Specific Knockdown of Oct4 and ?2-microglobulin Expression by RNA Interference in Human Embryonic Stem Cells and Embryonic Carcinoma Cells
http://www.100md.com
《干细胞学杂志》
a The Centre for Stem Cell Biology and the Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, U.K.;
b Axordia Ltd, Western Bank, Sheffield, U.K.;
c Section of Reproductive and Developmental Medicine, University of Sheffield, Royal Hallamshire Hospital, Jessop Wing, Sheffield, U.K.
Key Words. Human embryonic stem cells ? Embryonal carcinoma cells ? RNA interference Oct4 ? ?-2-microglobulin ? GFP ? Differentiation ? Gene expression
Correspondence: P. W. Andrews,B.Sc., D.Phil., M.B.A., The Centre for Stem Cell Biology and the Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, U.K. Telephone: +44 (0)114-222-4173; Fax: +44 (0)114-222-2399; e-mail: p.w.andrews@sheffield.ac.uk
ABSTRACT
Human embryonic stem (hES) cells offer the opportunity for the in vitro production of multiple cell types for use in regenerative medicine. A key to unlocking this potential is development of methods for controlling gene expression and, consequently, cell differentiation. One tool that might be exploited is RNA interference (RNAi) to manipulate specific signaling pathways in a transient manner and so influence the selection of specific pathways of differentiation by a pluripotent stem cell. To explore this possibility we have used RNAi to determine whether the transcription factor Oct4 is required to maintain the undifferentiated state of hES cells, as well as human embryonal carcinoma (hEC) cells, their malignant equivalent from teratocarcinomas, and whether forced knockdown of Oct4 expression results in differentiation toward trophectoderm. Murine ES cells have been shown to depend on the correct levels of Oct4 expression for their maintenance of an undifferentiated stem cell phenotype, and they differentiate to trophectoderm in its absence . However, various differences exist between mouse and human ES cells (e.g., the nonresponsiveness of hES cells to leukemia inhibitory factor and their different surface antigen phenotypes ), so that one cannot assume, a priori, that a regulatory factor active in mouse ES cells necessarily exhibits the same function in their human counterparts. Here we show that RNAi can be used effectively to downregulate genes in a specific manner in hEC and hES cells. Further, our results confirm the hypothesis that, indeed, Oct4 expression is required to maintain the undifferentiated state of human EC and ES cells.
MATERIALS AND METHODS
The hES lines H7 and H14 are pluripotent cell lines derived from early human embryos. Both 2102Ep and NTERA2 are hEC cell lines derived from teratocarcinomas ; they closely resemble hES cells and the inner cell mass of human blastocyst stage embryos . Although 2102Ep cells differentiate slightly when passaged at low density, NTERA2 but not 2102Ep cells differentiate, most notably in a neural direction, when exposed to retinoic acid . By contrast, the hES cultures always contained a significant level of spontaneously differentiated cells .
To establish conditions for the use of RNAi with these cells, and to ascertain whether the RNAi technique itself might induce nonspecific effects, we first used RNAi to knock down expression of eGFP and ?2M in stably transfected, eGFP-expressing NTERA2, 2102Ep, and H7 cells. After 3–5 days, the expression of eGFP and ?2M was specifically downregulated, 5- to 10-fold, only by their corresponding siRNA (Fig. 1a). At the same time, the expression of several developmentally regulated surface antigens characteristic of undifferentiated hEC and hES cells SSEA3, SSEA4, TRA-1-60, and TRA-2-54 was unaffected, while expression of SSEA1, which is not expressed by the undifferentiated cells but is expressed by some of their differentiated derivatives, was not induced. Thus, siRNA treatments do not appear to induce hEC or hES differentiation in a nonspecific way (Fig. 1b). However, expression of cell surface ?2M and the heavy chain of class 1 HLA (which is dependent on ?2M expression ), was specifically downregulated after treatment with siRNA to ?2M. RNAi efficiency varied with cell density (2 x 104 cells per cm2 was optimal) and different transfection reagents (Oligofectamine was the most efficient tested).
