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2010 116: 5762-5772
Prepublished online September 30, 2010;
doi:10.1182/blood-2010-04-280719

Efficient differentiation of human pluripotent stem cells into functional
CD34 + progenitor cells by combined modulation of the MEK/ERK and
BMP4 signaling pathways
Sang-Wook Park, Young Jun Koh, Jongwook Jeon, Yun-Hee Cho, Mi-Jin Jang, Yujung Kang,
Min-Jeong Kim, Chulhee Choi, Yee Sook Cho, Hyung-Min Chung, Gou Young Koh and Yong-Mahn
Han

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VASCULAR BIOLOGY

Efficient differentiation of human pluripotent stem cells into functional CD34⫹
progenitor cells by combined modulation of the MEK/ERK and BMP4 signaling
pathways
Sang-Wook Park,1 Young Jun Koh,2 Jongwook Jeon,2 Yun-Hee Cho,1 Mi-Jin Jang,1 Yujung Kang,3 Min-Jeong Kim,4Chulhee Choi,3 Yee Sook Cho,4 Hyung-Min Chung,5 Gou Young Koh,2 and Yong-Mahn Han1
1Department of Biological Sciences and Center for Stem Cell Differentiation, 2Graduate School of Medical Science & Engineering, and 3Department of Bio andBrain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea; 4Development & Differentiation Research Center,
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea; and 5Department of Biomedical Science, CHA University,
Seoul, Republic of Korea

Differentiation of human pluripotent stem
cells (hPSCs) into functional cell types isa crucial step in cell therapy. In the present
study, we demonstrate that functional
CD34ⴙ progenitor cells can be efficiently
produced from human embryonic stem
cells (hESCs) and induced pluripotent
stem cells (hiPSCs) by combined modulation of 2 signaling pathways. A higher
proportion of CD34ⴙ cells (⬃ 20%) could

be derived from hPSCs by inhibition of
mitogen-activated protein kinase (MAPK)
extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated
kinase (ERK) signaling and activation ofbone morphogenic protein-4 (BMP4) signaling. hPSC-derived CD34ⴙ progenitor
cells further developed to endothelial andsmooth muscle cells with functionality.
Moreover, they contributed directly to neo-vasculogenesis in ischemic mouse hindlimbs, thereby resulting in improved blood
perfusion and limb salvage. Our results
suggest that combined modulation of signaling pathways may be an efficientmeans of differentiating hPSCs into functional CD34ⴙ progenitor cells. (Blood.
2010;116(25):5762-5772)

Introduction
Human embryonic stem cells (hESCs) derived from an early
embryo can self-renew indefinitely and differentiate into a varietyof cell types.1 It has been reported that the “stemness” of hESCs islikely maintained through the harmonious actions of signaling
pathway networks.2 Basic fibroblast growth factor (bFGF) isessential for maintaining the stemness of hESCs by highly activating mitogen-activated protein kinase (MAPK) extracellular signalregulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling, which plays an important role in thestemness of hESCs.3 Stemness of hESCs is also supported by
bFGF-mediated regulation of transforming growth factor-␤ (TGF-␤)signaling4; activation of the TGF-␤/activin/nodal signaling pathway is required to maintain stemness in cooperation with the FGF
signaling pathway, whereas its inhibition results in differentiationof hESCs.5,6 The effect of Wnt signaling on stemness of hESCs is
still controversial. Activation of the Wnt pathway by 6-bromoindirubin-3-oxmie, a specific inhibitor of glycogen synthase-3, sustains
the undifferentiated status of hESCs.7 However, activation of Wnt
signaling is insufficient to maintain the undifferentiated status of
hESCs, because a canonical Wnt signaling is highly activatedduring differentiation.8
The stemness of human induced pluripotent stem cells(hiPSCs), like that of hESCs, seems to be maintained by coordinated networks of signaling molecules, although few differences
are observed in the gene expression profile.9,10 Thus, the stemnessand differentiation of hESCs and hiPSCs is regulated by complex
networks of signaling pathways. It is likely that the modulation ofthese signaling pathways can induce differentiation of hESCs into a
specialized cell type. In fact, bone morphogenic protein 2/4 (BMP2/4), a
member of the TGF␤ superfamily, could differentiate hESCs into
trophoblasts, primitive endoderm cells, and mesodermal cells.11-13
However, it has been reported that BMP4 is required for sustaining
stemness of mouse ESCs by blocking neural differentiation.14 hESCs
could be differentiated to definitive endoderm cells by activation ofactivin/nodal signaling and suppression of phosphoinositide 3-kinase(PI3K) signaling.15 Dual inhibition of SMAD signaling by treatment
with Noggin and SB431542 resulted in differentiation of hESCs andhiPSCs into neural cells.16 Many studies have tried to isolate specialized
cell types from spontaneously differentiated cells via formation ofhESC-derived embryoid bodies using antibodies against cell-typespecific surface markers.17,18 However, spontaneous differentiation
remains inefficient, in that hPSCs cannot be guided toward a specialized
lineage at the initial commitment step.
The hPSCs provide a possibility that degenerative or damaged
tissues can be replaced with hPSC-derived functional cells. A
paucity of number and activity of endothelial progenitor cells iscorrelated with cardiovascular diseases such hypercholesterolemia,
hypertension, and diabetes mellitus.19 Therefore, endothelial progenitor cells are able to be used for curing such cardiovasculardiseases. In fact, endothelial progenitor cells derived from bonemarrow or cord blood were effective on vasculogenesis in ischemic
diseases.20,21 Therefore, it is suggested that transplantation ofvascular cells into ischemic regions may enhance restoration oftissue revascularization.22 Recently, injection of hESC-derived

Submitted April 19, 2010; accepted September 20, 2010. Prepublished online
as Blood First Edition paper, September 30, 2010; DOI 10.1182/blood-201004-280719.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.

