Hematopoiesis and immunity of HOXB4-transduced embryonic stem cell–derived hematopoietic progenitor cells

The remarkable ability of hematopoietic stem cells (HSCs) to develop multilineage hematopoietic cell engraftment after transplantation is the most common clinical cell-based therapy today. Bone marrow, umbilical cord blood, and mobilized peripheral blood are sources of hematopoietic cells, the application of which is hampered by the need for human leukocyte antigen matching. In contrast, because of their low degree of immunogenicity and high propensity to proliferate, embryonic stem (ES) cells have emerged as a likely and more suitable alternative cell source for generating hematopoietic cells.^1-8  ES cells are generated from the inner cell mass of the blastocyst and are capable of developing into tissues of all 3 germinal layers. However, in vitro, differentiation of ES cells into specialized cells and tissues including hematopoietic cells has remained a challenge. Interestingly, data from our group and by others have shown that ES cells might be immune privileged because of their low expression of major histocompatibility complex (MHC) antigens.^9-11  Therefore, ES cells may provide an alternative source of hematopoietic cells if reliable protocols for their differentiation can be established.

However, technical problems and the inability of the ES cell–derived cells to survive long term have made it difficult to use ES cells as a source of hematopoietic cells that can reconstitute bone marrow. In our previous studies in the rat, we found that ES cell–like cells engrafted in allogeneic recipients and created long-term chimeras.¹²  In contrast, subsequent studies in the mouse by us with established ES cell lines revealed that ES cell–derived cells in chimeric animals disappeared from peripheral blood after 3 to 4 weeks because of their apoptosis within the lymphoid organs. Because the results were the same in both syngeneic and allogeneic recipients, we concluded that this lack of engraftment was not attributable to immunologic rejection, but rather to lack of the ability of ES cells and their progenitors to self-renew after transplantation.¹¹  Indeed, we failed to detect any ES cell–derived cells in the bone marrow of recipient mice. Therefore, there is a need for a better understanding of the requirements for successful differentiation of ES cells into hematopoietic progenitor cells (HPCs) that can engraft long term.

At least 2 protocols on the generation of multilineage hematopoietic cells from ES cells have been reported, namely embryoid body (EB) formation and cultivation of ES cells on stromal cells.^1-4,8,13,14  Apart from being difficult to reproduce, the data reported were mainly in vitro, providing little evidence on whether the newly derived cells were immunologically functional in vivo. A more appealing approach has been the use of HOXB4, a homeobox hematopoietic transcription factor that, when ectopically transfected in HSCs or in yolk sac–derived hematopoietic cells, allowed more than 100- to 1000-fold cell expansion.^15-17  This remarkable self-renewal property conferred by HOXB4 was further demonstrated in ES cells that developed into definitive hematopoietic cells and engrafted in mice.¹⁸  More recently, one of us (H.K.) analyzed genetic manipulation of ES cell–derived hematopoietic cells¹⁵  and demonstrated that ectopic expression of HOXB4 can mediate a significant expansion of differentiated ES cells in vitro and in vivo. The self-renewal properties of HOXB4-transduced cells have been further demonstrated in primate¹⁹  as well as in human ES cells, suggesting that the self-renewal properties of HOXB4 could potentially be exploited in ES cell–based cell therapy in humans. However, studies by Wang et al showed that ectopic expression of HOXB4 had no effect on repopulating capacity of human ES cell–derived cells.²⁰  These findings are in contrast to the study by Bowles et al, who claim an important role for HOXB4 in the differentiation of human ES cells into hematopoietic cells.²¹ 

Here, we took advantage of these unique properties of HOXB4 that led to a high expansion of differentiated progenitor cells in vivo and in vitro. By transplanting the HOXB4-transduced ES cell–derived HPCs into immunodeficient Rag2^−/−γ_c^−/− mice, high levels of mixed chimerism were established, leading to long-term engraftment. The HPC-derived newly generated T cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus (LCMV) in vivo and specifically secreted interleukin-2 (IL-2) and interferon-γ (IFN-γ) upon CD3 stimulation in vitro. More important, HPC-generated antigen presenting cells (APCs) in these chimeric mice were abundant and efficiently presented viral antigen to wild-type (WT) presensitized T cells. These results reveal that HPCs that derived from HOXB4-transduced ES cells reconstitute immunodeficient mice and restore partial immunologic function.

