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1.  Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells 
The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.
PMCID: PMC3659681  PMID: 23197756
Mesenchymal stem cells; Pluripotent stem cells; Differentiation; Induced pluripotent stem cells; Embryonic stem cells
2.  Utilization of IκB–EGFP Chimeric Gene as an Indicator to Identify Microbial Metabolites with NF-κB Inhibitor Activity 
Biological Procedures Online  2010;12:131-138.
NF-κB regulates several important expressions, such as cytokine release, anti-apoptosis, adhesion molecule expression, and cell cycle processing. Several NF-κB inhibitors have been discovered as an anti-tumor or anti-inflammatory drug. The activity of NF-κB transcription factor is negatively regulated by IκB binding. In this study, IκB assay system was established and IκB–EGFP fusion protein was used as an indicator to monitor the effects of substances on the IκB degradation. The results indicated that the chosen hydroquinone could inhibit the IκB degradation and cause the cell de-attachment from the bottom of culture plate. In addition, this system could also monitor the IκB degradation of microbial metabolite of natural mixtures of propolis. Thus, the IκB assay system may be a good system for drug discovery related to microbial metabolite.
PMCID: PMC3055915  PMID: 21406073
Microbial metabolite; Antioxidant; IκB; EGFP; Hydroquinone; Propolis
3.  ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿  
Journal of Virology  2008;82(19):9345-9358.
The small hepatitis delta virus (HDV) antigen (SHDAg) plays an essential role in HDV RNA double-rolling-circle replication. Several posttranslational modifications (PTMs) of HDAgs, including phosphorylation, acetylation, and methylation, have been characterized. Among the PTMs, the serine 177 residue of SHDAg is a phosphorylation site, and its mutation preferentially abolishes HDV RNA replication from antigenomic RNA to genomic RNA. Using coimmunoprecipitation analysis, the cellular kinases extracellular signal-related kinases 1 and 2 (ERK1/2) are found to be associated with the Flag-tagged SHDAg mutant (Ser-177 replaced with Cys-177). In an in vitro kinase assay, serine 177 of SHDAg was phosphorylated directly by either Flag-ERK1 or Flag-ERK2. Activation of endogenous ERK1/2 by a constitutively active MEK1 (hemagglutinin-AcMEK1) increased phosphorylation of SHDAg at Ser-177; this phosphorylation was confirmed by immunoblotting using an antibody against phosphorylated S177 and mass spectrometric analysis. Interestingly, we found an increase in the HDV replication from antigenomic RNA to genomic RNA but not in that from genomic RNA to antigenomic RNA. The Ser-177 residue was critical for SHDAg interaction with RNA polymerase II (RNAPII), the enzyme proposed to regulate antigenomic RNA replication. These results demonstrate the role of ERK1/2-mediated Ser-177 phosphorylation in modulating HDV antigenomic RNA replication, possibly through RNAPII regulation. The results may shed light on the mechanisms of HDV RNA replication.
PMCID: PMC2546944  PMID: 18632853
4.  Nucleolar Targeting of Hepatitis Delta Antigen Abolishes Its Ability To Initiate Viral Antigenomic RNA Replication▿  
Journal of Virology  2007;82(2):692-699.
Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.
PMCID: PMC2224599  PMID: 17989182

Results 1-4 (4)