Generation of functional erythrocytes from human
embryonic stem cell-derived definitive hematopoiesis
Feng Ma*†, Yasuhiro Ebihara*‡, Katsutsugu Umeda§, Hiromi Sakai¶, Sachiyo Hanada*, Hong Zhang¶, Yuji Zaike?,
Eishun Tsuchida¶, Tatsutoshi Nakahata§, Hiromitsu Nakauchi†, and Kohichiro Tsuji*‡**
*Division of Cellular Therapy, Advanced Clinical Research Center,†Laboratory of Stem Cells Therapy, Center for Experimental Medicine, Institute of Medical
Science, and Departments of‡Pediatric Hematology/Oncology and?Laboratory Medicine, Research Hospital, Institute of Medical Science, University of
Tokyo, Tokyo 108-8639, Japan;§Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; and
¶Institute for Biomedical Engineering, Waseda University, Tokyo 162-0041, Japan
Edited by Tasuku Honjo, Kyoto University, Kyoto, Japan, and approved May 2, 2008 (received for review March 5, 2008)
A critical issue for clinical utilization of human ES cells (hESCs) is
liver-derived stromal cells. Large numbers of hESCs-derived erythroid
progenitors generated by the coculture enabled us to analyze the
development of erythropoiesis at a clone level and investigate their
function. The results showed that the globin expression in the
erythroid cells in individual clones changed in a time-dependent
manner. In particular, embryonic ?-globin-expressing erythroid cells
from individual clones decreased, whereas adult-type ?-globin-
expressing cells increased to ?100% in all clones we examined,
erythrocytes also appeared among the clonal progeny. A comparison
analysis showed that hESC-derived erythroid cells took a similar
differentiation pathway to human cord blood CD34?progenitor-
derived cells when examined for the expression of glycophorin A,
CD71 and CD81. Furthermore, these hESC-derived erythroid cells
could function as oxygen carriers and had a sufficient glucose-6-
an experimental model for exploring early development of human
erythropoiesis and hemoglobin switching and may help in the dis-
covery of drugs for hereditary diseases in erythrocyte development.
development ? erythropoiesis ? hemoglobin ? primitive hematopoiesis
blood islands in the yolk sac (YS) transiently generate nucleated
RBCs that exclusively express those globins that are components of
the embryonic Hbs: Gower-1 (composed of ?- and ?-globins),
Gower-2 (composed of ?- and ?-globins), and Hb Portland (com-
posed of ?- and ?-globins). The second definitive hematopoiesis
wave, which gives rise to transplantable hematopoietic stem cells,
enucleated RBCs, and various other hematopoietic cells, takes
place mainly in the fetal liver (FL) through midgestation, although
there is some contribution by the YS. Definitive hematopoiesis
finally shifts to bone marrow, the site of lifelong adult-type hema-
topoiesis. Fetal and adult-type definitive hematopoiesis exhibit
different patterns of Hb expression. In the former, primarily ?- and
?-globins, the components of fetal Hb (Hb F), are expressed, and
A), are expressed (1, 2).
Previously, however, early human hematopoiesis had been dif-
ficult to study because of ethical limitations in the use of human
embryos. Recently established human ES cells (hESCs) provided
an ideal tool for investigation of early human embryonic/fetal
hematopoiesis (3). The in vitro generation of hematopoietic cells
from hESCs has been reported by several studies (4–7). We also
reported a method of hESC coculture with mouse FL-derived
stromal cells (mFLSCs) that generated a large quantity of hema-
topoietic progenitors that could give rise to erythroid cells, pro-
viding a means to characterize hESC-derived erythropoiesis (8).
ematopoiesis in humans is a dynamic process regulated both
temporally and spatially. In the primitive hematopoiesis wave,
We show here that, in the coculture system, hESC-derived
differentiation at a clonal level demonstrates that most hESC-
gradually increases to 100% over time. In addition, over time in
culture, hESC-derived erythropoiesis generates erythrocytes that
are not only enucleated but also functionally mature. Thus, we
propose that hESC-derived erythroid cells can provide an experi-
switching. This model will also be useful for investigating patho-
genesis and testing drug therapies for hereditary erythropoiesis-
Generation of Erythroid Cells from hESCs by Coculture with mFLSCs.
