doi:10.1182/blood-2007-06-093609 Prepublished online Nov 21, 2007;2008 111: 1876-1884

Elefanty and Edouard G. Stanley Richard P. Davis, Elizabeth S. Ng, Magdaline Costa, Anna K. Mossman, Koula Sourris, Andrew G.

isolation of primitive hematopoietic precursorsstem cells identifies human primitive streak like cells and enables

locus of human embryonicMIXL1Targeting a GFP reporter gene to the

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HEMATOPOIESIS

Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cellsidentifies human primitive streak–like cells and enables isolation of primitivehematopoietic precursorsRichard P. Davis,1 Elizabeth S. Ng,1 Magdaline Costa,1 Anna K. Mossman,1 Koula Sourris,1 Andrew G. Elefanty,1 andEdouard G. Stanley1

1Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Australia

Differentiating human embryonic stemcells (HESCs) represent an experimentalplatform for establishing the relation-ships between the earliest lineages thatemerge during human development. Herewe report the targeted insertion in HESCsof sequences encoding green fluorescentprotein (GFP) into the locus of MIXL1, agene transiently expressed in the primi-tive streak during embryogenesis.1,2 GFPfluorescence in MIXL1GFP/w HESCs differ-

entiated in the presence of BMP4 re-ported the expression of MIXL1, permit-ting the identification of viable humanprimitive streak-like cells. The use of GFPas a reporter for MIXL1 combined withcell surface staining for platelet-derivedgrowth factor receptor alpha (PDGFR!)enabled the isolation of a cell populationthat was highly enriched in primitive he-matopoietic precursors, the earliest de-rivatives of the primitive streak. These

experiments demonstrate the utility ofMIXL1GFP/w HESCs for analyzing the previ-ously inaccessible events surroundingthe development of human primitivestreak-like cells and their subsequentcommitment to hematopoiesis. (Blood.2008;111:1876-1884)

© 2008 by The American Society of Hematology

Introduction

In vertebrate species, a prerequisite for the development of theprimary germ layers is the commitment of primitive ectoderm(epiblast) cells to gastrulation.3-6 In mammalian embryos, thisprocess is accompanied by the formation of the primitive streak, amorphologic structure initiating at the prospective embryonicposterior.6-8 In the mouse epiblast, cells ingressing through thestreak emerge as either definitive endoderm or mesoderm, the latterincluding the progenitors of the hematopoietic system.9

In the mouse, primitive streak cells are marked by expression ofthe transcription factor Mixl11,2 and mouse embryos deficient inMixl1 display multiple defects in the formation of mesodermal andendodermal derived structures.10 Consistent with this, more recentstudies have confirmed that Mixl1 expression marks precursors ofboth mesoderm11 and endoderm.12 These latter studies took advan-tage of embryonic stem cells (ESCs) or mice in which one Mixl1allele had been replaced by sequences encoding green fluorescentprotein (GFP), facilitating the identification and isolation of viableGFP! (Mixl1!) primitive streak-like cells. Analysis of Mixl1GFP/w

mouse ESCs showed that a GFP! (Mixl1!) population present atdifferentiation days 3 and 4 contained hematopoietic precursors,11

supporting previous data indicating that, in mouse embryos, suchprecursors arise directly from the primitive streak.13 The majorityof progenitors at this time were hemangioblasts, precursors withboth hematopoietic and endothelial potential which, in the embryo,contribute to the primary vascular plexus and primitive erythropoi-esis of the yolk sac. Thus, these progenitors as well as lineagerestricted primitive erythroid precursors represent the first differen-tiated mesodermal derivatives that arise after the onset of gastrula-tion at embryonic day (E) 6.5.9

Because of the scarcity of examples, events surroundinggastrulation in the human have largely been inferred from compara-tive embryology,8 a situation that has led to uncertainty surround-ing the relationship between the first mesodermal like cells(mesoblasts) documented from postovulation day 13 onward, theappearance of hematopoietic cells, and the overt manifestation ofthe primitive streak, a structure that is first visible in embryosrepresenting embryonic day 15 (E15).8,14-16

Differentiating human embryonic stem cells (HESCs) represent anexperimental platform for dissecting the relationship between specificlineages and the early differentiation events surrounding formation ofthe primary germ layers. To examine the correlation between mesodermformation in the human and the emergence of hematopoietic precursors,we targeted sequences encoding GFP to the MIXL1 locus usinghomologous recombination. We demonstrate that GFP fluorescencefaithfully reported expression of the endogenous MIXL1 gene and that amesodermal cell population defined by coexpression of GFP (MIXL1)and the platelet-derived growth factor receptor alpha (PDGFR") washighly enriched in primitive hematopoietic precursors, the earliestderivatives of the primitive streak.

