Prospective isolation of human embryonic stem
cell-derived cardiovascular progenitors that
integrate into human fetal heart tissue
Reza Ardehalia,b,1, Shah R. Alic, Matthew A. Inlayc, Oscar J. Abilezd, Michael Q. Chend, Timothy A. Blauwkampc,
Masayuki Yazawac, Yongquan Gongc, Roeland Nussec, Micha Drukkerc, and Irving L. Weissmanc,1
aDivision of Cardiology, Department of Internal Medicine, University of California Los Angeles School of Medicine, Los Angeles, CA 90095;bBroad Stem Cell
Research Center, University of California Los Angeles School of Medicine, Los Angeles, CA 90095; andcInstitute for Stem Cell Biology and Regenerative
Medicine, anddDepartment of Bioengineering, Stanford University School of Medicine, Stanford, CA 94305
Contributed by Irving L. Weissman, January 7, 2013 (sent for review May 2, 2012)
A goal of regenerative medicine is to identify cardiovascular progen-
itors from human ES cells (hESCs) that can functionally integrate into
the human heart. Previous studies to evaluate the developmental
potential of candidate hESC-derived progenitors have delivered
these cells into murine and porcine cardiac tissue, with inconclusive
evidence regarding the capacity of these human cells to physiolog-
ically engraft in xenotransplantation assays. Further, the potential
of hESC-derived cardiovascular lineage cells to functionally couple
to human myocardium remains untested and unknown. Here, we
have prospectively identified a population of hESC-derived ROR2+/
CD13+/KDR+/PDGFRα+cells that give rise to cardiomyocytes, endo-
thelial cells, and vascular smooth muscle cells in vitro at a clonal
level. We observed rare clusters of ROR2+cells and diffuse expres-
sion of KDR and PDGFRα in first-trimester human fetal hearts. We
then developed an in vivo transplantation model by transplanting
second-trimester human fetal heart tissues s.c. into the ear pinna of
a SCID mouse. ROR2+/CD13+/KDR+/PDGFRα+cells were delivered
into these functioning fetal heart tissues: in contrast to traditional
murine heart models for cell transplantation, we show structural
and functional integration of hESC-derived cardiovascular progeni-
tors into human heart.
engraftment|surface markers|Stem cells|mature cardiomyocytes|
cursors during development, but there is little evidence to
support a robust postnatal regenerative capacity (1, 2). As a con-
sequence, myocardial injury or disease in adult humans results in
irreversible cardiomyocyte loss that can lead to progressive heart
failure. Cell transplantation may be an effective therapy to com-
pensate for myocardial loss in an attempt to improve the pumping
ability of the damaged heart (3). The expected mechanism by
which the grafted cells may restore function is to couple with the
native host myocardium, thereby functionally replacing the dead
tissue, with the assumption that the grafted cells or tissue adopt
a cardiovascular fate in situ (4, 5). However, no study to date has
demonstrated the delivery of a pure population of tissue-specific
stem cells capable of generating functioning cardiomyocytes in the
injured or healthy myocardium. Although adult stem cells have
many reports that demonstrate the nonplasticity of adult organ-
specific stem cells (6, 7), human ES cells (hESCs) have proven to
be a potential and unlimited source for generating cardiomyocytes
in vitro as a result of their pluripotent nature (8).
Although several studies have reported efficient differentiation
of hESCs toward cardiovascular lineages, the two most significant
barriers to therapy remain the heterogeneity of the putative pro-
remains insufficiently characterized for prospective clinical appli-
cation (9–11). The current method to evaluate the in vivo de-
velopmental potential and functional properties of hESC-derived
uman cardiomyocytes are derived from proliferating pre-
[commonly murine (muridae includes mouse and rat) or porcine
heart models, it is unclear whether they functionally integrate or
whether demonstration ofsucha proof-of-principle conceptwould
have relevance to the human. A recent report has shown electro-
mechanical integration of hESC-derived cardiomyocyte grafts in
guinea pig hearts (14). A system to prospectively isolate cardio-
vascular stem cells/progenitors and to evaluate their in vivo de-
velopmental potential in functioning human hearts will be an
important step in clinical translation for myocardial regeneration.
