Stress-stiffening-mediated stem-cell commitment Stress ...10.1038... · Supplementary Information...

Supplementary Information

Stress-stiffening-mediated stem-cell commitment

switch in soft responsive hydrogels

Rajat K. Das, #*1

Veronika Gocheva,#2,3

Roel Hammink1, Omar F. Zouani,

*2,3 Alan E.

Rowan*1

1 Institute for 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.

* Corresponding author. E-mail: [email protected], [email protected] or [email protected]

# These authors contributed equally to this work.

Stress-stiffening-mediated stem-cell commitmentswitch in soft responsive hydrogels

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NMAT4483

NATURE MATERIALS | www.nature.com/naturematerials 1

© 2015 Macmillan Publishers Limited. All rights reserved

http://dx.doi.org/10.1038/nmat4483

Supplementary Figure 1. (a)-(f) Representative AFM images of dilute (1 µg / mL) chloroform

solution of polymers P1-P6, respectively, drop cast on freshly cleaved mica. Scale bar: 500 nm

for (a)-(e) and 1 µm for (f).

a b c

d e f

P1 P2 P3

P4 P5 P6


Supplementary Figure 2. (a)-(f) Histograms showing the length distribution of single polymer

chains in individual polymers (P1-P6), as obtained by observing at least 150 single polymer

chains in AFM for dilute (1 µg / mL) chloroform solution of the polymers, drop cast on freshly

cleaved mica. These data were used to obtain the mean polymer length for the polymers.


Supplementary Figure 3. (a) The non-linear rheology data of polymer gels P1-P6 in alpha-

MEM at 2 mg/ mL. The differential modulus Kʹ, scaled with storage modulus Go, plotted as a

function of applied stress σ. The experiments were repeated in triplicate and the mean plots are

shown. (b) Overlay of the plots of differential modulus Kʹ as a function of applied stress σ, for

the gels of shortest (P1) and the longest (P6) polymer. σC denotes the critical stress for the onset

of stress-stiffening.

ba


Supplementary Figure 4. Confocal microscopy 400µm Z-stack side views (X,Z projections)

images showing the top, middle and bottom distribution of gel-encapsulated hMSCs (after 96 h

of culture) stained with calcein-AM. Scale bar: 200µm.

top

bottom

Side view

x

z


Supplementary Figure 5. Representative micrographs showing cross-sections of hMSCs (DAPI

staining) 36 h after encapsulation into 3D soft hydrogel. On the right, hMSCs are shown in

Bright-field, DAPI, F-actin and Vinculin stainings. Left scale bar: 50 µm and right scale bar: 10

µm.

50 m

BF

D

AP

I F

-Actin

V

inculin

10 m

36h

DAPI


Supplementary Figure 6. Total DNA content for short and long polymers (P1, P3, P4 and P6)

after 48 h and 96 h of cell culture as determined by the PicoGreen assay. A non-functionalized

hydrogel served as a control. Error bars, s.e.m. (n ≥ 3). NS. not significant (P > 0.05), Student’s

t-test.

N.S

0

5

10

15

20

25

30

Co

ntr

ol

P1

P3

P4

P6

DN

A c

on

ten

t (n

g)

48h

96h


Supplementary Figure 7. Mean percentages of hMSC osteogenesis (a) and adipogenesis (b) in

different hydrogel materials from polymers P1-P6 after 96 h of culture in bipotential medium. A

non-functionalized hydrogel (cell culture in growth medium) served as a control. Error bars,

s.e.m. (n ≥ 3). NS. not significant (P > 0.05), *P < 0.05; Student’s t-test.

