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Stem Cell & Translational Investigation 2015; 2: e504. doi: 10.14800/scti.504; © 2015 by Ken-ichiro Seino, et al.
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REVIEW
New immunosuppressive strategies for transplantation based
on pluripotent stem cell (PSC)-derived immunoregulatory cells
Ken-ichiro Seino, Haruka Wada, Muhammad Baghdadi
Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15 Nishi-7, Sapporo 060-0815, Japan
Correspondence: Ken-ichiro Seino
E-mail: [email protected]
Received: January 03, 2015
Published online: February 11, 2015
Pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have
the potential to give rise to cells from all three germ layers that can be utilized as new reliable sources in
transplantation medicine. However, when allogeneic PSCs are used for transplant generation, an effective
immunosuppressive strategy is required to protect transplants from rejection induced by immune responses of
recipient to donor alloantigen after transplantation. Cell-based immunosuppressive strategies offer effective,
safe and specific methods to inhibit recipient’s immune response against donor transplant and help to avoid side
effects accompanied with conventional immunosuppressant drugs. In this context, we hypothesized that
allogeneic PSCs-derived transplants can be protected from immune rejection if accompanied with adoptive
transfer of immunosuppressive cells generated from the same PSCs. Indeed, we have developed several
differentiation protocols to generate immunosuppressive cells such as regulatory T cells (Tregs) or macrophages
from PSCs, which showed promising results to prolong same PSCs-derived graft survival. In this review, we
introduce new findings related to PSCs-based cellular immunosuppressive therapy and its applications in several
transplantation models.
Keywords: Pluripotent stem cells; embryonic stem cells; induced pluripotent stem cells; immunosuppressive cells;
allograft rejection; immuneregulation
To cite this article: Ken-ichiro Seino, et al. New immunosuppressive strategies for transplantation based on pluripotent stem
cell (PSC)-derived immunoregulatory cells. Stem Cell Transl Invest 2015; 2: e504. doi: 10.14800/scti.504.
Introduction
Recent progress in stem cell research has opened up the
potential to create organs and tissues for transplantation from
pluripotent stem cells (PSCs) such as embryonic stem cells
(ESCs) and induced pluripotent stem cells (iPSCs) [1,2]. This
promising technology can help to generate transplants from
autologous cells and thus overcome the problem of immune
rejection. However, it is difficult to obtain autologous ESCs
from all patients. The development of iPS technology has
facilitated the generation of stem cells by introducing a
specific set of reprogramming factors into adult somatic cells
which can be obtained anytime from the patient.
Theoretically, this excellent resource of autologous cells can
help to generate transplants with limited immunogenicity and
thus avoid the risk of immune rejection. Practically, the
generation of autologous iPSCs faces many difficulties
represented by the cost and time needed to evaluate safety,
stability and efficacy for each individual case. To overcome
such problems, a pioneer project of generating a bank of
ready-to-use PSCs collected from healthy donors has been
triggered for future therapeutic use [3]. In such cases,
transplants are expected to be derived from allogeneic PSCs,
and thus must be accompanied with an effective
immunosuppressive therapy to protect transplant from
undesired immune responses induced by mismatching in
histocompatibility between donor and recipient.
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Recent advances in cell-based therapies such as
donor-specific blood transfusion and bone marrow
transplantation have shown promising effects on
conditioning the immune response of recipients to donor
alloantigen after transplantation [4-6]. Additionally, newer
methods are being developed to facilitate the ex vivo
induction, expansion and purification of tolerance-promoting
immune cells [7-9]. These novel strategies offer more
effective,
safe
and
specific
immunosuppressive
methodologies to help protecting transplanted organ or tissue
from acute and chronic immune rejection, and minimizing
the side effects accompanied with conventional
immunosuppressant drugs [10].
From these backgrounds, we hypothesized that immune
protection of ESCs- or iPSCs-derived transplants can be
achieved by adoptive transfer of immune-regulatory cells
differentiated from the same cellular origin of the transplant.
Efforts in our laboratory focus on developing new protocols
to generate immune-regulatory cells from ESCs and iPSCs
for promoting transplant tolerance in clinical settings. In this
brief review, we introduce protocols used to differentiate
immunosuppressive cells from ESCs and iPSCs, and its
applications in allogeneic transplantation models.
Induction of immunosuppressive cells from PSCs
Regulatory T cells
Immune rejection of transplanted grafts is an adaptive
immune response mediated by killer T cells that induce
apoptosis in target cells. To prevent organ rejection,
immunosuppressive drugs such as cyclosporins have been
used to inhibit T cell activation, thus preventing T cells from
attacking the transplanted organ. However, the use of
immunosuppressant drugs is associated with a wide range of
side effects, including increased risk of infections, cancers,
diabetes, in addition to neurotoxicity and nephrotoxicity [9].
Thus, it is critically important to develop new methods to
limit or reduce T cells function in an alloantigen-specific
manner, while other effector functions are kept intact.
