Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Surface Applications in
Feeder-Independent Culture of
Human Embryonic Stem Cells
Deepa Saxena, Ph.D.
Research Scientist
BD Biosciences – Discovery Labware
Derivation of Human Embryonic Stem (hES) Cells
Slide 1
Embryonic stem cells are most fascinating area of cell
biology. Potential application of these cells in regenerative
medicine offers hope to several people suffering with many
disabling disorders. Research is needed to determine
ideal culture conditions and reliable ways to promote
differentiation of these cells into the desired cell types.
Today I will talk about surface application in feederindependent culture of human embryonic stem cells.
Slide 2
Human embryonic stems cells were first isolated in lab in
1998 by group of Thomson and paper was published in
Science. Mouse embryonic stem cells have been cultured
by various groups for so many years starting early 1980.
Embryonic stem cells can be derived post-fertilization at
the blastocyst stage where inner cell mass of the cells can
be cultured in the petri dish and they are pluripotent stem
cells.
http://stemcells.nih.gov/staticresources/info/scireport/PDFs/C.%20Chapter%201.pdf
Embryonic and Adult Stem Cells
Embryonic
(ESC)
Adult
ICM: Inner Cell Mass, PGC: Primordial Germ Cell, EGC: Embryonic Germ Cell
Schematic adapted from Wobus, A. M. and Boheler, K. R. Physiol. Rev. 85:635-678 (2005).
Slide 3
Stem cells can fall into two categories – embryonic and
adult stem cells. Adult and embryonic stems cells differ in
number and types of differentiated cells they can become.
Embryonic stems cells have capability to give rise to three
germ layers – endoderm, ectoderm, and mesoderm and
also primordial germ cells. Those are the founder of male
and female gametes. In adult tissue multipotent stem cells
exist in the tissue and organs to replace injured cells and
adult stem cells typically generate cell types of the tissue
in which they reside. For example, a blood-forming
hematopoietic stem cell can give rise to RBC, WBC
platelet and so forth. Large number of embryonic stem
cells are relatively easy to culture and adult stem cells are
rare and it’s difficult to isolate them so embryonic stem
cells are a good choice for purpose of regenerative
medicine.
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Characteristics of Embryonic Stem Cells
Slide 4
Embryonic stem cells have some characteristic properties
and they can be isolated from blastocyst stage. They have
property of self-renewal and that is what, when a cell
divides, they can make copies of themselves without
differentiation and this process can go indefinitely. These
cells can be cultured for a long, long period of time in the
culture without differentiation. These cells are pluripotent,
that’s when a cell can differentiate into different germ
types. As shown in this figure, the cells are differentiated
into neurons from ectoderm, blood cells from mesoderm
and liver cells from endoderm and that is the property that
makes them really useful for regenerative medicine.
http://stemcells.nih.gov/staticresources/info/scireport/PDFs/C.%20Chapter%201.pdf
The Promise of Stem Cell Research
Slide 5
There is a great promise for research in cell therapy but
there are several other uses of these cells. For example
embryonic stem cells can be used for understanding and
prevention of birth defect. Understanding growth and
development will allow treatment of birth defects. These
cells could be a useful tool in screening of new drugs and
also testing organ toxicity. Other than that, differentiation
potential of these cells into different organs or different cell
types definitely offers hope for treatment of several
diseases. For example Parkinson’s, heart disease,
diabetes, leukemia and so forth.
http://stemcells.nih.gov/staticresources/info/scireport/PDFs/C.%20Chapter%201.pdf
Slide 6
While ES cells may ultimately be used for therapies in
humans, considerable basic research is required to
achieve this goal and certainly this requires growing large
number of cells in the lab.
While ES cells may ultimately be
used for therapies in humans,
considerable basic research is
required to achieve this goal.
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
ES Cells are Typically Cultured on Mouse
Embryonic Fibroblasts Feeder Layer
or irradiate
Slide 7
Conventionally these cells are cultured on embryonic
fibroblast feeder layer and typically mouse embryonic
fibroblasts are used for this purpose. Pups from a
pregnant mother are isolated and they are dissected.
