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作者：Greet Cauffmana, Inge Liebaersa,b, Andr Van Steirteghema,c, Hilde Van de Veldea,c
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, Q) z: A5 o V8 L& U/ B- L 【摘要】0 P! U8 h% _! `# I
The contribution of the POU domain, class 5, transcription factor-1 (POU5F1) in maintaining totipotency in human embryonic stem cells (hESCs) has been repeatedly proven. In humans, two isoforms are encoded: POU5F1_iA and POU5F1_iB. So far, no discrimination has been made between the isoforms in POU5F1 studies, and it is unknown which isoform contributes to the undifferentiated phenotype. Using immunocytochemistry, expression of POU5F1_iA and POU5F1_iB was examined in hESCs and all stages of human preimplantation development to look for differences in expression, biological activity, and relation to totipotency. POU5F1_iA and POU5F1_iB displayed different temporal and spatial expression patterns. During human preimplantation development, a significant POU5F1_iA expression was seen in all nuclei of compacted embryos and blastocysts and a clear POU5F1_iB expression was detected from the four-cell stage onwards in the cytoplasm of all cells. The cytoplasmic localization might imply no or other biological functions beyond transcription activation for POU5F1_iB. The stemness properties of POU5F1 can be assigned to POU5F1_iA because hESCs expressed POU5F1_iA but not POU5F1_iB. However, POU5F1_iA is not the appropriate marker to identify totipotent cells, because POU5F1_iA was also expressed in the nontotipotent trophectoderm and was not expressed in zygotes and early cleavage stage embryos, which are assumed to be totipotent. The expression pattern of POU5F1_iA may suggest that POU5F1_iA alone cannot sustain totipotency and that coexpression with other stemness factors might be the key to totipotency.
9 Q r3 F. N4 \$ E 【关键词】 POUF OCT- Totipotency Stem cell marker Human embryonic stem cells Human preimplantation embryos7 x9 U4 K m2 c Q" g! ?) i. K/ \
INTRODUCTION8 ?5 m. P/ M! h* m7 v0 I9 p0 d: { t
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Totipotency and indefinite self-renewal are characteristic for human embryonic stem cells (hESCs). The key molecular control of these features is still ambiguous, but a continuous blocking of differentiation by intrinsic and extrinsic factors is certainly required. One of the main intrinsic determinants is the POU domain, class 5, transcription factor-1 (POU5F1, previously known as OCT-4). POU5F1 orchestrates the expression of stemness genes such as its own, NANOG, and SOX2, activates genes that encode components of key signaling pathways, and represses genes that promote developmental processes .
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The human POU5F1 gene consists of five exons and is located on chromosome 6 in the region of the major histocompatibility complex . However, POU5F1 expression studies are generally carried out without considering both isoforms.
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1 ~; @2 S8 I5 ZFigure 1. Structure of POU5F1_iA and POU5F1_iB. (A): Alternative splicing of POU5F1 generates two variants, namely POU5F1_vA and POU5F1_vB. The five exons are shown as boxes. POU5F1_vA and POU5F1_vB encode the isoforms POU5F1_iA and POU5F1_iB, respectively. POU5F1_iA and POU5F1_iB differ in sequence at their N termini. The regions that are recognized by the three antibodies (Abs) are indicated. (B): POU5F1_iA and POU5F1_iB consist of 360 and 265 amino acids, respectively, of which the 225 amino acids at their C termini are identical. POU5F1_iA and POU5F1_iB have different N-transactivation domains (N) but an identical DNA-binding domain that consists of the POU-specific domain (POUs), a linker region, and the POU homeodomain (POUh), and an identical C-transactivation domain (C). POU5F1_iA and POU5F1_iB contain a nuclear localization sequence (NLS) at the beginning of the POUh.
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. L; u- w9 B& q! J6 pIn this study, we examined the expression of POU5F1_iA and POU5F1_iB in hESCs, human preimplantation embryos, and differentiated cells such as oocytes, spermatozoa, cumulus cells, and lymphoblasts to look for possible differences in expression, biological activity, and relation to totipotency.- b: W4 x x$ P& A& @5 j
" P' O9 S# @" b# F1 hMATERIALS AND METHODS6 @- V: r# y9 O) O7 q' b8 _8 Q
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Samples
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hESCs. The hESC lines VUB01 and VUB03_DM1 were examined . VUB01 is not known to carry a genetic disease, whereas VUB03_DM1 is genetically affected with the autosomal dominant disorder myotonic dystrophy type 1 (DM1). Colonies were cultured on inactivated mouse embryonic fibroblasts feeder layers in 80% knockout Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 20% knockout-serum replacement (Invitrogen), 2 mM L-glutamin (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM ß-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and 4 ng/ml human recombinant basic fibroblast growth factor (Invitrogen). Passaging was done manually. Colonies were tested at several passages.
