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The Journal of Immunology, 2005, 175: 5095-5103.
Copyright © 2005 by The American Association of Immunologists

Human Embryonic Stem Cell-Derived NK Cells Acquire Functional Receptors and Cytolytic Activity1

Petter S. Woll*, Colin H. Martin*, Jeffrey S. Miller{dagger} and Dan S. Kaufman2,*

* Stem Cell Institute and Department of Medicine and {dagger} Department of Medicine and Cancer Center, University of Minnesota, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 progenitors 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 characterize 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hemopoietic cells can be derived from human embryonic stem cells (hESCs)3 allowed to differentiate either by stromal cell coculture or formation of embryoid bodies (1, 2, 3, 4). Analyses of transcription factor and cell surface Ag expression suggest that hematopoiesis from hESCs follows developmental kinetics similar to what is observed during normal human ontogeny (1, 2, 3, 5). To date, most studies have characterized myeloid, erythroid, and megakaryocytic cells derived from hESCs (1, 2, 3, 4, 5). Although the process of lymphopoiesis has been well studied starting from hemopoietic stem cell populations isolated from mouse or human bone marrow or human umbilical cord blood (UCB) (6, 7, 8, 9), considerably less is known about the ability of hESCs to differentiate 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 activating 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, 13, 14). Analysis of NK cells derived from mouse ESCs has been instructive. Mouse ESC-derived NK cells express CD94/NKG2 receptors in an orderly 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 hemopoietic 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 importantly, 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 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

The hESC line H9 (obtained from Wicell) was maintained as undifferentiated 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 acids (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) containing 10% FBS, 1% penicillin-streptomycin (P/S) (Invitrogen Life Technologies), 1% MEM-nonessential amino acids, and 0.1 mM 2-ME. Before coculture with hESCs, S17 cells were incubated with conditioned medium containing 10 µg/ml mitomycin C (Bedford Laboratories) before attachment onto gelatin (Sigma-Aldrich)-coated 6-well plates (Nalge Nunc International). 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 human tissue was approved by the Committee on the Use of Human Subjects 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 Technologies), 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 (1, 21).

Positive selection of CD34+ and CD34+CD45+ cells by magnetic sorting

Single-cell suspensions from days 14–17 H9/S17 cocultures were prepared as described above. Cell pellet was resuspended in Dulbecco’s PBS (Mediatech) supplemented with 2% FBS and 1 mM EDTA (Fisher Chemicals) before magnetic sorting. EasySep CD34 selection kit (StemCell Technologies) 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 cytometric 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 confluent monolayer of irradiated AFT024 cells in medium designed to maximize NK cell growth as described previously (22). Briefly, cells were cocultured in DMEM:Ham’s F-12 supplemented with 20% heat-inactivated human AB serum (Nabi), 5 ng/ml sodium selenite (Sigma-Aldrich), 50 µM ethanolamine (MP Biomedicals), 20 mg/L ascorbic acid (Sigma-Aldrich), 25 µM 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 exception 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 propidium 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 incubation, 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 culture (22).

Functional evaluation of hESC-derived NK cells

Direct cytotoxicity assays were performed by standard 4-h 51Cr release assay using the K562 (American Type Culture Collection) and Raji (American 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 µg/ml anti-CD20 Ab (IgG1{kappa} isotype, rituximab; Genentech) for 30 min. As a negative control, Raji cells preincubated with 4 µg/ml IgG1{kappa} isotype control Ab (BD Pharmingen) was used.

For evaluation of ability to up-regulate IFN-{gamma} cytokine production, hESC-derived NK cells were incubated in humidified atmosphere at 37°C and 5% CO2 with RPMI 1640 medium supplemented with 10% FBS alone as negative control, 50 ng/ml PMA (Sigma-Aldrich), and 500 ng/ml calcium ionophore III (Sigma-Aldrich), as a positive control, or 10 µg/ml IL-12 and 100 µg/ml IL-18 (R&D Systems). After overnight stimulation, cells were incubated with 10 µg/ml brefeldin A (Sigma-Aldrich) for 5 h. Cell surface Ags were first stained for CD56-PE and CD45-allophycocyanin, in addition to isotype controls, cells were then fixed and permeabilized (Cytofix/Cytoperm kit; BD Pharmingen), followed by intracellular staining for IFN-{gamma}-FITC (BD Pharmingen). Flow cytometric analysis was performed 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 previously (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 Taqman probes specific for 13 different KIRs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Hematopoiesis and NK cell development from hESCs

The ability of hESCs to give rise to lymphoid cells was investigated 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 heterogeneous population of H9/S17 cells. Consistent with previous findings, 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. Sorting 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).



