Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.definitive endoderm ͉ function ͉ hepatocyte ͉ drug metabolism ͉ high throughput H uman embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos and demonstrate pluripotency in vitro and in vivo (1, 2). Such attributes allow hESCs to be differentiated down all germ lineages in large numbers and offer significant advantages over their adult stem cell counterparts, which are generally limited in their capacity to differentiate and proliferate (3). Although these hESCs provide a valuable source of adult differentiated cells, homogeneous cellular differentiation to specific germ layers has proven difficult to achieve. One potential explanation for this failure is that cells do not receive sequential developmental cues that they do in vivo.Wnt signaling has been shown to play an important role in hESC self-renewal and differentiation and stimulates numerous intracellular signal transduction cascades, including the canonical pathway regulating gene expression in the nucleus and what seems to be a network of noncanonical pathways regulating many other aspects of cell biology [reviewed by Cadigan and Liu (4)]. In the absence of Wnt signaling, ␤-catenin is targeted for degradation; however, active Wnt signaling inhibits ␤-catenin destruction, resulting in its nuclear translocation (5, 6). After nuclear localization, ␤-catenin dimerizes with the nuclear proteins from the T cell factor/lymphoid enhancer factor (TCF/ LEF) family and transactivates gene expression. TCF/LEFs are not only present in transcriptional activator complexes; they also play a role in corepressor complex assembly (6).The important role played by Wnt signaling during gastrulation in vivo is evidenced by gene knockouts or dominant negatives (7, 8). Wnt3-mediated Brachyury expression is also important for migration of precursor cells through the anterior region of the primitive streak (PS). The subsequent specification of the anterior region of the PS to mesoderm or endoderm is likely to depend on the duration and magnitude of Nodal signaling (...

With the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient "off the shelf" models of human liver disease. Here, we show the "proof of concept" that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%-90%, using a method mimicking physiological relevant condition. The iPSC-derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E-cadherin, as assessed by immunohistochemistry. They also expressed alpha-fetoprotein, hepatocyte nuclear factor-4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC-derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha-fetoprotein, an essential feature for functional HE. Additionally iPSC-derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing. Conclusion: This work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC-derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models. (HEPATOLOGY 2010;51:329-335.) H uman induced pluripotent stem cells (iPSCs) are reprogrammed mature somatic fibroblasts which represent a pluripotent cell population able to generate all primary cell types in vitro. [1][2][3] The ability to derive iPSCs from an indefinite range of genotypes makes them an attractive resource on which to model liver function reflecting the complexity of polygenic influences on metabolism in vitro. Another facet of iPSC technology is the ability to study the impact of gene polymorphisms in a native chromatin setting and model gene interactions with precision. Therefore iPSC-derived models hold great potential to develop a detailed understanding of human liver disease and metabolism including drug toxicity (for a review, see Dalgetty et al. 4

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Background-Stem/progenitor cell niches in tissues regulate stem/progenitor cell differentiation and proliferation through local signalling.

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A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte-colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD341 cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real-time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 6 2.7 vs. 6.6 6 3.7 vessels, respectively, n 5 9). Bone marrow-derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34 CD1461 mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrowderived circulating precursor for endothelial cells. STEM

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal–epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover.

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