Functional immobilization of signaling proteins enables control of stem cell fate.

Abstract

The mode of ligand presentation has a fundamental role in organizing cell fate throughout development. We report a rapid and simple approach for immobilizing signaling ligands to maleic anhydride copolymer thin-film coatings, enabling stable signaling ligand presentation at interfaces at defined concentrations. We demonstrate the utility of this platform technology using leukemia inhibitory factor (LIF) and stem cell factor (SCF). Immobilized LIF supported mouse embryonic stem cell (mESC) pluripotency for at least 2 weeks in the absence of added diffusible LIF. Immobilized LIF activated signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling in a dose-dependent manner. The introduced method allows for the robust investigation of cell fate responses from interface-immobilized ligands.

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Functional immobilization of signaling proteins
enables control of stem cell fate
Kristin Alberti1,7, Ryan E Davey2,7, Kento Onishi2, Sophia George3, Katrin Salchert1, F Philipp Seib1,4,
Martin Bornha ¨user4,5, Tilo Pompe1, Andras Nagy3, Carsten Werner1,2,5& Peter W Zandstra2,6
The mode of ligand presentation has a fundamental role in
organizing cell fate throughout development. We report a
rapid and simple approach for immobilizing signaling ligands
to maleic anhydride copolymer thin-film coatings, enabling
stable signaling ligand presentation at interfaces at defined
concentrations. We demonstrate the utility of this platform
technology using leukemia inhibitory factor (LIF) and stem
cell factor (SCF). Immobilized LIF supported mouse embryonic
stem cell (mESC) pluripotency for at least 2 weeks in the
absence of added diffusible LIF. Immobilized LIF activated
signal transducer and activator of transcription 3 (STAT3)
and mitogen-activated protein kinase (MAPK) signaling in
a dose-dependent manner. The introduced method allows for
the robust investigation of cell fate responses from interface-
immobilized ligands.
The importance of exogenously added or endogenously produced
soluble signal-transducing ligands in the regulation of cell fate is
intensively studied. Although it is widely recognized that a sub-
stantial number of these ligands act at interfaces in vivo (that is,
constrained to surfaces rather than in solution), studies on the
effects of cell exposure to the non–freely diffusible forms of these
molecules are just emerging1–5. The design principles associated
with bound ligand presentation are fundamental to many tissue-
engineeringefforts6, andstrategiestocontrolthe bindingofligands
to surfaces and to present immobilized cytokines and growth
factors have been an area of active development7. Although these
systems have provided evidence that the spatially constrained
presentation of proteins can modulate cell responses, only recently
has a quantitative analysis of the effects of ligand presentation on
signalingactivationandcellfatestartedtobeexplored8,9.Generally,
limitations in the technologies used to date include difficulties
in controlling the amount, stability and cellular accessibility of
surface-immobilized proteins.
To systematically examine the signaling properties of immo-
bilized cytokines, we developed and tested a method for quantita-
tively immobilizing functional proteins, with or without added
extracellular matrix (ECM), to reactive polymer surfaces. Our
method permits straightforward long-term culture of cells, includ-
ing stem cells, on culture plates with defined signaling-protein
concentrations. Our approach relies on generation of thin films
( B5 nm) of alternating maleic acid anhydride co-polymers to
which bioactive molecules are immobilized according to different
binding schemes10–12. Detailed studies on physicochemical proper-
ties of the films underpin the use of these substrates to control the
function of the attached biomolecules10,13,14. We demonstrated the
immobilization strategy for the cytokines LIF and SCF.
As LIF signaling in mESCs is well-characterized, it is an ideal
platform to test the robustness and versatility of our technology.
Furthermore, the mESC system is demanding in terms of culture
requirements and thus puts the described platform to the test.
LIF is an interesting molecule to investigate for the cellulareffects of
immobilized ligand presentation. LIF activates the STAT3 path-
way, which supports the self-renewal of mESCs in vitro17,18, as well
as MAPK, the activity of which has been shown to induce differ-
entiation in mESCs19–21. There is some evidence that the ratio of
thesepathwaysmaybeimportantinmESCdifferentiationcontrol22.
