Method of producing enteric neurons and uses thereof

A method of producing an in vitro model of the enteric nervous system comprising:

• i. contacting pluripotent stem cells to a first hydrogel disposed in a first culture vessel;
• ii. applying a first culture medium into the first culture vessel in a volume sufficient to cover the pluripotent stem cells in contact with the first hydrogel;
• iii. incubating the pluripotent stem cells for a first time and under conditions sufficient to grow a confluent layer of pluripotent stem cells;
• iv. inducing the pluripotent stem cells for a second time and under conditions sufficient to differentiate the induced pluripotent stem cells into enteric neural crest cells (ENCs);
• v. transferring the ENCs to a second culture vessel;
• vi. culturing the ENCs for a third time and under conditions for the ENCs to grow into enteric neural crest spheroids;
• and
• vii. contacting the enteric neural crest spheroids to a second hydrogel disposed in a third culture vessel;
• viii. applying a second culture medium into the third culture vessel in a volume sufficient to cover the enteric neural crest spheroids in contact with the second hydrogel;
• and
• ix. incubating the enteric neural crest spheroids for a third time and under conditions sufficient to differentiate the enteric neural crest spheroids into enteric neurons;
• wherein the ENCs comprise expression of about 5% CD49D and/or sex determining region of the Y chromosome-like high-mobility box transcription factor 10 (SOX10) higher than that expressed by pluripotent stem cells;
• wherein the enteric neurons comprise expression of about 5% class III beta-tubulin (TUJ1) and tyrosine-protein receptor kinase C (TRKC) higher than that expressed by ENCs;
• and
• wherein the enteric neurons comprise less than about 60% flat myofibroblast-like cells comprising expression of smooth muscle actin.

A system comprising:

• a culture vessel comprising a hydrogel;
• enteric neurons, wherein the enteric neurons are disposed in a two-dimensional layer on the hydrogel;
• and
• a culture medium, wherein the culture medium is free of any Smad signaling inhibitor,
• wherein the enteric neurons are in culture for 5-20 days;
• and
• wherein the enteric neurons comprise less than 60% of cells comprising expression of smooth muscle actin.

A method of differentiating one or a plurality of stem cells into one or a plurality of enteric neuronal cells in a culture vessel comprising a solid substrate, said method comprising:

• (a) contacting one or a plurality of stem cells with the solid substrate, said substrate comprising at least one exterior surface, at least one interior surface, and at least one interior chamber defined by the at least one interior surface and accessible from a point exterior to the solid substrate through at least one opening;
• (b) applying a first cell medium into the culture vessel for a time period sufficient to differentiate the one or plurality of cells into one or a plurality of neural crest cells;
• (c) removing the first cell medium from the culture vessel;
• and
• (d) applying a second cell medium into the culture vessel for a time period sufficient to differentiate the neural crest cells into enteric neurons.