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Stem Cells 2004;22:1191-1204 www.StemCells.com
© 2004 AlphaMed Press

Enforced Activation of STAT5A Facilitates the Generation of Embryonic Stem–Derived Hematopoietic Stem Cells That Contribute to Hematopoiesis In Vivo

Jan Jacob Schuringaa, Kaida Wua, Giovanni Morronea,b, Malcolm A.S. Moorea

a Laboratory of Developmental Hematopoiesis, Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA;
b Department of Experimental and Clinical Medicine, University of Catanzaro "Magna Graecia," Catanzaro, Italy

Key Words. Definitive hematopoiesis • Embryonic stem cells • STAT5A • Hematopoietic stem cells • Self-renewal

Correspondence: Malcolm A.S. Moore, D.Phil., Laboratory of Developmental Hematopoiesis, Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA. Telephone: 212-639-7090; Fax: 212-717-3618; e-mail: m-moore{at}ski.mskcc.org


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Little is known about the molecular mechanisms that direct the transition from primitive to definitive hematopoiesis. In this study, we cocultured murine embryonic stem (ES) cells on OP9 stroma to induce hematopoietic differentiation as a model to study factors involved in the generation of adult hematopoietic stem cells (HSCs). Overexpression of the constitutively activated mutant signal transducer and activator of transcription (STAT) 5A(1*6) in ES cells facilitated the generation of cells that expressed the endothelial-hemangioblast marker Flk-1 within 5 days of coculture on OP9. The first CD41+/ CD45+/c-Kit+/Flk-1– hematopoietic cells arose in our culture conditions between days 5 and 7. Persistent activation of STAT5A greatly enhanced the generation of hematopoietic progenitors compared with controls, as determined by colony assays in methylcellulose. Moreover, whereas controls generated only a short transient wave of hematopoiesis lasting less than 3 weeks, expression of STAT5A(1*6) resulted in the generation of hematopoietic cobblestone area–forming cells (CAFCs) on OP9 that could be serially passaged onto new OP9, giving rise to second and third CAFCs that generated hematopoietic progenitors for ≥5 weeks, indicating a role for STAT5A in HSC self-renewal in vitro. Several definitive hematopoietic genes were upregulated by STAT5A (1*6), as well as Runx1/AML1, vascular endothelial growth factor, oncostatin M receptor, HoxB4, Wnt5A, Delta-like-1, and Bmi-1. Furthermore, ES-derived hematopoietic cells expressing STAT5A(1*6) contributed to myeloid-lymphoid hematopoiesis in primary and secondary nonobese diabetic–severe combined immunodeficiency recipients, although no donor-derived cells could be detected after 7 weeks in the secondary recipients. These data indicate that a persistent activation of STAT5A allows the generation of ES-derived HSCs that can, at least for an intermediate period, contribute to hematopoiesis in vivo.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During mouse embryogenesis, hematopoiesis begins with the generation of primitive nucleated erythroid cells in the yolk sac (YS) beginning at 7.5 dpc [1]. This wave of primitive hematopoiesis is followed by a wave of definitive hematopoiesis and the development of hematopoietic clusters on the floor of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region at 10.5 dpc [2–4]. Hematopoietic cells from the AGM region and YS colonize the fetal liver and ultimately the adult bone marrow [5]. Initially, the YS lacks spleen colony-forming cells (CFU-Ss) and long-term repopulating hematopoietic stem cells (LTR-HSCs). HSCs capable of long-term multilineage engraftment in irradiated recipients are first observed in the AGM region at 10.5 dpc [3–5]. Hematopoietic cells derived from embryonic stem (ES) cells, like early YS progenitors, are ineffective in reconstituting hematopoiesis in irradiated recipients [6]. These observations possibly reflect the importance of an appropriate microenvironment to provide instructive signals for HSC maturation and homing to the bone marrow, because YS progenitors can contribute to lymphomyeloid hematopoiesis in adults when grown on AGM stroma [7] or when injected into sublethally myeloablated newborn mice [8, 9].

