The mouse mesentery is a viable location for the transplantation of human intestinal organoids, yielding grafts of reproducible size and quality. This improved model serves to advance functional and translational studies of human intestinal organoids.
Gastrointestinal organoids are an exciting new tool for modeling human development, physiology, and disease in human tissue. Derived from pluripotent stem cells (PSCs), gastrointestinal organoids consist of epithelial and mesenchymal cells organized in an intricate, three-dimensional structure that recapitulates the physiology and microscopic anatomy of the human GI tract. In vitro derivation of gastrointestinal organoids from Definitive Endoderm has permitted an exploration of the complex signaling pathways required for the initial maturation of each individual gastrointestinal organ. Further maturation beyond an early fetal state currently requires transplantation into an immunocompromised host. Transplantation induced maturation provides an opportunity to functionally interrogate the key mechanisms underlying development of the human GI tract. Gastrointestinal organoids can also be used to model human diseases, and ultimately may serve as the basis for developing novel, personalized therapies for human intestinal diseases.
Our group has developed two transplantation models for the engraftment of Human Intestinal Organoids (HIOs): the renal subcapsular space (RSS) and the mesentery each with specific benefits for study. While engraftment at both sites generates laminated intestinal structures, a direct comparison between models has not yet been performed. Embryonic stem cells were differentiated into HIOs, as previously described. HIOs from the same batch were transplanted on the same day into either the RSS or mesentery. 10 weeks were allowed for engraftment and differentiation, at which time they were harvested and assessed. Metrics for comparison included: mortality, engraftment rate, gross size, number and grade of lumens, and expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism. Mortality was significantly increased when undergoing mesentery transplantation, however engraftment was significantly higher. Graft sizes were similar between groups. Morphometric parameters were similar between groups, however m-tHIOs presented with significantly fewer lumens than k-tHIO. Transcript and protein level expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism were similar between groups. Transplantation into both sites yields viable tissue of similar quality based on our assessments with enhanced engraftment and a dominant lumen for uniform study benefiting the mesenteric site and survival benefiting RSS.
The in vitro differentiation of pluripotent stem cells into human intestinal organoids (HIOs) has served as a powerful means for creating complex three-dimensional intestinal structures. Due to their diverse cell populations, transplantation into an animal host is supported with this system and allows for the temporal formation of fully laminated structures, including crypt-villus architecture and smooth muscle layers which resemble native human intestine. While the endpoint of HIO engraftment has been well described, here, we aim to elucidate the developmental stages of HIO engraftment and establish if it parallels fetal human intestinal development. We analyzed a timecourse of transplanted HIOs histologically at two, four, six and eight weeks post-transplantation and demonstrated that HIO maturation closely resembles key stages of fetal human intestinal development. We also utilized single nuclear RNA sequencing to determine and track the emergence of distinct cell populations over time and validated our transcriptomic data through in situ protein expression. These observations suggest that transplanted HIOs indeed recapitulate early intestinal development, solidifying their value as a human intestinal model system.
The ability to maintain living articular cartilage tissue in long-term culture can serve as a valuable analytical research tool, allowing for direct examination of mechanical or chemical perturbations on tissue behavior. A fundamental challenge for this technique is the recreation of the salient environmental conditions of the synovial joint in culture that are required to maintain native cartilage homeostasis. Interestingly, conventional media formulations used in explanted cartilage tissue culture investigations often consist of levels of metabolic mediators that deviate greatly from their concentrations in synovial fluid (SF). Here, we hypothesize that the utilization of a culture medium consisting of near-physiologic levels of several highly influential metabolic mediators (glucose, amino acids, cortisol, insulin, and ascorbic acid) will maintain the homeostasis of cartilage explants as assessed by their mechanical properties and extracellular matrix (ECM) contents. Results demonstrate that the aforementioned mediators have a strong effect on the mechanical and biochemical stability of skeletally immature bovine cartilage explants. Most notably, (1) in the absence of cortisol, explants exhibit extensive swelling and tissue softening and (2) in the presence of supraphysiologic levels of anabolic mediators (glucose, amino acids, insulin), explants exhibit increased matrix accumulation and tissue stiffening. In contrast, the administration of physiologic levels of these mediators (as present in native SF) greatly improves the stability of live cartilage explants over one month of culture. These results may have broad applicability for articular cartilage and other musculoskeletal tissue research, setting the foundation for important culture formulations required for examinations into tissue behavior.
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