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First published online December 6, 2007
Stem Cells Vol. 26 No. 2 February 2008, pp. 445 -454
doi:10.1634/stemcells.2007-0553; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Pluripotential Reprogramming of the Somatic Genome in Hybrid Cells Occurs with the First Cell Cycle

Dong Wook Hana,b, Jeong Tae Doa, Luca Gentilea, Martin Stehlinga, Hoon Taek Leeb, Hans R. Schölera

aDepartment of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany;
bDepartment of Bioscience and Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul, South Korea

Key Words. Oct4 • Reprogramming • Fusion • DNA methylation • Cell cycle

Correspondence: Correspondence: Hans R. Schöler, Ph.D., Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany. Telephone: 49-251-70365-300; Fax: 49-251-70365-399; e-mail: schoeler{at}mpi-muenster.mpg.de

Received on July 11, 2007; accepted for publication on November 19, 2007.

First published online in STEM CELLS EXPRESS  December 6, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The fusion of pluripotent embryonic cells with somatic cells results in reprogramming of the somatic cell genome. Oct4-green fluorescent protein (GFP) transgenes that do not contain the proximal enhancer (PE) region are widely used to visualize reprogramming of the somatic to the pluripotent cell state. The temporal onset of Oct4-GFP activation has been found to occur 40–48 hours postfusion. We asked whether activation of the transgene actually reflects activation of the endogenous Oct4 gene. In the current study, we show that activation of an Oct4-GFP transgene that contains the PE region occurs within 22 hours of fusion. In addition, demethylation of the Oct4-GFP transgene and that of the endogenous Oct4 and Nanog genes was found to occur within 24 hours of fusion. As this timing corresponds with the timing of cell cycle completion in embryonic stem cells and fusion hybrids (~22 hours), we postulate that pluripotential reprogramming of the somatic cell genome begins during the first cell cycle after the fusion of a somatic cell with a pluripotent cell and has been completed by day 2 postfusion.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Reprogramming of the genome of somatic cells can be accomplished by a variety of methods, contributing to an enhanced understanding of early embryonic development and, ultimately, to an improvement of therapies for early developmental disorders. Somatic cell nuclear transfer (SCNT), a well-known and widely used method of reprogramming, involves replacement of the nucleus of an oocyte with that of a somatic cell [1]. Another reprogramming method involves the fusion of a somatic cell with a pluripotent cell to produce a fusion hybrid cell, obviating the use of oocytes and the attendant ethical considerations [2, 3]. By this method, fusion hybrids acquire various features of pluripotency from the embryonic stem cell fusion partner, including prolonged self-renewal ability, expression of pluripotency-specific marker genes, lack of expression of tissue-specific genes, developmental potential to contribute to the development of all three germ layers of the soma as well as to the germ cell lineage, and an undifferentiated epigenetic cell state [2].

The reprogramming status of somatic cells can be determined by monitoring the activation of Oct4-green fluorescent protein (GFP) transgene expression [4–7]. Oct4, a member of the pit.oct.unc transcription factor family, is required for the maintenance of embryonic cell pluripotency and self-renewal [8, 9]. Since the Oct4 gene is inactive in somatic cells but is active in pluripotent cells, activation of Oct4-GFP transgene expression (visualized by the presence of a GFP fluorescence signal) has been considered to reflect the reprogramming of somatic cells [10, 11]. In our previous study, reactivation of Oct4-GFP transgene expression was first observed on day 2 (around 44–48 hours) after fusion of embryonic stem (ES) cells with neurosphere cells [5]. This finding is consistent with previous studies, wherein Oct4-GFP-positive colonies were first detected at around 40–48 hours postfusion [7, 12]. In mouse cloning, we also detected the Oct4-GFP fluorescence signal as early as at the four- to eight-cell stage, corresponding to day 2 (48 hours) after nuclear transfer, consistent with the onset of Oct4 expression in normally fertilized embryos [4]. Taken together, these findings indicate that the Oct4-GFP transgene could be reactivated at approximately 44–48 hours following the inception of SCNT or cell fusion. However, reactivation of an Oct4-GFP transgene may not reflect the reprogramming status of the endogenous Oct4 gene, and, hence, of the entire genome of somatic cells during fusion-induced reprogramming and cloning. We know that fusion hybrids normally divide every 24 hours, as do their pluripotent fusion partner cells, and that epigenetic modifications required for reprogramming, including DNA methylation as well as histone acetylation and methylation [13], occur concomitant with or prior to DNA replication and subsequent to cell division [14–16]. It is therefore entirely possible that the reprogramming of somatic cell genome occurs earlier than previously anticipated.

The aim of the current study was to determine (a) the temporal onset of somatic cell genome reprogramming following fusion of somatic cells with pluripotent cells, and (b) whether reactivation of Oct4-GFP transgene expression indeed reflects the reprogramming status of the endogenous Oct4 gene from the somatic cell partner in the fusion hybrids. To this end, we generated F9 embryonal carcinoma (EC)-neural stem fusion hybrids, using two different neural stem cell (NSCs) lines that contain different Oct4-GFP transgenes, and conducted a temporal analysis of somatic cell genome reprogramming. We found that an Oct4-GFP transgene containing all the Oct4 regulatory elements—including the distal enhancer (DE), proximal enhancer (PE), and proximal promoter (PP)—could be reactivated within 22 hours of fusion of F9 EC cells with neural stem cells. We also compared the reprogramming of endogenous Oct4 gene with that of exogenous Oct4-GFP transgene following fusion.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mice
All mouse strains were bred and housed at the mouse facility of the Max Planck Institute (MPI) or were bought directly from Harlan (Indianapolis, http://www.harlan.com) or Jackson Immunoresearch Laboratories (West Grove, PA, http://www.jacksonimmuno.com). Animal handling was performed in accordance with the animal protection guidelines of the MPI.

