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Stress-stiffening-mediated stem-cell commitment

switch in soft responsive hydrogels
Rajat K. Das1*†, Veronika Gocheva2,3†, Roel Hammink1, Omar F. Zouani2,3* and Alan E. Rowan1*

Bulk matrix stiffness has emerged as a key mechanical cue in stem cell differentiation. Here, we show that the commitment
and differentiation of human mesenchymal stem cells encapsulated in physiologically soft (∼0.2–0.4 kPa), fully synthetic
polyisocyanopeptide-based three-dimensional (3D) matrices that mimic the stiffness of adult stem cell niches and show
biopolymer-like stress stiffening, can be readily switched from adipogenesis to osteogenesis by changing only the onset of
stress stiffening. This mechanical behaviour can be tuned by simply altering the material’s polymer length whilst maintaining
stiffness and ligand density. Our findings introduce stress stiffening as an important parameter that governs stem cell fate
in a 3D microenvironment, and reveal a correlation between the onset of stiffening and the expression of the microtubule-
associated protein DCAMKL1, thus implicating DCAMKL1 in a stress-stiffening-mediated, mechanotransduction pathway that
involves microtubule dynamics in stem cell osteogenesis.

ellular interactions with the extracellular matrix (ECM) restricts the focal contact maturation. In addition, the 2D nature of
mediate important cell functions such as survival, prolif- the micropost culture system limits its further applications to mimic
eration, migration and differentiation1–5 . The extracellular a 3D physiological condition.
stimuli include soluble and adhesive ligands that provide chemical We present a new class of cell culture systems based
and mechanical cues which have been shown to govern cell physiol- on fully synthetic biomimetic physically crosslinked soft
ogy1 . It has recently been demonstrated that physical cues from the (∼0.2–0.4 kPa) hydrogels derived from helical oligo(ethylene)glycol
matrix, especially the matrix stiffness and topography, can initiate polyisocyanopeptides (PICs; ref.24 ). We demonstrate the application
intracellular biochemical signals through mechanotransduction of this novel 3D matrix for the study of stem cell functions. These
and thus dictate the cell differentiation pathways6–8 . Understanding soft thermoresponsive hydrogels are formed at extremely low
the role of these microenvironmental physical cues has profound polymer concentrations (99.95% water) and possess pore sizes of
implications for realizing the full therapeutic potential of stem cell nanometre dimension (∼100–150 nm; ref. 25). Their mechanical
research in tissue engineering applications9–17 . properties are similar to those of gels prepared from filamentous
Three classical cell culture systems have been widely utilized to biopolymers, such as microtubules, F-actin, fibrin and collagen,
underpin the effect of the extracellular matrix mechanical properties and exhibit nonlinear stress response beyond a critical stress
on stem cell fate. First, natural ECM-protein-derived hydrogels σC (stress stiffening)25 . Thus, when the stress is increased beyond
prepared at different densities have been shown to significantly this value, the matrices become stiffer with increasing applied stress.
influence cell adhesion, cell shape and various cell functions18 . Janmey et al. demonstrated that different types of cells, for
However, changing density of these proteins not only changes example, fibroblasts and human mesenchymal stem cells (hMSCs),
the gel stiffness, but also alters the surface ligand concentration. adopt a stretched morphology when cultured on soft fibrin gels
Thus, in this type of culture systems, it is difficult to de-couple (2D substrates), suggesting that the cells can deform these gels,
and interpret the effect of the matrix stiffness and ligand density allowing access to the high strain moduli in the strain stiffening
on cellular response. The second class of culture systems employs regime26 . It is important to note that the onset of stiffening for
synthetic polymer gels as ECM mimetic scaffolds for stem cell fate the ECM proteins occurs at extremely small applied stresses. Our
control17,19,20 . In this case, the gel stiffness can be modulated by synthetic PIC hydrogels show stress stiffening in the biologically
altering the amount of a crosslinker; however, the porosity and relevant stress regime, as demonstrated in Fig. 1, which has been
the polymer surface chemistry also change as a function of the constructed by adapting data from literature27 and overlaying the
crosslinker amount21,22 . Thus, the stem cells are likely to interpret the data for the PIC gel obtained in the present work. It was therefore
combined effect of these parameters for their fate control, although of interest to investigate how this biologically relevant parameter
it has recently been suggested that substrate porosity in 2D cell influences 3D stem cell differentiation as part of the mechanisms
culture condition does not affect stem cell fate23 . A third class underlying the physiology of adult stem cell niches.
of culture systems has been recently introduced, which is based Here we show that for hMSCs encapsulated in these biomimetic
on elastomeric micropost arrays, where different post heights are gels (functionalized with cell-adhesive GRGDS peptides),
interpreted as different rigidities by the cells16 . This system thus preferential osteogenesis over adipogenesis can be induced in
provides an approach to de-couple the effect of the substrate stiffness an extremely soft microenvironment by increasing the polymer
from alterations in the surface chemical properties. However, the length, thereby increasing σC , without altering the bulk gel stiffness
micropost diameter dictates a focal adhesion limiting size, which and ligand density of the matrix. Thus, this fully synthetic culture

1 Institutefor Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands. 2 Histide, Chaltenbodenstrasse 8,
8834 Schindellegi, Switzerland. 3 Histide Lab, Accinov, 317, avenue Jean Jaurès, 69007 Lyon, France. †These authors contributed equally to this work.


© 2016 Macmillan Publishers Limited. All rights reserved

104 Polyisocyanopeptide materials, and also because they allow us to deconstitute some of
Biologically Polyacrylamide
relevant Actin
the key parameters of the generally complex biological systems
Differential modulus, K’ (Pa)

stress regime Collagen (enzyme degradability, lack of simultaneous control on ligand

