Quantitative Oct4 Overproduction in Mouse Embryonic Stem Cells Results in Prolon - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:03 |只看该作者 |倒序浏览 |打印

作者：Valrie Camara-Clayette, Franoise Le Pesteur, William Vainchenker, Franoise Sainteny作者单位：INSERM U, IFR, Institut Gustave Roussy, Villejuif cedex, France




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      【摘要】" W, K. W5 b' z* N* q( a7 a) }* G
   The Oct4 transcription factor is essential for the self-renewal and pluripotency of embryonic stem cells (ESCs). Oct4 level also controls the fate of ESCs. We analyzed the effects of Oct4 overproduction on the hematopoietic differentiation of ESCs. Oct4 was introduced into ESCs via a bicistronic retroviral vector, and cells were selected on the basis of Oct4 production, with Oct4  and Oct42  displaying twofold and three- to fourfold overproduction, respectively. Oct4 overproduction inhibited hematopoietic differentiation in a dose-dependent manner, after the induction of such differentiation by the formation of day 6 embryoid bodies (EB6). This effect resulted from defective EB6 formation rather than from defective hematopoietic differentiation. In contrast, when hematopoiesis was induced by the formation of blast colonies, the effects of Oct4 depended on the level of overproduction: twofold overproduction increased hematopoietic differentiation, whereas higher levels of overproduction markedly inhibited hematopoietic development. This increase or maintenance of Oct4 levels appears to alter the kinetics and pattern of mesoderm commitment, thereby modifying hemangioblast generation. These results demonstrate that Oct4 acts as a master regulator of ESC differentiation.
      【关键词】 Embryonic stem cell Hematopoiesis Cell differentiation Transcription factor Mesoderm Gene expression. X! A8 y0 ~/ z  ~1 y$ V% q' p
   INTRODUCTION

Self-renewal is one of the major properties of stem cells. This property is required for continuous tissue regeneration and for the persistence of long-lived cells capable of promoting tissue homeostasis and repair.
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However, the different stem cells in the human body differ considerably in their self-renewal capacities. Hematopoietic stem cells (HSCs), the best characterized adult stem cells, have considerable but limited self-renewal capacities and cannot be grown as permanent cell lines. They therefore cannot be propagated and expanded in vitro, limiting their use in clinical practice. It remains unclear whether this limitation is an intrinsic feature of HSCs or whether it results from a lack of knowledge of the growth factors regulating self-renewal and differentiation potential. In contrast, embryonic stem cells (ESCs) display extensive self-renewal potential in vitro and can be propagated as cell lines, through symmetric mitosis. They can also be induced to differentiate into multiple lineages in vitro. ESCs are derived from the inner cell mass (ICM) and can generate all embryonic cell types. Our understanding of the molecular mechanisms governing ESC self-renewal and differentiation has recently increased substantially. Two endogenous transcription factors, Oct4 and Nanog, have been shown to play key roles in ESC self-renewal .# M. ?9 }( z/ {  E; l* P
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Oct4, a POU transcription factor, is produced in ESCs and epiblast and primordial germline cells .
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The mechanisms controlling the pluripotency and differentiation of ESCs are unclear, and Oct4 is just one of the molecules affecting the fate of ESCs. The presence of Oct4 in ESCs, but not in their differentiated derivatives, suggests that downregulation of Oct4 gene is a prerequisite for the induction of differentiation. A doubling in Oct4 production leads to the induction of mesodermal markers, suggesting that hematopoietic differentiation should also be modified . We studied the effects of Oct4 overexpression in ESCs on their hematopoietic differentiation of these cells. We demonstrated that the effects of Oct4 overproduction in ESCs depended on Oct4 levels, as previously reported during primordial germ layer specification, and on the differentiation pathways used to induce hematopoiesis from ESCs in vitro.

MATERIALS AND METHODS/ v/ r+ Y4 W* \, B
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ESC Lines
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The 129/Sv-derived wild-type (WT) D3 ESC line was kindly provided by F. de Sauvage (Genentech, Inc., South San Francisco, CA, http://www.gene.com).
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Growth and Differentiation of ESCs
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Cells were cultured on a monolayer of mitomycin C-inactivated mouse embryonic fibroblasts (MEFs) derived from day-14 Swiss mouse embryos. ESCs were maintained in an undifferentiated state by culture in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Cergy Pontoise, France, http://www.invitrogen.com) supplemented with recombinant LIF (1,000 U/ml; ESGRO; Abcys, Paris, http://www.abcys.fr), 15% fetal bovine serum (FBS; Perbio Science, Brebieres, France, http://www.perbio.com), 150 µM monothioglycerol (MTG; Sigma, Saint Quentin Fallavier, France, http://www.sigma-aldrich.com), 1% L-glutamine (Invitrogen), and 1% penicillin-streptomycin (PS; Invitrogen). ESCs were passaged every 2 days, and the culture medium was changed daily. Cultures were maintained at 37¡ãC in an atmosphere containing 5% CO2 and O2 (7% O2).7 d7 L3 i7 W& ?0 S3 \5 y6 v5 K! L

