Stem Cells and Tissue Engineering

Stem cells are undifferentiated cells capable of proliferation, self-renewal and differentiation towards specific phenotypes. The differentiation of cells is controlled by a variety of cues, including the various mechanical forces at play in their surrounding microenvironments. In particular, the nature of the substrate on which these cells lie and its innate stiffness are known to be important in determining cell fate. Research at the MBI aims to elucidate the currently poorly understood effects of mechanical factors on stem cell biology.

Mouse embryonic fibroblasts stained for actin (green) and beta-catenin (red) highlighting cell-cell junctions. This was of particular interest because in fibroblasts, unlike epithelial cells, punctate and streak-like adherens junctions (AJs) are formed, with actin cables associated perpendicularly to the AJ membrane (as seen in this image). In contrast, actin filaments run parallel to the AJ membrane in epithelial cells. Image captured by Megha Vaman Rao of the Zaidel-Bar lab.

Multicellular Matters

Stem cells hold great potential in tissue engineering. Future hopes for the regeneration of diseased or damaged tissue lie in part with the use of intricate combinations of scaffolds, growth factors and stem cells. Mimicking natural tissue development in vitro requires appropriate biochemical and topographical cues to be provided in a spatially controlled manner. Our studies utilize nanotopography to influence cellular behaviors, ranging from attachment to proliferation and differentiation – all of which are key processes in tissue engineering.

Much of our research focuses on understanding how cell adhesion, biomechanical and biochemical signals cooperate to allow cells, tissues and ultimately living organisms to adapt to changes in the environment. Mechanical forces are transduced in a multicellular context through multiple means. These include, but are not limited to, mechanosensing through a compliant substratum, cell proliferation, cell-cell and cell-matrix adhesions. By controlling the mechanical and physical properties of the cellular environment, through the use of different micro- and nano-fabrication tools, we aim to understand how these multiple processes allow cells to respond appropriately to their ever-changing environments.

Mouse embryonic fibroblasts of the NIH 3T3 cell line stained for the DNA (blue) and chromosomes 15 (green) and 18 (red). Image captured by Shovamayee Maharana of the Shivashankar lab.

Nuclear Control

Nuclear morphology and size are emerging as potential mechanistic regulators of genome function and as such forms one of our key areas of investigation. Physical connections bridging the nucleus and cytoplasm govern the size and shape of a pre-stressed eukaryotic nucleus. During the process of cellular differentiation mechanical properties of the nucleus are known to change. Differentiation is known to be governed in part by physical aspects of the microenvironment, which is increasingly linked to gene expression and protein organization, as we and others have shown.

Human embryonic stem cells (hESCs) stained for the transcription factor Nanog (red), the oligodendrocyte marker O4 (green), actin (magenta) and the nucleus (blue). In order to induce neuronal differentiation, the hESCs were plated on a soft substrate of polydimethylsiloxane (PDMS) with neuronal supplements and cultured for 7 days before being imaged. Image captured by Ankam Soneela of the Yim lab.

Protein players

Numerous regulatory interactions occur from the point of detection of a mechanical force to the transduction of this information along a biochemical signaling pathway. The proteins responsible for detecting these forces, such as integrins and cadherins, play a key role in transmitting the information through to the necessary effector molecules that can then elicit a mechanoresponse. We utilize a range of different model systems including bacteria, yeast, C.elegans and human cells to study the various interactions between proteins involved in this cascade of events at cell-cell and cell-matrix adhesions. The results of these studies feed into the development of new paradigms in tissue engineering methods.

Rat embryonic fibroblasts of the REF52 cell line stained for actin (green) and the nucleus (blue) illustrating the presence of stress fibers. Image captured by Leong Man Chun of the Lim lab.

Tools of the Trade

The development of several new tools and protocols for measuring cell forces at the molecular level has revolutionized our understanding of how cells can both generate and respond to external forces. This is exemplified by our application of advanced microfluidics to explore the frequency response of single cells subjected to periodically changing environments. Advances in micro- and nanotechnology have also led us to study changes in the mechanical properties of not only individual cells but also molecules. Results from such work will help us better understand the relationship between mechanical properties and cellular functions.