In vivo, LIF is produced in two forms through expression of
alternative transcripts: a diffusible form and a form that associates
with the ECM23,24. The expression patterns of diffusible and ECM-
associatedLIFinthedevelopingembryoarenotidentical,suggestive
of distinct biological roles24. In support of this, forced expression of
diffusible and ECM-associated LIF result in different embryological
consequences25,26. However, it is unclear whether these alternative
modes of LIF presentation have distinct signaling properties, or if
expression of the different isoforms serves simply to localize and
concentrate LIF to a particular region of the embryo.
Although it has been previously reported that LIF can be retained
in a gelatin matrix by photochemical cross-linking, and that this
approachcanbeusedtoelicitLIF-mediatedsignalingactivationand
short-term maintenance of some mESC properties15, this system
appears limited in its ability to quantitatively control immobilized
RECEIVED 27 MARCH; ACCEPTED 16 MAY; PUBLISHED ONLINE 15 JUNE 2008; DOI:10.1038/NMETH.1222
1Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Hohe Str. 6, D-01069 Dresden, Germany.2Institute of Biomaterials and
Biomedical Engineering, 164 College Street, University of Toronto, Toronto, M5S 3G9, Canada.3Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600
University Avenue, Toronto, M5G 1X5, Canada.4University Hospital Carl Gustav Carus, Dresden University of Technology, Fetscherstr. 74, D-01307, Dresden, Germany.
5Center for Regenerative Therapies Dresden, Tatzberg 47/49, D-01307 Dresden, Germany.6Chemical Engineering and Applied Chemistry, University of Toronto, 200
College Street, Toronto, M5S 3E5, Canada.7These authors contributed equally to this work. Correspondence should be addressed to P.W.Z. (peter.zandstra@utoronto.ca)
or C.W. (werner@ipfdd.de).
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ligand presentation. Also, the use of ultraviolet irradiation has been
reported to affect the structure and function of proteins16.
We found that immobilized LIF surfaces were capable of long-
term (7 d) activation of signaling molecules downstream of LIF,
and that the immobilized protein supported mESC pluripotency
for at least 6–8 passages (42 weeks of culture). Immobilized
LIF activated STAT3 and MAPK signaling in mESCs in a dose-
dependent manner. The mode of ligand presentation did not
dramatically affect the differential activation of STAT3 and
MAPK, suggesting that in vivo presentation of immobilized versus
diffusible LIF may act primarily to localize and concentrate the
cytokine. Finally, the immobilization method allows the ligand to
be available in combination with ECM coatings, which may
facilitate the design of experiments to investigate factor-ECM
interactions in a combinatorial manner.
RESULTS
Quantification and stability testing of immobilized LIF
We examined three approaches for LIF immobilization (Fig.1a–c):
covalent attachment to poly(octadecene-alt-maleic anhydride)
(POMA), covalent attachment to flexible PEG7 spacer arms
tethered to POMA (POMA-PEG7) and noncovalent binding
to ECM precoatings deposited on top of hydrolyzed POMA
(POMA-matrix). To determine whether LIF could be immobilized
at different surface densities in these three configurations we used
different solution concentrations of cold LIF that was spiked with
2–6% of125I-labeled cytokine. Both covalent attachment and
matrix physisorption increased with increasing solution concentra-
tion of LIF (Fig. 1d). For covalently immobilized LIF on
POMA-PEG7, saturation was reached at solution concentra-
tions above 5 mg/ml (achieving a maximum surface density of
B92 ± 4 ng/cm2). In contrast, covalent immobilization directly to
POMAyieldedagreateramountofimmobilizedLIFandsaturation
was not achieved at the solution concentrations tested. PEG7
decoration generates a more hydrophilic, dynamic interface with
a lower density of binding sites as compared to unmodified
POMA11and would therefore be expected to have diminished
cytokine binding. For POMA-matrix, LIF physisorbed to matrix
protein precoatings in quantities comparable to those for covalent
attachment directly to POMA(we did not observe saturationatthe
concentrations tested). Quantification of immobilized LIF demon-
strated that on all three substrates similar amounts of cytokine
could be immobilized, at nominal grafting densities of 2, 8 or
75 ng/cm2. However, for each substrate it was necessary to use
different overlying solution concentrations to achieve those
nominal grafting densities (Supplementary Table 1 online).
To study the stability of immobilized LIF under cell culture
conditions, we incubated the surfaces with tissue culture medium
for 1–6 d, changing medium every 2 d. At day 3, the amount of
surface immobilized LIF was reduced for all samples (Fig. 1d). The
total amount of LIF released was greater from surfaces generated
with higher solution concentrations, though the relative amount
lost from each substrate was similar at lower nominal grafting
densities. The average protein retention over 3 d was 90 ± 8%
(POMA), 79 ± 4% (POMA-PEG7) and 75 ± 15% (POMA-matrix).