Methods

Generation and identification of targeted MIXL1GFP/w HESCs

The MIXL1 targeting vector comprised a 9.4 kb 5# homology arm, GFP,loxP flanked PGK-promoter-neomycin resistance gene and a 1.9 kb 3#homology arm. The homology arms were derived from previously de-scribed genomic clones of the human MIXL1 locus2 and spanned sequences

Submitted June 4, 2007; accepted November 3, 2007. Prepublished online as BloodFirst Edition paper, November 21, 2007; DOI 10.1182/blood-2007-06-093609.

The online version of this article contains a data supplement.

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from a PacI site situated 9466 bp 5# of the ATG to an HpaI site located 2242bp 3# of the ATG. The vector was digested with the restriction enzymes PacIand NotI before electroporation into HESCs as described elsewhere.17

HESC clones with a putative targeted MIXL1 allele were identified using apolymerase chain reaction (PCR)-based screening strategy using theprimer, Neo4, in conjunction with MIXL1 ScreenRev (primer b in Figure1A), a primer located immediately 3# of the genomic sequences encom-passed by the targeting vector (Table S1, primer details, available on theBlood website; see the Supplemental Materials link at the top of the onlinearticle). Using this criterion, several clones were identified in which thevector appeared to be correctly integrated into the MIXL1 locus. Twoindependent HES3-derived MIXL1GFPNeoR/w clones (clone 7 and clone 10)were expanded and transiently transfected with a pEFBOS-cre-IRESPurovector using Fugene 6 transfection reagent according to the manufacturer’sinstructions (Roche, Mannheim, Germany). This vector was designed toexpress a single transcript encoding cre recombinase and puromycinresistance, the latter translated from an internal ribosomal entry site (IRES).At 24 to 32 hours after transfection, cells were selected in 2 $g/mLpuromycin for 2 days and subsequently allowed to form colonies for afurther 7 days. Several colonies representing each primary clone werepicked and screened for the loss of the neomycin resistance cassette and forthe absence of the cre expression plasmid using a PCR based approach(Table S1, primer details and PCR conditions). Southern blot analysis wasperformed as described elsewhere.18 The 5# external DNA probe included amixture of fragments corresponding to human genomic sequences flankedby primer pairs listed in Table S1. The GFP probe used to verify the

presence of a single integration event encompassed the coding sequences ofEGFP (Invitrogen). The DNA fragment generated by PCR using the primersGFP1 (primer a in Figure 1A) and MIXL1 3# probe #1 was cloned andsequenced to establish that the 3# arm of the targeting vector had correctlyintegrated into the locus.

Cell culture and differentiation

HESC lines were passaged as reported elsewhere17,19 and differentiated asspin EBs according to previously established protocols.20 Serum freedifferentiation medium (SFM),20 containing recombinant human albuminand 0.05% to 0.25% polyvinylalcohol in some experiments was supple-mented with the following growth factors at the concentrations indicated:10 to 100 ng/mL BMP4, 50 ng/mL Activin A (R&D Systems, Minneapolis,MN), 50 to 100 ng/mL FGF2, 10 to 50 ng/mL VEGF, 20 to 100 ng/mL SCF,30 ng/mL IL3, 30 ng/mL IL-6, 30 ng/mL Tpo, 3 U/mL erythropoietin(PeproTech, Haifa, Israel). EBs were dissociated using either 0.25% w/vTrypsin-EDTA (Invitrogen, Carlsbad, CA) or TrypLE Select (Invitrogen).Preparation and analysis of methylcellulose cultures and cytocentrifugepreparations were conducted according to Ng et al.20 Karyotype analysisand teratoma assays were performed as described previously.19

Flow cytometric analysis

Intracellular flow cytometry with anti-Mixl1 and anti-Oct4 antibodies wasperformed as described previously.21 For analysis and sorting of live cells,HESCs were dissociated to give a single-cell suspension and labeled withantibodies as described previously.20 The antibodies used in this study werephycoerythrin (PE)-conjugated mouse anti–human CD34 (BD Bio-sciences), mouse anti–human E-CADHERIN (Zymed, South San Fran-cisco, CA), mouse anti–human PDGFR" (BD Biosciences), mouse anti–human CD43 (BD Biosciences), PE-conjugated mouse anti–human CD45(BD Biosciences), allophycocyanin (APC)-conjugated mouse anti–humanglycophorin A (BD Biosciences), and mouse anti–human Tra-1-60 (Chemi-con, Temecula, CA). Unconjugated primary antibodies were detected witheither PE or APC-conjugated goat anti–mouse IgG (BD Biosciences). Flowcytometry gates were set using control cells (HES3) and MIXL1GFP/w

HESCs labeled with the appropriate isotype control antibody. Alternatively,gates were set relative to MIXL1GFP/w HESCs differentiated in FGF2, whichdo not express MIXL1. Single-cell cloning was performed using thesingle-cell deposition function of a FACSaria FACS station to placesingle-cells into each well of 10 96-well trays preseeded with irradiatedprimary mouse embryonic fibroblasts (PMEFs) and containing HESCculture media.19 For reaggregation/reculture experiments, cells obtainedfrom flow cytometric sorting were aggregated using the spin EB protocol(104/well), in SFM supplemented with 30 ng/mL BMP4, 30 ng/mL VEGF,and 40 ng/mL SCF.