Figure 1. Short interfering RNA (siRNA) can specifically downregulate targeted gene expression in human embryonal carcinoma (EC) and embryonic stem (ES) cells without affecting their undifferentiated state. NTERA2 and 2102Ep human EC and H7 human ES cells, stably transfected with an enhanced green fluorescent protein (eGFP) expression vector, were treated with siRNA, targeting either eGFP or ?2-microglobulin (?2M), using several different transfection reagents: Lipofectin (Invitrogen), Superfect (Qiagen), Fugene 6 (Roche Diagnostics), ExGen500 (MBI Fermentas), GeneJuice (Novagen), and Oligofectamine (Invitrogen). Of these, Oligofectamine was judged the most effective for RNA interference. (A): Fluorescence-activated cell sorter (FACS) analysis of eGFP expression in cells treated with eGFP siRNA (red histograms) or ?2M siRNA (blue histograms) or untreated (green histograms). The analysis was carried out 3 days after RNAi in NTERA2 and H7 cells and 5 days after RNAi in 2102Ep cells. The fluorescence intensity is recorded on a log scale, and there is a 5- to 10-fold reduction in eGFP expression after eGFP siRNA. (B): Surface marker antigen expression by green fluorescent NTERA2 EC cells, either untreated (NT2cgB1) or 6 days after exposure to siRNA to eGFP (NT2cgB1-GFP) or ?2M (NT2cgB1-?2M). No effect on surface antigens characteristic of human EC cells was seen; however, ?2M siRNA caused a marked downregulation of cell surface expression of ?2-microglobulin (detected by BBM1) and HLA-A,B,C heavy chain (detected by W6/32), as expected.
Using the same protocol, we then treated NTERA2 and 2102Ep hEC cells and H7 hES cells in parallel with Oct4 or ?2M siRNA. Initially, we designed two siRNAs, corresponding to different regions of the Oct4 gene to target Oct4 expression (Oct4-A and Oct4-B; see Materials and Methods). Both did cause downregulation of Oct4 mRNA and protein expression. However, Oct4-A siRNA proved more efficient than Oct4-B, as judged by western blotting (data not shown). Therefore, Oct4-A was used in the further studies.
To determine the optimal time for RNA interference of Oct4 expression, 2102Ep cells were treated with Oct4 and ?2M siRNAs, and the expression of Oct4 was assayed by western blotting 3, 5, and 7 days after RNAi. The level of Oct4 protein expression remained constant in the cultures treated with siRNA to ?2M. By contrast, it was substantially downregulated 3 and 5 days after treatment with Oct4 siRNA, while levels began to recover by 7 days (Fig. 2).
Figure 2. siRNA directed to Oct4 causes downregulation of Oct4 protein expression. Western blot for Oct4 expression in 2102Ep cells treated with siRNA targeting either?2M (Lanes 1, 3, and 5) or Oct4 (Lanes 2, 4, and 6) after 3 days (Lanes 1, 2), 5 days (Lanes 3, 4), and 7 days (Lanes 5, 6). Note the substantial reduction in Oct4, 3 and 5 days after treatment with Oct4 siRNA and then its partial recovery after 7 days, due to unaffected cells beginning to overgrow the culture. The lanes were each loaded with lysate containing the equivalent of 1.5 x 105 cells. Following electrophoresis and blotting, membranes were stained with Ponceau-S to determine equal loading and transfer (lower panel).
The knockdown of Oct4 expression was accompanied by a marked reduction in cell growth, in comparison with cells treated with ?2M siRNA (Fig. 3). The cells exhibited a markedly different morphology and, when left for over 7 days, syncytial giant cells could be found in the Oct4 siRNA–treated cultures. These treated cells had lost expression of the characteristic human EC and ES cell marker antigen, TRA-1-60 (Fig. 4). Many of these large flat cells, including syncytial cells, could be found in treated cultures several weeks later, although the remaining, initially small, population of unaffected cells began to overgrow the culture and mask the apparently differentiated cells that did not proliferate. Similar effects on morphology and expression of cell surface TRA-1-60 were seen in ES cultures (data not shown).
