Human Pluripotent Stem Cells

Stem Cell Reports, Volume 11

Supplemental Information

DSG2 Is a Functional Cell Surface Marker for Identiﬁcation and Isolation of Human Pluripotent Stem Cells Jongjin Park, Yeonsung Son, Na Geum Lee, Kyungmin Lee, Dong Gwang Lee, Jinhoi Song, Jaemin Lee, Seokho Kim, Min Ji Cho, Ju-Hong Jang, Jangwook Lee, Jong-Gil Park, Yeon-Gu Kim, Jang-Seong Kim, Jungwoon Lee, Yee Sook Cho, Young-Jun Park, Baek Soo Han, Kwang-Hee Bae, Seungmin Han, Byunghoon Kang, Seungjoo Haam, Sang-Hyun Lee, Sang Chul Lee, and Jeong-Ki Min

Supplementary Information

DSG2 is a Functional Cell Surface Marker for Identification and Isolation of Human Pluripotent Stem Cells

Jongjin Park,1,2,7 Yeonsung Son,1,7 Na Geum Lee,1,2 Kyungmin Lee,1 Dong Gwang Lee,1 Jin Hoi Song,1 Jaemin Lee,3 Seokho Kim,3 Min Ji Cho,1,2 Ju-Hong Jang,1,2 Jangwook Lee,1 Jong-Gil Park,1 Yeon-Gu Kim,1 Jang-Seong Kim,1 Jungwoon Lee,4 Yee Sook Cho,4 Young-Jun Park,5 Baek Soo Han,5 Kwang-Hee Bae,5 Seungmin Han,6 Byunghoon Kang,6 Seungjoo Haam,6 Sang-Hyun Lee,1* Sang Chul Lee,5* and Jeong-Ki Min1,2,*

SUPPLEMENTAL EXPERIMENTAL PROCEDURES Cell culture hESC lines H1 and H9, mESC line J1 (Li et al., 1992), and embryonic carcinoma NTERA-2 cells (Andrews, 1982) were cultured as previously described. For feeder-independent hESC culture, hESC clumps were transferred onto Matrigel (354234, BD Bioscience, New Jersey, USA)-coated plates and treated with mTeSR (#05850, STEMCELL Technologies, Vancouver, Canada). In vitro differentiation was performed via treatment with 10 µM RA (R2625, SigmaAldrich, Ohio, USA) without the basic fibroblast growth factor from the culture medium. EB formation was examined using an AggreWell plate (#27845, STEMCELL Technologies) according to the manufacturer’s instructions. Briefly, hESCs were dissociated into single cells, and 2000 cells were transferred to each microwell. After 2 days, EBs were transferred to UltraLow attachment culture plates (3471, Corning, New York, USA) and cultured with EB differentiation medium lacking the basic fibroblast growth factor. The newborn HFFs (GSC3002, Globalstem, Maryland, USA) and CF-1 MEFs (CEFO, Korea) were cultured with DMEM containing 10% FBS (FBS-BBT, RMBIO, Montana, USA). Somatic reprogramming was performed using a previously described method (Okita et al., 2011). Generation, purification, and biotinylation of mAbs To produce MAbs, BALB/c mice were immunized with H9 hESCs as described previously (Son et al., 2005). Hybridomas were screened to select MAbs that bound to hESCs via flow cytometry. Each positive clone was isolated after two steps of subcloning. Isotyping of each MAb was performed using a mouse immunoglobulin isotyping ELISA kit (BD Biosciences) according to the supplier’s protocol. MAbs were purified from the culture supernatant of the hybridoma cell line via Protein G-Sepharose column chromatography as described previously. Biotinylation of the purified antibody was performed using the ECL protein biotinylation

