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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Reprogramming of the epigenetic state from differentiated to pluripotent cells can be attained by cell fusion of differentiated somatic cells with embryonic stem (ES) cells or transfer of the nucleus of a differentiated cell into an enucleated oocyte. Activation of Akt signaling is sufficient to maintain pluripotency of ES cells and promotes derivation of embryonic germ (EG) cells from primordial germ cells (PGCs). Here we analyzed the effects of Akt signaling on somatic cell nuclear reprogramming after cell fusion and nuclear transfer. We found that forced activation of Akt signaling stimulated reprogramming after cell fusion of ES cells with thymocytes or mouse embryonic fibroblasts. These hybrid cells showed ES cell characteristics, including in vitro and in vivo differentiation capacity. In contrast, Akt signaling significantly reduced the efficiency of reprogramming with nuclear transfer. Our results demonstrate that Akt signaling plays important roles on the nuclear reprogramming of somatic cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The epigenetic memories are inherited by daughter cells of the same lineage in vivo, but may be gradually modified during mammalian development. The differentiation capacity of cells is progressively restricted during development and, therefore, only cells at the earliest stages of development show totipotency or pluripotency. However, the restricted potential of differentiated cells can be reversed by three ways, that is, nuclear transfer into enucleated oocytes (Wilmut et al. 1997; Wakayama et al. 1998), fusion with pluripotent stem cells (Tada et al. 1997, 2001; Do & Scholer 2004; Kimura et al. 2004; Cowan et al. 2005), or ectopic expression of several transcription factors (Takahashi & Yamanaka 2006; Meissner et al. 2007; Okita et al. 2007; Wernig et al. 2007; Yu et al. 2007; Nakagawa et al. 2008). Nuclear transfer studies clearly demonstrate that epigenetic modification accumulated during cell differentiation can be fully reprogrammed into the pluripotent state of early preimplantation embryos. However, in contrast to the extensive study on the molecular basis for sustaining embryonic stem (ES) cells pluripotency, the mechanisms underlying the nuclear reprogramming of somatic cell remain poorly understood.

Phosphoinositide-3 kinase (PI3K) signaling is implicated in the regulation of cell proliferation, growth, death and adhesion, as well as tumorigenesis (Cantley 2002). Activation of PI3K generates the second messenger molecules, phosphatidylinositol (3, 4, 5)-triphosphate (PtdIns(3, 4, 5)P3) from PtdIns(4, 5)P2. PtdIns(3, 4, 5)P3 transmits the signals through downstream effectors including Akt, a serine/threonine kinase (Brazil et al. 2004). The physiological and pathological effects of PI3K/Akt signaling are counteracted by the tumor-suppressor PTEN, which dephosphorylates PtdIns(3, 4, 5)P3 into PtdIns(4, 5)P2 (Kishimoto et al. 2003). PI3K/Akt signaling regulates both tumorgenic potential and pluripotency in stem cells. In previous work, we found that Akt signaling is sufficient to maintain the pluripotency of mouse and primate ES cells without leukemia inhibitory factor (LIF) and feeder cells, respectively (Watanabe et al. 2006). We also demonstrated that the derivation of pluripotent embryonic germ (EG) cells from primordial germ cells (PGCs) is enhanced in PGC-specific Pten-deficient mice and Akt–Mer transgenic mice (Kimura et al. 2003b, 2008). In this study, we analyzed the effect of Akt signaling on reprogramming of somatic cell nucleus after cell fusions and nuclear transfers, and found that activation of Akt signaling promoted the nuclear reprogramming efficiency after cell fusions but not nuclear transfers.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Conditional activation of Akt signaling in ES cells