Transduction of the CCE ES cell line with HOXB4 was described previously by Klump el al.²²  Transduced cells were grown on gelatinized flasks in feeder cell–free ES cell culture medium consisting of Dulbecco modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 15% fetal calf serum (FCS), 0.1 mM of l-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, and 1000 U/mL leukemia inhibitory factor. The culture medium was changed daily, and the cells were passaged every 2 to 3 days to avoid overgrowth and differentiation.

To differentiate ES cells into HPCs, a 2-stage culture strategy was adopted as described previously by Pilat et al.¹⁵  Initially, ES cells were subjected to EB formation over 6 days. At this stage, ES cells were plated to an ultra-low attachment Petri dish at a concentration of 2000 cells/mL in a methycellulose-based differentiation medium containing Iscove modified Dulbecco medium (Invitrogen), 15% FCS, 300 μg/mL transferrin, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 5% protein-free hybridoma medium (Invitrogen), 4 × 10⁻⁴ M monothioglycerol, and 50 μg/mL ascorbic acid. At second stage, EBs were dissociated into single-cell suspension using trypsin (2.75%), and 3.5 × 10⁶ cells/mL cells were replated on another ultra-low attachment Petri dish in a serum-free hematopoietic differentiation medium that contained StemPro34 plus nutrient supplement (Invitrogen) and a cocktail of hematopoietic cytokines including murine stem cell factor (mSCF; 100 ng/mL; R&D Systems, Minneapolis, MN), mIL-3 (2 ng/mL), mIL-6 (5 ng/mL), Flt3-L (10 ng/mL), insulin-like growth factor-1 (IGF-1; 40 ng/mL; Promega, Madison, WI), and dexamethasone (1 μM; Sigma-Aldrich, St Louis, MO). Culture medium was changed every other day and cell density maintained below 4 × 10⁶ cells/mL.

Six- to 8-week-old C57BL/6 (B6), green fluorescent protein (GFP) transgenic B6 and 129/SvJ mice were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA), and Rag2^−/−γ_c^−/− mice on the B6/B10 mixed inbred background were obtained from Taconic Farms (Germantown, NY).²³  All mice were bred in the animal facility at the Veteran Affairs Medical Center, Iowa. Animal procedures were approved previously by the institutional animal care and use committee and in accordance with National Institutes of Health guidelines.

For HPC transplantation, 8- to 10-week-old mice were lethally irradiated (950 cGy) 24 hours before transplantation, and 2.5 × 10⁶ HPCs in addition to 5 × 10⁵ Rag2^−/−γ_c^−/− autologous bone marrow cells were coinfused through the supra-orbital venous plexus as we described previously.¹¹  Altogether, more than 70 mice have received HPC transplants. For bone marrow transplantation (BMT), 5 × 10⁶ bone marrow cells derived from either GFP transgenic or Rag2^−/−γ_c^−/− mice were transplanted into Rag2^−/−γ_c^−/− mice in similar fashion.

The differentiation of HPCs was monitored using flow cytometry. All antibodies, including rat anti–mouse immunoglobulin G (IgG), anti-CD3, anti-CD8, anti-CD45R/B220, anti-CD45, Thy1.2 (CD90.2), and the anti–Gr-1, were phycoerythrin (PE) conjugated and purchased from BD Biosciences (San Jose, CA). In addition, PE-conjugated streptavidin, allophycocyanin-conjugated IFN-γ, and biotin-conjugated anti-NK1.1 were also purchased from BD Biosciences. For analyzing peripheral blood, lysis solution (R&D Systems) was added for the removal of RBCs after antibody staining. To monitor the HPCs, the recipient mice were killed at predetermined time points, and cells from peripheral blood, spleen, and bone marrow were stained with the above-mentioned antibodies and analyzed by flow cytometry. Cell fluorescence measurement was performed on a fluorescence activated cell sorter (FACS) Calibur and data analyzed using Cellquest Software (BD Biosciences).

