Probes for INS

Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.

We previously identified an up-regulation of specific Wnt proteins in the cholangiocyte compartment during cholestatic liver injury and found that mice lacking Wnt secretion from hepatocytes and cholangiocytes showed fewer proliferating cholangiocytes and high mortality in response to a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, a murine model of primary sclerosing cholangitis. In vitro studies demonstrated that Wnt7b, one of the Wnts up-regulated during cholestasis, induces proliferation of cholangiocytes in an autocrine manner and increases secretion of proinflammatory cytokines. We hypothesized that loss of Wnt7b may exacerbate some of the complications of cholangiopathies by decreasing the ability of bile ducts to induce repair. Wnt7b-flox mice were bred with Krt19-cre mice to deplete Wnt7b expression in only cholangiocytes (CC) or with albumin-Cre mice to delete Wnt7b expression in both hepatocytes and cholangiocytes (HC + CC). These mice were placed on a DDC diet for 1 month then killed for evaluation. Contrary to our expectations, we found that mice lacking Wnt7b from CC and HC + CC compartments had improved biliary injury, decreased cellular senescence, and lesser bile acid accumulation after DDC exposure compared to controls, along with decreased expression of inflammatory cytokines. Although Wnt7b knockout (KO) resulted in fewer proliferating cholangiocytes, CC and HC + CC KO mice on a DDC diet also had more hepatocytes expressing cholangiocyte markers compared to wild-type mice on a DDC diet, indicating that Wnt7b suppression promotes hepatocyte reprogramming. Conclusion: Wnt7b induces a proproliferative proinflammatory program in cholangiocytes, and its loss is compensated for by conversion of hepatocytes to a biliary phenotype during cholestatic injury.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

Neurons in the cortex are heterogenous, sending diverse axonal projections to multiple brain regions. Unraveling the logic of these projections requires single-neuron resolution. Although a growing number of techniques have enabled high-throughput reconstruction, these techniques are typically limited to dozens or at most hundreds of neurons per brain, requiring that statistical analyses combine data from different specimens. Here we present axonal BARseq, a high-throughput approach based on reading out nucleic acid barcodes using in situ RNA sequencing, which enables analysis of even densely labeled neurons. As a proof of principle, we have mapped the long-range projections of >8000 mouse primary auditory cortex neurons from a single brain. We identified major cell types based on projection targets and axonal trajectory. The large sample size enabled us to systematically quantify the projections of intratelencephalic (IT) neurons, and revealed that individual IT neurons project to different layers in an area-dependent fashion. Axonal BARseq is a powerful technique for studying the heterogeneity of single neuronal projections at high throughput within individual brains.