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Cells Acquire Functional Receptors and
Human Embryonic Stem Cell-Derived NK
Petter S. Woll, Colin H. Martin, Jeffrey S. Miller and Dan S.
2005; 175:5095-5103; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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The Journal of Immunology
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Human Embryonic Stem Cell-Derived NK Cells Acquire
Functional Receptors and Cytolytic Activity1
Petter S. Woll,* Colin H. Martin,* Jeffrey S. Miller,†and Dan S. Kaufman2*
Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little
is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture
method to demonstrate efficient generation of functional NK cells from hESCs. The CD56?CD45?hESC-derived lymphocytes
express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity
receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic pro-
genitors at a clonal frequency similar to CD34?cells isolated from cord blood. The hESC-derived NK cells acquire the ability to
lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated
hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to charac-
terize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells
may also provide a source for innovative cellular immune therapies. The Journal of Immunology, 2005, 175: 5095–5103.
(1–4). Analyses of transcription factor and cell surface Ag expres-
sion suggest that hematopoiesis from hESCs follows developmen-
tal kinetics similar to what is observed during normal human on-
togeny (1–3, 5). To date, most studies have characterized myeloid,
erythroid, and megakaryocytic cells derived from hESCs (1–5).
Although the process of lymphopoiesis has been well studied start-
ing from hemopoietic stem cell populations isolated from mouse or
human bone marrow or human umbilical cord blood (UCB) (6–9),
considerably less is known about the ability of hESCs to differ-
entiate into the lymphoid lineage.
NK cells form a central component in the immune defense
against pathogens and various tumors (10). Putative NK cells and
B cells have been identified in cultures of differentiated hESCs (3,
11). However, these NK cells were characterized solely on basis of
CD56 expression, without functional analysis. In addition to CD56
expression, mature NK cells typically express inhibitory and acti-
vating receptors, and the balance of signals derived from these
receptors regulate NK cell activity. Killer cell Ig-like receptors
(KIRs) and CD94/NKG2 heterodimers are two major classes of
receptors that interact with MHC class I molecules on target cells
as their ligands to specify NK cell activity (12–14). Analysis of
NK cells derived from mouse ESCs has been instructive. Mouse
emopoietic cells can be derived from human embryonic
stem cells (hESCs)3allowed to differentiate either by
stromal cell coculture or formation of embryoid bodies
ESC-derived NK cells express CD94/NKG2 receptors in an or-
derly and nonstochastic manner; however, they do not express the
Ly49 receptors, which are analogous to the KIRs found in humans
(15). In contrast, mature NK cells isolated from adult mice express
both CD94/NKG2 receptors and Ly49 (16). For human hemopoi-
etic cells derived from more mature sources, acquisition of KIR
expression in vitro appears to be dependent on the stromal cell line
used to support NK differentiation. NK cells cocultured on MS-5
stromal cells require IL-21 for KIR expression, whereas NK cells
cocultured on AFT024 cells do not share this requirement (17, 18).
In these studies, we report that hESCs can efficiently give rise to
NK cells that express both KIRs and CD94/NKG2a, similar to
what is observed for mature NK cells found in vivo. More impor-
tantly, the hESC-derived NK cells exhibit appropriate functional
characteristics as displayed by ability to lyse cells by two separate
mechanisms: direct cell-mediated cytotoxicity and Ab-dependent
cell-mediated cytoxicity (ADCC). These hESC-derived NK cells
also can be induced to produce cytokines, another hallmark of NK
Materials and Methods
The hESC line H9 (obtained from Wicell) was maintained as undifferen-
tiated cells as described previously (1, 19). Briefly, undifferentiated hESCs
were cocultured with mouse embryonic fibroblasts in DMEM:Ham’s F-12
(Invitrogen Life Technologies) supplemented with 15% knockout serum
replacer (Invitrogen Life Technologies), 1% MEM-nonessential amino ac-
ids (Invitrogen Life Technologies), 1 mM L-glutamine (Mediatech), 0.1
mM 2-ME (Sigma-Aldrich), and 4 ng/ml basic fibroblast growth factor
(Invitrogen Life Technologies). The mouse bone marrow stromal cell line
S17 (kindly provided by Dr. K. Dorshkind, University of California, Los
Angeles, CA) was grown in DMEM (Invitrogen Life Technologies) con-
taining 10% FBS, 1% penicillin-streptomycin (P/S) (Invitrogen Life Tech-
nologies), 1% MEM-nonessential amino acids, and 0.1 mM 2-ME. Before
coculture with hESCs, S17 cells were incubated with conditioned medium
containing 10 ?g/ml mitomycin C (Bedford Laboratories) before attach-
ment onto gelatin (Sigma-Aldrich)-coated 6-well plates (Nalge Nunc In-
ternational). The mouse fetal liver cell line AFT024 (kindly provided by
Drs. K. Moore and I. Lemischka, Princeton University, Princeton, NJ) (20)
was grown at 33°C in DMEM containing 20% FBS, 1% P/S, and 0.05 mM
2-ME. AFT024 cells were irradiated with 2000 rad before coculture with
hESC-derived hemopoietic progenitor cells. UCB was obtained from units
that were unacceptable for storage in cord blood banks. The use of all
*Stem Cell Institute and Department of Medicine and†Department of Medicine and
Cancer Center, University of Minnesota, Minneapolis, MN 55455
Received for publication May 26, 2005. Accepted for publication August 4, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work supported in part National Institutes of Health Grant HL-72000 (to
D.S.K.) and an American Society of Hematology Scholars Award (to D.S.K.).