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  		FIGURE 1. In vitro hematopoiesis from hESCs. A, Schema to derive NK cells from hESCs. hESCs are first allowed to differentiate on S17 stromal cells 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.

 
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 lymphoid differentiation are CD34+CD7+CD45+ and CD34+CD10+CD45+ cells (9). Indeed, these can also be identified 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-derived AFT024 stromal cell line in medium supplemented with IL-15, IL-3, IL-7, SCF, and Flt3L (referred to as "NK cell conditions"). 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 conditions, 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 conditions (Fig. 2A). CD34+ H9/S17 cells demonstrated less expansion, although hemopoietic-like clusters of cells growing in a similar 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 hemopoietic progenitors responsive to proliferation by cytokines, as compared with the CD34+ cell population derived from hESCs. 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 heterogeneous than CD34+ cells isolated from UCB (of note, most CD34+ UCB cells also coexpress CD45).



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  		FIGURE 2. Proliferation of hESC-derived cells cultured in NK conditions. A, Proliferation of sorted CD34+ ({circ}) and CD34+CD45+ ({triangleup}) H9/S17 cells was analyzed after 13, 20, and 30 days in NK cell culture. CD34+ UCB cells were used as positive control ({square}) (results are mean ± SD of three experiments). B, Images of cell development and proliferation after 21 days in NK cell culture (top row: x20 original magnification; bottom row: x100 original magnification). White arrows indicate hemopoietic cell clusters.

 
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 population 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 progenitors with NK cell potential.



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  		FIGURE 3. Generation of NK cells from hESCs. 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 conditions) (B) for the indicated number of days. Representative 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 quadrants. Exclusion of propidium iodide was used for gating on live cells.

 
As these cultures also led to development of a significant population of CD45+CD56– cells, we also analyzed the cells cultured in NK cell conditions for expression of markers of other hemopoietic 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 expression of CD15+ cells (granulocytes) (Fig. 4) or glycophorinA+ (CD235a) cells (erythrocytes) (data not shown) was observed after 30 days of culture.



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  		FIGURE 4. Cell surface expression of myeloid and lymphoid Ags. 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), granulocytes (CD15), B lymphocytes (CD19), T lymphocytes (CD3), and myeloid 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.

 
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 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%).


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  		Table I. NK cell clonal frequency

 
hESC-derived NK cells express inhibitory and activating receptors

As initial phenotypic analysis suggested that hESCs can differentiate 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 involved in the regulation of cytolytic activity is the KIRs. Initially, we analyzed the expression of four KIRs using a mixture of Abs specific for three inhibitory KIRs: KIR2DL1 (CD158a), 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 investigating 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 cytometry 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, because the Ab used to detect CD158b does not specifically distinguish 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.



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  		FIGURE 5. hESC-derived NK cells express KIRs, CD94/NKG2a, and NCRs. A, Flow cytometric analysis of KIR acquisition (pooled Abs against CD158a, CD158b, CD158e1, and CD158i) in sorted CD34+CD45+ H9/S17 cells cultured in NK cell conditions 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 cytometry for expression of individual KIRs and the C-type lectin-like receptors CD94 and NKG2A, in addition to expression of CD16 (Fc{gamma}RIIIA) and NCRs. Representative results from two separate experiments 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. Exclusion of propidium iodide was used for gating on live cells. Similar results were found using sorted CD34+ H9/S17 cells.

 
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, 28, 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). Interestingly, 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 CD16, as compared with the CD56+ cells derived from UCB progenitors.



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  		FIGURE 6. hESC-derived NK cells express the lymphoid associated Ags CD2 and CD7. Sorted CD34+ and CD34+CD45+ H9/S17 cells cultured 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.

 
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-h 51Cr release assay. Cells derived from UCB after 17 days of NK cell culture on AFT024 cells supplemented with defined cytokines are able to effectively kill K562 cells. As expected, CD34+ H9/S17-derived cells did not display any significant cytolytic 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 population, 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-derived cells reside in the CD56+ population, as sorting for these cells after 30 days of culture markedly enhances the cytolytic activity compared with the unsorted cell population (Fig. 7C). Furthermore, 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).



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  		FIGURE 7. Functional NK cells derived from hESCs. A, Cytolytic activity in sorted CD34+ H9/S17-derived cells (•) or CD34+ UCB-derived cells ({blacksquare}) cultured in NK cell conditions for 17 and 32 days were analyzed for ability to lyse K562 cells by standard 4-h 51Cr release assay. B, CD34+CD45+ H9/S17-derived cells ({blacktriangleup}) or CD34+ UCB-derived cells ({blacksquare}) cultured in NK conditions for 30 days evaluated for ability to lyse K562 cells (results are mean ± SD of three separate experiments). C, CD56+ (x) and CD56– ({diamondsuit}) 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 ({blacktriangleup}). 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 ({alpha}CD20) or with 4 µg/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.

