Establishment of Embryonic Stem Cell
Lines from Adult Somatic Cells by
Usually, embryonic stem (ES) cells are established
from fertilized embryos. Recent studies have shown
that it is possible to produce cloned embryos from adult
somatic cell nuclei; even with a low success rate, some
of those embryos can develop to full term (Wakayama et al
., 1998, 2000a). Now, using this technique, it has
become possible to create a new embryonic stem (ES)
cell line via nuclear transfer (ntES cell line) from adult
somatic cells (Munsie et al
., 2000; Wakayama et al
These ntES cells differentiate not only all three embryonic
germ layers in vitro, but also germ cells in vivo, and
some ntES cell nuclei can develop to full term after a
second nuclear transfer (Wakayama et al
., 2001). The
ntES cell techniques could be useful for research tools
used in reprogramming, imprinting, and gene modification
(Wakayama et al
., 2002). "Whereas the ntES technique
is expected to have applications in regenerative
medicine without immune rejection, we demonstrated
that it is also applicable to the preservation of genetic
resources of mouse strains instead of an embryo,
oocyte, or spermatozoon. At present, this technique is
the only one available for the preservation of valuable
genetic resources from mutant mouse without germ
cells (Wakayama et al., 2005a,b)."
II. MATERIALS AND
A. Mouse Cloning Medium
KSOM (long-term culture medium) from Specialty
media (#MR-106-D). All others are obtained from
Sigma unless otherwise mentioned. NaCl (S7653),
KCl (P9327), CaCl2
O (C5080), MgSO4
(P5379), D-glucose (G8769), NaHCO3
($5761), sodium lactate 60% (ml/liter) (L1375),
HEPES-Na (H3784), EDTA-2Na (ED2SS), phenol
red (P0290), cytochalasin B (C6762), SrCl2
polyvinylpyrrolidone (PVP; 360kD) (PVP-360),
polyvinyl alcohol (PVA) (P8136), bovine serum
albumin (BSA, A3311), dimethyl sulfoxide (DMSO,
D8779), hyaluronidase (H4272), and mineral oil
B. ES Cell Establishment and
Acidic tyrode solution (MR-004-D), DMEM (SLM-
120-B), phosphate-buffered saline (PBS) Ca/Mg free
(BSS-1006-B), 0.1% gelatin (ES-006-B), nucleosides
(ES-008-CD), nonessential amino acid (TMS-001-C), 2-mercaptoethanol (ES-007-E), penicillin-streptomycin
solution (TMS-AB2-C), trypsin/EDTA solution (SM-
2000-C), mitomycin C (Sigma, 0503): fetal calf serum
(FCS, ES-009-B), KSR (invitrogen, 10828-028) and Lif
(GIBCO #13275-029); Specialty media (http://www.
specialitymedia.com) can provide most of these
Inverted microscope with Hoffman optics is from
Olympus (IX71) or Nikon (TE2000). Micromanipulator
set is from Narishige (MMO-202ND) (Fig. 1A). Piezo
impact drive system is from Prime Tech (PMM-150FU)
(Fig. 1B). Pipette puller is from Sutter Instrument Company (P-97). CO2
incubator, centrifuge, pipettes,
and tissue culture dishes/flasks are also needed.
|FIGURE 1 Micromanipulator and piezo system. (A) Equipment includes an inverted microscope with
Hoffman optics, two injectors, air cushion, and warming plate on the stage of the microscope. (B) Piezo impact
unit attached to the injection pipette holder (arrow).
A. Preparation of Medium
|FIGURE 2 Injection pipette and storage. (A) Breaking
the tip of a
pipette using a microforge. The inner
diameter of the tip depends on
donor cell size.
(B) Inject a small mount of mercury into the pipette
using a 1-ml syringe with a 26-gauge needle.
(C) Storage of the
pipettes in a 10-cm dish.
All pipettes are attached softly on sticky tape.
|FIGURE 3 Manipulation chamber. Right above the line is CZBPVP
medium for washing pipettes. Right under the line is CZB-CB
medium for oocyte enucleation. Left above the line is CZB-PVP
medium. Top two drops and pick up nucleus are washing and last
drop is donor cell diffusion. Left under the line is CZB-HEPES
medium for donor nuclei injection into enucleated oocytes.