103 Fibrin density and stiffness and so on.). Frequency sweep measurements
up to very low frequencies (10−3 rad s−1 ) on hydrogels of this class
of polymers have shown that these gels have slow relaxation on the
Go timescale of hours25 . These synthetic polymers were functionalized
with the basic cell-adhesive peptide GRGDS to promote stem cell
101 adhesion to the matrix. This peptide ligand can be homogeneously
distributed and allows direct sensing of the substrate mechanical
σc properties as there is a single anchoring point per short ligand.
100 This is a major advantage of our system compared to previously
10−1 100 101 102 103
Stress, σ (Pa) developed substrates, for example, with anchoring collagen28 , and
contributes towards the clear interpretation of the effect of stress
Figure 1 | Differential modulus, K’, as a function of stress, σ , for stiffening. The peptide ligand has been grafted to the polymer
intracellular and extracellular filamentous biopolymer gels that show via a short spacer, which is 24 atoms long and equivalent to
stress stiffening. For comparison, the rheology data for a representative a short PEG chain of approximately 8 units. This spacer was
synthetic gel (polyacrylamide) and the biomimetic stress-stiffening optimized according to molecular modelling so as not to play
polyisocyanopeptide hydrogel are included. Go indicates the equilibrium a role in the cell–substrate interactions. However, the length of
bulk stiffness and σC denotes the critical stress for the onset of stress the peptide tether was not varied to investigate its influence
stiffening of the polymer gel. on the cell–substrate interaction, although it may play a role29 .
Accordingly, polyisocyanopeptides (P10 –P60 ) were synthesized
system presents a model to study the effect of this physiologically by a nickel(II)-catalysed co-polymerization of triethylene glycol
important parameter (stress stiffening) on cells encapsulated functionalized isocyano-(D)-alanyl-(L)-alanine monomer 1 and the
in a 3D microenvironment. We also demonstrate that stress- azide-appended monomer 2 (Fig. 2a), with the molar ratio of
stiffening sensing implicates the microtubule-associated protein 1/2 = 100, resulting in polymers with one azide functionality every
DCAMKL1 in the mechanotransduction pathway, suggesting a role 14–18 nm of the polymer chain, as determined by reacting a strained
of microtubule dynamics in hMSCs fate control. rhodamine dye with the azides (Table 1 and Methods).
The catalyst to monomer molar ratio was varied from
Synthesis and characterization of polymers 1:1,000 to 1:8,000, to obtain polymers of increasing molecular
To construct a 3D cell culture matrix for the study of the effect of weight (determined by viscosity measurements, Table 1)
stress stiffening on stem cell fate, we chose polyisocyanopeptide- (P10 –P60 ). These azide-functionalized polymers were then
based hydrogels, as they demonstrate stress-stiffening behaviour25 in subjected to strain-promoted click reaction with BCN–GRGDS
the biologically relevant stress regime (Fig. 1), unlike other synthetic (BCN: Bicyclo[6.1.0]non-4-yn-9-ylmethyl) to obtain cell-adhesive

a b
O x y
N O N N Borate buffer
N O3 X X
H O O N pH = 8.4
Ni(ClO4)2 . 6 H2O O O HN–GRGDS
O Toluene
N O N3 O O
N O3
H O c
Polymer-N3 N HN GRGDS

Acetonitrile N H
P1′−P6′ P1′−P6′ P1−P6

d e 500 f
1,000 20
Critical stress, σ (Pa)
Gel stiffness, Go (Pa)

400 18
Stiffness, G’ (Pa)

100 350 16
250 14
200 12
1 100 10
50 8
0.1 0
5 10 15 20 25 30 35 40 150 200 250 300 350 400 450 150 200 250 300 350 400 450
Temperature (°C) Mean polymer length (nm) Mean polymer length (nm)

Figure 2 | Synthesis of polymers of different chain lengths and characterization of polymer hydrogels. a, Co-polymerization of unfunctionalized
monomer 1 and azide-functionalized monomer 2 produces polymers of different chain lengths (P1’–P6’) at different catalyst to monomer molar ratios.
b,c, Functionalization of azide-functionalized polymers P1’–P6’ with GRGDS peptide. d, Representative variable temperature rheology experiment of a
polymer gel. The onset of gelation temperature was observed to be ∼15 ◦ C. e, The storage modulus (Go ) remains fairly constant (0.2–0.4 kPa) at 37 ◦ C, as
a function of polymer length. f, The critical stress varies linearly as a function of polymer length. Error bars represent standard errors of the mean (n = 3).


© 2016 Macmillan Publishers Limited. All rights reserved

Table 1 | Characterization of the synthesized polymers and the gels in alpha-MEM.

Catalyst/monomer Viscosity derived Average spacing of −N3 on Mean (GRGDS functionalized) Mean critical stress (σc , Pa)
molecular weight the polymer chain polymer length from AFM in alpha-MEM gels at
(N3 -polymer; kg mol−1 ) (nm) (nm) 2 mg ml−1 concentration
1/1,000 307 14 182 9.4
1/2,500 426 14 226 9.9
1/3,000 491 18 250 12.8
1/4,000 571 15.6 309 14.6
1/6,000 591 14 367 16.6
1/8,000 685 17 434 19.3