Cell Culture
F9 EC cells were grown on gelatin-coated (0.1% in phosphate-buffered saline [PBS]) dishes in standard EC cell media, high-glucose Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) containing 15% fetal calf serum (Gibco-BRL), 1 x penicillin/streptomycin/glutamine, and 1 x nonessential amino acids (Gibco-BRL). For the derivation of NSCs, brain tissue was collected from 12.5- to 16.5-days post coitum (dpc) OG2/ROSA26 and GOF18 heterozygous female mice according to a previous protocol [17]. Isolated NSCs were grown in DMEM-Ham's F-12 medium (Gibco-BRL) supplemented with 20 ng/ml epidermal growth factor, 20 ng/ml basic fibroblast growth factor, B27 supplement (Gibco-BRL), 8 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin.

Cell Fusion and Detection of GFP-Positive Cells
Pluripotent EC cells and NSCs were fused according to our previous protocol [17]. Briefly, F9 EC cells were mixed with NSCs in a 1:1 ratio and then washed in PBS. The mixture was centrifuged at 130g for 5 minutes, and 1 ml of a prewarmed solution of 50% polyethylene glycol (PEG) 1500 (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) was added to the cell pellet over 1 minute. An additional 20 ml of DMEM was added to the cell suspension over 5 minutes, with constant stirring. The cells were centrifuged at 130g for 5 minutes to remove PEG, washed gently with DMEM, and cultured in EC cell medium containing leukemia inhibitory factor. The GFP-positive cells in culture dishes were visualized as green cells under fluorescence microscopy (Nikon, Tokyo, http://www.nikon.com).

RNA Extraction, cDNA Synthesis, and Real-Time Reverse Transcription-Polymerase Chain Reaction
Cells that were sorted by fluorescence-activated cell sorting (FACS) for real-time reverse transcription (RT)-polymerase chain reaction (PCR) were processed according to the manufacturer's instructions. RNA purity and concentration were determined using the Bioanalyzer RNA 6000 (Agilent Technologies, Palo Alto, CA, http://www.agilent.com). Complementary DNA synthesis was performed using the High Capacity cDNA Archive Kit (Applied Biosystems GmbH, Darmstadt, Germany, http://www.appliedbiosystems.com) according to the manufacturer's instructions. Transcript levels were determined using the ABI Prism Sequence Detection System 7900HT (Applied Biosystems) and the ready-to-use 5'-nuclease Assays-on-Demand. Amplification curves and gene expression were normalized to the internal standard beta-actin (BAct), whose expression did change upon cell fusion. Oligos for real-time RT-PCR are indicated in the supplemental online Materials and Methods. Three replicates were used for each real-time PCR; an RT– blank and a no-template blank served as negative controls. Quantification was normalized to the endogenous BAct gene within the log-linear phase of the amplification curve obtained for each probe/primer set using the {Delta}{Delta}CT method (ABI Prism 7700 Sequence Detection System, user bulletin 2). The theoretical background of the real-time PCR is described extensively in a previous publication [18].

Bisulfite Sequencing Analysis
To distinguish the methylated from the unmethylated CG dinucleotides, genomic DNA was treated with sodium bisulfite to convert all unmethylated cytosine residues to uracil residues using the One Day MSP kit (In2Gen, Seoul, Korea, http://www.in2gen.com/English/main.htm) according to the manufacturer's protocol. Briefly, purified genomic DNA (0.5–1 µg) was denatured with 3 N sodium hydroxide (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at 37°C for 10 minutes. Sodium bisulfite (5 M) was then added to the DNA, and the mixture was incubated at 50°C for 16 hours in the dark. Following desulfonation, neutralization, and desalting, the modified DNA was diluted with 20 µl of distilled water. Subsequently, bisulfite PCR amplification was carried out using aliquots of 1 µl of modified DNA for each PCR.

