium (AECGM), dual-SMAD inhibitor (DSI)-enriched AECGM, and PneumaCult Ex plus (PEx+). Analysis through RNA sequencing, immune assays, and impedance measurements revealed that PEx+ media significantly drove cell proliferation and a broad proinflammatory phenotype in BCs. In contrast, BCs expanded in AECGM and displayed increased expression of structural and extracellular matrix components at higher passage. AECGM increased expression of some cytokines at high passage, whereas DSI suppressed inflammation implicating the involvement TGF-β in BC inflammatory processes. Differentiation capacity of BCs declined with time in culture irrespective of expansion media. This was associated with an increase in PLUNC expressing secretory cells in AECGM and PEx+ media consistent with the known immune modulatory role of PLUNC in the airways. These findings highlight the profound impact of media conditions on inflammatory niche established by, and function of, in vitro expanded BCs. The broad proinflammatory phenotype driven by PEx+ media, in particular, should be considered in the development of cell-based models for airway diseases and therapeutic applications.NEW & NOTEWORTHY Airway basal cells, vital for airway regeneration and potential therapies, show significant changes based on culture conditions. Our study reveals that media composition and culture duration greatly affect basal cell properties with profound changes in the proinflammatory phenotype and extracellular matrix deposition driven by changes in growth conditions. These results underscore the critical impact of culture conditions on BC phenotype, influencing cell-based models for airway disease research and therapy.
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Affiliation(s)
- Shubha Murthy
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
| | - Denise A Seabold
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
| | - Lalit K Gautam
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
| | - Adrian M Caceres
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
| | - Rosemary Sease
- Department of Medicine, Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, California, United States
- Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, California, United States
| | - Ben A Calvert
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
- Department of Medicine, Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, California, United States
| | - Shana M Busch
- Department of Medicine, Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, California, United States
| | - Aaron Neely
- Department of Integrative Translational Sciences, Beckman Research Institute, City of Hope, Duarte, California, United States
| | - Crystal N Marconett
- Department of Medicine, Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, California, United States
- Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, California, United States
- Department of Integrative Translational Sciences, Beckman Research Institute, City of Hope, Duarte, California, United States
- Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
| | - Amy L Ryan
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States
- Department of Medicine, Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, California, United States
- Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, California, United States
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Wen TZ, Li TR, Chen XY, Chen HY, Wang S, Fu WJ, Xiao SQ, Luo J, Tang R, Ji JL, Huang JF, He ZC, Luo T, Zhao HL, Chen C, Miao JY, Niu Q, Wang Y, Bian XW, Yao XH. Increased adrenal steroidogenesis and suppressed corticosteroid responsiveness in critical COVID-19. Metabolism 2024; 160:155980. [PMID: 39053691 DOI: 10.1016/j.metabol.2024.155980] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Revised: 07/01/2024] [Accepted: 07/19/2024] [Indexed: 07/27/2024]
Abstract
BACKGROUND The effect of coronavirus disease 2019 (COVID-19) on adrenal endocrine metabolism in critically ill patients remains unclear. This study aimed to investigate the alterations in adrenal steroidogenic activity, elucidate underlying mechanisms, provide in situ histopathological evidence, and examine the clinical implications. METHODS The comparative analyses of the adrenal cortices from 24 patients with fatal COVID-19 and 20 matched controls were performed, excluding patients previously treated with glucocorticoids. SARS-CoV-2 and its receptors were identified and pathological alterations were examined. Furthermore, histological examinations, immunohistochemical staining and ultrastructural analyses were performed to assess corticosteroid biosynthesis. The zona glomerulosa (ZG) and zona fasciculata (ZF) were then dissected for proteomic analyses. The biological processes that affected steroidogenesis were analyzed by integrating histological, proteomic, and clinical data. Finally, the immunoreactivity and responsive genes of mineralocorticoid and glucocorticoid receptors in essential tissues were quantitatively measured to evaluate corticosteroid responsiveness. FINDINGS The demographic characteristics of COVID-19 patients were comparable with those of controls. SARS-CoV-2-like particles were identified in the adrenocortical cells of three patients; however, these particles did not affect cellular morphology or steroid synthesis compared with SARS-CoV-2-negative specimens. Although the adrenals exhibited focal necrosis, vacuolization, microthrombi, and inflammation, widespread degeneration was not evident. Notably, corticosteroid biosynthesis was significantly enhanced in both the ZG and ZF of COVID-19 patients. The increase in the inflammatory response and cellular differentiation in the adrenal cortices of patients with critical COVID-19 was positively correlated with heightened steroidogenic activity. Additionally, the appearance of more dual-ZG/ZF identity cells in COVID-19 adrenals was in accordance with the increased steroidogenic function. However, activated mineralocorticoid and glucocorticoid receptors and their responsive genes in vital tissues were markedly reduced in patients with critical COVID-19. INTERPRETATION Critical COVID-19 was characterized by potentiated adrenal steroidogenesis, associated with increased inflammation, enhanced differentiation and elevated dual-ZG/ZF identity cells, alongside suppressed corticosteroid responsiveness. These alterations implied the reduced effectiveness of conventional corticosteroid therapy and underscored the need for evaluation of the adrenal axis and corticosteroid sensitivity.
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Affiliation(s)
- Tian-Zi Wen
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Tian-Ran Li
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Xin-Yu Chen
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - He-Yuan Chen
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Shuai Wang
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Wen-Juan Fu
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Shi-Qi Xiao
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Jie Luo
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Rui Tang
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Jia-Le Ji
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Jia-Feng Huang
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Zhi-Cheng He
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Tao Luo
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Hong-Liang Zhao
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Cong Chen
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Jing-Ya Miao
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Qin Niu
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China
| | - Yan Wang
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China; Jinfeng Laboratory, Chongqing, China
| | - Xiu-Wu Bian
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China; YuYue Laboratory, Chongqing, China.
| | - Xiao-Hong Yao
- Institute of Pathology, Southwest Hospital, Third Military Medical University (Army Medical University), and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Chongqing, China.
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Murthy S, Seabold DA, Gautam LK, Caceres AM, Sease R, Calvert BA, Busch S, Neely A, Marconett CN, Ryan AL. Culture Conditions Differentially Regulate the Inflammatory Niche and Cellular Phenotype of Tracheo-Bronchial Basal Stem Cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.04.611264. [PMID: 39282256 PMCID: PMC11398510 DOI: 10.1101/2024.09.04.611264] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 09/21/2024]
Abstract
Human bronchial epithelial cells (HBECs) derived from the tracheo-bronchial regions of human airways provide an excellent in vitro model for studying pathological mechanisms and evaluating therapeutics in human airway cells. This cell population comprises a mixed population of basal cells (BCs), the predominant stem cell in airways capable of both self-renewal and functional differentiation. Despite their potential for regenerative medicine, BCs exhibit significant phenotypic variability in culture. To investigate how culture conditions influence BC phenotype and function, we expanded three independent BC isolates in three media, airway epithelial cell growth medium (AECGM), dual-SMAD inhibitor (DSI)-enriched AECGM, and Pneumacult Ex plus (PEx+). Extensive RNA sequencing, immune assays and electrical measurements revealed that PEx+ media significantly drove cell proliferation and a broad pro-inflammatory phenotype in BCs. In contrast, BCs expanded in AECGM, displayed increased expression of structural and extracellular matrix components at high passage. Whereas culture in AECGM increased expression of some cytokines at high passage, DSI suppressed inflammation altogether thus implicating TGF-β in BC inflammatory processes. Differentiation capacity declined with time in culture irrespective of expansion media except for PLUNC expressing secretory cells that were elevated at high passage in AECGM and PEx+ suggestive of an immune modulatory role of PLUNC in BCs. These findings underscore the profound impact of media conditions on inflammatory niche and function of in vitro expanded BCs. The broad pro-inflammatory phenotype driven by PEx+ media, in particular, should be considered in the development of cell-based models for airway diseases and therapeutic application.
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Affiliation(s)
- Shubha Murthy
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, IA
| | - Denise A. Seabold
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, IA
| | - Lalit K. Gautam
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, IA
| | - Adrian M. Caceres
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, IA
| | - Rosemary Sease
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, CA
- Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA
| | - Ben A. Calvert
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, IA
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, CA
| | - Shana Busch
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, CA
| | - Aaron Neely
- Department of Integrative Translational Sciences, Beckman Research Institute, City of Hope, Duarte, CA
| | - Crystal N. Marconett
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, CA
- Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA
- Department of Integrative Translational Sciences, Beckman Research Institute, City of Hope, Duarte, CA
- Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA
| | - Amy L. Ryan
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, IA
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, University of Southern California, Los Angeles, CA
- Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA
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Shi ZN, Zhang X, Du CY, Zhao B, Liu SG. Effects of pulmonary surfactant combined with noninvasive positive pressure ventilation in neonates with respiratory distress syndrome. World J Clin Cases 2024; 12:5366-5373. [PMID: 39156082 PMCID: PMC11238696 DOI: 10.12998/wjcc.v12.i23.5366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/22/2024] [Revised: 05/25/2024] [Accepted: 06/12/2024] [Indexed: 07/05/2024] Open
Abstract
BACKGROUND Neonatal respiratory distress syndrome (NRDS) is one of the most common diseases in neonatal intensive care units, with an incidence rate of about 7% among infants. Additionally, it is a leading cause of neonatal death in hospitals in China. The main mechanism of the disease is hypoxemia and hypercapnia caused by lack of surfactant. AIM To explore the effect of pulmonary surfactant (PS) combined with noninvasive positive pressure ventilation on keratin-14 (KRT-14) and endothelin-1 (ET-1) levels in peripheral blood and the effectiveness in treating NRDS. METHODS Altogether 137 neonates with respiratory distress syndrome treated in our hospital from April 2019 to July 2021 were included. Of these, 64 control cases were treated with noninvasive positive pressure ventilation and 73 observation cases were treated with PS combined with noninvasive positive pressure ventilation. The expression of KRT-14 and ET-1 in the two groups was compared. The deaths, complications, and PaO2, PaCO2, and PaO2/FiO2 blood gas indexes in the two groups were compared. Receiver operating characteristic curve (ROC) analysis was used to determine the diagnostic value of KRT-14 and ET-1 in the treatment of NRDS. RESULTS The observation group had a significantly higher effectiveness rate than the control group. There was no significant difference between the two groups in terms of neonatal mortality and adverse reactions, such as bronchial dysplasia, cyanosis, and shortness of breath. After treatment, the levels of PaO2 and PaO2/FiO2 in both groups were significantly higher than before treatment, while the level of PaCO2 was significantly lower. After treatment, the observation group had significantly higher levels of PaO2 and PaO2/FiO2 than the control group, while PaCO2 was notably lower in the observation group. After treatment, the KRT-14 and ET-1 levels in both groups were significantly decreased compared with the pre-treatment levels. The observation group had a reduction of KRT-14 and ET-1 levels than the control group. ROC curve analysis showed that the area under the curve (AUC) of KRT-14 was 0.791, and the AUC of ET-1 was 0.816. CONCLUSION Combining PS with noninvasive positive pressure ventilation significantly improved the effectiveness of NRDS therapy. KRT-14 and ET-1 levels may have potential as therapeutic and diagnostic indicators.
