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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2283-2295
Natural Killer and B-Lymphoid Potential in CD34+ Cells
Derived From Embryonic Stem Cells Differentiated in the Presence of
Vascular Endothelial Growth Factor
By
Naoki Nakayama,
Inghwa Fang, and
Gary Elliott
From the Department of Cell Biology, Amgen Inc, Thousand
Oaks, CA.
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ABSTRACT |
Differentiation of totipotent mouse embryonic stem (ES) cells to
various lymphohematopoietic cells is an in vitro model of the
hematopoietic cell development during embryogenesis. To understand this
process at cellular levels, differentiation intermediates were
investigated. ES cells generated progeny expressing CD34, which was
significantly enhanced by vascular endothelial growth factor (VEGF).
The isolated CD34+ cells were enriched for myeloid
colony-forming cells but not significantly for erythroid colony-forming
cells. When cultured on OP9 stroma cells in the presence of
interleukin-2 and interleukin-7, the CD34+ cells
developed two types of B220+ CD34
lymphocytes: CD3 cytotoxic lymphocytes and
CD19+ pre-B cells, and such lymphoid potential was highly
enriched in the CD34+ population. Interestingly, the
cytotoxic cells expressed the natural killer (NK) cell markers, such as
NKR-P1, perforin, and granzymes, classified into two types, one of
which showed target specificity of NK cells. Thus, ES cells have
potential to generate NK-type cytotoxic lymphocytes in vitro in
addition to erythro-myeloid cells and pre-B cells, and both myeloid and
lymphoid cells seem to be derived from the CD34+
intermediate, on which VEGF may play an important role.
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INTRODUCTION |
DURING DEVELOPMENT of the hematopoietic
system, certain splanchnopleuric mesodermal cells, originated from
pluripotent epiblasts, become committed to one of several developmental
pathways, thereby giving rise to precursors of the hematopoietic as
well as the endothelial lineage.1 However, the molecular
and cellular events leading to the generation of multipotential
hematopoietic stem cells (HSCs) from the mesoderm are still unresolved.
Our approach to studying these issues is based on the capacity of
totipotent embryonic stem (ES) cells, isolated from the inner cell mass
of the mouse preimplantation embryo, to differentiate in vitro into cells of the hematopoietic lineage.2 Upon induction of
differentiation, ES cells from a cell aggregate called the embryoid
body (EB), which contains germ layer-like structures that mimic, to
some extent, the early postimplantation embryo.3 Although a
variety of cell types derived from three germ layers are generated,
some EBs develop clustered, nucleated erythroid cells (primitive
erythrocytes) resembling a blood island in the E7.5-day yolk sac,
followed by the generation of macrophages, mast cells, and definitive
erythrocytes.2,4-9 Myeloid progenitor cells are also
detected in the yolk sac at about the same time of development, which
are also followed by the appearance of macrophages and mast
cells.10-12 In this manner, the transition from ES cells to
hematopoietic cells in vitro parallels the in vivo events observed in
the early yolk sac.5
The hematopoietic potential of ES cells is not restricted to the
erythro-myeloid lineages. Using two different methods of in vitro
differentiation (the EB formation method as described and the coculture
method with hematopoietic stroma cell lines), B- as well as
T-lymphocyte progenitors are generated from ES cells, although
derivation of the third lymphoid lineage, natural killer (NK) cell, has
not been reported.13-17 Marrow reconstituting activity (MRA) is generated with both methods as well.18,19 The
lymphocyte progenitors and MRA derived from ES cells may represent the
earliest lymphocyte progenitors and the earliest HSC developed during
embryogenesis. Although the first site of lymphocyte development in the
mammalian embryo remains to be definitively allocated,20-25
recent experiments have provided strong evidence that this may be an
intraembryonic site.26 Likewise, HSC for the entire
definitive hematopoietic system seems to be generated spontaneously at
an intraembryonic site designated as aorta-gonad-mesonephros
(AGM).27-29 Thus, the in vitro differentiation of
ES cells may represent not only the extraembryonic (yolk sac),
primitive hematopoietic cell development, but also certain fundamental
aspects of the intraembryonic, definitive hematopoietic cell
development in vivo.
Epiblasts isolated from postimplantation mouse embryos also generate
hematopoietic progenitor cells in vitro.30,31 Therefore, dissecting the differentiation pathway from ES cells to
lymphohematopoietic cells by clarifying intermediate cell types using
various cell-surface markers may shed light on the sequence of events
that occurs in vivo during the differentiation of epiblasts to
primitive and definitive hematopoietic progenitor cells. In this
report, we show that CD34+ progeny are produced from ES
cells in vitro and that vascular endothelial growth factor (VEGF)
markedly enhances the generation of this cell population. More
importantly, we report, for the first time, that (1) cytotoxic
lymphocytes with characteristics of natural killer (NK) cells, the
third lymphoid lineage, are generated in vitro from ES cells; and (2)
the CD34+ cell population is highly enriched for both
myeloid potential and NK as well as B-lymphoid potential.
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MATERIALS AND METHODS |
Cells and reagents.
The ES cell line, A3-1, was established as described
before.32 The J7 ES cell was kindly provided by K. Stark
(Amgen Inc, Thousand Oaks, CA), and 32Dcl3 myeloid progenitor cell line
by C. Saris (Amgen). The OP9 stroma cell line was obtained from H. Kodama (Ohu University, Fukushima, Japan), and Yac-1 lymphoma and P815
mastocytoma cell lines were from G. Trail (Amgen). Iscove's modified
Dulbecco's medium (IMDM),  -minimum essential medium (-MEM) and
Dulbecco's phosphate-buffered saline without Mg2+ and
Ca2+ (PBSA) were purchased from GIBCO-BRL (Gaithersburg,
MD). All tissue culture flasks and plates were from Falcon (Franklin
Lakes, NJ).
