Molecular imaging of human embryonic stem cells
Human embryonic stem cells (hESCs) are a renewable source of differentiated cell types that may be employed in various tissue regeneration strategies. However, clinical implementation of cell transplantation therapy is hindered by legitimate concerns regarding the in vivo teratoma formation of undifferentiated hESCs and host immune reactions to allogenic cells. Investigating in vivo hESC behaviour and the ultimate feasibility of cell transplantation therapy necessitates the development of novel molecular imaging techniques to longitudinally monitor hESC localization, proliferation, and viability in living subjects. An innovative approach to harness the respective strengths of various imaging platforms is the creation and use of a fusion reporter construct composed of red fluorescent protein (RFP), firefly luciferase (fluc), and herpes simplex virus thymidine kinase (HSV-tk). The imaging modalities made available by use of this construct, including optical fluorescence, bioluminescence, and positron emission tomography (PET), mat be adapted to investigate a variety of physiological phenomena, including the spatio-temporal kinetics of hESC engraftment and proliferation in living subjects. This chapter describes the applications of reporter gene imaging to accelerate basic science research and clinical studies involving hESCs through (1) isolation of a homogenous hESC population, (2) noninvasive, longitudinal tracking of the location and proliferation of hESCs administered to a living subject, and (3) ablation of the hESC graft in the event of cellular misbehavior.
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2748 Cell Cycle 2006; Vol. 5 Issue 23
[Cell Cycle 5:23, 2748-2752, 1 December 2006]; ©2006 Landes Bioscience
Molecular Imaging of Human Embryonic Stem Cells
Keeping an Eye on Differentiation, Tumorigenicity and Immunogenicity
Koen E.A. van der Bogt
Joseph C. Wu
Molecular Imaging Program at Stanford;
Laboratory of Cardiothoracic
Department of Medicine, Division of Cardiology; Stanford
University School of Medicine; Stanford, California USA
Department of Surgery, Leiden University School of Medicine; Leiden, The
*Correspondence to: Joseph C. Wu; Department of Medicine and Radiology;
Edwards Building, Room R-354; Stanford, California 94305-5324 USA; Tel.:
650.736.0234; Fax: 650.736.0234; Email: email@example.com
Original manuscript submitted: 10/13/06
Revised manuscript submitted: 10/20/06
Manuscript accepted: 10/22/06
Previously published online as a Cell Cycle E-publication:
embryonic stem cell, molecular imaging,
immunogenicity, tumorigenicity, teratoma,
Fluc firefly luciferase
PET positron emission tomography
HSV-ttk herpes simplex virus trun-
cated thymidine kinase
3hydroxy methylbutyl) guanine
SPECT single photon emission
hNIS human sodium/iodide
MRI magnetic resonance imaging
CCD charged coupled device
BLI bioluminescence imaging
Human embryonic stem cells (hESCs) are capable of differentiation into every cell
type of the human being. They are under extensive investigation for their regenerative
potential in a variety of debilitating diseases. However, the field of hESC research is still
in its infancy, as there are several critical issues that need to be resolved before clinical
translation. Two major concerns are the ability of undifferentiated hESCs to form teratomas
and the possibility of a provoked immune reaction after transplantation of hESCs into a
new host. Therefore, it is imperative to develop noninvasive imaging modalities that
allow for longitudinal, repetitive, and quantitative assessment of transplanted cell survival,
proliferation, and migration in vivo. Reporter gene‑based molecular imaging offers these
characteristics and has great potential in the field of stem cell therapy. Moreover, it has
recently been shown that reporter gene imaging can be combined with therapeutic
strategies. Here, we provide an outline of the current status of hESC research and discuss
the concerns of tumorigenicity and immunogenicity. Furthermore, we describe how
molecular imaging can be utilized to follow and resolve these issues.
Adult stem cells have great promise as potential treatments for a variety of intractable
diseases. However, these cells are generally limited in their plasticity. Therefore, it would
be ideal to obtain or create a cell line that is truly able to differentiate into every cell of the
body. The first such cell line was derived in the 1960’s and originated from teratomas that
developed spontaneously in male mice of the 129 strain. These “embryonal carcinoma”
(EC) cells were capable of teratoma formation after transplantation of single cells into a
confirming their ability to differentiate into progeny of all three germ layers
(ectoderm, mesoderm and endoderm)-a phenomenon known as ‘pluripotency’.
research led to the first isolation of murine embryonic stem cells (mESCs) in 1981 from
the epiblast of blastocyst-stage mouse embryos,
followed by the establishment of the
first human embryonic stem cell (hESC)-line in 1998.
