Lung Injury/Repair Studies
Objective: To examine AEC2 (type 2 alveolar epithelial cell) ablation and clonal expansion, and to investigate repair mechanisms through high-resolution imaging of intact lungs and in vitro culture studies
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Equipment2
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Protocol Steps
Genetic lineage-tracing experiments
Perform genetic lineage-tracing experiments to track SFTPC-positive AEC2s and monitor their self-renewal and differentiation patterns
Note: This demonstrates that the AEC2 population contains long-term alveolar stem cells
View evidence from paper
“Genetic lineage-tracing experiments showed that surfactant protein C-positive (SFTPC-positive) AEC2s self renew and differentiate over about a year”
AEC2 ablation
Specifically ablate many AEC2s using the injury/repair system
Note: Multiple injury/repair systems were used in combination with lineage analysis
View evidence from paper
“if many AEC2s were specifically ablated, high-resolution imaging of intact lungs showed that individual survivors undergo rapid clonal expansion”
High-resolution imaging of intact lungs
Perform high-resolution imaging of intact lungs to visualize individual AEC2 survivors and their clonal expansion and daughter cell dispersal
Note: This imaging reveals rapid clonal expansion and daughter cell dispersal patterns
View evidence from paper
“high-resolution imaging of intact lungs showed that individual survivors undergo rapid clonal expansion and daughter cell dispersal”
Isolation and purification of AEC2 populations
Isolate and purify lineage-labeled AEC2 cell populations for in vitro culture
Note: Cells are prepared for 3D culture experiments
View evidence from paper
“in vitro culture of purified cell populations to obtain new information about the contribution of AEC2s to alveolar maintenance and repair”
3D culture of AEC2s
Place individual lineage-labeled AEC2s into 3D culture to generate alveolospheres
Note: Alveolospheres are self-renewing structures containing both AEC2s and AEC1-like cells
View evidence from paper
“Individual lineage-labeled AEC2s placed into 3D culture gave rise to self-renewing alveolospheres”
Coculture with primary lung stromal cells
Coculture alveolospheres with primary PDGFR-alpha positive lung stromal cells to promote growth and differentiation
Note: This coculture condition is optimal for alveolosphere development and includes lipofibroblasts that may form a stem cell niche
View evidence from paper
“Growth and differentiation of the alveolospheres occurred most readily when cocultured with primary PDGFR-alpha + lung stromal cells”
Characterization of alveolosphere cell types
Analyze alveolospheres to identify both AEC2s and cells expressing multiple AEC1 markers, including HOPX
Note: HOPX is identified as a new marker for AEC1s in this study
View evidence from paper
“alveolospheres, which contained both AEC2s and cells expressing multiple AEC1 markers, including HOPX, a new marker for AEC1s”