Recombinant human erythropoietin (EPO), human leukemia inhibitory
factor (LIF), human granulocyte colony-stimulating factor (G-CSF) human
interleukin-2 (IL-2), mouse IL-3, human IL-6, rat stem cell factor
(SCF), and human transforming growth factor (TGF)- 1 were prepared at
Amgen (Thousand Oaks, CA). Recombinant mouse IL-4, mouse IL-7, human
IL-11, mouse IL-12, human IL-15, mouse VEGF, mouse granulocyte
macrophage colony-stimulating factor (GM-CSF) and mouse macrophage
colony-stimulating factor (M-CSF) were purchased from R&D Systems
(Minneapolis, MN). Monoclonal antibody (MoAb) for E-cadherin/uvomorulin
(clone DECMA1) was from Sigma (St Louis, MO). MoAb for CD34 (clone
RAM34) conjugated with biotin was purchased from Pharmingen (San Diego,
CA). For fluorescence-activated cell sorter (FACS) analysis, MoAb for
mouse CD16/CD32 (clone 2.4G2) from Pharmingen was added for blocking
nonspecific staining. Fluorescein isothiocyanate (FITC)-conjugated
streptavidin, and FITC-conjugated as well as phycoerythrin
(PE)-conjugated MoAbs for mouse B220 (clone RA3-6B2), BP-1 (clone 6C3),
CD3 (clone 145-2C11), CD4 (clone RM4-4), CD8 (clone 53-6.7), CD19
(clone 1D3), CD43 (clone S7), CD44 (clone IM7), CD45 (clone 30F11),
DX5, heat-stable antigen (HSA, clone M1/69), Mac-1 (clone M1/70), and
Sca-1 (clone E13-161.7) were also purchased from Pharmingen.
Maintenance of ES cells and induction of differentiation by the EB
formation method.
A3-1 as well as J7 ES cells were maintained as
described,3,32 except that 10 ng/mL LIF was included in the
medium. Differentiation of ES cells with the EB formation method was
according to Keller et al,5 with some modifications
according to Potocnik et al.16 Two days before initiation
of differentiation, 3 × 105 ES cells were passaged onto a
feeder-free, gelatinized 6-cm plate, and cultured in IMDM, 15% fetal
calf serum (FCS) (GIBCO), 10 ng/mL LIF, 0.15 mmol/L monothioglycerol
(MTG, Sigma). The day of differentiation, cells were harvested and
resuspended in the differentiation medium, consisting of IMDM 15% FCS
(Hyclone, Logan, UT), 100 µg/mL bovine transferrin (Sigma), 10 µg/mL bovine insulin (GIBCO), 0.45 mmol/L MTG, in the presence of
various cytokines with or without 1% methylcellulose (Fluka,
Ronkonkoma, NY), at 500 cells/mL for methylcellulose-containing culture, or at 5,000 cells/mL for liquid culture without
methylcellulose. The cell suspension was plated at 15 mL per 10-cm
bacterial grade dish and incubated in 5% CO2, 5%
O2, at 37°C. The standard concentration of SCF was at 100 ng/mL, VEGF at 20 ng/mL, IL-3 at 10 ng/mL, EPO at 1 U/mL, and TGF-1
at 1 ng/mL. For liquid culture, media was changed every day from day 5 to day 9, and then every other day. For methylcellulose culture, 10 mL
1% methylcellulose-containing differentiation medium was added on day
8 or day 9.
Harvesting and staining EB cells.
EBs were collected, washed, resuspended in 1 mL 1 mmol/L EDTA in PBSA,
incubated at 37°C for 3 min, and dissociated into single-cell suspension by passage through a 27-gauge needle, once. The remaining small aggregates were removed by filtration through a 40-µm filter (Falcon). The EB cells were spun and resuspended in 0.5% (vol/vol) Path-O-Cyte (purified bovine serum albumin solution from Miles, Kankakee, IL) in PBSA at 0.5 to 1 × 107 cells/mL. As a
control, undifferentiated ES cells were treated with the EDTA solution
for 10 minutes, and single-cell suspension was made in 0.5%
Path-O-Cyte solution at 0.5 × 107 cells/mL. The cell
suspensions, 50 to 100 µL/well, were transferred to a V-bottom
96-well microtiter plate (Nunc, Naperville, IL) and stained with 10 to
20 ng/mL of antibodies. The stained samples were analyzed on FACScan
(Becton Dickinson, San Jose, CA) or sorted for CD34 using FACStar plus
(Becton Dickinson).
Colonogenic cell assay.
Single-cell suspensions made from EBs, FACS-sorted CD34+ EB
cells, or CD34 EB cells were mixed with IMDM, 20% FCS
(Stem Cell Technology, Vancouver, Canada) 10% BSA (Stem Cell
Technology), 100 µg/mL bovine transferrin, 10 µg/mL bovine insulin,
0.1 mmol/L MTG, 1% methylcellulose, and distributed to four to six
35-mm bacterial-grade dishes at 104 to 105
cells/plate. For erythroid progenitors, 100 ng/mL SCF, 10 ng/mL IL-3,
and 1 U/mL EPO were added to the culture, and colony-forming unit
erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and a
combination of CFU-E Macrophage[M], CFU-n[neutrophil]E, CFU-mastE,
CFU-nEM, and CFU-mastEM) were counted. For myeloid colonies, 100 ng/mL
SCF, 10 ng/mL IL-3, 10 ng/mL GM-CSF, 50 ng/mL G-CSF, 50 ng/mL IL-6 were
included, and CFU-M, CFU-n, CFU-mast, CFU-Mmix (combination of CFU-nM
and CFU-mastM) were counted on day 8. By contrast, for pre-B cells, 100 ng/mL SCF and 25 ng/mL IL-7 with or without 50 ng/mL IL-11 were added,
and CFU-pre-B and CFU-M were counted on day 11. All the plates were
incubated under 5% O2 5% CO2 at 36°C.
