MicroRNA-221 regulates FAS-induced fulminant liver failure.

UNLABELLED: Death receptor-mediated apoptosis of hepatocytes contributes to hepatitis and fulminant liver failure. MicroRNAs (miRNAs), 19-25 nucleotide-long noncoding RNAs, have been implicated in the posttranscriptional regulation of the various apoptotic pathways. Here we report that global loss of miRNAs in hepatic cells leads to increased cell death in a model of FAS/CD95 receptor-induced apoptosis. miRNA profiling of murine liver identified 11 conserved miRNAs, which were up-regulated in response to FAS-induced fulminant liver failure. We show that ectopic expression of miR-221, one of the highly up-regulated miRNAs in response to apoptosis, protects primary hepatocytes and hepatoma cells from apoptosis. Importantly, in vivo overexpression of miR-221 by adeno-associated virus serotype 8 (AAV8) delays FAS-induced fulminant liver failure in mice. We additionally demonstrate that miR-221 regulates hepatic expression of p53 up-regulated modulator of apoptosis (Puma), a well-known proapoptotic member of the Bcl2 protein family. CONCLUSION: We identified miR-221 as a potent posttranscriptional regulator of FAS-induced apoptosis. miR-221 may serve as a potential therapeutic target for the treatment of hepatitis and liver failure.

Directed differentiation of murine-induced pluripotent stem cells to functional hepatocyte-like cells.

BACKGROUND & AIMS: Induced pluripotent stem (iPS) cells exert phenotypic and functional characteristics of embryonic stem cells even though the gene expression pattern is not completely identical. Therefore, it is important to develop procedures which are specifically oriented to induce iPS cell differentiation. METHODS: In this study, we describe the differentiation of mouse iPS cells to hepatocyte-like cells, following a directed differentiation procedure that mimics embryonic and fetal liver development. The sequential differentiation was monitored by real-time PCR, immunostaining, and functional assays. RESULTS: By sequential stimulation with cytokines known to play a role in liver development, iPS cells were specified to primitive streak/mesendoderm/definitive endoderm. They were then differentiated into two types of cells: those with hepatoblast features and those with hepatocyte characteristics. Differentiated hepatocyte-like cells showed functional properties of hepatocytes, such as albumin secretion, glycogen storage, urea production, and inducible cytochrome activity. Aside from hepatocyte-like cells, mesodermal cells displaying some characteristics of liver sinusoidal endothelium and stellate cells were also detected. CONCLUSIONS: These data demonstrate that a protocol, modeled on embryonic liver development, can induce hepatic differentiation of mouse iPS cells, generating a population of cells with mature hepatic phenotype.

Repopulation efficiencies of adult hepatocytes, fetal liver progenitor cells, and embryonic stem cell-derived hepatic cells in albumin-promoter-enhancer urokinase-type plasminogen activator mice.

Fetal liver progenitor cell suspensions (FLPC) and hepatic precursor cells derived from embryonic stem cells (ES-HPC) represent a potential source for liver cell therapy. However, the relative capacity of these cell types to engraft and repopulate a recipient liver compared with adult hepatocytes (HC) has not been comprehensively assessed. We transplanted mouse and human HC, FLPC, and ES-HPC into a new immunodeficient mouse strain (Alb-uPA(tg(+/-))Rag2(-/-)gamma(c)(-/-) mice) and estimated the percentages of HC after 3 months. Adult mouse HC repopulated approximately half of the liver mass (46.6 +/- 8.0%, 1 x 10(6) transplanted cells), whereas mouse FLPC derived from day 13.5 and 11.5 post conception embryos generated only 12.1 +/- 3.0% and 5.1 +/- 1.1%, respectively, of the recipient liver and smaller cell clusters. Adult human HC and FLPC generated overall less liver tissue than mouse cells and repopulated 10.0 +/- 3.9% and 2.7 +/- 1.1% of the recipient livers, respectively. Mouse and human ES-HPC did not generate HC clusters in our animal model. We conclude that, in contrast to expectations, adult HC of human and mouse origin generate liver tissue more efficiently than cells derived from fetal tissue or embryonic stem cells in a highly immunodeficient Alb-uPA transgenic mouse model system. These results have important implications in the context of selecting the optimal strategy for human liver cell therapies.

Hepatocyte transplantation: waiting for stem cells.

PURPOSE OF REVIEW: Hepatocyte transplantation has been proposed as an attractive therapeutic approach to a variety of liver diseases. Progress, however, in several areas is needed, before this form of therapy is broadly accepted and applied to patients with liver disease. The purpose of the review is to provide the reader with the latest scientific developments relevant for future liver cell therapies. RECENT FINDINGS: Clinical application of hepatocyte transplantation is limited by good quality donor livers for the isolation of cells. Recent publications thus focus on stem cells as suitable sources for hepatocytes and liver repopulation strategies that could reduce the number of transplanted cells. How to overcome host immune responses against allogeneic cells can potentially be learned from a new tolerance protocol. Recently discovered technologies for reprogramming of postnatal cells into pluripotent stem cells may pave the way towards the generation of patient-specific autologous cells. SUMMARY: The current focus of research aims to reduce the shortage of transplantable cells by the application of stem cell sources or by the conditioning of recipient livers. Therapies for severe chronic liver diseases based on adult (stem) cells are already beginning to move into clinical trials. However, many questions about safety and efficacy need to be answered, before fetal liver progenitor cells, embryonic stem cells and induced pluripotent stem cells can be applied in humans.

Induction of a mature hepatocyte phenotype in adult liver derived progenitor cells by ectopic expression of transcription factors.

BACKGROUND/AIMS: By ectopic expression of a distinct combination of transcription factors we aimed to induce a mature hepatocyte phenotype in an adult liver derived progenitor cell population (ALDPC). METHODS: The open reading frames encoding murine Foxa2, Hnf4α and C/ebpα were cloned into lentivirus vectors and sequentially expressed in target cells. After seven days of culture, cells were analysed for expression of liver specific genes, and functional assays were performed. Fresh primary hepatocytes, twenty four hours in culture, served as positive controls. RESULTS: Untransduced ALDPC under established differentiation conditions exhibited moderate signs of maturation, in particular in comparison with fresh hepatocyte controls. In transcription factor transduced cells, fifteen mRNA´s coding for secreted proteins, cytochrome p450 isoenzymes, liver metabolic enzymes were detected by RT-qPCR at levels close to controls. Albumin secretion increased incrementally in single (Foxa2), double (Foxa2, Hnf4α) and triple-transduced cells (Foxa2, Hnf4α, C/ebpα) and reached levels observed in primary hepatocytes. Glycogen storage as determined by PAS staining was detectable in double and triple transduced cells, comparable to controls. Ureagenesis was also induced in triple transduced cells, but at lower levels compared to primary hepatocytes. CONCLUSIONS: Sequential expression of Foxa2, Hnf4α and C/ebpα induces a mature hepatocyte phenotype in an expandable liver derived progenitor cell line.