Original Article

Yonago Acta medica 2016;59:126–134

Prion Protein and Stage Specific Embryo Antigen 1 as Selection Markers to
Enrich the Fraction of Murine Embryonic Stem Cell-Derived Cardiomyocytes
Nobuhito Ikeda,* Yuji Nakayama,† Natsumi Nakazawa,* Akio Yoshida,* Haruaki Ninomiya‡ and Yasuaki
Shirayoshi**Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Tottori University Graduate School of Medical Science, Yonago, 683-8503, Japan, †Division of Functional Genomics, Research Center for Bioscienceand Technology, Tottori University, Yonago, 683-8503, Japan and ‡Department of Biological Regulation, School of Health Sciences,
Tottori University Faculty of Medicine, Yonago, 683-8503, Japanproducing tumors.7 A sophisticated method is necessary to isolate cardiac progenitors from among ES cells
and achieve a fraction of high purity. The normal prion
protein (PrP) belongs to the glycosylphosphatidyl-inositol-anchored protein family, and is expressed in adulttissues.8–11 The PrP is anchored to the outer surface ofneurons, lymphocytes and other cells that express Nestin9, 12 and promotes the differentiation of ES cells into
neuronal progenitor cells.9, 12, 13 The PrP was reported to
be expressed in the intestine during embryonic development9, 14, 15 as well as in the heart and neurons.9, 14–16 The
PrP was reported to form a complex with stress induced
phosphoprotein 1 (STIP1) to regulate neuronal development.9, 17 The PrP is known to be involved in iron uptake9,18
and Ca2+ homeostasis9, 19 and also to play a pivotal rolein cellular responses against reactive oxygen species(ROS) to form superoxide dismutase (SOD) via binding
to copper.9, 11 Thus, the PrP is essential to responses tothe environment by cells of the three germ layers. An
abnormal PrP causes neurodegenerative diseases such
as Creutzfeldt-Jakob disease, fatal familial insomnia andGerstmann-Sträussler-Scheinker syndrome.10, 11
Hidaka et al. reported that the PrP was a marker ofcardiac progenitor cells in the early phase of EB formation.16 We also found that most of PrP+ cells differentiated into cardiomyocytes.20 Since the PrP is expressed invarious tissues, it is possible that PrP+ cells from ES cells
include cells different from cardiac progenitor cells. Recently, the PrP was reported to enhance the expressionof the transcription factor Nanog suggesting that PrP+

ABSTRACT
Background The prion protein (PrP) might be usefulas a tool to collect cardiac progenitor cells derived fromembryonic stem (ES) cells. It is also possible that PrP+
cells include undifferentiated cells with a capacity to develop into tumors.
Methods PrP+ cells isolated from embryoid bodies(EB) formed by mouse AB1 ES cells were examined using RT–PCR analysis and clonogeneic cell assay. To assess their potential to differentiate into cardiomyocytes,
Nkx2.5GFP/+ (hcgp7) cells, another ES cell line thatcarries the GFP reporter gene in the Nkx2.5 loci, wereused.
Results PrP+ cells isolated from EB of day 7 and 14
did not express pluripotency markers, but expressed cardiac cell markers, while PrP+ cells isolated from EB of
day 21 expressed pluripotency markers. Cultured PrP+
cells isolated from EB of day 21 expressed pluripotency
markers to form colonies, whereas those isolated from
EB of day 7 and 14 did not. To exclude proliferating cells
from PrP+ cells, stage specific embryo antigen 1 (SSEA1)
was employed as a second marker. PrP+/SSEA1– cells
did not proliferate and expressed cardiac cell markers,
while PrP+/SSEA1+ did proliferate.
Conclusion PrP+ cells isolated from EB included
undifferentiated cells in day 21. PrP+/SSEA1– cells included cardiomyoctes, suggesting PrP and SSEA1 may
be useful as markers to enrich the fraction of cardiomyocytes.
Key words

cell differentiation; embryonic stem cells;
prion protein; stage-specific embryonic antigens

