STAT3 Is Dispensable for Maintenance of Self-Renewal in Nonhuman Primate Embryon



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江边孤钓

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发表于 2009-3-5 10:37 |显示全部帖子

a Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences,/ L: V: a! A0 W8 F

b Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, and
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c Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
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Key Words. LIF ? STAT3 ? Phosphorylation ? Embryonic stem cells ? Primate ? Self-renewal ? Pluripotency. o8 l% l  \# n( z+ P' u

Correspondence: Hirofumi Suemori, Ph.D., Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: 81-75-751-3821; Fax: 81-75-751-3890; e-mail: hsuemori@frontier.kyoto-u.ac.jp
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ABSTRACT

Embryonic stem (ES) cells are pluripotent cells that can maintain unlimited and undifferentiated proliferation in vitro . ES cell lines were first established from the inner cell mass of mouse blastocysts and have been shown to retain the ability to differentiate into most specialized cells of the three embryonic germ layers in vitro and in vivo. Because of this developmental potential, ES cells have been used to study cell differentiation during embryogenesis. Moreover, ES cells injected into blastocysts can contribute to all tissues, including the germ cells in the resulting chimeric mice. Thus, murine ES cells have been used to introduce genetic modifications in the production of gene-disrupted mice. Human ES cell lines were established in 1998 and have been shown to have differentiation potency similar to those from mice . Their potential for multilineage differentiation could enable their use in generating an unlimited supply of various cell types and tissues for replacement therapy in human degenerative diseases . Despite the importance of ES cells in regenerative medicine, the molecular mechanisms involved in self-renewal and pluripotency are not completely understood. Furthermore, it is more difficult to maintain human ES cells stably in an undifferentiated state than murine ES cells, making them difficult to exploit for therapeutic use.

In mouse ES cells, several lines of evidence indicate that the leukemia inhibitory factor (LIF)– and interleukin-6 (IL-6)–related cytokines that signal through a common glycoprotein 130 (gp130) receptor play essential roles in maintaining self-renewal . LIF directs ES cell self-renewal through binding a complex comprised of the LIF receptor and gp130, allowing recruitment of JAK kinases that permit the activation of the signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) pathways. The gp130-mediated activation of STAT3 alone, and not MAPK, is sufficient to sustain self-renewal and retard differentiation of murine ES cells . In contrast, human ES cells lose pluripotency and rapidly differentiate under feeder layer–free culture conditions, even in the presence of LIF , suggesting that the LIF signaling pathway may not be fully functional in human ES cells. We have recently established cynomolgus monkey ES cell lines from blastocysts that can be propagated indefinitely in an undifferentiated state . It is intriguing that cynomolgus monkey ES cells show remarkably similar characteristics to human ES cells. Like human ES cells and those from other nonhuman primates , undifferentiated propagation of cynomolgus ES cells is dependent on the presence of a feeder layer, and LIF alone cannot support self-renewal. Thus, although important mechanisms controlling self-renewal in murine ES cells would be anticipated to be evolutionarily conserved in their primate homologues, these two cell types seem to differ in their requirement of LIF for self-renewal. Clarification of this distinction, however, requires closer examination of the downstream signals of LIF.8 S' w$ X7 d# }

In the present study, we investigated the contribution of the LIF/gp130/STAT3 pathway to the maintenance of self-renewal in cynomolgus monkey ES cells. Our findings indicate that these cells possess an active, intact LIF/gp130/STAT3 signaling pathway but that activation of STAT3 is not involved in maintenance of self-renewal. Furthermore, we suggest that self-renewal of nonhuman primate ES cells could be sustained via an LIF/gp130/STAT3–independent signaling pathway.