Adherent culture of mESCs requires the presence of ECM
components for cell attachment; this is relevant for the POMA
and POMA-PEG7 configurations for which matrix deposition on
0.25 2.5
0
20
40
60
80
100
**
Percentage antibody-accessible
immobilized LIF
Solution concentration
LIF (µg/ml)
**
POMA
a
d
ef
POMA
b
POMA-PEG7
c
POMA-matrix
CH2
O
OO
CH2
CH3
CH3
O
O
NH
OH
n
15
15
LIF
15
N
H
CH2
CH2
O
CH2
O
OO
CH2
CH3
N
OO
CH3
O
O
NH
R
O
n
15
15
15
7
7
LIF
LIF
CH2
O
OO
CH2
CH3
CH3
O
O
OH
OH
n
15
15
15
15
0.25 1 2.5510
0
20
40
60
80
100
120
140
160
180
200
Immobilized LIF (ng/cm2)
Solution concentration LIF (µg/ml)
Day 0
POMA
POMA-PEG7
POMA-matrix
*
**
**
**
2
Immobilized
LIF (ng/cm2)
8
0
1
2
3
4
5
6
Antibody-accessible
immobilized LIF (ng/cm2)
*
**
PEG7
Matrix
Cytokine
Glass
Figure 1 | Long-term stability and ligand accessibility for different LIF immobilization strategies.
(a–c) LIF was immobilized by covalent attachment to POMA (a), covalent attachment to flexible PEG7
spacer arms tethered to POMA (b) and noncovalent binding to ECM coating deposited on top of
hydrolyzed POMA (c). Red circles indicate covalent bond. Note that POMA was covalently bound to
amino-functionalized glass substrates and is the key component of the immobilization platform. POMA and POMA-PEG7 surfaces were coated with matrix for
cell-culture experiments (omitted from the figure for clarity). Only one covalent bond between the protein and the surface is shown, although more bonds are
possible. Chemical structures depict the POMA layer and the immobilization mode of LIF. Scheme is not drawn to scale. (d) Quantification and stability of
immobilized [125I]LIF after immobilization (day 0) on the three surfaces and after a 3-d incubation in cell culture medium (n ¼ 2, *P o 0.05, **P o 0.01).
(e,f) Assay of antibody-accessible LIF on matrix-overlaid slides prepared using the indicated amounts of LIF in solution (e; n ¼ 6) or of immobilized LIF
(f; n ¼ 6, *P o 0.05, **P o 0.01, POMA versus POMA-PEG7 or POMA-matrix at respective concentration). Samples were incubated for 3 d in cell culture
medium. Total amount of LIF was determined by radioassay of [125I]LIF and antibody-accessible LIF by an immunological assay. All error bars indicate s.d.
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topofimmobilizedLIFmay maskcytokinepresentationtomESCs.
To estimate the surface availability of immobilized LIF in the
presence of ECM components, we used an antibody to LIF
(Fig. 1e). We immobilized LIF at 0.25 mg/ml and 2.5 mg/ml
(solution concentrations), incubated the surfaces for 3 d and
washed them to remove any unbound LIF, and then measured
the percentage of antibody-detectible immobilized LIF. Although
radioisotope- and antibody-based measurements correlated well
for POMA-PEG7 and POMA-matrix configurations at the lower
concentration of immobilized LIF (76 ± 24and 71± 30% detected,
respectively), at the higher concentration, antibody to LIF detected
a considerably smaller fraction of immobilized LIF in comparison
to the radioisotope-based estimate (9.0 ± 1.5%, 20.3 ± 3.5% and
19.3 ± 3.5% for POMA, POMA-PEG7 and POMA-matrix, respec-
tively). We also determined the amount of antibody-accessible
immobilized LIF on the three surfaces generated with two different
nominalgraftingdensitiesofimmobilizedLIF(2and8ngLIF/cm2)
after conditioning in cell culture medium for 3 d (Fig. 1f). Using
the antibody, we detected a considerable fraction (up to 55%) of
immobilized LIF, with better accessibility of LIF on POMA-PEG7
and POMA-matrix than on POMA.