Thaw the freezing KSOM medium inside a refrigerator
overnight. After thawing, aliquot in 5-ml disposable
tube and keep at 4°C Make about 10 tubes and
use a new tube every day. Tubes can be used 2 weeks
2. CZB-HEPES (Embryo Handling and Nuclear
Basically, the CZB medium (Chatot et al
., 1990) is
used with slight modifications during in vitro
manipulations. As shown in Table I, all drugs must mix
into 99 ml distilled water (finally becoming 100 ml).
After everything is mixed adjust pH to about 7.4-7.6
HCl and 1N
NaOH. Sterilize with 0.45-µm
filters and aliquot into 5-ml tubes the same as KSOM.
3. CZB-PVP (Donor Cell Diffusion and
Add PVP in CZB-HEPES medium and keep in the
refrigerator overnight and then filtrate and aliquot in
a 1-ml tube.
4. CZB-CB (Oocyte Enucleation Medium)
Dissolve 1 mg cytochalasin B in 2 ml DMSO as stock
solution (100 times concentration) and store at-20°C Take 10 µl of this stock solution and mix with 990 µl of
5. CZB-Sr (Oocyte Activation Medium)
As shown in Table I, this medium is similar to CZB but
and BSA instead of calcium and HEPES.
After mixture, filtrate and aliquot in a 1-ml tube.
6. For ES Cell Establishment
All media can be obtained from Specialty media.
"After mixing all solutions, add FCS (for culture,
20%) or knockout serum (KSR; for ntES cell establishment,
20%) and filtrate."
B. Preparation of Micromanipulation Pipette
and Manipulation Chamber
|FIGURE 4 Oocyte enucleation. (A) Rotate and find the metaphase II spindle
and place it in the 8 to 10
o'clock position. Then hold this oocyte on the holding
pipette. (B) Cut the zona pellucida using an enucleation
pipette with piezo pulses.
(C) Insert enucleation pipette until it reaches the spindle. (D) Remove the
spindle by suction without breaking the plasma membrane and gently pull away
the pipette from oocytes.
(E) The stretched cytoplasmic bridge from oocytes to
pipette was pinched off. (F) Push out the spindle in
order to check enucleation,
which is harder than cytoplasm.
C. Collection of Oocytes and Enucleation
- Holding pipette: The diameter of the outer pipette
should be smaller than the oocyte. If the holding pipette
is larger than the oocyte, you will lose the oocyte
metaphase II spindle when the oocyte is caught by the
- Enucleation/injection pipette: Break the tip of the
pipette at flat, vertical and blunt ends using a microforge
(Narishige Cat. No. MF-900) (Fig. 2A). A notched
tip often kills the oocyte during enucleation and injection.
The inner diameter of the enucleation pipette is
about 8 btm and the injection pipette depends on the cell
type (e.g., for cumulus cell: 5-6 µm, for fibroblast or ES
cell: 6-7 µm, for G2/M phase cell: 7-8 µm).
- Bend the pipette very close to the tip at 15-20° by
- Load a small mount of mercury into the enucleation/
injection pipette, which is not essential but enhances the piezo impact power and control (Fig.
- Manipulation chamber: As shown in Fig. 3, different
media were put on the dish and then covered with
mineral oil. This chamber can be use for both enucleation
- Attach these pipettes to the pipette holder of
the piezo impact drive unit of the micromanipulator
- Expel the air, oil, and mercury from the enucleation/
injection pipette under CZB-PVP medium. Wash
inside/outside of the pipette with the PVP medium,
until inside wall of pipette becomes smooth. Without
this washing, the enucleation / injection pipette becomes
dirty quickly and needs to be changed.
D. Donor Cell Preparation
- Collect the oocyte/cumulus cell complex from
ampullae of the oviduct 14-15h after hCG injection
and move that complex into hyaluronitase (0.1%) containing
CZB-HEPES medium. About 10 min later, pick
up oocytes and keep in KSOM in the incubator.
- Place about 20 oocytes into CZB-CB (enucleation
medium) and keep in this medium at least 5 min before
- Find the metaphase II spindle inside the oocyte.
Without any staining, the spindle can be recognized
under Nomarski or Hoffman optics. Locate the spindle
between the 2 and 4 o'clock positions and then hold
on the holding pipette (Fig. 4A).
- Using a few piezo pulses, cut the zona pellucida
(Fig. 4B). To avoid damaging the oocyte by piezo
pulses, make a large space between the zona pellucida
and the oolemma.
