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a Whitehead Institute for Biomedical Research, Cambridge, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Division of Pediatric Hematology/Oncology, The Children’s Hospital and Dana Farber Cancer Institute, Boston, Massachusetts, USA;
b Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, United Kingdom;
c Technion University, Rambam Medical Center, Haifa, Israel
Key Words. Human embryonic stem cells • Leukemia inhibitory factor • STAT3 • Self-renewal
Correspondence: George Q. Daley, M.D., Ph.D., Children’s Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. Telephone: 617-919-2013; Fax: 617-730-0222; e-mail: george.daley{at}childrens.harvard.edu
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MATERIALS AND METHODS |
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Reverse Transcriptase Polymerase Chain Reaction Analysis
RNA was prepared using RNA STAT60 (Tel-test) according to the manufacturer’s instructions. RNA (1.5 µg) was reverse transcribed with superscript reverse transcriptase (RT) (Invitrogen). For polymerase chain reaction (PCR), 1 µl of cDNA was used in a 25-µl final volume using 1.25 units of Taq polymerase (Promega, Madison, WI) with the following primers: gp130 forward: 5'-ggagtgctgttctgctttaa-3', gp130 reverse: 5'-actgtgtaccacggtagaat-3', LIFR forward: 5'-ccta acagatggtggagtg-3', LIFR reverse: 5'-gctgatcgagtttccagaac-3', actin forward: 5'-tggcaccacaccttctacaatgagc-3', actin reverse: 5'-gcacagcttctccttaatgtcacgc-3', Rex1 forward: 5'-cagatcctaaacagctcgcagaat-3', Rex1 reverse: 5'-gcgtacgcaaat-taaagtccaga-3', Nanog forward: 5'-actaacatgagtgtggatcc-3', Nanog reverse: 5'-tcatcttcacacgtcttcag-3'. After 25 cycles,10µl of PCR product was separated on 1.5% agarose gel.
Immunoblot
hESCs or mES cells were plated on Matrigel or 0.2% gelatin, respectively, on 6-well plates (1 x 106 cells per well). One day later, the cells were washed with phosphate-buffered saline (PBS) and cultured with medium containing 0.1% serum replacement without cytokines. The next day, the cells were stimulated with 0.01 µg/ml murine LIF (mLIF) or human LIF (hLIF) (Chemicon, Temecula, CA) for 10, 20, 30, or 60 minutes, washed with PBS, and harvested with 0.25% trypsin-1 mM EDTA. Protein extracts were prepared by resuspending the cells in lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.4, 10 mM NaF, 1 mM EDTA, 1 mM ZnCl2, 1 mM MgCl2, 1% Non-idet P-40 [NP-40], 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM Na3VO4), and proteins were resolved on a 10% SDS polyacrylamide gel. The antibodies used were phospho STAT3 (Tyrosine 705), STAT3 antibody (Cell Signaling, Beverly, MA), phospho STAT3 (serine 727) antibody (Biosource, Camarillo, CA), oct 4 (BD Transduction Laboratories), Jak-2 (Upstate, Lake Placid, NY), and HDJ-2 (Lab Vision Corporation, Fremont, CA).
Assay for Differentiation
hESCs were plated at 1 x 105 on Matrigel or 0.2% gelatin and grown in the presence of MEF-CM, hLIF (10 ng/ml), or medium without hLIF. Expression of TRA-1-60 [13], a specific marker of undifferentiated hESCs, was checked after 8 and 16 days.
Surface-Antigen Expression
For antibody staining, hESCs were dissociated with Trypsin-EDTA. Cells were then stained with the primary antibody TRA-1-60 (provided by Dr. Peter Andrews). Phycoerythrin-conjugated goat anti-mouse IgM was used as secondary antibody. Samples were run through a FACScan cytometer (Becton, Dickinson, Franklin Lakes, NJ).