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Affiliation(s)
- Ze-Ning Shi
- Department of Pediatrics, Army Military Medical University Officer School Affiliated Hospital, Shijiazhuang 050000, Hebei Province, China
| | - Xin Zhang
- Department of Anesthesiology, Army Military Medical University Officer School Affiliated Hospital, Shijiazhuang 050000, Hebei Province, China
| | - Chun-Yuan Du
- Department of Gynecology and Obstetrics, Army Military Medical University Officer School Affiliated Hospital, Shijiazhuang 050000, Hebei Province, China
| | - Bing Zhao
- Department of Anesthesiology, Army Military Medical University Officer School Affiliated Hospital, Shijiazhuang 050000, Hebei Province, China
| | - Shu-Gang Liu
- Department of Pediatrics, Army Military Medical University Officer School Affiliated Hospital, Shijiazhuang 050000, Hebei Province, China
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Rouhani MJ, Janes SM, Kim CF. Epithelial stem and progenitor cells of the upper airway. Cells Dev 2024; 177:203905. [PMID: 38355015 DOI: 10.1016/j.cdev.2024.203905] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2023] [Accepted: 02/08/2024] [Indexed: 02/16/2024]
Abstract
The upper airway acts as a conduit for the passage of air to the respiratory system and is implicated in several chronic diseases. Whilst the cell biology of the distal respiratory system has been described in great detail, less is known about the proximal upper airway. In this review, we describe the relevant anatomy of the upper airway and discuss the literature detailing the identification and roles of the progenitor cells of these regions.
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Affiliation(s)
- Maral J Rouhani
- UCL Respiratory, Division of Medicine, University College London, London, UK
| | - Sam M Janes
- UCL Respiratory, Division of Medicine, University College London, London, UK
| | - Carla F Kim
- Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
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Ievlev V, Pai AC, Dillon DS, Kuhl S, Lynch TJ, Freischlag KW, Gries CB, Engelhardt JF, Parekh KR. Development and characterization of ferret ex vivo tracheal injury and cell engraftment model. Front Med (Lausanne) 2023; 10:1144754. [PMID: 37113613 PMCID: PMC10126424 DOI: 10.3389/fmed.2023.1144754] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2023] [Accepted: 03/15/2023] [Indexed: 04/29/2023] Open
Abstract
The field of airway biology research relies primarily on in vitro and in vivo models of disease and injury. The use of ex vivo models to study airway injury and cell-based therapies remains largely unexplored although such models have the potential to overcome certain limitations of working with live animals and may more closely replicate in vivo processes than in vitro models can. Here, we characterized a ferret ex vivo tracheal injury and cell engraftment model. We describe a protocol for whole-mount staining of cleared tracheal explants, and showed that it provides a more comprehensive structural overview of the surface airway epithelium (SAE) and submucosal glands (SMGs) than 2D sections, revealing previously underappreciated structural anatomy of tracheal innervation and vascularization. Using an ex vivo model of tracheal injury, we evaluated the injury responses in the SAE and SMGs that turned out to be consistent with published in vivo work. We used this model to assess factors that influence engraftment of transgenic cells, providing a system for optimizing cell-based therapies. Finally, we developed a novel 3D-printed reusable culture chamber that enables live imaging of tracheal explants and differentiation of engrafted cells at an air-liquid interface. These approaches promise to be useful for modeling pulmonary diseases and testing therapies. Graphical abstract1,2. We describe here a method for differential mechanical injury of ferret tracheal explants that can be used to evaluate airway injury responses ex vivo. 3. Injured explants can be cultured at ALI (using the novel tissue-transwell device on the right) and submerged long-term to evaluate tissue-autonomous regeneration responses. 4. Tracheal explants can also be used for low throughput screens of compounds to improve cell engraftment efficiency or can be seeded with particular cells to model a disease phenotype. 5. Lastly, we demonstrate that ex vivo-cultured tracheal explants can be evaluated by various molecular assays and by immunofluorescent imaging that can be performed live using our custom-designed tissue-transwell.
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Affiliation(s)
- Vitaly Ievlev
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, United States
| | - Albert C. Pai
- Department of Cardiothoracic Surgery, Carver College of Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA, United States
| | - Drew S. Dillon
- Protostudios, Carver College of Medicine, University of Iowa, Iowa City, IA, United States
| | - Spencer Kuhl
- Protostudios, Carver College of Medicine, University of Iowa, Iowa City, IA, United States
| | - Thomas J. Lynch
- Department of Cardiothoracic Surgery, Carver College of Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA, United States
| | - Kyle W. Freischlag
- Department of Cardiothoracic Surgery, Carver College of Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA, United States
| | - Caitlyn B. Gries
- Department of Cardiothoracic Surgery, Carver College of Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA, United States
| | - John F. Engelhardt
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, United States
| | - Kalpaj R. Parekh
- Department of Cardiothoracic Surgery, Carver College of Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA, United States
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A Kaleidoscope of Keratin Gene Expression and the Mosaic of Its Regulatory Mechanisms. Int J Mol Sci 2023; 24:ijms24065603. [PMID: 36982676 PMCID: PMC10052683 DOI: 10.3390/ijms24065603] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 03/07/2023] [Accepted: 03/10/2023] [Indexed: 03/17/2023] Open
Abstract
Keratins are a family of intermediate filament-forming proteins highly specific to epithelial cells. A combination of expressed keratin genes is a defining property of the epithelium belonging to a certain type, organ/tissue, cell differentiation potential, and at normal or pathological conditions. In a variety of processes such as differentiation and maturation, as well as during acute or chronic injury and malignant transformation, keratin expression undergoes switching: an initial keratin profile changes accordingly to changed cell functions and location within a tissue as well as other parameters of cellular phenotype and physiology. Tight control of keratin expression implies the presence of complex regulatory landscapes within the keratin gene loci. Here, we highlight patterns of keratin expression in different biological conditions and summarize disparate data on mechanisms controlling keratin expression at the level of genomic regulatory elements, transcription factors (TFs), and chromatin spatial structure.
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Ievlev V, Lynch TJ, Freischlag KW, Gries CB, Shah A, Pai AC, Ahlers BA, Park S, Engelhardt JF, Parekh KR. Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells. JCI Insight 2023; 8:e162041. [PMID: 36512409 PMCID: PMC9977304 DOI: 10.1172/jci.insight.162041] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2022] [Accepted: 12/07/2022] [Indexed: 12/15/2022] Open
Abstract
Keratin expression dynamically changes in airway basal cells (BCs) after acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In bronchiolitis obliterans (BO) after lung transplantation, BC clonogenicity declines, which is associated with a switch from keratin15 (Krt15) to keratin14 (Krt14). We investigated these keratins' roles using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs did not differentiate into club and ciliated cells but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo after injury. Krt14, but not Krt15, bound the tumor suppressor stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, whereas dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts with Krt14-KO phenotype and resembles the phenotype in BO with decreased clonogenicity, increased Krt14, and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior, which is relevant in chronic disease states like BO.
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Affiliation(s)
- Vitaly Ievlev
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Thomas J. Lynch
- Department of Cardiothoracic Surgery, University of Iowa Hospitals and Clinics, Carver College of Medicine, Iowa City, Iowa, USA
| | - Kyle W. Freischlag
- Department of Cardiothoracic Surgery, University of Iowa Hospitals and Clinics, Carver College of Medicine, Iowa City, Iowa, USA
| | - Caitlyn B. Gries
- Department of Cardiothoracic Surgery, University of Iowa Hospitals and Clinics, Carver College of Medicine, Iowa City, Iowa, USA
| | - Anit Shah
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Albert C. Pai
- Department of Cardiothoracic Surgery, University of Iowa Hospitals and Clinics, Carver College of Medicine, Iowa City, Iowa, USA
| | - Bethany A. Ahlers
- Department of Cardiothoracic Surgery, University of Iowa Hospitals and Clinics, Carver College of Medicine, Iowa City, Iowa, USA
| | - Soo Park
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - John F. Engelhardt
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Kalpaj R. Parekh
- Department of Cardiothoracic Surgery, University of Iowa Hospitals and Clinics, Carver College of Medicine, Iowa City, Iowa, USA
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Xu H, Pan G, Wang J. Repairing Mechanisms for Distal Airway Injuries and Related Targeted Therapeutics for Chronic Lung Diseases. Cell Transplant 2023; 32:9636897231196489. [PMID: 37698245 PMCID: PMC10498699 DOI: 10.1177/09636897231196489] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2023] [Revised: 08/04/2023] [Accepted: 08/07/2023] [Indexed: 09/13/2023] Open
Abstract
Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), involve progressive and irreversible destruction and pathogenic remodeling of airways and have become the leading health care burden worldwide. Pulmonary tissue has extensive capacities to launch injury-responsive repairing programs (IRRPs) to replace the damaged or dead cells upon acute lung injuries. However, the IRRPs are frequently compromised in chronic lung diseases. In this review, we aim to provide an overview of somatic stem cell subpopulations within distal airway epithelium and the underlying mechanisms mediating their self-renewal and trans-differentiation under both physiological and pathological circumstances. We also compared the differences between humans and mice on distal airway structure and stem cell composition. At last, we reviewed the current status and future directions for the development of targeted therapeutics on defective distal airway regeneration and repairment in chronic lung diseases.
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Affiliation(s)
- Huahua Xu
- State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
- Guangzhou Laboratory, Guangzhou International Bio Island, Guangzhou, China
| | - Guihong Pan
- Department of Pediatric Surgery, Guangdong Provincial Key Laboratory of Research in Structural Birth Defect Disease, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China
| | - Jun Wang
- Department of Pediatric Surgery, Guangdong Provincial Key Laboratory of Research in Structural Birth Defect Disease, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China
- The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China
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10
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Gautam LK, Harriott NC, Caceres AM, Ryan AL. Basic Science Perspective on Engineering and Modeling the Large Airways. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1413:73-106. [PMID: 37195527 DOI: 10.1007/978-3-031-26625-6_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/18/2023]
Abstract
The airway epithelium provides a physical and biochemical barrier playing a key role in protecting the lung from infiltration of pathogens and irritants and is, therefore, crucial in maintaining tissue homeostasis and regulating innate immunity. Due to continual inspiration and expiration of air during breathing, the epithelium is exposed to a plethora of environmental insults. When severe or persistent, these insults lead to inflammation and infection. The effectiveness of the epithelium as a barrier is reliant upon its capacity for mucociliary clearance, immune surveillance, and regeneration upon injury. These functions are accomplished by the cells that comprise the airway epithelium and the niche in which they reside. Engineering of new physiological and pathological models of the proximal airways requires the generation of complex structures comprising the surface airway epithelium, submucosal gland epithelium, extracellular matrix, and niche cells, including smooth muscle cells, fibroblasts, and immune cells. This chapter focuses on the structure-function relationships in the airways and the challenges of developing complex engineered models of the human airway.