We used slightly modified versions of two methods initially described by Kennedy et al.  to study hematopoietic differentiation from ESCs. Undifferentiated ESCs were treated with trypsin and cultured for 1 hour in DMEM without LIF in cell culture dishes to eliminate adherent embryonic fibroblasts. ESCs were then plated in Iscove's modified Dulbecco medium (IMDM; Invitrogen) supplemented with 15% FBS, 450 µM MTG, 50 µg/ml L-ascorbic acid (Sigma), and 200 µg/ml transferrin (Sigma). Under these conditions, ESCs give rise to embryoid bodies (EBs). EBs were generated on day 6 of differentiation (EB6) by plating 500 cells per milliliter in bacterial-grade dishes. ESC-derived blast cell colonies (BL) were generating by plating 4,500 cells per milliliter on these dishes. After 3 days of culture, EBs in their third day of differentiation (EB3) were rinsed twice with 1x phosphate-buffered saline (PBS; Invitrogen) and centrifuged. The pellet was disrupted by trypsin treatment, and the cells were replated in 1% methycellulose (Fluka, Buchs, Switzerland, http://www.emarketlabo.com) in IMDM supplemented with 1% L-glutamine, 1% PS, 10% FBS (Perbio Science), 450 µM MTG, 25 µg/ml ascorbic acid, 200 µg/ml transferrin, and 5 ng/ml vascular endothelial cell growth factor (VEGF). We plated 75,000 EB3 in a volume of 1.5 ml in 35-mm-diameter bacterial-grade dishes. In these conditions, BL were detected and scored on the fourth day of culture.

Hematopoietic Progenitor Assays
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We quantified myeloid progenitors among BL or in EB6 as follows. Cells were collected, rinsed, centrifuged, and mechanically dissociated in IMDM and filtered through Millipore filters (Millipore Corporation, Billerica, MA, http://www.millipore.com) with 40-µm pores. Cells were plated in 1% methylcellulose in IMDM containing six growth factors (5 U/ml erythropoietin ). Hematopoietic colonies were counted after 7 days.
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Endothelial Cell Culture7 O4 `$ Y$ i8 v1 j. W, e% ?

On day 4, BL were collected, rinsed, centrifuged, dissociated, and filtered as described above. We determined the number of BL-derived endothelial foci by plating 0.5¨C4 x 103 cells in 12-well gelatin-coated tissue culture plates containing IMDM supplemented with 10% FBS, 10% horse serum (Roche Diagnostics, Mannheim, Germany, http://www.roche-diagnostics.com), 5 ng/ml VEGF, 10 ng/ml basic fibroblast growth factor, 100 µg/ml endothelial cell growth supplement (Sigma), 100 µg/ml sodium heparin (Sigma), 450 µM MTG, and 0.5% L-glutamine. Endothelial foci were counted after 3 days of culture.% L) e- Y. B2 x  \5 B; @
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Growth Factors

Murine recombinant IL-3, recombinant IL-1, IL-6, and VEGF were purchased from Abcys. Human EPO was a gift from Cilag (Levallois-Perret, France, http://www.janssen-cilag.fr). Human G-CSF was obtained from Bellon Laboratory (Neuilly sur Seine, France). Murine BHK/MKL-SCF-producing cell lines were also used as a source of SCF.
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Vector Construction4 o  p: T$ A1 [+ T5 ?/ F
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Mig-R1, the murine stem cell virus (MSCV) retroviral vector containing the encephalomyocarditis virus (ECMV) internal ribosomal entry sequence (IRES) and the coding sequence for enhanced green fluorescent protein (eGFP), were kindly provided by Warren S. Pear (University of Pennsylvania, Philadelphia). The Mig-R2 vector was constructed by replacing the ECMV-IRES cassette with the VEGF-IRES cassette. The murine Oct4 cDNA was generated from embryonic murine stem cell mRNA amplified and inserted into Mig-R2 (Mig-R2-Oct4).. \& U6 v9 t5 ^& \& l! S
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For dual-luciferase assays (Dual-Luciferase Reporter Assay System; Promega, Madison, WI, http://www.promega.com), the reporter vectors were constructed by subcloning six repeats of the octamer sequence downstream from the thymidine kinase promoter in the pGL3 vector containing the firefly luciferase reporter gene (Promega) (i.e., :pGL3HSTK(6X)). The octamer sequence used was reported by Schöler et al. . The pGL3 control plasmid, containing the ubiquitous SV40 (simian virus 40) promoter and enhancer sequences, and pGL3HSTK (plasmid without the octamer sequence) were used as positive and negative controls, respectively.
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ESC Infection

