Uniaxial Force Induces Maturation of Human Intestinal Organoids in Vivo
Authors: Poling Holly M. , Brown Nicole , Wu David , Brink Michael , Huynh Nhan , Hogan Simon P. , Dunn James C.Y. , Wells James M. , Helmrath Michael A. , Mahe Maxime M. 
Published: November 2023

Background and Aims

In both directing and fostering the natural ability of stem cells to self‐organize, significant advances have been made in the generation of functional Human Intestinal Organoids (HIOs). These structures include both epithelial and mesenchymal components, intestinal architectural features, and the ability to engraft in vivo. Currently, HIOs are being used in the study of human specific gastrointestinal diseases. However, the ultimate goal of this methodology, as with most tissue engineering strategies, is its translation to humans in providing functional autologous tissue for transplantation. As of late, limited progress has been made in scaling and maturing these inherently immature tissues through transplanting them in vivo which results in grafts that approximate fetal intestinal tissue. Here, we aim to build upon this maturation strategy by incorporating a technique that has proven successful in eliciting an adaptive response from native intestine: the application of intraluminal uniaxial force.

Methods

As summarized in Fig. 1, HIOs are cultured prior to implantation within the mesentery of immunodeficient mice. They are allowed to grow, vascularize, and mature for 8–10 weeks before a second procedure is performed wherein a compressed nitinol spring is implanted within the lumen of the transplanted HIO (tHIO). Micro‐CT is used to visualize spring deployment in vivo. Histological and immunohistochemical techniques were used in order to assess morphometrics of the resulting tissues. TEM and SEM were used to visualize their internal and surface ultrastructures. To assess their functionality, ex vivo epithelial characteristics were assayed in an Ussing chamber and ex vivo muscle strength in an organ bath chamber. Next Generation RNA sequencing was performed across transplanted samples as well as on human surgical samples to highlight the transcriptional similarities and differences between groups.

Results

Upon harvest the tHIO+Spring (tHIO+S) displayed a dominant lumen and were larger than tHIOs. Histological analysis revealed increases in crypt depth, villus height, crypt fission, and muscle thickness and a shift in proliferation in tHIO+S compared to tHIO. The number of Chromogranin A positive enteroendocrine cells per crypt‐villus axis decreased in tHIO+S and approximated that of human tissue. No differences in other cell populations were observed, while some displayed trends towards that of adult human tissue. Functionally, a positive impact of stretch was observed with increases in short circuit current and transepithelial resistance. Transcriptionally, the tHIO+S samples were more similar to human tissues than the tHIO.

Conclusions

With these initial experiments, we conclude that the application of an intraluminal uniaxial force is a practical method to induce maturation of tHIOs in vivo without concomitant architectural disruptions. While our current system does lack certain complexities, we have demonstrated enterogenesis by means of biomechanical manipulation. This may prove to be applicable and advantageous in the engineering approaches of other organ systems. This methodology provides a platform which can be translated to bigger animal models to engineer more mature, larger scale human intestinal tissue segments for transplantation.

Support or Funding Information

1K99DK110414‐01 ‐ MMM and CCHMC ARC ‐ MAH