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Affiliation(s)
- Lalit K Gautam
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Noa C Harriott
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Adrian M Caceres
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - Amy L Ryan
- Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
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11
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Kelly NA, Shontz KM, Bergman M, Manning AM, Reynolds SD, Chiang T. Biobanked tracheal basal cells retain the capacity to differentiate. Laryngoscope Investig Otolaryngol 2022; 7:2119-2125. [PMID: 36544928 PMCID: PMC9764751 DOI: 10.1002/lio2.925] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2022] [Revised: 08/19/2022] [Accepted: 09/07/2022] [Indexed: 12/24/2022] Open
Abstract
Objective While airway epithelial biorepositories have established roles in the study of bronchial progenitor stem (basal) cells, the utility of a bank of tracheal basal cells from pediatric patients, who have or are suspected of having an airway disease, has not been established. In vitro study of these cells can enhance options for tracheal restoration, graft design, and disease modeling. Development of a functional epithelium in these settings is a key measure. The aim of this study was the creation a tracheal basal cell biorepository and assessment of recovered cells. Methods Pediatric patients undergoing bronchoscopy were identified and endotracheal brush (N = 29) biopsies were collected. Cells were cultured using the modified conditional reprogramming culture (mCRC) method. Samples producing colonies by day 14 were passaged and cryopreserved. To explore differentiation potential, cells were thawed and differentiated using the air-liquid interface (ALI) method. Results No adverse events were associated with biopsy collection. Of 29 brush biopsies, 16 (55%) were successfully cultured to passage 1/cryopreserved. Samples with higher initial cell yields were more likely to achieve this benchmark. Ten unique donors were then thawed for analysis of differentiation. The average age was 2.2 ± 2.2 years with five donors (50%) having laryngotracheal pathology. Nine donors (90%) demonstrated differentiation capacity at 21 days of culture, as indicated by detection of ciliated cells (ACT+) and mucous cells (MUC5B+). Conclusion Pediatric tracheal basal cells can be successfully collected and cryopreserved. Recovered cells retain the ability to differentiate into epithelial cell types in vitro. Level of Evidence Level 3.
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Affiliation(s)
- Natalie A. Kelly
- Department of OtolaryngologyNationwide Children's HospitalColumbusOhioUSA
| | - Kimberly M. Shontz
- Center for Regenerative MedicineAbigail Wexner Research Institute at Nationwide Children's HospitalColumbusOhioUSA
| | - Maxwell Bergman
- Department of Otolaryngology‐Head and Neck SurgeryThe Ohio State Wexner Medical CenterColumbusOhioUSA
| | - Amy M. Manning
- Department of OtolaryngologyNationwide Children's HospitalColumbusOhioUSA
- Department of Otolaryngology‐Head and Neck SurgeryThe Ohio State Wexner Medical CenterColumbusOhioUSA
| | - Susan D. Reynolds
- Center for Perinatal MedicineAbigail Wexner Research Institute at Nationwide Children's HospitalColumbusOhioUSA
| | - Tendy Chiang
- Department of OtolaryngologyNationwide Children's HospitalColumbusOhioUSA
- Center for Regenerative MedicineAbigail Wexner Research Institute at Nationwide Children's HospitalColumbusOhioUSA
- Department of Otolaryngology‐Head and Neck SurgeryThe Ohio State Wexner Medical CenterColumbusOhioUSA
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Zhou Y, Yang Y, Guo L, Qian J, Ge J, Sinner D, Ding H, Califano A, Cardoso WV. Airway basal cells show regionally distinct potential to undergo metaplastic differentiation. eLife 2022; 11:e80083. [PMID: 36178196 PMCID: PMC9578702 DOI: 10.7554/elife.80083] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2022] [Accepted: 09/29/2022] [Indexed: 02/07/2023] Open
Abstract
Basal cells are multipotent stem cells of a variety of organs, including the respiratory tract, where they are major components of the airway epithelium. However, it remains unclear how diverse basal cells are and how distinct subpopulations respond to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the adult murine trachea. Among these, we found two major subpopulations, collectively comprising the most uncommitted of all the pools, but with distinct gene expression signatures. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to self-renew and initiate a program of differentiation in response to environmental perturbations in primary cultures and in mouse injury models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are still being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of tracheal cartilage-deficient mouse mutants. Finally, we show that features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide novel insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.
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Affiliation(s)
- Yizhuo Zhou
- Columbia Center for Human Development, Columbia University Irving Medical CenterNew YorkUnited States
- Department of Medicine, Pulmonary Allergy Critical Care, Columbia University Irving Medical CenterNew YorkUnited States
| | - Ying Yang
- Columbia Center for Human Development, Columbia University Irving Medical CenterNew YorkUnited States
- Department of Genetics and Development, Columbia University Irving Medical CenterNew YorkUnited States
| | - Lihao Guo
- Department of Pharmacy Practice and Science, College of Pharmacy, University of ArizonaTucsonUnited States
| | - Jun Qian
- Columbia Center for Human Development, Columbia University Irving Medical CenterNew YorkUnited States
- Department of Medicine, Pulmonary Allergy Critical Care, Columbia University Irving Medical CenterNew YorkUnited States
| | - Jian Ge
- Columbia Center for Human Development, Columbia University Irving Medical CenterNew YorkUnited States
| | - Debora Sinner
- Neonatology and Pulmonary Biology Perinatal Institute, Cincinnati Children’s Hospital Medical Center and University of Cincinnati, College of MedicineCincinnatiUnited States
| | - Hongxu Ding
- Department of Pharmacy Practice and Science, College of Pharmacy, University of ArizonaTucsonUnited States
| | - Andrea Califano
- Departments of Systems Biology, Biochemistry & Molecular Biophysics, Biomedical Informatics, Medicine; JP Sulzberger Columbia Genome Center; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical CenterNew YorkUnited States
| | - Wellington V Cardoso
- Columbia Center for Human Development, Columbia University Irving Medical CenterNew YorkUnited States
- Department of Medicine, Pulmonary Allergy Critical Care, Columbia University Irving Medical CenterNew YorkUnited States
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13
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Hall-Stoodley L, McCoy KS. Biofilm aggregates and the host airway-microbial interface. Front Cell Infect Microbiol 2022; 12:969326. [PMID: 36081767 PMCID: PMC9445362 DOI: 10.3389/fcimb.2022.969326] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Accepted: 07/28/2022] [Indexed: 11/13/2022] Open
Abstract
Biofilms are multicellular microbial aggregates that can be associated with host mucosal epithelia in the airway, gut, and genitourinary tract. The host environment plays a critical role in the establishment of these microbial communities in both health and disease. These host mucosal microenvironments however are distinct histologically, functionally, and regarding nutrient availability. This review discusses the specific mucosal epithelial microenvironments lining the airway, focusing on: i) biofilms in the human respiratory tract and the unique airway microenvironments that make it exquisitely suited to defend against infection, and ii) how airway pathophysiology and dysfunctional barrier/clearance mechanisms due to genetic mutations, damage, and inflammation contribute to biofilm infections. The host cellular responses to infection that contribute to resolution or exacerbation, and insights about evaluating and therapeutically targeting airway-associated biofilm infections are briefly discussed. Since so many studies have focused on Pseudomonas aeruginosa in the context of cystic fibrosis (CF) or on Haemophilus influenzae in the context of upper and lower respiratory diseases, these bacteria are used as examples. However, there are notable differences in diseased airway microenvironments and the unique pathophysiology specific to the bacterial pathogens themselves.
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Affiliation(s)
- Luanne Hall-Stoodley
- Department of Microbial Infection and Immunity, The Ohio State University College of Medicine, Columbus, OH, United States
- *Correspondence: Luanne Hall-Stoodley,
| | - Karen S. McCoy
- Division of Pulmonary Medicine, Nationwide Children’s Hospital, Columbus, OH, United States
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14
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Reynolds SD, Hill CL, Alsudayri A, Lallier SW, Wijeratne S, Tan ZH, Chiang T, Cormet-Boyaka E. Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease. JCI Insight 2022; 7:e157380. [PMID: 35819850 PMCID: PMC9462471 DOI: 10.1172/jci.insight.157380] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Accepted: 07/06/2022] [Indexed: 11/17/2022] Open
Abstract
Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. Goblet and ciliated cell types are derived from tracheobronchial stem/progenitor cells via a Notch-dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and JAG2 is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex (GSC) inhibitors, neutralizing peptides/antibodies, or WNT/β-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent posttranslational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. GSC and glycogen synthase kinase 3 were implicated in these posttranslational events, but WNT agonist/antagonist studies and RNA-Seq indicated a WNT-independent mechanism. Collectively, these data suggest that posttranslational modifications create distinct assemblies of JAG1 and JAG2, which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.
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Affiliation(s)
| | | | | | | | | | - Zheng Hong Tan
- Center for Regenerative Medicine, Nationwide Children’s Hospital, Columbus, Ohio, USA
| | - Tendy Chiang
- Center for Regenerative Medicine, Nationwide Children’s Hospital, Columbus, Ohio, USA
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15
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Lin Y, Wang D, Zeng Y. A Maverick Review of Common Stem/Progenitor Markers in Lung Development. Stem Cell Rev Rep 2022; 18:2629-2645. [DOI: 10.1007/s12015-022-10422-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/26/2022] [Indexed: 10/16/2022]
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Wu M, Zhang X, Lin Y, Zeng Y. Roles of airway basal stem cells in lung homeostasis and regenerative medicine. Respir Res 2022; 23:122. [PMID: 35562719 PMCID: PMC9102684 DOI: 10.1186/s12931-022-02042-5] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Accepted: 05/01/2022] [Indexed: 11/10/2022] Open
Abstract
Airway basal stem cells (BSCs) in the proximal airways are recognized as resident stem cells capable of self-renewing and differentiating to virtually every pseudostratified epithelium cell type under steady-state and after acute injury. In homeostasis, BSCs typically maintain a quiescent state. However, when exposed to acute injuries by either physical insults, chemical damage, or pathogen infection, the remaining BSCs increase their proliferation rate apace within the first 24 h and differentiate to restore lung homeostasis. Given the progenitor property of airway BSCs, it is attractive to research their biological characteristics and how they maintain homeostatic airway structure and respond to injury. In this review, we focus on the roles of BSCs in lung homeostasis and regeneration, detail the research progress in the characteristics of airway BSCs, the cellular and molecular signaling communications involved in BSCs-related airway repair and regeneration, and further discuss the in vitro models for airway BSC propagation and their applications in lung regenerative medicine therapy.
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Affiliation(s)
- Meirong Wu
- Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, People's Republic of China.,Stem Cell Laboratory, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, People's Republic of China.,Respiratory Medicine Center of Fujian Province, Quanzhou, Fujian Province, People's Republic of China
| | - Xiaojing Zhang
- Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, People's Republic of China.,Stem Cell Laboratory, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, People's Republic of China.,Respiratory Medicine Center of Fujian Province, Quanzhou, Fujian Province, People's Republic of China
| | - Yijian Lin
- Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, People's Republic of China.,Stem Cell Laboratory, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, People's Republic of China.,Respiratory Medicine Center of Fujian Province, Quanzhou, Fujian Province, People's Republic of China
| | - Yiming Zeng
- Department of Respiratory and Critical Care Medicine, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, People's Republic of China. .,Stem Cell Laboratory, Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, People's Republic of China. .,Respiratory Medicine Center of Fujian Province, Quanzhou, Fujian Province, People's Republic of China.
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17
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Naeimi Kararoudi M, Alsudayri A, Hill CL, Elmas E, Sezgin Y, Thakkar A, Hester ME, Malleske DT, Lee DA, Neal ML, Perry MR, Harvilchuck JA, Reynolds SD. Assessment of Beta-2 Microglobulin Gene Edited Airway Epithelial Stem Cells as a treatment for Sulfur Mustard Inhalation. Front Genome Ed 2022; 4:781531. [PMID: 35199100 PMCID: PMC8859869 DOI: 10.3389/fgeed.2022.781531] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Accepted: 01/10/2022] [Indexed: 11/29/2022] Open
Abstract
Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient’s immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.
