University of California San Francisco and University of California Berkeley 1 article published in JoVE Bioengineering Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics Jonathon M. Muncie1,2, Roberto Falcón-Banchs1, Johnathon N. Lakins2, Lydia L. Sohn1,3, Valerie M. Weaver2,4,5,6 1Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, 2Center for Bioengineering and Tissue Regeneration, Department of Surgery, University of California San Francisco, 3Department of Mechanical Engineering, University of California Berkeley, 4Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 5UCSF Comprehensive Cancer Center, Helen Diller Family Cancer Research Center, University of California San Francisco, 6Department of Anatomy, Department of Bioengineering and Therapeutic Sciences, and Department of Radiation Oncology, University of California San Francisco Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.