Activin-A and Bmp4 Levels Modulate Cell Type Specification during CHIR-Induced Cardiomyogenesis

The use of human pluripotent cell progeny for cardiac disease modeling, drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success, recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR, which in turn induces the Activin-A and Bmp pathways, is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis, and to ultimately promote myocyte maturation, we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq, induction with CHIR during Day 1 (Days 0–1) was followed by immediate expression of Nodal ligands and receptors, followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50–100 ng/ml) during Day 0–1 efficiently induced definitive endoderm, whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency, even when CHIR alone was ineffective. Moreover, co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis, as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast, co-induction with CHIR plus low levels (3–10 ng/ml) of Bmp4 during Day 0–1 consistently and strongly inhibited cardiomyogenesis. These findings, which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression, are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages, while Bmp levels regulate subsequent allocation into mesodermal cell types

Super-Enhancers at the Nanog Locus Differentially Regulate Neighboring Pluripotency-Associated Genes

Super-enhancers are tissue-specific cis-regulatory elements that drive expression of genes associated with cell identity and malignancy. A cardinal feature of super-enhancers is that they are transcribed to produce enhancer-derived RNAs (eRNAs). It remains unclear whether super-enhancers robustly activate genes in situ and whether their functions are attributable to eRNAs or the DNA element. CRISPR/Cas9 was used to systematically delete three discrete super-enhancers at the Nanog locus in embryonic stem cells, revealing functional differences in Nanog transcriptional regulation. One distal super-enhancer 45 kb upstream of Nanog (−45 enhancer) regulates both nearest neighbor genes, Nanog and Dppa3. Interestingly, eRNAs produced at the −45 enhancer specifically regulate Dppa3 expression by stabilizing looping of the −45 enhancer and Dppa3. Our work illustrates that genomic editing is required to determine enhancer function and points to a method to selectively target a subset of super-enhancer-regulated genes by depleting eRNAs

Stress-Induced Cell-Cycle Activation in Tip60 Haploinsufficient Adult Cardiomyocytes

Background: Tat-interactive protein 60 (Tip60) is a member of the MYST family of histone acetyltransferases. Studies using cultured cells have shown that Tip60 has various functions including DNA repair, apoptosis and cell-cycle regulation. We globally ablated the Tip60 gene (Htatip), observing that Tip60-null embryos die at the blastocyst stage (Hu et al. Dev.Dyn.238:2912;2009). Although adult heterozygous (Tip60 +/2) mice reproduce normally without a haploinsufficient phenotype, stress caused by Myc over-expression induced B-cell lymphoma in Tip60 +/2 adults, suggesting that Tip60 is a tumor suppressor (Gorrini et al. Nature 448:1063;2007). These findings prompted assessment of whether Tip60, alternative splicing of which generates two predominant isoforms termed Tip60a and Tip60b, functions to suppress the cell-cycle in adult cardiomyocytes. Methodology/Principal Findings: Western blotting revealed that Tip60a is the predominant Tip60 isoprotein in the embryonic heart, transitioning at neonatal stages to Tip60b, which is the only isoprotein in the adult heart wherein it is highly enriched. Over-expression of Tip60b, but not Tip60a, inhibited cell proliferation in NIH3T3 cells; and, Tip60haploinsufficient cultured neonatal cardiomyocytes exhibited increased cell-cycle activity. To address whether Tip60b suppresses the cardiomyocyte cell-cycle in the adult heart, hypertrophic stress was induced in Tip60 +/+ and Tip +/2 littermates via two methods, Myc over-expression and aortic banding. Based on immunostaining cell-cycle markers an

RNA-seq heat map revealing endogenous gene expression patterns during cardiomyogenesis induced with CHIR alone.

<p>Pluripotent H1 ESCs expanded on Matrigel in mTeSR1 medium were induced to differentiate by changing the medium to RPMI/B27 (without insulin) including the small MW inhibitors CHIR (12 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5; insulin (4,000 ng/ml) was included after Day 7. RNA was purified from duplicate cultures on the indicated days, converted to cDNA, and processed to RNA-seq libraries as described in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118670#pone.0118670.s011" target="_blank">S1 Methods</a></b>. Colors indicate the range of each gene’s expression during the 14 day period, with least expression shown in green and highest expression shown in red (see inset). The number in each panel indicates the expression of each gene in transcripts per million (TPM) within each culture dish.</p

Wnt-modulated cardiomyogenesis is enhanced by Matrigel overlay.

<p>Pluripotent H1 ESCs expanded on Matrigel in mTeSR1 medium were treated with Matrigel overlays on Day -3 or -1. Differentiation was induced by changing medium to RPMI/B27 (no insulin) containing the small MW inhibitors CHIR (12 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5; insulin (4,000 ng/ml) was included after Day 7. <b>A</b>, scheme of cardiomyogenic induction using Matrigel overlay and small MW inhibitors; arrows denote days when medium was changed, before (green) and after (red) induction. <b>B</b>, typical flow cytometry results showing percentages of cardiac troponin-T (cTnT)-positive cells at Day 14. <b>C</b>, αMHC (MF20) immunostaining at Day 14. <b>D</b>, bar graph showing averaged flow cytometry results obtained in three independent determinations. Vertical lines depict ± SEM. Size bars in C = 200 μm. The p-value in D was calculated using Student’s t-test.</p

Activin-A levels during Day 0–1 modulate CM vs. DE differentiation.