Signal transducer and activator of transcription (STAT) 5 belongs to a family of transcription factors that fulfill key functions in hematopoiesis [10, 11]. STAT5 is activated in response to various hematopoietic cytokines, including interleukin-2 (IL-2), IL-3, IL-5, IL-7, GM-CSF, erythropoietin, and colony-stimulating factor-1 (CSF-1) [12]. Stat5a–/–5b–/– knockout mice are characterized by fetal anemia and increased apoptosis of fetal liver erythroid progenitors [13, 14]. Furthermore, in competitive repopulation assays, bone marrow and fetal liver cells of stat5a–/–b–/– mice display a decreased repopulating activity in granulocyte, macrophage, erythroid, and B-lymphocyte populations, with no detectable engraftment of T lymphocytes [15]. In a similar study, Snow et al. [16] also demonstrated that STAT5-null HSCs have a profound impairment in repopulating potential. These data suggest that STAT5 is required to sustain a robust hematopoietic reserve that contributes to host viability by promoting survival of early progenitor cells. Transduction of the constitutively activated mutant STAT5A(1*6) in human HSCs demonstrated that a persistent activation of STAT5A results in enhanced HSC self-renewal and induces expansion of the HSC pool [48]. These results prompted us to study the effects of STAT5A(1*6) on ES-derived hematopoiesis. In this study, we describe a persistent activation of STAT5A that facilitates hematopoietic differentiation and generates ES-derived HSCs that display long-term self-renewal characteristics in vitro and contribute to hematopoietic reconstitution in vivo in primary and secondary irradiated adult recipients.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Cell Lines, Constructs, and Generation of Stable ES Lines
The 129/Sv ES cell line R1 (a generous gift from Dr. Andras Nagy, Samuel Lunenfeld Research Institute, University of Toronto, Toronto) was propagated on murine embryonic fibroblasts (MEFs) in knockout Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA) supplemented with 15% fetal bovine serum (FBS; Hyclone. Logan, UT); penicillin and streptomycin, 200 mM glutamine; and 103 U/ml murine leukemia inhibitory factor (ESGRO, Chemicon International, Temecula, CA). The bone marrow stromal cell line OP9 [17] was propagated in {alpha}-minimal essential medium (MEM; Gibco Life Technologies) supplemented with 20% FBS (Hyclone); penicillin and streptomycin, 200 mM glutamine; 57.2 µM ß-mercaptoethanol; and 50 µg/ml vitamin C. The same medium was used for ES differentiation. 32D-c3 cells were propagated in Iscove’s modified Dulbecco’s medium supplemented with 10% FBS, 10 ng/ml IL-3, penicillin and streptomycin, and 200 mM Glutamine. Luciferase assays were performed as described previously using luciferase reporters containing three repetitive STAT5 binding sites from the ß-casein promoter [18]. Cells were transiently transfected by electroporation and 12 hours before harvest cells were depleted of IL-3 or grown in the presence of IL-3 as indicated. The constitutively active mutant murine STAT5A(1*6) [19, 20] was subcloned from pMXpuro-STAT5(1*6) into the EcoRI-SalI sites of the pIRES2-EGFP vector (Clontech, Palo Alto, CA), and this construct was verified by sequencing. For the generation of stable clones, ES cells were electroporated (Bio-Rad Gene Pulser [Hercules, CA], set at 270 V, 970 µF, 3 x 106 cells in 250 µl medium) with either pIRES2-EGFP (control) or pSTAT5A(1*6)-IRES2-EGFP (STAT5A[1*6]) vectors, plated on neomycin-resistant MEFs, and grown for 5–7 days, after which stable clones were selected in the presence of 500 µg/ml G418 (Gibco Life Technologies).

Hematopoietic Differentiation Studies
A total of 6 x 104 ES control and STAT5A(1*6) cells were differentiated on OP9 stroma in {alpha}-MEM as described above. The medium was changed at day 3. After 5 days of coculture, the ES cells and OP9 monolayer were trypsinized, and a single-cell suspension was preplated on tissue culture flasks for 30 minutes to remove the stromal cells. Nonadherent cells (2 x 106) were transferred to a new OP9 monolayer flask. At day 7, nonadherent cells were harvested for analysis and mice engraftment studies, because the adherent cells did not contain hematopoietic CD41+ cells (data not shown and [21]). Cocultures were propagated for up to 5 weeks, with replating on fresh OP9 at days 14 and 25.

Colony-Forming Cell Assays
Colony-forming cell (CFC) assays were performed as described previously [22]. Briefly, cells were plated in 1.2% methylcellulose containing 30% FBS, 57.2 µM ß-mercaptoethanol, 2 mM glutamine, 0.5 mM hemin (Sigma, St. Louis), and 20 ng/ml of each of the following growth factors: murine IL-3 (mIL-3), hIL-6, hIL-11, hG-CSF, mGM-CSF, mKL, and hEPO.

Immunoblotting, Histochemistry, and Cytospins
A total of 5 x 105 cells were lysed on ice in lysis buffer, and whole-cell extracts were boiled for 5 minutes in Laemmli sample buffer before separation on 12% SDS-acrylamide gels. Proteins were transferred to nitrocellulose filters (Millipore, Bedford, MA) in Trisglycine buffer at 9 V for 1.5 hours using a semidry electroblotter from Bio-Rad. Membranes were blocked in phosphate-buffered saline (PBS) containing 5% nonfat milk before incubation with antibodies. Binding of antibodies was detected by enhanced chemiluminescence according to the manufacturer’s instructions (Roche Diagnostics, Indianapolis). Antibodies against STAT5 (C20), Oct4, HoxB4, and GFP (B2) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and were used in dilutions of 1:2,000. For histochemistry, cytospins were fixed in 4% paraformaldehyde, permeabilized in PBS containing 0.1% Tween-20, and stained with antibodies in dilutions of 1:100. Secondary FITC-conjugated antibodies were obtained from Jackson Immuno Research (West Grove, PA) and were used in 1:200 dilutions. Cells were visualized using a Zeiss inverted fluorescence microscope. Max-Grünwald-Giemsa staining was used to analyze cytospins.

Polymerase Chain Reaction and Gene-Array Analysis
For reverse transcription–polymerase chain reaction (RT-PCR), total RNA was isolated from 1 x 106 cells using the RNeasy kit from Qiagen (Valencia, CA) according to the manufacturer’s recommendations. RNA, 2 µg, was reverse transcribed with M-MuLV reverse transcriptase (Roche Diagnostics). For PCR, 2 µl of cDNA was amplified using primers as indicated in the text (sequences and conditions are available on request) in a total volume of 50 µl using 2 units of Taq polymerase (Roche Diagnostics). As a negative control RNA minus reverse transcriptase, prepared cDNA was used in PCR reactions. Ten-µl aliquots were run on 1.5% agarose gels. For microarray analyses, ES cells were differentiated on OP9, and at day 5, the culture was trypsinized and stromal cells were depleted by preplating for 30 minutes on tissue culture flasks. Total RNA was isolated using the RNeasy kit from Qiagen, and 4 µg of RNA was used for labeling reactions according to the manufacturer’s instructions and was hybridized to the Affymetrix Mouse Expression Set 430. Control and STAT5A(1*6) transcripts were hybridized independently, and gene expression profiles were compared. Differences in gene expression were considered significant when the fold change was >1.87, with a detection p value < .0025.

