Promoting Human Intestinal Organoid Formation and Stimulation Using Piezoelectric Nanofiber Matrices.

Human organoid model systems have changed the landscape of developmental biology and basic science. They serve as a great tool for human specific interrogation. In order to advance our organoid technology, we aimed to test the compatibility of a piezoelectric material with organoid generation, because it will create a new platform with the potential for sensing and actuating organoids in physiologically relevant ways. We differentiated human pluripotent stem cells into spheroids following the traditional human intestinal organoid (HIO) protocol atop a piezoelectric nanofiber scaffold. We observed that exposure to the biocompatible piezoelectric nanofibers promoted spheroid morphology three days sooner than with the conventional methodology. At day 28 of culture, HIOs grown on the scaffold appeared similar. Both groups were readily transplantable and developed well-organized laminated structures. Graft sizes between groups were similar. Upon characterizing the tissue further, we found no detrimental effects of the piezoelectric nanofibers on intestinal patterning or maturation. Furthermore, to test the practical feasibility of the material, HIOs were also matured on the nanofiber scaffolds and treated with ultrasound, which lead to increased cellular proliferation which is critical for organoid development and tissue maintenance. This study establishes a proof of concept for integrating piezoelectric materials as a customizable platform for on-demand electrical stimulation of cells using remote ultrasonic waveforms in regenerative medicine.

Specific binding sites on Rhesus rotavirus capsid protein dictates the method of endocytosis inducing the murine model of biliary atresia.

Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV) induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated blocking dynamin decreased the infectivity of RRV whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV while late endosome inhibition had no effect. Following infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human BA patients experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that utilize Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10, and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction.

WNT2B Deficiency Causes Enhanced Susceptibility to Colitis Due to Increased Inflammatory Cytokine Production.

BACKGROUND & AIMS: Humans with WNT2B deficiency have severe intestinal disease, including significant inflammatory injury, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. METHODS: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon, and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue. RESULTS: Mice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased, epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6, and biopsy tissue from humans showed increased neutrophils. CONCLUSIONS: WNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation, particularly in the colon.

Topical everolimus therapy for epidermal nevi associated with woolly hair nevus in a patient with a mosaic HRAS mutation.

A patient with woolly hair nevus syndrome, presented with epidermal facial nevi by the age of 12 years. Despite transient improvement with topical 1% sirolimus cream, the facial nevus grew larger. The patient was then treated with topical 1% everolimus cream resulting in a reduction in the size of the nevus. This case highlights a novel use of topical 1% everolimus cream, which previously has not been used to treat epidermal nevi.

WNT2B Deficiency Causes Increased Susceptibility to Colitis in Mice and Impairs Intestinal Epithelial Development in Humans.

Background and aims: WNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. Methods: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3χ antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses. Results: Mice with WNT2B deficiency had significantly decreased Lgr5 expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3χ antibody was similar in Wnt2b KO and wild type (WT) mice. In contrast, the colonic response to DSS in Wnt2b KO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature. Conclusion: WNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine.WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization. Data Transparency Statement: All RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

Transplanted human intestinal organoids: a resource for modeling human intestinal development.

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. Owing to their diverse cell populations, transplantation into an animal host is supported with this system and allows the temporal formation of fully laminated structures, including crypt-villus architecture and smooth muscle layers that resemble native human intestine. Although the endpoint of HIO engraftment has been well described, here we aim to elucidate the developmental stages of HIO engraftment and establish whether it parallels fetal human intestinal development. We analyzed a time course of transplanted HIOs histologically at 2, 4, 6 and 8 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 do indeed recapitulate early intestinal development, solidifying their value as a human intestinal model system.

Evaluation of transplantation sites for human intestinal organoids.

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.

Gastrointestinal organoids: a next-generation tool for modeling human development.

Gastrointestinal organoids are an exciting new tool for modeling human development, physiology, and disease in human tissue. Derived from pluripotent stem cells, 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 gastrointestinal (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.

Physiologic Medium Maintains the Homeostasis of Immature Bovine Articular Cartilage Explants in Long-Term Culture.

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.

Transplantation of human intestinal organoids into the mouse mesentery: A more physiologic and anatomic engraftment site.

BACKGROUND: We previously described the development of human intestinal organoids from pluripotent stem cells, as well as their in vivo maturation when transplanted into the mouse kidney capsule. While sufficient for certain aspects of study, this model has limitations. Herein, we describe an alternative model of human intestinal organoids transplantation into the mouse mesentery. We hypothesize that efficient engraftment and marked differentiation of human intestinal organoids will be similar to our kidney model yet in a more anatomically appropriate location allowing for improved in vivo modeling. METHODS: Human intestinal organoids were generated by directed differentiation of H1 embryonic stem cells. Human intestinal organoids were then transplanted into the mesentery of immunosuppressed mice. Gross and histologic analysis of tissue was performed. RESULTS: Human intestinal organoids were transplanted into the mouse mesentery and allowed to grow for 10 weeks. Mouse survival was 85%, and among the surviving mice, 82% of transplanted human intestinal organoids successfully engrafted. Upon graft harvest, transplanted HIOs were larger than in vitro human intestinal organoids (1.75 mm vs 6.27 mm, P < .0001) and grew along a vascular pedicle, allowing for interventions and reconstructive surgeries to access the human intestinal organoid lumen. Histologic analyses of transplanted human intestinal organoids confirmed the presence of major cell types, as well as stem cell activity. CONCLUSIONS: 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.