Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

<p>Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.</p

Cellular determinants of HCV susceptibility.

<p>(<i>A</i>) Induction of microRNA miR-122 expression by FGF-10 during hepatic specification. Equal amounts of total cellular RNA from various cells at the indicated days were subjected to a real-time RT-PCR assay for detection of miR-122 expression. (<i>B</i>) Microarray heat map of gene expression levels in day-10 versus day-7 cells. Two independent RNA samples were processed for each time point. The numbers represent the average values and standard deviations. The conventional color spectrum with green representing downregulation and red representing upregulation was adopted. Fold of changes were also listed next to the name of the gene. (<i>C</i>) Quantitative RT-PCR results of EGFR and EphA2 induction. (<i>D</i>) Upregulation of PI4KIIIα protein during the differentiation process. The levels of CyPA and DDX-3 remained unchanged in the same samples. (<i>E</i>) Quantitative RT-PCR results of IFITM1 and IFI30 expression induction.</p

Genetic modification of hESCs and HCV-resistant DHHs.

<p>(<i>A</i>) Suppression of CyPA expression by shRNA in WA09 cells and day-21 DHHs. (<i>B</i>) CyPA knockdown did not affect the expression of pluripotency marker Oct-4 in WA09 cells. (<i>C</i>) Modified DHHs were resistant to wildtype HCV infection. Infection of both the wildtype and CyPA-KD (LA) DHHs were done at day 13 and allowed to proceed for 48 h. Luciferase in the culture supernatant for monitored. Wildtype HCVcc (Jc1/GLuc2A) infected unmodified DHHs but not CyPA-KD DHHs (redlines), and the DEYN mutant infected both cell types (blue lines). Error bars represent standard deviations of replicate experiments.</p

Hepatic differentiation from human embryonic stems cells (hESCs).

<p>(<i>A</i>) Representative images of cell morphology and protein marker expression of hESCs (day 0), definitive endoderm (day 4), hepatic progenitor cells (days 8–10), and hepatocyte-like cells (both immature and mature, days 11–21). For day-10 cells, double-staining of AFP and CK-7 (middle panel, 40×) showed mutually exclusive expression in the cell population. (<i>B</i>) Reciprocal expression of pluripotent marker Nanog and liver-specific marker AFP during differentiation. RT: reverse transcriptase. (<i>C</i>) Expression of mRNAs of ALB and AAT during differentiation. PHH: primary human hepatocytes; (<i>D</i>) Albumin secretion by differentiated human hepatocyte-like cells (DHHs). Culture media were collected at the indicated time points during differentiation and subjected to albumin detection with an ELISA kit. Error bars represent standard deviation from replicate experiments. (<i>E</i>) Periodic acid-Schiff staining of stem cells (WA09), DHHs, and PHHs.</p

Time course of infection for determination of the transition point at which the differentiating cells became permissive for HCV.

<p>(<i>A</i>) List of growth factors in media used in the various stages of differentiation. (<i>B</i>) Time course of DHH infection. Cells were exposed for 6 h on the indicated days before the inoculum was removed. The cells were then cultured in the appropriate medium for an additional 48 h before the cell lysates were collected for detection of NS3 expression. (<i>C</i>) Secreted luciferase activities were monitored in the same experiments described in (B). Error bars represent standard deviation of triplicate experiments. (<i>D</i>) Hepatic maturation was not required for HCV infection of day-10 cells. Day-10 DHHs were infected and then either kept in medium D (hepatic specification medium) or changed to HGF-containing Medium E (hepatic maturation medium) until day 21, when all cells were collected for western blotting. The anti-NS3 antibody also recognized a nonspecific band in the mock-infected sample. (<i>E</i>) A diagram indicating the time point for transition of DHHs to HCV permissiveness on the basis of results shown in (<i>B</i>) and (<i>C</i>).</p

Persistent and productive infection of DHHs by HCVcc.

