Hepatic differentiation of canine and
human induced pluripotent stem cells
(iPS cells)
Author: Nikita de Grauw
Instructor: Dr. B. Spee
Date: 10-04-2013
Appendix: Used protocols
1
Hepatic differentiation of canine and human induced
pluripotent stem cells (iPS cells)
Introduction
The liver is one of the largest organs in the body. It has a wide regeneration
capacity and it plays an important role in vascular, metabolic and secretory
pathways. The liver maintains nearly every organ of the body, with the removal
of waste materials as an important function. However, the location and the
many features make it a sensitive organ for multiple diseases. Malfunctioning
of the liver is mainly seen in chronic diseases due to decreasing regenerative
capacity of the liver [1].
Within the field of regenerative medicine stem cells hold great promise to
recover the regeneration capacity from the liver in the case of end-stage liver
diseases. One of the cells, which can play a role within this recovery, is the
induced pluripotent stem cell. Induced pluripotent stem cells (iPS cells) are a
type of pluripotent stem cells that are artificially derived from adult somatic
cells by a forced expression of specific genes. Yamanaka and colleagues were
the first who described iPS cells derived from mice and man [2,3]. Nowadays it
has also been described from the dog [4-7]. It is possible for iPS cells to
differentiate into all kind of cell types in vitro, including hepatocytes [8]. These
iPS-derived hepatocytes are very useful for (autologous) cell transplantation,
drug toxicity testing and disease modelling. iPS-derived hepatocytes are able to
repair mouse models of liver damage [9]. Until now it is not yet possible to
apply this technique to larger animal species. This is the reason why this study
investigates the potential of canine iPS cells to recover liver diseases. Therefore
cell transplantation will be applied with iPS cell-derived hepatocytes to dogs
with copper-storage inside the liver. These results will give an indication
whether iPS cell technology in dogs and man is safe and practicable.
2
Materials and methods
In order to determine whether
human- and dog iPS cells can
contribute to the recovery of liver
diseases it is necessary to start with
the culture of these cells. During the
culture and experimental process
we;
- Checked
the
possibility
whether canine and human
iPS cells can be differentiated
into hepatocytes
- Looked if the cultured
hepatocytes are functionally
similar
to
primary
hepatocytes.
culture, but any type of somatic cell
can be used for these purposes.
The fibroblasts from the skin biopsy
are cultured and transformed into
pluripotent stem cells by lentiviral
transduction. Due to this lentivirus it
is
possible
to
insert
four
transcription factors (also known as
the Yamanaka factors) that can
establish pluripotency in stem cells;
OCT4, SOX2, KLF4, c-MYC that
ensure reprogramming [2,3] (figure
1).
By mimicking the embryonic
development these cells will be
differentiated into hepatocytes in
vitro [7,11].
Potential use of iPS cells
It is a great benefit that the derived
cells are from the patient itself. In
the case of cell transplantation,
there will not be an immune
response when placing back the
cultured cells.
The patient specific iPS cells can be
used for several purposes, namely
for cell transplantation but also as
disease modelling to test newly
developed medication [8].
Obtaining cells
The iPS cells can be obtained in a
low-invasive way, which is from a
skin biopsy from a patient.
Fibroblast cells from skin, is chosen
because they are relatively easy to
Figure 1: Reprogram cells [2,3]
3
Culturing cells
The iPS cells are cultured on 0,1%
gelatin-coated plates with a feeder
layer of irradiated mouse embryonic
fibroblasts (MEFs). For human iPS
cells it is necessary to plate out 1
million MEFs on a 10-cm culture
dish, canine iPS cells need a density
of 4 million MEFs per 10-cm culture
dish. MEFs need to be in MEF media
(DMEM L-glutamine included, 10%
FBS, 1% P/S) for at least 4 hours to
attach prior to the plating of the iPS
cells in order to attach [10].
There are two iPS cell lines used for
this experiment. The canine cells,
also known as Cibelli, which come
from the Department of Animal
Science, Michigan State University,
USA and the human cells, H9, which
come from the Hubrecht institute.
