Collagen IV Induces Trophoectoderm Differentiation of MouseEmbryonic Stem Cells

KATJA SCHENKE-LAYLAND,a EKATERINI ANGELIS,a KATRIN E. RHODES,b SEPIDEH HEYDARKHAN-HAGVALL,c

HANNA K. MIKKOLA,b W. ROBB MACLELLANa

aDepartment of Medicine and Physiology, Cardiovascular Research Laboratory, bDepartment of Molecular, Cellularand Developmental Biology, cDepartment of Surgery, Regenerative Bioengineering and Repair Laboratory,University of California Los Angeles, Los Angeles, California, USA

Key Words. Collagen IV • Embryonic stem cells • Extracellular matrix • Trophoblast • Placenta

ABSTRACT

The earliest segregation of lineages in the developingembryo is the commitment of cells to the inner cell massor the trophoectoderm in preimplantation blastocysts.The exogenous signals that control commitment to a par-ticular cell lineage are poorly understood; however, it hasbeen suggested that extracellular “niche” and extracellu-lar matrix, in particular, play an important role in deter-mining the developmental fate of stem cells. Collagen IV(ColIV) has been reported to direct embryonic stem (ES)cell differentiation to mesodermal lineages in both mouseand human ES cells. To define the effects of ColIV on EScell differentiation and to identify the resulting heteroge-neous cell types, we performed microarray analyses anddetermined global gene expression. We observed that Co-lIV induced the expression of mesodermal genes specificto hematopoietic, endothelial, and smooth muscle cells

and, surprisingly, also a panel of trophoectoderm-re-stricted markers. This effect was specific to collagen IV,as no trophoblast differentiation was seen on collagen I,laminin, or fibronectin. Stimulation with basic fibroblastgrowth factor (FGF) or FGF4 increased the number oftrophoectodermal cells. These cells were isolated underclonal conditions and successfully differentiated into avariety of trophoblast derivatives. Interestingly, differen-tiation of ES cells to trophoblastic lineages was only seenin ES cell lines maintained on embryonic feeder layersand was caudal-type homeobox protein 2 (Cdx2)-depen-dent, consistent with Cdx2’s postulated role in trophoec-toderm commitment. Our data suggest that, given theappropriate extracellular stimuli, mouse embryonic stemcells can differentiate into trophoectoderm. STEM CELLS2007;25:1529 –1538

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

INTRODUCTION

In the early mouse preimplantation embryo, two distinct cellfates are generated that give rise to two morphologically andfunctionally distinct cell lineages. One is the inner cell mass(ICM), and the other is the trophoectoderm (TE) of the blasto-cyst. The ICM goes on to differentiate into the epiblast andoverlying primitive endoderm. The epiblast gives rise to theembryo proper as well as extraembryonic mesoderm, whereasthe primitive endoderm gives rise to the entire endoderm layerof the yolk sac. The remaining extraembryonic tissues, includ-ing the trophoblast layers of the placenta, are derived from thetrophoectoderm [1]. Mouse embryonic stem (mES) cells derivedfrom the ICM have virtually unlimited self-renewal potential invitro and can give rise to all three germ layers of the embryo andto the extraembryonic mesoderm. However, when reintroducedinto early embryos to form chimeras, mES cells contributepoorly to the extraembryonic endoderm and rarely, if ever, totrophoblast layers of the placenta [2, 3]. This has led to thewidespread belief that mES cells have irreversibly committed toan epiblast lineage and rarely spontaneously differentiate intoTE derivatives [4, 5]. Only with manipulation of master regu-

latory factors, such as octamer-4 (Oct4) and caudal-type ho-meobox protein 2 (Cdx2), could mES cells be efficiently di-verted to trophoectoderm lineages. Deletion of Oct4 expressionin mES cells triggers trophoblast-like differentiation [6],whereas forced expression of the earliest trophoblast-specificregulator Cdx2 can also promote differentiation of mES cellsinto trophoblast-like cells [7]. In contrast, numerous reportshave documented the ability of human embryonic stem (hES)cells to differentiate into cells of trophoblast lineage upon ap-propriate stimulation without genetic manipulation [8, 9]. Thereduced trophoectodermal differentiation potential of mES cellshas been cited as one of the prime differences between mES andhES cells [10].

Although the genetic regulation of the ICM/TE lineagedecision has received much attention [1], the exogenous cuesthat regulate this determination or control stem cell fate duringin vitro differentiation, more generally, have not been welldefined. The role that the extracellular “niche” plays on thedevelopmental fate of pluripotent stem cells is determined notonly by soluble factors and cell-cell contacts but also by theextracellular matrix (ECM). Thus, we sought to determine theeffects of ECM modulation on mES cell differentiation. Wewere specifically interested in collagen IV (ColIV), as it has

Correspondence: W. Robb MacLellan, M.D., Cardiovascular Research Laboratory, UCLA School of Medicine, 675 C.E. Young Dr., MRL3-645, Los Angeles, California 90095-1760, USA. Telephone: 310-825-2556; Fax: 310-206-5777; e-mail: rmaclellan@mednet.ucla.eduReceived November 10, 2006; accepted for publication March 7, 2007; first published online in STEM CELLS EXPRESS March 15, 2007.©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2006-0729

EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

STEM CELLS 2007;25:1529–1538 www.StemCells.com

been reported to direct embryonic stem (ES) cell differentiationto mesoderm lineages, including hematopoietic (HC), endothe-lial (EC), and smooth muscle cells (SMC), in both mouse[11–13] and human [14] cells. It has been proposed that thisrepresents a model of lateral mesoderm [15]; however, othermesodermal compartments, such as the allantoic mesoderm thatforms parts of the developing placenta, can also give rise tothese three cell types [16, 17].

In this study, we sought to identify the effects of ColIV onmES cell differentiation. We observed that ColIV-differentiatedmES cells expressed mesodermal genes specific to hematopoi-etic, endothelial, and smooth muscle cells and, surprisingly, apanel of genes normally restricted to cells of the trophoectoder-mal lineage. The ability to induce TE differentiation was spe-cific to ColIV, Cdx2-dependent, and only seen in ES cell linesmaintained on embryonic feeder layers. Stimulation with basicfibroblast growth factor (bFGF) or fibroblast growth factor 4(FGF4) increased the number of trophoectodermal cells, whichwere isolated under clonal conditions and successfully differen-tiated into a variety of trophoblast derivatives. Taken together,our results suggest that mES cells retain the ability to differen-tiate into trophoectoderm when given the appropriate extracel-lular stimuli.