MATERIALS AND METHODS
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LIF-Induced Tyrosine Phosphorylation of STAT3 in Monkey ES Cells2 |$ y. y' K: f
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Although LIF treatment alone can maintain murine ES cells in an undifferentiated state, it cannot do so in primate ES cells, such as those from human and monkey. STAT3 activation induced by LIF is essential for self-renewal of murine ES cells, but the downstream signaling pathway in primate ES cells has not been well examined. It is generally thought that the molecular mechanisms underlying cell growth and differentiation are conserved among mammals; consequently, it is thought that the inability of primate ES cells to respond to LIF results from some downstream obstruction of the LIF/STAT3 signaling pathway. Therefore, we examined whether STAT3 is activated by phosphorylation at specific residues in response to LIF. To examine this, we constructed an expression plasmid for myc-tagged STAT3, which could be distinguished from endogenous STAT3, and transiently transfected it into CMK6 and CMK9 cynomolgus monkey ES cell lines using a lipofection reagent. Exogenous myc-tagged STAT3 was detected in cocultures of either CMK6 or CMK9 cells together with MEF or SL10 feeder cells, but not in cells transfected with empty vector (Mock) or in feeder cells alone (Figs. 1A, 1B, left), indicating that the plasmid for myc-tagged STAT3 was mainly introduced into ES cells. To evaluate whether cynomolgus monkey ES cells can respond to LIF signaling, we analyzed the phosphorylation state of STAT3 in transfected ES cells using a specific antibody that recognizes activated STAT3 that has been phosphorylated at the Tyr-705 residue. Western blotting of total cell extracts revealed that tyrosine phosphorylation of STAT3 was induced by LIF in both feeder cells alone and in ES cell/feeder cell cocultures (Figs. 1A, 1B, left). Immunoprecipitation of these extracts with an anti-myc antibody revealed that exogenous myc-tagged STAT3 expressed in ES cells was phosphorylated at the tyrosine residue in a LIF-dependent manner (Figs. 1A, 1B, right). These results indicate that exogenous STAT3 expressed in cynomolgus monkey ES cells is responsive to LIF signaling." a* P- h+ ~2 n. J7 j0 i/ }9 z5 z

