J. Cell. Mol. Med. Vol 8, No 4, 2004 pp.

465-473

Stem Cell Review Series

Embryonic stem cells:

a promising tool for cell replacement therapy

Michael Xavier Doss, Christoph I. Koehler, C. Gissel,

Jürgen Hescheler, Agapios Sachinidis *

University of Cologne, Center of Physiology and Pathophysiology, Institute of Neurophysiology,

Cologne, Germany

Received: October 26, 2004; Accepted: November 15, 2004

• Introduction – Transplant rejection

– Cell replacement therapy • Advances in CRT with ES cells
• ES cells – Cardiovascular therapy
– Multilineage differentiation in vitro – Haematopoiesis
– ES cells: A powerful source for cell replace- – Therapy for neurological disorders
ment therapy – Treatment of diabetes
• Problems encountered – Liver therapy
– Selection of desired cell phenotype • Future perspectives
– Tumor formation • Conclusion

Abstract

Embryonic stem (ES) cells are revolutionizing the field of developmental biology as a potential tool to understand
the molecular mechanisms occurring during the process of differentiation from the embryonic stage to the adult phe-
notype. ES cells harvested from the inner cell mass (ICM) of the early embryo can proliferate indefinitely in vitro
while retaining the ability to differentiate into all somatic cells. Emerging results from mice models with ES cells are
promising and raising tremendous hope among the scientific community for the ES-cell based cell replacement ther-
apy (CRT) of various severe diseases. ES cells could potentially revolutionize medicine by providing an unlimited
renewable source of cells capable of replacing or repairing tissues that have been damaged in almost all degenera-
tive diseases such as diabetes, myocardial infarction and Parkinson’s disease. This review updates the progress of ES
cell research in CRT, discusses about the problems encountered in the practical utility of ES cells in CRT and eval-
uates how far this approach is successful experimentally.

