Isolation and Expansion of Endothelial Progenitor Cells Derived from Mouse Embryonic Stem Cells

  • S. Bahram Bahrami
  • Mandana Veiseh
  • Nancy J. Boudreau
Part of the Methods in Molecular Biology book series (MIMB, volume 916)

Abstract

The unlimited differentiation and proliferation capacity of embryonic stem cells represents a great resource for regenerative medicine. Here, we describe a method for differentiating, isolating, and expanding endothelial cells (ECs) from mouse embryonic stem cells (mESCs). First, mESCs are expanded on a mouse embryonic fibroblast (mEF) feeder layer and partially differentiated into embryoid bodies (EBs) by growing the cells in an ultra-low attachment plate for up to 5 days. The EBs are then differentiated along the endothelial lineage using endothelial growth medium supplemented with 40 ng/mL vascular endothelial growth factor (VEGF). The differentiated endothelial population expresses both Fetal Liver Kinase 1 (Flk-1) and VE-Cadherin on the cell surface which can be further purified using a fluorescence-activated cell sorting (FACS) system and subsequently expanded on 0.1 % gelatin-coated plates. The differentiated cells can be analyzed by real-time PCR and flow cytometry to confirm enrichment of EC-specific genes and proteins.

Key words

Embryonic stem cells Differentiation FACS Endothelial cells 

Notes

Acknowledgment

This work is supported by grants from NIH/NCI TMEN grant (U54CA126552.) to Nancy Boudreau and Mina J Bissell and U.S. Department of Energy, Office of Biological and Environmental Research (DE-AC02-05CH1123), a Distinguished Fellow Award and Low Dose Radiation Program (03-76SF00098) to Mina J. Bissell. Mandana Veiseh was supported by a postdoctoral fellowship from the NCI of the NIH (F32 CA132491A). We thank Pamela Derish in the Department of Surgery at UCSF for editorial review of the manuscript.

References

  1. 1.
    Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler RJ (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morph 87:27–45PubMedGoogle Scholar
  2. 2.
    Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147PubMedCrossRefGoogle Scholar
  3. 3.
    Vittet D, Prandini MH, Berthier R, Schweitzer A, Martin-Sisteron H, Uzan G, Dejana E (1996) Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88:3424–3431PubMedGoogle Scholar
  4. 4.
    Feraud O, Cao Y, Vittet D (2001) Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis. Lab Invest 81:1669–1681PubMedCrossRefGoogle Scholar
  5. 5.
    Blancas AA, Lauer NE, McCloskey KE (2008) Endothelial differentiation of embryonic stem cells. Curr Protoc Stem Cell Biol Chapter 1. Unit 1 F 5.Google Scholar
  6. 6.
    Marchetti S, Gimond C, Iljin K, Bourcier C, Alitalo K, Pouyssegur J, Pages G (2002) Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo. J Cell Sci 115:2075–2085PubMedGoogle Scholar
  7. 7.
    Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H (1998) Progressive lineage analysis by cell sorting and culture identifies FLK1  +  VE-cadherin  +  cells at a diverging point of endothelial and hemopoietic lineages. Development 125:1747–1757PubMedGoogle Scholar
  8. 8.
    Hirashima M, Kataoka H, Nishikawa S, Matsuyoshi N (1999) Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis. Blood 93:1253–1263PubMedGoogle Scholar
  9. 9.
    Bahrami SB, Veiseh M, Dunn AA, Boundreau NJ (2011) Temporal changes in Hox gene expression accompany endothelial cell differentiation of embryonic stem cells. Cell Adhesion & Migration 5(2):133–141PubMedCrossRefGoogle Scholar
  10. 10.
    Martinez-Ceballos E, Chambon P, Gudas LJ (2005) Differences in gene expression between wild type and Hoxa1 knockout embryonic stem cells after retinoic acid treatment or leukemia inhibitory factor (LIF) removal. J Biol Chem 280:16484–16498PubMedCrossRefGoogle Scholar
  11. 11.
    Loring JF, Porter JG, Seilhammer J, Kaser MR, Wesselschmidt R (2001) A gene expression profile of embryonic stem cells and embryonic stem cell-derived neurons. Restor Neurol Neurosci 18:81–88PubMedGoogle Scholar
  12. 12.
    Lugus JJ, Park C, Choi K (2005) Developmental relationship between hematopoietic and endothelial cells. Immunol Res 32:57–74PubMedCrossRefGoogle Scholar
  13. 13.
    Yamaguchi TP, Dumont DJ, Conlon RA, Breitman ML, Rossant J (1993) flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors. Development 118:489–498PubMedGoogle Scholar
  14. 14.
    Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336:684–687PubMedCrossRefGoogle Scholar
  15. 15.
    Preffer F, Dombkowski D (2009) Advances in complex multiparameter flow cytometry technology: applications in stem cell research. Cytometry B Clin Cytom 76:295–314PubMedGoogle Scholar
  16. 16.
    Wang Y, Hammes F, De Roy K, Verstraete W, Boon N (2010) Past, present and future applications of flow cytometry in aquatic microbiology. Trends Biotechnol 28:416–424PubMedCrossRefGoogle Scholar
  17. 17.
    Hulspas R, O’Gorman MR, Wood BL, Gratama JW, Sutherland DR (2009) Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin Cytom 76:355–364PubMedGoogle Scholar
  18. 18.
    Lugli E, Roederer M, Cossarizza A (2010) Data analysis in flow cytometry: the future just started. Cytometry A 77:705–713PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • S. Bahram Bahrami
    • 1
  • Mandana Veiseh
    • 2
  • Nancy J. Boudreau
    • 1
  1. 1.Department of SurgeryUniversity of California San FranciscoSan FranciscoUSA
  2. 2.Life Science Division, Lawrence Berkeley National LaboratoryBerkeleyUSA

Personalised recommendations