In a previous study (8), we found that the production of erythroid
progenitors from hESCs was greatly enhanced by coculture with
mFLSCs. This experimental paradigm provides the opportunity to
conduct large-scale investigations of hESC-derived erythropoiesis.
In the cocultures, 1 ? 104undifferentiated hESCs routinely gen-
erate a total of 5 ? 105cells by day 6 and 1 ? 106cells by day 14,
with cell numbers decreasing thereafter [supporting information
(SI) Fig. S1A]. The hESC-derived cells were primarily nonfloating
cells, whereas floating cells were ?0.05% of the total at any time
during the coculture (data not shown). Flowcytometric analysis
revealed that CD34?cells increased concomitantly with the total
cell increase (Fig. S1 A and B). Although total cell numbers
decreased after day 14, the number of CD34?cells peaked on day
erythroid cells before day 6 of coculture (Fig. S1 C and D). GPA?
cells appeared at days 8 to 10 and did not coexpress CD45 but
contained a substantial proportion of CD71?cells.
We examined the expression of various globins in the cocultures
by using immunostaining (Fig. 1 A and B). Human Hb-expressing
cells were observed as early as days 8–10 of coculture, consistent
with the appearance of GPA?cells. This early wave of hESC-
?-globin-negative, indicating that they possessed the characteristics
of embryonic and fetal erythrocytes. ?-globin?cells only appeared
at days 11 and 12. Although the hESC-derived Hb?erythroid cells
cells gradually increased over time. By day 18, 50–60% of the
H.Z., Y.Z., and E.T. performed research; F.M., Y.E., K.U., E.T., T.N., and K.T. analyzed data;
and F.M. and K.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
**To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0802220105 PNAS ?
September 2, 2008 ?
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hESC-derived erythroid cells were strongly positive for ?-globin,
indicating a gradual increase in adult-type erythropoiesis in the
We investigated the expression of various genes regulating
early hematopoiesis and erythroid lineage differentiation over
the same time course as the analysis of globin expression by using
RT-PCR (Fig. 1C). Genes for initial development of endothelial/
hematopoietic cells were expressed early in the coculture, as
reported in our previous work (8). The expression of ?-globin
could be detected by day 10, consistent with the immunostaining
Generation of Clonal Erythroid Progenitors from hESCs by Coculture
with mFLSC. To further characterize hESC-derived erythropoiesis,
we conducted hematopoietic colony assays of hESC/mFLSC cocul-
ture-derived cells. Before day 8 of coculture, there were few
day 8, and various types of hematopoietic CFCs then rapidly
increased in number, reaching a peak on day 14. As shown in Fig.
2A, most of the erythroid cell colonies [including erythroid (E)
colonies, E bursts, and mixed-lineage (Mix) colonies] were in the
nonfloating cell fraction, whereas a gradual increase in myeloid
colonies among the floating cells was observed. At the peak (day
colonies, of which ?75% were E colonies and E bursts (E colonies,
312.8 ? 14.3; E bursts, 31.8 ? 13.1; Mix colonies, 11.0 ? 2.8;
myeloid colonies, 143.8 ? 11.9).
The size of the E colonies did not change with the time [132 ?
25.5 (n ? 6) and 130.8 ? 29.1 (n ? 6) cells per E colony on days
10 and 14, respectively, P ? 0.96; Fig. 2 B and C]. E burst-forming
in 100% of Hb?erythroid cells. (Scale bar: 25 ?m.) (B) Immunostaining of Hb, ?-, ?-, ?-, and ?-globins in cells from day-18 hESC/mFLSC coculture cells. ?-Globin?cells
of early hematopoiesis-related genes and the definitive hematopoiesis ?-globin gene during the hESC/mFLSC coculture detected by RT-PCR.