Corresponding author: Nobuhito Ikeda
nikeda@med.tottori-u.ac.jp
Received 2016 March 4
Accepted 2016 March 15
Abbreviations: DMEM, Dulbecco’s-modified Eagle’s medium;EB, embryoid bodies; ES cells, embryonic stem cells; FACS, fluorescence activated cell sorting; FBS, fetal bovine serum; GMEM,
Glasgow minimum essential medium; HBSS, Hank’s balanced
salt solution; LIF, leukemia inhibitory factor; PE, phycoerythrin;
PrP, prion protein; ROS, reactive oxygen species; SOD, superoxide
dismutase; SSEA1, stage specific embryo antigen 1; STIP1, stressinduced phosphoprotein 1

Embryonic stem (ES) cells are characterized by theircapacity for self-renewal and pluripotency. They spontaneously differentiate into cardiomyocytes through theformation of embryoid bodies (EB).1–4 ES cells-derived
cardiomyocytes are a potential source for cell-transplantation therapy in patients with a damaged heart.5, 6
A prerequisite for transplantation of these cells is theirpurity, because undifferentiated ES cells are capable of126

Markers to enrich cardiomyocyte

cells can proliferate and form tumors after transplantation.7, 9, 21 This might indicate the harmfulness of PrP+
cells as a cell source for transplantation. However, it has
never been tested whether PrP+ cells from EB include
undifferentiated cells. In the present study, we attempted
to characterize PrP+ cells derived from EB formed bymouse ES cells. We found that PrP+ cells from EB ofdays 21, but not those of day 7 and 14, expressed pluripotency markers and were capable of proliferation.
Combining the PrP with stage specific embryo antigen 1(SSEA1) as the second marker enabled us to enrich thefraction of cardiomyocytes that do not proliferate.

and 1,000 units/mL LIF (ESGRO; Merck KGaA, Darmstadt, Germany).
Dr. Morisaki (National Cerebral and CardiovascularCenter, Suita, Japan) provided ht7 ES cells. These cells
carry the hygromycin resistance gene in one of the Oct4loci.22 Derived from the ht7 cells, hcgp7 (Nkx2.5GFP/+)
ES cells carry the GFP reporter gene in one of the
Nkx2.5 loci.23 Both ht7 and hcgp7 cells were grown andmaintained on gelatin-coated dishes in Glasgow minimum essential medium (GMEM; Wako Pure Chemical)
supplemented with 10% heat-inactivated FBS (Corning), 1 × penicillin-streptomycin-L-glutamine solution(Wako Pure Chemical), 1 × MEM non-essential aminoacid solution (Wako Pure Chemical), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 1 mM sodium pyruvate(Wako Pure Chemical), and 1,000 units/mL LIF (Merck
KGaA), without feeder cells. Differentiation of ES cells
into cardiac progenitors was induced via formation of
EB. Briefly, EB were generated by plating 20 µL of cellsuspension (2.5–10 × 104 cells/mL) in DMEM (Wako
Pure Chemical) supplemented with 10–20% heat-inactivated FBS (Corning), 1 × penicillin-streptomycin-L-glutamine solution (Wako Pule Chemical), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich) (EB medium) on the lidof a dish, followed by incubation in hanging drops for 2days. EB were transferred into the medium and cultured
as floating EB or attached out-growth cells for indicateddays until analysis.

MATERIALS AND METODS
Cell culture and differentiation

AB1 ES cells derived from 129SV/EV mice were kindly
provided by Dr. Shimotsuke (Riken CDB, Kobe, Japan).
They were cultured on SNL feeder cells treated withmitomycin C (Sigma-Aldrich, St Louis, MO). SNL cells
were derived from STO mouse embryonic fibroblasts
with a forced expression of leukemia inhibitory factor(LIF) and neomycin resistance genes. AB1 cells weregrown and maintained in Dulbecco’s-modified Eagle’s
medium (DMEM; Wako Pure Chemical, Osaka, Japan)
supplemented with 20% heat-inactivated fetal bovine serum (FBS; Corning, Corning, NY), 1 × penicillin-streptomycin-L-glutamine solution (Wako Pure Chemical), 1
× MEM non-essential amino acid solution (Wako Pure
Chemical), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich)
Table 1. Primer list in gene expression analysis
Gene