In this study the iPS cells are already
obtained and stored in liquid
nitrogen. It is therefore necessary to
defrost the cells before plating. It is
important to get rid of the MEF
media on the culture dishes and
replace it with standard ES cell
media for human- or canine
embryonic stem cells (DMEM/F12
(1:1) media w/20% v/v KOSR, 2mM
Glutamax,
50μM
2mercaptoethanol,
non-essential
amino acids, 10ng/ml bFGF and in
case of canine culture an addition
with 10ng/ml LIF is needed). Not
required, but recommended is the
addition of ROCK inhibitor to the
plates with iPS cells in order to
increase the survival efficiency after
thawing.
The plates with the iPS cells are not
allowed to get too confluent. If
there is not enough growth space
left it is important to pass the cells
with collagenase type IV (1mg/ml in
DMEM/F12) for six minutes. The
cells can be disrupted mechanically
by pipetting. It is important to carry
this step out carefully to avoid single
cells. Single cells differentiate
quickly and can then no longer be
used for the hepatic differentiation
experiment [10].
Hepatic differentiation
experiment
As soon there are enough 10-cm
culture dishes with a confluence of
70% per cell line it is possible to
start the differentiation experiment.
During this study we created an
optimized, cost-effective, protocol
based on previous studies. This
protocol imitates the embryonic
development [11].
First of all it is important to obtain
the iPS cells without any MEFs. To
remove MEFs we plated the entire
fraction on gelatine-coated plates
and allowed the MEFs to attach
within 20 minutes. This procedure is
repeated
two
more
times.
4
Afterwards we spun down the
supernatant, took up the pellet and
placed the iPS cells on a different,
matrigel coated plate (12-well plate,
24-well plate and chamberslides)
[11].
The first day we fed them with the
standard media, Human ESC (+LIF in
case of canine iPS). The next day the
differentiation protocol started.
First we have 5 days of endodermal
differentiation, then 5 days of
hepatic specification, 5 days of
hepatocyte specification, and 5 days
of hepatocyte maturation.
After (each) 5 days cells are fixed
(chamberslides), RNA is isolated and
liver
transaminases
were
determined in the media.
Cells need to be fed daily. The
media of the cells changed every 5
days in order to stimulate the
differentiation
into
mature
hepatocytes.
The protocol for this experiment is
the same for both human and dog
iPS cells [11].
To demonstrate the differentiation
towards hepatocytes with all
intermittent steps gene-expression,
immunocytochemistry
and
functional tests were performed.
Detailed protocol can be found in
supplemental
file
1,
short
descriptions of the protocols are
listed below.
RNA
During the experiment we isolated
cells for RNA isolation in triplicate
after each differentiation step
according to protocol (4 steps in
total) [11]. We used the RNeasy mini
kit (Qiagen) to isolate the RNA,
following
the
manufacturer’s
instructions, and used the Nanodrop
for
RNA
concentration
measurement.
cDNA
The cDNA was made according to
iScript protocol [12] (BioRad). All
qPCRs were performed in 384-well
reaction plates in 20μl reaction
volume using SYBR Green PCR
Master Mix [13] (BioRad).
Primers
To demonstrate that the cells
possess the necessary genes we
selected primers for both canine
and human qPCR, which indicate
stem cell characteristics and
hepatocyte features.
For canine qPCR we used RPS5,
RPS19 and HPRT as reference genes.
Cyp3a12, Spp1, CSA, HNf4a, AFP,
HNf1b, SOX17, Mrp2, CK18, CK19,
FOXa1,
ONECUT1,
ONECUT2,
PROX1, CPS1, OCT4, and TERT as
marker genes.
5
For human qPCR we used RPL19,
GAPDH and RPS5 as reference
genes. Cyp3a4, Tjp1, ALB, HNF4a,
AFP, Hnf1b, SOX17, Mrp2, KRT18,
KRT19, FOXa1, FOXa2, CD44, FN14,
OCT4 and Vimentin as marker
genes.
