Stress-stiffening-mediated stem-cell commitment switch in soft responsive hydrogels.

Bulk matrix stiffness has emerged as a key mechanical cue in stem cell differentiation. Here, we show that the commitment and differentiation of human mesenchymal stem cells encapsulated in physiologically soft (∼0.2-0.4 kPa), fully synthetic polyisocyanopeptide-based three-dimensional (3D) matrices that mimic the stiffness of adult stem cell niches and show biopolymer-like stress stiffening, can be readily switched from adipogenesis to osteogenesis by changing only the onset of stress stiffening. This mechanical behaviour can be tuned by simply altering the material's polymer length whilst maintaining stiffness and ligand density. Our findings introduce stress stiffening as an important parameter that governs stem cell fate in a 3D microenvironment, and reveal a correlation between the onset of stiffening and the expression of the microtubule-associated protein DCAMKL1, thus implicating DCAMKL1 in a stress-stiffening-mediated, mechanotransduction pathway that involves microtubule dynamics in stem cell osteogenesis.

[1]  Ravi A. Desai,et al.  Mechanical regulation of cell function with geometrically modulated elastomeric substrates , 2010, Nature Methods.

[2]  E. Elson,et al.  Contraction due to microtubule disruption is associated with increased phosphorylation of myosin regulatory light chain. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Reine Bareille,et al.  Altered nanofeature size dictates stem cell differentiation , 2012, Journal of Cell Science.

[4]  Yifeng Lei,et al.  Modulation of lumen formation by microgeometrical bioactive cues and migration mode of actin machinery. , 2013, Small.

[5]  M. Rémy,et al.  Geometrical Microfeature Cues for Directing Tubulogenesis of Endothelial Cells , 2012, PloS one.

[6]  Brendon M. Baker,et al.  Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues , 2012, Journal of Cell Science.

[7]  Yu Suk Choi,et al.  Interplay of Matrix Stiffness and Protein Tethering in Stem Cell Differentiation , 2014, Nature materials.

[8]  C. Murphy,et al.  Hydrogels with well-defined peptide-hydrogel spacing and concentration: impact on epithelial cell behavior(). , 2012, Soft matter.

[9]  L. Addadi,et al.  Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates , 2001, Nature Cell Biology.

[10]  K. Anseth,et al.  Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells. , 2008, Nature materials.

[11]  R. Das,et al.  Influence of nanohelical shape and periodicity on stem cell fate. , 2013, ACS nano.

[12]  M. Mrksich,et al.  The microenvironment of immobilized Arg-Gly-Asp peptides is an important determinant of cell adhesion. , 2001, Biomaterials.

[13]  R. Haugland,et al.  Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. , 1997, Analytical biochemistry.

[14]  D. G. T. Strange,et al.  Extracellular-matrix tethering regulates stem-cell fate. , 2012, Nature materials.

[15]  G. Plopper,et al.  Osteogenic differentiation of mesenchymal stem cells in defined protein beads. , 2008, Journal of biomedical materials research. Part B, Applied biomaterials.

[16]  P. Sikorski,et al.  Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices , 2015, PloS one.

[17]  C. Dani,et al.  Stathmin-like 2, a developmentally-associated neuronal marker, is expressed and modulated during osteogenesis of human mesenchymal stem cells. , 2008, Biochemical and biophysical research communications.

[18]  F. MacKintosh,et al.  Ultra-responsive soft matter from strain-stiffening hydrogels , 2014, Nature Communications.

[19]  Tsung-Chuan Ho,et al.  Pigment epithelial-derived factor inhibits c-FLIP expression and assists ciglitazone-induced apoptosis in hepatocellular carcinoma. , 2011, Anticancer research.

[20]  G. Karsenty,et al.  Osf2/Cbfa1: A Transcriptional Activator of Osteoblast Differentiation , 1997, Cell.

[21]  Christopher S. Chen,et al.  Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. , 2004, Developmental cell.

[22]  Di Chen,et al.  Inhibition of Microtubule Assembly in Osteoblasts Stimulates Bone Morphogenetic Protein 2 Expression and Bone Formation through Transcription Factor Gli2 , 2008, Molecular and Cellular Biology.

[23]  S. Gygi,et al.  The microtubule-associated protein DCAMKL 1 regulates osteoblast function via repression of Runx 2 , 2013 .