GRGDS functionalized polymers P1–P6 (Fig. 2b,c and Methods) cell specific marker33 . A significant decrease in the average STRO-1
of increasing chain lengths as determined by AFM (Table 1 and expression was observed for the cells in all of the gels after 96 h of
Supplementary Figs 1 and 2). Solutions of these polymers in culture, indicating the onset of stem cell differentiation (Fig. 3e). The
alpha-MEM (minimum essential medium) at a fixed concentration expression of osteogenic and adipogenic differentiation markers was
(2 mg ml−1 ) formed transparent gels on warming above ∼15 ◦ C then examined. For cells cultured in the gel with the lowest critical
(temperature sweep rheology, Fig. 2d). The mechanical properties stress (σC ∼ 9.4 Pa, constructed from the shortest polymer P1)
of the GRGDS functionalized polymer gels were investigated by predominant adipogenic commitment was observed (Oil Red O
rheological analysis. Temperature sweep experiments (heating up staining, Fig. 3i and Supplementary Fig. 17). With increasing
to 37 ◦ C) followed by time sweep at 37 ◦ C revealed that all the critical stress (by increasing the polymer length), osteogenesis
gels P1–P6 were soft and exhibited similar stiffnesses (0.2–0.4 kPa was progressively favoured over adipogenesis, as demonstrated
at 37 ◦ C) (Fig. 2e). Recently, we reported that hydrogels of non- by immunofluorescent staining of Osterix, an osteogenic specific
functionalized polyisocyanopeptide polymers show a biomimetic marker (Fig. 3h), and as determined from the mean percentages
stress-stiffening behaviour25 . Using the same pre-stress protocol30 , of osteogenic and adipogenic commitments (Supplementary Fig. 7)
the critical stresses (σC ) of P1–P6 alpha-MEM gels were measured in the various polymers (P1–P6). hMSCs cultured in the gel with
(Supplementary Fig. 3). The value of σC for nonlinear rheology the highest critical stress (P6) exhibited preferential osteogenic
behaviour of these gels was found to increase linearly as a function commitment. The predominant osteogenesis for the cells in the
of the polymer chain length (Fig. 2f and Table 1)31 , from ∼9 Pa longer polymers (P4–P6) was further confirmed by differentiation
in the P1 gel (average polymer length: 182 nm) to ∼19 Pa in the tests after three weeks of culture (Supplementary Fig. 8).
P6 gel (average polymer length: 434 nm). Although it seems that Finally, the hMSCs osteogenic commitment was verified by
there is a 1.5-fold increase in the mean gel stiffness when the mean analysing the expression of the osteogenic biomarker Core-binding
polymer length is increased from ∼180 nm to ∼240 nm (Fig. 2e), factor alpha 1 (Cbfa-1), also called RUNX2 and the expression of the
this difference is small in the context of cellular perception of bulk adipogenic biomarker PPARγ , by RT-PCR. We observed an increase
stiffness17 . Regarding the critical stress values, the error range is in the RUNX2 gene expression with increasing critical stress after
smaller and there seems to be a linear relationship between this 96 h of culture (Fig. 3f) in agreement with the immunofluorescence
parameter and the mean polymer length (Fig. 2f), which is why we staining results. An increase in osteogenesis for the longer polymer
consider the increase to be significant. gels has been further confirmed by the observed decrease in
PPARγ gene expression as a function of the increasing critical stress
Stress-stiffening-mediated hMSC differentiation after 96 h of culture (Fig. 3g).
To investigate the effect of stress stiffening on stem cell fate, To investigate the role of hMSCs-adhesive ligand interactions
hMSCs were mixed with a cold polymer solution (∼10 ◦ C) in in the observed stem cell fate, we performed the cell commitment
alpha-MEM, which was then warmed to 37 ◦ C to form the studies for RGD-modified polymers P1, P3, P4 and P6 in
3D matrix with encapsulated hMSCs. The cells were homogeneously the presence of antibodies recognizing specific integrin subunits
distributed throughout the gel, as indicated by confocal microscopy (α1, 2, 3 and 5; β1 and 2) which block their interactions with the
(Supplementary Fig. 4). Investigation of hMSCs morphology substrate-bound RGD ligands. In the presence of these integrin-
after 36 h of culture for all of the gels (P1–P6) revealed that blocking antibodies, osteogenic commitment was suppressed.
the cells remained spherical (Fig. 3a). These cells exhibited However, adipogenic commitment was maintained for all the
only limited cortical F-actin protrusions into the surrounding polymers (Supplementary Figs 9 and 10). This result is in
microenvironment (Phalloidin staining, Supplementary Fig. 5) and agreement with recent literature19 and highlights the importance
showed no significant modifications in their nuclear morphology, of the interaction between integrin receptors and the RGD
as shown by a representative DAPI fluorescence image of the ligands for mediating the stress-stiffening-induced commitment
cell nucleus after 36 h of culture (Fig. 3b). These observations switch. Interestingly, the presence of blebbistatin (a small molecule
are consistent with recent reports of hMSCs 3D cell cultures19,32 . inhibitor of actomyosin contractility showing high affinity and
Live/dead assay (calcein-AM and MTT) performed after 36 h of selectivity towards myosin II) inhibited the hMSCs commitment,
culture in growth media for all of the gels indicated excellent viability with stemness maintenance observed for all the polymer gels,
(>95%) of the encapsulated cells (Fig. 3c,d), as also confirmed as revealed by the high levels of STRO-1 in the encapsulated
by confocal microscopy (Supplementary Fig. 4). In addition, no cells (Supplementary Fig. 11). This suggests that the inhibition of
significant cell proliferation could be detected for the various gels, actomyosin contraction interferes with the mechanisms of hMSCs
as determined by the PicoGreen assay (Supplementary Fig. 6). The commitment both towards adipogenesis and osteogenesis. This
lineage commitment of the gel encapsulated hMSCs after 96 h of is most likely owing to the fact that the cells could not apply
culture in bipotential differentiation medium (1:1 v/v osteogenic any traction force for the microenvironmental mechanical (stress-
and adipogenic media) was then investigated. Cells were first stiffening) sensing. These results are consistent with previously
stained (immunofluorescence) for STRO-1, a mesenchymal stem published studies17 . Finally, to demonstrate the direct interaction


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a Shortest polymer Longest polymer b c d Cell viability
P3 P3

MTT (% control)
10 μm 5 μm 100 μm

e STRO-1 f RUNX2 g PPARγ h Osterix i LIP
1.4 5 10 7 6
1.2 6 5
4 ∗ 8 ∗
∗ ∗∗
Relative mRNA level

Relative mRNA level

1.0 ∗∗ 5 ∗∗
Relative intensity

Relative intensity

Relative area
3 NS 6
0.8 4
∗∗ 5 3
0.6 NS 3
2 4
3 2
0.4 2
1 2 1
0.2 1
0.0 0 0 0 0












Figure 3 | Effect of stress stiffening on hMSC commitment. a, Representative micrographs showing cross-sections of hMSCs 36 h after encapsulation into
3D matrices made from the shortest (P1) and the longest polymer (P6) and constant GRGDS density, visualized by bright-field microscopy. b, Confocal
image of fluorescence staining (DAPI; blue) of the cell nucleus after 36 h of culture for the P3 polymer. c, Live/dead viability test (calcein-AM) of hMSCs
after 36 h of encapsulation. In the representative image, almost all cells in the matrix from polymer P3 seem to be alive (green). d, Cell viability was
assessed by MTT assay in all the matrices (from shortest to longest polymers). e, The STRO-1 protein expression in the hMSCs is expressed as average
fluorescence intensity, normalized by the number of cells. Compared to control, STRO-1 expression has decreased for all of the matrices, indicating that the
hMSCs have lost their ‘stemness’ and initiated differentiation. f,g, Quantitative PCR analysis for RUNX2 and PPARγ , respectively; significant differences
were observed between the higher critical stress matrices and the lower critical stress matrices. h, Total cellular Osterix protein immunofluorescence
intensity was quantified for hMSCs cultured for 96 h in the various matrices. i, Total cellular area of immunofluorescence intensity of neutral lipid
accumulation quantified for hMSCs cultured for 96 h in the various matrices. For all the experiments, a non-functionalized soft polymer gel (cell culture in
growth medium) served as the control (∗∗ P < 0.001, ∗ P < 0.01). e–i, Error bars represent standard error of the mean (n = 3). NS, not significant.