Histologic sections were assessed by hematoxylin and eosin (H&E) staining of paraffin-embedded splenic tissue. Immunofluorescence staining of GFP and cell lineage markers was performed by staining cryosections with an Alexa Fluor 488 (1:500; Invitrogen)–conjugated anti-GFP antibody, a biotin-conjugated Gr-1, or a B220 antibody, respectively, followed by incubation with an Alexa Fluor 568–conjugated streptavidin (1:500; Invitrogen). Nuclei counterstaining was performed with TO-PRO3 (Invitrogen) according to manufacturer instructions. The immunofluoresence stained sections were subsequently visualized under a Bio-Rad Radience 2100mp Multi photon/confocal microscope (Bio-Rad, Hercules, CA), and images were captured and edited using Image J v1.37 software.

Chimeric Rag2^−/−γ_c^−/−, WT C57BL6, and nonchimeric Rag2^−/−γ_c^−/− control mice were infected with 2 × 10⁵ plaque-forming units of LCMV through intraperitoneal injection and subsequently killed after 8 days to determine T-cell responses. Splenic cells were collected and used to determine the number of epitope NP396-404–specific CD8 T-cell responses by intracellular cytokine staining for IFN-γ as described previously.²⁴ 

APCs were isolated from splenocytes of chimeric mice by a 2-step selection method. First, single-cell suspensions of spleens from chimeric mice were prepared and GFP-positive cells sorted using a FACS Digital Vantage (DiVa; flow cytometry facility, University of Iowa). In a second step, the sorted GFP-positive cells were suspended in complete media (RPMI 1640, 10% FBS, 50 μM 2-mercaptoethanol, 100 U/mL penicillin, and100 g/mL streptomycin) in the presence of either the LCMV-derived NP396-404 (1 μM/mL; Biosynthesis, Lewisville, TX) or control peptide OVA257-264 (1 μM/mL; Biosynthesis). Additional controls were left with no peptide and allowed to adhere to tissue culture dishes at 37°C for overnight. Then, the nonadherent cells were removed by washing. The adherent cells, so-called adhesion-enriched APCs, were detached by 10 mM ethylenediaminetetraacetic acid and washed with phosphate-buffered saline (PBS) twice before use.

T cells were separated from single-cell suspension of spleens from LCMV-infected B6, 129/SvJ, and chimeric Rag2^−/−γ_c^−/− mice using positive selection of Thy1.2-positive cells by either FACS DiVa or magnetic bead cell separation (Miltenyi Biotec, Auburn, CA) according to the manufacturer instruction manual.

APCs (10⁵) that were either pulsed with peptide or were nonpulsed were coincubated with 5 × 10⁵ T cells isolated from LCMV-infected WT B6 mice at 37°C for 6 hours. The cell mixture was stained for intracellular IFN-γ and analyzed by FACS.

Because the genetic background of HOXB4-transduced ES cells is 129/SvJ, T cells prepared from 129/SvJ were used as positive control. Isolated T cells were stimulated with plate-bound anti-CD3 (2 μg/mL) and anti-CD28 (2 μg/mL) monoclonal antibodies at a cell concentration of 5 × 10⁵ cells/mL for 24 to 36 hours and the culture supernatants subsequently collected to allow measurement of IL-2 and IFN-γ using enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA).