Average number of each colony type was adjusted per 105
seeded cells.
Stroma coculture method for developing lymphokine-activated killer
cells and pre-B cells.
The stroma cell line, OP9, was maintained as described.33
Confluent culture of OP9 in a six-well plate was used for coculture. The coculture medium contained -MEM, 5% FCS (Hyclone), 0.1 mmol/L MTG. For direct differentiation of ES cells on OP9 to
lymphokine-activated killer (LAK) cells or to pre-B cells, we used a
modified two-step culture method originally described by Nakano et
al.17 Briefly, 4,000 ES cells/well were cultured on the
confluent OP9 cells in the coculture medium containing an elevated
level of FCS, 15%. On days 5 to 6, cells were harvested, and 5 × 104 live cells/well were transferred onto a new confluent
OP9 cells. Culture medium was changed from day 6, to the standard
coculture medium (containing 5% FCS), and 50 ng/mL IL-2 and 5 ng/mL
IL-7 were added on day 7. On days 12 to 14, the culture was
transferred, again, to a new OP9 well by scraping off the adherent cell
layer with a rubber policeman (Falcon), followed by filtration through a 40-µm filter to remove large colonies of various adherent cell types. By day 21, LAK cells were detected as hallow-forming cells on
the OP9 cell layer. Pre-B-like cell foci were also detected by this
time in a lower frequency. Removal of IL-2 allowed them to expand in
the culture.
For demonstration of lymphoid potential in the CD34+ EB
cells, CD34+ cells sorted from day 6 to 7 EBs (formed in
the presence of 100 ng/mL SCF and 20 ng/mL VEGF) were seeded on the OP9
stroma cells (103 to 105 cells/well of a 6-well
plate) and cultured in the coculture medium containing 50 ng/mL IL-2
and 5 ng/mL IL-7. Medium was changed every 3 days, and small adherent
LAK cells appeared in approximately 2 weeks. The LAK cells were always
the dominant cell type under this condition, and removal of IL-7 on day
7 of culture had only a minimal effect on LAK cell generation. However,
to select pre-B cells, IL-2 was removed on day 7 and maintained only in
IL-7 for another 1 to 2 weeks.
Maintenance of LAK cells and pre-B cells.
CD34+ EB cell-derived as well as ES cell-derived LAK cells
were initially cultured on OP9 cells in the coculture medium containing 50 ng/mL IL-2. Because these LAK cells do not need OP9 cells for proliferation, before performing biological assays or preparing DNA and
RNA, they were transferred to a stroma-free culture. The type I LAK
cells (Table 1) were cultured in the same
medium, and type II LAK cells (Table 1) were kept in the presence of 500 ng/mL IL-2. Spleen LAK cells were generated as
described.34 ES- and EB34-pre-B cells (Table 1) were
maintained on a confluent layer of OP9 in the coculture medium with 5 ng/mL IL-7. For primary pre-B cells from bone marrow, 3 × 105 mouse bone marrow cells/well were cultured on OP9 cells
under the same condition for 3 weeks. Resulting cells were
CD43+ B220+ CD19+ (data not shown).
Proliferation assay and killer cell assay.
Sensitivity of the LAK cells and of pre-B cells to various cytokines
was determined with alamar blue dye (Alamar Bioscience, Sacramento,
CA). Briefly, cells were harvested, washed twice with the coculture
medium and distributed at 5,000 to 20,000 cells/50 µL/well into a
96-well plate containing 50 µL/well of coculture medium with
different concentrations of cytokines. Plates were incubated for 36 to
48 hours at 37°C in 5% CO2. Then, 10 µL/well of alamar
dye was added and incubated further for 5 hours before fluorescence
measurement (excitation wavelength 530 nm/emission wavelength 590 nm)
by CytoFlor II fluorescence plate reader (PerSeptive Biosystems,
Bedford, MA). For pre-B cells, the adherent population was harvested
from the stroma layer with brief trypsin-EDTA treatment, followed by
30-minute incubation in a tissue culture flask in the corresponding
FCS-containing medium to selectively remove the adherent stroma cells.
Cytotoxicity was quantified by the ability of effector cells to rupture
the target cell membrane, which caused the release of 51Cr
from labeled target cells to the medium. Two target cell lines, Yac-1
and P815, were labeled overnight with 2 µCi/mL
Na251CrO4 (Amersham, Arlington
Heights, IL), and a fixed number of the labeled target cells
(8 × 104) were mixed with various numbers of effector
cells in 0.6 mL coculture medium in a 48-well plate. After incubation
for 4 to 18 hours at 37°C in 5% CO2, 0.3 mL each of the
culture supernatant was removed for counting radioactivity with COBRA
II gamma counter (Packard, Meriden, CT). For the 18-hour assay, the
medium contained 50 ng/mL IL-2. Net release of radioactivity was
divided by net total radioactivity in the target cells to obtain
percentage specific killing. The former was determined by radioactivity
in the medium subtracted with those caused by autonomous release from
the target cells. The latter was obtained by radioactivity released
from detergent-lysed target cells subtracted with the autonomous
release.
Gene expression and chromosomal rearrangement in LAK cells and pre-B
cells.
Total RNA was prepared from LAK cells, pre-B cells, 32Dcl3 cells, P815
cells, and OP9 cells according to Chirgwin et al.35 Oligo
(dT)-primed reverse-transcription of the isolated RNAs was performed
with Ready-To-Go T-prime reverse-transcription kit (Pharmacia, Piscataway, NJ) according to the manufacturer's recommendation. Balb/c
mouse-derived spleen cDNA was purchased from Clontech (Palo Alto, CA).
