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Center for Animal Transgenesis and Germ Cell Research, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA
Key Words. Oct4 • Reprogramming • Embryonic stem cells • Neurosphere cells • Fusion
Correspondence: Hans R. Schöler, Ph.D., Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Mendelstrasse 7, 48149 Münster, Germany. Telephone: 49-0251-980-2866; Fax: 49-0251-959-2992; e-mail: schoeler{at}mpi-muenster.mpg.de
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ABSTRACT |
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INTRODUCTION |
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The ooplasm of an enucleated mammalian oocyte has the capacity to recondition or reset the genetic program of a fully differentiated somatic cell nucleus to the point of producing a fully developed organism. However, the experimental difficulties inherent in handling oocytes render them unfeasible for conducting analyses on the underlying mechanisms of genetic reprogramming. In contrast, ESC lines are more amenable to experimental manipulation and present an equally valid tool with which to address the molecular basis of genetic reprogramming. In previous studies, we analyzed the reactivation of Oct4 and an Oct4–green fluorescent protein (GFP) transgene—markers of pluripotency—after transfer of somatic cell nuclei into oocytes [11,12]. In the present study, we examined whether enucleated cytoplasts of ESCs can also activate the Oct4-GFP transgene in somatic cells or whether nuclear components are required.
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MATERIALS AND METHODS |
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ESC Culture
E14 ESCs were grown on top of mouse embryonic fibroblast feeder cells that had been inactivated with 0.01 mg/ml mitomycin C (MMC) in standard ESC media, comprised of high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, http://www.invitrogen.com) containing 15% fetal bovine serum (Hyclone, Logan, UT, http://www.hyclone.com), 1 x penicillin/streptomycin, 1 x nonessential amino acids (Gibco BRL), 0.1 mM 2-mercaptoethanol; and 1,000 U/ml leukemia inhibitory factor (ESGRO; Chemicon, Temecula, CA, http://www.chemcon.com). To obtain larger ESCs, the cells were cultured in ESC media containing 10 µM cytochalasin B (CB) for 20 hours.
Neurosphere Culture
The ROSA26 strain was crossed with the OG2 transgenic strain [13] over several generations to produce compound homozygous mice for the neo/lacZ and Oct4-GFP transgenes. To derive NSCs, homozygous ROSA26 x OG2 male mice were crossed with ICR females (Taconic, Germantown, NY, http://www.taconic.com) to produce heterozygous pups. Brain tissue was collected from 1- to 5-day-old ROSA26 x OG2 heterozygous mice. The cortex was dissected from the rest of the brain of each mouse and enzymatically dissociated in Hanks’ balanced salt solution (HBSS) (with 2 mM glucose) containing 0.7 mg/ml hyaluronic acid, 0.2 mg/ml kynurenic acid, and 1.33 mg/ml trypsin at 37°C for 30 minutes. The dissociated cells were passed through a 70-µm nylon mesh (Falcon, Franklin Lakes, NJ, http://www.bd.com/labware) to remove large cell clusters. These were then centrifuged at 200 g for 5 minutes and collected by centrifugation in 0.9 M sucrose in 0.5 x HBSS at 750 g for 10 minutes. The cell pellet was resuspended in 2 ml of culture medium, placed on top of 10 ml of 4% bovine serum albumin in Earl’s basal salt solution, and centrifuged at 200 g for 7 minutes. The culture medium was supplemented with 20 ng/ml epidermal growth factor (Gibco BRL), 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 in DMEM-F12 medium (Gibco BRL). Primary neurospheres were cultured for 5–8 days and used for fusion. Animals were maintained and used for experimentation under the guidelines of the Institutional Animal Care and Use Committee of the University of Pennsylvania.
Separation of Karyoplasts and Cytoplasts
Separation of karyoplasts and cytoplasts was completed by centrifugation through a Ficoll density gradient in the presence of CB. A 50% (wt/vol) stock solution of Ficoll-400 was prepared by dissolving Ficoll-400 powder in water; it was sterilized by autoclaving and then stored at 4°C. Various concentrations of Ficoll-400 solution were prepared by diluting the 50% Ficoll-400 stock solution with DMEM; CB was added to a final concentration of 10 µg/ml. The discontinuous density gradients were prepared in 13 x 51 mm Polyallomer tubes (Beckman Coulter Inc., Fullerton, CA. http://www.beckmancoulter.com) by successively layering 0.8 ml of 30%, 25%, 22%, 18%, and 15% Ficoll-400 solution that had been prewarmed at 36°C. Finally, 0.5 ml of 12.5% Ficoll-400 solution containing 5 to 10 x 106 ESCs and NSCs were layered on top of each gradient. The gradients were centrifuged through an MLS-50 rotor in a Beckman Optima ultracentrifuge (Beckman Coulter Inc.) at 40,000 rpm at 36°C for 30 minutes. After centrifugation, the cytoplasts were collected from the 15% and 18% regions, while the karyoplasts were collected from the bottom of the 30% region (see Fig. 3). Each fraction was washed with 10 volumes of DMEM by centrifugation at 2,000 rpm for 10 minutes. The separated karyoplasts and cytoplasts were stained with 0.4% trypan blue to test the viability.
