Oct-4 Knockdown Induces Similar Patterns of Endoderm and Trophoblast Differentia - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian, Scotland, United Kingdom

Key Words. Endoderm ? ES cells ? Oct-4 ? Pluripotency ? Trophoblast% h1 v7 b6 t, ~9 {  T
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Tom Burdon, Ph.D., Roslin Institute, Roslin, Midlothian, EH25 9PS Scotland, United Kingdom. Telephone: 00-44-131-527-4270; Fax: 00-44-131-440-0434; e-mail: tom.burdon@bbsrc.ac.uk; website: www.ri.bbsrc.ac.

ABSTRACT
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Embryonic stem (ES) cell lines derived from the inner cell mass of blastocysts remain pluripotent and can be stably propagated indefinitely when cultured in conditions that suppress their differentiation . Despite extended periods in culture, they retain the capacity to differentiate into all fetal and adult cell types, a characteristic best demonstrated by the ability of mouse ES (mES) cells to reintegrate into host blastocysts and contribute to somatic and germ line tissues in chimeras . Current knowledge of the mechanisms that regulate ES cell self-renewal and differentiation is based largely on the study of mES cells . However, the derivation of pluripotent stem cell lines from human blastocysts has enabled investigations to be extended to another species .

Maintenance of ES cell pluripotency requires the constant suppression of differentiation by both extrinsic and intrinsic factors . The POU-domain transcription factor Oct-4 is highly expressed in ES cells  and has been shown to be essential for maintaining pluripotency in mES cells . In an elegant series of experiments, in which Oct-4 expression was controlled by a tetracycline-regulated transgene, Niwa and colleagues showed that self-renewal was exquisitely dependent on the level of Oct-4 . Whereas a twofold increase in Oct-4 promoted differentiation into embryonic and extraembryonic cell types typically produced upon withdrawal of the cytokine leukemia inhibitory factor (LIF), a reduction in the level of Oct-4 induced dedifferentiation into trophoblast, an extraembryonic lineage that mES cells do not normally generate . This observation led to the proposal that at least one of the functions of Oct-4 is to operate as a gatekeeper to prevent respecification and dedifferentiation into extraembryonic ectoderm .4 B# Q: |1 p# ~- c6 Z/ ~4 N. I
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Establishment and maintenance of pluripotency in stem cells is a central issue in stem cell biology. In order to compare the role of Oct-4 in mouse and human ES cells, we employed RNA interference (RNAi) to knock down the transcription factor in both types of ES cells . A particularly attractive feature of RNAi is that we could directly compare the effects of Oct-4 downregulation in multiple lines of both mouse and human ES cells. Although RNAi cannot completely eliminate Oct-4 function, we reasoned that, if a threshold level of Oct-4 is required for self-renewal, a relatively modest knockdown would allow us to compare Oct-4 functions in ES cells from both species.; R- H: m4 R' P3 d- E