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Human Preimplantation Embryos. Human preimplantation embryos were obtained for research at our Center for Reproductive Medicine with the couples' informed consent and the approval of the institutional ethical committee. Embryos were obtained after conventional in vitro fertilization or intracytoplasmic sperm injection (ICSI) . The embryos used were assessed as unsuitable for transfer or cryopreservation at the day of transfer (day 3 or day 5 of the preimplantation development according to the transfer policy for the couple) or were obtained by applying ICSI on oocytes donated for research. These research embryos derived from normally fertilized oocytes and showed a normal morphology and a normal developmental timing at the moment of use.
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Differentiated Cells: Oocytes, Spermatozoa, Cumulus Cells, and Lymphoblasts. Oocytes and spermatozoa were obtained with the couples' informed consent and the approval of the institutional ethical committee. Female patients underwent controlled ovarian stimulation .
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& L$ C# D8 _7 j$ QIndirect Immunocytochemistry/ M- ]' v( y! n
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Immunocytochemistry was performed using three antibodies: antibody 1 (Ab1), a mouse monoclonal IgG2b antibody (sc-5279; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) the epitope of which corresponds to amino acids 1¨C134 of POU5F1_iA and therefore only recognizes POU5F1_iA; antibody 2 (Ab2), an affinity-purified goat polyclonal antibody (sc-8630; Santa Cruz Biotechnology, Inc.) that maps near the N terminus of POU5F1_iB and therefore recognizes POU5F1_iB only; and antibody 3 (Ab3), an affinity-purified goat polyclonal antibody (sc-8629; Santa Cruz Biotechnology, Inc.) that maps at the C terminus and therefore recognizes POU5F1_iA as well as POU5F1_iB (POU5F1_iA B) (Fig. 1A). Samples were incubated in the primary antibody solutions overnight at 4¡ãC at concentrations of 10 µg/ml, 2 µg/ml, and 2 µg/ml for Ab1, Ab2, and Ab3, respectively. Control reactions for nonspecific binding of the primary antibodies were included in each experiment and carried out by replacing Ab1 with a mouse monoclonal IgG2b antibody against human leukocyte antigen-DR (eBiosciences, San Diego, http://www.ebiosciences.com) or with mouse IgGs in the case of lymphoblasts and by replacing Ab2 and Ab3 with goat IgGs (Sigma-Aldrich) at the same concentrations as the primary antibodies. Fluorescein isothiocyanate-conjugated goat anti-mouse (sc-3699; Santa Cruz Biotechnology, Inc.) and rabbit anti-goat F(ab')2 fragments (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) were used as secondary antibodies. Samples were incubated in a 1:200 dilution for 2 hours at 4¡ãC in the dark. Control reactions for the secondary antibodies were performed by omitting the primary antibodies. All antibody solutions were prepared in phosphate-buffered saline (PBS) (Sigma-Aldrich) supplemented with 2% bovine serum albumin (BSA) (Sigma-Aldrich). Extensive washing with PBS was done between all steps. Apart from the incubation conditions of the antibodies, the procedure differed for the distinct samples.
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hESCs. Colonies were grown in a four-well Multidish Nunclon (NUNC A/S, Roskilde, Denmark, http://www.nuncbrand.com). One day after plating, the colonies were fixed with 3.7% formaldehyde (Merck, Darmstadt, Germany, http://www.merck.de) for 30 minutes at 4¡ãC and permeabilized by incubation in a detergent solution containing 0.1% Triton X-100 (Sigma-Aldrich) and 0.1% Igepal (Sigma-Aldrich) for 1 hour at room temperature. Both solutions were made in PBS. Every experiment was done in duplicate. After incubation with the antibodies, one series of wells was covered with SlowFade Light (Invitrogen) and the duplicates with Vectashield Mounting Medium with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Before examination, the colonies were put at 4¡ãC in the dark for at least 30 minutes.
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Preimplantation Embryos. Single embryos were placed in droplets of acidic Tyrode's solution pH 2.4 for zona pellucida thinning or complete removal. Subsequently, they were washed in droplets of PBS supplemented with 2% BSA and then in droplets of PBS without BSA. They were fixed and permeabilized by incubation in cold (¨C20¡ãC) methanol for 5 minutes. The following washing steps were performed in PBS with 2% BSA. Immunostaining took place in 50-µl droplets. After staining, embryos were put on glass coverslips (24 x 50 mm) in 2 µl of SlowFade Light and covered with a slide (25 x 75 mm). Acrytol mounting medium (Surgipath Medical Ind., Inc., Richmond, IL, http://www.surgipath.com) was put at several points between the coverslip and the slide to prevent squeezing of the embryo. Samples were immediately examined or stored at 4¡ãC in the dark until examination.