Mice
Nonobese diabetic–severe combined immunodeficiency (NOD-SCID) mice (also referred to as NOD/LtSz-SCID) were obtained from Jackson Laboratory (Bar Harbor, ME) and were maintained in germ-free conditions. Mice matched for age (8–10 weeks), weight (>20 g), and sex and were injected with 1 to 2 x 106 cells via tail-vein injection.

Flow Cytometry Analysis
All antibodies were obtained from Pharmingen (San Diego). Cells were incubated with antibodies at 4°C for 30 minutes. For blocking nonspecific binding to Fc{gamma} receptors, cells were blocked with anti-Fc{gamma} antibodies for 5 minutes at 4°C. All fluorescence-activated cell sorter (FACS) analyses were performed on a FACScalibur (Becton, Dickinson, San Jose, CA), and data was analyzed using FlowJo (Tree Star, Inc., San Carlos, CA).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of Constitutively Active STAT5 Facilitates Hematopoietic Differentiation of ES Cells
To evaluate the effects of a persistent activation of STAT5A on ES-derived hematopoiesis, murine ES-R1 lines were generated by electroporation with a pSTAT5A(1*6) IRES2-EGFP vector with neomycin selection for clones that stably express the constitutively active mutant STAT5A(1*6) [19, 20]. As a control, ES-R1 cells were electroporated with the empty pIRES2-EGFP vector. Single clones were expanded under continuous neomycin selection and tested for genomic insertion of the IRES2-EGFP cassette by genomic PCRs (Fig. 1AGo). Clones were also tested for STAT5 expression by Western blot analysis (Fig. 1BGo). Control clone 1 and STAT5A (1*6) clone 3 were selected for further analysis. ES cells expressing STAT5A(1*6) maintained an undifferentiated phenotype on MEF feeder cells (data not shown) and stained positive for OCT4 in immunostains (Fig. 1CGo). The activity of the STAT5A(1*6) mutant was tested by transient transfection assays in 32D cells using a luciferase reporter construct containing three STAT5 binding sites in the promoter in which STAT5A(1*6) induced a fivefold increase in luciferase expression (Fig. 1DGo). As a control, cells were also stimulated with IL-3, which resulted in a 4.5-fold induction of the reporter.



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  		Figure 1. Generation of ES (R1) lines expressing STAT5A(1*6). (A): ES (R1) cells were transiently electroporated with pSTAT5A(1*6)-IRES2-EGFP or the empty vector pIRES2-EGFP, and neomycin-positive clones were selected in the presence of 500 µg/ml G418 on neomycin-resistant MEFs. Genomic DNA was isolated from several clones and subjected to polymerase chain reaction with primers for the IRES2-EGFP cassette. (B): Western blot analysis of several G418-resistant clones using antibodies against STAT5. As a loading control, lysates were Western blotted using antibodies against OCT4. Control clone 1 and STAT5A(1*6) clone 3 were selected for further analysis. (C): Cytospin of undifferentiated ES-STAT5A(1*6) clone 3 maintained on MEFs. All cells stained positive for OCT4. (D): 32D-c3 cells were transiently transfected with luciferase reporters containing STAT5 response elements in the promoter. Cells were cotransfected with STAT5A(1*6) or stimulated with 10 ng/ml IL-3 for 24 hours, after which cell lysates were analyzed for luciferase activity. Abbreviations: ES, embryonic stem; IL, interleukin; MEF, murine embryonic fibroblast.

 
ES-R1 cells were cocultured with OP9 stroma to induce hematopoietic differentiation, and the appearance of hematopoietic cells was monitored by flow cytometry and CFC assays. At day 5, the complete culture was harvested for analysis, and 4 x 106 cells from control or STAT5A(1*6) cultures were replated on fresh OP9 (Fig. 2AGo). At day 7, nonadherent cells were used for analysis, and cultures were continuously expanded for up to 5 weeks by replating on fresh OP9 stroma on day 14 and day 25, as indicated in Figure 2AGo. Over-expression of constitutively active STAT5A(1*6) was verified by Western blotting. As depicted in Figure 2BGo, STAT5A (1*6) was overexpressed on day 0 and day 5 of differentiation on OP9 stroma over low endogenous STAT5A expression levels. Interestingly, endogenous STAT5A levels were significantly increased at day 7, suggesting a role for STAT5A in the differentiation process. As loading controls, blots were stripped and reprobed with anti-STAT3, anti-OCT4, and anti-GATA2 antibodies (Fig. 2BGo).