MATERIALS AND METHODS: w% y) W* s6 g$ Z) `. s$ S

RNAi-Mediated Oct-4 Knockdown in mES and hES Cells: g" Z  _) d7 g' h: |9 C4 j$ G
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To examine Oct-4 knockdown in ES cells, we transfected mouse and hES cells with Oct-4-specific siRNAs. The oligonucleotides were designed in accordance with Tuschl Lab guidelines  and derived from an orthologous region in mouse and human Oct-4 mRNAs differing in two nucleotides. Transfection with an EGFP-specific siRNA served as a negative control. Preliminary experiments performed in mES cells established that, 20 hours after transfection with the Oct-4-specific siRNA oligonucleotide, the level of Oct-4 was lower by more than 80% compared with EGFP siRNA-transfected controls (data not shown). However, due to an appreciable recovery of Oct-4 expression by 48 hours, we routinely employed a second transfection at 24 hours to ensure continuous inhibition over the 48-hour time course. Whereas transfection with species-specific siRNAs dramatically reduced the level of Oct-4 protein in mES and hES cells, cross-species oligonucleotides only exhibited a slightly lower level compared with control EGFP siRNA (Fig. 1A). Immunohistochemistry confirmed this selective knockdown of Oct-4 at the cellular level (Fig. 1B and 1C). Oct-4 protein was detected in >90% of the nuclei of mES and hES cells transfected with EGFP or the cross-species Oct-4 siRNA, compared with 30%-35% of mES and hES cells treated with the species-specific siRNA (Fig. 1D). Concomitant with the decrease in Oct-4 protein, we also observed a decrease in Oct-4-dependent transcription, as measured by activation of the Oct-4-regulated FGF4enh5' luciferase reporter gene . Whereas cotransfection of the reporter with cross-species Oct-4 siRNAs produced negligible reduction in luciferase activity, the species-specific Oct-4 siRNAs dramatically reduced Oct-4-dependent transcription by 50%-65% within 24 hours (Fig. 1E). The rapid knockdown and loss of Oct-4-dependent transcription demonstrated that our siRNAs could be used effectively to study Oct-4 function in both mES and hES cells.- c9 _6 ]) d/ |: a# ?8 B+ l

Figure 1. RNAi-mediated Oct-4 inhibition in mES and hES cells. A) Western blot analysis of Oct-4 protein levels in ES cells following siRNA transfection. Cell lysates from HM1 and H9 cells were prepared 48 hours after transfection with EGFP (E), mouse Oct-4 (m), or human Oct-4 (h) siRNAs and fractionated by SDS-PAGE, immunoblotted, and probed with Oct-4 and SHP2 (control) antibodies. B and C) Oct-4 protein expression in siRNA-transfected ES cells. HM1 (B) and H9 (C) cells were transfected for 48 hours, fixed, immunostained with a monoclonal antibody specific for Oct-4, and counterstained for DNA with DAPI. D) Quantitation of Oct-4 expression in situ. Oct-4-positive nuclei were counted in three fields of view (each >100 cells) for transfections in B and C. E) Inhibition of Oct-4-dependent transcription in siRNA-transfected ES cells. D027 mES and H9 hES cells were transfected with siRNA, EF1 Renilla control and the FGF4enh5'-dependent luciferase reporter plasmid. Transfections were performed in triplicate, and luciferase activities were normalized relative to the cotransfected EF1-Renilla control. Values represent means ± SE. Results of a representative transfection are shown.* Z3 P& p& o0 U+ z. X+ ~

