In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility

In Vitro Modeling of Paraxial and Lateral MesodermDifferentiation Reveals Early Reversibility

HIDETOSHI SAKURAI,a,b TAKUMI ERA,a LARS MARTIN JAKT,a,c MITSUHIRO OKADA,a,c SHIGERU NAKAI,b

SATOMI NISHIKAWA,a SHIN-ICHI NISHIKAWAa

aLaboratory for Stem Cell Biology, RIKEN Center for Development Biology, Kobe, Japan; bScience of In-Home

Medicine, Health and Community Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan;cPrefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, Japan Science and

Technology Corporation, Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Osaka, Japan

Key Words. Embryonic Stem cell • Mesoderm • Reversibility

ABSTRACT

Endothelial cells (ECs) are thought to be derived mainly fromthe vascular endothelial growth factor receptor 2 (VEGFR-2)�

lateral mesoderm during early embryogenesis. In this study, wespecified several pathways for EC differentiation using a mu-rine embryonic stem (ES) cell differentiation culture systemthat is a model for cellular processes during early embryogen-esis. Based on the results of in vitro fate analysis, we show that,in the main pathway, committed ECs are differentiatedthrough the VEGFR-2� platelet-derived growth factor recep-tor � (PDGFR-�)� single-positive (VSP) population that isderived from the VEGFR-2�PDGFR-�� double-positive (DP)population. This major differentiation course was also con-firmed using DNA microarray analysis. In addition to this

main pathway, however, ECs also can be generated fromthe VEGFR-2�PDGFR-�� single-positive (PSP) population,which represents the paraxial mesodermal lineage and is alsoderived from the DP population. Our results strongly suggestthat, even after differentiation from the common progenitorDP population into the VSP and PSP populations, these twopopulations continue spontaneous switching of their surfacephenotype, which results in switching of their eventual fates.The rate of this interlineage conversion between VSP and PSPis unexpectedly high. Because of this potential to undergo fateswitch, we conclude that ECs can be generated via multiplepathways in in vitro ES cell differentiation. STEM CELLS 2006;24:575–586

INTRODUCTIONThe formation of the vascular system is an essential processfor the neogenesis of all tissues during embryogenesis andpostnatal development [1, 2]. Because this process requiresthe recruitment of endothelial cells (ECs), how a sufficientnumber of ECs is enlisted to the region in need is one of thefundamental questions facing vascular biology. It is widelyaccepted that ECs are generated from vascular endothelialgrowth factor receptor 2 (VEGFR-2)� lateral mesodermalcells at the initial phase of vasculogenesis [3], and theirsubsequent recruitment is supported by the proliferation ofdifferentiated ECs. Recent studies have also suggested alter-native sources for these cells [4 – 6].

Our studies have focused on the earliest process of mouseEC differentiation [7–9], for which murine embryonic stem (ES)cell differentiation culture provides an ideal tool, as it allowsdetailed analysis of the cell specification process from thepluripotent stage [7, 10, 11]. We have attempted to define the

process of EC differentiation in terms of the expression ofsurface markers and showed that VEGFR-2, an essential mole-cule for all neoangiogenic processes [12], is the key markerrequired for a detailed examination of this process [7]. In ourprevious studies, we proposed that ECs are derived from anE-cadherin (ECD)�VEGFR-2� population [7, 13]. In this con-cept, ECD expression is used to monitor the process of exfoli-ation, as its expression is downregulated during the exfoliationof mesoderm cells from the primitive streak [14]. In vitro fateanalysis demonstrated that ECD�VEGFR-2� cells derived fromES cells have the potential to produce endothelial cells, hema-topoietic cells, and � smooth muscle actin–positive pericytes [7,9, 15], indicating that ECD�VEGFR-2� cells do indeed corre-spond to lateral mesodermal cells. In contrast to these findings,previous studies have shown that platelet-derived growth factorreceptor � (PDGFR-�) is expressed in paraxial mesoderm dur-ing mouse embryogenesis [16, 17]. Although PDGFR-�� cellswere observed in ES cell differentiation culture together with

Correspondence: Takumi Era, M.D., Ph.D., Laboratory for Stem Cell Biology, RIKEN Center for Development Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan. Telephone: 81-78-306-1893; Fax: 81-78-306-1895; e-mail: [email protected] ReceivedJune 7, 2005; accepted for publication September 6, 2005; first published online in STEM CELLS EXPRESS December 9, 2005.©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0256

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STEM CELLS 2006;24:575–586 www.StemCells.com

VEGFR-2� cells [7], the question of whether PDGFR-� expres-sion specifies the paraxial mesodermal lineage remains unan-swered, as does the potential of the ECDlow PDGFR-�� popu-lation to give rise to ECs during in vitro ES cell differentiation.

A recent study has interestingly demonstrated that cartilagecan be differentiated not only from PDGFR-�� but also fromVEGFR-2� cells [18]. A lineage analysis by Sato and hiscolleagues further showed that Cre-recombinase expressed un-der the promoter of the VEGFR-2 gene marked, in addition tothe endothelial cell lineages, skeletal muscle cells, which aresupposed to be derived from paraxial mesoderm [19]. In contrastto this, mounting evidence suggests that a portion of ECs can bederived from somites [6].

These observations, taken together, are a strong argumentagainst our previous simple model, which specified VEGFR-2�

lateral mesoderm as the sole pathway for EC differentiation [7,13]. There are two possibilities that may account for the expres-sion of VEGFR-2 in the non-EC differentiation pathway. Onepossibility is that VEGFR-2 is expressed in uncommitted me-soderm that is subsequently able to give rise to paraxial meso-dermal lineages. Alternatively, lineage switching may occurbetween the early stages of lateral and paraxial mesoderm.

The current research shows that both these possibilities doindeed contribute to the case for EC differentiation. Here, wepresent a new differentiation pathway in which the PDGFR-��VEGFR-2� double-positive (DP) population gives rise toboth the VEGFR-2� single-positive (VSP) population and thePDGFR-�� single-positive (PSP) population. By both DNAmicroarray analysis and in vitro fate analysis, we demonstratethat the ES cell-derived VSP and PSP populations correspond tothe lateral and paraxial mesodermal lineages, respectively. Al-though the PSP population represents the paraxial mesodermallineage, it is able to give rise to ECs; our results show that thisphenomenon is due to interlineage conversion between the VSPand PSP populations following divergence.