Immobilized LIF activates mESC signaling
To determine whether our approaches present LIF in a biologically
active form, we examined the signaling responses of mESCs to
immobilized LIF by quantitatively examining STAT3 and MAPK
phosphorylation using immunocytochemistry and single-cell
image analysis26(Fig. 2a). Both in the presence and the absence
of PEG7 spacer, immobilized LIF induced a dose-dependent
activation of STAT3 signaling (Fig. 2b). Although the amount of
antibody-accessibleimmobilizedLIFwasgreaterwhenthecytokine
was immobilized through PEG7 spacer (Fig. 1f), the presence of
the spacer did not appear to alter signaling properties and was also
not necessary to enable signaling through an ECM-overlayed
matrix. This result, in combination with the observation that
both the amount (Fig. 1d) and efficiency (Supplementary
Table 1) of immobilization was greater on POMA without PEG7,
motivated our in-depth analysis of STAT3 and MAPK signaling on
this surface. LIF immobilized on this surface resulted in dose-
dependent STAT3 activation (Fig. 2c). For LIF immobilized on the
same surface we observed a similar dose-dependent increase in the
signaling activation of phosphorylated MAPK (Fig. 2d). To com-
pare the mode of LIF presentation on the activation ratio of these
two signaling pathways, we normalized the values for phosphory-
latedSTAT3andMAPKtothemaximumresponsesandplottedthe
ratio (Fig. 2e). Our results suggest that the mode of LIF presenta-
tion (diffusible versus immobilized) does not differentially affect
the STAT3 and MAPK activation ratio.
mESC maintenance on immobilized LIF
We next examined the ability of LIF to support self-renewal of
mESCs, using different surface densities of LIF covalently immo-
bilized (POMA) or absorbed to POMA (POMA-matrix) and
overlaid with ECM to allow cell attachment. We used histograms
of mESC expression of the pluripotency marker Oct-4 (ref. 27) to
calculate the percentage of Oct-4+and Oct-4–cells under each
condition (Fig. 3a). We determined the percentage Oct-4–cells
generated after 3 d of culture in cells exposed to diffusible LIF, no
LIF or different concentrations of immobilized LIF. As expected
from the STAT3 activation studies, immobilized LIF inhibited the
generationofOct-4–cellsover72hinadose-dependentfashion. In
contrast, matrix-physisorbed LIF was incapable of maintaining
Oct-4 expression, and differentiation ensued similar to that in
no-LIF controls (Fig. 3b). We also tested the effectiveness of our
surfacestosupportmESCpluripotency.First,weseeded cellsatlow
cell densities and examined Oct-4 expression for 7 d (Fig. 3c). As
predicted from our signaling analysis, only the higher concentra-
tionsofimmobilizedLIFsupportedOct-4expressionoverthistime
period, with even the intermediate immobilized LIF concentration
yielding low numbers of Oct-4+cells by day 7. Although Oct-4
activation is associated with undifferentiated mESCs, we wanted to
test the ability of immobilized LIF to support mESC pluripotency
dbac
e
10028 75
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
**
25.72.70.702LIF (ng/well)
Diffusible LIF
(ng/ml)
Diffusible LIF
(ng/ml)
pSTAT3
(fold increase over control)
Immobilized LIF
(ng/cm2)
Immobilized LIF
(ng/cm2)
Diffusible LIF
(ng/ml)
Immobilized LIF
(ng/cm2)
Diffusible LIF
(ng/ml)
Immobilized
LIF
(ng/cm2)
POMA
POMA-PEG7
**
*
02875
0
100
200
300
400
pSTAT3
10
02875
0
200
400
600
800
1,000
pMAPK
10
0 75
0
1
2
3
pMAPK/pSTAT3 ratio
10
Figure 2 | Immobilized LIF activates STAT3 and MAPK in a dose-dependent manner. (a) Immunocytochemistry and single-cell
image analysis of mESCs cultured on 75 ng/cm2LIF immobilized covalently to POMA. Hoechst nuclear staining with (top left)
and without (top right) image algorithm cell mask. Blue masks represent objects fitting selection criteria and orange
represents objects excluded from analysis. Nuclear masks were applied to a second channel (bottom left) to quantify
phosphorylated STAT3 fluorescence (bottom right). Scale bar, B20 mm. (b) STAT3 activation in mESCs cultured for 12 h on
surfaces of LIF immobilized to POMA or POMA-PEG7. Diffusible LIF was used as control on hydrolyzed surfaces (3 ? 103–1 ?
