Human microvascular endothelial cell cultures.

Primary human cortex microvascular endothelial cells (MECs; Cell Systems, Rockland, ME; ACBRI 376 lots 2648 and 0537) were originally isolated from the human brain cortex of a healthy 24-year-old in the United States. The cells were elutriated from dispase-dissociated neurons and cultured on gelatin-coated tissue culture dishes in M199 medium supplemented with 20% newborn calf serum (NCS) (both from Invitrogen-Gibco, Carlsbad, CA), 5% human serum (BioCell, Rancho Dominguez, CA), 0.1 g/ml heparin, 0.05 g/ml ascorbic acid (Sigma-Aldrich), 1.6 mM l-glutamine (Invitrogen-Gibco, Carlsbad, CA), Sigma endothelial cell growth factor (Sigma-Aldrich), bovine brain extract (Clonetics BioWhittaker, Walkersville, MD), and 0.05 U/ml penicillin with 0.05 g/ml streptomycin (Invitrogen/Gibco, Carlsbad, CA) to maintain their differentiation state (16). All experiments were performed 6 to 8 days after passage of the cells, and only the early passages (11 to 24) were used. Nucleoside labeling experiments used approximately 4 × 10⁷ cells in 12 150-mm flasks. One flask of cells (approximately 4 × 10⁶ cells) was used for flow cytometric analysis.

R-NSC cultures.

H9 human embryonic stem cells (WA09) were differentiated toward R-NSCs as described previously (15). Mechanical isolation of rosettes was performed at day 16 of differentiation, followed by culture in N2 medium supplemented with sonic hedgehog (SHH) (50 ng/ml), FGF8 (100 ng/ml), brain-derived neurotrophic factor (BDNF; 20 ng/ml) (all R&D Systems), and ascorbic acid (100 μM; Sigma-Aldrich). At day 23 of differentiation, about 2 × 10⁷ cells were grown in the presence of the halogenated nucleosides and, prior to analysis, enriched for R-NSCs via FACS for expression of Hes5::eGFP (44) and N-cadherin (15). A control set of cells from the batch used for analysis of the replication program was replated under R-NSC conditions, and the percentage of R-NSC marker-positive cells was determined at day 27 of differentiation.

Immunohistochemistry for cell line characterization of hESCs, MECs, and R-NSCs. (i) Human embryonic stem cells.

Cells were stained with antibodies to Oct4, Nanog, SSEA-4, TRA1-60, and TRA1-81. Cells were fixed in 4% paraformaldehyde and 0.015% picric acid for 20 min at room temperature, followed by three 5-min washes in phosphate-buffered saline (PBS). Fixed cells were blocked in PBS, 0.5% bovine serum albumin (BSA), and 0.3% Triton X-100 for 50 min at room temperature, followed by three 5-min washes in PBS. Cells were incubated overnight at 4°C with primary antibodies diluted in PBS-1% BSA (mouse anti-Oct3/4, 1:200 [Santa Cruz Biotechnology, Santa Cruz, CA]; goat anti-Nanog, 1:50 [R&D Systems, Minneapolis, MN]). For cell surface markers (mouse anti-SSEA-4, 1:50 [DSHB, Iowa City, IA]; TRA1-60 and TRA1-81, 1:50 [Chemicon-Millipore, Billerica, MA]), the PBS-BSA blocking solution did not contain Triton X-100. Cells were washed with PBS, incubated with secondary antibodies (Alexa Fluor 488 anti-mouse antibody for SSEA-4, TRA1-60, and TRA1-81, Alexa Fluor 555 anti-mouse antibody for Oct3/4, and Alexa Fluor 488 anti-goat antibody for Nanog [Invitrogen/Molecular Probes]; all dilutions, 1:400) and diluted in PBS-1% BSA for 1 h at room temperature. This was followed by a 4′,6-diamidino-2-phenylindole (DAPI) counterstain (100 ng/ml for 5 min at room temperature) and then followed by a PBS wash. Fluorescent images were acquired with an Olympus 1X71 inverted microscope, using IPLab software. The same protocol was used for fluorescence-activated cell sorting (FACS) analysis, with the exception of maintaining the cells in suspension and constantly on ice and maintaining the secondary antibody dilutions at 1:1,000.