MATERIALS AND METHODS

Mouse ES Cells and Cell CulturesThe murine ES cell lines D3 (CRL-1934; American Type CultureCollection [ATCC], Manassas, VA, http://www.atcc.org), R1(SCRC-1011; ATCC), v6.5 C57BL/6 � 129/sv (MES1402; OpenBiosystems, Huntsville, AL, http://www.openbiosystems.com), andCdx2-deficient v6.5 C57BL/6 � 129/sv [18] (a kind gift fromRudolph Jaenisch) were cultured on mitomycin-C-treated (M0503;Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) primarymouse embryonic fibroblasts (MEF) in mES cell medium (knockoutDulbecco’s modified Eagle’s medium [Invitrogen, Carlsbad, CA,http://www.invitrogen.com] supplemented with 15% ES cell-qual-ified fetal calf serum [ES-FCS; Invitrogen], 0.1 mM �-mercapto-ethanol [Sigma], 2 mM glutamine [Invitrogen], 0.1 mM nonessen-tial amino acids [Invitrogen], and 1,000 U/ml recombinant leukemiainhibitory factor [LIF; Chemicon, Temecula, CA, http://www.chemicon.com]) at 37°C, 5% CO2. CCE-ES cells [19] (00300; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) were maintained MEF-free on 0.1% gelatin (Sigma) in mEScell culture medium at 37°C, 5% CO2. The medium was changed ona daily basis. Cells were passaged every second day using 0.05%trypsin-EDTA (Invitrogen).

Differentiation AssaysFor differentiation assays, the MEF-dependent mES cells were initiallyplated for 2 � 30 minutes on 0.1% gelatin-coated plastic flasks at 37°C,5% CO2 to remove fibroblasts. All mES cells, including CCE-ES cells,were then transferred to either collagen type I-, collagen type IV-,laminin-, or fibronectin-coated flasks (BD BioCoat; BD Biosciences,San Diego, http://www.bdbiosciences.com) and cultured in �-mini-mum essential medium (�-MEM; Invitrogen) (supplemented with 10%ES-FCS, 0.1 mM �-mercaptoethanol, 2 mM glutamine, and 0.1 mMnonessential amino acids) without LIF at 37°C, 5% CO2. After 4 daysthe cells were either harvested for analysis, or trypsinized and culti-vated for an additional 7 days on fibronectin-coated flasks and cultureslides (BD Biosciences), in either �-MEM; vascular endothelial growthfactor (VEGF) medium (endothelial growth medium-2; Cambrex,Walkersville, MD, http://www.cambrex.com, supplemented with 50ng/ml VEGF [R&D Systems Inc., Minneapolis, http://www.rndsystems.com]); or platelet-derived growth factor-BB medium (PDGF-BB)(smooth muscle growth medium-2; Cambrex, supplemented with 10ng/ml PDGF-BB [R&D Systems Inc.]), at 37°C, 5% CO2.

Isolation, Maintenance, and Differentiation of TECells Under Clonal ConditionsTo determine whether TE cells respond to growth factors, D3-EScells were additionally cultured on collagen type I and type IV ineither �-MEM, �-MEM supplemented with 10 ng/ml bFGF (In-vitrogen) [5], or trophoblast stem cell (TS) medium, containing 25ng/ml FGF4 (Peprotech, Rocky Hill, NJ, http://www.peprotech.com) and 1 �g/ml heparin (Sigma), with 70% of the medium beingMEF-preconditioned [20]. After 1, 2, 3, or 4 days, the cells wereharvested for analysis. Cloning of TE cells was carried out bylimited dilution. Briefly, D3-ES cells were cultured for 2 days onColIV in TS medium, trypsinized, resuspended to a density of 10cells per milliliter, and recultured in TS medium on 0.1% gelatin-coated 96-well plates (BD Biosciences). Cell clonality was con-firmed by phase-contrast microscopy using a Zeiss Axiovert 200microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com).The cloned cells were subcultured in triplicate, and, 4 days aftersubculturing, immunofluorescence analysis with an anti-Cdx2 anti-body was carried out (refer to Antibodies and Immunocytochemis-try for more detailed information). The cloned Cdx2-expressingcells were expanded in TS medium and either analyzed as undif-ferentiated TE cells or cultured for an additional 6 days in differ-entiation medium (TS medium without supplement of FGF4, hep-arin, and MEF-conditioned medium) prior to analysis.

AntibodiesPrimary antibodies used include (a) rabbit polyclonal antibodies:anti-�-smooth muscle actin (SMA) (ab5694 [1:400]; Abcam, Cam-bridge, U.K., http://www.abcam.com), anti-vimentin (ab7783[1:500]; Abcam), anti-mouse connexin 31 (Cx31) (CX31-A [1:100];Alpha Diagnostics, San Antonio, http://www.4adi.com), and anti-cow cytokeratin (Z0622, 1:1,000; Dako North America Inc.,Carpinteria, CA, http://www.dakousa.com); (b) mouse monoclonalantibodies anti-Cdx2 (MU392-UC [1:20]; BioGenex, San Ramon,CA, http://www.biogenex.com) and anti-Cadherin 3/P-Cadherin(clone 56C1 [1:20]; Lab Vision, Fremont, CA, http://www.labvision.com); as well as (c) a rat monoclonal antibody anti-mouseCD31 (PECAM-1, catalog number 550274 [1:50]; BD Pharmingen,San Diego, http://www.bdbiosciences.com/index_us.shtml). Sec-ondary antibodies included Alexa Fluor 488- and Alexa Fluor594-conjugated goat-anti-mouse IgG (H�L); Alexa Fluor 488-con-jugated goat-anti-rabbit IgG (H�L); Alexa Fluor 488-conjugatedgoat-anti-rat IgG (H�L) (1:250; all from Molecular Probes, Carls-bad, CA, http://probes.invitrogen.com). To visualize the F-actincytoskeleton, cells were stained using Alexa Fluor 594 phalloidin(Molecular Probes). For counterstaining of cell nuclei, 4,6-dia-midino-2-phenylindole (DAPI) (Sigma) was added to the finalphosphate-buffered saline (PBS) washing. Cell staining withoutprimary antibodies served as controls. Bright-field images wereacquired using the Zeiss Axiovert 200 microscope.

ImmunocytochemistryPrior to immunocytochemical staining, all cells were washed with1� PBS (Invitrogen), fixed for 10 minutes in 4% paraformaldehyde(Sigma), and rinsed twice in PBS. Cells were then permeabilizedusing 1% Triton X-100 (Sigma) for 30 minutes and subsequentlyincubated in serum blocking buffer (2% goat serum [Chemicon],1% bovine serum albumin [Sigma], 0.1% cold fish skin gelatin[Sigma], 0.1% Triton X-100, and 0.05% Tween 20 [Sigma] in PBS).After 3� rinsing with 0.05% Tween 20, cells were incubated withthe primary antibody (diluted as specified earlier) overnight at 4°Cfollowed by 2� washing with 0.05% Tween 20. For secondaryantibody detection, the appropriate Alexa Fluor-conjugated antibod-ies were incubated at a 1:250 dilution in PBS for 30 minutes at roomtemperature in the dark, either alone or in combination with AlexaFluor 594 phalloidin (1:40). After secondary antibody incubation,the cells were washed 3� in PBS and incubated in a DAPI-PBSsolution followed by 3� washes with PBS. In a final step, the cellculture chambers were removed from the slides (according to themanufacturer’s protocol), and slides and coverslips were mountedusing ProLong Gold antifade mounting medium (MolecularProbes). The slides were then stored in the dark overnight at 4°C

1530 Collagen IV Induces Trophoectoderm Differentiation

prior to imaging. Images were acquired using a confocal TCS SP2AOBS laser-scanning microscope system (Leica Microsystems Inc.,Exton, PA, http://www.leica.com) with 40� (1.3 numerical aperture[NA]) and 63� (1.4 NA) oil-immersion objectives. Images wereprocessed with Adobe Photoshop 7.0 (Adobe Systems Inc., SanJose, CA, http://www.adobe.com).

