substrate stiffness and cell fate

Cell and Tissue

Engineering

Diana Santos nº72460

Maike Gomes nº72459

Biomedical

Engineering

18-05-2012 2 Instituto Superior Técnico - Cell and Tissue Engineering 2

Stem cell niche

Factors involved in cell fate

Tissue's stiffness

Substrates used for cell culture

Cell mechanosensing process

Remarkable studies

Studies involving embryonic stem cells

Studies involving mesenchymal stem cells

Conclusions

References

Outline

18-05-2012 3 Instituto Superior Técnico - Cell and Tissue Engineering

Stem Cell Niche

Determination of cell fate

Stimuli affecting Stem cell differentiation

Soluble Factors

Chemical Properties

Morphology Mechanical

“Stimuli”

18-05-2012

4 Instituto Superior Técnico - Cell and Tissue Engineering

Tissue elastic modulus (E) is given by the resistance offered by the tissues to deformation effects, i.e. the tissue stiffness.


Tissue’s Young Modulus

18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 6

Polystyrene (Plastic) Optically clear Amenable to many different surface treatments Limitations Poor chemical resistance E = [3000-3600] MPa

Polyacrylamide (PAAm) gel

Highly water-absorvent Insoluble Tunable mechanical properties Limitations Citotoxity E=[1-100]kPa

Polydimethylsiloxane (PDMS)

Optically clear Flexible Inert Insoluble in medium culture non-toxic non-flammable Good biocompatibility Gas permeability E = [-] MPa

Substrates for cell culture

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Cell mechanosensing process

Instituto Superior Técnico - Cell and Tissue Engineering

Integrins are inactive

Cell binds to the substrate

Activation and clustering of

integrins – focal adhesion

formation (FA)

FA maturation due to

mechanical stimuli

Cytoskeletal and signaling proteins (FAK) are recruited

Cell shape, migration,

growth, differentiation,

apoptosis

Hard substrate – maturation of FA – stress fibers formation

Soft substrate – not enough forces to form FA

MSC’s


Disher, A.E., et al (2005)

MSCs in PDMS become:

Soft substrate – adipogenic profile; Intermediate stiffness - myogenic profile; Hard substrate - osteogenic profile

Chen, C.S., et al,(1997) McBeath R, et al.(2004)

Small islands of ECM – adipogenic fate

Bigger islands of ECM – ostogenic fate

Boonen, K.J., et al (2009)

Increasing the matrix stiffness the cellular proliferation will also increase, in muscle skeletal stem cells

Saha K.,et al (2008)

Neuronal adult stem cells:

Stiff matrix – glial cells

Soft matrix - neurons

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].


Remarkable studies

MSC differentiate or migrate first?

Pathological:

Myocardial infarction

8.761.5 kPa/mm

Normal tissue variation:

Myocardium

0.660.9 kPa/mm


MSCs differentiate or migrate first?

• Elasticity is static

In vitro

• Elasticity is dynamic

In vivo • In vitro

elasticity gradients

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?

Hydrogel characterization

Human MSCs were cultured on a collagen I-coated photopolymerized polyacrylamide (PA) hydrogel of varying stiffness.


Migration and proliferation of MSCs on hydrogels.


Spatial distribution of mitomycin C-treated MSCs on gradient hydrogels.


(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.

A B


(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.

A C B


(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.


Hypothesis: Grow embryonic stem cells (ESCs) on hydrophobic PDMS substrate with varying stiffness (0.041-2.7MPa) can influence ESCs differentiation.

18-05-2012 Source: http://www.gghjournal.com/volume22/4/ab03.cfm

http://www.gghjournal.com/volume22/4/ab03.cfm

http://www.gghjournal.com/volume22/4/ab03.cfm


Cell attachment after 24h in PDMS and TCP Cell morphology after 24h in PDMS and TCP

0.041 MPa <PDMS< 2.7MPa, Tricalcium Phosphate (TCP)


Cell perimeter after 24 in PDMS and TCP Phalloidin staining of cytoskeletal acin after 2 hours


Western Blots for pFAK in cells adherent in PDMS, TCP and fibronectin after 1hour

Total cell number per well vs time


Gene expression in day 6:

Primitive ectoderm

Primitive streak and mesendoderm precursors

Anterior mesendoderm

Neuroepithelium

Primitive endoderm

Cadherins

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.


