Scanning probe-enabled nanocombinatorics define the relationship between fibronectin feature size and stem cell fate

We report the development of a powerful analytical method that utilizes a tilted elastomeric pyramidal pen array in the context of a scanning probe lithography experiment to rapidly prepare libraries having as many as 25 million features over large areas with a range of feature sizes from the nano- to microscale. This technique can be used to probe important chemical and biological processes, opening up the field of nanocombinatorics. In a proof-of-concept investigation of mesenchymal stem cell (MSC) differentiation, combinatorial patterns first enabled a rapid and systematic screening of MSC adhesion, as a function of feature size, while uniform patterns were used to study differentiation with statistically significant sample sizes. Without media containing osteogenic-inducing chemical cues, cells cultured on nanopatterned fibronectin substrates direct MSC differentiation towards osteogenic fates when compared to nonpatterned fibronectin substrates. This powerful and versatile approach enables studies of many systems spanning biology, chemistry, and engineering areas.

[1]  Mark Schvartzman,et al.  Nanolithographic control of the spatial organization of cellular adhesion receptors at the single-molecule level. , 2011, Nano letters.

[2]  D E Ingber,et al.  Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver. , 1997, Experimental cell research.

[3]  C. Wilkinson,et al.  The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. , 2007, Nature materials.

[4]  Seunghun Hong,et al.  Carbon nanotube monolayer cues for osteogenesis of mesenchymal stem cells. , 2011, Small.

[5]  Tal Dvir,et al.  Nanotechnological strategies for engineering complex tissues. , 2020, Nature nanotechnology.

[6]  S. Bhatia,et al.  An extracellular matrix microarray for probing cellular differentiation , 2005, Nature Methods.

[7]  A. Boskey,et al.  Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells. , 2007, Experimental cell research.

[8]  M. Lutolf,et al.  Artificial niche microarrays for probing single stem cell fate in high throughput , 2011, Nature Methods.

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

[10]  Milan Mrksich,et al.  Geometric cues for directing the differentiation of mesenchymal stem cells , 2010, Proceedings of the National Academy of Sciences.

[11]  G. Csucs,et al.  Microcontact Printing of Macromolecules with Submicrometer Resolution by Means of Polyolefin Stamps , 2003 .

[12]  Matthias P. Lutolf,et al.  Designing materials to direct stem-cell fate , 2009, Nature.

[13]  Kenneth M. Yamada,et al.  Transmembrane crosstalk between the extracellular matrix and the cytoskeleton , 2001, Nature Reviews Molecular Cell Biology.

[14]  Chad A. Mirkin,et al.  Polymer Pen Lithography , 2008, Science.

[15]  Xing Liao,et al.  "Force-feedback" leveling of massively parallel arrays in polymer pen lithography. , 2010, Nano letters.

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

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

[18]  Seema M. Jadhav,et al.  Dip Pen Nanolithography , 2012 .

[19]  Benjamin Geiger,et al.  TRANSMEMBRANE EXTRACELLULAR MATRIX – CYTOSKELETON CROSSTALK , 2001 .

[20]  D. A. Hanson,et al.  Focal adhesion kinase: in command and control of cell motility , 2005, Nature Reviews Molecular Cell Biology.

[21]  Andre Levchenko,et al.  Biomimetic Nanopatterns as Enabling Tools for Analysis and Control of Live Cells , 2010, Advanced materials.

[22]  L. Kiessling,et al.  Spatial control of cell fate using synthetic surfaces to potentiate TGF-β signaling , 2011, Proceedings of the National Academy of Sciences.

[23]  C. S. Chen,et al.  Geometric control of cell life and death. , 1997, Science.

[24]  Chad A. Mirkin,et al.  Multiplexed protein arrays enabled by polymer pen lithography: addressing the inking challenge. , 2009, Angewandte Chemie.

[25]  Xing Liao,et al.  Force- and time-dependent feature size and shape control in molecular printing via polymer-pen lithography. , 2009, Small.

[26]  H. Ryoo,et al.  FGF2-activated ERK Mitogen-activated Protein Kinase Enhances Runx2 Acetylation and Stabilization* , 2009, The Journal of Biological Chemistry.

[27]  Buddy D Ratner,et al.  Biomaterials: where we have been and where we are going. , 2004, Annual review of biomedical engineering.

[28]  Chad A Mirkin,et al.  Cantilever-free scanning probe molecular printing. , 2011, Angewandte Chemie.

[29]  C. Seelos,et al.  Affinity binding of distinct functional fibronectin domains to immobilized metal chelates. , 1995, Archives of biochemistry and biophysics.

[30]  Ying Mei,et al.  Combinatorial Development of Biomaterials for Clonal Growth of Human Pluripotent Stem Cells , 2010, Nature materials.

[31]  J. Suh,et al.  Hes1 stimulates transcriptional activity of Runx2 by increasing protein stabilization during osteoblast differentiation. , 2008, Biochemical and biophysical research communications.

[32]  G. Stein,et al.  Expression of the Osteoblast Differentiation Factor RUNX2 (Cbfa1/AML3/Pebp2αA) Is Inhibited by Tumor Necrosis Factor-α* , 2002, The Journal of Biological Chemistry.

[33]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[34]  S. Bruder,et al.  Osteogenic differentiation of purified, culture‐expanded human mesenchymal stem cells in vitro , 1997, Journal of cellular biochemistry.