Time-associated changes in expression of globins and hematopoiesis-related genes in hESC/mFLSC-derived erythroid cells. (A) Immunostaining of Hb, ?-, ?-,
cocultures. (B–E) Micrographs of E colonies derived from day-8 (B) and day-16 (C) cocultures and E bursts from day-12 (D) and day-16 (E) cocultures. (F) Photo
of harvested E burst cells from day-16 coculture, showing the red color of human erythroid cells. A total of 2 ? 105(Right) and 1 ? 106(Left) erythroid cells were
collected from one and five E-bursts, respectively.
Generation of erythroid progenitors in hESC/mFLSC coculture. (A) Generation of CFCs from floating and nonfloating cells over time in hESC/mFLSC
www.pnas.org?cgi?doi?10.1073?pnas.0802220105 Ma et al.
cells (E-BFCs) appeared later that E-CFCs, on day 10, and the size
of E bursts was relatively small at that time (Fig. 2D). However,
through day 16 (Fig. 2E). On day 14, single large E-bursts had
1.97 ? 1.0 ? 105cells (n ? 6). Fig. 3F shows 2 ? 105and 1 ? 106
erythroid cells collected from one and five hESC-derived E bursts,
respectively, on day 16.
Thus, the large number of erythroid progenitors generated by
erythropoiesis at the clonal level.
Clonal Analysis of Globin Expression in hESC-Derived Erythropoiesis.
colony cultures of E-BFCs transferred from hESC/mFLSC cocul-
culture, E bursts were randomly and individually picked from
colony cultures started from the hESC/mFLSC cocultures, and
(n ? 6) derived from day-12 cocultures to 98.1 ? 1.1% in E bursts
derived from day-18 cocultures, whereas the opposite trend was
observed for ?-globin expression, 97.1 ? 4.3% of E bursts derived
from day-12 cocultures decreasing to 62.4 ? 16.0% of E bursts
derived from day-18 cocultures (Fig. 3). Thus, an up-regulation of
?-globin expression and a down-regulation of ?-globin over time in
hESC/mFLSC coculture was observed in all individual hESC-
derived E bursts. Because at all coculture time points all E bursts
simultaneously expressed ?- and ?-globins at a rate of 100% (data
not shown), these results indicate that at least one-third of the
day-18 coculture-derived E bursts contained erythroid cells ex-
pressing adult-type Hb A and fetal-type Hb F, but not the embry-
onic-type Hbs Gower-1 and Gower-2.
To examine changes in globin expression in individual erythroid
clones over time, we traced globin expression in single E bursts
individual E bursts were randomly selected, and 20% of the
individual E burst cells were centrifuged onto glass slides while the
remaining 80% were transferred into suspension cultures for an
additional 6 days (referred to as 12?6 cultures) and ?-globin
expression was then examined. The 12 E bursts examined exhibited
a significant increase in ?-globin expression (from 26.4 ? 17.0% to
99.8 ? 0.6%, P ? 0.001) and a corresponding decrease in ?-globin
expression (from 95.6 ? 7.7% to 49.5 ? 15.4%, P ? 0.001) (Table
as measured by globin switching from the embryonic to adult type.
Interestingly, a substantial number of ?-globin-expressing enu-
cleated erythrocytes existed in day-12?6 erythroid cells (Fig. 4 A
and B), whereas no enucleated erythrocytes were observed in
day-12 clonal erythroid cells. We observed clusters of hESC-
derived disk-shaped erythrocytes in the day-12?6 suspension cul-
ture (Fig. 4C).
We also randomly selected 12 E colonies derived from day-10
hESC/mFLSC cocultures and 10 E-colonies derived from day-14
cocultures. All erythroid cells in the E colonies expressed ?- and
?-globins (data not shown), whereas ?-globin was expressed only in
eight day-14 coculture-derived E-colonies (26.8 ? 26.2%, n ? 10)
[Table S1]. In contrast, ?-globin was expressed in 100% of the cells
in all E colonies from both coculture time points (data not shown).
We further examined globin expression in Mix colonies derived
from hESC/mFLSC cocultures. Using the clone-tracing method by
which we analyzed E bursts, we confirmed that erythroid cells in
Mix colonies underwent progressive maturation with increasing
?-globin expression and development of enucleated erythrocytes
and Mix colonies by RT-PCR (Fig. S2). When day-15 hESC/
mFLSC coculture-derived E bursts (n ? 6) and Mix colonies (n ?