FoxP3-positive regulatory T cells (Tregs), also known as
suppressor T cells, are a subset of T cells that play important
roles in the regulation of the immune response, and maintain
tolerance to self-antigens [11]. The utilization of
alloantigen-primed Tregs has been considered as a promising
non-toxic immunosuppressive strategy to inhibit transplant
rejection reactions. Indeed, the adoptive transfer of exvivo
expanded Tregs effectively prevents graft versus host disease
after allogeneic bone marrow transplantation in mice and
human. Tregs have also the potential to establish transplant
tolerance to MHC molecules of cellular origin used for Tregs
generation. In this regard, we investigated Tregs
differentiation from iPSCs in vitro as a possible cellular
therapy for the induction of tolerance to iPSCs-derived
transplants.
Tregs are classified into two distinct subsets: naturally
occurring Tregs and induced Tregs. Naturally occurring
Tregs develop in the thymus, while induced Tregs develop in
the periphery under the influence of transforming growth
factor β (TGFβ) [9]. TGFβ can be used to induce generation of
Tregs from peripheral T cells in in vitro culture [11]. To
generate Tregs from mouse iPSCs, we developed a culture
protocol based on the induction of hematopoietic cells from
iPSCs using Flt3L, followed by induction of T cells from
these hematopoietic cells using IL-7, and finally induction of
Tregs from T cells using IL-2 and TGFβ (figure. 1A) [12].
Using this protocol, we could generate FoxP3 positive T cells
at an efficiency reached up to 10%. These Tregs induced
from iPSCs have immunosuppressive features as shown by
effective inhibition of T cell proliferation [12]. Thus, adoptive
transfer of iPSCs-derived Tregs may contribute to decrease
the risk of allogeneic immune rejection of same
iPSCs-derived transplants, which should be examined in
details in future studies.
As indicated above, Tregs play essential roles in
transplantation tolerance, and the therapeutic effects of Tregs
have been reported in several animal models [13]. Human
Tregs can be isolated from peripheral blood or cord blood,
and expanded in ex vivo cultures as a therapeutic product [14].
For PSCs-derived transplants, Tregs can be generated from
the same PSCs and thus are expected to suppress allogeneic
immune responses. The efficiency of Treg induction from
iPSCs in our protocol is still unsatisfying; however,
accumulating knowledge of Tregs cell biology will
contribute to the improvement of this culture protocol using
several cytokines and their cocktails.
Immunosuppressive macrophages
Allograft rejection results from immune attack against the
transplanted graft, and is characterized by multiple
inflammatory features. Macrophage accumulation has been
considered as a prominent feature of allograft rejection [15].
Recent progress in macrophage biology has improved our
understanding of the roles of macrophages in allogeneic
immune response [16]. Macrophages may contribute to
allograft injury by inducing both innate and adaptive immune
responses during the process of acute and chronic allograft
rejection [15]. On the other hand, macrophages may also
promote tissue repair mechanisms and suppression of
immune responses [17]. Thus, macrophages serve as an
attractive and effective target to improve outcomes in organ
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Figure. 1 Generation of immunosuppressive cells from PSCs. A scheme describes culture protocol used to generate
regulatory T cells from iPSCs (A) or immunosuppressive macrophage-like cells from ESCs (B).
transplantation. Indeed, several approaches based on
macrophages are being examined in preclinical and clinical
models [18-22]. For example, in clinical renal transplantation,
adoptive transfer of donor-derived regulatory macrophages
was effective to maintain excellent graft functions in patients
who were minimized to low-dose tacrolimus monotherapy
[21,22]
. In this context, we examined the ability to generate
macrophages with immunosuppressive features from PSCs,
in order to suppress allogeneic immune responses against
transplants derived from the same PSCs [23].
Arginase-1, Nos-2 and Tgfβ1 (table.1) [23]. Indeed, this
fraction of cells showed significant abilities to inhibit T cell
proliferation, and thus refer to as ES-derived
immunosuppressive cells (ES-SCs). ES-SCs efficiently
suppressed T cell proliferation in allogeneic lymphocyte
reaction (MLR), which was related to iNOS expression23.
More importantly, systemic administration of ES-SCs was
effective to delay allogeneic immune rejection of ES-derived
embryoid bodies or cardiomyocytes [23], indicating that
ES-SCs have the potential to prolong survival of allogeneic
transplanted grafts.