For LAK cells, expression of NKR-P1A (gene 2)36 NKR-P1B (gene 34),36 perforin-1,37 granzyme
A,38 and granzyme B39,40 genes was measured by
reverse transcriptase-polymerase chain reaction (RT-PCR). For EB34- and
ES-pre-B cells, expression of the VpreB, Ig- (B29),
Rag-1 and Rag-2 genes, and the
VHDJH-Cµ transcript
was examined. The standard RT-PCR condition was 30-cycle amplification
with incubation for 1 minute at 94°C, followed by 1-minute incubation
at 60°C, and finally, by 1.75-minute incubation at 72°C per cycle.
For the NKR-P1 family, perforin, the granzyme family, and
VpreB, we used the nested PCR method to ensure the sequence
specificity of the products. The sense-1 and antisense-1 primers were
used for the initial amplification for 20 cycles, and the second
amplification (20 cycles) was performed with the 1,000-fold diluted
initial product, using the sense-2 and antisense-2 primers.
Oligonucleotide primers for NK/CTL-specific genes synthesized are as
follows (expected length of the individual PCR product is indicated in
parentheses).
Primers for pre-B-cell-specific genes, VpreB (sense-1 and
antisense-1), and Ig- were according to Rolink et al,41
and those for VpreB (sense-2 and antisense-2), Rag-1 and
Rag-2 were based on Li et al.42 The
VHDJH-Cµ transcript
was detected by the nested RT-PCR method according to Nakano et
al.17
Chromosomal DNAs from adherent as well as nonadherent pre-B cells, LAK
cells, and OP9 cells were prepared by the standard SDS-protease K
method.43 The DNA rearrangements at the immunoglobulin µ heavy chain locus (DJH and
VHDJH) and at the  light chain locus were, then, detected by PCR using the same primers
(DSF, V7183 VQ52,
JH4, V and J2) described by Potocnik et al.16
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RESULTS |
Transient generation of CD34+ cell population is
facilitated by exogenous VEGF.
In an attempt to dissect the differentiation process of ES cells into
lymphohematopoietic lineages, surface marker analysis was carried out
by FACS phenotyping. Undifferentiated ES cells were CD34
CD44, and expressed
E-cadherin/uvomorulin.44,45 Upon differentiation, the
E-cadherin expression diminished in five days, which was accompanied by
a gain of CD44 expression (Fig 1A). The first sign of colony-forming cell (CFC) generation was observed at this time (data not shown). This
finding is consistent with the fact that, during gastrulation, E-cadherin mesodermal cells are differentiated from
E-cadherin+ epiblasts.46 From day 7, some of
the EB cells start expressing the hematopoietic stem/progenitor cell
marker, CD34,47 followed by the appearance of leukocyte
common antigen (CD45)+ cells (Fig
1A). Because CD34+ cells from
yolk sac contain erythromyeloid colony forming activity,48 we further characterized these CD34+ progeny.
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| Fig 1.
VEGF enhances transient appearance of CD34+
cells during differentiation of ES cells in liquid culture. (A) Surface
marker analysis of differentiating ES cells. Undifferentiated A3-1 ES cells (day 0), as well as cells from EBs cultured for 5 days and 7 days
in the presence of 100 ng/mL SCF, 10 ng/mL IL-3, and 1 U/ml EPO were
stained with anti-E-cadherin, CD34, CD44, and CD45 monoclonal
antibodies. Results of the corresponding isotype control are shown in
gray. Positive cell population (region indicated with   )
on day 0, day 5, and day 7 for E-cadherin are 98.4%, 26.4%, and
0.3%, for CD34 are 0.2%, 1.4%, and 6.7%, for CD44 are 4.4%,
58.4%, and 68.7%, and for CD45 are 0.4%, 0.1%, and 1.0%, respectively. (B) Effect of VEGF on the CD34+ cell
generation. The day 8 EBs derived from A3-1 ES cells cultured in the
absence of cytokine ( ), and in the presence of 20 ng/mL VEGF (+)
were harvested and analyzed for CD34 expression. Staining results of
the gate R1 (top dot plot) are shown in histograms. Background staining
is indicated in gray. Positive cell population () is
3.9% () and 8.5% (+). (C) Kinetics of CD34+ cell
generation in EBs cultured in various growth factors. A3-1 ES cells (1)
and J7 ES (2, 3) cells were induced to differentiate in liquid culture
in the absence of cytokine ( ), and in the presence of 100 ng/mL SCF
( ), 20 ng/mL VEGF ( ), 100 ng/mL SCF, and 1 ng/mL TGF-1 ( ),
100 ng/mL SCF, 10 ng/mL IL-3, and 1 U/mL EPO ( ), and 100 ng/mL SCF
and 20 ng/mL VEGF ( ). From day 6 to day 18, EBs were harvested and
analyzed for CD34 expression. For A3-1, only day 6 and day 8 data were
average values of duplicated samples, and for J7, all but "no
cytokine" and "SCF + TGF-1" were average values of
duplicated samples. The vertical bar indicates standard deviation.
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To optimize the level of CD34+ population, we searched
extensively for protein factors to be included in the differentiation medium, focusing on those known to affect embryonic hematopoiesis in
vivo such as SCF,49,50 VEGF,51-53 and
TGF-1.54 Although various hematopoietic cytokines used
in the previous reports,4,5 including IL-3, IL-11, and EPO
were also tested, VEGF gave the strongest stimulatory effect as a
single factor on the enhancement of the CD34+ cell
population in EBs (Fig 1B and C-1). SCF showed a weaker but significant
effect, whereas TGF-1 demonstrated only an antagonistic effect to
SCF (Fig 1C). VEGF showed a strong additive effect to SCF (Fig 1C-1).
The addition of IL-3 + EPO to SCF also exhibited some enhancement (Fig
1C-3), albeit at lower levels than that achieved with VEGF (Fig 1C-2).