PCR Amplification and Sequencing of the Bisulfite-Treated Genomic DNA
In the current study, the PP and PE regions of the Oct4 gene, including the PP regions of the endogenous and exogenous Oct4 loci, and the promoter region of Nanog were analyzed to monitor changes in the methylation status of fusion hybrids. All genomic regions in the fusion hybrids were amplified after bisulfite treatment using a nested primer approach. PCR amplifications were performed using SuperTaq polymerase (Ambion, Austin, TX, http://www.ambion.com) in a total volume of 25 µl. All PCR amplifications consisted of a total of 50 cycles of denaturation at 94°C for 30 seconds, annealing at the appropriate temperature for each target region for 30 seconds, and extension at 72°C for 30 seconds, with an initial denaturation at 94°C for 5 minutes and a final extension at 72°C for 10 minutes. Three microliters of product from the first round of PCR was used in the second round of PCR. The amplified products were verified by electrophoresis on 1% agarose gel. PCR products were subcloned using the PCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's protocol. Reconstructed plasmids were purified using the QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany, http://www1.qiagen.com), and individual clones were sequenced (GATC-Biotech, Konstanz, Germany, http://www.chemeurope.com). Clones were accepted only if (a) there was ≥90% cytosine conversion, and (b) all possible clonalities were excluded based on the criteria from BiQ Analyzer software (Max-Planck Society, München, Germany, http://biq-analyzer.bioinf.mpi-sb.mpg.de/). At least 10 replicates were performed for each selected genomic region in the fusion hybrids, and at least three separate bisulfite treatments were conducted to confirm each result.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Gradual Demethylation of the Oct4 Proximal Promoter
NSCs were isolated from OG2+/–/ROSA26+/– double transgenic female embryos (14.5–16.5 dpc) and fused with F9 EC cells using PEG. As a fusion control, we generated hybrids with two different kinds of F9 EC cells: one with a cytomegalovirus-GFP transgene and the other with a Pgk-neo/lacZ transgene. The phenotype of control hybrids was typical of hybrid cells, including the expression of both transgenes (GFP and neo/lacZ) and the unaltered epigenetic status of Oct4 following fusion, indicating that the epigenetic modifications observed in EC-somatic cell hybrids (Figs. 2, 3, 5, and 6) are not an artifact of the experimental conditions (supplemental online Fig. 1). The Oct4-GFP transgene used was under the control of the Oct4 promoter lacking the PE region, and the neo/lacZ transgene used was under the control of a ubiquitously active promoter. In previous studies, Oct4 expression from the somatic Oct4-GFP transgene had been detected 40–48 hours after the fusion of somatic cells with embryonic stem cells [5, 6, 12]. In the current study, GFP-positive cells were also first detected around 44–48 hours after the fusion of F9 EC cells with OG2+/–/ROSA26+/– NSCs (OG2 hybrids). The OG2 hybrid cells were morphologically similar to F9 EC cells and stained positive for 5-bromo-4-chloro-3-indolyl-β-D-galactoside (Fig. 1A). We also analyzed alterations in gene expression of OG2 hybrids by real-time RT-PCR. GFP-positive OG2 hybrids were sorted by FACS at different time points of growth—days 2, 4, 6, and 9 postfusion—to eliminate contamination with cells that had not been reprogrammed. Expression of the pluripotency-specific marker genes Oct4 and Nanog, which is not detected in NSCs, was increased in the OG2 hybrids comparable to that in F9 EC cells. In contrast, expression of the neural stem and progenitor cell marker Nestin, which is highly expressed in NSCs but not expressed in F9 EC cells, was reduced from day 2 postfusion onward and was subsequently comparable to that in F9 EC cells. Sox2 expression is comparable in F9 EC cells and NSCs and unchanged in OG2 hybrids. Based on these findings, we concluded that following fusion of NSCs with F9 EC cells, the genome state of the former could be reprogrammed to that of the latter (Fig. 1B). The effects of epigenetic modifications during genome reprogramming were examined by monitoring the methylation status of the PP of Oct4 in the OG2 hybrids. In our previous study, we had analyzed the reprogramming pattern of the PE and found that this locus was completely demethylated by 48 hours postfusion (unpublished data). In mammalian cloning, the onset of Oct4 expression (visualized by Oct4-GFP fluorescence signal) coincides with the four- to eight-cell stage [4], although further demethylation of the Oct4 PP is gradual and is only comparable to that of the naturally produced embryo at the blastocyst stage [19, 20]. One of the objectives of the current study was to determine whether the Oct4 PP in OG2 hybrid cells has been completely demethylated by day 2 postfusion, as is the case for the PE, or becomes gradually demethylated from the onset of Oct4-GFP reactivation onward, as is the case for reconstructed embryos. We therefore compared the reprogramming profile of the Oct4 PP with that of Oct4 PE by performing bisulfite sequencing analysis using OG2 hybrid cells from the onset of Oct4-GFP reactivation onward—days 2, 4, 6, 9, and 15. The CpG sites of the Oct4 PP in F9 EC cells, where Oct4 is highly expressed, were completely unmethylated (0%). However, the CpG sites of the Oct4 PP in NSCs, where Oct4 is repressed, were partially methylated (male) or even highly methylated (female). The DNA methylation pattern of Oct4 is both variable and heterogeneous among the somatic cell population [21]. The methylation rate on the PE is also slightly higher in female (41.8%) than in male NSCs (32%) (unpublished data); however, this is an insignificant finding, as the opposite is found in certain female versus male somatic cell populations (data not shown). To our surprise, the Oct4 PP in OG2 hybrid cells was gradually demethylated by day 2 postfusion and completely demethylated by day 9 postfusion. There was a gradual reduction in DNA methylation following fusion: mean rates were 21.4% on day 2, 16.9% on day 4, 12.7% on day 6, and 0% and 1.8% on days 9 and 15, respectively (F9 level) (Fig. 2). According to our unpublished data, at least two copies of Oct4-GFP transgenes have been integrated into OG2 transgenic mice. Therefore, if the OG2 hybrid cells are not reprogrammed by day 2 postfusion, at least half of clones should have a somatic form of DNA methylation on the Oct4 PP region. However, only 6 of 14 clones remained as methylated on day 2 postfusion, indicating that the demethylation of Oct4 PP region of OG2 hybrids had already begun from day 2 postfusion (Fig. 2).