Selected Publications

Lim, C.T., S.J. Tan S, W.T. Lim, M.H. Tan. Biomechanics Based Microfluidic Biochip for the Label-free Isolation and Retrieval of Circulating Tumour Cells. European Journal of Cancer; 47, S48(2011)

Hou, H. W., W. C. Lee, M. C. Leong, S. Sonam, S. R. K. Vedula, C.T. Lim .Microfluidics for applications in cell mechanics and mechanobiology. Cellular and Molecular Bioengineering, (2011) [in press]

Cheng-han Yu, Jaslyn Bee Khuan Law, Mona Suryana, Hong Yee Low, Michael P. Sheetz. Early Integrin Binding to RGD Activates Actin Polymerization and Contractile Movement that Stimulates Outward Translocation. PNAS (2011) [in press]

Zhang X., Jiang G., Cai Y., Monkley S.J., Critchley D.R., Sheetz M.P. Talin depletion reveals independence of initial cell spreading from integrin activation and traction. Nat Cell Biol. Sep; 10(9); 1062-8(2008)

Y. Cai, O. Rossier, N. Gauthier, N. Biais, L. W. Miller, M. Fardin, B. Ladoux, V. W. Cornish & M. P. Sheetz. Cytoskeletal coherence depends on myosin IIA contractility. Journal of Cell Science, 123; 413-423 (2010)

B. Ladoux, E. Anon, M. Lambert, A. Rabodzey, P. Hersen, A. Buguin, P. Silberzan & R-M. Mège. Strength dependence of cadherins mediated adhesions. Biophysical Journal , 98, 534-542 (2010)

Evelyn K.F. Yim, Eric M. Darling, Karina Kulangara, Farshid Guilak and Kam W. Leong. Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells. Biomaterials, 31: 1299-1306 (2010)

Lim C.T., Bershadsky A., Sheetz M.P. Mechanobiology. J. R. Soc. Interface, 2010, 7, Suppl 3:S291-3.

del Rio A., Perez-Jimenez R., Liu R, Roca-Cusachs P., Fernandez J.M., Sheetz M.P. Stretching single talin rod molecules activates vinculin binding. Science, 323(5914):638-41(2009)

Zhang, C., Zhao, Z., Abdul Rahim, N.A., van Noort, D., and Yu, H. Towards a Human-on-Chip: Culturing Multiple Cell Types on a Chip with Compartmentalized Microenvironments. Lab on a chip, 9(22):3185-3192(2009)

G.V.Shivashankar. Mechanosignaling to cell nucleus and genome regulation. Annual Reviews of Biophysics; 40, 361-378(2011)

Zaidel-Bar R., Joyce M.J., Lynch A.M., Witte K., Audhya A., Hardin J.D. The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis. J. Cell. Biol. 191(4);761-769(2010)

Zaidel-Bar R. Evolution of Complexity in the Integrin Adhesome. J. Cell. Biol. 186(3); 317-21(2009)

Yukai Zeng , Tanny Lai , Cheng Gee Koh , Philip R. LeDuc, K.H. Chiam Investigating Circular Dorsal Ruffles through Varying Substrate Stiffness and Mathematical Modeling. Biophysical Journal, 101; 9, 2122-2130, (2011)

C Meghana, Nisha Ramdas, Feroz Hameed, Madan Rao, G.V. Shivashankar & Maithreyi Narasimha. Integrin adhesion drives the emergent polarisation of active cytoskeletal stresses to pattern cell delamination. PNAS, 108; 9107-9112(2011)

le Digabel J., Biais N., Fresnais J., Berret J.F., Hersen P., Ladoux B. Magnetic micropillars as a tool to govern substrate deformations. Lab Chip, 11(15):2630-6 (2011)