Thus, SCF + VEGF was the optimal factor combination for generating
CD34+ population in EBs. Neither SCF + VEGF nor SCF + IL-3 + EPO changed the transient nature of the appearance of
CD34+ EB cells. Commonly, they peaked around day 8 (J7 ES
cells) to day 9 (A3-1 ES cells) and disappeared by day 14, suggesting
that these cytokines were unable to prolong the viability of, or block differentiation of the CD34+ EB cells.
Erythro-myeloid CFC potential in the CD34+ EB cell
population.
Hematopoietic progenitor cell activity within the CD34+ EB
cell fraction was analyzed. We used two combinations of factors, SCF + IL-3 + EPO and SCF + VEGF, for differentiating ES cells, both of which
allowed significant levels of CD34+ cells to be generated
(Fig 1C). CD34+ EB cells were sorted from day 7 EBs (Fig
2A), and subjected to CFC analysis. The
isolated CD34+ cells contained erythro-myeloid CFCs but
were not necessarily enriched for early (bipotential or multipotential)
progenitor cell types (Fig 2B). Rather, they were preferentially
enriched for myeloid CFCs (CFU-M, CFU-mast [data not shown], and
bipotential CFU-Mmix), and not significantly for erythroid CFCs (CFU-E,
BFU-E, and bipotential, as well as tripotential CFU-Emix). The total myeloid CFCs represented approximately 0.6% to 0.7% of total EB cells
(740 ± 459 [standard deviation] CFCs/105
SCF + IL-3 + EPO EB cells, and 653 ± 308 CFCs/105
SCF + VEGF EB cells). Interestingly, the occurrence of myeloid CFCs
in the CD34+ cell population was consistently higher when
EBs were formed under SCF + VEGF (approximately 7%, 7,361 ± 1,563
CFCs/105 CD34+ cells) than when cultured in SCF + IL-3 + EPO (approximately 3%, 2,692 ± 648 CFCs/105
CD34+ cells). Nevertheless, considering the population of
CD34+ cells to be approximately 7% to 10% of total EB
cells (Figs 1C and 2A), most of the myelomonocytic progenitors seemed
to be CD34+, especially when developed in SCF + VEGF, and
cells with erythroid potential were probably heterogeneous for the CD34
expression.
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| Fig 2.
Types of colony-forming cells enriched in the
CD34+ EB cell fraction. A3-1 ES cells were induced to
differentiate in methylcellulose culture in the presence of 100 ng/mL
SCF, and 20 ng/mL VEGF (A, B2), or 100 ng/mL SCF, 10 ng/mL IL-3, and 1 U/mL EPO (B1). EBs were obtained on day 7, stained with RAM34, and
subjected to cell isolation by FACS. (A) One of the staining results of
SCF + VEGF EB cells is shown: (1) the scatter pattern of the RAM34
stained sample, (2) staining pattern of isotype control, and (3) that of RAM34, in dot plots. Regions for sorting CD34+ (right)
and CD34 (left) cell populations are indicated in boxes
(2 and 3). Number in each box indicates percentage of total EB cells.
Similar scatter pattern and staining pattern were obtained from EBs
developed with SCF + IL-3 + EPO. (B) Erythro-myeloid CFC in the
CD34+ and CD34 cell populations. Average
numbers of CFU-M, CFU-Mmix, BFU-E, CFU-Emix, and CFU-E in
105 presorted EB cells, 105 sorted
CD34+ EB cells, and 105 sorted
CD34 EB cells were determined. The average colony
numbers from CD34+ cells and CD34 cells
were divided by corresponding numbers from presorted EBs to obtain
enrichment factors for individual CFCs. Such enrichment factors
obtained from three (SCF + VEGF) to four (SCF + IL-3 + EPO) independent experiments were then averaged to obtain average enrichment factor. Hatched bar indicates results for CD34+ EB cells,
and open bar is for CD34 EB cells. Factor 1 means no
enrichment. Vertical line indicates standard deviation.
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Generation of two different B220+ lymphocytes from
CD34+ EB cells in vitro.
Since the ability of ES cells to differentiate into B- and T-lymphocyte
progenitors in vitro had been demonstrated,14,16,17 we
examined lymphoid potential of the CD34+ cell population.
However, the sorted CD34+ cells obtained from day 6 and day
7 EBs in the same way as shown in Fig 2A did not give rise to any pre-B
colonies in the presence of IL7 and SCF,55 nor in the
presence of SCF, IL-7, and IL-11.56 Therefore, we examined
a coculture method similar to what was previously described by Nakano
et al,17 except that two cytokines that affect B
lymphopoiesis in vivo, IL-257 and IL-7,58 were added. First, ES cells were cultured directly on OP9 in the presence of
50 ng/mL IL-2 and 5 ng/mL IL-7. In 2 to 3 weeks, B220+
cells and CD43+ cells emerged, suggesting the successful
generation of lymphoid cells. Removal of IL-7 leaving IL-2 in culture
led to accumulation of B220+ CD43 large
granular lymphocytes (LGLs) (LAKa and LAKj, Table 1), morphologically
similar to LAK cells from spleen (spleen-LAK, Table 1). Interestingly,
these LGL showed IL-2-dependent cytotoxicity to OP9 stroma cells. By
contrast, removal of IL-2 enriched for B220+
CD43+ BP-1 HSA+
CD19+ small round pre-B-like cells (ES-pre B, Table 1).
Cells of the same surface phenotype were also obtained when bone marrow
cells were cultured for 2 to 3 weeks on OP9 with IL-7 (BM-pre-B, Table 1).