We infected 293T Phoenix-Eco cells stably transfected with the GAG-polymerase and ecotropic envelope sequence with an Oct4 retroviral supernatant containing vesicular somatitis virus glycoprotein particles. The supernatant was collected, filtered (0.45-µm pores), and concentrated by a factor of 100 by centrifugation through a Centricon 30 (Millipore Corporation). ESCs were washed, treated with trypsin, and plated in culture dishes for 1 hour in complete medium to remove adherent MEFs. They were then plated at a density 106 cells per milliliter of complete medium containing LIF and MTG, in bacterial-grade Petri dishes, in the presence of 4 µg of Polybrene per milliliter. Cells were infected by two incubations with retroviral supernatant for 4 hours each at a multiplicity of infection (MOI) of 10.

Real-Time Quantitative Polymerase Chain Reaction! ~* r( Y; Q$ r! c& A
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Primers and internal probes for the amplification of Oct4 and Hprt sequences were designed using Primer Express Software (Perkin-Elmer Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Polymerase chain reaction (PCR) was carried out with the ABI Prism GeneAmp 5,700 Sequence Detection System (Perkin-Elmer Applied Biosystems) using TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosystems) containing specific primers (1.2 µM) and the specific probe (0.1 µM). For each Oct4 fraction (Oct4-GFP  and Oct4-GFP2 ), Oct4 gene expression levels were expressed with respect to levels of expression for the Hprt housekeeping gene.  h% w+ i6 I5 P( {0 C0 k5 ?" D+ y

Luciferase Assays! Z; j; J- L( n; @  j
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For the dual-luciferase assay, 1 x 106 EBs were electroporated, at 220 V and 500 µF, with 10 µg pGL3HSTK(6X) or control vectors in 250 µl of PBS on day 4. Cells were then cultured in EB culture medium and harvested 48 hours after transfection by lysis in 100 µl buffer. Cell lysate (20 µl) was mixed with 100 µl of luciferase assay reagent, and firefly luciferase luminescence, reflecting the activity of the pGL3HSTK(6X) promoter, was immediately quantified in a luminometer (Lumax Industries, Inc., Altoona, PA, http://www.lumaxlighting.com). This reaction was then quenched, and the Renilla luciferase reaction was simultaneously initiated by adding 100 µl of Stop and Glo reagent (Promega) to the mixture. Firefly luciferase luminescence was used to normalize the amount of protein in each cell lysate." |- D& Y  m3 ]) }: ?  @, q( u
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Gene Expression Analysis
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RNA was isolated with the SV total RNA isolation system (Promega), which includes DNaseI treatment to eliminate contaminating genomic DNA. Poly (A) RNA was reverse-transcribed with Superscript II RNase H reverse transcriptase (Invitrogen). The specific primers used were as follows : Hprt, forward, 5'CACAGGACTAGAACACCTGC3'; reverse, 5'GCTGGTGAAAAGGACCTCT3', 249 bp; Zfp42, forward, 5'CAGGTTCTGGAAGCGAGTTC3'; reverse, 5'TTGAAATCCAGGGAGAAACG3', 385 bp; Brachyury, forward, 5'TCCAGGTGCTATATATTGCC3'; reverse, 5'GCTGCCTGTGAGTCATAAC3', 947 bp; Scl/tal1, forward, 5'ATTGCACACACGGGATTCTG3'; reverse, 5'GAATTCAGGGTCTTCCTTAG3', 321 bp; Flk1, forward, 5'CACCTGGCACTCTCCACCTTC3'; reverse, 5'GATTTCATCCCACTACCGAAAG3', 239 bp; Fgf5, forward, 5'AAAGTCAATGGCTCCCACGAA3'; reverse, 5'CTTCAGTCTGTACTTCACTGG3', 376 bp; Gata4, forward, 5'CCGAGCAGGAATTTGAAGAGG3'; reverse, 5'GCCTGTATGTAATGCCTGCG3', 496 bp; CoupTFI forward 5'AGCCATCGTGCTATTCACG3'; reverse, 5'TTCTCACCAGACACGAGGTC3', 570 bp; Dab2 forward 5'GGCAACAGGCTGAACCATTAGT3'; reverse 5'TTGGTGTCGATTTCAGAGTTTAGAT3', 283 bp; Gata6 forward 5'GCAATGCATGCGGTCTCTAC3'; reverse 5'CTCTTGGTAGCACCAGCTCA.' ]& d( h3 A$ ?1 ~2 b2 R
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The specific primers used for quantitative PCR were the following: Hprt: forward primer 5'GCCCTTGACTATAATGAGTACTTCAGG3', reverse primer 5'TCAACAGGACTCCTCGTATTTGC3'; TaqMan probe 5'TCATTAGTGAAACTGGAAAAGCCAAATACAAAGCC3'; OCT4: forward primer 5'CTCACCCTGGGCGTTCTCT3', reverse primer 5'AGGCCTCGAAGCGACAGA 3', TaqMan probe 5'TGGAAAGGTGTTCAGCCAGACCACC3'; Nanog: forward primer 5'TGCTACTGAGATGCTCTGCACA3', reverse primer 5'TGCCTTGAAGAGGCAGGTCT3', TaqMan probe 5'AGGCTGCCTCTCCTCGCCCTTC3'.+ C! I$ L# t' ~" e5 N

