4. Determination of cell fate
Stimuli affecting
Stem cell differentiation
Soluble Chemical Mechanical
Morphology
Factors Properties “Stimuli”
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5. Tissue’s Young Modulus
Tissue elastic modulus (E) is given by the resistance offered by the
tissues to deformation effects, i.e. the tissue stiffness.
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6. Substrates for cell culture
Polystyrene (Plastic) Polydimethylsiloxane Polyacrylamide
(PDMS) (PAAm) gel
Optically clear Optically clear
Amenable to many Flexible Highly water-absorvent
different surface Inert Insoluble
treatments Insoluble in medium Tunable mechanical
culture properties
Limitations non-toxic
Poor chemical resistance non-flammable Limitations
Good biocompatibility Citotoxity
Gas permeability E=[1-100]kPa
E = [3000-3600] MPa E = [-] MPa
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7. Cell mechanosensing process
Integrins Cell binds to
are inactive the substrate
MSC’s
Hard substrate
Activation and – maturation of
FA maturation clustering of FA – stress
due to integrins – focal fibers formation
mechanical adhesion
stimuli formation (FA)
Soft substrate –
not enough
forces to form
FA
Cell shape,
Cytoskeletal
migration,
and signaling
growth,
proteins (FAK)
differentiation,
are recruited
apoptosis
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8. Remarkable studies
Disher, A.E., et al (2005) Chen, C.S., et al,(1997)
McBeath R, et al.(2004)
MSCs in PDMS become:
Small islands of ECM –
Soft substrate – adipogenic adipogenic fate
profile; Intermediate stiffness
- myogenic profile; Hard Bigger islands of ECM –
substrate - osteogenic profile ostogenic fate
Boonen, K.J., et al (2009) Saha K.,et al (2008)
Increasing the matrix Neuronal adult stem
stiffness the cellular cells:
proliferation will also
increase, in muscle Stiff matrix – glial cells
skeletal stem cells Soft matrix - neurons
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9. Remarkable studies
In 2005 Engler A.J., Disher A.E, et al, showed that mesenchymal stem
cells are differentiated into different cells due to the substrate stiffness
[1].
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10. MSCs differentiate or migrate first?
Pathological:
Myocardial infarction
8.761.5 kPa/mm
Normal tissue variation:
Myocardium
MSC differentiate or migrate first? 0.660.9 kPa/mm
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11. In vivo
• In vitro
• Elasticity is • Elasticity is elasticity
static dynamic gradients
In vitro Solution
Durotaxis –The movement of a cell along a rigidity gradient
What happen in a stiffness gradient of 1 kPa/mm in
the absence of other stimuli?
12. Human MSCs were cultured on a collagen I-coated photopolymerized
polyacrylamide (PA) hydrogel of varying stiffness.
Hydrogel characterization
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13. Migration and proliferation of MSCs on hydrogels.
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14. Spatial distribution of mitomycin C-treated MSCs on gradient hydrogels.
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15. A B
(A) Morphological changes of MSCs cultured on static 11 kPa hydrogels. (B) Quantification of MyoD intensity
for cells cultured on static 11kPa hidrogels over time. Gray circle represent the MyoD intensity of C2C12
myoblast cultured in the same conditions.
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16. A B C
(a) Morphological changes in cells stained with Hoescht 33342 (blue) and phalloidin (red) can be observed as a function of culture time
and stiffness in MSCs cultured on gradient hydrogels. (b) Immunofluorescent staining for MyoD (green) and phalloidin (red) observed
as a function of culture time and stiffness in MSCs cultured on gradient hydrogels. (c) Immunofluorescent staining for MyoD (green)
and phalloidin (red) in a C2C12 myoblast cultured for 1 day on a static 11 kPa hydrogel.
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17. (a) MSCs were cultured on 1 and 11 kPa static (top) and gradient (bottom) hydrogels and stained for b3
tubulin (red) and MyoD (green). Open arrowheads indicate cells expressing either b3 tubulin or MyoD
while filled arrowheads indicate doubly stained cells.
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18. Hypothesis: Grow embryonic stem cells (ESCs) on hydrophobic PDMS
substrate with varying stiffness (0.041-2.7MPa) can influence ESCs
differentiation.
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Source: http://www.gghjournal.com/volume22/4/ab03.cfm
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20. 0.041 MPa <PDMS< 2.7MPa, Tricalcium Phosphate (TCP)
Cell attachment after 24h in PDMS and TCP Cell morphology after 24h in PDMS and TCP
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20
21. Cell perimeter after 24 in PDMS and TCP Phalloidin staining of cytoskeletal acin
after 2 hours
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22. Western Blots for pFAK in cells adherent
in PDMS, TCP and fibronectin after 1hour
Total cell number per well vs time
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23. Gene expression in day 6:
Primitive Primitive streak and mesendoderm Anterior Neuroepithelium Cadherins
ectoderm precursors mesendoderm
Primitive
endoderm
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24. Conclusions
In embryonic stem cells it was verified that:
•Adhesion did not suffer significant alterations with the
increasing of the substrate stiffness
•There were an increasing in cell spreading and proliferation
increasing the stiffness
• Genes expressed in the primitive streak and nascent
mesendoderm (FOXA2. Brachury, MixlI, Cdh2 and Eomes) are
up-regulated in stiffer mediums with osteogenic differentiation
•These genes are down-regulated in soft surfaces.
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25. Conclusions
In mesenchymal stem cells it was verified that:
• MSCs migrate to stiffer matrix (durotaxis) and then differentiate into a more
contractile myogenic phenotype.
• phenotype is not completely determined by the stiff hydrogel as some cells retain
expression of a neural marker.
• stiffness variation, not just stiffness alone, can be an important regulator of MSC
behavior.
Limitations:
• MSC fate is directly affected by local hydrogel stiffness and gradient range, e.g. 1–
14 kPa
• The stiffness of healthy muscle only varies approximately between 8 and 15 kPa.
• In vivo gradient strength can range between 0.6 and 8.7 kPa/mm
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26. [1] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage
References specification. Cell 2006;126:677–689.
[2] Saha K, et al. Substrate modulus directs neural stem cell behavior. Biophys J 2008;95:4426–
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[13]Breuls, R.G.M., Jiya, T.U. & Smit, T.H. Scaffold Stiffness Influences Cell Behavior:
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[14] Evans, N.D., et al.(2009). “Substrate stiffness affects early differentiation events in
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[16] Choi, J. S. and B. A. C. Harley (2012). "The combined influence of substrate elasticity and
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