Reprogramming Somatic Nuclei and
Cells with Cell Extracts
|FIGURE 1 Strategy of in vitro cell
reprogramming. 293T cells grown
are reversibly permeabilised with SLO. The
cells are incubated in a nuclear and
derived from Jurkat TAg cells
for 1 h. The cells are resealed for 2h
medium containing 2mM CaCl2. The CaCl2-
medium is replaced by regular
complete culture medium, and the
reprogrammed cells are cultured for assessment
Genomic plasticity has generated considerable
interest in the past two decades; nevertheless the
mechanisms underlying nuclear programming remain
poorly understood. We report a method that allows the
processes of nuclear reprogramming to be investigated in vitro
Nuclear reprogramming occurs in a variety of
natural and experimental contexts. After fertilization,
epigenetic alterations of the embryonic genome take
place during successive stages of development. Epigenetic
changes and alterations in gene expression also
occur after the fusion of somatic cells with less differentiated
cell types (Blau and Blakely, 1999; Tada et al.
2001). The birth of clones and the production of embryonic
stem cells by transplantation of nuclei into
oocytes also provide evidence of complete reprogramming
of somatic nuclei (Cibelli et al.
, 1998; Gurdon
et at., 1979; Munsie et al.
, 2000; Wilmut et al.
Based on these examples, reprogramming can be
defined as an alteration of a differentiated nucleus into
a totipotent or mutipotent state. Additional studies
have shown that a somatic cell type could be, at least
partially, turned into another somatic cell type. This
was achieved in coculture conditions (Morrison, 2001)
and, more recently, by exposing somatic nuclei or cells
to an extract derived from another somatic cell type
(Håkelien et al.
, 2002; Landsverk et al.
, 2002). These
observations have led to the proposal of a simple definition
of nuclear reprogramming. Reprogramming
may not necessarily involve dedifferentiation or return
to a more pluripotent state, but may refer to the dominance
of the program of one cell type over another, resulting in "the transformation of the pliant nucleus
[in]to the dominant type" (Western and Surani, 2002).
We describe a procedure to redirect the nuclear
program of a transformed human fibroblast cell line
toward a T-cell program. The approach is outlined in
Fig. 1 and may, in principle, be applied to multiple cell
types. The procedure involves the use of a nuclear and
cytoplasmic extract derived from Jurkat T-cells in
which reversibly permeabilized fibroblasts are incubated.
At the end of incubation, the fibroblasts are
resealed and cultured to assess the expression of T-cellspecific
markers and the establishment of T-cellspecific
functions. As large numbers of cells or nuclei
can be processed simultaneously, and considering the
ability of cell extracts to be manipulated biochemically, in vitro
manipulation of nuclei and cells provides a
potentially powerful system for analyzing the mechanisms
of nuclear reprogramming.
II. MATERIALS AND
- 293T human fibroblasts cultured on glass
- Round, 12-mm glass coverslips, autoclaved
- Poly-L-lysine (Cat. No. P8920, Sigma-Aldrich Co.,
St. Louis, MO)
- Propidium iodide (Cat. No. P4170, Sigma). Make a
1-mg/ml stock solution in H2O and store at -20°C in the dark.
- RPMI 1640 medium (Cat. No. R0883, Sigma) supplemented
with 10% fetal calf serum
- Hanks balanced salt solution (HBSS; Cat. No.
14170-088, Gibco-BRL; Paisley, UK)
- Protease inhibitor cocktail (Cat. No. P2714, Sigma).
This is a 100× stock solution.
- Cell lysis buffer (50 mM NaCl, 5 mM MgCl2, 20 mM HEPES, pH 8.2, l mM dithiothreitol, 0.1mM phenylmethylsulphonyl fluoride and the protease
inhibitor cocktail) at 4°C
- Streptolysin O (SLO) (Cat. No. S5265, Sigma) at
100µg/ml in H2O, aliquoted, and stored at -20°C
- 1M CaCl2 (Cat. No. C4901, Sigma) in sterile H2O
- ATP (Cat. No. A3377, Sigma) at 200mM in H2O,
aliquoted, and stored at -20°C
- Creatine kinase (Cat. No. C3755, Sigma) at
5 mg/ml in H2O, aliquoted, and stored at -20°C
- Phosphocreatine (Cat. No. P7936, Sigma) at 2M in
H2O, aliquoted, and stored at -20°C
- GTP (Cat. No. G8752, Sigma) at 10mM in H2O,
aliquoted, and stored at -20°C
- Nucleotide triphosphate (NTP) set (Cat. No.