(ii) MECs.

Cells fixed in cold 70% ethanol were analyzed by double immunostaining with platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) (Sigma-Aldrich, St. Louis, MO) and Von Willebrand factor (vWF) (Sigma-Aldrich, St. Louis, MO) expression using the previously described protocol (16). Briefly, the cells were incubated in blocking solution (5 mM EDTA, 1% fish gelatin, 1% essentially immunoglobulin-free BSA, and 2% horse serum) for 30 min at room temperature and then incubated in diluted primary antibody (goat anti-PECAM-1 antibody or rabbit anti-vWF antibody, 1:500 or 1:800, respectively) overnight at 4°C. The cells were then washed with PBS, incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG F(ab′)₂ (Sigma-Aldrich, St. Louis, MO) or Cy3-conjugated anti-goat IgG (Sigma-Aldrich, St. Louis, MO) for 1 h at room temperature, followed by another wash in PBS for 1 h. Samples were examined by confocal microscopy with a Leica microscope. Specificity was confirmed by replacing the primary antibody with preimmune serum. For FACS analysis, goat PECAM-1, vWF, and CD99 were used as described above. In addition, the MECs were tested with antibodies to Oct4 and Nanog.

(iii) IGH control for endothelial cells.

Endothelial cells and Ramos cells (Igh-expressing cells) were also treated with goat anti-human IgM-FITC and goat anti-mouse IgM-FITC (Southern Biotechnology Associates Birmingham, Birmingham, AL). The immunohistochemistry was performed as described above. Ramos cells were used as a positive control because they are a human Burkitt's lymphoma germinal-center-like B-cell line that expresses IgM.

(iv) R-NSCs.

Cells were fixed and processed for immunocytochemistry under conditions identical to those for undifferentiated hESCs. Antibodies used for characterizing R-NSCs include monoclonal mouse antibodies Nestin (1:500; Neuromics, Edina, MN), PLZF (1:50; Calbiochem, San Diego, CA), Pax6 (1:75; DSHB), and Oct3/4 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and polyclonal rabbit antibodies Sox2 (1:200; Abcam) and ZO-1 (1:100; Zymed, San Francisco, CA).

Flow cytometric analysis.

The percentage of cells in S phase of the cell cycle was determined using propidium iodide. An aliquot of 10⁶ cells was centrifuged into a pellet, drained of liquid, and placed on ice for 15 min after the addition of propidium iodide.

Single-molecule analysis of replicated DNA (SMARD) (40, 41). (i) Nucleoside incorporation into the DNA of cultured human cells.

The nucleoside incorporation into the DNA of iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) does not require cell synchrony or large numbers of cells. To incorporate halogenated nucleosides into DNA in vivo, the cells must be cycling and ideally be in exponential phase. The number of cells in S phase (~40% for hESCs and ~26% for MECs) was large enough to allow the incorporation of the two halogenated nucleosides, IdU and CldU, into a sufficient number of DNA molecules for these studies.