Embryonic fibroblasts are isolated and expanded in the
petri dish these cells are made mitotically inactive either by
Mitomycin treatment or by gamma radiation. So these
cells after treatment with Mitomycin C or gamma radiation
can be frozen and when you are ready to make ES cell
culture a line of fibroblast feeder layer is made on the petri
dish and then on top of that, embryonic stem cells can be
cultured and they can be split on these lines, too.
http://stemcells.nih.gov/index.asp
MEF: Mouse Embryonic Fibroblasts
Limitations of Mouse Embryonic Fibroblast
(MEF) Feeder System
ES Cells are typically cultured on MEF Feeder Layer
• Labor intensive isolation and maintenance
• Variation of different MEF lots
• Undefined media composition
• Low transfection efficiency, also some “quenching” from
the feeder layers
• Potential contamination with animal pathogens
• Difficult to isolate pure DNA, RNA (cross-contamination
from feeder layers)
Key Challenges with Stem Cell Culture
• Lack of complete culturing environment with a
standard protocol
• Spontaneous differentiation
• Scaling up cultures
• Simulating physiological conditions in vitro
• Time required for full characterization of new
culturing condition: in vitro and in vivo
• Difficulty in comparing data from different
laboratories
Slide 8
This offers a good system for culture of embryonic stem
cells but there are certain disadvantages. First, the
process of isolation of fibroblast is tedious and
maintenance is also labor-intensive as well as timeconsuming. There are variations from lot to lot when you
use mouse embryonic feeder layer. Other than that this
layer secretes some undefined components in the media
so composition of media is not really defined. When you
are trying to do some genetic manipulations, we face low
transfection efficiency mainly because colony is tight on
the feeder layer and some quenching occurs due to
presence of feeder cells. And one big problem is that
growing these cells on animal source makes these cells a
source of possible animal pathogens, and those cells are
not definitely suitable for any clinical application. Other
than that, isolation of pure DNA, RNA is not really easy
because of cross-contamination of feeder layers.
Slide 9
We still face certain challenges with the stem cell culture.
The lack of complete culture environment with a standard
protocol is missing. Different labs are using different
surfaces and media. These cells tend to differentiate
spontaneously and that needs to be removed in order to
maintain healthy cultures. Scaling up of this culture is a
problem because what we are doing currently is in a small
petri dish and we have to monitor culture regularly and
take out spontaneously differentiated colonies. Simulating
physiological conditions in vitro are missing and time
required for full characterization of new culture condition is
a lot because first you need to make sure culture stays
undifferentiated for certain number of passages and then
culture retains its ability to differentiate into three germ
layers in vitro and in vivo. Also it’s very important that
culture maintains normal karyotype. And because of all
these reasons, it is difficult to compare data isolated from
different labs.
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Feeder-free hES Cell Culture
• First documented in 2001 (Nature Biotech. Xu, et al.)
• BD Matrigel™ Matrix-coated surface used with Mouse
Embryonic Fibroblast feeder layer conditioned media
(MEF-CM)
Slide 10
So feeder-free culture of human embryonic stem cells was
first documented in 2001 when a Nature Biotech paper
was published and BD Matrigel-coated surfaces with
mouse embryonic fibroblast feeder layer conditioned
media was used to culture these cells. And since then,
multiple media conditions, defined and undefined, have
been successfully used in combination with BD Matrigelcoated surface for culture of human ES cells.
• Multiple media conditions and defined media have
been used successfully in combination with
BD Matrigel Matrix-coated surface for culturing hES
cells
BD Matrigel™ Matrix Substrate for hES Culture
hES cells cultured on BD Matrigel™ Matrix:
• Maintain normal karyotype
Slide 11
Human ES cells cultured on BD Matrigel Matrix maintain
normal karyotype, demonstrate a stable proliferation rate
and high telomerase activity. They express characteristic
undifferentiated markers of human embryonic stem cells
and also form teratomas in SCID mice and differentiate
into cells from all three germ layers.