Figure 3. Effect of Oct4 RNAi on growth rate and the appearance of multinucleated cells in 2102Ep human EC cells. (A): Growth of 2102Ep cells after treatment with ?2M and Oct4 siRNAs (n = 3; ± standard deviation). (B): The morphology of (I) untreated 2102Ep cells or of multinucleated cells appearing (II) after Oct4 RNAi phase contrast and (III) after staining with DAPI and rhodamine-phalloidin, 7 days after RNAi.
Figure 4. Morphology and TRA-1-60 expression of human EC and ES cells after Oct4 RNAi. (A): Phase contrast (left) and TRA-1-60 staining (right) of 2102Ep cells 5 days after mock (top) and Oct4 RNAi (bottom) treatments. (B): Phase contrast (left) and TRA-1-60 staining (right) of H14 cells 3 days after mock (top) and Oct4 siRNA (bottom) treatments.
To investigate further the effects of Oct4 knockdown on the phenotype of human EC and ES cells, the expression patterns of several developmentally regulated antigens and genes were analyzed in NTERA2 and 2102Ep EC cells and H7 ES cells 5 days after siRNA knockdown of Oct4 expression (Fig. 5; Table 1). In each case, Oct4 protein levels were substantially reduced by siRNA for Oct4, in comparison with cells treated with siRNA targeting ?2M. Reciprocally, cell surface ?2M expression was reduced by ?2M siRNA, in comparison with cells treated with Oct4 siRNA. At the same time, in cultures treated with Oct4 siRNA, significant populations of cells appeared with substantially reduced expression of the stem cell marker antigens SSEA3 and TRA-1-60, compared with those treated with ?2M siRNA. Cells positive for SSEA1, which is not expressed by human EC and ES cells, also appeared in cultures of 2102Ep and H7, though not significantly in NTERA2, after treatment with Oct4 siRNA. These observations were quite clear in the case of the EC cell lines but were masked to some extent in the H7 hES line because of the initial heterogeneity of these cultures that results from uncontrolled spontaneous differentiation. Nevertheless, the results were consistent and were also observed, for example, in a second hES line, H14, in a separate experiment (Fig. 6a). In the latter case, the effect of Oct4 siRNA was compared with "mock-treated" control cultures, rather than in comparison with a ?2M siRNA control.
Figure 5. The effect of Oct4 knockdown by siRNA on the expression of surface antigens, and hCG, Gcm1, and Cdx2 by NTERA2 and 2102Ep hEC cells and H7 hES cells 5 days after treatment with Oct4 or ?2M siRNA. (A): Western blot for Oct4 in NTERA2 (lanes 1, 2), 2102Ep (lanes 3, 4), and H7 (lanes 5, 6); siRNA to Oct4 (lanes, 1, 3, and 5) and siRNA to ?2M (lanes 2, 4, and 6). Lysates containing the equivalence of 1.5 x 105 cells were loaded in each lane. Ponceau-S staining confirmed similar loading and transfer for lysates (not shown). (B): Surface antigen expression after treatment with Oct4 siRNA (red histograms) or ?2M (blue histograms). P3X63Ag8 is used as a negative first antibody control. Note the disappearance of markers of the undifferentiated state, SSEA3 and TRA-1-60, after treatment with siRNA to Oct4 and the appearance of SSEA1, especially in 2102Ep and H7 cells, indicating differentiation. The percentage of cells in each population is shown in Table 1. Also note that ?2-microglobulin was down-regulated after treatment with ?2M siRNA but not Oct4 siRNA in 2102Ep and H7 cells. The low and effectively undetectable level of ?2-microglobulin in the NTERA2 cells used in this experiment, in contrast to the experiment shown in Figure 1, reflects a commonly observed variability in MHC antigen expression by this particular hEC line in contrast to others . (C): RT-PCR analysis of hCG and Cdx2 expression after treatment with siRNA to Oct4. ?-actin PCR was used as a template loading control.