module (RPN2202, Amersham Biosciences, Buckinghamshire, UK) according to the supplier’s protocol. Target identification of mAb K6-1 To characterize target antigens recognized by K6-1, 1 × 109 H9 hESCs were immunoprecipitated as described below. Proteins immunoprecipitated by K6-1 were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie G250 (#1610406, Bio-Rad, California, USA) according to the supplier’s protocol. The protein band corresponding to approximately 160 kDa was excised, washed, and completely destained with 30% methanol. Then, the gel pieces were dehydrated in 100% acetonitrile for 10 min and dried for 30 min in a vacuum centrifuge. The protein was digested with modified porcine trypsin (V5117, Promega, Wisconsin, USA) in 50 mM ammonium bicarbonate (09830, Sigma-Aldrich) for 16 h at 37°C. The peptides extracted from the gel were concentrated using C18ZipTips (Millipore, Massachusetts, USA) and eluted with 50% (v/v) acetonitrile:water. Mass spectrometric analyses were performed using a Q-TOF MS (Micromass) equipped with a nano-ESI source. The peptide solution was sprayed at a potential of approximately 2 kV, leading to the production of molecular ions. To obtain fragment ions, the collision energy was increased from 10 to 30 eV for collision-induced dissociation experiments. Argon was introduced as a collision gas at a pressure of 10 psi. The Masslynx (Micromass) program was used for data processing, and the MS-Tag search program was employed to identify proteins based on the sequences of peptide fragments. Flow cytometry and cell sorting Cells were dissociated using Accutase (#07920, STEMCELL Technologies) for 10 min, and then, washed out using 3% bovine serum albumin in PBS. Then, 1 µg of Dsg2 antibody was added into the 1 × 106 cell suspension and incubated for 1 h at room temperature. After binding,

the remaining antibody was washed three times with the same buffer, and then, bound to a fluorescence-conjugated secondary antibody for 30 min on ice. Cell sorting was performed using a FACSAria Cell sorter (Becton Dickinson). The resultant histograms were drawn using the WinMDI program. Cells were incubated with fluorescence-conjugated antibodies including SSEA1 (#125606, BioLegend), SSEA3 (#330312, BioLegend), SSEA4 (#330406, BioLegend), Tra-1-60 (#330610, BioLegend), and Tra-1-81 (#330708, BioLegend) for 30min. The FACS control experiment was performed using each isotype control fluorescenceconjugated antibody (#401611 for SSEA1, Tra-1-60 and Tra-1-81, #400807 for SSEA3, #401319 for SSEA4, all from Biolegend, California, USA). shRNA targeting Dsg2 in hESCs shDsg2 (NM_001943.1-987s1c1) and non-target shRNA control (SHC002) encoded in pLKO.1 lentiviral vectors were purchased from Sigma-Aldrich. To generate stable transfectants, the lentiviral vector was co-transfected into Lenti-X-293T (#632180, Clontech, California, USA) cells with virus packaging mix (SHP001, Sigma-Aldrich) using Lipofectamine 2000 (#11668, Invitrogen, California, USA) according to the manufacturer’s instructions. Virus infection was performed by incubating 8 ng/ml polybrene with the culture medium for 5 hr. Fibroblast Reprogramming For somatic reprogramming, we referred previous study from Okita et al., 2011. Briefly, Human foreskin fibroblasts were cultured in DMEM supplemented with 10% FBS. When cells were confluent, they were dissociated via treatment with 0.05% trypsin-EDTA. Cells were transfected using a reprogramming episomal plasmid set including 2.5 µg pCXLE-hOCT3/4shp53-F (#27077), 2 µg pCXLE-hSK (#27078), and 2 µg pCLXE-hUL (#27080, all vectors were purchased from Addgene) via electroporation. Transduced cells were seeded in a culture

plate and cultured with medium. After 3 days, cells were transferred onto a Matrigel-coated cell culture plate and cultured with mTeSR medium. Colonies appeared 1 week after electroporation. Dsg2 Knockout via Crispr cas9 system GFP expressing SpCas9 vector (px458, #48138) was purchased from Addgene. Complementary primer sets for targeting Exon 3 of Dsg2 (Forward : 5’ CACCCTAAACATCCTCATTTAGTG

-

3’,

Reverse

:

5’

