Alveolar Regeneration: Role of p63+ Progenitor Cells Derived from Airway Secretory Cells

Background Introduction

Effective gas exchange in the lungs relies on the precise structure and function of various epithelial cells, distributed across two distinct structural regions: ductal airways and alveoli. When alveoli are damaged (e.g., due to inhaled toxins, viral infections, etc.), activation of epithelial stem or progenitor cells is required to restitute organ function. After alveolar damage, surfactant-secreting alveolar type II cells (AT2) get activated and differentiate into flattened alveolar type I cells (AT1), thus accomplishing alveolar repair. However, the source, fate, and differentiation mechanisms of various types of progenitor cells induced by lung injury remain unclear.

In recent years, research has discovered a rare type of basal-like cell expressing p63 that migrates to the injury site and participates in the repair process following severe lung injury. Particularly in alveolar damage caused by influenza virus infection, these p63+ cells proliferate and form tubular structures. However, whether these cells truly partake in alveolar epithelial regeneration and their relationship with other progenitor cell populations remain to be further studied.

Research Overview

A research team led by Zan Lv published the latest findings in “Cell Stem Cell,” which delved deeply into the generation of p63+ progenitor cells after lung injury and their role in alveolar regeneration. This research employed genetic lineage tracing and single-cell RNA sequencing to reveal that, after alveolar damage in mice, p63+ progenitor cells proliferate rapidly and differentiate into both alveolar type I and II cells. The study further found that these p63+ cells originate from airway secretory cells and require p63 activation for the alveolar repair process.

Research Process

The research team thoroughly explored the origin and function of p63+ progenitor cells through the following experimental steps:

1. Construction of Mouse Model and Lineage Tracing: Researchers constructed p63-CreER knock-in mice, induced Cre expression by tamoxifen injection, and subsequently injected bleomycin to induce alveolar damage. This design precisely marks and tracks changes in the lineage of p63+ cells.

2. Single-Cell RNA Sequencing Analysis: By employing single-cell RNA sequencing, the team analyzed p63+ cells at 14 days, 2 months, and 5 months after alveolar damage, revealing their diversity and differentiation trajectories. The results showed multiple cell subpopulations within p63+ progenitor cells, with some subpopulations specifically expressing genes related to epithelial cell proliferation and wound healing, indicating different differentiation stages of these cells.

3. Gene Tracking and Dual Recombinase System: The team used a dual recombinase tracking system to verify the origin of p63+ cells and found that these cells come from airway secretory cells. Experimental data also indicated that p63 activation after injury is critical for the proliferation and differentiation of p63+ progenitor cells.

4. Analysis of Cell Fate Trajectories: Through RNA velocity analysis, the researchers discovered that p63+ cells generate alveolar type I and II cells through two distinct differentiation pathways. Pathway 1 involves differentiation from p63-3 cells to AT1 cells, while Pathway 2 progresses from p63-3 cells through pre-AT2 cells to AT2 cells.

5. Function Verification Experiment: Further gene knockout experiments showed that removing the p63 gene significantly weakens the regenerative capacity of p63+ cells, leading to more severe fibrosis. This suggests that p63 activation is necessary for the differentiation of secretory cells into alveolar epithelial cells.

Main Research Findings

• Origin of p63+ Cells: The study confirms that after bleomycin-induced alveolar damage, p63+ progenitor cells are generated from airway secretory cells.
• Function of p63 Activation: p63 activation is crucial for the conversion of airway secretory cells to alveolar epithelial cells, and secretory cells lacking p63 show significantly reduced efficiency in lung regeneration.
• Dual Differentiation Pathways: p63+ progenitor cells can generate alveolar type I and II cells through two different differentiation pathways, providing diverse cellular sources for repair following alveolar damage.

Research Conclusion

The study provides new insights into the regeneration mechanism of the alveolar epithelium after lung injury. As a key repair cell type, p63+ progenitor cells can proliferate and convert to alveolar cells post-injury, with the process being strictly regulated by p63 activation. This discovery not only elucidates the molecular mechanisms of lung regeneration but also offers potential cellular and molecular targets for regeneration therapy following lung injury.

Research Significance

This study, through comprehensive genetic tracing and single-cell sequencing technologies, thoroughly describes the role of p63+ progenitor cells in alveolar regeneration and their dual differentiation pathways. This finding expands the understanding of the role of airway secretory cells in lung injury repair and highlights the potential therapeutic value of p63+ progenitor cells in pulmonary fibrosis diseases. Notably, the study suggests the possibility of enhancing lung repair ability post-injury by regulating p63 expression, providing an important theoretical basis for future lung regenerative medicine research.

Research Highlights

• Discovery of Novel Progenitor Cells: This study is the first to confirm that airway secretory cells can convert into p63+ progenitor cells after lung injury and participate in the regeneration of the alveolar epithelium.
• Application of Dual Lineage Tracing Technology: The innovative use of a dual recombinase system accurately traced the origin and fate of p63+ progenitor cells.
• Key Role of p63: The study reveals the necessity of p63 activation in the alveolar regeneration process, whose absence leads to impaired repair and exacerbated fibrosis.
• Clinical Potential: Provides new therapeutic targets for pulmonary fibrosis and other lung injury diseases, especially in enhancing the regenerative capacity of airway secretory cells.

Research Limitations

Although the study reveals the importance of p63+ progenitor cells, certain limitations exist. For instance, the high-dose tamoxifen treatment in the dual recombinase system may induce unexpected Cre-Rox cross-reactions, affecting experimental results. Moreover, the mechanisms and regulatory pathways of p63+ progenitor cell generation still require further study to better understand their role in lung injury repair.

Summary

Through systematic experimental design and advanced single-cell analysis, this study clarifies the origin, differentiation pathways, and critical role of p63+ progenitor cells in alveolar regeneration after lung injury. These research outcomes not only enrich the understanding of lung injury repair mechanisms but also provide potential scientific foundations for future lung regeneration therapies.