between the hMCSs and the polymer-bound RGD in our system, DCAMKL1 role in stem cell differentiation.
the cell commitment studies for RGD-modified polymers P1, P3, Several reports have implicated the cytoskeletal contractility
P4 and P6 were performed in the presence of soluble RGD ligands, and actin polymerization in the mechanotransduction pathway
which can block the interaction between the cells and the matrix by responsible for osteogenic differentiation on 2D substrates. In
competing for the integrin-binding sites. No significant osteogenic our study, a treatment with cytochalasin D (inhibitor of actin
or adipogenic commitment could be detected, indicating that polymerization) resulted in an overall decreased commitment of
integrin disengagement from the matrix-bound RGD is interfering the cultured stem cells towards both osteogenesis and adipogenesis
with the cell’s ability to sense stress stiffening (Supplementary (Supplementary Fig. 15a), suggesting a role of actin polymerization
Figs 12 and 13). These data also imply that the cells in these in the stress-stiffening-mediated hMSCs differentiation in our
gel culture systems need direct engagement with the bound RGD system. Alternatively we also observed a decrease in hMSCs
ligand, and not with the secreted ECM, for mediating the stress- commitment after treatment with Taxol, a well-characterized
stiffening-induced commitment switch. microtubule-stabilizing agent, which is known to inhibit tubulin
Although the macroscopic ligand density is kept constant in this depolymerization (Supplementary Fig. 15b). Taxol treatment did
study (one ligand every 14–18 nm of a polymer chain), the longer not affect cell viability, as indicated by a live/dead assay after 48 h
polymer chains (P4–P6) have almost twice as many ligands per and 96 h of culture (Supplementary Fig. 16). The effect of Taxol
chain (20–26), as compared to the corresponding shorter chains on the cell commitment outcome indicates that, in addition to
(P1–P3: 13–18). This could indeed impact the extent of cell- actin, the microtubule dynamics could also be involved in the
mediated local ligand clustering. To study the effect of ligand density mechanotransduction pathways underlying hMSCs differentiation
on the observed hMSC commitment switch, the commitment in our system.
study was performed as a function of ligand density (RGD every A recent report has indicated that the microtubule-associated
7 nm, 28 nm and 70 nm) for gels of the shortest (P1) and the protein DCAMKL1 represses RUNX2, an early osteogenesis
longest polymer (P6). Varying the ligand density for both of the marker, and thus regulates osteogenic differentiation in vitro
polymers was found not to interfere with the cell differentiation and in an in vivo rat model34 . DCAMKL1 is also known to
outcome (Supplementary Fig. 14). These results suggest that stress enhance microtubule polymerization. Furthermore, it has also
stiffening is the primary governing variable in our system, without been reported that microtubule depolymerization can alter the
excluding the possibility that cell-mediated ligand clustering is myosin mechanochemical activity through myosin regulatory side
occurring. Our data demonstrate that hMSCs fate can be switched chain phosphorylation, thus resulting in increased actomyosin
from adipogenesis to osteogenesis in a soft microenvironment contraction35 . We therefore investigated the role of DCAMKL1
(∼0.2–0.4 kPa), simply by increasing the critical stress for the onset in the stress-stiffening-mediated control of hMSCs differentiation
of stress stiffening. in our 3D culture system as a function of the gel critical


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a b Shortest polymer Longest polymer

P1 P2 P3 P4 P5 P6 N N
O H 3

α-DCAMKL1 82 kDa N
x N O
H 3

α-RUNX2 57 kDa High

Tubulin Str ess-stiff
Low 0.2−0.4 kPa

+ Critical stress −
RUNX2 relative
intensity (a.u.)

30 High
0 50 100 150
DCAMKL1 relative intensity (a.u.)
Adipogenesis Osteogenesis

Figure 4 | Stress-stiffening-mediated stem cell differentiation involves the microtubule-associated protein DCAMKL1. a, (Top) Western blot analysis of
DCAMKL1 and RUNX2 protein expression in hMSCs after 96 h of culture in all of the matrices (short and long polymers) (P1–P6). The western blot was
performed in triplicate. (Bottom) Plot of the relative protein expression intensities of RUNX2 versus DCAMKL1 for all the conditions (P1–P6), showing a
switch-like relationship between these two proteins with relevance for the mechanistic insights of stress-stiffening sensing. b, Schematic model illustrating
the overall trends of the mechanisms by which stress stiffening regulates hMSCs commitment and differentiation towards adipogenesis and osteogenesis
via modulation of the protein expression of DCAMKL1.

stress. Interestingly, western blot analysis revealed a negligible Altogether these results are the first report of a microtubule-
DCAMKL1 expression for the polymer gel with the highest critical associated protein DCAMKL1 being involved in a new stress-
stress (P6) and a significant increase in the expression of this stiffening-mediated mechanotransduction pathway involving
protein with decreasing critical stress for stress stiffening (Fig. 4a). microtubule dynamics for the control of hMSCs differentiation
Concomitantly, RUNX2 protein expression was not observed in the (Fig. 4b). These data indicate that stem cell fate is regulated by ECM
gels with lower critical stress (P1–P3), whereas the protein was stress stiffening via a different molecular mechanism than the one
clearly expressed in the higher critical stress polymers (P4–P6) in described for classical 2D substrate rigidity sensing.
correlation with the observed osteogenic commitment in these gels.
This is also in agreement with the observed overall increase in Outlook
the RUNX2 mRNA expression between the shorter (P1–P3) and This work introduces polyisocyanopeptide-based hydrogels as a
longer (P4–P6) polymers (Fig. 3f), although to a lesser extent, but new class of 3D cell culture systems for the study of the physical
still significant. These observations correlate well with preferential cues of the microenvironment involved in stem cell fate control.
osteogenesis in gels of higher critical stress and lack of osteogenic Despite being fully synthetic, the gels discussed in this work
commitment as the critical stress for stress stiffening is lowered. closely resemble natural biopolymer gels in that they exhibit stress-
A plot of the relative intensities (protein expression) of RUNX2 stiffening behaviour, a property that is widely observed in biological
versus DCAMKL1 for all the conditions (P1–P6) showed a switch- tissues and is believed to have important ramifications in the design
like relationship between these two proteins with the existence of artificial ECM mimetic scaffolds. In contrast to other synthetic
of a threshold value for the expression of DCAMKL1, which polymer matrices such as polyacrylamides or polyethylene glycol
antagonizes RUNX2 in adipogenic lineage commitment (Fig. 4a gels, the bulk stiffness of this new class of gels does not need
and Supplementary Fig. 18b). This observation has functional to be modulated to access different differentiation lineages of the
relevance for our mechanistic interpretations, as it correlates with encapsulated stem cells. Uniquely, despite being embedded in a
the observed stress-stiffening-mediated commitment switch. soft microenvironment, the hMSCs commitment can be switched
To further confirm the functional relationship between the from adipogenesis to osteogenesis simply by altering the polymer
two proteins in our stress-stiffening gel systems, DCAMKL1 gene chain length, and thus the critical stress, keeping the matrix stiffness
silencing (through shRNA) and overexpression (via transient and ligand density unaltered in the process. Such precise control
transfection) were performed for the hMSCs cultured in the P1 and of mechanical properties provides a platform to vary the stiffness
P6 polymer gels. The DCAMKL1 silencing resulted in the increased and ligand density independently in these gel materials to extract
expression of RUNX2 for the P1 polymer gel, as well as for the their individual effects on stem cell fate. These culture systems
P6 polymer gel, but to a lesser extent (Supplementary Fig. 19). In mimic the stiffness (0.2–0.4 kPa) of all the adult stem cell niches
contrast, DCAMKL1 overexpression did not significantly alter the (for example: bone marrow, brain and adipose tissue) present in
expression of RUNX2 in the P1 polymer gel, whereas a significant the human body, and hence provide a physiologically relevant soft
decrease was observed for P6 (Supplementary Fig. 19). These data 3D microenvironment for stem cells. Thus, these gels allow one
confirm the functional relationship between the two proteins in to assess the effect of stress stiffening on stem cell differentiation
our gel system with DCAMKL1 being ‘upstream’ of RUNX2 with a in a biologically relevant stress regime (Fig. 1) and in a soft
switch-like relationship, along with the existence of a threshold value 3D physiologically mimicking microenvironment independent of
for the expression of DCAMKL1, which inhibits the expression of matrix stiffness, ligand density and matrix porosity, improving
RUNX2. In addition, these data are in agreement with the previous on the traditional synthetic ECM mimetic scaffolds. Moreover,
in vivo and in vitro study34 . the thermoresponsive behaviour of these soft materials should be