Figure 1
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  		Figure 1. Reprogramming status of OG2 hybrid cells. (A): The reprogramming of somatic genome in OG2 hybrids was confirmed by morphology (phase contrast), activation of Oct4-GFP (green fluorescence), and verification of hybrid formation between F9 EC cells and NSCs by 5-bromo-4-chloro-3-indolyl-β-D-galactoside staining. (B): Real-time reverse transcription-polymerase chain reaction analysis of OG2 hybrid cells was performed to investigate the reprogramming status of OG2 hybrid cells. All data are normalized to Bact expression and calibrated on the F9 EC cells, whose expression is considered 1 for all genes. The red arrow indicates that no signal was detected after 45 cycles, but the software considers 45 as the effective CT. The y-axis value is on logarithmic scale, and minor gridlines are 1/10th the value of each major gridline.

 


Figure 2
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  		Figure 2. Methylation status of the Oct4 PP in the OG2 hybrid cells. The Oct4 PP region was analyzed by bisulfite sequencing, and the specific region examined for methylation is depicted as a gray box. Open and filled circles indicate unmethylated and methylated CpGs, respectively. Abbreviations: DE, distal enhancer; kb, kilobases; PE, proximal enhancer; PP, proximal promoter.

 
Distinct Reprogramming of Endogenous and Exogenous Oct4
Oct4 gene expression in normal embryos is under the control of at least three main regulatory elements: the DE, the PE, and the PP. The DE is active in undifferentiated cells of pre- and postimplantation embryos and is required for Oct4 expression in primordial germ cells, ES cells, and embryonic germ cells. In contrast, the PE is required for Oct4 expression in postimplantation embryos and in EC cells [10]. The PE, however, has been shown to mediate the repression of Oct4 expression upon retinoic acid-induced cell differentiation [21] and is needed for correct downregulation of Oct4 expression [22].

We postulated that the different demethylation patterns of the Oct4 PE and PP are due to the differential reprogramming susceptibility of the endogenous Oct4 gene and the exogenous Oct4-GFP transgene. Although the methylation status of the PE may only reflect that of the endogenous Oct4 gene in F9 EC and NSCs, the methylation status of the PP is a composite of that of the endogenous Oct4 gene (F9 EC and NSCs) and the exogenous Oct4-GFP transgene (NSCs). We also postulated that if the reprogramming patterns of the endogenous Oct4 gene and the exogenous Oct4-GFP transgene are indeed different, the PE regulatory region, which differs between the two, may be involved in the positive as well as negative regulation of Oct4 expression. To address this, we first compared the reprogramming profiles of the endogenous Oct4 gene and the exogenous Oct4-GFP transgene in OG2 hybrids. The PP regions of the endogenous Oct4 gene and the exogenous Oct4-GFP transgene are the same. However, the coding regions of these two genes are different, as the GFP region has been inserted between the PP and the Oct4 coding regions in the Oct4-GFP transgene (Fig. 3A). To discern any differences in the methylation patterns of the endogenous Oct4 gene and the exogenous Oct4-GFP transgene, two different sets of primers were designed: an endogenous primer set spanning the Oct4 PP and Oct4 exons and an exogenous primer set spanning the Oct4 PP and GFP (Fig. 3A). Using these two sets of primers, only the endogenous Oct4 gene could be detected in intact F9 EC cells, but both the endogenous Oct4 gene and the exogenous Oct4-GFP transgene could be detected in transgenic NSCs (Fig. 3B). We next compared the demethylation patterns of the PP regions in the endogenous Oct4 gene and the exogenous Oct4-GFP transgene from the onset of Oct4-GFP reactivation onward—days 2, 4, 6, 9, and 15. As expected, the endogenous Oct4 was completely unmethylated (0%) in F9 EC cells but partially methylated (46.0%; relatively high rate) in NSCs (Fig. 3C). The endogenous Oct4 was completely demethylated by day 2 postfusion, indicating that the endogenous Oct4 gene exhibited exactly the same demethylation pattern as the PE. In contrast, the exogenous Oct4-GFP transgene, which was highly methylated (85.9%) in the NSCs prior to fusion, became gradually demethylated postfusion: mean methylation rates were 84.6% on day 2, 51.3% on day 4, 39.4% on day 6, and 3.9% on day 9 (Fig. 3D). Therefore, the reprogramming profiles of the endogenous and exogenous Oct4 genes are very different. These results suggested that the difference in the demethylation pattern of the Oct4 PE and the Oct4 PP in OG2 hybrids is due to the discrepancy between the endogenous and exogenous Oct4 genes and that the reactivation of Oct4-GFP transgene, which does not contain the PE region, does not reflect the reprogramming of the somatic cell genome.


