How Much Do We Know About the Mouse Respiratory Epithelial Stem Cells? Characterization of a Doxycycline-Regulated Multi-Lineage Mouse Respiratory Epithelial Precursor Cell Line In Vitro And In Vivo

Main Article Content

Christian Elmshaeuser Ina Zoeller Darisuren Anhlan Ewald Beck Bruno Peault Olivier Tabary Una Chen

Abstract

Background-Purpose of this study: Tissue-specific stem cell lines are useful tools for cell biology studies. Information on respiratory tissue cell lines is limited. A doxycycline-regulated epithelial precursor cell line was established from the lung tissue of a tTAxSV40 Tag double transgenic mouse. In this study, we have characterized this cell line in vitro & in vivo, and found to mimic a rare subpopulation of club- and pneumocyte type II-dual cells.


Methods: It was partially characterized using cell viability and death assays, H3-thymidine incorporation assay, chloride efflux assay, Western blotting of proteins secreted, RT-PCR assays for RNA isolated. In addition, immune-deficient SCID mice were used as hosts for implantation of this precursor cell line, and feed with/without doxycycline containing water. Immunofluorescent typing using different antibodies were used to characterize the implanted lung.


Results: This cell line was found to mimic a rare subpopulation of club- and pneumocyte type II- dual cells with multiple phenotypes. Cell growth was doxycycline-regulated and observed only when doxycycline was omitted from the medium or present at concentrations up to 1 µg/ml, higher concentrations were inhibitory. ACT+ ciliated cells were found upon implantation into immune-deficient mice, in addition. Cell growth was doxycycline-regulated in vitro. When transplanted subcutaneously into immune-deficient mice, these cells migrated to the lung to form organized chimeric structures of donor and host origins, with club cells in the terminal bronchioles, ACT+ ciliated cells along the epithelial lining, and pneumocyte type II-cells in the alveolar interstices. No such homing of donor cells to the lung was observed when the implanted mice were fed doxycycline-containing water.


Discussions-Conclusions: This lung stem cell line might be able to provide us with an insight into the differentiation pathway of lung epithelial cells as well as with some understanding of the nature of air trophic-pulmonary epithelial cells. The results of this study underline the possibility of a future application for somatic (stem / precursor) cells in tissue replacement and tissue engineering of the damaged lung. Its ability to secrete and deliver soluble protein, might be a potential novel way for drug delivery. In addition, stem cells are thought to proliferate and differentiate in response to a deficiency or as a result of injury. Successful migration to the target organ and subsequent maturation of these precursors could be attributed to a requirement of lung stem cells to search for an aerated environment. Our findings challenge some current concepts of stem cell biology.This lung stem cell line may become a rich source of cells for tissue engineering and cell-based therapy for lung injury. The route and protocol established for cell introduction into the lung may provide a novel alternative to delivery of soluble protein substances through the airways. This lung stem cell line might also be modified to provide models for screening drugs against respiratory infection.

Keywords: Tet-off expression system, Lung stem cells, Lung disease, Lung viral infection, Drug screening and delivery

Article Details

How to Cite
ELMSHAEUSER, Christian et al. How Much Do We Know About the Mouse Respiratory Epithelial Stem Cells?. Medical Research Archives, [S.l.], v. 11, n. 10, oct. 2023. ISSN 2375-1924. Available at: <https://esmed.org/MRA/mra/article/view/4522>. Date accessed: 11 dec. 2023. doi: https://doi.org/10.18103/mra.v11i10.4522.
Section
Research Articles

