Scientists worldwide have been attempting to recreate miniature versions of organs, called organoids, with pluripotent stem cells (PSCs), such as embryonic stem and induced pluripotent stem cells, to model organ development or diseases and leverage them to identify new drugs. Depending on the organ, this often requires simulating natural in vivo conditions experienced by cells, such as growth factors, when developing into the organ of interest. Although organoids typically can self-organize into complex three-dimensional structures, in some cases, the mere presence of such signaling factors may be insufficient because interstitial flow produced by the blood supply in the developing embryo, for example, may help create a directional cue for cells to form polarized complex organ structures.

In this study, the group of researchers led by Takayama and Deguchi examined whether incorporating such interstitial flow into the construction of small intestinal tissues is vital to building in vivo-like structures. In addition, they successfully devised a way to stimulate PSC-derived cells simultaneously for endodermal and mesodermal differentiation on a microphysiological system (MPS)—small intestinal tissues grown on a microfluidic device—so that both the epithelial cell layer lining the small intestine and the mesenchymal cell layer under it can develop together. In this system, they deposited PSCs partially differentiated for a few days into endoderm and mesoderm lineages onto the MPS's top chamber, separated from the lower chamber by a semi-permeable membrane with microscopic pores, for further differentiation.

To first understand how much interstitial flow is created, the research team performed numerical simulations of the flow field generated in the upper chamber of the MPS by injecting culture medium into the lower chamber. Remarkably, calculations showed that the vertical flow velocity is dramatically—five orders of magnitude (i.e., 100,000 times)—lower than the injection velocity. The researchers thus believed that this accurately portrays interstitial flow powered by circulating plasma in the developing small intestines.

After establishing the system to reproduce interstitial flow in vitro, the research team examined how it affects gene expression as a measure to validate small intestinal tissues generated in this manner. They observed transient increases in endodermal and mesodermal markers, followed by increased expression of intestinal epithelial and mesenchymal cell markers. Notably, a single-cell RNA-sequencing comparison between small intestinal tissues generated in the MPS with interstitial flow and human fetal intestinal tissues revealed a remarkable resemblance between the two tissues. Furthermore, it indicated that the micro-small intestine system created by the research team can recapitulate the diversity of cell types found in naturally formed tissues.

Through histological and other RNA- or protein-based analyses, the researchers found that interstitial flow generated in their MPS created a polarity in the tissue and with primitive villi-like 3D structures on intestinal epithelial cells, absent otherwise when interstitial flow was not incorporated. Furthermore, as possible indicators of enhanced functionality, small intestinal tissues generated in the presence of interstitial flow showed increased barrier function and drug-metabolizing enzyme levels. In an effort to demonstrate the feasibility of using their micro-small intestine systems for virology research, the research team also showed that alanyl aminopeptidase, membrane (ANPEP)-the viral receptor of HCoV-229E, a human coronavirus known to infect the luminal side of organs-is expressed at a higher level and correctly localized to the apical side of the intestinal epithelium. Consequently, and consistent with expectations, apical HCoV-229E infections were more effective than basolateral infections. A detailed examination of infected tissues by RNA sequencing also suggests apically infected small intestinal tissues display the expected host response to viral infections.

Together, by incorporating interstitial flow and inducing endodermal and mesodermal differentiation from PSCs simultaneously, the group led by Takayama and Deguchi provides a significantly improved means to generate in vivo-like small intestinal tissues in vitro. The team hopes that with this advance, they and other scientists around the world could apply their newly generated system to study small intestinal development, model diseases affecting the small intestine, and development for new therapeutic agents for those diseases.

The results of this study were published online in Cell Stem Cell on July 11, 2024.

• doi: 10.1016/j.stem.2024.06.012