Figure 1. Tyrosine phosphorylation of overexpressed STAT3 induced by LIF and IL-6/sIL-6R in CMK6 (A and C) and CMK9 (B and D) cells. Embryonic stem cells cultured on MEFs or SL10 feeder cells were transfected with empty vector (Mock) or myc-tagged STAT3 and then stimulated with LIF or IL-6/sIL-6R for 15 minutes. The cell lysates were immunoprecipitated using anti-myc antibody and then subjected to immunoblotting. Immunoblots of cell lysates and IPs were sequentially probed with antibodies specific for phosphorylated (pY705, pS727), nonphosphorylated, and myc-tagged STAT3. Abbreviations: IL-6, interleukin 6; IP, immunoprecipitate; MEF, mouse embryonic fibroblast; sIL-6R, soluble interleukin 6 receptor.
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It has been reported that STAT3 is also activated by several different cytokines, including the IL-6 family of cytokines, which shares a common gp130 receptor . Furthermore, the self-renewal of murine ES cells is maintained by a combination of IL-6 and sIL-6R . To address whether STAT3 is also activated by IL-6/sIL-6R in cynomolgus monkey ES cells, transfected cells were treated with LIF or IL-6/sIL-6R, and the cell extracts were immunoprecipitated using an anti-myc antibody. Immunoprecipitation of myc-tagged STAT3 demonstrated a prominent increase in tyrosine phosphorylation in response to LIF (Figs. 1C, 1D, right). Likewise, an increase in tyrosine-phosphorylated STAT3 was also detected in cells stimulated with IL-6/sIL-6R (Figs. 1C, 1D, right). In addition to tyrosine phosphorylation, it has been reported that serine phosphorylation of STAT3 is definitely required for full transcriptional activity . Therefore, we analyzed the serine phosphorylation of STAT3 using a specific antibody that recognizes Ser-727–phosphorylated STAT3. As shown in Figures 1C and 1D, left, an increase in the level of Ser-727–phosphorylated STAT3 was observed after both LIF and IL-6/sIL-6R treatment in total cell extracts from ES cell/feeder cell cocultures. In contrast, when the cell extracts were immunoprecipitated using the anti-myc antibody, STAT3 expressed in ES cells was constitutively phosphorylated at Ser-727, independent of LIF and IL-6/sIL-6R treatment (Figs. 1C, 1D, right). Similar results were obtained when myc-tagged STAT3 was expressed in murine ES cells (data not shown). These data indicate that serine phosphorylation of STAT3 in cynomolgus monkey ES cells, distinct from feeder cells, does not significantly change upon the activation of gp130 signaling.4 G8 }. W$ W% b
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To further examine the phosphorylation of endogenous STAT3 in cynomolgus monkey ES cells, we developed a feeder-free culture system to eliminate contamination of MEF proteins. A recent report that undifferentiated human ES cells can be maintained on laminin or Matrigel, an extra-cellular matrix substrate, supplemented with MEF-conditioned media suggests that components of the ECM and soluble factors secreted by MEFs may be important for maintaining undifferentiated ES cells . Therefore, we attempted to culture ES cells on ECM secreted by MEFs. MEFs were detached with an EDTA solution, allowing the matrix proteins to remain firmly attached to the culture dish, and then ES cells were seeded onto these MEF-secreted matrix-coated dishes. As shown in Figure 2A, CMK6 cells cultured on ECM showed a typical undifferentiated morphology, with a high nuclear to cytoplasmic ratio, similar to cells cultured on MEFs. To assess the degree to which these cells were truly undifferentiated, we used immunocytochemical analysis to examine the expression of markers of undifferentiation, such as alkaline phosphatase and Oct-3/4, a key transcription factor for ES cell self-renewal . CMK6 cells cultured on ECM exhibited expression of alkaline phosphatase and nuclear Oct-3/4 at a level comparable to that of cells cultured on MEFs (Fig. 2A), and we could successfully maintain them in the undifferentiated state for limited numbers of passages (at least 2 weeks). In contrast, ES colonies developed a differentiated morphology, with markedly reduced expression of alkaline phosphatase and Oct-3/4, when ES cells were cultured on Matrigel without feeder cells (Fig. 2A). These data suggest that adhesion to the ECM or ECM-trapped soluble factors produced by MEFs is important for ES cell self-renewal. Under these culture conditions, we examined the state of endogenous STAT3 phosphorylation in undifferentiated CMK6 cells with or without LIF. Consistent with the data in Figure 1, tyrosine phosphorylation of endogenous STAT3 was induced by LIF treatment, whereas serine phosphorylation of STAT3 was not (Fig. 2B). Taken together, these results indicate that, in response to LIF, STAT3 in cynomolgus monkey ES cells is phosphorylated at the tyrosine residue that is required for dimerization and nuclear translocation.* A. g6 ?% g' M5 N0 C: G1 ^
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Figure 2. Tyrosine phosphorylation of endogenous STAT3 in CMK6 cells. (A): Expression of stem cell markers. CMK6 cells were cultured on MEFs, ECM, or Matrigel without feeder cells for 3 days and then stained for ALP and Oct-3/4. (B): Tyrosine phosphorylation of endogenous STAT3 induced by LIF. CMK6 cells cultured on ECM without feeder cells for 3 days were treated with or without LIF for 15 minutes, and then cell extracts were subjected to immunoblotting with antibodies against phosphorylated (pY705, pS727) and nonphosphorylated STAT3. Experiments were done in duplicate. Abbreviations:ALP, alkaline phosphatase; ECM, MEF-secreted matrix; MEF, mouse embryonic fibroblast.: ]6 d4 K" q* l# Y2 e
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LIF-Induced and IL-6/sIL-6R–Induced Nuclear Translocation of STAT3 in Monkey ES Cells