To determine B-cell responses to T-cell–dependent antigen, WT B6, chimeric Rag2^−/−γ_c^−/−, and nonchimeric Rag2^−/−γ_c^−/− mice were immunized intraperitoneally with 50 μg 2,4,6-Trinitropheny-Keyhole Limpet Hemocyanin-Biotin (TNP-KLH; Biosearch Technologies, Novato, CA) mixed in 50-μL complete Freund adjuvant (Sigma-Aldrich) and boosted with another dose of 50 μg TNP-KLH mixed in incomplete Freund adjuvant 21 days later. Serum was collected 28 days after initial immunization and analyzed for antibody formation. Similarly, T cell–independent responses were analyzed by immunizing mice intraperitoneally with 2,4,6-trinitropheny lipopolysaccharide (TNP-LPS; Biosearch Technologies) mixed in 50 μL PBS, and bled on day 21. Serum levels of anti–TNP-specific antibodies were obtained by running an anti-TNP ELISA kit (BD PharMingen, San Diego, CA) according to manufacturer instructions. At least 3 animals were used in each group.

To determine whether HOXB4 leads to a growth advantage of ES cell–derived HPCs, we first differentiated both HOXB4-tranduced and -nontransduced ES cells toward the hematopoietic lineage using the same protocol requiring a cocktail of hematopoietic cytokines. Briefly, EBs were formed over 6 days and subsequently dismantled to form single-cell suspensions that were further cultivated in hematopoietic growth medium containing mSCF, mIL-3, mIL-6, Flt3-L, IGF-1, and dexamethasone. By day 26, the cells had formed a heterogeneous cell population of large- and small-sized nonadherent cells, which we termed HPCs, that were predominantly CD45 positive (Figure 1A). In contrast, nontransduced ES cells failed to thrive under these conditions, and more than 90% were dead by day 20 (Figure 1B). This result is consistent with previous reports that HOXB4 enhances hematopoietic development in mouse ES cells,^15,17,18  and suggested that HOXB4 confers a growth advantage under these culture conditions. Therefore, the HPCs that we discuss in this article were derived from HOXB4-tranduced ES cells.

To determine the phenotype of the HPCs, the cells were analyzed by flow cytometry using a series of monoclonal antibodies against leukocyte and stem cell markers. The results of these studies are summarized in Table 1. A proportion of the newly generated HPCs also expressed Sca-1, c-kit, and CD34, markers of bone marrow resident stem cells. Typically, the HPCs had low expression of MHC class I antigens and poor expression of class II antigens.

To determine whether our newly generated HPCs engraft and induce multilineage mixed chimerism, CD45-positive cells were isolated from our HPC cultures by immunomagnetic bead separation to more than 99% purity. Subsequently, the purified HPCs were infused into previously irradiated Rag2^−/−γ_c^−/− mice. Because our HPCs express GFP, their green fluorescence was used for monitoring engraftment in peripheral blood, spleen, liver, and thymus at different time points. HPC-derived GFP-expressing hematopoietic cells were detected through 100 days after HPC transplantation (Table 2). The data indicate robust engraftment of the HPCs in peripheral blood, spleen, and bone marrow. A typical profile of the multilineage mixed chimerism detected in recipient mice is shown in Figure 2A, indicating that the GFP-positive HPC-derived cells were predominantly Gr-1 expressing. In contrast, mice-transplanted bone marrow cells from GFP transgenic mice showed that the donor cells predominantly became B cells (B220 expressing) in recipient mice (Figure 2B). Interestingly, however, there were no obvious differences between the NK1.1 and the CD3-positive cells in both groups.

