Mapping the first stages of mesoderm commitment
during differentiation of human embryonic stem cells
Denis Evseenkoa, Yuhua Zhua, Katja Schenke-Laylandb, Jeffrey Kuoa, Brooke Latoura, Shundi Gea, Jessica Scholesa,
Gautam Dravida, Xinmin Lia, W. Robb MacLellanb, and Gay M. Crooksa,1
aDepartment of Pathology and Laboratory Medicine, and Broad Stem Cell Research Center andbCardiovascular Research Laboratory, David Geffen School of
Medicine, University of California, Los Angeles, CA 90095
Edited* by Owen N. Witte, Howard Hughes Medical Institute, UCLA, Los Angeles, CA, and approved June 25, 2010 (received for review February 19, 2010)
Our understanding of how mesodermal tissue is formed has been
limited by the absence of specific and reliable markers of early
mesoderm commitment. We report that mesoderm commitment
from human embryonic stem cells (hESCs) is initiated by epithelial-
to-mesenchymal transition (EMT) as shown by gene expression
profiling and by reciprocal changes in expression of the cell surface
proteins, EpCAM/CD326 and NCAM/CD56. Molecular and func-
tional assays reveal that the earliest CD326−CD56+cells, generated
from hESCs in the presence of activin A, BMP4, VEGF, and FGF2,
represent a multipotent mesoderm-committed progenitor popula-
tion. CD326−CD56+progenitors are unique in their ability to gen-
erate all mesodermal lineages including hematopoietic, endothe-
lial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle,
and cardiomyocytes, while lacking the pluripotency of hESCs.
CD326−CD56+cells are the precursors of previously reported, more
lineage-restricted mesodermal progenitors. These findings present
a unique approach to study how germ layer specification is regu-
lated and offer a promising target for tissue engineering.
and tissue engineering research, studies with hESCs are generally
focused on the development of defined, fully differentiated tis-
sues rather than the earliest events of germ-layer specification.
It is well established that hESCs have the potential to generate
multiple mesodermal derivatives after several days in culture with
specific morphogens (1). Cardiac and hematoendothelial pro-
genitors can be identified during mouse and human ESC differ-
entiation using the expression of the cell surface markers KDR
(aka VEGFR2 or Flk-1) and CD34 (2–5). A primitive streak-like
population generated from human hESCs was also isolated using
simultaneous detection of the platelet-derived growth factor re-
ceptor alpha (PDGFR-α) protein and a Mixl-1–driven GFP re-
porter (6). The Mixl-1+PDGFR-α+cells were shown to possess
hematopoietic potential, whereas generation of cardiovascular
and mesenchymal cell potential was not tested. As KDR expres-
sion was not described, it remains unclear whether PDGFR-α and
KDR proteins are coexpressed on the emerging mesodermal pro-
genitors or mark different subsets of mesoderm-committed cells.
As yet, no population generated from hESC has been identified
marking a stage in which full mesodermal potential still exists.
Epithelial-to-mesenchymal transition (EMT) occurs through
a complex process that produces changes in tissue architecture,
cell morphology, adhesion, and migratory capacity (7). In early
embryogenesis, EMT plays a pivotal role during gastrulation and
formation of the germ layers. In amniotes, mesoderm is the pre-
dominant germ layer formed via EMT (8). Although there are
multiple genes associated with EMT progression, no reliable
nonepithelial surface markers that identify cells undergoing this
process have been reported to date. It was recently reported that
the loss of E-cadherin expression during EMT is associated with
he stages of early embryogenesis can be recapitulated during in
vitro differentiation of human embryonic stem cells (hESCs)
up-regulation of neuronal cell adhesion molecule (NCAM)/
CD56 in human epithelial breast carcinoma cells (9). Ablation of
CD56 expression inhibited cell spreading and EMT, whereas
forced expression of CD56 resulted in epithelial cell delami-
nation and migration (9).
Similar to many other epithelial systems, hESCs usually es-
tablish apical–basal polarity in relation to the feeder layers on
which they are cocultured (10, 11), and associate with each other
through adhesion molecules such as E-cadherin and EpCAM
(CD326) (11, 12), which have also been shown to be required for
maintenance of pluripotency (13, 14). We hypothesized that the
earliest stage of mesoderm commitment from pluripotent hESCs
would be represented by a coordinated up-regulation of CD56
expression during the loss of epithelial cell adhesion.
