Aim of this study was to investigate, for the first time, whether isolated newborn mouse enteric plexus could induce in vitro differentiation of the vagal neural crest-derived cells into enteric neuroblasts. Fragments of the myenteric plexus were isolated from the small intestine of 6-day-old Swiss mice and were collected and stored in DMEM-F12 medium, then cultured on polymerized human fibronectin layer. The vagal portion of the neural tube, isolated from a 9.5-day-old Swiss mouse embryo, was put in the same chamber slides where the isolated myenteric plexus had been cultured for 3 days. The vagal neural crest-derived cells migrated onto the polymerized human fibronectin layer and formed a crown of cells around the neural tube. After 6 days, the cultures were stopped and studied immunohistochemically for anti-NF160 KD, anti-TH, and RetR5 antibodies to analyse the differentiation stage of the cultured cells. Analysis of results included the comparison of two culture groups: Group 1, used as control, in which vagal neural crest-derived cells were put in DMEM-F12, supplemented only with 10 % of FCS; Group 2, in which vagal neural crest-derived cells were put in the same medium as Group 1, with the addition of myenteric plexus fragments isolated from newborn mice to form the co-culture. The following results were obtained: in Group 1 the neural tubes originated a cell population strongly positive for anti-NF160 and anti-TH Ab, but negative for RetR5 Ab. This positivity was found both in the cells adjacent to the neural tube and in those migrating from it distally. The Group 2 originated cells, which after migration were positive for anti-NF160 and for anti-TH antibodies. In addition, in this culture group, the cells which migrated from the neural tube were positive for anti-RetR5 antibody. The co-culture used in this study induces the differentiation of vagal stem cells into enteric neuroblasts, cells TH+ and RetR5+. These cells, after reaching the embryonic intestine, migrate to colonize the hindgut and form the ENS. Therefore this biotechnology seems a good method to obtain in vitro enteric precursors of ENS.
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