Next, the sorted CD34+ cells were cultured on the OP9
stroma cells with IL-2 and IL-7. A3-1 ES cells were primarily used
because the pre-B-cell potential was reproducibly higher than J7, and the SCF + VEGF condition was employed because IL-3 might be inhibitory for the pre-B-cell generation.59 Two types of
B220+ cells emerged from the CD34+ cell
fraction in approximately 2 weeks of coculture (Table
2). When IL-7 was removed on day 7, B220+ CD43 cells with LGL morphology and
cytotoxic characteristics to OP9 cells were expanded. We isolated such
LAK cells from three independent experiments and designated them
LAK34a-1, LAK34a-2, and LAK34a-3 (Table 1). The LAK34a-1 and LAK34a-3
were large LAK cells, morphologically similar to adherent LAK cells
derived directly from ES cells and from spleen (Fig
3A). However, LAK34a-2 was a relatively
small cell with typical LGL morphology (Fig 3A). The former group of CD34+ EB cell-derived and ES cell-derived LAK cells was
designated collectively as type I LAK cells, and the latter type II LAK
cells (Table 1). Removal of IL-2 on day 7 of coculture generated
B220+ CD43+ B lymphocyte like cells. These
cells, designated EB34-pre-B, were morphologically similar to ES-pre B
as well as BM-pre-B cells (Table 1; Fig 3A and C). Table 2 summarizes
the results of these coculture experiments. From all the experiments,
either or both LAK cells and pre-B cells were developed from
104 to 105 CD34+ EB cells. However,
no such lymphocytes were generated from the same numbers of
CD34 EB cells. Only in one case (experiment 4),
106 CD34 EB cells gave rise to type I-like
LAK cells. Thus, progenitor cells for both pre-B cells and LAK cells
were enriched in the CD34+ EB cell fraction.
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| Fig 3.
Two different B220+ lymphocytes derived
from the CD34+ EB cell fraction. (A) Morphological
characterization by Wright-Giemsa staining (Objective ×100 Oil). Type
II LAK (LAK34a-2) cells (left), type I LAK (LAK34a-3) cells (center),
and EB34-pre-B cells (right) were spun on slides and stained with
Wright-Giemsa. Large granules are present in both small LAK34a-2 cells
and larger LAK34a-3 cells, but not in EB34-pre-B cells. (B) Phenotypic
analysis of CD34+ EB cell- derived LAK cells. Type II
LAK cells (LAK34a-2, upper panels) and type I LAK cells (LAK34a-3,
lower panels) were stained with PE-conjugated anti-CD3 , Mac-1,
Sca-1, and B220 monoclonal antibodies. Positive cells for CD3, Mac-1,
Sca-1, and B220 (region ) are 0.1%, 23.0%, 40.5%, and
99.1%, respectively, in LAK34a-2, and 0.2%, 62.9%, 98.9%, and
98.5%, respectively, in LAK34a-3. Note the difference in the Sca-1
pattern between LAK34a-2 and LAK34a-3. Results of the corresponding
isotype control are indicated in gray. (C) Phenotypic analysis of
EB34-pre-B cells. Adherent EB34-pre-B cells were collected from the OP9
stroma layer, and stained with PE-conjugated anti-CD43, B220, BP-1, and
CD19 monoclonal antibodies. Positive cells for CD43, B220, BP-1, and
CD19 (region ) are 99.8%, 66.5%, 0%, and 99.5%,
respectively. Background staining is indicated in gray.
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B220+ CD43 LGL from
CD34+ EB cells contains LAK activity with target
specificity of NK cells.
NK1.1+ spleen LAK cells and NK progenitor cells in bone
marrow are known to be B220+.41,60 Therefore,
we sought to determine whether the CD34+ EB cell-derived
B220+ LAK cells could be categorized in the NK cell
lineage. C57BL/6 mouse NK cells are surface CD3
NK1.1+ LGLs.61 NK1.1 has been molecularly
defined as a member of the activating NK-receptor family,
NKR-P1.62 Because the NK1.1 marker is not available for NK
cells derived from the 129Sv mouse line,63 from which both
ES cells are originated, we tested another pan-NK cell marker,
DX5.64 All the CD34+ EB cell-derived and ES
cell-derived LAK cells were B220+ CD3
CD8 CD19 CD34
CD43 cells, suggesting that they were neither typical B
cells nor T cells (Fig 3B). However, expression of DX5 was not
detected. This may be attributable to the long-term culture of the LAK
cells in IL-2, because even spleen-LAK cells, initially containing
DX5+ cells, lost expression after a few weeks of culture in
IL-2 (data not shown). Weak expression of Mac-1 was found on type I LAK
cells and, in even lower levels, on type II LAK cells (Fig 3B).
Interestingly, Sca-1 differentiated the two types of LAK cells. It was
highly expressed on type I cells, whereas most type II cells expressed at very low levels. Therefore, type I LAK cells are B220+
CD3 CD8 CD19
CD34 CD43 Mac-1lo
Sca-1+, and type II LAK cells are B220+
CD3 CD8 CD19
CD34 CD43 Mac-1lo and
Sca-1lo.
Cytotoxicity of the LAK cells was quantified by 51Cr
release from two different target cells: NK-sensitive Yac-1 lymphoma
and NK-resistant p815 mastocytoma cell lines. Primary LAK cells from spleen showed strong killing activity to both target cells in 4 hours
(Fig 4A). However, type I LAK cells
required a much longer period (18 hours) to show appreciable
cytotoxicity, and they showed a similar degree of cytotoxicity to both
Yac-1 and P815 cells (Fig 4B and D). By contrast, type II cells,
LAK34a-2, exhibited strong cytotoxicity on Yac-1 cells, comparable to
spleen-LAK cells (Fig 4A and C) and did not kill NK-resistant P815
cells in the standard 4-hour assay. Thus, CD34+ EB
cell-derived as well as ES cell-derived LAK cells were cytotoxic, of
which type II LAK cells showed target specificity equivalent to that of
NK cells.
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| Fig 4.