The online version of this article contains a data supplement.

© 2010 by The American Society of Hematology

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BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

endothelial cells was found to salvage ischemic hind limbs andenhance blood perfusions.23,24 However, there are still limitationsof cell therapy using hESC-derived cells, including low efficiency
of differentiation into specialized progenitors and the use of animal
sources such as animal serum and feeder cells. In an effort toaddress these issues, we developed a simple and efficient methodby which hESCs and hiPSCs are directly differentiated to functional CD34⫹ progenitor cells through regulation of signaling
pathways. Both hESC- and hiPSC-derived CD34⫹ cells weredifferentiated into functional endothelial cells, and contributed toblood perfusion and limb salvage through the neovasculogenesis inhind limb ischemic mice, respectively.

Methods
Maintenance and feeder-free culture of hESCs and hiPSCs
The hESCs (CHA4-hES,25 supplemental Figure 1) and hiPSCs (supplemental Figure 2) were cultured in ESC medium on mitomycin C(Sigma-Aldrich)–treated STO (ATCC no. CRL-1503) feeders at 37°C, in air
containing 5% CO2. The ESC medium consisted of Dulbecco modified
Eagle medium (DMEM)/F12 medium containing 20% knockout serum
replacement, 1% nonessential amino acids, 0.1mM ␤-mercaptoethanol, and4 ng/mL of bFGF (all from Invitrogen). For feeder-free culture, hPSCs weremaintained on Matrigel (BD Biosciences)–coated culture dishes in STOconditioned medium.
RT-PCR and real-time RT-PCR
For extracting total RNA, hPSCs or mouse tissues were homogenized in
TRIzol (Invitrogen) according to the manufacturer’s protocol. Then, 1 ␮gof total RNA was used to generate first-strand cDNA using Superscript IIreverse transcriptase (RT) (Invitrogen), and cDNA was amplified bypolymerase chain reaction (PCR) using the PCR PreMix (Genet Bio,
Daejeon, Korea). The specific primers used in this study are listed insupplemental Tables 1 to 3. The RT-PCR reaction was performed with an
initial step at 95°C for 5 minutes, followed by 25 to 35 cycles of 30 seconds
at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C, and a final
elongation step at 72°C for 5 minutes. The PCR products were resolved
on a 2% agarose gel by electrophoresis. Relative expression levels
among respective genes were analyzed by using an iCycler iQ5 real-time
detection system (Bio-Rad Laboratories). All reactions were performed intriplicate. For comparative quantification, the expression level of respective
genes was normalized to that of GAPDH, and expressed as a fold change
relative to the expression level in undifferentiated hESCs. The sample ⌬Ct(S⌬Ct) value was calculated from the difference between the Ct values of
GAPDH and the target genes. The relative gene expression levels between
the sample and control were determined using the formula 2⫺(S⌬Ct⫺C⌬Ct).
Immunostaining
The cells were washed with phosphate-buffered saline (PBS) and fixed in
4% formaldehyde at room temperature for 15 minutes. For detection of
nuclear proteins, the hPSCs were permeabilized with 0.1% Triton X-100 in
PBS, and blocked with 4% normal goat serum or fetal bovine serum (FBS)
for 1 hour at room temperature. After that, antibodies against octamerbinding transcription factor 4 (OCT4; R&D Systems, 1:300), T (R&D
Systems, 1:100), GATA2 (R&D Systems, 1:100), SSEA-4 (R&D Systems,
1:300), PECAM-1 (R&D Systems, 1:100), Von Willebrand factor (VWF)(Abcam, 1:100), KDR (Cell Signaling Technologies, 1:100), VE-cadherin(R&D Systems, 1:100), ␣-smooth muscle actin (␣-SMA; R&D Systems,
1:100), and calponin (Abcam, 1:100) were diluted with blocking solution
and incubated with the prepared cells at 4°C overnight. Finally, the cells
were washed several times with PBST (0.1% Tween-20 in PBS) andincubated with Alexa Fluor 488– or 594–conjugated secondary antibodies(Invitrogen). Immunostained cells were observed on a fluorescence microscope (Olympus) or a Zeiss LSM 510 confocal microscope (Carl Zeiss).