2Address correspondence and reprint requests to Dr. Dan S. Kaufman, University of
Minnesota, Stem Cell Institute, 420 Delaware Street Southeast, MMC 716, Minne-
apolis, MN 55455. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: hESC, human embryonic stem cell; ADCC, Ab-
dependent cell-mediated cytotoxicity; CFC, colony-forming cell; ESC, embryonic
stem cell; Flt3L, Flt3 ligand; KIR, killer cell Ig-like receptor; NCR, natural cytotox-
icity receptor; P/S, penicillin and streptomycin; Q-RT-PCR, quantitative RT-PCR;
SCF, stem cell factor; UCB, umbilical cord blood.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
by guest on June 13, 2013
human tissue was approved by the Committee on the Use of Human Sub-
jects in Research at the University of Minnesota.
Hemopoietic differentiation of hESCs
H9 hESCs were cocultured with mouse bone marrow stromal cell line S17,
resulting in H9/S17 cells, as described previously (1, 21). Differentiation
medium composed of RPMI 1640 (Mediatech) supplemented with 15%
FBS (HyClone), 2 mM L-glutamine, 0.1 mM 2-ME, 1% MEM-nonessential
amino acids, and 1% P/S was changed every 2–3 days. After 14–17 days
of differentiation, the differentiated hESCs were harvested and made into a
single-cell suspension using collagenase type IV (Invitrogen Life Technol-
ogies), followed by trypsin/EDTA (0.05%; Mediatech) supplemented with
2% chick serum (Sigma-Aldrich). Cells were analyzed for hemopoietic
precursor cells by flow cytometry and colony-forming cell (CFC) assay
Positive selection of CD34?and CD34?CD45?cells by
Single-cell suspensions from days 14–17 H9/S17 cocultures were prepared
as described above. Cell pellet was resuspended in Dulbecco’s PBS (Me-
diatech) supplemented with 2% FBS and 1 mM EDTA (Fisher Chemicals)
before magnetic sorting. EasySep CD34 selection kit (StemCell Technol-
ogies) was used to isolate CD34?cells from differentiated hESCs and
UCB. For isolation of CD34?CD45?cells, the EasySep PE selection kit
(StemCell Technologies) was used on CD34?selected cells labeled with
CD45-PE (BD Pharmingen). Enrichment was confirmed by flow cytomet-
ric analysis and typically resulted in 70–90% positive population. Similar
results were obtained by flow cytometric sorting using FACSAria (BD
Biosciences) for CD34?and CD34?CD45?hESC-derived cells.
In vitro generation of NK cells
Hemopoietic precursor cells were transferred to 24-well plates with a con-
fluent monolayer of irradiated AFT024 cells in medium designed to max-
imize NK cell growth as described previously (22). Briefly, cells were
cocultured in DMEM:Ham’s F-12 supplemented with 20% heat-inacti-
vated human AB serum (Nabi), 5 ng/ml sodium selenite (Sigma-Aldrich),
50 ?M ethanolamine (MP Biomedicals), 20 mg/L ascorbic acid (Sigma-
Aldrich), 25 ?M 2-ME, 1% P/S, 10 ng/ml IL-15 (PeproTech), 5 ng/ml IL-3
(PeproTech), 20 ng/ml IL-7 (National Cancer Institute), 20 ng/ml stem cell
factor (SCF) (PeproTech), and 10 ng/ml Flt3 ligand (Flt3L) (PeproTech).
Medium containing fresh cytokines was changed weekly with the excep-
tion of IL-3 which was only included for the first week of culture. Wells
were harvested after 7–50 days of NK cell culture, counted for viable cells,
and assayed for phenotype and function.
Flow cytometric analysis
Single-cell suspension of differentiated H9/S17 and hESC-derived NK
cells were stained with allophycocyanin, PE-, and FITC-coupled control
Igs or specific Abs against human blood surface Ags: CD34-APC, CD45-
APC or -PE, CD56-APC or -PE, CD15-PE, CD19-PE, CD33-PE or -FITC,
CD3-FITC, CD158a-FITC, CD158b-FITC, CD158e1-FITC, CD16-FITC,
NKp30-PE, NKp44-PE, NKp46-PE, CD94-FITC, NKG2A-PE (all from
BD Pharmingen), CD158i-PE, and NKG2A-PE (Beckman Coulter). All
analyses were performed with a FACSCalibur (BD Biosciences) and
FlowJo analysis software (Tree Star). Live cells were identified by pro-
pidium iodide or 7-aminoactinomycin D exclusion.
