George R. Honig,1 Shi-Jiang Lu,2 Qiang Feng,2 Loyda N. Vida,1
Bao-Shiang Lee,3 and Robert Lanza2,4
1Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois, USA
2Stem Cell and Regenerative Medicine International, Worcester, Massachusetts, USA
3Protein Research Laboratory, Research Resources Center, University of Illinois at Chicago,
Chicago, Illinois, USA
4Advanced Cell Technology, Worcester, Massachusetts, USA
Under culture conditions that promote hematopoietic differentiation, human embryonic stem
cells (huESC) give rise to primitive erythroid cells that closely resemble the nucleated erythrocytes of
early-stage human embryos. The globin chain distribution of these cells is similar to that seen
during the embryonic and fetal stages of development. Here we show that huESC-derived erythroid
cells produce substantial quantities of homotetrameric hemoglobin (Hb) composed exclusively of
g-globin-containing subunits. The globin synthesis of these erythroid cells was also significantly
unbalanced, with a substantial decrease of a-like globin chain synthesis in relation to that of their
b-like globins, a pattern characteristically associated with a-thalassemia (a-thal). This pattern of
unbalanced globin synthesis appears to be an inherent feature of human erythroid cells that synthesize
predominantly embryonic-stage globins.
Keywords Human embryonic stem cells (huESC), Embryonic stem cells, Embryonic
hemoglobins (Hbs), Hb g4 globin synthesis
INTRODUCTION
Recent methodological advances in the directed hematological differentiation
of human embryonic stem cells (huESC) have provided efficient
means for generating substantial quantities of human erythrocytes (1,2),
Received 6 October 2009; Accepted 26 October 2009.
Address correspondence to George R. Honig, MD, PhD, Department of Pediatrics (MC 856),
University of Illinois at Chicago, 840 South Wood Street, Chicago, IL 60612, USA; Tel: +312-996-1788;
Fax: +312-413-5126; E-mail: ghonig@uic.edu
146 G.R. Honig et al.
sufficient to allow characterization of their cellular and functional properties
(2). These erythroid cells exhibit features similar to those seen during
the yolk sac stage of human embryogenesis: they are substantially larger
than adult erythrocytes; they are nucleated; z- and e-globins are their predominant
globin chain types; and the b-globin chains of adult hemoglobin
(Hb) are characteristically absent (1,2). In spite of these differences, within
the physiological range the oxygen affinity, Bohr effect, Hill’s n coefficient,
and 2,3-diphosphoglycerate interaction of these primitive erythroid cells
are similar to those of normal postnatal erythrocytes (2). Here we report
further characterization of these huESC-derived erythroid cells, including
their Hb composition and their pattern of globin chain synthesis.
MATERIALS AND METHODS
Procedures for the propagation and directed erythropoietic differentiation
of huESC were as previously described (2). For Hb determinations,
washed nucleated erythroid cells were lysed in three volumes of 3% saponin,
and the supernatants were clarified by centrifugation at 20,000 × g.
Electrophoresis was performed on cellulose acetate membranes in Tris/
EDTA/borate buffer at pH 8.6. The individual Hb bands were cut out from
the membranes, and the Hbs were eluted with distilled water. Globin chain
identification of the eluted Hb bands, as well as from each of the chromatographic
fractions recovered from the globin chain synthesis study, was by
matrix-assisted laser desorption-time of flight (MALDI-TOF) protein mass
spectrometry (MS) (2).
For the determination of the relative rates of synthesis of the individual
globin chains, washed nucleated erythroid cells derived from huESC line
MA-01 were incubated for 1 hour in medium (3) containing a mixture of
amino acids from which methionine was excluded. 35S-L-methionine (1175
Ci/mmol; MP Biochemicals, Solon, OH, USA) was added as a radio labeled
tracer. Globin chain fractionation was by carboxymethylcellulose column
chromatography (4).
RESULTS
Electrophoresis of Hb extracted from nucleated erythrocytes derived
from huESC lines MA-01 (2), H-1 and H-7, each demonstrated virtually
identical patterns. A prominent Hb band, migrating in a position slightly
more cathodal than Hb C [b6(A3)Glu→Lys], comprised ca. 70% of the
total (Figure 1); this Hb fraction was composed of z and e subunits, and
therefore represented Hb Gower-1 (5). The other major Hb band, which
migrated in a more anodal position than Hb A, accounted for 12-18% of
the total. This Hb was composed exclusively of g subunits, and therefore was
a-Thal-Like-Globin Synthesis by huESC-Derived RBCs 147
identical to Hb Bart’s (g4), a homotetramer characteristically present in
measurable amounts in erythrocytes of neonates with a-thalassemia (a-thal)
(6). The other Hb bands that could be identified included Hb F (a2g2), Hb
Portland (z2g2) and Hb Gower-2 (a2e2), each representing less than 5% of
the total.
To examine the relative rates of synthesis of the individual globin
chains, we incubated nucleated erythroid cells derived from huESC line
MA-01 in a medium containing 35S-L-methionine. The incorporation of
FIGURE 1 Hemoglobin electrophoresis and globin chain synthesis by nucleated erythroid cells derived
from huESC line MA-01. A) Electrophoresis in EDTA-Tris buffer, pH 8.6, on cellulose acetate. Lane 1:
Hbs A, F, S and C; lanes 2 and 3: Hb extracted from nucleated erythroid cells from huESC line MA-01. B)
Incorporation of 35S-L-methionine into globin chains by MA-01-derived erythroid cells. The identification
of the globin chains from each of the fractions isolated from both the electrophoresis and the globin
synthesis studies was by MALDI-TOF protein MS (2).