MATERIALS AND METHODS

Construction and Cell LineGreen fluorescent protein (GFP)-expressing vectors were con-structed by inserting an eGFP [20] cDNA downstream of theCAG promoter [21]. In this vector, the G418 resistance gene isdriven by the PGK promoter. The constructs were transfectedinto CCE ES cells by electroporation [22]. Stably transduced EScells were established as G418-resistant colonies. After thecolonies were picked up, the expression of GFP in undifferen-tiated and day 4 differentiated ES cells was confirmed byfluorescence-activated cell sorting (FACS) analysis. We estab-lished six clones that stably expressed GFP.

Cell Culture and In Vitro ES Cell DifferentiationMurine CCE, TT2 ES cells and OP9 stromal cells were main-tained as described previously [7, 23, 24]. To maintain theexpression of GFP, we continuously cultured the ES cells car-rying GFP in 200 �g/ml G418 (Gibco-BRL, Gaithersburg, MD,http://www.gibcobrl.com).

Induction of ES cell differentiation was performed as de-scribed previously [7]. For reculture studies, 1.0–5.0 � 105 cellspurified by FACS were recultured in a confluent OP9 cell layeron 24-well plates using � minimal essential medium (�-MEM)

(Gibco-BRL) supplemented with 10% fetal calf serum (FCS)and 50 �M 2-mercaptoethanol (2ME). Twenty-four hours later,the cells were harvested and collected for examination of thesurface markers and gene expression.

Antibodies, Cell Staining, and FACS AnalysisThe rat monoclonal antibodies (moAbs) APA5 (anti-PDGFR-�)[25], ECCD2 (anti-ECD) [26], AVAS12 (anti-VEGFR-2) [13],and VECD1(anti-VE-cadherin) [27] were prepared as reportedpreviously [7]. Phycoerythrin-conjugated streptavidin (Pharm-ingen, Franklin Lakes, NJ, http://www.bdbiosciences.com) wasused for detecting biotinylated-APA5 Ab and biotinylated anti-CD34 moAb (Pharmingen). ECCD2, AVAS12, and VECD1moAbs were directly conjugated using standard methods andAlexa488 and Alexa405 (Molecular Probes, Eugene, OR, http://probes.invitrogen.com), allophycocyanin (APC), and Alexa680(Molecular Probes), respectively.

Cultured cells were harvested and collected using either0.05% trypsin-EDTA (Gibco-BRL) or a dissociation buffer(Gibco-BRL), which was shown not to affect the surface ex-pression levels of ECD and VE-cadherin (VECD) [7]. Single-cell suspensions were stained as previously described [7] andanalyzed or sorted by FACSCalibur, FACSVantage, or FACS-Aria (Becton, Dickinson and Company, Franklin Lakes, NJ,http://www.bd.com).

Culture Conditions for Myogenesis, Osteogenesis,Chondrogenesis, Vasculogenesis, and HematopoiesisES cell-derived mesodermal cells purified by FACS were recul-tured in distinctive conditions specific to individual lineages.For myogenesis, sorted cells were cultured on a collagen typeI–coated dish in KnockOut Dulbecco’s modified Eagle’s me-dium (D-MEM; Gibco-BRL) supplemented with 5% horse se-rum and 2 ng/ml insulin-like growth factor-1 [28]. On day 14,expression of myocyte-specific markers was analyzed usingimmunohistochemistry. For osteogenesis, each fraction was re-cultured on a gelatin-coated dish in KnockOut D-MEM supple-mented with 10% FCS, 0.1 �M dexamethasone (Sigma-Aldrich,St. Louis, http://www.sigmaaldrich.com), 50 �M ascorbic-acid-2-phosphate (Sigma), 10 mM �-glycerophosphate (Sigma), and10 ng/ml BMP4 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) [29]. On day 28, Alizarin Red staining wasperformed as previously described [30]. For chondrogenesis, thecells were resuspended at a density of 8 � 106 cells/ml in�-MEM supplemented with 10% FCS, 0.1 �M dexamethasone,and 0.17 mM ascorbic-acid 2-phosphate [18]. Ten microliters ofthis cell suspension was spotted onto a 24-well culture plate andincubated for 30 minutes, after which 1 ml of the mediumcontaining 10 ng/ml transforming growth factor-�3 (R&D) wasadded to each well [18]. On day 7, the medium was replacedwith a medium containing 10 ng/ml BMP2 (R&D). On day 21,Alcian Blue staining was performed as previously described[30]. For vasculogenesis, 500 cells purified using a cell sorterwere recultured onto a confluent OP9 cell layer on each well ofa 24-well plate with �-MEM, 10% FCS, and 50 �M 2ME [8].Two days later, VECD was immunohistochemically stained byanti-VECD antibody, and the number of endothelial colonieswas counted. For hematopoiesis, the cells purified using FACSwere recultured on an OP9 cell layer in �-MEM supplementedwith 10% FCS and 10 ng/ml mouse recombinant erythropoietin

576 Early Reversibility of Mesoderm Differentiation

(R&D). Five days later, floating cells were gently harvested andused to analyze both morphology and gene expression. May-Giemsa staining was carried out for morphological analysis.Benzidine staining for confirming the presence of the erythro-cyte lineage was performed as previously described [31].

Real-Time Reverse Transcription-Polymerase ChainReaction AnalysisTotal RNA was isolated using TRIzol reagent (Invitrogen,Carlsbad, CA, http://www.invitrogen.com) according to the pro-tocol recommended by the manufacturer. Residual genomicDNA was digested and removed using DNase I (Roche Diag-nostics, Basel, Switzerland, http://www.roche-applied-science.com) treatment. First-strand cDNA was synthesized using theSuperscript First-Strand Synthesis System (Invitrogen) for re-verse transcription-polymerase chain reaction (RT-PCR). Weused a mixture of both oligo d(T)12–18 and random hexamers togenerate the first-strand cDNA. Ten microliters of cDNA (total,100 ng) was mixed with a reaction buffer consisting of 2.5 �lprimer mix (2 �M each) and 12.5 �l 2� SYBR Green ReactionMix (Applied Biosystems, Foster City, CA, http://www.applied-biosystems.com). Quantitative RT-PCR was performed usingthe ABI PRISM 7000 system (Applied Biosystems) accordingto the manufacturer’s instructions. We used GAPDH and Ubiq-uitin as the invariant controls. After being normalized by divi-sion to the value of the control, each value of the specific geneswas divided by the maximum value in the experiment forstandardization. The primers for the analyses are shown inSupplemental Table S1.