Immunofluorescence analysis

Dissociated cells from day (d)4 EBs were resuspended in SFM and allowedto adhere to a poly-L-lysine–coated glass slide for 20 minutes. The cellswere fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) andlabeled with the anti-MIXL1 antibody, 6-G2,21 or a rat IgG controlantibody. Primary antibodies were detected with a Texas Red conjugatedgoat anti–rat immunoglobulin G (IgG; Jackson ImmunoResearch, WestGrove, PA) and confocal images were captured using an OlympusFluoView 1000 (Olympus, Tokyo, Japan).

Gene-expression analysis

RNA was prepared using RNAeasy according to manufacturer’s instruc-tions (QIAGEN, Valencia, CA). RNA samples were reverse transcribed andnormalized as described previously.20 PCR was performed under standardconditions (30 cycles of 94°C, 20 seconds; 60°C, 30 seconds; 68°C,60 seconds) using the primer sets listed in Table S2. For PCR with PAX6specific primers, an annealing temperature of 55°C was used. Real-timePCR was performed using Taqman gene expression probes and expressionlevels calculated as described previously.22

Figure 1. Targeting of GFP to the MIXL1 locus in HESCs. (A) Structure of the genetargeting vector used to insert sequences encoding GFP into exon 1 of the MIXL1locus using homologous recombination. PacI and NotI are restriction enzyme sitesused to linearize the vector before electroporation. NeoR is the PGKNeo cassetteencoding G418 resistance, flanked by loxP sites (black triangles). The positions ofMfeI sites used to map the structure of the modified locus are shown, as are theposition of primers (a, b) used to identify correctly targeted clones. (B) Southern blotanalysis of MfeI digested genomic DNA shows that a 5# external probe detects afragment of 17 kb representing the endogenous locus from both parental HES3 cellsand HESCs with a targeted MIXL1 locus. An additional fragment of 14.4 kb is alsodetected in the genetically modified cells, representing the distance from the 5#external MfeI site to the 3# end of GFP. (C) Probing this same DNA with GFPsequences indicates that these cells contain a single copy of the GFP geneconsistent with a single genetic modification at the MIXL1 locus. (D) The integrity ofsequences 3# of the GFP gene was validated using a PCR-based approach (withprimers a and b) to amplify DNA representing the junction of the targeting vector withthe chromosomal DNA. Sequence analysis of this fragment confirmed that therelationship between the vector DNA and adjacent chromosomal sequences were asexpected (data not shown).

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Results

The MIXL1-GFP targeting vector (Figure 1A) was electroporatedinto HESCs and G418 resistant colonies isolated as describedelsewhere.17 Correctly targeted clones were identified using aPCR-based strategy with the primers indicated (Table S1). Afterremoval of the G418 resistance cassette (see “Methods”), thestructural integrity of the targeted locus was verified by Southernblot analysis (Figure 1B,C) and sequencing of the PCR productrepresenting the 3# junction between the vector and flankinggenomic DNA (Figure 1D and data not shown). In addition, oneMIXL1GFP/w HESC line was cloned by single-cell deposition usinga flow cytometer into 96-well trays (cloning efficiency of % 5%).The parental line and subclones were phenotypically indistinguish-able (data not shown). MIXL1GFP/w HESCs had normal karyotypes,formed teratomas, and expressed markers of undifferentiatedHESCs (Figure S1 and data not shown).

To examine the temporal association between expression ofGFP and the endogenous MIXL1 allele, the gene expression profileof MIXL1GFP/w HESCs differentiated in response to BMP4 wasanalyzed over a 12-day period using RT-PCR (Figure 2A). Theexpression profile of GFP transcripts mirrored that of MIXL1, and

as previously reported,20 MIXL1 expression was contemporaneouswith that of BRACHYURY, a transcription factor also present in theprimitive streak. The decline in the level of expression of theseprimitive streak genes between days 6 to 8, overlapped with theexpression of mesodermal (GATA2, CD34) and endodermal(FOXA2, ALPHA FETOPROTEIN, ALBUMIN) genes. This tran-sient wave of MIXL1 expression was consistent with our previousdata demonstrating the kinetics of differentiation using the “spinembryoid body” (spin EB) system in SFM supplemented withBMP4.20 Generally, these kinetics paralleled those reported byothers for BMP4 dependent HESC differentiation in serum freemedia, with transient expression of BRACHYURY and a gradualdiminution in the levels of OCT4.23 Also, in agreement with thestudies of Kennedy et al,23 substantial CD34 expression was notobserved until after day 4 (Figure 2A). As evidenced by the weakexpression of PAX6, BMP4 did not promote neurectodermaldifferentiation.

