A Novel In Vitro Model to Study Alveologenesis

Abstract

Many pediatric pulmonary diseases are associated with significant morbidity and mortality due to impairment of alveolar development. The lack of an appropriate in vitro model system limits the identification of therapies aimed at improving alveolarization. Herein, we characterize an ex vivo lung culture model that facilitates investigation of signaling pathways that influence alveolar septation. Postnatal Day 4 (P4) mouse pup lungs were inflated with 0.4% agarose, sliced, and cultured within a collagen matrix in medium that was optimized to support cell proliferation and promote septation. Lung slices were grown with and without 1D11, an active transforming growth factor-b-neutralizing antibody. After 4 days, the lung sections (designated P4 1 4) and noncultured lung sections were examined using quantitative morphometry to assess alveolar septation and immunohistochemistry to evaluate cell proliferation and differentiation. We observed that the P4 1 4 lung sections exhibited ex vivo alveolarization, as evidenced by an increase in septal density, thinning of septal walls, and a decrease in mean linear intercept comparable to P8, age-matched, uncultured lungs. Moreover, immunostaining showed ongoing cell proliferation and differentiation in cultured lungs that were similar to P8 controls. Cultured lungs exposed to 1D11 had a distinct phenotype of decreased septal density when compared with untreated P4 1 4 lungs, indicating the utility of investigating signaling in these lung slices. These results indicate that this novel lung culture system is optimized to permit the investigation of pathways involved in septation, and potentially the identification of therapeutic targets that enhance alveolarization.

Secondary crest myofibroblast PDGFRα controls the elastogenesis pathway via a secondary tier of signaling networks during alveologenesis

“…Application of a supraphysiological dose of TGFβ, or TGFβ induced via hyperoxia, arrests alveolar formation (Vicencio et al, 2004). In contrast, one study has shown that antibodyblockade of TGFβ disrupts alveologenesis in lung explants (Pieretti et al, 2014). We found TGFβ signaling in SCMFs to be important in regulating elastic fiber formation.…”

Can We Understand the Pathobiology of Bronchopulmonary Dysplasia?

“…Similarly, the use of decellularized lungs permits the unique opportunity to define the ECM-derived signals that direct lung cell fate during development, and how these signals are disrupted during injury 197 . Along these lines, the initial flurry of interest in the application of precision-cut lung sections to study lung alveolarization 198 has not yet found widespread application. Both organoids and lung sections present a wonderful opportunity for advanced molecular analyses to understand cell-cell and cell-matrix interactions and developmental pathways pertinent to BPD pathobiology.…”

Lysophosphatidic Acid Signaling through the Lysophosphatidic Acid-1 Receptor Is Required for Alveolarization

“…For the quantitative morphometry, multiple plastic-embedded 1-mm sections of the entire mouse lung were stained with toluidine blue according to established protocols (20). For assessment of elastin morphology, 5-mm lung sections were stained with Hart's resorcin-fuchsin solution, without and with prior oxidation with potassium permanganate and clearing with oxalic acid, and counterstained with tartrazine as described previously (21). Bright-field microscopy was used to generate the lung images; the elastin images were constructed from a z-stack of lung images using extended depth-of-field focusing (22).…”