NK cell cloning frequency
For analysis of frequency of hemopoietic precursor cells with NK cell
potential, CD34?and CD34?CD45?hESC-derived cells, or CD34?cells
isolated from UCB, were plated in limiting dilutions in 96-well plates with
a confluent monolayer of irradiated AFT024 cells (22). Cells were exposed
to the same NK cell culture conditions as described above. Wells were
monitored weekly for visual observation of growth. After 30 days of in-
cubation, NK cell development was assessed from all wells showing visual
evidence of growth by flow cytometric analysis for CD56?CD45?cells.
Frequency of NK-potent cells was calculated by Poisson distribution based
on number of wells with confirmed growth of NK cells after 30 days of
Functional evaluation of hESC-derived NK cells
Direct cytotoxicity assays were performed by standard 4-h51Cr release
assay using the K562 (American Type Culture Collection) and Raji (Amer-
ican Type Culture Collection) cell lines as target cells (22). Effector cells
were added in limiting dilution starting at 10:1 E:T ratio unless noted
otherwise. ADCC was analyzed by preincubating Raji cells with 4, 1, 0.25,
and 0.062 ?g/ml anti-CD20 Ab (IgG1? isotype, rituximab; Genentech) for
30 min. As a negative control, Raji cells preincubated with 4 ?g/ml IgG1?
isotype control Ab (BD Pharmingen) was used.
For evaluation of ability to up-regulate IFN-? cytokine production,
hESC-derived NK cells were incubated in humidified atmosphere at 37°C
and 5% CO2with RPMI 1640 medium supplemented with 10% FBS alone
as negative control, 50 ng/ml PMA (Sigma-Aldrich), and 500 ng/ml cal-
cium ionophore III (Sigma-Aldrich), as a positive control, or 10 ?g/ml
IL-12 and 100 ?g/ml IL-18 (R&D Systems). After overnight stimulation,
cells were incubated with 10 ?g/ml brefeldin A (Sigma-Aldrich) for 5 h.
Cell surface Ags were first stained for CD56-PE and CD45-allophycocya-
nin, in addition to isotype controls, cells were then fixed and permeabilized
(Cytofix/Cytoperm kit; BD Pharmingen), followed by intracellular staining
for IFN-?-FITC (BD Pharmingen). Flow cytometric analysis was per-
formed as described above on the lymphocyte cell population. CD34?
UCB-derived NK cells were again used as positive control.
Quantitative real-time PCR analysis of KIR expression
Quantitative real-time PCR (Q-RT-PCR) was preformed as described pre-
viously (23, 24). Briefly, total RNA from CD34?CD45?H9/S17 and
CD34?UCB cells cultured for 30 days in NK conditions was isolated by
RNeasy Micro kit (Qiagen), and KIR expression was evaluated using Taq-
man probes specific for 13 different KIRs.
Hematopoiesis and NK cell development from hESCs
The ability of hESCs to give rise to lymphoid cells was investi-
gated using a two-step in vitro differentiation scheme (Fig. 1A).
Initially, the hESCs (H9 cell line) were cocultured with the murine
bone marrow-derived stromal cell line S17 to derive a heteroge-
neous population of H9/S17 cells. Consistent with previous find-
ings, these H9/S17 cells contain myeloid progenitor cells (1, 4).
After 14–17 days of coculture with S17 cells, myeloid CFCs can
be demonstrated within the differentiated hESC population. Sort-
ing for CD34?and CD34?CD45?cells results in a significant
enrichment in the myeloid CFCs as compared with unsorted H9/
S17 cell population (Fig. 1B).
Both the CD34?and CD34?CD45?hESC-derived cells remain
heterogeneous, and we hypothesized that these cell populations
also contained lymphoid progenitor cells. Two cell populations
identified in UCB and bone marrow as being skewed toward
CD34?CD10?CD45?cells (9). Indeed, these can also be identi-
fied in hESCs differentiated on S17 stromal cells (Fig. 1C). To test
for lymphocyte development, we used a system that has been
shown previously to support proliferation and differentiation of
CD34?cells isolated from bone marrow, peripheral blood, and
UCB cells into NK cells (18, 25). In the present study, sorted
CD34?and CD34?CD45?cells derived from H9/S17 cells were
transferred to a secondary culture with the murine fetal liver-de-
rived AFT024 stromal cell line in medium supplemented with IL-
15, IL-3, IL-7, SCF, and Flt3L (referred to as “NK cell condi-
tions”). Proliferation of the hESC-derived progenitor cells from the
distinct starting cell populations was monitored by harvesting and
counting cells cultured in NK cell conditions. Under these condi-
tions, UCB-derived CD34?cells expanded over 1000-fold,
whereas CD34?CD45?H9/S17 cells demonstrated a significant
expansion of ?40-fold when cultured in these same NK cell con-
ditions (Fig. 2A). CD34?H9/S17 cells demonstrated less expan-
sion, although hemopoietic-like clusters of cells growing in a sim-
ilar pattern and displaying similar morphology to what was seen
for CD34?CD45?hESCs and CD34?UCB cells consistently is
found within this cell population (Fig. 2B). These results suggest
that the CD34?CD45?cell population is more enriched in hemo-
poietic progenitors responsive to proliferation by cytokines, as
compared with the CD34?cell population derived from hESCs.