Table 1. Changes in antigen expression following ?2M and Oct4 siRNA–induced knockdown
Figure 6. The response of H14 hES cells to Oct4 siRNA, in comparison with a mock-treated (dsRNA) control, 3 days after treatment. (A): Expression of surface antigens SSEA1, TRA-1-60, and SSEA3 3 days after treatment with Oct4 siRNA (red histograms), or mock transfected cells, using transfection reagents and conditions but no RNA (blue histograms). (B): hCG expression in the same cells, analyzed by reverse transcription poly-merase chain reaction (RT-PCR).
These results indicate that the expression of Oct4 is required for the maintenance of hEC and hES cells in an undifferentiated state and that knockdown of Oct4 expression in human EC and ES cells results in their differentiation, as in the case of mouse ES cells . Further, at least for the 2102Ep human EC cells and the H7 and H14 human ES cells, the induction of SSEA1 would be consistent with differentiation toward trophectoderm, as in mouse ES cells, since SSEA1 is strongly expressed by human trophectoderm . To test this hypothesis, the expression of Cdx2, hCG, and Gcm-1 was assayed by RT-PCR (Fig. 5c). The transcription factor Cdx2 has been reported to mark trophectodermal stem cells in the mouse . In the case of the hEC and hES cells, we found that untreated or control cells treated with ?2M siRNA expressed low levels of Cdx2, which was upregulated in 2102Ep and downregulated in NTERA2 but remained unchanged in H7 cells after Oct4 siRNA treatment. At the same time, hCG and Gcm1, also markers of trophectoderm, were upregulated after Oct4 siRNA in 2102Ep and H7 cells, though they were not detected in NTERA2 cultures. In a separate experiment, hCG was also upregulated in H14 hES cells after Oct4 siRNA (Fig. 6b).
DISCUSSION
We are grateful to JA Thomson for providing H7 and H14 ES cells, to Christine Pigott for her assistance in cell culture, and to Chee Gee Liew for providing NTERA2 cells transfected with eGFP. We thank Victoria Fox for her technical assistance and Nick Jenkins for his great help in taking pictures. This work was supported by grants from the Wellcome Trust, Yorkshire Cancer Research, Biotechnology and Biological Sciences Research Council, and Axordia Ltd.
REFERENCES
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Elbashir SM, Harborth J, Lendeckel W et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–498.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–376.
Draper JS, Pigott C, Thomson JA et al. Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat 2002;200:249–258.
Andrews PW, Goodfellow PN, Shevinsky L et al. Cell surface antigens of a clonal human embryonal carcinoma cell line: morphological and antigenic differentiation in culture. Int J Cancer 1982; 9:523–531.
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b Axordia Ltd, Western Bank, Sheffield, U.K.;
c Section of Reproductive and Developmental Medicine, University of Sheffield, Royal Hallamshire Hospital, Jessop Wing, Sheffield, U.K.
Key Words. Human embryonic stem cells ? Embryonal carcinoma cells ? RNA interference Oct4 ? ?-2-microglobulin ? GFP ? Differentiation ? Gene expression
Correspondence: P. W. Andrews,B.Sc., D.Phil., M.B.A., The Centre for Stem Cell Biology and the Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, U.K. Telephone: +44 (0)114-222-4173; Fax: +44 (0)114-222-2399; e-mail: p.w.andrews@sheffield.ac.uk
ABSTRACT
Human embryonic stem (hES) cells offer the opportunity for the in vitro production of multiple cell types for use in regenerative medicine. A key to unlocking this potential is development of methods for controlling gene expression and, consequently, cell differentiation. One tool that might be exploited is RNA interference (RNAi) to manipulate specific signaling pathways in a transient manner and so influence the selection of specific pathways of differentiation by a pluripotent stem cell. To explore this possibility we have used RNAi to determine whether the transcription factor Oct4 is required to maintain the undifferentiated state of hES cells, as well as human embryonal carcinoma (hEC) cells, their malignant equivalent from teratocarcinomas, and whether forced knockdown of Oct4 expression results in differentiation toward trophectoderm. Murine ES cells have been shown to depend on the correct levels of Oct4 expression for their maintenance of an undifferentiated stem cell phenotype, and they differentiate to trophectoderm in its absence . However, various differences exist between mouse and human ES cells (e.g., the nonresponsiveness of hES cells to leukemia inhibitory factor and their different surface antigen phenotypes ), so that one cannot assume, a priori, that a regulatory factor active in mouse ES cells necessarily exhibits the same function in their human counterparts. Here we show that RNAi can be used effectively to downregulate genes in a specific manner in hEC and hES cells. Further, our results confirm the hypothesis that, indeed, Oct4 expression is required to maintain the undifferentiated state of human EC and ES cells.
