Linghuan Zhang1,2, Wenping Luo2,3, Jiang Liu1, Maozhu Xu1, Qi Peng4, Wenjing Zou1, Jingyi You1, Yi Shu1,2, Piao Zhao2,5, William Wagstaff2, Guozhi Zhao2,5, Kevin Qin2,6, Rex C Haydon2, Hue H Luu2, Russell R Reid2,7,8, Yang Bi1,2, Tianyu Zhao9, Tong-Chuan He10,11, Zhou Fu12. 1. Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, and the Department of Respiratory Diseases, The Children's Hospital of Chongqing Medical University, Chongqing, 400014, China. 2. Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, 5841 South Maryland Avenue, MC3079, Chicago, IL, 60637, USA. 3. Laboratory Animal Center, Southwest University, Chongqing, 400715, China. 4. University-Town Hospital, Chongqing Medical University, Chongqing, 401331, China. 5. Departments of Orthopaedic Surgery and Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400046, China. 6. Rosalind Franklin University of Medicine, North Chicago, IL, 60064, USA. 7. Department of Surgery, The University of Chicago Medical Center, Chicago, IL, 60637, USA. 8. Laboratory of Craniofacial Suture Biology and Development, Department of Surgery Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL, 60637, USA. 9. Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, the Stomatological Hospital of Chongqing Medical University, Chongqing, 401147, China. 10. Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, 5841 South Maryland Avenue, MC3079, Chicago, IL, 60637, USA. tche@uchicago.edu. 11. Department of Surgery, The University of Chicago Medical Center, Chicago, IL, 60637, USA. tche@uchicago.edu. 12. Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, and the Department of Respiratory Diseases, The Children's Hospital of Chongqing Medical University, Chongqing, 400014, China. fu_zhou79@126.com.
Abstract
BACKGROUND: A healthy alveolar epithelium is critical to the gas exchange function of the lungs. As the major cell type of alveolar epithelium, alveolar type 2 (AT2) cells play a critical role in maintaining pulmonary homeostasis by serving as alveolar progenitors during lung injury, inflammation, and repair. Dysregulation of AT2 cells may lead to the development of acute and chronic lung diseases and cancer. The lack of clinically relevant AT2 cell models hampers our ability to understand pulmonary diseases. Here, we sought to establish reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2) and investigate their potential in forming alveolar organoids to model pulmonary diseases. METHODS: Primary mouse pulmonary alveolar cells (mPACs) were isolated and immortalized with a retroviral expression of SV40 Large T antigen (LTA). Cell proliferation and survival was assessed by crystal violet staining and WST-1 assays. Marker gene expression was assessed by qPCR, Western blotting, and/or immunostaining. Alveolar organoids were generated by using matrigel. Ad-TGF-β1 was used to transiently express TGF-β1. Stable silencing β-catenin or overexpression of mutant KRAS and TP53 was accomplished by using retroviral vectors. Subcutaneous cell implantations were carried out in athymic nude mice. The retrieved tissue masses were subjected to H & E histologic evaluation. RESULTS: We immortalized primary mPACs with SV40 LTA to yield the imPACs that were non-tumorigenic and maintained long-term proliferative activity that was reversible by FLP-mediated removal of SV40 LTA. The EpCAM+ AT2-enriched subpopulation (i.e., imPAC2) was sorted out from the imPACs, and was shown to express AT2 markers and form alveolar organoids. Functionally, silencing β-catenin decreased the expression of AT2 markers in imPAC2 cells, while TGF-β1 induced fibrosis-like response by regulating the expression of epithelial-mesenchymal transition markers in the imPAC2 cells. Lastly, concurrent expression of oncogenic KRAS and mutant TP53 rendered the imPAC2 cells a tumor-like phenotype and activated lung cancer-associated pathways. Collectively, our results suggest that the imPAC2 cells may faithfully represent AT2 populations that can be further explored to model pulmonary diseases.
BACKGROUND: A healthy alveolar epithelium is critical to the gas exchange function of the lungs. As the major cell type of alveolar epithelium, alveolar type 2 (AT2) cells play a critical role in maintaining pulmonary homeostasis by serving as alveolar progenitors during lung injury, inflammation, and repair. Dysregulation of AT2 cells may lead to the development of acute and chronic lung diseases and cancer. The lack of clinically relevant AT2 cell models hampers our ability to understand pulmonary diseases. Here, we sought to establish reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2) and investigate their potential in forming alveolar organoids to model pulmonary diseases. METHODS: Primary mouse pulmonary alveolar cells (mPACs) were isolated and immortalized with a retroviral expression of SV40 Large T antigen (LTA). Cell proliferation and survival was assessed by crystal violet staining and WST-1 assays. Marker gene expression was assessed by qPCR, Western blotting, and/or immunostaining. Alveolar organoids were generated by using matrigel. Ad-TGF-β1 was used to transiently express TGF-β1. Stable silencing β-catenin or overexpression of mutant KRAS and TP53 was accomplished by using retroviral vectors. Subcutaneous cell implantations were carried out in athymic nude mice. The retrieved tissue masses were subjected to H & E histologic evaluation. RESULTS: We immortalized primary mPACs with SV40 LTA to yield the imPACs that were non-tumorigenic and maintained long-term proliferative activity that was reversible by FLP-mediated removal of SV40 LTA. The EpCAM+ AT2-enriched subpopulation (i.e., imPAC2) was sorted out from the imPACs, and was shown to express AT2 markers and form alveolar organoids. Functionally, silencing β-catenin decreased the expression of AT2 markers in imPAC2 cells, while TGF-β1 induced fibrosis-like response by regulating the expression of epithelial-mesenchymal transition markers in the imPAC2 cells. Lastly, concurrent expression of oncogenic KRAS and mutant TP53 rendered the imPAC2 cells a tumor-like phenotype and activated lung cancer-associated pathways. Collectively, our results suggest that the imPAC2 cells may faithfully represent AT2 populations that can be further explored to model pulmonary diseases.
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