Infant and Adult Human Intestinal Enteroids are Morphologically and Functionally Distinct.

Background & Aims: Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. Methods: We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium. Results: RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. Conclusions: HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population.

Human intestinal organoids from Cronkhite-Canada syndrome patients reveal link between serotonin and proliferation.

Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.

A DLG1-ARHGAP31-CDC42 axis is essential for the intestinal stem cell response to fluctuating niche Wnt signaling.

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling.

Single-cell sequencing of rotavirus-infected intestinal epithelium reveals cell-type specific epithelial repair and tuft cell infection.

Intestinal epithelial damage is associated with most digestive diseases and results in detrimental effects on nutrient absorption and production of hormones and antimicrobial defense molecules. Thus, understanding epithelial repair and regeneration following damage is essential in developing therapeutics that assist in rapid healing and restoration of normal intestinal function. Here we used a well-characterized enteric virus (rotavirus) that damages the epithelium at the villus tip but does not directly damage the intestinal stem cell, to explore the regenerative transcriptional response of the intestinal epithelium at the single-cell level. We found that there are specific Lgr5+ cell subsets that exhibit increased cycling frequency associated with significant expansion of the epithelial crypt. This was accompanied by an increase in the number of immature enterocytes. Unexpectedly, we found rotavirus infects tuft cells. Transcriptional profiling indicates tuft cells respond to viral infection through interferon-related pathways. Together these data provide insights as to how the intestinal epithelium responds to insults by providing evidence of stimulation of a repair program driven by stem cells with involvement of tuft cells that results in the production of immature enterocytes that repair the damaged epithelium.

Use of human tissue stem cell-derived organoid cultures to model enterohepatic circulation.

The use of human tissue stem cell-derived organoids has advanced our knowledge of human physiological and pathophysiological processes that are unable to be studied using other model systems. Increased understanding of human epithelial tissues including intestine, stomach, liver, pancreas, lung, and brain have been achieved using organoids. However, it is not yet clear whether these cultures recapitulate in vivo organ-to-organ signaling or communication. In this work, we demonstrate that mature stem cell-derived intestinal and liver organoid cultures each express functional molecules that modulate bile acid uptake and recycling. These organoid cultures can be physically coupled in a Transwell system and display increased secretion of fibroblast growth factor 19 (FGF19) (intestine) and downregulation of P450 enzyme cholesterol 7 α-hydroxylase (CYP7A) (liver) in response to apical exposure of the intestine to bile acids. This work establishes that organoid cultures can be used to study and therapeutically modulate interorgan interactions and advance the development of personalized approaches to medical care.NEW & NOTEWORTHY Interorgan signaling is a critical feature of human biology and physiology, yet has remained difficult to study due to the lack of in vitro models. Here, we demonstrate that physical coupling of ex vivo human intestine and liver epithelial organoid cultures recapitulates in vivo interorgan bile acid signaling. These results suggest that coupling of multiple organoid systems provides new models to investigate interorgan communication and advances our knowledge of human physiological and pathophysiological processes.

A Millifluidic Perfusion Cassette for Studying the Pathogenesis of Enteric Infections Using Ex-Vivo Organoids.

To generate physiologically-relevant experimental models, the study of enteric diarrheal diseases is turning increasingly to advanced in vitro models that combine ex vivo, stem cell-derived "organoid" cell lines with bioengineered culture environments that expose them to mechanical stimuli, such as fluid flow. However, such approaches require considerable technical expertise with both microfabrication and organoid culture, and are, therefore, inaccessible to many researchers. For this reason, we have developed a perfusion system that is simple to fabricate, operate, and maintain. Its dimensions approximate the volume and cell culture area of traditional 96-well plates and allow the incorporation of fastidious primary, stem cell-derived cell lines with only minimal adaptation of their established culture techniques. We show that infections with enteroaggregative E. coli and norovirus, common causes of infectious diarrhea, in the system display important differences from static models, and in some ways better recreate the pathophysiology of in vivo infections. Furthermore, commensal strains of bacteria can be added alongside the pathogens to simulate the effects of a host microbiome on the infectious process. For these reasons, we believe that this perfusion system is a powerful, yet easily accessible tool for studying host-pathogen interactions in the human intestine.

Protein-Functionalized Poly(ethylene glycol) Hydrogels as Scaffolds for Monolayer Organoid Culture.

Stem cell-derived, organotypic in vitro models, known as organoids, have emerged as superior alternatives to traditional cell culture models due to their unparalleled ability to recreate complex physiological and pathophysiological processes. For this reason, they are attractive targets of tissue-engineering efforts, as constructs that include organoid technology would be expected to better simulate the many functions of the desired tissue or organ. While the 3D spheroidal architecture that is the default architecture of most organoid models may be preferred for some applications, 2D monolayer arrangements remain the preferred organization for many applications in tissue engineering. Therefore, in this work, we present a method to create monolayer organoid cultures on poly(ethylene glycol) (PEG) hydrogel scaffolds, using intestinal epithelial organoids (IEOs) as a proof-of-concept. Our process involves two steps: the hydrogel is first functionalized with a layer of poly(D-lysine) (PDL), which then allows the adsorption of pristine, unmodified basement membrane proteins. This approach successfully mediates the formation of IEO monolayer unlike conventional approaches that rely on covalent modification of the hydrogel surface with cell-adhesive peptides and basement membrane proteins. We show that these IEO monolayers recreate important physiological functions of the native intestinal epithelium, including multilineage differentiation, apical-basal polarization, and the ability to model infections with human norovirus. We also show coating of a scaffold mimicking intestinal villous topography, resulting in a 3D IEO monolayer. We expect that this protocol will be useful to researchers attempting to leverage the increased physiological relevance of organoid models to elevate the potential of their tissue-engineered constructs. Impact statement While organoids are physiologically superior models of biological functions than traditional cell cultures, their 3D spheroidal architecture is an obstacle to their incorporation in many tissue-engineering applications, which often prefer 2D monolayer arrangements of cells. For this reason, we developed a protocol to establish monolayer cultures of organoids on poly(ethylene glycol) hydrogels and demonstrate its utility using intestinal epithelial organoids as a proof-of-concept. We expect that this protocol will be of use to researchers creating engineered tissues for both regenerative medicine applications, as well as advanced in vitro experimental models.

Preclinical Development of Oncolytic Immunovirotherapy for Treatment of HPVPOS Cancers.

Immunotherapy for HPVPOS malignancies is attractive because well-defined, viral, non-self tumor antigens exist as targets. Several approaches to vaccinate therapeutically against HPV E6 and E7 antigens have been adopted, including viral platforms such as VSV. A major advantage of VSV expressing these antigens is that VSV also acts as an oncolytic virus, leading to direct tumor cell killing and induction of effective anti-E6 and anti-E7 T cell responses. We have also shown that addition of immune adjuvant genes, such as IFNß, further enhances safety and/or efficacy of VSV-based oncolytic immunovirotherapies. However, multiple designs of the viral vector are possible-with respect to levels of immunogen expression and method of virus attenuation-and optimal designs have not previously been tested head-to-head. Here, we tested three different VSV engineered to express a non-oncogenic HPV16 E7/6 fusion protein for their immunotherapeutic and oncolytic properties. We assessed their profiles of efficacy and toxicity against HPVPOS and HPVNEG murine tumor models and determined the optimal route of administration. Our data show that VSV is an excellent platform for the oncolytic immunovirotherapy of tumors expressing HPV target antigens, combining a balance of efficacy and safety suitable for evaluation in a first-in-human clinical trial.