RESULTS
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Retroviral Expression in ESCs) m! O7 o( w# m: w6 }; ]

We generated an ecotropic supernatant for the transduction of ESCs with Oct4. A vector containing a VEGF IRES and the Oct4 gene under the control of an MSCV promoter was constructed, giving levels of Oct4 gene expression proportional to GFP fluorescence intensity. A vector containing the GFP gene alone was used as a control (Fig. 1A). ESCs were reproducibly efficiently infected, as shown by flow cytometry 48 hours after infection (Fig. 1B). With an MOI of approximately 10, we obtained more than 35% GFP  cells. We characterized the effects of Oct4 by sorting ESCs into two fractions based on GFP fluorescence intensity: one with low to moderate levels of GFP (Oct4-GFP  ESCs, 36%) and the other with high levels of GFP (Oct4-GFP2  ESCs, 3.5%). Two ESC populations transduced with the control vector were also sorted in a similar manner on the basis of GFP fluorescence intensity, giving the GFP -only (45%) and GFP2 -only (7%) fractions (Fig. 1B). We could not maintain ESC lines overproducing Oct4 in continuous culture in standard conditions (feeder, serum, and LIF), because they spontaneously differentiated and lost their self-renewal capacities. We therefore transduced and sorted new ESCs for each experiment investigating the hematopoietic potential of these cells.
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Figure 1. Retroviral construct and GFP detection in embryonic stem cells (ESCs). (A): Retroviral construct. The retroviral vector was derived from the Migr retrovirus in which the encephalomyocarditis virus (ECMV) IRES has been replaced by the IRES from vascular endothelial cell growth factor. (B): GFP levels in ESCs. ESCs were infected with either a control retrovirus or an Oct4-GFP retrovirus. GFP levels were assessed by flow cytometry 48 hours after infection. Approximately 36% of ESCs infected with the Oct4-GFP retrovirus were GFP , and 3.5% were GFP2 . With the GFP control retrovirus, 45% of cells were GFP  and 7% GFP2 . Solid peak (gray): control ESCs (wild-type); solid line: control virus containing GFP alone; dotted line: Oct4-GFP retrovirus. Abbreviations: GFP, green fluorescent protein; IRES, internal ribosomal entry sequence; LTR, long terminal repeat.
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Oct4 Gene Expression in ESCs During the Induction of Differentiation