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Differentiated Cells. Oocytes were handled as embryos. Spermatozoa were washed three times in homemade HEPES-buffered Earle's medium, and cumulus cells and lymphoblasts were washed in PBS. One million cells were isolated and put on a slide (25 x 75 mm) using the Cytospin2 Cytocentrifuge (ThermoElectron Corporation, Waltham, MA, http://www.thermo.com) during 5 minutes at 1,200 rpm. Spermatozoa were fixed and permeabilized in cold (¨C20¡ãC) methanol for 5 minutes. Cumulus cells and lymphoblasts were fixed with 3.7% formaldehyde for 30 minutes at 4¡ãC and permeabilized with 0.1% Triton X-100 and 0.1% Igepal for 1 hour at room temperature. Both solutions were made in PBS. Immunostaining took place in a wet dark chamber. Experiments were done in duplicate. After staining, 20 µl of SlowFade Light was put on one series of slides, and 20 µl of Vectashield Mounting Medium with DAPI was put on the duplicates. A glass coverslip (24 x 50 mm) was put on top. Before examination, the slides were put at 4¡ãC in the dark for at least 30 minutes.
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& K- F9 j. {4 k% PConfocal scanning microscopy with an argon-krypton laser (488/568) (IX70 Fluoview 200; Olympus, Aartselaar, Belgium, http://www.olympusmicro.com) was performed to record the fluorescent images. All images for test antibodies and controls were collected using identical confocal settings and processed identically after collection.
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# [' d5 [5 Y5 _ z# q# a0 JRESULTS
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& a8 g6 D% a5 C8 D/ PhESCs9 @$ O2 _( Z/ P. W
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The hESC lines VUB01 and VUB03_DM1 showed a bright POU5F1_iA and POU5F1_iA B staining in the nuclei of their cells. The nucleoli were not stained. No staining was detected for POU5F1_iB (Fig. 2).
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Figure 2. POU5F1_iA, POU5F1_iB, and POU5F1_iA B expression in human embryonic stem cells. Indirect immunocytochemistry was performed using a mouse monoclonal IgG2b antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse F(ab')2 fragments for POU5F1_iA and two different affinity-purified goat polyclonal antibodies and FITC-conjugated rabbit anti-goat F(ab')2 fragments for POU5F1_iB and POU5F1_iA B. Each image represents a section throughout a colony.
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Human Preimplantation Embryos
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Representative images of the staining are shown in Figure 3. An overview of the results is shown in Table 1.
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Figure 3. POU5F1_iA, POU5F1_iB, and POU5F1_iA B expression throughout human preimplantation development. Indirect immunocytochemistry was performed using a mouse monoclonal IgG2b antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse F(ab')2 fragments for POU5F1_iA and two different affinity-purified goat polyclonal antibodies and FITC-conjugated rabbit anti-goat F(ab')2 fragments for POU5F1_iB and POU5F1_iA B. Each image represents a section throughout the examined material. Abbreviations: c, cell stage; TE, trophectoderm.8 [3 P2 ]6 K/ r# A& |: A7 r
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Table 1. POU5F1_iA, POU5F1_iB, and POU5F1_iA B expression in human preimplantation embryos
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/ y, d' Z' N0 n/ [; _3 uPOU5F1_iA expression was examined in 2 zygotes, 11 cleavage stage embryos, 4 compacting embryos, 4 compacted embryos, and 8 blastocysts. No expression of POU5F1_iA could be detected in zygotes and cleavage stage embryos with less than six cells. From the six-cell stage until the eight-cell stage, a very weak staining was detected in the cytoplasm of all cells. After the eight-cell stage, a very weak staining also appeared in all the nuclei, resulting in a faint diffuse POU5F1_iA staining. During compaction, a brighter staining appeared in some nuclei of the embryos. These nuclei were located at random. In compacted embryos, all nuclei displayed a bright POU5F1_iA staining. In blastocysts, the unequal distribution of POU5F1_iA within a cell became more explicit: a strong nuclear staining and no or a very weak cytoplasmic staining. The blastocysts showed a similar intensity of staining in the ICM and the TE at all stages of blastocyst expansion.