Carboxyfluorescein Diacetate Succinimidyl Ester Analysis
ESCs were washed twice in phosphate-buffered saline (PBS) to remove free protein. These were then stained with 5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, http://www.probes.com) at room temperature for 8–10 minutes, and the reaction was terminated by blocking with fetal calf serum (2% final). Stained ESCs were washed twice in PBS and centrifuged through a Ficoll gradient. CFSE remains in the cytoplasm, with diminishing amounts noted as the cytoplasm was progressively removed.
Cell Fusion and Subsequent Culture
ESCs were mixed with NSCs in a 1:1 ratio and washed in PBS. The mixture was centrifuged in conical tubes (Falcon) at 130 g for 5 minutes. The supernatant was thoroughly removed, and 1 ml of a prewarmed solution of 50% polyethylene glycol (PEG) 1500 (Roche, Indianapolis, http://www.biochem.roche.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 130 g for 5 minutes to remove the PEG, washed gently with DMEM, and cultured in ESC medium. Cells were selected in ESC medium containing 300 µg/ml G418 the next day after PEG fusion to eliminate nonfused ESCs.
Determination of GFP Expression
Oct4-GFP expression was examined in living embryonic stem (ES) hybrid cells on culture dishes under a fluorescent microscope (NiKon Instruments Inc., Melville, NY, http://www.nikonusa.com).
KaryotypeAnalysis
A 6-cm dish at 50% cell confluence was treated with 0.3 mg/ml Nocodazole for 3 hours. Cells were recovered by trypsinization and replated on gelatin-coated (0.1% in PBS) dishes for 30 minutes for adherence of feeder cells. Nonadherent cells were recovered and treated with hypotonic (0.56% wt/vol) KCl solution for 15 minutes. The cells were centrifuged at 500 rpm, fixed by washing three times in fresh fixative (3:1 methanol: acetic acid), and dropped onto clean glass slides. The slides were air dried, stained with 3% Giemsa, and observed under a microscope.
Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from ESCs, NSCs, and ESC-NSC hybrids using the To TALLY RNA kit (Ambion, Austin, TX, http://www.ambion.com). The resultant RNA was subjected to DNase treatment and removal to remove any contaminating DNA (Ambion). For reverse transcription–polymerase chain reaction (PCR), 1 µg of total RNA was used with an RETROscript kit (Ambion) following the manufacturer’s protocol. PCR was carried out in a Gene Amp 9700 PCR thermal cycler (Perkin-Elmer, Norwalk, CT, http://www.perkinelmer.com). Oligonucleotide primers used, along with PCR conditions, are described in Table 1
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RESULTS |
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). We then fused either kaESCs or cyESCs with NSCs. NSCs were prepared from OG2/ROSA26 heterozygous transgenic mice carrying GFP under the control of the Oct4 promoter (Oct4-GFP) and a neo/lacZ transgene that is expressed ubiquitously. The Oct4 gene is expressed in the pluripotential cells of the embryo up to the gastrulation stage [14,15] and is restricted to germ cells thereafter [16]. Because Oct4 is inactive in NSCs, Oct4-GFP can only be expressed in these cells if it becomes reactivated. The reprogramming capacity of somatic cells in ESC-NSC hybrid cells induced by PEG can be estimated by examining the GFP-positive signal under a fluorescence microscope. These ES hybrid clones were subcultured in G418-containing ES media every 2 or 3 days after fusion. Oct4-GFP expression was examined to assess reprogramming of a pluripotency marker in the ESC-NSC hybrid cells (Fig. 2). The first GFP-positive cells were observed at days 2 after PEG-induced fusion (Figs. 2A, 2B). The GFP-positive ESC-NSC fusion hybrids formed ESC-like colonies, which were, along with GFP expression, stably maintained for at least 20 passages after fusion and could be recovered from cryopreservation. The reprogramming rate (Oct4-GFP–positive/lacZ-positive colony) at passage 5 was approximately 95%, and the reprogramming rate was stable over subsequent passages (in preparation). To determine whether these were hybrid cells, we examined the karyotype of the cell fusion culture as well as the morphology of the nuclei. The fused Oct4-GFP–positive cells had tetraploid karyotypes (Fig. 2C) and enlarged nuclei with multiple nucleoli (Fig. 2D). The fusion hybrids expressed markers that characterize undifferentiated ESCs, including Oct4, Rex-1, and nanog, but did not express the ectoderm marker gene glutamate receptor 6 (GluR6) (Fig. 2E). These results suggest that the ESC-NSC hybrids have ESC-like potency and that the NSC hybrid counterparts lose neuronal characteristics.