The cells were grown at 37°C for 3.5 h in the presence of 25 μM 5-iodo-2′-deoxyuridine (Sigma-Aldrich, St. Louis, MO). The medium was withdrawn, and medium with 25 μM 5-chloro-2′-deoxyuridine (Sigma-Aldrich, St. Louis, MO) was added to the cultures, followed by another 3.5 h of incubation. The cultures were rinsed with room temperature Dulbecco's phosphate-buffered saline (DPBS), and the cells were lifted with either Accutase or trypsin. Following centrifugation, the cells were resuspended at 3 × 10⁷ cells per ml in DPBS. Equal volumes of melted 1% InCert agarose (Lonza Rockland, Inc., Rockland, ME) in DPBS was added to the cells at 42°C. The cell solution was pipetted into a chilled plastic mold with 0.5- by 0.2-cm wells with a depth of 0.9 cm for preparing DNA gel plugs. The gel plugs were allowed to polymerize on ice for 30 min and were then pushed out of the plastic mold into a 50-ml centrifuge tube containing lysis buffer (1% n-lauroylsarcosine [Sigma-Aldrich, St. Louis, MO], 0.5 M EDTA [pH 8], and 20 mg/ml proteinase K). The gel plugs remained at 50°C for 64 h and were treated with 20 mg/ml proteinase K, recombinant PCR grade (Roche Diagnostics, Mannheim, Germany), every 24 h.

(ii) Enzymatic digestion of genomic DNA.

The gel plugs were rinsed several times with Tris-EDTA (TE) and once with phenylmethanesulfonyl fluoride (Sigma-Aldrich, St. Louis, MO). The plugs were rinsed with 10 mM MgCl₂ and 10 mM Tris-HCl (pH 8.0). The genomic DNA in the gel plugs was digested with 50 units of PmeI (New England BioLabs Inc., Ipswich, MA) per plug in ~150 μl of digestion buffer containing 2× BSA (New England BioLabs Inc., Ipswich, MA) and 400 μM spermidine (New England BioLabs Inc., Ipswich, MA) at 37°C overnight.

(iii) Pulsed-field gel electrophoresis (PFGE).

The digested gel plugs were rinsed with TE and cast into a 0.7% gel (SeaPlaque GTG agarose [Lonza Rockland, Inc., Rockland, ME]). A gel lambda ladder PFG marker and yeast chromosome PFG marker (both from New England BioLabs Inc., Ipswich, MA) were cast next to the gel plugs.

(iv) Southern transfer and hybridization.

A Southern transfer was performed to determine the location of the DNA fragment on the gel. The region of the gel containing the segment of interest was excised and set aside, while the rest of the DNA (which includes the chromosome ladders) was transferred to a membrane (Hybond-XL [Amersham Biosciences, Piscataway, NJ]) and hybridized with probes for either the POU5F1, Nanog, or IGH segments. Radiography was used to determine the location of the appropriate DNA segment or locus from the gel transfer. The remainder of the saved DNA gel slice was then cut into sequential 0.5-mm pieces of DNA and stored at 4°C in 50 mM EDTA and 10 mM Tris-HCl (pH 8.0).

(v) Stretching the DNA molecules.

The DNA molecules were stretched and fixed on glass slides and then hybridized with biotinylated probes that specify sequences on the molecule. Samples of gel slices from the appropriate positions in the pulsed-field electrophoresis gel were separately melted, and aliquots of the resulting DNA solutions were separately stretched on microscope slides. For each segment analyzed, the results for multiple slides did not show any apparent differences among melted DNA solutions, and the results of several experiments were in agreement (the results were pooled). The gel section containing the segment of interest was sliced into 1-mm sections, rinsed with TE several times, and melted at 72°C for 15 min. GELase enzyme (1 unit per 50 μl of agarose suspension) (Epicentre Biotechnologies, Madison, WI) was carefully added to digest the agarose. The DNA remained at 45°C for a minimum of 2 h, and the DNA strands were incubated with YOYO-1 iodide (Molecular Probes, Eugene, OR) for at least 1 h prior to stretching on 3-aminopropyltriethoxysilane (Sigma-Aldrich, St. Louis, MO)-coated glass slides. The DNA was pipetted along one side of a coverslip that had been placed on top of a silane-treated glass slide and allowed to enter by capillary action. The DNA was denatured with sodium hydroxide in ethanol and then fixed with glutaraldehyde.

In vivo incorporation of nucleoside analogs into replicated DNA determined by SMARD.