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  		Figure 2. Enforced expression of STAT5A(1*6) facilitates the generation of ES-derived hematopoietic cells. (A): Schematic representation of hematopoietic differentiation protocol on OP9 stroma. (B): Western blot analysis of control and STAT5A(1*6) cells on OP9 day 0, day 5, and day 7. Lysates were Western blotted using antibodies against STAT5, STAT3, OCT4, and GATA2. (C): Expansion on OP9 cocultures. A representative experiment out of three independent experiments is shown. A total of 6 x 104 ES(R1) cells were plated on OP9 stroma on day 0, and cocultures were propagated for up to 5 weeks, with replating on fresh OP9 as indicated in Materials and Methods . (D): Phase-contrast images of control and STAT5A(1*6) cultures on OP9 on days 7, 25, and 35, as indicated. (E): CFC assays from cocultures on OP9. Data indicate CFCs per 10,000 plated cells of a representative experiment out of three independent experiments. (F): CFC assays as in (E), but now data represent total amounts of CFCs generated by control and STAT5A(1*6) cultures. (G): Cytospins of control and STAT5A(1*6) cultures on OP9 at day 14. Abbreviations: CFC, colony-forming cell; CFU-GEMM, colony-forming unit–granulocyte, erythrocyte, megakaryocyte, macrophage; CFU-GM, colony-forming unit–granulocyte-macrophage; ES, embryonic stem; FACS, fluorescence-activated cell sorter; NOD-SCID, nonobese diabetic–severe combined immunodeficiency; RT-PCR, reverse transcription–polymerase chain reaction.

 
At day 7, STAT5A(1*6) ES cells had generated many more nonadherent hematopoietic-like clusters than controls (a representative experiment out of three independent experiments is shown in Figs. 2C–GGo). At day 14, in STAT5A(1*6) cultures, we detected many more nonadherent cells, as well as the appearance of phase-dark cobblestone areas growing beneath the stroma. Although control cells only generated a short wave of hematopoiesis lasting less than 3 weeks, STAT5A(1*6) cells continued to expand for up to 5 weeks, giving rise to nonadherent hematopoietic cells and adherent cobblestone areas (CAs) that could be serially replated onto fresh OP9 (Figs. 2C, DGo).

The presence of hematopoietic progenitors was evaluated in colony assays in methylcellulose. Control ES cells generated hematopoietic progenitors at days 5 and 7 on OP9, but expression of STAT5A(1*6) resulted in the generation of significantly more progenitors at these days (Fig. 2EGo). At day 5, the STAT5A(1*6) generated predominantly BFU-Es and some CFU-granulocyte-macrophage (CFU-GM) colonies, whereas controls gave rise to both types. At day 7, the balance had shifted toward CFU-GM progenitors and some BFU-E and CFU-mix progenitors in the STAT5A(1*6) cultures. Most of the hematopoietic progenitors in control cultures appeared at day 14, whereas these cultures failed to generate hematopoietic progenitors after 2 weeks on OP9. In contrast, STAT5A(1*6) cultures continued to generate hematopoietic progenitors (mostly CFU-GM, and fewer BFU-E and mixed colonies) for up to 5 weeks (Fig. 2EGo). Because the STAT5A (1*6) ES cells generated many more hematopoietic cells, the total amount of progenitors per culture was also greatly enhanced in STAT5A(1*6) cultures (Fig. 2FGo).

Extensive immunophenotypical analysis revealed that expression of CD31 and Flk-1 was significantly elevated in STAT5A(1*6) cells at day 5 (Table 1Go and Fig. 3Go). In addition, CD41, which has recently been identified as a gene that marks the initiation of definitive hematopoiesis in the mouse embryo, was expressed in a higher percentage of STAT5A (1*6) cells compared with controls at day 5 (Table 1Go and Fig. 3Go). The CD41+ population was also positive for c-Kit and CD31 but negative for Flk-1, suggesting that the first hematopoietic cells arise in our culture conditions at day 5 and are CD41+/c-Kit+/CD31+ but have lost expression of Flk-1. Expression of the hematopoietic markers CD45, Mac-1, and Ter119 was still low in both control and STAT5A(1*6) cells at day 5. Approximately 65% of the cells stained positive for c-Kit at day 5, which is probably a reflection of the R1 ES cells that are c-Kit+. At day 7, 4.3% of control cells and 6.6% of STAT5A(1*6) cells had acquired CD45 expression, and these cells were also CD41+ and c-Kit+ but Flk-1–. Almost 50% of the cells expressed Ter119 at day 7, probably representing the first wave of primitive erythropoiesis. Interestingly, expression of STAT5A(1*6) prevented apoptosis, because significantly fewer cells were annexinV+ at days 5 and 7 compared with controls, suggesting that the STAT5A (1*6)-induced expansion might depend at least in part on the antiapoptotic effects of activated STAT5A. This antiapoptotic effect does not seem to involve known STAT5A target genes, such as Bcl2 and Bcl-xL, because we did not observe an upregulation of these genes in our RT-PCR and microarray studies (Fig. 4Go). Taken together, these data suggest that a persistent activation of STAT5 in ES cells differentiated on OP9 stroma accelerates the generation of primitive hematopoietic cells.


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  		Table 1. Phenotypical analysis of embryonic stem–derived hematopoietic cells cocultured on OP9
 


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  		Figure 3. FACS analysis of embryonic stem–derived hematopoietic cells cocultured on OP9. A total of 6 x 104 ES(R1) control and STAT5A(1*6) cells were plated on OP9 stroma, and day-5 cells were analyzed using FACS as indicated. Representative data out of two to five independent experiments are shown. Abbreviations: FACS, fluorescence-activated cell sorter; Ig, immunoglobulin.

 


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  		Figure 4. Differential gene expression induced by STAT5A (1*6) in ES-derived hematopoietic cells on OP9 stroma. Control and STAT5A(1*6) ES cells were differentiated on OP9 stroma, and at day 0, day 5, and day 7, total RNA was isolated for reverse transcription–polymerase chain reaction as indicated. Data are representative for two independent experiments. Abbreviation: ES, embryonic stem.