© 2016 Macmillan Publishers Limited. All rights reserved

advantageous for tissue engineering applications, as the material can recently very well characterized in a detailed in vitro and in vivo
be injected as a fluid, which then can form a gel in vivo (37 ◦ C). study, which has identified for the first time DCAMKL1 as a
In recent years, after the seminal study of Engler and co-workers, novel regulator of osteogenesis. For instance, depleting this protein
bulk matrix stiffness has emerged as the most important mechanical with specific shRNAs has increased the osteogenic differentiation
cue to direct stem cell differentiation17 , especially on 2D hydrogels. of hMSCs in vitro and Dcamkl1−/− mice exhibit an increase
However, recent works on stem cells in 3D hydrogels have iden- in osteoblast numbers, elevated bone mass, and increased rates
tified other crucial factors, such as cellular-traction-induced local of bone formation in vivo. Overexpressing DCAMKL1 in vitro
deformation of matrix (leading to integrin–ligand clustering)19 , cell- has further demonstrated that the ability of the protein to inhibit
mediated matrix degradation20 and mechanical constraints, as some bone growth occurs through antagonism of RUNX2 and requires
of the key regulators, acting in concert or independent of bulk its microtubule-binding domains. Moreover, the introduction of a
matrix stiffness to control cell fate in these materials. This has Dcamkl1-null allele in Runx2+/− mice has reversed the mutant
indicated that the actual mechanism of MSC lineage selection is phenotype, which has further confirmed in vivo that DCAMKL1
likely to be considerably more complex than solely being the result of is a negative regulator of RUNX2. Our work implicates this
a response to tissue matrix elasticity. For instance, altering the degree recently identified role of DCAMKL1 in the mechanism underlying
of cell spreading in 2D was shown to impact MSC osteogenic differ- stress-stiffening-mediated mechanotransduction. Moreover, the
entiation9 . This is not the case in vivo, where the process of spreading expression of RUNX2 in mesenchymal precursors is indeed known
in 3D is mechanically or physically constrained. In addition, in to be essential for their osteogenic commitment, and variations
most of the 3D studies, when comparing with 2D, the 3D cultures in the endogenous levels of DCAMKL1 were previously shown to
resulted in enhanced MSCs osteogenesis with increased osteogenic regulate osteoblast differentiation34 . In the present article, we have
gene expression, matrix formation and upregulated autocrine BMP2 established a correlation between the critical stress for the onset of
signalling36–39 . Our 3D culture system mediates MSCs osteogenesis stress stiffening of our 3D gels, the expression of DCAMKL1 and the
in a very soft microenvironment, similar to that of the bone marrow expression of RUNX2 (mRNA and protein). The protein expression
niche but different from the one reported in the study of Engler of DCAMKL1 indeed decreased with increasing critical stress of
et al.17 . Our results identify stress stiffening as a new key regulator the gels and the protein expression of RUNX2 was not detectable
of stem cell differentiation, and suggest that mechanical response for the gels with the lowest critical stress. The effect on RUNX2
(stiffening) of a matrix to cellular traction may be as important as expression was also observed on the mRNA level, although to a
bulk matrix stiffness in cellular fate determination. On the basis lesser extent, but still with a significant increase between the short
of the present data, although the stem cell commitment in our polymers (P1–P3) and the long polymers (P4–P6) (Fig. 3f). In fact
polymer gels was found to be independent of macroscopic ligand RUNX2 gene and protein expressions as well as its function were
density, a synergy between cell-mediated ligand clustering and stress shown to be very complex and regulated on multiple levels40–42 . In
stiffening cannot be ruled out at this point. particular, translational regulation was already identified as a major
Regarding the precise mechanism of how cells sense the substrate control point in RUNX2 gene expression in cells differentiating
mechanical properties, two main concepts have been proposed in along the osteoblastic lineage43 . Moreover, RUNX2 mRNA was
the literature. First, Engler and co-workers have put forward the shown to be expressed but dormant, with no protein expression
stiffness-dependence hypothesis, which highlights the importance in osteoblast precursors43 . We also probed the effect on RUNX2
of the bulk substrate stiffness in driving stem cell lineage specifica- mRNA expression after 9 h of culture for short and long polymers
tion17 . A second concept has been introduced more recently, which (P1 and P6), with no significant increase in the expression,
is the so-called ECM tethering hypothesis28 . In this case the cells indicating that osteogenesis was not initiated at this shorter time
do not directly sense the bulk stiffness of the underlying substrate point (Supplementary Fig. 20). Finally, the functional relationship
but instead respond to the mechanical feedback presented by cova- between DCAMKL1 and RUNX2 was further confirmed in our
lently anchored ECM molecules such as collagen. Alterations in the system via DCAMKL1 gene silencing and overexpression. Overall,
anchoring point distance may induce local stiffness modifications, these results could be related to the observed switch in hMSCs
which were indeed shown to guide stem cell fate determination as differentiation from adipogenesis to osteogenesis with increasing
the cell’s sensitivity to the substrate bulk stiffness was considerably critical stress in our 3D gels.
diminished. This issue has been avoided in our stress-stiffening DCAMKL1 contains indeed a conserved Doublecortin (DC)
hydrogels with the grafting of a short adhesion ligand via a single domain that can bind to tubulin and enhance microtubule
anchoring point. This enables direct mechanical sensing of the polymerization. Other proteins associating with the microtubules
matrix and direct application of traction forces—which is related to were also shown to play a role in hMSCs osteogenic differentiation.
the first concept, as it allows a more straightforward interpretation of This is the case of Stathmin-like 2 (Stmn2), which is upregulated
the mechanical properties sensing. However, in our case the cells can during osteoblast differentiation44 . In contrast to DCAMKL1,
deform the gels and respond to stress stiffening with modulation of stathmins are a class of microtubule-associated proteins that
the mechanical feedback to govern cell lineage commitment—which inhibit microtubule polymerization. In addition, other different
is related to the second concept. Stress-stiffening hydrogels may thus inhibitors of microtubule assembly were identified as stimulators
be seen as a new class of culture systems for mechanical sensing of BMP-2 transcription that increase osteogenic differentiation45 .
studies that allows us to unify the previously reported concepts, as This indicates the importance of the precise regulation of
the cells initially directly probe the matrix mechanical properties microtubule dynamics (polymerization and depolymerization) for
to apply traction forces which then trigger stress stiffening, the stem cell fate control. In fact, another Doublecortin-containing
response to which directs stem cell fate. microtubule-associated protein, DCX-EMAP, was shown to be
The exact sequence of events and molecular mechanisms involved in mechanotransduction of extracellular stimuli mediated
leading to stress-stiffening-mediated stem cell response still remain by the microtubule cytoskeleton46 . Moreover, a nanometre-
to be unravelled. In this article we have shown a potential scale cytoskeletal analysis has revealed that microtubules are
role of the microtubule-associated protein DCAMKL1 in the essential structures for transmitting stresses to activate signalling
mechanotransduction of stress stiffening. DCAMKL1 was shown cytoplasmic proteins. Strong activation sites of the Src kinase indeed
to regulate osteogenesis by antagonizing RUNX2, the master co-localized with microtubule large displacements and deformation
transcription factor for the osteoblast lineage34 . In fact, the sites. This suggested that signalling protein activation depends on
functional relationship between DCAMKL1 and RUNX2 has been the degree of microtubule deformation, which is inducing sufficient