- Insert the enucleation pipette into the oocyte
without breaking the oolemma (Fig. 4C) and remove
the metaphase II spindle by aspirating with a minimal
volume of oocyte cytoplasm. Oocyte membrane and
spindle must be pinched off slowly, do not apply piezo
pulses to cut the membrane (Figs. 4D and 4E).
- Wash the enucleated oocytes several times to
remove cytochalasin B completely and keep in KSOM medium in the incubator until used for donor cell injection.
- Choose the cell type: Cumulus cell (Wakayama et
al., 1998; Wakayama and Yanagimachi, 2001a), tail tip
cell (probably fibroblast; Wakayama and Yanagimachi,
1999b; Ogura et al., 2000a), Sertoli cell (Ogura et al.,
2000b), fetus cell (Wakayama and Yanagimachi, 2001b;
Ono et al., 2001), and ES cell (Wakayama et al., 1999)
have been successful in producing cloned mice. The
methods are slightly different among those cell types,
as shown in Table II. Cumulus cells are easy to prepare
as donors. However, tail tip cells must be prepared at
least 1 week before nuclear transfer, and then single
cell treatment is required.
- To make single cells, remove cells from culture
flask or dish by trypsin treatment. Because trypsin is
very toxic at the time of nuclear injection, donor cells
must be centrifuged at least three times to remove
trypsin completely. However, cumulus cells can be
used immediately without any treatment.
- For diffusing donor cells, pick up 1-3µl donor cell
suspension and introduce into a CZB-PVP drop on the
micromanipulation chamber (Fig. 3). Mix the donor
cells and PVP medium gently using sharp tweezers. If
there is not enough mixture, donor cells stick to each
other and it becomes difficult to pick up a single cell
(Fig. 5A). Do not scratch the bottom of the chamber.
E. Donor Cell Nucleus Injection
|FIGURE 5 Donor nucleus injection. (A and B) Donor nuclei are aspirated in and out of the injection pipette
gently until their nuclei are largely devoid of visible cytoplasmic material. (C) Hold the enucleated oocyte and
cut the zona pellucida using piezo pulses. (D) Insert the injection pipette into enucleated oocyte. (E) Apply a single
piezo pulse to break the membrane and immediately inject the donor nucleus. (F) Pull the pipette away gently.
- Place about 20 enucleated oocytes into
CZB-HEPES (injection medium, Fig. 3).
- Remove donor nuclei from cells and gently
aspirate in and out of the injection pipette until the
nucleus is devoid of visible cytoplasmic material (Figs.
5A and 5B). Put a few nuclei within the injection
- Hold an enucleated oocyte using the holding
- Cut the zona pellucida using a few piezo pulses
as described in the enucleation method (Fig. 5C).
- Push one nucleus forward until it is near the tip
of the pipette and then advance the pipette through the
enucleated oocyte until it almost reaches the opposite
side of the oocyte cortex (Fig. 5D).
- Apply one piezo pulse to puncture the oolemma
at the pipette tip, which is indicated by a rapid relaxation
of the oocyte membrane (Fig. 5E). Expel a donor
nucleus into the enucleated oocyte cytoplasm immediately
with a minimal amount of PVP medium.
Gently withdraw the injection pipette from the oocyte
- After injection, leave the injected oocyte for at
least 10min at room temperature. Then transfer the
oocytes into KSOM medium and store in the incubator
until they are activated with strontium.
F. Oocyte Activation and Culture
|FIGURE 6 Reconstructed oocyte activation medium. Above the
line is CZB-Sr medium, which activate the oocytes. The top two
drops are used for washing oocytes to remove calcium from
medium. Under the line is KSOM medium. The top two drops are
used for washing activated oocytes and to remove A strontium
G. Establishment of ntES Cell Line from
- Nuclear transferred oocytes should be cultured
in KSOM medium for at least 30min before activation.
During this time, donor nuclei may be reprogrammed
under oocyte cytoplasm (Wakayama et al.,
- Activate the oocytes in CZB-Sr medium for 6 h
with medium containing 2-10mM strontium chloride
and 5µg/ml cytochalasin B (Fig. 6). Strontium activates
oocytes, and cytochalasin B prevents extrusion of
the donor chromosomes as a polar body (Wakayama et al., 1998).