Immunofluorescence analysis of MIXL1GFP/w HESCs differenti-ated for 4 days in BMP4 revealed a correlation between GFPexpression and MIXLl protein. In the example shown, 5 of 6 intactcells coexpress MIXL1 and GFP (Figure 2B top panels). Specificlocalized staining was not observed in GFP! cells labeled with anisotype control antibody (middle panels), nor in the uniformly

Figure 2. GFP marks MIXL1" cells during the early stages of HESC differentiation. (A) PCR analysis indicates that GFP expression mirrors the wave of expression ofendogenous MIXL1. This analysis also shows the progressive down-regulation of the stem-cell marker, OCT4, the transient expression of the primitive streak genes, MIXL1and BRACHYURY, and activation of genes expressed in endodermal (FOXA2, AFP-alpha fetoprotein, ALBUMIN) and mesodermal (GATA2, CD34) cell types. –RT indicates &reverse transcriptase. This panel is a composite of images for individual ethidium bromide-stained agarose gels for each set of genes. (B) Immunofluorescent images of day 4MIXL1GFP/w HESCs differentiated in SFM in the presence of either 30 ng/mL BMP4 (top and middle panels) or 100 ng/mL FGF2 (bottom panel). The bottom panel shows thatMIXL1GFP/w HESCs differentiated in FGF2 expressed neither GFP nor MIXL1. (C) Sorting and reanalysis experiments examining the relationship between expression of GFPand MIXL1 protein by flow cytometric analysis. The top panel shows the profile of GFP expressing cells in day 4 MIXL1GFP/w EBs. The division between GFP! (red) and GFP&

(black) fractions was based on gates set using MIXL1w/w (HES3) control EBs (inset). The middle panel shows the reanalysis of the sorted populations with the distribution ofGFP! cells and GFP& cells indicated. Endogenous MIXL1 protein (bottom panels), as determined by intracellular flow cytometry with an anti-MIXL1 antibody, is largelyrestricted to GFP! cells and excluded from the GFP& cells. The position of gates for intracellular flow cytometry were set with MIXL1GFP/w day 4 EBs differentiated in SFMcontaining 100 ng/mL FGF2 and stained with the anti-MIXL1 antibody (inset, bottom panel). (D) Graphic representation showing the distribution of MIXL1! cells between theGFP! and GFP& sorted populations from 4 separate experiments using 2 independent MIXL1GFP/w HESC lines (P ' .001). Error bars represent the SEM. Calculations areshown in Table S3A-C. (E) PCR analysis of the GFP-sorted fractions from C showing both GFP and MIXL1 transcripts are essentially restricted to the GFP! population.

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Figure 3. BMP4 induces a wave of GFP expression in differentiating MIXL1GFP/w HESCs. (A) Time course of GFP expression determined by flow cytometric analysis ofdifferentiating MIXL1GFP/w HESCs shows the transient appearance of mesendodermal progenitors in response to 50 ng/mL BMP4. Note the absence of GFP! cells in culturesdifferentiated in SFM alone (top panel). The proportion of GFP! cells for each time point is indicated. (B) GFP expression is also induced in day 5 MIXL1GFP/w EBs formed inSFM supplemented with either 100 ng/mL BMP4 or 50 ng/mLActivin A (Act A) but not with 100 ng/mL FGF2. BF indicates bright field. (C) Flow cytometric analysis substantiatesthe capacity of BMP4 and Activin A, but not FGF2, to induce GFP expression in MIXL1GFP/w EBs (left panels). Intracellular flow cytometric analysis of endogenous MIXL1protein shows that GFP mirrors MIXL1 expression (right panels). (D) Time course analysis of GFP (MIXL1), E-CAD, and PDGFR" expression in MIXL1GFP/w HESCsdifferentiated in SFM containing BMP4, VEGF, and SCF, shows the transit of cells from undifferentiated E-CAD!GFP&PDGFR"& HESCs toward GFP!PDGFR"! mesoderm.This latter population gives rise to CD34! cells (bottom panel). As expected, cells differentiated in FGF2 did not express GFP or PDGFR". Region statistics relating to eachpopulation were calculated as described in Figure S4A. GFP! cells are shown in red in all plots. The proportion of the population expressing E-CAD or PDGFR" is shown abovethe line, and percentages of negative cells are shown below the line. In all instances, the proportion of cells expressing GFP is shown in red. CD34! cells are boxed with theGFP! and GFP& portions indicated with red and black type, respectively.

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GFP& cells derived from FGF2 differentiated cultures that werelabeled with anti-MIXL1 antibodies (bottom panels).