• Demonstrate a stable proliferation rate and high
telomerase activity
• Express characteristic undifferentiated hES cell
markers
• Form teratomas in severe combined
immunodeficient (SCID) mice and differentiate into
cells from all three germ layers
Advantages of BD BioCoat™ Matrigel™ Matrix
Plates for ES Cell Culture
• Ready-to-Use Convenience
• Optimized BD Matrigel™ Matrix coating process
• Patent pending
• Stable, when stored at -20°C
• Lot-to-lot consistency
• Eliminates tedious maintenance of MEF feeder layers
• Surface compatible with multiple hES cell culturing
media and hES cell lines
• Quality assurance testing to ensure functional
performance reproducibility of experiments
Slide 12
Matrigel is offered by BD Biosciences and it’s ready to use.
It’s really convenient. You don’t have to make embryonic
feeder layers, fibroblast feeder layers, and coating process
is optimized. Product is stable. It can be aliquoted and
stored and BD offers lot-to-lot consistency. This
eliminates need for maintenance of feeder layer and
surface is compatible with multiple culture media and
multiple human ES cell lines and quality assurance testing
is done to ensure functional performance and
reproducibility of the experiments. It’s easy to isolate
DNA, RNA, and protein for further studies and optimized
protocol enables standardization of the cultures.
• Easier DNA, RNA, and protein isolation
• Optimized protocol enables standardization of cultures
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Serum-free Defined Media: mTeSR™1
Maintenance Medium
• A defined media for feeder-independent and serum-free
culture of hES cells
Slide 13
Recently scientists at WiCell Research Institute have
demonstrated culture of human ES cells in defined animalfree media. It supports long-term undifferentiated
expansion of human ES cell and mTeSR is the modified
version of this media.
• Developed at the WiCell Research Institute in 2006
References:
1. Derivation of human embryonic stem cells in defined conditions, Ludwig T.E., et al., Nat. Biotechnol.
24(2):185-7, 2006
2. Feeder-independent culture of human embryonic stem cells, Ludwig T.E., et al., Nat. Methods.
3(8):637-46, 2006
Complete Culture Environment
Feeder-free Surface + Defined Media
Strategic collaboration by BD Biosciences, StemCell Technologies, and WiCell™
Research Institute to develop optimized, feeder-independent cell culture
environments for hES cell research
Slide 14
And here we offer a complete environment where human
ES cell-qualified Matrigel by BD Biosciences can be used
with multiple media along with mTeSR that is marketed by
StemCell Technologies.
+
BD Matrigel™ hESC-qualified Matrix
from BD Biosciences
mTeSR™1 Maintenance Medium
from StemCell Technologies
BD Matrigel™ hESC-qualified Matrix
Slide 15
So BD Matrigel Matrix, the human ES cell-qualified matrix
is optimized surface for human ES cell culture and it is
compatible with mTeSR along with other defined and
conditioned media.
• Optimized surface for hES cell culture
• Qualified as mTeSR™1-compatible
Available at bdbiosciences.com/stemcellsource
Xu, C., et al., Feeder-free growth of undifferentiated human embryonic stem cells,
Nature Biotechnology, 19:971-4 (2001).
Xu, C., et al., Immortalized fibroblast-like cells derived from human embryonic stem cells
support undifferentiated cell growth, Stem Cells 22:972-80 (2004).
Ludwig, T.E., et al., Derivation of human embryonic stem cells in defined conditions,
Nature Biotechnology, 24:185-7 (2006).
