Rapid DNA replication origin licensing protects stem cell pluripotency

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance

Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes

When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells

HNF4A Regulates the Formation of Hepatic Progenitor Cells from Human iPSC-Derived Endoderm by Facilitating Efficient Recruitment of RNA Pol II

Elucidating the molecular basis of cell differentiation will advance our understanding of organ development and disease. We have previously established a protocol that efficiently produces cells with hepatocyte characteristics from human induced pluripotent stem cells. We previously used this cell differentiation model to identify the transcription factor hepatocyte nuclear factor 4 α (HNF4A) as being essential during the transition of the endoderm to a hepatic fate. Here, we sought to define the molecular mechanisms through which HNF4A controls this process. By combining HNF4A chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) analyses at the onset of hepatic progenitor cell formation with transcriptome data collected during early stages of differentiation, we identified genes whose expression is directly dependent upon HNF4A. By examining the dynamic changes that occur at the promoters of these HNF4A targets we reveal that HNF4A is essential for recruitment of RNA polymerase (RNA pol) II to genes that are characteristically expressed as the hepatic progenitors differentiate from the endoderm

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

Most H3P-Positive Nuclei are in Cardiomyocytes.

<p>Sections from hearts processed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031569#pone-0031569-g005" target="_blank">Figure 5</a> were double-immunostained for phosphorylated H3P and Nkx2.5 to determine cardiomyocyte identity. <b>A</b> & <b>D</b> show Nkx2.5 staining (nucleus-specific brown DAB reaction product) in cardiomyocyte nuclei, as distinct from smaller non-myocyte nuclei in which only (blue) hematoxylin counter-stain is seen. <b>B</b> & <b>E</b> show H3P fluorescent green signal in the same nuclei. <b>C</b> and <b>F</b> are merged images of A–B and D–E, respectively. In <b>C</b> & <b>F</b>, dark arrows denote nuclei double-stained for Nkx2.5 and H3P; white arrows denote nuclei expressing only Nkx2.5. <b>G</b> summarizes results from enumerating a minimum of 150 H3P-positive nuclei per heart for co-localization of Nkx2.5. Error bars  =  ±SEM; statistical significance was determined by Student's t-Test (two-tailed, unpaired). Scale bars  = 20 µM.</p

Over-Expression of Tip60β, but not Tip60α, Inhibits Cell Proliferation in NIH/3T3 Cells.

<p>Cultured NIH/3T3 cells were transfected with plasmid p3xFLAG-CMV-7.1 (empty vector), or with the same vector containing cDNA encoding either Tip60α or Tip60β. <b>A</b> shows the average cell number in each well of a 12-well plate, five days after transfecting the cells with plasmid. Error bars  =  ±SEM; p-values were calculated using Student's t-test (two-tailed, unpaired). <b>B</b> shows western blots confirming exogenous expression of Tip60α and Tip60β isoproteins.</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 Expression during Heart Development & Maturation.

<p>Total RNA and protein was purified at the indicated stages of development and subjected to semi-quantitative (<b>A</b>) RT/PCR and (<b>B</b>) western blotting analysis. <b>A</b>, the 184 bp band in the upper panel is amplified from all known isoforms of Tip60, whereas the 402 and 558 bp bands in the lower panel are respectively amplified from the Tip60 β and α isoforms. <b>B</b>, the upper panel is a western blot sequentially probed with antibodies recognizing Tip60 (α & β isoproteins were detected with the same antibody) and GAPDH; protein from individual hearts was evaluated at each postnatal day, whereas pools of three hearts each were evaluated at each embryonic day. The lower panel shows densitometric analysis in which each point indicates the mean of three (N = 3) independent western blot determinations. Error bars indicate ±SEM.</p

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