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Module 1: Murine ES cell derivation and testing pluripotency
Hubert Schorle, Department of Developmental Pathology
The module will focus on early murine development and cell culture and surgical techniques
used at this stage. Further, the methodology of testing pluripotency of ES cells and iPS cells
by in vitro and in vivo techniques will be shown.
During the module you will isolate preimplantation embryos in order to collect blastocysts for
ES-cell injection and for derivation of novel ES-lines. Superovulation of female mice, choice
of mousestrain and general handling techniques will be shown and discussed. The handling of
ES (freezing-thawing and splitting) cells will be taught. Techniques for mitotic inactivation of
primary feeder- (fibroblast) cells will be discussed. Requirements for cell culture media and
sera (ES-grade) will be outlined. You will learn how to inject ES/iPS cells in blastocyst cavity
followed by in utero transplantation. In parallel, you will learn how to setup an embryoid
body differentiation system using microdrops in order to test the in vitro differentiation
capabilities of ES and iPS cells. Demonstrations will include the system of trophectoderm
stem cells (TSC) and we will discuss the factors governing inner cell mass and trophectoderm
specification and maintenance.
Module 2: Generation and characterization of human iPS cells
Simone Haupts, Oliver Brüstle, Institute of Rekonstruktive Neurobiologie, Life & Brain
Center
Reprogramming of adult human fibroblasts using defined factors – OCT4, SOX2, KLF4, and
MYC – to an induced pluripotent state has become a widely used technique. The so-called
induced pluripotent stem cells (iPSCs) resemble human embryonic stem cells (hESCs) with
respect to morphology, gene expression and functionality. IPS cells can differentiate into cell
types of all three germ layers in vitro and form teratomas in vivo, demonstrating multilineage
differentiation potential. IPS cells from patients carrying an inherited disease offer the
opportunity to generate disease-affected cell types of otherwise inaccessible tissues such as
the central nervous system. To establish suitable cell culture models individual primary hiPSC
colonies must be initially harvested and further characterized by a rigorous validation process
to select for optimal hiPSC clones.
In this module we will demonstrate and discuss critical steps for the generation and validation
of proper hiPSC clones. The module includes practical trainings on the identification,
initiation of in vitro differentiation and cryopreservation of hiPSCs. Additionally, we will
demonstrate the use of a new enabling technology (CellCelectorTM) for the automated
isolation of pluripotent hiPSC colonies in a highly selective manner at the phase contrast,
bright field or immunofluorescence level.
Module 3: Generation and validation of human pluripotent stem cell-derived neural
stem cells
Jerome Mertens, Philipp Koch, Barbara Steinfarz, Anke Leinhaas, Oliver Brüstle,
Institute of Reconstructive Neurobiology, Life & Brain Center
Human pluripotent stem cells (hPSC) are expected to have far reaching applications for
regenerative medicine and cell culture-based disease modeling. A key prerequisite for the use
of patient-specific somatic cells in the study of neurological disorders is the generation of
homogeneous and mature neuronal cultures, their phenotypic characterization and functional
validation.
This module will cover methods for in vitro differentiation and validation of human
pluripotent stem cell-derived neural precursors, neurons and glia. Topics will include cell
culture techniques for efficient neural conversion of hPSCs, key steps relating to patterning of
neural precursors into region-specific neuronal subtypes as well as in vitro and in vivo models
for validating differentiation, survival and network integration of hPSC-derived neurons in
CNS tissue. In this context the attendants will be introduced to the establishment and
maintenance of rodent hippocampal slice cultures and their use as ‘in vitro transplantation’
system. The module also covers fundamental training in stereotactic transplantation of neural
precursors into the rodent brain.
Module 4: Transgene removal from hiPS cells by DNA recombinase transduction
Frank Edenhofer, Stem Cell Engineering Group, Institute of Reconstructive Neurobiology
Embryonic stem (ES) cells being able to proliferate indefinitely and to differentiate into any
cell type of the body are highly attractive for biomedical applications. Recent studies
demonstrated that somatic cells like fibroblasts can be dedifferentiated to ES-like cells by
retroviral transduction of the four factors Oct4, Sox2, c-Myc, and Klf4. Such artificially
induced pluripotent cells (‘iPS’) would represent an appealing option for the derivation of
human pluripotent, patient-specific cells, as no embryos or oocytes are required for their
generation. However, various obstacles have to be overcome in order to adapt this procedure
for clinical use in humans. Some genes required for reprogramming (Oct4, c-Myc and Klf4)
play a role in tumor formation. Moreover the retroviral transduction method as such harbors
the risk of random insertional mutagenesis.
