doi:10.1182/blood-2007-06-093609Prepublished online November 21, 2007;   

 and Edouard G StanleyRichard P Davis, Elizabeth S Ng, Magdaline Costa, Anna K Mossman, Koula Sourris, Andrew G Elefanty of primitive hematopoietic precursorsstem cells identifies human primitive streak-like cells and enables isolation

locus of human embryonicMIXL1Targeting a GFP reporter gene to the

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1

Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem

cells identifies human primitive streak-like cells and enables isolation of

primitive hematopoietic precursors

Running Title: Genetic tagging of MIXL1 in HESCs

Richard P. Davis, Elizabeth S. Ng, Magdaline Costa, Anna K. Mossman, Koula

Sourris, Andrew G. Elefanty and Edouard G. Stanley*

Monash Immunology and Stem Cell Laboratories, Level 3, Building 75, Monash

University, Clayton, Victoria, Australia, 3800.

* Corresponding Author:

Edouard Stanley

Monash Immunology and Stem Cell Laboratories

Level 3, Building 75

Monash University

Clayton, Victoria, 3800

Australia

Phone: +61-3-9905-0651

Fax: +61-3-9905-0680

e-mail: ed.stanley@med.monash.edu.au

Blood First Edition Paper, prepublished online November 21, 2007; DOI 10.1182/blood-2007-06-093609

Copyright © 2007 American Society of Hematology

2

Abstract

Differentiating human embryonic stem cells (HESCs) represent an experimental

platform for establishing the relationships between the earliest lineages that

emerge during human development. Here we report the targeted insertion in

HESCs of sequences encoding GFP into the locus of MIXL1, a gene transiently

expressed in the primitive streak during embryogenesis1,2. GFP fluorescence in

MIXL1GFP/w HESCs differentiated in the presence of BMP4 reported the

expression of MIXL1, permitting the identification of viable human primitive

streak-like cells. The use of GFP as a reporter for MIXL1 combined with cell

surface staining for PDGFRα enabled the isolation of a cell population that was

highly enriched in primitive hematopoietic precursors, the earliest derivatives of

the primitive streak. These experiments demonstrate the utility of MIXL1GFP/w

HESCs for analysing the previously inaccessible events surrounding the

development of human primitive streak-like cells and their subsequent

commitment to hematopoiesis.

3

Introduction

In vertebrate species, a prerequisite for the development of the primary germ layers is

the commitment of primitive ectoderm (epiblast) cells to gastrulation3-6. In

mammalian embryos, this process is accompanied by the formation of the primitive

streak, a morphological structure initiating at the prospective embryonic posterior6-8.

In the mouse epiblast, cells ingressing through the streak emerge as either definitive

endoderm or mesoderm, the latter including the progenitors of the hematopoietic

system9.

In the mouse, primitive streak cells are marked by expression of the transcription

factor Mixl11,2 and mouse embryos deficient in Mixl1 display multiple defects in the

formation of mesodermal and endodermal derived structures10. Consistent with this,

more recent studies have confirmed that Mixl1 expression marks precursors of both

mesoderm11 and endoderm12. These latter studies took advantage of embryonic stem

cells (ESCs) or mice in which one Mixl1 allele had been replaced by sequences

encoding green fluorescent protein (GFP), facilitating the identification and isolation

of viable GFP+ (Mixl1+) primitive streak-like cells. Analysis of Mixl1GFP/w mouse

ESCs showed that a GFP+ (Mixl1+) population present at differentiation days 3 and 4

contained hematopoietic precursors11, supporting previous data indicating that, in

mouse embryos, such precursors arise directly from the primitive streak13. The

majority of progenitors at this time were hemangioblasts, precursors with both

hematopoietic and endothelial potential which, in the embryo, contribute to the

primary vascular plexus and primitive erythropoiesis of the yolk sac. Thus, these

progenitors as well as lineage restricted primitive erythroid precursors represent the

4

first differentiated mesodermal derivatives that arise following the onset of

gastrulation at embryonic day (E) 6.59.

Because of the scarcity of examples, events surrounding gastrulation in the human

have largely been inferred from comparative embryology8, a situation which has led

to uncertainty surrounding the relationship between the first mesodermal like cells

(mesoblasts) documented from post-ovulation day 13 onwards, the appearance of

hematopoietic cells, and the overt manifestation of the primitive streak, a structure

that is first visible in embryos representing embryonic day 15 (E15)8,14-16.

Differentiating human embryonic stem cells (HESCs) represent an experimental

platform for dissecting the relationship between specific lineages and the early

differentiation events surrounding formation of the primary germ layers. In order to

examine the correlation between mesoderm formation in the human and the

emergence of hematopoietic precursors, we targeted sequences encoding GFP to the

MIXL1 locus using homologous recombination. We demonstrate that GFP

fluorescence faithfully reported expression of the endogenous MIXL1 gene and that a

mesodermal cell population defined by co-expression of GFP (MIXL1) and the

platelet derived growth factor receptor α (PDGFRα) was highly enriched in primitive

hematopoietic precursors, the earliest derivatives of the primitive streak.

