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Human embryonic stem (hES) cells can self-renew, which enables them to have considerable expansion potential, and are pluripotent. If their differentiation can be controlled, they can offer promise for clinical programs in cell therapies. A novel strategy has been developed to derive early hepatocytic lineage stages from hES cells using four sequential inducing steps lasting 16 days. First, embryoid bodies (EBs) were generated by growing hES cells in suspension for 2 days; second, EBs were lineage restricted to definitive endoderm with 3 days of treatment with human activin A; third, cells were differentiated further by coculturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourth, treating them for 6 days with soluble signals comprised of hFLSC-derived bFGF, hepatocyte growth factor, oncostatin M, and dexamethasone. Induced cells displayed morphological, immunohistochemical, and biochemical characteristics of hepatocytic committed progenitors and of early lineage stage hepatocytes found in zone 1 of the liver acinus. They expressed alpha-fetoprotein, albumin, cytokeratin 18, glycogen, a fetal P450 isoform, and CYP1B1, and demonstrated indocyanine green uptake and excretion. In conclusion, we have developed a novel method to lineage restrict hES cells into early lineage stages of hepatocytic fates.

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1These authors contributed equally to this work.Abbreviations used: CART, cocaine-and amphetamine-regulated transcript; ChIP, chromatin immunoprecipitation assay; FBS, fetal bovine serum; HDAC, histone deacetylase; INS-1, insulin-secreting cells; NRSE, neuron-restrictive silencer element; NRSF, neuron-restrictive silencer factor; PC12, pheochromocytoma cells; REST, repressor element-1 silencing transcription factor; shNRSF, short hairpin RNA for NRSF; TSA, trichostatin A. AbstractCocaine-and amphetamine-regulated transcript (CART) peptide is an endogenous peptide which is widely expressed in the CNS and PNS as well as in endocrine cells. Despite the functional knowledge about CART, the mechanisms that regulate CART gene transcription are poorly characterized. Here, we showed that neuron-restrictive silencer factor (NRSF) functions as a negative regulator of CART gene expression in neuroendocrine cells. A putative neuron-restrictive silencer element (NRSE) conserved between the rodent and human CART promoter was identified and demonstrated to bind to NRSF in sequence-specific manner by the electrophoretic mobility shift and chromatin immunoprecipitation assays. Ectopic expression of NRSF in pheochromocytoma cells (PC12) and insulin-secreting cells (INS-1) induced a marked reduction in the level of CART mRNA and the activity of CART promoter or NRSE reporter. The CART promoter showed very low activity in endogenous NRSF-expressing HeLa cells. When expression of NRSF was down-regulated in HeLa cells using a RNA interfering technique, the transcriptional activity of the CART promoter or a NRSE reporter was significantly increased. Taken together, our data suggested that CART gene expression in neuroendocrine cells is strictly controlled by NRSF, via a mechanism dependent upon the CART NRSE.

Background: Cocaine-and amphetamine-regulated transcript (CART) plays a pivotal role in neuroprotection against stroke. Results: Transcriptional repressor neuron-restrictive silencer factor (NRSF) represses CART through binding to two NRSEs in the CART promoter and intron. Conclusion: NRSF represses CART transcription and antagonizes cAMP-response element-binding protein signaling through a dual NRSE mechanism. Significance: This is the first evidence to reveal the repression mechanism of CART transcription. NRSF serves as a therapeutic target for stroke treatment.