Keywords: embryonic stem cells • cell replacement therapy

* Correspondence to: Prof. Dr. A. SACHINIDIS Robert Koch Strasse 39, 50931 Cologne, Germany.
University of Cologne, Center of Physiology and Tel.: +49 221 478 73 73, Fax: +49 221 478 6965
Pathophysiology, Institute of Neurophysiology, Email: a.sachinidis@uni-koeln.de
Introduction
Extensive investigation of embryonic stem (ES)
cells for the last two decades resulted in the better
understanding of the molecular processes
involved during the differentiation process of ES
cells to the adult mature phenotype. Under specif-
ic in vitro culture conditions, ES cells can prolif-
erate indefinitely without senescence and are able
to differentiate into almost all tissue specific cell
lineages, if the appropriate extrinsic and intrinsic
stimuli are provided in the culture. These proper-
ties make ES cells an attractive candidate for the
so called cell replacement therapy (CRT) of vari-
ous degenerative diseases that are associated with
a loss of functional cells. It is hoped that with the
ES-based cell replacement therapy of the degen-
erative disorders such as myocardial infarction,
diabetes and Parkinson’s disease, normal function
of the respective organs will be restored signifi-
cantly. Still, exploitation and practical utility of
the ES cells are in their infancy. Further extensive
experimental investigations and clinical trials are
necessary for an optimal ES-based treatment of
degenerative disorders. This review explores how
well the ES cells are going to be manipulated to
serve as a renewable source of functional cells to
be used in the CRT to treat the degenerative dis-
orders, updates the progress in this regard, dis-
cusses about the problems encountered in the
practical utility of the ES cells and outlines how
far this approach will be successful in the near
future.
Cell replacement therapy
Degenerative disorders like myocardial infarction,
Parkinson’s disease, diabetes are due to the pro-
gressive (acute/chronic) loss of functional cells
due to disease or injury to the cells or ageing.
Replacing the worn out or injured cells by func-
tional cells to restore the normal function of the
tissues or organs is the underlying principle of
CRT, otherwise also called as regenerative
medicine. It is hoped that the organs or tissues
treated by this approach can perform their normal
function more efficiently than the ones treated by
conventional therapies like transplantation and
pharmocological therapy like treatment with
466
recombinant proteins such as recombinant insulin
or therapies based on mechanical devices. The
practical utility of this approach mainly depends
upon the availability of a versatile source of the
rightly chosen cells and is being currently delayed
for a long time due to the non-availability and/or
limited supply of a perfect source of cells
required. Much of the research has been drained in
order to find out a possible candidate to serve as a
bank of cells needed for the cell based therapeutic
applications. Several strategies are being currently
evaluated and include, cell therapies derived from
autologoues primary cell isolates, cell therapies
derived from established cell lines, cell therapies
derived from a variety of stem cells, including
bone marrow and mesenchymal stem cells, cord
blood stem cells, ES cells, as well as cells, tissues
and organs from genetically modified animals
[1,2]. The upcoming experimental results with ES
cells are encouraging and quite promising [3-6].
They give added impetus for this approach.
ES cells
ES cells have been in vitro derived in mouse and
human in a similar manner from the inner cell
mass (ICM) of the blastocysts. In 1981, ES cells
were isolated for the first time in mice and it was
a major breakthrough in the field of developmen-
tal biology [7,8]. ES cells can proliferate indefi-
nitely without senescence in in vitro culture in an
undifferentiated state in the presence of leukemia
inhibitory factor (LIF) or on top of a layer of
mitotically inactivated mouse embryonic fibrob-
lasts (MEFs) [9]. They can give rise to cell lin-
eages of any type of body tissues / organs, when
specific stimuli are provided in the culture (Fig.
1). The latter property of ES cells is called pluripo-
tency. ES cells are not totipotent because they fail
to develop a whole functional organism. To date,
ES cell lines are available from rodents, rabbits,
pigs and primates [7,8,10-12]. Murine ES cell
lines remain undifferentiated when grown either
on mitotically inactivated MEFs or in the presence
of relatively high concentrations of leukemia
inhibitory factor (LIF) whereas human ES cell
lines remain undifferentiated only when grown on
mitotically inactivated MEFs [13].
 J. Cell. Mol. Med. Vol 8, No 4, 2004

Fig. 1 Overview of the derivation of ES cells and their pluripotency. ES cells are derived from the ICM of the
early blastocyst and explanted into culture. The ESCA/Embryoid Body is an intermediate stage during in vitro dif-
ferentiation of ES cells into all three different germ layers and their derivates.

mation of ESCAs initiates signalling and sponta-

Multilineage differentiation in vitro
In the absence of LIF or when removed from feed-
er layers and transferred into suspension culture,
ES cells differentiate spontaneously into multicel-
lular ES cell aggregates (ESCAs), which resemble
early post implantation embryos (Fig. 2) (for
review see [14]). In general, it is believed that for-
neous differentiation of ES cells to the three
embryonic germ layers, the ectoderm, mesoderm
and endoderm [13,15]. Murine ES cell lines are
able to differentiate in vitro into a variety of embry-
onic and adult cell types from all three embryonic
germ layers. These include cardiomyocytes,
hematopoitic progenitors, yolk sac, skeletal
myoctes, smooth muscle cells, adipocytes, hepato-

467
Fig. 2 In vitro differentiation of ES cells into cardiomyocytes. A: outlines one of the protocols used for generating
ES cell derived cardiomyocytes. B: (I) Es cell derived beating cardiomyocytes in culture. (II) Beating cardiomyocytes
expressing GFP under the control of the cardiac α-actin promotor. (III) and (IV) Immunostaining of the developing
cardiomyocytes in plated EBs with monoclonal antibodies against sarcomeric α-actin. The white arrows indicate beat-
ing cardiac cell clusters. Figure (B II) taken with courtesy of Sachinidis A, Kolossov E, Fleischmann BK, Hescheler J.
Generation of cardiomyocytes from embryonic stem cells experimental studies. Herz. 2002 Nov;27(7):589-97
(Copyright Urban & Vogel. Reproduced with permission). Figure (B III and IV) taken with courtesy of Sachinidis A,
Gissel C, Nierhoff D, Hippler-Altenburg R, Sauer H, Wartenberg M, Hescheler J. Identification of plateled-derived
growth factor-BB as cardiogenesis-inducing factor in mouse embryonic stem cells under serum-free conditions. Cell
Physiol Biochem. 2003;13(6):423-9. (Copyright S. Karger AG, Basel. Reproduced with permission).

