Department of Pediatrics, David Geffen School of Medicine at UCLA
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Hegab, A. E., Luan Ha, V., Attiga, Y. S., Nickerson, D. W., Gomperts, B. N. Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea. J. Vis. Exp. (67), e3731, doi:10.3791/3731 (2012).
The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.
Outline of Steps
1. Dissection of trachea
2. Cleaning trachea and cutting it
3. Enzyme digestion and processing into single cell suspension
4. Staining for FACS and sorting
5. Processing sorted cells for in vivo and in vitro models
1. Dissection of Trachea
2. Cleaning Trachea and Cutting It
3. Enzyme Digestion and Processing into Single Cell Suspension (SCS)
4. Staining for FACS and Sorting
5. Processing Sorted Cells for: in vitro Culture and the in vivo Model
5.1 In vitro sphere forming 5-9 culture
5.2 In vivo model
6. Representative Results
Stripping of the mouse tracheas will result in a denuded trachea that is visible by light microscopy as shown in Figure 1A. Figure 1B shows the brightfield view of a trachea after epithelial stripping with submucosal glands being released from the tissues and resembling bunches of grapes.
Pronase removes the ITGA6 epitope and this is why the TROP2 duct cells cannot be separated into ITGA6+ and - populations. However, dispase, which preserves the ITGA6 epitope, digests all the surface epithelium and the duct cells even with very short incubation times, and therefore cannot be used to separate duct from basal cells3. Flow cytometry of the single cell suspensions should show forward scatter and side scatter plots that are represented in Figure 2A. Good separation of duct cells from the rest if the tracheal cells should be seen with the Trop2 antibody in flow cytometry for fluorescent activated cell sorting (FACS) as seen in Figure 2B. Spheres should be visible in the matrigel after about 1 week in culture and are dense in appearance as shown in Figure 3. The efficiency of this process is the order of 1-2% and single cells will still be present in the matrigel and likely represent cells from the submucosal gland ducts that do not possess the capacity for self-renewal. Injection of duct stem cells into the mouse fat pad results in the formation of submucosal gland-like structures that are shown Figure 4. These are seen in the blocks even without H&E staining. These are spherical and possess a central lumen. Many cross sections need to be made through the fat pad in order not to miss these epithelial structures.
Figure 1. Enzymatic digestion of tracheal epithelium. Removal of surface epithelial cells is shown with black arrows, removal of cells in the submucosal gland ducts is shown with green arrows. Denuded tracheas that are visible by light microscopy are shown after 30 min of dispase digestion and after 4 hr of pronase digestion.
Figure 2A. i. Flow cytometry of the single cell suspensions showing representative forward scatter and side scatter plots from mouse tracheas after surface epithelial stripping. ii. Representative FACS plots for TROP2 expression in duct cells from mouse tracheas.
Figure 2B. i. Flow cytometry of the single cell suspensions from stripped surface epithelium of the lower two thirds of the mouse trachea showing representative forward scatter and side scatter plots. ii. Typical FACS plot of ITGA6 expression in surface epithelium. iii. Another representative FACS plot of forward and side scatter from surface epithelium showing back gating of the basal cell population in green. iv. Typical ITGA6 and TROP2 expressing cell population in green.
Figure 3. Time course of development of spheres. A. Duct spheres. Spheres should be visible in the matrigel after about 1 week in culture and almost all are dense in appearance. Single cells that don't form spheres are also seen. (scale bar = 50 μm). B. Basal spheres are larger and luminal in appearance. Single cells that don't form spheres are also seen. (scale bar = 50 μm).
Figure 4. Injection of duct stem cells into the mouse fat pad results in the formation of submucosal gland-like structures. Representative structures are shown in the fat pad and some are shown producing mucin (light blue) with the Alcian blue Periodic Acid Schiff stain.
This technique to isolate duct and basal cells from the airways is important for improving our understanding of airway repair and regeneration and airway diseases. The techniques described here include a few critical steps. The first is the optimized enzymatic digestion period. The second is the creation of a single cell suspension through serial passaging with progressively higher gage needles to prevent cell shearing but to break up cell clumps. The third is the FACS analysis, and gating of cells with appropriate antibodies.
As some of the enzymes strip surface epitopes, like ITGA6 and NGFR, one possible modification is to alter the enzyme digestion protocols to spare the epitopes. This protocol could also be used to isolate tracheal endothelial cells or other mesenchymal cells by altering the specific antibodies to surface markers on cell types.
The limitations of the separation of duct and basal cells is related to our current lack of a surface marker to distinguish duct cells from basal cells and the need for surface epithelial stripping and the possibility of leaving some basal cells behind. Future identification of surface markers specifically for duct cells will make this process easier and obviate the need for surface epithelial stripping. It will also negate the need to use the lower two thirds of the trachea for pure basal cell preparations.
No conflicts of interest declared.
We would like to acknowledge the Broad Stem Cell Research Center FACS and especially thank Jessica Scholes and Felicia Codrea for their help with cell sorting. The work was funded by CIRM RN2-00904-1, K08 HL074229, American Thoracic Society/COPD Foundation ATS-06-065, The Concern Foundation, The UCLA Jonsson Comprehensive Cancer Center Thoracic Oncology Program/Lung Cancer SPORE, the University of California Cancer Research Coordinating Committee and the Gwynne Hazen Cherry Memorial Laboratories (BG).
|Complete medium 10:
DMEM-F-12 , 50/50, 1X)
|Hepes (15 mM)||Invitrogen||15630|
|Sodium bicarbonate (3.6mM or 0.03%)||Invitrogen||25080|
|L-glutamine (4 mM)||Mediatech||25-005-Cl|
|Penicillin (100 U/ml)||Mediatech||30-001-CI|
|Streptomycin (100 μg/m)||Mediatech||30-001-CI|
|Amphotericin B (0.25 μg/ ml)||Lonza||17-836R|
|Insulin (10 μg/ml)||Sigma||I6634|
|Transferrin (5 μg/ml)||Sigma||T1147|
|Cholera toxin (0.1 μg/ml)||Sigma||C8052|
|Epidermal Growth Factor (25 ng/ml)||BD||354001|
|Bovine Pituitary Extract (30 μg/ml)||Invitrogen||13028-014|
|Fetal Bovine Serum (5%)||Fisher||SH3008803HI|
|Retinoic acid (0.05 μM)||Sigma||R2625|
|Growth Factor Reduced Matrigel||BD||354230|
Table 1. Complete media components.
|Pronase||Roche||10165921001||Used at 0.15%:
-o/n at 4 °C digestion to isolate total tracheal cells (for ALI culture)
-4 hr digestion 4 °C to isolate SMG
|Dispase||BD Biosciences||354235||Used at 16 Units: 30 min at RT|
|DNase I||Sigma||DN25||Used at 0.5 mg/ml:
20-30 min at RT
Table 2. Enzymes used for enzymatic digestion of the trachea.