Biomedical   Engineering                       Cell and Tissue                        EngineeringDiana Santos nº72460Maike...
Outline             Stem cell niche             Factors involved in cell fate             Tissues stiffness             Su...
Stem Cell Niche18-05-2012                                                              3             Instituto Superior Té...
Determination of cell fate                                        Stimuli affecting                                     St...
Tissue’s Young Modulus             Tissue elastic modulus (E) is given by the resistance offered by the             tissue...
Substrates for cell culture               Polystyrene (Plastic)        Polydimethylsiloxane             Polyacrylamide    ...
Cell mechanosensing process                     Integrins                 Cell binds to                    are inactive   ...
Remarkable studies             Disher, A.E., et al (2005)        Chen, C.S., et al,(1997)                                 ...
Remarkable studies              In 2005 Engler A.J., Disher A.E, et al, showed that mesenchymal stem               cells ...
MSCs differentiate or migrate first?                                                   Pathological:                      ...
In vivo                                               • In vitro   • Elasticity is        • Elasticity is        elasticit...
Human MSCs were cultured on a collagen I-coated photopolymerizedpolyacrylamide (PA) hydrogel of varying stiffness.        ...
Migration and proliferation of MSCs on hydrogels.Instituto Superior Técnico - Cell and Tissue Engineering   13
Spatial distribution of mitomycin C-treated MSCs on gradient hydrogels.Instituto Superior Técnico - Cell and Tissue Engine...
A                                                   B    (A) Morphological changes of MSCs cultured on static 11 kPa hydro...
A                                                   B                                                      C(a) Morphologi...
(a) MSCs were cultured on 1 and 11 kPa static (top) and gradient (bottom) hydrogels and stained for b3tubulin (red) and My...
Hypothesis: Grow embryonic stem cells (ESCs) on hydrophobic PDMS                substrate with varying stiffness (0.041-2....
18-05-2012   Instituto Superior Técnico - Cell and Tissue Engineering   19
0.041 MPa <PDMS< 2.7MPa, Tricalcium Phosphate (TCP)             Cell attachment after 24h in PDMS and TCP   Cell morpholog...
Cell perimeter after 24 in PDMS and TCP     Phalloidin staining of cytoskeletal acin                                      ...
Western Blots for pFAK in cells adherent             in PDMS, TCP and fibronectin after 1hour                             ...
Gene expression in day 6:             Primitive   Primitive streak and mesendoderm   Anterior          Neuroepithelium   C...
Conclusions             In embryonic stem cells it was verified that:             •Adhesion did not suffer significant alt...
Conclusions             In mesenchymal stem cells it was verified that:             •   MSCs migrate to stiffer matrix (du...
[1] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineageReferences   specification. Cell ...
[12] Lutolf, M. P., P. M. Gilbert, et al. (2009). "Designing materials to direct stem-cell fate."References           Natu...
18-05-2012   Instituto Superior Técnico - Cell and Tissue Engineering   28
Upcoming SlideShare
Loading in …5
×

Substrate stiffness and cell fate

3,754 views

Published on

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
3,754
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
60
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Substrate stiffness and cell fate

  1. 1. Biomedical Engineering Cell and Tissue EngineeringDiana Santos nº72460Maike Gomes nº72459
  2. 2. Outline Stem cell niche Factors involved in cell fate Tissues stiffness Substrates used for cell culture Cell mechanosensing process Remarkable studies Studies involving embryonic stem cells Studies involving mesenchymal stem cells Conclusions References18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 22
  3. 3. Stem Cell Niche18-05-2012 3 Instituto Superior Técnico - Cell and Tissue Engineering
  4. 4. Determination of cell fate Stimuli affecting Stem cell differentiation Soluble Chemical Mechanical Morphology Factors Properties “Stimuli”18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 4
  5. 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.18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 5
  6. 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 = [-] MPa18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 6
  7. 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 apoptosis18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 7
  8. 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 - neurons18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 8
  9. 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].18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 9
  10. 10. MSCs differentiate or migrate first? Pathological: Myocardial infarction 8.761.5 kPa/mm Normal tissue variation: MyocardiumMSC differentiate or migrate first? 0.660.9 kPa/mm Instituto Superior Técnico - Cell and Tissue Engineering 10
  11. 11. In vivo • In vitro • Elasticity is • Elasticity is elasticity static dynamic gradients In vitro SolutionDurotaxis –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. 12. Human MSCs were cultured on a collagen I-coated photopolymerizedpolyacrylamide (PA) hydrogel of varying stiffness. Hydrogel characterization Instituto Superior Técnico - Cell and Tissue Engineering 12
  13. 13. Migration and proliferation of MSCs on hydrogels.Instituto Superior Técnico - Cell and Tissue Engineering 13
  14. 14. Spatial distribution of mitomycin C-treated MSCs on gradient hydrogels.Instituto Superior Técnico - Cell and Tissue Engineering 14
  15. 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. Instituto Superior Técnico - Cell and Tissue Engineering 15
  16. 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 timeand stiffness in MSCs cultured on gradient hydrogels. (b) Immunofluorescent staining for MyoD (green) and phalloidin (red) observedas 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. Instituto Superior Técnico - Cell and Tissue Engineering 16
  17. 17. (a) MSCs were cultured on 1 and 11 kPa static (top) and gradient (bottom) hydrogels and stained for b3tubulin (red) and MyoD (green). Open arrowheads indicate cells expressing either b3 tubulin or MyoDwhile filled arrowheads indicate doubly stained cells. Instituto Superior Técnico - Cell and Tissue Engineering 17
  18. 18. 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
  19. 19. 18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 19
  20. 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 TCP18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 20
  21. 21. Cell perimeter after 24 in PDMS and TCP Phalloidin staining of cytoskeletal acin after 2 hours18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 21
  22. 22. Western Blots for pFAK in cells adherent in PDMS, TCP and fibronectin after 1hour Total cell number per well vs time18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 22
  23. 23. Gene expression in day 6: Primitive Primitive streak and mesendoderm Anterior Neuroepithelium Cadherins ectoderm precursors mesendoderm Primitive endoderm18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 23
  24. 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.18-05-2012 24 Instituto Superior Técnico - Cell and Tissue Engineering
  25. 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/mm18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 25
  26. 26. [1] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineageReferences 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. www.themegallery.com [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]
  27. 27. [12] Lutolf, M. P., P. M. Gilbert, et al. (2009). "Designing materials to direct stem-cell fate."References 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. 18-05-2012 27
  28. 28. 18-05-2012 Instituto Superior Técnico - Cell and Tissue Engineering 28

×