ES cell to cardiac progenitor cell differentiation is disturbed by <i>Mst1/Mst2</i> depletion.

<p>(A) Heatmap of the expression of non-canonical Wnt signaling ligands (Wnt2, Wnt2b and Wnt5a) and canonical Wnt ligands (Wnt1, Wnt3a, Wnt8a and Wnt11) in day 4 and day 8 wild type EBs and <i>Mst-/</i>- EBs. (B) Relative mRNA levels of β-catenin in wild type and <i>Mst-/</i>- EBs at day 4 and day 8 during EB formation. <i>Actin</i> was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Immunoblotting analysis with antibodies against Active β-catenin and total β-catenin to check its expression in day 4 and day 8 wild type EBs and <i>Mst-/</i>- EBs. Gapdh1 was analyzed as an internal control. (D) Relative mRNA levels of Wnt5a during EB formation from day0 to day10. <i>Actin</i> was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (E) Recombinant Wnt5 were supplemented to the <i>Mst-/-</i>EB culture from day 2 and day 10. Wild type EBs and <i>Mst-/</i>- EBs were grown in non-Wnt5a supplemented medium as controls. The percentage of beating EBs was profiled on day 8 and day 10 after initiating EBs culture. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).</p

Characterization of Mst-/- ES cells.

<p>(A) Quantitative real-time PCR to examine the mRNA level of pluripotent markers <i>Pou5f1</i>, <i>Sox2</i> and <i>Nanog</i> in wild type ES cells and <i>Mst-/</i>- knockout ES cells. <i>Actin</i> was analyzed as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (B) Immunofluorescence staining of the pluripotent protein Oct4 and SSEA1 expression in wild type ES cells and <i>Mst-/</i>- knockout ES cells. Neuclei were stained with DAPI. Scale bar, 200μm. (<i>C</i>) Immunoblotting and densitometric analysis of Nanog and Oct4 in wild type ES cells and <i>Mst-/</i>- ES cells. Gapdh1 was analyzed as an internal control. The data are shown as the mean ± S.D (n=2). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting and densitometric analysis of the expression of Yap and phosphorylated Yap (YapS127) in wild type ES cells and <i>Mst-/</i>- ES cells. Gapdh1 was analyzed as an internal control. The data are shown as the mean ± S.D (n=2). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).</p

<i>Mst-/-</i> ES cells proliferate faster than wild type ES cells.

<p>(A) Morphology of 1x10⁵ wild type ES cells or <i>Mst-/</i>- ES cells grown in 2i+LIF ES medium for 2 days, 3 days and 4 days respectively. Scale bar, 200 μm. (B) Statistical analysis of the growth rate of wild type ES cells and <i>Mst-/</i>- ES cells on day 3 and day 4 culture. The data were shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Immunofluorescence staining with BrdU antibodies to examine BrdU integration in wild type ES cells and <i>Mst-/</i>- ES cells after serum starvation for 12 hours. Cells are pulsed labeled with BrdU for 45 minutes. The nuclei were stained with DAPI. Scale bar, 200 μm. (D) Representative histograms of cell cycle distribution in <i>Mst-/</i>- ES cells and wild type ES cells. (E) Table of the cell cycle distribution in <i>Mst-/</i>- ES cells and wild type ES cells from two independent experiments. (F) Statistical analysis of cell cycle distribution in <i>Mst-/</i>- ES cells and wild type ES cells from two independent experiments. (*, P<0.05).</p

Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation

<div><p>The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived <i>Mst1/Mst2</i> double knockout (<i>Mst-/-</i>) ES cells to completely perturb Hippo signaling. We found that <i>Mst-/-</i> ES cells express higher level of Nanog than wild type ES cells and show differentiation resistance after LIF withdrawal. They also proliferate faster than wild type ES cells. Although <i>Mst-/-</i> ES cells can form embryoid bodies (EBs), their differentiation into tissues of three germ layers is distorted. Intriguingly, <i>Mst-/-</i> ES cells are unable to form teratoma. <i>Mst-/-</i> ES cells can differentiate into mesoderm lineage, but further differentiation to cardiac lineage cells is significantly affected. Microarray analysis revealed that ligands of non-canonical Wnt signaling, which is critical for cardiac progenitor specification, are significantly repressed in <i>Mst-/-</i> EBs. Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation.</p> </div

Isolation of <i>Mst-/-</i> ES cells.

