© 2012 Nature America, Inc. All rights reserved.
Generation of induced pluripotent stem cells from
a small amount of human peripheral blood using a
combination of activated T cells and Sendai virus
718 | VOL.7 NO.4 | 2012 | nature protocols
Generating iPSCs is a prominent recent advance in stem cell
biology1. iPSCs have become cell sources for genetic disease
models and are expected to provide important new cell sources
for clinical therapies. Initial studies generated human iPSCs
from human fibroblasts obtained from dermal biopsy samples2,3.
However, although further studies successfully reprogrammed
several types of human somatic cells into iPSCs4–6, the methods
and cell sources most suitable for iPSC applications in humans
remain undetermined. In particular, the generation of iPSCs for
disease research should ideally avoid invasive tissue sampling,
which markedly reduces the number of patients who consent to
cell donations. In this regard, peripheral blood is an appealing cell
source because of the noninvasive collection and easy accessibility
of blood cells compared with skin fibroblasts and other types of
cells from adult tissues.
We recently demonstrated that transgene-free iPSCs can be effi-
ciently generated from a small amount of human peripheral blood
within 1 month of the blood sampling using a combination of
activated T cells and temperature-sensitive (TS) mutants of SeV
encoding human OCT3/4, SOX2, KLF4 and c-MYC (Fig. 1)7. We
named these T cell–derived iPSCs TiPS cells (TiPSCs). Recombinant
SeVs that replicate in the cytoplasm of infected cells in the form of
negative-sense single-stranded RNA were originally used to gen-
erate iPSCs from human fibroblasts8. These recombinant SeVs do
not integrate into the host genome9, and have already been used
in human iPSC generation using CD34 + cells from human cord
blood10. Introducing TS mutations also successfully erased residual
genomic RNA of the SeV vectors from the target cells10, thus gen-
erating transgene-free iPSCs with high efficiency. T cells are also
an appealing cell source because they are easily proliferated in vitro
using a plate-bound anti-CD3 monoclonal antibody and inter-
leukin (IL)-2 (ref. 11). Although it was reported that T cells are not
efficiently reprogrammed using only four factors in the mouse12,13,
SeV vectors were efficiently transduced into human activated T cells
to express exogenous genes14. Thus, the combination of activated
T cells and TS SeV mutants made it possible to generate TiPSCs
from patients effectively, easily and less invasively.
Advantages of the method
The initial methods for generating human iPSCs used a skin
biopsy2,3, requiring local anesthesia and suturation. In our pro-
tocol, iPSCs can be generated from patients without such inva-
sive tissue sampling. Sufficient patient-specific iPSCs can be
generated from 1 ml or less of peripheral blood, which contains
sufficient terminally differentiated T cells7. Our method might
therefore decrease the likelihood of patients refusing cell sam-
pling and therefore potentially increase the number of patients
who consent to generating iPSCs. In addition, TiPSCs can still
be generated from whole blood samples stored at room tem-
perature (20–25 °C) for 24 h and from mononuclear cells stored
at − 150 °C. Therefore, transported samples can be easily used
for generating iPSCs in any clinical situation. SeV also has the
possibility to be used for generating iPSCs from other human
blood cells such as monocytes, which, on the basis of existing
reports of iPSCs that were generated successfully using SeV from
T cells and CD34 + cells7,10, do not harbor T cell receptor (TCR)
or immunoglobulin gene rearrangements.
Comparison with other methods
In the first report of iPSC generation from human peripheral
blood cells6, mobilized CD34 + human peripheral blood cells were
successfully reprogrammed into iPSCs. However, this method
required extremely large amounts (~300 ml) of blood, an apher-
esis machine and drug administration before blood sampling
to mobilize the CD34 + blood cells, all of which should ideally
be avoided because of the possible associated side effects (e.g.,
bone pain), despite these effects being infrequent. Less invasive
methods using peripheral blood have also been reported for
Tomohisa Seki1, Shinsuke Yuasa1,2 & Keiichi Fukuda1
1Department of Cardiology, Keio University School of Medicine, Tokyo, Japan. 2Department of Cardiology, Center for Integrated Medical Research, Keio University School
of Medicine, Tokyo, Japan. Correspondence should be addressed to K.F. (email@example.com) or S.Y. (firstname.lastname@example.org).