Forward

ReverseAnnealing No. cycles
temperature

Zfp42

TTCACGGAGAGCTCGAAACTCCATCCCCTTCAATAGCTCA

6030

CACCCACCCATGCTAGTCTT

ACCCTCAAACTCCTGGTCCT60

28

ATTCTCGAACCTGGCTAAGCT

ATGGTGGTCTGGCTGAACACCTTT60

28

CAGAAGACTTTAACCAAGGGC

TGGGCTATTTTGCTTACAACTT

60

30

AAGCCATGAATGCAGAGGAGGACT

AGCTGCAGGCAGCCGGCGACC

6030ACATCAGTGTCTGCTGGCAC

AGCGAGTTTCCTTGGCAACAC

60

30

AAGAAACCCTAAACACAAACAGCG

TTTGTGGGAAGTGGGATCAAGAC

60

30

GTCTTCTGGTTCTCCGATGTCCAGGTGCTATATATTGCCT6030CAGAATCGTGGGACCCATCGGCGTCCAGGTTTCTAG

6030

ATGGTCCGTAACTGGCAAGTTTCGTCTGCTTTCACGATG

6030

ACCGTCGCTACAAGTGCAA

CCATAGGCATTGAGACCCA6030TGGGCTTCTTCCTCGTCTTTTCTACCGGCATCTTCTCCT6030TGACCCAGGCAGACAAGTTCCGTGGGTGATGATGTAGCAG

6030

AAGGTGTTTGATCCCGAGGGGGGAAAGGCTGCGAACATCT60

30

CTGAAGCATTCTGCCTTCCTGCCGACATCAGTCCACATAG

60

30

TGAACGGGAAGCTCACTGG

TCCACCACCCTGTTGCTGTA

60

25

Nanog
Oct4

Pax6

NeuroD1
Afp

Sox17
T

Mesp1
Tbx5

Nkx2.5
Anp

Myl7

Myl2
Prnp

Gapdh

127

N. Ikeda et al.

A

B

Fig. 1. PrP+ cells isolated from EB
involved the cardiac myocytes.
A: Flow cytometry analysis of cellsurface PrP in EB derived fromAB1 ES cells. Ordinate indicatesthe prevalence of DRAQ7-posisitve
cells as dead cells and abscissa indicated the prevalence of PrP+ cells.
The cells within a gated area show
the population of living PrP+ cells.
B: Summary of the flow cytometry
analysis. The graph shows the mean
and the standard deviation of the
PrP+ rate in Day 7, 14 and 21 after adifferentiation induction (n = 3).
C: Prevalence of Nkx2.5/GFP-positive cell in PrP+ cells isolated from
EB of hcgp7 at Day 7, 14 and 21.
The ordinate showed the prevalenceof PrP+ cells and the abscissa indicated the expression level of Nkx2.5/
GFP-positive cells. The upper panel
showed the flow cytometry analysis
of ht7 cells, as negative control andthe lower panel showed the flow cytometry analysis on hcgp7 cells.
EB, embryoid body; ES cells, embryonic stem cells; GFP, green fluorescent protein; PE, phycoerythrin;
PrP+, prion protein positive.

C

128

Markers to enrich cardiomyocyte
Day 0
PrP

Day 7

Day 14

Clonogenic cell assay

Day 21

PrP+ cells were isolated from EB at day 7, 14 and 21
by FACS. 1,000 or 10,000 cells were seeded on gelatin-coated dish and cultured in EB medium for 7 to
17 days. Colonies fixed with 100% ethyl alcohol were
stained with Giemsa.

(–) (+) (–) (+) (–) (+)

Zfp42
Nanog
Oct4
Pax6
NeuroD1
Afp
Sox17
T
Mesp1
Tbx5
Nkx2.5
Anp
Myl7
Myl2
Prnp
Gapdh RT (+)
Gapdh RT (-)

Reverse transcriptase-polymerase chain reaction

Total RNA was isolated from EB using an RNeasy
Mini Kit (Qiagen, Hilden, Germany), according to the
manufacturer’s instructions. RNA samples were treatedwith DNaseI (Promega Corporation, Fitchburg, WI) toeliminate genomic DNA and cDNA was synthesizedusing the PrimeScript RT reagent Kit with gDNA Eraser(Takara Bio, Kusatsu, Japan). PCR amplifications were
performed using Emerald Amp Max polymerase (TakaraBio) with primers listed in Table 1.
Statistical analysis

Data are expressed as mean ± SD.