–

AAACCACTAAATGAGGATGTTTAG – 3’) hybridized by incubating the mixture at 95°C for 5min and cooling slowly to room temperature. px458 vector was digested with BbsI restriction enzyme (NEB). Dsg2 guide sequence was inserted into the vector via T4 ligase treatment. Both human fibroblasts and human embryonic stem cells were transfected using a recombination vector via electroporation. After 3days incubation, cells were dissociated into single cell and sorted into two populations depending on GFP expression using FACSAriaII cell sorter. To evaluate the genome editing, genomic DNA extracted from GFP negative and positive cells, and target region was amplified with customized two primer set (Forward : 5’ AGACAATGAAGCCTCATAGG - 3’, Reverse : 5’ – ATGATGCTGCATCTTCCGGA – 3’). Amplified PCR products were analyzed by the sanger’s methods. Immunofluorescence staining Cells were transferred onto gelatin-coated coverslips plated with feeder cells. Samples were washed with PBS and fixed in 4% paraformaldehyde (Biosesang, Korea) in PBS for 15 min. After removing the fixative and washing with PBS, the cell membrane was permeabilized with 0.1% Triton X-100 (#0694, Amresco, Ohio, USA) for 20 min. Samples were blocked with 5% BSA in PBS for 1 h at room temperature and incubated with rabbit anti-EpCAM (#ab32392, clone E144, Abcam, 1:200 dlution), anti-Dsg2 (#ab150372, clone EPR6768, Abcam, 1:400

dlution), mouse anti-SSEA1 (#sc-21702, clone 480, Santa Cruz Biotechnology, 1:200), goat anti-Slug (#sc-10437, clone D-19, Santa Cruz Biotechnology, 1:100 dilution), rabbit anti-Ecadherin (#3195, clone 24E10, Cell Signaling Technology, 1:200 dilution), goat anti-Oct4 (#AF1759, R&D Systems, 1:50 dilution), goat anti-Nanog (#AF1997, R&D Systems, 1:50 dilution), and rabbit beta-catenin (#06-734, Millipore, 1:200 dilution) for 16 h at 4°C or 2 h at room temperature. After serial washes to remove residual antibody, samples were incubated with the appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature in the dark. Nuclei were stained with 1 µg/ml DAPI (#D9542, Sigma-Aldrich) for 5 min. Coverslips were mounted on the slide glass with a mounting medium (#S3023, Dako, California, USA). Fluorescence images were visualized using a Zeiss 510LSM META laserscanning microscope (Carl Zeiss AG). Image data were trimmed using the MetaMorph program (Molecular Devices). BrdU incorporation assay Cells were incubated in the presence of 20 μM BrdU (#B5002, Sigma-Aldrich) for 3 hr at 37°C and dissociated into single cells using Trypsin–EDTA (Invitrogen). After washing in PBS, the cell pellet was fixed in 70% ice-cold ethanol overnight. The supernatant was removed via centrifugation at 4000 RPM for 2 min, and the genomic DNA of the cells was denatured with 2N hydrogen chloride/0.5% Triton X-100 for 30 min at room temperature. After acid neutralization with 0.1 M sodium tetraborate (pH 8.5), cells were resuspended in PBS containing 1% BSA and incubated with mouse anti-BrdU antibody (#555627, BD Bioscience) for 30 min at room temperature. After washing with 1% BSA/PBS, 1 µg of anti-mouse FITC antibody (#A11001, Invitrogen) was added for every 1 × 106 cells, and the cells were incubated for 30 min at room temperature. Cells were transferred into an FACS tube, after which the pellet was dissolved in 0.5 ml PBS containing 10 µg RNase A (#R6148, Roche) and 5 µg

propidium iodide (#P4170, Sigma-Aldrich). Fluorescence was analyzed using a FACSCalibur flow cytometer (Becton Dickinson). AP staining Samples were fixed with 10% formalin solution (#HT501128) and then stained using an AP staining kit (#86R, all from Sigma-Aldrich) including Napthol and Fast Red Violet for 20 min in the dark as per the manufacturer’s manual. Images were taken using an HP Scanjet (HewlettPackard), and then, AP-positive colonies were manually counted. Teratoma formation NSG (NOD scid gamma) mice were obtained from Jackson Laboratory. All mice used in the study were 6 weeks of age. Mouse care followed the guidelines of the Animal Care Committee of the Korea Research Institute of Bioscience and Biotechnology. hESCs were dissociated using Accutase and harvested into tubes, and the pellets were suspended in 1:1 (vol:vol) DMEM/F12 and Matrigel. Approximately 1 cm of abdominal skin was incised with fine scissors under isoflurane inhalation anesthesia. The testes were removed from the abdominal cavity using sterilized forceps. The Dsg2-positive or –negative cells (2 × 105) were injected into each side of the testes using a 31-gauge Ultra-Fine™ syringe (BD Bioscience). After 8 weeks, the teratomas were excised and measured weight. Samples were fixed with 4% paraformaldehyde in PBS overnight at 4°C. Paraffin embedding was performed using a tissue automatic sample preparation system (SAKURA). Tissue including the paraffin block was sliced into 4-µm-thick sections using a Rotary microtome (SAKURA). Slices were fixed onto (3-aminopropyl) triethoxysilane (#390143, Sigma)-coated slide glass. After ethanol dehydration, slides were stained with hematoxylin and eosin. RT-PCR and real-time PCR

