1. Chromosomes and Gene Expression
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Rapid DNA replication origin licensing protects stem cell pluripotency

  1. Jacob Peter Matson
  2. Raluca Dumitru
  3. Philip Coryell
  4. Ryan M Baxley
  5. Weili Chen
  6. Kirk Twaroski
  7. Beau R Webber
  8. Jakub Tolar
  9. Anja-Katrin Bielinsky
  10. Jeremy E Purvis
  11. Jeanette Gowen Cook  Is a corresponding author
  1. The University of North Carolina, United States
  2. The University of Minnesota, United States
  3. University of Minnesota, United States
  4. University of North Carolina, United States
Research Article
Cite this article as: eLife 2017;6:e30473 doi: 10.7554/eLife.30473
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements
Pluripotent stem cells load MCMs faster than differentiated cells.

(a) DNA-loaded MCM levels increase in G1 and decrease in S phase, whereas total MCM protein levels are constant throughout the cell cycle. (b) Flow cytometric analysis of DNA-loaded and total MCM in asynchronously proliferating RPE1-hTERT cells. Cell cycle phases are defined by DNA content and DNA synthesis. Left: Cells were labeled with EdU, extracted with nonionic detergent to remove unbound MCM, fixed, and stained with anti-MCM2 (a marker for the MCM2-7 complex), DAPI (total DNA), and for EdU incorporation (active DNA synthesis). Orange cells are S-phase with DNA-loaded MCM, blue cells are G1-phase with DNA-loaded MCM, and grey cells are G1/G2/M phase cells without DNA-loaded MCM. Right: Cells were treated as on the left except that they were fixed prior to extraction to detect total MCM2. (c) T98G cells were synchronized in G0 by contact inhibition and serum deprivation, then released into G1 for 10 or 12 hr, or re-synchronized in early S with hydroxyurea (HU), and released into S for 6 or 8 hr. MCM3 in chromatin-enriched fractions (Loaded) or whole cell lysates (Total) was detected by immunoblotting. (d) Chromatin flow cytometry of the indicated asynchronous cell lines measuring DNA content (DAPI), DNA synthesis (EdU incorporation), and loaded MCM (anti-MCM2). Blue Cells are G1-MCMDNA-positive and EdU-negative, orange are S phase-MCMDNA-positive; grey are G1/G2/M-MCMDNA-negative. (e) Stacked bar graph of cell cycle phase distribution from cells in (d); mean with error bars ± SD (n = 3 biological replicates). The percentage of G1 cells in each population is reported in the green sectors. The doubling times were calculated experimentally using regression analysis in GraphPad Prism.

https://doi.org/10.7554/eLife.30473.003
Figure 1—figure supplement 1
Flow cytometry gating.

(a) Example flow cytometry gating with chromatin extracted ARPE-19 (RPE) cells from Figure 1d, measuring loaded MCM (anti-MCM2), DNA Synthesis (EdU incorporation) and DNA content (DAPI). Gating to discriminate cells from debris was on FS-area vs SS-area, singlets (individual cells) from clumps of cells/doublets was on DAPI height vs DAPI area, cell cycle phases were determined on DAPI vs EdU Non-specific background staining by the secondary antibody was measured using a negative control sample stained without primary antibody, only secondary antibody. This background threshold gate was applied to experimental samples; G1-MCMDNA-positive cells were identified first by DNA content and EdU negative status (G1) and then by MCM2 signal. (b) Example flow cytometry color gating with chromatin extracted ARPE-19 cells from Figure 1d. Left: Negative control to define background staining with neither EdU detection nor MCM2 primary antibody. Cells were stained with DAPI, subjected to EdU detection chemistry, and stained with secondary antibody. Background thresholds were set using these controls and applied to experimental samples. Right: Experimental samples in main Figure 1d showing the gating to identify DNA synthesis (EdU), and loaded MCM (anti-MCM2). Gates define color schemes for color dot plots.

https://doi.org/10.7554/eLife.30473.004
Figure 1—figure supplement 2
Validation of chromatin flow cytometry.

