EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Lack of ABCG2 Expression and Side Population Properties in Human

Pluripotent Stem Cells

HUI ZENG,a,b

JUNG WOO PARK,aMIN GUO,

c,dGE LIN,

aLEANN CRANDALL,

aTIWANNA COMPTON,

a

XIAOFANG WANG,a XUE-JUN LI,c,e FANG-PING CHEN,b REN-HE XUa,e

aDepartment of Genetics and Developmental Biology, University of Connecticut Stem Cell Institute,bDepartments of Hematology, Xiang-Ya Hospital, Central South University, Changsha, Hunan, China;cDepartment of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut, USA;dDepartments of Geriatrics, Xiang-Ya Hospital, Central South University, Changsha, Hunan, China;eUniversity of Connecticut Stem Cell Institute, Farmington, Connecticut, USA

Key Words. Pluripotent stem cells • Side population • Trophoblast

ABSTRACT

The multidrug transporter ABCG2 in cell membranes ena-bles various stem cells and cancer cells to efflux chemicals,including the fluorescent dye Hoechst 33342. The Hoechst2

cells can be sorted out as a side population with stem cellproperties. Abcg2 expression in mouse embryonic stem cells

(ESCs) reduces accumulation of DNA-damaging metabolitesin the cells, which helps prevent cell differentiation. Surpris-ingly, we found that human ESCs do not express ABCG2and cannot efflux Hoechst. In contrast, trophoblasts andneural epithelial cells derived from human ESCs are

ABCG21

and Hoechst2. Human ESCs ectopically express-

ing ABCG2 become Hoechst2, more tolerant of toxicity of

mitoxantrone, a substrate of ABCG2, and more capable ofself-renewal in basic fibroblast growth factor (bFGF)-freecondition than control cells. However, Hoechstlow cells sorted

as a small subpopulation from human ESCs express lowerlevels of pluripotency markers than the Hoechsthigh cells.

Similar results were observed with human induced pluripo-tent stem cells. Conversely, mouse ESCs are Abcg21 andmouse trophoblasts, Abcg22. Thus, absence of ABCG2 is a

novel feature of human pluripotent stem cells, which distin-guishes them from many other stem cells including mouse

ESCs, and may be a reason why they are sensitive to subop-timal culture conditions. STEM CELLS 2009;27:2435–2445

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

The superfamily of ATP-binding cassette (ABC) cell mem-brane transporters was initially characterized on the basis oftheir role in multidrug resistance, as a result of their ability toefflux various chemotherapeutic agents in an energy-depend-ent manner [1]. ABCG2, also named MXR, BCRP, or ABCP,is a half-transporter of this family, expressed in a variety ofnormal and malignant cells to efflux chemotherapeutic agents,for example, mitoxantrone, anthracyclines, and campothecins[2], as well as the fluorescent dye Hoechst 33342 from thecells [3]. Via fluorescence-activated cell sorting (FACS) ofmouse bone marrow cells incubated with the dye, a subset ofHoechst� cells can be identified and sorted out as a side pop-ulation (SP), which possesses hematopoietic stem cell proper-ties [4]. During differentiation of hematopoietic stem cells,Abcg2 expression sharply declines, suggesting Abcg2 as astem cell marker [5]. Abcg2-null mice almost completely lost

lin�/c-Kitþ/Sca-1þ SP cells, the residual SP cells lacked repo-pulating ability, and the Abcg2� hematopoietic cells weresensitive to mitoxantrone treatment in mice that underwenttransplantation [6].

SP cells can also be isolated from many other tissues suchas the liver, blood, lung, heart, gonad, intestine, and cornea[7], as well as from cancers such as acute myeloid leukemia,breast cancer, liver cancer, glioma, and lung cancer [8]. Inthese cases, the SP cells all express ABCG2 and associatewith normal or cancer stem cell properties. However, anincreasing number of facts challenge the association betweenABCG2, SP, and stem cells. For example, ABCG2 expressionis correlated neither to SP nor to hematopoietic progenitorfunction in human umbilical cord blood [9]. During mousedevelopment, the embryo remains efflux inactive until theblastocyst stage when the inner cell mass becomes effluxactive [10]. Mouse embryonic stem cells (ESCs), which areusually derived from the inner cell mass, also express Abcg2and efflux Hoechst like SP cells [10].

Author contributions: H.Z.: research conception and design, collection and/or assembly of data, data analysis, manuscript writing; J.W.P.and M.G.: collection and/or assembly of data, data analysis; G.L.: collection and/or assembly of data; L.C., T.C. and X.W.: provision ofstudy material; X.-J.L.: research design; F.-P.C: research conception and design; R.-H.X.: research conception and design, data analysis,manuscript writing, final approval of manuscript.