Total RNA was extracted using TRIZOL (Intron Biotechnology, Korea). After incubation for 5 min at room temperature, the solution was mixed with chloroform (#C2432, Sigma-aldrich) to isolate the RNA fraction from the other cell composite. The aqueous phase was isolated from the mixture, and then, samples were precipitated with isopropyl alcohol. After RNA pellet centrifugation at 14,000 RPM for 10 min, the supernatant was removed and the pellet was washed with 70% ethyl alcohol in distilled water. The dried pellet was dissolved in distilled water. Target RNA was converted to cDNA according to a previously described process (Son et al., 2005). The primers used in this study were described as follows; DSG2 (Sense : 5’ – AGGTATGGCCAAGGAAGCCACGA–

3’,

Anti

Sense

:

5’

–

ATAGCGCCTGTGGCCCCTGTAA– 3’), E-CADHERIN (Sense : 5’ –– 3’, Anti Sense : 5’ – – 3’), EpCAM (Sense : 5’ –GGGCCCTCCAGAACAATGAT– 3’, Anti Sense : 5’ – AGCCACATCAGCTATGTCCAC–

3’),

OCT4

(Sense

:

5’

–

TCGTGCAGGCCCGAAAGAGA– 3’, Anti Sense : 5’ –TGGCGCCGGTTACAGAACCA– 3’), NANOG (Sense : 5’ –ACCTGGAGCAACCAGACCCA– 3’, Anti Sense : 5’ – AGCTTCCAAGGCAGCCTCCA–

3’),

SOX2

(Sense

:

5’

–

AAAAACAGCCCGGACCGCGT– 3’, Anti Sense : 5’ –TCCGCCGGGGCCGGTATTTA– 3’), UTF1 (Sense : 5’ –ACCAGCTGCTGACCTTGA– 3’, Anti Sense : 5’ – CTGGAGAGGGGAGACTGG– 3’), GDF3 (Sense : 5’ –TGGTGACTCTCAACCCTGAT– 3’, Anti Sense : 5’ –ATGGTCAGTGAGAAGGGACA– 3’), DPPA2 (Sense : 5’ – TTTCTGTTGAGGCCTTTTTG– 3’, Anti Sense : 5’ –AAATGCAGGCAGGTAACAAG– 3’), DPPA3 (Sense : 5’ –AACCTACATCCCAGGGTCTC– 3’, Anti Sense : 5’ – CCTGCTGTAAAGCCACTCAT–

3’),

DPPA4

(Sense

:

5’

–

GACACATCCTCCTGAAGTGG– 3’, Anti Sense : 5’ –TGTCTGCAGGGAGACTTTTC– 3’), DPPA5 (Sense : 5’ –CCTTGGATGAAGTGAACCAG– 3’, Anti Sense : 5’ – AGACTCAGAGCCAAGGGTTT–

3’),

SOX17

(Sense

:

5’

–

CGCTTTCATGGTGTGGGCTAAGGACG–

3’,

TAGTTGGGGTGGTCCTGCATGTGCTG–

3’),

TGGACCTCCAGAACAAGCCCCA–

3’,

AGGAGACAGCCGCCTTCGCT–

3’),

GCGAGGGAGCGGCTGACATTAT–

Anti EOMES Anti

AFP

3’,

Sense

Anti

:

(Sense

Sense (Sense Sense

–

5’ :

5’

–

:

5’

–

:

5’

–

:

5’