Identification of surface markers that are uniquely expressed on
hESC-derived cardiovascular progenitors allows for their pro-
spective isolation. Here, we prospectively isolate an enriched pop-
surface markers. We then demonstrate the structural and func-
tional integration of the hESC-derived cardiovascular progenitors
in beating human fetal heart tissues.
Improved Culture Conditions to Promote Differentiation of hESCs to
Primitive Streak, Mesoderm, and Cardiac Mesoderm Progenitors. To
promote efficient cardiovascular differentiation from hESCs, it is
necessary to elucidate the signaling pathways that regulate cardio-
genesis during embryonic development and to manipulate them in
vitro. To this end, we established a protocol based on stage-specific
activation and then inhibition of the canonical Wnt/β-catenin
pathway: we generated embryoid bodies (EBs) from the hBCL2-
in this transgenic line greatly improves the survival of hESCs in
culture and experiments (15)] in serum-free media and temporally
exposed them to activin A, BMP4, Vascular Endothelial Growth
Factor (VEGF), and Fibroblast Growth Factor 8 (FGF8) (Fig. S1)
(16). Gene expression analysis of the populations generated at
mesodermal genes initially (T or Brachyury and Mix-Like Homeo-
box), followed by cardiac mesodermal genes (MESP1), and car-
with noncardiovascular developmental fates remained in the final
hESC-Derived Cardiovascular Progenitors Express a Unique Surface
Marker Expression Signature. We recently screened a large panel
Author contributions: R.A., M.A.I., T.A.B., M.D., and I.L.W. designed research; R.A., S.R.A.,
O.J.A., M.Q.C., M.Y., and Y.G. performed research; R.A. contributed new reagents/analytic
tools; R.A., T.A.B., R.N., M.D., and I.L.W. analyzed data; and R.A., S.R.A., and I.L.W. wrote
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or irv@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| February 26, 2013
| vol. 110
| no. 9
from differentiating hESCs (17). Several markers were shown to be
expressed individually or in combination on mesodermal cells. A
ROR2, and aminopeptidase-N (i.e., CD13) were individually
shown to allow FACS to enrich for mesodermal progenitors. Be-
cause cardiac cells develop from a Flk-1 (KDR) (11)-expressing
population, and the embryonic heart expresses platelet-derived
growth factor receptor-α (PDGFR-α) (18), we added these mAbs
to the screening protocol.
To determine whether cardiovascular progenitors develop from
a subpopulation of differentiating hESCs that expresses one or
more of these surface markers, EBs were analyzed for expression
ofROR2,CD13,KDR, and PDGFR-α after5 dofdifferentiation.
As shown in Fig. 1A and Fig. S2A, a distinct population marked by
coexpression of ROR2 and CD13 emerges temporally as hESCs
differentiate. This population exhibited a transcriptional profile
similar to primitive streak/mesodermal cells (Fig. 1B and Fig. S2).
Induction of genes such as T (Brachyury), MIXL, FOXA2, and
SOX17 revealed patterns consistent with generation of cells of
mesodermal as well as endodermal lineages.
The ROR2+/CD13+population was sorted, and expression of
KDR and PDGFRα was examined and confirmed. We then eval-
uated the lineage commitment of the ROR2+/CD13+/KDR+/
PDGFRα+population [hereafter referred to as the quadruple-
positive (QP) population]. The QP population expressed high
levels of cardiac mesoderm and cardiac developmental genes,
including mesoderm posterior 1 (MESP1), the earliest known
marker for cardiogenesis, and key cardiac transcription factors
of the primary and secondary heart fields, including TBX5,
GATA4, MEF2C, NKX2.5, and ISL1 (Fig. 1B and Fig. S2) (19, 20).
four markers had the highest expression of pluripotency genes,
indicative of residual undifferentiated hESCs. Although the QP
genes corresponding to the primitive streak and endoderm, al-
though to a much lesser degree (Fig. S2). Enrichment for cardiac
level detection of NKX2-5, MEF2C, and GATA4 (Fig. 1C).
