Generation of hepatocytes from human embryonic stem cells (hESCs) could represent an advantageous source of cells for cell therapy approaches as an alternative to orthotopic liver transplantation. However, the generation of differentiated hepatocytes from hESCs remains a major challenge, especially using a method compatible with clinical applications. We report a novel approach to differentiate hESCs into functional hepatic cells using fully defined culture conditions, which recapitulate essential stages of liver development. hESCs were first differentiated into a homogenous population of endoderm cells using a combination of activin, fibroblast growth factor 2, and bone morphogenetic protein 4 together with phosphoinositide 3-kinase inhibition. The endoderm cells were then induced to differentiate further into hepatic progenitors using fibroblast growth factor 10, retinoic acid, and an inhibitor of activin/nodal receptor. After further maturation, these cells expressed markers of mature hepatocytes, including asialoglycoprotein receptor, tyrosine aminotransferase, ␣1-antitrypsin, Cyp7A1, and hepatic transcription factors such as hepatocyte nuclear factors 4␣ and 6. Furthermore, the cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, cytochrome activity, and low-density lipoprotein uptake. After transduction with a green fluorescent protein-expressing lentivector and transplantation into immunodeficient uPA transgenic mice, differentiated cells engrafted into the liver, grew, and expressed human albumin and ␣1-antitrypsin as well as green fluorescent protein for at least 8 weeks. In addition, we showed that hepatic cells could be generated from human-induced pluripotent cells derived from reprogrammed fibroblasts, demonstrating the efficacy of this approach with pluripotent stem cells of diverse origins. Conclusion: We have developed a robust and efficient method to differentiate pluripotent stem cells into hepatic cells, which exhibit characteristics of human hepatocytes. Our approach should facilitate the development of clinical grade hepatocytes for transplantation and for research on drug discovery. (HEPATOLOGY 2010;51:1754-1765

The pluripotent status of embryonic stem cells (ESCs) confers upon them the capacity to differentiate into the three primary germ layers, ectoderm, mesoderm and endoderm, from which all the cells of the adult body are derived. An understanding of the mechanisms controlling pluripotency is thus essential for driving the differentiation of human pluripotent cells into cell types useful for clinical applications. The Activin/Nodal signalling pathway is necessary to maintain pluripotency in human ESCs and in mouse epiblast stem cells (EpiSCs), but the molecular mechanisms by which it achieves this effect remain obscure. Here, we demonstrate that Activin/Nodal signalling controls expression of the key pluripotency factor Nanog in human ESCs and in mouse EpiSCs. Nanog in turn prevents neuroectoderm differentiation induced by FGF signalling and limits the transcriptional activity of the Smad2/3 cascade, blocking progression along the endoderm lineage. This negative-feedback loop imposes stasis in neuroectoderm and mesendoderm differentiation, thereby maintaining the pluripotent status of human ESCs and mouse EpiSCs.

The histidine-rich amphipathic peptide LAH4 has antibiotic and DNA delivery capabilities. The peptide has a strong affinity for anionic lipids found in the outer membrane of bacterial membranes. A role for anionic lipids in release of cationic plasmid-containing complexes has been proposed previously, and disruption of membrane asymmetry and presentation of phosphatidylserine (PS) in the membrane outer leaflet is a general feature observed in diseased mammalian cells. Therefore, to understand the peptide-lipid interactions in more detail, solid-state NMR experiments on model membranes have been performed. 31P MAS NMR on mixed phosphatidylcholine (PC)/PS and PC/phosphatidylglycerol (PG) membranes has been used to demonstrate a strong interaction between LAH4 and anionic lipids. By using deuterated lipids and wide-line 2H NMR when probing lipid chain order, it is demonstrated that LAH4 preferentially interacts with PS over PC and effectively disorders the anionic PS lipid fatty acyl chains. In addition, we demonstrate that the efficiency of gene transfer in vitro to different cell lines is closely related to the degree of disruption of PS acyl chains for four isomers of LAH4. This work suggests a mechanism of selective destabilization by LAH4 of anionic lipids in the membranes of cells during transfection with implications for nucleic acid delivery in vivo.

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