Long‐term culture ofXenopuspresumptive ectoderm in a nutrient‐supplemented culture medium

Abstract

Animal cap assay is a useful experimental model for investigating the activity of inducers in amphibian development. This assay has revealed that activin A is a potent mesoderm-inducing factor. However, it has been very difficult to induce highly differentiated tissues such as cartilage in a 3-4 day culture period. It was recently reported that jaw cartilage was induced in vitro in an animal cap that had been cultured for 14 days in Steinberg's solution using the sandwich culture method and activin A. Under these conditions, necrosis was occasionally observed in the explants. In this study, we have achieved long-term animal cap cultures in a nutrient-supplemented culture medium designated RDX. This medium was made by modifying the saline concentration of the RD medium previously developed as a basal medium for the serum-free culture of various kinds of mammalian cells. The explants cultured in RDX grew more vigorously compared with those in Steinberg's solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinberg's solution, and also increased the frequency of cartilage induction. Therefore, the supplemental nutrients may support and promote the differentiation of cartilage. This long-term culture method using RDX medium is useful for studying the differentiation of tissues or organs such as cartilage in vitro.

Cell Differentiation of Pluripotent Tissue Sheets Immobilized on Supported Membranes Displaying Cadherin-11

“…Here, the inner blastocoelic surface of the animal caps was facing to the supported membrane. After seeding, the animal caps were cultured in RDX medium [44] supplemented with 1.5 mM Ca 2+ at 14°C. For more information on animal caps, see Supporting Information S2.…”

Programming Pluripotent Precursor Cells Derived from Xenopus Embryos to Generate Specific Tissues and Organs

“…They can be cultivated in simple saline solution for up to one or two weeks [11], corresponding to tadpole stages of development, when the primary organs have been formed. Manipulation of the corresponding explants can be performed in various ways:

ACC can be exposed to extracellular signaling molecules like growth factors or chemical agents affecting signal transduction.

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Bidirectional multiciliated cell extrusion is controlled by Notch driven basal extrusion and Piezo 1 driven apical extrusion

“…Importantly, in the absence of mesoderm, MCCs are maintained in these caps for a considerable time (Fukui et al, 2003). Strikingly, we find that at 15 dpf (ST50 of embryos) there are still a substantial number of MCCs (Figure 4A).…”

“…This cap of cells balls up into a rough sphere that develops into a functional mucociliary organoid (Sokol and Melton, 1991; Walentek and Quigley, 2017; Weber et al, 1996). Importantly, in the absence of mesoderm, MCCs are maintained in these caps for a considerable time (Fukui et al, 2003). Strikingly, we find that at 15 dpf (ST50 of embryos) there are still a substantial number of MCCs (Figure 4A).…”

“…After healing, caps were treated with 0.5ng/mL of Activin A (A4941-10, Sigma-Aldrich) in 0.5x MMR + gentamicin for one hour to promote development to epidermis (Ariizumi et al, 1991). Caps were transferred to Leibovitz L-15 cell culture medium+ Steinberg+1%BSA+antibiotics for long term culture (Ariizumi et al, 2017; Fukui et al ., 2003). Uncapped embryos were used as a stage reference.…”