To further document the relationship between MIXL1 expres-sion and GFP, MIXL1GFP/w HESCs were differentiated for 4 daysand flow cytometrically purified GFP! and GFP& fractions ana-lyzed by intracellular flow cytometry using MIXL1 antibodies(Figure 2C). This analysis demonstrated that, at day 4, cellsexpressing MIXL1 protein were enriched in the GFP! fraction andthat MIXL1 was essentially absent from the GFP& fraction. Theconcordance between presence of MIXL1 protein and GFP expres-sion at day 4 or day 5 was such that, on average, 83.3 (( 7.7,mean ( SEM, n ) 4) of the MIXL1! cells resided in the GFP!

fraction (Table S3, primary data and explanation of frequencycalculations). This correlation was also reflected in the distributionof MIXL1 and GFP transcripts between the sorted populations(Figures 2E, S2A). At later times, GFP expression persisted beyondthe point when MIXL1 protein levels had substantially diminished(Figure S2B), suggesting that the half-life of GFP, which is greaterthan 20 hours,24 exceeds that of MIXL1. In this regard, expressionof GFP also functioned as a lineage tracer, identifying cells that hadpreviously passed through a stage of being MIXL1!.

Consistent with previous studies where the expression ofMIXL1 has been analyzed,22 GFP expression was absolutelydependent on the inclusion of an inducing growth factor, in thiscase BMP4 (Figure 3A). This analysis revealed a wave of GFPexpression that mirrored that of MIXL1 and GFP RNA (Figure 2A).In this experiment, the frequency of GFP! cells was maximal atdays 4 to 6 (% 50%) and declined gradually thereafter, becomingnegligible by d12. Although the precise time point of peak GFPinduction varied between individual experiments (related to theconcentration or activity of the BMP4 preparation used as aninducer), the transient nature of this expression was a commonfeature of all differentiations performed with both independentlyderived MIXL1GFP/w HESC lines (Figure S3).

Apart from the more prolonged kinetics of differentiation, theseresults are analogous to those we obtained with mouse Mixl1GFP/w

ESCs differentiated under similar conditions.11 The enhancedviability associated with BMP4 treatment seen in differentiatingmouse ESCs11 also occurred with HESCs (data not shown). Inaddition to BMP4, GFP expression was also induced by activin A(Figure 3B), consistent with previous studies showing that MIXL1expression is up-regulated during activin-induced endodermaldifferentiation.25 Similarly, in line with its known role in blockingHESC differentiation,26 FGF2 failed to induce GFP expression(Figure 3B). In all cases, the induction of GFP correlated with theexpression of MIXL1 protein, as determined by intracellular flowcytometry (Figure 3C). The results of experiments in whichMIXL1GFP/w HESCs were differentiated in the presence of BMP4,activin A, or FGF2 paralleled those obtained with mouse Mixl1GFP/w

ESCs.11 The similar behavior of mouse and human ESCs differenti-ated under comparable conditions suggests that the signalingpathways underlying MIXL1 induction are probably to be con-served between these 2 species.

During mouse embryogenesis, epiblast cells entering the primi-tive streak retain E-cadherin (E-cad) before passing through atransition during which E-cad expression is down regulated andexpression of early mesodermal genes, including the receptors forvascular endothelial growth factor (Flk1) and platelet-derivedgrowth factor (PDRFR") are increased.27,28 We have previouslyshown that, in differentiating mouse Mixl1GFP/w ESCs, GFP expres-sion spanned the interval during which cells transit from E-cad!

epiblast to E-cad& Flk1! mesoderm.11 We sought to determine

whether a similar transition period could be discerned during thecourse of HESC differentiation. However, because we and othershave observed that the human homologue of Flk1, KDR, isexpressed on undifferentiated HESCs23 (E.S.N., E.G.S., and A.G.E.,unpublished data, January 2004), we instead examined the relation-ship between the expression of GFP, E-CAD, and PDGFR" (Figure3D). Flow cytometric analysis of MIXL1GFP/w HESCs differentiatedin BMP4, VEGF, and SCF (BVS) showed that the GFP! cells wereregularly seen from day 3. These cells were E-CAD!, and somealready expressed PDGFR". By day 5, the proportion of E-CAD!

cells started to fall while the frequency of cells expressingPDGFR" had increased to approximately 40% (Figure S4A, detailsof how percentages were calculated). In this experiment, the peakin the frequency of GFP! cells occurred at day 7 (33%), and themajority of these were PDGFR"!. At day 10, the proportion ofE-CAD! cells had fallen to below 40% and almost all of the GFP!

cells were also PDRFR"!. Previous studies in our laboratoryshowed that cell-surface expression of the hematopoietic andendothelial marker, CD34, was virtually absent prior to differentia-tion at day 4 (Figure S4B).22 Examination of differentiatingMIXL1GFP/w HESCs from day 8 onwards revealed a population ofCD34! cells, which appeared to derive from the preexisting GFP!

PDGFR"! fraction, as evidenced by the coexpression of thesemarkers on a proportion of CD34! cells (Figure 3D and data notshown).