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Affiliation(s)
| | | | | | - Ezgi Elmas
- Nationwide Children’s Hospital, Columbus, OH, United States
- Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, United States
| | - Yasemin Sezgin
- Nationwide Children’s Hospital, Columbus, OH, United States
| | - Aarohi Thakkar
- Nationwide Children’s Hospital, Columbus, OH, United States
| | - Mark E. Hester
- Nationwide Children’s Hospital, Columbus, OH, United States
| | | | - Dean A. Lee
- Nationwide Children’s Hospital, Columbus, OH, United States
| | | | - Mark R. Perry
- Battelle Memorial Institute, Columbus, OH, United States
| | | | - Susan D. Reynolds
- Nationwide Children’s Hospital, Columbus, OH, United States
- *Correspondence: Susan D. Reynolds,
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18
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Foote AG, Lungova V, Thibeault SL. Piezo1-expressing vocal fold epithelia modulate remodeling via effects on self-renewal and cytokeratin differentiation. Cell Mol Life Sci 2022; 79:591. [PMID: 36376494 PMCID: PMC9663367 DOI: 10.1007/s00018-022-04622-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 10/30/2022] [Accepted: 10/31/2022] [Indexed: 11/16/2022]
Abstract
Mechanoreceptors are implicated as functional afferents within mucosa of the airways and the recent discovery of mechanosensitive channels Piezo1 and Piezo2 has proved essential for cells of various mechanically sensitive tissues. However, the role for Piezo1/2 in vocal fold (VF) mucosal epithelia, a cell that withstands excessive biomechanical insult, remains unknown. The purpose of this study was to test the hypothesis that Piezo1 is required for VF mucosal repair pathways of epithelial cell injury. Utilizing a sonic hedgehog (shh) Cre line for epithelial-specific ablation of Piezo1/2 mechanoreceptors, we investigated 6wk adult VF mucosa following naphthalene exposure for repair strategies at 1, 3, 7 and 14 days post-injury (dpi). PIEZO1 localized to differentiated apical epithelia and was paramount for epithelial remodeling events. Injury to wildtype epithelium was most appreciated at 3 dpi. Shhcre/+; Piezo1loxP/loxP, Piezo2 loxP/+ mutant epithelium exhibited severe cell/nuclear defects compared to injured controls. Conditional ablation of Piezo1 and/or Piezo2 to uninjured VF epithelium did not result in abnormal phenotypes across P0, P15 and 6wk postnatal stages compared to heterozygote and control tissue. Results demonstrate a role for Piezo1-expressing VF epithelia in regulating self-renewal via effects on p63 transcription and YAP subcellular translocation-altering cytokeratin differentiation.
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Affiliation(s)
- Alexander G. Foote
- Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Wisconsin-Madison, Wisconsin, USA
| | - Vlasta Lungova
- Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Wisconsin-Madison, Wisconsin, USA
| | - Susan L. Thibeault
- Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Wisconsin-Madison, Wisconsin, USA
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Cao F, Wang C, Long D, Deng Y, Mao K, Zhong H. Network-Based Integrated Analysis of Transcriptomic Studies in Dissecting Gene Signatures for LPS-Induced Acute Lung Injury. Inflammation 2021; 44:2486-2498. [PMID: 34462829 PMCID: PMC8405180 DOI: 10.1007/s10753-021-01518-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2021] [Accepted: 07/07/2021] [Indexed: 10/26/2022]
Abstract
Acute lung injury (ALI) is a type of serious clinical syndrome leading to morbidity and mortality. However, the precise pathogenesis of ALI remains elusive. Here, we implemented an integrative meta-analysis of six GEO microarray studies with 76 samples in the ALI mouse model. A total of 958 differentially expressed genes (DEGs) were identified in LPS relative to normal samples. Then, a network-based meta-analysis was used to mine core DEGs and to unfold the interactions among these genes. We found that Ebi3 was the top upregulated genes in the LPS-induced ALI. GO, KEGG, and GSEA analyses were performed for functional annotation. qRT-PCR revealed augmented expression of six candidate genes (Stat1, Syk, Jak3, Rac2, Ripk1, and Traf6) in the established ALI mouse model with LPS exposure. Taken together, our study investigated comprehensively hub DEGs and their networks for LPS-stimulated ALI, which might afford an additional approach to determine biomarkers and therapeutic targets and explore the molecular pathophysiology toward ALI.
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Affiliation(s)
- Fang Cao
- Department of Cerebrovascular Disease, Affiliated Hospital of Zunyi Medical University, Huichuan District, 149 Dalian Road, Zunyi, Guizhou, 563003 China
| | - Chunyan Wang
- Department of Gastroenterology, Sichuan Provincial Peoples Hospital, University of Electronic Science and Technology, Chengdu, 610000 Sichuan China
| | - Danling Long
- Department of Stomatology, Taihe Hospital, Hubei University of Medicine, Shiyan, 442000 Hubei China
| | - Yujuan Deng
- School of Computer Science and Engineering, Shijiazhuang University, Shijiazhuang, Hebei China
| | - Kaimin Mao
- Department of Critical Care Medicine, School of Medicine, Renji Hospital, Shanghai Jiaotong University, Shanghai, 200127 China
| | - Hua Zhong
- College of Life Sciences, Wuhan University, Wuhan, 430072 Hubei China
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Ghosh M, Hill CL, Alsudayri A, Lallier SW, Hayes D, Wijeratne S, Tan ZH, Chiang T, Mahoney JE, Carraro G, Stripp BR, Reynolds SD. Repeated injury promotes tracheobronchial tissue stem cell attrition. Stem Cells Transl Med 2021; 10:1696-1713. [PMID: 34546001 PMCID: PMC8641087 DOI: 10.1002/sctm.21-0032] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Revised: 07/19/2021] [Accepted: 07/28/2021] [Indexed: 12/20/2022] Open
Abstract
Chronic lung disease has been attributed to stem cell aging and/or exhaustion. We investigated these mechanisms using mouse and human tracheobronchial tissue-specific stem cells (TSC). In mouse, chromatin labeling and flow cytometry demonstrated that naphthalene (NA) injury activated a subset of TSC. These activated TSC continued to proliferate after the epithelium was repaired and a clone study demonstrated that ~96% of activated TSC underwent terminal differentiation. Despite TSC attrition, epithelial repair after a second NA injury was normal. The second injury accelerated proliferation of previously activated TSC and a nucleotide-label retention study indicated that the second injury recruited TSC that were quiescent during the first injury. These mouse studies indicate that (a) injury causes selective activation of the TSC pool; (b) activated TSC are predisposed to further proliferation; and (c) the activated state leads to terminal differentiation. In human TSC, repeated proliferation also led to terminal differentiation and depleted the TSC pool. A clone study identified long- and short-lived TSC and showed that short-lived TSC clones had significantly shorter telomeres than their long-lived counterparts. The TSC pool was significantly depleted in dyskeratosis congenita donors, who harbor mutations in telomere biology genes. The remaining TSC had short telomeres and short lifespans. Collectively, the mouse and human studies support a model in which epithelial injury increases the biological age of the responding TSC. When applied to chronic lung disease, this model suggests that repeated injury accelerates the biological aging process resulting in abnormal repair and disease initiation.
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Affiliation(s)
- Moumita Ghosh
- Department of MedicineUniversity of Colorado‐DenverDenverColoradoUSA
| | - Cynthia L. Hill
- Center for Perinatal ResearchNationwide Children's HospitalColumbusOhioUSA
| | - Alfahdah Alsudayri
- Center for Perinatal ResearchNationwide Children's HospitalColumbusOhioUSA
| | - Scott W. Lallier
- Center for Perinatal ResearchNationwide Children's HospitalColumbusOhioUSA
| | - Don Hayes
- Department of PediatricsThe Ohio State University College of MedicineColumbusOhioUSA
| | - Saranga Wijeratne
- Department of PediatricsThe Ohio State University College of MedicineColumbusOhioUSA
| | - Zhang Hong Tan
- Center for Regenerative MedicineNationwide Children's HospitalColumbusOhioUSA
| | - Tendy Chiang
- Center for Regenerative MedicineNationwide Children's HospitalColumbusOhioUSA
| | - John E. Mahoney
- Cystic Fibrosis Foundation TherapeuticsLexingtonMassachusettsUSA
- Cystic Fibrosis FoundationBethesdaMarylandUSA
| | - Gianni Carraro
- Department of Medicine, Cedars‐Sinai Medical CenterLung and Regenerative Medicine InstitutesLos AngelesCaliforniaUSA
| | - Barry R. Stripp
- Department of Medicine, Cedars‐Sinai Medical CenterLung and Regenerative Medicine InstitutesLos AngelesCaliforniaUSA
| | - Susan D. Reynolds
- Center for Perinatal ResearchNationwide Children's HospitalColumbusOhioUSA
- Department of PediatricsThe Ohio State University College of MedicineColumbusOhioUSA
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Huang L, Liang H, Liu L, Lin Y, Lin X. Effects of Pulmonary Surfactant Combined with Noninvasive Positive Pressure Ventilation on KRT-14 and ET-1 Levels in Peripheral Blood and Therapeutic Effects in Neonates with Respiratory Distress Syndrome. BIOMED RESEARCH INTERNATIONAL 2021; 2021:4117800. [PMID: 38617025 PMCID: PMC11015946 DOI: 10.1155/2021/4117800] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/21/2021] [Revised: 07/24/2021] [Accepted: 07/26/2021] [Indexed: 04/16/2024]
Abstract
This study is aimed at exploring the effect of pulmonary surfactant (PS) combined with noninvasive positive pressure ventilation on the levels of Keratin-14 (KRT-14) and Endothelin-1 (ET-1) in peripheral blood and the therapeutic effect of neonatal respiratory distress syndrome (NRDS). Altogether 137 cases of neonates with respiratory distress syndrome treated in our hospital from April 2016 to July 2018 were collected. Among them, 64 cases treated with noninvasive positive pressure ventilation were considered as the control group, and 73 cases treated with PS combined with noninvasive positive pressure ventilation were considered as the observation group. The expression of KRT-14 and ET-1 in the two groups was compared. The therapeutic effect, death, complications, and blood gas indexes PaO2, PaCO2, and PaO2/FiO2 in the two groups were compared. Receiver operating characteristic curve (ROC) was applied to analyze the diagnostic value of KRT-14 and ET-1 in the therapeutic effect of NRDS. The effective rate of the observation group was higher than that of the control group. After treatment, PaO2 and PaO2/FiO2 in both groups were notably higher than that before treatment, while PaCO2 was notably lower than that before treatment. And after treatment, the levels of PaO2 and PaO2/FiO2 in the observation group were remarkably higher than that in the control group; PaCO2 was notably lower than that in the control group. After treatment, the levels of KRT-14 and ET-1 in the two groups were remarkably lower than those before treatment, and the levels of KRT-14 and ET-1 in the observation group were considerably lower than those in the control group after treatment. ROC curve showed that the area under the curve (AUC) of KRT-14 was 0.791, and the AUC of ET-1 was 0.816. PS combined with noninvasive positive pressure ventilation can notably improve the therapeutic effect of NRDS. KRT-14 and ET-1 levels may be potential therapeutic diagnostic indicators.