Oct4 plays an important role in self-renewal and cell fate determination in ESCs and the embryo. Oct4 gene expression is downregulated by retinoic acid in ESCs upon differentiation . In contrast, the pattern of Oct4 gene expression during the induction of hematopoietic differentiation from ESCs has not been defined. We used real-time quantitative PCR to track changes in Oct4 levels during the differentiation of ESCs into the hematopoietic lineage. We used two methods to induce differentiation: the formation of day-3 EBs (EB3) followed by BL (three-step method) and the formation of day-6 EBs (EB6) (two-step method). The three-step method was used to study primitive bipotent progenitors (hemangioblasts), whereas the two-step method targets more committed progenitors with hematopoietic potential only. In both cases, levels of the Oct4 transcript decreased and remained at the detection threshold in BL and EB6 (Fig. 2). Endogenous Oct4 gene expression decreased, the kinetics of this decrease depending on the method used to induce differentiation (two- or three-step methods).7 E) z% G$ u- I# K$ f
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Figure 2. Schematic representation of two methods used to induce hematopoietic differentiation. Quantitative endogenous Oct4 mRNA produced during embryonic stem cell (ESC) hematopoietic differentiation. Three-step method: 0 day: wild-type ESCs on feeder cells; 3 days: EB3; 7 days: BL; 14 days: BL-derived hematopoietic colonies. Two-step method: 6 days: EB6; 13 days: EB6-derived hematopoietic colonies. Oct4 mRNA level was determined by quantitative reverse transcription-polymerase chain reaction, using TaqMan oligonucleotides, and was expressed with respect to mRNA levels for the Hprt housekeeping gene (mean of three independent experiments). Abbreviations: BL, blast colonies; EB3, embryoid bodies in their third day of differentiation; EB6, embryoid bodies in their sixth day of differentiation; LIF, leukemia inhibitory factor; N, mRNA not detectable; VEGF, vascular endothelial cell growth factor.7 [1 E+ ]9 [4 j/ Y/ _
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We evaluated transduction efficiency by comparing total Oct4 levels during cell differentiation in control ESCs and cell lines transduced with the retroviral vector, by real-time quantitative PCR (Fig. 3A). Oct4-GFP  and Oct4-GFP2  ESCs had twice as many and three to four times as many Oct4 transcripts, respectively, as did ESC control cells (GFP only). During differentiation, the level of Oct4 transcription decreased in all cases, but differentiated cells derived from Oct4-GFP  and Oct4-GFP2  ESCs had higher Oct4 transcript levels than did control cells. Indeed, Oct4-GFP  and Oct4-GFP2  BL had twice as many and three to four times as many transcripts, respectively, as did control (WT, GFP , and GFP2 -only). However, in the two-step method, retroviral expression was strongly silenced at the EB6 stage and Oct4 transcript levels were similar in cells derived from Oct4-GFP  and Oct4-GFP2  ESCs. During hematopoietic differentiation, weak Oct4 levels were detected (approximately 10% that in ESCs) but retroviral transcripts were consistently higher than endogenous levels at the same stage. Endogenous Oct4 transcripts were undetectable in hematopoietic cells derived from control ESCs.
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Figure 3. Detection of Oct4 during ESC differentiation after transduction with retrovirus. (A): Total amount of Oct4 mRNA in infected cells during hematopoietic differentiation. (a) ESCs, (b) three-step hematopoietic differentiation method, and (c) two-step hematopoietic differentiation method. Control cells correspond to the mean of wild-type, GFP , and GFP2 -only cells. (B): Expression of Zfp42/Rex1, an Oct4 target gene, during differentiation, as assessed by reverse transcription-polymerase chain reaction. One profile representative of three independent experiments is shown. (C): Luciferase activity: Embryoid bodies on day 4 were transiently electroporated with pGL3HSTK or pGL3HSTK(6X). After 48 hours, luciferase activities were evaluated in cells corresponding to day-6 embryoid bodies and expressed in arbitrary units. The amount of protein in each cell lysate was normalized based on Renilla luciferase activity, following co-transfection with the pRL-TK vector. pGL3HSTK(6X) was three times more active than pGL3HSTK. Three independent experiments were carried out in duplicate. Abbreviations: ESC, embryonic stem cell; GFP, green fluorescent protein; ND, not determined; BL, blast colonies.
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We investigated whether Oct4 mRNA was efficiently translated into protein, by investigating the expression of Zfp42, a direct target gene of Oct4, encoding the transcription factor Rex1, during hematopoietic differentiation. Rex-1 levels decrease rapidly during ESC-induced differentiation . Reverse transcription-PCR showed that FGF4 mRNA was present in ESCs, its level gradually decreasing until the formation of day-4 EBs in GFP -only control cells. In contrast, FGF4 mRNA continued to be detected in Oct4-GFP  cells until the formation of day-5 EBs (Fig. 3B, right). Real-time quantitative PCR showed that Nanog mRNA was present in ESCs, its level decreasing during hematopoietic differentiation in GFP-only control cells. Oct4-GFP  and Oct4-GFP2  cells displayed a kinetic pattern similar to GFP-only control cells but with lower mRNA levels in ESCs and EB3 cells. The reason for this difference is unclear but may involve a negative effect of Oct4 on the Nanog promoter as a function of the ratio between Sox2, Oct4, and/or perhaps another transcription factor. These data suggest that in our conditions Oct4 protein is overproduced at least until EB3 formation, and this overproduction modifies the expression of target genes. We then tried to detect Oct4 transcriptional activity directly, using an artificial promoter containing the octamer binding site (pGL3HSTK(6X)) upstream from the luciferase gene. We used pGL3HSTK as a negative control (background). In EB6 derived from Oct4-GFP  ESCs, pGL3HSTK(6X) activity was three times higher than pGL3HSTK plasmid, with a p value of .008. In contrast, no specific activity was detected in control EB6 (Fig. 3C). These observations strongly suggest that the Oct4 mRNA was efficiently translated into a functional protein during differentiation, until the formation of EB6, derived from ESCs overexpressing Oct4.

