Abstract
Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.
Highlights
In the respiratory system, the developing embryonic lung epithelium shows dramatic alterations associated with branching morphogenesis, cellular differentiation, and functional maturation
Recent studies by Miller et al [16, 25] showed a similar explant culture experiment using isolated epithelium of embryonic mouse lung treated with fibroblast growth factor 7 (FGF7), FGF10, BMP4, retinoic acid, and a GSKβ3 inhibitor to identify conditions that maintain epithelial tip progenitors in vitro and further apply lung bud tip progenitor cells derived from human pluripotent stem cells
We have described an easy, reliable, and economical method to make alveolus-like organoids from isolated epithelium of embryonic mouse lung tip in serumfree condition using Matrigel, ITS supplement, and FGF7
Summary
The developing embryonic lung epithelium shows dramatic alterations associated with branching morphogenesis, cellular differentiation, and functional maturation. Originated cells of developing lung epithelium, including thyroid transcription factor-1 (TTF1), β-catenin, Forkhead orthologs (FOX), GATA, Sox, and ETS family members, are necessary to establish normal lung morphogenesis and cell differentiation [6]. Embryonic isolated epithelial explants can survive and differentiate with an extracellular matrix such as Matrigel substratum, and growth factors can modify branching morphogenesis [17,18,19,20]. Recent studies by Miller et al [16, 25] showed a similar explant culture experiment using isolated epithelium of embryonic mouse lung treated with FGF7, FGF10, BMP4, retinoic acid, and a GSKβ3 inhibitor to identify conditions that maintain epithelial tip progenitors in vitro and further apply lung bud tip progenitor cells derived from human pluripotent stem cells. Easy, and reproducible method to make alveolus-like organoids, and here introduced this method for future studies in the field of lung biology and medicine
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