Expression of ROR2, KDR, and PDGFRα During Early Human Fetal
Heart Development. To elucidate the expression of ROR2, CD13,
KDR, and PDGFRα during in utero heart development, human
fetal hearts were sectioned and immunostained for these proteins.
KDR and PDGFRα were broadly expressed in 9- to 10-wk-old
human fetal cardiac tissue, including in the vasculature. Rare,
distinct areas of ROR2 expression were detected in the myocar-
diumandinterventricularseptum,but notinthe epicardium(Fig.1
D and E). In contrast, we could not detect CD13 expression. Ex-
pression of ROR2 was limited to the human fetal hearts from the
ROR2. Furthermore, examination of adult human myocardial
tissues revealed no expression of ROR2 proteins.
QP Progenitor Population Gives Rise to Cardiomyocytes and Endothelial
and VascularSmoothMuscle Cells.To further characterize the in vitro
developmental potential of the QP progenitor population, freshly
sorted QP cells were cultured as aggregates in suspension for 7 to
10 d in the presence of Wnt11 and FGF8 in serum-free media.
Consistent with the gene expression profile described earlier, the
QP population gave rise to cells of the cardiovascular lineage based
on immunostaining and gene expression (Fig. 2 A and B and Fig.
S3A). We consistently detected a high frequency of cardiomyocytes
beating spontaneously as a synchronous mass (Movie S1). When
plated on Matrigel-coated dishes and treated with a high concen-
tration of VEGF, the QP cells acquired the morphology of endo-
thelial cells and formed a lattice (Fig. S3B). These cells expressed
CD31 and von Willebrand factor and efficiently incorporated Dil-
ROR2 DapiPDGFR Dapi
ROR2 NKX2-5 Dapi
fetal hearts. (A) Flow cytometric analysis of EBs at different time points of differentiation. On day 5, a distinct population defined by coexpression of ROR2
and CD13 (II) appeared, which was further analyzed for expression of KDR and PDGFRα. (B) Quantitative RT-PCR gene expression analysis of the QP (III), ROR2+
CD13+(II), and QN (I) cells isolated from day-5 EBs. The average expression is normalized to GAPDH. Data represent mean ± SD for three biologically in-
dependent experiments (P < 0.05, one-way ANOVA, populations III vs. I and II vs. I). (C) Presence of NKX2-5 (Left), MEF2C (Middle), and GATA-4 (Right)
immunostaining in the QP population 24 h after sorting (Fig. S2). (Scale bar, 50 μm.) (D) Immunofluorescence staining of first trimester human hearts revealed
pockets of ROR2-positive cells and diffuse KDR and PDGFRα staining in the left ventricle (arrows). (Scale bar, 120 μm.) (E) An area of the left ventricle with
a cluster of ROR2+cells that also costain with NKX2-5. (Scale bar, 120 μm.)
Identification of a cardiac mesoderm population marked by four surface markers—ROR2, CD13, KDR, and PDGFRα—and their expression in human
| www.pnas.org/cgi/doi/10.1073/pnas.1220832110Ardehali et al.
labeled acetylated LDL (Dil-Ac-LDL), confirming their endothe-
lial phenotype functionally (Fig. 2 C and D).