104single-cell measurements n ¼ 4 ± s.e.m., *P o 0.05, ** P o 0.01; diffusible LIF served as reference for Dunnett’s
post-hoc test). (c) STAT3 activation in mESCs cultured for 72 h in the presence of of POMA-immobilized LIF and diffusible LIF.
(d) Activated (phosphorylated) MAPK was measured for the same cells and conditions as in c. (e) The ratio of phosphorylated
MAPK to phosphorylated STAT3. Error bars, s.d. (c–e)
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over many passages. Thus, we compared mESCs passaged
on POMA-immobilized LIF surfaces in media devoid of diffusible
LIF for greater than 2 weeks (6–8 passages) to mESCs passaged
in media containing diffusible LIF, in chimera formation assays
using diploid embryos. Note that mESC passaged without
any LIF rapidly differentiate (after 2–3 passages) and cannot
be extensively propagated. This experiment demonstrates that
mESC propagated on covalently immobilized LIF can contribute
to embryonic development and support development to term
(adulthood) (Fig. 3d and Supplementary Table 2 online).
Our immobilization approach is broadly applicable
To test whether our approach is applicable to cytokines other than
LIF, we immobilized defined amounts of bioactive SCF on POMA
andPOMA-PEG7(Fig.4a).Comparabletothetrendsobservedfor
LIF, POMA substrates allowed greater surface immobilization of
SCF compared to covalent binding of SCF through a PEG spacer
(Fig. 4b) and both immobilized SCF systems were biologically
active and induced the expansion of the erythroleukemia cell line
Mo7E (a model system for blood progenitor cell growth) in a
concentration-dependent manner (Fig. 4c,d). Considering the
geometrically restricted accessibility of the immobilized cytokine
for Mo7E cells, which are typically suspension-grown cells and
therefore attach only temporarily or loosely to the matrix, our data
suggest high signaling efficacy of the immobilized SCF.
DISCUSSION
Strictliganddosagecontrolis anecessitygiven thethresholdnature
of cell fate signals28. In the case of LIF, specific amounts of ligand
and consequently of STAT3 activation have been shown to be
necessary to support mESC self-renewal29. Our measurements of
STAT3 and MAPK activation in response to diffusible and immo-
bilized LIF demonstrate that as one moves from differentiation-
permissive to differentiation-inhibitory concentrations of LIF, the
ratio of two activated downstream signaling molecules (STAT3 and
MAPK) remains relatively constant. Even in the absence of exo-
genous LIF (wherein locally produced STAT3 activation is signifi-
cant22), this ratio is not substantially different from that measured
at high concentrations of diffusible or immobilized LIF. Together,
these data support the hypothesis that it is the extent of activation
(the signaling threshold) of the signaling intermediates, not their
relative activation level, which determines stem cell fate.
c
Percentage of Oct-4+ cells
d
LIF
POMA and LIF
POMA and immobilized LIF
ab
Percentage Oct-4 negative
1002875
0
10
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50
60
70
120
60
50
40
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20
10
0
100
Cell number
Cell number
80
60
40
20
0
4.5 5.06.0
Oct-4
7.08.05.5
*
Diffusible LIF
(ng/ml)
cells
Immobilized LIF (ng/cm2)
POMAPOMA-matrix
*
25.72.70.702
LIF (ng/well)
POMA POMA-matrix
20
0357
30
40
50
60
70
80
90
100
Days in culture
0 ng/cm2
2 ng/cm2
8 ng/cm2
75 ng/cm2
10 ng/ml
6.57.5
4.5 5.0 6.0
Oct-4
7.08.0 5.5 6.57.5
Figure 3 | Immobilized LIF promotes Oct-4 expression and chimera formation in a dose-dependent manner. (a) Representative
histograms showing Oct-4 expression for cells cultured for 72 h on 75 ng/cm2immobilized LIF on POMA or POMA-matrix. Red lines
on each plot represent Gaussian fits of Oct-4+(right peak) and Oct-4–(left peak) subpopulations. The blue lines are the overall fit
for both populations. (b) Concentration-dependent expression of Oct-4 in mESCs cultured for 72 h on POMA-immobilized LIF, or matrix-physisorbed LIF.