6) were examined after 16 days in colony culture, none expressed
OCT-4, nestin, ?-fetoprotein, or brachyury. They all expressed ?-,
?-globin was expressed only in five of six E bursts and three of six
Mix colonies. These data indicate that the early embryonic Hbs
Gower-1 and Hb Portland were completely absent from a fraction
of the hESC-derived erythroid progenitors.
Thus, hESC-derived erythroid cells progressively matured in a
time-dependent manner at the clonal level.
Flowcytometric Analysis of CD81, GPA, and CD71 Coexpression in
hESC-Derived Erythroid Cells. CD81 is a widely expressed surface
protein that exists in almost all human tissues, with the exception
of erythrocytes and megakaryocytes (9). Our previous report
never expressed CD81 (10). To investigate the expression of CD81
in hESC-derived erythroid cells, we performed flowcytometric
analysis and cell sorting to examine coexpression of CD81 with
GPA and CD71.
As shown in Fig. S1D, GPA?CD45?erythroid cells appeared on
over time. All of the cells in this fraction coexpressed CD81, but
most were negative for CD71 at days 10–14. However, CD81 was
different amounts of time in coculture (n ? 6 in each time point). Each E burst
was individually picked from colony culture medium, and the globin expres-
the ratio of ?- or ?-globin?/human Hb?cells.*, P ? 0.01 when compared with
the average expression of ?- and ?-globins in day-12 cocultures. (B) Represen-
tative micrographs of Hb and ?- and ?-globin immunostaining of E burst cells
derived from E-BFCs at days 12 and 18 of coculture. (Scale bar: 20 ?m.)
Clonal analysis of time-associated changes in globin expression
Ma et al.
September 2, 2008 ?
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no. 35 ?
gradually down-regulated by days 16–18 concomitant with up-
regulation of CD71.
We then analyzed expression of CD81 on erythroid cells in
day-15 hESC/mFLSC coculture-derived E bursts on day 12 of
colony culture. CD81 and CD71 were coexpressed in most GPA?
the expression of CD81 considerably decreased, and most GPA?
cells did not express CD81, whereas half of the GPA?cells still
expressed CD71. This expression pattern mimics that of human
CB-derived E burst cells (Fig. S3B). Considering the increase in
?-globin?erythroid cells over the same time course, down-
regulation of CD81 expression may mark the progression of mat-
uration of hESC-derived erythroid cells. Therefore, we isolated
GPA?cells from E bursts on days 12 and 16 of clonal culture and
analyzed their globin expression (Fig. S3C). On day 12 of clonal
culture, fewer GPA?CD81highE burst cells than GPA?CD81low
cells expressed ?-globin (72.9% and 95.7%, respectively). On day
16, all GPA?CD81?cells expressed ?-globin, whereas 6.1% of
GPA?CD81?cells still did not express ?-globin (Table 2). These
results indicate that the gradual down-regulation of CD81 on
hESC-derived erythroid cells is correlated with progressive matu-
ration to adult-type erythroid cells.
Functional Assays of hESC-Derived Erythroid Cells. The large-scale
generation of hESC-derived erythroid cells permitted us to exam-
ine their function in detail. We measured oxygen dissociation in
hESC-derived E burst cells. As shown in Fig. 5A, hESC-derived
of human CB, whereas human adult peripheral blood (PB) exhib-
ited a slightly right-shifted curve. These data indicate that hESC-
than adult RBCs.
Because the oxygen dissociation curve showed that hESC-
derived erythroid cells are functional oxygen carriers, we exam-
ined glucose-6-phosphate dehydrogenase (G6PD) activity to
confirm their ability to defend against oxidative stresses (Fig.
5B). Adult PB (5 ? 106RBCs per 5 ?l) was used as a control.
The hESC-derived erythroid cells (5 ? 106) had higher G6PD
activity than the control. Human CB-derived erythroid cells (5 ?
106) also showed high G6PD activity, but comparatively lower
than hESC-derived erythroid cells.
