Growing evidence suggests that physical microenvironments and mechanical stresses, besides soluble factors, help direct mesenchymal stem cell fate. However, biological responses to a local force in embryonic stem (ES) cells remain elusive. Here we show that a local cyclic stress via focal adhesions induced spreading in mouse ES (mES) cells but not in mES cell-differentiated (ESD) cells that were 10-fold stiffer. This response was dictated by the cell material property (cell softness), suggesting that a threshold cell deformation is the key setpoint for triggering spreading responses. Traction quantification and pharmacological or shRNA intervention revealed that myosin II contractility, F-actin, Src, or Cdc42 were essential in the spreading response. The applied stress led to Oct3/4 gene downregulation in mES cells. Our findings demonstrate that cell softness dictates cellular sensitivity to force, suggesting that local small forces might play far more important roles in early developments of soft embryos than previously appreciated.

It is widely postulated that mechanotransduction is initiated at the local force-membrane interface by inducing local conformational changes of proteins, similar to soluble ligand-induced signal transduction. However, all published reports are limited in time scale to address this fundamental issue. Using a FRET-based cytosolic Src reporter in a living cell, we quantified changes of Src activities as a local stress via activated integrins was applied. The stress induced rapid (<0.3 s) activation of Src at remote cytoplasmic sites, which depends on the cytoskeletal prestress. In contrast, there was no Src activation within 12 s of soluble epidermal growth factor (EGF) stimulation. A 1.8-Pa stress over a focal adhesion activated Src to the same extent as 0.4 ng/ml EGF at long times (minutes), and the energy levels for mechanical stimulation and chemical stimulation were comparable. The effect of both stress and EGF was less than additive. Nanometer-scale cytoskeletal deformation analyses revealed that the strong activation sites of Src by stress colocalized with large deformation sites of microtubules, suggesting that microtubules are essential structures for transmitting stresses to activate cytoplasmic proteins. These results demonstrate that rapid signal transduction via the prestressed cytoskeleton is a unique feature of mechanotransduction.cytoskeleton ͉ growth factor ͉ mechanical force ͉ prestress ͉ microtubule T he sensing and response of living cells and tissues to mechanical forces and physical microenvironments are critical for their functions and survival (1-3). However, the underlying mechanisms remain largely elusive. Various models of mechanotransduction have been proposed (2, 4, 5); the most straightforward model involves force-induced local conformational changes of proteins (6). It is generally believed that like soluble ligand-induced signal transduction, mechanotransduction initiates at the local force-membrane interface (e.g., at focal adhesions) by inducing local conformational changes or unfolding of membrane-bound proteins, followed by a cascade of diffusion-based or translocation-based signaling in the cytoplasm. Recent reports demonstrate force-induced dynamic changes in Src activity (7), mechanical extension of the Src family kinase substrate p130Cas (8), and forced unfolding of proteins in living cells (9). However, all published reports, including past studies with the reporter-type of construct extended here (7), are limited in time scale. Therefore, it has not been possible to compare early dynamics of mechanotransduction with that of soluble ligand-induced signal transduction. Here, we applied a local stress of physiologic magnitude and simultaneously imaged changes in Src activity in living cells by using a CFP-YFP Src reporter and fluorescence resonance energy transfer (FRET) technology. We show that stress-induced Src activation occurs rapidly in the cytoplasm and depends on the integrity of the microfilaments and microtubules, substrate rigidity, and the cytoskeletal prestress, d...

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.

Stem cells derived from adult tissues or from the inner cell mass of blastocyst-stage embryos can self-renew in culture and have the remarkable potential to undergo lineage-specific differentiation. Extensive studies have been devoted to achieving a better understanding of the soluble factors and the mechanism(s) by which they regulate the fate decisions of these cells, but it is only recently that a critical role has been revealed for physical and mechanical factors in controlling self-renewal and lineage specification. This review summarizes selected aspects of current work on stem cell mechanics with an emphasis on the influence of matrix stiffness, surface topography, cell shape and mechanical forces on the fate determination of mesenchymal stem cells and embryonic stem cells.