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Affiliation(s)
- Lihan Huang
- Department of Neonatology, Women and Children's Hospital, School of Medicine, Xiamen University, Xiamen 361003, China
- Xiamen Key Laboratory of Perinatal-Neonatal Infection, Xiamen, 361003 Fujian Province, China
| | - Hong Liang
- Department of Neonatology, Women and Children's Hospital, School of Medicine, Xiamen University, Xiamen 361003, China
- Xiamen Key Laboratory of Perinatal-Neonatal Infection, Xiamen, 361003 Fujian Province, China
| | - Longbin Liu
- Department of Neonatology, Women and Children's Hospital, School of Medicine, Xiamen University, Xiamen 361003, China
- Xiamen Key Laboratory of Perinatal-Neonatal Infection, Xiamen, 361003 Fujian Province, China
| | - Yucong Lin
- Department of Neonatology, Women and Children's Hospital, School of Medicine, Xiamen University, Xiamen 361003, China
- Xiamen Key Laboratory of Perinatal-Neonatal Infection, Xiamen, 361003 Fujian Province, China
| | - Xinzhu Lin
- Department of Neonatology, Women and Children's Hospital, School of Medicine, Xiamen University, Xiamen 361003, China
- Xiamen Key Laboratory of Perinatal-Neonatal Infection, Xiamen, 361003 Fujian Province, China
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Jensen-Cody CW, Crooke AK, Rotti PG, Ievlev V, Shahin W, Park SY, Lynch TJ, Engelhardt JF. Lef-1 controls cell cycle progression in airway basal cells to regulate proliferation and differentiation. Stem Cells 2021; 39:1221-1235. [PMID: 33932322 PMCID: PMC8785221 DOI: 10.1002/stem.3386] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Accepted: 04/08/2021] [Indexed: 11/10/2022]
Abstract
The mammalian airways are lined by a continuous epithelial layer that is maintained by diverse populations of resident multipotent stem cells. These stem cells are responsible for replenishing the epithelium both at homeostasis and following injury, making them promising targets for stem cell and genetic-based therapies for a variety of respiratory diseases. However, the mechanisms that regulate when and how these stem cells proliferate, migrate, and differentiate remains incompletely understood. Here, we find that the high mobility group (HMG) domain transcription factor Lef-1 regulates proliferation and differentiation of mouse tracheal basal cells. We demonstrate that conditional deletion of Lef-1 stalls basal cell proliferation at the G1/S transition of the cell cycle, and that Lef-1 knockout cells are unable to maintain luminal tracheal cell types in long-term air-liquid interface culture. RNA sequencing analysis revealed that Lef-1 knockout (Lef-1KO) results in downregulation of key DNA damage response and cell cycle progression genes, including the kinase Chek1. Furthermore, chemical inhibition of Chek1 is sufficient to stall basal cell self-renewal in a similar fashion as Lef-1 deletion. Notably, the cell cycle block imposed by Lef-1KO in vitro is transient and basal cells eventually compensate to proliferate normally in a Chek1-independent manner. Finally, Lef-1KO cells were unable to fully regenerate tracheal epithelium following injury in vivo. These findings reveal that Lef-1 is essential for proper basal cell function. Thus, modulating Lef-1 function in airway basal cells may have applications in regenerative medicine.
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Affiliation(s)
- Chandler W Jensen-Cody
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Adrianne K Crooke
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Pavana G Rotti
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Vitaly Ievlev
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Weam Shahin
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Soo-Yeun Park
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - Thomas J Lynch
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
| | - John F Engelhardt
- Department of Anatomy & Cell Biology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, USA
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23
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Tilston-Lunel A, Mazzilli S, Kingston NM, Szymaniak AD, Hicks-Berthet J, Kern JG, Abo K, Reid ME, Perdomo C, Wilson AA, Spira A, Beane J, Varelas X. Aberrant epithelial polarity cues drive the development of precancerous airway lesions. Proc Natl Acad Sci U S A 2021; 118:e2019282118. [PMID: 33903236 PMCID: PMC8106308 DOI: 10.1073/pnas.2019282118] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Molecular events that drive the development of precancerous lesions in the bronchial epithelium, which are precursors of lung squamous cell carcinoma (LUSC), are poorly understood. We demonstrate that disruption of epithelial cellular polarity, via the conditional deletion of the apical determinant Crumbs3 (Crb3), initiates and sustains precancerous airway pathology. The loss of Crb3 in adult luminal airway epithelium promotes the uncontrolled activation of the transcriptional regulators YAP and TAZ, which stimulate intrinsic signals that promote epithelial cell plasticity and paracrine signals that induce basal-like cell growth. We show that aberrant polarity and YAP/TAZ-regulated gene expression associates with human bronchial precancer pathology and disease progression. Analyses of YAP/TAZ-regulated genes further identified the ERBB receptor ligand Neuregulin-1 (NRG1) as a key transcriptional target and therapeutic targeting of ERBB receptors as a means of preventing and treating precancerous cell growth. Our observations offer important molecular insight into the etiology of LUSC and provides directions for potential interception strategies of lung cancer.
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Affiliation(s)
- Andrew Tilston-Lunel
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118
| | - Sarah Mazzilli
- Department of Medicine, Boston University School of Medicine, Boston, MA 02118
| | - Nathan M Kingston
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118
| | | | - Julia Hicks-Berthet
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118
| | - Joseph G Kern
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118
| | - Kristine Abo
- Department of Medicine, Boston University School of Medicine, Boston, MA 02118
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118
| | - Mary E Reid
- Department of Medicine, Roswell Park Comprehensive Cancer Center, Buffalo, NY 14203
| | - Catalina Perdomo
- Department of Medicine, Boston University School of Medicine, Boston, MA 02118
| | - Andrew A Wilson
- Department of Medicine, Boston University School of Medicine, Boston, MA 02118
- Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118
- Pulmonary Center, Boston University School of Medicine , Boston, MA 02118
| | - Avrum Spira
- Department of Medicine, Boston University School of Medicine, Boston, MA 02118
- Pulmonary Center, Boston University School of Medicine , Boston, MA 02118
- Lung Cancer Initiative (LCI), Johnson and Johnson, Cambridge, MA 02142
| | - Jennifer Beane
- Department of Medicine, Boston University School of Medicine, Boston, MA 02118
| | - Xaralabos Varelas
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118;
- Pulmonary Center, Boston University School of Medicine , Boston, MA 02118
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24
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TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds. NPJ Regen Med 2021; 6:12. [PMID: 33674599 PMCID: PMC7935966 DOI: 10.1038/s41536-021-00124-4] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2020] [Accepted: 02/01/2021] [Indexed: 12/24/2022] Open
Abstract
The use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.
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25
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Thorsen AS, Khamis D, Kemp R, Colombé M, Lourenço FC, Morrissey E, Winton D. Heterogeneity in clone dynamics within and adjacent to intestinal tumours identified by Dre-mediated lineage tracing. Dis Model Mech 2021; 14:dmm046706. [PMID: 33093165 PMCID: PMC7823168 DOI: 10.1242/dmm.046706] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2020] [Accepted: 10/12/2020] [Indexed: 11/20/2022] Open
Abstract
Somatic models of tissue pathology commonly use induction of gene-specific mutations in mice mediated by spatiotemporal regulation of Cre recombinase. Subsequent investigation of the onset and development of disease can be limited by the inability to track changing cellular behaviours over time. Here, a lineage-tracing approach based on ligand-dependent activation of Dre recombinase that can be employed independently of Cre is described. The clonal biology of the intestinal epithelium following Cre-mediated stabilisation of β-catenin reveals that, within tumours, many new clones rapidly become extinct. Surviving clones show accelerated population of tumour glands compared to normal intestinal crypts but in a non-uniform manner, indicating that intra-tumour glands follow heterogeneous dynamics. In tumour-adjacent epithelia, clone sizes are smaller than in the background epithelia, as a whole. This suggests a zone of ∼seven crypt diameters within which clone expansion is inhibited by tumours and that may facilitate their growth.
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Affiliation(s)
- Ann-Sofie Thorsen
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
| | - Doran Khamis
- University of Oxford, Center for Computational Biology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK
| | - Richard Kemp
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
| | - Mathilde Colombé
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
| | - Filipe C. Lourenço
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
| | - Edward Morrissey
- University of Oxford, Center for Computational Biology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK
| | - Douglas Winton
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
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Parekh KR, Nawroth J, Pai A, Busch SM, Senger CN, Ryan AL. Stem cells and lung regeneration. Am J Physiol Cell Physiol 2020; 319:C675-C693. [PMID: 32783658 PMCID: PMC7654650 DOI: 10.1152/ajpcell.00036.2020] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2020] [Revised: 08/03/2020] [Accepted: 08/03/2020] [Indexed: 12/20/2022]
Abstract
The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease.
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Affiliation(s)
- Kalpaj R Parekh
- Department Surgery, Division of Cardiothoracic Surgery, University of Iowa, Iowa City, Iowa
| | - Janna Nawroth
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Southern California, Los Angeles, California
| | - Albert Pai
- Department Surgery, Division of Cardiothoracic Surgery, University of Iowa, Iowa City, Iowa
| | - Shana M Busch
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Southern California, Los Angeles, California
| | - Christiana N Senger
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Southern California, Los Angeles, California
| | - Amy L Ryan
- Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Southern California, Los Angeles, California
- Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, California
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27
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Yoshie S, Omori K, Hazama A. Airway regeneration using iPS cell-derived airway epithelial cells with Cl - channel function. Channels (Austin) 2020; 13:227-234. [PMID: 31198082 PMCID: PMC6602574 DOI: 10.1080/19336950.2019.1628550] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
induced pluripotent stem (iPS) cells can be differentiated into various cell types, including airway epithelial cells, since they have the capacity for self-renewal and pluripotency. Thus, airway epithelial cells generated from iPS cells are expected to be potent candidates for use in airway regeneration and the treatment of airway diseases such as cystic fibrosis (CF). Recently, it was reported that iPS cells can be differentiated into airway epithelial cells according to the airway developmental process. These studies demonstrate that airway epithelial cells generated from iPS cells are equivalent to their in vivo counterparts. However, it has not been evaluated in detail whether these cells exhibit physiological functions and are fully mature. Airway epithelial cells adequately control water volume on the airway surface via the function of Cl− channels. Reasonable environments on the airway surface cause ciliary movement with a constant rhythm and maintain airway clearance. Therefore, the generation of functional airway epithelial cells/tissues with Cl− channel function from iPS cells will be indispensable for cell/tissue replacement therapy, the development of a reliable airway disease model, and the treatment of airway disease. This review highlights the generation of functional airway epithelial cells from iPS cells and discusses the remaining challenges to the generation of functional airway epithelial cells for airway regeneration and the treatment of airway disease.