In our previous report (8), hESC/mFLSC cocultures generated a
large number of human hematopoietic progenitors, particularly
erythroid and multipotential cells. In the current study, using these
clonal erythroid cells, we demonstrated that most hESC-derived E
colonies, E bursts, and Mix colonies contained adult-type ?-globin-
expressing erythroid cells, and the percentages of ?-globin-
Table 1. Progressive maturation of hESC/mFLSC
coculture-derived erythroid cells
E bursts no.
Day 12Day 12?6 Day 12Day 12?6
95.6 ? 7.7
99.8 ? 0.6
Total 49.5 ? 15.426.4 ? 17.0
Progressive maturation of hESC/mFLSC coculture-derived clonal erythroid
cells. Cells were harvested on day 15 of hESC/mFLSC coculture and transferred
to colony cultures. On day 12 of colony culture, 12 E bursts were randomly
selected, and 20% of the individual clonal cells were centrifuged onto glass
slides, and the remaining 80% were transferred to suspension culture. After
an additional 6 days of suspension culture, globin expression in individual
colonies was examined. The percentages were calculated from the ratio of ?-
or ?-globin?/human Hb?cells.
derived erythroid cells. (A) Cytospin sample of hESC-derived erythroid cells from
a day-12?6 suspension culture (May-Grunwald-Giemsa staining). Arrows indi-
cate enucleated erythrocytes. (B) Immunostaining for ?-globin expression in
hESC-derived erythroid cells from the same suspension culture shown in A.
in A and B.
Clonal analysis of progressive maturation of hESC/mFLSC coculture-
Table 2. Time-related analysis of globin expression of sorted
hESC-derived E burst cells defined by CD81 expression
Day 12 hESC-E burst
Day 16 hESC-E-burst
CD81L NSCD81L CD81?
Expression of various globins in the sorted hESC-derived erythroid cell
fractions defined by the gates shown in Fig. S2C. Note that hESC-derived
E-burst cells on day 16 of colony culture were 100% ?-globin?within the
GPA?CD81?fraction. NS, no sorting.
www.pnas.org?cgi?doi?10.1073?pnas.0802220105Ma et al.
expressing cells increased over time in culture to ?100%, whereas
embryonic ?-globin-expressing cells decreased concomitantly.
Although it is not known whether primitive and definitive
hematopoietic cells are derived from the same source, progenitors
committed to primitive hematopoiesis are hypothesized to express
only embryonic globins, and we did not find E bursts or Mix
colonies that expressed only embryonic globins. There are several
colony culture conditions do not support primitive progenitors or
that mFLSCs are not able to stimulate the generation of primitive
progenitors. Alternatively, primitive progenitors may be able to
synthesize definitive globins in some hematopoietic environments.
This hypothesis is supported by the present finding that embryonic
(?), fetal (?), and adult-type (?) globins were coexpressed not only
in single colonies but also in single cells, suggesting that switching
of globin expression from primitive to definitive hematopoiesis is a
sequential event occurring in individual cells. Therefore, an hESC-
derived definitive hematopoiesis-fated erythroid cell may first
coexpress both primitive and definitive globins and finally change
to 100% definitive properties over time in culture. In murine
experiments, successful transplantation of YS hematopoietic cells
directly into adult recipients is difficult, indicating that the YS cells
differ from adult-type hematopoietic cells. However, YS-derived
hematopoietic stem cells transplantable to adult recipients can be
generated both by in vitro coculture with murine aorta-gonad-
mesonephros region-derived stromal cells (11) and in vivo trans-
plantation into fetuses or FL (12–15). These data suggest that
further stimulation of embryonic hematopoietic cells by the fetal
hematopoiesis. Our hESC/mFLSC coculture system may mimic
such an environment.
