David R. Hill

Gastrointestinal Organoids: Understanding the Molecular Basis of the Host–Microbe Interface

In recent years, increasing attention has been devoted to the concept that microorganisms play an integral role in human physiology and pathophysiology. Despite this, the molecular basis of host–pathogen and host–symbiont interactions in the human intestine remains poorly understood owing to the limited availability of human tissue, and the biological complexity of host–microbe interactions. Over the past decade, technological advances have enabled long-term culture of organotypic intestinal tissue derived from human subjects and from human pluripotent stem cells, and these in vitro culture systems already have shown the potential to inform our understanding significantly of host-microbe interactions. Gastrointestinal organoids represent a substantial advance in structural and functional complexity over traditional in vitro cell culture models of the human gastrointestinal epithelium while retaining much of the genetic and molecular tractability that makes in vitro experimentation so appealing. The opportunity to model epithelial barrier dynamics, cellular differentiation, and proliferation more accurately in specific intestinal segments and in tissue containing a proportional representation of the diverse epithelial subtypes found in the native gut greatly enhances the translational potential of organotypic gastrointestinal culture systems. By using these tools, researchers have uncovered novel aspects of host–pathogen and host–symbiont interactions with the intestinal epithelium. Application of these tools promises to reveal new insights into the pathogenesis of infectious disease, inflammation, cancer, and the role of microorganisms in intestinal development. This review summarizes research on the use of gastrointestinal organoids as a model of the host–microbe interface.

Response to Intervention in Secondary Schools: Considerations for Administrators

Secondary school administrators are increasingly finding themselves in the position of implementing Response to Intervention (RTI). This system of providing progressively intensive levels of intervention for the purposes of preventing academic failure and identifying children with learning disability may be useful at the secondary level. However, many aspects of RTI are based on research conducted in elementary schools. The purpose of this article is to provide a basic description of RTI, to summarize research conducted at the secondary level, and to provide a set of considerations for secondary administrators regarding RTI implementation.

Morphogenesis and maturation of the embryonic and postnatal intestine

The intestine is a vital organ responsible for nutrient absorption, bile and waste excretion, and a major site of host immunity. In order to keep up with daily demands, the intestine has evolved a mechanism to expand the absorptive surface area by undergoing a morphogenetic process to generate finger-like units called villi. These villi house specialized cell types critical for both absorbing nutrients from food, and for protecting the host from commensal and pathogenic microbes present in the adult gut. In this review, we will discuss mechanisms that coordinate intestinal development, growth, and maturation of the small intestine, starting from the formation of the early gut tube, through villus morphogenesis and into early postnatal life when the intestine must adapt to the acquisition of nutrients through food intake, and to interactions with microbes.

Natural spline block implicit methods

Two classes of one-step methods for the solution of the ordinary initial value problem are treated. The schemes of orderm give blocks ofm approximate solutions at each step and are constructed fromm integration formulas. Since each formula is obtained by the integration of an interpolatory natural spline, it is best in the sense of Sard. Sufficient conditions for the convergence of the iterative techniques used in each block and of the discrete variable solutions are given. The notion of block stability is introduced and the regions of block stability are given for two methods. Finally, eight block methods are compared by means of some numerical data.

Performance of Students with Significant Cognitive Disabilities on Early-Grade Curriculum-Based Measures of Word and Passage Reading Fluency

Alternate assessments have been used for the last 10 years to evaluate schools efforts to teach children with significant cognitive disabilities. However, few studies have examined the reading skills of children who participate in these assessments. The purpose of this study was to extend understanding of the reading skills of this population by administering early-grade word and passage reading fluency curriculum-based measures to a sample of 7,440 students in Grades 3 through 8 and 11. Overall, the performance on curriculum-based measures and the relationship with alternate assessment performance varied based upon disability, grade, and level of alternate assessment. The authors discuss implications for test developers and teachers along with future directions for research.

Integration Formulas and Schemes Based on g -Splines

Numerical integration formulas of interpolatory type are generated by the inte- gration of g-splines. These formulas, which are best in the sense of Sard, are used to con- struct predictor-corrector and block implicit schemes. The schemes are then compared with Adams-Bashforth-Adams-Moulton and Rosser schemes for a particular set of proto- type problems. Moreover, an improved error bound for linear multistep formulas based on g-splines and a comparison of L2 norms of Peano kernels for Adams and natural spline formulas are given. 1. Introduction. This paper is a sequel to (5), where a systematic technique for constructing linear multistep formulas based on g-splines was given. Here, we shall give examples of g-spline based formulas, construct numerical integration schemes with them, and present some numerical results for the solution of problems of the type

In Vitro Induction and In Vivo Engraftment of Lung Bud Tip Progenitor Cells Derived from Human Pluripotent Stem Cells

Summary The current study aimed to understand the developmental mechanisms regulating bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified cues that maintained isolated human fetal lung epithelial bud tip progenitor cells in vitro and induced three-dimensional hPSC-derived organoids with bud tip-like domains. Bud tip-like domains could be isolated, expanded, and maintained as a nearly homogeneous population. Molecular and cellular comparisons revealed that hPSC-derived bud tip-like cells are highly similar to native lung bud tip progenitors. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed, maintained multilineage potential, and successfully engrafted into the airways of immunocompromised mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.

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.

LGR4 and LGR5 Function Redundantly During Human Endoderm Differentiation

Background & Aims The Lgr family of transmembrane proteins (Lgr4, 5, 6) act as functional receptors for R-spondin proteins (Rspo 1, 2, 3, 4), and potentiate Wnt signaling in different contexts. Lgr5 is arguably the best characterized of the Lgr family members in a number of adult and embryonic contexts in mice. However, the function of LGR family members in early embryonic development is unclear, and has not been explored during human development or tissue differentiation in detail. Methods We interrogated the function and expression of LGR family members using human pluripotent stem cell–derived tissues including definitive endoderm, mid/hindgut, and intestinal organoids. We performed embryonic lineage tracing in Lgr5-GFP-IRES-CreERT2 mice. Results We show that LGR5 is part of the human definitive endoderm (DE) gene signature, and LGR5 transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show that LGR4 and 5 are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observe Lgr5 reporter (eGFP) activity in the embryonic day 8.5 mouse endoderm, and show the ability to lineage trace these cells into the adult intestine. However, gene expression data also suggest that there are human–mouse species-specific differences at later time points of embryonic development. Conclusions Our results show that LGR5 is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process.

Real-time measurement of epithelial barrier permeability in human intestinal organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.