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Recent seminal studies have rapidly advanced the understanding of intestinal epithelial stem cell (IESC) biology in murine models. However, the lack of techniques suitable for isolation and subsequent downstream analysis of IESCs from human tissue has hindered the application of these findings toward the development of novel diagnostics and therapies with direct clinical relevance. This study demonstrates that the cluster of differentiation genes CD24 and CD44 are differentially expressed across LGR5 positive "active" stem cells as well as HOPX positive "facultative" stem cells. Fluorescence-activated cell sorting enables differential enrichment of LGR5 (CD24-/CD44+) and HOPX (CD24+/CD44+) cells for gene expression analysis and culture. These findings provide the fundamental methodology and basic cell surface signature necessary for isolating and studying intestinal stem cell populations in human physiology and disease.

Ischemic damage to the intestinal epithelial barrier, such as in necrotizing enterocolitis or small intestinal volvulus, is associated with higher mortality rates in younger patients. We have recently reported a powerful pig model to investigate these age-dependent outcomes in which mucosal barrier restitution is strikingly absent in neonates but can be rescued by direct application of homogenized mucosa from older, juvenile pigs by a yet-undefined mechanism. Within the mucosa, a postnatally developing network of enteric glial cells (EGCs) is gaining recognition as a key regulator of the mucosal barrier. Therefore, we hypothesized that the developing EGC network may play an important role in coordinating intestinal barrier repair in neonates. Neonatal and juvenile jejunal mucosa recovering from surgically induced intestinal ischemia was visualized by scanning electron microscopy and the transcriptomic phenotypes were assessed by bulk RNA sequencing. EGC network density and glial activity were examined by Gene Set Enrichment Analysis, three-dimensional (3-D) volume imaging, and Western blot and its function in regulating epithelial restitution was assessed ex vivo in Ussing chamber using the glia-specific inhibitor fluoroacetate (FA), and in vitro by coculture assay. Here we refine and elaborate our translational model, confirming a neonatal phenotype characterized by a complete lack of coordinated reparative signaling in the mucosal microenvironment. Furthermore, we report important evidence that the subepithelial EGC network changes significantly over the early postnatal period and demonstrate that the proximity of a specific functional population of EGC to wounded intestinal epithelium contributes to intestinal barrier restitution following ischemic injury. [ABSTRACT FROM AUTHOR]

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the [Apc.sup.Min] mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling-induced proliferation, particularly during regeneration and adenoma formation., Introduction Deorphanization of GPCRs remains an active area of research, especially considering that approximately 40% of all approved drugs for humans target only a small fraction of the GPCRome (1, [...]

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic [...]

The inability to identify, isolate, and culture intestinal epithelial stem cells (IESCs) has been prohibitive to the study and therapeutic utilization of these cells. Using a [Sox9.sup.EGFP] mouse model, we demonstrate that [Sox9.sup.EGFP] fluorescence signatures can be used to differentiate between and enrich for progenitors ([Sox9.sup.EGFPsubLo]) and multipotent IESCs ([Sox9.sup.EGFPlo]). [Sox9.sup.EGFPlo] cells generate 'organoids' in a recently defined culture system that mimics the native IESC niche. These organoids possess all four differentiated cell types of the small intestine epithelium, demonstrating the multipotent capacity of [Sox9.sup.EGFPlo] cells. Our results are consistent with the previously reported observation that single IESCs generate cryptlike units without a detectable mesenchymal cell component. A prospective search revealed that CD24 is expressed in the [Sox9.sup.EGFPlo] population and marks IESCs that form organoids in culture. CD24 represents the first cell surface marker that facilitates fluorescence-activated cell sorting enrichment of IESCs with widely available antibodies without requiring a specialized fluorescent reporter gene mouse model. SOX factors; intestinal epithelial culture doi: 10.1152/ajpgi.00470.2009.

The transcription factor nuclear factor-[kappa]B (NF-[kappa]B) is activated during liver regeneration after partial hepatectomy. However, the physiological role and cellular localization of NF-[kappa]B activation are unresolved. In this study, we used an adenoviral vector expressing a mutated form of I[kappa]B[alpha] to inhibit NF-[kappa]B activity during liver regeneration. After partial hepatectomy in mice, introduction of Ad5I[kappa]B, but not a control virus (Ad5GFP), resulted in increased liver injury and decreased hepatocyte proliferation. Hepatocyte apoptosis was not observed. To investigate the kinetics and cellular localization of NF-[kappa]B-induced transcription during liver regeneration, we generated a transgenic mouse expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-[kappa]B cis elements (cis-NF-[kappa]B-EGFP). During liver regeneration, EGFP expression was detected within 12 h and was primarily located in Kupffer cells. Our data demonstrate that activation of NF-[kappa]B initially occurs in Kupffer cells after partial hepatectomy in mice. liver regeneration; nuclear factor-[kappa]; [kappa]B; Kupffer cell