Immunohistochemical Analyses of Cultured CellsFor histochemical analysis, cultured cells were fixed with 2%paraformaldehyde in phosphate-buffered saline (PBS). Antibod-ies were diluted in 1.5% skim milk and 0.1% Triton X-100 inPBS as follows: rabbit anti-skeletal myosin (Sigma) at 1:500,mouse anti-myogenin (Pharmingen) at 1:500, and rat moAbVECD1 (anti-VECD) at 1:500. horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Biosource, Camarillo, CA, http://www.biosource.com), HRP-conjugated anti-mouse IgG (ZymedLaboratories, San Francisco, http://www.invitrogen.com), andHRP-conjugated anti-rat IgG (Zymed) were used as secondaryantibodies. The substrate used was DAB-Ni for the detection ofsignals as previously described [25].

DNA Microarray Data ProcessingProbe intensity data were taken from Affymetrix CEL files andinternally normalized by subtracting the minimum intensityfound on the array and dividing by the adjusted median probeintensity. Those data were stored in a Postgresql database(http://www.postgresql.org/) and analyzed and visualized usingour own analysis system [32] (eXintegrator, http://www.cdb.riken.jp/scb/documentation/index.html). Individual probe pairdifferences were normalized across an experimental series byadjusting their means and variances to the median values for thatprobe set. Expression values were then estimated by taking themean value of the normalized probe pair differences for a givenexperimental point. Alternatively, we used the affy suite of theBioconductor package (http://www.bioconductor.org) to calcu-late expression values using the robust multi-array average(RMA) method as recommended. As the RMA method produces

expression values in log space these values were first trans-formed to the linear space using ex.

Probe sets with relevant expression patterns were identifiedas described in the text using the tools in the eXintegrator suite.Similarities to specified profiles were calculated as the mean ofEuclidean distances to the set of probe pair profiles in the probesets. Selections were made either by choosing appropriatethresholds based on the distribution of Euclidean distances or byinspecting the raw data for some number of probe sets orderedby their distances. In addition, the reliability of expressionprofiles was gauged by the extent of covariation shown byindividual probe profiles of given probe sets. This was calcu-lated using the anova score for variation between samples asopposed to variation within samples, and thresholds determinedas above for Euclidean distances.

Triangle PlotThe relative expression of genes in the three studied populationswere visualized by calculating positions within an equilateraltriangle formed from the points of three vectors of unit lengthradiating from the origin at 120-degree intervals. In this plot,each vector (0:1, �cos(30):�sin(30), cos(30):�sin(30)) repre-sents expression in one of the populations. The three expressionmeasurements (calculated as described above) were first trans-formed to have a sum of one. If any of the expression measure-ments was negative, then this value was subtracted from all themeasurements for that probe set resulting in a minimum value of0. The resulting expression measurements were then mappedonto their respective vectors and the vector sums were calcu-lated. This results in a set of points within an equilateral triangle,in which genes expressed in only one sample are located on oneof the three points of the triangle, genes expressed in only twosamples locate on the edges, and genes expressed equally in allthree samples are located in the center of the triangle. Thetriangle was arbitrarily divided into seven sections representingthe seven possible binary combinations, and genes were classi-fied by which area they fell into. Software implementing thismapping is available on request.

RESULTS

The Diverging Point to Paraxial and LateralMesodermal Cells in ES Cell Differentiation CultureIn our previous study, we proposed that ECs are derived fromthe VEGFR-2� population in ES cell culture [7]. However, therelationship between the DP population and the VSP populationthat are present together was left undetermined. Moreover, thefate of the PSP population, in particular, its potential to give riseto ECs, was not addressed. We thus wanted to complete a mapof the differentiation pathway of the VSP and PSP populationsin relation to the DP population.

First, we investigated the time course of generation of theDP, VSP, and PSP populations (Fig. 1). Differentiation of CCEES cells was induced according to the method previously de-scribed [7] and the proportions of the DP, VSP, and PSPfractions were assessed by FACS on various culture days. Underour culture conditions, the DP and PSP populations appearedalmost simultaneously on day 3. The proportion of the DPfraction increased to a peak on day 4, then rapidly decreasedover the next 2 days, whereas the VSP and PSP fractions

577Sakurai, Era, Jakt et al.

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reached their peaks later. The peak of each fraction was reachedsuccessively: DP, VSP, and PSP on culture days 4, 5, and 6,respectively. Along with this process, ECD expression progres-sively decreased.

In Vitro Fate AnalysisWe next attempted to determine whether the fate of each popula-tion was consistent with our hypothesis that the VSP and PSPpopulations represent lateral and paraxial mesoderm, respectively.

The DP, PSP, and VSP fractions were purified using FACS(Supplemental Fig. S1) and cultured under conditions for inducingmyocytes, osteocytes, and chondrocytes, as well as conditions forECs and hematopoietic cells. In contrast to our expectations, allfractions produced descendants of paraxial mesodermal lineages,such as myocytes, osteocytes, and chondrocytes, when they werecultured under conditions suited for the induction of these lineages(Fig. 2A–D). Although not based upon a quantitative assay, we hadthe impression that more osteocytes and myocytes were present inDP and PSP cultures than in VSP cultures. To confirm this im-pression, we performed quantitative RT-PCR analyses [33] oflineage markers for myocytes [34], osteocytes, and chondrocytes[35], derived from paraxial mesoderm (Fig. 2E–G). In agreementwith our impression, the highest expression level of markers forlineages derived from somites was observed in PSP cultures,whereas medium and low expression levels of these markers weredetected in DP cultures.

Similar to the osteochondrogenic and myogenic results, ECsthat we presumed to be derived from the lateral mesoderm wereinduced from all three aforementioned fractions, but not fromthe ECD�PDGFR-��VEGFR-2� double-negative (DN) popu-lation (Fig. 2H).

To assess the EC potential of each fraction in a morequantitative manner, we measured the frequency of endothelialprogenitors in each fraction (Fig. 2I). Using this method, the DPand VSP fractions contain nearly the same number of ECprogenitors, whereas the PSP fraction contained one fourthfewer EC progenitors than the other two fractions. In order toexamine hematopoietic potential, we harvested floating cells forcytological analysis and RT-PCR measurement of the expres-sion level of �H1 embryonic-type globin (Fig. 2J, K). Althoughmature hematopoietic cells including both erythrocytes andmegakaryocytes were observed in the cultures of all fractions,the VSP fraction had a higher potential to generate maturehematopoietic cells than the other fractions. Likewise, expres-sion of �H1 was detected in all fractions, whereas the VSPfraction exhibited the highest expression level among them.