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POU5F1_iB expression was examined in two zygotes, 13 cleavage stage embryos, 5 compacting embryos, 4 compacted embryos, and 12 blastocysts. A very weak staining restricted to the periphery of the cell could be detected in the zygotes. In the two-cell embryo, no staining was detected. In the other cleavage-stage embryos, compacting and compacted embryos and blastocysts, expression of POU5F1_iB was clearly seen in the cytoplasm of all cells. No expression was detected in the nuclei. In blastocysts, the staining was detected in the ICM and the TE but with a slight tendency to be lower in the ICM. Throughout the preimplantation development, a remarkable POU5F1_iB staining was noticed in the granules on the outside of the cell membranes.
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POU5F1_iA B staining was examined in two zygotes, five cleavage stage embryos, two compacting embryos, two compacted embryos and two blastocysts. The zygotes displayed a staining restricted to the cell periphery. A negligible staining was detected at the two-cell stage. In the other cleavage stage embryos, a bright staining was detected in the cytoplasm of all cells but not in the nuclei. From compaction onwards, staining appeared at random in the nuclei until all nuclei were stained at a similar intensity as the cytoplasm. This diffuse staining pattern continued throughout the blastocyst stage at comparable levels in the ICM and the TE. The results equalized to the sum of the separate POU5F1_iA and POU5F1_iB staining.: z/ L) @* Q' V! [0 X& v* k! Q0 `
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Differentiated Cells
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$ }" z1 q/ C- [, y8 O' NOocytes were used only to examine POU5F1_iB expression because the expression of POU5F1_iA and POU5F1_iA B had already been described in oocytes . Of the 10 oocytes used, 4 were GV, 3 MI, and 3 MII. A very weak staining for POU5F1_iB was found in the periphery of six oocytes, of which three were GV, two MI, and one MII. Here, staining was also noticed in the granules on the outside of the oolemma (Fig. 4). In spermatozoa, no staining could be detected for POU5F1_iA and POU5F1_iB. A negligible staining was seen for POU5F1_iA B (data not shown). No staining was detected in cumulus cells and lymphoblasts (data not shown).: x2 [1 E- ]% D0 L( i
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Figure 4. POU5F1_iB expression in human oocytes. Indirect immunocytochemistry was performed using a purified goat polyclonal antibody against POU5F1_iB and fluorescein isothiocyanate-conjugated rabbit anti-goat F(ab')2 fragments. Each image represents a section throughout an oocyte. Abbreviations: ( ), positive staining; (¨C), negative staining; GV, germinal vesicle stage; MI, metaphase I stage; MII, metaphase II stage.4 T! ]% i4 I' ~! O. b" l
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Control reactions for nonspecific and background staining gave no fluorescent signals (data not shown). Unlike Ab1 (POU5F1_iA) and Ab3 (POU5F1_iA B), Ab2 (POU5F1_iB) has not been cited before. The specificity of Ab2 has been confirmed by competition experiments on embryos with the peptide (sc-8630 P; Santa Cruz Biotechnology, Inc.) that was used to generate the Ab2 (Fig. 5)./ [5 M- s" d2 I3 q! N: T7 H3 g
4 I6 l+ n3 `, [; BFigure 5. Characterization of antibody 2 (Ab2) (POU5F_iB) by competition studies on human blastocysts with the blocking peptide that was used to generate Ab2. (A): POU5F1_iB staining. (B): Competition during POU5F1_iB staining. (C): Competition during staining with goat IgG (negative control). Each image represents a section throughout a blastocyst." w1 g+ ~( H' X# I% R
9 L) W/ G% L4 J$ l& @DISCUSSION
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- @- t; h' k. O% J" FThe involvement of POU5F1 in maintaining stem cell totipotency is beyond doubt. Upon differentiation of hESCs, POU5F1 expression is lost .! x( x8 g6 _/ J7 D1 Y4 s8 l0 Z) B% [
, {( E! U- ]: k( VIn human preimplantation embryos, POU5F1_iA and POU5F1_iB displayed temporal and spatial differences in expression. POU5F1_iA expression was seen for the first time in an embryo at the six-cell stage as a very weak cytoplasmic staining, but a significant POU5F1_iA expression was seen only in compacted embryos and blastocysts in which the staining was prominent in all the nuclei. POU5F1_iB expression, on the other hand, was weakly detected in the periphery of zygotes. The zygotic expression resembled, at a weaker level, the one found in some of the oocytes. At the two-cell stage, no POU5F1_iB proteins were found. This pattern represents the degrading pool of maternal POU5F1_iB proteins. At the four-cell stage, a clear cytoplasmic staining was seen. This probably embryonic POU5F1_iB expression continued throughout the other preimplantation stages. The most striking discrepancy between POU5F1_iA and POU5F1_iB expression is the localization within a cell: POU5F1_iA is rather a nuclear protein and POU5F1_iB a cytoplasmic protein. The cytoplasmic localization of POU5F_iB, being a transcription factor, is quite odd. Transcription factors are targeted to the nucleus by a nuclear localization sequence, which has been identified in murine POU5F1 as "RKRKR" . The absence of POU5F1_iB in hESCs is unlikely to be due to the polymorphism, given that a total of four different hESC lines (data not shown) were tested and found negative.6 g" v5 [ M* K; O6 l5 r