(a) Chromatin flow cytometry of RPE cells stained for loaded MCM2 (anti-MCM2), loaded MCM3 (anti-MCM3), and DNA content (DAPI). All three plots are the same sample. Cells stained with monoclonal MCM2 antibody for loaded MCM2 co-stain equally well for a polyclonal MCM3 antibody for loaded MCM3. (b) Chromatin flow cytometry of RPE cells treated with 100 nM siControl or a 100 nM pool of siMCM3 for 72 hr, measuring DNA content (DAPI), DNA synthesis (EdU incorporation), and loaded MCM3 (anti-MCM3). Loaded MCM3 levels decrease with siMCM3 treatment, demonstrating antibody specificity. (c) Immunoblot of cells from (b). (d) RPE cells synchronized in G0 by contact inhibition. Left: Total protein flow cytometry of G0 cells measuring total MCM (anti-MCM2), DNA synthesis (EdU incorporation) and DNA content (DAPI). Right: Chromatin flow cytometry of G0 cells measuring loaded MCM (anti-MCM2), DNA synthesis (EdU incorporation) and DNA content (DAPI). G0 cells lack loaded MCM, demonstrating antibody specificity. (e) Immunoblot of cells for (d) demonstrating G0 synchronization. (f) Immunoblots of cells fractionated into soluble protein and pellet (chromatin associated protein) using a normal-salt CSK buffer (100 mM NaCl) or high-salt CSK buffer (300 mM NaCl). High salt 300 mM NaCl strips away weakly chromatin-associated proteins, including Cdc6. (g) Chromatin flow cytometry of cells lysed with normal 100 mM NaCl CSK or high salt 300 mM NaCl CSK. (h) Histograms of only the G1-MCMDNA-positive cells from (g). The loaded MCM in G1 remains constant with high salt, demonstrating the chromatin flow cytometry measures DNA-loaded MCM. Note the harsh 300 mM salt alters nuclear morphology and scatter by flow cytometry; for this reason, we used the 100 mM NaCl extraction for all other flow cytometry experiments.

https://doi.org/10.7554/eLife.30473.005
Figure 1—figure supplement 3
Characterization of pluripotent and differentiated cells.

(a) Representative phase contrast images in greyscale of indicated cell lines from Figure 1d. Scale bar is 50 μm. (b) Immunofluorescence analysis of iPS cells visualizing TRA-1–60, TRA-1–81, SSEA or OCT4 (in green) and Nanog, SSEA3 or SOX2 (in red). DAPI counterstaining for DNA indicates the nuclei of individual cells in each colony. Live alkaline phosphatase (AP) staining was also performed as a positive indicator of stem cells. (c) Bisulfite sequencing analysis of the NANOG and OCT4-2 promoter regions in ARPE-19 and ARPE-19-iPS cells is indicated. Methylated CpGs are indicated with a closed circle, and unmethylated CpGs are indicated with an open circle. An ‘X’ indicates a mismatch or gap in the bisulfite sequence. The CpG position relative to the downstream transcription start site is shown above each row. (d) Quantitative RT-PCR analysis showing the relative gene expression of OCT4, SOX2, NANOG, KLF4, MYC, LIN28, REXO1, ABCG2 and DNMT3B in ARPE-19 and ARPE-19-iPS cells (normalized to GAPDH). Error bars indicate standard error. (e) Hematoxylin and eosin staining of teratoma sections from immunodeficient mice injected with ARPE-19-iPS cells show cartilage, as well as endoderm and ectoderm teratoma formed by ARPE-19-iPS.

https://doi.org/10.7554/eLife.30473.006
Figure 2 with 1 supplement
Quantification of MCM loading rate by ergodic rate analysis.

(a) Gating scheme for chromatin flow cytometry of iPSCs measuring DNA content (DAPI), DNA synthesis (EdU incorporation), and loaded MCM (anti-MCM2); this sample is from Figure 1d. (b) Histograms of only the G1-MCMDNA-positive cells from the four chromatin flow cytometry samples in Figure 1d. (c) Calculated mean MCM loading rate per hour by ergodic rate analysis; mean with error bars ± SD. (n = 3 biological replicates), unpaired two tailed t-test. **p=0.0049. ***p=0.001. See Materials and methods for details. See also Figure 2—source data 1.

https://doi.org/10.7554/eLife.30473.007
Figure 2—figure supplement 1
Ergodic rate analysis binning.

(a) Histograms from G1-MCMDNA-positive samples in Figure 2B, binned into 10 regions for ergodic rate analysis (see Materials and methods). (b) Ergodic rate equation based on Kafri et al. (2013). wn is the rate in each bin n, α is a constant accounting for doubling time, F is the fraction of cells in G1-MCMDNA positive out of all cells, fn is the fraction of cells in each bin n out of G1-MCMDNA positive (see Materials and methods). (c) Value of F for three biological replicates for each cell line, used to calculate Figure 2c. (d) Doubling time and alpha values used to calculate rates in Figure 2c.

https://doi.org/10.7554/eLife.30473.008
Figure 3 with 1 supplement
Differentiation universally decreases MCM loading rate.

(a) Chromatin flow cytometry of hESCs induced to differentiate toward mesoderm (BMP4), neuroectoderm, mesoderm (GSK3βi), or endoderm for 24 or 48 hr. Histograms show only G1-MCMDNA cells positive as in Figure 2b. See methods for differentiation protocols. Cell counts for 24 hr and 48 hr were normalized relative to corresponding hESC samples. (b) Stacked bar graphs of cell cycle distribution for cells in (a). (c) Gene expression analysis of differentiation markers by quantitative PCR of the samples in (a); log2 expression is relative to the undifferentiated cells. Data are mean ±SD of two biological replicates.

https://doi.org/10.7554/eLife.30473.010
Figure 3—figure supplement 1
Stem cell differentiation.

