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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;8 D$ o. J" g; ` r' M' |
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b Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, United Kingdom;
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c Technion University, Rambam Medical Center, Haifa, Israel" w& K- m$ Z7 V. Z
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Key Words. Human embryonic stem cells ? Leukemia inhibitory factor ? STAT3 ? Self-renewal
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7 C: Q! Z" Y. VCorrespondence: 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@childrens.harvard.edu. o1 b. O* @/ f2 D" n' G! \* X
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ABSTRACT5 U! D H! @9 A, }% |
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Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of blastocysts . Previous studies have shown that hESCs can be stably maintained in culture for longer than 1 year and have the capacity to differentiate in vitro and in vivo into cell types from the three germ layers . These characteristics make them an invaluable resource for studies of cell differentiation and development. A major challenge is to determine the conditions that allow convenient and efficient large-scale culture of hESCs and their directed differentiation into therapeutic cells. hESCs are routinely cultured on feeder fibroblasts to maintain their undifferentiated state , but feeder-free culture systems have recently been developed . hESCs cultured on Matrigel supplemented by conditioned media from mouse embryonic fibroblasts (MEF-CM) remain positive for markers of the undifferentiated state (e.g., oct-4, hTERT, alkaline phosphatase, TRA-1-60, and SSEA-4), retain the morphology of undifferentiated cells, and retain the potential to differentiate into numerous cell types. This work suggests that one or more factors secreted by MEFs are required to maintain hESC self-renewal. Murine embryonic stem (mES) cells can be kept undifferentiated in culture under feeder-free conditions by adding leukemia inhibitor factor (LIF) to the medium . LIF is a cytokine that belongs to the interleukin-6 (IL-6) family, which includes IL-6, oncostatin M, cardiotrophin-1, IL-11, and ciliary neurotrophic factor. The LIF receptor (LIFR) consists of the following two subunits: gp130, which is common to all the cytokines from the IL-6 family, and LIFR? (or gp190), specific for LIF . Both signal transducer and activator of transcription (STAT) and Ras/mitogen-activated protein kinase pathways are activated downstream of gp130. The activation of STAT3 appears both necessary and sufficient for mES cell self-renewal . Overexpression of a dominant-negative form of STAT3 in mouse embryonic stem (ES) cells inhibits their self-renewal and enhances their differentiation , whereas a conditionally active form of STAT3 is sufficient to maintain the undifferentiated state of mouse ES cells . Although the central role of the LIF-STAT3 signaling has been well documented in mES, the function of this pathway has remained unclear in hESCs. While anecdotal reports suggest that LIF is not required for maintenance of hESCs , others claim that "LIF helps retain the hES cells in an undifferentiated state" . In this work, we investigated whether LIF was necessary and sufficient for maintaining hESCs in an undifferentiated state and whether pathways activated by LIF in hESCs are functionally conserved between mouse ES and hESCs.9 M6 ?; n. `4 f1 h9 I
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MATERIALS AND METHODS$ ]! g, l7 V! a) e- Z
- ]/ l+ n# r1 w1 x" GEffect of LIF on the Maintenance of hESCs in an Undifferentiated State' w1 k" S' E9 p; \, }3 k" N' E
- J( N* K$ G6 @; \ i) nWe first assessed the potential of hLIF to maintain hESCs in an undifferentiated state. WA09 (H9) cells were grown on Matrigel or gelatin in the presence or absence of hLIF or with a combination of hLIF and bFGF. To assess the state of differentiation, we measured the level of expression of the TRA-1-60 antigen that selectively recognizes undifferentiated hESCs . WA09 cells grown on MEFs or on Matrigel supplemented with MEF-CM maintained consistent and high-level expression of the TRA-1-60 antigen (Fig. 1A). 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 . 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.( ^% T( n9 D! A T& W0 g
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Figure 1. hESC differentiation assay. (A): hESCs were plated on MEF, Matrigel MEF-CM, gelatin, gelatin hLIF, Matrigel hLIF, or Matrigel hLIF bFGF. At days 7 and 14, the state of differentiation of these cells was evaluated by quantifying TRA-1-60 expression by fluorescence-activated cell sorter analysis. (B): Total cell number was counted at days 3, 7, and 14 for each condition. (C): WA09 and UC06 hESC lines were plated on Matrigel (M) MEF-CM, hLIF, or – hLIF. After 7 days in culture, total RNA was prepared from these cells and the expression of Nanog and Rex-1, two markers of pluripotency, was assessed by reverse transcriptase–polymerase chain reaction. Abbreviations: CM, conditioned medium; FGF, fibroblast growth factor; hESC, human embryonic stem cell; hLIF, human leukemia inhibitory factor; MEF, mouse embryonic fibroblast.2 G& X# l% y. D @# K8 a
0 B* G Y7 E v6 fExpression of LIFR and gp130 in hESCs6 [; {: N2 p6 X. M6 O: F% ~
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Given the lack of response of hESCs to hLIF, we investigated whether the components of LIF receptor signaling were expressed on the cells. The receptor for the cytokine LIF consists of two subunits, the LIF receptor LIFR? (gp190) and the signal transducer gp130. After depleting the WA09 and UC06 hESCs of contaminating MEFs, we prepared total RNA, generated cDNA by RT, and performed PCR amplification using primers for LIFR or gp130. Both LIFR? and gp130 were detected in WA09 and UC06 cells (Figs. 2A, B). 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., [8 y& S x+ P1 g3 |0 n
% R* k) Z6 D' o h6 z& Z- bFigure 2. Expression of gp130 and LIFR? in hESCs. (A): Total RNA was reverse transcribed, and the cDNA was subjected to PCR using primers for gp130, LIFR? , and actin (as a loading control). (B): Total RNA from WA09 and UC06 human embryonic stem cell lines was reverse transcribed, and gp130 and LIFR were amplified by PCR. (C): Total cell extracts from hESCs, MEF, and mES cells were analyzed by immunoblotting using an antibody directed against murine and human gp130. Abbreviations: hESCs, human embryonic stem cells; M, Matrigel; MEF, mouse embryonic fibroblast; mES, murine embryonic stem; PCR, polymerase chain reaction./ @# K# @$ ~9 k- K