5096 NK CELLS DERIVED FROM hESCs
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However, because the proliferation of hESC-derived progenitors is
less than that of the UCB cells, it is likely that even this hESC-
derived CD34?CD45?cell population remains more heteroge-
neous than CD34?cells isolated from UCB (of note, most CD34?
UCB cells also coexpress CD45).
Phenotype and clonal frequency of NK cells derived from hESCs
To determine phenotype of the hESC-derived cells cultured in NK
cell conditions we analyzed cells after 14, 21, and 28 days by flow
cytometry for cell surface expression of CD56, a cell surface
marker expressed on human NK cells. Although CD34?H9/S17
cells have a limited expansion when cultured in NK conditions,
they demonstrate a robust ability to differentiate into NK cells.
After 14 days of culture, ?90% of the cells express CD45, a pan-
hemopoietic cell marker, but few CD56?cells are observed (Fig.
3A). By 21 days of culture, a distinct CD56?CD45?cell popula-
tion is observed (14.9%), which increases to 37.5% after 28 days
of culture. Similar results are observed for CD34?CD45?cells
derived from H9/S17 cells (Fig. 3B), suggesting that both CD34?
and CD34?CD45?cell populations contain hemopoietic progen-
itors with NK cell potential.
As these cultures also led to development of a significant pop-
ulation of CD45?CD56?cells, we also analyzed the cells cultured
in NK cell conditions for expression of markers of other hemo-
poietic lineages (Fig. 4). For CD34?and CD34?CD45?H9/S17
cells, no significant expression of CD34 (immature cells), CD19
(B cells), or CD3 (T cells) was observed after 30 days in NK cell
conditions. A population of the cells do express CD33 a marker of
immature and mature myeloid cells. However, no detectable ex-
pression of CD15?cells (granulocytes) (Fig. 4) or glycophorinA?
(CD235a) cells (erythrocytes) (data not shown) was observed after
30 days of culture.
To determine the clonal frequency of NK cell progenitors that
give rise to the CD56?cells, CD34?and CD34?CD45?H9/S17
cells were plated in limiting dilutions and cultured for 30 days in
tions. A, Proliferation of sorted CD34?(E) and CD34?CD45?(‚) H9/S17
cells was analyzed after 13, 20, and 30 days in NK cell culture. CD34?
UCB cells were used as positive control (?) (results are mean ? SD of
three experiments). B, Images of cell development and proliferation after
21 days in NK cell culture (top row: ?20 original magnification; bottom
row: ?100 original magnification). White arrows indicate hemopoietic cell
Proliferation of hESC-derived cells cultured in NK condi-
for 14–17 days to derive hemopoietic progenitor cells. These progenitors are then sorted based on CD34 or CD34/45 surface expression and cultured on
AFT024 cells with defined cytokines. Development of NK cells was analyzed at specific time points after culture on AFT024 cells. B, Generation of
erythroid and myeloid progenitors was analyzed by hemopoietic CFC assay with H9 hESCs allowed to differentiate on S17 stromal cells for 14–17 days.
In the present study, colonies produced by unsorted or sorted hESC-derived cell populations were quantified after 14 days (results are mean ? SD of five
separate experiments). C, Flow cytometric analysis of unsorted, CD34?sorted and CD34?CD45?sorted H9/S17 cells, in addition to CD34?UCB cells
isolated by magnetic sorting.
In vitro hematopoiesis from hESCs. A, Schema to derive NK cells from hESCs. hESCs are first allowed to differentiate on S17 stromal cells
5097 The Journal of Immunology
by guest on June 13, 2013
NK cell conditions. After 30 days, wells with visual growth were
harvested and analyzed by flow cytometry for presence of CD56?
cells. We found CD34?H9/S17 cells to have a NK cell frequency
of 0.16% (Table I). However, sorting for CD34?CD45?H9/S17
cells significantly increased the NK cell cloning frequency (1.1%)
to a level comparable to the frequency observed for CD34?UCB
cells cultured in the same manner (2.4%).