(a) Immunoblots of neuroectoderm differentiation, as in Figure 3a. (b) Representative phase contrast images during the indicated differentiation protocols from Figure 3a. Scale bar is 50 μm.

https://doi.org/10.7554/eLife.30473.011
Figure 4 with 1 supplement
Cyclin E overproduction uncouples MCM loading and G1 length.

(a) Immunoblots of a stable derivative of RPE1-hTert cells bearing an integrated doxycycline-inducible cyclin E construct treated with 100 ng/mL doxycycline for 72 hr to overproduce Cyclin E1 (↑Cyclin E1) or with vehicle control. (b) Stacked bar graphs of cell cycle distribution measured by flow cytometry for cells shown in (a); mean with error bars ± SD (n = 3 biological replicates). (c) Chromatin flow cytometry of control or cyclin E-overproducing cells measuring DNA content (DAPI), DNA synthesis (EdU incorporation), and loaded MCM (anti-MCM2). (d) S phase-MCMDNA-positive cells from samples in (c) divided into populations that began S phase with high or low MCMDNA. Early S cells are S phase cells with G1 DNA content. (e) The percentage of MCMDNA positive, but-low MCM signal intensity S phase cells out of all S-MCMDNA-positive cells from three biological replicates; mean with error bars ± SD, unpaired two tailed t-test. ***p=0.002. (f) Mean loaded MCM in early S phase, (S-MCMDNA positive, G1 DNA content) from three biological replicates; mean with error bars ± SD, unpaired two tailed t-test. ***p=0.0004. (g) Histogram of G1-MCMDNA-positive cells from samples shown in (c). Counts for ↑Cyclin E1 are normalized to the control. (h) Histogram of early S cells from samples shown in (d). Counts for ↑Cyclin E1 are normalized to the control. (These data are one of the replicates quantified in (f).). (i) EdU intensity from ↑Cyclin E1, MCM-high or MCM-low cells from (d) as box-and-whiskers plots. Center line is median, outer box edges are 25th and 75th percentile, whiskers edges are 1st and 99th percentile, individual data points are lowest and highest 1%, respectively. Median EdU incorporation of MCM-high ↑Cyclin E1 cells is 1.8 fold greater than MCM-low, mean EdU incorporation is 1.6-fold greater in MCM-high than MCM-low, average of three biological replicates. Samples compared by unpaired, two tailed t-test, **p=0.0027, **p=0.0033, respectively.

https://doi.org/10.7554/eLife.30473.012
Figure 4—figure supplement 1
G1 Cdt1 levels are unaffected by Cyclin E overproduction.

(a) Immunoblots of whole cell lysates of asynchronous cells treated as in (Figure 4a). (b) Total Cdt1 detected by flow cytometry of cells treated as in Figure 4a measuring DNA content (DAPI), DNA synthesis (EdU incorporation), and Cdt1 (anti-Cdt1). Green are G1 cells, Purple are S phase cells (EdU positive), Grey are G2/M DNA content. (c) Box-and-whiskers plots of G1 Cdt1 concentration per cell from (b). Center line is median, outer box edges are 25th and 75th percentile, whiskers edges are 1st and 99th percentile, individual data points are lowest and highest 1%, respectively. G1 Cdt1 intensity in controls is 1.1-fold greater mean and 1.2-fold greater median than G1 ↑Cyclin E1. Samples were not significantly different, compared by two-tailed, unpaired t test, p=0.1907, p=0.3525, respectively; average of three biological replicates. (d) Immunoblot of whole cell lysate from RPE1-htert cells treated with or without 20 J/m2 of UV irradiation, collected 1 hr after irradiation. UV irradiation induces DNA repair coupled PCNA loading, subsequently targeting Cdt1 for degradation (Arias and Walter, 2005). (e) Flow cytometry of control and UV irradiated cells as in (a), measuring DNA content (DAPI), DNA synthesis (EdU incorporation), and Cdt1 (anti-Cdt1). Plots demonstrate decreased DNA synthesis after UV irradiation. (f) The same samples as in (e). G1 Cdt1 is degraded upon UV-induced DNA repair, demonstrating Cdt1 antibody specificity for immunofluorescence flow cytometry. Black line indicates background defined by controls staining with the secondary antibody alone. Cells below the line (such as the S phase cells) are Cdt1 negative. Cells above the line are Cdt1 positive.

https://doi.org/10.7554/eLife.30473.013
hESCs have high levels of Cdt1 in G1.

