International Immunopharmacology 5 (2005) 13 – 21

www.elsevier.com/locate/intimp

Embryonic stem cells: a novel source of dendritic cells

for clinical applications
Paul J. Fairchild*, Kathleen F. Nolan, Siân Cartland, Herman Waldmann
The University of Oxford, Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK

Abstract

As arbitrators of the immune response, dendritic cells (DC) are uniquely placed to negotiate the balance between the
opposing forces of tolerance and immunity, making them attractive candidates for clinical applications. Accordingly, DC have
been used successfully in the treatment of cancer, enhancing immune responses to tumour-associated antigens (TAA) in
experimental animal models and phase I clinical trials. A novel source of DC that has recently been described is the embryonic
stem (ES) cell whose differentiation in vitro may be directed along multiple lineage pathways. Such pluripotency offers
unparalleled opportunities for the treatment of chronic and degenerative disease states by the replacement of affected tissues, a
vision which has inspired the emerging field of regenerative medicine. By sharing the genotype of therapeutic cell types, such as
cardiomyocytes and dopaminergic neurons derived from the same ES cell line, so-called esDC may offer prospects for
reprogramming the immune system to tolerate the grafted tissues. Here, we describe how the unique properties of esDC and the
ES cells from which they derive, make them eminently suited to clinical applications, overcoming many of the issues that
currently limit the effectiveness of DC-based immune intervention.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Dendritic cells; Embryonic stem cells; Regenerative medicine; Tolerance