The similarity between our culture system and hematopoietic
further maturation was suspended. These results mimic human
early YS hematopoiesis phenotypically and genetically (6). There-
fore, because the environment lacked properties required to pro-
mote definitive hematopoiesis, these EB-derived erythroid cells
may not progress toward further maturation. However, our hESC/
mFLSC coculture system may stimulate the maturation of early
hematopoietic cells. The present results in hESCs and nonhuman
primate ESCs (16) show that ESC-derived erythroid progenitors
capable of producing ?-globin-expressing erythroid cells were
stromal cells, indicating that continuous association with the stro-
mal layer is needed for the maturation of definitive progenitors.
be confirmed not only by globin switching, but also by changes in
surface marker expression. In our previous observations of human
CB cells, CD81 was never expressed on GPA?cells (10). In this
study, however, CD81 was expressed exclusively on hESC-derived
GPA?erythroid cells at early times, but expression was gradually
down-regulated by the time the colonies were 100% ?-globin
positive. Thus, coexpression of GPA and CD81 on hESC-derived
erythroid cells may represent an early stage in development and
suggests that CD81 could be used as a developmental marker in
In addition to providing insight into the mechanism of erythro-
poiesis in hESCs, the current study has important implications for
clinical use of hESC-derived erythrocytes. The high G6PD activity
represent younger populations, with a higher percentage of reticu-
locytes and young RBCs that are known to express higher levels of
G6PD. hESC-derived erythoid cells were also able to function as
oxygen carriers, although they exhibited an oxygen dissociation
pattern similar to CB rather than adult PB. In addition, hESC-
derived erythroid cells showed no expression of genes associated
with retention of ESC characteristics, indicating little possibility of
their oncogenicity or differentiation into cells other than RBCs.
setting, but significant advances in bioprocess engineering are still
needed to make clinical applications feasible. According to our in
vitro culture system, 104hESCs roughly amplify to 106mature
erythrocytes. Of note, a simple transfusion needs 1–2.5 ? 1012
require a bulk cell culture of some 104plates of hESCs. There will
be interest in considering the further scale-up of the culture system
for clinical transfusion.
Taken together, we have demonstrated the potential of hESC-
derived erythroid cells to progressively mature to synthesize adult-
type Hb, generate enucleated erythrocytes, and function as oxygen
carriers. The large quantity and high purity of hESC-derived
definitive erythroid cells generated in our coculture system can
provide an experimental model for investigation of the early stages
of human erythropoiesis, especially globin switching, and for ex-
amination of pathogenesis and therapeutic drugs for hereditary
disorders of erythropoiesis. Because transplantable hematopoietic
our system may also help in exploring the mechanism controlling
the generation of these stem cells.
Materials and Methods
hESC Cultures. The hESCs (line H1) were maintained and passaged weekly on
irradiated mouse embryonic fibroblast feeder cells as described (3).
cells at day 16 of colony culture, human CB, and adult PB. (B) G6PD activity of
Functional assays of hESC-derived erythroid cells. (A) Oxygen disso-
Ma et al.
September 2, 2008 ?
vol. 105 ?
no. 35 ?
Establishment of Murine FL-Derived Stromal Cells. mFLSCs were prepared by as Download full-text
described (8, 10). Briefly, FLs were removed from embryonic day 15 (E15) BCL/
Black 6 mice. After trituration, free FL cells were washed once with PBS and
washed and plated in a 75-cm2flask at a density of four FLs per flask. After 48 h
0.05% trypsin/EDTA and replated. Cells thus maintained were harvested and
stored in liquid nitrogen. Before use in coculture with hESCs, the frozen mFLSCs
days to reach confluence, and then irradiated (25 Gy).