Immunocytochemistry (ICC)
The
immunocytochemistry
is
performed according to protocol on
PFA fixed cells in chamberslides
[16]. With the experiment we
started plating out the iPS cells on
glass chamberslides. In the progress
of the differentiation the cells
detached from the slides. This was
tried again on plastic chamberslides
including a coating with matrigel,
but unfortunately all without
success. Because the use of
chamberslides is not possible a new
experiment on a 24-well plate is
started for ICC results (only canine).
We stained with the following
antibodies for demonstrating the
presence of antigens (species of the
antibody was generated in between
brackets):
ALB (mouse), ARG1 (rabbit), BCAT
(rabbit), CD133 (mouse), PROX1
(rabbit), CPS1 (rabbit), CK19
(mouse), CK7 (mouse), FN14
(rabbit), GGT (mouse), HNF1B
(rabbit), HNF4A (rabbit), MRP2
(mouse), OCT4 (rabbit), SOX17/SRY
(mouse), SOX9 (rabbit), STRO1
(mouse) and VIM (mouse).
Indocyanine green (ICG)
Indocyanine green (ICG) is an
organic anion, which is clinically
used as a test-chemical to control
liver function. The substance is nontoxic and eliminated only by
hepatocytes. After differentiation it
is needed to test if we had cultured
mature
good
functioning
hepatocytes. The ICG test gives us a
good indication whether the cells
meet
(one
of
the
many)
requirements. After the media is
added to the hepatocytes cells were
given maximum six hours to
eliminate the ICG.
Periodic Acid Schiff (PAS)
staining
Periodic Acid Schiff (PAS) staining is
used for the detection of glycogen in
tissue, like formalin- fixed liver
tissue, but can also be used for
cardiac and skeletal muscle.
Glycogen, fungi and mucin will turn
purple and nuclei will be stained
blue. Glycogen is a sugar
(carbohydrate), which is normally
stored in the liver and plays an
important role within the glycogen
metabolism [15].
Liver transaminase
determination
During the experiment we collected
media and cell pellets for enzyme
determination. This was collected
three times within every isolation
step (4 steps in total).
6
After both the canine and human
experiment was finished we sent
the samples (250 μl) to the UVDL.
Cytochrome P450
Cytochrome P450 3a4 (human) and
3a12 (canine), also known as
Cyp3a4/3a12, is situated within the
liver and plays an important role in
decomposing toxins and foreign
substances. For having wellfunctioning liver cells, it is important
to have an active Cyp3a4 enzyme.
This activity in hepatocytes can be
measured by BFC-assay. The
material converted due to the BFC
assay is fluorescent and can thereby
be measured fluor-metrically [17].
After the BFC assay is performed a
fluorescamine assay is done to
determine the amount of proteins in
each well for normalisation
purposes [18].
Micro albumin
Albumin is a protein created in the
liver. It plays an important role in
maintaining blood pressure and
fluid balance. It is also responsible
for the transport of all kind of
substances, for example vitamins
and medication [19]. After both the
canine and human experiment was
finished we sent samples with
media (60 μl) collected during the
experiment to the UVDL to find out
if our cultured hepatocytes-like cells
express albumin.
Ammonia tolerance
The liver converts ammonia to urea,
which is otherwise toxic. Ammonia
is neutralized by the urea cycle and
glutamin synthesis. With the
ammonia tolerance test an urea
assay is performed to find out
whether the cells are able to break
down the toxic ammonia and
convert it into urea. This test is
performed at the end of the
experiment [19, 20]. After the urea
assay is performed, a CyQUANT
assay is done to determine the
density of the cells for normalisation
purposes. Both are performed
according to the manufacturer’s
instructions [21].
Results
Quantitative PCR
Gene expression profiles were
determined by the ΔCt method. The
technical replicates are indicated
with the same symbol.
Gene-expression measurements in
the canine iPS cells indicate that
ONECUT1,
a
liver
specific
transcription factor, is increased
during differentiation, with the
highest expression in the hepatic
maturation stage.
PROX1 is an early hepatic marker.