RNA Extraction, cDNA Synthesis, andSemiquantitative Reverse Transcription-PolymeraseChain ReactionTotal RNA was extracted from placenta tissues at day 12.5 ofgestation (positive control) as well as from harvested cells by amodification of the acid-guanidinium-phenol-chloroform method(TRIzol Reagent; Sigma) as per manufacturer’s instructions. Pre-cipitated RNA was resuspended in RNase-free water and subjectedto an additional RNA purification step to remove possible genomicDNA contamination (RNeasy Plus Mini Kit; Qiagen, Valencia, CA,http://www.qiagen.com) before final storage at �80°C. First strandcDNA was generated from 2 �g of total RNA by using the Om-niscript Reverse Transcriptase (RT) Kit (Qiagen) as per manufac-turer’s instructions. All samples, along with the corresponding “no-RT” control (RNA) to confirm the absence of contaminatinggenomic DNA, were subjected to polymerase chain reaction (PCR)and carried out using 2.5 units of Taq DNA polymerase, 10� PCRbuffer, 2.5 mM MgCl2, 200 �M dNTP, Q-solution (Qiagen), 0.2�M gene-specific forward, and 0.2 �M reverse PCR primers. Thesequences of each specific primer set, including their annealingtemperatures and cycles, are listed in the supplemental online Table1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAwas used as an internal control. PCR reactions were performedunder the following conditions: 94°C denaturation for 30 seconds,specific primer annealing temperature (Supplemental Table 1) for45 seconds, and primer extension at 72°C for 45 seconds (all except

Eomesodermin [Eomes]); 94°C denaturation for 30 seconds, 50°Cfor 30 seconds, 68°C for 2 minutes, and primer extension at 68°Cfor 7 minutes (Eomes). The Oct4, Acta2, Cald1, Cdh5, and VwfRT-PCR primer sets were obtained from SuperArray Bioscience

Table 1. Summary of upregulated (�1.5-fold) cardiovascular-, hematopoietic-, and trophoectoderm-specific genes in D3-embryonic stemcells cultured on collagen IV

Gene symbol Gene names In vivo location Fold changeDifferential

score

Cardiovascular lineageActg2 Actin, gamma 2, smooth muscle Smooth muscle 23.1 277.1Mef2C Myocyte enhancer factor 2C Cardiac mesoderm 20.8 143.6Angpt2 Angiopoietin 2 Vascular endothelium 15.1 149.9Acta2 Actin, alpha 2, smooth muscle Smooth muscle 10.2 313.5Cnn2 Calponin 2 Smooth muscle 8.4 162.8Cald1 Caldesmon 1 Smooth muscle 7.6 153.4Vcam1 Vascular cell adhesion molecule 1 Vascular endothelium 6.1 208.7Actc1 Actin, alpha, cardiac Cardiac muscle 3.9 52.9Smtn Smoothelin Smooth muscle 2.2 262.7Edn1 Endothelin 1 Vascular endothelium 2.0 57.1

Hematopoietic lineageCxcl12 Stromal cell derived factor 1 Hematopoietic stem cells 22.7 371.3Runx1 Runt related transcription factor 1 Hematopoietic stem cells 8.3 93.0Lmo2 LIM domain only 2 Early hematopoiesis and T cells 7.9 73.4Alcam Activated leukocyte cell adhesion

moleculeHematopoietic progenitors 6.7 202.8

Trophoblast lineagePL-2/Csh2 Placental lactogen 2/ Chorionic

somatomammotropin hormone 2Giant cells �100 63.6

Psx1 Placenta-specific homeobox 1 Extraembryonic tissue 25.2 24.3Psx2 Placenta-specific homeobox 2 Extraembryonic tissue 8.4 35.1Plac8 Placenta-specific protein 8 Spongiotrophoblast 5.3 23.7Plac1 Placenta-specific protein 1 Ectoplacental cone; giant cells;

cytotrophoblast4.5 20.8

Dlx3 Distal-less homeobox 3 Ectoplacental cone; chorionic plate;cytotrophoblast

3.4 14.8

Cdh3 P-Cadherin, Cadherin 3 Ectoplacental cone; giant cells;trophoectoderm

2.0 36.3

Tpbg Trophoblast glycoprotein Trophoectoderm 1.8 241.1Idb2 Inhibitor of DNA binding 2 Cytotrophoblast 1.8 55.1Gcm1 Glial cell missing homolog 1 Chorionic trophoblast cells 1.6 20.3

Figure 1. Phase-contrast images show D3-embryonic stem cells cul-tured for 4 days on collagen IV in �-minimal essential medium (MEM)(A) followed by culture for 7 days on fibronectin in �-MEM (B),vascular endothelial growth factor-supplemented endothelial growthmedium (C), or platelet-derived growth factor-BB-supplementedsmooth muscle growth medium (D). White arrows point to cells withgiant cell characteristics.

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Corporation (Frederick, MD, http://www.superarray.com) and usedwith the ReactionReady HotStart PCR master mix (including aninternal normalizer) following the manufacturer’s instructions. Theresultant PCR products were resolved through 2% agarose gelsstained with ethidium bromide.

Real-Time RT-PCRReal-time PCR was conducted using the ABI PRISM 7700 Se-quence Detection System, TaqMan (Applied Biosystems, FosterCity, CA, http://www.appliedbiosystems.com). The Cdx2 andGAPDH primer sets utilized for real-time quantification were ob-tained from Qiagen (QuantiTect Primer Assay) and used followingthe manufacturer’s instructions. PCR amplicons were detected byfluorescent detection of SYBR Green (QuantiTect SYBR GreenPCR Kit, Qiagen). Cycling conditions were as follows: 95°C for 15minutes followed by 40 cycles at 94°C for 15 seconds, 55°C for 30seconds, and 72°C for 30 seconds. For statistical analysis, all dataare presented as mean � SD. Significant differences between thesamples were assessed by analysis of variance with Tukey’s mul-tiple comparison test. We defined p values less than .05 as statisti-cally significant.

