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Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACSisolating a specific crypt-based epithelial population (EpCAM+/ CD44+) from murine small intestine. Genetic biomarkers for stem/ progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium. [ABSTRACT FROM AUTHOR]

Many advances have been reported in the long-term culture of intestinal mucosal cells in recent years. A significant number of publications have described new culture media, cell formations, and growth patterns. Furthermore, it is now possible to study, e.g., the capabilities of isolated stem cells or the interactions between stem cells and mesenchyme. However, at the moment there is significant variation in the way these structures are described and named. A standardized nomenclature would benefit the ability to communicate and compare findings from different laboratories using the different culture systems. To address this issue, members of the NIH Intestinal Stem Cell Consortium herein propose a systematic nomenclature for in vitro cultures of the small and large intestine. We begin by describing the structures that are generated by preparative steps. We then define and describe structures produced in vitro, specifically: enterosphere, enteroid, reconstituted intestinal organoid, induced intestinal organoid, colonosphere, colonoid, and colonic organoid. [ABSTRACT FROM AUTHOR]

Unlike during development, blood vessels in the adult are generally thought not to require VEOF for normal function. However, VEGF is a survival factor for many tumor vessels, and there are clues that some normal blood vessels may also depend on VEGF. In this study, we sought to identify which, if any, vascular beds in adult mice depend on VEGF for survival. Mice were treated with a small-molecule VEGF receptor (VEGFR) tyrosine kinase inhibitor or soluble VEOFRs for 1–3 wk. Blood vessels were assessed using immunohistochemistry or scanning or transmission electron microscopy. In a study of 17 normal organs after VEGF inhibition, we found significant capillary regression in pancreatic islets, thyroid, adrenal cortex, pituitary, choroid plexus, small-intestinal villi, and epididymal adipose tissue. The amount of regression was dose dependent and varied from organ to organ, with a maximum of 68% in thyroid, but was less in normal organs than in tumors in RIP-Tag2-transgenic mice or in Lewis lung carcinoma. VEOF-dependent capillaries were fenestrated, expressed high levels of both VEOFR-2 and VEGFR-3, and had normal pericyte coverage. Surviving capillaries in affected organs had fewer fenestrations and less VEGFR expression. All mice appeared healthy, but distinct physiological changes, including more efficient blood glucose handling, accompanied some regimens of VEGF inhibition. Strikingly, most capillaries in the thyroid grew back within 2 wk after cessation of treatment for 1 wk. Our findings of VEGF dependency of normal fenestrated capillaries and rapid regrowth after regression demonstrate the plasticity of the adult microvasculature. [ABSTRACT FROM AUTHOR]

The vasculature of the embryo requires vascular endothelial growth factor (VEOF) during development, but most adult blood vessels lose VEGF dependence. However, some capillaries in the respiratory tract and selected other organs of adult mice regress after VEGF inhibition. The present study sought to identify the sequence of events and the fate of endothelial cells, pericytes, and vascular basement membrane during capillary regression in mouse tracheas after VEGF signaling was blocked with a VEGF-receptor tyrosine kinase inhibitor AG-013736 or soluble receptor construct (VEGF Trap or soluble adenoviral VEGFR-1). Within I day, patency was lost and fibrin accumulated in some tracheal capillaries. Apoptotic endothelial cells marked by activated caspase-3 were present in capillaries without blood flow. VEGF inhibition was accompanied by a 19% decrease in tracheal capillaries over 7 days and 30% over 21 days. During this period, desmin/NG2- immunoreactive pericytes moved away from regressing capillaries onto surviving vessels. Empty sleeves of basement membrane, left behind by regressing endothelial cells, persisted for about 2 wk and served as a scaffold for vascular regrowth after treatment ended. The amount of regrowth was limited by the number of surviving basement membrane sleeves. These findings demonstrate that, after inhibition of VEGF signaling, some normal capillaries regress in a systematic sequence of events initiated by a cessation of blood flow and followed by apoptosis of endothelial cells, migration of pericytes away from regressing vessels, and formation of empty basement membrane sleeves that can facilitate capillary regrowth. [ABSTRACT FROM AUTHOR]