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
BD Matrigel™ hESC-qualified Matrix
Summary of hES Culture on BD Matrigel™ Matrix Surface
Slide 16
Enough literature is out there that demonstrates
successful culture of human ES cells on Matrigel-coated
surface and here is a table summarized from Mallon et al.
where they demonstrated several human ES cell lines
were successfully cultured on Matrigel in presence of
different growth factors or other components and they
were cultured for long-term. For example, six months,
seven months or multiple passages – and they all
maintained characteristic stem cell markers. They
retained their pluripotency, differentiated into embryoid
bodies, also formed teratomas demonstrating their
potential for differentiation.
HES-df-CM: human ES cell-derived fibroblast conditioned media, EB: Embryoid body, TER: Teratoma formation, DF: in vitro differentiation
Modified from Mallon, et al., (2006) International Journal of Biochemistry and Cell Biology, 38:1063-1075
Induced Human Pluripotent Stem (iPS)
Cells Successfully Cultured on Matrigel
• Induced pluripotent stem cell Lines derived from
human somatic cell. (Junying, Y. et al Science 2007)
OCT-4, SOX2, NANOG, LIN28 reprogrammed human somatic
cells to pluripotent stem cells exhibiting characteristics of
embryonic stem cells
Slide 17
Recently two papers were published where pluripotency
was induced in somatic cells and a paper in Science
where somatic cells were induced to express OCT4,
SOX2, NANOG and LIN28. This reprogramming made
induced pluripotent stem cells and these cells were also
successfully cultured on Matrigel. Similarly, another paper
from Cell where pluripotency was induced in adult human
fibroblast also grew well on Matrigel.
• Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. (Takahashi, K. et al Cell 2007)
Oct-3/4, Sox2, Klf4, c-Myc in adult human fibroblast could
generate pluripotent stem cells similar to embryonic stem cells
Undifferentiated hES Cell Colony
MEF feeder layer
hES media
BD Matrigel™ hESC-qualified Matrix
MEF-conditioned media
Slide 18
In our lab, we tested conventional ES cell culture and used
feeder-free culture to compare defined media and
conditioned media. As you see here, the colony is
compact and dense when grown on feeder layer. Well
when it is grown on Matrigel either in conditioned media or
in presence of defined media, the mTeSR, they are nicely
spread out.
BD Matrigel™ hESC-qualified Matrix
mTeSR™1
• Compact and dense H9 colonies on MEF feeders
• Spread-out and monolayer-like colonies on BD Matrigel™ Plates
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Spontaneous Differentiation
Slide 19
Spontaneous differentiation is something that we typically
see in the culture and that’s how a colony looks like when
it is differentiated. The first panel is a published paper
from Nature Biotechnology where it demonstrated
spontaneous differentiation and next two pictures we got in
the lab and certainly to maintain healthy cultures, these
differentiated portion of the colonies need to be removed.
1
Differentiated colonies
Nature Biotechnology18, 399 - 404 (2000)
Markers of Undifferentiated hES Cells
on MEF-CM & mTeSR™1
H9 on BD Matrigel™ hESC-qualified Matrix
MEF-CM
Slide 20
After repeated culture on Matrigel in conditioned media
and defined media, we tested expression of OCT4, a
marker of undifferentiated ES cells. And as you see in this
picture, whether cells were grown in conditioned media or
in presence of TeSR on BD Matrigel, the human ES cellqualified matrix, they maintained expression of this marker
and OCT4 was expressed in all the cells. The only
difference could be seen here is MEF conditioned media
has something that makes cells scatter towards the
periphery and with defined media, borders are welldefined.
mTeSR™1
OCT-4 + Hoechst33342
Markers of Undifferentiated hES Cells
OCT-4
Slide 21
Cells grown on BD Matrigel also express marker of
undifferentiation that is SSEA4 and here you see a picture
for OCT4 again and from both the pictures you can
conclude the cells grown on Matrigel expressed markers of
undifferentiated human ES cells.
