This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Generation of stem/progenitor cultures from mouse lungs
Juan-Jose Ventura
CNIO (Spanish National Cancer Centre)
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Cell renewal is essential to maintain tissue homeostasis and repair throughout the life of multicellular organisms. These functions rely on the presence of stem cells that have the potential to differentiate into specialized cell types as well as the capacity for long-term self-renewal. Adult organs therefore need long-life reservoirs of somatic stem cells to ensure the turnover of the tissue. These stem cells are localized in specific areas known as “niches”, which provide the right environment for the viability and function of the stem cell.
The adult lung is an organ with slow turnover. There are different regions in the respiratory system, such as large airways and small distal bronchioles, which house different stem cell types that contribute to the maintenance and repair of lung tissue. Recently, a population of stem cells referred to as bronchioalveolar stem cells, has been identified at the bronchioalveolar duct junctions and has been shown to be important in maintaining bronchiolar Clara cells and alveolar cells of the distal lung. However, very little is known about the regulation of these lung stem/progenitor cells and the signals that induce their conversion into cancer cells.
We have now used a modified version of previous protocols to isolate and maintain in culture lung stem/progenitor cells.
DMEM media
PBS
Fetal Bovine Serum
FibrOutTM (CHI-SCIENTIFIC)
FGF, EGF, Insulin
Antibodies: Sca1, CD45, PECAM, SP-C, CC-10
96 well, 6 well, 35 mm and 100 mm culture plates
Flow Cytometer
40 and 70 microns strainers
Tissue culture incubators at 37C, 5% CO2
Mechanical disruption of mice lungs passing the homogenates through a 70 μm strainer to obtain single cells.
- Plate the cells in DMEM supplemented with 20% FBS and 30 μg/ml Insulin (Sigma). Incubate at 37C in a 5% CO2 atmosphere.
- After 4 h to allow the cells to attach to the plate, FibrOutTM (CHISCIENTIFIC) was added to eliminate fibroblasts.
- Keep he cells under these conditions for 2 weeks, changing the media every 3 days and splitting them when needed.
- After that time, the cells are changed to serum free DMEM supplemented with FGF (10 ng/ml) and EGF (20 ng/ml).
- After 2 or 3 weeks the resulting cell population must be analyzed by flow cytometry. An aliquot of these cells fixed, permeabilized and labeled with SPC and CC-10 antibodies confirming the percentage of the double positive cells (SPC+/CC10+).
- The remaining cells are to be sorted for Sca-1+/CD45–/PECAM– using a FACS Aria cytometer (BD Bioscience).
- After re-plating, the sorted cells are maintained in serum free media (plus FGF and EGF). An aliquot of these cells must be analyzed again by flow cytometry and confirmed to be SPC+/CC10+. Thus, the final population in culture should be SP-C+/CC-10+/Sca1+/CD45–/PECAM–.
- To isolate clones, serial dilutions of the cells were plated in 96 well plates to obtain wells with single cells. Six of these clones obtained can be expanded in serum-free media and then tested for their potential to differentiate. The stem/progenitor cells tend to aggregate when maintained in serum free media, but in the presence of 20% FBS the cells expand to form single cell layers and start differentiating.
5 to 7 weeks
- This is a critical step. A good quality population of cells is needed to success. If the cells have been damaged too much during isolation the culture could not progress. Try not to be too harsh during isolation and do not stress the cells. Allow them to recover from the stressing procedure before going on.
- Avoid the proximal lung tissue (trachea and bronchus) and use exclusively tissue from the distal lung (alveolar epithelia), where the SPC+/CC10+ double positive stem cells are localized.
- Allow the cells from the lung homogenates to set and attach to the plates properly before adding the FibOut.
- Do not split the cells when they are at low confluency.
You should obtain a culture of cells forming clusters or aggregates when maintained in serum free media plus FGF and EGF. These cells should be able to slowly proliferate under these conditions.
Kim, C.F. et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121, 823-35 (2005).
Basseres, D.S. et al. Respiratory failure due to differentiation arrest and expansion of alveolar cells following lung-specific loss of the transcription factor C/EBPalpha in mice. Mol Cell Biol 26, 1109-23 (2006).
Nystul, T.G. & Spradling, A.C. Breaking out of the mold: Diversity within adult stem cells and their niches. Curr Opin Genet Dev 16, 463-468 (2006).
Fuchs, E., Tumbar, T. & Guasch, G. Socializing with the neighbors: stem cells and their niche. Cell 116, 769-78 (2004).
commented on 24 January, 2011
Just wondering what CC10 antibody did you use for flow cytometry? Thanks a lot.
commented on 24 January, 2011
Just wondering what CC10 antibody did you use for flow cytometry? Thanks a lot.
Version History
CURRENT STATUS: Posted
Version 1
Posted 02 May, 2007
Community comments: 2
Metrics
Comments: 2
HTML Views: ...