First, we established mouse ES cells in which Akt activation can be conditionally obtained. An expression plasmid encoding Akt-Mer, which is a fusion protein consisting of the active form of Akt and the modified ligand binding domain of the estrogen receptor (Mer) (Kohn et al. 1998), was co-introduced into a feeder-free mouse ES cell line, E14tg2a, with a puromycin resistance gene. ES clones that exhibited high expression of Akt-Mer fusion proteins were selected and used for the following analysis. The expression level of Akt-Mer was similar to that of endogenous Akt (Fig. 1A). Enzymatic activity of Akt-Mer can be achieved by the addition of the Mer ligand 4-hydroxytamoxifen (4OHT) in clones, as shown in Fig. 1A. Phosphorylation of Akt-Mer was detectable 5 min after the addition of 4OHT and plateaued at 2 h, whereas it was undetectable in the absence of 4OHT after 72 h (Fig. 1B).

image

Figure 1. Activation of Akt signaling in Akt-Mer expressing ES cells. (A) Level of Akt activation in control and Akt-Mer-expressing ES cells. Expression and phosphorylation levels were analyzed by Western blotting using anti-Akt (upper panel) and anti-phospho-Akt antibodies (lower panel), respectively. Open and closed arrowheads is endogenous Akt and introduced Akt-Mer, respectively. The closed arrow is β-actin. (B) Level of Akt activation after withdrawal 4OHT in Akt-Mer-expressing ES cells. Expression and phosphorylation levels were analyzed as described in (A).

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Effects of Akt signaling on the nuclear reprogramming by cell fusion

To analyze the effect of Akt signaling on the cell fusion- mediated nuclear reprogramming, we fused ES cells expressing Akt-Mer with thymocytes (T cells) using polyethylene glycol (PEG). Hybrid cells were divided into two aliquots after cell fusion; one aliquot was cultured with 4OHT and one without 4OHT (Fig. 2A). T cells were obtained from transgenic mice carrying two transgenes, that is, enhanced green fluorescent protein (EGFP) under the control of an Oct4 promoter (Yoshimizu et al. 1999) and a neomycin resistance gene under the control of a PGK promoter (Kimura et al. 2003a). Therefore, neomycin resistance and EGFP expression were used for the selection of fused cells and as a marker of reprogramming, respectively. Oct4-driven EGFP can be an efficient indicator of the acquisition of pluripotency after cell fusion because the Oct4 transgene is active only in pluripotent and germ-line cells (Yeom et al. 1996; Yoshimizu et al. 1999; Ying et al. 2002).

image

Figure 2. Reactivation of Oct4-EGFP in hybrid cells and their proliferation. (A) Schematic diagram of the experiments generating ES × somatic hybrid cells. 2 × 107 Akt-Mer-expressing ES cells and 3 × 107 somatic cells (thymocytes or MEFs) were fused with PEG. Fused cells were divided into two aliquots, and 10 µm 4OHT was added to one aliquot immediately after cell fusion. Selection was applied after 24 h using 200 µg/mL G418. (B) Reactivation of Oct4-EGFP in hybrid cells generated in the presence of 4OHT. EGFP-positive cells emerged as early as 48 h after cell fusion (left). The hybrid cells expressing EGFP formed round and multi-layered colonies, which is consistent with the characteristics of undifferentiated ES cells (middle). The hybrid cells retained Oct4-EGFP expression at least until day 30 (right). Scale bars are 50 µm (left) and 200 µm (middle and right). (C) Growth curves of hybrid and ES cells. ES cells and ES × T hybrid cells were passed every 3 days.

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EGFP-positive cells emerged as early as 48 h after cell fusion (Fig. 2B). Hybrid cells expressing EGFP showed a low cytoplasm-to-nucleus ratio and formed round and multi-layered colonies, which is consistent with the characteristics of undifferentiated ES cells (Fig. 2B). In addition, these hybrid cells possessed ES cell-like proliferation properties (Fig. 2C). Five days of Akt signaling activation enhanced the efficiency of reprogramming by approximately 9- to 10-fold (Table 1). Next we examined the effect of shorter-duration Akt activation after cell fusion and found that activation of Akt signaling for 24 h was sufficient to induce efficient reprogramming (Table 1). These data show that the effect of Akt signaling on reprogramming should be an early event for somatic cells.

Table 1. Effect of Akt signaling on the efficiency of cell fusion-mediated nuclear reprogramming
Fusion partnersDuration of 4OHT treatmentExperiment numberNumber of colonies*Fold increasePaired t-test
4OHT (–)4OHT (+)
  • *

    Number of neomycin resistant EGFP-positive colonies 10 days after fusion.

Thymocyte5 days1  9  9310.3 
215135 9.0
311  96 8.7
Total11.7 ± 3.1107.7 ± 23.7  P < 0.01
Thymocyte24 h1 5  5511.0
2 4  4812.0
3 9  62 6.9
424175 7.3
518166 9.2
Total12.0 ± 8.7101.2 ± 63.5  P < 0.025
MEF24 h13945711.7
23242813.4
33237811.8
Total34.3 ± 4.0421.0 ± 40.0  P < 0.0025
ES cell24 h121702140 0.99
217801820 1.02
314801510 1.02
Total1810 ± 3461823 ± 315  P > 0.3

The same experiment was conducted using ES cells expressing Akt-Mer and mouse embryonic fibroblasts (MEFs) carrying the Oct4-EGFP and neomycin resistance transgenes. In this case, approximately 12-fold increase was achieved with 24 h of Akt activation (Table 1). 4OHT treatment had no significant effect on the yield of hybrid colonies in the cell fusion experiment with Akt-Mer-expressing ES cells and ES cells (Table 1). Meanwhile, activation of Akt-Mer did not alter cell adhesion or proliferation of ES cells, Akt-Mer expressing ES cells and the T cell-Akt-Mer ES cell hybrid cells (Fig. 2C). Taken together, nuclear reprogramming of somatic cells was significantly enhanced by Akt signaling, and the effect did not occur through increased cell proliferation or altered adhesion ability.

Differentiation capacities of the hybrid cells

We verified the differentiation capacity of ES × T hybrid cells generated in the presence of 4OHT. First, the reprogrammed hybrid cells were seeded at low density and cultured in the absence of LIF. The differentiation status was examined by cell morphology and two specific undifferentiated ES cell markers, namely, the activity of alkaline phosphatase (ALP) and the expression of Oct4-EGFP. After the removal of LIF, the hybrid cells differentiated and vast majority of colonies had completely differentiated by day 5 in the absence of 4OHT (Fig. 3A). The differentiated cells exhibited a high cytoplasm to nuclei ratio and were negative for ALP activity and Oct4-EGFP (Fig. 3A). In contrast, the Akt-Mer-expressing hybrid cells maintained undifferentiated phenotypes in the presence of 4OHT, even after the removal of LIF (Fig. 3A). There were no significant differences in differentiation capacity between the hybrid cells generated in the presence and absence of 4OHT (data not shown).

image

Figure 3. Multipotent differentiation capacity of hybrid cells obtained with 4OHT. (A) Differentiation states of hybrid cells generated in the presence of 4OHT. The cells were seeded at low density and cultured under the indicated conditions. After 5 days, the colonies were stained with the ALP substrate Fast Red. Scale bar, 200 µm. (B) In vitro EB formation. Scale bar, 200 µm. (C) Expression of differentiation markers in the EB formation assay. RT-PCR analysis of EBs generated from hybrid cells, which were generated in the presence or absence of 4OHT. Expression of marker genes for each of the three germ layers was analyzed by RT-PCR. The PCR products were run on an agarose gel and visualized by ethidium bromide staining. Mixl1, mesoderm marker; Collagen IV and AFP, endoderm markers; Nestin, ectoderm marker; Rex-1, pluripotent marker; GAPDH, internal control. U; undifferentiated ES cells, D; differentiated EBs. (D) Teratomas generated from hybrid cells. Hybrid cells generated in the presence or absence of 4OHT were injected under the skin of nude mice. After 3 weeks, the teratomas contained various tissues, including cartilage (left), mucosal gland (middle) and squamous epithelium (right). Scale bar, 500 µm.

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Second, to determine the differentiation capacities in more detail, we examined the marker gene expression in a long-term embryoid body (EB) differentiation system. The reprogrammed hybrid cells were applied to suspension cultures for 7 days to induce EBs, the EBs were adhered to gelatin coated plates, and differentiated for 5 days. As shown in Fig. 3B, there was no significant difference in the efficiency of production, size and morphology of EBs between ES cell-like hybrid cells generated in the presence and absence of 4OHT (data not shown). Expression of the marker genes for the three germ layers, Mixl1 (mesoderm marker), Collagen IV and AFP (endoderm markers) and Nestin (ectoderm marker), were efficiently induced in the EBs (Fig. 3C). In contrast, the pluripotent marker Rex-1 was repressed after differentiation.

Finally, we analyzed the multilineage differentiation ability of hybrid cells in vivo. When transplanted into nude mice, hybrid cells generated in the presence of 4OHT produced teratomas consisting of various tissues, including cartilage, mucosal gland and epithelium, as did hybrid cells generated in the absence of 4OHT (Fig. 3D). Taken together with the in vitro differentiation assays, our data indicate that the hybrid cells generated in the presence of 4OHT possessed pluripotent differentiation ability.

Effect of Akt signaling on the reprogramming after somatic cell nuclear transfer (SCNT)

Based on the cell fusion experiments, we entertained the idea that Akt signaling increases the efficiency of nuclear reprogramming after SCNT. To examine this possibility, we conducted nuclear transfer experiments using oocytes microinjected with EGFP or an active mutant of Akt mRNA before enucleation as recipient cells. The nuclear-transferred embryos were cultured in vitro for 4 days and their developmental stage was examined. As shown in Table 2, more than half of the reconstructed embryos developed to the two-cell stage in both the EGFP and Akt mRNA-injected groups. The percentage of SCNT embryos reaching the two-cell stage was not significantly different between these two groups (P > 0.1 by χ2 test). Of the two-cell stage SCNT embryos derived from EGFP mRNA-injected oocytes, 63% developed to the morula/blastocyst stage. However, only 14% of those from the active mutant Akt mRNA-injected oocytes developed to the morula/blastocyst stage; this difference was significant (P < 0.005 by χ2 test, Table 2). The majority of cloned embryos derived from the Akt mRNA-injected oocytes arrested at the transition from the two- to eight-cell stage (86%; Table 2), which was significantly higher than in nuclear transfer using the oocytes injected with EGFP mRNA (P < 0.005, χ2 test).

Table 2. Effect of Akt signaling on nuclear reprogramming after somatic cell nuclear transfer
Injected mRNANumber of culturedNumber of embryos reaching the two-cell stage (%)Number of embryos reaching the morula/ blastocyst (% per cleaved)Number of arrested at the two- to eight- cell stage (% per cleaved)
  • *

    Number of embryos that developed to the morula/blastocyst stage was significantly lower than with EGFP. P < 0.005 by χ2 test.

  • Number of embryos that arrested at two- to eight-cell stage was significantly higher than with EGFP. P < 0.005 by χ2 test.

EGFP3927 (69.2)17 (63.0)10 (37.0)
Akt7243 (59.7)6 (14.0)*37 (86.0)

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we analyzed the effect of Akt signaling on the nuclear reprogramming of somatic cells in cell fusion and nuclear transfer. Activation of Akt signaling dramatically enhanced the yield of pluripotent hybrid colonies after cell fusions between ES cells and somatic cells (Table 1). Multipotent differentiation capacities of the hybrid cells, which are generated in the presence of 4OHT, were shown by in vitro differentiation assays and teratoma formation in nude mice (Fig. 3A–D). In contrast, Akt signaling significantly reduced the efficiency of nuclear reprogramming by nuclear transfer (Table 2).

Activation of Akt signaling promotes the de-differentiation of PGCs into EG cells and maintains pluripotency in ES cells (Watanabe et al. 2006; Kimura et al. 2008). It is likely that Akt signaling has similar effect(s) on nuclear reprogramming after cell fusion. There are many molecules downstream of Akt activation and some can be regarded as candidates for efficient reprogramming by Akt activation. Glycogen synthase kinase 3 (GSK-3), whose activity is inhibited by Akt-mediated phosphorylation, is a candidate involved in enhancing reprogramming because inhibition of GSK3 maintains ES cell pluripotency (Sato et al. 2004) and improves the efficiency of ES cell derivation (Umehara et al. 2007). However, in contrast to the activation of Akt signaling, treatment with 6-bromoindirubin-3′-oxime (BIO), a GSK-3 specific inhibitor, did not enhance nuclear reprogramming after cell fusion (Table S1 in the Supporting information). A previous study reported that the introduction of an active form of Akt into ES cells induces hyperphosphorylation of GSK-3 but does not activate Wnt/β-catenin signaling. Akt activation does not inactivate a fraction of GSK-3 within the β-catenin destruction complex in ES cells (Watanabe et al. 2006). Thus, the function of Akt signaling is independent of GSK3 inhibition in cell fusion-mediated nuclear reprogramming.

Nanog is another candidate because it can maintain the pluripotency of ES cells and can promote efficient nuclear reprogramming after cell fusion (Silva et al. 2006). Recently, it was reported that the PI3K activity had some effects on the expression of Nanog (Storm et al. 2007). The report has shown that inhibition of PI3 K activity results in decreased expression of Nanog and that the effect is via GSK-3 activity. Considering our data that GSK-3 inhibitor did not show any effects on the efficiency of reprogramming after cell fusion, as discussed above, GSK-3 should not be involved in the reprogramming. Thus, it is unlikely that Nanog is involved in the efficient nuclear reprogramming after cell fusion.

It is generally believed that DNA and chromatin modifications, which are critically important in cell-type-specific gene expression, take place during nuclear reprogramming. A previous study indicated that the epigenetic states of somatic cells could be reprogrammed in ES × T hybrid cells (Kimura et al. 2004). It is likely that activation of Akt signaling facilitates epigenetic modification of fused somatic cells. Along this line, we examined the function of two molecules that can be phosphorylated by Akt. One is histone acetyl transferase p300 (Huang & Chen 2005) and the other is histone methyl transferase enhancer of zeste homologue 2 (Ezh2) (Cha et al. 2005). However, neither the knockdown of p300 nor the introduction of an Ezh2 mutant that cannot be phosphorylated by Akt had any effect on the efficiency of nuclear reprogramming (data not shown). Considering that Akt signaling regulates other downstream molecules, it is likely that Akt signaling enhanced the yield of pluripotent hybrid colonies after cell fusions between ES cells and somatic cells through multiple downstream molecules.

In contrast to cell fusion-mediated nuclear reprogramming, activation of Akt signaling during SCNT dramatically diminished their developmental potential. The cloned embryos injected with an active form of Akt mRNA were arrested at the transition from two- to eight-cell stage (Table 2). However, the developmental defects were not attributable to the toxic effects of Akt hyper-activation. First, it has been reported that microinjection of active mutant of Akt mRNA into fertilized eggs did not induce the developmental arrest but instead promoted the cell division and survival (Feng et al. 2007). Similarly, we also demonstrated that activation of Akt signaling from two-cell embryos increased the number and the size of inner cell mass cells (Umehara et al. 2007). Thus, it is likely that the developmental arrest observed in the Akt-injected clones is caused by the abrogated reprogramming during SCNT.

Epigenetic modification should be important in nuclear reprogramming after SCNT (Hochedlinger & Jaenisch 2006), as well as after cell fusion. For example, treatment with trichostatin A, an inhibitor of histone deacetylase, after SCNT significantly improves cloning efficiency (Kishigami et al. 2006). Genomic and histone lysine hypomethylation of donor cells also improve the reprogramming efficiency (Baxter et al. 2004; Blelloch et al. 2006). However, our data clearly demonstrate that Akt signaling brings about opposite effects on the efficiency of nuclear reprogramming in cell fusion and SCNT. There should be different sets of Akt downstream molecule(s) involved in the enhancement or the inhibition of nuclear reprogramming. Further study would not only provide efficient means for nuclear reprogramming but also give valuable insights into the molecular mechanisms of nuclear reprogramming.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Mice

Oct4-EGFP mice were mated with transgenic mice expressing a neomycin resistance gene to produce mice expressing Oct4-EGFP (Yoshimizu et al. 1999) and the neomycin resistance gene (Kimura et al. 2003a). The genotype of each individual was checked by polymerase chain reaction (PCR) as described previously (Kimura et al. 2003a). Animal care was in accordance with the guidelines of Osaka University.

Cell culture

Feeder-free mouse ES cells, E14tg2a, and their derivatives were maintained as described previously (Niwa et al. 1998). MEFs were prepared from day 13.5 embryos and grown in Dulbecco's modified Eagle medium (DMEM) that was supplemented with 10% fetal bovine serum. MEFs were used for cell fusion experiments within three passages to avoid the replicative senescence.

Plasmids

cDNA encoding myristoylated-human Akt lacking the PH domain fused to Mer in-frame was cloned into a pCAGGS vector to produce the Akt-Mer expression plasmid.

cDNA encoding myristoylated-human Akt lacking the PH domain was cloned into pBluescript KS + (Stratagene) to produce active form of Akt mRNA.

Western blot analysis

Western blotting was carried out as previously described (Nakamura et al. 2007). The primary antibodies used were anti-Akt (1 : 2000; #9272, Cell Signaling Technology), anti-phospho(Ser473)-Akt (1 : 2000; #9271, Cell Signaling Technology) and anti-β-actin (1 : 10 000; AC-15, Sigma).

Isolation of thymocytes

Thymocytes were obtained from 6- to 12-week-old mice carrying the Oct4-EGFP and the neomycin resistance gene. After dissection, the thymuses were squirted in and out of a 2.5-mL syringe through a 18-gauge needle several times to obtain a single cell suspension. Thymocytes were used for cell fusion experiment immediately after the harvest.

Cell fusion

To induce cell fusion, 2 × 107 ES cells and 3 × 107 somatic cells were combined in serum-free DMEM in a conical tube and pelleted, and the supernatant was aspirated. The pellet was broken by gentle tapping, and 1 mL of 50% w/v PEG 1500 (Roche) prewarmed to 37 °C was gently added over a period of 1 min with continual stirring. Cells were left in the 50% PEG solution for 3 min with continual stirring. Four milliliters of DMEM were then added over a period of 4 min with continual stirring. After 10 mL of medium were added, cells were incubated for 5 min at 37 °C. After incubation, the cells were spun down and the supernatant was discarded. The pellet was resuspended in complete ES cell medium and plated onto two 150-mm dishes. To activate Akt, 10 µm 4OHT were added to one of two dishes immediately after cell plating, for 5 days or 24 h. Selection was applied after 24 h using G418 (200 µg/mL), puromycin (2 µg/mL) or hygromycin (160 µg/mL), as appropriate. For cell fusion between Akt-Mer expressing ES cells and normal ES cells, hygromycin-resistant ES cells were used.

Reverse transcription-PCR

Total RNA was isolated using an RNeasy mini kit (Qiagen), and 1 µg of total RNA was used for cDNA synthesis. Reverse transcription was carried out using the ThermoScript RT-PCR system (Invitrogen) as previously described (Umehara et al. 2007). PCR reactions were optimized to allow semiquantitative comparisons within the log phase of amplification. The primer sequences and cycle conditions are listed in Table 3.