Cytotoxicity of the CD34+ EB cell-derived
LAK cells to NK-sensitive Yac-1 cells and NK-resistant P815 cells. The
cytotoxicity of spleen LAK (A), type I LAK (LAK34a-1) (B), LAK34a-3
(D), and type II LAK (LAK34a-2) (C) effector cells was compared by the 51Cr release assay. A fixed number of
51Cr-labeled target cells, Yac-1 (, ) and P815 (,
 ), were mixed with different numbers of effector cells, and
incubated for 4 hours (, ), or 18 hours (, ). Averaged
values of percent specific killing are shown with the corresponding
standard deviation (vertical line).
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Effects of IL-2 and IL-12 on the CD34+ EB
cell-derived LAK cells.
Both CD34+ EB cell-derived and ES cell-derived LAK cells
were able to be maintained in a stroma-free condition in the presence of 50 ng/mL IL-2. However, quantitative survival/proliferation assay
showed that type I LAK cells strongly responded to IL-2, whereas type
II LAK cells showed only a weak response to IL-2 (Fig
5A). IL-12 and IL-15 stimulate the
generation of LAK cells and cytotoxic T lymphocytes (CTLs), as well as
the proliferation of NK cells.65-69 IL-15, which shares its
receptor subunits with the IL-2 receptor,69-71 showed a
weak effect on both types of LAK cells in a high concentration range
(50 to 250 ng/mL). In addition, IL-12, which acts as a co-stimulator
with IL-2 and IL-15,65-68 synergized with a low
concentration of IL-2 (3 ng/mL) to stimulate survival/proliferation of
type II LAK cells (Fig 5B). To a lesser degree, SCF also showed a
synergistic effect. However, IL-12 exhibited no synergy with IL-2 or
IL-15 in type I LAK cells (data not shown). Therefore, type II LAK
cells had cytokine dependency equivalent to the NK/CTL cell-types.
Individually added IL-3, CSF-GM, CSF-M, IL-4, IL-7, IL-12, or SCF had
no appreciable effects on both types of LAK cells.
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| Fig 5.
Sensitivity of the CD34+ EB cell-derived
LAK cells to various cytokines. (A) Difference in response to IL-2.
Type I LAK cells (LAK34a-1; , ,  ; and LAK34-a-3: , ,
), and type II LAK cells (LAK34a-2: , , ) were cultured at
5,000 cell/well/0.1 mL coculture medium for 48 hours in the presence of
different concentration of IL-2 (, , ), IL-12 (), IL-15
(, , ), and SCF (). The 1 (100) × dilution
corresponds to 0.5 µg/mL of added cytokine except that, in the case
of SCF, 1× dilution is 10 µg/mL. The cell viability was measured
with alamar blue dye. Note that singly added IL-12 and SCF do not show
any signal. (B) Effect of IL-12 and SCF on the IL-2-activated type II
LAK cells. LAK34a-2 cells were cultured in a fixed concentration of
IL-2 (3 ng/mL) (), with various concentrations of IL-12 (0.5 µg/mL
at 1× dilution) (), and SCF (10 µg/mL at 1× dilution) ().
The cell viability was measured with alamar blue dye.
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Thus, type I LAK cells were IL-2-dependent B220+
CD3 Sca-1+ cells with weak nonspecific
cytotoxicity, and type II LAK cells were IL-2 + IL-12-dependent
B220+ CD3 Sca-1lo cells with
strong cytotoxicity to NK-sensitive Yac-1 cells. These observations
provide additional support to the similarity of type II LAK cells to
IL-2-activated NK cells.
The CD34+ EB cell-derived LAK cells express
NK-specific genes.
The mouse NK cell can be molecularly defined as a CD3
NKR-P1+ lymphocyte.72 The activating NK
receptor (NKR-P1) family is composed of NKR-P1A, NKR-P1B, and NKR-P1C
(NK1.1) in mice and is the specific marker for NK
cells.36,62,73 Furthermore, upon activation by IL-2, NK
cells increase the expression level of a set of proteins essential for
eliciting cytotoxicity. These include pore-forming protein, perforin,
and a serine protease family, granzyme, which are also specific markers
for CTLs and NK cells.74,75 We first determined if the NK
receptor gene was expressed in the CD34+ EB cell-derived
and ES cell-derived LAK cells by RT-PCR. Since the NK1.1 (NKR-P1C)
gene is known to be highly polymorphic,63 and sequence
information of 129Sv mouse-derived NK1.1 cDNA was unavailable, we
examined expression of the NKR-P1A and NKR-P1B genes. As shown in Fig
6, mRNA for NKR-P1A was detected
specifically in all the LAK cells we tested, and NKR-P1B mRNA
expression was found in spleen-LAK and type II LAK cells.
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| Fig 6.
Expression of NK cell-specific mRNAs in the
CD34+ EB cell-derived and ES cell-derived LAK cells.
RT-PCR analysis was performed with total RNAs extracted from the
C57BL/6 mouse spleen-LAK cells, type I LAK (LAKa, LAKj, LAK34a-1, and
LAK34a-3) cells, type II LAK (LAK34a-2) cells, ES-pre-B cells,
EB34-pre-B cells, P815 cells, 32Dcl3 cells, and OP9 cells, and analyzed
on 1.2% agarose gels. The first lane is for size standard, and
the rightmost lane is a negative control for cDNA. Results of the 2×
20-cycle amplification with different set of primers for NKR-P1A (top),
NKR-P1B, granzyme A (gzm A), granzyme B (gzm B), perforin, and
-actin (bottom) genes are shown.
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Next, expression of perforin, granzyme A, and granzyme B mRNAs was
examined. These mRNAs were expressed in all the LAK cells we tested
(Fig 6). Among non-LAK cells, 32Dcl3 myeloid cells expressed granzyme B
mRNA, and P815 mastocytoma expressed both perforin and granzyme B mRNA
as described.76,77 However, OP9 cells, and ES- as well as
EB34-pre-B cells did not show any sign of expression of these genes.