 
STAT5A(1*6) Promotes Long-Term Expansion of Primitive Hematopoietic Cells and Diverts Hematopoietic Differentiation toward Erythropoiesis
Although control cells only generated a short wave of hematopoiesis in less than 3 weeks in OP9 cocultures, STAT5A (1*6) cells continued to expand for up to 5 weeks (Fig. 2CGo), giving rise to nonadherent hematopoietic cells and adherent CAs. STAT5A(1*6)+ CAs could be serially replated onto fresh OP9 stroma, giving rise to second and third cobblestones, indicating that the HSCs generated by STAT5A (1*6) fulfill the criteria of in vitro stem cell self-renewal (Fig. 2DGo). Moreover, whereas control cultures failed to produce hematopoietic progenitors after 2 weeks on OP9, STAT5A (1*6) cultures continued to generate progenitors for up to 5 weeks that were mostly CFU-GM but also contained some BFU-E and few mixed colonies (Figs. 2E, FGo). Intriguingly, however, STAT5A(1*6) seemed to enhance terminal differentiation toward the erythroid lineage, because many more differentiated suspension cells at day 14 were positive for the erythroid marker Ter119 compared with control cells (48.4% for STAT5A[1*6] compared with 24.2% for controls; Table 1Go). In contrast, granulocyte/macrophage differentiation was strongly reduced in the presence of constitutively active STAT5A(1*6) (Table 1Go). These results were additionally underscored by cytospins of suspension cells at day 14, in which many erythroid but few myeloid cells were found in the STAT5A(1*6) cells (Fig. 2GGo). Although the cultures contained significant amounts of GM progenitors, these cells apparently did not mature toward granulocytes and macrophages, a phenotype that was strikingly similar to phenotypes we have observed in CB CD34+ cells transduced with STAT5A(1*6) [48]. The erythroid differentiation was even further pronounced at days 25 and 35, when 85.7% and 77.1% of the differentiated suspension cells were positive for Ter119, respectively (Table 1Go), whereas the adherent population at days 25 and 35 consisted of immature cells that were mostly c-Kit+ and CD45+ (Table 1Go, Fig. 2DGo). Taken together, these results indicate that overexpression of STAT5A(1*6) in ES cells results in long-term self-renewal in vitro and favors maturation toward the erythroid lineage at the expense of myeloid differentiation.

Expression of STAT5A(1*6) Confers Engraftability on ES-Derived Hematopoietic Stem Cells
Day-7 ES-derived cells were injected into sublethally irradiated NOD-SCID mice, and donor-derived hematopoietic cells in the peripheral blood (PB) were analyzed at weeks 5 and 7. Although stable genomic insertion of the STAT5 (1*6)-IRES2-EGFP cassette into ES cells resulted in appropriate mRNA expression (data not shown) and high expression of STAT5A(1*6) (Figs. 1BGo, 2BGo), the IRES2 sequence did not drive efficient expression of EGFP, a phenomenon that was observed in all stable clones and has been noted by others as well in some cases (Dr. T. Barberi, personal communication). Therefore, we resolved to use the H2Kb marker to distinguish H2Kb+ 129/Sv (R1) donor-derived cells from H2Kb–NOD-SCID hematopoietic cells (Fig. 5AGo). As indicated in Figure 5BGo, STAT5A(1*6) recipients showed donor-derived engraftment, whereas only very low engraftment levels were found in mice injected with control cells. A representative analysis of the PB is shown for a STAT5A(1*6) mouse 2 at week 5 (Fig. 5CGo). Engraftment of donor-derived STAT5A (1*6) cells was additionally confirmed by genomic PCRs for the STAT5A(1*6)-IRES2-EGFP cassette (data not shown) and neomycin-phosphotransferase II (Fig. 5DGo). At week 7, STAT5A(1*6) mice were euthanized for analysis and secondary engraftment studies. As indicated in Fig. 5EGo, recipients showed STAT5A(1*6) donor-derived cells of the myeloid and lymphoid lineages in the bone marrow, although the engraftment levels were somewhat lower in bone marrow than in PB (Fig. 5EGo). No extramedullary hematopoiesis in the spleens was observed (data not shown). The bone marrows from 4 STAT5A(1*6) NOD-SCID mice at week 7 were injected into eight sublethally irradiated secondary NOD-SCID recipients. We observed engraftment of donor-derived H2Kb+ (Fig. 5FGo) and neomycin-phosphotransferase II–positive cells (Fig. 3FGo) after 5–7 weeks in the PB in four out of eight mice, although we failed to detect donor-derived cells thereafter. These data suggest that the STAT5(1*6)-expressing ES-derived HSCs can initially engraft secondary recipients but are not capable of sustaining a long-term contribution to hematopoiesis in secondary hosts.