Subcellular Fractionation
Nuclear and cytoplasmic fractions were prepared as previously described [14]. Briefly, cells were harvested in CE buffer (10 mM HEPES [pH 7.6], 60 mM potassium chloride, 1 mM EDTA, 1 mM dithiothreitol) and supplemented with 0.275% NP-40 and protease inhibitors. After centrifugation at 2,000 rpm for 5 minutes at 4°C, the supernatant (cytoplasmic fraction) was stored on ice and the nuclear pellet was washed twice with CE buffer, resuspended in 60 µl of norepinephrine buffer (20 mM Tris [pH 8.0], 420 mM NaCl, 1.5 mM MCl2, 0.2 mM EDTA, 25% glycerol), and supplemented with protease inhibitors. After 10 minutes on ice, both fractions were centrifuged at 13,000 rpm for 10 minutes at 4°C, and supernatants were transferred to fresh tubes.
Retroviral Transduction of hES with a Constitutively Active Form of STAT3
Murine STAT3C cDNA (provided by Daniel C. Link) was cloned as an EcoR1 fragment into the MSCViresGFP (MIG) retroviral vector [15]. Retroviruses were produced by transient cotransfection of 293T cells with the following three plasmids: the viral vector MIG-STAT3C or MIG-empty, the gag-pol genes encoding retroviral packaging plasmid pUMVC3, and a plasmid encoding the vesicular stromatitus virus G protein [16]. Viruses containing supernatants were collected 48 hours after transfection and concentrated by ultracentrifugation. The mES cells and hESCs were transduced with retroviruses by a single-round infection (for 24 hours) at a multiplicity of infection (MOI) of 1, 10, and 50.
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RESULTS |
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). In contrast, when WA09 cells were cultured on gelatin or Matrigel supplemented with hLIF, marked downregulation of TRA-1-60 expression was seen after 7 days and was almost complete by 15 days (Fig. 1A). The same pattern of TRA-1-60 downregulation was found for UC06 (HSF-6) cells grown under comparable conditions (data not shown). Downregulation of TRA-1-60 expression in both hESC lines correlated with overt signs of morphological differentiation (not shown). We counted the number of cells for each condition (Fig. 1B) and showed that hESCs grown on Matrigel with MEF-CM expand in culture whereas cells grown on Matrigel and gelatin (with or without LIF) stopped proliferating after 3 to 5 days and assumed a differentiated morphology. We also assayed by RT-PCR the expression of two markers of ES cell pluripotency, Nanog and Rex1 [17–19]. Both Nanog and Rex-1 are expressed at high levels in WA09 and UC06 cells grown for 7 days on Matrigel and MEF-CM (Fig. 1C). Corroborating the downregulation of TRA-1-60 expression and morphologic differentiation, Nanog and Rex-1 expression likewise decreased when the cells were cultured on Matrigel alone, with or without hLIF. Unlike WA09 cells, UC06 still expressed low levels of Rex-1 after 7 days with or without hLIF. This difference is likely attributable to the lower growth rate of UC06 compared with WA09 and therefore slower kinetics of differentiation. These data confirmed that hLIF is not sufficient to maintain hES in the pluripotent, undifferentiated state.
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). No amplification was obtained from MEF cDNA, which confirms that the primers were specific for the human sequence. As positive controls, cDNA from K562 and TF1, two human cell lines known to express the LIF receptor, were amplified. Furthermore, expression of gp130 protein was confirmed by immunoblotting WA09 cells, mES, and MEFs with an antibody that recognizes both mouse and human gp130 (Fig. 2C). These data indicate that the components of the LIF receptor complex are expressed in hESCs.
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). The signal can be detected after 10 minutes, reaches a peak at 20 minutes, and decreases after 30 minutes, in concordance with previous reports [22, 23]. In contrast, no STAT3 Y705 phosphorylation was observed in hESCs in response to mLIF, although STAT3 was expressed (Fig. 3A). We repeated the same experiment with hLIF, and phosphorylation of STAT3 on Y705 was detected in both mES and hESCs (Fig. 3B). These results show that hLIF is able to induce STAT3 phosphorylation in hESCs, whereas mLIF fails to activate hLIF receptor signaling.