cytes, chondrocytes, endothelial cells, melano-
cytes, neurons, glia, pancreatic islet cells, primitive
endoderm and so on (for review see ref. [15], [16-
27]). These experiments clearly demonstrate that
ES cells can be induced to form the desired cell
phenotype in vitro when appropriate culture condi-
tions are provided and they reflect many of the crit-
ical developmental stages found in the normal
embryo. Figure 2 shows an example for generation
of cardiac cells.

468

ES cells: A powerful source for cell
replacement therapy
If the appropriate stimuli and/or carefully chosen
combination of extrinsic and intrinsic signal fac-
tors are available in culture, ES cells will differen-
tiate into almost all cell lineages of body tissues
like cardiomyocytes, oligodendrocytes, astrocytes,
beta cells of the pancreas, hepatic cells and so on
[28]. Several transcription factors have been
demonstrated to regulate differentiation of ES
cells to specific cell types [29]. Ectopic overex-
pression of such factors stimulates ES cells to dif-
ferentiate selectively into certain cell types [30-
32]. Also several other substances like ascorbic
acid, butyric acid and DMSO enhance the differ-
entiation of ES cells to specific mature phenotypes
[33,34].
Problems encountered
Selection of desired cell phenotype
The routinely used differentiation protocols give
rise to a mixture of different cell populations.To
harvest a single population of cells of our interest,
several strategies have been developed. Magnetic
bead tagged antibodies directed against the unique
cell surface marker of the desired cell population
can be directed and the desired population can be
harvested by running through a magnetic column
where the magnet tagged antibodies bound with the
desired cell popuation will be retained, allowing
other cell population washed out. Fluorescence
labelled antibodies raised against unique cell sur-
face markers of the desired cell population can be
lifted up by the fluorescence -activated cell sorting
(FACS). Some protocols employ selective culture
conditions that promote the growth of one particu-
lar cell type at the expense of the other. ES cells
may be genetically engineered to have selection
markers or fluorescent markers under the control of
a lineage/tissue specific promoter, for example a
transcription factor that is switched on early during
lineage specific differentiation. When the tissue/lin-
eage specific promoter is activated during the par-
ticular lineage differentiation, the selection marker
or the fluorescent marker will be expressed under
J. Cell. Mol. Med. Vol 8, No 4, 2004
the control of this promoter. Expression of selection
markers (antibiotics resistant genes) makes the
desired population resistant to that particular antibi-
otic when applied to kill the other cell population.
These antibiotics block the endogenous translation
or transcription machineries if cells do not express
the particular enzyme/ protein which antagonizes
the blocking activity of these antibiotics.
Expression of the fluorescent marker allows the
desired cell phenotype to be harvested by FACS.
The drawback of these two approaches is that the
genetically manipulated ES cell derivatives might
be immunologically rejected since they express the
foreign proteins -the selection and the fluorescent
markers.
Tumor formation
Implantation of undifferentiated ES cells leads to
formation of benign teratoma in the recipients
[12,13]. This demands a pure population of termi-
nally differentiated cell phenotype. Negative selec-
tion of Oct4 expressing cells might be a solution for
this issue. New strategies and methodologies need
to be developed to isolate the terminally differenti-
ated cells. ES cell implants can be tagged with some
kind of death signals in such a way that when they
start to form tumors by accident, or when they start
to cause severe complications, they will be cleared
off from the body leaving the host unaffected. New
methodologies and the forthcoming new discover-
ies will find a possible way for this approach.
Transplant rejection
ES cell derived cellular grafts might be rejected due
to immunogenicity of the transplanted cells. ES
cells can be easily manipulated genetically to over-
come the problem of immune rejection so that life-
long pharmocologic immunosuppression will not
be needed. One attractive approach is that the MHC