5

Materials and 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 described genomic clones of the human MIXL1

locus2 and spanned sequences from a PacI site situated 9466 bp 5' of the ATG to an

HpaI site located 2242 bp 3' of the ATG. The vector was digested with the restriction

enzymes PacI and NotI prior to electroporation into HESCs as described elsewhere17.

HESC clones with a putative targeted MIXL1 allele were identified using a PCR based

screening strategy utilising the primer, Neo4, in conjunction with MIXL1 ScreenRev

(primer b in Figure 1a), a primer located immediately 3' of the genomic sequences

encompassed by the targeting vector (see Supplementary Table 1 for primer details).

Using this criterion, a number of clones were identified in which the vector appeared

to be correctly integrated into the MIXL1 locus. Two independent HES3-derived

MIXL1GFPNeoR/w clones (Clone 7 and Clone 10) were expanded and transiently

transfected with a pEFBOS-cre-IRESPuro vector using Fugene 6 transfection reagent

according to the manufacturers instructions (Roche). This vector was designed to

express a single transcript encoding cre recombinase and puromycin resistance, the

latter translated from an internal ribosomal entry site (IRES). 24-32 hours post

transfection, cells were selected in 2µg/ml puromycin for 2 days and subsequently

allowed to form colonies for a further 7 days. Several colonies representing each

primary clone were picked and screened for the loss of the neomycin resistance

cassette and for the absence of the cre expression plasmid using a PCR based

approach (see Supplementary Table 1 for primer details and PCR conditions).

Southern blot analysis was performed as described elsewhere18. The 5' external DNA

6

probe included a mixture of fragments corresponding to human genomic sequences

flanked by primer pairs listed in Supplementary Table 1. The GFP probe used to

verify the presence of a single integration event encompassed the coding sequences of

EGFP (Invitrogen). The DNA fragment generated by PCR using the primers GFP1

(primer a in Figure 1A) and MIXL1 3' probe #1 was cloned and sequenced to establish

that the 3' arm of the targeting vector had correctly integrated into the locus.

Cell culture and differentiation. HESC lines were passaged as reported

elsewhere17,19 and differentiated as spin EBs according to previously established

protocols20. Serum free differentiation medium (SFM)20, containing recombinant

human albumin and 0.05-0.25% Polyvinylalcohol in some experiments, was

supplemented with the following growth factors at the concentrations indicated: 10-

100ng/ml BMP4, 50 ng/ml Activin A (R&D Systems), 50-100ng/ml FGF2, 10-50

ng/ml VEGF, 20-100ng/ml SCF, 30ng/ml IL3, 30 ng/ml IL6, 30ng/ml Tpo, 3U/ml

EPO (PeproTech). EBs were dissociated using either 0.25% w/v Trypsin-EDTA

(Invitrogen) or TrypLE SelectTM (Invitrogen). Preparation and analysis of

methylcellulose cultures and cytocentrifuge preparations were conducted according to

Ng et al (2005)20. Karyotype analysis and teratoma assays were performed as

described previously19

Flow cytometric analysis. Intracellular flow cytometry with anti-Mixl1 and anti-

Oct4 antibodies was performed as described previously21. For analysis and sorting of

live cells, HESCs were dissociated to give a single cell suspension and labeled with

antibodies as described previously20. The antibodies used in this study were

phycoerythrin (PE)-conjugated mouse anti-human CD34 (BD Biosciences, cat

7

#348057), mouse anti-human E-CADHERIN (Zymed, cat #13-1700), mouse anti-

human PDGFRα (BD Biosciences, cat #556001), mouse anti-human CD43 (BD

Biosciences, cat# 555474), PE-conjugated mouse antihuman CD45 (BD Biosciences,

cat# 555483), allophycocyanin (APC)-conjugated mouse anti-human glycophorin A

(BD Biosciences, cat# 551336) and mouse anti-human Tra-1-60 (Chemicon, cat

#MAB4360). Unconjugated primary antibodies were detected with either PE or APC-

conjugated goat anti-mouse IgG (BD Biosciences, cat #550589 and #550826). Flow

cytometry gates were set using control cells (HES3) and MIXL1GFP/w HESCs labelled

with the appropriate isotype control antibody. Alternatively, gates were set relative to

MIXL1GFP/w HESCs differentiated in FGF2, which do not express MIXL1. Single cell

cloning was performed using the single cell deposition function of a FACSaria FACS

station to place single cells into each well of ten 96 well trays pre-seeded with

irradiated primary mouse embryonic fibroblasts (PMEFs) and containing HESC

culture media19. For re-aggregation/re-culture experiments, cells obtained from flow

cytometric sorting were aggregated using the spin EB protocol (104/well), in SFM

supplemented with 30ng/ml BMP4, 30ng/ml VEGF and 40ng/ml SCF.

