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
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
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.
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.
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 vivo
teratomas and in vitro
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,
II. MATERIALS AND
: Adult mice, ES cells, and neo r feeder cells (see
: 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
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
: 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),
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
(Sigma S8636), 7.5% sodium bicarbonate
(Sigma S8761), Ca2+
saline (PBS) (GIBCO 10010-023), 0.25% trypsin/
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
= 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.
- ES medium: Mix 500ml of DMEM/F12, 75ml of FBS,
5ml of 200mM glutamine, 5ml of penicillinstreptomycin
(100×), 5 ml of 100 mM sodium pyruvate,
8ml of 7.5% sodium bicarbonate, 4µl of 10-4M 2-mercaptoethanol, and 0.05 ml of 107U/ml LIF
(final 1000U/ml). Store at 4°C.
- PEF medium: Mix 500ml of DMEM, 50ml of FBS,
5ml of 200mM glutamine, 5ml of 10,000IU/ml
penicillin, and 10 mg/ml streptomycin. Store at 4°C.
- 0.25% trypsin/1 mM EDTA.4Na: Dispense into
aliquots and store at-20°C.
- Ca2+/Mg2+-free phosphate-buffered saline (PBS)
- lOlag/ml mytomycin C: 0.2mg/ml in PBS, dispense
into aliquots, and store at-20°C.
- 0.1% gelatin: 0.1% gelatin in distilled water. Sterilize
by autoclaving and store at 4°C.
B. Pretreatment of ES and Somatic Cells for
Fresh nonelectrolyte solution; 0.3 M mannitol buffer
- Coat 60-mm culture dishes with 0.1% gelatin for at
least 30min at room temperature.
- Prepare mouse primary embryonic fibroblasts
(PEFs) produced from day 13 embryos of ROSA26
transgenic mice carrying the ubiquitously expressed neo/lacZ gene (Friedrich and Soriano
1991). Treat neor PEFs with 10µg/ml mitomycin C
(MMC) and incubate at 37°C in a CO2 incubator for
2h to produce mitotically inactivated feeder cells.
Prepare frozen stocks of the MMC-treated PEFs at a
concentration of 5 × 106 cells/ml and store in a cryotube
in liquid nitrogen. The inactivated neo r PEFs
are routinely used as feeder cells (1 × 106 cells/60-
mm culture dish and 2.5 × 106 cells/100-mm culture
dish) for culture of ES and hybrid cells, and also
for selection of hybrid cell colonies with G418.
For establishment of PEFs, see Abbondanzo et al. (1993).
- Culture exponentially growing ES cells on the inactivated
PEFs with changes of culture medium once
or twice a day. Carry out subculturing of the ES cells
every 2 days by a 1:4 split. ES cells at early passages
are used for experiments. Before cell fusion, it
should be verified that the karyotype of the ES cells
- Prepare gelatin-coated 30-mm culture dishes (6-
well-culture plates) containing the inactivated PEFs
(4 × 105 cells/well) in 3ml of ES medium 1 day
before cell fusion experiments.
make 50ml, dissolve 2.74g of mannitol in distilled
water. Filter through a 0.2-µm filter. Store at 4°C.
- Trypsinize ES cells and remove excess trypsin
quickly. Add 3ml of ES medium to inactivate the
trypsin and dissociate the cells into a single-cell
suspension by gentle pipetting. Plate them on a new
gelatin-coated 60-mm culture dish. Incubate the ES
cells in a CO2 incubator for 30min to separate feeder
cells from ES cells.
- Collect unattached ES cells and harvest them by
centrifugation at 1500rpm for 5min. Resuspend the
cell pellet in 10ml of DMEM, transfer them into a
15-ml conical tube, and place at room temperature.
- Sacrifice a 6- to 8-week-old adult mouse
humanely and dissect out the thymus in a clean room
if a clean bench is not available. All of the dissection
instruments should be sterilized by immersion in 70%
ethanol, followed by flaming.
- Wash the tissues with sterilized PBS twice in
60-mm petri dishes and place one lobe of the thymus
in the barrel of a sterile 2.5-ml syringe with a sterile
- To dissociate the thymus into a single-cell suspension,
gently expel the thymus through the tip of
the needle into 2ml of DMEM in a 50-ml conical
tube. Draw up and expel the suspension several
times. Allow to stand for several minutes at room
- Transfer the supernatant excluding cell clumps to
a 15-ml conical tube and add 10ml of DMEM.