Fig. 2. mRNA level of pluripotent, ectodermal, endodermal, mesodermal and cardiac cell markers in PrP+ cells from EB at day
7, 14 and 21. Cells were isolated by FACS, and then mRNA wasextracted from them. Each set corresponds to the transcript amplified using the indicated primers. (+) : PrP+ cell fraction, (–) : PrP–
cell fraction. PCR of Gapdh from RNA sample without reverse
transcriptase [Gapdh RT (–) ] did not show any fragment, indicating no contamination of cDNA by genomic DNA.
EB, embryoid body; FACS, fluorescence activated cell sorting;mRNA, messenger RNA; PrP−, prion protein negative; PrP+, prion
protein positive; RT, reverse transcription.

RESULTS

PrP+ cells differentiated into cardiac myocytes

Figure 1A shows representative flow cytometry data indicating the prevalence of PrP+ cells in EB of AB1 cells.
And Fig. 1B shows the summary of the flow cytometry
analysis. The PrP+ rate were 22.9, (s = 5.8. n = 3), 13.7, (s= 1.9. n = 3) and 18.3, (s = 12.7. n = 3) (%) in EB of day
7, 14 and 21, respectively. PrP+ cells were not detected
in EB before day 4 of differentiation induction (data notshown). The prevalence of PrP+ cells expressing Nkx2.5/
GFP was studied using mouse Nkx2.5GFP/+ ES cells. As
shown in Fig. 1C, the prevalence of PrP+ cells expressing
Nkx2.5/GFP on days 7, 14 and 21 was 1.3, 0.3 and 0.1%,
respectively, indicating a decline in the number of cardiac progenitor cells among PrP+ cells.

Flow cytometry

Cells were dissociated from EB at day 7 ± 1, 14 ± 1 and21 ± 1 by Collagenase type Ⅱ (Worthington, Lakewood,NJ) with gentle pipetting, followed by a treatment with
Cell Dissociation Buffer (enzyme-free, Hanks’-based;
Thermo Fisher Scientific, Waltham, MA) for 5–8 min.
Cells were stained with phycoerythrin (PE) -conjugatedanti-PrP (mouse monoclonal clone SAF83; Funakoshi,
Tokyo, Japan) labeled with the PE Labeling Kit-NH2(Dojindo Laboratories, Kumamoto, Japan) according to
the manufacturer’s instructions. Dead cells were excluded with Draq7 (Biostatus, Shepshed, England).
The percentage of cells positive for PrP or GFP wasdetermined by flow cytometry (BD FACS Canto II; BD,
Franklin Lakes, NJ). They were resuspended in Hank’sbalanced salt solution (HBSS, Wako Pure Chemical)
containing 2% FBS and Draq7, diluted 100 times, andsubjected to cell sorting (Moflo XDP, Beckman Coulter,
Brea, CA) with Summit software to collect either PrP+
or GFP+ cells.20

Heterogeneity of PrP+ cells isolated from EB atvarious periods post differentiation induction

To verify whether PrP+ cells contained undifferentiatedcells, mRNAs levels of pluripotency markers were examined in PrP+ cells isolated from EB. Figure 2 shows
mRNA levels of pluripotency markers (Zfp42, Nanog
and Oct4), ectoderm cell markers (Pax6, NeuroD1), endoderm cell markers (Afp, Sox17), mesoderm cell markers (T, Mesp1) and cardiac cell markers (Tbx5, Nkx2.5,
Anp, Myl7 and Myl2) expressed in PrP+ cells isolated
from EB of days 7, 14 and 21. PrP+ cells from EB of
day 7 predominantly expressed mRNAs of cardiac cellmarkers. PrP+ cells from EB of day 14 expressed mRNAs of ectodermal and endodermal cell markers besides
mesodermal cell and cardiac cell markers. Surprisingly,
129

