Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

This method provides step-by-step instructions on how to generate human-induced pluripotent stem cell-derived intestinal organoids by differentiation into definitive endoderm, then hind gut epithelium, and, finally, the transfer into 3D culture conditions. Results obtained from human-induced pluripotent stem cell-derived intestinal organoids demonstrate greater translatability than data from monocellular cultures, while also being more practical than primary tissue-derived organoids, which can be difficult to keep in culture long term. Upon the generation of human intestinal cells from differentiated iPSCs, use a 5 milliliter serological pipette to detach the cell monolayer from each well of the 6-well plate.

And pool the cells in a single 15 milliliter tube for centrifugation. Re-suspend the pellet in intestinal growth medium supplemented with growth factors. And add the appropriate volume of extracellular matrix according to the number of wells being plated.

Add 30 microliters of the cell suspension to the center of the appropriate number of wells of a 48-well plate. Ensuring that the ECM is placed in the center of the well is very important to the long-term stability of that sample. If the ECM touches the wall of the well, it may collapse and the samples can be lost.

Place the plate in the cell culture incubator for at least 5 minutes. When the extracellular matrix has set, add 300 microliters of fresh intestinal growth medium supplemented with growth factors to each well, and return the plate to the cell culture incubator. After 48 hours, use a light microscope to check the wells for organoid formation.

After 7 days of culture, aspirate the culture media and replace it with ice cold DPBS. Use a 5 milliliter serological pipette to mechanically detach the organoids and extracellular matrix sphere from the plate. Then, pool the organoids into a single 15 milliliter centrifuge tube.

Collect the organoids by centrifugation and aspirate the supernatant down to the top of the visible ECM layer. Re-suspend the pellet in 15 milliliters of ice cold DPBS and centrifuge the cells again. After aspirating the supernatant as demonstrated, re-suspend the pellet in 1 milliliter of ice cold PBS and use a P200 pipette to manually disrupt the intact organoids.

Confirm a complete dissociation of the organoids. Spin the dissociated organoids and re-suspend in the required volume of the extracellular matrix before plating onto a fresh 48-well plate, as demonstrated. Then, return the plate to the incubator for 5 minutes.

After 5 minutes, remove the plate from the incubator and add intestinal basal media with growth factors and ROCK inhibitor. Change media every 2-4 days. To trigger an inflammatory response in the intestinal organoids, replace the supernatant in each well of the organoid culture with 300 microliters of freshly prepared basal medium supplemented with 40 nanograms per milliliter of TNF alpha.

Then, return the plate to the cell culture incubator for 48 hours to replicate a pro-inflammatory environment. Gene expression can be monitored over the course of human IPSC differentiation using pluripotency markers that are highly expressed on day 0 and are rapidly down-regulated during the process of definitive endoderm differentiation. On day 2 of differentiation, definitive endoderm genes should begin to be expressed, and expression should peak at day 3.

During hind gut specification, CDX2 and HNF4alpha expression should be induced and increase over time. 48 hours after 2D cell sheet transfer, the sheets of cells should begin to auto-organize into more compacted 3D spheroid structures that are initially small, but that gradually increase in size and complexity over the next 7-10 days of culture. The organoids should not be passaged until they have achieved a clear organoid spheroid morphology with obvious epithelium and with the lumen facing toward the center of the structure.

When the structures reach this stage, immunocytochemistry can be performed to confirm the expression of intestinal markers, such as villin and CDX2. To model inflammation, TNF alpha can be added to the tissue culture medium for 24-48 hours, which typically results in the expression of pro-inflammatory markers in conjunction with the downregulation of intestinal epithelial markers. Human-induced pluripotent stem cell-derived intestinal organoids can be used for drug discovery, disease modeling, gene editing, a study of the tumor microenvironment, and transcriptomic and proteomic, and epigenetic profile of any disease of interest.

This method can help many labs establish intestinal organoids as a model system, providing them an additional method for their research.