- Spin down the ES cells and thymocytes
separately in 15-ml conical tubes at 1500 rpm for
- Wash them with 10ml of DMEM and spin down
at 1500rpm for 5min and repeat again to remove FBS
- Add 5-10ml of DMEM and adjust the density of
ES cells and thymocytes each to 1 × 106 cells/ml.
- Pellet a 1:5 mixture of ES cells and thymocytes
(1 ml of the ES cell suspension and 5 ml of the thymocyte
suspension made in step 9). Keep the remaining
cells for control experiments.
- Spin down and resuspend the cell pellet in
0.3M mannitol buffer at 6 × 106 cells/ml. Usually, 1 ml
of the mixture of ES cells and thymocytes is sufficient
for the following fusion experiment. Use the cells
immediately for electrofusion (Fig. 1).
|FIGURE 1 Schematic representation of the electrofusion system. A mixture of ES cells and somatic cells
suspended in nonelectrolytic mannitol solution is placed in a 1-mm gap between electrodes on a microslide.
Set parameters (1-6) of the AC/DC pulse generator and press the automatic start button. AC electric pulses
induce pearl chain formation, and the subsequent DC electric pulse induces cell fusion. ES cells (ES) and
ES×ES hybrid cells are nonviable in the selection medium, whereas thymocytes (T) and T×T hybrid cells
are nonadherent. ES hybrid cells (ES×T) rescued by the thymocyte genome are capable of surviving and
proliferating in the selection medium.
C. Operation of ECM 2001 Pulse Generator
and Electrofusion Protocol
D. Fusion Examples: Selection System for
- Sterilize the microslides by immersion in 70%
ethanol, followed by flaming.
- Set a microslide in a 100-mm plastic dish
- For each electrofusion, apply 40 gl of cell mixture
between the electrodes with a 1-mm gap on the
- Connect the microslide in the 100-mm plastic
dish chamber with the ECM 2001 AC/DC pulse
generator by electric cables. Set the chamber on an
inverted microscope to allow for observation of cell
alignment and the fusion processes. It is important to
monitor the fusion process microscopically each time.
The electrofusion may proceed somewhat differently
depending on the density of cell preparations and on
the cell type (cell size).
- Set the optimized electrical parameters to fuse ES
cells and thymocytes (Fig. 1): 10V alternating current
(AC), 99s AC duration, and 250-300V direct current
(DC). Adjust the DC voltage according to the size of
the gap between the electrodes. The appropriate electric
field strength is 2.5-3.0 kV/cm. When microslides with a 2-mm gap are used, the DC voltage should be
almost 600 V.
DC pulse length: 10 µs
Number of DC pulses: 1
Postfusion AC duration: 8s
- Use the automatic operation switch to initiate AC
followed by DC. AC is utilized to induce an inhomogeneous,
or divergent electric field, resulting in cell
alignment and pearl chain formation. DC is utilized to
produce reversible temporary pores in the cytoplasmic
membranes. When juxtaposed pores in the physically
associated cells reseal, cells have a chance to be
hybridized via cytoplasmic membrane fusion. AC
application after the DC pulse induces compression of the cells, which helps the process of fusion between the
- Add 40µl of DMEM to the fusion mixture
between the electrodes to induce the recovery of membrane
- Place the cell mixture at room temperature for
10 min and transfer the cells to a 30-mm culture dish containing
inactivated PEFs with 3 ml of the ES medium.
- Repeat the cell fusion procedure sequentially
using several microslides. Usually, cells recovered
from three microslides (40µl × 3) are plated into one
30-mm culture dish.
- As a control, plate the untreated cell mixture
and culture under the same conditions.
- Change the medium to ES medium with appropriate
supplements for selecting ES hybrid cells 24h
after cell fusion. The selection medium should be
changed once a day (see Section III,D). During the 7-
day selection treatment, unfused ES cells and hybrid
cells between ES cells are killed and hybrid cells
between thymocytes are nonadherent. Thus, only the
hybrid cells between ES cells and somatic cells survive,
proliferate, and form colonies. Several colonies of
hybrid cells per 104 host ES cells are obtained under
appropriate cell fusion and culture conditions.