Consequences of Enforced Oct4 Production for ESC Hematopoietic Differentiation
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Previous studies showed that artificially increasing Oct4 levels in ESCs led to an increase in the expression of endodermal and mesodermal markers . We were interested in the effects of Oct4 on hematopoietic differentiation, so we first determined whether the overproduction of Oct4 in ESCs influenced the formation of EBs, the first step in the mesodermal differentiation of ESCs, and the content of EBs in hematopoietic progenitor cells. The cloning efficiency of ESCs in EB6 decreased with increasing Oct4 expression (Fig. 4A). Oct4-GFP  and Oct4-GFP2  ESCs were associated with 25% and 50% fewer EB6, respectively, than the controls. In contrast, the production of GFP alone had no effect on EB formation (data not shown). We determined the progenitor cell content of these EB6 in methylcellulose in the presence of six growth factors. Similar numbers of GFP-only and Oct4-GFP overproducing EBs were replated for hematopoietic differentiation. Myeloid progenitor content was identical in EB6 derived from control and Oct4  ESCs but was significantly lower (40% lower) in Oct4-GFP2  ESCs (Fig. 4B). However, this decrease was not associated with qualitative changes in the type of hematopoietic progenitor (data not shown). Thus, Oct4 overproduction modifies the fate of ESCs, because high Oct4 levels (associated with prolonged Oct4 production) slightly inhibited hematopoietic differentiation.
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Figure 4. Consequence of enforced Oct4 production for hematopoietic differentiation. (A): Effect of Oct4 overproduction on EB6 cloning efficiency. Oct4  and Oct42  ESCs displayed 25% and 50% decreases, respectively, in EB6 cloning efficiency. (B): Oct4 overproduction modified the hematopoietic potential of EB6 (expressed as a percentage of control including GFP  and GFP2 -only, GFP = 100% ¡À SEM). The number of myeloid progenitors in Oct4-GFP  cells was identical to that of the control, whereas that in Oct4-GFP2  cells was 25% lower. (C): A doubling of Oct4 levels in ESCs resulted in an increase in the cloning efficiency of BL-CFC, whereas higher levels of Oct4 expression (increase by a factor of three to four) led to 50% of BL-CFC colony formation. (D): The hematopoietic potential of BL-CFC was affected only by the larger increase in Oct4 levels. All results are expressed with respect to control cells (mean of wild-type, GFP -only, and GFP2 -only cells). *, significant difference compared with control. Abbreviations: BL-CFC, blast colony-forming cell; EB6, embryoid bodies in their sixth day of differentiation; ESC, embryonic stem cell; GFP, green fluorescent protein.& {8 P7 Y- e2 }( W# W$ q7 f

We then determined the consequences of Oct4 overproduction for blast colony-forming cells (BL-CFC). BL-CFC-derived colonies were obtained from EB3 in methylcellulose cultures containing VEGF. Cultures from Oct4-GFP  ESCs yielded 2.5 times as many BL as did control cells (GFP-only) (Fig. 4C). Surprisingly, higher levels of Oct4 production (Oct4-GFP2  ESCs) inhibited BL-CFC colony formation by 35% with respect to control GFP-only cells (Fig. 4C). The number of BL-CFC therefore differed by a factor of 3.5 between Oct4-GFP  and Oct4-GFP2  ESCs, suggesting that the development of this progenitor is strongly regulated by Oct4 levels. The hematopoietic progenitor content of BL did not differ markedly between control ESCs, Oct4-GFP , and Oct4-GFP2  ESCs, but slightly fewer (25% fewer) hematopoietic progenitors were observed for Oct4-GFP2  ESCs (Fig. 4D). In contrast, EBs from Oct4 and control ESCs had similar secondary EB formation capacities (data not shown). BL derived from Oct4-GFP  and Oct4-GFP2  ESCs were unable to generate secondary colonies.