Correspondence: Dr. Ren-He Xu, M.D., Ph.D., University of Connecticut Stem Cell Institute; Department of Genetics andDevelopmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut, 06030, USA.Telephone: 860-679-3363; Fax: 860-679-8345; e-mail: renhexu@uchc.edu; or Fang-Ping Chen, M.D., Department of Hematology,Xiang-Ya Hospital, Central South University, 87 Xiang-ya Road, Changsha, Hunan, 410008, China. Telephone: 86-731-84327330; Fax:86-731-84327332; e-mail: xychenfp@2118.cn Received June 8, 2009; accepted for publication July 31, 2009; first published online inSTEM CELLS EXPRESS August 7, 2009. VC AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.192

STEM CELLS 2009;27:2435–2445 www.StemCells.com

Surprisingly, when we counterstained live human ESCsfor their nuclei with Hoechst 33342, we observed that onlyundifferentiated human ESCs were Hoechstþ, whereas somespontaneously differentiated human ESCs were Hoechst�. Wefollowed up with this observation to clarify whether humanESCs possess the SP property at all. Our results demonstratethat ABCG2 is not expressed in human ESCs, but is presentin their early stage derivatives such as trophoblasts and neuralepithelial cells. Human ESCs ectopically expressing ABCG2become more tolerant of the toxicity of mitoxantrone, anABCG2 substrate, and less dependent on bFGF to sustainself-renewal, than control cells. Similar results were observedwith human induced pluripotent stem (iPS) cells. In contrast,Abcg2 is present in mouse ESCs [5, 10], and we found that itis absent in mouse trophoblast stem (TS) cells as well as dif-ferentiated trophoblasts. These results highlight the lack ofABCG2 as a novel feature that distinguishes human pluripotentstem cells from many other stem cells including mouse ESCs.

MATERIALS AND METHODS

Reagents

Rat anti-ABCG2 (for Western blotting), rabbit anti-NANOG, andmouse anti-b-ACTIN antibodies were from Abcam (Cambridge,MA, http://www.abcam.com). Mouse anti-ABCG2 antibody (forimmunostaining) and anti-ABCG2 antibody conjugated with fluo-rescein isothiocyanate (FITC) (for FACS) were from Millipore(Billerica, MA, http://www.millipore.com); mouse anti-PAX6, ratanti-TROMA-1 was from Developmental Studies Hybridoma Bank(Iowa City, IA, http://www.uiowa.edu/~dshbwww); mouse antibodiesagainst OCT3/4, TRA-1-60, and TRA-1-81 were from Santa CruzBiotechnology (Santa Cruz, CA); and rabbit antibodies against AKTand phosphorylated AKT were from Cell Signaling Technology(Danvers, MA, http://www.cellsignal.com). Anti-OCT3/4 antibodyconjugated with Alexa-694 (for FACS) was from BD Biosciences(San Jose, CA, http://www.bdbiosciences.com). Bone morphogeneticprotein 4 (BMP4) was from R&D Systems (Minneapolis, http://www.rndsystems.com), and Hoechst 33342 was from Invitrogen(Carlsbad, CA, http://www.invitrogen.com). Mitoxantrone, verapamil,and leukemia inhibitory factor (LIF) were from Sigma-Aldrich(St. Louis, http://www.sigmaaldrich.com).

Cell Culture

Human ES cell lines H9, HUES1, and CT2, and human iPS celllines iPS(IMR90)-1 and iPS(foreskin)-1 were cultured on platescoated with Matrigel (BD Biosciences) in human ES medium,that is, Dulbecco’s modified Eagle’s medium (DMEM)/F12 con-taining 20% knockout serum replacer, 0.1 mM nonessentialamino acids, 1 mM L-glutamine (all from Invitrogen), and 0.1mM b-mercaptoethanol (Sigma-Aldrich), which was conditionedon mouse embryonic fibroblast (MEF) as feeders and then supple-mented with 4 ng/ml bFGF (Millipore) [11]. Some results wereobtained from human ESCs cultured directly on the feeders inunconditioned human ES medium supplemented with 4 ng/mlbFGF [12, 13] or on Matrigel in defined TeSR1 medium [14].