Gene Expression AnalysisRNA samples from three 75-cm2 flasks (each flask 3–5 � 106 cells)of (a) undifferentiated D3-ES cells and (b) D3-ES cells, cultured for4 days in �-MEM on collagen type IV, were analyzed at the UCLAIllumina Microarray Laboratory. Briefly, biotinylated cRNA wasprepared using the Illumina RNA Amplification Kit (Ambion Inc.,Austin, TX, http://www.ambion.com) starting with 100 ng of totalRNA. Samples were purified and used for hybridization on a SentrixMouseRef-8 Expression BeadChip System (Illumina Inc., San Di-ego, http://www.illumina.com) containing approximately 24,000reference-sequence-based probe sequences per array. Scanning wasperformed according to the Illumina BeadStation 500� manual.Microarray raw data were analyzed using BeadStudio version1.5.1.3. Software was provided by the manufacturer. Differentialexpression analysis was selected to quantify gene expression inten-sity values as well as to determine changes of the gene expressionlevels between undifferentiated ES cells (reference group) and Co-lIV-differentiated ES cells. To filter out nonspecific signal intensi-ties, local background subtraction was performed. Only genes withintensities �0.99 were selected for analysis. A differential score of�13.0 demonstrated that gene expression from ColIV-differentiatedES cells had changed significantly when compared with genes of

undifferentiated mES cells. A summary of the upregulated cardio-vascular-, HC-, and TE-specific genes in D3-ES cells cultured onColIV, with a more than 1.5-fold difference, is presented in Table 1.The full microarray analysis is provided as a supplemental dataExcel (Microsoft Corporation, Redmond, WA, http://www.microsoft.com) spreadsheet.

Fluorescence-Activated Cell Sorter AnalysisCells were detached using 0.05% trypsin-EDTA, pelleted by cen-trifugation, washed in PBS, and fixed using the 4% paraformalde-hyde-containing BD Cytofix solution (BD Pharmingen). For doublelabeling, cells were first processed for surface staining using ananti-Cadherin 3 antibody (see Antibodies), followed by permeabi-lization using BD Perm/Wash buffer (BD Pharmingen) according tothe manufacturer’s instructions and staining with the anti-Cdx2antibody as described earlier (see Antibodies). Nonspecific isotype-matched IgGs (Santa Cruz Biotechnology Inc., Santa Cruz, CA,http://www.scbt.com) served as controls. Secondary detection wasdone using appropriate Alexa Fluor 488-, and Alexa Fluor 647-conjugated antibodies. All analyses were performed using a BDLSR2 flow cytometer (BD Biosciences). FCS files were exportedand analyzed using the FlowJo 8.3.3 software (Tree Star Inc.,Ashland, OR, http://www.flowjo.com).

RESULTS

Induction of Hematopoietic, Endothelial, andSmooth Muscle Markers in ColIV-DifferentiatedmES CellsIt has been reported that mES cells cultured on ColIV andstimulated with growth factors differentiate into cultures of EC,SMC, and HC [11–13]. To confirm these results, we plated mEScells on ColIV for 4 days and then replated the disaggregatedcells in the presence or absence of VEGF and PDGF-BB. After48 hours, ColIV-differentiated mES cells developed a charac-teristic colony-like morphology that was visible in all cultures,independent of the culture medium (Figs. 1, 2). Clusters of cellswith a colony-like structure were surrounded by cuboidal cellswith a boundary layer of spindle-shaped cells (Fig. 2A). Tobetter characterize the identity of the cells within each of these

Figure 2. ColIV-differentiated embryonic stem cells express hematopoietic, endothelial, and smooth muscle cell markers. (A): Extracellularmatrix-induced differentiation of mouse embryonic stem cells leads to a highly heterogeneous cell population. F-actin staining (red) shows clearlythe different cytoskeleton dimensions of a variety of cells, which grow in a characteristic colony-like pattern: large cuboidal cells surround centralcell clusters of round cells, and in the adjacent regions of the cuboidal cells elongated, spindle-shaped cells can be found. Within the cuboidal cellssome large flattened cells with polyploid nuclei (�) are visible. (B): Spindle-shaped, fibroblast-like cells are positive when stained with antibodiesagainst �-SMA (green) (Ba–Bc) and vimentin (green) (Bd–Bf), indicating a smooth muscle cell phenotype, whereas the majority of cells situatedwithin the cell clusters are positive for the endothelial cell marker CD31 (green) (Bg–Bi). Cell nuclei are stained with 4,6-diamidino-2-phenylindole(blue). Abbreviations: cc, cuboidal cells; MEM, minimal essential medium; PDGF-BB, platelet-derived growth factor-BB; rc, round cells; sc,spindle-shaped cells; SMA, smooth muscle actin; VEGF, vascular endothelial growth factor.

1532 Collagen IV Induces Trophoectoderm Differentiation

three areas, we performed immunostaining with a panel ofcell-type-specific antibodies. Cells within the clusters were pre-dominantly positive for HC (data not shown) and EC markersincluding CD31 (Fig. 2Bg–2Bi); however, some of the inner,more compact cells continued to express markers such as Nanogand Oct4 (data not shown), likely representing a small fractionof undifferentiated mES cells. The spindle-shaped cells at theperiphery expressed vimentin (Fig. 2Bd–2Bf) as well as SMCmarkers including basic calponin, h-caldesmon, smooth muscle-myosin (data not shown), and �-SMA (Fig. 2Ba–2Bc), indicat-ing a SMC phenotype. The flattened cuboidal-shaped cells in theborder zone did not express markers consistent with EC, SMC,or HC, and some of them showed an enlarged morphology (Fig.1B–1D) with multiple nuclei (Fig. 2A).

Mouse ES Cells Cultured on ColIV Differentiateinto Trophoblast LineagesTo ascertain the identity of the cuboidal cells induced by ColIV,microarrays were used to characterize global gene expression. In

addition to the expected expression of cardiovascular- and HC-specific genes, ColIV-differentiated D3-ES cells expressed apanel of genes normally restricted to trophoblast cells (Table 1).Although mES cells have been thought to be incapable ofdifferentiating into trophoblasts, the large flattened cuboidal-shaped cells with enlarged and multiple cell nuclei (Fig. 1B–1D;Fig. 2A) demonstrated morphological characteristics that areclassic for trophoblast giant cells and were reminiscent of thosepreviously reported in studies using trophoblast stem cells [20]or forced Cdx2 expression in mES cells [7].