References

1. Katsura, H, Sontake V, Tata A, et al. Human lung stem cell-based alveolospheres provide insights into SARS-CoV-2-mediated interferon responses and pneumocyte dysfunction. Cell Stem Cell 2020;27:890-904.e8. doi: 10.1016/j.stem.2020.10.005
2. Mou H, Vinarsky V, Tata PR, et al. Dual SMAD signaling inhibition enables long-term expansion of diverse epithelial basal cells. Cell Stem Cell 2016;19:217-231. doi: 10.1016/j.stem.2016.05.012
3. Kim CF, Jackson EL, Woolfenden AE, et al. Identification of Bronchioalveolar Stem Cells in Normal Lung and Lung Cancer. Cell 2005;121:823-835.
doi: 10.1016/j.cell.2005.03.032
4. Louie SM, Moye AL, Wong IG, et al. Progenitor potential of lung epithelial organoid cells in a transplantation model. Cell Reports 2022;39:110662-110692. doi: 10.1016/j.celrep.2022.110662
5. Muth H, Elmshauser C, Broad S, et al. Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line. J Gene Med 2001;3:585-597. doi: 10.1002/jgm.221.
6. Elmshaeuser C, Bechtel J, Motta I, et al. Characterization of a mouse tet-on glia precursor cell line in vitro and in vivo using the electrophysiological measurement. J Physiol Paris 2002;96:329-338. doi: 10.1016/S0928-4257(02)00024-4.
7. Chen U. Some properties and applications of cell lines and clones established from tet-responsive-SV40 Tag mice and mES cell lines. Scand. J Immunology 2011;73:531-535. doi: 10.1111/j.1365-3083.2011.02551.x
8. Milo-Landesman D, Efrat S. Growth factor-dependent proliferation of the pancreatic β-cell line βTC-tet: An assay for β-cell mitogenic factors. Int J Exp Diabetes Res 2002;3:69–74. doi: 10.1080/15604280212526
9. Efrat S, Fusco-DeMane D, Lemberg H, et al. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 1995;92:3576-3580.
doi: 10.1073/pnas.92.8.3576

