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a The Islet Research Laboratory, Whittier Institute for Diabetes, Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, California, USA;4 ]" _/ X: X/ d ~5 i d$ R
8 A3 x6 S% y8 m- X) |% b: sb UCSF Stem Cell Research, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California at San Francisco, San Francisco, California, USA;/ Q3 ~& ~. r5 s, c0 H0 a. E
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c Department of Biochemistry, Christian-Albrechts-Universit?t zu Kiel, Kiel, Germany. _& h! A8 _' x1 a+ N4 t* p
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Key Words. Human embryonic stem cells ? Pluripotency ? STAT3 ? gp130 ? LIF
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& m4 t/ _* r: JCorrespondence: Alberto Hayek, M.D., Whittier Institute for Diabetes, Department of Pediatrics, University of California San Diego, 9894 Genesee Ave, La Jolla, California 92037, USA. Telephone: 858-622-7298; Fax: 858-558-3495; e-mail: ahayek@ucsd.edu1 a$ K9 b4 h0 i8 R. o
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ABSTRACT# z1 l. C' F& ]
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Maintenance of the undifferentiated state and pluripotency in mouse embryonic stem (mES) cells requires the presence of mouse embryonic fibroblast (mEF) feeder layers or leukemia inhibitory factor (LIF). LIF is a member of the interleukin-6 (IL-6) family of cytokines, which also includes Oncostatin M, ciliary neurotropic factor, IL-6, IL-11, cardiotrophin-1, and cardiotrophin-like cytokine (CLC) . LIF is known to bind to its transmembrane receptor, LIFR, which heterodimerizes with the signal-transducing receptor gp130. The pluripotency of mES depends on the intracellular signaling events that follow, including phosphorylation by the Janus family of tyrosine kinases (JAK), which leads to activation of the signal transducer protein STAT3 . The combination of IL-6 and soluble IL-6 receptor also interacts with and activates a homodimer of gp130 and has been used to maintain mES cells without involvement of LIFR . STAT3 activation is sufficient to maintain mES in the undifferentiated state, and inhibition of the gp130-triggered mitogen-activated protein (MAP) kinase (Erk1/2) pathway enhanced this effect . Activation of Erk1/2, in turn, leads to the loss of pluripotency and the onset of differentiation, such that a balance between these opposing signaling pathways determines the fate of the cells . Recently it was demonstrated that gp130 signaling has a physiologic role in the process of diapause that occurs naturally in lactating female mice. Mouse embryos arrest at the late blastocyst stage when implantation is prevented and gp130–/–embryos are unable to resume development after the end of diapause. The responsiveness of embryonic stem cells to gp130 signaling most likely has its origin in this reaction .' Y |6 O- N2 }& Y. F
% k3 S3 j+ {. h0 ^2 O' D" QHuman embryonic stem (hES) cells require mEFs to maintain pluripotency. Neither LIF nor IL-6 secreted by the mEFs is responsible for this effect since neither murine LIF nor murine IL-6 acts on human receptors . Additionally, human LIF was not sufficient to maintain the hES cells in the undifferentiated state . One possible explanation of the inability of LIF to maintain pluripotency might relate to deficient cell surface expression of the appropriate cytokine receptors . While gp130 is known to be present on all cells in the body, the LIFR protein is not ubiquitously expressed .7 p7 \' H. |8 y$ ?3 x( x* \
