Derivation of Vasculature from Embryonic Stem Cells

Jeffrey N. Lindquist1, David A. Cheresh1, Evan Y. Snyder2

1 UCSD Moores Cancer Center, La Jolla, 2 The Burnham Institute for Medical Research, La Jolla
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.9
DOI:  10.1002/9780470151808.sc01f09s12
Online Posting Date:  March, 2010
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Abstract

The formation of the multicellular vascular system is critical to the growth, development, and viability of an organism, and many embryonic lethal mouse knockouts are due to vascular defects. Unfortunately, the complex nature, and many cell types involved in vasculogenesis and angiogenesis has stymied in vitro models of vascular formation. This unit describes a system that allows human embryonic stem cells to differentiate and spontaneously form vascular networks via both vasculogenesis and angiogenesis in the context of the three germ layers. Curr. Protoc. Stem Cell Biol. 12:1F.9.1‐1F.9.6. © 2010 by John Wiley & Sons, Inc.

Keywords: endothelial cells; vascular patterning; differentiation; angiogenesis; embryoid body

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: hESC Dissection, Embryoid Body Formation, and Seeding
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: hESC Dissection, Embryoid Body Formation, and Seeding

  Materials
  • hESCs, starting from macroscopic colonies (<1‐mm diameter) grown on MEFs or human fibroblast feeders
  • hESC growth medium (see recipe)
  • Enzymatically passaged hESC
  • Type I collagen (Sigma, cat. no. C3867)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 14040‐133)
  • Differentiation medium (see recipe)
  • Reagents for detecting endothelial markers (Table 1.9.1)
  • 6‐well low‐binding tissue culture plates (Corning, cat. no. 3471)
  • 20‐ to 200‐µl pipets and tips, sterile
  • 12‐well tissue culture–treated culture plates
  • 37°C incubator
  • 15‐ and 50‐ml tubes
  • Stereomicroscope (preferably with a heated stage)
    Table 1.0.1   MaterialsEndothelial Markers and Related Detection Reagents

    Endothelial marker Antibody Supplier
    CD‐31 Mouse‐anti‐CD31 Zymed, cat. no. 37‐0700
    VE‐Cadherin Goat‐anti‐VE‐Cad Santa Cruz, cat. no. SC‐6458
    Ulex Europa Lectin Vector Laboratories, cat. no. RL1062

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Figures

Videos

Literature Cited

Literature Cited
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   Ellerstrom, C., Strehl, R., Noaksson, K., Hyllner, J., and Semb, H. 2007. Facilitated expansion of human embryonic stem cells by single‐cell enzymatic dissociation. Stem Cells 25:1690‐1696.
   Krenning, G., Dankers, P.Y., Jovanovic, D., van Luyn, M.J., and Harmsen, M.C. 2007. Efficient differentiation of CD14+ monocytic cells into endothelial cells on degradable biomaterials. Biomaterials 28:1470‐1479.
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   Lee, G., Kim, H., Elkabetz, Y., Al Shamy, G.,. Panagiotakos, G., Barberi, T., Tabar, V., and Studer, L. 2007. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat. Biotechnol. 25:1468‐1475.
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   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145‐1147.
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