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Affiliation(s)
- Susumu Yoshie
- a Department of Cellular and Integrative Physiology, School of Medicine , Fukushima Medical University , Fukushima , Japan
| | - Koichi Omori
- b Department of Otolaryngology Head and Neck Surgery, Graduate School of Medicine , Kyoto University , Kyoto , Japan
| | - Akihiro Hazama
- a Department of Cellular and Integrative Physiology, School of Medicine , Fukushima Medical University , Fukushima , Japan
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28
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Sivakumar A, Frank DB. Paradigms that define lung epithelial progenitor cell fate in development and regeneration. CURRENT STEM CELL REPORTS 2019; 5:133-144. [PMID: 32587809 DOI: 10.1007/s40778-019-00166-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Purpose of Review Throughout the lifespan, lung injury impedes the primary critical function essential for life-respiration. To repair quickly and efficiently is critical and is orchestrated by a diverse repertoire of progenitor cells and their niche. This review incorporates knowledge gained from early studies in lung epithelial morphogenesis and cell fate and explores its relevance to more recent findings of lung progenitor and stem cells in development and regeneration. Recent Findings Cell fate in the lung is organized into an early specification phase and progressive differentiation phase in lung development. The advent of single cell analysis combined with lineage analysis and projections is uncovering new functional cell types in the lung providing a topographical atlas for progenitor cell lineage commitment during development, homeostasis, and regeneration. Summary Lineage commitment of lung progenitor cells is spatiotemporally regulated during development. Single cell sequencing technologies have significantly advanced our understanding of the similarities and differences between developmental and regenerative cell fate trajectories. Subsequent unraveling of the molecular mechanisms underlying these cell fate decisions will be essential to manipulating progenitor cells for regeneration.
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Affiliation(s)
- Aravind Sivakumar
- Division of Cardiology, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA
- Penn-CHOP Lung Biology Institute, University of Pennsylvania, Philadelphia, PA, 19104, USA
- Penn Cardiovascular Institute, University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - David B Frank
- Division of Cardiology, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA
- Penn-CHOP Lung Biology Institute, University of Pennsylvania, Philadelphia, PA, 19104, USA
- Penn Cardiovascular Institute, University of Pennsylvania, Philadelphia, PA, 19104, USA
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29
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Schwartz CM, Stack J, Hill CL, Lallier SW, Chiang T, Johnson J, Reynolds SD. Electrospun scaffolds limit the regenerative potential of the airway epithelium. Laryngoscope Investig Otolaryngol 2019; 4:446-454. [PMID: 31453356 PMCID: PMC6703117 DOI: 10.1002/lio2.289] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2019] [Accepted: 06/20/2019] [Indexed: 01/28/2023] Open
Abstract
Objective Significant morbidity and mortality are associated with clinical use of synthetic tissue‐engineered tracheal grafts (TETG). Our previous work focused on an electrospun polyethylene terephthalate and polyurethane (PET/PU) TETG that was tested in sheep using a long‐segment tracheal defect model. We reported that graft stenosis and limited epithelialization contributed to graft failure. The present study determined if the epithelialization defect could be attributed to: 1) postsurgical depletion of native airway basal stem/progenitor cells; 2) an inability of the PET/PU‐TETG to support epithelial migration; or 3) compromised basal stem/progenitor cell proliferation within the PET/PU environment. Study Design Experimental. Methods Basal stem/progenitor cell frequency in sheep that underwent TETG implantation was determined using the clone‐forming cell frequency (CFCF) method. A novel migration model that mimics epithelial migration toward an acellular scaffold was developed and used to compare epithelial migration toward a control polyester scaffold and the PET/PU scaffold. Basal stem/progenitor cell proliferation within the PET/PU scaffold was evaluated using the CFCF assay, doubling‐time analysis, and mitotic cell quantification. Results We report that TETG implantation did not decrease basal stem/progenitor cell frequency. In contrast, we find that epithelial migration toward the PET/PU scaffold was significantly less extensive than migration toward a polyester scaffold and that the PET/PU scaffold did not support basal stem/progenitor cell proliferation. Conclusions We conclude that epithelialization of a PET/PU scaffold is compromised by poor migration of native tissue‐derived epithelial cells and by a lack of basal stem/progenitor cell proliferation within the scaffold. Level of Evidence NA
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Affiliation(s)
| | - Jacob Stack
- Center for Perinatal Research Nationwide Children's Hospital Columbus Ohio U.S.A
| | - Cynthia L Hill
- Center for Perinatal Research Nationwide Children's Hospital Columbus Ohio U.S.A
| | - Scott W Lallier
- Center for Perinatal Research Nationwide Children's Hospital Columbus Ohio U.S.A
| | - Tendy Chiang
- College of Medicine The Ohio State University Columbus Ohio U.S.A.,Center for Regenerative Medicine Nationwide Children's Hospital Columbus Ohio U.S.A.,Department of Otolaryngology Nationwide Children's Hospital Columbus Ohio U.S.A
| | | | - Susan D Reynolds
- Center for Perinatal Research Nationwide Children's Hospital Columbus Ohio U.S.A
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Abstract
INTRODUCTION Lifelong maintenance of a healthy lung requires resident stem cells to proliferate according to tissue requirements. Once thought to be a quiescent tissue, evolving views of the complex differentiation landscape of lung stem and progenitor cells have broad implications for our understanding of how the lung is maintained, as well as the development of new therapies for promoting endogenous regeneration in lung disease. AREAS COVERED This review collates a large body of research relating to the hierarchical organization of epithelial stem cells in the adult lung and their role in tissue homeostasis and regeneration after injury. To identify relevant studies, PubMed was queried using one or a combination of the terms 'lung', 'airway', 'alveoli', 'stem cells', 'progenitor', 'repair' and 'regeneration'. EXPERT OPINION This review discusses how new technologies and injury models have challenged the demarcations between stem and progenitor cell populations.
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Affiliation(s)
- Jonathan L McQualter
- a School of Health and Biomedical Sciences , RMIT University , Melbourne , Australia
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31
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Abstract
Epithelial stem cells reside within multiple regions of the lung where they renew various region-specific cells. In addition, there are multiple routes of regeneration after injury through built-in heterogeneity within stem cell populations and through a capacity for cellular plasticity among differentiated cells. These processes are important facets of respiratory tissue resiliency and organism survival. However, this regenerative capacity is not limitless, and repetitive or chronic injuries, environmental stresses, or underlying factors of disease may ultimately lead to or contribute to tissue remodeling and end-stage lung disease. This chapter will review stem cell heterogeneity among pulmonary epithelia in the lower respiratory system, discuss recent findings that may challenge long-held scientific paradigms, and identify several clinically relevant research opportunities for regenerative medicine.
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32
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Swatek AM, Lynch TJ, Crooke AK, Anderson PJ, Tyler SR, Brooks L, Ivanovic M, Klesney-Tait JA, Eberlein M, Pena T, Meyerholz DK, Engelhardt JF, Parekh KR. Depletion of Airway Submucosal Glands and TP63 +KRT5 + Basal Cells in Obliterative Bronchiolitis. Am J Respir Crit Care Med 2018; 197:1045-1057. [PMID: 29236513 PMCID: PMC5909161 DOI: 10.1164/rccm.201707-1368oc] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2017] [Accepted: 12/12/2017] [Indexed: 12/24/2022] Open
Abstract
RATIONALE Obliterative bronchiolitis (OB) is a major cause of mortality after lung transplantation. Depletion of airway stem cells (SCs) may lead to fibrosis in OB. OBJECTIVES Two major SC compartments in airways are submucosal glands (SMGs) and surface airway p63 (also known as TP63 [tumor protein 63])-positive/K5 (also known as KRT5 [keratin 5])-positive basal cells (BCs). We hypothesized that depletion of these SC compartments occurs in OB. METHODS Ferret orthotopic left lung transplants were used as an experimental model of OB, and findings were corroborated in human lung allografts. Morphometric analysis was performed in ferret and human lungs to evaluate the abundance of SMGs and changes in the expression of phenotypic BC markers in control, lymphocytic bronchiolitis, and OB airways. The abundance and proliferative capacity of proximal and distal airway SCs was assessed using a clonogenic colony-forming efficiency assay. MEASUREMENTS AND MAIN RESULTS Ferret allografts revealed significant loss of SMGs with development of OB. A progressive decline in p63+/K5+ and increase in K5+/K14+ and K14+ BC phenotypes correlated with the severity of allograft rejection in large and small ferret airways. The abundance and proliferative capacity of basal SCs in large allograft airways declined with severity of OB, and there was complete ablation of basal SCs in distal OB airways. Human allografts mirrored phenotypic BC changes observed in the ferret model. CONCLUSIONS SMGs and basal SC compartments are depleted in large and/or small airways of lung allografts, and basal SC proliferative capacity declines with progression of disease and phenotypic changes. Global airway SC depletion may be a mechanism for pulmonary allograft failure.
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Affiliation(s)
| | | | | | | | | | | | | | - Julia A. Klesney-Tait
- Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Michael Eberlein
- Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Tahuanty Pena
- Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa
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Ghosh M, Miller YE, Nakachi I, Kwon JB, Barón AE, Brantley AE, Merrick DT, Franklin WA, Keith RL, Vandivier RW. Exhaustion of Airway Basal Progenitor Cells in Early and Established Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med 2018; 197:885-896. [PMID: 29211494 PMCID: PMC6020409 DOI: 10.1164/rccm.201704-0667oc] [Citation(s) in RCA: 82] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2017] [Accepted: 12/01/2017] [Indexed: 12/17/2022] Open
Abstract
RATIONALE Up to 40% of smokers develop chronic obstructive pulmonary disease (COPD) over a period that spans decades. Despite the importance of COPD, much remains to be learned about susceptibility and pathogenesis, especially during early, prediagnostic stages of disease. Airway basal progenitor cells are crucial for lung health and resilience because of their ability to repair injured airways. In COPD, the normal airway epithelium is replaced with increased basal and secretory (mucous) cells and decreased ciliated cells, suggesting that progenitors are impaired. OBJECTIVES To examine airway basal progenitor cells and lung function in smokers with and without COPD. METHODS Bronchial biopsies taken from smokers at risk for COPD and lung cancer were used to acquire airway basal progenitor cells. They were evaluated for count, self-renewal, and multipotentiality (ability to differentiate to basal, mucous, and ciliated cells), and progenitor count was examined for its relationship with lung function. MEASUREMENTS AND MAIN RESULTS Basal progenitor count, self-renewal, and multipotentiality were all reduced in COPD versus non-COPD. COPD progenitors produced an epithelium with increased basal and mucous cells and decreased ciliated cells, replicating the COPD phenotype. Progenitor depletion correlated with lung function and identified a subset of subjects without COPD with lung function that was midway between non-COPD with high progenitor counts and those with COPD. CONCLUSIONS Basal progenitor dysfunction relates to the histologic and physiologic manifestations of COPD and identifies a subset that may represent an early, prediagnostic stage of COPD, indicating that progenitor exhaustion is involved in COPD pathogenesis.