MATERIALS AND METHODS
The hES lines H7 and H14 are pluripotent cell lines derived from early human embryos. Both 2102Ep and NTERA2 are hEC cell lines derived from teratocarcinomas ; they closely resemble hES cells and the inner cell mass of human blastocyst stage embryos . Although 2102Ep cells differentiate slightly when passaged at low density, NTERA2 but not 2102Ep cells differentiate, most notably in a neural direction, when exposed to retinoic acid . By contrast, the hES cultures always contained a significant level of spontaneously differentiated cells .
To establish conditions for the use of RNAi with these cells, and to ascertain whether the RNAi technique itself might induce nonspecific effects, we first used RNAi to knock down expression of eGFP and ?2M in stably transfected, eGFP-expressing NTERA2, 2102Ep, and H7 cells. After 3–5 days, the expression of eGFP and ?2M was specifically downregulated, 5- to 10-fold, only by their corresponding siRNA (Fig. 1a). At the same time, the expression of several developmentally regulated surface antigens characteristic of undifferentiated hEC and hES cells SSEA3, SSEA4, TRA-1-60, and TRA-2-54 was unaffected, while expression of SSEA1, which is not expressed by the undifferentiated cells but is expressed by some of their differentiated derivatives, was not induced. Thus, siRNA treatments do not appear to induce hEC or hES differentiation in a nonspecific way (Fig. 1b). However, expression of cell surface ?2M and the heavy chain of class 1 HLA (which is dependent on ?2M expression ), was specifically downregulated after treatment with siRNA to ?2M. RNAi efficiency varied with cell density (2 x 104 cells per cm2 was optimal) and different transfection reagents (Oligofectamine was the most efficient tested).
Figure 1. Short interfering RNA (siRNA) can specifically downregulate targeted gene expression in human embryonal carcinoma (EC) and embryonic stem (ES) cells without affecting their undifferentiated state. NTERA2 and 2102Ep human EC and H7 human ES cells, stably transfected with an enhanced green fluorescent protein (eGFP) expression vector, were treated with siRNA, targeting either eGFP or ?2-microglobulin (?2M), using several different transfection reagents: Lipofectin (Invitrogen), Superfect (Qiagen), Fugene 6 (Roche Diagnostics), ExGen500 (MBI Fermentas), GeneJuice (Novagen), and Oligofectamine (Invitrogen). Of these, Oligofectamine was judged the most effective for RNA interference. (A): Fluorescence-activated cell sorter (FACS) analysis of eGFP expression in cells treated with eGFP siRNA (red histograms) or ?2M siRNA (blue histograms) or untreated (green histograms). The analysis was carried out 3 days after RNAi in NTERA2 and H7 cells and 5 days after RNAi in 2102Ep cells. The fluorescence intensity is recorded on a log scale, and there is a 5- to 10-fold reduction in eGFP expression after eGFP siRNA. (B): Surface marker antigen expression by green fluorescent NTERA2 EC cells, either untreated (NT2cgB1) or 6 days after exposure to siRNA to eGFP (NT2cgB1-GFP) or ?2M (NT2cgB1-?2M). No effect on surface antigens characteristic of human EC cells was seen; however, ?2M siRNA caused a marked downregulation of cell surface expression of ?2-microglobulin (detected by BBM1) and HLA-A,B,C heavy chain (detected by W6/32), as expected.
Using the same protocol, we then treated NTERA2 and 2102Ep hEC cells and H7 hES cells in parallel with Oct4 or ?2M siRNA. Initially, we designed two siRNAs, corresponding to different regions of the Oct4 gene to target Oct4 expression (Oct4-A and Oct4-B; see Materials and Methods). Both did cause downregulation of Oct4 mRNA and protein expression. However, Oct4-A siRNA proved more efficient than Oct4-B, as judged by western blotting (data not shown). Therefore, Oct4-A was used in the further studies.
