(J-372) Engineering Large-Scale and Innervated Functional Human Gut for Transplantation

Introduction:: Human pluripotent stem cell derived organoid technologies have catapulted scientists and engineers into more sophisticated realms of personalized medicine, human disease modeling and translational science. However, significant limitations still exist within the aspects of scalability, throughput, cellular complexity, and functionality. Here, we aim to create a novel methodology for organoid generation that can simultaneously address these challenges, creating the next generation of gastrointestinal organoids. We hypothesized that a geometric confinement strategy would positively influence the collective behavior of the cells and provide spatial cues needed to define a specific three-dimensional conformation while promoting the natural processes of organogenesis.


Materials and Methods:: Commercially available and in-house made human pluripotent stem cell lines were utilized to generate gastrointestinal organoids in this study. A scaffolding tray was designed for the in vitro culture and maturation of a critical mass of conventionally generated mid-hindgut spheroids. Soft lithography was used to fabricate the scaffolding trays. Spheroids were seeded and grown in vitro as a small intestinal groove (SI Groove) using the scaffolding trays. SI Grooves were grown in vitro for 14 days, with the first 6 days utilizing the scaffolding tray. Bulk RNA sequencing was performed on the SI Groove and conventionally generated human intestinal organoids (HIOs). SI Grooves and HIOs were also transplanted into immunocompromised rat hosts. After allowing 10 weeks for engraftment, tissues were harvested and analyzed ex vivo. Histology and immunohistochemistry were performed to characterize the tissue grafts. Single nucleus RNA sequencing was performed to define the organoid’s cellular complexity. Organ bath chamber assays were performed to assess neuromuscular function. Experimentation was then expanded and repeated to generate colonic and gastric tissues with the scaffolding tray.


Results, Conclusions, and Discussions:: SI Grooves were observed to be enriched for biological processes relating to neurogenesis when compared to conventionally generated HIOs. After analyzing engrafted SI Grooves, they were significantly larger than conventionally generated HIOs and exhibited enhanced morphometric features (Figure). A robust enteric nervous system developed as demonstrated by neuronal cell type presence in snRNA sequencing and protein expression. Organ bath chamber experiments demonstrated that the enteric nervous system (ENS) fired in response to electrical stimulation. Similar results were observed in generating colonic and gastric tissues.



With this study, we have leveraged the ease of soft lithography to manufacture scaffolds that were incorporated into organoid culture to generate an improved organoid for transplantation. The simplicity of this experimental approach makes this methodology widely accessible and straightforward to execute. Using this technique, we have established a more reliable and scalable method for generating innervated transplantable gastrointestinal tissues. Clinically, this is relevant because the ENS controls various functions within the gastrointestinal tract such as motility, regulation of fluid exchange and localized blood flow, and defense reactions. The capability of an organoid to recapitulate such critical physiologic functions is essential in not only modeling native organs, but their downstream clinical translation. While further experimentation and subsequent studies must be performed to fully identify and describe the origins of the ENS in groove-based organoids, they may represent a unique tool to study ENS-related conditions such as Hirschsprung's disease, which is characterized by the absence of functional ENS ganglion cells. It may also embody a secondary source of generating neuroglial cells in contexts where deficiencies exist in the traditionally thought of pathway. By taking advantage of cell-cell interactions and fostering the co-development of cell types through confinement, we continue to optimize how organoid cultures affect the in vitro and subsequent in vivo development of our engineered tissue systems.


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