Mouse ESCs derived from a 129 strain mouse blastocystwere cultured on MEF feeders in mouse ES medium, that is,DMEM (Invitrogen) containing 20% fetal bovine serum (FBS)(Hyclone, Logan, UT, http://www.hyclone.com), 0.1 mM nones-sential amino acids, 1 mM L-glutamine, and 0.1 mM b-mercapto-ethanol, in the presence of 1,000 unit/ml LIF. Mouse TS cells(courtesy of Dr. Janet Rossant) were cultured on gelatin-coatedplates in 70% MEF-conditioned mouse ES medium, 30% TS me-dium, that is, RPMI 1640 (Invitrogen) supplemented with 20%FBS, 1 mM sodium pyruvate, 0.1 mM b-mercaptoethanol, 2 mML-glutamine. Human recombinant fibroblast growth factor 4

(FGF-4, 25 ng/ml; Sigma-Aldrich) and heparin (1 mg/ml) wereadded to aliquots of TS cell medium and used immediately.

Cell Differentiation

For trophoblast differentiation [15], human ES or iPS cells weretreated with 100 ng/ml BMP4 for up to 7 days. The cells wereharvested at various times of the treatment and subjected to anal-yses by immunostaining, reverse-transcription polymerase chainreaction (RT-PCR), or Western blotting. The differentiation ofneural epithelial cells from human ESCs was carried out asdescribed previously [16]. Briefly, human ES cell colonies weredetached from MEF feeder cells at day 0 and suspended inhuman ES cell medium (without bFGF) for 4 days. Then thesehuman ES cell aggregates were cultured in a neural medium con-sisting of DMEM/F12, N2 supplement, and 2 lg/ml heparin inthe absence of growth factors. After adherence to plastic surfaceon day 6, human ES cell aggregates flattened down and devel-oped to columnar cells that were organized to rosette structure atdays 8-10. Subsequently, these early rosette cells (primitive neuralepithelial cells) formed neural tube-like rosettes with lumens (defini-tive neural epithelial cells) at days 14-17 of differentiation [17].

Differentiation of mouse ESCs was induced by culturing thecells on gelatin-coated plates in mouse ES medium, in the ab-sence of LIF and presence of 100 ng/ml BMP4 for 5 days [18].Differentiation of mouse TS cells was induced by culturing theTS cells in the mouse ES medium, in the absence of FGF4 andheparin for 5 days [19].

Lentiviral Transduction of ABCG2 to Human ESCs

A 1.8-kb BamH1-EcoRI fragment coding for the full-lengthABCG2 gene was subcloned into the BamHI and EcoRI sites byreplacing EGFP in the pSIN4-EF2-EGFP-IRES-Neo plasmid(courtesy of Dr. James Thomson), resulting in pSIN4-EF2-ABCG2-IRES-Neo. Alternatively, EGFP cassette was deleted byBamHI to generate pSIN4-EF2-IRES-Neo as a negative control.Each of the two plasmids was used to prepare lentivirus in 293FTcell line (Invitrogen) with Superfect (Qiagen, Valencia, CA,http://www1.qiagen.com). The lentiviral supernatants were har-vested at 48 hours and 72 hours after transfection, pooled, andsupplemented with 6 lg/ml polybrene (Sigma-Aldrich) for infec-tion of human ESCs. H9 cells at 2-3 days after split were trans-duced with the lentiviral supernatants derived from pSIN4-EF2-ABCG2-IRES-Neo to express ABCG2. Supernatants derivedfrom pSIN4-EF2-IRES-Neo were used to transduce sibling H9cells as a mock control. Forty-eight hours after transduction, 300 lg/mlneomycin was added daily for selection of cell clones expressing thetransgenes. Stable clones expressing ABCG2 or the mock controlwere obtained 12-15 days after transduction and confirmed.

Hoechst Dye Efflux Assay

Human ES or iPS cells and mouse ES or TS cells cultured beforereaching 75% confluence were live stained with 5 lg/ml Hoechst33342 at 37�C under swirling motion for 10 minutes. For specificinhibition of ABCG2, some cells were simultaneously treatedwith 50 lM verapamil. Subsequently, the cells were washed threetimes with cold PBS, and examined under fluorescence micro-scope (Olympus, CKX41, Tokyo, Japan, http://www.olympus-global.com) with a digital camera (15.2, 64 Mp, Shifting Pixel;Diagnostic Instruments, Inc., Sterling Heights, MI, http://www.diaginc.com), which captures images with 360/40 and 457/20 filters for excitation and emission, respectively.

Immunofluorescence Analysis

Cells were fixed with 4% paraformaldehyde for 10 minutes andincubated in PBS containing 5% rabbit serum and 0.4% TritonX-100 for blocking and permeablization, respectively. PBS con-taining 0.5% Tween 20 (PBS-T) was used to dilute antibodiesand wash the cells in the following procedures. The cells wereincubated with antibodies against ABCG2 (diluted 1:50), OCT4,TRA-1-60, TRA-1-81 (all diluted 1:1000), or TROMA-I (without

2436 ABCG2 and Human Pluripotent Stem Cells

dilution) at 37�C for 1 hour, followed by washing with PBS-Tthree times. Afterward, the cells were incubated with fluoro-chrome-conjugated, corresponding secondary antibodies at 37�Cfor 45 minutes and washed with PBS-T three times. Finally, thecells were examined under fluorescence microscope to captureboth phase and fluorescent images.