To confirm that the cuboidal cells within the highly heter-ogeneous population of differentiating D3-ES cells were indeedtrophoblast-like cells, we surveyed them for expression ofknown TE markers, including cytokeratin [21], Cx31 [22], P-Cadherin (Cadherin 3), and Cdx2 [23] by immunofluorescencestaining (Figs. 3, 4A). Double immunocytochemical labelingusing these markers demonstrated cells positive for cytokeratinand Cadherin 3, cytokeratin and Cdx2, and Cx31 and Cdx2within the zone of cuboidal cells and at the outer edge of the cellclusters (Fig. 4A). It is possible that trophoblast cells closer to

Figure 4. ColIV-differentiated cells expressmarkers of trophoblast lineages. (A): Immuno-labeling of differentiating mES cells identifiesdouble-positive cells for cytokeratin (green)/Cadherin 3 (red) (Aa, Ab); cytokeratin (green)/Cdx2 (red) (Ac, Ad); and Cx31 (green)/Cdx2(red) (Ae, Af). (B): D3-embryonic stem cellsexpress a panel of trophoectoderm-related genesduring the course of extracellular matrix-induceddifferentiation. Abbreviations: ColIV, collagenIV; d, day; GAPDH, glyceraldehyde-3-phos-phate dehydrogenase; MEF, mouse embryonicfibroblast; MEM, minimal essential medium;mES, mouse embryonic stem; PDGF-BB, plate-let-derived growth factor-BB; VEGF, vascularendothelial growth factor.

Figure 3. Collagen IV-differentiated embry-onic stem cells cultured in �-MEM (A–E),VEGF-supplemented endothelial growth me-dium (F–J), or PDGF-BB-supplementedsmooth muscle growth medium (K–O) expressmarkers of trophoectodermal cells. Imagesshow cytoskeleton-staining (red) and positivestaining (green) for cytokeratin (B, G, L),Cx31 (C, H, M), Cadherin 3 (D, I, N), andCdx2 (E, J, O). Staining using the secondaryantibody alone (A, F, K) serves as control.Staining with 4,6-diamidino-2-phenylindolewas performed to show cell nuclei (blue). Ab-breviations: MEM, minimal essential medium;PDGF-BB, platelet-derived growth factor-BB;VEGF, vascular endothelial growth factor.

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the compact cell clusters represent the more primitive tropho-blast stem cells, since TE cells with a more differentiatedphenotype such as giant cells appeared to grow out and werefurther away from the cell clusters, a phenomenon also seen inprevious studies using trophoblast stem cells [20].

Several groups have proposed detailed gene profiles thatallow identification of specific trophoblast subtype lineages [24,25]. Thus, we sought to identify the expression of trophoblast-restricted genes in ColIV-differentiated mES cells using semi-quantitative RT-PCR (Fig. 4B). Murine placenta at day 12.5 ofgestation was included as positive control for most of themarkers along with MEF and undifferentiated D3-ES cells asnegative controls. mES cells cultured for 4 days on ColIVshowed a downregulated expression of the pluripotency-associ-ated genes Nanog and Oct4 (data not shown). Expression of thebrachyury gene, a transcription factor essential for the genesisand maintenance of mesoderm and notochord [26], as well asNodal, a growth factor implicated in regulating trophoblastdifferentiation and placental development, were increased. Ex-pression of a panel of TE-restricted genes that identify tropho-blast subtypes was upregulated in ColIV-differentiated ES cells.These included markers for trophoblast stem cells (Cdx2,Eomes, estrogen-related receptor [Esrr�, Err2], spongiotropho-blast cells (mammalian achaete-scute homologous protein-2[Mash2], trophoblast specific protein alpha [Tpbp/-4311]), tro-phoblast giant cells (placental lactogen [PL-1], Homo sapiensheart and neural crest derivatives expressed 1 [Hand1]), andlabyrinthine trophoblasts (extraembryonic spermatogenesis ho-meobox 1 [Esx1], glial cells missing homolog 1 [Gcm1], anddistal-less homeobox 3 [Dlx3]) (Fig. 4B).

When the ColIV-differentiated mES cells were replated inthe presence of VEGF- or PDGF-BB-supplemented media, asimilar expression pattern was observed, although relative dif-ferences were seen (Fig. 3; Fig. 4B). Expression levels of themesodermal marker brachyury and early trophoblast markersCdx2 and PL-1 declined, accompanied by a corresponding in-crease in expression of the spongiotrophoblast-specific markerTpbp/-4311 and labyrinthine trophoblast marker Dlx3. Further-more, cells that were cultured in PDGF-BB-supplemented me-dium showed enhanced expression of Gcm1 and Mash2, whichwas not detectable in cells cultured in VEGF-supplemented

medium. Nodal was expressed in all cells but seemed to beupregulated in cells cultured in VEGF- and PDGF-BB-supple-mented medium. The expression levels of Eomes, Esrr�,Hand1, and Esx1 were constant across the different cultureconditions.

TE Differentiation Is Specific to ColIV and IsRestricted to MEF-Dependent mES CellsTo determine if TE differentiation of mES cells is specific toColIV, we compared trophoblast gene expression in ColIV-differentiated cells with D3-ES cells cultured on various ECMproteins including collagen type I, laminin, and fibronectin (Fig.5A, 5B). Expression of Cdx2, Eomes, PL-1, and Tpbp/-4311 wasonly seen in ColIV-differentiated mES cells. Expression ofCdx2 was over sixfold higher on ColIV when compared withundifferentiated mES cells or ES cells differentiated on otherECM proteins (p � .05; Fig. 5B). In contrast, expression of lessspecific markers such as Esrr�, Esx1, and Gcm1 was seen in allsamples, although Esx1 and Gcm1 were decreased on collagenI (Fig. 5A). These results indicate that TE differentiation of mEScells is indeed specific to collagen type IV.

One potential explanation for the lack of previous reports ofmES cells cultured on ColIV differentiating into TE cells maybe that feeder-free mES cell lines were used, which may have amore limited differentiation potential [11, 12]. Data from aprevious study showed that the heterogeneous population ofdifferentiated J1 and 210-58 ES cells that were maintained onMEFs contained a small population of cells with TE potential[5]. To determine whether the ability of mES cells to differen-tiate into cells of the TE lineage is a general phenomenon orindeed restricted to specific cell lines, we examined the abilityof a second MEF-dependent cell line, R1-ES cells, as well asMEF-free CCE-ES cells, to differentiate into trophoblast cells(Fig. 5C). As shown, only mES cells that had been maintainedon MEF layers (D3 and R1) could be induced to differentiateinto TE cell types. CCE-ES cells, differentiated on ColIV andfurther on fibronectin, appeared to be morphologically distinctwhen compared with ColIV-differentiated D3-ES and R1-EScells (data not shown), showing no expression of TE markersincluding Cdx2 or Tpbp/-4311 (Fig. 5C). In contrast, markers for

Figure 5. Trophoectoderm differentiation isspecific to ColIV and is restricted to embry-onic stem (ES) cells that were maintained onMEF feeder layers. (A): Gene expressionanalysis of D3-ES cells cultured on a panel ofextracellular matrix (ECM)-proteins. The ex-pression of trophoblast lineage genes includ-ing Cdx2, Eomes, PL-1, and Tpbp/4311 isexclusively seen in cells cultured for 4 days onColIV. (B): Real-time reverse transcription-polymerase chain reaction analysis of Cdx2expression in D3-ES cells cultured on ColIVcompared with undifferentiated ES cells aswell as ES cells cultured on other ECM pro-teins (�, p � .05 vs. mouse ES cells, MEF,Coll I, laminin, and Fibro.). (C): Expressionof TE-restricted markers is observed in bothMEF-dependent ES cell lines D3 and R1, butis not seen in the MEF-free CCE-ES cells.Abbreviations: ColIV, collagen IV; Coll I,collagen I; d, day; Fibro., fibronectin;GAPDH, glyceraldehyde-3-phosphate dehy-drogenase; MEF, mouse embryonic fibro-blast; mESC, mouse embryonic stem cell.

