FIG. 2.

HLOs grown in alginate express airway epithelial makers. (A) Epithelial structures were observed as early as 43 days in culture and were still observed at day 67. Scale bar represents 200 μm. (B) HLOs cultured in Matrigel have epithelial structures labeled by β-Catenin (red) that are lined with P63⁺ basal cells (green) along the basal lamina with few multiciliated cells labeled by acetylated tubulin (ACTTUB, white). At day 65 (middle panel), the HLOs only have ACTTUB staining toward the apical side of the epithelial structures but no visible cilia. At day 80, HLOs have few multiciliated cells where the cilia are labeled with ACTTUB (white). (C) HLOs cultured in 2% alginate for 49 days and 75 days have ACTTUB⁺ (white) multiciliated cells coating the epithelial structures. The P63⁺ (green) cells labeling the basal cells were tucked in toward the HLO. Epithelial progenitor marker and proximal mesenchymal marker SOX9 (red) was scattered in the epithelial structures and the body of the HLOs. Scale bar is the same for all panels in the subfigure. Color images are available online.