To quantify the extent of cardiomyocyte generation, a cardiac
troponin-GFP reporter hESC line was used and the QP pop-
ulation was maintained in culture for more than 30 d. More than
55% of the derived cells developed into cardiomyocytes, based on
troponin expression, reflecting efficient enrichment of progenitors
in the QP population (Figs. S3C and S4 A and B; Tables S1 and
S2).To determine the nature oftheothercelltypesthat arisefrom
QP cells, we performed immunostaining for endodermal, ecto-
dermal, and hematopoietic (i.e., mesodermal) lineages. The ma-
jority of these cells stained positive for vimentin, a marker
expressed on many cell types, including fibroblasts (Fig. S3D).
These cells appear to expand rapidly over time (Fig. S3C).
evaluated 10 d after sorting, using field potential measurements, as
well as whole-cell patch-clamp recording (Fig. 3A). Synchronous
multifocal field potential recordings performed on microelectrode
arrays showed homogeneous spread of electrical activity through-
out the adherent cultures (Fig. S5 A–C). Additionally, action po-
tential (AP) recordings from single cells revealed the presence of
pacemaker-, atrial-, and ventricular-like patterns characterized
predominantly by a fast phase 1 depolarization. More than 90% of
the single cells studied exhibited a ventricular-like AP morphology.
These results confirm that the QP population can differentiate to
contractile cardiomyocytes with a fetal-like AP phenotype (21).
To further understand the mechanism by which QPcells commit
to the cardiovascular fate, we tested whether the addition of con-
ditioned media from cultured QP cells to control (i.e., unsorted)
EBs could recapitulate the cardiac specification of QP cells. The
daily addition of the QP conditioned media did not promote
cardiac differentiation in control cells, indicating that QP cells do
not secrete soluble proteins that enhance cardiovascular specifi-
cation, arguing against a cell-nonautonomous mechanism. We
next confirmed the specification of the QP population to a car-
diomyocyte fate by transferring freshly sorted QP cells derived
from a GFP-expressing hESC line into a synchronous EB derived
from unlabeled hESCs. The majority of the GFP+cells in the
chimeric EB developed into contracting foci (Fig. S5 D–F and
Movie S2). These results suggest that QP cells are a distinct
population with commitment to cardiovascular fate, most likely
through a cell-autonomous mechanism.
Single QP Cell Is Multipotent for Cardiovascular Lineages. To de-
termine further whether the QPcells are multipotential in a clonal
manner, a single QP cell from a GFP-expressing hESC line was
sorted directly into each well of a 96-well plate containing 1,000
WT QP cells. This approach was taken as a result of the poor
survival of single QP cells when grown individually compared with
their growth in clusters, in which they have better survival. After
a few days in culture, colonies emerged that includedfoci of GFP+
cells, which were analyzed to determine the differentiation po-
tential of a single QP cell (Fig. S6 A–D). Immunostaining of these
colonies confirmed the presence of GFP+cardiomyocyte, endo-
thelial, and vascular smooth muscle cells in these culture clones
(Fig. 3B and Fig. S6D). Taken together, the clonal analysis shows
cardiomyocytes, endothelial, and vascular smooth muscle cells in
vitro, as would be expected of an embryonic cardiac progenitor.
VE Cadherin Dapi
of QP cells 6 d after sorting for markers of cardiomyocytes and smooth muscle
and endothelial cells. (Scale bar, 25 μm.) (B) Quantitative RT-PCR analysis of QP
cells grown in culture after 13 d after sorting for cardiac genes.Datarepresent
to VEGF after sorting, QP cells (derived from hBCL2-hESC line and therefore
expressing GFP) formed a lattice of tubular structures. (Scale bar, 100 μm.) (D)
Endothelial phenotype was further confirmed by Dil-labeled acetylated LDL
uptake. (Scale bar, 50 μm.)