Oct-4 Knockdown Induces Rapid Differentiation in mES Cells
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To establish whether Oct-4 knockdown induced differentiation in mES cells, we transfected two ES cell lines carrying wild-type Oct-4 alleles: an E14Tg2A (E14-IA3) cell line that contributes efficiently to the coat color and germ line of chimeric animals and is, therefore, pluripotent (data not shown), and an HM1 subclone . E14-IA3 cells were transfected with either Oct-4 or EGFP siRNA in the presence of LIF or cultured in the absence of the cytokine, and their responses were analyzed over 72 hours. Whereas EGFP siRNA-transfected colonies remained largely undifferentiated for up to 62 hours, withdrawal of LIF produced ES cell mixed colonies containing morphologically differentiated cell types and residual stem cells. In contrast, cells transfected with mOct-4 siRNA began to flatten at 24 hours, and by 48 hours, the majority of colonies were composed of overtly differentiated cells (Fig. 2A). These cells continued to proliferate rapidly, and by 62 hours they had spread out to form a monolayer comprising cells that were morphologically similar to a subset formed during LIF withdrawal or background differentiation (Fig. 2A, compare inset panels). At the molecular level, Oct-4 knockdown in E14-IA3 cells correlated with the upregulation of a trophoblast stem cell marker, Cdx2 , in line with previously reported results . In addition, however, we also observed increased expression of the endoderm-associated genes Gata6 and -fetoprotein (AFP). Negligible induction of FGF5, (a primitive ectoderm marker), Brachyury (mesoderm), or Pax6 (embryonic ectoderm, data not shown) was observed on Oct-4 knockdown. Expression of placental lactogen (PL-1), a marker of differentiated trophoblast, was below the level of detection in E14-IA3 cells. FGF5 and Brachyury (T) were upregulated when cells were cultured in the absence of LIF, confirming that E14-IA3 cells differentiated appropriately in our culture conditions (Fig. 2B). Oct-4 knockdown in the HM1 cell line induced a pattern of morphological and molecular differentiation similar to that observed with E14-IA3 cells (Fig. 2C). Differentiated HM1 cells exhibited coordinate induction of Cdx2, Gata6, and AFP expression, but also no detectable upregulation of PL-1 transcription (Fig. 2D). Induction of Gata6 and AFP expression as well as the trophoblast-associated gene Cdx2 is, therefore, a consistent feature of Oct-4 knockdown in wild-type mES cells.' H& C4 |3 v8 q9 R4 m& u
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Figure 2. Oct-4 knockdown induces epithelial-like differentiation in mES cells. A) Morphology of an E14-IA3 mES cell line after transfection with siRNAs or withdrawal of LIF. E14-IA3 cells were seeded overnight in ES cell medium and then transfected with siRNA or switched to medium without LIF. Cell morphology was recorded at intervals during 62 hours after transfection. The higher magnification of differentiated cells (inset) shows the similar morphologies of cells following mOct-4 siRNA transfection and LIF withdrawal. The white arrow highlights cell morphology typical of undifferentiated mES cells. B) RT-PCR analysis of gene expression in E14-IA3 cells. cDNA was prepared from samples collected at 24, 48, and 72 hours posttransfection and amplified with primer pairs specific for the genes shown. Samples EB and PL correspond to control amplifications with day-10 embryoid body-and mouse placenta-derived cDNA, respectively. C) HM1 cells were seeded overnight in ES cell medium and then transfected with siRNAs. Cell morphology was recorded 48 hours posttransfection. D) RT-PCR analysis of gene expression in HM1 cells. cDNA was prepared from samples collected at 48 hours posttransfection and amplified with primer pairs specific for the genes shown.

Induction of an Endoderm-Specific ?-Galactosidase Gene-Trap Reporter$ [  s6 E1 N! N9 t8 i$ x# U

To explore further the possible association between Oct-4 knockdown and endoderm differentiation, we examined the response of a unique ?-galactosidase gene-trap reporter within the Gtar gene of the I114 cell line  that is exclusively expressed in primitive embryonic liver endoderm and yolk sac in vivo and coexpressed with liver marker genes in vitro . We found that transfection of mOct-4 siRNA, in contrast to control siRNAs, produced a markedly (~ninefold) greater level of ?-galactosidase expression in I114 cells (Fig. 3A). X-gal staining of transfected cultures revealed that ?-galactosidase-positive cells were first evident 3 days after transfection, and their number increased until day 5, when they represented a small, but significant, minority of cells within the culture (Fig. 3B). To exclude the possibility that the I114 lacZ-positive cells might be generated indirectly from untransfected stem cells induced by paracrine signaling from differentiating neighbors, we cocultured I114 cells with Oct-4 siRNA-transfected HM1 cells. ?-galactosidase activity was not induced in these cocultures, demonstrating that induction of the Gtar gene-trap reporter by Oct-4 knockdown in I114 cells was cell autonomous (data not shown).
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Figure 3. Induction of an endoderm-specific ?-galactosidase gene trap reporter. A) Biochemical analysis of gene-trap reporter activity in siRNA-transfected ES cells. I114 cells were transfected with siRNAs or grown in the absence of LIF. Five days after transfection, ?-galactosidase activity was measured by ONPG assay and normalized against the protein concentration in cell lysates. Values are the mean of triplicate transfections ± SE. B) Morphology of cells that expressed the gene-trap reporter following Oct-4 knockdown. I114 cells transfected with mouse Oct-4 siRNA were cultured for 5 days, fixed, and stained for ?-galactosidase activity with X-gal. A representative field of view containing X-gal-positive cells is shown.