genes in ES cells can be replaced with host MHC
genes so that the host immune system will recog-
nise the implanted cells as ‘self’ [35,36]. This
approach is practically possible with help of homol-
ogous recombination techniques. An alternative
approach will be the insertion of genes for the
immunosuppressive molecules such as Fas- ligand
469
into the ES cells [37,38]. Additionally the immuno-
logically-reactive molecules such as B7 antigens, or
CD40 ligand present on the surface of the ES cells
could be abrogated from ES cells [37,38]. One
interesting approach in this regard is the application
of nuclear transplantation technology in ES cells. In
this approach, the nucleus from a normal somatic
cell of the recipient, say from a skin biopsy is
extracted and is then injected into an enucleated
oocyte. The cytoplasm of this oocyte has the
required potential to reprogram the differentiated
nucleus injected and re-establishes an embryonic
gene expression pattern in the chromatin of the
somatic cell nucleus. The blastocyst formed from
this oocyte would be the source for the derivation of
new ES cell lines which would be genetically
matched for each nuclear gene of the recipient
[39,40]. In this case, whole MHC regions in ES
cells and other immune molecules will be identical
to that of the patient, except minor molecules
derived from mitochondrial genes. The immune
rejection in this case will be the least of all the
above-mentioned approaches. However, although
the therapeutical cloning approach appears to be
promising for avoiding possible immunological
problems by the recipients, this approach bears sev-
eral ethical problems that should be solved by
social consensus before clinical studies in this
direction can be initiated.
Advances in CRT with ES cells
Despite the pitfalls discussed above, there are
more interesting results coming out with ES cells
for the therapy of several degenerative disorders
as follows.
Cardiovascular therapy
Several cardiovascular diseases are associated
with myocardial cell death resulting from
ischemic heart disease, viral infections or
immunopathological conditions. The only current
treatment for the patients with end stage heart fail-
ure is the cardiac transplantation. Often, this is
limited by the availability of a perfectly matched
organ. The demand for organ supply is increasing
470
steadily, necessitating the vital need for the devel-
opment of new therapeutic options. In the last
decade, several animal studies demonstrated that
transplantation of isolated cardiomyocytes might
offer an alternative approach for the treatment of
cardiac degenerative disorders. Several animal
studies demonstrate that engraftment of cardiac
myocytes into the adult heart can be achieved suc-
cessfully. The emerging results with ES cells have
the potential to revolutionize Medicine and could
find a cure for the cardiac degenerative disorders
obviating the need for the allogeneic organ trans-
plantation. Several factors alone or in combination
have been shown to enrich cardiac differentiation
such as hepatocyte growth factor (HGF), epider-
mal growth factor (EGF), basic fibroblast growth
factor (bFGF), transforming growth factor b1
(TGFb1), platelet derived growth factor (PDGF),
sphingosine-1-phosphate, retinoic acid, 5-azacyti-
dine, vitamin C, and overexpression of GATA-4
(For review see [41], [28,30,33,42,43] The so
called CRT where the damaged cardiac cells will
be replaced by cells derived from the ES cells to
restore the normal function of the heart could be a
boon for heart patients in near future.
Haematopoiesis
Hematopoiesis in ESCAs develops spontaneous-
ly in the absence of exogenous cytokines demon-
strating that the microenvironment of the devel-
oping ESCAs is enough to supply the necessary
inductive signals for commitment of mesodermal
precursors to haematopoietic cell fate, and also to
provide the differentiation signals for blood cell
maturation. The pattern of haematopoiesis in the
ESCAs mimics yolk sac development and is
dominated by primitive erythroid cells that
express embryonic forms of hemoglobin (bH1)
[21,44].
Therapy for neurological disorders
Neuroectodermal differentiation is also relatively
easy to achieve from murine ES cells. High yields
of neurons, astrocytes and oligodendrocyte like
cells have been achieved with several different
optimised protocols. These protocols have since