Western Blotting

Cells were lysed in RIPA lysis buffer supplemented with phenyl-methylsulphonyl fluoride, sodium orthovanadate, and protease in-hibitor cocktail solutions (all from Santa Cruz Biotechnology) at10 ll/ml each. The cell lysates were stored at �80�C before use.Proteins in the lysates were separated in a 10% SDS-polyacryl-amide gel and transferred electrophoretically to polyvinylidenefluoride membranes (Bio-Rad Laboratories, Irvine, CA, http://www.bio-rad.com). The membranes were blocked with 5% nonfatmilk and incubated with antibodies against ABCG2 (diluted1:50), NANOG (1:500), AKT (1:2,000), phosphorylated AKT(1:2,000) or b-ACTIN (1:2,000) at room temperature for 1 hourfollowed by PBS-T washing three times. Subsequently, the mem-branes were incubated with horseradish peroxidase-conjugatedcorresponding secondary antibodies (diluted 1:5,000) at roomtemperature for 30 minutes followed by washing with PBS-Tthree times. Finally, target protein bands on the membranes werevisualized using Immobilon Western Chemiluminescent HRPSubstrate (Millipore).

Flow Cytometry Analysis and Cell Sorting

For Hoechst staining analysis, human ESCs cultured at approxi-mately 75% confluence were dissociated by incubation withAccutase (Innovative Cell Technologies, San Diego, http://www.innovativecelltech.com) at 37�C for 10 minutes, then centri-fuged and resuspended in 400 ll cold PBS containing 2% FBS,10 mM HEPES buffer, and 2 lg/ml propidium iodide (PI)(Sigma-Aldrich). The cells were then stained with 5 lg/mlHoechst 33342 for 10 minutes at 37�C while swirling. Flow cyto-metric analysis and cell sorting were performed on BD FACSAriaCell-Sorting System (BD Biosciences). Live cells were gated inby excluding PI-positive cells in both detectors. The sorting ofHoechsthigh and Hoechstlow cells was then decided on the basis ofHoechst fluorescence emission of the live cells in both the blueand red wavelengths.

For bFGF withdrawal analysis, human ESCs were split toMatrigel-coated plates, cultured in TeSR1 medium without bFGFfor 3 days, and dissociated with Accutase. The dissociated cellswere washed with FACS buffer (PBS supplemented with 0.1%sodium azide and 2% FBS), fixed with 4% paraformaldehyde for10 minutes, and permeabilized with 0.1% Triton X-100. Thencells were incubated with anti-OCT3/4 antibody conjugated withAlexa-694 and anti-ABCG2 antibody conjugated with FITC for60 minutes, washed with the FACS buffer, and proceeded to flowcytometry analysis with CellQuest Pro (BD Biosciences).

Low-Density Array Analysis

Hoechsthigh and Hoechstlow cells sorted through flow cytometrywere subjected to RNA isolation and reverse transcription usingHigh Capacity cDNA Reverse Transcription Kit (Applied Biosys-tems, Foster City, CA, http://www.appliedbiosystems.com)according to the manufacturer’s protocol. cDNA derived fromapproximately 100 ng RNA per sample was applied to TaqManHuman Stem Cell Pluripotency Low-Density Array card for real-time PCR on ABI 7900HT Fast System. The samples were testedin triplicate and the data analyzed with RQ2.1 software and dis-played as change in cycle threshold (DCt) in a scatter plot. Allthe array cards, real-time PCR system, and software were fromApplied Biosystems.

RT-PCR Analysis

RNA was isolated from cells using TRIzol reagent (Invitrogen),and cDNA was synthesized from the RNA using ThermoScript

(Invitrogen), according to the manufacturer’s instructions. Geneexpression was assessed through PCR with primers for specificgenes (supporting information Table 1) under the following con-ditions: an initial 5-minute denaturation at 95�C; followed by 30cycles of 45 seconds of denaturation at 95�C, 45 seconds ofannealing at 55�C, and 45 seconds of extension at 72�C; andcompleted with a final extension at 72�C for 10 minutes.