Immunofluorescence analysis. Dissociated cells from day (d)4 EBs were

resuspended in SFM and allowed to adhere to a poly-L Lysine coated glass slide for

20 minutes. The cells were fixed and permeabilized with Cytofix/Cytoperm™ (BD

Biosciences) and labelled with the anti-MIXL1 antibody, 6-G221, or a Rat IgG control

antibody. Primary antibodies were detected with a Texas Red conjugated (TR) goat

anti-rat immunoglobulin G (IgG; Jackson Immuno-Research) and confocal images

were captured using an Olympus FluoView 1000.

8

Gene expression analysis. RNA was prepared using RNAeasyTM according to

manufacturers instructions (Qiagen). RNA samples were reverse transcribed and

normalised as described previously20. PCR was performed under standard conditions

(30 cycles of 94ºC, 20 seconds; 60ºC, 30 seconds; 68ºC, 60 seconds) using the primer

sets listed in Supplementary Table 2. For PCR with PAX6 specific primers an

annealing temperature of 55ºC was used. Real time PCR was performed using

Taqman gene expression probes and expression levels calculated as described

previously22.

Results

The MIXL1-GFP targeting vector (Figure 1A) was electroporated into HESCs and

G418 resistant colonies isolated as described elsewhere17. Correctly targeted clones

were identified using a PCR based strategy with the primers indicated (Supplementary

Table 1). Following removal of the G418 resistance cassette (see Methods), the

structural integrity of the targeted locus was verified by Southern blot analysis (Figure

1B, C) and sequencing of the PCR product representing the 3' junction between the

vector and flanking genomic DNA (Figure 1D and data not shown). In addition, one

MIXL1GFP/w HESC line was cloned by single cell deposition using a flow cytometer

into 96 well trays (cloning efficiency of ~5%). The parental line and subclones were

phenotypically indistinguishable (data not shown). MIXL1GFP/w HESCs had normal

karyotypes, formed teratomas and expressed markers of undifferentiated HESCs

(Supplementary Figure 1 and data not shown).

9

In order to examine the temporal association between expression of GFP and the

endogenous MIXL1 allele, the gene expression profile of MIXL1GFP/W HESCs

differentiated in response to BMP4 was analysed over a 12 day period using RT-PCR

(Figure 2A). The expression profile of GFP transcripts mirrored that of MIXL1, and,

as previously reported20, MIXL1 expression was contemporaneous with that of

BRACHYURY, a transcription factor also present in the primitive streak. The decline

in the level of expression of these primitive streak genes between days (d) 6-8,

overlapped with the expression of mesodermal (GATA2, CD34) and endodermal

(FOXA2, ALPHA FETOPROTEIN, ALBUMIN) genes. This transient wave of MIXL1

expression was consistent with our previous data demonstrating the kinetics of

differentiation using the 'spin embryoid body' (spin EB) system in SFM supplemented

with BMP420. By and large, these kinetics paralleled those reported by others for

BMP4 dependent HESC differentiation in serum free media, with transient expression

of brachyury and a gradual diminution in the levels of OCT423. Also in agreement

with the studies of Kennedy et al23, substantial CD34 expression was not observed

until after d4 (Figure 2A). As evidenced by the weak expression of PAX6, BMP4 did

not promote neurectodermal differentiation.

Immunofluorescence analysis of MIXL1GFP/w HESCs differentiated for 4 days in

BMP4 revealed a correlation between GFP expression and MIXLl protein. In the

example shown, five of six intact cells co-express MIXL1 and GFP (Figure 2B, upper

panels). Specific localized staining was not observed in GFP+ cells labelled with an

isotype control antibody (middle panels), nor in - the uniformly GFP- cells derived

from FGF2 differentiated cultures that were labelled with anti-MIXL1 antibodies

(lower panels).

10

To further document the relationship between MIXL1 expression and GFP,

MIXL1GFP/w HESCs were differentiated for 4 days and flow cytometrically purified

GFP+ and GFP- fractions analysed by intracellular flow cytometry using MIXL1

antibodies (Figure 2C). This analysis demonstrated that, at d4, cells expressing

MIXL1 protein were enriched in the GFP+ fraction and that MIXL1 was essentially

absent from the GFP- fraction. The concordance between presence of MIXL1 protein

and GFP expression at d4 or d5 was such that, on average, 83.3±7.7 (Mean ± SEM,

n=4) of the MIXL1+ cells resided in the GFP+ fraction (see Supplementary Table 3 for

primary data and explanation of frequency calculations). This correlation was also

reflected in the distribution of MIXL1 and GFP transcripts between the sorted

populations (Figure 2E and Supplementary Figure 2A). At later times, GFP

expression persisted beyond the point when MIXL1 protein levels had substantially

diminished (see Supplementary Figure 2B), suggesting that the half-life of GFP,

which is greater than 20 hours24, exceeds that of MIXL1. In this regard, expression of

GFP also functioned as a lineage tracer, identifying cells that had previously passed

through a stage of being MIXL1+.