Oct-4 Knockdown Parallels Conditional Suppression of the Oct-4 Transgene in the ZHBTc4 mES Cell Line3 ]* b$ j; w+ V% q- |
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To confirm that induction of the endoderm-associated genes by RNAi was due to suppression of Oct-4, we compared siRNA-mediated knockdown with inactivation of Oct-4 expression in the ZHBTc4 cell line . In this cell line, both endogenous alleles are mutated, and Oct-4 expression is maintained by a tetracycline-regulated Oct-4 transgene at 60% of the level in wild-type ES cells . After 24 hours of transfection with mOct-4 siRNA, cells formed small compact colonies that by 48 hours began to flatten out into coherent groups of differentiated epithelial-like cells (Fig. 4A). This paralleled the changes in ES cell morphology induced by treating ZHBTc4 cells with doxycycline and was similar to the pattern described previously for inactivation of the Oct-4 transgene . In contrast, ZHBTc4 cells transfected with EGFP siRNA remained morphologically undifferentiated and were indistinguishable from control cells. RT-PCR analysis confirmed that expression of Oct-4 was significantly reduced by Oct-4 siRNA transfection and completely eliminated by doxycycline treatment. Despite the different methods used to downregulate Oct-4, expression of the trophoblast stem cell-associated transcription factor Cdx2 was induced similarly by both treatments. In contrast to the other mES cells tested, we also detected induction of the trophoblast differentiation marker PL-1 (Fig. 4B). Upregulation of Gata6 and AFP in both doxycycline-treated and Oct-4 siRNA-transfected cells, however, confirmed that increased expression of these endoderm-associated genes in mES cells was induced by downregulation of Oct-4." f- T3 W1 h4 u
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Figure 4. Oct-4 knockdown in the ZHBTc4 mES cell line. A) Morphology of ZHBTc colonies 48 hours post-siRNA transfection or posttreatment with doxycycline (200 ng/ml). Both mOct-4 siRNA- and doxycycline-treated ZHBTc4 cultures contained smaller coherent colonies that often contained areas with amorphous tightly packed cells. B) Expression of endoderm and trophoblast genes in ZHBTc4 cells. PCR analysis was performed on cDNA prepared from cell cultures collected 48 hours posttransfection with siRNAs or 48 hours postinduction with doxycycline (dx) or untreated cell cultures (–).