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Affiliation(s)
- Moumita Ghosh
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, National Jewish Health, Denver, Colorado
| | - York E. Miller
- COPD Program, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, and
- Veterans Affairs Eastern Colorado Healthcare System, Denver, Colorado
| | - Ichiro Nakachi
- Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine, Tokyo, Japan; and
| | - Jennifer B. Kwon
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, National Jewish Health, Denver, Colorado
| | - Anna E. Barón
- Department of Biostatistics and Bioinformatics, University of Colorado School of Public Health, Aurora, Colorado
| | - Alexandra E. Brantley
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, National Jewish Health, Denver, Colorado
| | - Daniel T. Merrick
- Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado
| | - Wilbur A. Franklin
- Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado
| | - Robert L. Keith
- COPD Program, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, and
- Veterans Affairs Eastern Colorado Healthcare System, Denver, Colorado
| | - R. William Vandivier
- COPD Program, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, and
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Barkauskas CE, Chung MI, Fioret B, Gao X, Katsura H, Hogan BLM. Lung organoids: current uses and future promise. Development 2017; 144:986-997. [PMID: 28292845 DOI: 10.1242/dev.140103] [Citation(s) in RCA: 286] [Impact Index Per Article: 35.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Lungs are composed of a system of highly branched tubes that bring air into the alveoli, where gas exchange takes place. The proximal and distal regions of the lung contain epithelial cells specialized for different functions: basal, secretory and ciliated cells in the conducting airways and type II and type I cells lining the alveoli. Basal, secretory and type II cells can be grown in three-dimensional culture, with or without supporting stromal cells, and under these conditions they give rise to self-organizing structures known as organoids. This Review summarizes the different methods for generating organoids from cells isolated from human and mouse lungs, and compares their final structure and cellular composition with that of the airways or alveoli of the adult lung. We also discuss the potential and limitations of organoids for addressing outstanding questions in lung biology and for developing new drugs for disorders such as cystic fibrosis and asthma.
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Affiliation(s)
- Christina E Barkauskas
- Department of Medicine, Division of Pulmonary and Critical Medicine, Duke Medicine, Durham, NC 27710, USA
| | - Mei-I Chung
- Department of Cell Biology, Duke Medicine, Durham, NC 27710, USA
| | - Bryan Fioret
- Department of Cell Biology, Duke Medicine, Durham, NC 27710, USA
| | - Xia Gao
- Department of Cell Biology, Duke Medicine, Durham, NC 27710, USA
| | - Hiroaki Katsura
- Department of Cell Biology, Duke Medicine, Durham, NC 27710, USA
| | - Brigid L M Hogan
- Department of Cell Biology, Duke Medicine, Durham, NC 27710, USA
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Ghosh M, Ahmad S, White CW, Reynolds SD. Transplantation of Airway Epithelial Stem/Progenitor Cells: A Future for Cell-Based Therapy. Am J Respir Cell Mol Biol 2017; 56:1-10. [PMID: 27632244 DOI: 10.1165/rcmb.2016-0181ma] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.
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Affiliation(s)
- Moumita Ghosh
- 1 Department of Pediatrics, National Jewish Health, Denver, Colorado
| | - Shama Ahmad
- 2 Department of Anaesthesiology and Perioperative Medicine, University of Alabama, Birmingham, Alabama
| | - Carl W White
- 3 Department of Pediatric Pulmonology, University of Colorado, Aurora, Colorado; and
| | - Susan D Reynolds
- 4 Center for Perinatal Research, Nationwide Children's Hospital, Columbus, Ohio
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Abstract
Keratin 24 (K24) is a new kind of keratin genes, which encodes a novel keratin protein, K24 that bears high similarity to the type I keratins and displays a unique expression profile. However, the role of K24 is incompletely understood. In our study, we investigated the localization of K24 within the epidermis and possible functions. Keratin 24 was found to be modestly overexpressed in senescent keratinocytes and was mainly restricted to the upper stratum spinosum of epidermis. The protein was required for terminal differentiation upon CaCl2-induced differentiation. In vitro results showed that increased K24 in keratinocytes dramatically changed the differentiation of primary keratinocytes. It also inhibited cell survival by G1/S phase cell cycle arrest and induced senescence, autophagy and apoptosis of keratinocytes. In addition, K24 activated PKCδ signal pathway involving in cellular survival. In summary, K24 may be suggested as a potential differentiation marker and anti-proliferative factor in the epidermis.
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37
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Deng M, Li J, Gan Y, Chen P. [Advances in Classification and Research Methods of Lung Epithelial Stem
and Progenitor Cells]. ZHONGGUO FEI AI ZA ZHI = CHINESE JOURNAL OF LUNG CANCER 2017; 20:130-137. [PMID: 28228225 PMCID: PMC5972970 DOI: 10.3779/j.issn.1009-3419.2017.02.08] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
Abstract
分离和鉴定肺上皮干/祖细胞，深入了解他们在肺脏生理病理条件下的具体作用机理，对于防治包括肺癌在内的肺脏疾病有重要意义。本综述介绍了已鉴定的肺上皮干/祖细胞种类和肺上皮干/祖细胞研究方法的最新进展，前者具有区域特异性，主要包括位近端气道的基底细胞和导管细胞，位细支气管的Clara细胞、变异Clara细胞、细支气管肺泡干细胞和诱导出的krt5+细胞及位肺泡的Ⅱ型肺泡上皮细胞和Ⅱ型肺泡上皮祖细胞；后者主要包括肺损伤模型、谱系示踪技术、三维培养技术、移植、慢性标记细胞法及单细胞转录组学分析等。最后简述了肺上皮干/祖细胞与肺癌的关系以及肺癌干细胞靶向药物治疗进展。
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Affiliation(s)
- Minhua Deng
- Department of Respiratory Medicine, PLA Rocket Force General Hospital, Beijing 100088, China;Department of Respiratory Medicine, Second Xiangya Hospital, Central South University, Changsha 410011, China
| | - Jinhua Li
- Department of Respiratory Medicine, Second Xiangya Hospital, Central South University, Changsha 410011, China
| | - Ye Gan
- Department of Rehabilitation, Second Xiangya Hospital, Central South University, Changsha 410011, China
| | - Ping Chen
- Department of Respiratory Medicine, Second Xiangya Hospital, Central South University, Changsha 410011, China
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Lynch TJ, Anderson PJ, Xie W, Crooke AK, Liu X, Tyler SR, Luo M, Kusner DM, Zhang Y, Neff T, Burnette DC, Walters KS, Goodheart MJ, Parekh KR, Engelhardt JF. Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands. Stem Cells 2016; 34:2758-2771. [PMID: 27341073 PMCID: PMC5809158 DOI: 10.1002/stem.2443] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Revised: 05/08/2016] [Accepted: 05/24/2016] [Indexed: 12/19/2022]
Abstract
Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.
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Affiliation(s)
- Thomas J. Lynch
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | | | - Weiliang Xie
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
- Molecular and Cellular Biology Program, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | | | - Xiaoming Liu
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
- Center for Gene Therapy, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - Scott R. Tyler
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
- Molecular and Cellular Biology Program, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - Meihui Luo
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - David M Kusner
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - Yulong Zhang
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - Traci Neff
- Department of Obstetrics and Gynecology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - Daniel C. Burnette
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - Katherine S. Walters
- Central Microscopy Research Facility, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - Michael J. Goodheart
- Department of Obstetrics and Gynecology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - Kalpaj R. Parekh
- Department of Cardiothoracic Surgery, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
| | - John F. Engelhardt
- Department of Anatomy & Cell Biology, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
- Center for Gene Therapy, the University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
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Choi J, Iich E, Lee JH. Organogenesis of adult lung in a dish: Differentiation, disease and therapy. Dev Biol 2016; 420:278-286. [PMID: 27713058 DOI: 10.1016/j.ydbio.2016.10.002] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2016] [Revised: 09/29/2016] [Accepted: 10/02/2016] [Indexed: 12/26/2022]
Abstract
The remarkable regenerative capacity of the lung suggests that stem cells could be of therapeutic importance in diverse lung diseases; however, the successful exploitation of lung stem cell biology has long been hampered by our inability to maintain and expand adult lung stem cells while retaining their multi-lineage potential in vitro. Recently, advances in our understanding of stem cell niches and the role of key signalling modulators in controlling stem cell maintenance and differentiation have fuelled the development of new in vitro three-dimensional (3D) culture technologies that sustain the stem cell-driven formation of near-physiological, self-organizing structures called organoids. Here we review basic approaches to organoid model systems and highlight recent achievements in the generation of organoids from adult stem and progenitor cells of both the murine and human lungs. We evaluate current applications in studying cellular changes in proliferation, differentiation, plasticity, and cell polarity, and cellular and molecular crosstalk of epithelial cells with stroma. Advantages and limitations of organoids for clinical use are also discussed.
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Affiliation(s)
- Jinwook Choi
- Wellcome Trust/Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Elhadi Iich
- Wellcome Trust/Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Joo-Hyeon Lee
- Wellcome Trust/Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Physiology, Development and Neutabroscience, University of Cambridge, Cambridge CB2 3DY, UK.
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40
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Bou Saab J, Bacchetta M, Chanson M. Ineffective correction of PPARγ signaling in cystic fibrosis airway epithelial cells undergoing repair. Int J Biochem Cell Biol 2016; 78:361-369. [DOI: 10.1016/j.biocel.2016.07.035] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Revised: 07/27/2016] [Accepted: 07/29/2016] [Indexed: 12/28/2022]
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41
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Bertoncello I. Properties of Adult Lung Stem and Progenitor Cells. J Cell Physiol 2016; 231:2582-9. [PMID: 27062064 DOI: 10.1002/jcp.25404] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2016] [Accepted: 04/06/2016] [Indexed: 12/13/2022]
Abstract
The last decade has seen significant progress in understanding the organisation of regenerative cells in the adult lung. Cell-lineage tracing and in vitro clonogenic assays have enabled the identification and characterisation of endogenous lung epithelial stem and progenitor cells. Selective lung injury models, and genetically engineered mice have revealed highly conserved gene networks, factors, signalling pathways, and cellular interactions important in maintaining lung homeostasis and regulating lung regeneration and repair following injury. This review describes the current models of lung epithelial stem and progenitor cell organisation in adult mice, and the impediments encountered in translational studies aiming to identify and characterise their human homologs. J. Cell. Physiol. 231: 2582-2589, 2016. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Ivan Bertoncello
- Lung Health Research Centre, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria, Australia
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42
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Fate Mapping Mammalian Corneal Epithelia. Ocul Surf 2016; 14:82-99. [PMID: 26774909 DOI: 10.1016/j.jtos.2015.11.007] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Revised: 11/16/2015] [Accepted: 11/18/2015] [Indexed: 02/07/2023]
Abstract
The anterior aspect of the cornea consists of a stratified squamous epithelium, thought to be maintained by a rare population of stem cells (SCs) that reside in the limbal transition zone. Although migration of cells that replenish the corneal epithelium has been studied for over a century, the process is still poorly understood and not well characterized. Numerous techniques have been employed to examine corneal epithelial dynamics, including visualization by light microscopy, the incorporation of vital dyes and DNA labels, and transplantation of genetically marked cells that have acted as cell and lineage beacons. Modern-day lineage tracing utilizes molecular methods to determine the fate of a specific cell and its progeny over time. Classically employed in developmental biology, lineage tracing has been used more recently to track the progeny of adult SCs in a number of organs to pin-point their location and understand their movement and influence on tissue regeneration. This review highlights key discoveries that have led researchers to develop cutting-edge genetic tools to effectively and more accurately monitor turnover and displacement of cells within the mammalian corneal epithelium. Collating information on the basic biology of SCs will have clinical ramifications in furthering our knowledge of the processes that govern their role in homeostasis, wound-healing, transplantation, and how we can improve current unsatisfactory SC-based therapies for patients suffering blinding corneal disease.