N. Ikeda et al.

A
A

AB1
d0

EB EB EB d7 EB d7
d7 d7 PrP− PrP+
+d7
PrP− PrP+ +d7

Zfp42

B
B

EB
AB1
d14
d0
PrP−

Zfp42
Nanog

Nanog

Oct4

Oct4
Pax6

Pax6

NeuroD1
Sox17
Afp

NeuroD1

T
Mesp1
Tbx5

T
Mesp1

Nkx2.5Nkx2.5

Sox17
Afp

Tbx5

Anp

Anp

Myl7

Myl7

Myl2

Myl2

Prnp

Prnp

Gapdh(+)

Gapdh(+)
Gapdh(-)

Gapdh(-)

CC
Zfp42
Nanog
Oct4
Pax6
NeuroD1
Sox17
Afp
T
Mesp1
Tbx5
Nkx2.5
Anp
Myl7
Myl2
Prnp
Gapdh(+)
Gapdh(-)

EB EB d14 EB d14
d14 PrP− PrP+
+d7
PrP+ +d7

EB
AB1
d21
d0
PrP−

EB EB d21 EB d21
PrP+
d21 PrP−+d7
PrP+ +d7

Fig. 3. Changes in mRNA level of pluripotent stem, ectodermal,
endodermal, mesodermal and cardiac cell markers expressed incultured PrP+ cells from EB at various period.
A: PrP+ cells were sorted from EB at day 7 after differentiation
by FACS and were cultured for another 7 days, and then mRNAwas extracted from them. Each band corresponds to the transcript
amplified using the indicated primers.
B: PrP+ cells were sorted from EB at day 14 by FACS and were
subsequently cultured for another 7 days, and then their mRNAwas extracted.
C: PrP+ cells were sorted from EB at day 21 by FACS and were
subsequently cultured for another 7 days, and then their mRNAwas extracted. AB1 d0: undifferentiated AB1 cells, (+) : PrP+ cell
fraction, (–) : PrP− cell fraction. PCR of Gapdh from RNA sample without reverse transcriptase [Gapdh RT (–) ] did not show
any fragment, indicating no contamination of cDNA by genomicDNA.
d, day; EB, embryoid body; FACS, fluorescence activated cellsorting; mRNA, messenger RNA; PrP−, prion protein negative;
PrP+, prion protein positive; RT, reverse transcription.

130

Markers to enrich cardiomyocyte
Day 7 + 7

Day 14 + 7

Use of PrP and SSEA1 as selection markers to enrich the fraction of differentiated cardiomyocytes

Day 21 + 7

Stage specific embryo antigen 1 (SSEA1) is a surface
marker of pluripotent stem cells.24 Figure 5A shows
expression of SSEA1 in PrP+ cells isolated from EBof days 7, 14 and 21. The prevalence of PrP+/SSEA1+
cells was 0.3%, 0.1% and 0.3%, respectively. Figure 5Bshows colony formation by PrP+ /SSEA1– cells and PrP+
/SSEA1+ cells from EB of day 21 cultured for 10 and 17days, respectively. Cultured PrP+ /SSEA1– cells did notform any colony, but cultured PrP+ /SSEA1+ cells did,indicating SSEA1 is a useful marker to exclude proliferating cells from among PrP+ cells. Figure 5C showscardiac cell markers (Tbx5 and Myl2) expressed in PrP+
/SSEA1– cells isolated from EB of day 21. The level ofmRNA corresponding to cardiac cell markers was comparable between PrP+ cells and PrP+ /SSEA1– cells.

PrP−

PrP+

Fig. 4. Proliferation of cultured PrP+ cells derived from EB at day 7,
14 and 21.
PrP+ cells were sorted from EB at day 7, 14 and 21 by FACS, andwere subsequently cultured for 7 days. Each cell was stained by
Giemsa’s solution. The upper panels: colony formation of PrP−
cells derived from EB at the various periods. The lower panels:colony derived of PrP+ cells derived from EB at the various periods.
EB, embryoid body; FACS, florescence activated cell sorting;
PrP−, prion protein negative; PrP+, prion protein positive.

