Human embryonic stem cells (hESCs) may provide a cell source for functional hepatocytes for clinical applications and drug development. Initially, the hESC population was enriched to be more than 85% definitive endoderm (DE) as assessed by the expression of CXCR4, SOX17, and FOXA2. We then successfully converted DE into hepatic progenitors with 93% of the cells being positive for a-feto protein within 9 days. The percentage of albumin positive cells gradually increased to 90% at days 20-22 after differentiation. Moreover, our hESC-derived hepatocytes (hEH) developed a complete biotransformation system including phase I and II metabolizing enyzmes and phase III transporters. Nuclear receptors, which are critical in regulating the expression of metabolizing enzymes, were also expressed by our hEH. Using ultraperformance liquid chromatography-tandem mass spectrometry technology, we identified seven metabolic pathways of the drug bufuralol including four newly-reported ones in our hEH, which are the same as those in freshly isolated human primary hepatocytes (hPH). In addition, the results of the metabolism of four drugs indicate that our hEH have the capacity to metabolize these drugs at levels that are comparable to hPH. In conclusion, we have generated a relatively homogenous population of hepatocytes from hESCs, which appear to have complete metabolic function that is comparable to primary liver cells. These results represent a significant step towards the efficient differentiation of mature hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies. STEM CELLS 2010;28:674-686 Disclosure of potential conflicts of interest is found at the end of this article.
RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair.
This article considers a decentralized supply chain in which a single manufacturer is selling a perishable product to a single retailer facing uncertain demand. It differs from traditional supply chain contract models in two ways. First, while traditional supply chain models are based on risk neutrality, this article takes the viewpoint of behavioral principal-agency theory and assumes the manufacturer is risk neutral and the retailer is loss averse. Second, while gain/loss (GL) sharing is common in practice, there is a lack of analysis of GL-sharing contracts in the supply chain contract literature. This article investigates the role of a GL-sharing provision for mitigating the loss-aversion effect, which drives down the retailer order quantity and total supply chain profit. We analyze contracts that include GL-sharing-and-buyback (GLB) credit provisions as well as the special cases of GL contracts and buyback contracts. Our analytical and numerical results lend insight into how a manufacturer can design a contract to improve total supply chain, manufacturer, and retailer performance. In particular, we show that there exists a special class of distribution-free GLB contracts that can coordinate the supply chain and arbitrarily allocate the expected supply chain profit between the manufacturer and retailer; in contrast with other contracts, the parameter values for contracts in this class do not depend on the probability distribution of market demand. This feature is meaningful in practice because (i) the probability distribution of demand faced by a retailer is typically unknown by the manufacturer and (ii) a manufacturer can offer the same contract to multiple noncompeting retailers that differ by demand distribution and still coordinate the supply chains.
Human induced pluripotent stem cells (hiPSCs) hold great potential for use in regenerative medicine, novel drug development, and disease progression/developmental studies. Here, we report highly efficient differentiation of hiPSCs toward a relatively homogeneous population of functional hepatocytes. hiPSC-derived hepatocytes (hiHs) not only showed a high expression of hepatocyte-specific proteins and liver-specific functions, but they also developed a functional biotransformation system including phase I and II metabolizing enzymes and phase III transporters. Nuclear receptors, which are critical for regulating the expression of metabolizing enzymes, were also expressed in hiHs. hiHs also responded to different compounds/inducers of cytochrome P450 as mature hepatocytes do. To follow up on this observation, we analyzed the drug metabolizing capacity of hiHs in real time using a novel ultraperformance liquid chromatography-tandem mass spectrometry. We found that, like freshly isolated primary human hepatocytes, the seven major metabolic pathways of the drug bufuralol were found in hiHs. In addition, transplanted hiHs engrafted, integrated, and proliferated in livers of an immune-deficient mouse model, and secreted human albumin, indicating that hiHs also function in vivo. In conclusion, we have generated a method for the efficient generation of hepatocytes from induced pluripotent stem cells in vitro and in vivo, and it appears that the cells function similarly to primary human hepatocytes, including developing a complete metabolic function. These results represent a significant step toward using patient/disease-specific hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies. STEM CELLS TRANS- LATIONAL MEDICINE 2013;2:409 -419
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