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  		Figure 5. Expression of STAT5A(1*6) enables engraftment of embryonic stem–derived hematopoietic cells in sublethally irradiated NOD-SCID mice. (A): FACS analysis controls of the H2Kb antibody. As a positive control, H2Kb FACS analysis of the PB leukocytes of a 129/Sv mouse is shown, and as a negative control, H2Kb FACS analysis of the PB leukocytes of a noninjected NOD-SCID mouse is shown. (B): A total of 1 to 2 x 106 cells from OP9 cocultures on day 7 were tail-vein injected into sublethally irradiated NOD-SCID mice. The percentage of ES(R1)-derived H2Kb+ donor cells was determined in the PB at week 5 and week 7. (C): Representative example of H2Kb+ FACS analysis of PB of STAT5A(1*6) mouse 2 and control mouse 1 at week 5. (D): Genomic PCR for neomycin-phos-photransferase II in PB of control mouse 1 and STAT5A(1*6) mice 1 through 3. (E):At week 7, mice were euthanized and bone marrow was analyzed for H2Kb, Ter119, Mac1, and B220 expression. Data represent FACS analysis of STAT5A(1*6) mouse 3. (F): The femoral content of each STAT5A(1*6) mouse at week 7 was injected into two sublethally irradiated NOD-SCID recipients for secondary engraftment studies (m1 was injected into m1.1 and 1.2, etc.). H2Kb+ FACS analysis of PB of STAT5A(1*6) mice 1.2 and 2.1 at week 5 is shown. Neo+ genomic PCRs (G) were observed up until 5–7 weeks in approximately 50% of the mice in PB samples, but both H2Kb and Neo signals were lost in analyses thereafter. Abbreviations: FACS, fluorescence-activated cell sorter; Ig, immunoglobulin; NOD-SCID, nonobese diabetic–severe combined immunodeficiency; PB, peripheral blood; PCR, polymerase chain reaction.

 
Molecular Analysis of Differential Gene Expression Induced by STAT5A(1*6) in ES-Derived Hematopoietic Cells
To further characterize ES-derived hematopoiesis on OP9 and to study the effects of STAT5A(1*6) on the differential expression of relevant genes, RT-PCR analysis was performed on ES cells as well as on day-5 and day-7 cells from OP9 cocultures in two independent experiments. Flk-1, which is expressed on hemangioblasts and endothelial cells, was first observed at day 5, and its expression was transient, because no Flk-1 RNA was detected at day 7 (Fig. 4AGo). mRNA for the transcription factor Runx-1/AML1, which has been shown to be expressed in hemangioblasts and is required for definitive hematopoiesis, was observed in all samples, but STAT5A(1*6) significantly enhanced its expression. The marker of primitive erythropoiesis ß-H1 globin was strongly expressed at days 5 and 7 in both control and STAT5A(1*6) cells, whereas the definitive marker ß-major globin was absent in ES cells and expressed at its highest levels on day 7. Similarly, the definitive hematopoiesis marker GATA1 was first observed in day-5 cells but was expressed most strongly at day 7 in both control and STAT5A(1*6) cells. GATA2 RNA was expressed in all samples, particularly at day 5, and was slightly upregulated by STAT5A(1*6) at day 7 compared with controls. Transcripts for the homeobox transcription factor HOXB4 were first observed at day 5, with slightly elevated RNA levels at day 7. Vascular endothelial growth factor (VEGF) was significantly upregulated by constitutively activated STAT5 at days 0 and 5. BMP4, Bcl2, and Bcl-xL were expressed in all samples, but the expression levels were not changed by STAT5A(1*6). As PCR controls, all samples were subjected to RT-PCR using primers for ß-actin as well as neomycin-phosphotransferase II.

These results were confirmed by comparative Affymetrix microarray A430 analysis of control and STAT5A (1*6) day-5 cells, and these studies additionally revealed a STAT5A(1*6)-induced upregulation of SCL/Tal1, Runx2, embryonic and adult hemoglobins, glycophorin A, IL-6, Fli1, Bmi1, Wnt5A, Delta-like-1, Wasp, Sox18, ß1-integrin, CXCR4, CD44, and oncostatin M receptor (OSM-R), amongst several other genes (Table 2Go). Cytokine-inducible SH2 protein 3 was also upregulated by STAT5A(1*6) and has been implicated in a STAT-induced negative-feedback pathway that negatively regulates STAT5 activity. Many of the genes upregulated by STAT5A(1*6) have been associated with hematopoiesis.


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  		Table 2. Differential gene expression in ES(R1)-derived hematopoietic cells on OP9 day 5, STAT5A(1*6) versus controls
 

   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
STAT5 fulfills a variety of key functions in the hematopoietic system, ranging from maintenance of the stem cell pool to the initiation of hematopoietic differentiation. In this study, we have used murine ES cells differentiated toward hematopoietic cells on OP9 stroma as a model to study the effects of STAT5A activation in the generation of adult HSCs. The coculture of ES cells on OP9 bone marrow stroma has been widely used as a model to induce hematopoietic differentiation without the need for exogenous growth factors [17]. However, these in vitro–generated hematopoietic cells were generated for a relative short duration and did not reconstitute long-term hematopoiesis in irradiated hosts. In agreement with previously published studies, we found that ES cells generated a transient wave of hematopoiesis that lasted less than 3 weeks of coculture on OP9 stroma, resulting in the production of erythroid and myeloid progenitors without repopulating potential. In contrast, enforced activation of STAT5A resulted in a dramatic increase in the generation of hematopoietic progenitors, as well as in HSCs with long-term self-renewal activity in vitro that also contributed to hematopoiesis in vivo in irradiated hosts.