: I& C5 F! Y& J- @3 }) [9 mPOU5F1 has been suggested as a candidate lineage marker gene in human preimplantation embryos . During human preimplantation development, cell allocation in the earliest stages has not yet been proven, and totipotency of blastomeres in these early stages is believed. At compaction, a polarity of cells within the embryo is established, which through subsequent cell divisions leads to the formation of the undifferentiated ICM and the differentiated TE. In compacting embryos, expression of POU5F1_iB was detected equally in all the cells, and expression of POU5F1_iA was brightly seen in some nuclei. These nuclei were localized at random. In compacted embryos, POU5F1_iA and POU5F1_iB were expressed in all the cells at a similar intensity. Therefore, neither POU5F1_iA nor POU5F1_iB seems to induce a specific allocation of the dividing blastomeres, giving rise to the ICM or the TE.: r7 x$ c% }$ [' h+ L" `% y
% n8 V3 p: U; O7 o/ u" \We emphasize the use of immunocytochemistry in gene expression studies, as it is possible not only to detect but also to localize a specific antigen in a cell. Although the precise quantification of expression levels is limited and at best semiquantitative in contrast with quantitative RT-PCR, the additional information about the localization within a cell can be crucial. Nevertheless, we wish to stress the restrictions associated with a confocal microscope. Embryos are very dense specimens, and so, part of the laser light might already be absorbed by the embryo structure before the light reaches the point of interest, and, vice versa, part of the emission light of the excited point might be absorbed before it reaches the detector. Subsequently, it is possible that some blastomeres give false-negative results. Unawareness of these technical limitations has led to misinterpretation of the result in eight dense cleavage stage embryos , whereas at protein level, the intensity of the POU5F1_iA and POU5F1_iB staining was not lower in the TE as compared with the ICM of blastocysts, in which the TE and the ICM displayed a comparable quality. Proteins are more related to the biological activity of a gene than transcripts. Therefore, we recommend looking for the localization of a protein rather than looking for the presence or amount of the transcripts when the material is scarce such as human oocytes and preimplantation embryos donated for research.. O% v) @# M- }8 E: A% w( P
$ h' i+ M! ^ W! iCONCLUSION# x2 K+ F3 A6 p% ?
, U9 C J' n. O' w r+ \The isoforms of POU5F1, POU5F1_iA and POU5F1_iB, display different temporal and spatial expression patterns during human preimplantation development. The main difference is their localization in a cell: POU5F1_iA behaves as a nuclear protein and POU5F1_iB as a cytoplasmic protein. The cytoplasmic localization of POU5F1_iB, assumed to be a transcription factor, implies other or no biological functions for POU5F1_iB. Hence, it is of great importance to define which of the isoforms is examined when performing POU5F1 expression studies. Regarding totipotency, the stemness properties of POU5F1 can be assigned to POU5F1_iA because hESCs do not express POU5F1_iB. Because POU5F1_iA is expressed in the TE and in oocytes and is not expressed in totipotent zygotes and blastomeres of early cleavage stage embryos, POU5F1_iA on its own is not an appropriate marker to identify totipotent cell populations in the human.
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4 L9 _+ t$ i' c! W" _, pDISCLOSURES; B' N6 E" r, ]
5 i* [7 q9 B$ _1 B' oThe authors indicate no potential conflicts of interest.
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: v$ L1 W! j' f$ y' V& N# WACKNOWLEDGMENTS
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We thank I. Mateizel, N. De Temmerman, and U. Ullmann of our Embryonic Stem Cell Laboratory, the laboratory staff of the Center for Reproductive Medicine, the Hematopoietic Stem Cell Laboratory, and the Follicle Biology Laboratory for putting the confocal microscope at our disposal. We also thank Prof. Dr. K. Sermon and Dr. M. De Rycke for their support in this project and M. Whitburn of the Language Education Center for proofreading the manuscript. The work was supported by grants from a Concerted Action of the "Vrije Universiteit Brussel" (Free University of Brussels).' z) X1 _. B2 N1 W, f3 T
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