SSEA-4
H9 in mTeSR™1 on
BD Matrigel™ hESC-qualified Matrix
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Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Spontaneous Differentiation
OCT-4
Slide 22
Spontaneous differentiation, is something we routinely see
and OCT4 is a good marker to judge that. Here in this
picture the batch of differentiated cells was seen where
OCT4 expression is completely diminished and on an
overlay you see DAPI but OCT4 is still missing so
expression of OCT4 is diminished upon differentiation.
DAPI
Merge
Expression of OCT4 is diminished upon differentiation
Undifferentiated Marker Expression
qRT-PCR Analysis
Raw R Q (Relative Quantification)
SOX2 gene expression in hESC
2.500
2.000
1.500
1.000
0.500
0.000
Control
Lot1, p2
Lot2, p5
Lot3, p5
Raw RQ (Relative Quantification)
OCT4 gene expression in hESC
Slide 23
We also compared cells grown on conventional embryonic
fibroblast layer and Matrigel and we tested three different
lots of Matrigel for this purpose. Cells were grown for
multiple passages and RNA was isolated and we analyzed
expression of SOX2 and OCT4 gene. Those are again
markers for undifferentiated cells by quantitative real-time
PCR and in both the graphs, expression of SOX2, as well
as expression of OCT4 is comparable to control, that is
human ES cells grown on feeder layer.
3.000
2.500
2.000
1.500
1.000
0.500
0.000
Control
Lot1, p2
hESC media on
MEF feeder layer
Lot2, p5
Lot3, p5
mTeSR™1 on
BD Matrigel™ hESC-qualified Matrix
Undifferentiated Marker Expression
FACS Analysis
Isotype
Oct-3/4
98% OCT4 positive cells
Isotype
SSEA4
Slide 24
We also quantified expression of undifferentiated markers
OCT4 and SSEA4 by flow cytometry analysis and 98%
cells stayed positive for OCT4 expression and 94% of the
cells showed SSEA4 expression by flow cytometry
analysis and this experiment was conducted at passage
five when cells were grown in defined media on BD human
ES cell-qualified Matrigel Matrix.
94% SSEA4 positive cells
H9 cells grown on BD Matrigel™ hESC-qualified Matrix in mTeSR™1 for 5 passages
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Undifferentiated Marker Expression
FACS Analysis
Isotype
SSEA4
SSEA4 (PE)
BD Pharmingen 560128
IgG3 isotype (PE)
BD Pharmingen 559926
Isotype
Tra-1-60
Tra-1-81
Tra-1-60 (PE)
BD Pharmingen 560193
Tra-1-81 (PE)
BD Pharmingen 560161
IgM isotype (PE)
BD Pharmingen 555584
Isotype
OCT-3/4
Slide 25
We grew cells for many more passages and this analysis
was performed again and as you see here, a big shift for
SSEA4 and two more markers of undifferentiated ES cells,
Tra-1-60, Tra-1-81 and OCT4. In the red is shown isotype
control. So cells grown for multiple passages maintained
undifferentiated marker expression.
OCT-3/4
BD Pharmingen 611202
IgG1 isotype
BD Pharmingen 557273
H9 cells grown on BD Matrigel™ hESC-qualified Matrix in mTeSR™1
Embryoid Body (EB) Formation
Slide 26
Then we wanted to look at differentiation potential of these
cells. Cells were grown for multiple passages on Matrigel
and differentiation potential was demonstrated by
embryoid body formation.
Phase contrast image of EBs formed by H9 cells grown on
BD BioCoat™ Matrigel™ Plates for ES Cell Culture
Beating Cardiomyocytes from EBs
Slide 27
So embryoid bodies were further transferred to a gelatincoated dish in differentiation media and they were allowed
to further grow and differentiate and they differentiated into
beating cardiomyocytes.
H9 Cells grown on BD Matrigel™ hESC-qualified Matrix in mTeSR™1 for
>10 passages formed EBs and differentiated to beating cardiomyocytes
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Neurons from EBs
Slide 28
These embryoid bodies could also be differentiated into
neurons and there are the images captured from the petri
dish.