Coculture of hESCs with mFLSCs. Undifferentiated hESC colonies consisting of
mFLSCs prepared in gelatin-coated six-well culture plates in 3 ml of culture
medium [15% FBS (HyClone), 1 mM glutamine, 1% nonessential amino acid
solution (100?; Invitrogen), and ?-MEM (Invitrogen)]. Plates were incubated at
37°C in a humidified atmosphere containing 5% CO2. The culture medium was
exchanged every 3 days by removing all of the supernatant from the culture
dishes and adding new medium. On certain days, floating cells in the coculture
were collected, and nonfloating cells were harvested by treating them with
Hematopoietic Colony Culture and Suspension Culture. Hematopoietic colony
mixture containing 1.2% methylcellulose (Shin-etsu Chemical), 30% FBS, 1%
deionized fraction V BSA, 0.1 mM 2-mercaptoethanol (ME), ?MEM, a human-
cytokine mixture (100 ng/ml stem cell factor, 10 ng/ml IL-3, 100 ng/ml IL-6, 10
ng/ml thrombopoietin, 10 ng/ml granulocyte colony-stimulating factor, and 4
units/ml erythropoietin), and cells (hESC/mFLSC coculture cells or human CB
mononuclear cells) was plated in a 35-mm culture dish (Becton Dickinson Lab-
CO2at 37°C under an inverted microscope. Erythroid cell colonies including E
colonies, E bursts, and Mix colonies were characterized as containing bright red
types; E bursts, colonies that consist of ?200 erythroid cells, or exhibited two or
more subcolonies, without other types of cells; Mix colonies, colonies that con-
determined on days 7–10 of the culture. The numbers of E bursts, Mix colonies,
and myeloid colonies including granulocyte, macrophage, and granulocyte-
macrophage colonies were determined on days 12–14 of the culture. In some
experiments, individual colonies were picked from the methylcellulose culture,
FBS, 0.1 mM 2-ME, ?MEM, and the cytokine mixture, similar to the previous
method (8). For clonal analysis of Hb expression and RT-PCR, individual colonies
slides by using a Cytospin instrument (Shandon) or lysed in RNA-preparation
buffer and frozen at ?80°C.
Flowcytometry Analysis, Cell Sorting, and Antibodies. Cocultured hESC/mFLSCs
with normal rabbit serum to block nonspecific binding and then stained with
various mAbs conjugated to FITC, phycoerythrin (PE), or allophycocyanin (APC).
Stained cells were washed with PBS and analyzed by using a FACSCalibur flow-
(BD Pharmingen), CD71 (Beckman Coulter), and CD81 (BD). Recorded data were
analyzed by using the Flowjo software (Tomy Digital Biology). In some experi-
and anti-CD45-APC and fractionated by sorting on a FACSAria sorter (BD).
sion, RT-PCR was used. Total RNA was prepared from hESC/mFLSC cocultures,
individual hESC-derived E burst and Mix colony cells, and human CB-derived E
burst cells by using the RNA subtract kit (Promega). Single-stranded cDNA was
synthesized from total RNA using a SuperScript first-strand synthesis system for
RT-PCR (Invitrogen). PCR conditions were the same as reported (8, 16). Human
gene-specific primers were used throughout our experiments to avoid interfer-
ence from mFLSCs (8, 16, 19). For semiquantitative comparisons of gene expres-
sion, amounts of cDNA template were standardized against the relative expres-
sion of GAPDH in each sample.
Morphological Observation and Immunochemical Staining. For morphological
slides and stained with a May-Grunwald-Giemsa solution. For immunochemical
staining using fluorescently labeled Abs, glass slide samples of hESC/mFLSC co-
cultures, hESC-derived colony cells, suspension cultures, and human CB-derived
colony cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with
incubated with primary anti-human Abs [goat anti-human Hb polyclonal Ab
(pAb); Bethyl Laboratories; rabbit anti-human Hb pAb; MP Biomedicals; mouse
anti-human ?-, ?-, and ?-globin mAbs; Santa Cruz Biotechnology; and mouse
with PBS containing 5% skim milk, and incubated with FITC- or carbocyanin (Cy)
3-conjugated secondary Abs (Jackson ImmunoResearch) for 30 min at room
temperature. Nuclei were labeled with Hoechst 33342 (Molecular Probes). After
three washes with PBS, samples were observed with a fluorescence microscope.
With the exception of E colony cells, the percentage of positive cells was deter-
mined from examination of 200–400 cells.
Functional Assays for hESC-Derived Erythroid Cells. To detect G6PD activity, an
manufacturer’s protocol (Dojindo) (20). The oxygen binding ability of hESC-
derived erythroid cells, human CB, and adult PB was measured with a Hemox
analyzer, as reported (21, 22).
determined by using Student’s t test. P ? 0.05 is considered significant.
ACKNOWLEDGMENTS. We thank Drs. Y. Cui, N. Watanabe, and Y. Ishii for
their help. This work was supported by Japan Society for the Promotion of
Science Grants 18591217 (to F.M.), 19591277 (to Y.E.), and 17390297 (to K.T.).
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