To examine the relationship between GFP and PDGFR"expression, we isolated cells expressing combinations of thesemarkers by flow cytometric sorting and recultured each fraction for3 days in SFM supplemented with BVS (Figures 4A, S5, gatingstrategy). This analysis demonstrated that GFP! PDGFR"! cellsarose from the GFP! PDGFR"& population and implied that cellssequentially acquired the expression of GFP (MIXL1), PDGFR",and subsequently CD34, reflecting the sequential commitment toprimitive streak, mesoderm, and hematopoietic development.PDGFR" expression was quite stable, as evidenced by the highproportion of cells (% 85%-95%) from both GFP& PDGFR"! andGFP! PDGFR"! populations that retained PDGFR" expression3 days after sorting and reculturing. Conversely, GFP expressionwas only retained in approximately 40% to 60% of GFP!

PDGFR"& or GFP! PDGFR"! cells over this same time period.These experiments also showed that GFP& PDGFR"! cells did notgive rise to GFP! cells and that little new GFP or PDGFR"expression was induced after day 4 from GFP& PDGFR"&

(Figure 4A).Quantitative PCR analysis confirmed that the mesendodermal

and hematopoietic markers BRACHYURY, GSC, FOXA2, GATA2,and RUNX1 were expressed in day 4 EBs (Figure S6A). Thedistribution of transcripts representing these markers varied be-tween the different GFP and PDGFR" subfractions, perhapsreflecting difficulties in analyzing dynamic populations. Neverthe-less, it was generally found that expression of these markers washigher in the GFP! PDGFR! than in the GFP& PDGFR& fractions(Figure S6B), as would be predicted from the hypothesis that GFP!

PDGFR! cells represent primitive streak and nascent mesodermwhile the GFP& PDGFR& cells include less differentiated cells orectodermal precursors.

We and others have observed that the earliest hematopoieticprecursors that develop from HESCs, termed blast colony formingcells (Bl-CFCs), are seen after 3 to 4 days of differentiation23,29

(E.S.N., E.G.S., and A.G.E., unpublished data, May 2006). Experi-ments with HESCs indicated that some Bl-CFCs have the capacityto form both hematopoietic and endothelial lineages,23,29 indicating

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this population contains hemangioblasts similar to those identifiedduring the early phases of mouse ESC differentiation and mousedevelopment.13,30 In this regard, Bl-CFCs most probably corre-spond to progenitors that give rise to hematopoietic cells that havebeen documented in the yolk sac of the human embryos atapproximately embryonic day 15.8,14

In view of previous data showing that Bl-CFCs arose during theearly phases of HESC differentiation, we examined the methylcellulosecolony forming ability of day 4 subpopulations isolated on the basis oftheir GFP and PDGFR" expression. In the 5 consecutive experimentsshown in Figure 4B,C, the first 2 used a batch of BMP4 with a 3- to

5-fold lower specific activity (batch 1) than the batch used in the last3 experiments (batch 2). This led to a lower percentage of GFP! andPDGFR"! expressing cells in the first 2 experiments that could becorrelated with the low frequency of Bl-CFCs in the unsorted day 4 EBs.For example, 7 to 12 Bl-CFCs per 2 * 104 cells were observed in theday 4 EBs differentiated in BMP4 from batch 1, while 112 to 349Bl-CFCs per 2 * 104 cells were seen in the day 4 EBs cultured in BMP4from batch 2. Despite these differences, hematopoietic Bl-CFCs werehighly enriched in the GFP! PDGFR"! fraction in all 5 experiments(Figure 4B-D), demonstrating that, as in the mouse, the earliest humanhematopoietic progenitors arose within the primitive streak and nascent

Figure 4. Hematopoietic progenitors are enrichedin the MIXL1"PDGFR!" fraction of differentiatingHESCs. (A) Cell sorting and reculture experimentshowing that day 4 GFP! PDGFR"& cells give rise toGFP!PDGFR"! cells when cultured in SFM supple-mented with BVS. Some GFP! cells can still developfrom the GFP& PDGFR"& fraction, but they remainedPDGFR"& at the time points examined. The fraction ofMIXL1! (red) cells is indicated, as is the proportion ofcells in each quadrant (black text). (B) Low-powerimages of methylcellulose cultures showing that com-pared with the GFP! PDGFR"! fraction, approxi-mately 5-fold fewer CFCs were present in cell popula-tions expressing only GFP (G!P&) or PDGFR" (G&P!)(original magnification, *12). (C) Results from 5 inde-pendent experiments (experiments 1-5) confirmed thatthe frequency of blast (Bl)-CFCs was highest in theGFP!(MIXL1!) PDGFR"! fraction. The proportion ofeach subpopulation present at the time of sorting isshown across the top of the panel. (D) Summary of datain panel C showing that on average the GFP!