The presence of this gene increases
throughout the experiment. The
highest
concentrations
are
7
Relative gene expression
60
40
30
20
10
iPS
ENDO
SPEC
CYTE
HEPA
PROX1
1400
1200
1000
800
600
400
200
0
iPS
ENDO
SPEC
CYTE
HEPA
FOXA1
Results canine:
Relative gene expression
1000
3000
Relative gene expression
50
0
Relative gene expression
measured
at
the
moment
hepatocytes are formed.
FOXA1 is also an early hepatic
marker. This graph shows us a
positive result, although we would
expect slightly lower relative gene
expression at HEPA stage because it
is an early marker.
KRT19 is a progenitor cell marker,
present within hepatocytes that are
not 100% mature yet. The relative
gene expression comes up at the
CYTE stage of the experiment and is
at the highest concentration within
HEPA stage.
ALB is a hepatic marker. The relative
gene expression comes up at HEPA
stage, as we would expect.
HNF4a is an early hepatic marker,
which maintains a high relative gene
expression.
2500
2000
800
600
400
200
0
1500
iPS
1000
ENDO
SPEC
CYTE
HEPA
KRT19
500
0
iPS
ENDO
SPEC
CYTE
HEPA
ONECUT1
8
Relative gene expression
different in comparison with the
canine results of FOXA1.
HNF1B is a bile duct marker. The
graph shows us high relative gene
expression during SPEC stage. This is
a positive result because at SPEC
stage the cells are not only
hepatocytes yet. Beyond the SPEC
stage the relative gene expression
decreases which shows that there
are no bile duct cells cultured.
ALB is a hepatic marker and should
have the highest relative gene
expression within HEPA stage.
CYP3a4 is a hepatic marker. See ALB
HNF4a is an early hepatic marker,
which maintains a high relative gene
expression.
ALB
40
30
20
10
0
iPS
ENDO
SPEC
CYTE
HEPA
Results Human
HNF4a
AFP is an early hepatocyte marker,
which maintains a high relative gene
expression. This gene is present
within CYTE and HEPA stage.
OCT4 is a pluripotency marker. The
relative gene expression is high at
iPS and ENDO stage and low at the
moment hepatocytes are formed.
This is a positive result because
pluripotency of the cells should
decrease
throughout
the
experiment.
FOXA1 is an early hepatic marker
(see results canine). The relative
gene expression is already low
within HEPA stage, which is
6000
Relative gene expression
Relative gene expression
50
5000
4000
3000
2000
1000
0
iPS
ENDO SPEC
CYTE
HEPA
AFP
9
18000
16000
50000
Relative gene expression
Relative gene expression
60000
40000
30000
20000
10000
14000
12000
10000
8000
6000
4000
2000
0
0
iPS
ENDO SPEC
CYTE
iPS
HEPA
Relative gene expression
Relative gene expression
HEPA
7
10
8
6
4
2
0
6
5
4
3
2
1
0
iPS
ENDO SPEC
CYTE
iPS
HEPA
ENDO SPEC
CYTE
HEPA
CYP3a4
FOXA1
12
Relative gene expression
800
Relative gene expression
CYTE
ALB
OCT4
600
400
200
0
10
8
6
4
2
0
iPS
HNF1B
ENDO SPEC
ENDO SPEC
CYTE
iPS
HEPA
ENDO SPEC
CYTE
HEPA
HNF4a
Immunocytochemistry (ICC)
The ICC results allow us to see
whether or not the cells express the
antigen in question. Because
pictures are taken at different times
we are able to say whether a
10
particular type of antigen increases
or decreases as time passes.
OCT4 is a pluripotency marker,
which should be present at the
beginning of the experiment (within
iPS cells or ENDO stage cells). Over
time there is little or no expression
of OCT4.
SOX17 is an endodermal liver
marker and shows expression at the
beginning of the experiment. After
endodermal stage is over we see a
decreasing of SOX17.
KRT7 is a progenitor cell marker and
marks hepatocytes, that are not
100%, mature yet. The expression of
this marker is low at the beginning
of the experiment and is increasing
over time.
HNF1B is a bile duct marker. In the
beginning of the experiment
expression of this marker is present,
due to the fact that this stage does
not exist of hepatocytes only.
Within the HEPA stage of HNF1B
there is little or no expression,
showing us there are no bile duct
cells cultured.