Oct-4 Knockdown Induces Differentiation of hES Cells
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To examine the requirement for Oct-4 in human ES cells, we transfected two independently derived lines of ES cells, H1 and H9, under feeder-free conditions routinely used for long-term propagation of undifferentiated hES cells . Transfection with hOct-4 siRNA produced diffuse colonies of cells with a flattened, squat morphology, indicative of differentiation (Fig. 5A). In contrast, cells transfected with either EGFP or mouse-specific Oct-4 siRNA formed dense overgrowing colonies typical of undifferentiated hES cell cultures. Analysis of gene expression by RT-PCR confirmed the selective downregulation of Oct-4 in hOct-4 siRNA-transfected cells (Fig. 5B). A slight but reproducible decrease in expression of telomerase catalytic subunit with reverse transcriptase activity (Tert) was observed upon Oct-4 knockdown, consistent with the differentiation of stem cells. Interestingly, there was a marked increase in the level of Gata6 and Gata4 transcripts upon Oct-4 knockdown. The increase in Gata6 expression was confirmed at the cellular level by immunofluorescence (Fig. 5C). In contrast to Oct-4 knockdown in mES cell transfections, however, increased expression of AFP expression was not detected. Moreover, with the exception of Gata3, little induction was observed for either early (Gata2, Cdx2) or late (chorionic gonadatropin , CG?, PL-1) markers of trophoblast differentiation. Since Gata6 precedes AFP expression in some endoderm cell types, we considered the possibilities that these Oct-4-depleted hES cultures might contain immature endodermal precursors or that the particular culture conditions might restrict the differentiation of ES cell derivatives. We, therefore, accelerated the maturation of differentiated cells by transfecting cells cultured in a modified N2B27 medium  that lacks serum supplements, fibroblast-conditioned medium, or FGF2. In this medium, the majority of hES cells transfected with hOct-4 siRNA had clearly differentiated by 96 hours, in comparison with the mOct-4 siRNA control cultures (Fig. 5D). The morphology of these differentiated cells was strikingly similar to the epithelial-like cells produced by Oct-4 knockdown in mouse ES cells. Moreover, RT-PCR analysis showed a dramatic induction of AFP as well as Gata6, paralleling the pattern of endoderm marker gene expression observed in mouse ES cells. Interestingly, increased expression of CG and Cdx2 was also detected in these cultures, consistent with the induction of trophoblast differentiation.
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Figure 5. Oct-4 knockdown induces differentiation of hES cells. A) Morphology of H9 and H1 hES cells 48 hours after siRNA transfection. H9 and H1 cells maintained in medium supplemented with mouse embryonic fibroblast-conditioned medium (MEF-CM) and basic FGF were transfected with EGFP, mOct-4, and hOct-4 siRNAs. Images of representative colonies were recorded approximately 48 hours post-transfection. The white arrows highlight examples of flattened cell morphology typical of differentiated hES cells. B) PCR analysis was performed on cDNA prepared from cell cultures (maintained in MEF-CM) 48 hours posttransfection, and cDNA prepared from day 6 H9 EB served as a positive control. C) Immunohistochemical detection of Gata6 protein in siRNA-transfected H9 hES cells. H9 cultures were fixed 48 hours after siRNA transfection, permeabilized, and incubated with Gata6 polyclonal antibody. Immobilized antigen/antibody complexes were detected using an antirabbit TR-conjugated secondary antibody. Quantitation of Gata6-positive nuclei in three fields of view (total >100 cells) revealed at least a fivefold greater level of Gata6-expressing cells in hOct-4 siRNA-transfected cells (28%) compared with EGFP siRNA (5%) controls. D) Morphology of H9 hES cells after Oct-4 knockdown in N2B27 medium. After seeding cells overnight in standard hES cell culture conditions, the medium was changed to N2B27, and cells were transfected with hOct-4 or mOct-4 siRNAs. The morphology of representative colonies was recorded 96 hours after starting transfections. E) RT-PCR analysis of differentiation markers in H9 cells cultured in N2B27 medium. cDNA prepared from cells treated as described in (D) was analyzed for induction of endoderm and trophoblast marker expression by PCR. EB cDNA was prepared from day-6 H9 EBs.# R1 m1 z0 U' t. u; X; L: s

DISCUSSION. V) D$ I! d# b: B; T
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This work was supported by the Biotechnology and Biological Sciences Research Council (T.B., L.S., J.C.) and the Geron Corporation (D.C.H., J.C.). We are grateful to Melany Jackson for advice on RT-PCR and providing control RNA and primers, Virginie Sottile for control hES RNA, Kinchi Nakashima for EF1-Renilla plasmid, Hitoshi Niwa for FGF4enh5' plasmid, Austin Smith for ZHBTc4 cells, Lesley Forrester for I114 cells, and Lesley Gerrard for HM1 and H9 sublines. We also thank Michael Clinton, Joseph Mee, and Joshua Brickman for critical comments during development of the manuscript. This work is dedicated to the memory of Dr. Michael Burdon.1 k5 `% d" a% D1 O. F4 A& l

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