DISCUSSION
In the present study, we found that PrP+ cells isolated
from EB of day 7 and 14 did not express pluripotency
markers but expressed cardiac cell markers, and that
PrP+ cells isolated from EB of day 21 expressed pluripotency markers as well as those of the three germ layers;that PrP+ cells isolated from EB of day 21 formed colonies and expressed pluripotency markers, whereas thosefrom EB of day 7 and 14 did not form colonies; and that
PrP+/SSEA1– cells expressed cardiac cell markers anddid not proliferate, while PrP+/SSEA1+ did proliferate.
Pluripotent stem cells such as ES or induced pluripostent stem cells are a potential cellular source forcell transplantation therapy of damaged hearts.16, 20, 25
However, pluripotent stem cells-derived cells include
undifferentiated cells. Several groups tried to purify
cardiomyocytes among differentiated ES cells by marker gene transduction or fluorescence-based purification
methods. Yet, for clinical use, fast, effective and scalable
purification methods with no genetic modification are
essential. A few surface markers that can be used for
isolation of cardiomyocytes have been reported.16, 23
Flk1 is a marker of cardiovascular progenitors: common
progenitors for cardiac, smooth muscle and endothelial
cells.5 c-Kit is reported to be a cardiovascular stem cellmarker of adult and embryonic heart.26, 27 Pdgfra is
widely expressed in the mesoderm, including the cardiac
lineage.26 Vascular cell adhesion molecule-1 (VCAM-1)
has been reported as useful to isolate embryonic cardiomyocytes with 98% purity. VCAM-1 positive cells havebeen reported to specify committed cardiomyocytes,28and have been confirmed in human ES cells.29 Recently,
Hidaka et al. reported the PrP served to predominantly
enrich the fraction of cardiomyocytes expressing cTnI,

PrP+ cells from EB of day 21 expressed mRNAs of pluripotency markers as well as those of cells of the threegerm layers.
PrP+ cells from EB included undifferentiated cells
capable of proliferation

Expression of pluripotency markers suggested the prevalence of undifferentiated cells among PrP+ cells from
EB of day 21. Since undifferentiated cells proliferate ina short-term culture, we examined the expression of pluripotency markers in PrP+ cells cultured for 7 days after
isolation from EB. As shown in Fig. 3A, PrP+ cells from
EB of day 7 expressed mesodermal, cardiac cell markers. But, they also expressed pluripotent, ectodermal andendodermal cell markers, slightly. PrP+ cells from EBof day 14 did not express pluripotency markers, ectodermal or endodermal cell markers after a 7-day culture,
but they expressed cardiac cell markers in Fig. 3B. PrP+
cells from EB of day 21 expressed pluripotency markers
after a 7-day culture without changes in mRNA levels of
mesodermal, cardiac, ectodermal and endodermal cellmarkers in Fig. 3C. Next, we examined the capability of
PrP+ cells from EB to proliferate by means of clonogenic
cell assay. As shown in Fig. 4, PrP+ cells isolated from
EB of day 21 formed colonies, while cultured PrP+ cells
from EB of day 7 and 14 did not. These results indicatedthat PrP+ cells from EB of day 21 contained undifferentiated cells.
131

A

PE
(PrP)

A

Day 7

Day 14

Day 210.1

0.0

N. Ikeda et al.
0.1

99.9

0.0

99.9

0.0

99.7

0.0

37.5

0.3

15.4

0.1

11.6

0.3

53.3

8.9

81.5

3.0

73.8

14.3

0.0

0.3

0.0

Negative
control

Antibody
stainedAlexa488(SSEA1)B
B

PrP+/SSEA1−
10,000 cells

C
C

PrP+
/SSEA1+1,000 cells

PrP− PrP+

PrP+
SSEA1−

Prnp
Tbx5
Myl2
Gapdh RT (+)
Gapdh RT (−)

