Dr Suzanne Kadereit

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Leist et aL.
Altex 25, Suppl. 1/0893
Human embryonic stem cells (heSC) are 
presently being cultured in many labora-
tories, and differentiation protocols are 
available for a large variety of cell types. 
the most immediate use of these cells 
may not be therapeutic applications, but 
more immediately, the design of test sys-
tems for toxicological and pharmacologi-
cal research (UKtI, 2005; Bremer and 
Hartung, 2004; Vogel, 2005). the use of 
such human-based test systems would 
contribute to a bottom-up test strategy for 
new chemicals, where initially the mode 
of action is explored, and animal experi-
ments are only used as a last resort and 
in special situations (leist et al., 2008a). 
One obstacle to the broad use of heSC 
for experimental test systems are ethical 
issues that have different legal implica-
tions in different countries (MBBNet, 
2008).
What are embryonic stem cells? Before 
entering a bioethical debate it is important 
to create a common platform of biologi-
cal facts important for such a debate. We 
will first take a look at natural conception 
and embryo development as basis for the 
overall understanding of the technology. 
After fertilization of the oocyte (“egg”) 
in the oviduct by a sperm, a zygote is 
formed. this cell, which contains genetic 
information from two parental gametes 
(i.e. the oocyte and sperm) starts divid-
ing while migrating down the oviduct, 
and gives rise to a tiny (less than 0.1 mm), 
blackberry-shaped, compact cell clump 
termed the morula, which eventually en-
ters the uterus at around day 4. By day 5, 
a cavity is formed in the “ball” and the 
resulting structure of embryonic cells is 
now termed early blastocyst. the blas-
tocyst is initially surrounded by a trans-
lucent structure (the zona pellucida) that 
protects it and prevents it from attaching 
to incorrect structures. In the uterus, the 
blastocyst begins to form different cell 
types - the outer cells (about 200), which 
will later become the placenta, and the 
inner cell mass (ICM) of about 30 cells 
which will later develop the embryo. 
eventually it “hatches” i.e. it breaks out of 
the zona pellucida and attaches to the wall 
of the uterus. this process of “implanta-
tion” of the late blastocyst occurs around 
day 9 post-fertilization and represents the 
first physical connection between the ear-
ly embryo and the fertilized woman. this 
step, also termed “nidation”, is crucial for 
the development of embryo polarity (body 
axes/up and down-definition). Finally, at 
day 12-14, a dramatic morphological re-
structuring occurs when the embryonic 
cells form a double-layered structure – 
the gastrula. this is the start of primitive 
tissue formation (primitive streak) in the 
embryo proper (the real embryo). At day 
14, therefore, pregnancy is established, 
the embryo has a close connection to the 
womb, it has developed polarity as well 
as “inside” and “outside” directionality, 
and, importantly, the three germ layers 
(primordial tissues) begin to form early 
organs such as the primitive gut and neu-
ral system. In parallel the placenta forms 
from formerly external cells of the blas-
tocyst.
Couples facing problems in conceiving 
naturally (in some countries also women 
that can get anonymous sperm) now have 
the option to increase their chances of 
pregnancy by undergoing an in vitro fer-
tilization (IVF) procedure. This process 
differs from a naturally-occurring preg-
nancy only in the initiation phase where 
sperm and oocyte meet. Oocytes are har-
vested from the woman, and this process 
is facilitated by ovarian stimulation with 
hormones. then they are fertilized with 
a sperm sample from the potential father 
to form zygotes. typically, 10-15 early 
pre-implantation embryos are generated 
in such a process and propagated in vit-
ro up to the blastocyst stage (day 5). In 
some countries (e.g. Germany), national 
regulations forbid selection of the embryo 
after nuclear fusion and preclude the cul-
tivation of more than three embryos at a 
time (Zollner et al., 2003). After positive 
selection of the best blastocysts, 1-3 (de-
pending on the country) are re-implanted 
directly into the uterus, where they have 
the chance to attach to the uterine wall 
and form an embryo just as it occurs in a 
natural conception. IVF procedures result 
in a successful pregnancy in about 15-
25% of the procedures. If the first attempt 
is unsuccessful, a second and third round 
of re-implantation of blastocysts may be 
initiated, since the initially superfluous 
blastocysts are typically cryopreserved, 
i.e. stored in a liquid nitrogen tank where 
they can be maintained for several years. 
It is not known when a blastocyst under 
conditions of cryopreservation loses its 
potential to form an embryo. Albeit one 
case study reported a successful preg-
nancy after implantation of a blastocyst 
stored for 12 years (Revel et al., 2004), 
it is generally observed that the quality 
is strongly falling after 5-10 years. Many 
supernumerary blastocysts accumulate in 
fertility clinics and will be ultimately de-
stroyed. It is estimated that 400,000 ferti-
lized oocytes were stored in 450 fertility 
clinics in the USA alone in 2003. tens 
of thousands are also frozen in Germany, 
The biological basis of the use  
of human embryonic stem cells for  
in vitro test systems
Introduction to the roundtable “Embryonic or adult stem cells: scientific  
and ethical considerations”
Marcel Leist1, Hannes Hentze2 and Suzanne Kadereit1
1 Doerenkamp-Zbinden Chair for Alternative in vitro Methods, University of Konstanz, Germany; 2 S*BIO Pte ltd, Singapore
090-092-LinzLeist.indd   93 25.8.2008   14:31:53 Uhr
Leist et aL.