Apoptosis Detection by TUNEL Assays

Forty-eight hours after mitoxantrone treatment of cells, nuclearfragmentation in the cells was analyzed using the terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick-end labeling(TUNEL) Assay Kit (Roche Applied Science, Indianapolis, IN,http://www.roche-applied-science.com) according to the manufac-turer’s instructions. The numbers of apoptotic cells (TUNEL posi-tive) and total cells (40,6-diamidino-2-phenylindole positive) werecounted in five views per well of cells under fluorescent micro-scope at �10 magnification. The percentages of the apoptoticcells over the total cells from the five views were averaged, andarcsine was transformed for statistical analysis using analysis ofvariance. Three independent experiments were repeated. The datawere expressed as mean � standard deviation.

RESULTS

ABCG2 is Absent in Human Pluripotent Stem Cellsbut Present in Their Derivative Trophoblasts andNeural Progenitor Cells

As introduced above, this study started with an incidental ob-servation when we counterstained live human ESCs for theirnuclei with Hoechst 33342. We observed that undifferentiated(OCT4þ) human ESCs (H9) were Hoechstþ, whereas somespontaneously differentiated H9 cells were Hoechst� (Fig.1A). We have previously demonstrated that BMPs can inducehuman ES cell differentiation to trophoblasts [15], and BMPactivities present in the Serum Replacer (Invitrogen) used forhuman ES cell culture contribute to the spontaneous differen-tiation to mixed cell lineages including trophoblasts [20]. Wethen stained live trophoblasts differentiated from BMP4-treated H9 cells with Hoechst 33342 and found that they wereindeed Hoechst�, whereas undifferentiated H9 cells wereHoechstþ and ABCG2� (Fig. 1B).

As ABCG2 mediates the efflux of the Hoechst dye [3],we tested whether ABCG2 is expressed in these cells. BothABCG2 transcripts and protein were detected highly in theBMP4-treated H9 cells and slightly in spontaneously differen-tiated H9 cells, but absent in untreated, undifferentiated con-trol cells (Figs. 1B and 2A–2D). Similar results (supportinginformation Fig. 1A–1C) were observed with other humanpluripotent stem cell lines. Human ES cell lines CT2 (derivedin our laboratory) and HUES1 (derived in Melton laboratory)[21], and human iPS cell lines iPS(IMR90)-1 and iPS(foreskin)-1 (derived in Thomson laboratory) [22] were ABCG2�/low andHoechstþ, whereas trophoblasts differentiated from these celllines following BMP4 treatment were ABCG2þ and Hoechst�.Reanalysis of microarray database data from our previousstudies [15] revealed that the transcript levels of ABCG2 andsome other ABC family members such as ABCA4, ABCC1,ABCC2, ABCD1, and ABCD3 in BMP4-treated H1 humanESCs increase to 6.0-, 2.1-, 2.4-, 2.0-, 2.6-, and 3.4-fold,respectively, on day 7 of treatment, compared with paired,untreated H1 cells, where ABCG2 expression increased themost steadily and was the highest.

To exclude the possibility that different ABCG2 tran-scripts are expressed in human ESCs cultured in various con-ditions, we tested three ABCG2 transcripts termed a, b, and c,

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which are generated by alternative usage of exons 1a, 1b, and1c, respectively [23]. We detected only the transcripts a andb, but not c, in BMP4-treated H9 cells cultured on not onlyMatrigel in mouse embryonic fibroblast-conditioned medium[11] a system used throughout this study but also on the fibro-blast feeders directly [13] or on Matrigel in the defined me-dium TeSR1 [24] (Fig. 2E). However, ABCG2c could bedetected in human KG-1 dendritic-like cells (data not shown).

Since neural stem and progenitor cells can be isolated asAbcg2þ cells from mouse forebrain [25], we tested whetherhuman ES cell-derived neural epithelial cells also expressABCG2. Indeed, ABCG2 was detected among neural epithelialcells (PAX6þ) differentiated from H9 cells (Fig. 3). This sug-gests that ABCG2 is not exclusively expressed in trophoblasts,but also in human ES cell-derived neural epithelial cells.

Abcg2 Is Present in Mouse ESCs, but Absent inMouse Trophoblasts

The differential expression of ABCG2 in human ESCs andtrophoblasts prompted us to test whether this difference is alsopresent between mouse ES and trophoblasts cells. We con-firmed that mouse ESCs were indeed Abcg2þ and Hoechst� asreported [5, 10], and also found that randomly differentiatedmouse ESCs (which occurred upon withdrawal of leukemia in-hibitory factor and addition of BMP4) remained largelyAbcg2þ and Hoechst� (Fig. 4A). Next we tested Abcg2 expres-sion in mouse trophoblasts. Mouse TS cells can be isolatedfrom mouse blastocyst and maintained in mouse embryonicfibroblast-conditioned medium containing 25 ng/ml FGF4 and1 lg/ml heparin, and they differentiate to trophoblasts whencultured in unconditioned medium in the absence of FGF4 and

Figure 1. Hoechst staining of human ESCs. (A): H9 cells cultured in mouse embryonic fibroblast-conditioned medium (CM) with spontaneousdifferentiation were live stained with Hoechst before immunostaining for OCT4. White arrows point to a spontaneously differentiated area. (B):H9 cells cultured in CM � bone morphogenetic protein 4 at 100 ng/ml (same dose hereafter) for 5 days were live stained with Hoechst beforeimmunostaining for ABCG2. Scale bars ¼ 20 lm each. Abbreviation: DPI, dots per inch.