(a) Immunoblots of whole cell lysates from the indicated asynchronous cell lines. (b) Expected changes in total protein levels of Cdt1 and Cdc6 during the human cell cycle. (c) Total Cdt1 detected in asynchronous cells by flow cytometry measuring DNA content (DAPI), DNA synthesis (EdU incorporation), and Cdt1 (anti-Cdt1). Green cells are G1, purple cells are S phase (EdU positive), grey cells are G2/M. (d) Box-and-whiskers plots of G1 Cdt1 concentration per cell from (C). Center line is median, outer box edges are 25th and 75th percentile, whiskers edges are 1st and 99th percentile, individual data points are lowest and highest 1%, respectively. Median G1 Cdt1 in hESCs is 2.9-fold greater, mean is 2.2-fold greater than G1 Cdt1 in NPCs, mean p=0.0504 median p=0.0243, average of three biological replicates. Flow plots are G1 cells only (green) from (c).

https://doi.org/10.7554/eLife.30473.014
Figure 6 with 1 supplement
Manipulating MCM loading factors alters MCM loading rates.

(a) Chromatin flow cytometry for hESCs treated with 25 nM siCdt1 or 100 nM siControl for 24 hr and labeled with EdU for 30 min prior to harvest. (b) Immunoblot of total protein from cells in (a). (c) Stacked bar graph of cell cycle distributions for samples in (a); representative of two biological replicates. The percentage of G1 cells in each population is reported in the green sectors. (d) Histograms of loaded MCM in G1-MCMDNA cells. Counts for siCdt1 are normalized to the corresponding siControl sample. (e) Immunoblots of Cdt1 and Cdc6 in RPE cells with combinations of the following: constitutive production of 5Myc-Cdc6-wt or 5myc-Cdc6-mut (not targeted for degradation by APCCDH1: R56A, L59A, K81A, E82A, N83A) and an integrated doxycycline-inducible Cdt1-HA construct treated with 100 ng/mL doxycycline for 48 hr to overproduce Cdt1-HA. (f) Stacked bar graphs of cell cycle distribution measured by flow cytometry for cells shown in (e); mean with error bars ± SD (n = 3 biological replicates). The percentage of G1 cells in each population is reported in the green sectors. (g) Histogram of loaded MCM in G1-MCMDNA-positive cells from (e). Counts of Cdc6-wt and Cdc6-mut are normalized to parent RPE controls.

https://doi.org/10.7554/eLife.30473.015
Figure 6—figure supplement 1
Manipulating MCM loading factors alters MCM loading rates.

(a) Validation of the 5myc-Cdc6-mut. Immunoblots of total cell lysates of RPE, RPE +5myc-Cdc6-wt or RPE +5myc-Cdc6-mut. Cells were synchronized in G0 by contact inhibition. Asynchronous cells were treated with 10 ug/mL cycloheximide for 4 or 8 hr. Cdc6-mut is more stable in cycloheximde and is resistant to APCCDH1-induced degradation in G0 cells. (b) Histogram of loaded MCM in G1-MCMDNA positive cells in RPE cells expressing ectopic Cdt1 compared to control. There is little increase in MCM loading rate. Counts of Cdt1-expressing cells (blue,+Cdt1) are normalized to parent RPE controls (grey). (c) Histogram of loaded MCM in G1-MCMDNA-positive cells in RPE cells from (Figure 6e) with the indicated combinations of Cdc6 and Cdt1 expression. Counts in experimental samples (green and blue) are normalized to their respective controls (grey).

https://doi.org/10.7554/eLife.30473.016
Figure 7 with 3 supplements
Slow MCM loading promotes differentiation.

(a) Immunofluorescence microscopy of hESCs treated with 100 nM of siControl or 100 nM of siCdt1 for 20 hr and then treated with BMP4 as indicated. Cells were fixed and stained with DAPI (blue), Cdx2 antibody (green), and Oct4 antibody (red). Images are one region of 18 fields-of-view per condition; scale bar is 100 μm (see Materials and methods). (b) Density scatterplots of mean fluorescence intensity (arbitrary units) of Oct4 and Cdx2 staining for each cell in each condition, >18,000 cells were quantified per condition. See also Figure 7—source data 1. (c) Diagram of the relationship between Oct4 and Cdx2 in pluripotent and differentiated cells as plotted in (b); color bar for scatterplots in (b). (d) Histogram of mean fluorescence intensity ratio Cdx2/Oct4 for all cells in siControl and siCdt1 treated with 10 ng/mL BMP4 for 48 hr. Rightmost histogram bin contains all values greater than 3.5. The inset is a box-and-whiskers plot of the same data, center line is median, outer box edges are 25th and 75th percentile, whiskers edges are 1st and 99th percentile, individual data points are lowest and highest 1%, respectively. Medians are 0.3722, 0.9319, and means are 0.4285, 1.194 for siControl and siCdt1, respectively. Samples compared by two tailed Mann-Whitney test, ****p<0.0001. See also Figure 7—source data 1. (e) Immunoblot for Cdt1 in whole cell lysates at 20 hr of siRNA treatment, prior to BMP4 treatment. (f) Illustration of the relationship between differentiation and MCM loading rate changes.

https://doi.org/10.7554/eLife.30473.017
Figure 7—figure supplement 1
Complete microscopy dataset and endoderm differentiation.