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The study reported here was undertaken to explore the possibility that "stemness" could be maintained in hES cells using the designer cytokine hyper-IL-6 (a complex of IL-6 and its soluble receptor sIL-6R), which is a potent activator of gp130 that does not require the presence of LIFR or IL-6R to activate signal transduction . Although hyper-IL-6 has recently been shown to block differentiation in mES cells , we demonstrate that this is not the case for hES cells. LIFR, IL-6R, and gp130 were expressed on hES cells and, when stimulated with either LIF or IL-6, activated STAT3. However, augmented levels of gp130-stimulated STAT3 activation failed to maintain hES cells in the undifferentiated state, with the progressive loss of TRA-1-60, Nanog, and Oct-4 expression. Culture of hES cells on mEF feeder layers or in the presence of conditioned media from mEF cells also failed to induce STAT3 phosphorylation, although high levels of TRA-1-60, Nanog, and Oct-4 expression were observed. This is the first evidence demonstrating that gp130 can activate STAT3 in hES cells and that this pathway is not activated in the undifferentiated state. Taken together, these results suggest that the maintenance of hES cell "stemness" is STAT3 independent.# n: H7 p& G) l. u0 G! o3 D
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MATERIALS AND METHODS& Z3 b- a. D }0 c. I {( W- Z g3 q
. {2 j' H: n' m) }HSF6 Cells Differentiate in the Presence of the IL-6 Family of Cytokines8 W B3 G* @$ h' k
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To remain in an undifferentiated state, hES cells require either a feeder layer of mouse fibroblasts or plating on laminin and CM from mouse fibroblasts . The morphology of the hES cell line HSF6 grown on feeder layers (Fig. 1A) or in CM on laminin-coated plates (Fig. 1B) was similar. Under both conditions, cells grew as undifferentiated colonies that stained uniformly for the stem cell markers TRA-1-60 (Fig. 1C–E) and Oct-4 (Fig. 1E), with rare cells staining for nestin, a marker of differentiation, at the edge of the colonies (Fig. 1C, D). In contrast, cells grown on laminin in DSR medium with or without hLIF (Fig. 1F), IL-6 (data not shown), or hyper-IL-6 (Fig. 1G, H) differentiated rapidly, as determined by the loss of TRA-1-60 and the appearance of nestin-positive cells. The rate of differentiation was similar at all concentrations of hyper-IL-6 (data not shown). After 24–48 hours, cytokine-treated cells increased in volume, flattened, and migrated from the colonies. After 1 week, the stem cell colonies had almost disappeared, leaving behind spreading monolayers of single cells. Immunofluorescence of these cells indicated that most cells were nestin positive (Fig. 1G), while TRA-1-60 (Fig. 1F, G) and Oct-4 staining was much reduced (Fig. 1F). After 2 weeks, TRA-1-60 expression was not detected (Fig. 1H). No difference was observed between cells grown with or without the cytokines, either morphologically or in the expression of differentiation and stem cell markers (data not shown).
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Figure 1. Differentiation of human embryonic stem (hES) cells in the presence of the interleukin-6 (IL-6) family of cytokines. Morphology of undifferentiated HSF6 cells cultured on mouse embryonic fibroblast (mEF) feeder layers (A) or on laminin in the presence of conditioned media (CM) from feeder layers (B) was similar when viewed under phase contrast microscopy. Immunohistochemical analysis of undifferentiated HSF6 cells cultured on mEF feeder layers (C) or on laminin in the presence of CM from feeder layers (D) was also similar. Uniform staining for the human stem cell marker TRA-1-60 was observed, with only rare cells at the edges of the colonies staining for nestin, a marker for differentiating cells. Nuclear staining for the stem cell marker Oct-4 was also observed in undifferentiated cells on feeder layers (not shown) and on laminin with CM (E). After 1 week of culture in the absence of CM and feeder layers and in the presence of either human leukemia inhibitory factor (hLIF) (F) or hyper-IL-6 (G), staining for TRA-1-60 was rarely seen (F, G), and Oct-4 staining was much reduced (G), while differentiating cells stained for nestin (G). After 2 weeks culture with hyper-IL-6 in the absence of CM, no stem cell markers were observed and only nestin-positive cells could be seen (H). Magnification, bar = 25 μMA–D and 12.5 μM in E–F.