These results suggest that the VSP and PSP populationsrepresent the lateral and paraxial mesodermal lineages, respec-tively, and that the differentiation potential of the DP populationis inherently different from those of the PSP and VSP popula-tions, though the functional capacity for generating EC coloniesis similar to that of the VSP population. However, the fate ofeach population has not yet been fully determined.

Gene-Expression Profiles During Differentiation ofES Cell–Derived MesodermIn order to further characterize the DP, PSP, and VSP popula-tions, in a comprehensive manner, we examined their geneexpression using the Affymetrix DNA microarray [32]. In orderto visualize the expression of a number of genes in the threepopulations, we used a simple algorithm that converts the rela-tive expression levels of genes into coordinates within a triangle.To obtain sensible data, it is necessary to select sets of genes thatare expressed in at least one of the three cell types, but are notgenerally expressed. This can be achieved in many differentways, using both arbitrary thresholds for parameters that arerelated to these properties and semiautomated methods. Thegenes displayed in Figure 3A were selected by searching forgenes that are selectively expressed in at least one of the threepopulations studied, compared to ES cells. The raw data fromthese lists were then inspected, and probe sets with high-qualityexpression data (as evidenced by covariation in individual probepair profiles) were selected by manual inspection. In order toestablish that this did not result in a bias in the overall pattern,we also selected probe sets using several different automatedmethods as well as using a different method of calculatingexpression values. Although these methods do not select exactlythe same set of genes, and the calculated coordinates are notidentical, they all essentially display the same overall pattern asthe semiautomated method used here (data not shown).

In Figure 3A, the position close to the apex of the triangleindicates expression specifically in the DP population and theposition close to the left point indicates expression exclusivelyin the PSP population. The expression data reveal that almost allthe genes expressed in the DP population are coexpressed ineither the VSP or PSP population, although the DP-specific

Figure 1. Mesodermal differentiation in in vitro embryonic stem (ES)cell differentiation. CCE ES cells were cultured on collagen type IV–coated dishes with differentiation medium in the absence of leukemiainhibitory factor. Three to six days after induction, differentiated EScells were harvested and the expression of E-cadherin (ECD), vascularendothelial growth factor receptor 2 (VEGFR-2), and platelet-derivedgrowth factor receptor � (PDGFR-�) were investigated using fluores-cence-activated cell sorting. A few ECDlowPDGFR-��VEGFR-2�

(PDGFR-� single-positive [PSP]) and ECD�PDGFR-��VEGFR-2�

(PDGFR-� and VEGFR-2 double-positive [DP]) colonies appearedon days 3–3.5. ECD�PDGFR-��VEGFR-2� (VEGFR-2 single-pos-itive [VSP]) colonies appeared one half day later than the DP andPSP populations. The DP fraction increased to a peak on day 4 andthen dramatically decreased on day 5. The VSP fraction increased toa peak on day 5. The PSP fraction increased over time at least untilday 6. Almost all differentiated ES cells expressed ECD on day 3,and the percentage of ECD� cells gradually decreased as differen-tiation progressed.


genes, which distribute in the apex, are few. Moreover, theVSP-specific group consists of genes such as VECD, Flt4,GATA2, Tal1, and Ikaros that are known to be expressed indifferentiated ECs or hematopoietic cells [36–40], whereas thePSP-specific group contains genes such as Tbx6, Mesp2, Fol-listatin (Fst), Fgf8, and Dll1, which are well known to beexpressed in paraxial mesoderm and somites [41–45] (right andleft corners of Fig. 3A and Table 1). This result suggests that thegenes that are expressed exclusively in either the VSP or PSPpopulations display the prospective fate of the two populations.An almost complete lack of genes that are expressed equally inboth the PSP and VSP populations indicates that the VSP andPSP are fully separated.

To confirm the results of the DNA microarray analysis, wecompared them with the results of a quantitative (Q)-PCRanalysis (Fig. 3B). From microarray data, we selected GATA2,VECD, and Tal1, which are known to be expressed in bloodcells and ECs [46, 47], for lineage markers of lateral mesoderm.

Figure 2. Fate of the embryonic stem (ES) cell–derived mesodermalcells in vitro to paraxial and lateral mesodermal descendants. All threepopulations—the platelet-derived growth factor receptor � (PDGFR-�)single-positive (PSP) population, the vascular endothelial growth factorreceptor 2 (VEGFR-2) single-positive (VSP) population, and thePDGFR-� and VEGFR-2 double-positive (DP) population—showed theability to differentiate into myocytes, osteocytes, and chondrocytesunder specific conditions (A–D). (A, B): Myogenic potential of meso-dermal subsets. Myogenin-positive (A) and skeletal myosin–positive(B) cells (dark brown) were derived from all mesodermal populations.(C): Osteocytic differentiation of ES cell–derived mesoderm. Alizarinred–positive calcium matrixes, shown as orange-colored areas, were