Consistent with previous studies where the expression of MIXL1 has been analysed22,

GFP expression was absolutely dependent on the inclusion of an inducing growth

factor, in this case BMP4 (Figure 3A). This analysis revealed a wave of GFP

expression that mirrored that of MIXL1 and GFP RNA (see Figure 2A). In this

experiment, the frequency of GFP+ cells was maximal at d4-6 (~50%) and declined

gradually thereafter, becoming negligible by d12. Although the precise time point of

11

peak GFP induction varied between individual experiments (related to the

concentration or activity of the BMP4 preparation used as an inducer), the transient

nature of this expression was a common feature of all differentiations performed with

both independently derived MIXL1GFP/w HESC lines (Supplementary Figure 3).

Apart from the more prolonged kinetics of differentiation, these results are analogous

to those we obtained with mouse Mixl1GFP/w ESCs differentiated under similar

conditions11. The enhanced viability associated with BMP4 treatment seen in

differentiating mouse ESCs11 also occurred with HESCs (data not shown). In addition

to BMP4, GFP expression was also induced by Activin A (Figure 3B), consistent with

previous studies showing that MIXL1 expression is up-regulated during Activin

induced endodermal differentiation25. Similarly, in line with its known role in

blocking HESC differentiation26, FGF2 failed to induce GFP expression (Figure 3B).

In all cases, the induction of GFP correlated with the expression of MIXL1 protein, as

determined by intracellular flow cytometry (Figure 3C). The results of experiments in

which MIXL1GFP/w HESCs were differentiated in the presence of BMP4, Activin A or

FGF2 paralleled those obtained with mouse Mixl1GFP/w ESCs11. The similar

behaviour of mouse and human ESCs differentiated under comparable conditions

suggests that the signalling pathways underlying MIXL1 induction are likely to be

conserved between these two species.

During mouse embryogenesis, epiblast cells entering the primitive streak retain E-

cadherin (E-cad) before passing through a transition during which E-cad expression is

down regulated and expression of early mesodermal genes, including the receptors for

vascular endothelial growth factor (Flk1) and platelet derived growth factor

12

(PDRFRα) are increased27,28. We have previously shown that in differentiating mouse

Mixl1GFP/w ESCs, GFP expression spanned the interval during which cells transit from

E-cad+ epiblast to E-cad-Flk1+ mesoderm11. We sought to determine if a similar

transition period could be discerned during the course of HESC differentiation.

However, because we and others have observed that the human homologue of Flk1,

KDR, is expressed on undifferentiated HESCs (ref23 and ESN, EGS and AGE,

unpublished data), we instead examined the relationship between the expression of

GFP, E-CAD and PDGFRα (Figure 3D). Flow cytometric analysis of MIXL1GFP/W

HESCs differentiated in BMP4, VEGF and SCF (BVS) showed that the GFP+ cells

were regularly seen from d3. These cells were E-CAD+ and some already expressed

PDGFRα. By d5, the proportion of E-CAD+ cells started to fall whilst the frequency

of cells expressing PDGFRα had increased to ~40% (see Supplementary Figure 4A

for details of how percentages were calculated). In this experiment, the peak in the

frequency of GFP+ cells occurred at d7 (33%) and the majority of these were

PDGFRα+. At d10, the proportion of E-CAD+ cells had fallen to below 40% and

almost all of the GFP+ cells were also PDRFRα+. Previous studies in our laboratory

showed that cell surface expression of the hematopoietic and endothelial marker,

CD34, was virtually absent prior to differentiation d4 (Supplementary Figure 4B)22.

Examination of differentiating MIXL1GFP/w HESCs from d8 onwards revealed a

population of CD34+ cells which appeared to derive from the pre-existing GFP+

PDGFRα+ fraction, as evidenced by the co-expression of these markers on a

proportion of CD34+ cells (Figure 3D and data not shown).

To examine the relationship between GFP and PDGFRα expression, we isolated cells

expressing combinations of these markers by flow cytometric sorting and re-cultured

13

each fraction for 3 days in SFM supplemented with BVS (Figure 4A and

Supplementary Figure 5 for gating strategy). This analysis demonstrated that

GFP+PDGFRα+ cells arose from the GFP+PDGFRα- population and implied that cells

sequentially acquired the expression of GFP (MIXL1), PDGFRα and subsequently

CD34, reflecting the sequential commitment to primitive streak, mesoderm and

hematopoietic development. PDGFRα expression was quite stable, as evidenced by

the high proportion of cells (~85-95%) from both GFP-PDGFRα+ and

GFP+PDGFRα+ populations that retained PDGFRα expression 3 days after sorting

and re-culturing. Conversely, GFP expression was only retained in ~40-60% of

GFP+PDGFRα- or GFP+PDGFRα+ cells over this same time period. These

experiments also showed that GFP-PDGFRα+ cells did not give rise to GFP+ cells and

that little new GFP or PDGFRα expression was induced after d4 from GFP-PDGFRα-

(Figure 4A).