In vitro characterization of QP cells. (A) Immunofluorescence analysis
ADP90 (ms) Peak Amplitude (mV) Resting Potential (mV) Frequency of AP (per min)
500 ± 39 37 ± 1.3 -43 ± 1.4 21 ± 2.4
ventricular atrial nodal
(n=16) (n=1) (n=1)
recordings of spontaneous APs demonstrate ventricular-, atrial-, and nodal-
like APs in the cultured QP population (Fig. S5). (B) Immunostaining of cells
grown from a single GFP-QP cell indicates the presence of cardiomyocytes and
endothelial and smooth muscle cells. (Top Left) Corresponding GFP cells (Fig.
S6). (Scale bar, 50 μm.).
Developmental potential of QP cells. (A) Whole-cell current-clamp
Ardehali et al.PNAS
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QP Cells Transplanted into Mouse Hearts Mature to Cardiomyocytes
but Fail to Integrate. To test their in vivo developmental potential,
day-5 EB-derived QP cells from a GFP-hESC line were trans-
planted into healthy or injured hearts of nonobese diabetic/SCID
mice with common γ-chain KO. Approximately 5 to 10 × 105QP
cells were sorted and immediately transplanted by direct injection
into the left ventricle of healthy mice or into the periinfarct area of
mice following occlusion of the left anterior descending artery. As
controls, quadruple-negative (QN) cells from the same EB culture
as described earlier were also sorted and transplanted in similar
areas in healthy and injured nonobese diabetic/SCID mice with
common γ-chain KO. The animals were euthanized after 8 wk, and
histological analyses of the explanted QP-transplanted hearts
showed clusters of GFP-positive cells throughout the injected area
(Fig. 4A). Although no teratomas were observed in any of the
animals transplanted with the QP cardiovascular progenitors, one
of the seven mice transplanted with the QN cells developed ter-
atomas in the heart (Fig. 4D and Fig. S7D), demonstrating that
even EB cells exposed to as much as 5 d of potent differentiation
basis of the QP markers. The GFP-positive QP cells were detected
only as clusters in the injection sites without significant migration,
but exhibited cardiac differentiation as evidenced by expression of
troponin and myosin heavy chain (Fig. 4 B and C). Despite their
engraftment and differentiation into mature cells, detailed histo-
formation between hESC-derived cardiovascular progenitors and
the host myocardium: the human QP cells failed to integrate with
derived cardiomyocytes to structurally and functionally integrate
into the adult mouse host may be a result of several factors, in-
cluding: (i) interspecies differences that prevent the coupling of
optimal environment for maturation and integration of the cardiac
progenitors, and/or (iii) an inherent inability of QP-derived car-
diovascular progenitors to functionally integrate.
Structural and Functional Integration of QP Cells in Viable Fetal Human
Heart. To address these issues, we developed a transplantation
model that allows us to assess the functional development of
23). The ventricular tissues from a second-trimester human fetal
heart (15 wk gestation) were implanted s.c. into a pouch formed in
the ear pinna of an SCID mouse (Fig. S7A and Movie S3). Graft
viability was confirmed by the presence of autonomous beating
determinedbyvisualinspectionandelectrocardiography∼7to 10 d
after implantation (Fig. S7B and Movie S3). Two weeks later, ∼5 ×
105freshly sorted QP cardiovascular progenitors (and QN cells as
control) from a GFP-hESC line were transplanted into the heart
graft. The animals were euthanized after 8 wk, and confocal mi-
croscopy of the explanted heart receiving QP cells revealed clusters
of GFP-positive cells spread throughout the myocardium, including
areas distant from the injection site (Fig. 5A). Such a distribution of
the grafted cells is most likely a result of migration or simple
spreading, in contrast to the possibility of the cells passively
spreading along the injection site (5). The GFP-positive cells
coexpressed troponin, α-actinin, or CD31, which suggests in vivo
differentiation of the progenitors into cardiomyocyte and endo-
thelial lineages (Fig. 5 B and C and Fig. S7C). No teratoma for-
mation was observed in animals receiving QP cells (n = 4). In
contrast, transplantation of QN cells resulted in teratoma forma-
tionin one ear–heartmodel(n = 4), and transplantedQNcellsdid
not differentiate into cardiomyocyte or endothelial lineages.