hESC-derived NK cells express inhibitory and activating
As initial phenotypic analysis suggested that hESCs can differen-
tiate into NK cells, we next characterized the hESC-derived cells
for surface expression of other NK cell Ags. NK cell cytolytic
activity is regulated by signals initiated by specific activating and
inhibitory receptors (16). One important family of receptors in-
volved in the regulation of cytolytic activity is the KIRs. Initially,
we analyzed the expression of four KIRs using a mixture of Abs
specific forthree inhibitory
KIR2DL2/DL3/DS2 (CD158b), and KIR3DL1 (CD158e1) and
one activating KIR, KIR2DS4 (CD158i). During NK culture of
CD34?CD45?H9/S17 cells, CD56?NK cells start to express
KIRs after 18 days of culture (Fig. 5A). After 50 days, 40% of cells
are CD56?KIR?cells, suggesting a time-dependent up-regulation
of KIR expression (Fig. 5A). Interestingly, the KIR expression is
consistently higher in the hESC-derived NK cells as compared
with NK cells derived from CD34?UCB cells. When investigat-
ing the expression of the individual KIRs, hESC-derived NK cells
express CD158b, CD158e1, and CD158i but do not express
CD158a (Fig. 5B). This is different from UCB-derived NK cells
that express only low levels of CD158e1 and do not express
CD158i. The KIR protein expression as analyzed by flow cytom-
etry was further resolved by a Q-RT-PCR method to better define
the expression of 13 individual KIR genes (23, 24). This Q-RT-
PCR analysis showed hESC-derived NK cells express transcripts
for KIR2DS1, KIR2DL4, KIR2DL5, KIR2DS5, KIR3DS1, and
KIR3DL2, in addition to KIRs mentioned above, as determined by
flow cytometry (our unpublished observations). Furthermore, be-
cause the Ab used to detect CD158b does not specifically distin-
guish between KIR2DL2, KIR2DS2, and KIR2DL3, the Q-RT-
PCR assay resolved that the hESC-derived NK cells expressed
only KIR2DL3 to account for the CD158b surface expression.
The hESC-derived NK cells also express the C-type lectin-like
receptors CD94 and NKG2A (Fig. 5C), which upon dimerization
form an inhibitory receptor that primarily interacts with HLA-E
(26). Another group of activating receptors, collectively termed
natural cytotoxicity receptors (NCRs), can be identified by their
exclusive expression on NK cells (27–29). These include NKp30,
NKp44, and NKp46, all of which are expressed on the hESC-
derived NK cells (Fig. 5C). In addition, we found hESC-derived
NK cells express lymphoid associated markers CD7 and CD2 (Fig.
6). In vivo, most mature NK cells express CD16. However, in vitro
studies of UCB-derived NK cell differentiation by coculture with
the MS-5 stromal cell line depends on the addition of IL-21 to
differentiate to fully mature CD56?CD16?NK cells (17). Inter-
estingly, this is not a requirement of these studies using the
AFT024 stromal cell line because hESC- and UCB-derived NK
cells express CD16 after 30 days of culture (Fig. 5C). Notably, a
greater percentage of the hESC-derived CD56?cells coexpressed
Flow cytometric analysis of sorted CD34?H9/S17 cells
and CD34?cells isolated from UCB (positive control)
(A), and CD34?CD45?H9/S17 cells (B) cocultured
with AFT024 stromal cells in medium supplemented
with SCF, Flt3L, IL-3, IL-7, and IL-15 (NK cell condi-
tions) (B) for the indicated number of days. Represen-
tative results from three separate experiments are
shown. Quadrant markers were set based on controls in
which ?0.5% of the cells were included in the quad-
rants. Exclusion of propidium iodide was used for gat-
ing on live cells.
Generation of NK cells from hESCs.
5098 NK CELLS DERIVED FROM hESCs
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CD16, as compared with the CD56?cells derived from UCB
Cytolytic activity of hESC-derived NK cells
The above phenotypic results suggest that hESCs can be efficiently
induced to differentiate into NK cells in vitro. We next examined
functional cytolytic activity of the hESC-derived NK cells. One
hallmark of NK cells is their ability to target and lyse human tumor
cells (30). Thus, hESC-derived NK cells were harvested and tested
for their cytolytic activity toward K562 erythroleukemia cells in a
standard 4-h51Cr release assay. Cells derived from UCB after 17
days of NK cell culture on AFT024 cells supplemented with de-
fined cytokines are able to effectively kill K562 cells. As expected,
CD34?H9/S17-derived cells did not display any significant cyto-
lytic activity at this time (Fig. 7A) due to the few CD56?CD45?
NK cells present at this early time point (Fig. 3A). However, at day
32, the CD56?CD45?cells have developed into a significant pop-
ulation, and these cells demonstrate cytolytic activity similar to
UCB-derived NK cells. CD34?CD45?H9/S17-derived NK cells
also demonstrate cytolytic activity after 30 days of NK culture
comparable to the cytolytic activity observed for UCB-derived NK
cells (Fig. 7B). The cytolytic activity observed for the hESC-de-
rived cells reside in the CD56?population, as sorting for these
cells after 30 days of culture markedly enhances the cytolytic ac-
tivity compared with the unsorted cell population (Fig. 7C). Fur-
thermore, no significant cytolytic activity was observed in the
CD56?cell population, even at higher E:T ratios. Similar results
were observed for the UCB-derived NK cells (data not shown).