Thus, prolonged Oct4 overproduction in ESCs primarily disrupts the development of BL-CFC, having only a moderate effect on later stages of hematopoietic differentiation. High levels inhibited hematopoietic differentiation (25%; Fig. 4D), whereas moderate overproduction had no effect. We then analyzed the endothelial potential of BL. We compared the endothelial potential of Oct4/GFP BL cells with that of control cells with similar levels of GFP fluorescence. In three independent experiments, GFP-only and Oct4-GFP BL gave rise to endothelial cells in limiting dilution assays, in the same way (Fig. 5). Similar numbers of endothelial cells foci for cells with moderate and high levels of Oct4 were counted. High levels of GFP production in the control also seemed to inhibit endothelial cell differentiation, especially if plates were seeded with 4,000 BL cells. Thus, increases in Oct4 levels influenced hematopoietic progenitors in the same way (20%¨C25% inhibition) regardless of the method used to induce cell differentiation (three- or two-step method). No effect on endothelial differentiation from hemangioblasts was observed.

Figure 5. Consequences of enforced Oct4 production for the endothelial differentiation potential of BL-CFC. No difference was observed between Oct4 and GFP control. Error bars indicate SE of three experiments performed in triplicate. Abbreviations: BL-CFC, blast colony-forming cell; GFP, green fluorescent protein.. K; ?) n' ]0 p( |0 h9 E

Gene Expression Analysis During Hematopoietic Differentiation5 P  j3 J) c$ O4 H2 H( b& h
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We examined the expression profiles of two genes involved in the earliest stages of endothelial and hematopoietic development (Flk1 . During the differentiation of Oct4-GFP  ESCs, Flk1 gene expression levels were higher in day-3 EBs than in the control whereas SCL expression was not affected, suggesting that Oct4 overproduction initially favors mesodermal differentiation (Fig. 6). We were unable to study Oct42  ESCs before day 6, because too few such cells were available after retroviral infection. In EB6 and in hematopoietic colonies derived later from Oct4-GFP2  ESCs, we observed no change in SCL or Flk1 gene expression.) A# u( Q8 J2 E) ?7 Y5 j
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Figure 6. Analysis of gene expression by reverse transcription-polymerase chain reaction, during the differentiation of ES cells overproducing Oct4. Two controls (GFP -only and GFP2 -only) were used corresponding to the GFP levels similar to those of Oct4  and Oct42  cells. We obtained polymerase chain reaction products of the following sizes: Hprt, 249 bp; SCL/tal-1, 321 bp; Flk1, 239 bp; Gata4, 496 bp; Fgf5, 376 bp; Brachyury, 947 bp; and Gata6, coupTfI, 570 bp. Results from one of three independent experiments are shown. Abbreviations: BL, blast colonies; EB, embryoid body; ES, embryonic stem; GFP, green fluorescent protein.2 z- s4 u8 e. _9 E/ r
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We analyzed the expression profile of marker genes for the various germ layers to confirm this effect on mesodermal differentiation. We detected no change in the expression pattern of genes restricted to the primitive ectoderm and parietal/visceral endoderm germ layers (Fig. 6) . Brachyury mRNA was more abundant in EB3 derived from Oct4-GFP  ESCs than in EB3 derived from the control. However, the kinetic profile of Brachyury expression did not differ markedly between the two types of cell; this mRNA was not detected in day-6 EBs derived from either type of ESC. In contrast, during the differentiation of Oct4-GFP2  ESCs, Brachyury gene expression persisted in EB6 and BL (Fig. 6). These expression kinetics for brachyury suggest that Oct4 can slow down the differentiation of mesoderm cells.* j7 V+ N7 n# y8 u# y
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Thus, the marked differences in BL-CFC colony formation between Oct4-GFP  and Oct4-GFP2  cells and controls may result from differences in differentiation kinetics. We therefore investigated the pattern of BL-CFC development from EBs. It has been shown that the potential to form BL-CFC is limited to a very short period of EB development. Kinetic analysis of the developmental potential of BL-CFC confirmed that EBs derived from Oct4-GFP  ESCs gave rise to two to three times as many BL as the control, whereas Oct4-GFP2  ESCs gave rise to only half as many BL as the control (Fig. 7). No marked differences in the kinetics of BL-CFC development were found between the three types of ESCs, although EBs began to form slightly later (up to day 3.5) for Oct4-GFP  ESCs. In contrast, BL-CFC developed from Oct4-GFP2  ESCs with kinetics similar to the control. Thus, low levels of Oct4 overproduction slightly prolonged the window of development of BL-CFC (day 3.8, 1 GFP-only control BL against 11 Oct4-GFP  BL), whereas higher levels of overproduction had no effect. Finally, we showed that the number of secondary EBs was strictly similar in the different conditions (Oct4-GFP  or Oct4-GFP2  vs. GFP-only control).