1. Introduction shows no indication of having reached a plateau [1].

Recent decades have witnessed a progressive
change in emphasis in the healthcare needs of
developed countries. Due partly to improved living
conditions and significant advances in the treatment of
infectious microorganisms, life expectancy in Western
societies has risen, unabated, since the 1840s and
* Corresponding author. Tel.: +44 1865 275606; fax: +44 1865
275501.
E-mail address: Paul.Fairchild@path.ox.ac.uk (P.J. Fairchild).
1567-5769/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2004.09.005
While increased longevity is undoubtedly one of the
successes of modern medicine, it has been accom-
panied by a dramatic increase in incidence of chronic
and degenerative disease states which threaten to
dominate healthcare resources in the future [2]. Since
many conditions, such as diabetes and Parkinson’s
disease, may be amenable to cell replacement therapy
(CRT), the advent of pluripotent human embryonic
stem (ES) cells [3] has received considerable acclaim
by promising to meet the growing demand for
replacement cell types and tissues [4]. Indeed, current
14 P.J. Fairchild et al. / International Immunopharmacology 5 (2005) 13–21
estimates suggest that upwards of 3000 Americans die
each day of conditions that may ultimately be treated
through the panacea of regenerative medicine [5].
Although various animal models of disease have
proven amenable to treatment by such an approach
[6–8], translation to the clinic will require an effective
strategy for addressing the immunological barriers of
histoincompatibility between the recipient of CRT and
the ES cell line from which the tissues are derived.
A promising approach to this problem lies in the
dual capacity of ES cells to act as a source of
therapeutic cell types to ameliorate the progression of
disease and of leukocyte subsets to ensure their
acceptance through the induction of transplantation
tolerance [9]. Given the emerging role of dendritic
cells (DC) in the maintenance of self-tolerance under
steady state conditions and their ability to reestablish
tolerance in both autoimmune and alloimmune set-
tings [10–12], the description of protocols for their
derivation from mouse ES cells [13,14] represents an
important step towards realising this goal.
While the tolerogenicity of DC has been appreciated
for some years, an understanding of the molecular basis
of this phenomenon remains less well defined than the
mechanisms that underlie immunogenicity. Conse-
quently, the use of DC in a therapeutic context has so
far been largely restricted to strategies for vaccination
to tumour-associated antigens (TAA) [15]. Such an
approach to cancer immunotherapy has enjoyed con-
siderable success in animal models of disease [16–18]
and has inspired a number of small-scale clinical trials
for the treatment of melanoma, lymphoma, myeloma,
as well as prostate and renal cancer [19,20]. The
expansion of tumour-specific cytotoxic T lymphocytes
(CTL) in these studies has correlated with regression of
established tumours and secondary metastases in a
proportion of patients, although clinical outcomes have
varied considerably between trials. This inconsistency
has highlighted the need to optimise and standardise
protocols for the derivation, culture and administration
of DC; indeed, in the 8 years since the first trial was
performed, various issues have been identified that
must be addressed before DC-based treatment regimes
may be offered as reliable alternatives to conventional
therapies [21,22].
Firstly, identification of the most appropriate source
of DC remains to be resolved, the main criteria for their
use in vivo being their availability, yield, stability of
phenotype and propensity for long-term storage. While
CD34+ stem cells may be stored frozen over prolonged
periods of time, access to such progenitors is largely
constrained by the limited availability of umbilical cord
blood; conversely, DC differentiated from the patient’s
own peripheral blood monocytes are more readily
available but may show less phenotypic stability with
time, displaying an inherent tendency for spontaneous
maturation in culture. Secondly, an issue which has
proven to be the rate-limiting step in cancer immuno-
therapy has been the identification of appropriate target
antigens and the most effective form for their delivery
to DC; synthetic peptides, recombinant TAA and
unfractionated tumour lysates have each been used
with varying success. While endogenous expression of
TAA is widely believed to be the most appropriate form
of delivery by virtue of the access it provides to both
MHC class I and II pathways of antigen processing, the
approach is limited by the need for effective protocols
for genetic modification of the DC inoculum, which
remains a significant obstacle. Although the develop-
ment of retroviral and adenoviral vectors has begun to
fulfill this need, their use in a clinical context
introduces a significant element of risk which may be
difficult to justify on ethical grounds [23].
These considerations help illustrate the pressing
need for quality control of preparations of DC intended
for clinical use, so as to ensure both safety and efficacy
of the procedure. Clearly, any source of DC that permits
consistency and reproducibility between batches, while
providing ample opportunities for quality control,
would offer significant advantages over the current
art. Although efforts are presently underway in a
number of laboratories to secure the differentiation of
DC from human ES cells, we discuss below how our
own experiences of esDC in the mouse suggest that
they fulfil many of these requirements, auguring well
for their ultimate application to the clinic.
2. Materials and methods
2.1. ES cell lines and mice
ESF116 is an ES cell line derived from CBA/Ca
mice which is karyotypically male and has been
described in detail previously [13]. ESF121 is likewise
of CBA/Ca origin but is karyotypically female. Both
 P.J. Fairchild et al. / International Immunopharmacology 5 (2005) 13–21
lines display germline competence and were cultured
according to standard protocols [14]. C57Bl/10 mice
were used as a source of allogeneic T cells.
A1xRAG1 / mice transgenically express a T cell
receptor (TCR) specific for the 479–493 epitope of the
male specific antigen, Dby, in the context of the MHC
class II determinant, H2Ek. Female A1xRAG1 /
mice were, therefore, used as a source of T cells
specific for this well-defined minor histocompatibility
(mH) antigen.
2.2. Directed differentiation of ES cells
Protocols for directing the differentiation of pluri-
potent ES cells towards the DC lineage have been
described in detail elsewhere [14]. Briefly, ES cells
were cultured on mitotically inactivated embryonic
fibroblast feeder cells in DMEM (Gibco) supple-
mented with 15% FCS, 2 mM l-glutamine, 1 mM
sodium pyruvate and 510 5 M 2-mercaptoethanol.
Genetic modification of the parent ES cell lines was
achieved by lipofection as described [14]. Stable
transfectants were selected by neomycin resistance
and were cloned at the single-cell level to produce