Replication of the DNA segments containing the genes of interest was studied using a previously established technique, single-molecule analysis of replicated DNA (SMARD), for fluorescent visualization of the DNA replication program across DNA segments (40, 41). Cells in exponential phase are pulsed with halogenated nucleotides: IdU followed by a second pulse with CldU. At the end of the labeling periods, DNA segments containing the loci studied here were either substituted along the whole length of the segment (totally substituted) with one nucleotide analog (see Items S3 to S11 in the supplemental material) or both nucleotide analogs, substituted along part of the length of the segment, or not substituted at all.

The cells were then suspended in agarose plugs and lysed and cleaved in the plugs to avoid DNA breakage. We then used pulsed-field gel electrophoresis to enrich for the specific segment containing the locus of interest and excised a slice of agarose that contained this segment. The agarose was melted and digested with agarase, and an aliquot of the DNA solution was allowed to slide under a coverslip; the molecules stretched as they attached to the surface of an amino silane-treated microscope slide. A red fluorescent antibody was used to detect IdU, while a green fluorescent antibody was used to detect CldU. Multiple biotinylated DNA probes were used for FISH to produce a distinctive blue “bar code” to distinguish and tag the DNA molecules containing the loci of interest and determine the orientation of these molecules. Photomicrographic images of tagged molecules that are totally substituted and contain both nucleotide analogs (red plus green along the full length) were aligned using the boundaries of the blue FISH signals. This was done in a nonsubjective manner in accordance with increasing incorporation of IdU (red stain) in randomly selected representative stretched DNA strands from the cells. Aligning each group of molecules in this manner effectively eliminates any potential bias in the arrangement of the molecules. Thus, if replication forks progress in a single direction through the examined region, the DNA molecules will have increasing red stain from one end (Fig. ​(Fig.11 Ai and ii). Replication initiation is represented by a patch of red stain (IdU incorporation) flanked on both sides by green stain (CldU incorporation) (Fig. ​(Fig.1Aiii).1Aiii). Termination of replication is represented by a patch of green stain flanked by red stain on both sides (Fig. ​(Fig.1A1Aiv).

Replication of the IGH segment is very similar in hESCs and MECs.

The human IGH locus (~2 Mb) is a gene locus transcriptionally active only in B-lineage cells. We analyzed the replication of an ~161-kb PmeI DNA segment containing part of the IGH locus (IGH segment, including part of the V region, D, J, Cμ, and Cδ genes). We found that replication forks proceed exclusively in the 3′-to-5′ direction in all DNA molecules fully substituted with IdU and CldU (red-green molecules), lacking any internal initiation or termination sites (Fig. ​(Fig.1B,1B, molecules 1 to 32, and 1C, molecules 1 to 23) in the hESCs and MECs. As shown in Fig. 1D and E, the replication profiles of the IGH segment show very similar patterns (r = 0.962; P < 0.001) which decrease progressively in a 3′-to-5′ direction. Thus, the replication programs for the transcriptionally silent IGH segment are very similar in hESCs and MECs; external replication initiation sites downstream of the segment were used to replicate the IGH segment.

Fork direction and location of the center of the replication initiation zone for the POU5F1 segment are different in the hESCs compared to in the differentiated MECs.

