Lung damage causes proliferation of P63-expressing cells in bronchioles, migration to damaged tissue, and regeneration of alveoli.

Scientists have identified a subset of stem cells in rodents and in humans that appears to be responsible for regenerating alveoli in response to the extensive lung damage caused by diseases such as deadly forms of flu. A team led by researchers at A-STAR’s Genome Institute of Singapore and Harvard Medical School claim that lung infection triggers massive proliferation of bronchiolar p63-expressing stem cells and their migration to damaged regions of the lungs, where they assemble into foci, or ‘pods,’ that eventually lead to the formation of new alveoli.

Reporting in Cell, Frank McKeon, Ph.D., and colleagues, suggest their findings have thrown up a number of questions about how the new alveoli are formed and connect with airways and capillary network but could ultimately lead to new regenerative therapies for serious lung diseases.

Research has provided valuable insights into the pathology of Acute respiratory distress syndrome (ARDS), but much less is known about lung regenerative processes or even how much of the improvement seen in recovering ARDS patients is due to lung remodeling rather than regeneration.

Bronchioalveolar stem cells (BASCs), which express both Clara cell markers (CC10) and alveolar type II (AT2) cell markers, have been found at terminal bronchioles and are proposed as stem cells for both bronchiolar and alveolar epithelia. However, the role of BASCs in alveolar regeneration is uncertain, the team continues, and lineage tracing of Scgb1a1+ (CC10) Clara cells has demonstrated their role as progenitors in the repair of terminal bronchiolar epithelium but not of the alveolar epithelium.

To further investigate the cellular basis of alveolar regeneration in response to ARDS, the authors looked specifically at mice infected with a murine-adapted H1N1 (PR8) influenza. In these animals tissue damage appeared to peak at 11 days post infection (dpi) but was reduced at 21 dpi and had for the most part cleared across the lung by 60 dpi. At the cellular level there was widespread destruction of all airway epithelial cells at 7 dpi, which resulted in a significant loss of markers for Clara cells (CC10) and ciliated cells (acetylated a-tubulin, TAp73) in the bronchiolar epithelia as well as AT2 cells (SPC+) of the alveolar epithelium.

The most remarkable thing, the researchers state, is that despite the widespread cytopathic effects and extreme weight loss demonstrated by these animals, they recovered without evidence of lung fibrosis, a pathology which occurs in mice with lung damage due to bleomycin administration.

Their hunt for a cell population responsible for lung regeneration in response to ARDS followed on from previous published research suggesting that basal cells expressing the stratified epithelial stem cell transcription factor p63, represent a pool of stem cells in the nasal and tracheal epithelia of upper airways. Dr. McKeon et al.’s latest research found that while there was little sign of p63-expressing basal cells in bronchioles of normal mice, these cells became evident in the bronchioles and bronchiolar epithelium following infection and were also present in large numbers in damaged lung parenchyma.

Further analysis showed that the highly proliferating p53-expressing cells appeared to be clustered in small groups that formed clusters, or pods. These keratin 5 (Krt5)-expressing pods moved from the intrabronchiolar regions from about 11 dpi and began to increase in number in the interbronchiolar regions. “The appearance of Krt5+ pods in the peribronchiolar regions of lung parenchyma coincides with the pinnacle of influenza-induced lung damage,” the authors state. Interestingly, such Krt5+ pods were not found in the bleomycin-dependent lung fibrosis model.

The investigators moved on to try and identify distal airway stem cells and evaluate their relationship to stem cells found in the upper airways. To this end they used single cell cloning methods to propagate human epithelial cells derived from nasal turbinate, tracheobronchial epithelia, and distal airway tissue including bronchioles and alveoli. The resulting clones were designated according to their site of origin; nasal epithelial stem cells (NESCs), tracheal airway stem cells (TASCs) and distal airway stem cells (DASCs).

Interestingly, the immature clones derived from the different tissues all stained uniformly for p63 and for Krt5, were indistinguishable by morphology and staining with basal cell markers and were 99% equivalent in terms of gene expression patterns. The majority of colonies could also be propagated while maintaining an immature phenotype.

However, when NESC-, TASC-, and DASC-derived clones were differentiated using the air-liquid interface (ALI) model, dissimilarities began to emerge. The NESC and TASC lines showed robust differentiation into ciliated cells and mucin-producing goblet cells during the 21-day period of ALI culture. In contrast, DASC cells showed very little indication of mucin expression, rare ciliated cell formation, and only occasional CC10 expression indicative of Clara cells.

And while TASCs generated a multilayered epithelium with p63-positive basal cells underneath differentiated goblet and ciliated cells, the DASC pedigrees retained a monolayer appearance over the differentiation period. “Gene expression analysis of the ALI cultures of TASC and DASC lineages confirmed these observations, with the differentiated TASC cultures showing high expression of genes involved in ciliogenesis, mucin production, and epithelial stratification compared to DASCs grown in ALI culture,” the authors write.