10. Hook GE, Brody AR, Cameron GS, et al. Repopulation of denuded tracheas by Clara cells isolated from the lung of rabbits. Exp Lung Res 1987;12:311-329.
doi: 10.3109/01902148709062843
11. Engelhardt JF, Zepeda M, Cohn JA, et al. Expression of the cystic fibrosis gene in adult human lung. J Clin Invest 1994;93:737-749. doi: 10.1172/JCI117028
12. Engelhardt JF. Stem cell niches in the mouse airway. Am J Respir Cell Mol Biol 2001;24:649-652.
doi: 10.1165/ajrcmb.24.6.f206
13. Tirouvanziam R, Desternes A, Puchelle E, et al. Bioelectric properties of human cystic fibrosis and non-cystic fibrosis fetal respiratory xenografts in SCID mice. Am J Physiol 1998;274: C875-882. doi: 10.1152/ajpcell.1998.274.4.C875
14. Singh G, Katyal SL. Clara cell proteins. Ann New York Acad Sci 2000;923:43-58. doi: 10.1111/j.1749-6632.2000.tb05518.x
15. Singh G, Katyal SL. An immunologic study of the secretory products of rat Clara cells. J Histochem Cytochem 1984;32:49-54. doi: 10.1177/32.1.6418790
16. Bedetti CD, Singh J, Singh G, et al. Ultrastructural localization of rat Clara cell 10 kd secretory protein by the immunologic technique using polyclonal and monoclonal antibodies. J Histochem Cytochem 1987;35:789-794.
doi: 10.1177/35.7.2438324
17. Reynolds SD, Malkinson AM. Clara cell: progenitor for the bronchiolar epithelium. Int J Biochem Cell Biol 2010;42:1-4. doi: 10.1016/j.biocel.2009.09.002
18. Stripp BR, Maxon K, Mera R. Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol 1995;269: L791-L799. doi: 10.1152/ajplung.1995.269.6.L791
19. Hong KU, Reynolds SD, Watkins S, et al. Basel cells are a multipotent progenitor capable of renewing the bronchial epithelium. J Pathol 2004;164:577-588. doi: 10.1016/S0002-9440(10)63147-1
20. Borthwick DW, Shahbazian M, Krantz TQ, et al. Evidence for stem-cell niches in the tracheal epithelium. Am J Respir Cell Mol Biol 2001;24;662-670.
doi: 10.1165/ajrcmb.24.6.4217
21. Fehrenbach H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2001;2:33-46. doi: 10.1186/rr36
22. Otto WR. Lung epithelial stem cells. J Pathology 2002;197:527-535. doi: 10.1002/path.1160
23. Kim CF. Intersections of lung progenitor cells, lung disease and lung cancer. Eur Respir Rev 2017;26:170054-170064. doi: 10.1183/16000617.0054-2017
24. Ohle SJ, Anandaiah A, Fabian AJ, et al. Maintenance and Repair of the Lung Endothelium Does Not Involve Contributions from Marrow-Derived Endothelial Precursor Cells. Am J Respir Cell Mol Biol 2012;47:11–19. doi: 10.1165/rcmb.2011-0180OC
25. Reynolds SD, Malkinson AM. Clara cell: progenitor for the bronchiolar epithelium. Int J Biochem Cell Biol 2010;42:1–4. doi: 10.1016/j.biocel.2009.09.002
26. Roomans GM. Pharmacological treatment of the basic defect in cystic fibrosis. Cell Biol Int: 2014;38:1244-1246.
doi: 10.1002/cbin.10312
27. Huang SXL, Islam MN, O’Neill J, et al. Highly efficient generation of airway and lung epithelial cells from human pluripotent stem cells. Nat Biotechnol 2014;32:84–91. doi: 10.1038/nbt.2754
28. Lynch TJ, Anderson PJ, Rotti PG, et al. Submucosal gland myoepithelial cells are reserve stem cells that can regenerate mouse tracheal epithelium. Cell Stem Cell 2018;22:653–667.e5. doi: 10.1016/j.stem.2018.04.007
29. Basil MC, Morrisey EE. Lung regeneration: a tale of mice and men, Sem in Cell Dev Biol 2020:100:88-100.
doi: 10.1016/j.semcdb.2019.11.006
30. Dibattista M, Al Koborssy D, Genovese F, Reisert J. The functional relevance of olfactory marker protein in the vertebrate olfactory system: a never-ending story. Cell and Tissue Res 2021:383:409-427. doi: 10.1007/s00441-020-03349-9
31. Riccetti M, Gokey JJ, Aronow B, Perl AT. The elephant in the lung: Integrating lineage-tracing, molecular markers, and single cell sequencing data to identify distinct fibroblast populations during lung development and regeneration. Matrix Biol 2020;91-92:51-74. doi:10.1016/j.matbio.2020.05.002
32. Perl AK, Wert SE, Nagy A, et al. Early restriction of peripheral and proximal cell lineages during formation of the lung. Proc Natl Acad Sci USA 2002;99:10482-10487. doi: 0.1073/pnas.152238499
33. Perl AK, Riethmacher D, Whitsett JA. Conditional depletion of airway progenitor cells induces peribronchiolar fibrosis. Am J Respir Crit Care Med 2011;183:511-521. doi: 10.1164/rccm.201005-0744OC