Quantitative PCR analysis confirmed that the mesendodermal and hematopoietic

markers BRACHYURY, GSC, FOXA2, GATA2 and RUNX1, were expressed in d4

EBs (Supplementary Figure 6A). The distribution of transcripts representing these

markers varied between the different GFP and PDGFRα subfractions, perhaps

reflecting difficulties in analysing dynamic populations. Nevertheless, it was

generally found that expression of these markers was higher in the GFP+PDGFR+ than

in the GFP-PDGFR- fractions (Supplementary Figure 6B), as would be predicted from

the hypothesis that GFP+PDGFR+ cells represent primitive streak and nascent

mesoderm whilst the GFP-PDGFR- cells include less differentiated cells or ectodermal

precursors.

14

We and others have observed that the earliest hematopoietic precursors that develop

from HESCs, termed blast colony forming cells (Bl-CFCs), are seen after 3-4 days of

differentiation (ref23,29 and ESN, EGS and AGE, unpublished data). Experiments with

HESCs indicated that some Bl-CFCs have the capacity to form both hematopoietic

and endothelial lineages23,29, indicating this population contains hemangioblasts

similar to those identified during the early phases of mouse ESC differentiation and

mouse development13,30. In this regard, Bl-CFCs most likely correspond to

progenitors that give rise to haematopoietic cells that have been documented in the

yolk sac of the human embryos at approximately embryonic day 158,14.

In view of previous data showing that Bl-CFCs arose during the early phases of

HESC differentiation, we examined the methylcellulose colony forming ability of d4

sub-populations isolated on the basis of their GFP and PDGFRα expression. In the

five consecutive experiments shown in Figure 4B and C, the first two utilised a batch

of BMP4 with a 3- to 5-fold lower specific activity (batch 1) than the batch used in

the last three experiments (batch 2). This led to a lower percentage of GFP+ and

PDGFRα+ expressing cells in the first two experiments that could be correlated with

the low frequency of Bl-CFCs in the unsorted d4 EBs. For example, 7-12 Bl-CFC per

2 x 104 cells were observed in the d4 EBs differentiated in BMP4 from batch 1, whilst

112-349 Bl-CFC per 2 x 104 cells were seen in the d4 EBs cultured in BMP4 from

batch 2. Despite these differences, hematopoietic Bl-CFCs were highly enriched in

the GFP+PDGFRα+ fraction in all 5 experiments (Figure 4B - D), demonstrating that,

as in the mouse, the earliest human hematopoietic progenitors arose within the

primitive streak and nascent mesoderm13. Although the GFP+PDGFRα- and GFP-

PDGFRα+ populations also contained hematopoietic CFCs, 90.5±2.2% of CFCs were

15

present in the GFP+PDGFRα+ fraction (Figure 4E and Supplementary Tables 4-7).

This conclusion is reinforced by the strong correlation (R2=0.8497) between the

frequency of Bl-CFCs and the percentage of GFP+PDGFRα+ cells (Figure 4F). Bl-

CFCs were essentially absent from the GFP-PDGFRα- populations.

Although the frequency of hematopoietic colonies varied between the different sorted

populations, a similar spectrum of colony morphologies was detected in each case. At

early stages of blast colony formation, blood cells emerged from a dense core of cells

that was morphologically similar to the mesodermal core observed in mouse

hemangioblast colonies31 (Figure 5A). In the presence of erythropoietin (EPO),

developing blast colonies became overtly hemoglobinised (Figure 5B, C). Where

colonies contacted the plate surface, hematopoietic cells developed in association with

adherent cells with a morphology resembling hemangioblast derived endothelial cells

recently reported23,29 (Figure 5D). Although erythroid colonies, occasionally with a

halo of migrating myeloid cells, comprised the most frequent colony types (~95%)

(Figure 5E, F and data not shown), colonies wholly composed of migrating myeloid

cells were also routinely observed (~5%)(Figure 5G).

Examination of May-Grünwald-Giemsa stained cytospin preparations revealed that

most colonies were composed of primitive nucleated erythrocytes, although the

presence of small numbers of enucleated erythrocytes was frequently observed

(Figure 5H). Myeloid colonies contained macrophages, often in combination with

mast cells or neutrophils (Figure 5I, J). Many erythroid colonies, even those without

an obvious myeloid component (such as Figure 5E), contained macrophages, mast

cells, neutrophils and megakaryocytes at a low frequency, indicating that these CFCs

16

were multipotent (Figure 5K-M). The proportions of cells representing the erythroid

and myeloid lineages that were present in d15 methylcellulose cultures was

determined by flow cytometric analysis. As expected, the majority of cells were

glycophorin A+ (GlyA+) whilst a substantial proportion were CD45+ (Figure 5O).

Expression of CD45 and GlyA were essentially mutually exclusive, consistent with

down regulation of the former as cells committed to erythroid differentiation (GlyA+).