When grown in 3-D matrigel cultures, DASC pedigree lines assembled into spheres, which became hollow and by day 21 collapsed into multispherical structures. Interestingly, the entities formed from DASC lines didn’t stratify but rather seemed to be comprised of unilaminar cellular assemblies that expressed the alveolar type 1 marker PDPN and could be labeled with an antibody that recognizes a 300 kDa protein specific to human alveoli.

Gene Set Enrichment Analysis (GSEA) confirmed that while the squamous metaplasia generated by TASCs in Matrigel structures expressed genes associated with epidermal development, the DASC-derived structures showed high expression of genes associated with angiogenesis regulation, monocation transport, and synaptic transmission, which possibly reflects the  importance of neurogenic control over alveolar function, the team notes.

The investigators were similarly able to generate equivalent p63-expressing stem cells from the distal airways of experimental rats. As with the human cells, the rat DASC lines formed uniform solid spheres after growth in 3-D Matrigel, and these subsequently hollowed by day 21. Structures formed by the differentiated rat DASCs could be stained by two mAb that are specific to rat alveoli.

“Further, the DASCs we describe here are fully committed to alveolar lineages with additional potential to form Clara, ciliated, and mucin-producing cells and therefore distinct from the TASCs and NESCs that are committed to ciliated cells, goblet cells, and as well can undergo squamous metaplasia,” the researchers note.

To look more closely at basal-like cells taken from infected lungs, the researchers plated dissociated distal airway tissues from flu-infected mice, in clonogenic assays. Resulting colonies were composed of small, p63-expressing immature cells and expressed Krt5. Again, when the colonies were grown in Matrigel cultures, cells formed solid spheres that hollowed out to yield unilaminar structures akin to those generated by human DASCs, and which stained with antibodies to the alveolar marker aquaporin 5 (Aqp5). Notably, as the cells differentiated in Matrigel and the spheres formed, they upregulated markers of alveoli, including Aqp5 and the surfactant proteins Sftpa1, Sftpb, and Sftpc.

Whole genome microarray analysis confirmed that cells cloned from distal lung tissues of infected and normal tissues displayed differences in over 350 genes including keratin 6A (Krt6a), a known marker of migrating keratinocytes during wound healing in the epidermis. GSEA of the expression data sets derived from the distal airway colonies confirmed that cells from the infected tissue demonstrated a bias for genes involved in wound healing, tissue development, and regulation of growth.

“The ability of human distal airway stem cells to form alveoli-like structures in vitro made it tempting to predict that the Krt5 pods of basal-like cells seen in the influenza-damaged lung were components of a regenerative process,” the authors admit.

Indeed, when they looked at the whole of an influenza-infected lung, there were three distinct areas: normal appearing lung parenchyma with histologically normal alveoli; damaged regions containing semi-dense infiltrate and Krt4 pods, and damaged regions with dense infiltrates but without Krt5 pods. Significantly, the Krt5 pods were never seen in regions of histologically normal lung tissue.

They then used laser capture microdissection to isolate RNA for expression analysiss from frozen sections from 25 dpi lung regions that were rich in Krt5 pods, normal or repaired lung, and two regions of high-density infiltrates without obvious repair, those with SPC (a marker of alveolar type II cells), and those without SPC. Overall gene expression analysis indicated that the regions rich in Krt5 pods were the most closely related to the regions of normal or repaired lung tissue, and indicated that the Krt5 pods were involved in alveolar regeneration.

“On the broadest level, these data support the notion that regions with Krt5 pods express genes similar to apparently normal or repaired lung and very different from regions marked by severe damage,” Dr. McKeon et al, state. “They also suggest a dynamic process involving a host of pathways whose significance for the recovery from ARDS will require extensive empirical validation.”

Finally, the researchers used lineage tracing to confirm that parenchymal Krt5 pods developing as a result of lung infection do, indeed, originate from the pool of Krt5 cells in the bronchioles. Using this approach they demonstrated that Krt5 cells proliferate in the bronchioles as a result of infection until about day 11, when there appears to be a concerted migration of the cells to the interstitial lung, and the subsequent appearance of Krt5 pods.

The role of p63-expressing basal cells as stem cells for distal lung has in the past been largely discounted because such cells are rare in the small bronchioles compared with the upper airways, the authors remark. As a result they admit they were surprised at how easy it was to generate immature clones of p63-expressing cells from populations of human distal airway epithelial cells.

Questions do remain, however, they admit. “What, for instance, are the signals that trigger the proliferation and radiation of p63-expressing cells from bronchioles to sites of influenza-damaged lung? How do incipient alveolar structures assembled in damaged lung link up to the conducting airway? What are the mechanisms that merge microvasculature assemblies with the nascent alveoli? Why is SPC expression, a marker of AT2 cells, absent from the alveolar-like structures formed by the Krt5 pods?”

The answers to questions such as these will remain an immense challenge, they conclude, “but one that is likely to generate new therapies for an array of presently incurable airway diseases.”

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