(a) Complete immunofluorescence microscopy data for Figure 7. White boxes mark the areas shown in Figure 7b; scale bars are 100 μM. (b) Immunoblots for Cdt1 in whole cell lysates of hESCs treated with 100 nM siControl or siCdt1 for 24 hr prior to initiating differentiation toward endoderm, two biological replicates. Bar graph indicates the percentage of Sox17-positive cells, n > 2,300 cells per condition. See also Figure 7—source data 1.

https://doi.org/10.7554/eLife.30473.018
Figure 7—figure supplement 2
Slow MCM loading promotes differentiation.

(a) Histogram of mean fluorescence intensity ratio Cdx2/Oct4 for all cells in siControl and siCdt1 treated with 50 ng/mL BMP4 for 36 or 48 hr. Rightmost histogram bin contains all values greater than 3.0 or 4.0, respectively. The inset is a box-and-whiskers plot of the same data, center line is median, outer box edges are 25th and 75th percentile, whiskers edges are 1st and 99th percentile, individual data points are lowest and highest 1%, respectively. Medians for 50 ng/mL 36 hr are 0. 0.5418, 0.9465, and means are 0. 0.5993, 1.05 for siControl and siCdt1, respectively. Medians for 50 ng/mL 48 hr are 0.916, 1.368, and means are 1.055, 1.387 for siControl and siCdt1, respectively. Samples compared by two tailed Mann-Whitney test, ****p<0.0001. See also Figure 7—source data 1. (b) Immunofluorescence microscopy of hESCs treated with 100 nM of siControl or 100 nM of siCdc6 for 32 hr and then treated with BMP4 as indicated. Cells were fixed and stained with DAPI (blue), Cdx2 antibody (green), and Oct4 antibody (red). Images are one region of 27 fields of view per condition; scale bar is 100 μm. (see Materials and methods). (c) Density scatterplots of mean fluorescence intensity (arbitrary units) of Oct4 and Cdx2 staining for each cell in each condition,>7,900 cells were quantified per condition. See also Figure 7—source data 1. (d) Diagram of the relationship between Oct4 and Cdx2 in pluripotent and differentiated cells as plotted in (b); color bar for scatterplots in (b). (e) Histogram of the mean fluorescence intensity ratio Cdx2/Oct4 for all cells in siControl and siCdc6 treated with 100 ng/mL BMP4 for 36 hr. Rightmost histogram bin contains all values greater than 5.0. the inset is a box-and-whiskers plot of the same data, center line is median, outer box edges are 25th and 75th percentile, whiskers edges are 1st and 99th percentile, individual data points are lowest and highest 1%, respectively. Medians are 1.296, 1.58, and means are 1.433, 1.728 for siControl and siCdc6, respectively. Samples compared by two tailed Mann-Whitney test, ****p<0.0001. See also Figure 7—source data 1. (e) Immunoblot for Cdc6 in whole cell lysates at 32 hr of siRNA treatment, prior to BMP4 treatment.

https://doi.org/10.7554/eLife.30473.019
Figure 7—figure supplement 3
Reducing MCM loading rate by an alternative siRNA targeting Cdc6 instead of Cdt1.

(a) Immunoblot of total protein for hESCs treated with 100 nM siControl for 32 hr or 100 nM siCdc6 pool for 32 hr and pulse-labeled with EdU for 30 min prior to harvest. (b) Chromatin flow cytometry of cells from (a) stained with DAPI and anti-MCM2 and subjected to EdU detection. (c) Histogram MCMDNA positive intensity in G1 cells. Counts for siCdc6 are normalized to the corresponding siControl sample. (d) Stacked bar graph of cell cycle distributions for samples in (a).