detected in cultures derived from all mesodermal populations. (D):Chondrogenic potential of the DP, PSP, and VSP populations. To detectthe sulfated glycosaminoglycans that are one of the major componentsof chondrocytes, Alcian Blue staining was performed. Positive chon-drocytes expressing a blue color were generated from the three EScell–derived mesodermal populations. (E–G): The expression profile ofmyogenesis-related (E), osteogenesis-related (F), and chondrogenesis-related genes (G) in the progeny of ES cell–derived mesoderm. Thethree populations were cultured under distinct conditions that allow thedifferentiation of myocytes, osteocytes, or chondrocytes. After differ-entiation, RNA was purified and the expression levels of the individualspecific markers were measured using quantitative reverse transcription-polymerase chain reaction (RT-PCR). (E): Culture cells derived fromthe PSP fraction showed the highest expression levels of myogenesis-related genes, such as Myf5, MyoD, and myogenin. (F, G): Culturedcells derived from the PSP and DP fractions also expressed osteogen-esis-related (Bglap1 and Bglap2) and chondrogenesis-related (col2a1and col10a1) genes at higher levels than those from the VSP population.(H–K): The DP, PSP, and VSP populations bear the ability to differ-entiate into vascular endothelial cells and hematopoietic cells. (H):Colony assay of endothelial cells (ECs). Five hundred sorted cells werecultured on a confluent OP9 cell layer for 3 days. EC colonies werevisualized using VE-cadherin (VECD) immunostaining. The left panelin each figure shows colonies of ECs (arrow heads). VECD� colonieswere present in all cultures of the three mesodermal fractions. The rightpanel in each figure displays a high-magnification view of a single ECcolony. (I): Number of EC colonies derived from different mesodermalpopulations. ES cell–derived mesodermal cells were cultured as de-scribed in (H). The number of VECD� colonies was counted in eachwell of the 24-well plates. (Error bars represent standard deviation.) Thefrequency of the DP fraction is almost the same as that of the VSPfraction. The frequency of endothelial progenitors in the PSP fractionwas one fourth of those of the DP and the VSP fractions. (J): Hema-topoietic cell differentiation of each fraction. Sorted cells (2.0 � 105)were cultured on a confluent OP9 cell layer in six-well plates for 5 dayswith erythropoietin. The morphology of floating cells was shown usingGiemsa staining (large panels). (arrow head, erythroblast; arrow, eryth-rocyte; red arrow head, megakaryocyte.) The presence of erythroid cellswas confirmed using Benzidine staining (blue cells in inset panels). (K):Expression level of �H1 in the culture of each sorted fraction. Theexpression level of �H1 was measured by quantitative RT-PCR andnormalized by GAPDH expression level. While �H1 was detected in allfractions, except for the ECD�PDGFR-��VEGFR-2� double-negativepopulation, VSP cultures exhibited the highest expression of �H1. Scalebars: 500 �m (C); 100 �m (A, B, D, H right); 5 mm (H left); 20 �m(J).


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Likewise, Tbx6, Fst, and Mesp2 were selected as markers forparaxial mesoderm because they are widely accepted to beexpressed in the paraxial mesoderm, presomitic mesoderm, andsomites [42, 43, 48]. Indeed, the three genes isolated from themicroarray data of the VSP fraction are selectively expressed inthe VSP fraction using Q-PCR analysis (Fig. 3B). On the otherhand, Tbx6, Fst, and Mesp2 were also detectable in the DP andVSP fractions, at lower levels than in the PSP fraction (Fig. 3B).This is not a result of contamination of unwanted fractions in theFACS-purified samples because the expression levels ofPDGFR-� and VEGFR-2 mRNA, respectively, in the FACS-sorted VSP and PSP fractions were negligible (Fig. 3B). Actu-ally, Tbx6 and Fst are also known to be expressed in nascentmesoderm in the primitive streak during mouse embryogenesis[41, 43], and a weak expression of Mesp2 has been shown formost mesodermal cells during early embryogenesis [42]. Weconsider that this discrepancy is a result of the difference indetection sensitivity between the DNA microarray and Q-PCR.All these results indicate that: (a) the microarray data are con-sistent with the Q-PCR data and (b) the lateral and paraxialmesodermal markers are specifically expressed in the VSP andPSP populations, respectively.

Evidence for Interlineage Conversion as theUnderlying Mechanism for Phenotype SwitchingBetween VSP and PSPGene-expression profiling suggests that the PSP and VSP frac-tions represent distinctive populations corresponding to theparaxial and lateral mesoderm, respectively. However, our invitro fate analysis suggests that their fates are not fully re-stricted. Given that both the VSP and PSP fractions generated inday 4 ES cell culture maintain some flexibility in their fate, it isexpected that they first undergo a switch in surface phenotypefrom VSP to PSP, or vice versa, before completing an irrevers-ible fate determination.

Because an OP9 cell layer can support clonogenic prolifer-ation of single EC progenitors [8], we used this to investigatewhether switching of surface phenotype between VSP and PSPcan occur. Because this experimental setting requires a methodfor distinguishing inoculated cells from OP9 cells, we preparedthe ES cell lines harboring eGFP cDNA driven by chicken�-actin promoter (GFP-ES).

GFP-ES cells were cultured for 4 days to induce mesodermcells, and the DP, PSP, and VSP fractions were purified andrecultured with OP9 stromal cells. After additional incubation for24 hours, the cells were harvested and the surface expression ofVEGFR-2 and PDGFR-� in the GFP� population was analyzed(Fig. 4A). Under this condition, the DP culture produced equalnumbers of VSP and PSP cells, confirming that the PSP and VSPpopulations diverge from the DP population. Interestingly, a highlevel of phenotype switching in both directions was observedbetween the VSP and PSP cultures within 24 hours (Fig. 4A). Thepurified PSP culture at day 4 produced 44% PSP cells and 20%VSP cells, and cells the VSP culture differentiated into 12% PSP

Figure 3. Gene-expression profile of embryonic stem (ES) cell–de-rived mesoderm. (A): DNA microarray analysis of ES cell–derivedmesoderm. The position close to the apex of the triangle indicatesexpression only in the platelet-derived growth factor receptor �(PDGFR-�) and vascular endothelial growth factor receptor 2(VEGFR-2) double-positive (DP) population (light blue dots; group 1),and the positions close to the left point and to the right point indicateexpression only in the PDGFR-� single-positive (PSP) population(black dots; group 5) and the VEGFR-2 single-positive (VSP) popula-tion (green dots; group 7), respectively. The center area of the triangleindicates expression in all three mesodermal populations (red dots;group 3). The left middle trapezoidal area and the right middle trape-zoidal area indicate expression in both the DP and PSP populations(light green dots; Group 2) and in both the DP and VSP populations(purple dots; Group 4), respectively. The bottom trapezoidal area indi-cates expression in both the PSP and VSP populations (blue dots; group6). Some sample genes located in the PSP-specific area (black dotsnumbered 1–5) and in the VSP-specific area (green dots numbered6–10) are shown in Table 1. The total number of genes in each area isindicated in brackets. All the gene names are shown in SupplementalFigure S2 and Supplemental Table S2. (B): Gene-expression profile oflineage-specific markers in ES cell–derived mesoderm. Each mesodermfraction was isolated and purified by fluorescence-activated cell sortingon day 4. The expression levels of lineage-specific markers were inves-tigated using quantitative reverse transcription-polymerase chain reac-tion (error bars indicate standard deviation; n � 3). Vascular endothelialgrowth factor receptor 2 (VEGFR-2) and platelet-derived growth factorreceptor � (PDGFR-�) expression levels in the three mesodermal pop-ulations indicate that the purities of these samples were reliable enough

for quantitative analyses. Paraxial mesodermal markers, such as Tbx6,Mesp2, and Fst, were highly expressed in the PSP fraction. In contrast,lateral and extraembryonic mesodermal markers, such as GATA2 andTal1, were selectively expressed in the VSP fraction.


cells and 51% VSP cells. It should be noted that DP cells could notbe detected in cultures of any population, including the DP cultureitself. Thus, the phenotypic switch between VSP and PSP occursdirectly rather than via an immature DP stage. The same switch

between VSP and PSP was also observed in the cultured TT2 EScell line (data not shown).

