Generation of Budding-Like Intestinal Organoids from Human Induced Pluripotent Stem Cells

Abstract

Human induced pluripotent stem (iPS) cell-derived intestinal organoids have low invasiveness; however, the current differentiation method does not reflect the crypt-villus-like structure due to structural immaturity. Here, we generated budding-like organoids that formed epithelial tissue-like structures and had the characteristics of the mature small intestine from human iPS cells. They showed a high expression of drug transporters and induced the expression of cytochrome P450 3A4 and P-glycoprotein. When treated with tumor necrosis factor-a and/or transforming growth factor-b, the budding-like organoids replicated the pathogenesis of mucosal damage or intestinal fibrosis. Upon dissociation and seeding on cell culture inserts, the organoids retained intestinal characteristics, forming polarized intestinal folds with approximately 400 V £ cm 2 transepithelial electrical resistance. This novel method has great potential for disease modeling and drug screening applications.

Protective Effect of Irsogladine against Aspirin-Induced Mucosal Injury in Human Induced Pluripotent Stem Cell-Derived Small Intestine

“…Human iPS cells (Windy) were cultured as reported previously [ 24 , 25 ]. A modified version of a previously described differentiation protocol for induction at day 7 was used.…”

“…Hence, 3D-organoids are not suitable research tools for drug absorption and transport studies. To overcome this drawback, we recently established a two-dimensional (2D) monolayer culture system from human iPSC-derived intestinal organoids [ 25 ]. Cells seeded in a cell culture insert from iPSC-derived intestinal organoids formed a villus structure toward the upper air-side, which improved the versatility for experimental manipulation.…”

Human Intestinal Organoids: Promise and Challenge

“…Therefore, they will potentially model human tissue functionality and physiology more accurately than animal models or 2D models ( Lancaster and Knoblich, 2014 ; Kim J. et al, 2020 ). For instance, when sufficiently matured, human intestinal organoids recapitulate budding crypt and villi domains, harbouring proliferative ISCs and progenitors, as well as differentiated enterocytes, goblet cells and Paneth cells, respectively ( Onozato et al, 2021 ). Although organoids contain differentiated cell types, they can be cryopreserved and expanded relatively rapidly ( Clinton and McWilliams-Koeppen, 2019 ).…”

“…Nevertheless, the directed differentiation process of PSCs towards intestinal organoids takes quite some time (2–3 months), after which hIOs still largely resemble fetal intestinal homeostasis ( McCracken et al, 2011 ; Frum and Spence, 2021 ). Though recently, Onozato and colleagues described a method to enhance organoid maturation in vitro , generating crypt-like budding and expression of villin1 ( Onozato et al, 2021 ). Conversely, enteroids do not have to undergo sequential differentiation steps and can therefore be generated much more rapidly.…”

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

“…Potentially significant advantages of this approach include (1) bypassing the reliance on the highly variable spontaneous spheroid production and detachment step during the early stages of organoid formation, and (2) the potential for progenitors to be cryopreserved before aggregation and organoid formation, facilitating the production of banks of organoid precursors that can be accessed without maintaining hPSCs in culture. Several studies have reported utilization of the patterned MHE monolayer to generate HIOs ( Forbester et al., 2015 ; Onozato et al., 2018 , 2021 ; Yoshida et al., 2020 ). However, none of these studies compared the resulting HIOs to those arising spontaneously from multiple hPSC lines, investigated whether MHE can be cryopreserved, banked, and thawed for subsequent HIO generation, nor translated forced aggregation more broadly to other gastrointestinal (GI) organoid types.…”