We next compared the replication program for an ~350-kb DNA segment containing the POU5F1 gene encoding the pluripotency regulator Oct4 (POU5F1 segment). Unlike the IGH segment, which is passively replicated by origins located downstream of the segment, several internal replication initiations were observed in the POU5F1 segment (Fig. ​(Fig.2).2). The identification of the positions of initiation sites determined by SMARD is based on the following two assumptions. First, the replication fork proceeds bidirectionally from the initiation sites. Second, the replication forks progress at an equal speed in both directions. Thus, the initiation site will be located at the center of a red patch surrounded by green, assuming there is only one initiation site in the IdU-incorporated (red-stained) patch in the molecule. It should be noted that these assumptions likely apply to most but not necessarily all replication initiations in mammalian cells. In the H9 and H14 hESCs, most of the replication initiation sites (asterisks in Fig. 2A and B) were close to the POU5F1 gene, exhibiting a peak centered at the POU5F1 gene in the replication profile (Fig. 2D and E). Forks originating from external replication origins both upstream and downstream of the segment also replicate the POU5F1 segments. For all of the red-green DNA molecules without initiations and terminations, 20% and 39% proceeded from the 5′-to-3′ direction in H9 and H14 hESCs, respectively (Fig. ​(Fig.2A,2A, molecules 16 to 20, and B, molecules 21 to 31, respectively), and 80% and 61% proceeded from the 3′-to-5′ direction in H9 and H14 hESCs, respectively (Fig. ​(Fig.2A,2A, molecules 21 to 40, and B, molecules 32 to 48, respectively). There were 8 molecules and 12 molecules with replication termination events that randomly occurred in the DNA segment for the H9 and H14 hESCs, respectively (Fig. ​(Fig.2A,2A, molecules 41 to 48, and B, molecules 49 to 60, respectively). These results showed that the DNA replication programs for the POU5F1 segment are very similar in the two hESC lines which exhibited very similar DNA replication profiles (r = 0.818; P < 0.001) (Fig. 2D and E).

In the MECs, DNA replication initiation sites were also detected within the POU5F1 segment (Fig. ​(Fig.2C,2C, molecules 1 to 27). There were 17 termination sites in the segments, and many of these termination sites were located in the molecules that also have initiation sites (molecules 15 to 27, 44 to 47). With regard to fork direction, there were almost twice as many molecules with replication forks proceeding from 5′ to 3′ (Fig. ​(Fig.2C,2C, molecules 28 to 37) than those with replication forks proceeding from 3′ to 5′ through the entire segment containing the POU5F1 gene (Fig. ​(Fig.2C,2C, molecules 38 to 43). In particular, replication forks progressed predominantly in the 5′-to-3′ direction through the POU5F1 gene (Fig. ​(Fig.2C).2C). This was reflected in the replication profile of the MECs (Fig. ​(Fig.2F),2F), where there was a decrease in the percentage of molecules containing incorporated IdU from 5′-to-3′ near the POU5F1 gene. Importantly, this revealed a significant difference between hESCs and MECs in the direction of replication fork movement through the POU5F1 gene. In hESCs, forks proceeded through the gene predominantly in the 3′-to-5′ direction, in contrast to fork movement in the MECs, which was predominantly in the 5′-to-3′ direction (Fig. ​(Fig.2).2). Thus, replication fork progression through the POU5F1 gene in hESCs was clearly different from that in MECs.

An additional important finding was that the replication initiation program of the POU5F1 segment in H9 and H14 hESCs was clearly different from that of MECs (Fig. ​(Fig.2).2). In DNA molecules from different cells, initiation can occur at different sites. For the POU5F1 segment, as in many other loci, these initiation sites are located within a region called the initiation zone. DNA replication initiated within an ~100-kb region centered around the POU5F1 gene in H9 and H14 hESCs (Fig. 2A and B), whereas most of the initiation sites in MECs were located in a more distal region from the gene (Fig. ​(Fig.2C).2C). The differences in the location of replication initiation sites in hESCs and MECs were evident in the replication profiles (Fig. 2D to F). The peaks in the profiles indicated the center of the initiation zone. To better visualize initiation events in DNA molecules, we determined the replication profile for DNA molecules that had replication initiations (initiation profile). We calculated the distribution of red-stained areas that are surrounded by green stain (initiation region) in DNA molecules and plotted this at intervals of 5 kb along the POU5F1 segment. Assuming the center of the red signal is the initiation site, the midpoint of the peak of the initiation profile will reflect the location of preferential initiation sites. Profiles of initiation for both hESC lines showed a peak midpoint near the POU5F1 gene (Fig. ​(Fig.33 A and B), indicating that most initiation originated near the gene. In contrast, the peak midpoint of the replication initiation profile for MECs was ~70 kb upstream of the POU5F1 gene (Fig. ​(Fig.3C).3C). These results thus indicated that there is a clear difference in the position of initiation sites for the POU5F1 segment; initiations are proximal to the gene in hESCs but distal to the gene in MECs.