https://doi.org/10.7554/eLife.30473.020

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Escherichia coli)E. coli: DH5αInvitrogenCat#11319019
Genetic reagent (Homo sapiens)Rc/CMV cyclin EHinds et al. (1992) PMID: 1388095Addgene 8963
Genetic reagent (Homo sapiens)pInducer20Meerbrey et al., 2011PMID: 21307310Addgene 44012
Genetic reagent (Homo sapiens)ΔNRFDr. J. BearN/A
Genetic reagent (Homo sapiens)VSVGDr. J. BearN/A
Genetic reagent (Homo sapiens)pInducer20-Cyclin E1This PaperN/Asee Materials and methods
Genetic reagent (Homo sapiens)pDONR221InvitrogenCat#12536017
Genetic reagent (Homo sapiens)pENTR221-Cyclin E1This PaperN/A
Genetic reagent (Homo sapiens)PCR4-TOPOInvitrogenCat# 450030
Genetic reagent (Homo sapiens)pInducer20-blastThis PaperN/Asee Materials and methods
Genetic reagent (Homo sapiens)pInducer20-blast-Cdt1-HAThis PaperN/Asee Materials and methods
Genetic reagent (Homo sapiens)CLXSN-5myc-Cdc6-wtThis PaperN/Asee Materials and methods
Genetic reagent (Homo sapiens)CLXSN-5myc-Cdc6-mutThis PaperN/Asee Materials and methods
Cell line (Homo sapiens) maleT98GATCCCat#CRL-1690
Cell line (Homo sapiens) femaleRPE1-hTERTATCCCat#CRL-4000
Cell line (Homo sapiens) maleARPE-19ATCCCat#CRL-2302
Cell line (Homo sapiens) femaleH9 hESC (WA09)WiCellhPSCReg ID: WAe009-A
Cell line (Homo sapiens) maleNPCThis PaperN/Asee Materials and methods
Cell line (Homo sapiens) femaleHEK293TATCCCat# CRL-3216
Cell line (Homo sapiens) maleARPE-iPSCThis PaperN/Asee Materials and methods
AntibodyAnti-Mcm2, mouse monoclonal (BM28)BD BiosciencesCat#610700;RRID: AB_21419521:10,000 (IB) 1:200 (FC)
AntibodyAnti-Mcm3, rabbit polyclonalBethyl LaboratoriesCat#A300-192A; RRID: AB_1627261:10,000 (IB) 1:200 (FC)
AntibodyAnti-Cdt1, rabbit monoclonal (D10F11) (immunoblots)Cell Signaling TechnologiesCat#8064S; RRID: AB_108968511:10,000 (IB)
AntibodyAnti-Cdt1, rabbit monoclonal (EPR17891) (flow cytometry)AbcamCat#ab202067; RRID:AB_26511221:100 (FC)
AntibodyAnti-Cdc6, mouse monoclonal (180.2)Santa Cruz BiotechnologyCat#sc-9964; RRID: AB_6272361:2000 (IB)
AntibodyAnti-Oct4, rabbit polyclonal (immunoblots)AbcamCat#ab19857; RRID: AB_4451751:4000 (IB)
AntibodyAnti-Oct4, mouse monoclonal (9B7) (microscopy)MilliporeCat#:MABD76; RRID: AB_109191701:1000 (IF)
AntibodyAnti-Cdx2, rabbit monoclonal (EPR2764Y)AbcamCat#ab76541; RRID: AB_15233341:1000 (IF)
AntibodyAnti-Sox17, goat polyclonalR and D SystemsCat#AF1924; RRID: AB_3550601:500 (IF)
AntibodyAnti-Cyclin E1, rabbit polyclonalSanta Cruz BiotechnologyCat#sc-198; RRID: AB_6313461:2000 (IB)
AntibodyAnti-Orc1, rabbit polyclonalBethyl LaboratoriesCat#A301-892A; AB_15241031:1000 (IB)
AntibodyAnti-Orc6, rat monoclonal (3A4)Santa Cruz BiotechnologyCat#sc-32735; RRID: AB_6702951:5000 (IB)
AntibodyAnti-geminin, rabbit polyclonalSanta Cruz BiotechnologyCat#sc-13015; RRID: AB_22633941:3000 (IB)
AntibodyAnti-Histone H3, rabbit monoclonal (D1H2)Cell Signaling TechnologiesCat#4499S; RRID: AB_105445371:10,000 (IB)
AntibodyAnti-TRA-1–60, mouse monoclonal (cl.A)InvitrogenCat#41–1000; RRID: AB_6053761:5000 (IB)
AntibodyAnti-nestin, mouse monoclonal (10 C2)AbcamCat#ab22035; RRID: AB_4467231:10000 (IB)
AntibodyAnti-TRA-1–60 mouse (immunofluorescence)Millipore/ChemiconCat# MAB4360; RRID: AB_21191831:400 (IF)
AntibodyAnti-TRA-81 mouse (immunofluorescence)Millipore/ChemiconCat# MAB4381; RRID:AB_1776381:400 (IF)