1534 Collagen IV Induces Trophoectoderm Differentiation

endothelial and smooth muscle differentiation were unaffected(supplemental online Fig. 1).

ColIV-Induced Trophoblast Differentiation IsCdx2-DependentTo determine if TE differentiation of mES cells on ColIV isCdx2-dependent, we cultured Cdx2-deficient ES cells [18] andthe wild-type (WT) parental ES cell line for 4 days on ColIVand analyzed trophoblast gene expression and cell morphology(Fig. 6). No expression of Cdx2, Eomes, or Tpbp/4311 wasobserved in Cdx2-deficient cells, and PL-1 was only weaklyexpressed (Fig. 6A). The morphological characteristics of WTcells cultured on ColIV were distinct from those of Cdx2-deficient ES cells (Fig. 6B). ColIV-differentiated WT ES cellsshowed a more heterogeneous cell population (Fig. 6Ba) whencompared with ColIV-differentiated Cdx2-deficient ES cells(Fig. 6Bb). Double immunocytochemical labeling of WT EScells revealed the presence of cells positive for cytokeratin andCadherin 3, cytokeratin and Cdx2, and Cx31 and Cdx2 (Fig.6Ca, 6Cc, 6Ce). Although a very limited number of cells pos-itive for Cadherin 3, cytokeratin, or Cx31 were detectable withinthe ColIV-differentiated Cdx2-deficient ES cells (Fig. 6Cb, 6Cd,6Cf), these cells showed a very different morphology whencompared with the WT cells. Cells positive for Cdx2 were neverobserved within ColIV-differentiated Cdx2-deficient ES cells(Fig. 6Cd, 6Cf).

ColIV-Differentiated TE Cells Are FGF-Responsiveand Can Be Grown Under Clonal ConditionsTo determine if the TE differentiation on ColIV is modulated bygrowth factors known to promote trophoblast cells, cells werecultured for 1, 2, 3, and 4 days on collagen type I and type IVin either �-MEM, bFGF-supplemented �-MEM, or TS mediumcontaining FGF4 and heparin. Fluorescence-activated cell sorter(FACS) analysis showed significantly higher numbers of Cdx2-expressing cells in the ColIV-differentiated cultures, grown ineither bFGF-supplemented �-MEM or in TS medium, whencompared with �-MEM cultures without supplements (Fig. 7A).

In ColIV-�-MEM cultures, the Cdx2-positive population repre-sented 1.07% of cells on day 1 and increased to 2.62% by day4. Addition of bFGF to the medium significantly increased thenumber of Cdx2-positive cells from 3.01% to 6.51% (day 1 today 4), whereas the FGF4-/heparin-supplemented TS mediumresulted in a further increase (3.83% to 15.71% [day 1 to day 4])of Cdx2-expressing cells (Fig. 7A). No Cdx2-positive cells andno FGF-responsiveness was observed in cultures grown oncollagen type I (Fig. 7A). Analysis of gene expression in Co-lIV-/TS medium-differentiated mES cells revealed a progressivedownregulation of Oct4 accompanied by an upregulation oftrophoblast stem cell markers including Cdx2, Eomes, andEsrr� (Fig. 7B).

To ensure that we had committed but undifferentiated TEcells, Cdx2-positive clones were isolated after 2 days in cultureon ColIV by limited dilution. Isolated clones were expanded andsubsequently analyzed (Fig. 7C–7E). FACS analysis demon-strated that 99.7% and 98.7% of the clonally expanded popula-tions retained Cdx2 and Cadherin 3 expression, respectively,suggesting that these clones most likely represent trophoblaststem cells (Fig. 7C). These FACS results were confirmed byimmunocytochemical staining (Fig. 7Da–7Dc). Gene expressionanalysis of the clonally derived, undifferentiated Cdx2 cellsshowed expression of trophoblast stem cell genes includingCdx2, Eomes, and Esrr�, whereas markers for differentiated TEcells were absent (Fig. 7E). However, when these clones werecultured in differentiation-promoting conditions by withdrawalof FGF4 and heparin, they differentiated into a variety of TEderivatives, as showed by increased expression of markers forgiant cells, spongiotrophoblast, and labyrinth and a concomitantdecrease in expression of trophoblast stem cell genes (Fig. 7E).Further analysis of these cultures demonstrated the presence ofcells with trophoblast giant cell morphology (Fig. 7Dd). Immu-nocytochemistry showed a downregulated expression of Cdx2;however, differentiated TE cells were still positive for cytoker-atin (data not shown) and Cadherin 3 (Fig. 7Dd–7Df). Expres-sion of markers for endothelial or smooth muscle differentiationwas not observed in these cultures at any time point, suggesting

Figure 6. ColIV-induced differentiation intotrophoectoderm cells is Cdx2-dependent.(A): Semiquantitative reverse transcription-polymerase chain reaction showing that tro-phoblast-restricted markers are exclusivelyexpressed in WT cells when cultured onColIV. No expression of trophoectoderm-specific markers could be detected in Cdx2-deficient embryonic stem (ES) cells. (B):Bright-field microscopy reveals morphologi-cal differences between WT (A) and Cdx2-deficient (B) ES cells cultured for 4 days onColIV. White arrows point to flattened cuboi-dal-shaped cells seen in the WT but not in theCdx2-deficient ES cell cultures. (C): Immu-nocytochemical staining of ColIV-differenti-ated WT (Ca, Cc, Ce) and Cdx2-deficient(Cb, Cd, Cf) ES cells. WT ES cells showdouble-positive cells for cytokeratin (green)and Cadherin 3 (red) (Ca); cytokeratin(green) and Cdx2 (red) (Cc); as well as Cx31(green) and Cdx2 (red) (Ce). In contrast,Cdx2-deficient ES cells are only positive forCadherin 3 (red) (Cb). Some single cells arepositive for cytokeratin (green) (Cb, Cd) andCx31 (green) (Cf). No Cdx2-positive cells(red) (Cd, Cf) were detectable. Abbrevia-tions: ColIV, collagen IV; d, day; GAPDH,glyceraldehyde-3-phosphate dehydrogenase;WT, wild-type.

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that the progenitors that give rise to TE derivatives do notproduce vascular cells (supplemental online Fig. 2).

