Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation

• Published in: Stem cell reports 2022-08, Vol.17 (8), p.1889-1902
• Main Authors: Pitstick, Amy L., Poling, Holly M., Sundaram, Nambirajan, Lewis, Phillip L., Kechele, Daniel O., Sanchez, J. Guillermo, Scott, Melissa A., Broda, Taylor R., Helmrath, Michael A., Wells, James M., Mayhew, Christopher N.
• Format: Article
• Language: English
• Subjects: aggregation; antral stomach organoid; Cell Differentiation; colonic organoid; cryopreservation; Endoderm; human pluripotent stem cell; Humans; intestinal organoid; Intestine, Small; mid-hindgut endoderm; Organoids; Pluripotent Stem Cells; Resource; spheroid
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2022.06.011

A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production. •hPSC-derived HIO production requires highly variable spontaneous spheroid formation•hPSC-derived MHE can be aggregated to reliably increase HIO production ∼10-fold•MHE can be cryopreserved before aggregation and subsequent HIO production•Antral stomach and colonic organoids can also be generated by aggregation Generation of human small intestinal organoids (HIOs) requires de novo hPSC differentiation and highly variable spontaneous spheroid production and detachment. Pitstick and colleagues demonstrate that the aggregation of cryopreservable mid-hindgut endoderm results in significantly more reliable HIO production. Aggregation can also be used to generate stomach and colonic organoids. This method will enhance the dissemination and accessibility of hPSC-derived GI organoid technologies.