Here we provide a method for generation of human enteric neurons from human pluripotent stem cells under fully defined conditions. And we use these cells in order to understand human enteric nervous system development and function. I think human enteric nervous system development, physiology and pathophysiology, has been a great challenge and it's because of the rarity of the enteric nervous system cells and the transient nature of the neural crest progenitors and difficulty in access to tissue and isolation of the tissue.
This is a robust platform for generating authentic and functional human enteric neurons from HPSCs. And by overcoming longstanding technical challenges in tissue access, it provides scalable cultures of enteric nervous system components for disease modeling, drug discovery, and studying ENS developments and function. To induce neural crest formation, replace the maintenance medium of the human pluripotent stem cell culture with 200 microliters of Medium A, per square centimeter of the well surface.
Incubate the culture under 5%carbon dioxide supplementation at 37 degrees Celsius for two days. Gently aspirate the medium from the well containing 100%confluent cells. Then pipette about 200 microliters of Medium B, per square centimeter of the well surface area.
On day four, feed the cells with 200 microliters of Medium B, per square centimeter of the well surface. Incubate the cells for two more days, then replace the Medium B with 400 microliters per square centimeter of Medium C.To begin, take 12 day cultures of human pluripotent stem cells that have been subjected to neural crest induction. Gently aspirate Medium C from the cells.
Add 100 microliters of PBS per square centimeter of well surface area to wash the cells, and then remove the PBS. Add an equal volume of cell detachment solution to the cells and incubate for 30 minutes under 5%carbon dioxide supplementation at 37 degrees Celsius. Add an equal volume of NCC Medium into the plate.
Then use a serological pipette to harvest the cell suspension and transfer it to a 15 milliliter conical tube. Spin the cells at 290 G for two minutes between 20 to 25 degrees Celsius. Carefully discard the supernatant without disturbing the cell pellet.
Use a 10 milliliter serological pipette to pipette 12 milliliters of NCC medium to the cell pellet. Mechanically pipette the suspension up and down to create a suspension. Then transfer the cell suspension to an ultra-low attachment plate.
On day 14, gently swirl the cell culture plates to gather small spheroids at the center of each well. Use a P1000 Micropipette to slowly aspirate the spent medium from the circumference of each well. Re-feed the cells with the original volume of NCC medium.
On day 15, carefully remove the medium. Add PBS to the wells to wash the spheroids. Then remove as much PBS as possible, ensuring spheroids are not disturbed.
Next, add 100 microliters of cell detachment solution per square centimeter of well surface area. Incubate the cells in the solution for 30 minutes under 5%carbon dioxide at 37 degrees Celsius. With a 10 milliliter serological pipette, add an equal volume of ENC Medium to each well.
Mechanically pipette the solution to break any remaining spheroids. Then transfer the cells to a 50 milliliter conical tube. Discard the supernatant after centrifuging as before.
Then resuspend the cells in five to 10 milliliters of ENC Medium. Count the concentration of viable cells using trypan blue and hemocytometer. Now, add enough volume of ENC Medium to plate about 300, 000 to 400, 000 viable cells per square centimeter of surface area at a density of about 1 million cells per milliliter.
Then aspirate the solutions from the wells. Next, add the cells to a plate with PO/FN Laminin Coated wells. Feed cells every other day with 200 microliters of ENC Medium per square centimeter of well surface area, until days 30 to 40.
High-quality free-floating spheres with smooth surfaces were observed after neural crest induction. The spheroids expressed the neural crest marker SOX10. Neurites were observed between days 25 to 30, during the enteric neuron induction.