As the hESC-derived NK cells express CD16 (Fc?RIII), a re-
ceptor that binds the Fc region of IgG molecules (Fig. 5C), we also
tested their ability to mediate ADCC. The cytolytic activity of
hESC-derived NK cells was targeted against the NK-resistant Raji
cell line incubated with anti-CD20 Ab or isotype control Ab. Our
results show that the hESC-derived NK cells can mediate lysis of
Raji cells in an anti-CD20 dose-dependent manner, whereas Raji
cells incubated without Ab or isotype control Ab were not effec-
tively lysed (Fig. 7D).
Cytokine production from hESC-derived NK cells
Another means to analyze functional characteristics of NK cells is
their ability to up-regulate IFN-? production in response to IL-12/
IL-18 stimulation. Consistent with this capacity, hESC-derived
NK cells stimulated overnight with IL-12 and IL-18 up-regulated
IFN-? production in the same manner as when these cells are stim-
ulated by PMA and calcium ionophore, similar to what seen in
UCB-derived NK cells used as positive controls (Fig. 8).
In the present study, we demonstrate that hESCs can develop into
mature, functional NK cells. These hESC-derived NK cells acquire
KIR expression similar to what is observed for mature NK cells in
vivo (16). CD94/NKG2A, NCRs, and CD16 are also expressed by
the hESC-derived NK cells. Sorting for specific cell surface phe-
notypes to isolate hESC-derived hemopoietic progenitors results in
a significant increase in NK cell cloning frequency. More impor-
tantly, hESC-derived NK cells exhibited cytokine production and
cytolytic activity against human tumor cells by both direct cell-
mediated cellular cytotoxicity and ADCC. These results suggest
that not only can functional lymphoid cells be routinely and effi-
ciently derived from undifferentiated hESCs but also specific phe-
notypic cell populations derived from hESCs can be effectively
and efficiently selected, expanded, and induced to mature into spe-
cific blood lineages.
Although recent reports suggest that phenotypic NK cells can be
derived from hESCs, prior analyses have relied solely on CD56
Sorted CD34?and CD34?CD45?H9/S17 cells were cultured in NK cell
conditions for 30 days and analyzed by flow cytometry for cell surface
expression of markers for hemopoietic precursor/progenitors (CD34), gran-
ulocytes (CD15), B lymphocytes (CD19), T lymphocytes (CD3), and my-
eloid cells (CD33). CD34?cells isolated from UCB were used as a positive
control. Quadrant markers were set based on controls in which ?0.5% of
the cells were included in the quadrants. Exclusion of propidium iodide
was used for gating on live cells.
Cell surface expression of myeloid and lymphoid Ags.
Table I. NK cell clonal frequency
CD34?or CD34?CD45?cells derived from hESCs allowed to differentiate on
S17 cells or CD34?cells isolated from UCB were cultured in limiting dilution in NK
cell conditions for 30 days. Wells were then evaluated for cell proliferation, and
presence of CD56?CD45?NK cells was confirmed by flow cytometry. Poisson dis-
tribution was used to calculate frequency of NK progenitor cells (22).
5099The Journal of Immunology
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expression as a marker of NK cells (3, 11). CD56 alone is insuf-
ficient to identify NK cells because it is promiscuously expressed
on neuronal (31) and pancreatic cells (32), as well as in low levels
on undifferentiated hESCs (P. S. Woll and D. S. Kaufman, unpub-
lished observations). CD56 is also found on myeloid cells in some
patients with chronic myeloid leukemia (33). Thus, to more clearly
demonstrate generation of NK cells, characterization of additional
receptors and NK cell activity is needed. In the present study, we
demonstrate hESC-derived NK cells acquire CD94, KIR, and
CD16 expression to generate functional cytolytic and cytokine-
producing NK cells. CD56 and KIR expression was acquired in a
time-dependent manner, that agrees with current models of NK
cell maturation (16).
The molecular and cellular events that regulate development of
NK cell precursors to mature NK cells are not well characterized.
However, a sequential pattern of cell surface Ag expression has
been identified. The earliest cells committed to the NK cell lineage
can be identified as CD161?CD56?, which subsequently give rise
to CD56?NK cells. CD94 expression is acquired before KIR and
CD16 expression, generating cytolytic and cytokine-producing
NK cells (16). Furthermore, NK cells can be classified either by
CD56 expression pattern or cytokine production. CD56brightcells
have low expression of CD16 and KIRs, poor cytolytic activity,
and high levels of cytokine production (34, 35). In contrast, most
cytolytic activity is found in the CD56dimcells, which also have
high surface expression of CD16 and KIRs. Recent reports suggest
that CD56brightcells are less mature than CD56dimcells (36, 37).