heart tissues revealed typical punctate staining for connexin-43
along the regions of intimate cell-to-cell contact between hESC-
derived cardiomyocytes (GFP+) and host cardiomyocytes (Fig. 5 B
and C). These results indicate that, when transplanted into a hu-
man fetal heart, hESC-derived cardiovascular progenitors not only
mature to cardiomyocytes, they can also migrate and couple
structurally to their neighboring allogeneic cells. We seldom ob-
served phosphohistone H3-positive QP cells in explanted sections
(phosphohistone H3 is a marker for mitotic activity), suggesting
that, at some point within 8 wk after transplantation, the QP cells
lose the robust proliferative activity they exhibit in vitro (Fig. S7E).
Although junctional proteins connecting QP cells to the sur-
rounding cells were readily detected, it is important to determine
whether the transplanted cells were also electrically connected to
the host myocardium. Human fetal heart tissues with QP
transplantation were removed from the mouse ear and immedi-
ately sectioned, and real-time Ca2+transients were measured in
areas with QP-derived GFP-positive cells. GFP-positive cells dem-
onstrated periodic Ca2+oscillations similar to—and synchronized
with—the host cells (24). The Ca2+oscillations responded to in-
creasing frequencies of external electrical stimulation. These
recordings showed conduction of Ca2+signals from the host myo-
cardium into areas of GFP-positive transplanted cells resembling
a continuous electrical propagation (Fig. 6A and Movie S4). Al-
though these results indicate functional integration of the trans-
planted QP cells into the human myocardium, the present model
has limitations. We cannot rule out the possibility that the graft can
be transilluminated by calcium transients in the surrounding tissue.
As some studies havereportedfusion between transplantedcells
andhost myocardium(25, 26),wesoughtto determine whetherthe
extent of cell fusion was significant enough to explain our findings.
Because the H9 ESC line is of female origin and the transplanted
by evaluating for the presence of the Y chromosome in the GFP
transplanted cells (derived from XX H9 cells). We traced donor
GFP-positive cells for FISH with X- and Y- chromosome paints to
GFP DapiGFP Dapi
GFP DapiTroponin Dapi Merge
mouse heart explanted 8 wk after injection of GFP+QP cells. The dotted circle
indicates the approximate location of the infarct zone. (Scale bar, 0.5 mm.)
(Middle and Right). (Scale bar, 120 μm.) (B and C) Immunofluorescence
staining of mouse hearts 8 wk after QP transplantation reveals presence
of GFP-hESC–derived QP cells that costain with troponin (B) and with
human cardiomyocyte-specific β-myosin heavy chain (C). (Scale bar, 100 μm.)
(D) Immunohistochemical evidence for teratoma formation after 8 wk upon
transplantation of QN cells. The QN-derived cells gave rise to all three germ
layers including columnar epithelium (Left), cartilage (Center), and neural
rosette (Right). (Scale bar, 100 μm.)
Engraftment of QP cells in mouse myocardium. (A) (Left) Whole
| www.pnas.org/cgi/doi/10.1073/pnas.1220832110Ardehali et al.
assess karyotype (Fig. 6B). Fewer than 3.8% of the transplanted
cells expressed the Y in addition to the X chromosome, which may
be an overestimate for degree of cell fusion as a result of the
overlap of nuclei of unique cells on 6-μm sections contributing to
this value. This experiment indicates that, although rare fusion
the transplanted cardiovascular progenitors into the host tissue.
PDGFRα, that mark early cardiovascular progenitors among dif-
ferentiating hESCs. These cells, designated QP cells, can give rise
to three distinct cell populations, namely cardiomyocytes, endo-
thelial cells, and vascular smooth muscle cells.