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). To this end, we centrifuged cells through a Ficoll gradient and collected cytoplasts from the 15% and 18% regions and karyoplasts from the bottom of the gradient (30% region). To confirm successful separation, the collected cytoplasts and karyoplasts were stained with Hoechst and examined under a fluorescence microscope (Figs. 3E, 3G). More than 95% of the cells were successfully enucleated, as determined by Hoechst staining. The size of cytoplasts was smaller than that of karyoplasts (Figs. 3D, 3F). The cell viability of cytoplasts and karyoplasts estimated by trypan blue exclusion test was 99.6% and 96.7%, respectively. A karyoplast is defined as a nucleus isolated from a eukaryotic cell that is surrounded by a very thin layer of cytoplasm enclosed within a plasma membrane. The remainder of the cell is called a cytoplast. In CFSE analysis, we found that purified karyoplasts contained only trace amounts of cytoplasm (compare Fig. 3J with Fig. 3M). These observations suggested that the cytoplasts and karyoplasts purified by centrifugation through Ficoll gradient were suitable for fusion with somatic cells.
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). The kaESC-NSC hybrids formed ESC-like colonies and expressed the ESC markers Oct4, Rex-1, and nanog, just like ESC-NSC hybrids and E14 ESCs (data not shown). This result indicates that karyoplasts of ESCs contain factors that are required for reprogramming.
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), and some survived a few days longer. Expression of GFP after fusion of ESCs or kaESCs with NSCs was detected after 2 days. In contrast, even after 3 days or more, cyESCs could not reactivate Oct4-GFP in NSCs, indicating that cyESCs either lacked factors crucial for reactivation or that these factors were present but were below a critical level.
MMC-Treated ESCs Can Reprogram NSCs Pretreated with 5-aza C
To investigate whether DNA replication is important for reprogramming of the somatic cell genome, we performed fusion experiments between NSCs and MMC-treated ESCs. MMC inhibits DNA replication and cell division but does not affect gene transcription and protein synthesis. We observed that MMC-treated ESCs did not reactivate the Oct4-GFP of intact NSCs after fusion. However, treatment of NSCs with 5-aza C to facilitate reprogramming of NSCs indeed resulted in reactivation of Oct4-GFP. Figures 5A and 5B show a hybrid cell that was attached to the feeder layer but did not form a colony, remaining as a single cell even 3 days after fusion. Many Oct4-GFP–expressing cells were identified in fusion colonies (data not shown). However, only Oct4-GFP expression in a single nondividing cell indicates that a marker of pluripotency can be reactivated in NSCs without replication. In contrast, unfused control NSCs, treated with 5-aza C and PEG, did not re-express Oct4-GFP (Figs. 5C, 5D). These results indicate that ESCs that are incapable of DNA replication and cell division still retain their reprogramming ability. These results suggest that the reprogramming of Oct4-GFP in differentiated cells requires neither DNA replication nor cell division.
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DISCUSSION |
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We cannot fully exclude the possibility that trace amounts of cytoplasmic factors contaminated the karyoplast fractions during their preparation, thereby influencing Oct4-GFP reactivation. However, we consider it unlikely for the following reasons. After separation of karyoplasts through Ficoll gradient, their size was reduced because of the removal of cytoplasm (Fig. 3). In CFSE analysis, we found that purified karyoplasts contained only trace amounts of cytoplasm (compare Fig. 3J with Fig. 3M). In addition, we observed approximately two to six GFP-expressing colonies at days 2 through 4 after kaESC-NSC fusion (in each experiment). At 2–4 days after fusion, the plates were subcultured. The number of ESC-like colonies expressing Oct4-GFP was almost the same in the ESC-NSC fusion culture on days 2 through 4 after fusion. If only intact ESCs were capable of reprogramming NSCs, the Oct4-GFP–expressing colonies would be observed much less frequently. These observations suggest that NSCs can be reprogrammed by kaESCs and not by contaminating intact ESCs. Therefore, we can suggest that the ESC cytoplasm is not essential for reprogramming NSCs.