2438 ABCG2 and Human Pluripotent Stem Cells

Figure 2. ABCG2 expression in humanESCs. (A–C): H9 cells cultured in CMwith BMP4 or spontaneous differentiationwere subjected to reverse-transcription po-lymerase chain reaction (RT-PCR) (A, B)

or Western blotting analysis (C). (D): RT-PCR analysis of ABCG2 expression in H9cells cultured on MEFs or in TeSR1 me-dium � BMP4 for 5 days. (E): RT-PCRanalysis of expression of three ABCG2 iso-forms in H9 cells cultured in three condi-tions � BMP4 for 5 days. Abbreviation: d,days; BMP4, bone morphogenetic protein4; CM, conditioned medium; DPI, dots perinch; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; MEF, mouse embryonicfibroblast; Spont Diff, spontaneous differ-entiation; TP, trophoblasts.

Figure 3. Immunostaining for PAX6 and ABCG2 of neural epithelial cells differentiated from H9 cells. The cell nuclei were counterstained byDAPI. Scale bars ¼ 20 lm each. Abbreviations: DAPI, 40,6-diamidino-2-phenylindole; DPI, dots per inch.

heparin [19]. Interestingly, neither mouse TS cells nor theirdifferentiated trophoblasts expressed Abcg2, thus both wereHoechstþ (Fig. 4B). The absence of Abcg2 in mouse TS cells

was confirmed by Western blotting (Fig. 4C). These data sug-gest that human and mouse ESCs and trophoblasts have com-pletely opposite patterns of ABCG2/Abcg2 expression.

Figure 4. Abcg2 expression in mouse ESCs and TS cells. (A): Undifferentiated or randomly differentiated mouse ESCs (mESCs) were livestained with Hoechst before immunostaining for Abcg2. (B): Undifferentiated or differentiated mouse TS cells (mTSCs) were live stained withHoechst before immunostaining for Abcg2. Scale bars ¼ 20 lm each. (C): Western blotting for Abcg2 in hESCs, mESCs, and mTSCs. Abbrevia-tions: DPI, dots per inch; mTSC, mouse trophoblast stem cells.

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Ectopic ABCG2 Expression Renders Human ESCsMore Resistant to Mitoxantrone, and Less Depend-ent on bFGF

Recently Abcg2 has been reported to help sustain self-renewalof mouse ESCs by pumping out DNA-damaging metabolites

such as protoporphyrin [26]. We decided to test whether ec-topic expression of ABCG2 would confer similar actions tohuman ESCs. We first transduced H9 cells with ABCG2 lenti-viral particles or control viral particles. Stable cell cloneswere established following drug selection (see Materials and

Figure 5. Analyses of ABCG2-expressing or mock-transduced H9 human ESCs. (A): Hoechst staining and immunostaining for ABCG2. Scalebar ¼ 20 lm. (B): Detection of apoptotic (TUNELþ) cells following treatment with various concentrations (0, 1, and 100 nM) of mitoxantrone� 50 nM verapamil. * p < .01. (C): Western blotting for AKT and phosphorylated AKT. (D): Immunostaining for OCT4 and NANOG (scalebar ¼ 20 lm). (E): Fluorescence-activated cell sorting analysis for OCT4 and ABCG2 in the cells cultured in TeSR1 minus basic fibroblastgrowth factor for 3 days. Abbreviations: DPI, dots per inch; p-AKT, phosphorylated AKT; TUNEL, terminal deoxynucleotidyl transferase-medi-ated dUTP-biotin nick-end labeling; þVera, with verapamil.

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Methods). The ABCG2-expressing cells became ABCG2þ

and Hoechst�, whereas H9 cells derived from the mock trans-duction (negative control) remained ABCG2� and Hoechstþ

(Fig. 5A). Like control cells, the ABCG2-expressing cellsremained positive for pluripotency markers (supporting infor-mation Fig. 2A). Following mitoxantrone treatment, the per-centage of apoptotic (TUNELþ) cells was much lower inABCG2-expressing H9 cells and H9-derived trophoblasts thanin untreated or mock-transduced H9 cells, and the ABCG2 in-hibitor verapamil diminished the differences (Fig. 5B). Thesedata suggest that ABCG2 can protect human ESCs and troph-

oblasts from cytotoxic substrates. Nevertheless, their ability todifferentiate to trophoblasts (supporting information Fig. 2B)and neural epithelial cells (supporting information Fig. 2C)was not compromised.