© 2016 Macmillan Publishers Limited. All rights reserved

conformational changes of this protein for its activation47 . To extend 10. McNamara, L. E. et al. The role of microtopography in cellular
the role of the microtubule-associated proteins, a recent report mechanotransduction. Biomaterials 33, 2835–2847 (2012).
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two proteins are not interacting together, as revealed by the absence 15. Cheng, Z. A., Zouani, O. F., Glinel, K., Jonas, A. M. & Durrieu, M. C. Bioactive
of association in coimmunoprecipitation experiments and given chemical nanopatterns impact human mesenchymal stem cell fate. Nano Lett.
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required for the antagonism34 . 16. Fu, J. et al. Mechanical regulation of cell function with geometrically
Altogether, our results and the previous reports have established modulated elastomeric substrates. Nature Methods 7, 733–736 (2010).
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Methods 29. Wilson, M. J., Liliensiek, S. J., Murphy, C. J., Murphy, W. L. & Nealey, P. F.
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Received 30 October 2014; accepted 20 October 2015; biopolymer gels. Soft Matter 6, 4120–4127 (2010).
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41. Xiao, Z.-S., Simpson, L. G. & Quarles, L. D. IRES-dependent translational Acknowledgements
control of Cbfa1/Runx2 expression. J. Cell. Biochem. 88, 493–505 (2003). This work was supported by NWO grant 728.011.102 (R.K.D.), Gravitation grant
42. Galindo, M. et al. The bone-specific expression of Runx2 oscillates during the Functional Molecular System (A.E.R.), NanoNext grant 3D.12 (R.H.), Histide AG and
cell cycle to support a G1-related antiproliferative function in osteoblasts. Histide Lab. We acknowledge P. Kouwer for discussions on stress stiffening. We also
J. Biol. Chem. 280, 20274–20285 (2005). acknowledge K. Blank for useful discussions on biofunctionalization. We also thank
R. Nolte for his interest in the work and useful general suggestions.
43. Sudhakar, S., Li, Y., Katz, M. S. & Elango, N. Translational regulation is a
control point in RUNX2/Cbfa1 gene expression. Biochem. Biophys. Res.
Commun. 289, 616–622 (2001). Author contributions
44. Chiellini, C. et al. Stathmin-like 2, a developmentally-associated neuronal R.K.D., O.F.Z. and A.E.R. conceived and initiated the project. R.K.D. and R.H.
marker, is expressed and modulated during osteogenesis of human synthesized the polymers and did the rheological characterization. V.G. and O.F.Z.
mesenchymal stem cells. Biochem. Biophys. Res. Commun. 374, 64–68 (2008). performed the stem cell experiments. R.H. performed the AFM experiments. O.F.Z.,
45. Zhao, M. et al. Inhibition of microtubule assembly in osteoblasts stimulates R.K.D., V.G., R.H. and A.E.R. analysed the data. R.K.D., V.G., A.E.R. and O.F.Z. wrote the
bone morphogenetic protein 2 expression and bone formation through manuscript. A.E.R. and O.F.Z. supervised the project.
transcription factor Gli2. Mol. Cell. Biol. 29, 1291–1305 (2009).
46. Bechstedt, S. et al. A doublecortin containing microtubule-associated protein is
implicated in mechanotransduction in Drosophila sensory cilia. Nature Additional information
Commun. 1, 1–11 (2010). Supplementary information is available in the online version of the paper. Reprints and
permissions information is available online at For
47. Na, S. et al. Rapid signal transduction in living cells is a unique feature of
polyisocyanopeptide-based hydrogel materials, contact
mechanotransduction. Proc. Natl Acad. Sci. USA 105, 6626–6631 (2008). Correspondence and requests for materials should be addressed to R.K.D., O.F.Z.
48. Terenna, C. R. et al. Physical mechanisms redirecting cell polarity and cell or A.E.R.
shape in fission yeast. Curr. Biol. 18, 1748–1753 (2008).
49. Balaban, N. Q. et al. Force and focal adhesion assembly: A close relationship
studied using elastic micropatterned substrates. Nature Cell Biol. 3, Competing financial interests
466–472 (2001). The authors declare no competing financial interests.


