Stimuli-Specific Senescence of Primary Human Lung Fibroblasts Modulates Alveolar Stem Cell Function
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethic Statement
2.2. Cell Culture
2.3. Induction of Cellular Senescence
2.4. Organoid Assay
2.5. RT-qPCR
2.6. ELISA
2.7. Senescence Associated β-Galactosidase Staining
2.8. Immunofluorescence Staining
2.9. Bulk-RNA Sequencing
2.10. Analysis of Human Lung COPD Single Cell RNA Sequencing Data
2.11. Analysis of Human Lung IPF Single Cell RNA Sequencing Data
2.12. Microarray Analysis
2.13. Data Collection and Analysis
3. Results
3.1. Senescent Fibroblasts Accumulate in IPF and COPD Lungs
3.2. Senescence Program in phLF Derived from Different Diseases Backgrounds
3.3. Disease Relevant Stimuli Can Induce Senescence in phLF
3.4. Senescence Induction in phLF Is Not Impacted by Disease Background
3.5. Senescence Induction in phLF Is Stimuli-Specific
3.6. Senescent Fibroblasts Disrupt Progenitor Potential of Distal Alveolar Epithelial Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sex | Age | Smoker | Diagnosis |
---|---|---|---|
female | 72 | ex-smoker | Ctrl Donor |
female | 67 | smoker | Ctrl Donor |
male | 84 | ex-smoker | Ctrl Donor |
female | 57 | smoker | Ctrl Donor |
female | 56 | ex-smoker | IPF |
male | 72 | n/a | IPF |
female | 54 | never smoker | IPF |
male | 73 | n/a | IPF |
male | 63 | ex-smoker | IPF |
male | 67 | ex-smoker | COPD |
male | 73 | ex-smoker | COPD |
female | 69 | ex-smoker | COPD |
male | 62 | ex-smoker | COPD |
female | n/a | n/a | COPD |
male | 64 | smoker | COPD |
male | 58 | smoker | COPD |
female | 60 | smoker | COPD |
female | 61 | smoker | COPD |
unknown | n/a | n/a | Ctrl Donor |
unknown | n/a | n/a | Ctrl Donor |
unknown | n/a | n/a | Ctrl Donor |
unknown | n/a | n/a | Ctrl Donor |
male | 58 | ex-smoker | IPF |
male | 63 | ex-smoker | IPF |
male | n/a | never smoker | IPF |
male | 61 | n/a | IPF |
female | 29 | never smoker | Ctrl Donor |
female | 71 | ex-smoker | Ctrl Donor |
male | 59 | ex-smoker | Ctrl Donor |
male | 49 | never smoker | Ctrl Donor |
male | 62 | ex-smoker | COPD |
male | 58 | ex-smoker | COPD |
male | 60 | ex-smoker | COPD |
male | 65 | ex-smoker | COPD |
male | 57 | ex-smoker | IPF |
female | 40 | never smoker | IPF |
male | 52 | ex-smoker | IPF |
male | 62 | ex-smoker | IPF |
male | 59 | ex-smoker | IPF |
Primer | Sequence (5′-3′) |
---|---|
ACTA2_fw | CGAGATCTCACTGACTACCTCATGA |
ACTA2_rv | AGAGCTACATAACACAGTTTCTCCTTGA |
FN-1_fw | CCGACCAGAAGTTTGGGTTCT |
FN-1_rv | CAATGCGGTACATGACCCCT |
COL1a1_fw | CAAGAGGAAGGCCAAGTCGAG |
COL1a1_rv | TTGTCGCAGACGCAGATCC |
PAI-1 fw | GACATCCTGGAACTGCCCTA |
PAI-1 rv | GGTCATGTTGCCTTTCCAGT |
CDKN2A_fw | ACCAGAGGCAGTAACCATGC |
CDKN2A_rev | CCTGTAGGACCTTCGGTGAC |
CDKN1A_fw | GTCAGTTCCTTGTGGAGCCG |
CDKN1A_rev | TGGGTTCTGACGGACATCCC |
TP53_fw | CGCTTCGAGATGTTCCGAGA |
TP53_rv | CTTCAGGTGGCTGGAGTGAG |
HPRT_fw | AAGGACCCCACGAAGTGTTG |
HPRT_rv | GGCTTTGTATTTTGCTTTTCCA |
Target Protein | Host | Company | Ref. No |
---|---|---|---|
P21 | rabbit | Abcam | ab109520 |
Phospho-histone H2A.X | mouse | Millipore | 05–636 |
ACT | mouse | Abcam | ab24610 |
SP-C | rabbit | Abcam | ab3786 |
Krt8 | rat | DSHB | TROMAI |
Anti-rat-488 | donkey | Invitrogen | A21208 |
Anti-rabbit-647 | donkey | Invitrogen | A31573 |
Anti-mouse-568 | donkey | Invitrogen | A10037 |
Anti-mouse-555 | goat | Invitrogen | A21424 |
Anti-rabbit-488 | goat | Invitrogen | A11008 |
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Melo-Narváez, M.C.; Bramey, N.; See, F.; Heinzelmann, K.; Ballester, B.; Steinchen, C.; Jain, E.; Federl, K.; Hu, Q.; Dhakad, D.; et al. Stimuli-Specific Senescence of Primary Human Lung Fibroblasts Modulates Alveolar Stem Cell Function. Cells 2024, 13, 1129. https://doi.org/10.3390/cells13131129
Melo-Narváez MC, Bramey N, See F, Heinzelmann K, Ballester B, Steinchen C, Jain E, Federl K, Hu Q, Dhakad D, et al. Stimuli-Specific Senescence of Primary Human Lung Fibroblasts Modulates Alveolar Stem Cell Function. Cells. 2024; 13(13):1129. https://doi.org/10.3390/cells13131129
Chicago/Turabian StyleMelo-Narváez, Maria Camila, Nora Bramey, Fenja See, Katharina Heinzelmann, Beatriz Ballester, Carina Steinchen, Eshita Jain, Kathrin Federl, Qianjiang Hu, Deepesh Dhakad, and et al. 2024. "Stimuli-Specific Senescence of Primary Human Lung Fibroblasts Modulates Alveolar Stem Cell Function" Cells 13, no. 13: 1129. https://doi.org/10.3390/cells13131129
APA StyleMelo-Narváez, M. C., Bramey, N., See, F., Heinzelmann, K., Ballester, B., Steinchen, C., Jain, E., Federl, K., Hu, Q., Dhakad, D., Behr, J., Eickelberg, O., Yildirim, A. Ö., Königshoff, M., & Lehmann, M. (2024). Stimuli-Specific Senescence of Primary Human Lung Fibroblasts Modulates Alveolar Stem Cell Function. Cells, 13(13), 1129. https://doi.org/10.3390/cells13131129