In response to cytokines and growth factors, phosphorylated STAT3 dimerizes, translocates to the nucleus, and binds specific DNA promoter sequences . Therefore, we analyzed the changes in subcellular localization of STAT3 in cynomolgus monkey ES cells in response to cytokine treatment. In untreated cells, STAT3 was distributed diffusely in the cytoplasm and the nucleus, whereas after LIF treatment STAT3 was concentrated in the nucleus in both CMK6 and CMK9 cells (Figs. 3A, 3B). Similar nuclear translocation of STAT3 was observed when the cells were stimulated by IL-6/sIL-6R (Fig. 3A). Furthermore, tyrosine-phosphorylated STAT3 was only observed in the nucleus after LIF and IL-6/sIL-6R stimulation (Figs. 3A, 3B). These results indicate that STAT3 in cynomolgus monkey ES cells is phosphorylated and translocates to the nucleus after gp130 activation.* f9 L% V6 W7 a& c; V. w

Figure 3. Nuclear translocation of STAT3 induced by LIF and IL-6/sIL-6R in CMK6 (A) and CMK9 (B) cells. ES cells cultured on feeder cells were treated with or without LIF or IL-6/sIL-6R for 15 minutes, and then cells were fixed and stained with antibodies against phosphorylated (pY705) and nonphosphorylated STAT3. Dotted lines show the boundary between ES cells and feeder cells. Bar = 100 μm. Abbreviations: ES, embryonic stem; IL-6, interleukin 6; sIL-6R, soluble interleukin 6 receptor.- `6 J0 L' y, t3 p, X1 d' Q5 I

STAT3 Is Not Essential for the Maintenance of Self-Renewal in Monkey ES Cells/ {' h* |6 f, F, W/ N" C
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On the basis of the above observations, we reasoned that activation of STAT3 signaling might maintain cynomolgus monkey ES cells in an undifferentiated state, as it does for murine ES cells. When CMK6 cells were cultured on Matrigel-coated dishes without the feeder layer for 3 days, however, they uniformly differentiated, adopting a flattened, epithelial morphology with reduced expression of alkaline phosphatase and Oct-3/4, even in the presence of LIF or IL-6/sIL-6R (Fig. 4). To additionally investigate whether STAT3 can contribute to the self-renewal signaling of monkey ES cells, we attempted to compete against endogenous STAT3 by overexpressing a dominant interfering mutant of STAT3 (STAT3F), in which Tyr-705 was replaced with a phenylalanine residue. High levels of STAT3F expression have been shown to strongly induce differentiation in murine ES cells . We first tested the effect of overexpressed STAT3F on proliferation of undifferentiated murine ES cells. Vectors encoding wild-type STAT3 and STAT3F were transfected into R1 cells, a murine ES cell line, and stable transfectants were recovered by selection in G418 for 7 days. The differentiation state of these cells was assessed by morphology and alkaline phosphatase activity. As shown in Figure 5, more than 80%–95% of colonies expressing either empty vector (Mock) or wild-type STAT3 were undifferentiated. In contrast, cells expressing STAT3F underwent morphological differentiation and exhibited reduced alkaline phosphatase activity, even in the presence of LIF. These data are consistent with previous observations  and confirm that STAT3 is a key factor in the maintenance of self-renewal in murine ES cells.