H9 Cells grown on BD Matrigel™ hESC-qualified Matrix in mTeSR™1 for
>10 passages formed EBs and differentiated to Neurons
Expression of 3-Germ Layer Markers in
Embryoid Bodies by QRT-PCR
Relative gene Expression
10000
1000
HAND1
TUBB3
100
10
1
0.1
H9 Cells
EB Day9
AFP (alpha fetoprotein) - Endoderm (CT 21.28 vs. Not Detected in H9 cells)
HAND1 (heart and neural crest derivatives expressed 1) - Mesoderm
TUBB3 (tubulin beta 3) - Ectoderm
Potential Alternative Surfaces to
BD Matrigel™ Matrix
• Single defined ECM protein
• Defined ECM combinations
• Chemically modified surfaces
Slide 29
We also demonstrated expression of three-germ layer
markers in embryoid bodies by quantitative RT-PCR and
as seen here, HAND1 heart and neural crest derivative
expressed and TUBB3 tubulin 3 markers of mesoderm
and ectoderm were highly expressed as compared to H9
cells and endoderm marker alpha fetoprotein was not
detected in H9 cells but it was detected in embryoid bodies
at Day 9 and the CT value was 21.28. So here we
demonstrate differentiation potential of these cells after
multiple passage on BD Matrigel Matrix and they
differentiate into all three germ layers.
Slide 30
Matrigel is relatively undefined because it’s a complex
mixture of certain extracellular matrix protein but what
could be potential alternative surfaces to BD Matrigel
Matrix. It could be single defined ECM protein or maybe a
combination of ECM proteins or a chemically modified
surface but definitely critical considerations for a surface
would be functionality, cost, as well as complexity.
• Critical considerations
• Functionality
• Cost
• Complexity
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Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Comparison of BD Matrigel™ Matrix
vs. other ECM Proteins
• BD Matrigel™ Matrix is equivalent or better than most
single extracellular matrices (ECMs) tested:
• Laminin equivalent to BD Matrigel Matrix (Xu, et al. 2001)
• Laminin, collagen, fibronectin, and vitronectin were combined for
optimal ECM complex for culturing hES cells (Ludwig, et al. 2006)
• Fibronectin and collagen combination is required to match the
performance of BD Matrigel Matrix (Lu, et al. 2006)
• Pure ECM often much more expensive to use
• ECM combination requires more steps and time to coat
New Surface for ES Cell Culture
BD™ Laminin/Entactin complex
• Major component of basement membrane in
Engelbreth-Holm-Swarm (EHS) mouse tumors
Slide 31
BD Matrigel Matrix is equivalent or better than most of the
single extracellular matrices tested so far. Laminin was
found to be equivalent to Matrigel. Laminin, collagen,
fibronectin and vitronectin combination was required to
match performance of Matrigel by another group and
fibronectin and collagen combination is required to match
performance of BD Matrigel Matrix. That was reported by
another group. But pure ECMs are often much more
expensive to use and a combination of ECM’s definitely
requires more steps to code and it is time-consuming.
Slide 32
In an attempt to test alternative surfaces, we tested BD
Laminin/Entactin complex and that is a major component
of basement membrane in EHS mouse tumor. It provides
a 3D environment that more closely models the cellular
microenvironment in vivo and this is a step further from
Matrigel and that’s a further purified complex.
• Provides a 3D environment that more closely
models the cellular microenvironment in vivo
Comparison of MEF Conditioned
Media and mTeSR™1
A
A & B:
BD Matrigel™
hESC-qualified
Matrix
B
Slide 33
Cells maintained undifferentiated morphology as they did
on human ES cell-qualified BD Matrigel Matrix and again
we tested this Laminin/Entactin complex for conditioned
media as well as defined media. And as you see here,
undifferentiated colonies were maintained in presence of
either conditioned or defined media, both on Matrigel and
Laminin/Entactin complex.