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43
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Pardo-Saganta A, Law BM, Tata PR, Villoria J, Saez B, Mou H, Zhao R, Rajagopal J. Injury induces direct lineage segregation of functionally distinct airway basal stem/progenitor cell subpopulations. Cell Stem Cell 2015; 16:184-97. [PMID: 25658372 DOI: 10.1016/j.stem.2015.01.002] [Citation(s) in RCA: 152] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2014] [Revised: 10/17/2014] [Accepted: 01/06/2015] [Indexed: 12/21/2022]
Abstract
Following injury, stem cells restore normal tissue architecture by producing the proper number and proportions of differentiated cells. Current models of airway epithelial regeneration propose that distinct cytokeratin 8-expressing progenitor cells, arising from p63(+) basal stem cells, subsequently differentiate into secretory and ciliated cell lineages. We now show that immediately following injury, discrete subpopulations of p63(+) airway basal stem/progenitor cells themselves express Notch pathway components associated with either secretory or ciliated cell fate commitment. One basal cell population displays intracellular Notch2 activation and directly generates secretory cells; the other expresses c-myb and directly yields ciliated cells. Furthermore, disrupting Notch ligand activity within the basal cell population at large disrupts the normal pattern of lineage segregation. These non-cell-autonomous effects demonstrate that effective airway epithelial regeneration requires intercellular communication within the broader basal stem/progenitor cell population. These findings have broad implications for understanding epithelial regeneration and stem cell heterogeneity.
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Affiliation(s)
- Ana Pardo-Saganta
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Departments of Internal Medicine and Pediatrics, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Brandon M Law
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Departments of Internal Medicine and Pediatrics, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Purushothama Rao Tata
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Departments of Internal Medicine and Pediatrics, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Jorge Villoria
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Departments of Internal Medicine and Pediatrics, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Borja Saez
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Hongmei Mou
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Departments of Internal Medicine and Pediatrics, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Rui Zhao
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Departments of Internal Medicine and Pediatrics, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Jayaraj Rajagopal
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Departments of Internal Medicine and Pediatrics, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.
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Pan JH, Adair-Kirk TL, Patel AC, Huang T, Yozamp NS, Xu J, Reddy EP, Byers DE, Pierce RA, Holtzman MJ, Brody SL. Myb permits multilineage airway epithelial cell differentiation. Stem Cells 2015; 32:3245-56. [PMID: 25103188 DOI: 10.1002/stem.1814] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2014] [Accepted: 07/14/2014] [Indexed: 12/12/2022]
Abstract
The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63(+) basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps are poorly defined. Here, we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb(+) cells were identified as p63(-) and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63(-) population with failed maturation of Foxj1(+) ciliated cells as well as Scbg1a1(+) and Muc5ac(+) secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb(+) cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63(-) Myb(+) population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease.
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Affiliation(s)
- Jie-Hong Pan
- Department of Medicine, Washington University, St. Louis, Missouri, USA
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45
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Di Girolamo N, Bobba S, Raviraj V, Delic NC, Slapetova I, Nicovich PR, Halliday GM, Wakefield D, Whan R, Lyons JG. Tracing the fate of limbal epithelial progenitor cells in the murine cornea. Stem Cells 2015; 33:157-69. [PMID: 24966117 DOI: 10.1002/stem.1769] [Citation(s) in RCA: 112] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2013] [Accepted: 05/24/2014] [Indexed: 12/15/2022]
Abstract
Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation.
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Affiliation(s)
- N Di Girolamo
- School of Medical Sciences, University of New South Wales, Sydney, Australia
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Watson JK, Rulands S, Wilkinson AC, Wuidart A, Ousset M, Van Keymeulen A, Göttgens B, Blanpain C, Simons BD, Rawlins EL. Clonal Dynamics Reveal Two Distinct Populations of Basal Cells in Slow-Turnover Airway Epithelium. Cell Rep 2015; 12:90-101. [PMID: 26119728 PMCID: PMC4518462 DOI: 10.1016/j.celrep.2015.06.011] [Citation(s) in RCA: 117] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2015] [Revised: 04/25/2015] [Accepted: 06/02/2015] [Indexed: 01/06/2023] Open
Abstract
Epithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells.
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Affiliation(s)
- Julie K Watson
- Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK; Wellcome Trust-MRC Stem Cell Institute University of Cambridge, Cambridge CB2 3EG, UK; Department of Pathology, University of Cambridge, Cambridge CB2 3EG, UK
| | - Steffen Rulands
- Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK; Wellcome Trust-MRC Stem Cell Institute University of Cambridge, Cambridge CB2 3EG, UK; Cavendish Laboratory, Department of Physics, J. J. Thomson Avenue, Cambridge CB3 0HE, UK
| | - Adam C Wilkinson
- Wellcome Trust-MRC Stem Cell Institute University of Cambridge, Cambridge CB2 3EG, UK; Department of Haematology, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK; Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK
| | - Aline Wuidart
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels 1070, Belgium
| | - Marielle Ousset
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels 1070, Belgium
| | - Alexandra Van Keymeulen
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels 1070, Belgium
| | - Berthold Göttgens
- Wellcome Trust-MRC Stem Cell Institute University of Cambridge, Cambridge CB2 3EG, UK; Department of Haematology, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK; Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK
| | - Cédric Blanpain
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Brussels 1070, Belgium; Welbio, Université Libre de Bruxelles, Brussels 1070, Belgium
| | - Benjamin D Simons
- Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK; Wellcome Trust-MRC Stem Cell Institute University of Cambridge, Cambridge CB2 3EG, UK; Cavendish Laboratory, Department of Physics, J. J. Thomson Avenue, Cambridge CB3 0HE, UK
| | - Emma L Rawlins
- Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK; Wellcome Trust-MRC Stem Cell Institute University of Cambridge, Cambridge CB2 3EG, UK; Department of Pathology, University of Cambridge, Cambridge CB2 3EG, UK.
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Lynch TJ, Engelhardt JF. Progenitor cells in proximal airway epithelial development and regeneration. J Cell Biochem 2015; 115:1637-45. [PMID: 24818588 DOI: 10.1002/jcb.24834] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2014] [Accepted: 05/08/2014] [Indexed: 12/15/2022]
Abstract
Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma.
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Affiliation(s)
- Thomas J Lynch
- Department of Anatomy & Cell Biology, University of Iowa Carver College of Medicine, Iowa City, Iowa, 52242
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Ghosh M, Dwyer-Nield LD, Kwon JB, Barthel L, Janssen WJ, Merrick DT, Keith RL. Tracheal dysplasia precedes bronchial dysplasia in mouse model of N-nitroso trischloroethylurea induced squamous cell lung cancer. PLoS One 2015; 10:e0122823. [PMID: 25860262 PMCID: PMC4393296 DOI: 10.1371/journal.pone.0122823] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2014] [Accepted: 02/17/2015] [Indexed: 01/01/2023] Open
Abstract
Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US and has a 5-year survival rate of only 16%. Histological changes in the bronchial epithelium termed dysplasia are precursors to invasive SCC. However, the cellular mechanisms that cause dysplasia are unknown. To fill this knowledge gap, we used topical application of N-nitroso-tris chloroethylurea (NTCU) for 32 weeks to induce squamous dysplasia and SCC in mice. At 32 weeks the predominant cell type in the dysplastic airways was Keratin (K) 5 and K14 expressing basal cells. Notably, basal cells are extremely rare in the normal mouse bronchial epithelium but are abundant in the trachea. We therefore evaluated time-dependent changes in tracheal and bronchial histopathology after NTCU exposure (4, 8, 12, 16, 25 and 32 weeks). We show that tracheal dysplasia occurs significantly earlier than that of the bronchial epithelium (12 weeks vs. 25 weeks). This was associated with increased numbers of K5+/K14+ tracheal basal cells and a complete loss of secretory (Club cell secretory protein expressing CCSP+) and ciliated cells. TUNEL staining of NTCU treated tissues confirmed that the loss of CCSP+ and ciliated cells was not due to apoptosis. However, mitotic index (measured by bromodeoxyuridine incorporation) showed that NTCU treatment increased proliferation of K5+ basal cells in the trachea, and altered bronchial mitotic population from CCSP+ to K5+ basal cells. Thus, we demonstrate that NTCU-induced lung epithelial dysplasia starts in the tracheal epithelium, and is followed by basal cell metaplasia of the bronchial epithelium. This analysis extends our knowledge of the NTCU-SCC model by defining the early changes in epithelial cell phenotypes in distinct airway locations, and this may assist in identifying new targets for future chemoprevention studies.
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Affiliation(s)
- Moumita Ghosh
- Department of Pediatrics, National Jewish Health, Denver, Colorado, United States of America
- * E-mail:
| | - Lori D. Dwyer-Nield
- Department of Pharmaceutical Sciences, University of Colorado, Aurora, Colorado, United States of America
| | - Jennifer B. Kwon
- Department of Pediatrics, National Jewish Health, Denver, Colorado, United States of America
| | - Lea Barthel
- Department of Medicine, National Jewish Health, Denver, Colorado, United States of America
| | - William J. Janssen
- Department of Medicine, National Jewish Health, Denver, Colorado, United States of America
| | - Daniel T. Merrick
- Department of Pathology, University of Colorado, Aurora, Colorado, United States of America
| | - Robert L. Keith
- Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, Denver Veteran Affairs Medical Center, Denver, Colorado, United States of America
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Ghosh M, Smith RW, Runkle CM, Hicks DA, Helm KM, Reynolds SD. Regulation of trachebronchial tissue-specific stem cell pool size. Stem Cells 2015; 31:2767-78. [PMID: 23712882 DOI: 10.1002/stem.1440] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2012] [Revised: 02/22/2013] [Accepted: 03/18/2013] [Indexed: 02/05/2023]
Abstract
Tissue-specific stem cell (TSC) number is tightly regulated in normal individuals but can change following severe injury. We previously showed that tracheobronchial epithelial TSC number increased after severe naphthalene (NA) injury and then returned to normal. This study focused on the fate of the supernumerary TSC and the signals that regulate TSC pool size. We used the Keratin 5-rTA/Histone 2B:green fluorescent protein (GFP) model to purify basal cells that proliferated infrequently (GFP(bright) ) or frequently (GFP(dim) ) after NA injury. Both populations contained TSC but TSCs were 8.5-fold more abundant in the GFP(bright) population. Interestingly, both populations also contained a unipotential basal progenitor (UPB), a mitotic basal cell subtype whose daughters were terminally differentiated basal cells. The ratio of TSC to UPB was 5:1 in the GFP(bright) population and 1:5 in the GFP(dim) population. These data suggested that TSC proliferation in vivo promoted TSC-to-UPB differentiation. To evaluate this question, we cloned TSC from the GFP(bright) and GFP(dim) populations and passaged the clones seven times. We found that TSC number decreased and UPB number increased at each passage. Reciprocal changes in TSC and UPB frequency were more dramatic in the GFP(dim) lineage. Gene expression analysis showed that β-catenin and Notch pathway genes were differentially expressed in freshly isolated TSC derived from GFP(bright) and GFP(dim) populations. We conclude that (a) TSC and UPB are members of a single lineage; (b) TSC proliferation in vivo or in vitro promotes TSC-to-UPB differentiation; and (c) an interaction between the β-catenin and Notch pathways regulates the TSC-to-UPB differentiation process.
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Affiliation(s)
- Moumita Ghosh
- Department of Pediatrics, National Jewish Health, Denver, Colorado, USA
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