Enforced Activation of STAT5A Facilitates the Generation of Embryonic Stem–Deriv - 《stemcells》2004-2005文献干细胞之家



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a Laboratory of Developmental Hematopoiesis, Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA;' N+ H! u8 ^  A2 ~  M0 D1 J
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b Department of Experimental and Clinical Medicine, University of Catanzaro "Magna Graecia," Catanzaro, Italy- x8 g$ o  l& |) H& T; S& t' U
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Key Words. Definitive hematopoiesis ? Embryonic stem cells ? STAT5A ? Hematopoietic stem cells ? Self-renewal
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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@ski.mskcc.org

ABSTRACT

During mouse embryogenesis, hematopoiesis begins with the generation of primitive nucleated erythroid cells in the yolk sac (YS) beginning at 7.5 dpc . 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 . Hematopoietic cells from the AGM region and YS colonize the fetal liver and ultimately the adult bone marrow . 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 . Hematopoietic cells derived from embryonic stem (ES) cells, like early YS progenitors, are ineffective in reconstituting hematopoiesis in irradiated recipients . 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  or when injected into sublethally myeloablated newborn mice .

Signal transducer and activator of transcription (STAT) 5 belongs to a family of transcription factors that fulfill key functions in hematopoiesis . 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) . Stat5a–/–5b–/– knockout mice are characterized by fetal anemia and increased apoptosis of fetal liver erythroid progenitors . 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 . In a similar study, Snow et al.  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 . 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

Overexpression of Constitutively Active STAT5 Facilitates Hematopoietic Differentiation of ES Cells0 w1 E# H; b" T% ~" b6 }
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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) . 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. 1A). Clones were also tested for STAT5 expression by Western blot analysis (Fig. 1B). 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. 1C). 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. 1D). As a control, cells were also stimulated with IL-3, which resulted in a 4.5-fold induction of the reporter.' y: J1 t: c  m2 a6 ~+ D/ B

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. 2A). 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 2A. Over-expression of constitutively active STAT5A(1*6) was verified by Western blotting. As depicted in Figure 2B, 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. 2B).  \  ^# {$ ?: T3 ?. ]

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.# l# T0 N  g9 W$ A" S8 @

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–G). 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, D).
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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. 2E). 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. 2E). 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. 2F).

Extensive immunophenotypical analysis revealed that expression of CD31 and Flk-1 was significantly elevated in STAT5A(1*6) cells at day 5 (Table 1 and Fig. 3). 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 1 and Fig. 3). 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. 4). 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.1 k! W9 f  X* h1 {

Table 1. Phenotypical analysis of embryonic stem–derived hematopoietic cells cocultured on OP9' k: k$ P( J& J
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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.

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.
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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. 2C), 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. 2D). 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, F). 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 compared with 24.2% for controls; Table 1). In contrast, granulocyte/macrophage differentiation was strongly reduced in the presence of constitutively active STAT5A(1*6) (Table 1). 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. 2G). 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) . 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 1), whereas the adherent population at days 25 and 35 consisted of immature cells that were mostly c-Kit  and CD45  (Table 1, Fig. 2D). 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.. d5 f9 }8 Q( k% l
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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. 1B, 2B), 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. 5A). As indicated in Figure 5B, 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. 5C). 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. 5D). At week 7, STAT5A(1*6) mice were euthanized for analysis and secondary engraftment studies. As indicated in Fig. 5E, 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. 5E). 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. 5F) and neomycin-phosphotransferase II–positive cells (Fig. 3F) 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.' R  E% H" ]- j1 ]3 _

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.: \  S% j2 z; {8 @3 R

Molecular Analysis of Differential Gene Expression Induced by STAT5A(1*6) in ES-Derived Hematopoietic Cells5 L  I0 ?0 }" z0 }8 r; e
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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. 4A). 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 ?-H1 globin was strongly expressed at days 5 and 7 in both control and STAT5A(1*6) cells, whereas the definitive marker ?-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 ?-actin as well as neomycin-phosphotransferase II.3 z! S9 u; L7 L: _  H1 ]) w
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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, ?1-integrin, CXCR4, CD44, and oncostatin M receptor (OSM-R), amongst several other genes (Table 2). 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.% V" M6 K2 |3 e/ K* @
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Table 2. Differential gene expression in ES(R1)-derived hematopoietic cells on OP9 day 5, STAT5A(1*6) versus controls6 p% u) b% a* h
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DISCUSSION" o( m' d8 p3 G; Q. v1 H# B

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.2 w# b7 \; B- O: g3 {

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

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