DISCUSSION

Our data suggest that mES cells retain the capacity to differen-tiate into cells of the TE lineage given the correct extracellularsignals. Previous dogma held that mES cells were incapable ofdifferentiating into TE derivatives [1, 27]; however, this infor-mation was based on in vivo data, where it was found that mEScells rarely give rise to the trophoblast layers of the placenta [2,3]. This lack of contribution to TE cells in the developingembryo may simply represent a preferred or default differenti-ation pathway of mES cells. Consistent with this hypothesis,recent reports have documented that genetic manipulation candivert mES cells to TE lineages, which suggests that there mightbe some plasticity to the commitment of mES cells to epiblast-restricted lineages [5–7]. Thus, reported differences in the dif-ferentiation potential among species of mES cells may be morerelated to limitations in determining the correct extracellularmicroenvironment than true biological distinctions.

Although relative differences have been reported betweenmouse and human ES cells, including morphology, signaling path-ways mediating self-renewal, and expression of certain genes [27–29], the major purported difference of developmental significancebetween human and mouse ES cells was that hES cells can giverise to TE-like cells spontaneously[8, 29] or with bone morphoge-netic protein 4 (BMP-4) treatment [9]. This has led to the belief thatthe regulatory pathways for differentiation of early embryonicstructures such as the placenta and extraembryonic membraneswere distinct in mouse and human embryos. Although the specificrole of Oct4, Cdx2, and other lineage-associated genes in regulatingTE differentiation in human ES cells remains to be determined,similarities to mouse developmental programs have already beenseen, as downregulation of Oct4 in human ES cells leads to up-regulation of Cdx2 and trophoblast markers [30]. It will be impor-tant to determine if hES cells spontaneously differentiate into TEcells on ColIV. If so, this would provide an in vitro model todirectly compare mouse and human ES-differentiation potentialand resolve whether the differences reported in developmentalusage of some factors in early lineage specification [31] representtrue biological differences versus specious differences related totechnical disparities.

Figure 7. ColIV-differentiated TE cells areFGF-responsive and can be grown underclonal conditions. (A): Fluorescence-acti-vated cell sorting (FACS) analysis of Cdx2-expressing cells within the ColIV- and CollI-differentiated cultures (�, p � .05 vs.�-MEM; †, p � .05 vs. bFGF). (B): Geneexpression pattern of the heterogeneous pop-ulation of ColIV-differentiated D3-embry-onic stem cells, cultured for 1, 2, 3, and 4days in TS medium. (C): Histogram showingthe FACS profile of clonally derived TE cellsthat were cultured in TS medium. (D):Bright-field and immunofluorescence imagesof clonally derived, undifferentiated TE cellsin TS medium (Da–Dc) and after 6 days ofculture in differentiation medium (Dd–Df).Immunolabeling demonstrates that the major-ity of the undifferentiated TE cells are posi-tive (red) for Cdx2 (Db) and Cadherin 3 (Dc),whereas only a few Cdx2-positive cells weredetectable within the differentiated cells (De).Cadherin 3 was expressed by most of thedifferentiated cells (Df). Cell nuclei arestained with 4,6-diamidino-2-phenylindole(blue). (E): Reverse transcription-polymerasechain reaction analysis of three isolated TEcell clones at day 0 of differentiation in TSmedium and after a 6-day exposure to differ-entiation medium. Abbreviations: APC, allo-phycocyanin; bFGF, basic fibroblast growthfactor; ColIV, collagen IV; Coll I, collagen I;d, day; Diff., differentiation; FITC, fluores-cein isothiocyanate; GAPDH, glyceralde-hyde-3-phosphate dehydrogenase; MEF,mouse embryonic fibroblast; MEM, minimalessential medium; mES, mouse embryonicstem; TE, trophoectoderm; TS, trophoblaststem cell.

1536 Collagen IV Induces Trophoectoderm Differentiation

Cells of the trophoblast lineage are essential for the estab-lishment and maintenance of the placenta, and a number ofspecialized trophoblast subtypes have evolved to address spe-cific physiological needs. Using a lineage-specific gene profile,we were able to identify markers of all lineages of placentaltrophoblast cells including trophoblast stem cells (Cdx2, Id2,Eomes, Esrr�), trophoblast giant cells (PL-1, Hand1), spongio-trophoblasts (Tpbp/-4311), and cytotrophoblasts (Esx1, Gcm1,and Dlx3) [24, 25]. A similar potential to differentiate intomultiple trophoblast lineages was also seen in human ES cells[9]. Interestingly, our results and others have documented thepresence of HC, as well as EC and SMC, in mES cells differ-entiated on ColIV [11–13]. These cell types are all present in thedeveloping placenta along with ColIV [32, 33]. The importanceof the placenta in hematopoietic development has been recentlyappreciated, as placental hematopoietic stem cell (HSC) local-ization appears in parallel with the aorta-gonad-mesonephrosregion before HSCs are found in circulation or have colonizedthe fetal liver [34]. This placental HS cell colonization culmi-nates in a rapid expansion of a definitive HSC pool. TheColIV-based ES cell differentiation model presented here couldprovide a unique opportunity to study the HSC-promoting prop-erties of the placental niche and the role of trophoblasts in thisprocess specifically. Furthermore, if trophoblast cells indeedsupport self-renewal of HSC, and if hES cells demonstrate asimilar response on ColIV, this in vitro differentiation modelmight represent a novel culture system to generate HSCs forstem cell-based therapies in the future.

ColIV is detected in the mouse embryo as early as the32– 64-cell stage [35] and is highly expressed in the devel-oping placenta in both fetal and maternal layers [32]. ColIVlikely plays a dual role in embryonic development both as aninducer of differentiation and as a structural protein. ColIV isessential for basement membrane stability and provides ascaffold that integrates other components such as laminins,nidogens, and perlecan into a highly organized supramolecu-lar structure. Consistent with this role, ColIV-null mutantmice die at midgestation with placental defects [36]. The

mechanisms whereby ColIV might be directing ES cells todifferentiate into trophoblast are speculative; however, inte-grin-dependent mechanisms are known to regulate BMP-4expression in ES cells. BMP-4, a member of the transforminggrowth factor-beta superfamily, was capable of inducing thedifferentiation of human ES cells to trophoblast [9].

SummaryThe data we have provided challenge the widely held notion thatmouse ES cells are incapable of spontaneously differentiatinginto TE without genetic manipulation. We could identify theexact extracellular stimuli and the appropriate microenviron-ment that are necessary for mES cells to successfully differen-tiate into TE cells. This in vitro model of ColIV-induced TEdifferentiation should prove useful, both as a tool for studyingthe differentiation and function of early trophoblasts as well asfurther elucidating the specific developmental role of the largenumber of factors on TE differentiation that, when geneticallydeleted, result in abnormal placental development and earlyembryonic lethality [25].