The possibility that progenitors marked by QP markers repre-
sent only a distinct, transitory state of differentiating hESCs can-
not be formally excluded. We have provided evidence that ROR2/
CD13 cells may resemble a population of primitive-streak/meso-
dermal cells. This claim is based on several lines of evidence from
our work and that of others: (i) the similarity of the gene expres-
sion profiles of ROR2/CD13 cells and primitive streak cells (i.e.,
expression of MIXL, T, GSC, Mesp1, KDR, GATA4); (ii) in vitro
phenotype of a subpopulation of ROR/CD13 cells that differen-
tiate to cardiomyocytes, vascular smooth muscle cells, and endo-
thelial cells, which are mesoderm-derived progeny; (iii) expression
of ROR2 in the first-trimester human fetal heart; (iv) transplanta-
tion into the human fetal heart of a subpopulation of ROR2/CD13
cells resulting in robust commitment toward cardiovascular line-
ages; and (v) expression of ROR2 in the entire region of the
primitive streak in embryonic day 7.5 mouse embryo and sub-
sequent ROR2 mutation in mice leading to defects in somito-
genesis and cardiogenesis (27, 28). To formally show that QP cells
also exist in utero is ethically impermissible, as it would require
access to early human embryos to comprehensively screen for
ROR2 and CD13 expression in early human development (i.e.,
during primitive streak and lateral plate mesoderm formation).
Nonetheless, the identification of cells that can generate three
major cell types in the heart provides a unique opportunity to
investigate the mechanisms that regulate the onset of human
cardiac development as well as those that control their specifica-
tion to the cardiac and vascular cells.
processes, cell migration, and polarity (29, 30). It has been shown
mouse embryonic development and later in the developing limbs,
brain, heart, and lungs (27). In humans, mutations in the ROR2
gene have been associated with autosomal-recessive Robinow
syndrome, characterized by short stature, mesomelic limb short-
ening, abnormal craniofacial features, and distinct cardiac anom-
alies affecting the myocardium (31). The observed phenotypes
arising from deficient or mutated ROR2 emphasizes its essential
roles in morphogenetic and developmental processes.
Although the potential of hESCs to generate cardiovascular
cells is indisputable, a set of challenges remains that limit the
therapeutic application of hESC-derived cells. A major concern
GFP DapiGFP Dapi GFP DapiGFP Dapi
in a human fetal heart. (A) Myocardial
sections from human fetal heart tissues 8
wk after transplantation s.c. into mouse
ear and delivery ofQPcells shows clusters
of GFP+cells spread throughout the
ventricle. The site of injection of QP cells
in the first micrograph is in the left upper
corner. (Right, Inset) High-magnification
image of sarcomeric structure. (Scale bar,
100 μm.) (B and C) Coexpression of GFP
withcardiac-specific markerstroponin (B)
or α-actinin (C) and Connexin43 staining
between host and transplanted GFP+
cells. (Right) Overlay with Connexin43
staining depicted in red and troponin or
α-actinin in white. (Scale bar, 100 μm.)
Structural Integration of QP cells
show evoked calcium signals when paced electrically ex vivo. Fluo-4 calcium dye
wasaddedtotissue, whichwasthen electricallypacedat 2Hz.(Right)Samearea
after treatment with anti-GFP antibody reveals a GFP+area. This region was
analyzed for dye intensity changes (f) and results are plotted normalized to the
intensity of the initial movie frame (f0). Real-time Ca2+flux through the tissue
indicate functional integration of GFP+cells into the host tissue. (B) GFP+cells,
derived from XX H9 ESCs, were traced for FISH analyses to reveal XX karyotype
(white arrowhead, Inset), whereas the host myocardium, from a male donor,
expresses XY karyotype (white arrowhead, Inset). (Scale bar, 50 μm.)