1277057, Roche; Basel, Switzerland). Prepare a
stock solution by mixing 20µl of each NTP provided
in the set at a 1:1:1:1 ratio on ice. Aliquot
in 10 µl and store at-20°C This makes an NTP mix
at 25 mM of each NTP. Prepare more stock solution
- Sonicator fitted with a 2-mm-diameter probe
(Model Labsonic M, B. Braun Biotech International;
- Regular atmosphere incubator set at 37°C (for
- 5% CO2 incubator set at 37°C
- 50- and 15-ml conical tubes (Corning; Corning, NY)
- 1.5-ml centrifuge tubes
- 24-well cell culture plates (Costar Cat. No. 3524,
- Refrigerated centrifuge with swinging buckets
suited for 15- and 50-ml conical tubes
The methods describe (1) the preparation of
"donor" cells to be reprogrammed, (2) the preparation
of the reprogramming extract, (3) the permeabilisation
of the donor cells, (4) the reprogramming reaction, (5)
the resealing of the reprogrammed cells, and (6) examples
of assessments of nuclear and cell reprogramming.
The procedures described are based on the
reprogramming of a human fibroblast cell line (293T)
in an extract derived from the human Jurkat TAg cell
line (Håkelien et al.
A. Seeding 293T Cells
On the day prior to reprogramming reaction, plate
293T cells onto 12-mm round, sterile, poly-L-lysinecoated
glass coverslips at a density of 50,000 cells per
coverslip. Each coverslip is placed in individual wells
of a 24-well culture plate. Overlay coverslips with
500µl of complete RPMI 1640 medium and place in a
incubator at 37°C.
B. Preparation of the Reprogramming Extract
1. Cell Harvest
2. Cell Swelling
- Transfer the Jurkat TAg cell suspension culture into
50-ml conical tubes and sediment the cells at 800g for 10 min at 4°C.
- Wash the cells twice in ice-cold phosphate-buffered
saline (PBS) by suspension and sedimentation at
800g for 10min at 4°C. Cells can be pooled into a
single tube after the first wash.
3. Extract Preparation
- Resuspend the cells in 10 ml ice-cold cell lysis buffer
(CLB). It is preferable to use a graduated 15-ml
conical tube to estimate the cell volume after
- Centrifuge at 800g for 10min at 4°C.
- Estimate the volume of the cell pellet. Resuspend
the pellet into 2 volumes of ice-cold CLB.
- Hold the cells on ice for 45 min to allow swelling.
This makes it easier to lyse the cells during sonication.
Keep the cells well suspended by occasional
tapping of the tube during swelling. Note that the
cells can be allowed to swell for longer than 45 min.
This swelling step can be omitted for Jurkat TAg or
primary T-cells as these cell types lyse promptly
4. Extract Toxicity Assay
- Aliquot the cell suspension into 200µl in 1.5-ml
centrifuge tubes previously chilled on ice. Sonicate
each tube one by one (on ice) until all cells and nuclei
are lysed. Lysis is assessed by complete disruption of
the cells and nuclei, as judged by the sole appearance
of cell "debris" by phase-contrast microscopy examination
of 3-µl samples. Once lysis is achieved in a tube,
keep the tube on ice and proceed with all other tubes.
Power and duration of sonication varies with each cell
type. For Jurkat TAg cells, sonication of each tube at
25% power and 0.5-s pulse cycle over 1rain 40sec is
- Pool all the lysates into one (or multiple, if
needed) chilled 1.5-ml centrifuge tube. Sediment
the lysate at 15,000g for 15rain at 4°C in a fixedangled
rotor. Note that a swing-out rotor can also be
- Carefully collect the supernatant with a 200-µl
pipette and transfer it into a new 1.5-ml tube chilled
on ice. This is the reprogramming extract.
- It is possible to aliquot the extract into 200-µl
tubes such as those used for polymerase chain reaction,
with 100 µl extract per tube. Snap-freeze each tube
in liquid N2 and store at -80°C. However, we recommend
carrying out reprogramming with freshly made extract as the stability of the extract at -80°C may vary
with cell types and batches.
- Following sedimentation in step 3, remove 20µl
of extract to determine protein concentration and pH.
The protein concentration should be between 20 and
25mg/ml. The pH should be between 6.7 and 7.0 (see
|FIGURE 2 Results from toxicity
assay. Rat embryo fibroblasts
were exposed to a eprogramming
extract for 1 h as described in the
text. Cells were examined by
phase-contrast microscopy. (A)
cells. (B) Nonviable cells.
discarded. (C) Control cells
exposed for 30min to the cell
lysis buffer used to prepare the
extract. Bar: 20 µm.
Each new batch of Jurkat TAg cell extract requires a
cell toxicity test.