Representative pictures have been
taken at the beginning (ENDO stage)
of the experiment and at the end
(HEPA stage) of the experiment.
OCT 4 ENDO Canine
OCT4 HEPA Canine
SOX17 ENDO Canine
Results Canine:
11
SOX17 HEPA Canine
HNF1B ENDO Canine
KRT7 ENDO Canine
HNF1B HEPA Canine
Indocyanine green (ICG)
KRT7 HEPA Canine
The results of the indocyanine green
test (ICG) are positive for both
species. The green test chemical we
applied to the cells is incorporated.
As the time goes by, the cells
eliminate the green test chemical,
which
is
shown
in
the
representative pictures, cells turn
less green (after six hours) then at
the start [14]. It must be noted that
some of the cells did not eliminate
the green test chemical. These are
apoptotic cells
wherein
the
substance can flow into the cell, but
is not able to be eliminated actively
because of cell death.
12
Representative pictures have been
made at the start and after six hours
[13] (figure 2-5).
Figure 5: Human ICG test after six hours
Periodic
staining
Figure 2: Start Canine ICG test
Figure 3: Canine ICG test after six hours
Acid
Schiff
(PAS)
Periodic Acid Schiff (PAS) staining
results shows us purple staining
within the cells and some blue
staining within the nuclei [15]. The
staining is positive, but to confirm
we stained glycogen an additional
test is needed. In this case the
purple staining could come from
mucin, fungi and glycogen. In order
to show we stained glycogen,
amylase pre-treatment is needed as
a negative control. Based on the
representative pictures below, we
can say we did not stained mucin or
fungi, and therefore we can say that
this staining is another positive
result.
Representative pictures have been
taken before staining (bright field)
and after staining (figure 6-9).
Figure 4: Start Human ICG test
13
Figure 9: PAS staining Human
Figure 6: Bright field Canine
Liver transaminase
determination
Figure 7: PAS staining Canine
Figure 8: Bright field Human
Determination of the liver enzymes
is done by the UVDL. Previous
studies showed enzymes could be
determined in the media. After we
sent in 250μl media of each sample,
we received some disappointing
results and we decided to hand in
250μl cell pellet of each sample. The
results were much better this time,
so we used the determination in the
cell pellet.
When we look at the results we
would expect to find the highest
concentration of enzymes within
hepatic maturation, except for GGT,
because at this point cells should
function like primary hepatocytes.
GGT is a bile duct enzyme, which we
would not expect within primary
hepatocytes.
For canine cells we see real high
enzyme
concentrations
within
endodermal differentiation for ALT
and AST. It is difficult to say whether
this is abnormal because there is no
comparable data available. If we
14
look at the measurement of other
differentiation steps we see what
would be expected. The enzyme
concentration increases as the
differentiation
experiment
progresses with the highest
concentration
within
hepatic
maturation. GGT is high at the
specification step, which make
sense because in this stage bile duct
cells can be present.
For human cells we did not see real
high enzyme concentration within
the endodermal differentiation
except for GLDH. In general all the
enzymes
show
the
highest
concentration in the hepatic
maturation stage. What is abnormal
is the concentration of GGT during
the different steps of the
experiment. These results suggest
there are, besides hepatocytes, also
bile duct cells.
The results per enzyme are
corrected for cell count. An
overview is made for the enzymes
ALT, AF, AST, GGT and GLDH for
both canine and human (figure 1014).
15
140
ALT U/L
120
100
80
Canine
60
Human
40
Figure 10: ALT U/L Canine and human
20
0
-20
ENDO
SPEC
CYTE
30
HEPA
AF U/L
25
20
15
Canine
10
Human
Figure 11: AF U/L Canine and human
5
0
-5
ENDO
1000
900
800
700
600
500
400
300
200
100
0
SPEC
CYTE
HEPA
AST U/L
Canine
Human
ENDO
SPEC
CYTE
Figure 12: AST U/L Canine and human
HEPA
16
40
GGT U/L
35
30
25
20
Canine
15
Human
Figure 13: GGT U/L Canine and human
10
5
0
-5
ENDO
SPEC
CYTE
HEPA
GLDH U/L
160
140
120
100
Canine
80
Figure 14: GLDH U/L Canine and human
Human
60
40
20
0
ENDO
SPEC
CYTE
HEPA
Cytochrome P450
The presence of an active
cytochrome 3a12 for canine and
cytochrome 3a4 for human is of
great
importance
for
wellfunctioning liver cells. In the graph
we can read the activity of both
species.