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FIG. 3.

Locations of the centers of the replication initiation zones in the POU5F1 segments differ in hESCs, MECs, and R-NSCs. Initiation profiles for the POU5F1 segments in hESCs, MECs, and R-NSCs were compared. A region of the molecule that is red and surrounded by green contains an initiation site, and we assume that the centers of the red patches are initiation sites. Thus, the distribution of these red regions (in each 5-kb interval) in DNA molecules reflects the initiation profile, and the midpoint of the peak indicates the location of the preferential initiation site. The x axis represents the POU5F1 segment, divided into 70 intervals (5 kb each); the y axis indicates the percentage of molecules in each interval that had replication initiations (red signal in the initiation region) at that location on the segment. The map of the POU5F1 segment is diagrammed above the graphs. The graphs for H9 hESCs (A), H14 hESCs (B), MECs (C), and R-NSCs (D) are displayed beneath the POU5F1 segment map. The initiation profile of MECs displays a curve with a peak at ~70 kb upstream of the POU5F1 gene (interval 36). The profiles of H9 and H14 hESCs show a peak position at interval 47, proximal to the POU5F1 gene (indicated by the gray bar), located at interval 49.

We examined whether these differences in the fork directions and origin locations could be caused by significant variations in DNA sequences, for example, large DNA insertions or deletions or rearrangements in the examined segments. We did not detect any large duplications or deletions for the PmeI segment in the hESCs and MECs based on the segment sizes measured by pulsed-field gel electrophoresis (at a resolution of 5 kb). Additionally, on a more sensitive level, PCR results did not reveal any significant differences between hESCs and MECs when the lengths of the products (see Items S12 and S13 in the supplemental material) obtained from the POU5F1 and Nanog segments (where the differences in replication were detected) were compared. Therefore, it is highly unlikely that the differences in replication at the POU5F1 segment for hESCs and MECs could be attributed to detectable insertions or deletions. Since the directions of replication forks through the POU5F1 gene and locations of origins were conserved in the two hESCs (and also in the H1 hESCs [data not shown]), it indicates that they are an intrinsic epigenetic property of hESCs.

Fork direction and replication initiation for the POU5F1 segment are distinct in the hESCs compared to H9 hESC-derived multipotent R-NSCs.

To further determine whether the DNA replication program used by the two hESC lines to replicate the POU5F1 segment was specific to these lines, we examined replication in R-NSCs (Fig. ​(Fig.4)4) . R-NSCs are a novel neural stem cell type with broad differentiation potential toward central nervous system and peripheral nervous system fates and capable of in vivo engraftment (15). Furthermore, these cells share a genotype with the H9 hESCs studied here. Additional important features that distinguish R-NSCs from MECs include the broad differentiation repertoire (multipotency), expression of several markers shared with pluripotent cells, such as SOX2 (Fig. ​(Fig.4A)4A) and LIN28, and the early embryonic nature of the cells emerging shortly after the loss of hESC pluripotency. Similar to MECs, R-NSCs do not express POU5F1 (Fig. ​(Fig.4A)4A) or Nanog.