In addition, cytokine production has been used to classify NK cell
populations. A linear developmental progression from IL-
13?IFN-??stage cells (type 2) to an intermediate IL-13?IFN-??
stage (type 0), followed by IL-13?IFN-??cells (type 1), has been
The development of hESC-derived NK cells closely recapitu-
lates normal NK cell developmental kinetics. This correlation
strongly suggests that the hESC system provides an accurate de-
velopmental model to evaluate specific cellular and genetic mech-
anisms that regulate NK cell maturation. One unique aspect of the
ESC system is that the differentiation process follows distinct se-
quential steps of hemopoietic maturation that can be monitored at
very early developmental stages. Initial differentiation of hESCs
can generate CD34?cells that give rise to myeloid and lymphoid
progenitors, which in turn produce mature blood cells. Unlike
UCB and bone marrow, hESCs do not initially contain mature
Ags CD2 and CD7. Sorted CD34?and CD34?CD45?H9/S17 cells cul-
tured in NK cell conditions for 30 days were analyzed by flow cytometry
for cell surface expression of the lymphoid-associated Ags CD7 and CD2.
CD34?cells isolated from UCB were used as positive control. Quadrant
markers were set based on controls in which ?0.5% of the cells were
included in the quadrants. Exclusion of propidium iodide was used for
gating on live cells.
hESC-derived NK cells express the lymphoid associated
cells express KIRs, CD94/NKG2a,
and NCRs. A, Flow cytometric anal-
ysis of KIR acquisition (pooled Abs
against CD158a, CD158b, CD158e1,
and CD158i) in sorted CD34?CD45?
H9/S17 cells cultured in NK cell con-
ditions for the indicated number of
days. B and C, Sorted CD34?CD45?
H9/S17 cultured in NK conditions for
30 days were analyzed by flow cy-
tometry for expression of individual
KIRs and the C-type lectin-like re-
ceptors CD94 and NKG2A, in ad-
(Fc?RIIIA) and NCRs. Representa-
tive results from two separate exper-
iments are shown. CD34?UCB cell
were used as a positive control.
Quadrant markers were set based on
controls in which ?0.5% of the cells
were included in the quadrants. Ex-
clusion of propidium iodide was used
for gating on live cells. Similar re-
sults were found using sorted CD34?
5100NK CELLS DERIVED FROM hESCs
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hemopoietic cells, reducing the possibility that contaminating ma-
ture cells might obscure in vitro analysis of differentiation path-
ways. Thus, following the multistep hemopoietic differentiation
process from hESCs allows for an unbiased and reproducible anal-
ysis of transcriptional regulation of differentiation and maturation.
As far as the models of NK cell maturation based on levels of
CD56 expression and cytokine production (38, 39), our results do
not yet provide enough information to evaluate if one pathway
predominates for hESC-derived NK cells. However, the cytolytic
activity and IFN-? cytokine production demonstrated in the hESC-
derived NK cells suggest that the hESCs can now serve as a model
system to distinguish these two models of NK cell maturation.
Interestingly, the KIR expression on the hESC-derived NK cells
was higher than on UCB-derived NK cells. Because of the highly
polymorphic nature of the KIR locus on chromosome 19, this could
be explained by differences in inherited KIR genes (23). However,
genetic heterogeneity is unlikely to be the sole explanation for this
phenomenon, as the same difference in KIR expression was ob-
served when NK cells derived from multiple UCB-donors were
compared with that of hESC-derived NK cells. Instead, our results
support previous findings where KIR acquisition on developing
NK cells in vitro inversely correlated with the ontogeny of the
stem cell source (18). This has been explained previously by the
relatively higher proliferation capabilities of the more immature
source. However, this is not a likely explanation to describe our
results, as the proliferation observed for hESCs-derived NK cells
was lower compared with the proliferation observed for UCB cells
(Fig. 2A). This may be due to the hESC-derived CD34?CD45?
cell population remaining more heterogeneous and containing
quantitatively fewer lymphocyte progenitors than CD34?cells
isolated from UCB. Certainly, this heterogeneity between hESC-
and UCB-derived progenitors needs to be compared because these
studies so far have been incomplete. Although phenotypic analysis
of hESCs differentiated on OP9 stromal cells suggests that CD34?
hESC-derived cells resemble primitive bone marrow and intraembry-
onic hemopoietic precursors by expression of CD90, CD117, and
CD164, the functional relevance of this remains unknown (3). Indeed,
we can identify CD34?CD45?CD7?and CD34?CD45?CD10?
cells from differentiated hESCs, corresponding to more mature
common lymphoid progenitor cell populations identified in UCB
and bone marrow (9) (Fig. 1C). Future studies will determine the
NK cell cloning frequency and proliferative potential of these
hESC-derived cell populations, and comparison to similar popu-
lations isolated from UCB and bone marrow will be instructive for
establishing a hemopoietic maturation scheme from hESC-derived
duction following stimulation with IL-12 and IL-18. Sorted CD34?CD45?