Day 21 + 10

Day 21 + 10 +7

Fig. 5. Characterization of PrP+/ SSEA1− cells isolated from EB at various periods.
A: Prevalence of PrP+/ SSEA1+ cells derived from EB at the various periods. PrP+ cells were sorted from EB at Day 7, 14 and 21 by
FACS. The ordinate showed the prevalence of PrP+ cells and the abscissa indicated the prevalence of SSEA1+ cells. The upper control
board showed the flow cytometry analysis as negative control and the lower control board showed the flow cytometry analysis of PrP+/
SSEA1+ cells.
B: Colongenic cell assay of cultured PrP+/ SSEA1− cells from EB at day 21. Either PrP+/ SSEA1+ or PrP+/ SSEA1− cells of 10,000 cells
were isolated from EB at day 21 by FACS and were subsequently cultured for another 10 and 17 days. Upper panels: colonies of cultured
PrP+/ SSEA1+ or PrP+/ SSEA1− cells from EB at day 21 for another 10 days stained with Giemsa’s dye. Lower panels: colonies of cultured
PrP+/ SSEA1+ or PrP+/ SSEA1− cells from EB at day 21 for another 17 days stained with Giemsa’s dye.
C: Enrichment of cardiac myocytes in PrP+/ SSEA1− cells from EB at day 21. PrP− cells, PrP+ cells and PrP+/ SSEA1− cells were sorted
from EB at day 21 by FACS and were subjected to RT−PCR using indicating primers. PCR of Gapdh from RNA sample without reverse
transcriptase [Gapdh RT (−) ] did not show any fragment, indicating no contamination of cDNA by genomic DNA.
EB, embryoid body; FACS, fluorescence activated cell sorting; mRNA, messenger RNA; PE, phycoerythrin; PrP−, prion protein negative;
PrP+, prion protein positive; RT, reverse transcription, SSEA1−, SSEA1 negative; SSEA1+, SSEA1 positive.

132

Markers to enrich cardiomyocyte
Acknowledgments: I would like to thank Prof. Takayuki Morisaki
and Prof. Kyoko Hidaka for their useful suggestion. I also would
like to thanks Ms. Yumi Miyauchi and Ms. Yoshimi Kobayashi
for their excellent technical support.

and cultured PrP+ cells from EB have been reported todifferentiate into atrial and ventricular myocytes.16 Hidaka et al. also demonstrated that PrP+ cells from EB could
proliferate, because PrP+ cells included cardiac progenitors.16 Nevertheless, in the present study, PrP+ cells from
EB of days 21 expressed pluripotency markers and PrP+
cells from EB of day 21 proliferated in vitro. In contrast,
PrP+ cells from EB of day 14 did not express pluripotency markers and did not proliferate. The mechanismof re-expression of pluripotency markers in PrP+ cells
from EB of day 21 remains unclear. The PrP has beenreported to activate Nanog expression through Integrin
signaling.9, 25 PrP is known to influence the fate of cells
and cell cycle besides their pluripotency, suggestingthat prolonged expression of PrP in cultured cells may
reactivate pluripotency9, 30 markers leading thereby tocell proliferation. Further experiments are necessary to
examine this possibility.
Since individual markers are commonly expressedby cells of multiple lineage, a single marker may not
be sufficient to distinguish cells of the cardiac lineage.
Combination of two surface markers has been reported
to be useful for isolation of cells of the cardiac lineage.
Hidaka et al. used PDGFRa and PrP as markers to improve the purity of the cardiomyocyte fraction.16 The
present data indicated that PrP+ cells from EB of days
21 included undifferentiated cells. Thus, for clinical use,
it is necessary to exclude undifferentiated cells. SSEA1
is expressed in mouse pluripotent stem cells and is theirauthentic marker. In the present study, PrP+ /SSEA1–
cells isolated from EB of day 21 included cardiomyocytes but not undifferentiated cells. Taken together, useof the PrP with SSEA1 might enable us to enrich thefraction of differentiated cardiomyocytes.
Clinical implication of the present study might beobvious. The transplantation of both c-Kit+ cells isolated
from the right atrial appendage31 and cardiac progenitor
cells from endomyocardial biopsies obtained from theright ventricular septum32 had been reported to improvethe cardiac function in patients with ischemic heart disease, whereas other stem-cell based therapies including
human bone marrow stem cells or mesenchymal stem
cells failed to do so.33 This indicated the importanceof enriching cardiac progenitor cells to exert positiveeffects in the clinical setting. The present provided anovel method to collect the cardiac progenitor cells frompluripotent stem cells and to exclude the undifferentiated cells, ensuring the feasibility as well as safety on theimplantation of the cardiac progenitor cells from pluripotent stem cells.

The authors declare no conflict of interest.

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