The replication of the POU5F1 segment in R-NSCs (Fig. 4B and C) was clearly different from that in the H9 ESCs from which they were derived as well as another independent hESC line, H14 (Fig. 2A and B), and was also different from that in the MECs (Fig. ​(Fig.2C).2C). In H9 hESCs, replication mainly initiated within (showing a peak in the replication profile near the POU5F1 gene) (Fig. ​(Fig.2A)2A) or downstream of the segment (predominance of 3′-to-5′ replication forks), while in the R-NSCs, replication initiated predominantly upstream of the segment (5′-to-3′ replication forks). As shown in Fig. ​Fig.4B,4B, the replication profile decreased in the 5′-to-3′ direction, indicating that most of the replication forks proceeded 5′ to 3′ through the segment. Forks proceeded predominantly 5′ to 3′ through the POU5F1 gene in R-NSCs, distinct from hESCs, in which the forks proceeded predominantly 3′ to 5′ through the gene. Furthermore, in the H9 hESCs, most of the replication initiation sites were found near the POU5F1 gene (no more than 50 kb upstream of the POU5F1 gene), while in R-NSCs, most replication initiations were located at positions more than 50 kb upstream of the gene (Fig. ​(Fig.3D3D and ​and4B).4B). These findings demonstrate that DNA replication for the DNA segment containing the pluripotent locus POU5F1 in the H9 derivative R-NSCs is clearly distinct from that in the hESCs and the somatic MECs.

DNA replication forks in H14 hESCs proceed in different directions through the segment containing the NANOG gene compared to replication forks in somatic MECs and R-NSCs.

We analyzed the replication of the DNA segment containing another pluripotency gene, NANOG (Nanog segment, ~110 kb). In the H9 hESCs, most of the replication forks proceeded from 3′ to 5′. Three initiation and three termination sites were observed near or upstream of the NANOG gene. In the H14 hESCs, similar numbers of molecules (Fig. ​(Fig.5B,5B, molecules 1 to 26) exhibited forks progressing from 5′ to 3′ and 3′ to 5′. Twenty-three percent of the DNA molecules exhibited replication initiation sites (Fig. ​(Fig.5B,5B, molecules 27 to 35 and 40). There were five termination sites upstream and downstream of the NANOG gene for the H14 hESCs (Fig. ​(Fig.5B,5B, molecules 36 to 40). Comparison of the replication profiles of H9 and H14 hESCs showed that the replication program of the segment was not completely conserved in the two hESCs analyzed (Fig. 5A and B).

In contrast to H14, in the MECs and R-NSCs, almost all of the molecules had replication forks proceeding only in the 3′-to-5′ direction (Fig. 5C, D, G, and H). We did not detect any DNA molecules that proceeded 5′ to 3′ through the segment in the MECs (Fig. ​(Fig.5C),5C), and only one did so in the R-NSCs (Fig. ​(Fig.5D,5D, molecule 1). Calculation of the directions of replication forks proceeding through the NANOG gene showed that replication forks proceeded predominantly 3′ to 5′ through the gene in H9 hESCs, MECs, and R-NSCs but showed similar numbers of 5′-to-3′ and 3′-to-5′ forks proceeding through the NANOG gene in H14 hESCs.

Like the POU5F1 segment, we did not detect any difference in the lengths of the Nanog segments for all cell types examined in this study based on pulsed-field gel electrophoresis and PCR analysis (20 pairs of primers were designed to span a 100-kb region [see Item S13 in the supplemental material]); hence, major sequence modifications did not account for the differences we detected.

The replication programs in the DNA segments containing pluripotent regulatory genes.

We found that the replication program for the POU5F1 segment was remarkably similar in the H9 and H14 hESC lines presented here as well as in the H1 hESC line (data not shown). These similarities included the region in which replication initiation occurred, the average number of initiation events, the average number of replication forks, and the average speed per replication fork. However, significant differences were detected when the replication program of pluripotent regulatory gene POU5F1 examined here in hESCs was compared to that in MECs and R-NSCs. In the DNA segment containing the POU5F1 gene, the location of the center of the replication initiation zone in hESCs was clearly different from the center of the initiation zone in somatic cells. The predominant direction of fork progression through the gene was also significantly different in hESCs compared to that in the somatic cells, indicating a differential origin usage in the region flanking the gene or outside the whole segment. Hence, our findings reveal that, in the cells studied here, there are characteristic differences in replication initiation and fork direction between hESCs and somatic cells.