Several strategies have been used to derive factor-free iPS cells, including the application of
non-integrating viruses, small molecules and cell-permeant proteins. However, these methods
are thus far extremely inefficient and thus not useful for routine derivation of iPS cells. The
delivery of reprogramming transgenes and subsequent removal by site-specific recombinases
represent an attractive alternative. The application of cell-permeant versions of recombinases
such as Cre (Nolden et al., Nat. Methods. 2006) or FLP (Patsch et al., Stem Cells 2010)
allows highly efficient transgene deletion without plasmid transfection. This module aims at
providing hand-on practice to use cell-permeant recombinases for deleting reprogramming
transgenes from human iPS cells. The module will include preparation and application of cellpermeant recombinases, cultivation of hiPS cells and confirmation of transgene removal by
PCR. The overall aim is to deliver factor-free iPS cells.
Module 5: Engineering recombinant cell-permeant transcription factors for cell
(re)programming
Frank Edenhofer, Stem Cell Engineering Group, Institute of Reconstructive Neurobiology
Induced pluripotent cells (‘iPS’) would represent an appealing option for the derivation of
human pluripotent, patient-specific cells for disease modeling and regenerative medicine.
However, in order for iPS technology to become clinically relevant, various issues have to be
addressed. Methods need to be developed that improve the safety profile while increasing the
overall efficiency of the production of the cells. We reported the generation of recombinant
cell-permeant variants of Oct4 and Sox2 employing protein transduction technology. For this,
proteins are fused to cell penetrating peptides enabling the direct delivery into cells. Cellpermeant Oct4 and Sox2 proteins are biologically active as confirmed by DNA binding
analysis and by an RNAi-based rescue scenario. This module will provide hand-on practice
for the engineering and purification of cell-permeant transcription factors. Recombinant
proteins will be expressed in E. coli and purified using Ni-affinity chromatography. SDSPAGE and Western blot analysis will be used to analyze the protein fractions. Purified protein
will be directly delivered into cultured cells. This module provides a basis for the application
of protein transduction to non-genetically manipulate mammalian cells.
Module 6: Identification and therapy of cardiac diseases with functional characterization
and transplantation of iPS and ES cells
P. Sasse, M. Hesse, Institute for Physiology I, W. Röll, Clinic for Cardiac Surgery
The participant of this module will gain insights in the current state of the art methods for
functional characterization, application and analysis of proliferation of ES and iPS cell
derived cardiomyocytes. In detail the module will cover the following topics:
Application of iPS cells for the analysis and therapy of cardiac diseases:
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Generation and application of cardiac disease-specific iPS cells for modelling the long
QT syndrome.
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Electrophysiological investigation of disease-specific and healthy cardiomyocytes by
patch-clamp and multi-electrode array analysis.
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Strategies for purification of cardiomyocytes from various iPS and ES cell lines for
safe transplantation.
Clinical application of stem cells in a murine infarction model and in vivo postoperative
functional diagnosis:
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Demonstration of murine cardiac anatomy and myocardial lesion models (cryolesion,
coronary artery occlusion) and transplantation of purified ES-cell derived
cardiomyocytes.
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Introduction into basic principles of cardiac hemodynamics. Live demonstration of left
ventricular catheterization and analysis of pressure-volume-loops.
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Introduction into basic principles of the murine ECG recording and
electrophysiological investigation of arrhythmogeneis. Live demonstration of ECG
monitoring via peripheral leads as well as intracardial ECG monitoring via right heart
catheterization.