C & D:
Laminin/Entactin
Complex
C
MEF-Conditioned media
D
mTeSR™1
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Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Comparison of Different Surfaces and
Media by IHC
MEF-CM
Slide 34
We also tested expression of OCT4 in these cells and
again, when cells were grown on Matrigel or BD
Laminin/Entactin complex in presence of either
conditioned media or TeSR media, they expressed plenty
of OCT4 and as seen earlier in presence of conditioned
media, cells are scattered at the periphery.
mTeSRTM1
BD Matrigel™ hESC-qualified Matrix
BD™ Laminin/Entactin complex
OCT-3/4 expression in H9 cells
Comparison of Different Surfaces
and Media by IHC
Slide 35
Cells grown on Laminin/Entactin complex expressed
SSEA4 as well and expression was similar when
compared to Matrigel.
MEF-CM
mTeSRTM1
BD Matrigel™ hESC-qualified Matrix
BD™ Laminin/Entactin complex
SSEA4 expression in H9 cells
H9 Cells Expressing Markers Specific
for Undifferentiated Cells
OCT-4
Slide 36
Here is a comparison again showing cells cultured on
Laminin/Entactin complex or BD Matrigel Matrix and
expression of SSEA4 and OCT4 and cells were grown in
defined media here and as you see, both the surfaces,
Laminin/Entactin as well as Matrigel, performed really well
for culture of human embryonic stem cells.
SSEA-4
BD™ Laminin/Entactin complex
BD Matrigel™ hESC-qualified Matrix
H9 colony grown in mTeSR™1 medium
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Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Comparison of BD Matrigel™ Matrix and
Laminin/Entactin by BD FACS™ Analysis
SSEA-4
83% on BD Matrigel™ hESC-qualified Matrix
91% on BD™ Laminin/Entactin Complex
Isotype control
SSEA-4
Slide 37
We also quantified expression of those two markers by
flow cytometry and 83% of the cells stayed SSEA4positive on human ES cell-qualified matrix, BD Matrigel,
and 91% of the cells expressed this marker when grown
on Laminin/Entactin complex and in black is the isotype
control. Similarly for OCT4, 95% of the cells stayed
positive for OCT4 expression when grown on
Laminin/Entactin complex so again, these two BD surfaces
are good feeder-free culture alternative for culture of
human embryonic stem cells.
OCT-4
98% on BD Matrigel™ hESC-qualified Matrix
95% on BD™ Laminin/Entactin Complex
Isotype control
OCT-4
Embryoid Body (EB) Formation
from hES Cells
BD™ Laminin/Entactin complex
Slide 38
We also tested differentiation potential of H9 cells when
they were cultured on Laminin/Entactin complex for
multiple passages and they were differentiated into
embryoid bodies.
BD Matrigel™ hESC-qualified Matrix
Phase contrast image of Embryoid bodies formed by H9 cells grown on different surfaces.
Expression of 3-Germ Layer Markers in
Embryoid Bodies by QRT-PCR
Relative Gene Expression
Differentiation Markers in Embryoid Bodies
1000000
100000
10000
1000
100
HAND1
TUBB3
Slide 39
We also monitored expression of three-germ layer markers
in embryoid body and on the Y axis it is represented as a
log scale and when you compare expression of three-germ
layer markers between H9 cells and embryoid body, you
see all these genes are highly upregulated upon
differentiation.
FOX2A
10
1
0.1
H9 Cells
Embryoid Bodies
H9 cells maintained on BD Laminin/Entactin complex in mTeSR1
media expressed 3-germ layer marker upon differentiation
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Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Beating Cardiomyocytes
H9 –Derived Cardiomyocytes and
Neuronal Cells
GATA-4 + DAPI
(BD Pharmingen coming soon)
Slide 40
So after demonstrating expression of all three-germ layers
in the embryoid bodies, embryoid bodies were transferred
to gelatin-coated dishes and allowed to differentiate further
and again we saw beating cardiomyocytes as seen in this
movie.
