From Clones to Buds and Branches: The Use of Lung Organoids to Model Branching Morphogenesis Ex Vivo

Abstract

Three-dimensional (3D) organoid culture systems have rapidly emerged as powerful tools to study organ development and disease. The lung is a complex and highly specialized organ that comprises more than 40 cell types that offer several region-specific roles. During organogenesis, the lung goes through sequential and morphologically distinctive stages to assume its mature form, both structurally and functionally. As branching takes place, multipotent epithelial progenitors at the distal tips of the growing/bifurcating epithelial tubes progressively become lineage-restricted, giving rise to more differentiated and specialized cell types. Although many cellular and molecular mechanisms leading to branching morphogenesis have been explored, deeper understanding of biological processes governing cell-fate decisions and lung patterning is still needed. Given that these distinct processes cannot be easily analyzed in vivo, 3D culture systems have become a valuable platform to study organogenesis in vitro. This minireview focuses on the current lung organoid systems that recapitulate developmental events occurring before and during branching morphogenesis. In addition, we also discuss their limitations and future directions.

Keywords: branching morphogenesis; cell-fate decisions; lung development; lung organoids; stem cells.

Figure 1

In vitro branching lung organoid models. Schematic representation of the current lung organoids models showing proximodistal specification and bud/branching formation in vitro derived from hPSCs and murine adult stem cells. (A) Lung bud organoids (LBO) are derived from hPSCs and have several cells expressing AECII markers SFTPC, SFTPB, and ABCA3 while airway goblet cell markers MUC5AC and MUC5AB are rare. While most cells co-expressed SOX9⁺ the pheripheral luminal cells expressed only mature AECII markers (MUC1, SPTPC, SFTPB, ABCA3) containing lamellar bodies (B) Human lung organoids (HLO) are derived from hPSCs and display proximal airways, expressing proximal cell type-specific markers for basal cells (P63), ciliated cells (FOXJ1) and club cells (SCGB1A1) co-expressing SOX2; distal-like structures expressing SFTPC/SFTPB AECII and PDPN/HOPX AECI cell-type markers mostly co-stained with SOX9. Lamellar bodies are found inside and outside the SFTPC⁺ AECII-like cells while SMA⁺ PDGFRα⁺ (myofibroblasts) and SMA⁺ PDGFRα⁻ [smooth muscle cells (SMCs)] are observed in close proximity to the epithelial tube-like structures. (C) Patterned lung organoid (PLO) are derived from hPSCs and have peripheral regions that contain cells co-expressing SOX9/SOX2, some also SFTPC and ID2 while internal regions only express SOX2 with some cells expressing the club cells marker SCGB1A1 and goblet cells marker MUC5AC. (D) Bronchioalveolar lung organoids (BALO) are derived from BASC isolated from the lung of adult mice and possess proximal tubular structures containing club/secretory cells (SCGB1A1), basal cells (P63), ciliated cells (β-4 tubulin) and distal alveolar-like structures are composed of AECII (SFTPC) and AECI (PDPN). Lamellar bodies are observed in AECII and in the lumen. Myofibroblasts (PDGFRα^highαSMA⁺) are observed close to BALO's branching points while lipofibroblasts (PDGFRα^lowLipidTOX⁺) are observed in close proximity to AECII.