Histologic examination of the spleen, liver, and thymus by immunofluorescence staining showed extensive engraftment of HPCs in the spleen and thymus but not in the liver (Figure 3A). More interestingly, a large population of HPC-derived cells was detected in the thymic cortex (Figure 3B). These results indicate the ability of HPCs to migrate and populate the thymus of recipient mice, an important observation because these cells might shape the T-cell repertoire in immunocompetent mice. As studied by H&E staining, we found that the dystrophic splenic structure of recipient mice nearly normalized after transplantation of HPCs, becoming almost identical to the spleens of control mice transplanted with B6 bone marrow cells (Figure 4A). However, spleens of control WT mice showed an even denser cell population, suggesting that the microenvironment in the Rag2^−/−γ^−/− mice might negatively influence full restoration of normal splenic structure. The data were further confirmed by immunofluorescence staining for donor cells in the spleen (Figure 4B). HPC-derived GFP-expressing cells restored the splenic structure of recipient mice. To further investigate the cell lineages comprising donor cells in the spleen, we stained for B220- and Gr-1–expressing cells. The restored follicular structures in HPC chimeric mice comprised mainly Gr-1 positive cells, with only a few B220-positive cells, whereas the restored splenic follicular structure in bone marrow transplanted mice were mainly B220 positive (Figure 4C). These findings were consistent with the flow cytometric data in Figure 2B. Therefore, the data revealed that HPC transplantation leads to near normalization and restoration of the follicular architecture, but lineage commitment appears to be heavily in favor of myeloid cells.

The data so far showed evidence of good HPC engraftment in lymphoid tissues and peripheral blood but did not provide information on the immunity of these chimeric and otherwise immunodeficient mice. To determine the ability of HPCs to restore the adaptive immunity of recipient mice to viral antigen, mice were infected with LCMV, and peptide-specific cytotoxic T lymphocytes were analyzed. Studies in mice have revealed a remarkable expansion of CD8 effector T cells that kill infected target cells during the acute phase of LCMV infection.²⁵  Eight days after infection of the WT B6 mice, viral specific effector CD8-positive T-cell responses were detected by intracellular staining for IFN-γ after short-term in vitro incubation with LCMV peptide NP396-404. Chimeric but not nonchimeric Rag2^−/−γ_c^−/− mice (Figure 5A) showed specific secretion of IFN-γ. To confirm that the IFN-γ–secreting cells were HPC derived, we showed that these cytokine-secreting T cells were GFP positive (Figure 5B). However, the cytotoxic T lymphocytes detected in the chimeric mice were much lower than those in WT mice, which relates to the low cell numbers of newly developed T cells in the recipient mice of HPCs, as shown in Figure 2.

Because we noted that the HPCs predominantly contributed to the development of myeloid cells, we wondered whether APCs derived from the HPCs were functional. To investigate this, GFP-positive cells were first isolated by flow cytometric sorting. Adhesion-enriched APCs from these cells were pulsed with the LCMV-specific peptide (NP396-404) followed by coincubation with T cells sorted from LCMV-infected WT mice. T-cell responses were determined by quantitating IFN-γ–producing cells. As shown in Figure 5C, these APCs successfully presented the LCMV peptide, but not control peptide, suggesting that ES cell–derived APCs functionally present antigen to T cells.

Our data so far showed robust engraftment of HPCs and functional T cells and APCs in recipient mice. To further test the capability of newly generated HPC-derived T cells to be a mediator and regulator of adaptive immunity that play important roles in the activation phase of T cell–mediated immune responses, we enriched the HPC-derived T cells from chimeric mice by immunomagnetic bead separation and stimulated them with plate-bound anti-CD3/anti-CD28 antibodies in vitro. Indeed, HPC-derived T cells secreted IL-2 and IFN-γ upon CD3 stimulation (Figure 6). The results suggested that these de novo HPC-derived T lymphocytes in the Rag2^−/−γ_c^−/− mice produced cytokines critical for the activation and regulation of T cell–mediated immune reactions in response to stimulation.