DIFFERENTIATION OF hPSCs TO FUNCTIONAL CD34⫹ CELLS

5763

Immunohistochemistry
For histologic analysis, mice were anesthetized by intramuscular injection(80 mg/kg ketamine and 12 mg/kg xylazine), and then fixed by vascular
perfusion of 1% paraformaldehyde in PBS. Tissues were harvested andembedded in cryofreezing medium. Cryosections (12 ␮m thickness) wereincubated in PBST (0.3% Triton X-100 in PBS) containing 5% donkey
serum (Jackson ImmunoResearch Laboratories) at room temperature for
1 hour. After blocking, the samples were incubated at 4°C overnight with amouse anti–human CD31 antibody (Abcam, 1:200) combined with ahamster anti–mouse CD31 antibody (Chemicon International, 1:200). Afterseveral washes in PBST, the samples were incubated for 1 hour at room
temperature with fluorescent-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories, 1:500). Nuclei were stained with
4,6-diamidino-2-phenylindole, dihydrochloride (DAPI, 1 ␮g/mL in PBS;
Invitrogen) at room temperature for 30 minutes. Signals were visualized
on a confocal microscope equipped with argon and helium-neon lasers(model LSM 510, Zeiss). For determining functionality of blood vessels,
fluorescein Bauhinia purpurea lectin (2 mg/mL; FL-1281, Vector Laboratories) was intravenously injected into ischemic mice at a volume of
3.75 ␮L/g body weight 7 days after injection of hPSC-derived CD34⫹ cells.
Ten minutes after injection of lectin, the tissue samples were preparedand immunostained with anti–human CD31 antibody and anti–mouse
CD31 antibody, as described above.
Isolation of CD34ⴙ cells by magnetic sorting
To dissociate differentiated hPSCs into single cells, differentiated hPSCs
were treated with TrypLE Express (Invitrogen) for 10 minutes at 37°C and,after gentle pipetting, passed through 40-␮m cell strainers (BD Biosciences).
CD34⫹ cells were then isolated by MACS MagneticBead columns (Miltenyi
Biotec) using an antibody against CD34, according to the manufacturer’s
instructions.
Acetylated low-density lipoprotein uptake assay and vasculartube-like structure formation assay
For the low-density lipoprotein (LDL) uptake assay, endothelial cells derived
from CD34⫹ cells were incubated with 10 ␮g/mL of 1,1⬘-dioctadecyl-3,3,3⬘,3⬘tetramethylindocarbocyanine (Dil)–labeled acetylated LDL (Invitrogen)for 5 hours. Red fluorescent signals were detected with a fluorescencemicroscope. To assess the formation of vascular tube-like structures,1 ⫻ 105 endothelial cells were incubated on Matrigel matrix (BDBiosciences) in endothelial cell growth medium-2 (EGM-2) supplemented
with vascular endothelial growth factor-1 (VEGF-A) for 24 hours. The
vascular tube-like structures were observed with an inverted microscope.
Colony-forming unit assay
A colony-forming unit (CFU) assay was used to determine whether CD34⫹
cells could differentiate to hematopoietic cells. Approximately 1 ⫻ 105
CD34⫹ cells were placed on a 35-mm culture dish containing MethoCult
GF H4434 (StemCell Technologies), which is composed of 1% methylcellulose, 30% FBS, 1% BSA, 0.1mM 2-mercaptoethanol, 2mM L-glutamine,
50 ng/mL of recombinant human stem cell factor, 10 ng/mL of recombinanthuman granulocyte monocyte–colony-stimulating growth factor, 10 ng/mLof recombinant human interleukin-3, and 3 U/mL of recombinant humanerythropoietin. The various hematopoietic colonies were enumerated andidentified at days 14 to 21.
Flow cytometry
Cells were labeled with antibodies against CD105-APC, CD31-PE, CD45FITC, CD73-PE, CD90-APC, and CD34-APC (all from BD Pharmingen)at 4°C for 30 minutes. After being washed twice with PBS containing
1% FBS, the antibody-labeled cells were analyzed with a flow cytometer(LSRII, Becton Dickinson), according to the manufacturer’s instructions.
The data were analyzed using FlowJo Version 7.2.5 software (TreeStar).

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5764

BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

PARK et al

Figure 1. Enhanced expression of mesoderm marker
genes in hESCs and hiPSCs by treating PD98059 and
BMP4. (A) and (B) Relative expression levels of mesoderm (T and WNT3), endoderm (FOXA2 and FOXQ1),
and ectoderm(ZIC1, SOX1, and PAX6) marker genes
between experimental groups in hESCs and hiPSCs.
The values are the mean ⫾ SD of 3 independent experiments. A P value ⬍ .05 was considered to be statisticallysignificant (*P ⬍ .05; n ⫽ 3). (C) Western blot analysis
for phosphorylation of ERK1/2 and SMAD1/5/8 andexpression of a stem cell marker (OCT4) and mesoderm
markers (T, GATA2) in undifferentiated hESCs (control)and PDB4-treated hESCs. (D) Immunostaining for astem cell marker (OCT4) and a mesoderm marker (T) in
undifferentiated hESCs (control) and PDB4-treated
hESCs. Scale bar is 200 ␮m. Abbreviations: Control,
untreated hESCs; PD, PD98059; B4, BMP4; PDB4,
hESCs treated with PD98059 and BMP4.

In vivo Matrigel assay
A total of 1 ⫻ 106 hESC-derived CD34⫹ cells were mixed with Matrigel(100 ␮L, BD Biosciences) and then subcutaneously injected into the flankregion of an 8-week-old BALB/cByJ athymic nude (CbyJ.Cg-Foxn1nu/J)
mouse. Immunohistochemistry was performed 10 days after implantation.
Preparation of ischemic hind-limb mice
BalB/cAnNCriBgi-nu nude male mice were purchased from Charles River
Japan (Yokohama, Japan). Seven- to 8-week-old mice (15-20 g body weight)
were used to make the ischemia model. Hind-limb ischemia was induced byligation and excision of the right femoral artery and vein under ketamine-xylazine
anesthesia. Animal care and experimental procedures were performed under theapproval of the animal care committees of KAIST. For therapeutic angiogenesis
studies, mice were divided into 3 groups after induction of ischemia for
intramuscular injection with culture medium (20 ␮L), CD34⫹ culture(1 ⫻ 106cells/20 ␮L medium) or CD34⫺ culture (1 ⫻ 106cells/20 ␮L medium).
Serial near-infrared fluorescence imaging was performed immediately after
surgery and then on postoperative days (POD) 3 and 7.
Analysis of near-infrared fluorescence imaging
To measure tissue perfusion, we performed indocyanine green perfusion
imaging using a near-infrared fluorescence imaging system (Vieworks), as

described previously.26 Detailed experimental procedures are described in
the supplemental Methods (available on the Blood Web site; see theSupplemental Materials link at the top of the online article).
Statistical analysis
The statistical significance of the real-time RT-PCR data rate was evaluatedby using one-way ANOVA and Bonferroni post-hoc tests, where values ofP ⬍ .05 were considered significant. For the analysis of the perfusion rates
and necrosis probability in the ischemic hind limbs, ANOVA and Scheffe
post-hoc test were performed.