In order to determine whether the switch in surface pheno-type reflects a simple fluctuation in the expression of VEGFR-2

Figure 4. Lineage conversion of embryonic stem (ES) cell–derivedmesodermal populations. A: Surface marker analyses of purified ES cell–derived mesodermal populations after additional incubation for 24 hours ona confluent OP9 cell layer. The platelet-derived growth factor receptor �(PDGFR-�) and vascular endothelial growth factor receptor 2 (VEGFR-2)double-positive (DP), PDGFR-� single-positive (PSP), and VEGFR-2 sin-gle-positive (VSP) populations were derived from green fluorescent pro-tein (GFP)-ES cells to allow them to be distinguished from OP9 feedercells using GFP expression. All three mesodermal populations retained theability to differentiate into both PSP and VSP cells. (B): The gene-expression patterns of the PSP and the VSP populations derived from day4 VSP and PSP cultures, respectively, were similar to those of the day 4PSP and VSP populations. RNA was isolated from day 4 ES cell–derivedmesoderm and mesodermal cells were recultured on an OP9 cell layer forone day. Gene-expression levels of specific markers were measured usingquantitative reverse transcription-polymerase chain reaction. The lateraland extraembryonic mesodermal markers, such as GATA2 and Tal1, wereexpressed in both the day 4 VSP population and the day 4 PSP culture–derived VSP population. Similarly, the day 4 PSP population and the PSPpopulation derived from the day 4 VSP population showed dominantexpression of paraxial markers, such as Tbx6 and Mesp2. (C): Vasculo-genic potentials of cells generated in secondary cultures. Day 4 mesoder-mal fractions were recultured on an OP9 cell layer, and the recultured PSP,VSP, and DP fractions were purified again using fluorescence-activatedcell sorting (FACS). One thousand cells of each population were culturedon an OP9 cell layer in one well of a 24-well plate to induce ECdifferentiation. The mean and standard deviation of endothelial colonynumber visualized by VE-cadherin (VECD) staining are shown in (C).VECD� colonies were observed in the VSP population derived from everymesodermal subset, but they were not observed in the other two popula-tions. (D): Hematopoietic potentials of cells generated in secondary cul-tures. Day 4 mesodermal fractions were recultured on an OP9 cell layer,and the PSP and VSP fractions were isolated using FACS. Cells (2.0 �105) from each population were cultured on an OP9 cell layer in one wellof six-well plate to induce hematopoietic differentiation. Four days afterinduction, floating cells were harvested and counted (upper panel). Thesame cells were subjected to quantitative RT-PCR analysis for �H1.Expression was observed exclusively in the VSP fraction, irrespective of itsorigin. (E): Osteogenic potential of lineage-converted populations. Eachlineage-converted population was recultured under conditions favoringosteocytic differentiation for 28 days. Progeny of the PSP cells derivedfrom the DP population and the VSP population had widely spread calciummatrixes (red area in upper left and lower right panels, Alizarin redstaining). In contrast, little calcium deposition was observed in the cellsdifferentiated from the VSP population. Scale bar � 500 �m.


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and PDGFR-� or a more global process involving other mole-cules, we analyzed the expression of the six aforementionedmarkers (Fig. 3B) that are expressed in either paraxial or lateralmesoderm cells (Fig. 4B). PSP and VSP cells were purified andrecultured on an OP9 stromal cell layer. After additional incu-bation for 24 hours, newly generated PSP and VSP cells werepurified again from the PSP and VSP cultures, respectively. Theexpression levels of the six genes in each population wereassessed by quantitative RT-PCR. During the phenotype switchbetween VSP and PSP, the expression profile of these markergenes changed completely from one type to the other (Fig. 4B).Thus, the phenotype switch between VSP and PSP is not theresult of a simple fluctuation in the expression levels ofVEGFR-2 and PDGFR-�.

In order to further determine whether the phenotype switchbetween VSP and PSP is accompanied by an actual switch infate, we evaluated the in vitro differentiation potential of eachfraction in the secondary culture. DP, VSP, and PSP cells wereprepared from ES cell differentiation cultures at day 4 andincubated on an OP9 feeder layer for 24 hours. Each PSP andVSP fraction was separately purified from the secondary cul-tures and cultured again on either an OP9 feeder layer, tomeasure the frequency of endothelial progenitors and the ex-pression level of embryonic-type globin, or under osteogenesis-inducing culture conditions. Regardless of the initial phenotypeof the cells in the secondary culture, only the VSP fraction could

Figure 5. Commitment point from embryonic stem (ES) cell–derivedmesoderm to specific lineage progenitors. A: Differentiation into plate-let-derived growth factor receptor � (PDGFR-�) single-positive (PSP)cells and vascular endothelial growth factor receptor 2 (VEGFR-2)single-positive (VSP) cells at various time points of in vitro ES cellculture. The PDGFR-� and VEGFR-2 double-positive (DP), PSP, andVSP fractions were purified from differentiated green fluorescent pro-tein (GFP)-ES cells on day 3.5, day 5, and day 6 of culture and wererecultured on a confluent OP9 cell layer. One day later, the expressionof surface markers in the GFP� population was analyzed by fluores-cence-activated cell sorting (FACS). Although the DP fraction de-creased dramatically from day 4 to day 6 (Fig. 1), the DP populationretained its ability to give rise to both PSP and VSP cells even on day6 (left column). The VSP population also retained its ability to differ-entiate into PSP cells through the time course of the experiment (rightcolumn). In contrast to these two populations, the PSP populationshowed a dramatic reduction in conversion potency through the pro-gression of differentiation, and cells in this population could no longerconvert to the VSP phenotype on day 6 (center column). (B): Lack ofsecondary switching capability in the population generated by lineageswitching. Day 4 mesodermal fractions were recultured on an OP9 celllayer, and the lineage-switched populations—the VSP fraction derivedfrom PSP cultures and the PSP fraction derived from VSP cultures—were purified again using FACS. Each switched population was recul-tured again on an OP9 cell layer for 24 hours. After secondary culture,the expression levels of PDGFR-� and VEGFR-2 were analyzed. TheVSP cells derived from the PSP fraction could no longer convert to thePSP phenotype (i). Similarly, the PSP cells derived from the VSPfraction could not convert to the VSP phenotype (ii). (C): VE-cadherin(VECD) expression in the VSP population implies a commitment to theendothelial lineage. i: Expression pattern of VECD in the VSP popula-tion on day 4. The VSP population contains both VECD� (10%) andVECD� (90%) cells. Each subpopulation was purified and recultured onan OP9 cell layer for 24 hours. ii: Expression of PDGFR-� andVEGFR-2 in GFP� cells after reculture. The VECD� subpopulation hadlost the potential for lineage conversion, whereas the VECD� VSPfraction could give rise to PSP cells.