AntibodyAnti-SSEA-4 mouse (MC-813–70) (immunofluorescence)Millipore/ChemiconCat# MAB4304; RRID:AB_1776291:200 (IF)
antibodyAnti-SSEA3 rabbit (MC-631) (immunofluorescence)Millipore/ChemiconCat# MAB4303; RRID:AB_1776281:200 (IF)
AntibodyAnti-Oct3/4 goat polyclonal (immunofluorescence)AbcamCat# ab27985; RRID:AB_7768981:200 (IF)
AntibodyAnti-NANOG goat polyclonal (immunofluorescence)Everest BiotechCat# EB068601; RRID:AB_21503791:200 (IF)
AntibodyAnti-p27 rabbit polyclonalSanta Cruz BiotechnologyCat#sc-528; RRID:AB_6321291:2000 (IB)
AntibodyAnti-α-tubulinSigma AldrichCat#90261:50000 (IB)
AntibodyGoat anti-Mouse-HRPJackson ImmunoResearchCat#115-035-146; RRID: AB_23073921:10000 (IB)
AntibodyDonkey anti-Rabbit-HRPJackson ImmunoResearchCat#711-035-152; RRID: AB_100152821:10000 (IB)
AntibodyBovine anti-Goat-HRPJackson ImmunoResearchCat#805-035-180; RRID: AB_23408741:10000 (IB)
AntibodyDonkey anti-Rat-HRPJackson ImmunoResearchCat#712-035-153; RRID: AB_23406391:10000 (IB)
AntibodyDonkey anti-Goat-Alexa 594Jackson ImmunoResearchCat#705-585-147; RRID: AB_23404331:1000 (IF)
AntibodyDonkey anti-Rabbit-Alexa 488Life TechnologiesCat#A21206; RRID: AB_25357921:1000 (IF) (FC)
AntibodyGoat anti-Mouse-Alexa 594Life TechnologiesCat#A11032; RRID: AB_25357921:1000 (IF)
AntibodyDonkey anti-Rabbit-Alexa 647Jackson ImmunoResearchCat#711-605-152; RRID: AB_24922881:1000 (FC)
AntibodyDonkey anti-Mouse-Alexa 488Jackson ImmunoResearchCat#715-545-150; RRID: AB_23408451:1000 (FC)
Sequence-based reagentsiCdt1- CCUACGUCAAGCUGGACAATTNevis et al. (2009) PMCID: PMC2972510N/A
Sequence-based reagentsiCdc6-2534- CACCAUGCUCAGCCAUUAAGGUAUUNevis et al. (2009) PMCID: PMC2972510N/A
Sequence-based reagentsiCdc6-2144- UCUAGCCAAUGUGCUUGCAAGUGUANevis et al. (2009) PMCID: PMC2972510N/A
Sequence-based reagentsiControl (Luciferase)- CUUACGCUGAGUACUUCGAColeman et al. (2015) PMID: 26272819N/A
Sequence-based reagentsiMCM3-2859 5’- augacuauugcaucuucauugThis papersynthesized by invitrogen
Sequence-based reagentsiMCM3-2936 5’- aacauaugacuucugaguacuThis papersynthesized by invitrogen
Sequence-based reagentPOU5F1-F: 5'-CCTGAAGCAGAAGAGGATCACC, Eton Bioscience
Sequence-based reagentPOU5F1-R 5'-AAAGCGGCAGATGGTCGTTTGG, Eton Bioscience
Sequence-based reagentCDX2-F 5'-ACAGTCGCTACATCACCATCCG, Eton Bioscience
Sequence-based reagentCDX2-R 5'-CCTCTCCTTTGCTCTGCGGTTC, Eton Bioscience
Sequence-based reagentT-F 5'-CTTCAGCAAAGTCAAGCTCACC, Eton Bioscience
Sequence-based reagentT-R 5'-TGAACTGGGTCTCAGGGAAGCA, Eton Bioscience
Sequence-based reagentSOX17-F 5'-ACGCTTTCATGGTGTGGGCTAAG, Eton Bioscience
Sequence-based reagentSOX17-R 5'-GTCAGCGCCTTCCACGACTTG, Eton Bioscience
Sequence-based reagentCDT1-F 5'-GGAGGTCAGATTACCAGCTCAC, Eton Bioscience
Sequence-based reagentCDT1-R, 5'-TTGACGTGCTCCACCAGCTTCT, Eton Bioscience
Sequence-based reagentSOX2-F 5'-CTACAGCATGATGCAGGACCA, Eton Bioscience
Sequence-based reagentSOX2-R 5'-TCTGCGAGCTGGTCATGGAGT, Eton Bioscience
Sequence-based reagentPAX6-F 5'-AATCAGAGAAGACAGGCCA, Eton Bioscience
Sequence-based reagentPAX6-R 5'-GTGTAGGTATCATAACTC, Eton Bioscience
Sequence-based reagentACTB-F 5'-CACCATTGGCAATGAGCGGTTC, Eton Bioscience
Sequence-based reagentACTB-R 5'-AGGTCTTTGCGGATGTCCACGTEton Bioscience
Sequence-based reagentCDC6-KEN-F: 5- ctccaccaaagcaaggcaaggcggccgcaggtccccctcactcacatacacEurofins
Sequence-based reagentCDC6-KEN-R: 5- GTGTATGTGAGTGAGGGGGACCTGCGGCCGCCTTGCCTTGCTTTGGTGGAGEurofins