Results
Synergistic differentiation of hPSCs into mesoderm-lineage
cells by regulation of MEK/ERK and BMP4 signaling

In this study, we have established a novel procedure for direct
differentiation of hESCs to mesoderm-lineage cells by combined
regulation of the MEK/ERK and BMP4 signaling pathways(supplemental Figure 3). When hESCs were treated with PD98059
and BMP4 (PDB4) for 3 days, expression of mesoderm marker

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BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

DIFFERENTIATION OF hPSCs TO FUNCTIONAL CD34⫹ CELLS

5765

Figure 2. Generation of CD34ⴙ cells from PDB4treated hESCs after treatment with VEGF-A and bFGF.
(A) and (B) Relative mRNA levels of CD34 between
experimental groups. Error bars indicate the mean ⫾ SDof 3 independent experiments. A P value ⬍ .05 wasconsidered to be statistically significant (*P ⬍ .05; n ⫽ 3).
(C) Proportion of CD34⫹ cells derived from CHA-hES4
and hiPSCs. PDB4-treated hESCs were cultured in
VEGF-A and bFGF-containing medium for 6 and
9 days. The percentage of CD34⫹ cells (n ⫽ 3) is indicated as mean ⫾ SD. (D) Coexpression of CD34
and CD31 in isolated CD34⫹ cells (n ⫽ 2). (E) Transcriptional expression of vascular-lineage genes (␣-SMA,
VE-CADHERIN, and CD31) and hematopoietic-lineage
genes (␤-GLOBIN, ⑀-GLOBIN, ␨-GLOBIN, RUNX1, andLMO2) in CD34⫹ cells. Abbreviations: Control, untreated
hESCs; VE, VEGF-A; bFGF, bFGF; Vb, VEGF-A andbFGF; B4Vb, BMP4, VEGF-A and bFGF.

genes (T and WNT3) were significantly increased compared withthat of PD98059 or BMP4 treatment alone (Figure 1A). However,definitive endodermal (FOXQ1 and FOXA2) and ectodermal(ZIC1, SOX1, and PAX6) genes were not significantly changed(Figure 1A). Similarly, hiPSCs could be also differentiated into
mesoderm lineage cells by treatment with PD98059 and BMP4
(Figure 1B). The expression of the mesoderm markers was
confirmed at the protein level by western-blot analysis (Figure 1C).
As expected, PDB4 treatment enhanced phosphorylation of
SMAD1/5/8 and reduced phosphorylation of ERK1/2 in hESCs.
Moreover, the mesoderm markers T and GATA2 were upregulated, whereas the expression of the stem-cell marker OCT4
was down-regulated in the PDB4-treated sample (Figure 1C). In
addition, the expression of T was observed in PDB4-treated hESCs(Figure 1D) and hiPSCs (supplemental Figure 4). These results
demonstrate that the mesoderm-lineage differentiation of hESCs
and hiPSs could be committed by coordinated regulation of the
MEK/ERK and BMP4 signaling pathways.

Differentiation of PDB4-treated hESCs and hiPSCs into CD34ⴙ
progenitor cells

To further develop the PDB4-treated hESCs into the CD34⫹ cells
downstream of mesodermal lineage, we first investigated theeffects of growth factors. When PDB4-treated hESCs were cultured in ESC medium supplemented with VEGF-A and bFGF (Vb)for 6 days, the expression of a vascular/hematopoietic progenitor
marker gene CD34 was remarkably enhanced compared with
VEGF-A (VE) or bFGF (bF) treatment alone (Figure 2A). It has
been determined that BMP4 is effective on the induction of hESCs
into hematopoietic and endothelial cells.18,27,28 However, in thisstudy, additional BMP4 did not facilitate differentiation of themesodermal cells into CD34⫹ cells (Figure 2A), although BMP4
was essential for mesoderm induction in hESCs, as shown in
Figure 1. Thus, bFGF and VEGF-A were effective in differentiating the PDB4-treated cells into CD34⫹ cells. To determine
whether the expression level of CD34 could be influenced by
upstream protocols for mesodermal induction, PD-, BMP4-, andFrom bloodjournal.hematologylibrary.org at YALE UNIVERSITY on February 18, 2012. For personal use only.
5766

PARK et al

BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

Figure 3. Expression of mature endothelial cell markers. (A) Relative expression of endothelial cell marker
genes (VWF, P-SELECTIN, and ANG2). The samples
were obtained at 3, 6, 9, 12 days of culture. The values
are the mean ⫾ SD of 3 independent experiments.
A P value ⬍ .05 was considered to be statistically significant (*P ⬍ .05; n ⫽ 3). (B-C) Flow cytometric analysis forthe endothelial markers CD31 and CD105 in hESCderived (B) and hiPSC-derived (C) ECs.

PDB4-treated hESCs were cultured in ESC medium supplemented with Vb for 6 days (Figure 2B). The PDB4 group
showed a higher transcriptional expression of CD34 than theother groups, demonstrating that the expression of CD34 may be
proportional to the strength of mesodermal induction. Other
vascular/hematopoietic progenitor marker genes such as RUNX1,
KDR (VEGFR2), and VE-CADHERIN were also highly transcribed in Vb-treated samples (supplemental Figure 5). There
was a minor difference in the proportion of CD34⫹ cells
generated from hESCs and hiPSCs using our protocols (Figure2C). The proportion of CD34⫹ cells was approximately 20% at
9 days of vascular induction in hESC group. A lower proportion(⬃13%) of hiPSC-derived mesodermal cells reached CD34⫹
cells at 9 days of vascular induction. Next, CD34⫹ cells wereisolated by a magnetic cell sorter (MACS) using an antibody
against CD34. Most of the CD34⫹ cells coexpressed CD31, andthe proportions of CD34⫹CD31⫺ and CD34⫺CD31⫹ cells wereapproximately 6.5% and 1.2%, respectively (Figure 2D). As
shown in Figure 2E, the CD34⫹ cells also expressed othervascular marker genes (␣-SMA, VE-CADHERIN, and CD31)
and hematopoietic marker genes (␤-, ⑀-, and ␨-GLOBIN,RUNX1, and LMO2) at the transcriptional level. The results
suggest that hESC-derived CD34⫹ cells can differentiate into
hematopoietic- and vascular-lineage cells.

