Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Abstract

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, selforganizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium. associated with impaired production and clearance of mucus which is often found in pulmonary disorders such as chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchiectasis, and primary ciliary dyskinesia 1 , 2 , 3 , 4. A recent advance in organoid technology, for instance, the basal cell derived lung organoid called tracheosphere that recapitulates the regeneration of mucociliary epithelium arise as a promising model with therapeutic potential 1 , 5 , 6. However, its use is currently limited, in part because of lack of the defined culture conditions and low efficiency in organoid productions. Mucociliary epithelium in the human airway and frog epidermis are remarkably similar in tissue morphology, cellular composition, and its function 7 , 8 , 9 , 10 , 11 , 12. In both organisms, mucociliary epithelium provides first-line defense by secreting mucus and antimicrobial substances and clears Copyright © 2020 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License jove.com July 2020 • 161 • e61604 • Page 2 of 11 harmful particles and pathogens through the synchronized action of cilia. Here, we describe a simple protocol to generate mucociliary epithelial organoids using the multipotent progenitors of Xenopus laevis embryos 13 , 14. Previously, we reported 14 that in the absence of exogenous growth factors and the extracellular matrix, the microsurgically isolated deep cells from the early gastrula stage ectoderm spontaneously assemble into aggregates, regenerate epithelium on its surface, and mature into mucociliary epithelium by intercalating multiciliated and other accessory cells within 24 h. In addition to the rapid development, this protocol offers a distinct opportunity for directly accessing the transitions of multipotent deep ectoderm cells into epithelial gob...

Revealing non-trivial information structures in aneural biological tissues via functional connectivity

“…Two mRNA's were co-injected in the reported work; GCaMP6s, a reporter of calcium activity [73,74], and the intracellular domain of Notch (Notch ICD), which is known to inhibit multiciliated cell induction in developing frog epidermis [50,75,76]. Multiciliated cells were molecularly inhibited in the current study as the presence of these motile structures causes the mucociliary organoid to move during observation, complicating image analysis [77][78][79].…”

Biological Robots: Perspectives on an Emerging Interdisciplinary Field

“…Additionally, this term should only be applied to the system where motility is rationally designed or used to complete human specified work, and an unmodified animal cap, or the differentiated mucociliary organoid, 24 , 26–29 should be labeled as such where applicable. Finally, while reconfigurable organism is the specific nomenclature used in the articles, this term is also problematic as biologists currently have no operational definition of the word organism, 30 , 31 with some arguing the term is without technical meaning (the current author does not consider the designs organisms by any definition).…”