Personalised versus non-individualised case-based CME: A randomised pilot study.

The PinPoint Case Platform (PPCP) offers independent online case-based CME. To align with personal learning needs, a functionality of needs assessments ("QuickScan") was developed, directing users to follow personalised case journeys. A randomised study was conducted, comparing its effectiveness, time efficiency and user experience with a format of non-individualised case-based learning. Forty-two residents in urology from five European countries were randomly assigned to follow non-individualised case-based learning (control group) or a needs assessment plus personalised case journeys on different topics in prostate cancer. After performing a pre- and post-assessment, both groups showed a similar increase in test scores (Mann-Whitney U = 247; p = .113), but the time needed for completing the learning exercise was significantly lower in the group with the personalised approach (median: 45 vs 90 minutes; Mann-Whitney U = 97.5; p = .0141). The quality of the two learning methods was similarly well received by both groups. In conclusion, learners who followed personalised case journeys learned similarly effective but more time efficient than non-individualised case-based learners. Future studies should determine if these findings can be extrapolated to board-certified physicians following CME activities.

Human stem cell-derived hepatocyte-like cells support Zika virus replication and provide a relevant model to assess the efficacy of potential antivirals.

Zika virus (ZIKV) infection during pregnancy has been extensively linked to microcephaly in newborns. High levels of ZIKV RNA were, however, also detected in mice and non-human primates in organs other than the brain, such as the liver. As ZIKV is a flavivirus closely related to the dengue and yellow fever virus, which are known to cause hepatitis, we here examined whether human hepatocytes are susceptible to ZIKV infection. We demonstrated that both human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) and the Huh7 hepatoma cell line support the complete ZIKV replication cycle. Of three antiviral molecules that inhibit ZIKV infection in Vero cells, only 7-deaza-2'-C-methyladenosine (7DMA) inhibited ZIKV replication in hPSC-HLCs, while all drugs inhibited ZIKV infection in Huh7 cells. ZIKV-infected hPSC-HLCs but not Huh7 cells mounted an innate immune and NFκß response, which may explain the more extensive cytopathic effect observed in Huh7 cells. In conclusion, ZIKV productively infects human hepatocytes in vitro. However, significant differences in the innate immune response against ZIKV and antiviral drug sensitivity were observed when comparing hPSC-HLCs and hepatoma cells, highlighting the need to assess ZIKV infection as well as antiviral activity not only in hepatoma cells, but also in more physiologically relevant systems.

Generation of hepatocyte- and endocrine pancreatic-like cells from human induced endodermal progenitor cells.

Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or ß-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14 transcription factors (TFs) in reprogramming MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic-like cells. We demonstrated that 14 TF-iENDO cells can be expanded for at least 20 passages, differentiate spontaneously to hepatocyte-, endocrine pancreatic-, gut tube-like cells as well as endodermal tumor formation when grafted in immunodeficient mice. Furthermore, iENDO cells can be differentiated in vitro into hepatocyte- and endocrine pancreatic-like cells. However, the pluripotency TF OCT4, which is not silenced in iENDO cells, may contribute to the incomplete differentiation to mature cells in vitro and to endodermal tumor formation in vivo. Nevertheless, the studies presented here provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiate to multiple endodermal cell types, might be a valid alternative for the use of PSCs for creation of endodermal cell types.

Dynamic regulation of EZH2 from HPSc to hepatocyte-like cell fate.

Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging.

Stem cell-derived hepatocytes: A novel model for hepatitis E virus replication.

BACKGROUND & AIMS: Yearly, approximately 20million people become infected with the hepatitis E virus (HEV) resulting in over 3million cases of acute hepatitis. Although HEV-mediated hepatitis is usually self-limiting, severe cases of fulminant hepatitis as well as chronic infections have been reported, resulting annually in an estimated 60,000 deaths. We studied whether pluripotent stem cell (PSC)-derived hepatocytes, mesodermal and/or neuroprogenitor cells support HEV replication. METHODS: Human PSC were differentiated towards hepatocyte-like cells, mesodermal cells and neuroprogenitors and subsequently infected with HEV. Infection and replication of HEV was analyzed by qRT-PCR, RNA in situ hybridization, negative strand RT-PCR, production of infectious virions and transfection with a transient HEV reporter replicon. RESULTS: PSC-derived hepatocytes supported the complete replication cycle of HEV, as demonstrated by the intracellular presence of positive and negative strand HEV RNA and the production of infectious virions. The replication of the virus in these cells was inhibited by the antiviral drugs ribavirin and interferon-α2b. In contrast to PSC-derived hepatocytes, PSC-derived mesodermal cells and neuroprogenitors only supported HEV replication upon transfection with a HEV subgenomic replicon. CONCLUSION: We demonstrate that PSC can be used to study the hepatotropism of HEV infection. The complete replication cycle of HEV can be recapitulated in infected PSC-derived hepatocytes. By contrast other germ layer cells support intracellular replication but are not infectable with HEV. Thus the early steps in the viral cycle are the main determinant governing HEV tissue tropism. PSC-hepatocytes offer a physiological relevant tool to study the biology of HEV infection and replication and may aid in the design of therapeutic strategies.

Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

Understanding the molecular mechanism of host-based statin resistance in hepatitis C virus replicon containing cells.

A number of statins, the cholesterol-lowering drugs, inhibit the in vitro replication of hepatitis C virus (HCV). In HCV-infected patients, addition of statins to the earlier standard of care therapy (pegIFN-α and ribavirin) resulted in increased sustained virological response rates. The mechanism by which statins inhibit HCV replication has not yet been elucidated. In an attempt to gain insight in the underlying mechanism, hepatoma cells carrying an HCV replicon were passaged in the presence of increasing concentrations of fluvastatin. Fluvastatin-resistant replicon containing cells could be generated and proved ∼8-fold less susceptible to fluvastatin than wild-type cultures. The growth efficiency of the resistant replicon containing cells was comparable to that of wild-type replicon cells. The fluvastatin-resistant phenotype was not conferred by mutations in the viral genome but is caused by cellular changes. The resistant cell line had a markedly increased HMG-CoA reductase expression upon statin treatment. Furthermore, the expression of the efflux transporter P-gp was increased in fluvastatin-resistant replicon cells (determined by qRT-PCR and flow cytometry). This increased expression resulted also in an increased functional transport activity as measured by the P-gp mediated efflux of calcein AM. In conclusion, we demonstrate that statin resistance in HCV replicon containing hepatoma cells is conferred by changes in the cellular environment.

Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation.

Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.