Reversing Epithelial Polarity in Pluripotent Stem Cell-Derived Intestinal Organoids

Abstract

The inner surface of the intestine is a dynamic system, composed of a single layer of polarized epithelial cells. The development of intestinal organoids was a major breakthrough since they robustly recapitulate intestinal architecture, regional specification and cell composition in vitro. However, the cyst-like organization hinders direct access to the apical side of the epithelium, thus limiting their use in functional assays. For the first time, we show an intestinal organoid model from pluripotent stem cells with reversed polarity where the apical side faces the surrounding culture media and the basal side faces the lumen. These inside-out organoids preserve a distinct apico-basolateral orientation for a long period and differentiate into the major intestinal cell types. This novel model lays the foundation for developing new in vitro functional assays particularly targeting the apical surface of the epithelium and thus offers a new research tool to study nutrient/drug uptake, metabolism and host-microbiome/pathogen interactions.

Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous ECM

“…In fact, both formulations result in the accumulation of ECM proteins at the surface of the organoids, leading to polarity establishment and rosette formation within 3 days. This is in contrast with other organoid systems in which both exECM presence and jellification are needed to establish apical-basal polarity ( Inman & Bissell , 2010; Kakni et al ., 2022; Plachot et al ., 2009). Furthermore, early morphological rearrangements are maintained in exECM +L organoids after Matrigel is removed from the culture medium, indicating that continuity of exposure is also unnecessary.…”

Hypoxia-tolerant apical-out intestinal organoids to model host-microbiome interactions

“…Recently, human intestinal organoid models with reversed polarity have been described in order to facilitate the access to the apical surface of the organoids. 28 , 29 Based on our existing reversed polarity intestinal organoid model, 29 here, we developed apical-out intestinal organoids in hypoxic conditions (5% O 2 ), aiming to create a more physiologically relevant in vitro model for host-microbiome and host-pathogen interaction studies. Briefly, human embryonic stem cells (H9 cells) were aggregated to create homogeneous EBs using polymer film-based microwell arrays ( Figure 1(a) , Supplemental Figure 1 ).…”

“…Previous reports have shown that pluripotent stem cell-derived organoids recapitulate closely the cellular composition of the in vivo intestine. 29 , 40 To assess the differentiation capacity and maturation level of apical-out organoids in hypoxic conditions, we performed gene expression analysis for both proliferation and differentiation of intestinal cell lineages after 30 days in culture and compared them with apical-in organoids grown embedded in Matrigel domes and with apical-out organoids grown in normoxic conditions (21% O 2 ) ( Figure 1(b) ). Specifically, we evaluated the expression of the leucine-rich repeat-containing G-protein-coupled receptor 5 ( LGR5 ), the sex determining region Y-box 9 ( SOX9 ), the Krueppel-like factor 5 ( KLF5 ) and the Achaete scute-like 2 ( ASCL2 ), all indicators of proliferation.…”

“…Directed differentiation of ESCs toward intestinal organoids was performed as previously described. 29 Briefly, to create embryoid bodies (EBs), ESC colonies were dissociated into single cells with TrypLE™ Express Enzyme (Thermofisher), resuspended in mTesR1 supplemented with Y-27632 (10 μΜ; Tocris) and then seeded in the microwell arrays at a density of 1000 cells/microwell. A 3-day incubation with Activin A (100 ng/mL; Cell guidance systems) in RPMI 1640 (Thermofisher) medium supplemented with increasing concentrations (0%, 0%, 2%, and 2%) of Hyclone defined fetal bovine serum (dFBS; Fisher scientific) promoted the definitive endoderm (DE) differentiation.…”

“…As an alternative method to gain access to the apical surface that faces the lumen, researchers developed ways to reverse epithelial polarity. By culturing organoids in suspension, human adult, 27 , 28 human pluripotent stem cell, 29 and chicken, 30 and porcine 31 adult stem cell-derived organoids reversed their polarity in a way that the apical surface is facing outwards to the culture medium. These organoids have been used to study infections by Salmonella Typhimurium, Listeria monocytogenes , influenza A virus strain PR8, Eimeria tenella , and transmissible gastroenteritis virus.…”