hESC-derived CD34ⴙ progenitors can differentiate intofunctional endothelial cells in vitro and in vivo

Vascular-lineage cells such as endothelial cells (ECs) and smoothmuscle cells (SMCs) are derived from vascular progenitors andcardiovascular progenitors.17,29 In this study, hESC-derived
CD34⫹cells were cultured in EGM-2 supplemented with VEGF-A
and bFGF to induce differentiation into ECs. Mature EC marker
genes (P-SELECTIN, VWF, and ANG2) were highly expressed atday 12 of culture (Figure 3A). Thus, it is likely that hESC-derived
CD34⫹cells are vascular lineage progenitors. FACS analysis showedthat a higher proportion of hPSC-derived CD34⫹cells could
differentiate into ECs expressing endothelial markers such as
CD31 and CD105 (Figure 3B,C). The ECs derived from hESCs
and hiPSCs showed typical morphologies (Figure 4A,Ci), andstrongly expressed endothelial cell-specific proteins, including
CD31, VWF, VE-cadherin, ANG2, and KDR (Figure 4A-C).
Furthermore, these cells formed vascular-like structures on
Matrigel (Figure 4B-D, I) and took up acetylated-LDL (Figure
4B-Dii). To determine whether hESC-derived CD34⫹ cells canform blood vessels in vivo, 1 ⫻ 106 cells of hESC-derived
CD34⫹ cells were mixed with Matrigel, and the mixture wasthen subcutaneously injected into a nude mouse. Humanspecific CD31 positive signals were observed in the vessel-likeFrom bloodjournal.hematologylibrary.org at YALE UNIVERSITY on February 18, 2012. For personal use only.
BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

DIFFERENTIATION OF hPSCs TO FUNCTIONAL CD34⫹ CELLS

5767

Figure 4. Endothelial cells from hESC-derived CD34ⴙ progenitor cells are functional in vitro and in vivo. (A,C) Immunostaining for EC markers (CD31, VWF, KDR, ANG2, and VE-cadherin)
in hESC- and hiPSC-derived ECs. hESC-derived ECs showed
typical endothelial cell morphology (A and Ci), and expressed
multiple EC markers (A and Cii-iv). (B) and (D) In vitro functional
assay for hESC- and hiPSC-derived ECs. hESC- and hiPSCderived ECs formed vascular tube-like structures on
Matrigel (B and Di), and took up Dil-labeled acetylated-LDL(B,Dii). (E) Matrigel plug assay for hESC-derived CD34⫹ cells.
Antibody for VWF was used to observe both mouse and humanblood vessels. Species-specific CD31 antibodies were used todiscriminate mouse and human blood vessels. Mouse and humanspecific CD31 antibodies are indicated as blue and red colors,
respectively. Scale bar is 20 ␮m.

structure at 10 days after injection of hESC-derived CD34⫹
cells, whereas mouse-specific CD31 positive signals were notdetected (Figure 4E). Our results indicate that hESC-derived
CD34⫹ progenitor cells have vasculogenic potential in vivo asFrom bloodjournal.hematologylibrary.org at YALE UNIVERSITY on February 18, 2012. For personal use only.
5768

PARK et al

BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

Figure 5. Differentiation of hESC-derived CD34ⴙ cells to vascularsmooth muscle cells and hematopoietic cells. (A) Morphology andexpression of SMC markers in hESC-derived SMCs (␣-SMA andCALPONIN). Cell nuclei were stained with DAPI (blue). Scale bar is
100 ␮m. B. Transcriptional expression of SMC (NG2, SM22␣,PDGFR-␤, CALDESMON, and ␣-SMA) and EC (VWF and ANG2)
marker genes. (C) Various hematopoietic colonies were formed fromhESC-derived CD34⫹ cells in methylcellulose medium: (i) BFU-E,(ii) CFU-GM, (iii) CFU-G, and (iv) CFU-GEMM. Scale bar is 100 ␮m.
(D) The proportion of various hematopoietic cell-type colonies in
CD34⫹ cells cultured in MethoCult.

well as in vitro. The hESC-derived CD34⫹ cells could also
differentiate to SMCs when they were cultured in EGM-2
supplemented with platelet-derived growth factor-BB (PDGFBB) and bFGF for 15 to 21 days. hESC-derived SMCs showed
spindle-like morphologies and coexpressed ␣-SMA andCALPONIN in their cytoplasm (Figure 5A). They also expressed SMC marker genes such as NG2, SM22␣, PDGFR-␤,CALDESMON, and ␣-SMA, whereas the endothelial marker
genes (VWF and Ang2) did not (Figure 5B). In addition,
hESC-derived SMCs were contracted within 30 minutes after
treatment with carbachol, an agonist against acetylcholine
receptors (supplemental Video 1), but hESC-derived ECs werenot (supplemental Video 2), indicating functionality of hESCderived SMCs.
In the next experiment, a CFU assay was used to determine thedifferentiation potential of hESC-derived CD34⫹ cells to hematopoietic lineages. Various hematopoietic cells, including granulocytes, granulomacrophages, erythroids, and megakaryocytes, could
be differentiated from hESC-derived CD34⫹ cells (Figure 5C), but
not from CD34⫺ cells. The CD34⫹ cells began to form colonies oferythroid-lineage cells, CFU-E (CFU-erythroid) at 6 days ofculture in methylcellulose medium. Around 11 days of culture,erythroid-lineage cells such as BFU-E (burst-forming uniterythroid) reached a maximum population (Figure 5C, I). Myeloidlineage colonies, including CFU-GM (CFU-granulocyte, macrophage), CFU-G (CFU-granulocyte), and CFU-GEMM (CFUgranulocyte, erythroid, macrophage, megakaryocyte) were also
observed between 14 and 21 days after plating on methylcellulose
medium (Figure 5C, II, III, and IV). Hematopoietic lineage
colonies were formed at an average of 200 CFUs per 105 CD34⫹

cells. Of these colonies, erythroid-lineage cells (eg, BFU-E and
CFU-E) were abundant (Figure 5D). Collectively, hESC-derived
CD34⫹ cells are bipotent in that they could differentiate into
vascular lineage cells and various hematopoietic cells.
hESC-derived CD34ⴙ progenitor cells improve perfusion andsubsequent prognosis of ischemic mouse hind limbs bycontributing to neovasculogenesis