Finally, to test whether HPC-derived B cells in the chimeric Rag2^−/−γ_c^−/− mice secrete antibodies upon antigen stimulation and possess the capability to class switch, we immunized mice with either the T cell–dependent antigen TNP-KLH or with the T cell–independent antigen TNP-LPS. As shown in Figure 7, IgG isotypes in both chimeric and nonchimeric Rag2^−/−γ_c^−/− mice were almost undetectable after either TNP-KLH (Figure 7A) or TNP-LPS (Figure 7B) stimulation, but modest IgM levels were present in chimeric mice. In contrast, as expected, WT mice robustly responded to both antigens (Figure 7C). Surprisingly, whereas we measured HPC-derived T and B cells in the vaccinated animals, TNP-KLH vaccination boosted the expansion of the HPC-derived T and B cells, especially in T cells, suggesting the ability of these de novo–derived lymphocytes to clonally expand in response to antigen stimulation (Figure 7D). These expanded T cells showed 2 subpopulations (GFP^high and GFP^dim), possibly suggesting DNA methylation and silencing of the GFP gene.

To our knowledge, these are the first in vivo functional studies on lymphoid and myeloid cells derived from HOXB4-transduced ES cells. Altogether, our data indicate the ability of HOXB4 to confer self-renewal properties to ESC-derived HPCs, enabling development of hematopoietic cells that are immunologically responsive, a property much desired for treatments of human disease.

The ability of ES cells to form tissues of all 3 germ layers has raised hope that ES cells can be applied in regenerative medicine to cure chronic degenerative diseases. However, before that goal can be achieved, there is a need to establish protocols that allow reproducible differentiation of ES cells. For example, successful differentiation of ES cells into hematopoietic cells has remained elusive and difficult. Our own previous studies indicated that after transplantation of nondifferentiated ES cells, only low levels of ES cell–derived cells were transiently detectable.¹¹  Here, by transducing ES cells with the transcription factor HOXB4, we were able to generate highly enriched progenitor cells that show characteristics of hematopoietic cells after engraftment in recipient mice. More important, we describe the functionality of the newly generated T and B cells to respond to antigen stimulation and that of HPC-derived APCs to robustly present antigen to T cells.

Currently, there are 2 major approaches that have been established for the derivation of hematopoietic cells from either human or murine ES cells, namely the cocultivation of ES cells with stromal cell lines^1,2,13,14  and secondly, formation of EBs followed by hematopoietic cytokine treatment.^3,4,8,15  In our hands, the differentiation of ES cells by either method failed to yield sufficient numbers of hematopoietic cells that could be used for transplantation. Here, we used the self-renewal properties of HOXB4 by transducing ES cells that were used to derive hematopoietic cells. We show successful expansion of hematopoietic precursor cells and their engraftment in immunoincompetent mice.

Interestingly, the ES cell–derived HPCs were predominantly Gr-1 expressing, suggesting that they are myeloid and not lymphocytic, which is consistent with previous observations and reports by others.^15,19  Because of our histologic data showing repopulation of lymphoid organs with these cells, one would predict robust T- and B-cell development. Surprisingly, both cell populations were poorly developed. However, several reports have suggested that HOXB4 blocks lymphopoiesis,^18,26  although no formal experimental data on how this occurs have been reported. Whether this is a result of the HOXB4 or that of the less developed thymus in the incompetent mice remains to be determined. To further explore this phenomenon, we transplanted Rag2^−/−γ_c^−/− mice with bone marrow cells from WT B6 GFP-transgenic mice and studied lymphocytic cell development. Interestingly, few mature T cells but abundant B cells were detected in recipient mice. These data seem to suggest that the poor T- and B-cell development after HPC transplants was probably attributable to the effect of HOXB4 on lymphocytic development because myeloid cell development was robust.

Despite these low T-cell numbers in our study, the HPC-derived T cells were clearly functional, as suggested by their ability to generate specificity to LCMV. Further, direct stimulation with anti-CD3/anti-CD28 antibodies led to the secretion of IL-2 and IFN-γ, critical cytokines in T-cell function, suggesting that signaling in these de novo generated T cells was normal. In contrast, B-cell function was low because we were able to measure very low antibody secretion after antigen stimulation.

