title: Embryonic stem cells growing in 3-dimensions shift from reliance on the substrate to each other for mechanical support.
abstract: Embryonic stem cells (ESCs) grow into three-dimensional (3D) spheroid structures en-route to tissue growth. In vitro spheroids can be controllably induced on a two-dimensional (2D) substrate with high viability. Here we use a method for inducing pluripotent embryoid body (EB) formation on flat polyacrylamide gels while simultaneously evaluating the dynamic changes in the mechano-biology of the growing 3D spheroids. During colony growth in 3D, pluripotency is conserved while the spheroid-substrate interactions change significantly. We correlate colony-size, cell-applied traction-forces, and expressions of cell-surface molecules indicating cell-cell and cell-substrate interactions, while verifying pluripotency. We show that as the colony size increases with time, the stresses applied by the spheroid to the gel decrease in the 3D growing EBs; control cells growing in 2D-monolayers maintain unvarying forces. Concurrently, focal-adhesion mediated cell-substrate interactions give way to E-cadherin cell-cell connections, while pluripotency. The mechano-biological changes occurring in the growing embryoid body are required for stabilization of the growing pluripotent 3D-structure, and can affect its potential uses including differentiation. This could enable development of more effective expansion, differentiation, and separation approaches for clinical purposes.
authors.affiliation: Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore.
authors.forename: Ailing
authors.initials: A
authors.lastname: Teo
authors.affiliation: Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore.
authors.forename: Mayasari
authors.initials: M
authors.lastname: Lim
authors.affiliation: Faculty of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa 3200003, Israel. Electronic address: daphnew@tx.technion.ac.il.
authors.forename: Daphne
authors.initials: D
authors.lastname: Weihs
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001696:Biomechanical Phenomena; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D058732:Embryoid Bodies / Q000166:cytology* / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D055595:Mechanical Phenomena; D051379:Mice; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D015820:Cadherins; D000099166:Polyacrylamides
keywords: Cell biomechanics; Mouse embryonic stem cells; Traction force microscopy