PDGFR"! fraction contained approximately 300 Bl-CFCs/20 000 cells plated. (E) Summary of the blastcolony distribution based on the data in panel Cshowing that approximately 90% of Bl-CFCs are presentin the GFP! PDGFR"! fraction. (F) Graph showingthat the frequency Bl-CFCs at day 4 correlates withproportion of cells that are GFP! PDGFR"!. Error barsrepresent SEM. G&P&, GFP& PDGFR"&; G!P&,GFP!PDGFR"&; G!P!, GFP! PDGFR"!; G&P!,GFP& PDGFR"!.

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mesoderm.13 Although the GFP! PDGFR"& and GFP& PDGFR"!

populations also contained hematopoietic CFCs, 90.5% plus or minus2.2% of CFCs were present in the GFP! PDGFR"! fraction (Figure 4E;Tables S4-S7). This conclusion is reinforced by the strong correlation(R2 ) 0.8497) between the frequency of Bl-CFCs and the percentage ofGFP! PDGFR"! cells (Figure 4F). Bl-CFCs were essentially absentfrom the GFP& PDGFR"& populations.

Although the frequency of hematopoietic colonies variedbetween the different sorted populations, a similar spectrum ofcolony morphologies was detected in each case. At early stagesof blast colony formation, blood cells emerged from a densecore of cells that was morphologically similar to the mesoder-mal core observed in mouse hemangioblast colonies31 (Figure

5A). In the presence of erythropoietin, developing blast coloniesbecame overtly hemoglobinized (Figure 5B,C). Where coloniescontacted the plate surface, hematopoietic cells developed inassociation with adherent cells with a morphology resemblinghemangioblast derived endothelial cells recently reported23,29

(Figure 5D). Although erythroid colonies, occasionally with ahalo of migrating myeloid cells, comprised the most frequentcolony types (% 95%) (Figure 5E,F and data not shown),colonies wholly composed of migrating myeloid cells were alsoroutinely observed (% 5%) (Figure 5G).

Examination of May-Grunwald-Giemsa stained cytospin prepa-rations revealed that most colonies were composed of primitivenucleated erythrocytes, although the presence of small numbers of

Figure 5. Blast colonies derived from day EBscontain primitive erythroid and myeloid cells.(A-D) After 8 days of methylcellulose culture, day 4MIXl1!PDGFR"! cells gave rise to hematopoieticcolonies representing different stages of blastcolony maturation. (A) Early stage colonies oftencontained a dense central core (white arrowhead)with a morphology distinct from the surroundinghematopoietic cells. (B,C) In more mature colo-nies, this feature was lost as cells within the colonyunderwent hemoglobinization. (D) Some coloniesalso contained adherent cells (black arrowheads).(E-G) Colonies arising from the day 4MIXL1!PDGFR"! fraction after 11 days of methyl-cellulose culture displayed phenotypes indicativeof erythroid, myeloid, and bipotential progenitors.(H-M) Cytocentrifuge preparations of day 4 colo-nies after 13 days of methylcellulose confirmed thepresence of nucleated primitive erythroid and my-eloid cells. Enucleated erythroid cells were alsoobserved (* in panel H) as well as cells with themorphologic appearance of neutrophils (n),megakaryocytes (mk), macrophages (mø), andmast cells (m). Panels K-M are derived from acytocentrifuge preparation of a single erythroidcolony similar to that shown in E. (N-P) Flowcytometric analysis of 15-day methylcellulose cul-tures showing that the majority of cells express theerythroid marker glycophorin A (GLYA) or CD45.Approximately 90% of cells also express the pan-hematopoietic marker CD43 and, of these, approxi-mately 20% also express CD34.

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enucleated erythrocytes was frequently observed (Figure 5H).Myeloid colonies contained macrophages, often in combinationwith mast cells or neutrophils (Figure 5I,J). Many erythroidcolonies, even those without an obvious myeloid component (suchas Figure 5E), contained macrophages, mast cells, neutrophils, andmegakaryocytes at a low frequency, indicating that these CFCswere multipotent (Figure 5K-M). The proportions of cells represent-ing the erythroid and myeloid lineages that were present in day 15methylcellulose cultures was determined by flow cytometric analy-sis. As expected, the majority of cells were glycophorin A!

(GLYA!), and a substantial proportion were CD45! (Figure 5O).Expression of CD45 and GlyA was essentially mutually exclusive,consistent with down-regulation of the former as cells committedto erythroid differentiation (GLYA!). The hematopoietic pheno-type of cells present in these methylcellulose cultures was furtherconfirmed by the high frequency of cells that expressed CD43,32 anantigen found on the surface of cells belonging to the erythroid,myeloid, and lymphoid lineages (Figure 5P). A sizable fraction ofcells also expressed CD34, suggesting, as well as mature cellsrepresenting the erythroid and myeloid lineages, these cultures alsocontained hematopoietic progenitors (Figure 5P).