0

10

20

30

40

50

60

P1

P2

P3

P4

P5

P6

Os

teo

ge

ne

sis

(%

)

0

5

10

15

20

25

30

P1

P2

P3

P4

P5

P6

Ad

ipo

ge

ne

sis

(%

)

a b

*

N.S

N.S N.S

N.S

*

Co

ntr

ol

Co

ntr

ol


Supplementary Figure 8. (a) Immunofluorescent staining for OPN (red), actin (green), and

nucleus (blue) in hMSCs encapsulated in different hydrogel materials from polymers P1-P6 after

3 weeks of culture in bipotential medium. Scale bar: 100 µm. (b) Total cellular

immunofluorescence intensity of osteopontin quantified for hMSCs cultured for 3 weeks in

different matrices. (c) Triglyceride detection on hMSCs after 3 weeks on cell culture. A non-

functionalized hydrogel (cell culture in growth medium) served as a control for all the

experiments. Error bars, s.e.m. (n ≥ 3). NS. not significant (P > 0.05), *P < 0.05; Student’s t-test.

a

b

P1 P2 P3 P4 P5 P6 Control

100 m

N.S

N.S

*

0

5

10

15

20

25

Co

ntr

ol

P1

P2

P3

P4

P5

P6

Tri

gly

ceri

de D

ete

cti

on

(M

) 0

2

4

6

8

10

12

14

Con

tro

l

P1

P2

P3

P4

P5

P6

Rela

tive I

nte

ns

ity

OPN

c

*

N.S

N.S


Supplementary Figure 9. hMSCs commitment studies after 96 h of culture in different polymer

gels in the presence of integrin antibodies α1, 2, 3, 5 and β1, 2. (a) The quantitative PCR analysis

for RUNX2. No significant differences were observed between the highest critical stress matrix

(P6) and the lower critical stress matrices. (b) Total cellular immunofluorescence area of neutral

lipid accumulation in hMSCs. For all the experiments, a non-functionalized hydrogel (cell

culture in growth medium) served as the control (*P < 0.01).

0

0,5

1

1,5

2

2,5

Co

ntr

ol

P1

P3

P4

P6

Re

lati

ve

mR

NA

le

ve

l

RUNX2

0

1

2

3

4

5

6

7

Co

ntr

ol

P1

P3

P4

P6

Re

lati

ve

Are

a

LIP

a

N.S N.S

N.S

*

b


Supplementary Figure 10. Mean percentages of hMSC osteogenesis (a) and adipogenesis (b) in

different polymer gels in the presence of integrin antobodies α1, 2, 3, 5 and β1, 2. A non-

functionalized hydrogel (cell culture in growth medium) served as the control. Error bars, s.e.m.

(n ≥ 3). NS. not significant (P > 0.05), *P < 0.01; Student’s t-test.

a b

N.S

0

2

4

6

8

10

12

14

16

18

20

Con

tro

l

P1

P3

P4

P6

Os

teo

ge

ne

sis

(%

)

0

5

10

15

20

25

30

35

40

45

50

Co

ntr

ol

P1

P3

P4

P6

Ad

ipo

ge

ne

sis

(%

)

N.S *

N.S


Supplementary Figure 11. The amount of STRO-1 present in the hMSCs is expressed as

average fluorescent density, normalized with the number of cells. Compared to control (non-

functionalized hydrogel), STRO-1 activity is present on matrices (+ blebbistatin), indicating that

the hMSCs have kept “stemness” after 96 h of culture in bipotential medium.

0

0,2

0,4

0,6

0,8

1

1,2

Co

ntr

ol

P1

P3

P4

P6

Re

lati

ve

ST

RO

-1 I

nte

ns

ity

N.S

+ Blebbistatin


Supplementary Figure 12. hMSCs commitment studies after 96 h of culture in different

polymer gels in the presence of soluble RGD ligands. (a) Quantitative PCR analysis for RUNX2.