34. Furth PA, St Onge L, Boger H, et al. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 1994:91:9302-9306. doi: 10.1073/pnas.91.20.9302
35. G Hagen, M Wolf, S L Katyal, et al. Tissue specific expression, hormonal regulation and 5'-flanking gene region of the rat Clara cell 10 kDa protein: comparison to rabbit uteroglobin. Nucleic Acids Res 1990:18:2939–2946. doi: 10.1093/nar/18.10.2939
36. Stripp BR, Maxson K, Mera R, Singh G. Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol 1995;269:L791-799. doi: 10.1152/ajplung.1995.269.6.L791
37. Wang G, Umstead TM, Phelps DS, et al. The effect of ozone exposure on the ability of human surfactant protein a variants to stimulate cytokine production. Environ Health Perspect 2002;11:79–84. doi: 10.1289/ehp.0211079
38. Schmidt R, Markart P, Ruppert C, et al. Time-dependent changes in pulmonary surfactant function and composition in acute respiratory distress syndrome due to pneumonia or aspiration. Respir Res 2007;8:55-66. doi: 10.1186/1465-9921-8-55
39. Perl AK, Wert SE, Nagy A, et al. Early restriction of peripheral and proximal cell lineages during formation of the lung. Proc Natl Acad Sci USA 99 (2002): 10482-10487. doi: 10.1073/pnas.152238499
40. Perl AK, Riethmacher D, Whitsett JA. Conditional depletion of airway progenitor cells induces peribronchiolar fibrosis. Am J Respir Crit Care Med 183 (2011): 511-521. doi: 10.1164/rccm.201005-0744OC
41. 41.Whitsett JA, Wert SE, Weaver TE. Alveolar Surfactant Homeostasis and the Pathogenesis of Pulmonary Disease. Annu Rev Med 61 (2010):105-119. doi: 10.1146/annurev.med.60.041807.123500
42. Rindler TN, Stockman CA, Filuta AL, et al. Alveolar injury and regeneration following deletion of ABCA3. JCI Insight 2 (2017): e97381-97395. doi: 10.1172/jci.insight.97381
43. Seeger W, Giinther A, Walmrath HD, et al. Alveolar surfactant and adult respiratory distress syndrome; Pathogenetic role and therapeutic prospects. Clinical Investigator 71 (1993):177-190. doi: 10.1007/BF00180100
44. Günther A, Ruppert C, Schmidt R, et al. Surfactant alteration and replacement in acute respiratory distress syndrome. Respiratory Research 2 (2001):353-364. doi: 10.1186/rr86
45. Schmidt R, Markart P, Ruppert C, et al. Time-dependent changes in pulmonary surfactant function and composition in acute respiratory distress syndrome due to pneumonia or aspiration. Respiratory Research 8 (2007):55-66. doi: 10.1186/1465-9921-8-55
46. Tabary O, Zahm JM, Hinnrasky J, et al. Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro. J Pathol 153 (1998): 921-930. doi: 10.1016/S0002-9440(10)65633-7
47. Fehrenbach H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2 (2001): 33-46. doi: 10.1186/rr36
48. Alison MR, Poulsom R, Otto WR, et al. Recipes for adult stem cell plasticity: fusion cuisine or readymade? J Clin Pathol 57 (2004):113–120. doi: 10.1136/jcp.2003.010074
49. Demello DE, Mahmoud S, Padfield PJ, Hoffmann JW. Generation of an immortal differentiated lung type-II epithelial cell line from the adult H-2KbtsA58 transgenic mouse. In Vitro Cell Dev Biol Anim 36 (2000):374–382. doi: 10.1290/1071-2690(2000)036<0374:GOAIDL>2.0.CO;2
50. Hashimoto S, Nakano H, Suguta Y, et al. Immunolocalization of Sprouty-1 and Sprouty-2 in developing rat lung. Pathobiology 79 (2012):34–44 doi: 10.1159/000332215
51. Singh G, Katyal SL. An immunologic study of the secretory products of rat Clara cells. J Histochem Cytochem 1984;32:49-54. doi: 10.1177/32.1.6418790
52. Furth PA, St Onge L, Boger H, et al. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci USA 1994:91:9302-9306. doi: 10.1073/pnas.91.20.9302
53. Muth H, Elmshaeuser C, Broad S, et al. Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line. J Gene Med 2001;3: 585-597. doi: 10.1002/jgm.221
54. Elmshaeuser C, Bechtel J, Motta I, et al. Characterization of a mouse tet-on glia precursor cell line in vitro and in vivo using the electrophysiological measurement. J Physiol Paris: 2002;96:329-338. doi: 10.1016/S0928-4257(02)00024-4
55. Wang G, Phelps DS, Umstead TM, Floros J. Human SP-A protein variants derived from one or both genes stimulate TNF-alpha production in the THP-1 cell line. Am J Physiol Lung Cell Mol Physiol 2000;278:L946-L954. doi: 10.1152/ajplung.2000.278.5.L946
56. Günther A, Ruppert C, Schmidt R, et al. Surfactant alteration and replacement in acute respiratory distress syndrome. Respir Res 2001;2:353-364. doi: 10.1186/rr86