The hematopoietic phenotype of cells present in these methylcellulose cultures was

further confirmed by the high frequency of cells that expressed CD4332, an antigen

found on the surface of cells belonging to the erythroid, myeloid and lymphoid

lineages (Figure 5P). A sizable fraction of cells also expressed CD34, suggesting, as

well as mature cells representing the erythroid and myeloid lineages, these cultures

also contained hematopoietic progenitors (Figure 5P).

Discussion

The in vitro analysis of lineage commitment from differentiating mouse ESCs has

been greatly facilitated by the availability of ESC lines containing reporter genes

inserted into loci whose expression marks critical developmental milestones. The

most reliable method of generating genetically tagged lines is by targeting the reporter

gene to the chosen locus using homologous recombination. Descriptions of gene

targeting in HESCs have paved the way for the application of this approach to genetic

tagging experiments in the human system33,34. However, these previous reports have

used a promoter trapping approach that takes advantage of expression from the target

locus33, or methods that rely on drug resistance resulting from disruption of the

targeted gene34. Since most genes are not amenable to targeting by such approaches,

17

we developed a generic strategy utilizing conventional gene targeting in which the

selectable marker is driven from a promoter within the vector and that does not

require expression of the target locus in undifferentiated ESCs17. Using this approach

we have obtained a number of HESC lines in which sequences encoding a fluorescent

protein have been targeted to developmentally significant loci17 (this study and EGS

and AGS unpublished results). In a similar fashion to genetically tagged mouse ESCs,

these lines are likely to prove useful in dissecting relationships between cell lineages

that emerge during the course of HESC differentiation.

In this study we have used gene targeting to insert sequences encoding GFP into the

locus of MIXL1, the human orthologue of a gene expressed in primitive streak cells of

other vertebrate species. During the early phases of HESC differentiation, GFP

reliably identified cells that expressed MIXL1 protein, indicating this reporter could

be used to isolate cells corresponding to a primitive streak-like stage of human

embryogenesis. Due to the long half-life of GFP24, at later time points GFP

fluorescence persisted in cells which had lost MIXL1 expression, enabling a

proportion of CD34+ cells to be identified as direct descendents of a pre-existing

MIXL1+PDGFRα+ population. This latter property of GFP may prove useful in

analysing the lineage relationships between MIXL1+ cells and cells representing other

mesodermal and endodermal derivatives.

Using these genetically tagged MIXL1GFP/w HESCs, we examined the relationship

between primitive streak-like MIXL1+ cells and the earliest hematopoietic

progenitors, blast colony forming cells (Bl-CFCs)23,29. The results of this analysis

provided a concrete example of how fundamental aspects of early ESC differentiation

18

are conserved between mouse and human. In both species, SFM supplemented by

BMP4 induces Mixl1+ cells that give rise to a mesoderm-committed sub-population

that harbours progenitors of primitive hematopoiesis11.

In the mouse, Mixl1 expression is localised to the primitive streak and emerging

mesendoderm, providing a molecular marker of this process which spans

approximately 3 days; beginning at E6.5 and ending with the generation of

mesodermal derivatives within the tail bud of the E9.5 embryo1,2. Similarly, in

differentiating mouse ESCs, Mixl1 expression also spans a 3 day period, from its

onset at ~d3 and extinction by d6. Thus, in the mouse, the time interval between the

blastocyst stage (E3.5) or undifferentiated ESC (d0) stages to the onset of Mixl1

expression is similar in the in vivo and in vitro systems. In the human, although a

primitive streak has been documented as early as E15, the appearance of

extraembryonic mesoderm from E12 suggests that gastrulation may have begun well

before it is apparent morphologically. Analysis of early human embryos suggest that

mesoderm continues to be generated at least until E19, suggesting that human

gastrulation may span up to 7 days8. Indeed, depending on the growth milieu, the

duration of MIXL1 expression in differentiating HESCS, from d2 up to d8, translated

into GFP expression between d3 and d12, and is consistent with the protracted

gastrulation stage in humans relative to mice. What is surprising is the relatively brief

period between the initiation of differentiation and the onset of MIXL1 expression

observed in this study and by others20,25. If HESCs correspond to the inner cell mass

of an E6 embryo, then expression of gastrulation markers would not be expected for

around 6 days following the initiation of HESC differentiation. The brevity of this

interval may suggest that HESCs represent a cell type which more closely resembles

19

the epiblast than the inner cell mass. 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 for generation and migration of mesodermal

like cells observed in the few examples of early stage human embryos that have been

examined8. Future comparative studies with the human and mouse MIXL1GFP/w ESCs

should enable a better understanding of the events surrounding this inaccessible but

critical period of human development.

Acknowledgments. We would like to thank Robyn Mayberry and Kathy Koutsis for

provision of HESCs and Andrew Fryga and Darren Ellemor for flow cytometric

sorting. This work was supported by the Australian Stem Cell Centre, the Juvenile

Diabetes Research Foundation and the National Health and Medical Research Council

of Australia.

Author contributions. RP Davis, ES Ng, M Costa, AK Mossman, K Sourris

performed the research and analyzed data. RP Davis, AG Elefanty and EG Stanley

designed the research, analyzed data and wrote the paper.

