Journal: Frontiers in Cell and Developmental Biology

Article Title: 3D Cell Culture Models Demonstrate a Role for FGF and WNT Signaling in Regulation of Lung Epithelial Cell Fate and Morphogenesis

doi: 10.3389/fcell.2020.00574

Figure Lengend Snippet: Lung epithelium contains LSPCs that form lungospheres with the capacity for self-renewal and differentiation. (A,B) Lungospheres formed from unsorted lung epithelial cells in non-adherent conditions with EGF and FGF2. (A) Representative photographs of primary and secondary lungospheres. Scale bar, 100 μm. (B) The efficiency of primary and secondary lungosphere formation with EGF and FGF2. The plots show mean + SD; n = 5. (C,D) Lungospheres formed from FACS-sorted EpCAM + , CD49f + , CD24 low , CD104 + cells in non-adherent conditions with EGF and FGF2 (C) Representative photographs of primary and secondary lungospheres. Scale bar, 100 μm. (D) The efficiency of primary and secondary lungosphere formation with EGF and FGF2. The plots show mean + SD; n = 3 ( n = 1 for tertiary lungospheres). The columns left from the dashed vertical line show LFE from cells cultured at thousands of cells per well; the column right from the line shows LFE from cells cultured individually – a single cell per well. (E,F) The efficiency of primary and secondary lungosphere formation of FACS-sorted EpCAM + , CD49f + , CD24 hi , CD104 + (E) and EpCAM + , CD49f + , CD24 neg , CD104 neg (F) cells in non-adherent conditions with EGF and FGF2. The plots show mean + SD; n = 1. (G,H) Primary lungospheres grown in non-adherent conditions form lung-like structures. (G) The photographs of hematoxylin/eosin-stained paraffin sections show alveolar-like structures (arrowheads) and airways-like structures (arrows). (H) Immunofluorescence staining of paraffin sections for E-cadherin, keratin 5 (K5), keratin 8 (K8), keratin 14 (K14), Pro-SPB, Pro-SPC, aquaporin 5 (AQP5), CC10, MUC5AC, lysozyme (Lys), and nuclei (DAPI). (I–K) Lungospheres embedded in 3D Matrigel and cultured with EGF and FGF2 proliferate and form large organoids with lung-like structure. (I) The photographs show morphogenesis of a lungosphere-derived organoid over 32 days. (J) Hematoxylin/eosin-stained section of lungosphere-derived organoid. (K) Immunofluorescence staining of paraffin sections of lungosphere-derived organoids for keratin 5 (K5), cytokeratin 8 (K8), Pro-SPC, CC10, AQP5, and nuclei (DAPI). (G–K) Scale bar, 100 μm.

Article Snippet: Briefly, for primary lungosphere assay, unsorted lung epithelial cells were seeded in polyHEMA-treated six-well plates at 2.5 × 10 4 to 5 × 10 4 cells in 2 ml/well of a lungosphere medium [1 × B-27 without vitamin A, 100 U/ml penicillin, 100 μg/ml streptomycin (all Thermo Fisher Scientific), 4 μg/ml heparin (Sigma/Merck), 20 ng/ml murine EGF (#315-09, Peprotech), 10 ng/ml FGF2-STAB (thermostable FGF2 based on human FGF2 sequence; Enantis), or 10 ng/ml human FGF2-wt (#100-18C, Peprotech), 10 μM Y-27632 (Merck) in phenol red-free DMEM/F12 (Thermo Fisher Scientific)]; or 500–1000 FACS-sorted cells were seeded in polyHEMA-treated 24-well plates in 1 ml/well of a lungosphere medium.

Techniques: Cell Culture, Staining, Immunofluorescence, Derivative Assay