FEATURE
Use of pluripotent stem cells and
their differentiated products in
pharmacological drug discovery
and safety testing
by
S
Suzanne Kadereit and Marcel Leist
ilently, at the end of last century,
a new age dawned: the biological
revolution. Without much notice,
scientists isolated the first embryonic stem
cells, from mice (1). Embryonic stem cells
are ‘pluripotent’ stem cells, that is they can
theoretically generate every cell type of the
body, and also self-amplify in culture dishes.
These cells transformed biology as we knew
it until then, and all of a sudden it became
possible to culture genetically normal, human
cells to large amounts. Until then, most cells
in long term culture were obtained from
tumors and were genetically abnormal. While
tumor cells can grow very fast under “in vitro”
conditions (in culture dishes), they usually
produce only one kind of cells, cells similar
to the tumor tissue they were isolated from.
Therefore, generation of heart cells from lung
tumors, or blood cells from colon tumor cells
is impossible. Accordingly, such cells never
raised much hope for regenerative medicine.
Instead, the generation of embryonic stem
cells opened a new world of possibilities. Not
surprisingly, scientists competed fiercely to
repeat the isolation of human equivalents.
Finally, almost 20 years later, they succeeded
in overcoming the cell culture problems raised
by the species differences between human
and mouse and isolated the first human
embryonic stem cells (2, 3). Recently, another
pluripotent stem cell type entered the arena
of regenerative medicine: induced pluripotent
stem cells (4). These cells are very similar to
embryonic stem cells as they can generate
large amounts of cells in culture and can
become almost every cell type of the body.
Most importantly, they can be generated
directly from cells derived from patients
and could thus, in contrast to embryonic
stem cells, be used for transplantation
without being rejected by the host immune
system. Needless to say, with an aging world
population and thus, increasing numbers of
patients suffering from degenerative diseases
in conjunction with an inadequate number of
organ donors, these cells are anticipated to
save countless lives, and also generate billions
of dollars in the regenerative medicine field
alone. But while scientists are still struggling
with the propensity of embryonic and induced
pluripotent stem cells to make every cell
type, and to direct them into cells that are
transplantable, a new field of applications has
emerged for these human stem cells.
Modern man lives surrounded by
synthetic chemicals and other compounds
and particles in air, water and soil. This
‘chemical’ universe in which we reside is
suspected to partly contribute to diseases
such as cancer, cardiovascular disease,
neurodegenerative disease, and behavioural
and learning deficits. Toxicologists are talking
about a silent pandemic caused by chronic
environmental and direct exposure to such
chemicals. Environmental pollution with
the heavy metal lead alone is estimated to
have caused economical loss to the USA due
to reduced IQ in the whole population, and
productivity of US$ 110-319 billion in each
year’s birth cohort between 1960 and 1980.
Today, after reduction of lead pollution, the
costs of lead poisoning are still estimated to
be $43 billion in each birth cohort. The costs
of low-level prenatal poisoning by methylmercury, another pollutant, are estimated to
amount to $8.7 billion yearly (5).
Regulations for mandatory testing
of consumer products have been in place
for almost 70 years. This regulatory safety
testing was started after two scandals shook
up the consumer world beginning of last
century. For example, more than a dozen
women were poisoned by a mascara called
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FEATURE
Lash Lure in 1933, leading to blindness for
some, and the death of one woman. Lash
Lure contained an irritating chemical that
was untested at the time, as there were no
regulations to ensure the safety of consumer
products. This incident was followed in 1937
by the Elixir Sulfanilamide scandal, killing 105
people in 15 states within a few months. As
a consequence of these cases, the Food and
Drug Administration (FDA) of the US passed
the Federal Food, Drug and Cosmetic Act in
1938 demanding from all manufacturers to
prove the safety of their products, and thus
opening the era of regulatory toxicity testing.
Since then, many countries have similar
requirements, and new drugs, consumer
products and chemicals are tested in animal
models and more recently also in cells,
mostly tumor-derived cells, in culture.
Animal models are however the mainstay
of regulatory toxicity testing and are widely
used to evaluate the safety of food additives,
household products, cosmetics, drugs and
vaccines, workplace chemicals, water and
air pollutants, and many other substances.
National regulatory agencies oversee these
testing processes which have to abide by
strict standards and regulations imposed by
the legislator.
In 2007, the European Union enacted
a new directive, REACH (Registration,
Evaluation, and Authorisation of CHemicals),
requiring manufacturers to test these
chemicals for their safety, prior to obtain
approval for their release. Rapidly it became
clear that the demand for test animals
would dramatically outstrip supply, and
logistically it would be impossible to breed
the required numbers of animals in the time
frame assigned by the European Commission
for compliance. The number of chemicals
to be tested has been estimated to be over
100,000 and more than 1000 animals would
be required for a full safety evaluation of one
single chemical (6).
Another reason to retreat from animal
models for human safety testing is that in
recent years it has become obvious that
animal models are not always predictive for
humans. Two of the more prominent examples
were the thalidomide scandal in the 60’s, and
the more recent example of the TGN1412
clinical trial. Thalidomide, a sedative drug, did
not reveal teratogenicity in extensive animal
testing. However, when used by pregnant
women severe limb malformations developed
in the babies. TGN1412, a drug developed
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against autoimmune disease and cancer,
had passed preclinical studies without any
problems. However, when injected into six
human healthy volunteers, all test persons
reacted within only 90 minutes with systemic
inflammatory response which escalated
within eight hours to life-threatening
multiorgan failure. Such dramatic examples
are fortunately relatively rare. Nevertheless,
particularly in drug development, cases
in which the developed drug revealed
unexpected side effects in human trials,
abound. This underscores the necessity of
developing safer test strategies for the future.