To determine the optimal time for RNA interference of Oct4 expression, 2102Ep cells were treated with Oct4 and ?2M siRNAs, and the expression of Oct4 was assayed by western blotting 3, 5, and 7 days after RNAi. The level of Oct4 protein expression remained constant in the cultures treated with siRNA to ?2M. By contrast, it was substantially downregulated 3 and 5 days after treatment with Oct4 siRNA, while levels began to recover by 7 days (Fig. 2).
Figure 2. siRNA directed to Oct4 causes downregulation of Oct4 protein expression. Western blot for Oct4 expression in 2102Ep cells treated with siRNA targeting either?2M (Lanes 1, 3, and 5) or Oct4 (Lanes 2, 4, and 6) after 3 days (Lanes 1, 2), 5 days (Lanes 3, 4), and 7 days (Lanes 5, 6). Note the substantial reduction in Oct4, 3 and 5 days after treatment with Oct4 siRNA and then its partial recovery after 7 days, due to unaffected cells beginning to overgrow the culture. The lanes were each loaded with lysate containing the equivalent of 1.5 x 105 cells. Following electrophoresis and blotting, membranes were stained with Ponceau-S to determine equal loading and transfer (lower panel).
The knockdown of Oct4 expression was accompanied by a marked reduction in cell growth, in comparison with cells treated with ?2M siRNA (Fig. 3). The cells exhibited a markedly different morphology and, when left for over 7 days, syncytial giant cells could be found in the Oct4 siRNA–treated cultures. These treated cells had lost expression of the characteristic human EC and ES cell marker antigen, TRA-1-60 (Fig. 4). Many of these large flat cells, including syncytial cells, could be found in treated cultures several weeks later, although the remaining, initially small, population of unaffected cells began to overgrow the culture and mask the apparently differentiated cells that did not proliferate. Similar effects on morphology and expression of cell surface TRA-1-60 were seen in ES cultures (data not shown).
Figure 3. Effect of Oct4 RNAi on growth rate and the appearance of multinucleated cells in 2102Ep human EC cells. (A): Growth of 2102Ep cells after treatment with ?2M and Oct4 siRNAs (n = 3; ± standard deviation). (B): The morphology of (I) untreated 2102Ep cells or of multinucleated cells appearing (II) after Oct4 RNAi phase contrast and (III) after staining with DAPI and rhodamine-phalloidin, 7 days after RNAi.
Figure 4. Morphology and TRA-1-60 expression of human EC and ES cells after Oct4 RNAi. (A): Phase contrast (left) and TRA-1-60 staining (right) of 2102Ep cells 5 days after mock (top) and Oct4 RNAi (bottom) treatments. (B): Phase contrast (left) and TRA-1-60 staining (right) of H14 cells 3 days after mock (top) and Oct4 siRNA (bottom) treatments.
To investigate further the effects of Oct4 knockdown on the phenotype of human EC and ES cells, the expression patterns of several developmentally regulated antigens and genes were analyzed in NTERA2 and 2102Ep EC cells and H7 ES cells 5 days after siRNA knockdown of Oct4 expression (Fig. 5; Table 1). In each case, Oct4 protein levels were substantially reduced by siRNA for Oct4, in comparison with cells treated with siRNA targeting ?2M. Reciprocally, cell surface ?2M expression was reduced by ?2M siRNA, in comparison with cells treated with Oct4 siRNA. At the same time, in cultures treated with Oct4 siRNA, significant populations of cells appeared with substantially reduced expression of the stem cell marker antigens SSEA3 and TRA-1-60, compared with those treated with ?2M siRNA. Cells positive for SSEA1, which is not expressed by human EC and ES cells, also appeared in cultures of 2102Ep and H7, though not significantly in NTERA2, after treatment with Oct4 siRNA. These observations were quite clear in the case of the EC cell lines but were masked to some extent in the H7 hES line because of the initial heterogeneity of these cultures that results from uncontrolled spontaneous differentiation. Nevertheless, the results were consistent and were also observed, for example, in a second hES line, H14, in a separate experiment (Fig. 6a). In the latter case, the effect of Oct4 siRNA was compared with "mock-treated" control cultures, rather than in comparison with a ?2M siRNA control.