Overexpression of constitutively activated STAT5A (1*6) in ES-R1 cells facilitates the generation of Flk-1+ cells, which is required for appropriate hematopoietic differentiation [23–25]. In addition to Flk-1 expression, we also find increased numbers of cells expressing CD31 within 5 days of OP9 coculture, and the expression of VEGF, vascular cellular adhesion molecule 1, IL-6, and OSM-R were also upregulated by expression of STAT5A(1*6). CD31 (platelet and endothelial cell adhesion molecule-1) is strongly expressed on the hemangioblast as well as on endothelial and hematopoietic cells, where it plays an important role in adhesion processes but also acts as a scaffold protein that enables activation of downstream molecules such as STATs and ß-catenin [26]. VEGF was upregulated by STAT5A(1*6), as determined by RT-PCR analysis, as well as by Affymetrix microarray, but because the p value was just above the cutoff value of .0025, it was therefore not included in Table 2Go. VEGF has been shown to act as a growth factor for hemangioblast formation, suggesting that the upregulation of both VEGF and its receptor Flk-1 might facilitate hemangioblast formation from ES (R1) cells expressing constitutively activated STAT5A. Indeed, we observed reduced numbers of CFCs at days 5 and 7 of OP9 coculture in the presence of antagonistic antibodies against VEGFR1 and VEGFR2 in both control and STAT5A(1*6) cell cultures (data not shown). It is of particular interest that OSM-R and IL-6 were also upregulated by STAT5A(1*6). OSM, OSM-R, and gp130 receptor, a common subunit shared by the receptors for OSM, IL-6, and LIF, are expressed in the AGM region of mouse embryos [27–29], the postulated site where engraftable HSCs first appear. OSM is absolutely required for the expansion of multipotential hematopoietic progenitors from the AGM region in vitro [27], and gp130 deficiency in the AGM region results in a failure of the expansion of early hematopoietic cells, which involves a lack of STAT3 activation [28]. Because STAT5A(1*6) upregulates the expression of IL-6 and the OSM-R, these data suggest that these molecules might contribute to the hemangioblast formation from ES cells expressing STAT5A(1*6). Further studies will elucidate whether these proteins are indeed involved in STAT5A-downstream mechanisms in the development of ES-derived hematopoiesis.

The first hematopoietic cells to arise in our culture conditions between day 5 and day 7 were CD41+/c-Kit+/CD31+/ Flk-1–. CD41 has recently been identified as a marker that defines the onset of definitive hematopoiesis in the mouse embryo [30–32], and the CD41+/c-Kit+ population from YS or d6 embryoid bodies (EBs) is enriched in definitive hematopoietic progenitors, whereas this population is absent in runx1/AML1–/– EBs that lack definitive hematopoiesis [31]. Because the expression of STAT5A(1*6) significantly increased the number of CD41+/c-Kit+ cells within 5 days of coculture on OP9 stroma, our data suggest that enforced activation of STAT5A facilitates the generation of early hematopoietic cells. Furthermore, the expression of the transcription factor Runx1/AML1 [33], which is essential for hematopoietic commitment at the hemangioblast stage during development, was upregulated by STAT5A(1*6), as determined by RT-PCR analysis. These observations are further underscored by progenitor assays, in which we found a 10-fold increase of hematopoietic progenitors at day 5 in STAT5A(1*6)-expressing cell populations, as determined by colony assays in methylcellulose. Upon further culture, control cells gave rise to only a short wave of hematopoietic cells, with maximum generation of progenitors at week 2. In contrast, STAT5A(1*6) cells continued to expand on OP9 stroma and generated significantly more progenitors in the 5-week period of culture. Taken together, these data indicate that a persistent activation of STAT5A results in a highly augmented and prolonged ES-derived hematopoietic differentiation.

Expression of STAT5A(1*6) resulted in the formation of CAs that demonstrated secondary and tertiary plating potential, suggesting that STAT5A(1*6) allows the generation of ES-derived HSCs with self-renewal characteristics in vitro. These cells could be cultured for at least 5 weeks, continuously generating nonadherent hematopoietic cells that contained progenitors of the erythroid and myeloid lineages. This phenotype is strikingly similar to phenotypes we have observed in human cord blood–derived CD34+/CD38– cells transduced with STAT5A(1*6). These cells also generated CAs that arose within 10 days of plating and could be serially passaged onto new stromal cells for up to 18 weeks, giving rise to nonadherent cells that contained progenitors as well as more differentiated cells [48]. These data, together with our new observations in ES-derived hematopoietic cells, suggest that enforced activation of STAT5A in the HSC compartment results in extensive HSC self-renewal in vitro. Recently, several genes have been implicated in HSC self-renewal by loss-of-function (gene targeting) or gain-of-function (ectopic expression) experiments, including HoxB4, Bmi-1, ß-catenin, and Notch1 [34–38]. Unfortunately, little information is currently available on the events downstream of these molecules in relation to HSC self-renewal. One of the genes that was overexpressed by STAT5A is Dlk1 (delta-like 1/preadipocyte factor-1), a cell-surface glycoprotein encoding EGF repeats related to Notch/delta/serrate. It was selectively expressed in stromal lines supportive of HSC proliferation. Its overexpression in stroma resulted in enhanced (fourfold to sixfold) numbers of early CAs in stem cell coculture, and these areas transiently contained progenitors and engraftable stem cells [39]. Furthermore, we found that HoxB4,ß-catenin, and Notch1 were expressed in day-5 cells on OP9 as determined by RT-PCR or microarray analyses, but their expression was not enhanced by STAT5A(1*6). In contrast, Bmi-1 was upregulated in the STAT5A(1*6) cells, as was Wnt5A, which is a known activator of ß-catenin. It has recently been demonstrated that Bmi-1 is required for the maintenance of adult self-renewing HSCs [35, 40]. Wnt3A has recently been reported to induce expansion of the HSC pool [37], but little is known about the effects of Wnt5A on HSCs, although it is expressed in the adult bone marrow CD34+/Lin– compartment [41] and has been shown to expand the multilineage progenitor pool [42]. Nevertheless, our findings raise the possibility that the effects of STAT5A(1*6) on self-renewal of the HSC pool involve activation of some, or all, of these pathways, a possibility that will certainly be the focus of further studies.