Figure 4. Differentiation of cynomolgus monkey embryonic stem cells in a feeder cell–free culture. CMK6 cells were cultured on feeder cells or Matrigel-coated dishes in the absence of feeder cells with or without LIF (1,000 U/ml) or IL-6/sIL-6R (200/250 ng/ml). After 3 days of culture, cells were stained for ALP and Oct-3/4. Abbreviations:ALP, alkaline phosphatase; IL-6, interleukin 6; MEF, mouse embryonic fibroblast; sIL-6R, soluble interleukin 6 receptor.
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Figure 5. Differentiation of murine ES cells induced by STAT3F expression. R1 cells were transfected with empty vector (Mock), wild-type STAT3, or STAT3F and cultured with ES medium containing 200 μg/ml G418 for 7 days. The surviving colonies were visualized by ALP staining, and the numbers of undifferentiated or differentiated colonies were scored as a percentage of the number formed in the presence of G418 (right). Experiments were repeated at least three times with similar results. Three colony types were observed as indicated (left): undifferentiated cells (top), partly differentiated cells (middle), and differentiated cells (bottom).Abbreviations:ALP, alkaline phosphatase; ES, embryonic stem.

Next, we exploited the above vectors to determine if STAT3 plays a role in maintaining self-renewal in monkey ES cells. Stable transfectants expressing either the wild-type STAT3 or the dominant-negative mutant STAT3F were established by G418 selection for 10 days, and then colonies were stained with alkaline phosphatase as a marker of undifferentiation. As shown in Figure 6, stable transfectants harboring either an empty vector or wild-type STAT3 formed G418-resistant undifferentiated ES colonies with similar frequencies. Unexpectedly, we were also able to establish undifferentiated ES cells in those colonies overexpressing STAT3F. Even after prolonged culture of greater than 2 months, these cells did not show any morphological signs of differentiation, as was observed for murine ES cells. No differentiated ES colonies were observed in any of three independent experiments.

Figure 6. Formation of stem cell colonies followed by transfection of STAT3 derivatives in cynomolgus monkey ES cells. CMK6 and CKM9 cells cultured on feeder cells were transfected with empty vector (Mock), wild-type STAT3, and STAT3F and cultured with ES medium containing 100 μg/ml G418 for 10 days. The surviving colonies were visualized by alkaline phosphatase staining (left), and the number of colonies was counted (right). No differentiated colonies were observed in three independent experiments. Data are the averages of three independent experiments; bars indicate standard deviations (n = 3). Abbreviation: ES, embryonic stem.* y2 V, s2 t. e3 _0 M& w& j* m

To further clarify these findings, we selected several transfectants expressing comparably high levels of exogenous STAT3, as assessed by immunoblotting, and used at least two independent clones for each transfectant in the following experiments. All of these clones had normal expression of stem cell markers, such as alkaline phosphatase, the cell-surface antigen SSEA-4, and the transcription factor Oct-3/4 (Figs. 7, 8B). To examine whether STAT3F overexpression competes with endogenous STAT3 function, LIF-induced STAT3 activation was determined by immunoblotting. In wild-type STAT3-and STAT3F-overexpressing cells, expression of the respective STAT3 proteins was approximately 10-fold higher than that of control cells transfected with empty vector (Mock) (Fig. 8A). In control cells and wild-type STAT3 clones, LIF induced tyrosine phoshorylation of STAT3, whereas the STAT3F clone had diminished levels of LIF-induced STAT3 phosphorylation (Fig. 8A). In contrast, a comparable level of Oct-3/4 expression was observed in all transformants. Furthermore, immunofluorescent visualization of phospho-STAT3 showed that nuclear translocation of phospho-STAT3 was induced by LIF in cells expressing either empty vector or wild-type STAT3 but was completely abolished in the STAT3F clones (Fig. 8B).) H* {" [- B5 u6 Z: @2 q
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Figure 7. Expression of stem cell markers in STAT3 transfectants. Recovered stable embryonic stem clones expressing empty vector (Mock), wild-type STAT3, and STAT3F were fixed and stained for ALP and a cell-surface antigen, SSEA-4. Typical data, with two independent clones, one of each type, are shown.Abbreviation:ALP, alkaline phosphatase.