Altex 25, Suppl. 1/08 94
Switzerland, Austria, the UK and many 
european and Asian countries. thus, it 
is reasonable to assume that by now over 
one million pre-implantation embryos are 
stored in the USA and elsewhere. A very 
small minority of these (less than 1%) is 
typically donated for research purposes, 
including heSC generation.
the in vitro generation of heSC starts 
with thawing and propagating a super-
fluous donated day five pre-implanta-
tion blastocyst. the ICM is isolated and 
placed in growth medium. these cells 
can be cultured in vitro, but they require 
a supportive “feeder” cell layer to provide 
them with nutrients and hormonal signals. 
Typically, mouse or human fibroblasts 
(connective tissue cells) are used as feed-
ers. If the procedure is successful, the cel-
lular outgrowth of this ICM will form the 
“passage 1” of a new heSC line, and once 
the cell culture dish is densely grown 
with heSC, the cells will be harvested 
and transferred to five fresh cell culture 
dishes. there they grow again to cover the 
whole dish (passage 2), and be transferred 
to five new dishes, and this procedure 
can be repeated continuously to generate 
more cells. It is now possible, in principle, 
to expand hESC indefinitely, and hESC 
have been cultured as stable cell lines up 
to passage numbers far beyond 150 using 
methods developed originally in 1998 in 
the laboratory of James thomson (thom-
son et al., 1998). All heSC harbour the 
potential to form a diversity of different 
cells – theoretically any cell type of the 
about 200 existing in the human body. 
therefore, they are called “pluripotent” 
cells. Pluripotent means that the cells can 
form any known cell type, but they cannot 
generate a whole organism, because they 
are lacking the capacity for implantation 
and formation of a placenta. this capacity 
is only found in the zygote and the cells up 
to the 8-cell stage of the morula, and these 
cells are called “totipotent”. It is important 
to emphasize here that heSC themselves 
are not totipotent and they are therefore 
unable to generate a new embryo.
the use of heSC in research requires 
the generation of heSC lines. the lines 
currently used were mostly produced 
from fertilized oocytes that had under-
gone about 7-8 divisions. the major 
objection to the use of eSC is that their 
generation is purported to involve an ‘act 
of killing’. To evaluate this point of view 
it is best to look at a typical example of 
cell line generation: A couple wishing to 
have children decides to try in vitro fer-
tilization (IVF) – oocytes are removed 
from the woman, fertilized with sperm, 
and two embryos are implanted into the 
woman. the remaining embryos are 
stored, frozen in liquid nitrogen, and the 
woman is lucky to become pregnant on 
the first attempt – possibly even giving 
birth to twins. life goes on and the couple 
is content with their two kids. After more 
than five years of storage, the quality of 
the stored blastocysts starts to deteriorate, 
and the storage is expensive as well. Pres-
ently, at least half a million such left-over 
zygotes are stored in the US alone. None 
of these blastocysts has any potential at 
all to ever develop to a human being with-
out a foster mother. After parental consent 
of our happy couple, the left-over mate-
rial will either be destroyed (moved to a 
trash can), or it may be donated for re-
search purposes (only in some countries). 
embryo adoption schemes as an alterna-
tive to discarding them were discussed, 
but this obviously does not appear to be 
a realistic option for all stored blastocysts 
at annual storage costs of several hundred 
million $. In the case of research donation 
for heSC research, the blastocyst will be 
allowed to be thawed, and heSC will be 
generated. When the procedure is suc-
cessful, this will result in the generation 
of a single continuously growing heSC 
line that can provide pluripotent cells in-
definitely. For instance the first such cell 
line, generated around 1998 (thomson et 
al., 1998) has now been spread to hun-
dreds of laboratories worldwide; its us-
age has generated a tremendous amount 
of novel scientific knowledge which may 
enable future therapies, and it is still being 
used extensively today. Most laboratories 
working with hESC nowadays use such 
cell lines for their research, but were nev-
er involved in the generation of cell lines, 
i.e. the use of any blastocyst.
Concerning the issue of the “use of hu-
man beings for research”, the definition 
of “human personhood” is the most cru-
cial issue, and a wide range of viewpoints 
exists:
• On one side of the spectrum one can 
find views that human life and personality 
begin and reach their full extent within a 
single moment, when the nuclei and chro-
mosomes of the gametes combine. 
• On the other side of the spectrum one 
finds views that the first days after the zy-
gote formation merely result in the forma-
tion of a “pile of cells” (morula/blastocyst 
stage) which cannot yet be assigned full 
human rights and therefore does not de-
serve the specific ethical consideration of 
a person (Schuklenk, 2008). 
• In between these extremes, there are al-
so views that acknowledge the zygote as 
being unique (as opposed to other cells) in 
its natural, but theoretical, potentiality to 
become a human being, but that full hu-
man personhood and the rights associated 
to it are acquired gradually. Here, meta-
phors are often evoked of the embryo 
being a walnut, a cherry or a caterpillar, 
which may or may not become a walnut 
tree, a cherry tree or a butterfly – but they 
certainly are none of the latter, and there-
fore they deserve a different status. No 
one would, for instance, argue that each 
walnut deserves the same protection as a 
full-grown walnut tree.
We have no authority of deciding who 
is right in the above issue, but we can look 
at the basic concepts associated with these 
different views. 
One line of thought builds on the con-
cepts of continuity, identity and potential-
ity. Obviously, a zygote does not fit our 
intuitive and accustomed picture of a hu-
man being. Still, it clearly belongs to the 
human race (biologically). In order to as-
sign the status of personhood to a micro-
scopic pile of cells, is has to pass at least 
the test of the following characteristics: 
• A defined succession of events leading 
invariably from the zygote to a human 
person (= continuity); 
• A situation in which one zygote only 
becomes one person and one person can 
only be derived from one single zygote (= 
identity); 
• The full capacity of a zygote to become 
a human person without further manip-
ulation or help (= potentiality). 
If one accepts that these three require-
ments are met, and if one assumes that 
fulfillment of these requirements is both 
necessary and sufficient for the person-
hood definition, then a zygote indeed is a 
human being. 
However, biological research in mam-
mals has shown that these assumptions 
090-092-LinzLeist.indd   94 25.8.2008   14:31:54 Uhr
Leist et aL.
Altex 25, Suppl. 1/08 95
may not hold true: the continuity crite-
rion is not fulfilled, because most zygotes 
(70%) never become persons (because of 
implantation failure), the identity crite-
rion is not met, because one zygote can 
produce twins, and vice versa some per-
sons are made up from two fused zygotes, 
and the full potentiality is not found in the 
zygote, as an embryo can never develop 
to a person in the absence of the mother’s 
womb, i.e. without maternal signals re-
ceived upon implantation. It may also be 
argued that these requirements are neces-
sary, but not sufficient (e.g. time as addi-
tional factor, see: gradual acquisition of 
personhood).
It has been argued that heSC can by 
no means be used to substitute animal 
experiments. this would mean that ex-
periments are instead performed on “hu-
mans”. However, this argument contains 
a misconception. the testing of toxicity 
on human cells allows man to take re-
sponsibility for himself without relying 
on animals and their suffering. It would 
not be a substitute, but an entirely differ-
ent, and possibly better approach. Hu-
man cell-based in vitro methods would 
fully correspond to the new vision of a 
mechanistically based toxicology (leist 
et al., 2008b; Hartung and leist, 2008) 
that implies higher financial efficiency 
of the procedure and improved safety 
for mankind. For instance the EU project 
eSNAtS has been started with the goal 
to develop novel heSC-based alterna-
tive test systems for drug profiling. This 
is a logical consequence of a line of re-
search that has shown that 3R (reduction, 
replacement, and refinement of animal 
experiments) methods have been aston-
ishingly successful and can sometimes 
yield better data than animal based tests 
(Hartung, 2001, 2007a, 2007b, 2008; 
leist et al., 2008b). heSC-based test sys-
tems could contribute to such a strategy 
of safety testing with fewer animals, and 
with data more relevant to humans, and 
more based on sound science. this con-
cept should by no means be mistaken (or 
distorted) to imply that animal experi-
ments would be substituted by embryo 
research. typical examples for the appli-
cation of heSC at the moment are in the 
field of developmental neurotoxicity. The 
thalidomide catastrophe showed that ani-
mal models can be very poor predictors 
of human developmental toxicity. Ac-
cordingly, a lot of focus has been on the 
development of eSC-based test systems, 
initially on murine cells, and in recent 
years also using human cells (Pellizer et 
al., 2005). Research on human neurons is 
extremely difficult due to the poor avail-
ability of the material. therefore, most 
experiments to test toxicity have relied 
on animals or animal material, and this 
has sometimes led to detrimental errone-
ous conclusions (see thalidomide). An-
other illustrative example is cardiotox-
icity (in particular toxicity of drugs that 
cause changes of the heart beat). this is 
frequently associated with drugs that in-
teract with the so-called heRG potassium 
channel on the heart. It is likely that in the 
near future, heSC-derived cells may be 
substituting the current assays performed 
with primary porcine or rabbit cardio-
myocytes (heart muscle cells). Such cells 
can now be generated from heSC with 
almost 100% purity, and these cells have 
been shown to function in the heRG test 
(xu et al.). 
Acknowledgement
We gratefully acknowledge funding from 
the European Community̓s Seventh 
Framework Programme (FP7/2007-2013; 
eSNAtS project), the land Baden-Würt-
temberg and the Doerenkamp-Zbinden 
Foundation.
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Correspondence to:
Dr. Suzanne Kadereit
University of Konstanz
Box M657
78457 Konstanz
Germany
e-mail:  
Suzanne.kadereit@uni-konstanz.de
tel.: +49-7531-885037
Fax: +49-7531-885039
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