No significant increase in RUNX2 gene expression was observed for all the matrices and no

significant differences could be detected between the higher critical stress matrices (P4 and P6)

and the lower critical stress matrices (P1 and P3) in the presence of soluble RGD. (b) Total

cellular immunofluorescence area of neutral lipid accumulation quantified for hMSC cultured for

96 h in different matrices. No significant increase in lipid accumulation was observed for all the

matrices and no significant differences could be detected between the different polymers. For all

the experiments, a non-functionalized soft polymer gel (cell culture in growth medium) served as

a control. NS. not significant (P > 0.05). Student’s t-test.

a b

0

0,5

1

1,5

2

2,5

Co

ntr

ol

P1

P3

P4

P6

Re

lati

ve

Are

a

LIP

N.S

0

0,4

0,8

1,2

1,6

2

Co

ntr

ol

P1

P3

P4

P6

Re

lati

ve

mR

NA

le

ve

l

RUNX2

N.S


Supplementary Figure 13. Mean percentages of hMSC osteogenesis (a) and adipogenesis (b)

for different polymer gels (P1, P3, P4 and P6) in the presence of soluble RGD ligands. A non-

functionalized hydrogel served as a control. Error bars, s.e.m. (n ≥ 3). NS. not significant (P >

0.05), Student’s t-test.

a b

0

2

4

6

8

10

12

14

16

Co

ntr

ol

P1

P3

P4

P6

Os

teo

ge

ne

sis

(%

)

N.S

0

2

4

6

8

10

12

14

16

Co

ntr

ol

P1

P3

P4

P6

Ad

ipo

ge

ne

sis

(%

) N.S


Supplementary Figure 14. Commitment of hMSCs at different ligand densities (7 nm, 28 nm

and 70 nm) in polymer gels of lowest and highest critical stress (P1 and P6, respectively). (a)

The quantitative PCR analysis for RUNX2, and (b) Total cellular immunofluorescence area of

neutral lipid accumulation quantified for hMSC cultured for 96 h in bipotential medium. For all

the experiments, a non-functionalized soft polymer gel (cell culture in growth medium) served as

the control (*P < 0.005).

0

0,5

1

1,5

2

2,5

3

3,5

4

7nm 28nm 70nm

Re

lati

ve

mR

NA

le

vel

RUNX2

0

0,5

1

1,5

2

2,5

3

3,5

4

7nm 28nm 70nm

Re

lati

ve

Are

a

LIP

P1

P6

a b

* * * * * *


Supplementary Figure 15. Immunofluorescent staining for MyoD, Tubulin β3 and Osterix in

hMSCs encapsulated in different hydrogel materials from polymers P1-P6 in presence of (a)

Cytochalasin D and (b) Taxol (inhibitor of actin and inductor of microtubule cytoskeleton

polymerization, respectively) after 96 h of culture in bipotential medium. Control condition

consists of naïve hMSCs encapsulated in non-functionalized soft hydrogel and cultured in

growth medium.

0

0,5

1

1,5

2

2,5

3

3,5

Contr

ol

P1

P2

P3

P4

P5

P6 R

ela

tiv

e I

nte

ns

ity

/Are

a

0

0,5

1

1,5

2

2,5

3

3,5

Contr

ol

P1

P2

P3

P4

P5

P6 R

ela

tiv

e I

nte

ns

ity

/Are

a

LIP

Tubulin 3

MyoD

Osterix

+ Cytochalasin D + Taxol

a b


Supplementary Figure 16. Calcein-AM live-dead viability test (live cells fluoresce green) at

different time points (48 h and 96 h) for hMSCs treated with Taxol and encapsulated in polymer

gel P3 in growth medium. Scale bar: 20 µm.

48h 96h

Taxol


Supplementary Figure 17. Representative bright-field images (Oil red O staining) of hMSCs

encapsulated in different gels from polymers P1-P6 after 96 h of culture in bipotential

(osteogenic/adipogenic) medium. Scale bar: 10 µm.