produce ECs (Fig. 4C). In order to confirm that the VSP cellsderived from the PSP cultures still maintained the characteristicsof lateral mesoderm, we assessed their erythropoietic potential.As shown in Figure 4D, erythropoiesis was observed only in theVSP culture, irrespective of its origin. Similarly, osteogenesiswas only induced in the PSP culture, regardless of the phenotypeof the initial population (Fig. 4E).

Time- and Stage-Dependent Restriction ofInterlineage Conversion CapabilityIn order to determine how long the capability of undergoing aphenotype switch is maintained in each fraction, we examinedphenotypic changes in VSP and PSP cultures until day 6 (Fig.5A). The incidence of phenotype switching from PSP to VSPdecreased remarkably with a longer incubation time. On day 6,the potential for switching from PSP to VSP was almost com-pletely lost. On the other hand, the potential for VSP cultures togive rise to PSP cells was preserved for longer.

We also compared the potential to undergo a phenotypeswitch in VSP and PSP cells derived directly from ES cells withthose generated by the phenotype switches. As shown in Figure5B, this switch potential was detected only in primary VSP andPSP cultures derived directly from ES cells and not in thosegenerated in the secondary culture.

As some of the VSP cells differentiate into VECD� ECsduring culture, we investigated whether or not the potential toswitch the surface phenotype to PSP is maintained after com-pleting EC differentiation (Fig. 5C). The VSP population wasdivided into VECD� VSP and VECD� VSP fractions. TheVECD� VSP population could no longer produce PSP cells,whereas this potential was maintained in the VECD� population(Fig. 5C). These results suggest that the potential for phenotypicswitching depends on the time and the stage of cell culture.

DISCUSSIONThe aim of this study was to dissect the early process ofdivergence of the EC differentiation pathway away from othermesodermal lineages. Combining PDGFR-�, as a marker for theparaxial mesodermal lineage, with VEGFR-2, as a marker forthe lateral mesodermal lineage, we have found multiple ECdifferentiation pathways in ES cell development.

An in vitro ES cell differentiation system is advantageous toanalyze the process of cell specification during early embryo-genesis, particularly when obtaining enough cells from the ac-tual embryo is difficult. Conversely, the lack of positionalinformation that is available for specifying cell types in theembryo is a disadvantage of ES cell culture. Consequently, for

analyzing the divergence point between lateral mesoderm andparaxial mesoderm in vitro, it is necessary to redefine the twolineages according to their individual features. Because ourprevious study in gastrulating embryos demonstrated thatVEGFR-2 and PDGFR-� are expressed in the lateral and ex-traembryonic mesoderm and the paraxial mesoderm, respec-tively [13], we speculated that surface expression of PDGFR-�and VEGFR-2 could define these mesodermal lineages in EScell culture. Differentiated ES cells that expressed VEGFR-2and PDGFR-� were divided into three populations—DP, VSP,and PSP. In the present study, we characterized these threepopulations in a comprehensive way using a DNA microarray.To track the dynamic change in gene-expression profiles duringdiversification, we developed a new method that can display thegenes according to their expression levels in three populations.Compared with the most popular method, which simply classi-fies genes into groups, in our method, each dot in Figure 3Acorresponds to a value reflecting the relative specificity of theexpression of a given gene among the three populations. Usingthis new method, the current study demonstrates two importantfindings. First, we found two groups of genes that are expressedexclusively by either the VSP or PSP population. The VSP-specific group consists of genes that are known to be expressedin differentiated ECs or blood cells, whereas the PSP-specificgroup contains genes that are known to be expressed in somites.Of note is that the expression of only a few genes overlaps inthese two populations, though many more genes are found to beexpressed in all three populations. Thus, the VSP and PSPpopulations are likely to represent fully segregated populations.Second, many genes expressed in the DP population are coex-pressed by either the VSP or PSP population. In contrast,DP-specific genes are few. This distribution pattern of genes fitswell into a model of lineage divergence in which a commonprogenitor population, that is, the DP population, separates intotwo more mature stages, PSP and VSP.

Based on the results of the DNA microarray analysis, wehypothesized that the DP population is located at the divergencepoint from which PSP paraxial and VSP lateral mesoderm aregenerated. The results of in vitro fate analyses demonstrated thatDP cells have the potential to differentiate into the lateralmesodermal lineage (ECs) and the paraxial mesodermal lineage(myocytes, osteocytes, and chondrocytes). DP cells can directlygive rise to both VSP and PSP cells after short-term reculture onan OP9 feeder layer. Taken together, our results lead us toconcluded that the VSP and PSP populations diverge from thecommon DP progenitor population, as illustrated in Figure 6.

Table 1. Genes indicated with a number in Figure 3A

No. Gene symbol Affymetrix ID No. Gene symbol Affymetrix ID

PSP-specific group VSP-specific group

1 Tbx6 93611_at 6 VE-cadherin 104083_at2 Fgf8 97742_s_at 7 Flt4 104417_at3 Fst 98817_at 8 Tal1 97973_at4 Dll1 92931_at 9 GATA2 102789_at5 MESP2 97092_at 10 Ikaros 112792_

Abbreviations: PSP, platelet-derived growth factor receptor � single-positive; VSP, vascular endothelial growth factor receptor 2 single-positive.