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# N7 w( k1 p3 u: PWe next wanted to examine the ability of hyper-IL-6 to prevent hES cell differentiation. Robust expression of the stem cell markers Oct-4 and Nanog was observed in undifferentiated cells grown on laminin with CM (Fig. 2, lane 1). Expression of these markers was decreased in hES cells grown on laminin without CM for 1 week (Fig. 2, lane 2) and absent after 2 weeks (Fig. 2, lane 4). Treatment of the cells with hyper-IL-6 did not prevent differentiation over the same 2-week time period (Fig. 2, lanes 3 and 5).
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2 ~3 W" M0 ?1 q1 D, DFigure 2. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) of hES cells for Oct-4 and Nanog cultured in the presence of conditioned media (CM) (lane 1), without CM (lanes 2, 4), or hyper-IL-6 (lanes 3, 5). Cells were maintained in CM for 2 weeks in parallel with cells grown without CM or in hyper-IL-6. Samples grown without CM or treated with hyper-IL-6 cells were collected at 1 week (lanes 2, 3) and at 2 weeks (lanes 4, 5). Both Nanog and Oct-4 expression decreased after 1 week, both in the absence of CM and in the presence of hyper-IL-6, and was absent at 2 weeks (lanes 4, 5). Abbreviation: RT, reverse transcriptase.
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4 ~; Z* d+ N) Y6 j6 AHSF6 Cells Express the Receptors for LIF, gp130, and IL-6
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' L! g+ ]. H5 vMouse ES cells require LIF to maintain an undifferentiated state. Binding of the cytokine to the LIFR results in heterodimerization of LIFR with the signaling receptor gp130 and activation of the JAK/STAT pathway. The self-renewal program has been attributed to STAT3-mediated transcription . In contrast to mES cells, LIF does not maintain hES cells in an undifferentiated state, and this has been attributed to the lack of the LIFR on hES cells . RT-PCR experiments showed that a high level of LIFR mRNA was expressed in both the hES and mES cells (Fig. 3A and B respectively), suggesting that LIFR alone does not explain the difference between mouse and human ES cells.
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Figure 3. Expression of leukemia inhibitory factor receptor (LIFR), gp130, and IL-6R (gp80). (A, B): RT-PCR was performaed on a master-mix of the same cDNA from either hES or mES cells. LIFR and gp130 were expressed at high levels in the mouse and human cells. In addition, the relative level of mouse gp130 compared with mouse LIFR was the same as the relative level of human gp130 compared with human LIFR. IL-6 (gp80) was present at lower levels than LIFR and gp130 expression in mouse and human cells. (C): Western blot for gp130 in the membrane fractions of both mES and hES cells. Abbreviations: cyt, cytoplasmic fraction; Mb, membrane fraction; /–, reverse transcriptase.
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9 D9 B2 Y( {. }LIFR is not the only receptor to dimerize with gp130. The IL-6 receptor (gp80) also binds gp130 to signal to the JAK/STAT pathway . Although gp80 was expressed in both hES and mES cells, it was at a lower relative level than LIFR and gp130 (Fig. 3A, B).$ t" V0 \' l1 v: w, b. T5 V* {
" c/ w; |2 W0 O8 N) JWe next wanted to determine whether hES cells expressed gp130 mRNA. RT-PCR analysis (Fig. 3A, B) and western blotting (Fig. 3C) indicate the presence of the receptor, suggesting that the signaling machinery required for the activation of the JAK/STAT pathway are present in both the human and mouse stem cells.2 i0 y: Z+ ^( y- G3 I* H
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Further, because RT-PCR for gp130, LIFR, and gp80 was performed on a master-mix of the same cDNA from either the human or mouse cells, it is possible to compare the relative levels of human gp130 to human LIFR, as it is also possible to compare the relative levels of mouse gp130 to mouse LIFR. This is an important observation, since efficient activation of STAT3 requires dimerization of gp130 and LIFR. Mouse gp130 and LIFR were expressed at the same relative level, which is sufficient to activate STAT3 (Fig. 4). Human gp130 was also expressed at a similar level to human LIFR. Hence, the ratio of mouse gp130 to LIFR required for STAT3 activation was the same as the ratio of human gp130 to LIFR.