Our data show that although HOXB4 strongly confers self-renewal properties to hematopoietic cells generated from ES cells and promotes myelopoiesis, it appears to have a negative influence on lymphopoiesis. In contrast to our findings, human ES cells transduced with HOXB4 failed to induce extensive growth or repopulation in mice.²¹  Thus, the role of HOXB4 in hematopoiesis may not be identical in all species, although data in nonhuman primates revealed that transduced ES cells indeed showed rapid cell growth similar to that observed in mice¹⁹  and in human hematopoietic stem cells.²⁷  These findings suggest that the influence of HOXB4 on lymphopoiesis and indeed on hematopoiesis requires further investigation to allow optimization of ES cell differentiation into multilineage hematopoietic cells. For example, data by Schiedlmeier et al appear to suggest that high levels of HOXB4 impaired myeloerythroid differentiation but also reduced lymphopoiesis in human cord blood stem cells.²⁸  Thus, ideal concentrations of HOXB4 need to be worked out that would allow normal hematopoiesis, including that of lymphocytes. Furthermore, Wang et al showed that ectopic Cdx4 enhanced hematopoietic progenitor formation from ES cells as well as increased lymphoid reconstitution in combination with HOXB4.²⁹  Therefore, other transcription factors that might influence or work in concert with HOXB4 remain to be investigated.

We also noted a small cell population of GFP-negative lymphoid cells in the chimeric animals. Clearly, these cells were HPC derived because the Rag2^−/−γ_c^−/− mouse lacks the gene required for lymphopoiesis and consequently has not much lymphoid tissue. A possible explanation for these GFP-negative lymphocytes might be the methylation of the HOXB4 gene in the chimeric Rag2^−/−γ_c^−/− mice leading to silencing of the GFP signal.^29-31  Typically, in our flow cytometric analyses, 2 populations of GFP^dim and GFP^high, respectively, were detected. The expression of GFP^dim might reflect this situation. Hence, in our transplantation model of Rag2^−/−γ_c^−/− mice, we assumed that the lymphoid cells in the GFP-negative quadrants of the flow cytometric plots derived from the HPCs.

Thus, as we move toward developing therapy-oriented protocols for deriving hematopoietic cells that could be used in clinical treatment, there is a need for optimization of protocols to establish ideal conditions for the derivation of leukocytes that could be used for the cure of hematologic diseases and for immunotherapy. The potential risk of tumor formation remains of concern. By purifying the CD45-positive cells from the HPC cultures, we virtually eliminated the danger of tumor formation in vivo because we failed to detect any tumors more than 200 days after transplantation. Moreover, we transplanted more than 70 mice in this series but have detected no tumors. In conclusion, we characterized the hematopoietic potential of HOXB4-tranduced ES cell–derived HPCs. Although the HPCs only partially restored the immunity of the immunodeficient mice, the functional data on T cells, B cells, and APCs indicate a huge potential for these cells. Thus, eventually, ES cells might be considered an alternative source to bone marrow cells, allowing their possible use in cell-based therapies.

We thank Simone Rollins and David Gordon, both at the University of Iowa, for technical assistance and also acknowledge the help of Aaruni Khanolkar in Dr John T. Harty's laboratory in establishing the LCMV infection model.

This study would not have been possible without the generous grants from the National Institutes of Health/National Heart, Lung, and Blood Institute (RO1 HL073015), Veterans Administration Merit Review, and Roche Organ Transplantation Research Foundation.

National Institutes of Health

Contribution: K.-M.C. performed most experiments, analyzed data, and wrote the manuscript. S.B. performed some critical experiments and analyzed data. H.K. contributed vital embryonic stem-cells lines and critically reviewed the manuscript. N.Z. designed the research and wrote and revised the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Nicholas Zavazava, MD, PhD, University of Iowa Hospitals and Clinics, Department of Internal Medicine, 200 Hawkins Dr, C51-F, Iowa City, IA 52242; e-mail: nicholas-zavazava@uiowa.edu.