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). In hESCs, we could detect a basal level of S727 phosphorylation in the absence of LIF and a slight increase of S727 phosphorylation after addition of hLIF Fig. 3C). Taken together, these results suggest that the transcription factor STAT3 can be phosphorylated at both Y705 and S727 by LIF receptor occupancy in hESCs.
Activation of STAT3 by MEF-CM
Because MEF-conditioned medium supplemented with FGF (4 ng/ml) can sustain hESC self-renewal on Matrigel, we asked whether this condition was accompanied by STAT3 phosphorylation. After 12 hours on Matrigel with 0.1% serum replacement, hESCs were stimulated with MEF-CM + bFGF (4 ng/ml) for 15, 30, and 60 minutes. Total protein extracts were prepared from these cells, and STAT3 phosphorylation was assessed by immunoblotting with antibodies directed against phosphotyrosine 705 and phosphoserine 727. The combination of MEF-CM + bFGF did not induce Y705 STAT3 phosphorylation in hESCs, whereas phosphorylation is induced by hLIF (Fig. 4). In contrast, S727 STAT3 phosphorylation is modestly increased after addition of MEF-CM + bFGF (Fig. 4). These data demonstrate that conditions critical for maintaining hESCs in an undifferentiated state (MEF-CM + bFGF) are not associated with STAT3 phosphorylation on the critical residue Y705.
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). Moreover, using an antibody against phosphotyrosine 705, we confirmed that the phosphorylated form of STAT3 is found exclusively in the nucleus (Fig. 5A). Effective subcellular fractionation was verified by the detection of the transcription factor Oct-4 protein within the nuclear fractions and the cytoplasmic Jak-2 or HDJ-2 proteins within the cytosolic fractions (Fig. 5A). We determined the localization of STAT3 in undifferentiated mES cells maintained in log-phase growth in the presence of LIF and undifferentiated hESCs grown on Matrigel with MEF-CM. Using an antibody to detect total protein, STAT3 was found predominantly in the cytoplasm of mES cells; however, using the more specific antibody directed against phosphotyrosine 705, the modification critical for nuclear translocation [21], we indeed detected phosphorylated STAT3 in the nuclear fraction (Fig. 5B). In contrast, phosphorylated STAT3 was not found in the nuclear extracts from hESCs cultured under conditions that maintain self-renewal. Taken together, these results indicate that nuclear localization of STAT3 is not associated with conditions that maintain hESCs self-renewal and apparently is not required for maintenance of hESCs in the undifferentiated state.
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), the percentage of WA09 cells infected with MIG-STAT3C decreased dramatically from 48% at day 2 to 4% at day 15. We attempted to isolate the low numbers of GFP+ cells by flow cytometry to select for clones that maintain STAT3C expression and self-renewal. However, these attempts failed repeatedly, because growth of STAT3C-expressing hESCs apparently could not be sustained. The level of GFP+ cells infected with MIG-empty remained constant for both mES cells and hESCs. We likewise attempted to express a conditionally activated fusion protein of STAT3 and the estrogen receptor that had been shown previously to support the self-renewal of mES cells [10] but again failed to obtain stable lines of hESCs that remained undifferentiated. Our experience suggests that constitutive activation of STAT3 is not sufficient to maintain hESCs in the undifferentiated state and may be associated with enhanced differentiation or apoptosis.