To determine the functionality of hESC-derived cells in vivo,
approximately 1 ⫻ 106 hESC-derived CD34⫹ cells were intramuscularly injected into an ischemic hind limb. A total of 23 mice weredivided into 3 groups; 9 mice were injected with CD34⫹ cells,
9 with medium alone, and 5 with CD34⫺ cells. The initial perfusion
rates of the ischemic limbs were not significantly different between
groups (ANOVA test, F2,20 ⫽ .761, P ⫽ .48). After injection ofhESC-derived CD34⫹ cells into ischemic limbs, follow-up indocyanine green perfusion imaging was subsequently performed on POD
3 and 7. As shown in the perfusion map in Figure 6A, mouseischemic hind limbs with hESC-derived CD34⫹ cells were recovered at POD 3 and further clearly at POD7, whereas those injectedwith the medium or hESC-derived CD34⫺ cells resulted in necrosis
at the end of the hind limbs. These differences between the groups
were visually obviously (Figure 6A photographs in “appearance”
panel). Intergroup comparisons of time-dependent increments inthe perfusion rates of the ischemic limbs clearly demonstrated thetherapeutic effects of CD34⫹ cells, especially on POD 7 (Figure
6B). The probability of necrosis of the ischemic region was
estimated by the relationship between the probability of regional
tissue necrosis on POD 7 and the initial tissue perfusion rate/

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BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

DIFFERENTIATION OF hPSCs TO FUNCTIONAL CD34⫹ CELLS

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Figure 6. Therapeutic effects of hESC-derived CD34ⴙ progenitor cells on neovasculogenesis in ischemic mouse hindlimbs. (A) Comparison of perfusion rates and prognosis in
ischemic limbs injected with CD34⫹, medium, or CD34⫺ cells. Leftpanel indicates indocyanine green perfusion maps obtained at
0, 3, and 7 days after surgery. The perfusion maps of day 0 show
tissue perfusion distribution of the entire lower half of the body, including normal limbs. Right panel shows photographs of the hindlimbs at 3 and 7 days after injection. (B) Average perfusion rates ofischemic hind limbs according to POD are indicated for each group.
*P ⫽ .014 vs POD 0 (ANOVA F2,24 ⫽ 5.311, P ⫽ .012). (C) Probabilityof necrosis; the relationship between the probability of regional tissue
necrosis on POD 7 and the tissue-perfusion rate of the corresponding region estimated immediately after surgery. The X axis shows
the regional perfusion rate; poor (lower than 15%/min), moderate(16%-120%/min), and (⬎ 120%/min). ANOVA and Scheffe posthoc test applied to the significant effect of groups on poor andmoderate perfusion rate, (ANOVA F2,3 ⫽ 27.993, P ⫽ .011.
*P ⫽ .015 vs media-treated group and P ⫽ .02 vs SC34⫺ cells-treatedgroup with poor perfusion rate; ANOVA F2,9 ⫽ 18.872, P ⫽ .001.
**P ⫽ .02 vs media-treated group and P ⫽ .001 vs SC34⫺ cellstreated group with a moderate perfusion rate. (D) Various types ofhESC-derived CD34⫹ cells involved in neovasculogenesis in ischemic
hind limbs. hESC-derived CD34⫹ cells could contribute indirectly (i),partially (ii-iii), or mainly (iv) to neovasculogenesis. Immunohistochemical analysis of ischemic hindlimb regions transplanted with hESCderived CD34⫹ cells. (E) Expression of angiogenic genes in theischemic region injected with hESC-derived CD34⫹ cells. Human- andmouse-specific primers were used for analyzing the expression ofvarious angiogenic genes: (1) normal hind limb tissue, (2) ischemic
hind-limb tissue injected with the hESC-derived CD34⫹ cells.

measured perfusion rate of the corresponding region estimatedimmediately after surgery. In the hESC-derived CD34⫹ cellsinjected group, regions with poor and moderate perfusion rate
ranging lower than 120%/min, which is approximately 20% of theperfusion rate of normal tissue, showed statistically significant
decreases of necrosis probability compared with that of media or
hESC-derived CD34⫺ cells-injected group (Figure 6C). To investigate how transplanted hESC-derived CD34⫹cells recover theischemic hind limb, tissues of the operated region were harvested
on POD 7, and the presence of hESC-derived ECs was assessed byimmunostaining using antibodies specific for human CD31. The
coexistence of human and mouse CD31⫹ cells was observed inischemic hind-limb regions injected with hESC-derived CD34⫹
cells. This implies that hESC-derived CD34⫹ progenitor cells candevelop into ECs in vivo, thereby participating in neovasculogenesis of the ischemic hind-limb region. In fact, hESC-derived
CD34⫹ cells are likely to contribute to neovasculogenesis in theischemic region within a week after injection. In most cases, humanCD31⫹ cells were localized outside of the mouse blood vessels(Figure 6Di) and inserted in part into hybrid blood vessels withmouse CD31⫹ cells (Figure 6Dii-iii). Rarely, blood vessels thatwere mainly composed of human CD31⫹ cells were also observed.