Discussion

The in vitro analysis of lineage commitment from differentiatingmouse ESCs has been greatly facilitated by the availability ofESC lines containing reporter genes inserted into loci whoseexpression marks critical developmental milestones. The mostreliable method of generating genetically tagged lines is bytargeting the reporter gene to the chosen locus using homolo-gous recombination. Descriptions of gene targeting in HESCshave paved the way for the application of this approach togenetic tagging experiments in the human system.33,34 However,these previous reports have used a promoter trapping approachthat takes advantage of expression from the target locus,33 ormethods that rely on drug resistance resulting from disruption ofthe targeted gene.34 Because most genes are not amenable totargeting by such approaches, we developed a generic strategyusing conventional gene targeting in which the selectablemarker is driven from a promoter within the vector and that doesnot require expression of the target locus in undifferentiatedESCs.17 Using this approach, we have obtained several HESClines in which sequences encoding a fluorescent protein havebeen targeted to developmentally significant loci17 (currentstudy; E.G.S. and A.G.E., unpublished results). In a similarfashion to genetically tagged mouse ESCs, these lines are likelyto prove useful in dissecting relationships between cell lineagesthat emerge during the course of HESC differentiation.

In this study, we have used gene targeting to insert sequencesencoding GFP into the locus of MIXL1, the human ortholog of agene expressed in primitive streak cells of other vertebratespecies. During the early phases of HESC differentiation, GFPreliably identified cells that expressed MIXL1 protein, indicat-ing that this reporter could be used to isolate cells correspondingto a primitive streak-like stage of human embryogenesis.Because of the long half-life of GFP,24 at later time points GFPfluorescence persisted in cells that had lost MIXL1 expression,enabling a proportion of CD34! cells to be identified as directdescendants of a preexisting MIXL1! PDGFR"! population.This latter property of GFP may prove useful in analyzing the

lineage relationships between MIXL1! cells and cells represent-ing other mesodermal and endodermal derivatives.

Using these genetically tagged MIXL1GFP/w HESCs, we exam-ined the relationship between primitive streak-like MIXL1! cellsand the earliest hematopoietic progenitors, blast colony formingcells (Bl-CFCs).23,29 The results of this analysis provided a concreteexample of how fundamental aspects of early ESC differentiationare conserved between mouse and human. In both species, SFMsupplemented by BMP4 induces Mixl1! cells that give rise to amesoderm-committed subpopulation that harbors progenitors ofprimitive hematopoiesis.11

In the mouse, Mixl1 expression is localized to the primitivestreak and emerging mesendoderm, providing a molecularmarker of this process, which spans approximately 3 days,beginning at E6.5 and ending with the generation of mesodermalderivatives within the tail bud of the E9.5 embryo.1,2 Similarly,in differentiating mouse ESCs, Mixl1 expression also spans a3-day period, from its onset at approximately day 3 andextinction by day 6. Thus, in the mouse, the time intervalbetween the blastocyst stage (E3.5) or undifferentiated ESC(day 0) stages to the onset of Mixl1 expression is similar in the invivo and in vitro systems. In the human, although a primitivestreak has been documented as early as E15, the appearance ofextraembryonic mesoderm from E12 suggests that gastrulationmay have begun well before it is apparent morphologically.Analysis of early human embryos suggest that mesodermcontinues to be generated at least until E19, suggesting thathuman gastrulation may span up to 7 days.8 Indeed, dependingon the growth milieu, the duration of MIXL1 expression indifferentiating HESCS, from day 2 up to day 8, translated intoGFP expression between day 3 and day 12, and is consistentwith the protracted gastrulation stage in humans relative tomice. What is surprising is the relatively brief period betweenthe initiation of differentiation and the onset of MIXL1 expres-sion observed in this study and by others.20,25 If HESCscorrespond to the inner cell mass of an E6 embryo, thenexpression of gastrulation markers would not be expected foraround 6 days after the initiation of HESC differentiation. Thebrevity of this interval may suggest that HESCs represent a celltype that more closely resembles the epiblast than the inner cellmass. Alternatively, our results might suggest that, like mouse,human gastrulation begins around 3 days after implantation.Such a time line would then provide ample opportunity forgeneration and migration of mesodermal like cells observed inthe few examples of early stage human embryos that have beenexamined.8 Future comparative studies with the human andmouse MIXL1GFP/w ESCs should enable a better understandingof the events surrounding this inaccessible but critical period ofhuman development.

Acknowledgments

The authors thank Robyn Mayberry and Kathy Koutsis forprovision of HESCs and Andrew Fryga and Darren Ellemor forflow cytometric sorting.

This work was supported by the Australian Stem Cell Center,the Juvenile Diabetes Research Foundation, and the NationalHealth and Medical Research Council of Australia.

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Authorship

Contribution: R.P.D., E.S.N., M.C., A.K.M., and K.S. performedthe research and analyzed data; R.P.D., A.G.E., and E.G.S.designed the research, analyzed data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Edouard G. Stanley, Monash Immunologyand Stem Cell Laboratories, Level 3, Building 75, MonashUniversity, Clayton, Victoria, 3800, Australia; e-mail:ed.stanley@med.monash.edu.au.

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