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Our results also revealed that the VSP and PSP populationscorrespond to the lateral and paraxial mesodermal lineages,respectively. In vitro fate analyses revealed that the VSP pop-ulation has a greater capacity to produce ECs and hematopoieticcells than does the PSP population. Likewise, the PSP popula-tion has higher myogenic, osteogenic, and chondrogenic poten-tials than does the VSP population. The genes expressed exclu-sively by the VSP population contain markers for ECs andhematopoietic cells. Similarly, the genes that are specificallyexpressed by the PSP population contain the genes expressed inembryonic somites and paraxial mesoderm.

Though the expression of VEGFR-2 and PDGFR-� candefine the lateral and paraxial mesodermal lineages, respec-tively, in ES cell culture, we also demonstrated that VSP andPSP cells can give rise to progeny of the other lineage. Thisunexpected result is due to lineage switching between VSP andPSP cells. Our result shows that ECs are generated from notonly DP and VSP cells but also from PSP cells. It is remarkablethat the frequency of endothelial progenitors in the PSP fractionharvested from day 4 ES cell differentiation cultures is as highas one fourth of those in the VSP fraction from the same culture.Similarly, long-term in vitro fate analyses have revealed thatmyocytes, osteocytes, and chondrocytes, which are progeny ofparaxial mesoderm, are generated from VSP cells. With respectto the potential of the PSP population to give rise to ECs, it hasalready been reported that some committed somitic cells cangive rise to ECs [49, 50]. However, what we describe here is thetransition of PSP cells to multipotent VSP cells that can give riseto not only ECs but also to hematopoietic cells. Considering thatsomitic cells cannot give rise to hematopoietic cells, it is likelythat VSP cells derived from PSP cells correspond to lateralmesoderm.

It is generally considered that the decision of fate is such anambiguous process that the selected fate is reversible for a whileafter lineage diversion [51, 52]. Even so, it is remarkable that aphenotype switch of such magnitude—20% of the cells gener-ated in the PSP culture were VSP cells and 10% of the cellsgenerated in the VSP culture were PSP cells—occurs within 24hours. Because our culture conditions allow phenotype switch inboth directions between the VSP and PSP populations, it isunlikely that this is a result of selectivity of the culture condi-tions to expand a particular population. Thus, a considerableproportion of nascent VSP and PSP cells maintain flexibility intheir fate.

What then underlies this phenotype switch between PSP andVSP? The first possibility is that the switch is restricted to theexpression of VEGFR-2 and PDGFR-�, while the actual fateremains unaffected. Our result suggests that this is unlikely.VSP populations derived from PSP cultures contain nearly thesame number of EC progenitors as those derived from VSPcultures, or DP populations derived directly from ES cells.Likewise, PSP populations derived from DP and VSP culturesdo give rise to progeny of the paraxial mesoderm, such asosteocytes. Therefore, the switch in surface phenotype betweenPSP and VSP is associated with a switch in prospective fate. Inaccordance with the fate analysis, along with the switch fromPSP to VSP, the expression pattern of six markers for paraxialand lateral mesoderm shifted from one type to the other. Theseresults suggest that phenotype switching between PSP and VSPindeed represents a lineage conversion between lateral andparaxial mesodermal cells. Our results also suggest that thislineage conversion is not a result of dedifferentiation, becausethe DP population, which is located at the divergence point ofthe PSP and VSP populations in the normal differentiationcourse, was hardly detected during the process of phenotypeswitch in culture. Thus, what is being observed here is a directlineage conversion between VSP and PSP. Our findings suggestthat in the actual embryo, this lineage conversion may occurspontaneously after commitment of the lateral and paraxialmesoderm. Further investigation is necessary to resolve thisquestion.

Figure 6. Differentiation process of mesoderm in in vitro an embry-onic stem (ES) cell differentiation system. The analyses of differentiatedES cells revealed three types of mesodermal cells—an E-cadherin(ECD)�/platelet-derived growth factor receptor � (PDGFR-�)�/vascu-lar endothelial growth factor receptor 2 (VEGFR-2)� population (thePDGFR-� and VEGFR-2 double-positive [DP] population, green), anECDlowPDGFR-��VEGFR-2� population (the PDGFR-� single-posi-tive population [PSP] population, dark blue), and an ECD�PDGFR-��VEGFR-2� population (the VEGFR-2 single-positive [VSP] popu-lation, yellow). The DP population is the most immature and can giverise to both PSP and VSP cells. Although both the VSP and the PSPpopulations exhibit the specific properties of paraxial and lateral meso-derm, respectively, these populations can be converted into each other atan early stage. This conversion is dependent on the time course of theculture and the stage of the mesodermal cells. The VE-cadherin–positivesubpopulation, which is illustrated as though forming a drop that isderived from, but discontinuous with, the VSP population, has lost thispotential and has irreversibly committed to the endothelial lineage.


We have demonstrated that the ability to undergo lineageconversion is restricted to an early stage of mesodermal celldifferentiation. The capacity to undergo lineage conversion isquickly lost over time, as the PSP fraction harvested from day 6ES cell differentiation cultures is irreversibly committed to aparaxial mesoderm lineage. The ability to undergo a phenotypeswitch is maintained longer in the VSP population, but it iscompletely lost when VECD is expressed on the cell surfaceupon differentiation to ECs. These results suggest that the lin-eage conversion observed in this study is a phenomenon inher-ent to early mesodermal cells and may not be relevant to theplasticity of fully differentiated cells.

Taking our new results into account, we corrected our pre-vious model of EC differentiation to the one illustrated in Figure6. In this scheme, the DP population is placed at the divergencepoint of the VSP and PSP populations. While VSP and PSP cellsrepresent fully diverged populations in terms of their gene-expression profiles, some of them maintain the ability to un-

dergo interlineage conversion for a short period of time afterdivergence to VSP and PSP cells. Concerning EC differentiationpathways, ECs are generated from not only the DP populationbut also from the PSP population via the VSP population, andsurface expression of VECD coincides with the timing of irre-versible commitment.

ACKNOWLEDGMENTSWe thank Dr. K. Nakao for a kind gift of the TT2 ES cells. Wealso thank Dr. H. Yoshida and Dr. H. Kurata for technicalsupport. This work was supported by grants from the Ministry ofEducation and Science of Japan (No. 12219209 to N.S. and No.16606005 and No. 17045039 to E.T.; and the Project for Real-ization of Regenerative Medicine to N.S.).

DISCLOSURESThe authors indicate no potential conflicts of interest.

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In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility