Electrofusion: Nuclear Reprogramming of Somatic Cells by Cell Hybridization with Pluripotential Stem Cells
The technique of cell fusion, which was pioneered by Henry Harris (1965), has proved to be a powerful procedure with applications in cell biology, genetics, and developmental biology and in fields of practical concern such as medicine and agriculture. The spontaneous or induced cell fusion of two different types of cells (heterokaryons) generates intraspecific or interspecific hybrid cells. Genetically programmed spontaneous cell fusion is known to occur in the formation of polykaryons such as myotubes, osteoclasts, and syntrophoblasts in vivo. Under in vitro culture conditions, spontaneous cell fusion has been found to occur occasionally in some cell lines and malignant cells. Cell fusion due to membrane integrity between two different cells is induced by treatment with chemical agents such as calcium ions, lysolecithin, and polyethylene glycol; by mediation by viruses such as paramyxoviruses, including Sendai virus (HVJ), oncornavirus, coronavirus, herpesvirus and poxvirus; and by electrofusion.
In 1997, the successful production of the cloned sheep named Dolly demonstrated that committed animal somatic cell nuclei are able to reacquire totipotency as a result of nuclear transplantation into enucleated unfertilized oocytes and the subsequent embryonic development (Wilmut et al., 1997). This nuclear reprogramming results from the resetting of the somatic cell-specific epigenetic program by transacting factors present in unfertilized oocytes. Nearly 20 years ago, genomic plasticity had already been examined by cell fusion experiments between differentiated cell types (Blau et al., 1985; Baron and Maniatis, 1986; Blau and Baltimore, 1991). More recently, this approach has been used to study genomic reprogramming that occurs in X chromosome reactivation (Takagi et al., 1983; Tada et al., 2001; Kimura et al., 2003) and switching of parental origin-specific marks of imprinted genes (Tada et al., 1997).
An important finding is that pluripotential embryonic stem (ES) cells have an intrinsic capacity for epigenetic reprogramming of somatic genomes following cell fusion (Tada et al., 2001, 2003; Kimura et al., 2003). In hybrid cells between ES cells and adult thymocytes, nuclear reprogramming of the somatic genome has been demonstrated by (1) the contribution of ES hybrid cells to normal embryogenesis of chimeras, (2) reactivation of the silenced X chromosome derived from female somatic cells, (3) reactivation of pluripotential cell-specific genes, Oct4, Xist, and Tsix, which were derived from the somatic cell, (4) redifferentiation into a variety of cell types in teratomas, (5) tissue-specific gene expression from the reprogrammed somatic genome in addition to the ES genome in in vivodifferentiated teratomas and in vitro-differentiated neuronal cells, and (6) acquisition by reprogrammed somatic genomes of pluripotential cell-specific histone tail modifications. More interestingly, cell fusion experiments between somatic cells and embryonic germ (EG) cells derived from the gonadal primordial germ cells of mouse 11.5-12.5dpc embryos have demonstrated that EG cells possess an additional potential for inducing the reprogramming of somatic cell-derived parental imprints accompanied by the disruption of parental origin-specific DNA methylation of imprinted genes (Tada et al., 1997, 1998). Therefore, cell fusion with pluripotential stem cells is now recognized as a powerful approach for elucidating mechanisms of epigenetic reprogramming involving DNA and chromatin modifications.
More recent evidence has shown that neurosphere and bone marrow cells undergo nuclear reprogramming following spontaneous cell fusion with cocultured ES cells in vitro (Terada et al., 2002; Ying et al., 2002). Furthermore, experiments involving the in vivo transplantation of bone marrow cells have demonstrated that regenerated hepatocytes are derived from donor hematopoietic cells that undergo fusion with host hepatocytes, not from the transdifferentiation of hematopoietic stem cells or hepatic stem cells present in bone marrow (Vassilopoulos et al., 2003; Wang et al., 2003). Thus, the nuclear reprogramming of somatic cells by in vivo cell fusion is thought to play an important role in maintaining the homeostasis of some tissues by regeneration during defined self-renewal and following tissue damage.
This article describes a practical procedure for electrofusion to produce hybrid cells between pluripotential stem cells and committed somatic cells (mouse ES cells and lymphocytes isolated from the adult thymus) without the use of virus or chemicals to mediate the fusion. ES cells are adherent cells that undergo selfrenewal by rapid cell division, whereas thymocytes are nondividing and nonadherent cells. In order to select the hybrid cells effectively, either thymocytes carrying the neo transgene or male ES cells deficient for the Xlinked Hprt (hyoxanthine phosphoribosyl transferase) gene are used as the partner cells in the cell fusion. Consequently, only hybrid cell colonies are capable of surviving and growing in culture in the presence of antibiotic G418 or HAT (hypoxanthine, aminopterin, and thymidine).
II. MATERIALS AND INSTRUMENTATION
Cells: Adult mice, ES cells, and neo r feeder cells (see Section II,A)
Instruments: ECM 2001 AC/DC pulse generator (BTX), 1-mm gap microslide chambers (BTX P/N450- 10WG), inverted microscope with 10 and 20× objectives, humidified incubator at 37°C, 5% CO2, 95% air, 60-mm plastic tissue culture dishes, 60- and 100-mm bacterial dishes, 10- and 30-mm well plastic tissue culture plates, 15- and 50-ml conical tubes, 0.2-µm microfilters, 200- and 1000-µl capacity adjustable pipetters with autoclaved tips, forceps, scissors, 2.5-ml syringes, 18-gauge needles
Compounds: Dulbecco's modified Eagle's medium/ nutrient mixture F12 Ham (DMEM/F12) (Sigma D6421), Dulbecco's modified Eagle's medium (DMEM) (Sigma D5796), fetal bovine serum (FBS) (JRH Biosciences 12003-78P), recombinant leukemia inhibitory factor (LIF) (Chemicon ESG1107), 200 mM glutamine (GIBCO 320-5030AG), 2-mercaptoethanol (Sigma M7520), 10,000 IU/ml penicillin and 10mg/ml streptomycin (penicillin-streptomycin 100×) (Sigma P-0781), 100 mM sodium pyruvate (Sigma S8636), 7.5% sodium bicarbonate (Sigma S8761), Ca2+/Mg2+-free phosphate-buffered saline (PBS) (GIBCO 10010-023), 0.25% trypsin/ 1 mM EDTA. 4Na (GIBCO 25200-056), mytomycin C (Sigma M0503), gelatin from porcine skin, type A (Sigma G-1890), D-mannitol (Sigma M-9546), G418 (geneticin) (Sigma G-9516), HAT media supplement 50x (HAT) (Sigma H0262)
A. Mouse ES Cell and Feeder Cell Culture
One of the most important variables for cell fusion experiments is how stably ES cells (2n = 40) and hybrid cells (2n = 80) can be cultured without loss of the pluripotential competence and the full set of chromosomes derived from mouse ES cells and somatic cells through numerous cell divisions. The culture conditions are basically those described previously (Abbondanzo et al., 1993). A crucial point is quality control of the FBS, which is added to make the ES cell culture medium cocktail. FBS certified for ES cell culturing has become available commercially. We strongly recommend the use of a suitable production lot of FBS that can support effective cell growth without inducing differentiation equivalently to the ES cell-certified FBS.
B. Pretreatment of ES and Somatic Cells for Cell Fusion
Fresh nonelectrolyte solution; 0.3 M mannitol buffer: To make 50ml, dissolve 2.74g of mannitol in distilled water. Filter through a 0.2-µm filter. Store at 4°C.
C. Operation of ECM 2001 Pulse Generator and Electrofusion Protocol
D. Fusion Examples: Selection System for Hybrid Cells
This section describes one independent chemical selection system that can be used to select for hybrid cells between ES cells and somatic cells.
Selection with G418
Selection with HAT
Figure 2A shows representative ES hybrid cells in culture on feeder cells in the ES medium. Figure 2B shows representative neuronal cells differentiated from ES hybrid cells. The ES hybrid cells are pluripotential and can differentiate into a variety of tissues in vivo and in vitro. Tissue-specific transcripts derived from the reprogrammed somatic genomes can be identified based on genetic polymorphisms found in intersubspecific ES hybrid cells (Mus musculus domesticus× M. m. molossinus). The reprogrammed somatic cell genomes function similarly to the ES cell genomes in undifferentiated ES hybrid cells and also in ES hybrid cell derivatives differentiated in vivo and in vitro (Kimura et al., 2003; Tada et al., 2003).
ES hybrid cells can also be produced by 50% polyethylene glycol (PEG) treatment. Hybrid cells between embryonic carcinoma (EC) cells deficient for the Hprt gene and lymphocytes from the thymus or spleen are produced by cell fusion induced chemically by PEG (Takagi et al., 1983). To produce ES hybrid cells using PEG, wash a mixture of ES cells and thymocytes with DMEM and pellet the cells by centrifugation. Prewarm 1 ml of a 50% PEG mixture (PEG4000/DMEM = 1:1) at 37°C and then add the PEG mixture to the cell pellet gradually using the tip of a pipette. Add 9ml of DMEM gradually. Collect the cells by centrifugation, resuspend the cells in ES medium, and transfer them to a culture dish. Selection of hybrid cells is begun 1 day after the PEG-induced fusion treatment. Electrofusion has the following advantages over the PEGinduced cell fusion: (1) electrofusion is appropriate for in vivo applications of the hybrid cells, whereas PEGinduced fusion is not because PEG is toxic to cells; (2) electrofusion is more efficient and reproducible than PEG-induced cell fusion for producing ES hybrid cells; and (3) it is easier to produce hybrid cells by electrofusion than by PEG-induced fusion.
If there are problems with the AC procedure, you may be able to solve the problems as follows. Pellet the mixed cells by centrifugation and resuspend the cells in a suitable amount of fresh mannitol buffer.
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