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Methods and attains the final stiffness in 2–3 min. This favours the supporting of cells in 3D
Azide-functionalized polymer synthesis (General procedure)50 . A solution of rather than the cells settling at the bottom. After gel formation, the two cover slips
catalyst Ni(ClO4 )2 · 6H2 O (1 mM) in toluene/ethanol (9:1) was added to a solution were removed and alpha-MEM medium (without serum) was added. All cell
of non-functionalized monomer 1 and azide-appended monomer 2 in freshly culture experiments were carried out without any serum in the medium for the first
distilled toluene (50 mg ml−1 total concentration; molar ratio 1/2 = 100) in the 6 h of culture12 . Then, alpha-MEM medium with 10% serum was added. All cells
required amount and the reaction mixture was stirred at room temperature (20 ◦ C) were used at low passage numbers (≤passage 4), were subconfluently cultured and
for 72 h. The resultant polymer was precipitated three times from were seeded at 106 cells ml−1 for the purpose of the experiments and to avoid
dicholoromethane in di-isopropyl ether and dried overnight in air. The polymer cell–cell contacts. The lineage commitment and differentiation of the gel
was characterized by rheology, viscometry and AFM analysis. encapsulated hMSCs after 96 h and three weeks of culture, respectively, were
Synthesis of P10 : The catalyst to monomer (1 + 2) molar ratio used: 1/1,000; investigated with bipotential differentiation medium (1:1 v/v osteogenic and
Synthesis of P20 : The catalyst to monomer (1 + 2) molar ratio used: 1/2,500; adipogenic media, Lonza). For all the experiments, a non-functionalized soft
Synthesis of P30 : The catalyst to monomer (1 + 2) molar ratio used: 1/3,000; polymer gel (cell culture in growth medium) served as control. The live/dead
Synthesis of P40 : The catalyst to monomer (1 + 2) molar ratio used: 1/4,000; viability assay at three weeks in these control gels indicated excellent cell viability
Synthesis of P50 : The catalyst to monomer (1 + 2) molar ratio used: 1/6,000; (Supplementary Fig. 22). The pharmacological agents used were 50 µM Blebbistatin
Synthesis of P60 : The catalyst to monomer (1 + 2) molar ratio used: 1/8,000. (EMD Biosciences-Calbiochem), 1 µM cytochalasin D (Sigma) and 50 nM Taxol
(Abcam). The hMSCs were exposed to each pharmacological agent for 1 h, 24 h
Conjugation of azide-functionalized polymers with GRGDS peptide. The and 72 h, respectively, after seeding on a modified polymer. For antibody inhibition
GRGDS peptide was dissolved in borate buffer (pH 8.4) at a concentration of studies, cells were preincubated with 5 ng ml−1 anti-α1, 2, 3 and 5-β1, 2 (all from
2 mg ml−1 . A solution of BCN–NHS in DMSO was added to the peptide solution in Santa Cruz Biotechnology). For competition experiments with soluble RGD
borate buffer in 1:1 molar ratio and stirred on roller mixer for 3 h at room peptides, the cells were incubated in 1 ml of cell culture media containing 200 µg of
temperature (20 ◦ C). The formation of BCN–GRGDS conjugate was confirmed by RGDS peptides for 20 min on plastic and then transferred to the polymer gels. To
mass spectrometry. MS calc.: 910.4, obtained: 911.4 evaluate proliferation, total double-stranded DNA content was determined by
The azide-functionalized polymer (P10 –P60 ) was dissolved in acetonitrile at a using the PicoGreen assay as previously reported51 .
concentration of 3 mg ml−1 . To this solution, the appropriate volume of
BCN–GRGDS solution in borate buffer (based on the molar equivalent of azide Confocal microscopy. To assess the homogeneous distribution of cells in our
functions of the polymer) was added. The mixture was allowed to stir on roller hydrogels, very thin slices of the gel were cut transversely at various depths,
mixer for 72 h at room temperature (20 ◦ C). The resultant polymer–peptide including the two interfaces. The fluorescently labelled cells encapsulated in the gel
conjugates (P1–P6) were precipitated by adding the reaction mixture dropwise to slices were imaged by confocal microscopy with a Leica SP5 confocal microscope,
di-isopropyl ether. ×10 objective, 0,3 NA. 400 µm thick z-stacks were then acquired every 2.39 µm
and the 3D images were reconstructed by using the Imaris 7.0 software.
Determination of the amount of azides on the azide-functionalized polymer.
A dichloromethane solution of BCN-conjugated lissamine dye was added to a MTT assay. As described in literature52 , briefly, cell viability was determined by the
dichloromethane solution of the polymer (1 mg ml−1 ) in 1:1.2 molar ratio with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and
respect to the calculated amount of azides in an azide polymer. The reaction the data are presented as a percentage of control viability.
mixture was rotated at 15 r.p.m. in the dark for 12 h at room temperature (20 ◦ C).
The polymer–dye conjugate was precipitated four times from dichloromethane in Live/dead staining. Cell viability was determined with the live/dead
di-isopropylether, dried in air overnight, re-dissolved in dichloromethane, after viability/cytotoxicity kit (Molecular Probes), according to the manufacturer’s
which the absorption spectra were recorded. The extinction coefficient of protocol.
138,428 l mol−1 cm−1 was used at a wavelength of 559 nm to determine the amount
of dye attached to the polymer, and thus to calculate the amount of azide present on Real-time PCR analysis of gene expression. RT-PCR was performed as previously
the polymer (Table 1). described53,54 . Briefly, total RNA was extracted by using the RNeasy total RNA kit
from Qiagen in accordance with the manufacturer’s instructions. Purified total
Rheology analysis. The polymers were dissolved at a concentration of 2 mg ml−1 in RNA was used to make cDNA by reverse transcription reaction (Gibco BRL) by
alpha-MEM (without serum) by gentle rotation (7–8 r.p.m.) at 4 ◦ C on a 90◦ rotor using random primers (Invitrogen). Real-time PCR was performed by using SYBR
for 36 h. For determining the bulk stiffness of the gel, a variable temperature green reagents (Bio-Rad). The data were analysed by using the iCycler IQTM
rheology was performed (plate–plate geometry; 250 µm geometry gap), by heating software. The cDNA samples (1 µl in a total volume of 20 µl) were analysed for the
the solution from 5 to 37 ◦ C at a heating rate of 2 ◦ C min−1 at a constant strain of gene of interest and for the house-keeping gene GAPDH. The comparison test of
2% and constant frequency of 1 Hz. This experiment was immediately followed by the cycle-threshold point was used to quantify the gene expression level in each
a time sweep experiment (5 min) at 37 ◦ C at a constant frequency of 1 Hz; the G’ sample. The primers used for the amplification are listed in Supplementary Table 1.
observed at the end of the experiment was taken as the equilibrium bulk stiffness of
the gel at this temperature. For nonlinear rheology, the previously described Western blotting. After 96 h, the polymer gels were exposed to a cold environment
pre-stress protocol30 was employed immediately after the aforementioned time (around 10 ◦ C). The cell pellet was obtained by centrifugation. The cells were
sweep experiment. permeabilized (10% SDS, 25 mM NaCl, 10 nM pepstatin and 10 nM leupeptin in
distilled water and loading buffer), boiled for 10 min and resolved by reducing
Atomic force microscopy. To visualize individual polymer chains and determine PAGE (Invitrogen). Proteins were transferred onto nitrocellulose, blocked, and
the average length of the polymers, solutions (∼1 µg ml−1 in CHCl3 ) were spin labelled with HRP-conjugated antibodies (Invitrogen). The microtubule-associated
coated (300 r.p.m. for 20 s) on freshly cleaved mica substrates and imaged by using protein DCAMKL1 was blotted by using the monoclonal anti-DCAMKL1 antibody
AFM tapping mode. Polymer lengths were determined by using the ImageJ (Santa Cruz Biotechnology). The transcriptional factor RUNX2 was blotted by
software. The lengths of at least 150 polymer chains were counted to obtain the using the monoclonal anti-Runx2 antibody (Abcam). The western blots in these
distribution and the mean of the polymer chain length for any particular sample. experiments were run in triplicate, along with an additional blot for tubulin and
Coomassie Blue staining to ensure consistent protein load between samples. To
Cell culture. Human mesenchymal stem cells (hMSCs) (Poietics human construct the plot of the relative intensities of RUNX2 versus DCAMKL1 (Fig. 4a)
mesenchymal stem cells) were purchased from Lonza and verified to be free of and to illustrate the switch-like relationship between the two proteins, the ‘zero’ of
mycoplasma by the manufacturer. The cells were subsequently verified to be free of the RUNX2 relative intensity was set at the corresponding level of expression of
mycoplasma also by using the LookOut Mycoplasma PCR Detection Kit (Sigma). RUNX2 in hMSCs cultured on plastic (Supplementary Fig. 18a), which was set to 1.
Cells were then cultured in Alpha-MEM medium (Invitrogen) supplemented with
10% fetal bovine serum (FBS), 1% penicillin/streptomycin and incubated in a Immunostaining. After 96 h or three weeks of culture, the gels were exposed to
humidified atmosphere containing 5% (v/v) CO2 at 37 ◦ C. For the encapsulation of cold environment (∼10 ◦ C), the cell pellet was collected from the fluid by
cells in the gels, first, the cell pellets were obtained by centrifugation. Then 500 µl of centrifugation, transferred onto the well plate and allowed to adhere to the well
the cold polymer solution (∼10 ◦ C) was added directly to the pellet, followed by a plate surface by culturing in Alpha-MEM with serum for 16 h. The cells were then
gentle pipetting up and down three to four times to ensure a homogeneous mixture fixed for 20 min in 4% paraformaldehyde/PBS at ∼37 ◦ C. After fixation, the cells
that was directly put onto a cover slip in a six-well plate (also kept cold). Thereafter, were permeabilized in a PBS solution of 1% Triton X-100 for 15 min. The cells were
the solution was sandwiched between two cover slips and the well plate was then incubated with primary antibody (mouse anti-vinculin for adhesion, mouse
transferred to a 37 ◦ C incubator. The volume of the suspension was chosen (500 µl) anti-STRO-1 for differentiation) for 1 h at 37 ◦ C. After washing, cells were stained
to obtain hydrogel thickness in the range of 3 mm. The polymer solution forms a with Alexa Fluor 647 rabbit anti-mouse IgG secondary antibody for 30 min at
gel immediately after incubation at 37 ◦ C, as revealed by kinetic rheology ∼37 ◦ C. Cell cytoskeletal filamentous actin (F-actin) was visualized by treating the
experiments (Supplementary Fig. 21). Afterwards, the gel becomes stiffer with time cells with 5 U ml−1 Alexa Fluor 488 Phalloidin (Sigma, France) for 1 h at 37 ◦ C.