–

GTTTGCAGCGCTACACCCTGA– 3’), GFAP (Sense : 5’ –CCTCTCCCTGGCTCGAATG– 3’, Anti Sense : 5’ –GGAAGCGAACCTTCTCGATGTA– 3’), PAX6 (Sense : 5’ – AGCGGGAGTGCCCGTCCATC– 3’, Anti Sense : 5’ –GGTTGCCCTGGCACCGAAGT– 3’), NEUROD1 (Sense : 5’ –GCAGCGCTGGAGCCCTTCTT– 3’, Anti Sense : 5’ – GATCCGTGGCTTTGGGCCCC–

3’),

HAND1

(Sense

:

–

5’

TCCCTTTTCCGCTTGCTCTC– 3’, Anti Sense : 5’ –CATCGCCTACCTGATGGACG– 3’), LEF1 (Sense : 5’ –CGGACACGAGGTGGCCAGAC– 3’, Anti Sense : 5’ – ACCGCATGGGATGGCTGCAC–

3’),

COL2A1

(Sense

:

–

5’

GGAGATCCGGGCAGAGGGCA– 3’, Anti Sense : 5’ –CCGAATTCCTGCTCGGGCCC– 3’), MSI1 (Sense : 5’ –ACCCCCACATTCTCTCACTG– 3’, Anti Sense : 5’ – AAACCCAAAACACGAACAGC–

3’),

and

human

GAPDH

(Sense

:

5’

–

ACACGTTGGCAGTGGGGACA– 3’, Anti Sense : 5’ –TGCCTCCTGCACCACCAACT– 3’).

In

mouse

gene

expression

experiment,

Afp

(Sense

:

5’

–

GTGAGCATTGCCTCCACGTGCTG– 3’, Anti Sense : 5’ –AACTGGTGATGCATAGCCT– 3’), Sox17 (Sense : 5’ –CACAGCAGAACCCAGATCTGCA– 3’, Anti Sense : 5’ – CATGTGCGGAGACATCAGCGGAG–

3’),

Nestin

(Sense

:

5’

–

TCTGGAAGTCAACAGAGGTGG– 3’, Anti Sense : 5’ –ACGGAGTCTTGTTCACCTGC– 3’), Sox1 (Sense : 5’ –GGATCTCTGGTCAAGTCGGAG– 3’, Anti Sense : 5’ – CTGGCGCTCGGCTCTCCAGAG– GTTCCTGGTGCTGGCACCCTCTGC–

3’),

Brachyury 3’,

Anti

(Sense Sense

: :

5’

–

5’

–

CAGACCAGAGACTGGGATACTG–

3’),

Oct4

(Sense

:

5’

–

CACGAGTGGAAAGCAACTCA– 3’, Anti Sense : 5’ –AGATGGTGGTCTGGCTGAAC– 3’), Nanog (Sense : 5’ –AAGTACCTCAGCCTCCAGCA– 3’, Anti Sense : 5’ – GTGCTGAGCCCTTCTGAATC– 3’), Dsg2 (Sense : 5’ –GAAGGCATTCATTTCAAGAG– 3’, Anti Sense : 5’ –GACCCAGTTGTCAGTGTCTT– 3’), and mouse Gapdh (Sense : 5’ – GAGACCCCACTAACATCAAA– 3’, Anti Sense : 5’ –TTGCTGACAATCTTGAGTGA– 3’). Dual-luciferase reporter gene assay To evaluate Slug and TCF promoter activity following the loss of Dsg2 in hESCs and HFFs, stable cells were dissociated into single cells using Accutase or 0.05% trypsin/EDTA treatment. After washing in PBS, 4 × 105 cells were resuspended in 100 µl of resuspension buffer, and then, 2 µg of pGL4.1_Slug (−981 to +1 or +174), TOP, or FOP FLASH vector (Addgene) were mixed with 0.2 µg of pRenilla-TK. Vector transduction into cells was conducted using a Neon electroporator (ThermoFisher Scientific) following the manufacturer’s cell type-specific instructions. Transduced cells were cultured for 2 days. Luciferase activities were measured using a fluorescence spectrophotometer (FlOUstar omega, BMG Labtech, Ortenberg, Germany) in triplicate according to the manufacturer’s instructions for the Dual-Luciferase Reporter Assay kit (Promega). Western blotting Cell lysates or immunoprecipitates were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blocked membranes were then incubated with the indicated antibodies, and the immunoreactive bands were visualized by developing medical Xlay film (AGFA).

Statistical Analysis Data from in vitro and in vivo experiments were expressed as the mean ± SEM. Differences between groups were analyzed using Student’s t-test. p < 0.05 denoted statistical significance. Data are representative of at least three independent experiments.

Supplementary Figure S1

Figure S1. K6-1 recognizes human DSG2. (A) Sequence analysis of DSG2 expressed in hESCs. The MS/MS spectrum of DSG2 obtained after trypsin digestion was analyzed by QTOF mass spectrometry (data was not shown). Of the 1117 amino acids encoding human DSG2, 15 cleaved peptides covered 418 amino acids (37.4% coverage). Cleaved peptides that matched DSG2 are written in red. (B) K6-1 could not detect mouse embryonic stem cells. FACS analysis of K6-1 and SSEA1 expression in mouse embryonic fibroblasts and mouse ESCs.


Figure S2. DSG2 is rapidly downregulated during the differentiation of mESCs. (A) To confirm mouse ESC differentiation, SSEA1 expression in undifferentiated J1 and differentiated 3–9-day EBs was compared to mESC by FACS analysis. (B) And the Expression of three germ layer markers in mESCs and EBs as assessed by RT-PCR. (C and D) DSG2 expression in mESC and differentiated EBs were determined by immunoblotting and qPCR compared with that of pluripotent markers. Error bars indicate ±SEM. (E) DSG2 expression was more abundant than well-known PSC marker. Immunostaining was performed on fixed cells with anti-DSG2 and E-Cadherin antibody at 9 and 12 days from reprogramming. Scale bars, 50 µm.


Figure S3. Among desmosome components, DSG2 is uniquely expressed in undifferentiated stem cells. (A) To compare DSG2 expression on undifferentiated hPSCs, Desmosome component expression in hESC was compared to differentiated 3–9-day EBs by RT-PCR. (B) Moreover, Expression of desmosome composition in hPSCs was compared to body-forming variable tissues via meta-analysis (based on human public transcriptome data, Amazonia; probesets 207324_s_at, 204750_s_at, 206033_s_at, 206642_at, 217901_at, 205595_at, 1561330_at, 221854_at, 207717_at, 209873_at, 201928_at, 201015_at, and 200606_at are presented for DSC1–3, DSG1–4, PKP1–4, JUP, and DSP, respectively). Sample description (PSC, pluripotent stem cell; Fib/Germ, fibroblasts and germ cells; other tissuespecific cells were divided into the three germ layers) was used.


Figure S4. Loss of DSG2 blocks somatic reprogramming. To evaluate the role of DSG2 in somatic reprogramming, shCtrl and shDSG2 human fibroblasts were generated via lentiviral infection. (A) Expression of DSG2 was determined by qPCR. Quantification from five independent assays is shown in the graph. *p < 0.05 versus shCtrl. Error bars indicate ±SEM. (B and C) Reprogramming efficacy was assessed by AP staining after 15 days of reprogramming. Quantification from five independent assays is shown in the graph. *p < 0.05 versus shCtrl. (D and E) shCtrl and shDSG2 reprogrammed human fibroblasts were fixed at day 12 of reprogramming and stained with anti-TRA-1-60 antibody. The graph represents five independent experiments in which 10 colonies were analyzed for each condition. *p < 0.01 versus shCtrl. (F) qPCR analysis of core PSC transcripts, including UTF1, REX1, DPPA2, and GDF3, was performed at late time points of reprogramming in shCtrl and shDSG2 cells. (G) Expression of PSC markers was determined by immunoblotting at the indicated time points.


Figure S5. DSG2 knockout disrupts pluripotency in PSC. To further evaluate the role of DSG2 in pluripotency, we generated Dsg2 knockout hESC by Crispr/Cas9 system. (A) Representative images indicate alkaline phosphatase activity in control and DSG2 knockout hESC. (B) Gene expression of pluripotency transcription factors was assessed by qPCR. In addition to gene expression, (C) pluripotency marker expression was analyzed by Western blotting.


Figure S6. DSG2 low population showed more mesenchymal state than DSG2 high population. To evaluate epithelial and mesenchymal state according to DSG2 expression, (A) Low, Middle and High population was isolated via DSG2 expression dependent FACS sorting. (B) E-Cadherin, Vimentin, and Slug expression was compared by immunoblotting.

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