The advent of pluripotent stem cells
now enables the development of culture
systems that will allow safer, and also
importantly for the economy, much faster
and cheaper testing of drugs, food and
cosmetic additives, chemicals, nanoparticles
and other compounds that require safety
testing. Importantly, the pluripotency of these
cells enables the generation of potentially
any cell type of the body. This will lead
to the design of more organ-specific test
systems and allow the development of test
batteries ‘recreating’ the entire human
system in culture, including metabolizing
components with liver cells to identify
toxicity of innocuous compounds that require
metabolic transformation (for example in
the liver) to become toxic. The possibility to
generate large amounts of specific cell types
will also allow the development of disease
specific models for disease targeted drug
discovery and development. Most exciting
is the possibility to derive pluripotent stem
cells from genetically affected embryos or to
derive induced pluripotent stem cells from
patients with genetic diseases, and thus to
develop specific disease models. This will not
only yield a wealth of knowledge but can also
be exploited for targeted, faster and cheaper
drug development. First steps in this direction
have already been taken.
Currently, however, such sophisticated
test systems are still a theme of the future.
The only pluripotent stem cell-based test
system validated for regulatory safety testing
is the Embryonic Stem Cell Test (EST). This
test system uses murine embryonic stem
cells and measures toxicity for embryos
with a focus on heart development (7).
The process of regulatory validation, i.e.
the rigorous standardization of the culture
system, and, importantly, acceptance by
regulatory bodies to be used instead of
animal testing, is extremely time consuming
and involves numerous iterative steps of
testing, improving, and standardization by
several laboratories. The regulatory validation
of the EST took roughly 10 years, and
although human embryonic stem cells have
been available since 1998, still no human
pluripotent stem cell test has been validated
by regulators.
Faced with the shear overwhelming load
of testing that the EU requires under REACH,
the European Commission has launched
several funding initiatives under FP6 and FP7
to support research projects and consortia
aiming at developing human stem cell-based
test systems. But only slowly are new test
systems emerging from research laboratories.
These cell systems show different sensitivities,
depending on at which stage the cells are
FEATURE
exposed, and to which chemicals. In some
instances the pluripotent stem cell tests
are very sensitive, in some other instances
sensitivity is low (8). Also, not unexpectedly,
there are sensitivity differences between
human and mouse embryonic stem cell-
derived test systems, reiterating the necessity
to test with human cells (9, 10).
Currently, while more complex human
pluripotent stem cell-based systems are being
feverishly developed in research laboratories
around the world, industry is already using
existing stem cells-based tests for the prescreenings of drugs. This eliminates the most
toxic molecules early in the drug development
process, and thus, pluripotent stem cells are
already contributing to a significant reduction
in drug development costs (11).
References
1.
Evans MJ, Kaufman MH (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154-6.
2. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998). Embryonic stem cell lines derived
from human blastocysts. Science 282:1145-7.
3. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 18:399-404.
4. Takahashi K, Yamanaka S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-76.
5. Grandjean P, Landrigan PJ (2006). Developmental neurotoxicity of industrial chemicals. Lancet 368:2167-78.
6. Hartung T, Rovida C (2009). Chemical regulators have overreached. Nature 460:1080-1.
7. Genschow E, Spielmann H, Scholz G, Pohl I, Seiler A, Clemann N, Bremer S, Becker K (2004) Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern Lab Anim. 32, 209-244
8. Zimmer B, Kuegler PB, Baudis B, Genewsky A, Tanavde V, Koh W, Tan B, Waldmann T, Kadereit S, Leist M (2011). Coordinated waves
of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing. Cell Death Differ 18:383-95.
9. Stummann TC, Hareng L, Bremer S (2009). Hazard assessment of methylmercury toxicity to neuronal induction in embryogenesis using human embryonic stem cells. Toxicology 257:117-26.
10. Zimmer B, Schildknecht S, Kuegler PB, Tanavde V, Kadereit S, Leist M (2011). Sensitivity of dopaminergic neuron differentiation from stem cells to chronic low-dose methylmercury exposure. Toxicol Sci 121:357-67.
11. Wobus AM, Löser P (2011). Present state and future perspectives of using pluripotent stem cells in toxicology research. Arch Toxicol. 85:79-117.
About the Authors
Suzanne Kadereit is a stem cell biologist who studied in Germany, and
obtained her PhD at the Pasteur Institute in Paris. She worked on umbilical
cord blood immune and stem cells in the US, prior to working with human
embryonic stem cells in Singapore. While in Singapore, she became founding
head of the Singapore Stem Cell Bank. She is now Group Leader of the
stem cell group at the Chair for in vitro Toxicology and Biomedicine at the
University of Konstanz, developing stem cell-based systems from mouse and
human embryonic stem cells for disease modelling and toxicological studies.
Marcel Leist is a full professor and holder of the Doerenkamp-Zbinden
Chair for in vitro Toxicology and Biomedicine at the University of Konstanz
(Germany). He obtained an MSc for Toxicology in the UK and a PhD
for Biochemical Pharmacology in Germany. For six years, he worked on
neuropharmacologic drug discovery and toxicology at the company H.
Lundbeck A/S in Denmark. He is the co-Director of the center for alternatives
to animal testing in Europe, a joint venture with the Johns-Hopkins University
Bloomberg School of Public Health in Baltimore (USA).
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