To evaluate this concept, we developed a culture protocol
based on the induction of hematopoietic cells from ESCs,
followed by stimulation with cytokines related to myeloid
cell differentiation including M-CSF, GM-CSF and IL-4, and
finally LPS (figure. 1B) [23]. Using this protocol, we obtained
floating cells with dendritic cells-like features and thus refer
to as ES-derived dendritic cells (ES-DCs), as previously
described [20]. On the other hand, adherent cells were
characterized by increased expression of macrophage
markers such as F4/80 and CD115, and showed potent
immunosuppressive capacities as indicated by the high
expression levels of immunosuppressive genes including
In summary, macrophages play important roles in the
determination of fate of transplanted grafts, and thus serve as
a promising therapeutic target for improving the outcome of
organ transplantation. Using our culture protocol,
macrophage-like cells with immunosuppressive features can
be generated from PSCs and contribute to the survival of
same PSCs-derived transplants. Further analysis of
characterization, differentiation and function of various
macrophages subsets may provide novel therapeutic targets
to improve transplant survival.
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Table 1. Comparison between ES-DCs and ES-SCs obtained from ESCs
Induction of hematopoietic stem cells from PSCs
Combination of hematopoietic stem cell (HSC)
transplantation and T cell inhibition has been reported to
induce indefinite allograft survival [24]. Therefore, it seems of
great interest to generate HSCs from PSCs.
A recent study has reported the ability to induce
hematopoietic progenitor cells from HOXB4-transduced
mouse ESCs25. HOXB4 is a hematopoietic transcription
factor, which contributes to the expansion of HSCs [26].
CD45+ hematopoietic progenitor cells isolated from
HOXB4-transduced ESCs show low expression of MHC
molecules [25]. This feature allows the long-term engraftment
of these CD45+ MHClow hematopoietic progenitor cells in
sublethally irradiated recipients across MHC barriers without
the need of immunosuppressive drugs [25]. Low levels of
chimerism were observed in the bone marrow of the
recipients over 100 days, and the recipients were protected
from rejection of donor-derived cardiac allografts [25].
serially transplantable in vivo [27]. It is anticipated that iHSCs
described above could be also derived from PSCs with
similar protocol, and might help to facilitate the engraftment
of PSCs-derived grafts.
In another recent study, HSCs were successfully induced
directly from human iPSCs by the manipulation of
Wntβ-catenin signal pathway during hematopoietic programs
transition [28]. The generation of KDR+ CD235a+ primitive
progenitors depends on stage-specific activin-nodal signaling
and inhibition of the Wnt–ß-catenin pathway, whereas
specification of KDR+ CD235a− definitive progenitors
requires Wntβ-catenin signaling during this same timeframe.
The manipulation of Wntβ-catenin signal pathway offers a
simple and effective differentiation strategy for the
development of PSC-derived hematopoietic progenitors [28].
Together, these differentiation protocols are expected to
enable the generation of HSCs from PSCs as a promising
future strategy for immune regulation.
Concluding remarks
More recent study has reported the ability to impart HSC
potential onto committed mouse blood cells by transient
expression of six transcription factors including Run1t1, Hlf,
Lmo2, Prdm5, Pbx1 and Zfp37 in committed lymphoid and
myeloid progenitors [27]. The efficacy of this direct
reprogramming protocol was further improved by addition of
Mycn and Meis1 and use of polycistronic viruses. These
induced-HSCs (iHSCs) showed a gene expression profile
similar to endogenous HSCs. Additionally, iHSCs
reconstituted stem/progenitor compartments, and were
The use of several immunosuppressive drugs over the past
decade has helped to overcome the problems of acute
rejection and improved short-term allograft survival.
However, in addition to their precautions and side effects, the
contribution of these immunosuppressive drugs on long-term
allograft
survival
remains
unsatisfying.
Current
immunosuppressive strategies are focusing on conditioning
immune responses of organ transplanted-recipients to
donor-derived alloantigen using various cell-based therapies.
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Figure. 2 PSCs-derived immunosuppressive cells as a promising strategy for future transplantation medicine.
Recent progress in the field of regenerative medicine has enabled the generation of tissues and organs for transplantation
from PSCs such as ESCs and iPSCs. The induction of recipient’s tolerance to donor alloantigen after transplantation can be
mediated by the adoptive transfer of immunosuppressive cells generated from the same allogeneic PSCs used for
transplants generation.
These novel strategies can help to specifically suppress
immune responses against alloantigens, and thus keep other
effector immune functions intact avoiding the nonspecific
immunosuppression
mediated
by
conventional
immunosuppressive drugs.
Current works in our laboratory focus on developing new
culture protocols that help to facilitate the ex vivo induction,
expansion, and purification of tolerance-promoting cells
from PSCs such as ESCs or iPSCs (figure.2). Compared to
other established techniques such as donor-specific blood
transfusion and bone marrow transplantation, theses
protocols offer the advantages of obtaining desired doses and
qualities of target cells in a short-term cell culture. In
addition to Tregs and immunosuppressive macrophages
described in this review, it is of great interest to examine if
other immunoregulatory cells such as tolerogenic dendritic
cells, mesenchymal stem cells can be generated from PSCs.
In
conclusion,
we
hope
that
PSCs-derived
immunosuppressive cells would be useful for the
development of safe and effective immunotherapy for
transplantation medicine, and that this experimental
technology will graduate to clinic in the near future.
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