Identification of a CD56+Subpopulation Generated from hESCs During
Early Mesoendodermal Induction. To explore the process of meso-
dermal commitment in the context of EMT, we used candidate
cell surface markers to identify populations generated from
hESCs during the earliest stages of differentiation. The epithelial
marker CD326 was uniformly expressed at high levels in un-
differentiated cells from three different human embryonic
stem cell lines (H9, H1, and HES3), whereas CD56 was not ex-
pressed in undifferentiated hESCs (Fig. 1A). By day 3.5 of dif-
ferentiation in mesoendoderm induction conditions, a population
marked by loss of CD326 expression and acquisition of CD56
cells were organized in clusters (Fig. S1A), and after FACS isola-
tion attached to Matrigel-coated plates and exhibited polygonal
morphology (Fig. S1B).
The efficiency and timing of the generation of CD326−CD56+
cells from hESCs was tested using various mesoendoderm-
inducing signals (Fig. S1 C and D). A small population (∼3%) of
CD326−CD56+cells was spontaneously produced without the
addition of any exogenous morphogens. The most efficient pro-
duction of CD326−CD56+cells was accomplished with the com-
bination of BMP4, VEGF, and bFGF and a transient (1 d)
exposure to activin A (“A-BVF conditions”) (n = 3, P < 0.001)
(Fig. S1 C and E). Activin/nodal signaling was critical for
the generation of CD326−CD56+cells; the presence of the ALK
4/5/7 inhibitor SB-431542 significantly reduced the number of
The optimal A-BVF conditions (Fig. S1C) were used for all
Author contributions: D.E. and G.M.C. designed research; D.E., Y.Z., K.S.-L., J.K., B.L., and
S.G. performed research; K.S.-L., J.S., and X.L. contributed new reagents/analytic tools;
D.E., K.S.-L., G.D., X.L., W.R.M., and G.M.C. analyzed data; and D.E. and G.M.C. wrote
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 3, 2010
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hESCs Undergo EMT During the Generation of CD326−CD56+Cells.
Microarray analysis confirmed that the population of cells that
down-regulate CD326 and acquire CD56 cell surface expression
at day 3.5 of culture had undergone the process of EMT. Sig-
nificant up-regulation of the key transcriptional regulators of
EMT, Snail-1, Snail-2, and Twist coincided with down-regulation
of E-cadherin and CD326 and tight junction-related genes such
as claudins, syndecans, and occludins (Fig. 1B). A significant re-
organization of cytoskeletal proteins was documented on the
basis of vimentin up-regulation (Fig. 1B). Up-regulation of fi-
bronectin expression was also seen (Fig. 1B), consistent with the
role this extracellular matrix protein plays in facilitating cell
migration. More global analysis of the microarray data showed
a total of 1,623 genes were differentially expressed fivefold or
more between the CD326−CD56+population and hESCs, (sig-
nal difference ≥ 100, P < 0.05), of which 851 genes were up-
regulated and 772 down-regulated (please see normalized data at
the publicly open database NCBI GEO, record GSE21668 at
Significantly enriched biofunctional groups of genes differen-
tially expressed between the hESCs and CD326−CD56+cells
are presented in Fig. S2A. The quality control analysis of the
microarray data are shown in Fig. S2 B–D.
Gene Expression and Functional Assays Demonstrate That the Day
3.5 CD326−CD56+Population Has Lost Pluripotency. Flow cytometry
demonstrated that CD9 and SSEA-4, cell surface markers often
used to identify undifferentiated cells, were down-regulated in
the CD326−CD56+population (Fig. 1C). Microarray analysis
of CD326−CD56+cells also demonstrated significant down-
regulation of Nanog (20-fold, P < 0.001), Sox-2 (15-fold, P =
0.012) and Oct-4/Pou5f1 (2-fold, P = 0.006) (Fig. 1B), three
key transcriptional factors associated with the pluripotency of
hESCs. Microarray data were additionally validated with real-
time PCR (Fig. S3A).
Evidence that the pluripotency of undifferentiated hESCs was
lost during generation of the CD326−CD56+population was
further verified using an in vivo teratoma formation assay. No
tumor formation was observed 2 mo after injection of day 3.5
CD326−CD56+sorted cells, whereas undifferentiated hESCs
produced teratomas in all tested animals (n = 6 animals in each
arm, in two independent experiments) (Fig. S3 B and C).
