ZFX Knockdown Human ESCs
H9 (WA-09) hESCs were maintained on MEFs (GlobalStem, Inc., Rockville, MD) before feeder-free expansion on Matrigel with conditioned media and 10 ng/ml FGF2 (3–5 days). To prepare cells for transduction, hESCs were exposed to Accutase (Innovative Cell Technologies, Inc., San Diego, CA) to create a single-celled suspension before plating at 18,000 cells/cm2 in the presence of 10 µM Y-27632. The following day, cells were transduced at a multiplicity of infection of 1 and 0.1. For each knockdown, phenotypes were observed at both multiplicities relative to the scrambled control. Puromycin selection began two days after transduction and no mock transduced cells remained two days after selection began. At this time, 5,000 puromycin resistant cells were replated per condition and were expanded in conditioned media +10 ng/ml FGF2 for seven days before crystal violet staining.
Cells were transduced at a multiplicity of ~0.1 as described above and puromycin selection was applied as described. Cells were fixed in 4% paraformaldehyde for 15 minutes before rinsing four times with PBS. Fixed cells were blocked/permeabilized with 1× Perm/wash buffer (BD Biosciences, San Jose, CA) for 20 minutes before exposure to ZFX primary antibody overnight at 4°C. The next day, cells were washed three times in Perm/wash buffer before exposure to secondary antibodies conjugated to Alexa 488 and Hoechst 33258. Cells were washed three times after the hour, and PBS was added to the cells for imaging.
Imaging was performed on an Operetta High Content Screening System. The Hoechst was exposed for 20 mSec and the Alexa 488 signal was captured by a 300 mSec exposure using 50% intensity from the light source. Harmony software version 3.0 was used to identify cell nuclei (Hoechst signal) and quantitated the Alexa 488 pixel intensity (ZFX protein) for each cell nucleus. The mean pixel intensity from 50 cell fields per sample were imaged from 3 independent experiments for quantitation. All knockdowns were significantly different from the scrambled control (p<0.0001 for pairwise t-tests). 11,803 (Scr), 10,296 (Z2), 4,765 (Z3) and 6,905 (Z4) nuclei were measured in these experiments.
ZFX Overexpressing Human ESCs
Human ESCs were maintained as previously described 
. BAC overexpressing human ESCs were made essentially as described for transgenic reporter BAC integration 
. Briefly, H9 (WA-09) hESCs were maintained on MEFs (GlobalStem, Inc., Rockville, MD) before feeder-free expansion on Matrigel with conditioned media and 10 ng/ml FGF2 (3–5 days). To prepare cells for nucleofection, Accutase (Innovative Cell Technologies, Inc., San Diego, CA) was used to create a single-celled suspension, and 5 million cells were used per nucleofection (solution V, protocol B-16; Lonza, Cologne, Germany) with 3 ug of human Zfx BAC (RP11-1107D4) retrofitted with pRetroES 
. Each nucleofection reaction was plated onto Neo-resistant MEFs (GlobalStem, Inc., Rockville, MD) in 10 µM Y-27632 for two days after nucleofection (Tocris Bioscience, Bristol, UK), and cells were allowed to recover for 4 days. Selection began with 25 ug/ml G418 (days 4–14; Invitrogen Life Science, Carlsbad, CA) before increasing selection to 40 ug/ml (days 14–20). Drug-resistant colonies were manually dissected before expansion into lines.
Quantitative PCR Analysis
Real-time PCR analysis was performed using the following QuantiTect primers: POU5F1, CXCR4, PAX6, GAPDH and ACTN2. Other gene expression levels were determined by qPCR using the following primers:
R: 5′- CTGGTGATGCAGGCTGACAA-3′
RNA was isolated using Trizol (Invitrogen, #15596-018) and reversed transcribed with the QuantiTect Reverse Transcription kit (Qiagen, #205313). SYBR green PCR reactions were performed using the PerfeCta SYBR Green SuperMix (Quanta Bioscience, #95054-500) on an Eppendorf Mastercycler Realplex2.
Stem Cell Marker Expression
Direct immunofluorescence was performed using SSEA-4 Alexa Fluor® 488 (BD Pharmingen, #560308, La Jolla, CA), TRA1-81 Alexa Fluor® 555 (BD Pharmingen, #560123, La Jolla, CA), and Oct3/4 Alexa Fluor® 647 (BD Pharmingen, #560307, La Jolla, CA) according to the manufacturers recommendations.
Protein samples of H9 hESC and ZFXOver
hESC clones were prepared in RIPA buffer plus protease inhibitors (Roche Diagnostics Ltd.). Protein concentration was determined using Bradford assay and 30 ug or 15 ug of each sample were separated by 10% polyacrylamide gel. Gels were transferred to PVDF membranes using the Bio-Rad mini-gel transfer apparatus. Membranes were blocked with 3% milk for 1 hour at room temperature and probed with affinity-purified polyclonal anti-Zfx antibody (in 3% Milk, 1
1000 overnight at 4°C). After washing 3X with TBST, membranes were next incubated with peroxidase-labeled goat anti-rabbit IgG (1
10,000 in 3% milk) for 1 hour at room temperature before visualization with ECL.