Figure 5. The effect of Oct4 knockdown by siRNA on the expression of surface antigens, and hCG, Gcm1, and Cdx2 by NTERA2 and 2102Ep hEC cells and H7 hES cells 5 days after treatment with Oct4 or ?2M siRNA. (A): Western blot for Oct4 in NTERA2 (lanes 1, 2), 2102Ep (lanes 3, 4), and H7 (lanes 5, 6); siRNA to Oct4 (lanes, 1, 3, and 5) and siRNA to ?2M (lanes 2, 4, and 6). Lysates containing the equivalence of 1.5 x 105 cells were loaded in each lane. Ponceau-S staining confirmed similar loading and transfer for lysates (not shown). (B): Surface antigen expression after treatment with Oct4 siRNA (red histograms) or ?2M (blue histograms). P3X63Ag8 is used as a negative first antibody control. Note the disappearance of markers of the undifferentiated state, SSEA3 and TRA-1-60, after treatment with siRNA to Oct4 and the appearance of SSEA1, especially in 2102Ep and H7 cells, indicating differentiation. The percentage of cells in each population is shown in Table 1. Also note that ?2-microglobulin was down-regulated after treatment with ?2M siRNA but not Oct4 siRNA in 2102Ep and H7 cells. The low and effectively undetectable level of ?2-microglobulin in the NTERA2 cells used in this experiment, in contrast to the experiment shown in Figure 1, reflects a commonly observed variability in MHC antigen expression by this particular hEC line in contrast to others . (C): RT-PCR analysis of hCG and Cdx2 expression after treatment with siRNA to Oct4. ?-actin PCR was used as a template loading control.
Table 1. Changes in antigen expression following ?2M and Oct4 siRNA–induced knockdown
Figure 6. The response of H14 hES cells to Oct4 siRNA, in comparison with a mock-treated (dsRNA) control, 3 days after treatment. (A): Expression of surface antigens SSEA1, TRA-1-60, and SSEA3 3 days after treatment with Oct4 siRNA (red histograms), or mock transfected cells, using transfection reagents and conditions but no RNA (blue histograms). (B): hCG expression in the same cells, analyzed by reverse transcription poly-merase chain reaction (RT-PCR).
These results indicate that the expression of Oct4 is required for the maintenance of hEC and hES cells in an undifferentiated state and that knockdown of Oct4 expression in human EC and ES cells results in their differentiation, as in the case of mouse ES cells . Further, at least for the 2102Ep human EC cells and the H7 and H14 human ES cells, the induction of SSEA1 would be consistent with differentiation toward trophectoderm, as in mouse ES cells, since SSEA1 is strongly expressed by human trophectoderm . To test this hypothesis, the expression of Cdx2, hCG, and Gcm-1 was assayed by RT-PCR (Fig. 5c). The transcription factor Cdx2 has been reported to mark trophectodermal stem cells in the mouse . In the case of the hEC and hES cells, we found that untreated or control cells treated with ?2M siRNA expressed low levels of Cdx2, which was upregulated in 2102Ep and downregulated in NTERA2 but remained unchanged in H7 cells after Oct4 siRNA treatment. At the same time, hCG and Gcm1, also markers of trophectoderm, were upregulated after Oct4 siRNA in 2102Ep and H7 cells, though they were not detected in NTERA2 cultures. In a separate experiment, hCG was also upregulated in H14 hES cells after Oct4 siRNA (Fig. 6b).
DISCUSSION
We are grateful to JA Thomson for providing H7 and H14 ES cells, to Christine Pigott for her assistance in cell culture, and to Chee Gee Liew for providing NTERA2 cells transfected with eGFP. We thank Victoria Fox for her technical assistance and Nick Jenkins for his great help in taking pictures. This work was supported by grants from the Wellcome Trust, Yorkshire Cancer Research, Biotechnology and Biological Sciences Research Council, and Axordia Ltd.
REFERENCES
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