individual ES cell lines which were stored frozen.
As a prelude to their differentiation, ES cells were
passaged twice in gelatinised flasks in medium
supplemented with 1000 U/ml of recombinant leukae-
mia inhibitory factor (rLIF; Chemicon) to remove the
feeder cells. ES cells were then cultured in suspension
at low density (4105 cells per 90-mm dish of
bacteriological plastic) so as to permit the formation
of embryoid bodies (EB). After 14 days, EB were
plated onto tissue culture plastic in medium containing
200 U/ml recombinant interleukin-3 (IL-3; R&D
Systems) and 25 ng/ml of granulocyte-macrophage
colony stimulating factor (GM-CSF) to support the
outgrowth of DC.
ES cell-derived DC were harvested by gentle
pipetting to release lightly adherent cells, and were
replated in fresh medium containing IL-3 and GM-
CSF, to permit the adherence of contaminating
stromal cells. After overnight culture, esDC were
again dislodged by gentle pipetting and used in
functional assays or stored frozen for future use.
Cryopreservation was achieved by resuspending
esDC at a density of 5106 cells/ml in medium
containing 10% DMSO. Cells were frozen rapidly on
15
dry ice before being stored long term under liquid
nitrogen.
To induce the maturation of esDC, replated cells
were exposed to 1 Ag/ml of lipopolysaccharide (LPS)
from E. coli Serotype 0127:B8 (Sigma). After over-
night culture, the medium was replaced and the cells
cultured for a further 24 h: mature esDC were
harvested the following day as the nonadherent
population. Populations of control bone marrow
derived DC (bmDC) were obtained by culturing bone
marrow cells from CBA/Ca mice in medium supple-
mented with 25 ng/ml of GM-CSF. Cultures were fed
by replacement of the medium on days 3 and 6 of
culture and maturation was specifically induced by
addition of 1 Ag/ml of LPS on day 6.
2.3. Proliferative assays
Mature or immature esDC (5104 cells per well of a
96-well round-bottomed plate) were cocultured with
2105 nylon wool purified T cells from either alloge-
neic C57Bl/10 or TCR transgenic A1xRAG1 / female
mice. After 2 days of culture, proliferation was assessed
by pulsing with 25 ACi/well of 3H-thymidine deoxy-
ribose (TdR; Amersham). Cultures were harvested and
3
H-TdR incorporation measured using a flat bed
scintillation counter.
2.4. Flow cytometry
The surface phenotype of DC was assessed by flow
cytometry using FITC-conjugated monoclonal anti-
bodies (mAb) to MHC class II (Clone OX6; mouse
IgG1) or biotinylated anti-CD86 (Clone GL-1; Rat
IgG2a), followed by ExtrAvidin-FITC (Sigma). Irrel-
evant, species- and isotype-matched mAb were used
as negative controls. Cells were stained on ice for 45
min, washed and fixed in 1% formaldehyde before
being analysed by means of a Becton Dickinson
FACSCalibur using CellQuest software.
3. Results
3.1. Yield and storage of esDC
Protocols developed in our laboratory for the
differentiation of DC from mouse ES cell lines make
16 P.J. Fairchild et al. / International Immunopharmacology 5 (2005) 13–21
use of EB as a microenvironment conducive to
hematopoiesis [13,14]. EB are macroscopic structures
derived from the sustained proliferation of single ES
cells maintained in suspension culture and mimic
development of the early embryo in all but their
propensity for pattern formation. Despite these limi-
tations, EB frequently develop cystic structures
analogous to the visceral yolk sac, the most primitive
of all hematopoietic tissues. When plated onto tissue
culture plastic, to which they may adhere, EB support
the outgrowth of colonies containing derivatives of
each of the three embryonic germ layers in an
inherently chaotic manner. The addition of GM-CSF
and IL-3 to cultures steers mesodermal differentiation
overwhelmingly towards the myeloid lineage, favour-
ing the appearance of almost limitless numbers of
terminally differentiated DC. We estimate that a single
EB is capable of sustaining the development of more
than 2107 esDC, which, once established in culture,
may be harvested periodically every 4–5 days before
eventually succumbing to replicative senescence.
Such a degree of expansion is unrivalled among
conventional sources of DC, ensuring the ready
availability of material for clinical applications.
Since a single passage of the parent ES cell line has
the potential to yield several million EB, each with the
capacity to spawn over 107 esDC, there is a significant
likelihood that cells may be generated that are surplus
to requirements. We have, therefore, investigated
whether esDC may be cryopreserved for subsequent
use. Significantly, snap-freezing of cells in medium
supplemented with 10% DMSO permits their storage
under liquid nitrogen for protracted periods of time
with no detectable loss of viability. Furthermore,
esDC stored in this way show no loss of functional
integrity, responding appropriately to maturation
stimuli and performing identically to freshly harvested
cells as stimulators of naRve, allogeneic T cell
responses (Fig. 1).
3.2. Phenotypic stability of esDC in culture
A feature of esDC which distinguishes them from
DC derived from bone marrow progenitors (bmDC),
is the stability of their phenotype and function over
time. Fig. 2 shows how bmDC are prone to sponta-
neous maturation in culture, as evidenced by the
progressive translocation of MHC class II to the cell
Fig. 1. Immunogenicity of esDC cryopreserved for 2 weeks in liquid
nitrogen. Embryonic stem-cell-derived DCs were frozen at 107/ml
in medium supplemented with 10% DMSO. Upon thawing, cells
were plated overnight and induced to mature by addition of 1 Ag/ml
of LPS before being used as stimulators of naRve, allogeneic T cells
.
( ). Freshly harvested mature (w) and immature esDC (n) were
included for comparison.
surface (Fig. 2A–C) and up-regulation of costimula-
tory molecules, such as CD86 (Fig. 2E–G), even in
the absence of exogenously added stimuli. In contrast,
esDC remain unchanged phenotypically during many
weeks in culture, appearing largely indistinguishable
from the most immature bmDC after 35 days in
culture (Fig. 2D and H). Such observations are
relevant in the light of recent studies showing that
administration to healthy volunteers of immature
monocyte-derived DC pulsed with peptides from
influenza virus matrix protein (MP) suppresses MP-
specific CD8+ CTL through the generation of IL-10-
secreting regulatory T cells [24]. These observations
are consistent with studies in the mouse suggesting
that antigen presentation by immature DC polarises
responding T cells towards a regulatory phenotype
and may contribute to the maintenance of peripheral
tolerance [25,26]. The arrest of esDC at an immature
stage of the DC life cycle may, therefore, favour a
protolerogenic phenotype, endorsing plans for their
use as a conditioning regimen for CRT.
3.3. Antigen processing and presentation
The need to identify appropriate antigens against
which to target the immune response has greatly
limited DC-based strategies for cancer immunother-
 P.J. Fairchild et al. / International Immunopharmacology 5 (2005) 13–21 17