Ardehali et al. PNAS
| February 26, 2013
| vol. 110
| no. 9
is their capacity to from teratomas after transplantation (32). Download full-text
The progenitor population we isolated based on the expression
of distinct surface markers did not form teratomas when trans-
planted into mouse or human hearts. On the contrary, the
population characterized by absence of the QP surface proteins
resulted in teratoma formation upon transplantation into mouse
or human hearts, which indicates that pluripotent stem cells can
remain in culture even after 5 d of differentiation.
Another significant challenge to clinical application relates to
the fate of the hESC-derived cardiac cells upon transplantation
(12). Several investigators have reported engraftment of hESC-
derived cells in infarcted mouse, rat, guinea pig, and pig hearts
(14, 33, 34). Aside from the recent report of electromechanical
integration of hESC-derived cardiomyocytes into guinea pig
hearts, careful examination of animal models has revealed that
the transplanted cells form islands of nascent myocardium within
the scar zone (12). Furthermore, the rapid heart rate of mice
(∼600 beats/min) may prevent the human cardiac cells, which
have an intrinsic rate of 60 to 100 beats/min, from keeping pace
with the mouse cardiomyocytes. In our experiments involving QP
cell transplantation, we failed to see coupling of these cells
within the mouse heart. In contrast, transplantation of QP cells
into the human fetal heart revealed maturation, migration,
coupling with resident cardiomyocytes, and electrophysiological
activity in concert with the host myocardium.
Our findings clarify the ambiguity regarding the fate of trans-
planted hESC-derived cardiovascular cells. We show here that
cardiovascular progenitors generated from hESCs engraft into
mouse hearts, but fail to integrate. However, when transplanted
into a human fetal heart tissue, they integrate into the host myo-
cardium. A major limitation of the described model is that it is not
amenable to a variety of physiological activities (i.e., hemody-
namics or sinoatrial and atrioventricular conduction), the lack of
which may influence the development of hESC-derived cardio-
vascular progenitors. Fetal hearts also have much less developed
connective tissue, which may promote integration of the QP cells
that would be absent in adult hearts. Additionally, the present
be a different environment for engraftment than an injured heart,
and, moreover, does not allow us to investigate whether the
engrafted cells could provide any functional improvement after
injury. Nevertheless, the data herein establish the transplantability
of hESC-derived cardiovascular progenitors into human fetal
hearts as a proof of concept of functional allogeneic integration.
Taken together, the data presented in this article suggest that
hESC-derived cardiovascular progenitors, defined by four surface
markers, can structurally and functionally integrate into the elec-
trical syncytia of human fetal heart tissue upon transplantation.
Additionally, our finding of ROR2 as an early marker for cardiac
lineage specification highlights a previously unknown role of ROR2
expression in cardiac development. Further research to delineate
the mechanism by which ROR2 is involved in early cardiovascular
progenitor formation is warranted. These valuable results provide
the basis for future hESC-based cardiac therapy by identification of
a progenitor population capable of engraftment and regeneration
without risk of teratoma formation.
Materials and Methods
Human ES cells were maintained in standard ES culture as described. EBs
were differentiated in the presence of Wnt 3a, BMP4, VEGF, activin A, sFz 8,
FGF8, and Wnt11 Tables S3 and S4. Fetal heart tissues were obtained from
Advanced Bioscience Resources. The Stanford institutional review board
approved the use of one fetal heart for transplantation into a mouse ear,
followed by injection of hESC-derived cardiovascular progenitors. A more
detailed discussion is provided in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. Yoav Soen for valuable advice, Dr.
Mirko Corselli for his help in immunostaining, Libuse Jerabek for excellent
laboratory management, and members of the I.L.W laboratory for advice
and assistance. This work was supported by California Institute for Re-
generative Medicine Grant RCI 00354 (to I.L.W); National Institutes of
Health/National Heart, Lung, and Blood Institute Fellowship 5T32 HL00708;
an American College of Cardiology/Pfizer Career Development Award (to
R.A.); the Howard Hughes Medical Institute (to S.R.A.); and the California
Institute for Regenerative Medicine (to R.N.).
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