Nuclear transfer is a well-established technology to reestablish the totipotent program of genes. As shown for various mammalian species, the ooplasm of an enucleated oocyte can alter the genetic program of a somatic cell to support full-term development. In contrast, as shown in this study, ESC cytoplasts cannot re-establish expression of Oct4-GFP. There is a likely explanation for the different reprogramming capacities of egg cytoplasts and ESC cytoplasts. Two kinds of cytoplast recipients have been used in mammalian cloning: oocytes arrested at metaphase II (MII) and pronuclear zygotes. It is possible that factors that are crucial for reprogramming events are removed during the process of enucleation [17]. However, in the MII oocyte, the nuclear envelope is absent, and during enucleation, chromosomes and the spindle are removed whereas nuclear factors responsible for reprogramming remain in the ooplasm. In contrast, it has been shown that zygotes are inappropriate recipients for the cloning of mice [18]; they apparently lack the activities required for reprogramming [19]. In the pronuclear zygote, the chromosome and nuclear factors are located in two pronuclei. Therefore, during enucleation of pronuclear zygotes, chromosomes and relevant nuclear factors may be removed with the pronuclei [2]. Collectively, the results obtained with oocytes suggest that nuclear factors are important for reprogramming. Enucleation of ESCs by centrifugation through Ficoll gradients to obtain ESC cytoplasts might be considered similar to enucleation of pronuclear zygotes. Therefore, ESCs and MII oocytes are not comparable in regard to their reprogramming capacity. We consider it likely that during enucleation of ESCs, factors necessary for successful reprogramming are removed with the nuclei.
Treatment of NSCs with 5-aza C, a cytidine analogue that inhibits DNA methylation [20], induced expression of the Oct4-GFP after fusion with MMC-treated ESCs. However, 5-aza C–treated NSCs without fusion—unfused control NSCs—did not lead to Oct4-GFP reactivation. This result is similar to the reactivation of muscle genes in 5-aza C–treated HeLa cells fused with muscle cells, which were never expressed in HeLa cell heterokaryons [21]. The authors of this study suggest that DNA methylation changes induced by 5-aza C are required for muscle gene reactivation in HeLa cells in response to putative transacting regulatory factors present in muscle cells. This result and our own observation suggest that epigenetic modifications before cell fusion could facilitate reprogramming of somatic genomes. MMC neither inhibits protein synthesis nor affects proteins already present in ESCs. Consequently, crucial factors may already exist in MMC-treated ESCs, or such factors may be synthesized. These factors may reprogram the NSCs that are pretreated with 5-aza C. In contrast, 5-aza C treatment may not suffice when cyESCs and NSCs are fused, because cyESCs lack sufficient amounts of reprogramming factors. In addition, the fact that 5-aza C treatment alone (control) and 5-aza C treatment followed by cytoplast fusion could not activate Oct4-GFP suggests that both the epigenetic modifications and the presence of reprogramming factors are required for somatic cell reprogramming.
Recently, it was suggested that somatic stem cells could regain pluripotency in vitro without the assistance of other pluripotent cells [22]. These cells, termed MAPCs, were self-reprogrammed by long-term culture in vitro. This finding raises the question of whether ESC factors are indispensable for somatic cell reprogramming or whether pluripotency, in this case, was regained during the process of cell culturing. It is noteworthy that these cells exhibited 1,000-fold less Oct4 expression than ESCs. This is an important point, because the Oct4 protein level is crucial for the maintenance of ESC pluripotency; a twofold difference in Oct4 protein level has a dramatic effect on ESC differentiation potential [23]. Therefore, it will be interesting to determine whether pluripotency can be re-established and maintained by different regulatory systems.
Additional experiments will show whether removal of mitotic chromosomes leads to cytoplasts that are more efficient at reprogramming or whether reprogramming activities can be maintained in other ways. In addition, specific nuclear factors from mammalian oocytes or ESCs can be added to cytoplasts of ESCs and NSCs to determine their effect on genetic reprogramming. It would be especially interesting to test whether the addition of specific factors from nonmammalian species is capable of reactivating Oct4 to re-establish pluripotency. The finding that nuclei from adult mouse thymocytes and those from adult human blood lymphocytes when injected into Xenopus oocytes are induced to extinguish a differentiation marker and to strongly express Oct4 encourages these types of experiments [24].
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ACKNOWLEDGMENTS |
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REFERENCES |
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