Importantly, fork direction has been found to play a major role in a developmental pathway of S. pombe (mating type switching) (10-12); thus, the implications of replication fork direction in pluripotency could be an important question to address in the future. Although the R-NSCs represent a multipotent stem cell type with several properties in common with the hESCs they derive from, the fact that there are differences in the replication program of the POU5F1 segment in the R-NSCs provides additional evidence for tissue-specific replication programs.

Heterogeneity of replication initiation over wide distances at the POU5F1 locus.

The potential role of replication timing, transcriptional activity, and chromatin structure in the heterogeneity of initiation in zones in mammalian cells is currently unclear. Although initiation sites have a heterogeneous frequency of usage in both Schizosaccharomyces cerevisiae and mammalian cells, there is a major difference. S. cerevisiae has initiation sites at defined DNA sequences that are used at different frequencies, in contrast to initiation in mammalian cells, which often occurs at a large number of sites within initiation zones. We present the first evidence for initiation zones in hESCs. We observed a DNA replication initiation zone within the 350-kb segment containing the POU5F1 gene and a zone within the 110-kb segment containing the NANOG gene in two hESC lines. Importantly, the major portion of initiations in the POU5F1 zone did not appear to occur at preferred sites in any of the cell lines examined here. Another example of heterogeneity of initiation in hESCs is also exemplified with the Nanog segment. When two different hESC lines are compared with respect to replication of the segment containing the NANOG gene, there were significant differences in positions of initiation sites. An additional point is that variations in NANOG expression have been observed among ESCs (47). The POU5F1 and Nanog results provide further evidence for heterogeneity in the selection of initiation sites.

In hESCs, RNA levels from POU5F1 are approximately 30-fold higher than the average levels from seven other genes in the flanking 100-kb region (23). However, the increased transcriptional activity does not result in preferred initiation sites within the initiation zone (Fig. ​(Fig.22 and ​and3).3). In the hMECs and R-NSCs, where the POU5F1 gene is not expressed, there is still a replication initiation zone, but the center of the zone is shifted from POU5F1 in the direction of the TCF19 gene. Interestingly, in vascular endothelial cells and neuronal cells, the TCF19 gene is also a highly transcribed gene in this region (Gene Expression Omnibus series GSE19090). The shift in the position of the center of the POU5F1 initiation zone is not due to a change in replication timing in hESCs relative to that in somatic cells, since the 350-kb segment containing the POU5F1 gene replicates relatively early in S phase in all the cell types studied here (14, 23, 26; and R. S. Hansen, personal communication) and (Gene Expression Omnibus series GSE19090). To assess replication timing, we also used the valuable Replication Domain website (http://www.replicationdomain.org) developed by David Gilbert. Thus, the results reported here support the heterogeneity of initiation sites and raise the possibility that transcriptional activity, chromatin structure, and other factors play a role in their selection; however, their direct role in establishing replication initiation sites appears to be very complex and is beyond the scope of this study (2).

We thank Eliseo Eugenin and Joan Berman for characterizing and providing the human endothelial cells, Prasanta Patel for experiments, data collection, and analysis of the IGH locus in the human embryonic stem cells, Paolo Norio for providing advice on the SMARD technique, and Zeqiang Guan, Scott Hansen, and William Drosopoulos for valuable suggestions during the writing of the manuscript. We thank former lab member Xiaohua Qi for information about the replication of the Oct4 locus in H1 hESCs.

This work was supported by the National Cancer Institute Immunology and Immunooncology training grant 5T32CA00173, National Institute of Health Molecular Neuropathology training grant NS 07098 (NIGMS P-20 GM 075037), National Institute of General Medicine Sciences grant 5R01-GM045751 (C.L.S.), and the Empire State Stem Cell Fund through NYS contract C024348 (C.L.S. and L.S.).