To date, there are more than
300 hESC lines, but only 22 hESC lines are commercially available and registered in the
“NIH Human Embryonic Stem Cell Registry”.
Although most studies using hESCs in disease models show auspicious results, there are
several concerns about hESC transplantation. First, the pluripotent character of hESCs is
somewhat a double-edged sword. They are an attractive candidate for cell based therapies,
but their pluripotency can also lead to risk of teratoma formation after transplantation.
Second, since it is presently impossible to transplant hESCs syngeneically, the possibility
that hESCs might provoke an immune reaction following allogeneic transplantation must
be considered. This review will discuss these issues and how molecular imaging can help
DERIVATION, MAINTENANCE AND DIFFERENTIATION
Traditionally, hESCs are isolated from the inner cell mass of the human blastocyst,
or as recently shown, can be derived from single blastomeres.
The isolated cells can be
expanded in vitro, with an average doubling time of 30–35 h.
However, strict homeo-
static culture conditions and the addition of inhibiting compounds are necessary to keep
the hESCs in an undifferentiated state, a condition required for maintaining a normal
karyotype and an unlimited capacity for self-renewal. This is possible by growing hESCs
on a cellular feeder layer. Inactivated murine embryonic fibroblasts prove to be an effective
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Molecular Imaging of Human Embryonic Stem Cells
feeder layer for the undifferentiated growth of hESCs because
they secrete differentiation-inhibiting factors.
Due to the risk of
cross-species retroviral infection, however, this is an unattractive
option in the long term. Recently, several reports have described
the culture of hESCs in animal-free conditions, using human feeder
cells consisting of foreskin,
pure human fibroblast populations,
or uterine endometrial cells and serum-free medium.
these undifferentiated hESCs express transcription factors OCT-3/4,
Sox-2, and NANOG; surface markers CD9, CD133, and SSEA-3/4;
proteoglycans TRA-1-60/81 and TRA-2-54; and enzyme alkaline
phosphatases and telomerase.
Following withdrawal of inhibitory factors, hESCs will aggregate
into three-dimensional clusters of cells in an early stage of differentia-
tion, thereby losing pluripotency. These clusters, named Embryoid
form the first step of further differentiation into any
type of progeny. Within the EBs, a microenvironment exists in which
various signals will promote differentiation into all three germ layers.
Although differentiation generally occurs spontaneously, much effort
currently focuses on stimulating directed differentiation to achieve
sufficiently large populations for clinical use. The generation of pure,
differentiated cultures is indispensable for developing cell based
therapies, and will help us better understand cellular developmental
processes and test pharmacological strategies.
DIFFERENTIATION INTO MESODERM LINEAGES
While reports have been published of hESCs differentiating into
various mesodermal lineages, including kidney, muscle, bone and
it is cardiomyogenesis that has received the most atten-
tion. Cardiomyogenesis typically manifests as a beating area within
the EBs around 5 days after EB-formation, the surface of which
increases gradually with time.
Kehat and colleagues were the first
to show that hESC-derived cardiomyocytes within these beating EBs
actually resemble the structural and functional properties of early
stage human cardiomyocytes.
Since then, several other methods
have been tested to improve the efficiency of in vitro differentiation
of hESCs into cardiomyocytes with moderate success.
DIFFERENTIATION INTO ECTODERM LINEAGES
Using different growth factors and stimulating environments,
hESCs can also be driven to differentiate into brain, skin, and adrenal
The potential of hESC-derived cultures for the treatment
of neurodegenerative disorders is under intensive investigation.
While many groups have described neuronal differentiation within
factor-induced neuronal differentiation seems to be
limited to the addition of retinoic acid (RA) and nerve growth factor
or the use of serum-free, conditioned medium.
coculture of hESCs with murine skull bone marrow-derived stromal
cells also seems to induce neuronal differentiation.
DIFFERENTIATION INTO ENDODERM LINEAGES
From the beginning, hESCs were shown to be capable of differ-
entiating in vitro into liver and pancreatic cells when exposed to
a variety of growth factors.
The creation of insulin-secreting
pancreatic cell populations has generated much interest, as this
might ultimately provide a cell-based therapy for patients with type
However, the identification of insulin-producing cells
has proven to be difficult and susceptible to artifacts.
Thus, the in
vitro pancreatic differentiation from hESCs remains a challenging
multi-step culturing procedure at present.