Table 3. Oligonucleotides primers used for RT-PCR analysis
GeneSenseAntisensePCR cycles
Mixl1 5′-ACTTTCCAGCTCTTTCAAGAGCC-3′5′-ATTGTGTACTCCCCAACTTTCCC-3′28
Collagen IV 5′-CAAGCATAGTGGTCCGAGTC-3′5′-AGGCAGGTCAAGTTCTAGCG-3′20
AFP 5′-TGCAGAAACACATCGAGGAGAG-3′5′-GCTTCACCAGGTTAAGAGAAGCT-3′25
Nestin 5′-AACTGGCACACCTCAAGATGT-3′5′-TCAAGGGTATTAGGCAAGGGG-3′25
Rex-1 5′-CACCGACAACATGAATGAACAAAAA-3′5′-CAATCTGTCTCCACCTTCAGCATTT-3′25
GAPDH 5′-GGGTGGAGCCAAACGGGTCATC-3′5′-GCCAGTGAGCTTCCCGTTCAGC-3′20

In vitro differentiation assays

Cells were harvested by tripsinization and transferred to bacterial culture dishes in the ES medium without LIF. Cells were refed every second day and cultured for 7 days in suspension. The resultant cystic EBs were then adhered to gelatin-coated dishes and cultured for an additional 5 days.

Teratoma formation

Hybrid cells (5 × 106) were injected subcutaneously into nude mice. After 3 weeks, the teratoma was excised, fixed in 4% PFA, and subjected to histological examination with hematoxylin and eosin (H&E) staining.

Somatic cell nuclear transfer

Nuclear transfer was carried out as described previously (Wakayama et al. 1998; Ogura et al. 2000; Inoue et al. 2006, 2007). Female B6D2F1 mice, 7–10 weeks old, were superovulated with 7.5 IU of pregnant mare serum gonadotropin and 7.5 IU of human chorionic gonadotropin (hCG) at 48-h intervals, then killed 16 h after the hCG injection. Mature meiosis stage II (MII) oocytes were collected from their oviducts. Cumulus cells were released in potassium-modified simplex-optimized medium (KSOM) containing 0.1% hyaluronidase and washed several times with fresh medium. Oocytes were cultured in KSOM at 37.5 °C in an atmosphere of 5.5% CO2 in air until mRNA injection. mRNA of EGFP or an active mutant of Akt were synthesized using mMESSAGE mMACHINE T7 Ultra (Ambion) according to the manufacturer's protocol. In vitro transcribed mRNA of EGFP or an active mutant of Akt (50 ng/µg) was injected into MII oocytes. The mRNA-injected oocytes were placed in HEPES-buffered KSOM including 7.5 µg/mL of cytochalasin B (Calbiochem) and nuclei were removed with a small amount of cytoplasm. Enucleated oocytes were cultured in KSOM in an incubator as above for 30–60 min to allow the cell membrane to recover. The cumulus cells were enucleated using glass micropipettes, and their nuclei were injected into the ooplasm using a Piezo-driven micromanipulator (PrimeTech). After nuclear transfer, reconstructed oocytes were cultured with KSOM for 1–2 h and transferred into Ca2+-free KSOM, including 3 mm SrCl2 and 5 µg/mL of cytochalasin B. One hour later, activated oocytes were transferred into KSOM containing only 5 µg/mL of cytochalasin B and cultured for 5 h longer. After washing, the oocytes were cultured in fresh KSOM at 37.5 °C in an atmosphere of 5.5% CO2 for 4 days.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Drs Y. Matsui and H. Niwa for kindly providing Oct4-EGFP mice and plasmids. We also thank N. Asada for assistance and A. Mizokami for secretarial assistance. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Support Program for Technology Development on the Basis of Academic Findings (NEDO); the Uehara Memorial Foundation; the Osaka Cancer Foundation; and the 21st Century COE “CICET”.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1 Effect of GSK inhibitor on the efficiency of cell fusion mediated nuclear reprogramming

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GTC_1243_sm_TableS1.doc26KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.