C. Permeabilisation of 293T Cells
- Add 50,000 293T cells (or, in principle, HeLa cells
or any other epithelial of fibroblast cell line growing
in the laboratory) to 35 µl of extract on ice in a 1.5-ml
centrifuge tube. The extract does not need to contain
any additives (unlike for a reprogramming reaction;
see Section III,E,1).
- Incubate for 1 h at 37°C in a water bath.
- Remove a 3-µl aliquot and assess cell morphology
by phase-contrast microscopy. Fig. 2 illustrates
primary rat fetal fibroblasts after a 30-min exposure to
reprogramming extracts. In our hands, the morphology
of the cells after a 30-min incubation in the extract
reflects their survival in culture as judged 24 h after the
toxicity assay. Cells shown in Fig. 2A survive the
extract exposure, whereas cells in Fig. 2B have been
damaged by the extract and do not survive in culture.
Extract batches producing such cells should be
- If so wished, replate the cells directly from the
extract in complete RPMI 1640 for an overnight culture
to assess survival further. There is no need to remove
the extract prior to replating.
In order for components from the reprogramming
extract to enter 293T cells, the cells must be reversibly
permeabilised. Permeabilisation is accomplished with
the Streptococcus pyogenes
toxin, streptolysin O. SLO
is a cholesterol-binding toxin that forms large pores
in the plasma membrane of mammalian cells (Walev et al.
1. Preparation of SLO Stock Solution
2. Cell Permeabilisation
- Dissolve SLO powder in sterile-filtered MilliQ H2O to 100 µg/ml. Keep on ice while dissolving the SLO.
- Aliquot 10µl in 200-µl tubes and store at-20°C
- Discard all tubes after 1 month of storage at-20°C and prepare a new stock. Stock aliquots should be
thawed only once. Note that because commercial
batches of SLO vary in specific activity, a range of
SLO concentrations (200, 500, 1000, 2000, and 4000ng/ml SLO) should be tested on the cell type
to be reprogrammed after a new stock is prepared.
D. Cell Permeabilisation Assay
- Dilute the SLO stock in ice-cold HBSS to 230ng/ml.
This is the working solution. Note that this concentration
is valid for 293T cells and should be adjusted
for other cell types using a cell permeabilisation
assay described in Section III,D.
- Keep the SLO on ice until addition to 293T cells.
- Remove the RPMI 1640 medium from wells containing
293T cells grown on coverslips and wash the
cells four times with PBS at room temperature to
remove all Ca2+ from the culture medium. This step
is essential as Ca2+ inhibits SLO activity.
- Add 250µl SLO working solution to each well.
- Incubate at 37°C in regular atmosphere for 50min.
Proceed to Section III,E,1.
This assay allows evaluation of the efficiency of the
SLO treatment. It is recommended to carry out this assay using four additional coverslips supporting 293T
cells as described in Section III,A, in addition to those
used for the reprogramming reaction. This assay is
based on the uptake of the fluorescent DNA stain,
propidium iodide, by permeabilised cells, but not
by intact cells.
E. Reprogramming Reaction
1. Extract Preparation
- Permeabilise cells on two coverslips with SLO as
described under Section III,C,2. However, in the first
step, add propidium iodide to 0.1 µg/ml to the SLO
dilution in HBSS on one of the coverslips. Propidium
iodide will be taken up as cells are being permeabilised.
The other coverslip receives 250µl SLO dilution
in HBSS without propidium iodide.
- Two additional coverslips should also be used
as controls for the absence of SLO. Add 250µl HBSS
containing propidium iodide to one of the control
coverslips as in step 1. The other coverslip receives
250µl HBSS without propidium iodide.
- Incubate at 37°C in regular atmosphere for
- For SLO-treated and control coverslips not containing
propidium iodide, remove HBSS and immediately
add 1.5ml of preheated (37°C) complete RPMI
1640 containing 2 mM Ca2+ added from a 1M stock (see
Section II,A). Incubate at 37°C for 2h to allow resealing
of the plasma membranes.
- For SLO-treated and control coverslips labelled
with propidium iodide, remove HBSS, rinse with PBS,
and add 250µl PBS.
- Assess propidium iodide labelling of the nuclei
by epifluorescence microscopy.
- After the 2-h membrane resealing step described
in step 4, remove the culture medium, rinse with PBS,
and add 250µl PBS containing 0.1µg/ml propidium
iodide; incubate for 10 min.
- Assess propidium iodide uptake, or lack thereof,
in the resealed cells as in step 6.
During SLO treatment, the extract should be prepared
2. Reprogramming Reaction
- Prepare the ATP-regenerating system: mix on ice
ATP:GTP:creatine kinase:phosphocreatine in a
1:1:1:1 ratio from each separate stock (described
in Section II,A) and keep on ice.
- Add 5µl of the ATP-regenerating system mix to
100 µl of extract on ice.