<p>(<i>A</i>) Continuous replication of HCVcc in DHHs. Day-10 DHHs were exposed to Jc1/GLuc2A for 9 h before the inoculum was removed and the cells were changed to medium E with or without cyclophilin inhibitor CsA at 1 µg/ml. Culture supernatants were collected daily for measurement of luciferase activity. The culture medium was replaced with thorough washing every 48 h, and CsA was included every time fresh medium was used. Error bars represent standard deviations from triplicate experiments. (<i>B</i>) Secretion of HCV core antigen into the culture medium by infected DHHs. Day-13 DHHs were exposed to HCVcc for 9 h before the inoculum was removed, and the cells washed and changed to medium E, then immediately collected as the 0-h samples. The infected cells were then incubated for an additional 48 h in medium E with or without IFN-α (50 units/ml) before the culture supernatants were collected as the 48-h samples. Error bars represent standard deviations from replicate experiments. (<i>C</i>) Reinfection of Huh-7.5 cells by HCV particles produced from DHHs. The 48-h media from (<i>B</i>) were used to infect Huh-7.5 cells, which were then fixed for NS3 staining four days after infection. The infectious titer of the HCVcc produced by DHHs is shown. FFU: focus-forming units.</p

Copy Number Variation Is a Fundamental Aspect of the Placental Genome

<div><p>Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication.</p></div

UR domains are heterochromatic.

<p>UR domains are enriched for repressive histone marks and depleted of active histone marks compared to what is expected by chance. Interestingly, while UR domains in TS cells are enriched for both the repressive mark H3K9me3 and H3K27me3, UR domains in TGCs are only enriched for the repressive mark H3K9me3, and are depleted of the repressive mark H2K27me3 (asterisk). The p-value for all enrichment/depletion values is <0.001.</p

UR domains have low gene content and expression both <i>in vivo</i> and <i>in vitro</i>.

<p><b>A</b>. <i>In vitro</i> TGCs produce the same UR domains as <i>in vivo</i>. Plot comparing position along chromosome 14 to the NLog2 Ratio of array intensity of TGC vs. embryo (e9.5) and TGC vs. TS cells (day 3, 5, and 7). Red: e9.5 (<i>in vivo</i>); blue: day 3 (<i>in vitro</i>); green: day 5 (<i>in vitro</i>); orange: day 7 (<i>in vitro</i>). Two biological replicates are plotted for each cell type. Dashed line: FDR = 0.0001. All autosomes shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004290#pgen.1004290.s008" target="_blank">Figure S8</a>. <b>B</b>. Location of UR domains on the autosomes of cultured TGCs compared to e9.5 <i>in vivo</i> TGCs. Summary of results from both biological replicates of aCGH of cultured TGCs differentiated 3, 5 and 7 days vs. TS cells, and of e9.5 <i>in vivo</i> TGCs vs. embryo (FDR = 0.0001). Darker green/longer bars indicate UR domains present in more replicates. Asterisks indicate the location of UR domains present in both replicates at e9.5. <b>C</b>. UR domains are gene poor. Histogram plotting number of Ensembl genes versus level of representation (NLog2 of TGCs vs. embryos (WGS)). UR domains boxed in pink. <b>D</b>. Low gene expression in UR domains <i>in vivo</i>. Plot of TGC normalized expression (NE) counts versus level of representation (NLog2 of e9.5 TGCs vs. embryos (WGS)). UR domains boxed in pink. <b>E</b>. Low gene expression in UR domains <i>in vitro</i>. Plot of TGC NE counts versus level of representation (NLog2 of day 7 TGCs vs. TS cells (aCGH)). Genes not present on the array were filtered out. UR domains boxed in pink.</p

TGC UR domains are a subset of late-replicating regions in TS cells.

<p><b>A</b>. UR domains correlate with late-replicating regions in TS cells. The Pearson correlation (R) between NLog2 values of TGC vs. embryo (WGS) and average TS replication timing. Data points represent 1 Mb windows in the genome. UR domains boxed in pink. <b>B</b>. UR domains are late-replicating. Screen shot from the UCSC genome browser of the second half of chromosome 14 (schematic shown above screen shot) depicting the following: aCGH and WGS data from e9.5 TGCs and replication timing data from cultured TS cells. UR domains boxed in pink. <b>C</b>. Box plot analysis shows that UR domains are smaller than the late-replicating regions that contain them. Asterisk marks the comparison that is statistically significant (p<0.01). <b>D</b>. UR domains form from a subset of late-replicating regions. Diagram depicting late-replicating regions that contain UR domains versus ones that do not contain UR domains. <b>E</b>. Box plot analysis shows that the late-replicating regions that contain UR domains are significantly larger, but not significantly more late-replicating, than those that do not. Asterisk marks the comparison that is statistically significant (p<0.01). <b>F</b>. Box plot analysis shows that the late-replicating regions that contain UR domains have significantly fewer genes than those that do not (double asterisks). “Shuffled” refers to a random set of regions that have the same length and chromosome distribution. Asterisks mark comparisons that are statistically significant (p<0.01).</p