148 G.R. Honig et al.
radioactivity into the individual globin chains is shown in Figure 1. After
correction for the numbers of methionine residues in each of the globin
chains (a = 2, z = 1, g = 2, e = 3)the calculated ratio of (a + z)/(g + e)
was 0.53, typical of that seen in reticulocytes of individuals with Hb H
disease (– –/–a) (7,8).
To look for evidence that one or more genetic determinants of a-thal
might have been present in this huESC line, the a2- and a1-globin genes
were amplified by polymerase chain reaction (PCR), the amplicons were
purified, and their nucleotide sequences were determined from 60 nts
(nucleotides) 5′ to the ATG initiation codon through 50 nt 3′ to the polyadenylation
signal (poly A) site. No mutation or evidence of a single nt
polymorphism (SNP) heterozygosity was detected in these sequences. Gap-
PCR tests designed to detect single a-globin gene deletions of the rightward
(–a3.7 ) and leftward (–a4.2) types, and also deletions of both a-globin genes
in cis of the (– –MED ), –(a)20.5, (– –SA), (– –SEA), (– –FIL) and (– –THAI) types
were done. None of these deletions was found. Polymerase chain reactionbased
testing to detect a-globin gene triplications of both the anti-3.7 and
anti-4.2 types was also performed (9) and neither abnormality was
detected.
The z2-globin gene of huESC line MA-01 was also examined for evidence
of a thalassemia mutation. The sequences from nt 13449 to 13890
(GenBank NT_000006.1) and from 15125 to 15476, corresponding to
exons 1 and 3 of the gene, were mutation-free. However, repeated
attempts to sequence the remainder of the sequences were not successful.
To look for evidence of a large deletion involving the HS-40 regulatory
sequences, multiplex ligation probe amplification (10) of the
a-globin gene cluster was also performed, from which no deletion was
detected.
Although the a- and b-like globin chains are encoded by genes that
reside on separate chromosomes, in mature erythroid cell lineages the synthesis
of these complementary globins proceeds in a highly coordinated
manner (11). The characterizing feature common to all of the various
pathological forms of thalassemia is an unbalanced pattern of a/b chain
synthesis, which appears to play a causative role in the disease manifestations
of these syndromes (12).
However, several lines of evidence now suggest that a significant imbalance
between the complementary globins, with a relative excess in the representation
of b-like chains, may be the normal pattern of globin chain
synthesis at the embryonic yolk sac stage of erythropoiesis. Studies of the
Hb composition of erythrocytes from early-stage human embryos (13,14)
demonstrated in virtually every case, the presence of significant quantities
of Hb Bart’s, an indication of a relative deficiency of a-like chains. Hb Bart’s
was seen most prominently in the earliest-stage embryos and diminished in
a-Thal-Like-Globin Synthesis by huESC-Derived RBCs 149
embryos of increasing size and maturation of Hb expression, in which
embryonic Hb types were also declining.
A similar pattern has also been observed from studies of K562 cells, a
line of human myeloid leukemia cells that can be induced to synthesize Hb.
Their Hb composition is composed of embryonic and fetal types, with a
characteristic absence of adult Hb A. These cells have been shown to produce
measurable quantities of Hb Bart’s (15), and correspondingly, K562
cells have also been observed to express an a-thal-like pattern of globin
chain synthesis (16).
Our observations from the present studies indicate that nucleated erythroid
cells derived from huESC, in addition to producing a pattern of globin
chain expression typical of the yolk sac stage of development, demonstrate
an unbalanced ratio of complementary a- and b-like globin chains, comparable
to that seen in individuals with moderately severe a-thal. These findings,
taken together with the reported observations from studies of human
embryonic erythrocytes and K562 cells, suggest that this thalassemia-like
pattern of globin synthesis may be a characteristic common to all erythroid
cells of human origin that synthesize predominantly embryonic Hbs. Moreover,
in light of the more extreme expression of this imbalance we observed
in this study, nucleated red cells derived from the differentiation in vitro of
huESCs would appear to correspond to a very early stage of erythropoietic
development.
We have previously demonstrated that nucleated erythroid cells produced
from huESC possess functional properties similar to those of erythrocytes
of normal adult blood (2). The directed erythroid differentiation of
human embryonic stem cells could therefore potentially be capable of generating
an inexhaustible and donorless supply of transfusable red cells, suitable
for clinical applications. However, in light of the adverse consequences
known to be associated with thalassemia-like globin chain synthesis imbalance,
a further maturation process to advance the developmental stage of
their globin production might well be essential if these erythroid cells are to
prove clinically useful.
Although culture conditions that promote the enucleation of huESCderived
erythroid cells also appear to stimulate the synthesis of Hb A in
these cells (2); thus far this effect has been observed only to a minimal
degree. Consequently, culture conditions that promote this transition with
considerably greater efficiency will be needed if this aim is to be achieved.
ACKNOWLEDGMENTS
We are greatly indebted to Dr. David H.K. Chui and Dr. Hong-Yuan Luo
(Boston University Hemoglobin Diagnostic Reference Laboratory, Boston,
MA, USA) for their analyses of the a- and z-globin genes.
150 G.R. Honig et al.
Declaration of Interest: The authors report no conflicts of interest. The
authors alone are responsible for the content and writing of this article.
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