Slide 41
And later the cells were fixed and stained for marker
GATA-4. Cardiomyocytes expressed this marker and
differentiation into neural cells was demonstrated by
Nestin and as you see here in the green, the Nestinpositive cells.
Nestin + DAPI
Nestin (BD Pharmingen 611658)
Differentiated to Cardiomyocytes and Neuronal cells after culture on
BD™ Laminin/Entactin complex in mTeSR1 media for 32 passages
Human ES Cells Cultured on Laminin/Entactin
Complex Demonstrated Karyotype Stability
Slide 42
After multiple passages, another concern was if
Laminin/Entactin also maintains karyotypic stability of the
cells and after 26 passages in defined media we send
these cultures for karyotype analysis and they turned out
to be normal so Laminin/Entactin also maintained
karyotype stability.
G banding chromosome analysis of H9 grown on
Laminin/Entactin in mTeSR1 for P26
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
Future Directions
• Next Step:
• Cost-effective human ECM surface to be
used with the animal-free TeSR1 media
• Ultimate goal:
Slide 43
Next step could be cost-effective human ECM surface that
is to be used with animal-free version of TeSR media but it
will be ideal to have chemically defined surfaces. While
research continues to search for animal-free surface and
animal-free media, there are many basic questions that
can be answered using BD Laminin/Entactin and BD
Matrigel surfaces with defined and conditioned media.
• Chemically-defined surface
References
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Slide 43
Here are some of the references that are used for this talk.
J. A. Thomson et al., Science, 282:1145 (1998).
A. G. Smith et al., Nature. 336:688 (1988).
R. L., Williams et al., Nature, 336:684 (1988).
C. Xu et al., Nat. Biotech.,9:971 (2001).
C. Xu, J et al., Stem Cell, 22:972 (2004).
P. Stojkovic et al., Stem Cells, 23:306 (2005).
E. Sjögren-Jansson et al., Dev.Dynamics, 233:1304 (2005).
M. Amit et al., Dev. Biology, 227:271 (2000).
C. Xu et al., Stem Cells, 23:315 (2005a).
L. Wang, L. Li, P. Menendez, C. Cerdan, M. Bhatia, Blood, 105:4598 (2005a).
M. E. Levenstein et al., Stem Cells, 24:568 (2006).
R. H. Xu et al., Nat. Biotech., 20:1261 (2002).
R. H. Xu, R. M. Peck, D. S. Li, T. Ludwig, J. A. Thomson, Nat. Methods, 2:185 (2005b).
G. Wang et al., Biochem. Biophys. Communications, 330:934 (2005b).
N. Sato et al., Nat. Med., 10:55 (2004).
T. E. Ludwig et al., Nat. Biotech,.Brief Communications, January (2006).
J. Lu, R. Hou, C. J. Booth, S.-H.Yang, M. Snyder, PNAS, 103:5688 (2006).
S. Yao et al., PNAS, 103:6907 (2006).
B. S. Mallon, Intl. J. Biochem. & Cell Biol., 38:1063 (2006).
U. Lakshmipathy et al., Stem Cells, 22:531 (2004).
L. Gerrard, D. Zhao, A. J. Clark, W. Cui, Stem Cells, 23:124 (2005).
C. P. Ren et al., Acta Biochim Biophys Sin (Shanghai), 37:68 (2005).
H. Zaehres et al., Stem Cells, 23:299 (2005).
A. R. Greenlee et al., Toxicol. In Vitro, 19:389 (2005).
Contact Information
Slide 44
If you have any question, you can contact our technical
support department at [email protected]. Thank you for
your attention.
BD Biosciences has a variety of products for stem cell research.
For a complete listing of products, please visit our website at:
bdbiosciences.com/stemcellsource
Technical Support
Tel: 877.232.8995 (US)
978.901.7300 (outside US)
Email: [email protected]
bdbiosciences.com/webinars
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Surface Applications in Feeder-Independent Culture of Human Embryonic Stem Cells – Deepa Saxena
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD