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Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Dr. Péter Balogh and Dr. Péter Engelmann
Transdifferentiation and regenerative medicine –
Lecture 5
GENOMIC AND OTHER
CELL TRACING
APPROACHES,
REPROGRAMMING
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Animal cloning
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1952:
1963:
1986:
1996:
2000:
2001:
2003:
2005:
Tadpole
Carp
Mice
Sheep
Monkey
Cattle, cat
Rat, horse, mule
Dog
• 2008: Human
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Stem Cell Potential
Type
Description
Examples
Totipotent
Cells develop into a
new individual
Cells of 1-4 days old
embryos
Pluripotent
Cells form any cell type
Some cells of blastocyst
(5-14 days old)
Multipotent
Differentiated cells, but Fetal tissue, cord blood,
can form other tissues and adult stem cells
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Origin of stem cells and
reprogramming
Zygote
Epiblast
Late embryo/
Blastocyst
(post-implantation)
early foetus
Fate
decision
Adult
Skin
Fate
decision
Inner cell mass Epiblast
Central nervous
system
Primordial
germ cells
Bone marrow
Embryonic
stem cells
Totipotent Pluripotent
Epiblast
stem cells
Pluripotent
Embryonic
germ cells
Adult
stem cells
Pluripotent
Multipotent or
unipotent
Induced pluripotent
stem cells
Pluripotent
Other
+ Oct4, Sox2,
Klf4, Myc
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Conventional Sources of
Stem Cells
1 Somatic stem cells
• Harvested from mature organs or tissues
(bone marrow)
• Multipotent, may be tissue specific,
pluripotent?
• Many established clinical uses
2 Embryonic stem cell
• Derived from ICM of blastocyst
• Pluripotent, differentiate to all cell
lineages
• Encumbered by technical and ethical issues
• May be induced from adult tissues
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Origins of ES Cell Lines
1 Excess IVF embryos
2 Therapeutic Cloning (somatic cell nuclear
transfer)
• Donor oocyte + somatic cell nucleus
• Cells have characteristics of nuclear donor
• Lines representing different diseases
• Individualized lines: non-immunogenic to
donor
Somatic Cell Nuclear
Transfer
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• Challenging: In cloned cell lines,
about 4% of genes function abnormally,
owing to departures from normal
activation or expression of certain
genes -Imprinting, methylation state
• Limited success: ~25 percent of
nuclear transfers led to a blastocyst;
35 percent of blastocysts led to
establishment of cell lines
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Micromanipulation equipment
• Inverted microscope (fluorescent)
• CO2 incubator
• Thermal / heatable stage
• Holding pipette (inner diameter 10
µm)
• Injection pipette ( inner diameter 7
µm)
Chromosome removal
(‘Enucleation’)
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• Chemical enucleation: using specific
inhibitors
• Mechanical enucleation:
1 The egg is immobilized on the holding
pipette with the chromosome–spindle.
2 The zona pellucida is penetrated by the
injection pipette and the injection
pipette is pushed against the chromosome–
spindle complex for aspiration.
3 Aspiration of the chromosome–spindle
complex.
4 Complete removal of the chromosome–spindle
complex and exit of injection pipette.
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Nuclear injection
• Electrofusion
•
Microinjection:
1 Penetration of the egg’s zona pellucida
by the injection pipette.
2 Aspiration of small amount of cytoplasm to
facilitate re-sealing of the egg’s plasma
membrane.
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Egg activation
• Mammalian eggs are arrested in metaphase
stage during ovulation.
• SCNT is unable to reinitiate / trigger the
cell cycle, PLCζ enzyme is missing,
resulting in abolished Ca2+ influx.
• During egg activation Ca2+ rise is
essential, which can be evoked by strontium
chloride (SrCl2).
• SrCl2 treatment is more effective than EtOH
or ionophores.
• One hour after nuclear injection happened,
egg activation can be performed in
specialized conditions.
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Blastocysts and ESC colony
formation
• ESCs can be derived from eight cell
embryos or from morula stage, however
the most efficien scenario, when
blastocysts are used.
• By the 5th or 6th day after plating,
an inner cell mass outgrowth is
usually observed.
• For the culture of ESC cells feeder
cells are essential.
• Four to five days later ESC colonies
should appear at the side of culture
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Stem cell characterization
I
• Characterization: test the cells to see
whether they exhibit the fundamental
properties that make them embryonic stem
cells
• Growing and subculturing the stem cells for
many months microscope inspection for the
healthy and undifferentiated of cells.
• Using specific techniques to determine the
presence of surface markers that are found
only on undifferentiated cells
• Presence of Oct4 a transcription factor,
which helps turn genes on and off at the
right time for the processes of cell
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Stem cell characterization
II
• Determining whether the cells can be subcultured
after freezing, thawing, and replating
• Testing whether the human embryonic stem cells are
pluripotent by:
– allowing the cells to differentiate spontaneously
in cell culture
– manipulating the cells so they will differentiate
to form specific cell types
– injecting the cells into an immunosuppressed mouse
to test for the formation of a benign tumor called
a teratoma
• Teratomas typically contain a mixture of many
differentiated or partly differentiated cell types
• An indication that the embryonic stem cells are
capable of differentiating into multiple cell types.
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Stem cell markers I
• Oct4: octamer-binding transcription factor 4
homeodomain tr. molecule is coded by POU5F1 gene and
marks ES cells and undifferentiated, maternal factor
active in oocyte and in embryos.
• Sox2: or SRY (sex determining region Y)-box 2 HMG
factor act as a transcriptional activator after
forming a protein complex with other proteins (Oct4,
Pax6). Essential for iPSc formation.
• SSEA3/4: stage specific embryonic antigens are of
five to six monosaccharides attached to a ceramide
lipid tail. Their presence rapidly increasing during
differentiation. SSEA-3 and SSEA-4 were recently
shown not to be essential for the maintenance of hESC
pluripotency
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Stem cell markers II
• TRA-1-60, TRA-1-81: tumor rejection antigens
widely used markers for stem cell
characterization. They can recognize a
keratan-sulfated proteoglycan (KSPG) in
neuraminidase-sensitive and neuraminidaseinsensitive fashion.
• Alkaline phosphatase is a hydrolase enzyme,
it is also essential to identify stem cells
and verify their functionality.
Cell tracing in stem cell
biology:
non genomic
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• BrdU (bromodeoxyuridine) incorporation
• Fluorescent dyes:
– CM-DiI
– CFSE
– Hoechst 33342
– PKH26
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Cell tracing in stem cell
biology: genomic I
1 GFP
• 27 kDa protein (isolated originally
from jellyfish)
• popular reporter system in tissue
after cloning gene of interest
• different GFP variant
2 Lac-Z
• lac operon gene from E. coli
• histochemical reporter using X-gal
substrate
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Cell tracing in stem cell
biology: genomic II
3 Y chromosome marker
• The detection is a relatively simple
process compared to gene cloning and
expression based methods (GFP, LacZ)
• FISH analysis
• High labeling efficiency
• Widely used stem cell
transplantation approaches (cardiac, intestine disease)
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In vivo imaging for cell
tracing
• New development of time lapse and twophoton microscopy gave boost for live
cell imaging including cell tracing.
• Stem cells can be imaged at various
time points and locations to generate
time-lapse movies, and automated image
analysis and statistical analyses are
used to quantify the dynamic cells’
behaviour.
• Together with cell migration, changes
in cell shape and changes in
proliferation kinetics can be
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Cell tracing in stem cell
biology
z
t1
x
y
t2
Automated
image analyses
and
statistical
analyses
tn
Migration
Proliferation
Single-cell fate analyses
Cell-shape change
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Reprogramming
Somatic cells can be dedifferentiated
into stem cells, so called induced
pluripotent stem (iPS) cells using
certain aprroaches.
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Cell-fusion based
Nuclear extract based
Transfection of pluripotent factors
Somatic cell nuclear transfer
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Molecular mechanisms of
self-renewal
Lif
PI3K
Grb2
Jak
Akt
MAPK
STAT3
Tbx3
Klf4
Nanog
Sox2
Oct3/4
Cdx
Gata4
2
Prevention of differentation
Cell-cycle regulation
S
G2
G1
M
b-Myb
c-Myc
Genes involved in
reprogramming
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• Nanog:The nanog cDNA consists of 2184 nucleotides
(nt) and contains a single open reading frame
encoding a poly-peptide of 305 amino acids.
– the role in pluripotency of both inner cell mass
(ICM) and embryonic stem (ES) cells
– the ability to maintain ES cell self-renewal.
• Klf4: Krüppel-like factor, interact with CREB
transcription factor, expressed in ESC and MSCs.
• Lin28: a cytoplasmic mRNA binding protein, binds to
IGF-2 mRNA, enhance the efficiency of the formation
of iPSc from human fibroblasts, marker of
undifferentiated human embryonic stem cells, able to
bind let-7 miRNA and inhibit it.
• Oct4: see previously
• Sox2: see previously
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Telomerase activity I
• Telomeres are ribonucleoprotein
heterochromatic structures at the ends of
chromosomes that protect them from
degradation and from being detected as
double-strand DNA breaks.
• When Dolly was cloned using SCNT, reliable
question was raised about the age of her
cells? Telomere was shorter, by
approximately 20%, when compared with agematched controls.
• After some conflicting results concluded
that shortened telomeres of somatic donor
cells could be indeed re-elongated during
reprogramming, although the degree of
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Telomerase activity II
Telomers in iPS cells
• High levels of Tert (reverse transcriptase
component of telomerase) and high telomerase
activity were described in iPS cells.
• iPS reprogramming of normal cells (mice and
human) results telomerase activation and
restoration of telomeres, like setting the
clock, to a length and chromatin state that is
similar to that found in ES cells.
• Telomerase activation during iPS reprogramming
is associated with upregulation of TERT, but
also TERC (Tel. Associated rNA component) become
activated. Moreover OCT4 and NANOG bind to the
TERC gene reg. element, which may explain why
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Summary
• ES and iPS cell fates can be monitored
with a branch of fluorescent vital
dyes (non-genomic/ genomic) using in
vivo imaging techniques.
• Restoration of pluripotency factors
and self-renewal specific genes (Oct4,
Sox2, Klf4) can exhibit the
reprogramming for iPS cells.
• iPS cell generation can be good
candidates for regenerative medicine,
however there are still several