The Day 3.5 CD326−CD56+Population Generates Cells Expressing KDR,
PDGFR-α, and CD34. The onset of expression of known mesodermal
markers relative to the generation of CD326−CD56+cells was
next examined. KDR, PDGFR-α, and CD34 are three markers
previously reported to identify hematoendothelial or cardiac cells
during early hESC differentiation (3, 4, 6). Flow cytometric
analysis indicated that undifferentiated cells of all three tested
hESC lines uniformly expressed low levels of KDR (Fig. 2A), but
had no detectable PDGFR-α or CD34 expression. At day 3.5 after
mesoderm induction, KDR expression was down-regulated (Fig.
2A) and protein expression levels of PDGFR-α (Fig. 2B) and
CD34 remained undetectable on the CD326−CD56+population.
Other markers of more differentiated mesodermal derivatives,
e.g., CD45, CD31, CD117, VE-cadherin, CD105, and CD73 were
also not detectable on the day 3.5 CD326−CD56+population.
After 4 d of mesoderm induction (day 5 cultures), KDR (Fig. 2A),
CD34, and PDGFR-α began to be detected on small subsets
within the CD326−CD56+population. By day 7, several different
subpopulations could be distinguished on the basis of KDR,
PDGFR-α, and CD34 expression. A total of 52.1 ± 6.3% of the
CD326−CD56+cells (∼20% of total cells) could be readily iden-
tified as KDRhi(Fig. 2A). Within the KDRhifraction, 15–20% of
cells (∼3–4% of total cells) coexpressed CD34 by day 7 (Fig. 2B).
A large subset of CD326−CD56+cells (47.9 ± 7.4%) showed no
up-regulation of either CD34 or KDR expression throughout all
7 d of culture, but did express PDGFR-α at high levels (Fig. 2B).
Another subset of PDGFR-α+cells expressed KDR at high levels
but did not express CD34. A subset of CD34+cells expressed low
but detectable levels of PDGFR-α, consistent with previous re-
ports (6). Thus, although the percentage of CD326−CD56+cells
peaked at day 14 of induction culture, from day 5 onward they
represented a highly heterogeneous pool of cells. It should be
noted that the differentiation conditions used did not involve
generation of embryoid bodies (EB). Thus differentiation kinetics
of CD56+cells may be different from those observed when dif-
ferentiation is induced by EB formation.
To confirm that the various KDR-, PDGFR-α–, and CD34-
expressing populations that appeared by day 7 were generated
from the CD326−CD56+population, CD326−CD56+cells were
isolated by flow cytometry at day 3.5 of A-BVF induction and
reaggregated for further differentiation (Fig. 2C). After an addi-
tional 4 d of culture in the presence of BMP4, VEGF, and bFGF,
the aggregates were dissociated and analyzed by FACS. Sub-
populations marked by increased KDR, PDGFR-α, and CD34
expression were generated from the day 3.5 CD326−CD56+pop-
ulation (Fig. 2C). Thus the day 3.5 CD326−CD56+population is
a precursor of other reported mesodermal phenotypes previously
identified with the surface markers KDR, PDGFR-α, and CD34. It
should be noted that although large numbers of CD326−CD56+
cells continued to be generated for at least 14 d of culture (Fig.
S1C), immunophenotypically they were otherwise dissimilar to day
3.5 cells as more than 90% of day 14 CD326−CD56+cells had high
expression of PDGFR-α and coexpressed mesenchymal stem cell
markers CD73 and CD105.
the process of EMT. (A) Flow cytometry analysis of CD326 and CD56 ex-
pression in undifferentiated hESCs (H9) at day 0, and cells generated from
hESCs after 3.5 d in A-BVF induction conditions. (B) Microarray analysis
demonstrates changes in gene expression typical of EMT during the gener-
ation of the day 3.5 CD32−CD56+population from undifferentiated hESCs.