We performed three
triplicate measurements, but did
not include a positive control.
Without this positive control it is
hard to give a conclusion based on
this data. For now we are able to say
that there is activity expressed for
both human and canine cells and
human cells seem to show a slightly
higher activity than canine cells.
The results per well are corrected
for the cell count made during the
fluorescamine assay. Each well
represents a triplicate measurement
(figure
15).
17
Fluorescent units/mg protein
200
Million
150
Canine
100
Figure 15: Fluorescent units/mg protein
for canine and human
Human
50
0
well 1
well 2
well 3
lines (figure 16 – 17).
Micro albumin
We also used the media collected
during the experiment for micro
albumin determination. Since micro
albumin is created within the liver
we expected to find the highest
concentration of albumin in the
hepatic maturation step. This step is
the closest to primary hepatocytes.
Surprisingly
the
highest
concentration of albumin was
measured
during
endodermal
differentiation.
The rest of the results are according
to our expectations. Human cells
seem to express more albumin than
canine cells.
The micro albumin results are
corrected for cell count for both cell
Ammonia tolerance test
The results of the ammonia
tolerance test were not usable and
were omitted from this study. We
100
80
60
40
20
0
ENDO
SPEC
CYTE
HEPA
Figure 16: Canine Micro albumin
120
100
80
60
40
20
0
ENDO
SPEC
CYTE
HEPA
Figure 17: Human Micro albumin
recommend future experiments to
be performed with higher amounts
of ammonia in order to increase the
amounts of urea after an overnight
incubation.
18
Discussion
During the experiment we had
noticed that there were some
differences between the cell lines.
The canine cells seem to grow less in
culture compared to the human
cells.
After culturing both cell lines we
performed the above tests. Overall
it seems that the canine cells show
less expression of hepatocyte
specific genes compared to the
human cells, but both seem to be
able
to
differentiate
into
hepatocyte-like cells. This difference
might be due to the confluence of
the cell lines at the beginning of the
differentiation experiment. Because
canine cells grow slower the
confluence may have been lower in
comparison with the human cells. It
is also possible that the human cells
grew faster during differentiation
compared to the canine cells.
For immunocytochemistry it is
necessary
to
change
the
differentiation protocol [11]. During
the experiment it has been found
that the cells do not stay attached
to the chamberslides, not even after
several adjustments (see above). For
now the use of 24 well plates seems
to be the solution. The surface
within one well of a 24 well plate is
bigger and contains more cells than
a well of a chamberslide. Perhaps
the cells detach from the
chamberslides because of a lack of
space or cells surrounding them.
The indocyanine green (ICG) test
would be more optimized if there
were bright field pictures taken
before starting the test and the
pictures taken should be from exact
the same area every time.
Periodic Acid Schiff (PAS) staining
needs an amylase pre-treatment as
a negative control to optimize the
results.
The determination of liver enzymes
in this experiment is performed
using two different methods;
determination of the media and
determination of the cell pellet. The
question remains whether it is
possible to say one of these
methods is the best in every
situation or that a distinction should
be made between different cell
lines.
For a good discussion about the
canine results of the liver enzyme
determination more comparable
data is needed.
The results of the micro albumin
determination
were
partly
surprising;
the
highest
concentration of albumin was
measured
during
endodermal
differentiation. The reason for this
high concentration is due to the
media we used within the
19
endodermal step. This media
contains Bovine Serum Albumin
(BSA). This supplement is only used
during endodermal differentiation
and not present in the other
differentiation
steps.
The
measurement
within
the
endodermal step should be
eliminated to have a good
interpretation of the results.
The rest of the results are according
to our expectations.