(Figure 6Div). When 3 sections of the ischemic tissue wererandomly taken and then examined for the contribution states,
proportion of outside (Figure 6Di), hybrid (Figure 6Dii-iii), andwhole (Figure 6Div) types was 45.5% (30/66 cases), 51.5% (34/66
cases), and 3% (2/66 cases), respectively. In addition to directcontribution of human CD31⫹ cells in neovasculargenesis, whether
the injected hESC-derived CD34⫹ cells can promote mouseangiogenesis indirectly, we addressed transcriptional expressionsof angiogenesis-related genes such as VEGF-A, bFGF, ANG1, andANG2 were examined from the ischemic tissue sample. As shownin Figure 6E, the human angiogenesis-related genes such as bFGF,
ANG-1, and ANG-2 were expressed in the ischemic tissue injectedwith hESC-derived CD34⫹ cells. Interestingly, mouse angiogenesisrelated genes such as Vegfa, Fbfb, and Ang-2 were also highly
expressed in the ischemic tissue. These data imply that angiogenic
cytokines secreted from hESC-derived CD34⫹ cells may indirectly
contribute to neovasculogenesis in the ischemic hind limbs. In
addition, this study demonstrates that hiPSC-derived CD34⫹ cells
can improve salvage of ischemic hind limbs (supplemental Figure6). Like hESCs, ischemic hind limbs injected with hiPSC-derived
CD34⫹ cells were recovered by POD 7 (supplemental Figure 6A).
The coexistence (yellow arrowhead) of human CD31⫹ cells andFrom bloodjournal.hematologylibrary.org at YALE UNIVERSITY on February 18, 2012. For personal use only.
5770

PARK et almouse lectin⫹CD31⫹ cells was observed in ischemic hind-limb
regions injected with hiPSC-derived CD34⫹ cells, representing
functional blood vessels (supplemental Figure 6B). Another type of
hiPSC-derived CD31⫹ cells surrounding the mouse blood vessels
was detected (supplemental Figure 6B). Necrosis probability ofischemic hind limbs injected with hiPSC-derived CD34⫹ cells at
POD 7 was diminished in the ischemic regions in a manner similar
to that of hESC derivatives (supplemental Figure 6C). Theseobservations indicate that hPSC-derived CD34⫹ progenitor cells
are involved, either directly or indirectly, in neovasculogenesis,
causing functionality in the ischemic model.

Discussion
Developing a simple and efficient protocol for the differentiation of
hPSCs into a specialized cell type is a key factor for future celltherapy. Reprogramming of human somatic cells to the pluripotent
state could also provide a breakthrough for clinical applications incell therapy using pluripotent stem cells.30 In this study, an efficientmethod for directly differentiating hESCs and hiPSCs into functional CD34⫹ progenitor cells was achieved by both inhibition of
MEK/ERK signaling and activation of BMP4 signaling. To date,vascular lineage cells have usually been isolated from spontaneously differentiated cells via formation of embryoid bodies or by
coculturing with mouse stromal cells in hESCs and hiPSCs.17,23,31-36 In the present study, vascular lineage cells could be
differentiated directly from hESC-derived CD34⫹ progenitors. ECs
originating from hPSC-derived CD34⫹ progenitors could form
vascular tube-like structure in vitro and de novo blood vessels invivo (Figure 4). Thus, it is likely that the hPSC-derived CD34⫹
progenitors may differentiate into vascular lineage cells withfunctionality. It has been known that CD34⫹ progenitor cells
isolated from peripheral blood, cord blood, and bone marrow ofanimals and human have therapeutic potential for cardiovasculardiseases such as ischemia and myocardial infarction.21,37-39 As analternative approach, therapeutic cells can be generated frompluripotent stem cells such as hESCs and hiPSCs. It has beenreported that hESC-derived endothelial cells are effective for the
restoration of the ischemic hind limb.24 Ischemic hind limbs
injected with hPSC-derived ECs could be salvaged around 4 weeks
after injection, representing a longer period of therapeutic effect.
VEGFR2⫹TRA-1-60⫺ vascular progenitor cells derived from hESCs
could be also differentiated into endothelial and smooth muscle
cells.23 These VEGFR2⫹TRA-1-60⫺ vascular progenitor cells were
not effective in recovering the ischemic hind limb, although thevascular progenitor cell-derived ECs could salvage the ischemic
hind limb 9 days after injection into the femoral artery.23 In
contrast, we showed that hPSC-derived CD34⫹ progenitor cells
recovered ischemic hind limbs within only a week of injection of
the CD34⫹ progenitors (Figure 6 and supplemental Figure 6).
Furthermore, hPSC-derived ECs were incorporated into mouseblood vessels (Figure 6D and supplemental Figure 6B), indicatingthat hPSC-derived CD34⫹ cells may directly contribute to theneovasculogenesis in the ischemic hind limb. Nonetheless, anotherresult (Figure 6E) offers a hint that angiogenic factors secreted
from hESC-derived ECs may facilitate the formation of mouseblood vessels in ischemic hind limbs. Collectively, our results
demonstrate that hPSC-derived CD34⫹ progenitors may be more
effective in curing ischemic disease than mature endothelial cells.
Apart from vasculogenic competence of hESC-derived ECs,
hESC-derived CD34⫹ cells can differentiate into a variety of

BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

hematopoietic cells in vitro.40,41 However, transplanted hESCderived CD34⫹ cells or CD34⫹CD38⫺ have limitations for engrafting into bone marrow in NOD/SCID/␥c⫺/⫺ mice and fetal sheep,
respectively.42,43 In contrast to hESC-derived CD34⫹ cells, human
CD34⫹ cells originated from umbilical cord blood could contribute to long-term bone marrow engraftment in NOD/SCID/IL2Rc2/2 mice.44 This functional discrepancy between hESC- andumbilical cord blood-derived CD34⫹ cells may be explained bytheir phenotypic differences. Unlike umbilical cord bloodderived CD34⫹ cells, hESC-derived CD34⫹ cells express CD31,
CD90, CD73, and FLK1, but not CD45 and CD117.43 Similarly,
hESC-derived CD34⫹ cells generated in the present study
coexpressed CD31, CD90, and CD73, but did not express CD45
(supplemental Figure 7A). In addition, only a small proportion(5%) of hESC-derived CD34⫹ cells expressed CD43, an early
hematopoietic progenitor marker (supplemental Figure 7B).
Because of both phenotypic differences and a paucity ofhematopoietic progenitors, hESC-derived CD34⫹ cells probably
have limitations for bone marrow engraftment.
It has been reported that overall gene expression profiles of
hiPSCs are similar to those of hESCs.9,10 In our study, however, a
few differences were observed in the differentiation processes
between hESCs and hiPSCs. First, responses to chemicals or
growth factors appeared to be slightly different. For induction to
the mesodermal lineage, hESCs were treated with MEK inhibitor
and BMP4 for 3 days, while hiPSCs were treated for 5 days; forsubsequent differentiation into CD34⫹ cells, it took 6 days in
hESCs and 9 days in hiPSCs (supplemental Figure 3). Secondly,
differentiation potentials into a specialized cell types seem to be
distinct; unlike hESC-derived CD34⫹ cells, hiPSC-derived CD34⫹
cells could not form hematopoietic colonies (supplemental Figure
8A), and the resultant cells did not express hematopoietic genes(supplemental Figure 8B). Therefore, it is conceivable that hiPSCderived CD34⫹ cells are likely to develop into endothelial cells, nothematopoietic cells. To clarify this biased differentiation potential,
further experiments should be done in several hiPSC lines.
The method for direct differentiation developed in this study has
several advantages for inducing hPSCs to specialized cell types.
First, inhibition of MEK/ERK signaling and activation of BMP4
signaling synergistically trigger the efficient production of mesoderm-lineage cells (Figure 1). hESCs could be induced to differentiate to extra-embryonic lineages without apoptosis by inhibition of
MEK/ERK signaling.3 BMP4 could trigger the differentiation of
hESCs to trophoblasts or primitive endoderm,11,12 whereas BMP4
treatment could facilitate mesoderm differentiation.13 In this study,
we showed that simultaneous inhibition of MEK/ERK signalingplus activation of BMP4 signaling synergistically facilitated theinduction of hESCs and hiPSCs to mesoderm-lineage cells. Second, the feeder- and serum-free systems used herein for hPSC
differentiation will be useful for the manipulation of hPSCs
destined for cell therapy. During our differentiation process from
hESCs and hiPSCs to CD34⫹ progenitor cells, animal supplements(eg, mouse embryonic fibroblast [MEF] or FBS) were not added tothe differentiation medium. The use of chemical agents versus
animal-derived factors is likely to be safer for inducing differentiation of hPSCs intended for cell therapy. In addition, our chemicalbased, feeder-free system will be helpful for studying the mechanisms of hPSC differentiation, because the side effects fromunknown (i.e., animal-derived) factors will be minimized. Third,
our stepwise protocol for differentiation of hPSCs may be usefulfor studies aimed at exploring the properties of lineage-specificprogenitors and understanding the differentiation process of stem

From bloodjournal.hematologylibrary.org at YALE UNIVERSITY on February 18, 2012. For personal use only.
BLOOD, 16 DECEMBER 2010 䡠 VOLUME 116, NUMBER 25

cells or progenitors to specialized cell types. In the present study,
mesoderm-lineage cells were first induced from hPSCs by PD98059
and BMP4. Then, a high proportion of CD34⫹ progenitor cells
were derived from mesoderm-lineage cells cultured in medium
supplemented with VEGF-A and bFGF. Finally, CD34⫹ progenitor
cells were differentiated into vascular and hematopoietic cells. The
specialized cell types generated in each experimental step could be
helpful for studying vasculogenesis and hematopoiesis in vitro.
In summary, this study demonstrates that functional CD34⫹
progenitor cells could be efficiently differentiated from hESCs andhiPSCs in animal serum- and feeder-free systems by combined theregulation of 2 signaling pathways. In addition, it is suggested thatthe hPSC-derived CD34⫹ progenitor cells produced by this novel
method have the potential to cure vascular diseases.

Acknowledgments
We thank Dr K. Jung for providing recombinant human VEGF-A.
This research was supported by a grant (no. SC-2210) from theStem Cell Research Center, a grant (no. 2009-0084073) from the
National Research Foundation of Korea (NRF) funded by the

DIFFERENTIATION OF hPSCs TO FUNCTIONAL CD34⫹ CELLS

5771

Ministry of Education, Science and Technology (MEST); and a
grant (no. A084697) from the Korea Healthcare Technology R&D
Project, Ministry for Health and Welfare, Republic of Korea.

Authorship
Contribution: S.-W.P. and Y.-M.H designed and organized theexperiments, analyzed data, and wrote the manuscript; S.-W.P. and
Y.-H.C. performed differentiation experiments to vascular andhematopoietic lineages, respectively; M.-J.J. maintained hESCs
and hiPSCs; Y.J.K. and G.Y.K. contributed to immunohistochemistry analysis and Matrigel plug assay; Y.K., J.J., and C.C. carried out
in vivo functionality of hESC-derived cells in hind-limb ischemic
mice via near-infrared fluorescence imaging; and M.-J.K., Y.S.C.,
and H.-M.C. generated and characterized hiPSCs and CHA4-hES.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Yong-Mahn Han, Department of Biological
Sciences, KAIST, 335 Gwahangno Yuseong-gu, Daejeon 305-701,
Republic of Korea; e-mail: ymhan@kaist.ac.kr.

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