It has been known that cooperation of the two branches ofFGF signaling, mitogen-activated protein kinase/extracellularsignal-related kinase (ERK) and phosphoinositide-3 kinase(PI3K)/AKT, is required to sustain self-renewal of humanESCs [27, 28]. We also found that the ABCG2-expressing H9cells had higher phosphorylation level of AKT (Fig. 5C), andthe majority of them remained pluripotent (OCT4þ and

Figure 5. (Continued)

2442 ABCG2 and Human Pluripotent Stem Cells

NANOGþ) in bFGF-free condition for up to 3 days, whereasthe control cells had already mostly differentiated or died off(Fig. 5D, 5E). This suggests that ABCG2 may partially com-pensate for the inhibition of FGF signaling through activationof the downstream effecter AKT. However, it does not appearthat inhibition of FGF signaling markedly affects expressionof ABC transporters in human ESCs, based on our previousmicroarray database [29].

The ‘‘Side Population’’ Among Human ESCs HasDecreased Expression of Pluripotency Markers

Although human ESCs appeared mostly Hoechstþ in pheno-type, we asked whether they also contain Hoechst� cells and,if yes, what would be their pluripotency status. To addressthese questions, we used FACS to sort H9 cells and obtainedtwo subsets of cells: a major Hoechsthigh population and aminor Hoechstlow population (Fig. 6A). Their gene expressionprofiles were analyzed using the Low-Density Stem CellArray card (Applied Biosystems). This card included real-time RT-PCR primers to detect expression of multiple well-defined genes validated as markers for pluripotency anddifferentiation as well as endogenous controls. As shown inFigure 6B, the Hoechsthigh cells had higher transcriptional lev-els (indicated by lower DCt values) of the pluripotencymarker genes such as OCT4 [30], TERT [31], FGF4, GABR3[32], and NR5A [33] than the Hoechstlow cells, nevertheless,almost no transcripts for differentiation marker genes weredetected in either subset of the cells. These results were con-firmed by testing some of the marker genes through regularRT-PCR, with trophoblasts used as a positive control forABCG2 expression (Fig. 6C). Therefore, the SP-associatedHoechst efflux inversely correlates to the pluripotency ofhuman ESCs, and the Hoechstlow cells may represent sponta-neously differentiating (or poised-to-differentiate) ESCs in thegiven culture.

DISCUSSION

Human and mouse ESCs share great similarities in cell mor-phology, gene expression profile, differentiation ability, andso on [34]. Many differentiation protocols for human ESCshave been adapted from those for mouse ESCs [35]. How-ever, dramatic differences in cell surface markers and growthfactor requirements have been discovered between ESCs fromthe two species. For examples, human ESCs express SSEA3and SSEA4 [13], whereas mouse ESCs express SSEA1 [36].Activation of FGF and transforming growth factor b (TGFb)signaling and inhibition of BMP signaling are required to sus-tain human ES cell self-renewal [37]. However, activation ofBMP and LIF signaling and inhibition of FGF/Erk signalingare required to sustain mouse ES cell self-renewal [37]. Inter-estingly, LIF/signal transducer and activator of transcription 3signaling is dispensable for human ESCs [38], and TGFb sig-naling is dispensable for mouse ESCs [39]. In addition,human ESCs differentiate to trophoblasts in response toBMPs [15], whereas mouse ESCs do so only when their Oct4expression is repressed [40]. This study revealed another sub-stantial difference between human and mouse ESCs. HumanESCs do not express ABCG2 and cannot efflux Hoechst, thuslacking SP property. In contrast, mouse ESCs are Abcg2þ

and Hoechst�, thus possessing SP property. Moreover, humanES cell-derived trophoblasts are ABCG2þ and Hoechst�,whereas mouse trophoblasts are Abcg2� and Hoechstþ.

Our observations are not cell line-dependent because simi-lar results were obtained from multiple human ES and iPS

Figure 6. Analyses of Hoechsthigh and Hoechstlow cells sorted fromhuman ESCs. (A): H9 cells were harvested for live staining with pro-pidium iodide (PI) and Hoechst. Live (PI�) cells were gated in.Hoechsthigh and Hoechstlow cells were then sorted. (B): Detection ofgene expression by Low-Density Array, which is displayed as changein cycle threshold (inverse to the gene expression levels) in a scatter-plot. Several pluripotency genes and two internal control genes ACTBand 18S are marked. (C): Reverse-transcription polymerase chainreaction confirmation of expression of representative genes in thesorted cells with H9-differentiated trophoblasts as a control. Abbrevi-ations: DPI, dots per inch; w/o, without.