Analysis of the proliferation potential of cardiomyocytes derived from ES cells and in vivo:
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Introduction to the eGFPanillin system for visualization of proliferating cells
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Identification of proliferating cardiomyocytes in-vitro by analysis of a double
transgenic ES cell line (αMHC-RFP; CAG-eGFPanillin)
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Identification of proliferating cardiomyocytes in-vivo by analysis of a double
transgenic mouse line (αMHC-mCherry; CAG-eGFPanillin)
Module 7: Mesodermal differentiation of embryonic and adult stem cells
D. Wenzel, C.Geisen, M. Breitbach, Institute for Physiology I
The participant of this module will gain insights into the differentiation and purification of
mesodermal tissues like endothelial cells and cardiomyocytes from murine embryonic stem
cells. Moreover, this module deals with the preparation of murine mesenchymal stem cells
from adult bone marrow and their differentiation into bone, cartilage and fat. In detail the
module will cover the following topics:
Generation of mesodermal tissues (endothelial cells, cardiomyocytes) from murine embryonic
stem cells:
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Conditions and cultivation protocols for mesodermal differentiation (embryoid body
system, hanging drops, mass culture)
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Genetic approaches for the visualization of differentiating endothelial cells and
cardiomyocytes
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Methods for purification of endothelial and cardiac tissues from embryoid bodies
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Monitoring of mesodermal differentiation by histological stainings and fluorescence
microscopy
Preparation, characterization and differentiation of mesenchymal stem cells from murine bone
marrow:
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Isolation of bone marrow cells and cultivation protocols for mesenchymal stem cell
enrichment
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Surface antigen profiling by flow cytometry
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Differentiation of mesenchymal stem cells into typical derivatives and verification by
histological stainings
Module 8: Heterogeneity of cancer (stem) cells
Björn Scheffler, MD; Roman Reinartz, PhD stud; Martin Glas, MD; Stem Cell Pathologies,
Institute of Reconstructive Neurobiology, University of Bonn
Heterogeneity is a quintessential feature of glioblastoma (GBM) - the deadliest brain tumor of
adulthood. There are, eminently, several aspects of heterogeneity known to this disease. First,
on genetic grounds, there is a notable inter-individual diversity: Even though specific genetic
aberrations are known to occur within the tumor cells (1), GBM signatures are generally
patient-specific. Second, cellular characteristics and functions can vary significantly within
the same patient’s tumor tissue (2).
Intriguingly, while heterogeneity of tumor cells is a generally accepted phenomenon, very
little information is available on the extent, the specifics, and the consequences of this
diversity. This module will thus focus on experimental strategies, tools, and techniques that
might be useful to elucidate the basis of cellular and molecular diversity in human cancer. We
will, among others, demonstrate evidence for cellular heterogeneity based on singlenucleotide polymorphism (SNP)- and fluorescence in situ hybridization (FISH)- analysis of
GBM cell and tissue samples, we will study the functional consequences of this diversity, and
we will put the findings in the context of the “cancer stem cell”- versus the “clonal
evolution”-hypothesis of tumorigenesis.
Module 9: Prospective isolation of hematopoietic stem cells
Viktor Janzen, University Hospital of Bonn, [email protected]
Hematopoietic stem cells (HSC) are the best characterized adult stem cells in the mammalian
system and have been used in clinical settings for many decades already. However, the
molecular mechanisms that regulate the unique ability of stem cells to self-renew and to
differentiate into different cell lineages are still poorly understood. Since stem cells are
defined by their function many attempts have been undertaken to identify the stem cells
prospectively to be able to study their regulation at the molecular level. The participants of the
current module will learn to understand the basics of hematopoietic stem cells characteristics,
different strategies of stem cell enrichment as well as the methods to isolate the most pure
stem cells population known so far by FACS soring them based on 6 colours surface staining
and sorting by fasc-sort. Subsequently the isolated stem and progenitor-populations will be
transplanted into lethaly irradiated mice in a competitive transplantation setting using
congenic mice strain. Also, the participants will undergo practical training in the analysis of
HSC and progenitor cells, including clonogenic assays in semisolid medium as well as long
term culture on feeder layer. This module will take place in the laboratories of the Department
of Medicine (Hematology/Oncology) in the BMZ-building of the University of Bonn.
Module 10: Genetic inducible fate mapping: linking neuronal identity with genetically
defined progenitor populations
Sandra Blaess, Neurodevelopmental Genetics, Institute of Reconstructive
Neurobiology
The coordinated generation of a vast number of diverse neuronal cell types and their
subsequent organization into neuronal networks during development is critical for the proper
functioning of the adult brain. To understand the underlying complex mechanism of these
developmental processes, it is important to gain insight into how the genetic identity of
progenitor cells is established and how their genetic identity relates to the final location and
function of the mature neurons derived from these progenitors. Over recent years, the
development of sophisticated transgenic and gene targeting techniques, combined with the use
of site-specific recombinases, has opened many new opportunities for fate mapping studies in
the mouse. Genetic fate mapping marks progenitor cells based on their gene expression
pattern and allows to determine the relationship between embryonic gene expression and cell
fate (genetic lineage) and the link between gene expression domains and anatomy (genetic
anatomy) (Joyner and Zervas, 2006).
In this module we will focus on genetic inducible fate mapping. This system allows for
temporally and spatially controlled fate mapping by utilizing an inducible form of Cre
recombinase expressed under control of gene-specific promoters and a ubiquitously expressed
reporter allele (Feil et al., 1996; Soriano, 1999). Upon induction of Cre, Cre-mediated
recombination of the reporter allele results in expression of the reporter gene (e.g. lacZ, GFP)
and the permanent marking of the recombined progenitors and their descendants.
The aim of this module is to give insight into applications and potential caveats of the genetic
inducible fate mapping system within the context of the developing nervous system. We will
provide hands-on training on administration of Cre inducing agents and on tissue dissection
and processing. We will analyze and compare fate maps from different Cre lines and different
reporter alleles in embryos and adult brains and discuss advantages and disadvantages of the
distinct experimental set-ups.
Module 11: Module: High-Resolution Genome Analysis
Thomas W. Mühleisen, Michael Alexander, Institute of Human Genetics, Department of
Genomics, Life & Brain Center University of Bonn
The Department of Genomics at the Life & Brain Center of the University of Bonn is a
leading laboratory in the field of genome research. In the last few years, technological
advances and the successive deciphering of the human genome‘s sequence and structure have
enabled the identification of genes which contribute to common, genetically complex
diseases. In this context, high-throughput microarray assays and novel statistical methods play
an important role.
In this module, we will give an overview of such high-resolution genome analyses. The topics
will cover genome-wide and sub-genome-wide genotyping, copy-number variant detection,
and global gene expression. The participants will be introduced to concepts and workflows of
these analyses using both the BeadArray technology platforms by Illumina (San Diego, USA)
and the MALDI-TOF mass spectrometry platform by Sequenom (San Diego, USA).
For instance, we will demonstrate genome-wide genotyping using Illumina‘s HumanOmni1Quad array. Using genetic variation data from the International HapMap Consortium
(http://hapmap.ncbi.nlm.nih.gov/) and the 1000 Genome Project
(http://www.1000genomes.org/page.php), the >1 Mio. Omni1 probes were designed to
analyze common single-nucleotide polymorphisms (SNPs) with minor allele frequencies of
>5% for genome-wide association studies (GWAS). The principle of GWAS has recently
been reviewed by McCarthy et al. (2008). Omni1 arrays also offer analysis of common and
rare structural variation, including copy number variants (CNVs) and copy neutral variants
like inversions and translocations (for review see Stankiewicz and Lupski, 2010).
To obtain insights into whole genome expression analyses and interpretation of signalling
cascades and their alterations in cells, tissue and specific organs, gene expression analyses via
microarrays will be a second topic in this module. By the use of gene expression analyses it is
possible to investigate the global RNA expression of cells simultaneously on one chip (for
review see Cookson et al., 2009). Thus one can identify potential RNA expression fingerprint
profiles of e.g. different stem cells which are at different time points of developmental stages.
As one example of the broad portfolio (human gene expression arrays, mouse gene expression
arrays, etc.) we will present the technology, the platform and the workflow for processing
Illumina BeadChips for gene expression analyses.
Module 12: Analysis of mitochondrial DNA: Determination of mitochondrial genotypes
Wolfram S. Kunz, Abt. Neurochemistry, Clinic for Epileptology and Life&Brain Center
Due to its highly variable nature, its high abundance and uniparental inheritance,
mitochondrial DNA is a frequently used tool to investigate the identity and maternal relation
of individuals, or the origin of cell lines. Mitochondrial DNA accumulates somatic mutations
at a high rate, which has been identified as one important genetic factor of ageing. The
abundance of somatic mitochondrial DNA mutations is therefore one factor which can limit
the proliferative potential of stem cells. Mitochondrial genotyping has therefore important
implications for nuclear transfer, since donor mitochondria might also be transferred along
with the nucleus. And finally, mitochondrial genotyping can be used in distinguishing feeder
cells from the cells of interest. Many of the highly variable mitochondrial DNA nucleotide
positions are located within the 1 kilobase long D-loop region (the only larger non-coding
region of the mitochondrial genome), which makes mitochondrial genotyping relatively
uncomplicated. In the module we will identify mitochondrial DNA mutations by using RFLP
and D-loop sequencing of the mitochondrial genome. We will use allele-specific PCR and
single-molecule PCR to identify and to quantify somatic mitochondrial DNA mutations as
rare mitochondrial genotypes in the presence of another prevalent genotype. Additionally,
bioinformatics tools to evaluate mitochondrial genotyping results will be introduced.
Module 13: Modern aspects of flow cytometry in stem cell research
Dr. Elmar Endl, Unit: Institute of Molecular Medicine, Flow Cytometry Core Facility
The training course will provide the opportunity to gain hands-on experience of modern flow
cytometric techniques. In addition to the hands-on component, the course will include
tutorials and lectures on the theory and practice of relevant topics in the field of stem cell
biology.
Flow cytometry itself is a versatile tool to analyse the biology of cells on a single cell level. It
can unravel the position of cells on their way to differentiation, their proliferative capabilities,
physiological state, and expression of membrane- and intracellular antigens of a cell within
cohorts of cells and cell communities. Furthermore flow cytometry enables the physical
separation of cells by cell sorting followed by further functional and genomic analysis on
purified cell populations. Knowledge about the current protocols and methods in flow
cytometry is therefore advantageous for having the most modern toolbox to address specific
scientific questions in the area of stem cell research.
An essential feature of the module will be the combination of practical, laboratory-based work
with the corresponding theory taught in classroom sessions. The Flow Cytometry Core
Facility is well equipped for modern flow cytometry, including three analysers, with 3 lasers
(405, 488 and 635nm) and up to 10 colour detectors. Participants will also have the
opportunity to get in touch with instruments, reagents, assays and software packages, which
represent the current state of the art. Topics will focus on the characterisation of cells
according to the expression of their surface antigens in a multicolour setup. Strategies for the
fixation and permeabilisation of cells for staining of intracellular antigens and current
protocols for the quantification and reporting of cell growth and apoptosis. Moreover,
strategies for the interpretation and presentation of data will be demonstrated.
The module is aimed at enabling the participants to genereate, trouble shoot and interpret flow
cytometry data, and perform flow cytometry on complex samples using multiple
fluorochromes. While topics can be focused on the interests of the participants, the trainees
are expected to learn the following:
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History and Basics of flow cytometry
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Theory of experimental design, chromophore selection and multicolour compensation
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Hands-on set up of multilaser flow cytometers, instrument quality control and
compensation of complex samples
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Multicolour analysis of the expression of cell surface molecules
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Cell proliferation and apoptosis
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Demonstration of cell sorting including discussions on the use of Fluorescent Proteins
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Analysis of complex data sets
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Special applications in Stem Cell Biology
By the end of this workshop, participants should be able to explain the fundamental aspects of
flow cytometry results, and should be confident in knowing what their data mean. The module
is also intended to train the attendees on how to work around difficulties that may arise using
flow cytometry by providing them with examples they are most likely to be faced with in real
life. It is also intended to help navigating the roadmap of flow cytometric methods and
incorporate them into the future directions of the individual research projects.
Module 14: In vivo experiments: useful surgical techniques in small animal models
J. Kalff, Sven Wehner, Clinic for Surgery
Commonly, small animal models are the only way to investigate complex scientific questions.
Participants of this module will learn the handling, anesthesia and surgical techniques for
fluid administration, blood/lymph sampling and in vivo modulation of nerval functions in rats.
After a training period all techniques are easily transferable into a mouse model. All
participants will be introduced into the animal anatomy, the use of the different surgical
instruments, needle types, sutures and catheter materials.
In the first part, we will prepare the external jugular vein and insert a permanent flexible
catheter for continuous application of fluids or repetitive blood sampling. Furthermore, we
will implant an osmotic pump for continuous intravenous injection. We also will prepare and
electrically stimulate the cervical part of the vagus nerve.
In the second part, we will perform a laparotomy with subsequent subdiaphragmal preparation
of the anterior and posterior branches of the vagus nerve, followed by a bilateral vagotomy.
Next, an intraduodenal catheter will be placed for continuous enteral nutrition. Additionally,
collection of visceral lymph from the thoracic duct/cysterna chyli will be demonstrated.
Participants will also learn the preparation, application of fluids or blood sampling via the
portal vein, mesenteric artery and the vena cava.
The third part focuses on organ harvesting. Herein, we will perform a transcardial whole body
perfusion and a selective intestinal perfusion via the mesenteric artery. Subsequently, we will
harvest mesenteric lymph nodes, the visceral organs, lung and brain and separate intestinal
tissues (tunica muscularis, tunica mucosa, payer patches) under microscopical observation.
Module 15: Regulatory Framework for Stem Cell Research and Translation
Tade Matthias Spranger, Institute for Science and Ethics (IWE)
The course aims at giving an in-depth overview on the relevant national, supranational and
international regulation with regard to stem cells and derived technologies. Notwithstanding
its legal focus, the course should also foster the interdisciplinary discussion on crucial facets
of stem cell research. In particular, the following topics will be addressed:
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The German Stem Cell Act and the Ordinance on Stem Cells
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German Court Decisions on Stem Cell Technologies
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Patentability of Stem Cells according to national, European and international law
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Stem Cells in European politics and law
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In particular: The ethical assessment of FP7 applications
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Stem Cell Regulations as barriers to trade? The EU and the WTO perspective
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Normative perspectives