57. Korfhagen TR, Bruno MD, Ross GF, et al. Altered surfactant function and structure in SP-A gene targeted mice. Proc Natl Acad Sci USA 1996;93:9594-9599. doi: 10.1073/pnas.93.18.9594
58. Schmidt R, Steinhilber W, Ruppert C, et al. An ELISA technique for quantification of surfactant apoprotein (SP)-C in bronchoalveolar lavage fluid. Am J Respir Crit Care Med 2002;165:470-474.
doi: 10.1164/ajrccm.165.4.2102080
59. Singh G, Katyal SL. Clara cell proteins. Ann New York Acad Sci 2000;923:43-58. doi: 10.1111/j.1749-6632.2000.tb05518.x
60. Bedetti CD, Singh J, Singh G, et al. Ultrastructural localization of rat Clara cell 10 kd secretory protein by the immunogold technique using polyclonal and monoclonal antibodies. J Histochem Cytochem 1987;35:789-794. doi: 10.1177/35.7.2438324
61. Reynolds SD, Malkinson AM. Clara cell: progenitor for the bronchiolar epithelium. Int J Biochem Cell Biol 2010;42:1-4. doi: 10.1016/j.biocel.2009.09.002
62. Green MD, Huang SX, Snoeck HW. Stem cells of the respiratory system: from identification to differentiation into functional epithelium. BioEssays. news and reviews in molecular, cellular and developmental biology. BioEssays 2013;35:261-270. doi: 10.1002/bies.201200090
63. Ruiz P, Schwaerzler C, Günthert U. CD44 isoforms during differentiation and development. BioEssays 1995;17:17-24. doi: 10.1002/bies.950170106
64. Hong KU, Reynolds SD, Watkins S, et al. Basal cells are a multipotent progenitor capable of renewing the bronchial epitelium. Am J Pathol 2004;164:577–588. doi: 10.1016/S0002-9440(10)63147-1
65. Günther A, Ruppert C, Schmidt R, et al. Surfactant alteration and replacement in acute respiratory distress syndrome. Respir Res 2001;2:353-364. doi: 10.1186/rr86
66. Brouard N, Chapel A, Neildez-Nguyen TMA, et al. Transplantation of stromal cells transduced with the human IL3 gene to stimulate hematopoiesis in human fetal bone grafts in non-obese, diabetic-severe combined immunodeficiency mice. Leukemia 1998;12:1128-1135.
doi: 10.1038/sj.leu.2401081
67. Delplanque A, Coraux C, Tirouvanziam R. et al. Epithelial stem cell-mediated development of the human respiratory mucosa in SCID mice. J Cell Sci 2000;113:767-778.
doi: 10.1242/jcs.113.5.767
68. Engelhardt JF. Stem cell niches in the mouse airway. Am J Respir Cell Mol Biol 2001;24:649-652. doi: 10.1165/ajrcmb.24.6.f206
69. Tirouvanziam R, Desternes A, Puchelle E, et al. Bioelectric properties of human cystic fibrosis and non-cystic fibrosis fetal respiratory xenografts in SCID mice. Am J Physiol 1998;274: C875-882. doi: 10.1152/ajpcell.1998.274.4.C875
70. Borthwick DW, Shahbazian M, Krantz TQ, et al. Evidence for stem-cell niches in the tracheal epithelium. Am J Respir Cell Mol Biol 2001;24:662-670. doi: 10.1165/ajrcmb.24.6.4217