Diffusible LIF was replaced during medium change every 24 h (results obtained from 5 ? 103–1 ? 104single-cell measurements; error bars, s.e.m.; *P o 0.05,
Student’s t-test for respective conditions). (c) Oct-4 expression in cells cultured in the absence of LIF, the presence of indicated amount of LIF immobilized
covalently to POMA and diffusible LIF over time. Error bars, s.d. (d) Chimeric mice generated from cells cultured and passaged 6–8 times on POMA substrates
with covalently attached LIF (8 ng/cm2), hydrolyzed POMA with diffusible LIF and tissue culture plastic with diffusible LIF (10 ng/ml each). All substrates were
overlaid with gelatin to facilitate cell attachment.
012
Time (d)
345
0
10
20
30
40
50
Immobilized SCF (ng/cm2)
02468
10
0
25
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75
Immobilized SCF (ng/cm2)
Solution concentration (µg/ml)
POMA
POMA-PEG7
abcd
0 20 4060 80
0
2
4
6
8
Cell count (104)
SCF (ng/well)
8
16
57
3
5
20
Figure 4 | Functional immobilization of SCF. (a) Amount of covalently bound SCF after a 1-d incubation under cell culture conditions. Graph indicates the
relationship between the solution concentration and the amount of covalently immobilized [125I]SCF on POMA and POMA-PEG7. (b) Longer-term behavior of
immobilized SCF on surfaces prepared using 2.5 mg/ml SCF in solution. (c) Analysis of proliferation induction of Mo7E cells by immobilized SCF. Mo7E cells were
cultured without medium change on POMA or POMA-PEG7 with immobilized SCF at 3.5–74 ng/well and counted on day 6. Numbers next to plot symbols
correspond to the respective surface concentration in ng/cm2. Samples were used after a 1-d preincubation as described in a. Dashed line corresponds to samples
with diffusible SCF at 2 ng/ml, dotted line to Mo7E proliferation without SCF. Graphs show typical datasets generated from triplicate samples ± s.d.
(d) Phase contrast image of Mo7E cells cultured on polymeric substrate at day 5. Both intimately associated Mo7E cells (open arrow) and suspended cells
(closed arrow) are present in culture. Scale bar, 50 mm.
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Surfaces with covalently immobilized LIF could be coated with
an ECM, and we did not detect temporal or dose-dependent
changes in ligand accessibility owing to ECM remodeling. ECM
itselfisimportantinmodulatingcellattachmentandfate30,andthe
possibility of independently controlling ECM and immobilized
cytokine exposure to cells should enable screening studies into the
interactive effects31of these two important cell-signaling compo-
nents. Notably, we observed differences in antibody accessibility
betweenPOMAandPOMA-PEG7immobilizedLIF,butfunctional
responses (signaling activation) on these surfaces were similar.
These results suggest that although matrix may interfere with
antibody access to immobilized LIF, cells efficiently accessed
immobilized protein through the matrix. In contrast to the condi-
tion where LIF was simply physisorbed, the cells did not appreci-
ably degrade immobilized LIF over the period studied (that is,
immobilized LIF was active for extended periods of time).
The approach we report here sets the groundwork for an in-
depth comparisonofotherdiffusibleand immobilized ligandssuch
as activin and nodal (known to be important morphogens in
development32) or Notch ligands33on cell signaling and fate. The
approach may be generally applicable to address the importance of
exogenouscuesinstemcellniches,asindicatedbyourobservations
with SCF. Notably, the polymer thin film coating can be applied to
manyamine-bearingcarriermaterials,forexample,glassorsilicone
wafers (after pre-treatment with aminosilanes) as well as different
bulk polymer materials such as polystyrene or polydimethylsilox-
ane (after ammonia plasma treatment). This allows adaptation of
the coatings to standard multiwell substrates and combination
with common techniques of microstructuring and upscaling
for surface modification of three-dimensional objects. The
versatility and robustness of the substrate functionalization
scheme can be expected to facilitate multifactorial cell fate
screening experiments.