Fig. 2. Comparison of phenotypic stability among esDC and DC derived from bone marrow progenitors. Histograms represent the levels of
expression of MHC class II (A–D) and CD86 (E–H) by bmDC at day 9 (A and E) and day 14 of culture (B and F), showing spontaneous
maturation of a significant proportion of cells over time. For comparison, (C) and (G) depict expression levels of the respective surface markers
by bmDC, induced to mature in response to overnight incubation with LPS. (D) and (H) represent levels of surface expression by esDC
maintained continuously in culture for 35 days, showing a similar phenotypic profile to the most immature populations of bmDC (A and E).

apy. Despite considerable efforts over the past decade
to identify TAA, the number of proteins truly
restricted in their distribution to transformed cells is
surprisingly small, even for melanoma, the best
defined of all tumours at the biochemical level. And
yet, targeting destructive immune responses to anti-
gens that are more widely disseminated than the
tumour itself may have potentially deleterious con-
sequences. Mice engineered to express a neoantigen
in pancreatic h cells that was shared by a tumour cell
line with which they had been inoculated, developed
fatal autoimmune responses when vaccinated with DC
presenting the same antigen in an immunogenic
fashion [27]. Although DC have been generally well
tolerated during clinical trials, autoimmune sequalae
in the form of vitiligo have been reported as a
consequence of vaccination against TAA expressed by
melanoma but shared by normal, untransformed
melanocytes, illustrating the need for caution [21].
Importantly, the issues of antigen identity and the
form and vehicle for its delivery to DC are rendered
irrelevant when exploiting esDC for the induction of
transplantation tolerance to tissues derived from the
same ES cell line. Having differentiated from a
common progenitor, esDC naturally share all relevant
major and minor histocompatibility antigens with the
therapeutic cell types, their endogenous expression
obviating the need for the provision of antigen in an
appropriate form for uptake by DC. Indeed, the
molecular identity of the transplantation antigens
may become little more than academic rather than
representing an insurmountable obstacle to the imple-
mentation of immunotherapy. The issues of MHC
restriction that greatly complicate the use of peptide
vaccines are also circumvented, since esDC may be
entrusted with the task of selecting appropriate
epitopes for presentation to the T cell repertoire in
the context of both MHC class I and class II
determinants. Furthermore, the risk of eliciting erro-
neous autoimmune responses is minimised by the lack
of alloantigen expression by the recipient’s own
somatic tissues and the tolerogenic context in which
any shared autoantigenic epitopes are presented.
In order to investigate the capacity of esDC to
present endogenously expressed mH antigens, we
have made use of A1xRAG1 / mice, transgenically
18 P.J. Fairchild et al. / International Immunopharmacology 5 (2005) 13–21