In summary, although much is being done to improve the efficacy
of in vitro differentiation systems, little is known about the cellular
interactions that occur during natural differentiation. Most of the in
vitro differentiation methods are to some extent dependent on EB
formation. The process of in vitro EB formation mimics the natural
transcriptional pathways occurring in the developing embryo, leading
not only to the differentiation into the desired cell type, but also to
the production of undesired cells. The most dangerous example of
the latter is undifferentiated hESCs that retain the ability to form
teratomas. Until we understand the precise pathways of pluripotent
differentiation, the acquisition of desired, transplantable cell types
can only rely on stimulating known pathways and the pretransplanta-
tion selection of the desired cell type.
At present no selection method exists that can yield a 100% pure
population, which is a major obstacle for clinical translation. When
transplanted in immunodeficient mice, hESCs form teratomas
consisting of human tissues from all germ layers.
and composition of teratomas seem to be influenced by several factors,
including graft site,
transplanted cell number, and developmental
phase of the host,
as described next.
One factor influencing hESC-based teratoma formation is the
site of transplantation, which affects both growth and composition
of the tumor. As recently shown by Cooke and colleagues, teratomas
rising from hESCs will grow faster and contain more undifferenti-
ated cells when transplanted in the liver of nude mice, as compared
to subcutaneous transplantation.
It is of major interest why hESCs
differentiate at the subcutaneous site but remain undifferentiated
in the liver. The authors hypothesize that this was due to the well
vascularized, growth factor-rich, immune-privileged porous structure
in the liver.
These findings are not only a stimulant for further
research on graft site and teratoma formation, but also indicate the
importance of in vivo experiments, as there may be factors present
in the liver that could help maintain hESCs in an undifferentiated
state in vitro.
Another factor influencing teratoma formation is the number
of undifferentiated hESCs that are viable after transplantation. As
discussed earlier, transplantation of pure undifferentiated hESC
inevitably leads to teratoma formation.
tion of selected hESC-derived cells in a more developed phase will
not automatically lead to teratomas, even when this population is
not 100% pure.
However, there are no extant studies that assess
the minimal cell number needed for teratoma formation, or stated
otherwise, the maximum percentage of contamination with undiffer-
entiated hESC. Our laboratory is actively investigating these issues.
Recently, Muotri and colleagues have studied undifferentiated
hESC after in utero transplantation into the lateral brain ventricle of
day-14 mouse embryos.
The results showed that hESCs integrated
into the brain, giving rise to neuronal and glial lineages, but not to
teratomas. These findings suggest that hESCs are susceptible to envi-
ronmental cues that can modulate its differentiation and tumorigenic
potential, as was suggested in earlier studies with hESCs in chick
Taken together, these observations and questions are not only a
stimulant for further research on teratoma biology, but also indicate
the importance of developing novel imaging techniques to track their
growth in vivo longitudinally, repetitively, and quantitatively.
Molecular Imaging of Human Embryonic Stem Cells
2750 Cell Cycle 2006; Vol. 5 Issue 23
Another hurdle facing clinical transplantation of hESCs is the
potential immunologic barrier. The immune response generated after
transplantation is directed towards alloantigens, which are antigens
presented on the cell surface that are considered non-self by the recip-
ient immune system.
The major system of alloantigens responsible
for cell incompatibility is the major histocompatibility complex
(MHC). In humans, MHC-I molecules are expressed on the surface
of virtually all nucleated cells and present antigens to CD8
T cells, while MHC-II molecules are normally more restricted to
antigen presenting cells such as dendritic cells and macrophages, and
are selectively recognized by CD4
helper T cells.
It has been shown that hESCs express low levels of MHC-I in
their undifferentiated state.
In one study, the MHC-I expression
increased two to four-fold when the cells were induced to spontane-
ously differentiate to EB, and an eight to ten-fold when induced to
differentiate into teratomas.
In contrast, a different group observed
MHC-I downregulation after differentiation induced with retinoic
acid, on Matrigel or in extended cultures.