Figure 3
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  		Figure 3. Reprogramming patterns of endogenous Oct4 and exogenous Oct4-GFP. (A): Schematic representation of both endogenous Oct4 and exogenous Oct4-GFP shows DE, PE, and PP in the 5' regulatory region, and the deleted region of exogenous Oct4-GFP transgene is boxed with the dotted line. The examined regions in endogenous Oct4 and in exogenous Oct4-GFP transgene are indicated with gray boxes. (B): Allelic-specific primers detect each endogenous and exogenous Oct4. Although only endogenous Oct4 was detected from F9 EC cells, both endogenous Oct4 and exogenous Oct4 were detected from OG2 NSCs. (C, D): Reprogramming patterns of endogenous and exogenous Oct4 of OG2 hybrids were compared from the onset of Oct4-GFP reactivation onward (days 2, 4, 6, and 9). Open and filled circles indicate unmethylated and methylated CpGs, respectively. Abbreviations: DE, distal enhancer; GFP, green fluorescent protein; PCR, polymerase chain reaction; PE, proximal enhancer; PP, proximal promoter.

 
Oct4-GFP Fluorescence Signal Detected Shortly After the First Cell Cycle of Fusion Hybrids
The Oct4-GFP from OG2+/–/ROSA26+/– double transgenic female NSCs, which does not contain the Oct4 PE regulatory sequences, exhibited delayed reprogramming compared with the endogenous Oct4 gene. To determine whether this delay is due to the absence of required regulatory elements, we generated hybrid cells by fusing F9 EC cells with NSCs from female embryos (14.5–16.5 dpc) carrying the GOF18 Oct4-GFP transgene (genomic Oct4 fragment, 18 kilobases), which contains the entire Oct4 upstream regulatory region, including the PE. GFP-positive cells were first detected in hybrid cells approximately 23 hours after the fusion of F9 EC cells with GOF18 NSCs. Furthermore, to determine the onset of reactivation of Oct4-GFP transgene expression, we performed a time-course analysis of GFP expression at 12, 15, 18, 22, and 24 hours postfusion. GFP-positive cells were first detected 22 hours postfusion (Fig. 4A). Since pluripotent embryonic cells normally divide every 24 hours, we postulated that reprogramming of the somatic cell genome may occur during the first cell cycle of the fusion hybrid cells. To confirm our hypothesis, we compared the doubling times of F9 EC cells, GOF18 NSCs, and the fusion hybrids (GOF18 hybrid) and found that the doubling time of GOF18 NSCs was approximately 30 hours, whereas that of F9 EC and hybrid cells was approximately 20 hours (Fig. 4C). Since the temporal onset of GFP fluorescence signal appearance (22 hours after fusion) is similar to the cell cycle duration of the hybrid cells, it appears that reactivation of Oct4-GFP expression is induced during or immediately after the first cell cycle of the fusion hybrids. In the current study, we used asynchronous NSCs for cell fusion. A recent study, however, showed that synchronization of fusion partner cells significantly increases the hybrid colony number [23]. We therefore synchronized NSCs into metaphase with nocodazole treatment. The results show a slight increase in the number of Oct4-GFP-positive cells in both OG2 and GOF18 hybrids (data not shown) but a nonsignificantly faster rate of Oct4-GFP transgene reactivation with synchronized cells (supplemental online Fig. 2). The faster reactivation rate of the GOF18 Oct4-GFP transgene, compared with that of the Oct4-GFP transgene, suggests that the transgene containing the PE can be used to accurately assess the reprogramming status of the somatic cell genome. Moreover, the PE region may be involved in the positive regulation of Oct4 re-expression during cell fusion-induced somatic cell genome reprogramming. Since, in our previous study, downregulation of Oct4-lacZ expression was observed in the presence, but not the absence, of the PE [24], we examined whether this negative effect of PE was also conserved in the fusion hybrids. We induced differentiation of both OG2 and GOF18 hybrids by forming embryoid bodies (EB) for 2 days. To our surprise, although the GOF18 hybrids properly lost Oct4-GFP expression with EB maturation, repression of Oct4-GFP signal was significantly protracted in OG2 hybrids (Fig. 4D), indicating that PE is also needed for appropriate downregulation of Oct4 in the differentiated fusion hybrids.


Figure 4
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  		Figure 4. Reprogramming status of GOF18 hybrid cells. (A): The reprogrammed cells were detected by 22 h postfusion. (B): Real-time reverse transcription-polymerase chain reaction analysis of GOF18 hybrid cells was performed to compare the reprogramming status of GOF18 hybrid cells with that of OG2 hybrids. (C): The doubling time was determined for each fusion partner cell, F9 EC and GOF18 NSCs, and their fusion hybrid cells. Cells (2 x 105) were plated on 6-cm dishes, and cell number was examined at 10, 20, and 30 h after seeding cells. (D): Cells from the OG2 and GOF18 hybrids were cultured to form EB, and the expression of Oct4-GFP transgenes was monitored under fluorescence microscope. The gradual downregulation of Oct4-GFP expression from GOF18 EB was observed on the 2-day EB; in the absence of PE (OG2 EB), downregulation of Oct4-GFP was not observed. Abbreviations: GFP, green fluorescent protein; h, hours; hrs, hours.