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cell lines (Fig. 1B and supporting information Figure 1A–1C).Neither were they caused by differences between various cul-ture conditions or by differential expression of the threeABCG2 transcripts (Fig. 2E). However, spontaneous differen-tiation among human ESCs can indeed cause elevated ABCG2expression in a mixed cell population (Fig. 2B). This mightbe accountable for the detection of ABCG2 in human ESCs(HUES1) in a previous report [23], in case the cells analyzedwere from suboptimal culture conditions.

As mentioned above, Abcg2 promotes mouse ES cell self-renewal by maintaining homoeostasis of intracellular metabo-lites, and inhibition of Abcg2 causes mouse ES cell differen-tiation accompanied by decline of Nanog expression [26].Consistently, human ESCs ectopically expressing ABCG2 hadincreased tolerance to the cytotoxicity of mitoxantrone (Fig.5B), and even could expand in a bFGF-free culture mediumwithout obvious differentiation for up to 3 days, whereas thecontrol cells had already mostly differentiated or died off bythen (Fig. 5D, 5E). These data indicate that lack of ABCG2may be a reason why human ES as well as iPS cells havehigher demand for optimal culture conditions and delicatecare to sustain self-renewal than mouse ESCs. Further studyis necessary to elucidate how the human pluripotent stem cellsmaintain their homeostasis in the absence of ABCG2 andpresence of bFGF, given the fact that none of the detectedABC transporters in human ESCs is remarkably regulated bybFGF [29].

It has been known that ABCG2 is expressed in late-stagetrophoblasts in the placenta, protecting the fetus by expellingdrugs, xenobiotics, and metabolites across the placental bar-rier at midgestational ages [41, 42]. However, no informationis available about the role of ABCG2 at early stages ofhuman embryos. Human ESCs provide us with a great oppor-tunity to address this question, as they may behave similarlyto the inner cell mass, whereas human ES cell-derived tropho-blasts may somehow mirror the early stage trophoblasts pres-ent in the trophectoderm of the blastocyst. The absence ofABCG2 in human ESCs and presence in their derivativetrophoblasts implicate that human trophoblasts might protectthe inner cell mass from detrimental substances in the micro-environment during blastocyst implantation. On the otherhand, Abcg2 is present in mouse ESCs as well as mouse innercell mass [10], but absent in mouse trophoblasts (Fig. 4).Abcg2-null mice develop normally without identifiable defectsduring gastrulation and placental formation [6]. These dataindicate that, during mouse blastocyst implantation, the troph-

oblasts might protect the inner cell mass through other ABCtransporters in the trophectoderm.

CONCLUSION

Our data suggest that the cell membrane transporter ABCG2is absent in human pluripotent stem cells including ES andiPS cells, but present in their derivative trophoblast and neuralepithelial cells. This is opposite to the expression pattern ofmouse Abcg2, which is present in mouse ESCs but absent inmouse trophoblasts. The absence of ABCG2 in human pluri-potent stem cells makes them a unique stem cell type thatdoes not have the property of side population cells, thus theyare less resistant to certain cytotoxic chemicals than side pop-ulation cells. Interestingly, ectopic expression of ABCG2 inhuman ESCs renders them more tolerant of bFGF-free cul-ture. The mechanism behind this observation awaits furtherinvestigations.

ACKNOWLEDGMENTS

We thank Dr. James Thomson for human ES cell lines H1 andH9, human iPS cell lines iPS(IMR90)-1 and iPS(foreskin)-1, andpSIN4-EF2-EGFP-IRES-Neo lentiviral plasmid; Dr. DouglasMelton for human ES cell line HUES1; Dr. Janet Rossant formouse TS cell line; and Dr. Zihai Li for human KG-1 dendritic-like cell line. We also thank Diane Gran for flow cytometricanalysis and cell sorting, Dr. Zhi-bo Wang for technical assis-tance for Western blotting, Drs. Gordon Carmichael and LixiaYue for critical reading of the manuscript, and all the membersof Xu laboratory for kind help and support. This work was sup-ported by Connecticut Stem Cell Research Grants 06SCB14 and06SCD02 to R.-H.X., and the fund from China ScholarshipCouncil to H.Z. The contents in this work are solely the responsi-bility of the authors and do not necessarily represent the officialviews of the State of Connecticut.

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

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