<p>Pluripotent H1 ESCs were induced by changing medium to RPMI/B27 (no insulin), including CHIR (7.5 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5. Activin-A was included at the indicated levels during Day 0–1. Insulin (4,000 ng/ml) was included after Day 7. <b>Panel A</b>, a-e shows cells double-immunostained on Day 5 for Oct4 (red) and Sox17 (green); <b>e</b> is a positive control wherein cells were induced to DE with Activin-A (50 ng/ml) and Bmp4 (10 ng/ml) during Days 0–5. <b>Panel A f-i</b> shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. <b>Panel B</b> depicts the effect of Activin-A levels during Day 0–1 on cardiomyocyte differentiation at Day 14, determined by flow cytometry using anti-cTnT. Cultures treated with 10 ng/ml Activin-A began to rhythmically contract at Day 6. Cells treated with 50 or 100 ng/ml Activin-A did not beat at any time. Bars indicate the average values combined from multiple experiments. Vertical lines = ±SEM. P-values were calculated by Student’s t-test. The p-value over the bar denoting 10 ng/ml Activin-A is relative to cells treated with CHIR only (0 ng/ml Activin-A), whereas the p-values over the bars denoting 50 and 100 ng/ml Activin-A are relative to cells treated with 10 ng/ml Activin-A. The size bar in Aa, which pertains to panels a-i, = 200 μm.</p

Spontaneous Development of Written Literacy

Poměrně delší dobu sleduji, jak děti zacházejí s písmem a jak se mu věnují, ať už se jedná o děti předškolní nebo školou povinné, ať už je dítě ve škole nebo doma a na školu se připravuje, nebo je mimo ni, v družině, v zájmovém kroužku, na táboře, na škole v přírodě, u prarodičů nebo s kamarády. Ve všech těchto a mnoha dalších situacích se často děti v tomto věku věnují psaní záměrně i nezáměrně, hrou nebo "prací", tedy činností, která je podmíněna svým dokončením (školní učební látka). Středem mého zájmu je vztah dítěte ve vývojovém období školní zralosti1 a jeho spontánního psaní jako komunikativní dovednosti ve vztahu k výchovněvzdělávacímu působení. Komunikativní psaní je součástí vzdělávací oblasti Český jazyk a literatura2 a je obsaženo ve všech jejich částech: Komunikační a slohové výchově, Jazykové výchově i Literární výchově

Increased Cell-Cycle Activity in Tip60^+/− Neonatal Cardiomyocytes.

<p>Cardiomyocytes were isolated from 2 day-old neonatal hearts and cultured on gelatin-coated dishes. When the cells were ∼60% confluent (72 hrs later) they were double-immunostained for phosphorylated histone-H3 (H3P) to detect M-phase cells (arrows in <b>A</b> & <b>C</b>) and for sarcomeric α-actin (not shown) to verify cardiomyocyte identity. Nuclei were stained with DAPI (<b>B</b>,<b>D</b>); H3P-labeled nuclei are encircled because DAPI is obscured DAB-stained nuclei. E shows percentages of H3P-positive neonatal cardiomyocytes, based on enumerating 1,000-2,000 cells in each dish; error bars  =  +/−SEM. Scale bars in <b>A</b>–<b>D</b> = 10 µm.</p

Tip60-Haploinsufficiency Augments 4-OHT-Induced Induction of Cell-Cycle Activity in MycER Transgenic Hearts.

<p>Eight week old MycER transgenic Tip60 wild-type (WT) and heterozygous (Het) mice were induced with 4-OHT for seven days and assessed for cell-cycle activity by immunostaining phosphorylated histone H3 (H3P; arrows in <b>A</b>,<b>B</b>). This was verified by immunostaining BrdU-incorporated nuclei (arrows in <b>D</b>,<b>E</b>). Percentages of labeled cells were determined by evaluating at least 5,000 (<b>C</b>) or 2,500 (<b>F</b>) hematoxylin-stained nuclei for H3P or BrdU antigen, respectively. (N)  =  number of hearts; vertical bars  =  ±SEM; p-values were calculated using Student's t-Test (two-tailed, unpaired). The scale bar in all images  =  10 µm. (Note: A control utilizing WT-MycER mice demonstrated that 4-OHT had no effect on these parameters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031569#pone.0031569-Xiao1" target="_blank">[26]</a>).</p

Tip60-Haploinsufficiency Increases 4-OHT-Induced Expression of Cyclin D2.

<p>Eight week old MycER transgenic Tip60 wild-type (WT) or heterozygous (Het) mice were induced with 4-OHT for seven days and assessed for cyclin D2 by western blotting. <b>A</b> is a western blot showing cyclin D2 and GAPDH levels in non-stressed (no 4-OHT) and stressed (+4-OHT) WT and Het myocardium. Each lane contained 10 µg total protein from separate hearts. <b>B</b> shows densitometry of the bands in <b>A</b>, with cyclin D2 normalized to GAPDH. Numbers (N) in parentheses indicate numbers of hearts evaluated. Vertical bars  =  ±SEM; p-values were calculated using Student's t-Test (two-tailed, unpaired).</p