[24]  M. Sheetz,et al.  Local force and geometry sensing regulate cell functions , 2006, Nature Reviews Molecular Cell Biology.

[25]  M. Doran,et al.  3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling. , 2012, Biochemical and biophysical research communications.

[26]  C. Broedersz,et al.  Measurement of nonlinear rheology of cross-linked biopolymer gels , 2010 .

[27]  Adam J. Engler,et al.  Matrix elasticity directs stem cell differentiation , 2006 .

[28]  C. Figdor,et al.  Therapeutic nanoworms: towards novel synthetic dendritic cells for immunotherapy , 2013 .

[29]  David J Mooney,et al.  Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. , 2014, Nature materials.

[30]  B. Guillotin,et al.  Differentiation of pre-osteoblast cells on poly(ethylene terephthalate) grafted with RGD and/or BMPs mimetic peptides. , 2010, Biomaterials.

[31]  Matthew J. Dalby,et al.  The role of microtopography in cellular mechanotransduction. , 2012, Biomaterials.

[32]  N. Elango,et al.  Translational regulation is a control point in RUNX2/Cbfa1 gene expression. , 2001, Biochemical and biophysical research communications.

[33]  Benjamin G. Keselowsky,et al.  Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[34]  S. Sen,et al.  Matrix Elasticity Directs Stem Cell Lineage Specification , 2006, Cell.

[35]  C. Walsh,et al.  DCAMKL1 Encodes a Protein Kinase with Homology to Doublecortin that Regulates Microtubule Polymerization , 2000, The Journal of Neuroscience.

[36]  Wesley R. Legant,et al.  Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels , 2013, Nature materials.

[37]  Thomas Boudou,et al.  A hitchhiker's guide to mechanobiology. , 2011, Developmental cell.

[38]  David P. Kreil,et al.  A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia , 2010, Nature communications.

[39]  Yifeng Lei,et al.  Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling , 2013, Biology Open.

[40]  Paul A. Janmey,et al.  Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation , 2009, PloS one.

[41]  Eduardo Mendes,et al.  Responsive biomimetic networks from polyisocyanopeptide hydrogels , 2013, Nature.

[42]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

[43]  David J. Mooney,et al.  Harnessing Traction-Mediated Manipulation of the Cell-Matrix Interface to Control Stem Cell Fate , 2010, Nature materials.

[44]  Michel Bornens,et al.  Physical Mechanisms Redirecting Cell Polarity and Cell Shape in Fission Yeast , 2008, Current Biology.

[45]  G. Stein,et al.  The Bone-specific Expression of Runx2 Oscillates during the Cell Cycle to Support a G1-related Antiproliferative Function in Osteoblasts* , 2005, Journal of Biological Chemistry.

[46]  P. Janmey,et al.  Nonlinear elasticity in biological gels , 2004, Nature.

[47]  S. Gygi,et al.  The microtubule-associated protein DCAMKL1 regulates osteoblast function via repression of Runx2 , 2013, The Journal of experimental medicine.

[48]  R. Das,et al.  A review of the effects of the cell environment physicochemical nanoarchitecture on stem cell commitment. , 2014, Biomaterials.

[49]  David J. Mooney,et al.  Growth Factors, Matrices, and Forces Combine and Control Stem Cells , 2009, Science.

[50]  D. Ingber.  Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. , 2002, Circulation research.

[51]  N. Gadegaard,et al.  Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency. , 2011, Nature materials.

[52]  L. Quarles,et al.  IRES‐dependent translational control of Cbfa1/Runx2 expression , 2003, Journal of cellular biochemistry.

[53]  J. Kalisky,et al.  Effect of BMP-2 from matrices of different stiffnesses for the modulation of stem cell fate. , 2013, Biomaterials.

[54]  R. Nolte,et al.  Stiffness versus architecture of single helical polyisocyanopeptides , 2013 .

[55]  Yuichiro Yamaguchi,et al.  Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential , 2014, BMC Biotechnology.

[56]  Ning Wang,et al.  Rapid signal transduction in living cells is a unique feature of mechanotransduction , 2008, Proceedings of the National Academy of Sciences.

[57]  Karine Glinel,et al.  Bioactive chemical nanopatterns impact human mesenchymal stem cell fate. , 2013, Nano letters.

[58]  A. Curtis,et al.  Osteogenesis of mesenchymal stem cells by nanoscale mechanotransduction. , 2013, ACS nano.