# R* y' Q5 u+ q2 N7 T/ rActivation of STAT3 Phosphorylation by LIF' |2 T& ]4 y3 ?
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In mES cells, the binding of LIF to its receptor triggers the activation of associated Jak tyrosine kinases, which in turn phosphorylate STAT3 . Therefore, we asked whether LIF could induce STAT3 phosphorylation in hESCs. Both mES cells (on gelatin) and hESCs (on Matrigel) were cultured for 12 hours without LIF in 0.1% serum replacement before stimulation with either mLIF or hLIF. We prepared whole-cell extracts from these cells and assayed for STAT3 phosphorylation using an antibody recognizing phosphotyrosine at position Y705, a residue critical for STAT3 dimerization and nuclear translocation . mLIF induced Y705 STAT3 phosphorylation in mES cells (Fig. 3A). The signal can be detected after 10 minutes, reaches a peak at 20 minutes, and decreases after 30 minutes, in concordance with previous reports . 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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Figure 3. STAT3 phosphorylation after stimulation of mES and hES with murine or human LIF. mES and hESCs were factor-deprived for 12 hours and then stimulated with mLIF (10 ng/ml) (A) or human LIF (10 ng/ml) (B) for indicated times. Immunoblots of whole-cell extracts were then probed with an antibody directed against phosphotyrosine 705 on STAT3 (Y705 P-STAT3) or total STAT3. (C): Immunoblots of whole-cell extracts prepared from mES and hESCs stimulated with mLIF and hLIF, respectively, for indicated times were probed with an antibody directed against phosphoserine 727 on STAT3 (S727 P-STAT). Abbreviations: hESC, human embryonic stem cell; hLIF, human leukemia inhibitory factor; LIF, leukemia inhibitory factor; mES, murine embryonic stem; mLIF, murine leukemia inhibitory factor; STAT3, signal transducer and activator of transcription 3.
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! w6 L" X& P8 ^STAT3 contains a second phosphorylation site on Serine 727 that is critical for optimal induction of STAT3 transcriptional activity . Therefore, we analyzed STAT3 phosphorylation at S727 upon LIF addition to mES and hESCs. As anticipated, S727 was phosphorylated upon addition of mLIF to mES cells (Fig. 3C). 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.7 X8 I1 }# l9 ^( v/ P0 q) O
$ t p# m& H% c: _Activation of STAT3 by MEF-CM
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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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Figure 4. STAT3 phosphorylation after stimulation of hESCs with MEF-CM bFGF. hESCs were plated on Matrigel. After 12 hours of factor deprivation, cells were stimulated with MEF-CM bFGF (4 ng/ml). Immunoblots of whole-cell extracts were probed with antibodies directed against phosphotyrosine 705 and phosphoserine 727 on STAT3 or total STAT3. Abbreviations: bFGF, basic fibroblast growth factor; CM, conditioned medium; hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast; STAT3, signal transducer and activator of transcription 3.
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6 L# p+ ]5 K! C" J7 ^, BSTAT3 Subcellular Localization8 p) K# k" H$ t4 m: F* i* G: b
) ~" F" K5 u* r& F0 a3 g1 P5 y2 hPhosphorylation of Y705 is known to promote dimerization of STAT3 and translocation to the nucleus, where it functions in transcriptional activation . We examined STAT3 sub-cellular localization in mES cells (CCE) and hESCs (WA09) after acute addition of LIF, as well as in mES and hESCs cultured under conditions that maintain the undifferentiated state. Cytosolic and nuclear fractions were prepared from WA09 and CCE cells treated for 10 minutes with hLIF (10 ng/μl) and mLIF (10 ng/μl), respectively. LIF induced the nuclear translocation of STAT3 in both WA09 and CCE cells (Fig. 5A). 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 th(Laurence Dah谷rona, Sarah ) |
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发表于 2009-3-5 10:36
发表于 2015-5-24 15:09