<p>(A) Genotyping of wild type (WT) ES cells and <i>Mst-/</i>- ES cells derived from blastocysts by PCR amplification of genomic DNA. Wild type ES cells showed a larger band while <i>Mst-/</i>- ES cells displayed a smaller band. <i>Actin</i> was used as an internal control. (B) Phase contrast microscopy of wild type (WT) and two independent <i>Mst-/</i>- knockout ES cell lines (<i>Mst-/-</i>1 and <i>Mst-/-</i>2) grown on 0.2% gelatin in 2i+LIF medium (Upper). These cells were stained for alkaline phosphatase (Lower). Scale bar, 200 μm. (C) mRNA level of <i>Mst1</i> and <i>Mst2</i> in wild type ES cells and <i>Mst-/</i>- ES cells examined by quantitative real-time PCR using primers flanking the deleted region of <i>Mst</i>1 and <i>Mst</i>2. The data are shown as the mean ± S.D (n=3). <i>Actin</i> was normalized as an internal control. Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunoblotting analysis of the expression of Mst1 and Mst2 in wild type ES cells and <i>Mst-/</i>- ES cells. Gapdh1 was used as a loading control.</p

Differentiation resistance of <i>Mst-/-</i> ES cells.

<p>(A) Morphology of wild type ES cells and <i>Mst-/</i>- ES cells initially and 24 hour after growing in ES cell differentiation medium supplemented with RA, but not 2i and LIF. Scale bar, 200 μm. (B) Quantitative real-time PCR to examine the mRNA level of <i>Yap</i>, <i>Pou5f1</i> and <i>Nanog</i> in wild type ES cells and <i>Mst-/</i>- ES cells during ES cell differentiation medium for 12 hours and 24 hours. <i>Actin</i> was analyzed as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Immunoblotting and densitometric analysis of Yap, YapS127, Oct4 and Nanog in wild type ES cells and <i>Mst-/</i>- ES cells in ES cell differentiation medium for 12 hours and 24 hours. Gapdh1 was analyzed as an internal loading control. The data are shown as the mean ± S.D (n=2). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).</p

ES cell to cardiac progenitor cell differentiation is disturbed by <i>Mst1/Mst2</i> depletion.

<p>(A) Phase contrast pictures of differentiated wild type EBs and <i>Mst-/</i>- EBs in cardiac differentiation medium. Scale bar, 200 μm. (B) Percentage of spontaneously beating EBs determined from day 6 to day 20 during differentiation (n>100 per time point). <i>Mst-/-</i>EBs showed a significant less beating EBs than wild type EBs. Experiments were performed in triplicate, and error bars represent SD. Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Relative mRNA levels of cardiac progenitor cell markers <i>Nkx2.5, Tbx5, Mesp1, Isl1</i> and <i>Baf60c</i> in wild type and <i>Mst-/</i>- EBs at day 4 and day 8 during EB formation. <i>Actin</i> was used as an internal control. The data are shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (D) Immunofluorescence stain with antibody against Nkx2.5 to examine cardiac progenitor marker <i>Nkx2.5</i> expression in the wild type EBs and <i>Mst-/</i>- EBs in cardiac differentiation medium for 8 days. Scale bar, 200 μm. (E) Immunoblotting and denstitometric analysis with antibody against Mesp1, Isl1 and Nkx2.5 to check their expression in wild type EBs and <i>Mst-/</i>- EBs in cardiac differentiation medium for 4 days or 8 days. Gapdh1 was analyzed as an internal control. The data are shown as the mean ± S.D (n=2). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).</p

Depletion of <i>Mst1/Mst2</i> affects proper EB differentiation.