Although control ES-derived hematopoietic cells did not significantly engraft sublethally irradiated NOD-SCID recipients, expression of STAT5A(1*6) resulted in the generation of hematopoietic cells that contributed to hematopoiesis in vivo. Hematopoietic reconstitution of erythroid, myeloid, and lymphoid lineages was observed, although engraftment in the bone marrow was typically somewhat lower than in the PB. It is conceivable that STAT5A(1*6) cells in the PB may expand outside of the bone marrow for a period of at least 7 weeks via a transient wave of hematopoiesis in the spleen. However, no extramedullary hematopoiesis was observed at week 7 (data not shown). Alternatively, it is possible that the more primitive HSCs in the bone marrow express less H2Kb. We have indeed observed that the more differentiated PB H2Kb+ cells stained much stronger than more primitive bone marrow cells in 129/Sv control mice (data not shown).

To investigate whether STAT5A(1*6) ES-derived HSCs were capable of long-term in vivo hematopoietic reconstitution, primary recipients were euthanized after 7 weeks, and bone marrow of each STAT5A(1*6) mouse was injected into two secondary sublethally irradiated NOD-SCID recipients. We observed secondary engraftment for up to 5–7 weeks in four out of eight recipients as determined by H2Kb FACS analysis (data not shown) and neomycin-phosphotransferase II RT-PCRs (Fig. 3FGo). However, we were unable to detect H2Kb+ or Neo+ cells thereafter, suggesting that although the STAT5A(1*6) cells were able to engraft secondary recipients, these cells could not fully sustain long-term hematopoiesis. Such a decline of STAT5A(1*6) ES-derived hematopoiesis, despite the engraftment of secondary recipients, could be attributable to silencing, a possibility that will be verified in future studies.

Recently, during the course of our studies, Kyba et al. [43] reported that expression of STAT5A(1*6) results in enhanced hematopoietic differentiation of ES cells using EB formation and a doxycycline-inducible system. In agreement with our data, the authors describe that enforced expression of STAT5A(1*6) promotes expansion of hematopoietic cells in vitro in OP9 cocultures and results in an increase in CFC frequency. In addition, the STAT5A(1*6) EB day-6 cells propagated on OP9 engrafted recipients for a transient period of 8 weeks, which depended on the continuous expression of STAT5A(1*6). The authors propose that persistent activation of STAT5A might not be sufficient to induce the transition from primitive to definitive hematopoiesis in ES-derived hematopoietic cells, as is instead observed in the presence of overexpressed HoxB4 [44], because STAT5A(1*6) cells less efficiently downregulate the expression of embryonic ß-H1 globin compared with HoxB4 upon differentiation [43]. In our experiments, both control and STAT5A(1*6) cells expressed HoxB4 RNA (Fig. 5Go and microarray analysis) as well as HoxB4 protein as determined by Western blot, and this expression was even further upregulated in STAT5A (1*6)-expressing cells at day 7 (data not shown). A persistent activation of STAT5A also resulted in the upregulation of various definitive hematopoietic genes, including Runx1, adultß-major globin, adult ß-globin, Wasp, and CD41. Furthermore, we observed a STAT5A(1*6)-induced upregulation of ß1-integrin and CD44, which are required for homing of P-Sp cells into fetal liver and bone marrow [45, 46], suggesting that these cells are indeed capable of migrating to the appropriate sites of definitive hematopoiesis. CXCR4, which is essential in stromal cell–derived factor-1–mediated homing of HSCs to the bone marrow [47], is also upregulated by STAT5A(1*6). One important difference between our strategy and that used by Kyba et al. [43] is the use of EBs as a differentiating system. Kyba et al. [43] indicate that the best engraftment results were obtained when EB day-6 ES cells were grown on OP9 stroma for a limited time [43]. We did not use the EB system but differentiated ES cells on OP9 directly and thus were able to generate HSCs in a significantly shorter time period. These differences in culture conditions might prove to be crucial for the generation of ES-derived HSCs with long-term engraftability.

In conclusion, the data presented in this paper demonstrate that enforced expression of STAT5A(1*6) in murine ES cells leads to enhanced production of cells capable of generating progeny with the molecular features typical of definitive hematopoiesis and endowed with repopulating ability in primary and secondary recipients. It will be of importance in the near future to determine whether a persistent activation of STAT5A in ES cells indeed results in the generation of HSCs with long-term hematopoietic reconstitution potential.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors would like to acknowledge Diane Domingo for flow cytometry assistance, Kang Zhang and Wei-Hong Yang for excellent assistance with mice studies, and Dr. Viale from the Genomics Core Facility of the Memorial Sloan-Kettering Institute for microarray analyses. J.J.S. was supported by a grant from the EMBO (ALTF-412-2001). M.A.S.M. was supported by P01 CA 59350, R01 HL 61401, and Leukemia and Lymphoma Society SCOR grants.

Jan Jacob Schuringa and Kaida Wu contributed equally to this study.


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 Introduction
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 Discussion
 References
 

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Received February 19, 2004; accepted for publication July 12, 2004.



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