been further simplified to remove the ESCAs
stage and differentiate murine and human ES cells
into neuroectodermal lineages in monolayer. This
is the only somatic cell type for which this is
known to be possible. Preliminary implantation
studies of ES derived neurons into mouse models
have reported that these cells are functionally
active, integrate into the brain and in one case
have corrected the phenotype of a neurodegenera-
tive disease [45,45-47].
Treatment of diabetes
β-cell replacement therapy via islet transplantation
through stem cells has gained more popularity due
to the recent upcoming results in the animal model
studies. Early investigations demonstrate that ES
cells are the promising sources for alleviating the
pathological conditions of diabetic patients [48]. It
has been reported that the insulin-expressing cells
that had spontaneously differentiated within
embryoid bodies were selected by an approach
where an introduced antibiotic resistance marker
under the control of the insulin promoter was
made use of [49].
Liver therapy
Hepatocytes or hepatocyte-like cells derived from
ES cells can be a promising approach in the therapy
of liver diseases. Several entities like chronic viral
hepatitis, alcoholic liver disease, haemochromatosis
etc. result in liver cirrhosis and finally organ failure
making a liver transplantation inevitable.
Differentiated ES cells could be used either for direct
transplantation into the diseased liver or for the gen-
eration of bioartificial liver (BAL) devices to bypass
situations of acute liver failure until an organ for
transplantation is available, for example acute fulmi-
nant viral hepatitis or severe intoxications. Studies
have shown the differentiation of murine and human
ES cells into hepatocyte-like cells and results have
also been achieved on the transplantation of differen-
tiated murine ES cell into the liver [50-54].
Especially BAL devices seem to be promising for the
next future. Main difficulties of CRT like tumor for-
mation or problematic engraftment of the cells are of
no relevance in this therapeutic approach.
J. Cell. Mol. Med. Vol 8, No 4, 2004
Future perspectives
To serve as a versatile renewable source of
healthier cells for the use in CRT, the problems
mentioned above should be overcome. That is,
new methodologies and protocols should be
developed for the derivation of every single cell
lineage population of body tissues/organs. New
strategies should be developed to harvest only
the terminally differentiated phenotype of the
cell or the phenotype of the cell which serves as
nearly an ideal substitute to be used for CRT.
This population of cells should be free from the
tumourigenic population or from the population
that may lead to severe complications during the
post transplantation period. The problem of the
immnogenicity of ES cell derived cellular trans-
plant in the host should be overcome by new
approaches. Once all these issues will be
addressed and a proper solution is found to over-
come these obstacles and hurdles, ES cells will
prove to be the potential versatile source of the
cellular transplants to be used in CRT to treat a
number of degenerative disorders. The upcoming
new discoveries and better understanding of the
molecular processes involved during the transi-
tion of embryonic stage to the adult phenotype
are opening up new avenues to overcome the
above mentioned hurdles and they will convert
our hopes into reality in the near future.
Conclusion
Utilization and practical application of ES cells in
cell replacement therapy are still in their infancy
and need further exploration and extensive inves-
tigations, experimental confirmations and clinical
trials before they will be made available as an
ideal cell substitute for the treatment of a number
of degenerative disorders. The daily upcoming
experimental observations are reinforcing the
solid hope that ES cells will be the potential
source for use in cell replacement therapy. Once
first successful clinical studies will be achieved
and evaluated, ES-based cell replacement therapy
will revolutionize medicine in the near future
offering therapeutical alternatives for treatment of
severe degenerative disorders.
471

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