(C) FACS analysis of day 3.5 CD326−CD56+cells showing down-regulation of
expression of SSEA4 and CD9, two cell surface markers associated with
Generation of day 3.5 CD326−CD56+cells from hESCs occurs during
Evseenko et al.PNAS
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CD326−CD56+Cells Generated by Day 3.5 of Culture Have Committed
to the Mesodermal Germ Layer. As the CD326−CD56+population
precedes and generates cells expressing the mesodermal markers
KDR, PDGFR-α, and CD34, we next explored the mesoderm
specificity of the CD326−CD56+immunophenotype. Microarray
analysis of gene expression in day 3.5 CD326−CD56+cells, in-
dicated consistent and dramatic up-regulation of genes essential
for the earliest stages of mesoderm specification, including T,
Tbx-6, Snail-1, and Mesp-1 and -2 (Table 1).
In contrast, no pattern of either ectodermal- or endodermal-
specific differentiation was found in gene expression analyses of
the CD326−CD56+cells (Table 1 and Fig. S4).
Using differentiation systems previously shown to be efficient
for ectodermal or endodermal specification (15, 16), the non-
mesodermal potential of the CD326−CD56+population was
next examined. Neither endoderm- (Fig. S4A) nor ectoderm-
(Fig. S4B) associated genes were up-regulated in cultures initi-
ated by day 3.5 CD326−CD56+cells. Similarly, cells expressing
endodermal proteins (FOXA-2, α-Feto-Protein [AFP]), and ec-
todermal proteins (Pax-6 and Sox-1) were not detectable in
cultures initiated by day 3.5 CD326−CD56+cells, whereas robust
generation of both endodermal and neuroectodermal cells was
achieved in the same conditions from undifferentiated hESCs
(Fig. S4C). Thus, consistent with the gene expression profile, the
CD326−CD56+phenotype produced by day 3.5 in A-BVF con-
ditions, identifies an early stage of hESC differentiation, specif-
ically committed to the mesoderm germ layer.
A subset of cells that retained the original CD326+CD56−
phenotype showed progressive down-regulation of the marker
SSEA4 beginning at day 3.5. The pluripotency genes Nanog,
sodermal markers KDR, PDGFR-α, and CD34 in the CD326−
CD56+population. (A) Time course of CD326 and CD56 ex-
pression in A-BVF induction conditions (Top). At day 3.5
of culture, CD326−CD56+cells were detectable as a distinct
population demonstrating lower levels of KDR (Bottom). The
control panel shows undifferentiated hESCs that have not
been stained with antibody. (B) PDGFR-α and CD56 expression
on ungated day 3.5 cells generated in A-BVF (Top). CD34,
KDR, and CD56 expression on ungated day 7 cells (Middle
row and Lower Left panel). PDGFR-α and KDR expression on
CD56+gated cells at day 7 (Lower Right). (C) Subpopulations
marked by increased KDR, CD34, and PDGFR-α expression
were generated from the CD326−CD56+population. Shown
are FACS analyses of H9 hESC-derived cultures, representing
one of three experiments conducted on different passages
of hESCs (using also HES3 and H1 lines). Controls are un-
Time course analysis of expression of the early me-
Table 1.Gene expression analysis demonstrates mesoderm commitment in CD326−CD56+cells
Mesoderm Ectoderm Endoderm
GeneFold changeP value Gene Fold changeP valueGeneFold changeP value
Microarray analysis of undifferentiated hESCs (H9) vs. CD326−CD56+cells isolated from A-BVF induction
conditions at day 3.5. Fold change represents the ratio of transcript levels in CD326−CD56+cells/day 0 hESCs.
Values shown are average fold changes of separate cell isolations from three independent experiments for each
cell type. P < 0.05 was considered statistically significant.
| www.pnas.org/cgi/doi/10.1073/pnas.1002077107 Evseenko et al.
OCT-4, and Sox-2 were significantly down-regulated by day 3.5
(Fig. S5A). By day 14 of A-BFV culture, the endodermal genes
HNF4-α, AFP, GATA-4, and GATA-6 were up-regulated in
cells that remained CD326+(Fig. S5C). In the same conditions,
CD326+cells showed either no change or down-regulation of
ectoderm- and mesoderm-associated genes (Fig. S5 B and D).
These data suggest that in the A-BVF conditions, the persistence
of CD326 most likely defines primitive and visceral endoderm.
The CD326−CD56+Population Can Generate All Mesodermal Lineages.