Figure 7. Analysis of the kinetics of blast colony-forming cell development. EBs were cultured for various times (2¨C4 days), dissociated, and replated on semisolid medium containing vascular endothelial cell growth factor. Blast colony (BL) and secondary EBs were counted. The number of BL was expressed per 105 EBs (three independent experiments performed in triplicate). Oct4-GFP  and Oct4-GFP2  were compared with GFP control (mixture of GFP -only and GFP2 -only cells). Abbreviations: EB, embryoid body; GFP, green fluorescent protein.

DISCUSSION3 ~% Z8 |" t3 S

In this report, we studied the effects of Oct4 overproduction on hematopoietic differentiation from ESCs. Oct4 overproduction was induced using a bicistronic Oct4 and eGFP-containing retroviral vector with high infection efficiency in ESCs. This made it possible to select cells with two different eGFP levels, corresponding to a doubling and tripling of Oct4 production. As previously reported, ESCs overproducing Oct4 lost their self-renewal capacity in the presence of LIF and underwent spontaneous differentiation. We had to infect new ESCs for each differentiation experiment because no system for inducing Oct4 overproduction was available. The retroviral vector used gave high levels of Oct4 production in ESCs, but Oct4 levels fell continually during differentiation. This decrease may be due to silencing of the MSCV promoter during differentiation, by methylation or by more profound changes to chromatin . However, Oct4 transcripts were detected in BL, in EB6, and in hematopoietic colonies during differentiation but at only 30%, 10%, and 5%, respectively, of the level in ESCs. In these conditions, retroviral transcripts were not constitutively produced, but they were consistently produced in larger amounts than endogenous Oct4 transcripts at the same stage. Retroviral Oct4 production during differentiation seemed to decrease with kinetics similar to those reported for the endogenous.+ E2 x2 Z$ G4 l$ T5 E7 Z
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In vitro experiments have shown that increasing endogenous Oct4 levels in ESCs by more than 50% leads to the expression of endodermal and mesodermal markers. These data were obtained with ESCs maintained in culture with LIF .* D% I. Q) R0 W7 n7 ?* g

With the technique by which ESCs were induced to undergo differentiation into hematopoietic colonies via the VEGF-driven generation of BL from EB3, marked differences were observed, depending on the level of Oct4 overproduction. A doubling of Oct4 production markedly increased BL formation, whereas BL formation was inhibited at higher levels of Oct4 production. BL-CFC, which give rise to BL, are mesenchymatous progenitors with a bipotent endothelial and hematopoietic potential theoretically corresponding to a hemangioblast . No modification of FLK1¨C cells was found that could lead to the generation of BL or the secretion of factors with the capacity to modulate FLK1  potential (data not shown). In addition, although EB3 secreted VEGF into the blast cell culture medium, the overproduction of Oct4 in these cells did not lead to BL formation unless exogenous VEGF was provided.
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We found that the window of BL Oct4  generation was extended (lasting until day 3.5), suggesting a change in embryonic lineage determination (Fig. 7). These data suggest that BL-CFC in Oct4-GFP  cell culture should be more than bipotent. Higher levels of Oct4 overproduction may inhibit BL-CFC generation by profoundly affecting mesodermal differentiation. During the differentiation of ESCs, Brachyury, an early mesoderm marker absent from ESCs, was detected in EB3 but not in BL ." G& z* _' d% [7 n3 J9 t8 N& Y' _
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CONCLUSION# ~: K6 s4 Z; h, P

Our study showed that the effects of Oct4 overproduction on mesodermal progenitors from ESCs differ according to the amount of Oct4 produced. High levels of overproduction markedly inhibit hematopoietic differentiation, whereas low levels of Oct4 production may increase the number of bipotent progenitors (BL-CFC) without affecting potency. In cells producing small amounts of Oct4, prolongation of the period of BL-CFC development should be seen as indicating a much larger change in potency. It would be interesting to determine which type of potency is affected.
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DISCLOSURES
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The authors indicate no potential conflicts of interest.& |; ?' k, P" V

ACKNOWLEDGMENTS$ d' c/ S5 X1 x/ W  y3 \

This paper is dedicated to F. Le Pesteur who died in 2003. We thank Philippe Rameau and Yann Lecluse for cell sorting experiments, Isabelle Godin for helpful criticism, and Lorna Saint Ange for editing. This work was supported by grants from the INSERM and la Ligue Nationale contre le Cancer (¨¦quipe labelis¨¦e 2004). V.C.-C. received a grant from the Fondation sur la Leuc¨¦mie.
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