- Pick up the colonies with a micropipette and
transfer each colony into a 10-mm well of a 24-well
culture plate containing 1 × 105 inactivated PEFs per
well and 0.8 ml of the ES medium with supplements
- Subculture the cells every 2 or 3 days and gradually
expand the number of cells in 30- and then 60-
mm culture dishes with the inactivated PEFs and ES
medium with supplements for selection. When the
cells become nearly confluent in a 60-mm culture dish,
it is considered that a hybrid cell line of passage 1 has
- Change the ES medium without selection supplements
once or twice a day and subculture the
hybrid cell line every 2 days under optimal culture
conditions by splitting 1:4.
- Subject hybrid cells to chromosome analysis
soon after they are established.
This section describes one independent chemical
selection system that can be used to select for hybrid
cells between ES cells and somatic cells.
- Normal ES cells are hybridized with thymocytes
containing the bacterial neomycin resistance (neor)
gene (Tada et al., 1997, 2001). Thymocytes are derived
from ROSA26 transgenic mice, which carry the ubiquitously expressed neo/lacZ transgene (Friedrich and
Soriano, 1991). Only ES hybrid cells with the thymocytes
can survive and grow in the ES medium supplemented
with the antibiotic G418, a protein synthesis
inhibitor. In this case, the ES hybrid cells and their
derivatives are identified visually by their positive
reaction with X-gal due to β-galactosidase activity,
allowing one to analyze their contribution to the development
of chimeric embryos and tissues (Tada et al.,
1997, 2001, 2003). Male ES cells deficient for the Hprt gene on the X chromosome are a powerful tool for
producing hybrid cells with wild-type somatic cells.
Electrofusion-treated cells are cultured in ES medium
with the HAT supplement. In DNA synthesis, purine
nucleotides can be synthesized by the de novo pathway
and recycled by the salvage pathway. Hprt is a purine
salvage enzyme, responsible for converting the purine
degradation product hypoxanthine to inosine monophosphate,
a precursor of ATP and GTP. In the
presence of aminopterin, the de novo pathway is inhibited
and only the salvage pathway functions. Consequently,
dysfunction of Hprt induces cell death in
cultures grown in HAT medium. Thus, the HAT
medium proves fatal to Hprt-deficient ES cells,
whereas ES hybrid cells, which are rescued by the
thymocytes-derived wild-type Hprt gene, are able to
survive and proliferate (Tada et al., 2003; Kimura et al.,
- ES medium with G418: Reconstitute G418 with
water (50mg/ml). Sterilize through a 0.2-µm filter and
store at 4°C. Add 50µl of the G418 solution to 10ml
of ES medium, yielding a final concentration of
- ES medium with HAT: Reconstitute the HAT
media supplement obtained from the supplier in a vial
with 10 ml of DMEM (50× solution) and store at -20°C. Each vial contains 5 × 10-3 M hypoxanthine, 2 × 10-5 M aminopterin, and 8 × 10-4 M thymidine. Add 200µl of
50× solution to 10ml of ES medium.
Selection with G418
- Perform electrofusion between normal ES cells and
thymocytes collected from the 6- to 8-week-old
ROSA26 transgenic mice carrying the neo/lacZ transgene according to the procedure described
- Culture the electrofusion-treated cells in ES
medium for 24 h.
- Change to ES medium supplemented with G418. ES
hybrid cell colonies can be detected by 7-10 days.
- ES thymocyte hybrid cells are positive for X-gal
staining and immunoreactive with the anti-β-
Selection with HAT
- Carry out electrofusion between ES cells (XY) deficient
for Hprt and thymocytes collected from 6- to
8-week-old female mice (XX) according to the procedure
- Culture electrofusion-treated cells in ES medium for
- Change to ES medium containing the HAT supplement.
ES hybrid cell colonies can be detected by
- ES thymocyte hybrid cells possess a karyotype of 4n = 80 with an XXXY sex chromosome constitution.
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.
|FIGURE 2 Pluripotential competence of ES hybrid cells with somatic cells. (A) Undifferentiated ES somatic
hybrid cell colonies in culture on mitotically inactivated PEFs. (B) Neuronal cells differentiated in vitro from
ES somatic hybrid cells on PA6 stromal feeder cells.
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.
- Adjust the cell density according to the size of the
cells used as the fusion partner. Remove cell debris
from the mixture of ES cells and somatic cells. Cell
debris, which is irregular in size, sometimes makes
the formation of pearl chains difficult.
- Increase the cell density if the pearl chains form
3. Decrease the cell density if the cell movement is
disturbed.Decrease the cell density if the cell movement is
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