 
Identical Reprogramming of the Endogenous Oct4 Gene and the GOF18 Oct4-GFP Transgene
If the status of the Oct4-GFP transgene containing all the Oct4 regulatory elements is truly representative of the in vivo status of the endogenous Oct4 gene, then the Oct4-GFP transgene of GOF18 hybrids should exhibit a reprogramming pattern more similar or even identical to that of the endogenous Oct4 gene. To test this, we first assessed the gene expression profiles of pluripotency- and tissue-specific genes in GOF18 hybrids, by real-time RT PCR, to determine whether gene expression could be reprogrammed to that of the pluripotent partner cell within 24 hours of fusion. On day 1 postfusion, the expression of pluripotency-specific markers Oct4, Nanog, and Sox2 was statistically indistinguishable from that at later time points—days 2, 4, 6, and 9. Moreover, the tissue-specific marker Nestin was also inactive from day 1 onward (Fig. 4B).

We then performed a time-course analysis—days 1, 2, 4, 6, and 9 postfusion—of the methylation status of the Oct4-GFP transgene in GOF18 hybrids by bisulfite sequencing PCR. As expected, the Oct4-GFP transgene of GOF18 NSCs, which do not express Oct4, was partially methylated (22.4%) prior to fusion. However, DNA methylation in GFP-positive cells on day 1 postfusion was similar to that of NSCs (rate, 28.9%) (Fig. 5C), indicating that by 22–23 hours postfusion, when GFP-positive cells were first observed, the exogenous Oct4-GFP transgene gene had not been fully demethylated. According to a previous report [25], DNA methylation and histone modification mediate the coordinate regulation of Oct4 transcription. Therefore, our data indicate that Oct4-GFP transgene is also regulated on both the DNA and histone levels, further indicating that DNA demethylation alone cannot account for the derepression of Oct4-GFP transgene. This can be explained by the fact that Oct4-GFP is still methylated on day 1 postfusion, even though this coincides with the onset of Oct4-GFP expression in the GOF18 hybrids. However, the exogenous Oct4-GFP transgene was completely demethylated by day 2 postfusion—the methylation rate was 3.1% on day 2, 0.6% on day 4, 0.1% on day 6, and 3.1% on day 9 (Fig. 5C). We also determined the methylation status of the PE, which may represent that of both the endogenous Oct4 gene and the exogenous Oct4-GFP transgene in GOF18 hybrids. The PE of GOF18 NSCs, like that of OG2 NSCs, was also highly methylated (67.3%). However, the PE of GFP-positive cells had been considerably demethylated by 24 hours postfusion (14.6%; Fig. 5B). Although the exogenous Oct4-GFP transgene of GOF18 hybrids does not appear to have been completely reprogrammed by day 1 postfusion, the PE region appears to have been significantly demethylated from day 1 onward.


Figure 5
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  		Figure 5. Methylation status of PE and exogenous Oct4-GFP in the GOF18 hybrid cells. (A): Schematic representation shows the physical maps of both endogenous Oct4 and exogenous Oct4-GFP in the GOF18 hybrid cells; all regions examined for methylation are depicted as gray boxes. (B, C): Reprogramming patterns of PE (B) and exogenous Oct4-GFP (C) of GOF18 hybrid cells were investigated from the onset of Oct4-GFP reactivation onward (days 1, 2, 4, 6, 9, and 15). Open and filled circles indicate unmethylated and methylated CpGs, respectively. Abbreviations: DE, distal enhancer; GFP, green fluorescent protein; kb, kilobases; PCR, polymerase chain reaction; PE, proximal enhancer; PP, proximal promoter.

 
NSCs Could Be Reprogrammed Within 24 Hours of Fusion
To determine whether pluripotential reprogramming of somatic cell genome begins within 24 hours of fusion, we analyzed the methylation status of two pluripotent-specific marker genes: the endogenous Oct4 gene and Nanog. The PP region of the endogenous Oct4 gene was highly methylated in GOF18 NSCs (63.3% vs. 67.3% for PE). However, the endogenous Oct4 gene in FACS-sorted hybrids at 24 hours postfusion exhibited considerable demethylation (methylation rate, 10.8%), with complete demethylation noted in 8 of 13 clones (as in F9 EC cells) (Fig. 6A). We next determined whether the reprogramming status of the Oct4 gene is similar to that of another pluripotency-specific marker by analyzing the methylation status of the Nanog gene in GOF18 hybrids. Nanog was highly methylated (82.5%) in NSCs, which do not express Nanog. However, Nanog was also dramatically demethylated by 24 hours postfusion; methylation rates were 17.5% on day 1, 2.5% on day 2, and 0.0% on day 4 (Fig. 6B). Taken together, our findings suggest that reprogramming of the pluripotency-specific marker genes Nanog and Oct4 begins within 24 hours of fusion and has been completed by day 2 postfusion. Furthermore, the reprogramming status of the endogenous Oct4 gene is consistent with that of the exogenous Oct4-GFP transgene (GOF18). Although pluripotential reprogramming of the somatic cell genome, as evidenced by the expression of pluripotency-specific genes, begins after the first cell cycle following fusion of NSCs with F9 EC cells, demethylation of these pluripotency-specific genes has been completed by day 2 postfusion in GOF18 hybrids, indicating that the Oct4-GFP transgene containing all Oct4 regulatory elements (GOF18 Oct4-GFP) is a suitable model with which to monitor the reprogramming status of the somatic cell genome.


Figure 6
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  		Figure 6. Methylation status of endogenous Oct4 and Nanog in GOF18 hybrid cells on day 1 postfusion. Methylation status of endogenous Oct4 (A) and Nanog (B) was analyzed using green fluorescent protein-positive cells that were sorted by fluorescence-activated cell sorting from day 1 postfusion onward. All examined regions are depicted as gray boxes, and open and filled circles indicate unmethylated and methylated CpGs, respectively. Abbreviation: E1, exon1 of Nanog; PCR, polymerase chain reaction.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
The reprogrammed fusion hybrid cells exhibit patterns of gene expression identical to those of the pluripotent fusion partner cells [6, 23, 24]. General epigenetic modifications, including DNA methylation and histone modifications, are required to reprogram the somatic cell genome and establish pluripotency in fusion hybrids. It has been suggested that the Oct4 gene is regulated epigenetically through DNA methylation [26]. Here, we also observed that the methylation of Nanog (Fig. 6B) and Nestin (data not shown) regulatory regions reflects the expression of these two genes. Hybrid cells, in fact, exhibited dramatic alterations in the methylation of Nanog. However, changes in the DNA methylation of the Nestin regulatory region are not so dramatic (data not shown). In the current study, we investigated whether the temporal onset of somatic cell genome reprogramming is associated with epigenetic modifications, and we also examined the temporal onset of activation of the Oct4 loci—the endogenous Oct4 gene and the exogenous Oct4-GFP transgene—in the fusion hybrids. A recent study has demonstrated that the reprogramming of somatic cells can be induced by four factors (Oct4, Sox2, c-Myc, and Klf4) but that it takes at least 20 days to convert the state of the somatic cell genome to the pluripotent state [27]. This discrepancy in the duration of reprogramming between the four-factor study (≥20 days) and the current cell fusion study (1–2 days) suggests that there may be an additional factor(s) that can boost genomic reprogramming in the cell-fusion-based reprogramming process. In this study, we also analyzed the expression of Klf4 and c-Myc, two of the four factors needed for reprogramming, in hybrid cells. Klf4, along with Oct4 and Nanog, was minimally expressed in NSCs; c-Myc was highly expressed (supplemental online Fig. 3). Following cell fusion, upregulation of Klf4 expression was observed in both hybrids, but c-Myc expression was not altered until day 6 postfusion (supplemental online Fig. 3B, 3C), indicating that the transcription pattern of Klf4 was reprogrammed to that of the pluripotent fusion partner cell (F9 EC cells) but that of c-Myc was not properly reprogrammed and still adheres to NSC-type expression. These data can be interpreted in two possible ways. First, the expression level of c-Myc may not be so critical for the reprogramming of NSCs into the pluripotent state. The second, and more feasible, explanation is that c-Myc may indeed be important, but if somatic cells highly express c-Myc, then no further reprogramming may be needed, as NSCs could be reprogrammed without any changes in c-Myc expression.

In a previous study, it was found that the somatic cell genome could be reprogrammed by day 2 postfusion, as evidenced by activation of somatic Oct4-GFP transgene expression, which was detected by visualization of the fluorescence signal, around 44–48 hours after fusion [17]. According to our unpublished data, reprogrammed Oct4-GFP-positive OG2 hybrids exhibited complete demethylation of the Oct4 PE locus by day 2 postfusion, whereas the hybrids exhibited gradual demethylation of the Oct4 PP locus, which was similar to that of the pluripotent fusion partner F9 EC cells by day 9 postfusion (Fig. 2). We postulated that the different demethylation patterns of the Oct4 PE and PP are due to the differential reprogramming susceptibility of the endogenous Oct4 gene and the exogenous Oct4-GFP transgene. Therefore, the Oct4-GFP transgenes that did not contain the PE region exhibited delayed reprogramming of the somatic cell genome. Although Oct4-GFP transgenic mice (OG2 mice) have been good models to study reprogramming, as both the pluripotent state of embryonic cells and the reprogramming status of somatic cells could be easily detected by simply visualizing the GFP fluorescence signal in viable cells, the Oct4-GFP construct does not contain the PE regulatory region. According to a previous report and the current study (Fig. 4D), the PE locus is important for repression of Oct4 expression during induction of differentiation via embryoid body formation, indicating a possible role of the PE in the positive regulation of Oct4 expression in fusion hybrids [22]. We therefore generated another type of hybrid cell (GOF18 hybrids) by fusing F9 EC cells with NSCs from GOF18 transgenic mice in which GFP expression is under the control of the entire Oct4 regulatory region, including the PE. We found no significant difference in gene expression between OG2 hybrids and GOF18 hybrids, but GFP-positive cells were first detected in the GOF18 hybrids 22–23 hours postfusion. Furthermore, the demethylation pattern of the exogenous Oct4-GFP transgene was identical to that of the endogenous Oct4 gene in GOF18 hybrids, indicating that the status of Oct4-GFP transgene containing the PE regulatory element reflects the epigenetic status of endogenous Oct4 gene. In fact, the Oct4 PE of GOFf18 hybrids, which could be derived from either the endogenous Oct4 gene of F9 EC cells and NSCs or the exogenous Oct4-GFP transgene of NSCs, was completely demethylated by day 2 postfusion. Taken together, these findings suggest that the status of the Oct4-GFP transgene containing the PE region (GOF18) better reflects the epigenetic status of the endogenous Oct4 gene compared with that of the Oct4-GFP transgene without the PE (OG2). Considering the difference in the extent of demethylation between OG2 and GOF18 hybrids (Figs. 3, 5), GOF18 hybrids appear to undergo an active demethylation, whereas OG2 hybrids clearly undergo a passive demethylation that is cell cycle-dependent. According to a previous report [28], Oct4 harbors a cis-specific demodification element that includes the PE sequence. This element is able to demethylate the initially methylated Oct4 loci. Moreover, when the PE is mutated or deleted, the ability of the PE to induce demethylation was almost completely abolished. Therefore, the different demethylation pattern of OG2 and GOF18 hybrids may be caused by the absence or presence of this putative cis-specific demodification element located within the PE of the Oct4 gene.

Moreover, GFP-positive cells observed 24 hours after fusion exhibited an expression of pluripotency- and tissue-specific marker genes that was almost identical to that observed at later time points, indicating that the pluripotential reprogramming of the somatic cell genome can occur within 24 hours of fusion. However, the methylation status of the exogenous Oct4-GFP transgene in GFP-positive cells observed 22 hours postfusion was not consistent with complete genomic reprogramming. Complete demethylation of the exogenous Oct4-GFP was not observed in any of the NSC clones; however, complete demethylation was noted in a few (2 of 12; 16.7%) GOF18 hybrid clones on day 1 postfusion (Fig. 5C), which was consistent with that of the endogenous Oct4 gene. This result indicates that more than one copy of the Oct4-GFP transgene had been integrated within the GOF18 genome of somatic cells. Moreover, some of the exogenous Oct4-GFP transgenes had already been demethylated by 22–23 hours postfusion. Methylation of the exogenous Oct4-GFP transgene in OG2 NSCs (rate, 85.9%; Fig. 3D) was almost three times higher than that of GOF18 NSCs (30.2%; Fig. 5C). Therefore, the Oct4-GFP transgene of GOF18 hybrids (with PE) is more amenable to demethylation than the transgene of OG2 hybrids (without PE). The gradual demethylation of the transgene in OG2 hybrids may be due to the relative inaccessibility of reprogramming factors to the Oct4 regulatory regions, as (hyper)methylation may block binding of reprogramming and transcription factors to the regulatory regions of the Oct4-GFP transgene.

It is possible that the degree of DNA methylation required for repression of Oct4-GFP transgene expression in OG2 hybrids may be different from that required for repression in GOF18 hybrids, and this may be related to the presence of the PE in the Oct4-GFP transgene. Although the Oct4-GFP transgene of GOF18 hybrids could not be completely demethylated, the PE region was significantly less methylated than that in GOF18 NSCs. The PE, in fact, was completely demethylated in 9 of 12 clones (75%). The PE region in these clones may have originated from three different alleles: the endogenous Oct4 gene of F9 EC cells, the endogenous Oct4 gene of GOF18 NSCs, and the exogenous Oct4-GFP transgene of GOF18 NSCs. Furthermore, taking the potential copy number of transgenes into consideration, we can see that if the exogenous Oct4-GFP copies had not been reprogrammed, there should have been at least a 33.3% methylation of the PE. However, methylation was noted in only 3 of 12 clones (25%), indicating that some Oct4-GFP transgene copies had already been reprogrammed by 24 hours postfusion. In particular, a large portion of the somatic cell genome (endogenous Oct4 and Nanog) in GOF18 hybrids had been already reprogrammed with respect to pluripotency. According to a previous study [7], in which the Oct4-GFP transgene containing the PE was used as a marker for reprogramming, reprogrammed cells were first detected within 38–48 hours of fusion, rather than 22 hours. This difference may be due to the different types of pluripotent fusion partner cells used—ES cells (in the previous study) and F9 EC cells (in the present study)—as different embryonic cells have been shown to have different reprogramming potentials (unpublished data). Taken together, our data suggest that although the exogenous Oct4-GFP transgene had not been fully reprogrammed by day 1 after fusion, pluripotential reprogramming of the somatic cell genome may still have begun concomitantly with the first cell cycle of the fusion hybrids and, eventually, been completed by day 2 postfusion. Moreover, although in the current study, the status of the Oct4-GFP transgene not containing the PE locus did not reflect the reprogramming status of the endogenous Oct4 gene, FACS-sorted GFP-positive cells exhibited complete pluripotential reprogramming on day 2 postfusion, indicating that the Oct4-GFP transgene without the PE (OG2) may be a better model system to select for fully reprogrammed cells. In contrast, the Oct4-GFP transgene containing the entire Oct4 upstream regulatory region, including the PE (GOF18), may be more appropriate to investigate earlier reprogramming events, as GFP-positive cells exhibiting partial genome reprogramming were detected early on in the cell fusion-based reprogramming process.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We are indebted to all members of Schöler laboratory for helpful discussions of the results. We are especially grateful to Drs. Nataila Tapia, Vittorio Sebastiano, and Kinarm Ko for valuable comments on the manuscript. This work was supported by the Federal Ministry of Education and Research initiative "Cell-Based Regenerative Medicine" (Grant 01GN0539). D.W.H. and J.T.D. contributed equally to this work.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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