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


Conclusions

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[1] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–689. [2] Saha K, et al. Substrate modulus directs neural stem cell behavior. Biophys J 2008;95:4426–4438. [PubMed: 18658232] [3] Boonen KJ, Rosaria-Chak KY, Baaijens FP, van der Schaft DW, Post MJ. Essential environmental cues from the satellite cell niche: optimizing proliferation and differentiation. Am J Physiol Cell Physiol 2009;296:C1338–C1345. [PubMed: 19321742] [4] Wei, W.-C., H.-H. Lin, et al. (2008). "Mechanosensing machinery for cells under low substratum rigidity." American Journal of Physiology - Cell Physiology 295(6): C1579-C1589. [5] Guilak, F., D. M. Cohen, et al. (2009). "Control of Stem Cell Fate by Physical Interactions with the Extracellular Matrix." Cell stem cell 5(1): 17-26. [6] Tabata, Y. (2011). "Biomaterials Design of Culture Substrates for Cell Research." Inflammation and Regeneration 31(2): 137-145. [7] Joy, A., D. M. Cohen, et al. (2011). "Control of Surface Chemistry, Substrate Stiffness, and Cell Function in a Novel Terpolymer Methacrylate Library." Langmuir 27(5): 1891-1899. [8] (2012). "Engineering Airway Epithelium." Journal of Biomedicine and Biotechnology 2012: 1. [9] Wang, P.-Y., W.-B. Tsai, et al. (2012). "Screening of rat mesenchymal stem cell behaviour on polydimethylsiloxane stiffness gradients." Acta Biomaterialia 8(2): 519-530. [10] Georges, P. C. and P. A. Janmey (2005). "Cell type-specific response to growth on soft materials." Journal of Applied Physiology 98(4): 1547-1553. [11]Peerani R, et al. Niche-mediated control of human embryonic stem cell self-renewal and differentiation. EMBO J 2007;26:4744–4755. [PubMed: 17948051]

References

[12] Lutolf, M. P., P. M. Gilbert, et al. (2009). "Designing materials to direct stem-cell fate." Nature 462(7272): 433-441.

[13]Breuls, R.G.M., Jiya, T.U. & Smit, T.H. Scaffold Stiffness Influences Cell Behavior: Opportunities for Skeletal Tissue Engineering. The open orthopaedics 2, 103-109 (2008).

[14] Evans, N.D., et al.(2009). “Substrate stiffness affects early differentiation events in embryonic stem cells”. Cells and Materials 18 : 1-14

[15] Yeung, T., P. C. Georges, et al. (2005). "Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion." Cell Motility and the Cytoskeleton 60(1): 24-34.

[16] Choi, J. S. and B. A. C. Harley (2012). "The combined influence of substrate elasticity and ligand density on the viability and biophysical properties of hematopoietic stem and progenitor cells." Biomaterials 33(18): 4460-4468.

[17] Dennis E. Discher, Paul Janmey, and Yu-li Wang. (2005). “Tissue Cells Feel and Respond to the Stiffness of Their Substrate” . Science 310 (5751) 1139-1143.

[18] Tse, J. R. and A. J. Engler (2011). "Stiffness Gradients Mimicking Tissue Variation Regulate Mesenchymal Stem Cell Fate." PLoS One 6(1): e15978.

[19] Zhang, X., M. Jaramillo, et al. (2012). "Analysis of Regulatory Network Involved in Mechanical Induction of Embryonic Stem Cell Differentiation." PLoS One 7(4): e35700.

[20] Li, D., J. Zhou, et al. (2011). "Role of mechanical factors in fate decisions of stem cells." Regenerative Medicine 6(2): 229-240.

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References

substrate stiffness and cell fate

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