Exploring the Link Between Human Embryonic Stem Cell Organization and Fate Using Tension-Calibrated Extracellular Matrix Functionalized Polyacrylamide Gels

  • Johnathon N. Lakins
  • Andrew R. Chin
  • Valerie M. Weaver
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 916)

Abstract

Human embryonic stem cell (hESc) lines are likely the in vitro equivalent of the pluripotent epiblast. hESc express high levels of the extracellular matrix (ECM) laminin integrin receptor α6β1 and consequently can adhere robustly and be propagated in an undifferentiated state on tissue culture plastic coated with the laminin rich basement membrane preparation, Matrigel, even in the absence of supporting fibroblasts. Such cultures represent a critical step in the development of more defined feeder free cultures of hESc; a goal deemed necessary for regenerative medical applications and have been used as the starting point in some differentiation protocols. However, on standard non-deformable tissue culture plastic hESc either fail or inadequately develop the structural/morphological organization of the epiblast in vivo. By contrast, growth of hESc on appropriately defined mechanically deformable polyacrylamide substrates permits recapitulation of many of these in vivo features. These likely herald differences in the precise nature of the integration of signal transduction pathways from soluble morphogens and represent an unexplored variable in hESc (fate) state space. In this chapter we describe how to establish viable hESc colonies on these functionalized polyacrylamide gels. We suggest this strategy as a prospective in vitro model of the genetics, biochemistry, and cell biology of pre- and early-gastrulation stage human embryos and the permissive and instructive roles that cellular and substrate mechanics might play in early embryonic cell fate decisions. Such knowledge should inform regenerative medical applications aimed at enabling or improving the differentiation of specific cell types from embryonic or induced embryonic stem cells.

Key words

Embryonic stem cell Early embryonic differentiation Epiblast Epithelial organization Apical constriction Gastrulation Rho kinase (ROCK) Polyacrylamide substrates Extracellular matrix Mechanics Visco elasticity Substrate stiffness 

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Johnathon N. Lakins
    • 1
  • Andrew R. Chin
    • 2
  • Valerie M. Weaver
    • 3
    • 4
    • 5
    • 6
  1. 1.Department of Surgery, Center for Bioengineering and Tissue RegenerationUniversity of California, San FranciscoSan FranciscoUSA
  2. 2.Center for Bioengineering and Tissue Regeneration, Department of SurgeryUniversity of CaliforniaSan FranciscoUSA
  3. 3.Department of Surgery and Center for Bioengineering and Tissue RegenerationUniversity of CaliforniaSan FranciscoUSA
  4. 4.Departments of Anatomy and Bioengineering and Therapeutic SciencesUniversity of California San FranciscoSan FranciscoUSA
  5. 5.Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell ResearchUniversity of California San FranciscoSan FranciscoUSA
  6. 6.Helen Diller Family Comprehensive Cancer CenterUniversity of California San FranciscoSan FranciscoUSA

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