<p>(A) Quantitative real time PCR to reveal the mRNA level of endoderm markers <i>Gata6</i> and <i>Sox17</i>, mesoderm markers <i>T</i> and <i>Gcs</i>, and ectoderm markers <i>Sox1</i> and <i>Nestin</i> in wild type EBs and <i>Mst-/</i>- EBs at day 4 and day 8 during EB formation. <i>Actin</i> was analyzed as an internal control. The data were shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (B) Immunoblotting and densitometric analysis to check the protein level of Yap, YapS127, Oct4 and Nanog in day 4 and day 8 wild type EBs and <i>Mst-/</i>- EBs. Gapdh1 was analyzed as an internal control. The data are shown as the mean ± S.D (n=2). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).</p

HMGB1/RAGE Signaling and Pro-Inflammatory Cytokine Responses in Non-HIV Adults with Active Pulmonary Tuberculosis

<div><p>Background</p><p>We aimed to study the pathogenic roles of High-Mobility Group Box 1 (HMGB1) / Receptor-for-Advanced-Glycation-End-products (RAGE) signaling and pro-inflammatory cytokines in patients with active pulmonary tuberculosis (PTB).</p><p>Methods</p><p>A prospective study was conducted among non-HIV adults newly-diagnosed with active PTB at two acute-care hospitals (n = 80); age-and-sex matched asymptomatic individuals (tested for latent TB) were used for comparison (n = 45). Plasma concentrations of 8 cytokines/chemokines, HMGB1, soluble-RAGE, and transmembrane-RAGE expressed on monocytes/dendritic cells, were measured. Gene expression (mRNA) of HMGB1, RAGE, and inflammasome-NALP3 was quantified. Patients’ PBMCs were stimulated with recombinant-HMGB1 and MTB-antigen (lipoarabinomannan) for cytokine induction e<i>x vivo</i>.</p><p>Results</p><p>In active PTB, plasma IL-8/CXCL8 [median(IQR), 6.0(3.6–15.1) <i>vs</i> 3.6(3.6–3.6) pg/ml, P<0.001] and IL-6 were elevated, which significantly correlated with mycobacterial load, extent of lung consolidation (<b><i>r</i></b>_{<b><i>s</i></b>} +0.509, P<0.001), severity-score (<b><i>r</i></b>_{<b><i>s</i></b>} +0.317, P = 0.004), and fever and hospitalization durations (<b><i>r</i></b>_{<b><i>s</i></b>} +0.407, P<0.001). IL-18 and sTNFR1 also increased. Plasma IL-8/CXCL8 (adjusted OR 1.12, 95%CI 1.02–1.23 per unit increase, P = 0.021) and HMGB1 (adjusted OR 1.42 per unit increase, 95%CI 1.08–1.87, P = 0.012) concentrations were independent predictors for respiratory failure, as well as for ICU admission/death. Gene expression of HMGB1, RAGE, and inflammasome-NALP3 were upregulated (1.2−2.8 fold). Transmembrane-RAGE was increased, whereas the decoy soluble-RAGE was significantly depleted. RAGE and HMGB1 gene expressions positively correlated with cytokine levels (IL-8/CXCL8, IL-6, sTNFR1) and clinico-/radiographical severity (e.g. extent of consolidation <i>r</i>_s +0.240, P = 0.034). <i>Ex vivo</i>, recombinant-HMGB1 potentiated cytokine release (e.g. TNF-α) when combined with lipoarabinomannan.</p><p>Conclusion</p><p>In patients with active PTB, HMGB1/RAGE signaling and pro-inflammatory cytokines may play important roles in pathogenesis and disease manifestations. Our clinico-immunological data can provide basis for the development of new strategies for disease monitoring, management and control.</p></div

Expressions of RAGE, HMGB1 and inflammasome in active PTB patients, compared with IGRA-positive and IGRA-negative asymptomatic individuals.

<p>Expressions of RAGE, HMGB1 and inflammasome in active PTB patients, compared with IGRA-positive and IGRA-negative asymptomatic individuals.</p