In both studies, MHC-I
expression was strongly upregulated after treatment of the cells with
interferon γ, a potent MHC expression-inducing cytokine known
to be released during the course of an immune response. MHC-II
antigens were not expressed on hESCs or hESC derivatives.
latter finding confirms that, in contrast to tissue allografts, hESC
transplants are devoid of highly immunogenic mature dendritic cells,
or any other type of specialized antigen presenting
cells. Thus, the transplanted cells may not
express MHC-II molecules required for effective
priming of alloreactive CD4
T cells through
Previously, our group tested allogeneic undif-
ferentiated mESCs for their ability to trigger
alloimmune response in a murine model of
We found progressive
intra-graft infiltration of inflammatory cells
mediating both adaptive (T cells, B cells, and
dendritic cells) and innate (macrophages and
granulocytes) immunity. Cellular infiltration
progressed from mild infiltration at two weeks
to vigorous infiltration at four weeks, leading to
rejection of the mESC allograft. Moreover, we
found an accelerated immune response against
mESCs that had differentiated in vivo for two
weeks, suggesting that mESC immunogenicity
increases upon their differentiation.
Although it was previously reported that
hESCs failed to elicit immune responses during
the first 48 hours after intramuscular injection of
a recent report using
a similar model found hESCs to be completely
eliminated at 1 month post-transplantation.
Thus, questions regarding the exact character and
intensity of immune responses towards allogeneic
hESCs and their derivatives remain. Solutions that
reduce or eliminate the potential immunological
response to transplanted allogeneic hESCs are
urgently needed. Possible strategies to minimize
rejection of hESC transplants have been exten-
sively reviewed elsewhere.
Examples of these
strategies include: (1) forming HLA isotyped
hESC-line banks; (2) creating a universal donor cell by genetic
modification; (3) inducing tolerance by hematopoietic chimerism;
or (4) generating isogeneic hESC lines by somatic nuclear transfer.
To optimize these techniques in the future, it is crucial to develop
sensitive and reliable imaging methods for monitoring the viability
of transplanted cells in vivo.
To date, most studies on stem cell therapy have relied on conven-
tional reporter genes such as GFP
and b-galactosidase (lacZ) to
monitor cell survival and differentiation. However, these reporter
genes cannot be used to reliably track in vivo characteristics of
transplanted cells due to poor tissue penetration and the need for
extrinsic excitation light, which produces an unacceptable amount
of background signal. Instead, GFP-labeled cells are typically
identified histologically, which provides only a single snapshot repre-
sentation rather than a complete picture of cell survival over time.
To solve these shortcomings, our group has been developing reporter
gene-based molecular imaging techniques.
Molecular imaging can be broadly defined as the in vivo char-
acterization of cellular and molecular processes.
The backbone of
reporter gene-based molecular imaging technique is the design of
a suitable reporter construct. This construct carries a reporter gene
linked to a promoter/enhancer, which can be inducible, constitutive,
Figure 1. Schematic overview of molecular imaging. Outline of a vector containing a DNA report‑
er construct with the reporter gene(s) driven by a promoter of choice. Transcription and translation
lead to production of mRNA and reporter protein, respectively. After administration of a reporter
probe systemically, the reporter probe will be catalyzed by specific cells that have the reporter
proteins. This amplification process can be detected by a sensitive imaging device. Examples of
reporter genes and their specific reporter probes are listed per imaging modality. Abbreviations:
Fluc, Firefly luciferase; PET, positron emission tomography; HSV‑ttk, herpes simplex virus truncated
thymidine kinase; [
F]‑fluoro‑3hydroxymethylbutyl) guanine; SPECT, single
photon emission computed tomography; hNIS, human sodium/iodide symporter; MRI, magnetic
resonance imaging; CCD, charged coupled device; BLI, bioluminescence imaging.
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Molecular Imaging of Human Embryonic Stem Cells
or tissue specific. The construct can be
introduced into the target tissue by molec-
ular biology techniques using either viral
or nonviral techniques. Transcription of
DNA and translation of mRNA lead to the
production of reporter protein. After admin-
istration of a reporter probe, the reporter
protein reacts with the reporter probe,
giving rise to signals that are detectable by
a charged-coupled device (CCD) camera,
positron emission tomography (PET), single
photon emission computed tomography
(SPECT), or magnetic resonance imaging
(MRI) (Fig. 1). For thorough review, please
refer to other relevant articles.
IMAGING ESC TRANSPLANTATION,
A major advantage of reporter gene
imaging is the incorporation of the reporter
construct into the cellular DNA. This
ensures that the reporter gene will only
be expressed by living cells and will be
passed on equally to the cell’s progeny.
Thus, this imaging modality can provide
significant insight into cell viability and
proliferation. As discussed earlier, moni-
toring cell viability is a critical requirement
to assess immunogenicity, as a provoked
immune reaction can kill transplanted cells.
Monitoring cell proliferation is another
important feature, considering the tumori-
genic potential of undifferentiated hESCs.