The authors declare no competing financial interests.

20

References

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19. Costa M, Dottori M, Ng E, et al. The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat Methods. 2005;2:259-260. 20. Ng ES, Davis RP, Azzola L, Stanley EG, Elefanty AG. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood. 2005;106:1601-1603. 21. Mossman AK, Sourris K, Ng E, Stanley EG, Elefanty AG. Mixl1 and oct4 proteins are transiently co-expressed in differentiating mouse and human embryonic stem cells. Stem Cells Dev. 2005;14:656-663. 22. Pick M, Azzola L, Mossman A, Stanley EG, Elefanty AG. Differentiation of Human Embryonic Stem Cells in Serum Free Medium Reveals Distinct Roles for BMP4, VEGF, SCF and FGF2 in Hematopoiesis. Stem Cells. 2007;25:2206-2214. 23. Kennedy M, D'Souza SL, Lynch-Kattman M, Schwantz S, Keller G. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood. 2007;109:2679-2687. 24. Corish P, Tyler-Smith C. Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 1999;12:1035-1040. 25. D'Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23:1534-1541. 26. Levenstein ME, Ludwig TE, Xu RH, et al. Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells. 2006;24:568-574. 27. Kataoka H, Takakura N, Nishikawa S, et al. Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells. Dev Growth Differ. 1997;39:729-740. 28. Tada S, Era T, Furusawa C, et al. Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development. 2005;132:4363-4374. 29. Lu SJ, Feng Q, Caballero S, et al. Generation of functional hemangioblasts from human embryonic stem cells. Nat Methods. 2007;4:501-509. 30. Kennedy M, Firpo M, Choi K, et al. A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature. 1997;386:488-493. 31. D'Souza SL, Elefanty AG, Keller G. SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast but not for its development. Blood. 2005;105:3862-3870. 32. Vodyanik MA, Thomson JA, Slukvin, II. Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures. Blood. 2006;108:2095-2105. 33. Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol. 2003;21:319-321. 34. Urbach A, Schuldiner M, Benvenisty N. Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells. Stem Cells. 2004;22:635-641.

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Figure Legends:

Figure 1. Targeting of GFP to the MIXL1 locus in HESCs. (A) Structure of the

gene targeting vector used to insert sequences encoding GFP into exon 1 of the

MIXL1 locus using homologous recombination. PacI and NotI are restriction enzyme

sites used to linearize the vector prior to electroporation. NeoR is the PGKNeo

cassette encoding G418 resistance, flanked by loxP sites (black triangles). The

positions of MfeI sites used to map the structure of the modified locus are shown, as

are the position of primers (a, b) used to identify correctly targeted clones. (B)

Southern blot analysis of MfeI digested genomic DNA shows that a 5' external probe

detects a fragment of 17 Kb representing the endogenous locus from both parental

HES3 cells and HESCs with a targeted MIXL1 locus. An additional fragment of 14.4

Kb is also detected 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 GFP

sequences indicates that these cells contain a single copy of the GFP gene consistent

with a single genetic modification at the MIXL1 locus. (D) The integrity of sequences

3' of the GFP gene was validated using a PCR based approach (with primers a and b)

to amplify DNA representing the junction of the targeting vector with the

chromosomal DNA. Sequence analysis of this fragment confirmed that the

relationship between the vector DNA and adjacent chromosomal sequences were as

expected (data not shown).

23

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 of endogenous MIXl1. This analysis also shows the progressive

downregulation of the stem cell marker, OCT4, the transient expression of the

primitive streak genes, MIXL1 and BRACHYURY, activation of genes expressed in

endodermal (FOXA2, AFP-alpha fetoprotein, ALBUMIN) and mesodermal (GATA2,

CD34) cell types. –RT, - 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 d4 MIXL1GFP/w HESCs differentiated in SFM in the

presence of either 30 ng/ml BMP4 (upper and middle panels) or 100 ng/ml FGF2

(bottom panel). The lower panel shows that MIXL1GFP/w HESCs differentiated in

FGF2 expressed neither GFP nor MIXL1. (C) Sorting and reanalysis experiments

examining the relationship between expression of GFP and MIXL1 protein by flow

cytometric analysis. The upper panel shows the profile of GFP expressing cells in d4

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 of GFP+ cells and

GFP- cells indicated. Endogenous MIXL1 protein (bottom panels), as determined by

intracellular flow cytometry with an anti-MIXL1 antibody, is largely restricted to

GFP+ cells and excluded from the GFP- cells. The position of gates for intracellular

flow cytometry were set with MIXL1GFP/w d4 EBs differentiated in SFM containing

100 ng/ml FGF2 and stained with the anti-MIXL1 antibody (inset, bottom panel). (D)

Graphical representation showing the distribution of MIXL1+ cells between the GFP+

and GFP- sorted populations from four separate experiments using two independent

MIXL1GFP/w HESC lines (P-value < 0.001). Error bars represent the standard error of

24

the mean. Calculations are shown in Supplementary Tables 3A-C. (E) PCR analysis

of the GFP-sorted fractions from C showing both GFP and MIXL1 transcripts are

essentially restricted to the GFP+ population.