- Following activation, transfer all embryos and
wash several times in KSOM medium to remove
cytochalasin B completely (Wakayama and
Yanagimachi, 2001b). Then culture until cloned
embryos develop to the blastocyst stage. "Rarely,
cytochalasin B seeps from activation drops and prevents
cloned embryo development. In this case, it is
better to culture all cloned embryo in another dish."
These embryos may develop to full term when transferred
into a pseudo-pregnant mother with about a
1-2% success rate (Wakayama and Yanagimachi,
- To make an embryonic feeder cell collect day 12.5
to 13.5 dpc fetuses from pregnant mother and then
remove the head and internal organs on a 10-cm petri
dish containing PBS. Place the embryos into a new 10-
cm dish and mince the embryos into very small pieces
with sterile scissors. Add 25 ml DMEM medium and
plate into a larger size (175-cm2) tissue culture flask.
One or 2 days later, split the cells 1:5 by trypsinization
and allow them to grow to confluence.
- Mitomycin C treatment. When the cells become
confluent, treat with 10µg/ml mitomycin C for 2h in
an incubator. Wash the flask several times with PBS to
remove mitomycin C and then collect the cells by
trypsinization. Pellet the cells by centrifugation
(1000rpm for 10min). Aspirate the supernatant and
gently resuspend the cell pellet in freezing medium
(final concentration about l× 106 cells/ml) and divide
into cryovials. Place the vials in a -80°C freezer
overnight; the next day, vials can be transferred to
liquid nitrogen for long-term storage.
- Thaw the vial quickly and transfer cells into a 15-
ml tube filled with ES medium. Pellet the cells by centrifuging
at 1000rpm for 10min and then aspirate the
supernatant and resuspend with fresh ES medium.
Plate on a 96-well multidish and culture in an incubator
until use. This feeder cell preparation should be
done at least 1 day before plating the cloned
- Remove the zona pellucida from the cloned blastocysts
using acidic tyrode solution. The zona pellucida
dissolves quickly within 30 s.
- Wash the cloned blastocysts several times in
KSOM medium and then plate them into a 96-well
multidish containing a performed feeder layer. Culture
the multidish about 10 days. During this period,
cloned blastocysts attach to the surface of the feeder
layer and the inner cell mass (ICM) can be seen to grow
(Figs. 7A and 7B).
- Some wells of the multidish may have clumps of
large ICM (Fig. 7C). If these clumps appear, treat with
trypsin and disaggregate the clump using a 200µl
pipettor (Nagy et al., 2003). Then replate the suspension
into another well of the same multidish. Several
days later, if ICM-like clumps appear on the dish,
replate again the same as before.
- When ES-like cell colonies dominate the well (Fig. 7D), the cells should be expanded gradually into
48-, 24-, and 12-wells and then into 12.5-, 25-, and 75-
cm2 flasks. After the cell number has increased greatly,
freeze and store the cells the same as feeder cells.
|FIGURE 7 Establishment of ntES cell line. (A) A cloned blastocyst was attached to the feeder cell in the
96-well dish. (B) Spreading the trophoblast cells and an inner cell mass (ICM) appearing. (C) ICM grew up
almost 5 to 10 times as large. (D) After trypsinization, some wells show a nearly established ntES cell line.
- So far many types of somatic cell have been used
as donors, but only a few cell types have demonstrated
the capacity of full-term developments.
- For nuclear transfer, only 100 to 150 cells were
used as donors. However, to pick up better cells, donor
cells must be prepared with high concentrations (more
than 105 cells) on the manipulation chamber.
- When using cumulus cells, these cells can be
picked up immediately after collection without washing,
as hyaluronidase is not toxic for oocyte injection.
- Nuclear injection by piezo is a very new method
and very difficult. However, this is almost the same
method as sperm injection into oocytes. You should
begin with the sperm injection experiment before starting
the nuclear transfer experiment. Sperm injection
will help you understand the mechanisms of the piezo
- Washing the pipette by PVP is very important.
This process affects not only the survival rate, but also
development after nuclear transfer.
- During strontium treatment, some eggs die and
the medium becames dirty. This is a normal process
and surviving oocytes suffer no damage by strontium
- It will probably take a few months to generate
data due to incomplete technical skills (Perry et al.,
2002). Without hard practice, the establishment of a
ntES cell line is impossible.
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