H9/S17-derived cells cultured in NK conditions for 30 days were stimu-
lated overnight with medium alone, PMA, and calcium ionophore III
(PMA?Ca/I) or IL-12 and IL-18. Cells were analyzed by intracellular flow
cytometric straining for IFN-?. Quadrant markers were set based on con-
trols in which ?0.5% of the cells were included in the quadrants. Repre-
sentative intracellular flow cytometric analysis for three separate experi-
ments is shown.
hESC-derived NK cells up-regulate IFN-? cytokine pro-
cultured in NK cell conditions for 17 and 32 days were analyzed for ability to lyse K562 cells by standard 4-h51Cr release assay. B, CD34?CD45?
H9/S17-derived cells (Œ) or CD34?UCB-derived cells (f) cultured in NK conditions for 30 days evaluated for ability to lyse K562 cells (results are mean
? SD of three separate experiments). C, CD56?(?) and CD56?(?) cells derived from CD34?CD45?H9/S17 cells after 35 days in NK cell conditions
were isolated and tested for ability to lyse K562 cells and compared with the unsorted cell population (Œ). Similar results were found for CD34?UCB
cells cultured in same conditions. D, ADCC was tested by preincubating Raji cells with indicated concentrations of anti-CD20 Ab (?CD20) or with 4 ?g/ml
IgG1k isotype control Ab. Cells derived from CD34?CD45?H9/S17 and CD34?UCB cultured in NK conditions for 30 days were used as effectors against
the Ab-treated Raji cells at a 20:1 E:T ratio.
Functional NK cells derived from hESCs. A, Cytolytic activity in sorted CD34?H9/S17-derived cells (F) or CD34?UCB-derived cells (f)
5101The Journal of Immunology
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As MHC class I molecules act as ligands for KIRs, some studies
suggest that MHC class I exposure might affect NK cell develop-
ment and KIR expression (40). Mice lacking MHC class I have a
higher expression of Ly49 receptors than wild-type littermates
(41). In humans, although HLA and KIR genes are not linked,
HLA class I appears to affect the frequency of KIR expression on
developing NK cells that reconstitute after hemopoietic cell trans-
plantation (40). Another possible explanation to account for varied
level of expression of NK cell surface receptors between different
cell populations is the epigenetic regulation of KIRs. Recently, the
expression of KIRs has been found to be regulated by the meth-
ylation status of the KIR locus (42, 43). As hESCs are associated
with a more open chromatin structure (44), it is possible that this
epigenetic regulation is less efficient in the hESC-derived NK
cells. Future analyses will be important to further investigate this
During embryonic development, hematopoiesis occurs in two
waves. The primitive hemopoietic cells are confined to the ex-
traembryonic yolk sac, generating primarily nucleated RBCs.
These cells lack in vitro lymphomyeloid cell potential, which is
found in later definitive hemopoietic cells (45). Demonstration of
lymphocytes from hESCs would indicate that these cells are ca-
pable of definitive hematopoiesis. However, as phenotypic NK
cells can be derived from yolk sac (46), the results presented here
cannot be solely used to distinguish primitive vs definitive hema-
topoiesis from the hESCs. The demonstration of CD19?B cells
derived from hESCs, a population not found to be derived from
yolk-sac progenitors (3, 46), and other recent studies that demon-
strate expression of ? and ? globin genes in hESC-derived eryth-
rocytes (5, 47) together more conclusively establishes definitive
hemopoietic cells can be derived from hESCs. Indeed, recent stud-
ies also more clearly delineate that a transition from hemopoietic-
endothelial cell precursors to primitive then definitive hemopoietic
cells can be modeled with hESCs (5, 48).
In addition to extensive in vitro characterization of the hemo-
poietic potential of hESCs, recent reports demonstrate in vivo en-
graftment of hemopoietic cells derived from hESCs when trans-
planted into immunodeficient mice or fetal sheep (49–51). Similar
results have also been obtained from transplantation of hemopoi-
etic cells derived from cynomolgus monkey ESCs transplanted
into fetal sheep (52). These studies of in vivo engraftment remain
the gold standard to evaluate the full hemopoietic potential for
specific precursor cell populations.
Future clinical application of hESCs may involve use of these
cells an alternative source of hemopoietic cells of various specific
lineages, including hemopoietic stem cells, mature erythroid cells,
platelets, and lymphocytes (53–55). NK cells provide important
cell-mediated antitumor activity, as clearly demonstrated in recent
clinical trials (56, 57). Because hESC-derived NK cells demon-
strate mature effector functions, these cells may prove to be useful
in clinical therapeutic applications
that require further
We gratefully acknowledge Jon Linehan and Julie Morris for help with
maintaining cells lines, Sarah McNearney and Valarie McCullar for tech-
nical support, Dr. Paul Leibson for expert advice, Paul Marker for
FACSAria sorting, Dr. Catherine Verfaille, and lab members for sharing
reagents and equipment.
The authors have no financial conflict of interest.
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