SUMMARYThe availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, we previously established a protocol that facilitates the differentiation of both human embryonic stem cells and induced pluripotent stem cells into cells that share many characteristics with hepatocytes. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation towards a hepatocyte-like fate appeared to recapitulate many of the developmental stages normally associated with the formation of hepatocytes in vivo. In the current study, we addressed the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation. We demonstrate (1) that human embryonic stem cells express a number of mRNAs that characterize each stage in the differentiation process, (2) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses and (3) that the nuclear hormone receptor HNF4A is essential for specification of human hepatic progenitor cells by establishing the expression of the network of transcription factors that controls the onset of hepatocyte cell fate.

Elevated levels of low density lipoprotein cholesterol (LDL-C) in plasma are a major contributor to cardiovascular disease (CVD), which is the leading cause of death worldwide. Genome–wide association studies (GWAS) have identified 95 loci that associate with control of lipid/cholesterol metabolism. Although GWAS results are highly provocative, direct analyses of the contribution of specific allelic variations in regulating LDL-C has been challenging due to the difficulty in accessing appropriate cells from affected patients. The primary cell type responsible for controlling cholesterol and lipid flux is the hepatocyte. Recently we have shown that cells with hepatocyte characteristics can be generated from human induced pluripotent stem cells (iPSC). This finding raises the possibility of using patient–specific iPSC–derived hepatocytes to study the functional contribution of GWAS loci in regulating lipid metabolism. To test the validity of this approach we produced iPSCs from a patient with mutations in the Low density lipoprotein receptor(LDLR) gene that result in familial hypercholesterolemia (FH). Conclusion: We demonstrate that 1) hepatocytes can be efficiently generated from FH iPSCs, 2) in contrast to control cells FH iPSC–derived hepatocytes are deficient in LDL–C uptake, 3) control but not FH iPS cell–derived hepatocytes increase LDL uptake in response to lovastatin, and 4) FH iPSC–derived hepatocytes display a marked elevation in secretion of lipidated ApoB-100. Cumulatively, these findings demonstrate that FH iPSC–derived hepatocytes recapitulate the complex pathophysiology of FH in culture. These results also establish that patient specific iPSC–derived hepatocytes could be used to definitively determine the functional contribution of allelic variation in regulating lipid and cholesterol metabolism and could potentially provide a platform for the identification of novel treatments of CVD.

The gastrointestinal (GI) tract, in simplest terms, can be described as an epithelial-lined muscular tube extending along the cephalocaudal axis from the oral cavity to the anus. Although the general architecture of the GI tract organs is conserved from end to end, the presence of different epithelial tissue structures and unique epithelial cell types within each organ enables each to perform the distinct digestive functions required for efficient nutrient assimilation. Spatiotemporal regulation of signaling pathways and downstream transcription factors controls GI epithelial morphogenesis during development to confer essential regional-specific epithelial structures and functions. Here, we discuss the fundamental functions of each GI tract organ and summarize the diversity of epithelial structures present along the cephalocaudal axis of the GI tract. Next, we discuss findings, primarily from genetic mouse models, that have defined the roles of key transcription factors during epithelial morphogenesis, including p63, SOX2, SOX15, GATA4, GATA6, HNF4A, and HNF4G. Additionally, we examine how the Hedgehog, WNT, and BMP signaling pathways contribute to defining unique epithelial features along the cephalocaudal axis of the GI tract. Lastly, we examine the molecular mechanisms controlling regionalized cytodifferentiation of organ-specific epithelial cell types within the GI tract, concentrating on the stomach and small intestine. The delineation of GI epithelial patterning mechanisms in mice has provided fundamental knowledge to guide the development and refinement of three-dimensional GI organotypic culture models such as those derived from directed differentiation of human pluripotent stem cells and those derived directly from human tissue samples. Continued examination of these pathways will undoubtedly provide vital insights into the mechanisms of GI development and disease and may afford new avenues for innovative tissue engineering and personalized medicine approaches to treating GI diseases.

A more thorough understanding of the genetic architecture underlying obesity-related lipid disorders could someday facilitate cardiometabolic risk reduction through early clinical intervention based upon improved characterization of individual risk. In recent years, there has been tremendous interest in understanding the endocannabinoid system as a novel therapeutic target for the treatment of obesity-related dyslipidemia. Aims N-arachidonylethanolamine activates G-protein-coupled receptors within the endocannabinoid system. Fatty acid amide hydrolase (FAAH) is a primary catabolic regulator of N-acylethanolamines, including arachidonylethanolamine. Genetic variants in FAAH have inconsistently been associated with obesity. It is conceivable that genetic variability in FAAH directly influences lipid homeostasis. The current study characterizes the relationship between FAAH and obesity-related dyslipidemia, in one of the most rigorously-phenotyped obesity study cohorts in the USA. Materials & methods Members of 261 extended families (pedigrees ranging from 4 to 14 individuals) were genotyped using haplotype tagging SNPs obtained for the FAAH locus, including 5 kb upstream and 5 kb downstream. Each SNP was tested for basic obesity-related phenotypes (BMI, waist and hip circumference, waist:hip ratio, fasting glucose, fasting insulin and fasting lipid levels) in 1644 individuals within these 261 families. Each SNP was also tested for association with insulin responsiveness using data obtained from a frequently sampled intravenous glucose tolerance test in 399 individuals (32 extended families). Results A well characterized coding SNP in FAAH (rs324420) was associated with increased BMI, increased triglycerides, and reduced levels of high-density lipoprotein cholesterol. Mean (standard deviation) high-density lipoprotein cholesterol level was 40.5 (14.7) mg/dl for major allele homozygotes, 39.1 (10.4) mg/dl for heterozygotes, and 34.8 (8.1) mg/dl for minor allele homozygotes (p < 0.01, Family-Based Association Test). This SNP was not associated with insulin sensitivity, acute insulin response to intravenous glucose, glucose effectiveness or glucose disposition index. Conclusion Genetic variability in FAAH is associated with dyslipidemia, independent of insulin response.

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