expressing a TCR specific for the immunodominant
479–493 epitope of the male-restricted antigen, Dby,
in the context of H2Ek. NaRve, splenic T cells from
female A1xRAG1 / mice responded vigorously to
mature esDC differentiated from a karyotypically
male ES cell line of CBA/Ca origin, but failed to
respond to female esDC (Fig. 3A). Nevertheless,
addition of the synthetic Dby peptide to cultures
restored maximal responses in either case (Fig. 3B).
These results demonstrate the ability of esDC to
process an endogenously expressed mH antigen and
present the immunodominant epitopes in the context
of MHC class II determinants, a necessary require-
ment for the induction of transplantation tolerance.
3.4. Genetic modification of esDC
The advantages that routine genetic modification of
DC would offer to strategies of immune intervention
extend far beyond the expression of appropriate TAA.
The ability to overexpress or functionally silence
genes involved in immune regulation would provide
welcome opportunities to subtly intervene in the
outcome of the immune response. Nevertheless, all
sources of DC, described to date, display significant
barriers to the introduction of heterologous genes and
constructs. While lipofection is entirely inadequate as
an approach to transfection (Fig. 4A), use of the
cationic peptide, CL22, as a vehicle for delivery of
plasmid DNA has enjoyed some measure of success,
up to 17% of mouse DC having been induced to
express the enhanced green fluorescent protein
(EGFP) reporter gene [28]. The use of mRNA for
the purpose of transfection has also proven surpris-
ingly effective [29], although expression of the
transcribed gene is necessarily transient and has yet
to exceed a transduction efficiency of 20%. Both of
these approaches, while adequate to secure priming to
TAA, are far too inefficient as a strategy for
modifying cellular phenotype and function. Indeed,
the only approach to genetic modification that comes
close to such a remit is the use of viral vectors, such as
adenovirus, through which the stable introduction of
transgenes may be achieved in up to 80% of cells.

Fig. 3. Presentation of the endogenously expressed male mH antigen, Dby, by mature esDC. (A) Primary proliferative responses of naRve,
female T cells from A1xRAG1 / mice, transgenically expressing a TCR specific for the 479–493 epitope of Dby in the context of H2Ek. T
.
cells were cultured with mature esDC differentiated from either karyotypically male ( ) or female (E) ES cells. (B) Stimulation of T cell
proliferation by both male and female esDC pulsed with 5 nM Dby479–493 peptide.
 P.J. Fairchild et al. / International Immunopharmacology 5 (2005) 13–21 19

Fig. 4. Comparison of bmDC (A) and esDC (B) genetically modified to express the EGFP reporter gene (filled histograms) under control of
the EF1a promoter. Open histograms show levels of autofluorescence among control, nontransfected cells. Lipofection was used in both
cases, either to modify terminally differentiated bmDC directly (A) or esDC indirectly by modification and differentiation of a standard ES
cell line (B).

