Priya H. Dedhia

Transcriptome-wide Analysis Reveals Hallmarks of Human Intestine Development and Maturation In Vitro and In Vivo.

Summary Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation.

A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro, and become more adult-like in their structure, cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al., 2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment, long-term survival, and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue, we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue, resulting in airway-like structures that were remarkably similar to the native adult human lung. DOI: http://dx.doi.org/10.7554/eLife.19732.001

Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis

Objective We tested the ability of Notch pathway receptors Notch1 and Notch2 to regulate stem and epithelial cell homoeostasis in mouse and human gastric antral tissue. Design Mice were treated with the pan-Notch inhibitor dibenzazepine (DBZ) or inhibitory antibodies targeting Notch1 and/or Notch2. Epithelial proliferation, apoptosis and cellular differentiation were measured by histological and molecular approaches. Organoids were established from mouse and human antral glands; growth and differentiation were measured after treatment with Notch inhibitors. Results Notch1 and Notch2 are the predominant Notch receptors expressed in mouse and human antral tissue and organoid cultures. Combined inhibition of Notch1 and Notch2 in adult mice led to decreased epithelial cell proliferation, including reduced proliferation of LGR5 stem cells, and increased apoptosis, similar to the response to global Notch inhibition with DBZ. Less pronounced effects were observed after inhibition of individual receptors. Notch pathway inhibition with DBZ or combined inhibition of Notch1 and Notch2 led to increased differentiation of all gastric antral lineages, with remodelling of cells to express secretory products normally associated with other regions of the GI tract, including intestine. Analysis of mouse and human organoids showed that Notch signalling through Notch1 and Notch2 is intrinsic to the epithelium and required for organoid growth. Conclusions Notch signalling is required to maintain gastric antral stem cells. Notch1 and Notch2 are the primary Notch receptors regulating epithelial cell homoeostasis in mouse and human stomach.

In vitro patterning of pluripotent stem cell-derived intestine recapitulates in vivo human development.

ABSTRACT The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3β inhibitor that stabilizes β-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research. Summary: Human embryonic stem cell-derived intestinal organoids can be patterned into duodenum-like or ileum-like tissue, recapitulating in vivo human development.

Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium

The human gastrointestinal tract is immature at birth, yet must adapt to dramatic changes such as oral nutrition and microbial colonization. The confluence of these factors can lead to severe inflammatory disease in premature infants; however, investigating complex environment-host interactions is difficult due to limited access to immature human tissue. Here, we demonstrate that the epithelium of human pluripotent stem-cell-derived human intestinal organoids is globally similar to the immature human epithelium and we utilize HIOs to investigate complex host-microbe interactions in this naive epithelium. Our findings demonstrate that the immature epithelium is intrinsically capable of establishing a stable host-microbe symbiosis. Microbial colonization leads to complex contact and hypoxia driven responses resulting in increased antimicrobial peptide production, maturation of the mucus layer, and improved barrier function. These studies lay the groundwork for an improved mechanistic understanding of how colonization influences development of the immature human intestine.

Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling

In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

Resident training in a new robotic thoracic surgery program.

BACKGROUND The volume of robot-assisted operations has drastically increased over the past decade. New programs have focused on training surgeons, whereas resident training has lagged behind. The objective of this study was to evaluate our institutional experience with resident participation in thoracic robotic surgery cases since the initiation of our program. METHODS The first 100 robotic thoracic surgery cases at our institution were retrospectively reviewed and categorized into three sequential cohorts. Procedure type, patient and operative characteristics, level of resident participation (primary surgeon [PS] or assistant), and postoperative variables were evaluated. RESULTS Of the first 100 cases, 38% were lung resections, 23% were esophageal operations, and 20% were sympathectomies. The distribution of cases changed over time with the proportion of pulmonary resections significantly increasing. Patient age (P < 0.05), body mass index (P = not significant [NS]), and comorbidities (P = NS) increased over time. Resident participation as PS increased from 33%-59% between the early and late cohorts (P < 0.05). A subset analysis of the 20 lobectomies (7 attending PS, 13 residents) showed similar patient characteristics (P = NS): age (67 versus 69), body mass index (29.5 versus 26.1), and American Society of Anesthesiologists category (2.8 versus 2.8). Operative and postoperative characteristics were also similar (P = NS) regardless of PS: operative time (260 versus 249 min), estimated blood loss (187 versus 203 mL), and length of stay (4.8 versus 4.7 d). CONCLUSIONS Residents can participate as the PS in a variety of thoracic operations during the implementation of a robotics program. Operative time, estimated blood loss, and length of stay were similar regardless of level of resident participation.

Identification, isolation, and characterization of human LGR5-positive colon adenoma cells

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis. Summary: Immunohistochemical and transcriptomic analyses of organoids generated from precancerous adenoma, colon adenocarcinoma and normal human tissue shows that the intestinal stem cell marker LGR5 is a colon cancer prognostic factor.

Chronic stress and intestinal barrier dysfunction: Glucocorticoid receptor and transcription repressor HES1 regulate tight junction protein Claudin-1 promoter

Chronic stress and elevated glucocorticoid hormone are associated with decreases in the intestinal epithelial tight junction protein claudin-1 (CLDN1). Human/rat CLDN1 promoters contain glucocorticoid response elements (GREs) and adjacent transcription repressor HES1 binding N-boxes. Notch signaling target HES1 expression was high and glucocorticoid receptor (NR3C1) low at the crypt base and the pattern reversed at the crypt apex. Chronic stress reduced overall rat colon HES1 and NR3C1 that was associated with CLDN1 downregulation. Chromatin-immunoprecipitation experiments showed that HES1 and NR3C1 bind to the CLDN1 promoter in rat colon crypts. The binding of NR3C1 but not HES1 to CLDN1 promoter significantly decreased in chronically stressed animals, which was prevented by the NR3C1 antagonist RU486. We employed the 21-day Caco-2/BBe cell model to replicate cell differentiation along the crypt axis. HES1 siRNA treatment early in differentiation increased CLDN1. In contrast, stress levels of cortisol decreased CLDN1 in late differentiation stage but not in the early stage. HES1 was high, whereas NR3C1 and CLDN1 were low in the early stage which reversed in the late stage, e.g. HES1/NR3C1 binding to CLDN1 promoter demonstrates a dynamic and reciprocal pattern. These results suggest that chronic stress impairs colon epithelium homeostasis and barrier function via different mechanisms along the crypt axis.

Strategic questioning in surgical education

In the complex and nuanced world of surgical education, oneway teaching – often in the form of directives from the attending physician to the resident (i.e. from specialist to trainee) – is insuffi cient for understanding the needs of trainees and developing safe opportunities for learner advancement. We propose a novel learnercentred approach to intraoperative teaching using questioning that integrates: (1) Socratic questioning and (2) Bloom ’ s Taxonomy, which respectively incorporates wait time and progressively complex questioning to stimulate higherorder thinking. 1–4 As surgical faculty members, surgical residents and medical educators, we collaborate on this actionoriented initiative to enhance trainee education. The need for this multidimensional team approach has become particularly important in the context of educational time constraints, as a side effect of trainee workhour restrictions and the exponential growth in surgical innovations, interventions and technologies.