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

of differentiating MIXL1GFP/w HESCs shows the transient appearance of

mesendodermal progenitors in response to 50 ng/ml BMP4. Note the absence of GFP+

cells in cultures differentiated in SFM alone (upper panel). The proportion of GFP+

cells for each time point is indicated. (B) GFP expression is also induced in d5

MIXL1GFP/w EBs formed in SFM supplemented with either 100ng/ml BMP4 or 50

ng/ml Activin A (Act A) but not with 100ng/ml FGF2. BF, bright field. (C) Flow

cytometric analysis substantiates the 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 MIXL1 protein shows that GFP mirrors MIXL1

expression (right panels). (D) Time course analysis of GFP (MIXL1), E-CAD and

PDGFRα expression in MIXL1GFP/w HESCs differentiated in SFM containing BMP4,

VEGF and SCF, shows the transit of cells from undifferentiated E-CAD+GFP-

PDGFRα- HESCs towards GFP+PDGFRα+ mesoderm. This latter population gives

rise to CD34+ cells (lower panel). As expected, cells differentiated in FGF2 did not

express GFP or PDGFRα. Region statistics relating to each population were

calculated as described in Supplementary Figure 4A. GFP+ cells are shown in red in

all plots. The proportion of the population expressing E-CAD or PDGFRα is shown

above the line and percentages of negative cells are shown below the line. In all

25

instances the proportion of cells expressing GFP are shown in red. CD34+ cells are

boxed with the GFP+ and GFP- portions indicated with red and black type

respectively.

Figure 4. Hematopoietic progenitors are enriched in the MIXL1+PDGFRα+

fraction of differentiating HESCs. (A) Cell sorting and re-culture experiment

showing that d4 GFP+ PDGFRα- cells give rise to GFP+PDGFRα+ cells when

cultured in SFM supplemented with BVS. Some GFP+ cells can still develop from the

GFP-PDGFRα- fraction, but they remained PDGFRα- at the time points examined.

The fraction of MIXL1+ (red) cells is indicated, as is the proportion of cells in each

quadrant (black text). (B) Low power images of methylcellulose cultures showing that

compared to the GFP+PDGFRα+ fraction, approximately 5 fold fewer CFCs were

present in cell populations expressing only GFP (G+P-) or PDGFRα (G-P+) (original

magnification x 12). (C) Results from five independent experiments (Expt. 1-5)

confirmed that the frequency of blast (Bl)-CFCs was highest in the

GFP+(MIXL1+)PDGFRα+ fraction. The proportion of each sub-population present at

the time of sorting is shown across the top of the panel. (D) Summary of data in (C)

showing that on average the GFP+PDGFRα+ fraction contained ~ 300 Bl-

CFCs/20,000 cells plated. (E) Summary of the blast colony distribution based on the

data in (C) showing that ~ 90% of Bl-CFCs are present in the GFP+PDGFRα+

fraction. (F) Graph showing that frequency Bl-CFCs at d4 correlates with proportion

of cells that are GFP+PDGFRα+. Error bars are standard error of the mean. G-P-, GFP-

PDGFRα-; G+P-, GFP+PDGFRα-; G+P+, GFP+PDGFRα+; G-P+, GFP-PDGFRα+.

26

Figure 5. Blast colonies derived from day EBs contain primitive erythroid and

myeloid cells. (A-D) After 8 days of methylcellulose culture, day 4 Mixl1+PDGFR+

cells gave rise to hematopoietic colonies representing different stages of blast colony

maturation. (A) Early stage colonies often contained a dense central core (white

arrowhead) with a morphology distinct from the surrounding hematopoietic cells. (B,

C) In more mature colonies, this feature was lost as cells within the colony underwent

hemoglobinisation. (D) Some colonies also contained adherent cells (black

arrowheads). (E-G) Colonies arising from the day 4 MIXL1+PDGFRα+ fraction after

11 days of methylcellulose culture displayed phenotypes indicative of erythroid,

myeloid and bipotential progenitors. (H-M) Cytocentrifuge preparations of day 4

colonies after 13 days of methylcellulose confirmed the presence of nucleated

primitive erythroid and myeloid cells. Enucleated erythroid cells were also observed

(* in H) as well as cells with the morphological appearance of neutrophils (n),

megakaryocytes (mk), macrophages (mø) and mast cells (m). Images K-M are derived

from a cytocentrifuge preparation of a single erythroid colony similar to that shown in

E. (N-P) Flow cytometric analysis of 15 day methylcellulose cultures showing the

majority of cells express the erythroid marker glycophorin A (GlyA) or CD45.

Approximately 90% of cells also express the pan-hematopoietic marker CD43 and of

these, approximately 20% also express CD34.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5