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Isolation, differentiation and characterization of vascular cells derived from human embryonic stem cells

Nature Protocols volume 5, pages 1115–1126 (2010)Cite this article

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Abstract

Herein, we describe a protocol for the isolation of human embryonic stem cells (hESCs)-derived vascular cells at various stages of development. The cells are isolated from 10 to 15-d-old human embryoid bodies (EBs) cultured in suspension. After dissociation, cells are labeled with anti-CD34 or anti-CD31 (PECAM1) antibody and separated from the cell mixture by magnetic-activated cell separation (MACS) or fluorescent-activated cell sorting (FACS). Isolated vascular cells are then cultured in media conditions that support specific differentiation and expansion pathways. The resulting vascular cell populations contain >80% endothelial-like or smooth muscle-like cells. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 1.5 months. Vascular cells isolated and differentiated under the described conditions may constitute a potential cell source for therapeutic application toward repair of ischemic tissues, preparation of tissue-engineered vascular grafts and design of cellular kits for drug screening applications.

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Figure 1: Characterization of hES-derived endothelial cells isolated from 13-d-old EBs by FACS sorting of CD31+ cells.
Figure 2: Immunofluorescent characterization of VPCs and VPC-derived endothelial and smooth muscle cells.
Figure 3

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Acknowledgements

We would like to acknowledge the financial support of NIH grants HL060435 and DE013023 (R.L.), Marie-Curie Reintegration Grants (S.L. and L.S.F.), MIT-Portugal program (L.S.F.), Crioestaminal/Associação Viver a Ciência (L.S.F.) and FCT (PTDC/SAU-BEB/098468/2008) (L.S.F.). TPK was supported by the Swiss National Science Foundation (grant number PBELP3-127902).

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Author notes

  1. Shulamit Levenberg and Lino S Ferreira: These authors contributed equally to this work.

Authors and Affiliations

  1. Faculty of Biomedical Engineering, Technion Institute of Technology, Haifa, Israel

    Shulamit Levenberg & Limor Chen-Konak

  2. Center of Neurosciences and Cell Biology, University of Coimbra, Portugal

    Lino S Ferreira

  3. Biocant-Biotechnology Innovation Center, Cantanhede, Portugal

    Lino S Ferreira

  4. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Thomas P Kraehenbuehl & Robert Langer

Authors
  1. Shulamit Levenberg
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  2. Lino S Ferreira
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  3. Limor Chen-Konak
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  4. Thomas P Kraehenbuehl
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  5. Robert Langer
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All authors wrote this paper.

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Correspondence to Robert Langer.

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The authors declare no competing financial interests.

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Levenberg, S., Ferreira, L., Chen-Konak, L. et al. Isolation, differentiation and characterization of vascular cells derived from human embryonic stem cells. Nat Protoc 5, 1115–1126 (2010). https://doi.org/10.1038/nprot.2010.31

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