Supplementary Figure 18. (a) Western blot showing RUNX2 protein expression for hMSCs

cultured on plastic for 24 h and 96 h. (b) Relative protein expression intensity for RUNX2 and

DCAMKL1 in different polymer gels (P1-P6) quantified from the Western blot presented in

Main Fig. 4a and showing a switch-like relationship between the two proteins.

a b

0

50

100

150

0 2 4 8

Pro

tein

qu

an

tity

(a

.u.)

RUNX2

DCAMKL1

P1 P2 P3 P4 P5 P6

RUNX2 - 57

- 42

24h 96h kDa

-actin


Supplementary Figure 19. (a) shRNA mediated DCAMKL1 silencing was induced, as shown

by western blot for hMSCs cultured on plastic for 24h. (b) Overexpression of DCAMKL1 was

enabled by transient transfection with a plasmid subcloned with the DCAMKL1 gene, as shown

by western blot for hMSCs cultured on plastic for 72h. (c) Non transfected cells or cells at 48 h

post-transfection (mock plasmid, shRNA or DCAMKL1) were incorporated into RGD-bound

hydrogels (P1 or P6) for 96h. Relative RUNX2 mRNA levels were then measured by using RT-

PCR. Non-transfected cells incorporated into non-functionalized hydrogels served as a control.

*P < 0.001; ** represents the comparison between RGD-Polymers and +DCAMKL1 for the P6

hydrogel and P < 0.05 ; Student’s t-test.

b c a

- 82

- 42

kDa

-actin DCAMKL1 - 82

- 42

kDa

Sh-DCAMKL1

- +

actin

DCAMKL1

+ DCAMKL1

- +

+

*

*

* *

0

1

2

3

4

5

6

7

P1 P6

Re

lati

ve

mR

NA

lev

el

RUNX2

Control

RGD-Polymer

RGD-Polymer Mock

RGD-Polymer

sh-DCAMKL1

RGD-Polymer

DCAMKL1

-


Supplementary Figure 20. hMSCs commitment studies after 9 h of culture in different polymer

gels by Quantitative PCR analysis for RUNX2. No significant increase in RUNX2 gene

expression was observed for all the matrices and no significant differences could be detected

between the higher critical stress matrix (P6) and the lower critical stress matrix (P1). NS. not

significant (P > 0.05). Student’s t-test.

0

1

Co

ntr

ol

P1

P6

Re

lati

ve

mR

NA

lev

el

RUNX2

N.S


Supplementary Figure 21. Representative time sweep rheology experiments after incubating

the cold polymer solution at 37 oC at an applied strain of 1% at a frequency of 1 Hz.

0 50 100 150 200 250 300 350 400 450 500 550 600

1

10

100

1000

Sto

rag

e (

G')

an

d L

oss (

G'')

mo

du

lus (

Pa)

Time (s)

P1

P4

G'

G''

Kinetic experiment @ 37 oC


Supplementary Figure 22. Live-dead viability test at different time points, for hMSCs

encapsulated in control polymer gels (non-functionalized) in growth medium by (a) calcein-AM

(live cells fluoresce green), and by (b) MTT assay. Scale bar: 20 µm.

0

10

20

30

40

50

60

70

80

90

100

Pla

stic

4

14

21

MT

T (

% c

on

tro

l)

Time (days)

a b

4d 14d 21d


Supplementary Table 1. Nucleotide sequences of primers used for quantitative RT-PCR

detection.

Gene Primer Sequences

Runx2 5'-GACGTGCCCAGGCGTATTTC-3'(Forward)

5'-AAGTCTGGGGTCCGTCAAGG-3'(Reverse)

PPAR 5'-GGCTTCATGACAAGGGAGTTTC-3‘'(Forward)

5'-AACTCAAACTTGGGCTCCATAAAG-3‘ (Reverse)

GAPDH 5'-GCAGTACAGCCCCAAAATGG-3'(Forward)

5'-ACAAAGTCCGGCCTGTATCCAA-3'(Reverse)


Stress-stiffening-mediated stem-cell commitment Stress ...10.1038... · Supplementary Information...

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