Sequence-based reagentCDC6-DBOX-F: 5- aagccctgcctctcagccccgccaaacgtgccggcgatgacaacctatgcaaEurofins
Sequence-based reagentCDC6-DBOX-R: 5- TTGCATAGGTTGTCATCGCCGGCACGTTTGGCGGGGCTGAGAGGCAGGGCTTEurofins
Sequence-based reagentAgeI-rta3-F: 5- gctcggatctccaccccgtaccggtcctgcagtcgaattcacEurofins
Sequence-based reagentAgeI-IRES-blast-R: 5-ACAAAGGCTTGGCCATGGTTTAAGCTTATCATCGTGTTTTTCAEurofins
Sequence-based reagentBlast-F:5- tgaAaaacacgatgataagcttaaaccatggccaagcctttgtEurofins
Sequence-based reagentBlast-AgeI-Ind-R: 5-GTTCAATCATGGTGGACCGG CTATTAGCCCTCCCACACATAACCAEurofins
Sequence-based reagentBP-cycE-F 5'GGGGACAAGTTTGTACAAAAAAGCAGGCTACCATGAAGGAGGACGGCGGCEurofins
Sequence-based reagentBP-cycE-R 5'GGGGACCACTTTGTACAAGAAAGCTGGGTTCACGCCATTTCCGGCCCGCTEurofins
Software, algorithmMATLABMathWorkshttps://www.mathworks.com/
Software, algorithmGraphPad Prism 7GraphPad Softwarehttps://www.graphpad.com/scientific-software/prism/
Software, algorithmNIS-Elements Advanced Research SoftwareNikonhttps://www.nikoninstruments.com/Products/Software/NIS-Elements-Advanced-Research
Software, algorithmCellProfilerCarpenter et al., 2006 PMC1794559http://cellprofiler.org/
Software, algorithmFCS Express 6De Novo Softwarehttps://www.denovosoftware.com/
Software, algorithmFCSExtract UtilityEarl F Glynnhttp://research.stowers.org/mcm/efg/ScientificSoftware/Utility/FCSExtract/index.htm
Software, algorithmQUMARIKENhttp://quma.cdb.riken.jp
Software, algorithmAdobe Photoshop CS6Adobehttp://www.adobe.com/products/photoshop.html
Commercial assay or kitCytoTune-iPS 2.0 Sendai reprogramming kitInvitrogenCat#A16517
Commercial assay or kitDNeasy Blood and Tissue kitQiagenCat#69504
Commercial assay or kitRNeasy Mini kitQiagenCat#74104
Commercial assay or kitEpitect Bisulfite kitQiagenCat#59104
Commercial assay or kitNorgen Biotek’s Total RNA Purification KitNorgen BiotekCat#37500
Commercial assay or kitApplied Biosystem’s High-Capacity RNA-to-cDNAApplied BiosystemCat#4387406
Commercial assay or kitAlkaline Phosphatase Detection KitMilliporeCat# SCR004
Commercial assay or kitQIAquick Gel Extraction kitQiagenCat# 28704
Chemical compound, drugDAPILife TechnologiesCat#D1306
Chemical compound, drugEdUSanta Cruz BiotechnologyCat#sc-284628
Chemical compound, drugPonceau SSigma AldrichCat#P7170-1L
Peptide, recombinant proteinBMP4 ProteinR and D SystemsCat#314 BP-010
Peptide, recombinant proteinActivin A ProteinR and D SystemsCat#338-AC-010
Chemical compound, drugY-27632 2HClSelleck ChemicalsCat#S1049
Chemical compound, drugCHIR-99021Selleck ChemicalsCat#S2924
Chemical compound, drugmTESR1Stem Cell TechnologiesCat#05850
Chemical compound, drugSTEMdiff Neural Induction MediumStem Cell TechnologiesCat#05835
Chemical compound, drugSTEMdiff Neural Progenitor MediumStem Cell TechnologiesCat#05833
Chemical compound, drugEssential 8 MediumLife TechnologiesCat#A1517001
Chemical compound, drugDoxycyclineCalBiochemCat#324385
Chemical compound, drugAlexa 647-azideLife TechnologiesCat#A10277
Chemical compound, drugAlexa 488-azideLife TechnologiesCat#A10266
Chemical compound, drugHydroxyureaAlfa AesarCat#A10831
Chemical compound, drugCorning Matrigel GFR Membrane MatrixCorningCat#CB-40230
Chemical compound, drugPoly-L-OrnithineSigma AldrichCat#P4957-50ML
Chemical compound, drugLamininSigma AldrichCat#L2020-1MG
Chemical compound, drugReLesRStem Cell TechnologiesCat#05872

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