Moreover, the ability to image the whole
body will allow us to track cell migration
in other organs. This is a major advantage when compared to tissue
biopsies using GFP-labeled cells.
In addition, multiple reporter genes can also be introduced into
the same cell for multimodality imaging. Recently, our group has
tested the efficacy of mESC with a self-inactivating lentiviral vector
carrying triple-fusion (TF) construct containing firefly luciferase
(Fluc), monomeric red fluorescent protein (mRFP), and herpes
simplex virus truncated thymidine kinase (HSV-ttk).
in the construct facilitates the imaging of single cells by fluorescence
microscopy and allows for the isolation of a stable clone population
by fluorescence activated cell sorter (FACS). The Fluc can be used to
perform high throughput bioluminescence imaging (BLI) for assess-
ment of cell survival, proliferation and migration at relatively low
costs. Finally, the HSV-ttk allows for deep-tissue PET imaging of
gene expression in small animals
as well as in patients.
transplantation into the hearts of athymic nude rats, mESCs could
be successfully followed for 4 weeks using BLI and PET imaging.
Between week 2 and 4, both BLI and PET reporter gene signals
increased rapidly, indicating teratoma formation. This was confirmed
by histological analysis.
Because of the risk of teratoma formation, it would be ideal to
have an in vivo imaging modality in combination with a fail-safe
suicide-gene mechanism. Using the antiviral drug ganciclovir, which
is toxic against cells expressing HSV-ttk, Cao and colleagues were
able to ablate teratoma formation and follow this progress non-
This study reveals the excellent potential of reporter gene
imaging for future use with hESC transplantation. In fact, prelimi-
nary studies in our lab suggest that as low as 100 undifferentiated
hESCs (H9 line) can cause teratoma formation after subcutaneous
injection (Fig. 2). Whether lower cell numbers (e.g., 1, 10, 50),
other graft sites (e.g., intramuscular, intravenous), or different cell
lines (e.g., federally and nonfederally approved) have similar kinetics
of teratoma formation will need to be determined carefully in the
Finally, a very critical question with regard to reporter genes is
whether they will affect ESC differentiation and hamper
for clinical applications. A previous study from our lab has shown
that the TF reporter genes affect <2% of total genes of mESC
using transcriptional profiling analysis.
A more recent follow up
study using proteomic analysis show that there were no significant
differences between control mESCs versus mESCs with reporter
Importantly, reporter probes such as D-Luciferin (for Fluc)
F]-FHBG (for HSV-ttk) had no adverse effects on mESC
viability and proliferation as well.
Ongoing studies are evaluating
the effects of reporter gene expression and reporter probes on various
hESC cell lines.
Figure 2. In vivo bioluminescence imaging of teratoma formation after transplantation of 100 hESCs.
(A) Bioluminescence image showing longitudinal follow up after transplantation of 100 hESCs stably
expressing a double fusion reporter gene (Fluc‑GFP). Faint imaging signals were seen as early as 2
hrs after transplant, which became progressively stronger over two weeks. Histology at eight weeks
confirmed teratoma formation. Note one of the hESC transplanted sites did not successfully engraft
(arrow) as there were no detectable signals by two weeks. (B) Histology from a representative explanted
teratoma showing hESCs that have differentiated into derivates from different germ layers. (I) squamous
cell differentiation with keratin pearl; (II) respiratory epithelium with ciliated columnar and mucin produc‑
ing goblet cells; (III) osteoid (nonmineralized bone) formation; (IV) cartilage formation; (V) gland cells;
and (VI) rosette consistent with neuroectodermal differentiation (400x magnification).
Molecular Imaging of Human Embryonic Stem Cells
2752 Cell Cycle 2006; Vol. 5 Issue 23
Clearly, the capacity of hESCs to differentiate into almost all
human cell types highlights their promising role in regenerative
therapies for the treatment of heart disease, Parkinson’s disease,
leukemia, diabetes, and other degenerative disorders. But the
pluripotency of hESCs may also pose major risks such as teratoma
formation. Likewise, hESCs might not be immunoprivileged and
could trigger host immune responses, leading to decreased cell
survival or acute rejection. These are issues that can become signifi-
cant barriers to future clinical application of hESC-based therapies.
To meet these challenges, researchers must gain a better understanding
of the in vivo behavior of transplanted hESCs. This review outlines
the burgeoning application of molecular imaging to track transplanted
hESCs in vivo. Continuing research merging molecular imaging and
hESC biology will likely lead to significant advances in the future,
both scientifically and medically.
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