71. Glasser SW, Burhans MS, Korfhagen TR, et al. Altered stability of pulmonary surfactant in SP-C-deficient mice. Proc Natl Acad Sci USA 2001;98:6366–6371. doi: 10.1073/pnas.101500298
72. Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science 2000;287:1427-1430. doi: 10.1126/science.287.5457.1427
73. Pitard B, Oudrhiri N, Lambert O, et al. Sterically stabilized BGTC-based lipoplexes: structural features and gene transfection into the mouse airways in vivo. J Gene Med 2001;3:478-487. doi: 10.1002/jgm.211
74. Clements JA, Avery ME. Lung surfactant and neonatal respiratory distress syndrome. Am J Respir Crit Care Med 1998;157:S59-66. doi: 10.1164/ajrccm.157.4.nhlb1-1
75. Avery ME. Surfactant deficiency in hyaline membrane disease: the story of discovery. Am J Respir Crit Care Med 2000;161:1074-1075. doi: 10.1164/ajrccm.161.4.16142
76. Huddleston CB, Bloch JB, Sweet SC, et al. Lung transplantation in children. Ann Surg 2002;236:270-276. doi: 10.1097/00000658-200209000-00003
77. Kotton DN, Summer RS, Sun X, et al. Stem cell antigen-1 expression in the pulmonary vascular endothelium. Am J Physiol Lung Cell Mol Physiol 2003;284:L990-L996. doi: 10.1152/ajplung.00415.2002
78. Hook GE, Brody AR, Cameron GS, et al. Repopulation of denuded tracheas by Clara cells isolated from the lung of rabbits. Exp Lung Res 1987;12:311-329.
doi: 10.3109/01902148709062843
79. Engelhardt JF, Zepeda M, Cohn JA, et al. Expression of the cystic fibrosis gene in adult human lung. J Clin Invest 1994;93:737-749. doi: 10.1172/JCI117028
80. Stripp B, Maxon K, Mera R, Singh G. Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol 1995;269:L791-L799. doi: 10.1152/ajplung.1995.269.6.L791
81. Fehrenbach H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2001;2:33-46. doi: 10.1186/rr36
82. Otto WR. Lung epithelial stem cells. J Pathology 2002;197:527-535. doi: 10.1002/path.1160
83. Glasser SW, Burhans MS, Korfhagen TR, et al. Altered stability of pulmonary surfactant in SP-C-deficient mice. Proc Natl Acad Sci USA 2001;98:6366-6371.
doi: 10.1073/pnas.101500298
84. Watson TM, Reynolds SD, Mango GW, et al. Altered lung gene expression in CCSP-null mice suggests immuno-regulatory roles for Clara cells. Am J Patho 2001;281:L1523-1530. doi: 10.1152/ajplung.2001.281.6.L1523
85. Rikimaru K, Moles JP, Watt FM. Correlation between hyperproliferation and suprabasal integrin expression in human epidermis reconstituted in culture. Exp Dermatol 1997;6:214-221. doi: 10.1111/j.1600-0625.1997.tb00165.x
86. Katsura, H, Sontake V, Tata A, et al. Human lung stem cell-based alveolospheres provide insights into SARS-CoV-2-mediated interferon responses and pneumocyte dysfunction. Cell Stem Cell 2020;27:890-904.e8.
Doi: 10.1016/j.stem.2020.10.005
87. 87. Mou, H, Vinarsky V, Tata PR, et al. Dual SMAD signaling inhibition enables long-term expansion of diverse epithelial basal cells. Cell Stem Cell 2016;19: 217-231.
doi: 10.1016/j.stem.2016.05.012
88. Kim CF, Jackson EL, Woolfenden AE, et al. Identification of Bronchioalveolar Stem Cells in Normal Lung and Lung Cancer. Cell 2005;121:823-835.
doi: 10.1016/j.cell.2005.03.032
89. Louie SM, Moye AL, Wong IG, et al. Progenitor potential of lung epithelial organoid cells in a transplantation model. Cell Reports 2022;39:110662-692. doi: 10.1016/j.celrep.2022.110662
90. Yorifuji T, Lemna WK, Ballard CF, et al. Molecular cloning and sequence analysis of the murine cDNA for the cystic fibrosis transmembrane conductance regulator. Genomics. 1991;10(3):547-550.
doi: 10.1016/0888-7543(91)90434-g.
91. Glasser SW, Korfhagen TR, Bruno MD, et al. Structure and expression of the pulmonary surfactant protein SP-C gene in the mouse. J Biol Chem. 1990; 265(35):21986-991. PMID: 2254341