Figure 8. Inhibition of endogenous STAT3 activity by expression of the STAT3F mutant. (A): Tyrosine phosphorylation of STAT3 induced by LIF. ES clones expressing empty vector (Mock), wild-type STAT3, and STAT3F were treated with or without LIF for 15 minutes, and then cell extracts were subjected to immunoblotting with antibodies against phosphorylated and nonphosphorylated STAT3 and against Oct-3/4. (B): LIF-induced nuclear translocation of phosphorylated STAT3. ES clones were treated with or without LIF for 15 minutes, and then cells were fixed and stained with antibodies against phosphorylated STAT3 and Oct-3/4. Bar = 100 μm. (C): RT-PCR analysis of SOCS-3 and Nanog expression in CMK-6 cells. ES clones expressing empty vector (Mock), wild-type STAT3, and STAT3F were cultured on extracellular matrix for 3 days. The cells were stimulated with or without LIF (1,000 U/ml) for 1 hour, and then total RNA was isolated from them. Expression levels of SOCS-3, Nanog, and GAPDH were analyzed by RT-PCR. Abbreviations: ES, embryonic stem; RT-PCR, reverse transcriptase polymerase chain reaction.* W0 G/ O( w! h* x$ }

We also examined whether overexpression of STAT3F blocks the transcriptional activity of endogenous STAT3. Using RT-PCR, we analyzed the induction of SOCS-3, a STAT3 target gene whose expression is induced by LIF . As shown in Figure 8C, SOCS-3 mRNA was highly induced by LIF in cells expressing empty vector or wild-type STAT3 but was completely inhibited in the STAT3F clones. In contrast, expression of Nanog, which has been recently identified as a homeodomain transcription factor that is essential for the LIF signaling–independent maintenance of self-renewal in murine ES cells , did not substantially change as a consequence of STAT3 expression. Likewise, GAPDH expression, examined as a control, was also not changed (Fig. 8C). These results indicate that the expression of STAT3F inhibits endogenous STAT3 in a dominant-negative manner.- k$ V( [2 I3 m. o7 h. k

Finally, we asked whether the blockage of STAT3 signaling affects the potential of ES cells to differentiate into all three embryonic germ layers. For this purpose, stable ES clones were cultured for 10 days in suspension to form EBs, at which point we analyzed the expression of several lineage-specific genes by RT-PCR (Fig. 9). EBs formed from each STAT3- or STAT3F-expressing clone were shown to express markers for all three germ layers: endoderm (albumin and -fetoprotein), mesoderm (-myosin heavy chain), and ectoderm (musashi1 and neurofilament 68KD). On the other hand, Oct-3/4, a marker of undifferentiated ES cells, was downregulated during differentiation. These results demonstrate that overexpression of either wild-type STAT3 or dominant-negative STAT3F does not affect the pluripotency of monkey ES cells. Taken together, these data clearly demonstrate that the LIF/gp130/STAT3 pathway is dispensable for the maintenance of self-renewal in cynomolgus monkey ES cells.
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Figure 9. In vitro differentiation of stable ES clones expressing STAT3 derivatives. ES clones expressing empty vector (Mock), wild-type STAT3, and STAT3F were cultured in suspension for 10 days without feeder cells to form EBs. Total RNA was isolated from undifferentiated ES cells and 10-day-old EBs and then analyzed by RT polymerase chain reaction for the expression of the following lineage-specific markers: endoderm, albumin (Alb), and -fetoprotein (AFP); mesoderm and -myosin heavy chain (MHC); and ectoderm, musashi1, and neurofilament 68KD (NF68KD). Oct-3/4 and GAPDH served as an undifferentiated ES cell marker and internal control, respectively. Abbreviations: EB, embryoid body; ES, embryonic stem; RT, reverse transcriptase.2 h) q) I2 H; X# a8 M- w1 c

DISCUSSION: n6 Y0 |& u9 ?8 p9 f+ y/ U" l

This work was supported in part by grants from The National Bio Resource Project and the Japan Society for the Promotion of Science.; l/ e9 `/ Y# v# ]& @  ^
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