Thus, LAK cells generated from CD34+ EB cells and ES cells
seemed to express the perforin/granzyme system for exerting
cytotoxicity similar to CTL and NK cells.
In summary, the IL-2-dependent B220+ CD3
Sca-1+ type I LAK cells were NKR-P1A+
perforin+ granzyme A/B+, and IL-2 + IL-12-dependent B220+ CD3
Sca-1lo type II LAK cells were NKR-P1A/B+
perforin+ granzyme A/B+. These results suggest
that CD34+ EB cell-derived as well as ES cell-derived LAK
cells are likely to be IL-2-activated NK cells.
B220+ CD43+ cells derived from
CD34+ EB cells show characteristics of pre-B cells.
Although EB34- as well as ES-pre-B cells responded to IL-7,
maintenance of these cells was strictly dependent on both OP9 and IL-7
analogous to the PA6 + IL-7 pre-B cells described by Hayashi et
al.78 Pre-B cells developed directly from ES cells have
been described.14,16,17 To confirm that the
CD34+ EB-derived pre-B-like cells were equivalent to
BM-pre-B cells as well as the previously described ES cell-derived
pre-B cells, we investigated expression of genes whose products are
involved in pre-B-cell development (Table
3). RT-PCR analysis clearly showed that
mRNAs coding for the pre-B-cell receptor proteins such as
VpreB, and Ig-, and those encoding proteins for the
immunogloblin gene rearrangement, Rag-1 and Rag-2, were expressed in
these cells, but not in LAK cells nor in other non-B cells tested. Only
spleen LAK cells showed some signal for Ig- expression, probably
because of contamination of spleen B cells. The adherent EB34-pre-B
cells isolated from the OP9 stromal layer expressed somewhat lower
levels of Rag-1 and Rag-2 than those expressed by the nonadherent
population.
Next, chromosomal rearrangement at the immunoglobulin heavy chain locus
was examined by PCR (Fig 7). Amplification
with DSF and JH4
primers generated products indicating that, as in BM-pre-B cells, the
four possible DJH recombinations, DJH1, DJH2,
DJH3, and DJH4, took
place in both EB34- and ES-pre-B cells (Fig 7 top). The spleen LAK
cells showed again a sign of B cell contamination. The result with
V7183 and JH4 primers indicated
that some of the EB34- as well as ES-pre-B cells underwent
VHDJH recombination as
well (Fig 7 middle). PCR with VQ52 and JH4 primers gave a similar result (data not
shown). Non-B cells such as type I LAK, type II LAK, and OP9 cells did
not give any signal for the DJH and
VHDJH recombination. The VHDJH-Cµ
transcript was also detected specifically in ES- and EB34-pre-B
cells, albeit at lower levels in the adherent population than in the
nonadherent population (Table 3). By contrast, all LAK cells and OP9
stroma cells were negative. Chromosomal rearrangement at the light
chain locus was also noted using V and J2
primers in BM-pre-B, ES-pre-B, and nonadherent EB34-pre-B cells
(data not shown).
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| Fig 7.
VHDJH as
well as DJH rearrangements in the Hµ-locus in
the EB34-pre-B and ES-pre-B cells. Large molecular weight DNAs from
adherent (ad) BM-pre B cells, nonadherent (non-ad) BM-pre-B cells,
ES-pre-B cells, adherent EB34-pre-B cells, nonadherent EB34-pre-B
cells, LAK34a-1 cells, LAK34a-3 cells, and OP9 cells were subjected to
PCR analysis using the DSF sense primer and the
JH4 antisense primer for
DJH recombinations (top), and the
V7183 sense primer and the JH4 primer for detecting
VHDJH recombinations
(middle), and the -actin primers (bottom), and separated on 1.2%
agarose gels. Specificity of the DJH as well as
VHDJH products were
further ensured by Southern blotting, followed by visualization with
the JH probe.16
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These observations suggest that the pre-B-like cells derived from
CD34+ EB cells on OP9 correspond to pre-B cells from bone
marrow, and thus, are similar to ES cell-derived pre-B cells described
previously.16,17 We have therefore concluded that the
CD34+ cells from EBs formed in the presence of VEGF contain
B-lymphoid potential.
| |
DISCUSSION |
We have shown that the CD34+ cell population appears during
the in vitro differentiation of ES cells, which becomes readily detectable in the presence of VEGF, and that ES cells have the potential to generate in vitro the third lymphoid lineage, NK cells,
through the CD34+ progeny. Furthermore, the
CD34+ cell fraction appears to represent the B-lymphoid
potential and part of the erythro-myeloid cell potential of ES cells as
well (Fig 8). Therefore, the
CD34+ cell appears to be a critical developmental
intermediate between ES cells and the lymphohematopoietic lineages.
The dramatic effect of VEGF on the elevation of the CD34+
cell population in EBs was curious because VEGF is known to be a specific mitogen for vascular endothelial cells.79,80
However, one of the VEGF receptors (VEGFR), flk-1, was originally
isolated from fetal liver HSC fraction,81 and phenotypes of
mutant mice that lack either the VEGF gene or the flk-1 gene indicate
that VEGF signaling is essential for both embryonic hematopoiesis and vasculogenesis.51-53 Therefore, it is conceivable that the
VEGF effect on the CD34+ EB cell generation may be a
reflection of the in vivo role of VEGF on embryonic hematopoiesis.
Direct target(s) of VEGF in EBs remain(s) unclear. It is possible that
proliferation of CD34+ lymphohematopoietic progenitor cells
in EBs is stimulated by VEGF (Fig 8). It is equally possible that VEGF
has a stimul |
Abstract
Introduction
Abstract