Spontaneous Differentiation Assay
Human ESCs were expanded on Matrigel-coated dishes in standard human ESC media containing 6 ng/ml FGF2 without conditioned media. After seven days, single-celled suspensions were made by dissociating cells with Accutase (Innovative Cell Technologies, Inc., San Diego, CA) for 45 minutes before being washed twice with human ESC media. Aliquots of the cells were stained with anti-SSEA-3 (SSEA-3 Alexa Fluor® 647; unpublished, BD Pharmingen, La Jolla, CA) and anti-SSEA-1 antibodies (SSEA-1 Alexa Fluor® 647; BD Pharmingen #560120, La Jolla, CA) before quantitation on a FACSAria. For this assay, a number of unrelated BAC transgenic cell lines were used as normal clone controls (see ). These lines do not overexpress ZFX but have undergone the same clonal selection as the ZFXOver
clones (data not shown). Control clones were made from BACs with: ID1::YFP (clone 2) 
, HES5::GFP (clone 10) and DLL1::GFP (clones 281 and 277) 
. All control clones were derived from H9 hESC and were made with the same nucleofection/selection protocol 
All control hESC lines and clones used in this manuscript.
For colony-forming cell assays, human ESC were dissociated with Accutase (Innovative Cell Technologies, Inc., San Diego, CA) for 45 minutes. Dissociated cells were washed twice with human ESC media. Serial dilutions of hESCs were plated onto Matrigel-coated 6 well dishes in conditioned media with 10 ng/ml FGF2. Cells were exposed to Y-27632 for 24 hours after plating, and colonies were visualized after 7 days by staining with crystal violet. The data presented are the number of colonies from the 1
100 dilution and represent the number of colonies derived from 2,666 cells in 9.6 cm2
Crystal violet staining was used because it is easier and cheaper and works as well for determining the number of colony forming units in a given culture. Figure S1A
shows the alkaline phosphatase staining (Sigma, 86R-1KT) in the colony-forming assay: nearly every colony stains positive. We confirmed this result by growing clonal colonies before performing Oct4 FACS analysis (Figure S1B
). Over 97.5% of the cells from all genotypes were Oct4+ demonstrating that these growth conditions strongly select for undifferentiated hESCs. This makes alkaline phosphatase detection a redundant feature of the colony growth assay.
To direct human ESCs to endoderm, we used a previously published protocol 
. Briefly, endoderm induction was performed by switching from hESC media to RPMI with glutamine, 0.5% Hyclone FBS and 100 ng/ml Activin A. Cells were fed daily and were assayed on days 1 and 3. ESCs were directed to neural cells through co-culture with MS-5 bone marrow stromal cells as previously described 
Gene Expression Array
RNA was isolated from the Tra1-81HI
fraction of ZFXOver1,2
and H9 hESCs using Trizol (Invitrogen). Samples were labeled and hybridized to Illumina human 6 oligonucleotide arrays. Normalization and model-based expression measurements were performed using the Illumina analysis package (LUMI) available through open-source Bioconductor project (www.bioconductor.org
) within the statistical programming language R (http://cran.r-project.org/
). Pairwise comparisons were performed using the Linear Models for Microarray Data package (LIMMA) available through Bioconductor. Genes found to have an adjusted p-value <0.05 were considered significant.
Statistical analysis was performed using Prism for Mac version 5.0a. Unpaired, two-tailed T tests were performed comparing control cell lines versus ZFXOver clones.
Sub-confluent cultures were treated with 0.1 ug/ml Colcemid (Karyomax, Invitrogen) for 60–90 minutes before harvesting according to standard cytogenetics procedures. Briefly, cells were trypsinized to a single cell suspension, pelleted at 250 g for 5 minutes and resuspended in warm 0.075M KCl. After 8 minutes incubation at 37°C, the hypotonic was diluted with approximately 1/4 volume of 3
1 methanol/glacial acetic acid fixative, gently mixed, and the cells pelleted as before. The supernatant was removed and the cell pellet loosened by gently flicking the base of the tube. The cells were then fixed in 3 changes of fixative. Fixed cell suspensions were stored at −20°C. Fixed metaphase preparations were dropped onto dry slides and the quality of spreading assessed by phase microscopy. Slides were then air-dried and aged at 37°C overnight.
DNA for human BAC clone RP11-1107D4 (BACPAC Resources, Children’s Hospital and Research Center at Oakland), spanning the ZFX locus, was labeled by nick translation with Red dUTP (Enzo Life Sciences Inc., supplied by Abbott Molecular Inc.), and hybridized to metaphase slides. Briefly, approximately 100 ng of labeled probe and 1 ug human Cot-1 DNA (Invitrogen) was mixed with hybridization buffer (50% formamide, 2×SSC,10% dextran sulfate, 0.1% SDS, 1×Denhardt’s solution, 40 mM sodium phosphate buffer, pH7) and applied to each slide before sealing under a coverslip with rubber cement. The slides were then placed in a HYBrite™ (Abbott Molecular), denatured at 72°C for 3°minutes, and then hybridized at 37°C overnight. After coverslip removal in 2×SCC, 0.1% Igepal CA 630 at room temperature, the slides were washed in 0.4×SSC, 0.3% Igepal at 73°C for 2°minutes, then in 2×SCC, 0.1% Igepal at RT, and rinsed briefly in 2×SSC. The slides were then stained in 0.08 µg/ml DAPI in 2×SSC for 3 minutes, rinsed, air-dried, then mounted in antifade solution (Vectashield, Vector Labs), and stored at 4°C. Slides were scanned using a Zeiss Axioplan 2i epifluorescence microscope equipped with a megapixel CCD camera (CV-M4+CL, JAI) controlled by Isis 5.2 imaging software (Metasystems Group Inc, Waltham, MA). At least 10 metaphases and 5 interphase nuclei were examined for each preparation.