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9 Z8 P( y# f7 z& NFigure 4. Chronic and acute stimulation of gp130 signal transduction in mES and hES cells. pSTAT3 (Tyr705), pErk1/2 (Thr202/Tyr204), pAkt (Thr308), and ?-actin were detected on the same blot that contained positive and negative controls for pSTAT3 only. Acute treatment was performed on undifferentiated ES cells for 15 minutes and included no treatment: ES cell media (mES: DMEM FBS; hES: DSR FGF); or either LIF, IL-6, or hyper-IL-6 supplemented ES cell media. Chronic treatment was performed for 24 hours and included the following: Undiff, undifferentiated mouse and human cells stimulated with ES cell media supplemented with LIF or CM, respectively; Diff, mouse and human cells stimulated with their respective ES cell media. Western blots are representative of three separate experiments done in duplicate.
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LIF and IL-6 Induce STAT3 Activation in HSF6 Cells) \8 C& Q, F4 k" _4 m! @; y; S
$ A# ]: \% U/ k0 V2 b" N& [7 m' d7 KThe ability of the IL-6 family of receptors to activate JAK/STAT signal transduction in response to their ligands was investigated in the hES cells using the mES cells as a positive control (Fig. 4). Consistent with the role of STAT3-mediated transcriptional activation of "self-renewal," undifferentiated mES cells were characterized by a robust level of phosphorylated STAT3, which was lost upon differentiation. Activation was observed 15 minutes after acute stimulation with LIF and was sustained for 24 hours. Hyper-IL-6 also activated STAT3 in the mES cells. In contrast, phospho-STAT3 was not observed in either undifferentiated or differentiated hES cells. Acute stimulation with CM did not activate STAT3, and phosphorylation was not observed in undifferentiated hES cells grown on feeder layers (data not shown). The lack of activated STAT3 in the hES cells was not due to missing or inactive receptors, since the addition of the hLIF resulted in a robust activation of STAT3. Hyper-IL-6, which served as a positive control, activated STAT3 to a higher level than hLIF, while IL-6 alone induced a lower level of STAT3 phosphorylation. The lower levels may relate to the lower level of IL-6R expression relative to LIFR and gp130. Furthermore, there was a larger relative difference between the expression levels of both mouse LIFR and gp130 compared with IL-6R. This may explain the lack of STAT3 activation in mES cells when stimulated with IL6 alone.+ T7 y5 g& \( o
, |, n& j+ O* V( C6 s5 LThe IL-6 family of receptors also activates other signal transduction pathways such as the MAP kinase Erk1/2 and Akt (Fig. 4). The presence or absence of LIF in the media for either mES (DMEM FBS) or hES (DSR FGF) had no effect on the acute activation of Erk1/2. However, when compared with differentiated cells, undifferentiated cells in the presence of LIF (mES) or CM (hES) maintained a higher level of pErk1/2 expression 24 hours after stimulation. This difference upon differentiation was more apparent in the mES cells and may be a result of the FGF-supplemented hES cell media. Similarly, the level of Akt activation in the presence of LIF over 24 hours was maintained at a higher level in the undifferentiated mES cells than in the differentiated cells. In contrast, Akt phosphorylation remained constant in both undifferentiated and differentiated hES cells, and following treatment with LIF, IL-6, or hyper-IL-6.
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These results are the first demonstration of a functional gp130 signal transduction pathway in hES cells. Furthermore, STAT3 activation via gp130 is not observed in undifferentiated hES, suggesting the self-renewal program is regulated independently of gp130 signal transduction.: e2 q+ f5 L0 m3 V4 C
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DISCUSSION) {. d+ U3 z3 n. N
m$ w/ z! D6 R, O- H8 ?The authors wish to thank Dr. Ulupi Jhala for her input during the preparation of the manuscript and Liora Newfield for her technical assistance. This work was funded by the Larry L. Hilblom Foundation.4 E( `" V1 b1 T5 O" F7 d0 G
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