Published online 15 March 2012; doi:10.1038/nprot.2012.015
Induced pluripotent stem cells (ipscs) have become important cell sources for genetic disease models, and they have the
potential to be cell sources for future clinical therapies. However, invasive tissue sampling reduces the number of candidates
who consent to donate cells for ipsc generation. In addition, integrated transgenes can potentially insert at inappropriate
points in the genome, and in turn have a direct oncogenic effect. technical modifications using a combination of activated
t cells and a temperature-sensitive mutant of sendai virus (seV) can avoid invasive tissue sampling and residual transgene
issues in generating ipscs. such advances may increase the number of consenting patients for cell donations. Here we present a
detailed protocol for the generation of ipscs from a small amount of human peripheral blood using a combination of activated
t cells and mutant seV encoding human oct3/4, soX2, KlF4 and c-MYc; t cell–derived ipscs can be generated within 1 month of
© 2012 Nature America, Inc. All rights reserved.
nature protocols | VOL.7 NO.4 | 2012 | 719
the successful reprogramming of mono-
nuclear blood cells15–17. In these methods,
mononuclear blood cells from donors or
frozen samples were infected using retro-
virus15 or lentivirus16,17 to express four
factors, human OCT3/4, SOX2, KLF4 and
c-MYC. In these studies, human T cell
reprogramming into iPSCs was achieved,
but the efficiency of reprogramming was
extremely low (approximately 0.0008–
0.01%). Although these methods used
less peripheral blood and did not require the pharmacological
pretreatment of patients, the problems of transgene genomic
insertion and low reprogramming efficiency remained, pre-
cluding their wide use in the clinical application of iPSCs.
Generating iPSCs with TS-mutated SeV easily erases residual
genomic viral RNA from the target cells10, and the method
is significantly more efficient (~0.1%) compared with those
protocols in which iPSCs were generated from T cells with
retrovirus or lentivirus.
Human keratinocytes derived from plucked human hair have also
been used as another less invasive method of obtaining iPSCs from
patient cells4,18. However, in some cases, these reported methods
require several hairs to obtain successful cell outgrowth of keratino-
cytes. Dental tissue has also been explored as a potential source of
iPSCs19. However, although teeth are routinely removed in many
clinics and no further procedures are required with respect to the
donor, it is generally difficult to routinely obtain patients’ dental
tissues—with specific genetic or nongenetic diseases—for the pur-
pose of iPSC studies. In comparison with these outlined methods,
our protocol involves harvesting only a small sample of peripheral
blood; in addition, T cell proliferation does not need stochastic cell
outgrowth. These are clear advantages for clinical application in
comparison with the methods reported in the past.
Blood sampling. Our protocol is focused on the simple procedure
of peripheral venous blood sampling to obtain the donor cells,
using a standard process. Patient somatic cells can then be easily
and aseptically obtained from the blood sample. In our protocol,
1 ml of whole blood is sufficient to generate TiPSCs (Fig. 2a).
Derivation of activated T cells. Peripheral blood mononuclear
cells (PBMCs) can be separated by a Ficoll gradient method from
heparinized whole blood samples (Fig. 2b). Although PBMCs
contain lymphocytes and monocytes, activation with plate-bound
anti-CD3 monoclonal antibody and IL-2 selectively proliferates
T cells, and clearly increases the proportion of T cells in the
cultured PBMCs11. CD3 protein exists in the complex of TCR
proteins on the surface of T cells, and can therefore be used
as a T cell–specific marker. Anti-CD3 antibody modulates the
TCR-CD3 complex to induce T cell proliferation and activation20,
whereas IL-2 also activates general T cell signaling pathways
and eventually promotes cytokine transcription, cell survival,
cell-cycle entry and growth21. At day 5 of culture with anti-CD3
monoclonal antibody and IL-2, CD3 + cells increased up to ~95%
of cultured PBMCs (Fig. 2c–e). With this culture method, users
can avoid using a fluorescence-activated cell sorter in which
Isolation of PBMCs
with cell count
IL-2 + anti-CD3 antibody +
ESC condition (bFGF +)
T cells before
on feeder cells
Day 5 Day 6Days 20–25
onto feeder cells
Figure 1 | Overview of the TiPSC generation
protocol. PBMCs are activated for 5 d with IL-2
and anti-CD3 antibody, and then transduced with
SeV expressing human OCT3/4, SOX2, KLF4 and
c-MYC. TiPSC colonies emerge at 20–25 d after
PBMCs day 0
500 µm500 µm
PBMCs day 5
PBMCs day 0
PBMCs day 5
Figure 2 | Isolation and activation of PBMCs. (a) Whole blood is collected into a 2.5-ml syringe by venipuncture. (b) Before
centrifugation, two distinct layers are distinguishable in the 15-ml tube. The upper, dark red layer is the diluted blood and the
lower layer is the Ficoll solution. After centrifugation, three distinct layers are apparent. The upper yellow layer contains platelet-
rich plasma, whereas the bottom clear layer is the Ficoll solution, and the thin white layer in between contains PBMCs (arrow).
(c) Morphology of PBMCs shortly after being seeded in the culture plate and activated using anti-CD3 antibody with IL-2 for 5 d.
Proliferated T cells show many clusters in the culture plate at day 5 of activation. (d,e) Flow cytometric analysis of isolated PBMCs
and PBMCs activated for 5 d with anti-CD3 antibody and IL-2 gated on the CD45 + cell population. CD3 surface expressions of these
populations were examined. The graph represents an average of three independent examinations. Error bars show means ± s.d.
© 2012 Nature America, Inc. All rights reserved.
728 | VOL.7 NO.4 | 2012 | nature protocols
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