Characterization of mesenchymal support required for in vivo engraftment and development of human PSC-derived organoids

Human gastrointestinal (GI) organoids derived from pluripotent stem cells have unique potential for studying organogenesis, physiology, and diseases. To this end, their transplantation into animal models to further their development into functional organs provides a valuable tool. However, organoids from different GI regions, for example, the esophagus or intestine, harbor different engraftment capabilities. Here, we developed a tissue-engineering approach and show that enhancing the mesenchyme-to-endoderm ratio enables reliable engraftment of GI organoids. However, endoderm/mesoderm recombinations reveal that only Human Intestinal Organoid (HIO) mesenchyme allows full development of GI epitheliums in vivo. Comparative mesenchyme analysis from esophageal, gastric, intestinal, and colonic organoids shows endothelial cell enrichment within the HIO mesenchyme, and we demonstrate by endothelial cell depletion or addition that these cells are required and sufficient for proper GI organoid development in vivo. Our findings highlight the optimal mesenchymal cellular composition needed to support GI organoid engraftment, tissue growth, and function. We performed a single-cell transcriptomic analysis of HEOs, HGOs, HCOs, and HIOs by profiling a total of 27 437 cells isolated from organoids at days 18 and 28 of culture, to analyze the composition of there respective mesenchymes.