Nevertheless, despite the opportunities that adenoviral
vectors may provide, their use exacts a heavy price by
provoking the premature maturation of DC, thereby
sabotaging aspects of their normal physiology [30].
Furthermore, the introduction of transgenes and their
subsequent expression is accompanied by the syn-
thesis of viral proteins whose presentation may elicit
vigorous CTL responses, rendering the DC them-
selves vulnerable to depletion [29,31,32].
Whereas esDC share with conventional sources an
intrinsic resilience to genetic modification, we have
shown that ES cells may serve as an efficient conduit
through which genes may be introduced into DC
without corrupting their function [33]. Since ES cells
are comparatively amenable to genetic manipulation
and their capacity for self-renewal permits cloning at
the single-cell level, lines may be produced that
express a desired mutant phenotype which is faithfully
transmitted to esDC, downstream of the differentia-
tion pathway [14,33]. Furthermore, the capacity for
cloning and expansion of individual cells as a means
of rescuing rare, mutant phenotypes helps counteract
the low transduction efficiency of lipid-based
approaches to transfection, thereby avoiding the need
for viral vectors and their far-reaching influence on
DC function. Fig. 4 illustrates the efficiency with
which esDC may be genetically modified through
lipofection of the parental ES cell line (Fig. 4B),
compared to terminally differentiated bmDC (Fig.
4A). In addition to paving the way for the rational
design of esDC uniformly expressing a desired mutant
phenotype, the properties of ES cells may be further
exploited for the production of lines functionally
deficient in both alleles of a target gene through
homologous recombination. Such an approach has
already been successfully pursued for the production
of lines of esDC deficient in expression of Notch 1
[34], an achievement which is far beyond the scope of
traditional strategies for genetic modification of
terminally differentiated DC.
4. Discussion
ES cells display a number of properties that make
them unique, their pluripotency in vitro, capacity for
self-renewal and tractability for genetic modification
readily distinguishing them from fully differentiated
cell types. By exploiting ES cells as a novel source of
DC, these features may be harnessed in addition to
those of the DC themselves, to enhance their
suitability as candidates for immunotherapy. Accord-
ingly, esDC may be obtained in unparalleled abun-
dance and with a stability of phenotype that eludes
DC differentiated from conventional sources, such as
bone marrow progenitors or peripheral blood mono-
cytes. Furthermore, the potential for genetic modifi-
cation of the parental ES cell line provides
opportunities for the rational design of esDC,
uniformly expressing a desired mutant phenotype,
aimed at directing the outcome of the immune
response upon readministration in vivo. The capacity
to obtain such lines without recourse to the use of
viral vectors offers a significant advantage over
current practice, since the corrupting influence of
viral infection on the physiology of DC is avoided, as
20 P.J. Fairchild et al. / International Immunopharmacology 5 (2005) 13–21
are confounding immune responses targeted at viral
antigens.
The production of a genetically modified ES cell
line generates a self-renewing resource that may be
cryopreserved long term and differentiated into esDC
on demand. Such a resource may be distributed to
multiple centres so as to ensure much-needed
consistency and reproducibility between clinical
trials. Furthermore, by dissociating, temporally, the
genetic modification of the parent ES cells from the in
vivo administration of the resulting esDC, ample
opportunity is provided for quality control in advance
of their clinical use. This may involve identification
of the copy number and site of integration of
transgenes, assessment of the associated risks of
transformation and screening for potential human
pathogens, thereby rigorously addressing fundamental
issues of safety.
Whereas esDC, genetically modified to express
various chemokines, have been successfully
employed to enhance vaccination to TAA [35], their
most likely application lies within the field of
regenerative medicine, for which an active role in
tolerance induction is essential. While the arrest of
esDC at an immature stage of the DC life cycle may
favour such a role, genetic modification may also be
required to prevent their acquisition of immunoge-
nicity. As our understanding of the molecular basis
of tolerogenicity yields to investigation, so it may
prove feasible to programme into ES cell lines a
default propensity for tolerance by their DC progeny.
While protocols developed in the mouse await
adaptation to human ES cells, the advent of esDC
of human origin will doubtless provide significant
advances over current approaches to DC-based
immunotherapy, heralding an exciting phase in the
realisation of cell replacement therapy within the clinic
[36].
Acknowledgements
We are grateful to Drs. Stephen Cobbold and Luis
Graça for helpful discussions and to Prof. Richard
Gardner and Dr. Frances Brook for the provision of ES
cell lines. Work in the authors’ laboratory was
supported by a programme grant from the Medical
Research Council (UK).
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