ACKNOWLEDGMENTS

The authors would like to thank Y. Bukshpun and E. Butylkovafor their technical assistance and Rudolph Jaenisch (WhiteheadInstitute, Cambridge, MA) for providing the Cdx2-deficient EScells. This work was supported by the Laubisch and GlazerFunds as well as the Deutsche Forschungsgemeinschaft(Sche701/2-1, 3-1 [K.S.-L.]), NIH R21DK069659 (H.K.M.),R01HL70748, and AHA 0340087N Grants (W.R.M.).

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

REFERENCES

1 Yamanaka Y, Ralston A, Stephenson RO et al. Cell and molecularregulation of the mouse blastocyst. Dev Dyn 2006;235:2301–2314.

2 Nagy A, Gocza E, Diaz EM et al. Embryonic stem cells alone are able tosupport fetal development in the mouse. Development 1990;110:815–821.

3 Beddington RS, Robertson EJ. An assessment of the developmentalpotential of embryonic stem cells in the midgestation mouse embryo.Development 1989;105:733–737.

4 Rossant J, Spence A, Rossant J. Chimeras and mosaics in mouse mutantanalysis. Trends Genet 1998;14:358–363.

5 Hemberger M, Nozaki T, Winterhager E et al. Parp1-deficiency inducesdifferentiation of ES cells into trophoblast derivates. Dev Biol 2003;257:371–381.

6 Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4defines differentiation, dedifferentiation or self-renewal of ES cells. NatGenet 2000;24:372–376.

7 Tolkunova E, Cavaleri F, Eckardt S et al. The caudal-related protein cdx2promotes trophoblast differentiation of mouse embryonic stem cells.STEM CELLS 2006;24:139–144.

8 Gerami-Naini B, Dovzhenko OV, Durning M et al. Trophoblast differ-entiation in embryoid bodies derived from human embryonic stem cells.Endocrinology 2004;145:1517–1524.

9 Xu RH, Chen X, Li DS et al. BMP4 initiates human embryonic stem celldifferentiation to trophoblast. Nat Biotechnol 2002;20:1261–1264.

10 Gerecht-Nir S, Itskovitz-Eldor J. The promise of human embryonic stemcells. Best Pract Res Clin Obstet Gynaecol 2004;18:843–852.

11 Nishikawa SI, Nishikawa S, Hirashima M et al. Progressive lineageanalysis by cell sorting and culture identifies FLK1�VE-cadherin� cells

at a diverging point of endothelial and hemopoietic lineages. Develop-ment 1998;125:1747–1757.

12 Yamashita J, Itoh H, Hirashima M et al. Flk1-positive cells derived fromembryonic stem cells serve as vascular progenitors. Nature 2000;408:92–96.

13 Xiao Q, Zeng L, Zhang Z et al. Stem cell-derived Sca-1� progenitorsdifferentiate into smooth muscle cells, which is mediated by collagenIV-integrin alpha1/beta1/alphav and PDGF receptor pathways. Am JPhysiol Cell Physiol 2007;292:C342–C352.

14 Gerecht-Nir S, Ziskind A, Cohen S et al. Human embryonic stem cells asan in vitro model for human vascular development and the induction ofvascular differentiation. Lab Invest 2003;83:1811–1820.

15 Sakurai H, Era T, Jakt LM et al. In vitro modeling of paraxial and lateralmesoderm differentiation reveals early reversibility. STEM CELLS 2006;24:575–586.

16 Zeigler BM, Sugiyama D, Chen M et al. The allantois and chorion, whenisolated before circulation or chorio-allantoic fusion, have hematopoieticpotential. Development 2006;133:4183–4192.

17 Corbel C, Salaun J, Belo-Diabangouaya P et al. Hematopoietic potentialof the pre-fusion allantois. Dev Biol 2007;301:478–488.

18 Meissner A, Jaenisch R. Generation of nuclear transfer-derived pluripo-tent ES cells from cloned Cdx2-deficient blastocysts. Nature 2006;439:212–215.

19 Robertson E, Bradley A, Kuehn M et al. Germ-line transmission of genesintroduced into cultured pluripotential cells by retroviral vector. Nature1986;323:445–448.

20 Quinn J, Kunath T, Rossant J. Mouse trophoblast stem cells. MethodsMol Med 2006;121:125–148.

21 Bulmer JN, Johnson PM. Antigen expression by trophoblast populationsin the human placenta and their possible immunobiological relevance.Placenta 1985;6:127–140.

22 Kibschull M, Winterhager E. Connexins and trophoblast cell lineagedevelopment. Methods Mol Med 2005;121:149–158.

1537Schenke-Layland, Angelis, Rhodes et al.

www.StemCells.com

23 Niwa H, Toyooka Y, Shimosato D et al. Interaction between Oct3/4 andCdx2 determines trophectoderm differentiation. Cell 2005;123:917–929.

24 Selesniemi KL, Reedy MA, Gultice AD et al. Identification of committedplacental stem cell lines for studies of differentiation. Stem Cells Dev2005;14:535–547.

25 Simmons DG, Cross JC. Determinants of trophoblast lineage and cellsubtype specification in the mouse placenta. Dev Biol 2005;284:12–24.

26 Kispert A, Herrmann BG. The Brachyury gene encodes a novel DNAbinding protein. EMBO J 1993;12:3211–3220.

27 Boiani M, Scholer HR. Regulatory networks in embryo-derived pluripo-tent stem cells. Nat Rev Mol Cell Biol 2005;6:872–884.

28 Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines fromhuman blastocysts: Somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.

29 Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem celllines derived from human blastocysts. Science 1998;282:1145–1147.

30 Hay DC, Sutherland L, Clark J et al. Oct-4 knockdown induces similarpatterns of endoderm and trophoblast differentiation markers in humanand mouse embryonic stem cells. STEM CELLS 2004;22:225–235.

31 Ralston A, Rossant J. Genetic regulation of stem cell origins in the mouseembryo. Clin Genet 2005;68:106–112.

32 Thomas T, Dziadek M. Differential expression of laminin, nidogen andcollagen IV genes in the midgestation mouse placenta. Placenta 1993;14:701–713.

33 Saylam C, Ozdemir N, Itil IM et al. Distribution of fibronectin, lamininand collagen type IV in the materno-fetal boundary zone of the devel-oping mouse placenta. Experimental study. Arch Gynecol Obstet 2002;266:83–85.

34 Gekas C, Dieterlen-Lievre F, Orkin SH et al. Placenta is a niche forhematopoietic stem cells. Dev Cell 2005;8:365–375.

35 Dziadek M, Timpl R. Expression of nidogen and laminin in basementmembranes during mouse embryogenesis and in teratocarcinoma cells.Dev Biol 1985;111:372–382.

36 Poschl E, Schlotzer-Schrehardt U, Brachvogel B et al. Collagen IV isessential for basement membrane stability but dispensable for initiationof its assembly during early development. Development 2004;131:1619–1628.

See www.StemCells.com for supplemental material available online.

1538 Collagen IV Induces Trophoectoderm Differentiation