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DISCUSSION |
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Why hES and mouse ES cells should differ in their mechanisms of self-renewal remains an interesting question. One principal function of LIF in murine development is to enable embryonic diapause [26], the temporary arrest of blastocyst development in lactating female mice that affords optimal timing of multiple and repetitive pregnancies in this highly fecund species. LIF acts as an antidifferentiation factor for cells of the inner cell mass, which can then be propagated as ES cells in what is arguably a fortuitous artifact of cell culture. Because human embryos are not susceptible to diapause, it is perhaps not surprising that blastocyst-derived ES cell lines from the human fail to respond similarly to human LIF, which nonetheless plays critical functions in the hematopoietic, reproductive, endocrine, and central nervous systems [27]. Although by nomenclature, hESCs and mouse ES cells represent a comparable in vitro facsimile of the inner cell mass, the differential response to LIF together with the fact that hESCs but not mouse ES cells show trophoblastic potential [28, 29] argue that hESCs may derive from a more developmentally primitive embryonic cell type that responds to different soluble and cell-associated factors. Because conditioned media from several murine and human cells can indeed sustain hESCs in an undifferentiated state, important self-renewal or antidifferentiation factors remain to be identified.
Our results confirm previous observations about the lack of cross-reactivity of murine and human forms of LIF for the LIF receptor [30]. Indeed, despite high sequence homology between hLIF and mouse LIF (78% amino acid identity) and between the human and mouse LIF receptors (76% identity), mLIF shows a species-restricted binding activity. In contrast, hLIF binds both mouse LIF and hLIF receptor and even shows a higher affinity to the mouse receptor than does mouse LIF itself [31]. In response to LIF cytokine stimulation, the LIF receptor common signaling subunit gp130 activates janus kinases to phosphorylate the transcription factor STAT3 on tyrosine residue 705. Y705 phosphorylation induces STAT3 dimerization, STAT3 translocation to the nucleus, and subsequent DNA binding [21]. The transcriptional activity of STAT3 is also regulated through phosphorylation of serine 727, because an alanine-727 mutant shows reduced transcription factor activity [24]. Both sites must be phosphorylated to achieve the full transcriptional activity of STAT3. In this report, we demonstrated that hLIF could stimulate phosphorylation of both STAT3 Y705 and S727 in hESCs. We also demonstrated that STAT3 translocates to the nucleus after addition of hLIF, and we have detected activation of STAT3 target genes after LIF stimulation (not shown). However, despite apparent functional activation of the STAT3 pathway, hESCs will differentiate despite the presence of hLIF. These results suggest that STAT3 activation is not sufficient to maintain hESCs in an undifferentiated state. This conclusion is additionally strengthened by our failure to isolate self-renewing hESC lines after transduction with either a conditional or a constitutive allele of STAT3. In using a conditionally active form of STAT3 to demonstrate the sufficiency of STAT3 signaling, Matsuda et al. [10] reported that there was a threshold of STAT3 activity required to maintain self-renewal of mES cells, and thus one might argue that hLIF does not achieve the required threshold of STAT3 activation in hESCs. However, we think this is unlikely, because even with methods that yield high gene transduction efficiency, our attempts to express a constitutively activated form of STAT3 in hESCs failed. Indeed, our collective experience suggests increased differentiation or induction of apoptosis in STAT3-expressing hESCs. Finally, we investigated the status of STAT3 activation in hESCs grown on Matrigel with MEF-CM and bFGF, conditions known to maintain these cells in a pristine and undifferentiated state. We found that STAT3 was not phosphorylated on Y705 and could detect only a modest modification at S727. Because Y705 phosphorylation provides the essential prerequisite for dimerization, nuclear translocation, and biological activity, these results suggest that STAT3 is not activated in hESCs cultured under maintenance conditions. Corroborating these observations, we showed that STAT3 was not found in the nucleus of hESCs grown in maintenance media, whereas it is transiently localized in the nucleus of mES cells cultured with LIF.
The work presented here documents the lack of participation of the LIF-STAT3 signaling pathway in the maintenance mechanisms of self-renewal of hESCs. Extensive efforts are underway to unveil the pathways and factors required for propagating ES cells without differentiation, because such agents will be profoundly useful for enhanced ES cell culture.
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ACKNOWLEDGMENTS |
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REFERENCES |
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L. Hyslop, M. Stojkovic, |
