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. 2020 Sep 28;39(21):e103476. doi: 10.15252/embj.2019103476

Figure 2. The BALO model mimics the 3D morphology and cellular composition of the bronchioalveolar compartment with proximo‐distal patterning and pulmonary surfactant secretion.

Figure 2

  • A
    mRNA expression analysis of epithelial cell differentiation markers Sftpc (AEC II), Hopx (AEC I), Foxj1 (ciliated cells), Muc5ac (secretory cells), and p63 (basal cells) in BALO at days 0, 10, and 21 of culture (n = 3 biological replicates with pooled cells from 4 cultures per replicate).
  • B
    Electron microscopy of BALO alveoli showing two cuboidal epithelial cells (AEC II) connected by tight junctions (red square) with numerous mitochondria (M) and lamellar bodies (LB). A LIF with numerous lipid droplets (*) and a MYO with cisterns of rough ER (red arrow) are located at the basal side (left). The alveolar lumen is filled with lamellar surfactant including tubular myelin (white arrow) (lower right). Exocytosis of a lamellar body (*) from an AEC II (upper right). Scale bars indicate 500 nm.
  • C
    Electron microscopy of bronchiolar‐like airway depicting columnar ciliated cells (red arrowhead) with basal bodies (black arrowhead) at the left side of the longitudinally sectioned lumen. The boxed area indicates an apical junctional complex between two ciliated cells: 1 = tight junction and 2 = adherens junction. Scale bar indicates 500 nm (in insert: 100 nm).
  • D
    Staining of lamellar bodies in BALO with RFP LysoTracker. Scale bars represent 100 μm.
  • E
    Western blot analysis of the surfactant proteins: pro‐SPC, mature SPC, pro‐SPB, mature SPB, and SPA in lung homogenate (LH), AEC, and day 21 BALO (n = 3 biological replicates).
  • F, G
    Alveolar diameter (F) and number of alveoli (G) in tdTomato+ BALO at days 15, 21, 30, and 40 of culture were measured from n = 5 BALOs in n = 3 biological replicates BALOs.
  • H, I
    Representative scheme and images of day 40 BALO alveoli (H) and airway (I). BALO alveolar‐like structures (H) are shown (h′) (red arrowheads) in semi‐thin section (0.5 μm) stained with Toluidine blue. Scale bar indicates 100 μm (left). Electron microscopy showing AEC I (h″) ultrastructure within BALO. Scale bars indicate 2,500 nm (center) and 1,000 nm (right). An airway‐like structure (i′) is shown with secretory and ciliated cells (red arrowheads) in semi‐thin sections (0.5 μm) longitudinally cut and stained with Toluidine blue. Scale bar indicates 50 μm (far left). Electron microscopy of a bronchiolar‐like airway (I) depicting pseudostratified epithelium (i″) with a basal‐like cell (BC), not reaching the lumen in which cilia (C) are seen, located between a secretory (SC) and a ciliated cell (Ci). Scale bars indicate 1,000 nm (left and right). Mature cilia (i‴) in BALO at higher magnification depicting the 9 × 2 + 2 structure with central doubled microtubules (insert, red arrowhead), a characteristic for motile cilia. Scale bar indicates 100 nm (far right).
Data information: Bar and dot charts presented as the mean ± SEM and probability determined using one‐way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).