- Add 4 µl of the 25 mM NTP mix (see Section II,A) to
100 µl of extract on ice.
- Vortex briefly and replace the extract on ice.
E Resealing Reprogrammed Cells
- Remove SLO from the cells by careful aspiration.
- Quickly add PBS to prevent drying.
- Immediately transfer each coverslip into a new
dry well of a 24-well plate and carefully lay 65µl of
extract (prepared as described in Section III, B,3) onto
each coverslip. Be careful that cells do not dry out
upon transfer of the coverslip(s) to the new wells and
prior to addition of the extract. It is important that the
coverslip be covered by the extract during the entire
incubation time. Should the extract spread out of the
coverslip, transfer the coverslip into a new well and
pipette the extract back onto the cells.
- Incubate at 37°C for 1h in regular atmosphere.
Note that reprogramming has also proven to be successful
upon incubation in a 5% CO2 incubator at 37°C.
- At the end of incubation, directly add to each well
1.5 ml of preheated (37°C) complete RPMI 1640 containing
2mM Ca2+ added from the 1M stock (see
Section II,A). Do not remove the extract before
adding the Ca2+-containing medium.
- Incubate for 2h in a 5% CO2 incubator at 37°C.
- Remove the Ca2+-containing medium by gentle
aspiration and replace with 250 µl of complete RPMI
1640 (Jurkat TAg cell culture medium).
- Place the cells back into the 5% CO2 incubator
and culture until reprogramming assessments are
G. Assessment of Nuclear Reprogramming
|FIGURE 3 Immunofluorescence detection of (A) T-cell
(TCR) α and β chain expression and (B) CD3
expression on the
surface of 293T cells reprogrammed in
a Jurkat TAg cell extract.
DNA is labelled with 0.1 µg/ml
propidium iodide. Analysis was
performed ~14 days after
the reprogramming reaction. Bars: 20µm.
Various assessments of nuclear and cell reprogramming
can be performed, depending on the purpose of
the experiment. Using the method described here, we
have reported changes in the gene expression profile
of the reprogrammed 293T cells using cDNA macroarrays
from R&D Systems (Abington, UK) (Håkelien et al.
, 2002). Expression of new proteins can also be
monitored at regular intervals after the reprogramming
reaction by immunofluorescence or flow cytometry
using standard protocols. We have shown the
expression of several antigens on the surface of the
reprogrammed cells, which are specific for hematopoietic
cells. These include CD3, CD4, CD8, CD45, and
components of the T-cell receptor (TCR) complex
(Håkelien et al.
, 2002). A variety of functional assays
can also be carried out, such as cytokine secretion in
response to stimulation of the T-cell receptor/CD3
complex in the reprogrammed cells, or expression of
additional cytokine receptors on the cell surface (Håkelien et al.
, 2002). Fig. 3 illustrates the expression
of the TCR α and β chains and of CD3 molecules on
the surface of 293T cells reprogrammed in a Jurkat TAg
- Commercially available SLO batches vary greatly
in activity. Thus, it is recommended to test a range of
SLO concentration on the cell type to be reprogrammed
prior to initiating reprogramming reactions.
The efficiency of SLO-mediated permeabilisation also
varies for various cell types.
- pH of the extract. We usually observe a drop of
1-1.5 pH unit upon extract preparation, which explains
the pH 8.2 of the CLB. Notably, raising the pH of CLB
to 8.7 with a HEPES buffer does not increase the pH of
the final extract. Other buffers with greater buffering
capacity have not been tested.
- The method described here can be used easily
with either purified cell nuclei (Landsverk et al., 2002)
or permeabilised cells (Hakelien et al., 2002). Procedures
for purifying intact (membrane-enclosed) nuclei
from interphase cultured cells have been reported
earlier (Collas et al., 1999; Landsverk et al., 2002;
Martins et al., 2000; Steen et al., 2000).
- It is currently difficult to objectively assess the
extent of sonication of Jurkat TAg cells or any other cell
type. It is important to sonicate until all cells and nuclei
are completely lysed. Whether extended sonication
after cell lysis is complete is detrimental or beneficial
is at present unknown.
- Variability in batches of reprogramming extracts
is seen, even among extracts that have been rated as
"nontoxic" in the toxicity assay described in Section
III,B,4. Variability is evident by the absence of markers
of cell reprogramming approximately I week after the
- Currently, the duration of expression of a reprogrammed
phenotype is limited to at least 2 months
for 293T cells reprogramming in Jurkat TAg extract
(Håkelien et al., 2002). The reprogrammed phenotype
may also last for shorter periods depending on the
Our work was supported by Nucleotech, LLC and
grants from the Research Council of Norway, the
Norwegian Cancer Society, and the Human Frontiers
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