Having established that the process of EMT defines the loss of
pluripotency and mesodermal commitment, an exhaustive anal-
ysis was conducted to test the full mesodermal potential of the
day 3.5 CD326−CD56+population. The TGF-β inhibitor SB-
431542 has been shown in recent studies to enhance the gener-
ation of endothelial cells from hESCs (17). In the current study
we found that TGF-β inhibition also enhanced generation of
hematopoietic cells (fivefold, compared with control) from the
day 3.5 CD326−CD56+population (Fig. S6). SB-914542 was thus
added in combination with cytokines and OP9 stroma coculture
to optimize the assay of hematoendothelial differentiation po-
tential (SI Materials and Methods).
The day 3.5 CD326−CD56+population generated robust cul-
tures of hematopoietic and endothelial cells on the basis of
both morphology and expression of hematopoietic cell surface
markers (CD45 and CD235) or endothelial markers (CD31 and
VE-cadherin) in stromal coculture and semisolid assays (Fig.
3 A–C, J and K and Fig. S6 B–E). The CD45+hematopoietic cells
coexpressed dim levels of surface CD31, whereas CD45 expres-
sion was largely absent on CD34+cells (Fig. S6 B, C, and E).
Endothelial differentiation was confirmed with LDL-Dil-Ac
uptake studies and immunohistochemical staining for e-NOS
and von Willebrand factor.
When cultured either on Matrigel-coated plates (Fig. 3D) or
cocultivated on OP9 stroma, CD326−CD56+cells could also ef-
ficiently generate cells that expressed the mesenchymal marker
CD73 and did not express CD34. In contrast to the endothelial
cells, CD73+cells expressed high levels of PDGFR-α as well as
the mesenchymal markers PDGFR-β, CD146, CD10, CD105, and
CD166, but had little or no KDR expression (Fig. S7). To test
further whether the CD73+CD34−population represented mes-
conditions and placed in specific differentiation conditions to test mesoderm lineage potential. (A–C) Hematopoietic and endothelial potential as shown by:
(A) CD45+and CD235+cells generated after 2 wk of culture on OP9 stroma. (B) Erythroid colony generated in semisolid culture. (C) Generation of CD31+CD45−
endothelial cells on OP9 stroma. (D) Generation of CD73+CD34−mesenchymal cells from day 3.5 CD326−CD56+cells after an additional 7-d culture in Matrigel.
(E–H) Mesenchymal potential as shown by histochemical staining of (E) cartilage (Alcian blue), (F) bone (von Kossa staining), and (G) adipose (Oil Red O)
tissues. (H) Fibroblastic cells generated in day 7 culture. (I) Proportion of GFP+cells, produced after 14 d from day 3.5 GFP+CD326−CD56+cells, which represent
hematopoietic, endothelial, or mesenchymal lineages based on flow cytometry. Data were compiled from limiting dilution analysis as described in Fig. S8.
(J and K) Endothelial potential was confirmed, shown on FACS plot (J) by generation of CD31+VE-cadherin+endothelial cells after 2 wk in culture. (K) The
formation of branching capillaries in Matrigel-coated chamber slides, expressing CD31 (green). (L) Smooth muscle potential shown as cells expressing α-SMA
(green). (M–P) Cardiac potential of CD326−CD56+cells. (M) Myosin heavy chain, (N) CTNT, and (O) α-actinin protein (all as green) expressing cells at day 14 of
culture. TOPRO3-staining (blue) showed cell nuclei. (P) Expression of genes involved in cardiomyocyte specification demonstrated by semiquantitative RT-PCR
at day 14 of culture. RPL-7 is a housekeeping gene. Shown are data from H9, HES3, and H1 lines. Magnification 200× in B–H and 400× in K, N, and O).
The CD326−CD56+population can generate all mesodermal lineages. CD326−CD56+cells were isolated at day 3.5 from hESC cultures in A-BVF
Evseenko et al.PNAS
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| vol. 107
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enchymal progenitors, we isolated the CD73+CD34−cells from
Matrigel or OP9 cultures and replated them into osteogenic,
chondrogenic, and adipogenic culture conditions. After an addi-
tional 3 wk of culture, generation of bone, cartilage, and adipo-
cytes was demonstrated, confirming the presence of a separate
mesenchymal branch of differentiation from the CD326−CD56+
population (Fig. 3 E–H).
