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.

Infant and adult human intestinal enteroids are morphologically and functionally distinct.

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. 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 then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. 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. 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. IMPORTANCE: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.

THE SMALL INTESTINAL MICROBIOME: VIBING WITH INTESTINAL STEM CELLS.

The epithelial lining of the small intestine mediates its absorptive and secretory function and thus is a critical component of human health. Regeneration and renewal of the epithelium is the result of proliferation of intestinal stem cells (ISCs). Many cell types and molecular factors are known to regulate the ability of ISCs to proliferate, including adjacent neighboring epithelial cells and the underlying, supportive stromal cells. The microbiome resides in the lumen of the small intestine and is in close contact with the epithelium. Due to its proximity to ISCs, it has been hypothesized that species within the microbiome have the capacity to regulate ISC proliferation and differentiation. This review highlights research that probes interactions between ISCs and the microbiome in the small intestine to detail the current understanding of microbial regulation of ISCs. Results from these studies provide important knowledge that can be exploited to identify therapeutic targets or develop novel preventative treatments to treat intestinal diseases.

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.

Two Distinct Genotypes of Spissistilus festinus (Say, 1830) (Hemiptera, Membracidae) in the United States Revealed by Phylogenetic and Morphological Analyses.

Spissistilus festinus (Say, 1830) (Hemiptera: Membracidae) is a frequent pest of leguminous crops in the Southern United States, and a vector of grapevine red blotch virus. There is currently no information on the genetic diversity of S. festinus. In this study, populations of S. festinus were collected in 2015-2017 from various crops and geographic locations in the United States, and fragments of the mitochondrial cytochrome C oxidase 1 (mt-COI) gene and the nuclear internal transcribed spacer 2 (ITS2) region were characterized by polymerase chain reaction and sequencing. Maximum-likelihood and Bayesian analyses of the mt-COI and ITS2 sequences yielded similar phylogenetic tree topologies, revealing two distinct genetic S. festinus lineages with all of the specimens from California comprising one phylogenetic clade, alongside a single GenBank entry from Arizona, and all specimens from the Southeastern United States comprising a statistically-supported distinct clade, regardless of host and year of collection. The mt-COI gene fragment showed up to 10.8% genetic distance between the two phylogenetic clades. These results suggest the existence of two genotypes within S. festinus in the United States. The only distinct morphological trait between the two genotypes was a less elevated pronotum in the representative specimens from California, compared to the representative specimens from the Southeastern United States. Since this phenotypic feature is inconspicuous, a diagnostic polymerase chain reaction targeting a variable region of the mt-COI fragment was developed to reliably distinguish between the specimens of the two genotypes of S. festinus and to facilitate their specific identification.