Subject Areas
Biological techniques
Associated Publications
p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation
by Juan José Ventura, Stephan Tenbaum, Eusebio Perdiguero, +4
Nature Genetics (12 April, 2007)
Method Article
Generation of stem/progenitor cultures from mouse lungs
Juan-Jose Ventura
CNIO (Spanish National Cancer Centre)
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 02 May, 2007
Community comments: 2
Metrics
Comments: 2
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Cell renewal is essential to maintain tissue homeostasis and repair throughout the life of multicellular organisms. These functions rely on the presence of stem cells that have the potential to differentiate into specialized cell types as well as the capacity for long-term self-renewal. Adult organs therefore need long-life reservoirs of somatic stem cells to ensure the turnover of the tissue. These stem cells are localized in specific areas known as “niches”, which provide the right environment for the viability and function of the stem cell.
The adult lung is an organ with slow turnover. There are different regions in the respiratory system, such as large airways and small distal bronchioles, which house different stem cell types that contribute to the maintenance and repair of lung tissue. Recently, a population of stem cells referred to as bronchioalveolar stem cells, has been identified at the bronchioalveolar duct junctions and has been shown to be important in maintaining bronchiolar Clara cells and alveolar cells of the distal lung. However, very little is known about the regulation of these lung stem/progenitor cells and the signals that induce their conversion into cancer cells.
We have now used a modified version of previous protocols to isolate and maintain in culture lung stem/progenitor cells.
DMEM media
PBS
Fetal Bovine Serum
FibrOutTM (CHI-SCIENTIFIC)
FGF, EGF, Insulin
Antibodies: Sca1, CD45, PECAM, SP-C, CC-10
96 well, 6 well, 35 mm and 100 mm culture plates
Flow Cytometer
40 and 70 microns strainers
Tissue culture incubators at 37C, 5% CO2
Mechanical disruption of mice lungs passing the homogenates through a 70 μm strainer to obtain single cells.
- Plate the cells in DMEM supplemented with 20% FBS and 30 μg/ml Insulin (Sigma). Incubate at 37C in a 5% CO2 atmosphere.
- After 4 h to allow the cells to attach to the plate, FibrOutTM (CHISCIENTIFIC) was added to eliminate fibroblasts.
- Keep he cells under these conditions for 2 weeks, changing the media every 3 days and splitting them when needed.
- After that time, the cells are changed to serum free DMEM supplemented with FGF (10 ng/ml) and EGF (20 ng/ml).
- After 2 or 3 weeks the resulting cell population must be analyzed by flow cytometry. An aliquot of these cells fixed, permeabilized and labeled with SPC and CC-10 antibodies confirming the percentage of the double positive cells (SPC+/CC10+).
- The remaining cells are to be sorted for Sca-1+/CD45–/PECAM– using a FACS Aria cytometer (BD Bioscience).
- After re-plating, the sorted cells are maintained in serum free media (plus FGF and EGF). An aliquot of these cells must be analyzed again by flow cytometry and confirmed to be SPC+/CC10+. Thus, the final population in culture should be SP-C+/CC-10+/Sca1+/CD45–/PECAM–.
- To isolate clones, serial dilutions of the cells were plated in 96 well plates to obtain wells with single cells. Six of these clones obtained can be expanded in serum-free media and then tested for their potential to differentiate. The stem/progenitor cells tend to aggregate when maintained in serum free media, but in the presence of 20% FBS the cells expand to form single cell layers and start differentiating.
5 to 7 weeks
- This is a critical step. A good quality population of cells is needed to success. If the cells have been damaged too much during isolation the culture could not progress. Try not to be too harsh during isolation and do not stress the cells. Allow them to recover from the stressing procedure before going on.
- Avoid the proximal lung tissue (trachea and bronchus) and use exclusively tissue from the distal lung (alveolar epithelia), where the SPC+/CC10+ double positive stem cells are localized.
- Allow the cells from the lung homogenates to set and attach to the plates properly before adding the FibOut.
- Do not split the cells when they are at low confluency.
You should obtain a culture of cells forming clusters or aggregates when maintained in serum free media plus FGF and EGF. These cells should be able to slowly proliferate under these conditions.
Kim, C.F. et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121, 823-35 (2005).
Basseres, D.S. et al. Respiratory failure due to differentiation arrest and expansion of alveolar cells following lung-specific loss of the transcription factor C/EBPalpha in mice. Mol Cell Biol 26, 1109-23 (2006).
Nystul, T.G. & Spradling, A.C. Breaking out of the mold: Diversity within adult stem cells and their niches. Curr Opin Genet Dev 16, 463-468 (2006).
Fuchs, E., Tumbar, T. & Guasch, G. Socializing with the neighbors: stem cells and their niche. Cell 116, 769-78 (2004).
commented on 24 January, 2011
Just wondering what CC10 antibody did you use for flow cytometry? Thanks a lot.
commented on 24 January, 2011
Just wondering what CC10 antibody did you use for flow cytometry? Thanks a lot.
Associated Publications
p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation
by Juan José Ventura, Stephan Tenbaum, Eusebio Perdiguero, +4
Nature Genetics (12 April, 2007)