To assess smooth muscle and cardiac potential, the day 3.5
CD326−CD56+cells were isolated by FACS and reaggregated in
large cell clumps and then transferred to gelatin in cardiovascular
differentiation conditions. After 10–14 d ofculture,smooth muscle
actin (α-SMA) (Fig. 3L) and H-caldesmon expressing smooth
muscle cells were generated. These cultures also contained endo-
thelial cellsthat expressed CD31 and VE-cadherin (Fig.3 J and K).
Cardiomyocyte differentiation was demonstrated by myosin heavy
chain (Fig. 3M), CTNT (Fig. 3N), and α-actinin expression (Fig.
in these cultures was ≈5–10%. Further confirmation of cardiac
differentiation was shown by detection of message for the tran-
for muscle-associated proteins myosin (MLC2A) and CTNT (Fig.
3P). Beating colonies of cardiomyocytes were generated from
CD326−CD56+cells by 11–14 d of culture (Movie S1).
The generation of endothelial and blood lineages was respec-
tively ∼30- and 50-fold more efficient from cultures initiated with
CD326−CD56+cells than the reciprocal CD326+CD56−pop-
ulation (Fig. S8A), confirming that mesodermal potential is highly
enriched in the CD326−CD56+population.
For a more quantitative assessment of hematopoietic, endothe-
lial, and mesenchymal cell potential, day 3.5 CD326−CD56+cells
were generated using an hESC subclone, which stably expresses
GFP. GFP+day 3.5 CD326−CD56+cells were seeded onto OP9
cells in serial dilution after reaggregation with GFP−CD326−
CD56+cells from the H9 parent line. High differentiation effi-
ciency was simultaneously demonstrated for all tested mesodermal
lineages (Fig. S8B). Of note, as maximal numbers of hemato-
poietic and mesenchymal cells are generated in different culture
conditions to those optimal for the endothelial lineage, simul-
taneous culture of all three lineages does not represent the
maximal efficiency for each. In addition, cardiomyocyte differ-
entiation conditions are sufficiently different to prevent simul-
taneous readout of this lineage using this assay. Nonetheless,
after 14 d in the conditions tested, more than 80% of all GFP+
cells generated from CD326−CD56+cells represented either
mesenchymal, hematopoietic, or endothelial cells (Fig. 3I).
We report the identification and characterization of a unique po-
pulation of human embryonic mesodermal progenitors (hEMP),
whicharise fromhESCs throughthe process ofEMT.Theseevents
can be tracked through the combined loss of the epithelial adhe-
sion marker EpCAM/CD326 and up-regulation of NCAM/CD56.
Several transcription factors that orchestrate EMT during
epiblast-to-mesoderm transition in vivo in the primitive streak
during gastrulation (18, 19), are also involved in the initial stages
of mesodermal differentiation of hESCs. The gene expression
profile of CD326−CD56+cells showed profound loss of the epi-
thelial genes encoding tight junctions and adherin junction pro-
teins responsible for belt-like structures at the lateral interface of
epithelial cells. The down-regulation of epithelial genes coincided
with a dramatic up-regulation of Snail-1, Snail-2, Twist, Eomes,
Mesp-1 and -2, and other transcriptional regulators of EMT.
It has recently been found that the loss of E-cadherin during
EMT is associated with up-regulation of expression of CD56 in
human epithelial breast carcinoma cells (9). The finding that
CD56 is also up-regulated during mesodermal differentiation of
hESCs suggests that CD56 is likely to be fundamental to the reor-
ganization of cell assemblyrather than specific to mesodermal com-
mitment per se. Expression of CD56 has also been documented in
neuroectodermal progenitors derived from hESCs (20). Thus, the
significance of surface markers is context specific and may differ
dramatically in different culture conditions.
The generation from hESCs of several progenitor populations
with limited mesodermal potential has been previously described
using combinations of markers of hematoendothelial (CD34 and
PDGFR-α), cardiovascular (KDR+CD117−cells), and mesen-
chymal (CD73) differentiation (3, 4, 6, 21). We now demonstrate
that day 3.5 CD326−CD56+cells represent the earliest multi-
potentmesodermal progenitors reportedtodate,beinggenerated
before the appearance of the previously described populations
and capable of giving rise to all of the above progenitor cell
phenotypes and related mesodermal lineages. On the basis of
these data, a schema can be proposed of the lineage relationships
between more restricted mesoderm progenitors that are gener-
ated later in culture (Fig. 4).