© 2016 Macmillan Publishers Limited. All rights reserved

Vinculin was visualized by treating the cells with 1% (v/v) monoclonal Super-fectamine (Qiagen) according to the manufacturer’s recommendations. The
anti-vinculin (clone hVIN-1 antibody produced in mouse) for 1 h at 37 ◦ C. The efficiency of the DCAMKL1 overexpression was assessed by western blot for
cells were then stained with Alexa Fluor 568 (F(ab0 )2 fragment of rabbit anti-mouse hMSCs cultured on plastic. A 180–200% increase in protein level was observed
IgG(H + L)) for 30 min at room temperature. After 96 h, Osterix was visualized by after 72 h.
treating the cells with 1% (v/v) rabbit monoclonal anti-Osterix (antibody produced
in rabbit) for 1 h at 37 ◦ C. The cells were then stained with Alexa Fluor 568 (F(ab0 )2 DCAMKL1 shRNA silencing. DCAMKL1 silencing has been performed by
fragment of mouse anti-rabbit IgG(H + L)) for 30 min at room temperature. transfecting hMSCs with a pool of three target-specific lentiviral vector plasmids
Tubulin (stained by Anti-Tubulin β3 (Sigma, France) was visualized by treating the each encoding 19–25 nt (plus hairpin) shRNAs designed to knock down gene
cells with 1% (v/v) monoclonal anti-Tubulin β3 (Abcam, Cambridge), for 1 h at expression (Santa Cruz Biotechnology). A mock plasmid was transfected as a
37 ◦ C and then with Alexa Fluor 588 (F(ab0 )2 fragment of goat anti-rabbit control. Transient transfection was performed by using Lipofectamine 2000
IgG(H + L)) for 30 min at room temperature. There was no detection of the muscle (Invitrogen) according to the manufacturer’s protocol. The efficiency of the
transcription factor MyoD1 (stained with anti-MyoD1 (Santa Cruz Biotechnology, DCAMKL1 silencing was assessed by western blot for hMSCs cultured on plastic.
USA)). To stain lipid fat droplets, the cells were fixed in 4% paraformaldehyde, The DCAMKL1 silencing decreased DCAMKL1 mRNA level by 50–60% (not
rinsed in PBS and 60% isopropanol, stained with 3 mg ml−1 Oil Red O (Sigma, shown) and DCAMKL1 protein level by 60–70% after 24 h.
France) in 60% isopropanol and rinsed in PBS at ∼37 ◦ C. For the purpose of
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