 
As the hESC-derived NK cells express CD16 (Fc{gamma}RIII), a receptor 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 effectively 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-{gamma} 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-{gamma} production in the same manner as when these cells are stimulated by PMA and calcium ionophore, similar to what seen in UCB-derived NK cells used as positive controls (Fig. 8).



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  		FIGURE 8. hESC-derived NK cells up-regulate IFN-{gamma} cytokine production following stimulation with IL-12 and IL-18. Sorted CD34+CD45+ H9/S17-derived cells cultured in NK conditions for 30 days were stimulated 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-{gamma}. Quadrant markers were set based on controls in which <0.5% of the cells were included in the quadrants. Representative intracellular flow cytometric analysis for three separate experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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 phenotypes to isolate hESC-derived hemopoietic progenitors results in a significant increase in NK cell cloning frequency. More importantly, 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 efficiently derived from undifferentiated hESCs but also specific phenotypic cell populations derived from hESCs can be effectively and efficiently selected, expanded, and induced to mature into specific blood lineages.

Although recent reports suggest that phenotypic NK cells can be derived from hESCs, prior analyses have relied solely on CD56 expression as a marker of NK cells (3, 11). CD56 alone is insufficient 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, unpublished 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. CD56bright cells 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 CD56dim cells, which also have high surface expression of CD16 and KIRs. Recent reports suggest that CD56bright cells are less mature than CD56dim cells (36, 37). In addition, cytokine production has been used to classify NK cell populations. A linear developmental progression from IL-13+IFN-{gamma}– stage cells (type 2) to an intermediate IL-13+IFN-{gamma}+ stage (type 0), followed by IL-13–IFN-{gamma}+ cells (type 1), has been suggested (38).

The development of hESC-derived NK cells closely recapitulates normal NK cell developmental kinetics. This correlation strongly suggests that the hESC system provides an accurate developmental model to evaluate specific cellular and genetic mechanisms that regulate NK cell maturation. One unique aspect of the ESC system is that the differentiation process follows distinct sequential 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 hemopoietic cells, reducing the possibility that contaminating mature cells might obscure in vitro analysis of differentiation pathways. Thus, following the multistep hemopoietic differentiation process from hESCs allows for an unbiased and reproducible analysis 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-{gamma} 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 observed 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 intraembryonic 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 populations isolated from UCB and bone marrow will be instructive for establishing a hemopoietic maturation scheme from hESC-derived hemopoietic progenitors.

As MHC class I molecules act as ligands for KIRs, some studies suggest that MHC class I exposure might affect NK cell development 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 transplantation (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 methylation 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 issue.

During embryonic development, hematopoiesis occurs in two waves. The primitive hemopoietic cells are confined to the extraembryonic 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 capable 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 hematopoiesis 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 demonstrate expression of {gamma} and {beta} globin genes in hESC-derived erythrocytes (5, 47) together more conclusively establishes definitive hemopoietic cells can be derived from hESCs. Indeed, recent studies 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 hemopoietic potential of hESCs, recent reports demonstrate in vivo engraftment of hemopoietic cells derived from hESCs when transplanted into immunodeficient mice or fetal sheep (49, 50, 51). Similar results have also been obtained from transplantation of hemopoietic 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, 54, 55). NK cells provide important cell-mediated antitumor activity, as clearly demonstrated in recent clinical trials (56, 57). Because hESC-derived NK cells demonstrate mature effector functions, these cells may prove to be useful in clinical therapeutic applications that require further investigation.


   Acknowledgments
 
We gratefully acknowledge Jon Linehan and Julie Morris for help with maintaining cells lines, Sarah McNearney and Valarie McCullar for technical support, Dr. Paul Leibson for expert advice, Paul Marker for FACSAria sorting, Dr. Catherine Verfaille, and lab members for sharing reagents and equipment.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This 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.). Back

2 Address correspondence and reprint requests to Dr. Dan S. Kaufman, University of Minnesota, Stem Cell Institute, 420 Delaware Street Southeast, MMC 716, Minneapolis, MN 55455. E-mail address: kaufm020{at}umn.edu Back

3 Abbreviations 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 cytotoxicity receptor; P/S, penicillin and streptomycin; Q-RT-PCR, quantitative RT-PCR; SCF, stem cell factor; UCB, umbilical cord blood. Back

Received for publication May 26, 2005. Accepted for publication August 4, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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