A mesoendodermal stage of mouse ES differentiation has
been previously proposed (22). Up-regulation of Sox-17 seen in
our analyses of CD326−CD56+cells may suggest that this pop-
ulation is derived from a stage that possesses both mesodermal
and endodermal potential. In addition to its role in endoderm
differentiation, Sox-17 has been shown to be essential for me-
ing mesodermal specification from hESCs. The initial stage of
mesoderm commitment is marked by the process of EMT during
which the CD326−CD56+population is generated. Subsequent
commitment to mesoderm populations with more restricted
potential is identified by day 7 of induction cultures by differ-
ential expression of the surface markers KDR, PDGFR-α, CD34,
and CD73. The phenotype of precursors to the day 7 populations
shown is yet to be delineated.
Proposed model of cell surface marker expression dur-
| www.pnas.org/cgi/doi/10.1073/pnas.1002077107 Evseenko et al.
soderm specification (23). Sox-17 has also been recently shown Download full-text
to play an important role in the maintenance of fetal and neo-
natal, but not adult hematopoietic stem cells (24). Thus, the
finding of up-regulation of Sox-17, but not other endodermal
genes in the CD326−CD56+population, (Table 1), and the lack
of functional evidence of endodermal potential (Fig. S4) is en-
tirely consistent with the assignment of mesoderm commitment
to this population.
The separation of hEMP from hESCs and other germ layers at
an early stage of differentiation allows their manipulation in
more defined culture conditions than those present within the
bulk colonies of differentiating hESCs or embryoid bodies. The
isolation of hEMP may also allow generation of functional tissue
units or niches, composed of, for example, hematopoietic cells,
endothelium, and supportive mesenchymal stroma, recapitulat-
ing the microenvironmental interactions present during normal
In conclusion, this study has demonstrated the existence of
a primitive population of hEMP, generated from hESCs by the
process of epithelial-to-mesenchymal transition. The CD326−
CD56+cells emerge before more lineage-restricted mesodermal
populations, when full mesodermal potential still exists, thereby
providing a unique opportunity for understanding the mecha-
nisms regulating mesoderm specification in humans. Isolation
and manipulation of this population in controlled and defined
culture conditions may also significantly improve existing pro-
tocols for tissue engineering.
Materials and Methods
hESC lines H1, H9, and HES3 obtained from WiCell were maintained on ir-
radiated primary mouse embryonic fibroblasts (MEFs) in medium recom-
mended by WiCell. hESCs were routinely characterized and found to have
a normal karyotype and expression of pluripotency markers SSEA4, CD9,
OCT4, and alkaline phosphatase. To induce mesoderm differentiation, col-
onies of H1, H9, or HES3 were cut into uniform-sized pieces using the
StemProEZPassage tool (Invitrogen), transferred into 6-well plates precoated
for 1 h with Matrigel (growth factor reduced, no phenol red; BD Biosciences),
and cultured initially in TESR medium (Stem Cell Technologies) until 50–60%
confluent (typically 2 d). To induce differentiation, TESR medium was
replaced with basal induction medium Stemline II (Sigma-Aldrich). Basal in-
duction medium was supplemented with optimal concentrations of human
VEGF, bFGF, BMP4, and activin A (all at 10 ng/mL; R&D Systems, see
SI Materials and Methods for details) (A-BVF conditions). After mesoderm
induction, CD326−CD56+cells were typically isolated by fluorescence acti-
vated cell sorting (FACS) at day 3.5 (unless otherwise described) and further
differentiated into mesodermal lineages in hematoendothelial, cardiac, or
mesenchymal stem cell conditions (SI Materials and Methods). Differentia-
tion potential was assessed with microarray analysis, PCR (Table S1), FACS,
morphology, and immunohistochemistry (SI Materials and Methods).
ACKNOWLEDGMENTS. We acknowledge the University of California Los
Angeles Clinical Microarray Core and Broad Stem Cell Research Center FACS
Core, Mr. Ben Van Handel, and Mr. Tan Ke Duong for their excellent
technical assistance and Dr. Don Kohn for his thoughtful contributions to
this manuscript. This research was made possible by California Institute for
Regenerative Medicine Grant RC1-00108-1 and support from a University of
California Los Angeles Broad Stem Cell Research Center award.
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Evseenko et al.PNAS
| August 3, 2010
| vol. 107
| no. 31