METHODS
LIF immobilization. We dissolved LIF (recombinant, mouse;
Chemicon) in Dulbecco’s PBS (pH 7.4), and covalently immobi-
lized it to POMA films directly or via PEG7 spacers (POMA-
PEG7). We prepared POMA films as described in Supplementary
Methods online. For LIF immobilization to plain POMA surfaces,
we incubated freshly annealed polymer films with LIF solutions
(0.25–10 mg/ml) overnight at room temperature (18–22 1C) and
rinsed them twice with PBS before use. LIF conjugation to POMA-
PEG7 was achieved with 50 mM 1-ethyl-3-(3-dimethylamino-
propyl) carbodiimide hydrochloride (EDC) (Sigma-Aldrich) and
25 mM N-hydroxysulfosuccinimide (sulfo-NHS) (Fluka) in phos-
phate buffer (67 mM, pH 7.4) for 4 h at room temperature. Then
we rinsed the surfaces twice with purified water. For noncovalent
LIF immobilization to ECM (POMA-matrix), we incubated the
POMA-coated coverslips overnight in PBS to hydrolyze the
anhydride moieties. Subsequently, we coated the polymer films
overnight at 37 1C with a solution of native collagen type I
(0.2 mg/ml, Vitrogen; Cohesion Technologies) and fibronectin
(12.5 mg/ml, purified from adult human plasma) in PBS34. Then
we gently rinsed POMA-matrix samples with PBS and physisorbed
LIF as described above. For cell culture experiments, we used
gelatin (type B, from lime-cured tissues; Sigma) instead of collagen
type I. For all cell culture experiments, we applied gelatin to all
other studied surface preparations and their respective controls
to facilitate cell attachment. Additional details are available in
Supplementary Methods.
Preparation of 96-well plates with POMA substrates. We
detected LIF by immunological assay and carried out cell-culture
experiments in glass-bottom 96-well plates (Greiner Bio-One). We
coated glass-bottom plates with POMA layers as described above
followed by fixation of the plates to polystyrene 96-well grids. To
regenerate anhydride groups, we annealed POMA-coated plates
for 18 h at 90 1C under vacuum. Then we attached PEG7, LIF or
ECM as described above.
Immobilization of predefined amounts of immobilized LIF.
We immobilized 2, 8 and 75 ng LIF/cm2on either POMA,
POMA-PEG7 or POMA-matrix as described above. The corre-
sponding solutionconcentrations
[125I]LIF calibration curves (see Supplementary Table 1 for
solution concentrations).
were extrapolatedfrom
Quantification of immobilized LIF and stability testing.
We quantified LIF using [125I]LIF (custom-ordered from GE
Healthcare), which was immobilized on the respective surface in
custom-made incubation chambers (2 cm2sample surface). We
made a dilution series of LIF (0.25–10 mg/ml) in 250 ml of PBS that
we spiked with 2–6% of125I-labeled cytokine (50 mCi/mg protein,
o2% free125I). We shook the samples and incubated them at
room temperature for 24 h. After the immobilization period, we
washed the samples three times with PBS and measured coverslip-
associated radioactivity (gamma counter UMo LB 123; Berthold
Technologies). To assess the stability of attachment of immobilized
LIF in cell culture medium (DMEM with 15% knockout serum
replacement and 2 mM NaN3) we repeated the measurements of
coverslip-associated radioactivity on day 3. We quantified the
amount of LIF using [125I]LIF standards.
Examination of accessibility of LIF. We used a LIF-specific
antibody to assay the accessibility of immobilized LIF. We pre-
pared samples in a 96-well format, rinsed them twice with PBS
and blocked them with 50 ml/well of 5% BSA in Tris-buffered
saline (TBS, pH 7.3) for 30 min at room temperature. Subse-
quently we incubated 50 ml/well of 1.5 mg/ml biotinylated goat
antibody to LIF (R&D Systems) in TBS for 2 h at room tempera-
ture. Then we rinsed the samples with TBS, added 100 ml/well of
horseradish peroxidase–conjugated streptavidin (75 ng/ml in TBS;
Rockland) and incubated the samples in the dark for 30 min. Next
we rinsed the wells, added 100 ml/well of tetramethylbenzidine
(TMB) peroxidase enzyme immunological assay substrate (BioR-
ad), and stopped the reaction after 8 min with 100 ml/well of 0.5 M
H2SO4. Optical densities were measured using a microplate reader
(Tecan) at 450 nm. We applied the matrix required for cell
attachment and determined the antibody accessibility of immobi-
lized LIF after 3 d in cell culture medium.
mESC culture, chimera production, immunocytochemistry and
single-cell image analysis. We used mouse R1 mESCs35for the
experiments; cell culture conditions, staining procedures and
analysis are described in Supplementary Methods. In vivo studies
were conducted in accordance with national regulations and
institutional guidelines. For chimera experiments we directly
NATURE METHODS | VOL.5 NO.7 | JULY 2008 | 649
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