Conclusion
During this study we have been able
to create and test an optimized,
cost-effective, protocol for the
hepatic differentiation of canine and
human iPS cells.
This protocol seems to work well
because we have been able to show
the
possibility
to
culture
hepatocyte-like-cells based on geneexpression
profiling,
immunocytochemistry as well as
functional assays.
In general the cultured hepatocytes
from human iPS cells seem to show
better test results in comparison
with the cultured hepatocytes from
canine iPS cells. The results indicate
that the cells are similar, only the
human hepatocytes seem to have a
higher expression of hepatocyte
specific genes.
The cultured iPS cell derived
hepatocytes seem to look similar in
function in comparison with primary
hepatocytes according to all the
tests we performed. Not all test
results are entirely convincing and
some test should be performed
once again to achieve better results
(cytochrome
test,
ammonia
tolerance test).
Generally we are able to say that all
the results together are certainly
positive. The clinical relevance of
culturing and differentiation of iPS
cells is therefore high. In order for
the iPS cells to play a role within the
solution of the shortage of donor
organs (not available for dogs) or
disease modelling it is important
there is a quick and safe way for
culturing. A patient with chronic
liver disease does not have years of
time to wait before there are
enough good functioning cells for
treatment. In a short period of time
a lot of cells need to be cultured.
It is also important that one can say
with certainty that the cultured cells
meet the requirements for primary
hepatocytes. Because of the amount
of cells required and the short
period of time there is a possibility
of culturing tumour cells.
It is not feasible in terms of time and
costs to perform all of the above
tests before a patient can be
treated. At this moment of time, the
clinical use of this technique is
therefore not possible.
20
Nevertheless, the above results are
encouraging enough for further
research about the use of iPS cells
for cell transplantation and disease
modelling in the future. The
veterinary relevance lies within the
disease modelling. For example
think about copper accumulation,
(Wilson’s disease in human),
wherein the excretion of copper
into the bile is reduced.
If this disease modelling is proven to
work in dogs, dogs may contribute
to solving complex diseases like
Wilson’s disease (mentioned above)
because these syndromes are quite
similar.
The gene that causes this disease is
located in the liver. Copper storage
disease has been demonstrated in
several different dog breeds. Until
now, dogs diagnosed with copper
accumulation need medical support
for the rest of their lives.
It can be possible that this life-long
medical support is not needed
anymore in the future. This study
shows the possibility to culture wellfunctioning hepatocyte-like cells
from the fibroblast cells of a patient.
This could mean that we are able to
cure the gene defect of this disease
by replacing it with a good working
cultured gene.
21
References
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medicine, 2003
2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic
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Induction of pluripotent stem cells from adult human fibroblasts by defined factors.
Cell. 2007
4. Whitworth DJ, Ovchinnikov DA, Wolvetang EJ. Generation and Characterization of
LIF-dependent Canine Induced Pluripotent Stem Cells from Adult Dermal Fibroblasts.
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therapies. Stem Cell Res. 2012
9. Wu G, Liu N, Rittelmeyer I, Sharma AD, Sgodda M, Zaehres H, Bleidissel M, Greber B,
Gentile L, Han DW, Rudolph C, Steinemann D, Schambach A, Ott M, Schöler HR, Cantz
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pluripotent stem cells. PLoS Biol. 2011
10. Standard operating procedures for Canine and Human induced pluripotent stem
cells
11. Hepatic differentiation protocol for iPS cells
12. iScript protocol
13. Protocol qPCR 384-well IQSYBRgreensuperMix
14. Cardiogreen / indocyanine green (ICG) uptake protocol
15. PAS (Periodic Acid SCHiff) staining Protocol for cell-culture
16. Two step nuclear immunocytochemistry (ICC) on PFA fixed cells in chamberslides.
STEMLITE kit (stemLite iPS cell Reprograming kit #9092)
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17. Cyp3a4 activity measurement with BFC assay
18. Fluorescamine assay
19. Syllabi course 15. Hepatobiliary system. Faculty of Veterinary Medicine, 2011
20. Urea assay for cell-culture
21. CyQUANT cell Proliferation Assay Kit. Invitrogen, 2006
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Appendix
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