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Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells

Nature Protocols volume 9, pages 1514–1531 (2014)Cite this article

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Abstract

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease, as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture, differentiation, isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2–3 weeks: this involves differentiation (10 d), isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes, respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs.

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Figure 1: Timeline of the protocol procedures.
Figure 2: Phase-contrast images of hPSCs at different stages of differentiation.
Figure 3: Flow cytometric analysis of VE-cadherin and CD31 expression at different time points of differentiation.
Figure 4: Characterization of isolated ECs and pericytes.
Figure 5: Coculture of hPSC-derived ECs with pericytes.
Figure 6: Zebrafish xenotransplantation assay.
Figure 7: Zebrafish xenograft as a model to assess mammalian EC functionality.

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Acknowledgements

We thank C. Freund (Department of Anatomy and Embryology and human iPS core facility, LUMC) for the establishment of the hPSC cultures, and D. Ward-van Oostwaard (Department of Anatomy and Embryology, LUMC) and S. van de Pas (human iPS core facility, LUMC) for their excellent technical assistance. We also thank J. Wiegant and A. Boonzaier-van der Laan (Department of Molecular Cell Biology, LUMC) for assistance with the imaging and confocal microscopy. We acknowledge K. Iwata (OSI Pharmaceuticals) for providing TGF-β3. The work was supported by the LUMC Gisela Thier Fellowship (V.V.O.); by the EU's Seventh Framework Programme for research, technical development and demonstration under grant agreement no. 602423 (Plurimes) (V.V.O. and F.E.v.d.H.); by the SWORO Foundation for Research on HHT (V.V.O. and F.E.v.d.H.); by the LeDucq VasculoMorph Transatlantic Consortium (P.t.D.); and the Netherlands Institute of Regenerative Medicine and the Netherlands Heart Foundation (2008B106; C.L.M.).

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Authors and Affiliations

  1. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands

    Valeria V Orlova, Francijna E van den Hil, Sandra Petrus-Reurer & Christine L Mummery

  2. Department of Molecular Cell Biology, Cancer Genomics Centre, Leiden University Medical Center, Leiden, The Netherlands

    Valeria V Orlova, Yvette Drabsch & Peter ten Dijke

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  1. Valeria V Orlova
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  2. Francijna E van den Hil
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  3. Sandra Petrus-Reurer
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Contributions

V.V.O. designed the protocol, supervised the project, wrote the manuscript, established the monolayer differentiation, performed isolation and expansion of ECs and pericytes, performed the coculture experiment and performed all of the cell experiments. F.E.v.d.H. performed all of the cell experiments and wrote part of the protocol. S.P.-R. characterized the monolayer differentiation, performed the coculture experiments and developed the pipeline for the quantification of the vascular sprouts with CellProfiler. Y.D. performed zebrafish xenotransplantation experiments and wrote the zebrafish protocol. P.t.D. supervised the project and edited the manuscript. C.L.M. designed the protocol, supervised the project and wrote the manuscript.

Corresponding authors

Correspondence to Valeria V Orlova or Christine L Mummery.

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Orlova, V., van den Hil, F., Petrus-Reurer, S. et al. Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. Nat Protoc 9, 1514–1531 (2014). https://doi.org/10.1038/nprot.2014.102

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