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Isolation of tissue-resident vascular endothelial stem cells from mouse liver

Nature Protocols volume 15, pages 1066–1081 (2020)Cite this article

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Abstract

Endothelial cells (ECs) are fundamental components of the blood vessels that comprise the vascular system; facilitate blood flow; and regulate permeability, angiogenesis, inflammatory responses and homeostatic tissue maintenance. Accumulating evidence suggests there is EC heterogeneity in vivo. However, isolation of fresh ECs from adult mice to investigate this further is challenging. Here, we describe an easy and reproducible protocol for isolation of different types of ECs and CD157+ vascular-resident endothelial stem cells (VESCs) by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting. The procedure was established on liver tissue but can be used to isolate ECs from other organs with minimal modification. Preparation of single-cell suspensions can be completed in 2.5 h. We also describe assays for EC clonal and network formation, as well as transcriptomic analysis of isolated ECs. The protocol enables isolation of primary ECs and VESCs that can be used for a wide range of downstream analyses in vascular research.

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Fig. 1: Schema for the mechano-enzymatic dissociation of liver.
Fig. 2: FACS gating strategy for liver ECs.
Fig. 3: EC network formation and colony-forming assays with CD157+ VESCs.
Fig. 4: qRT-PCR analysis of different types of liver cells.

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Acknowledgements

We thank N. Fujimoto and Y. Mori for technical assistance. This work was supported by the Japan Agency for Medical Research and Development (AMED)-PRIME grant no. 19gm6210009h0002, AMED grant nos. 19cm0106508h0004 and 19gm5010002s1103, and JSPS KAKENHI grant no. 19K22562, the Takeda Science Foundation, the Princess Takamatsu Cancer Research Foundation, the Daiichi-Sankyo Foundation of Life Science, and the SGH Foundation.

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Authors and Affiliations

  1. Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

    Hisamichi Naito, Taku Wakabayashi, Masako Ishida, Tomohiro Iba, Fitriana Nur Rahmawati, Shota Shimizu & Nobuyuki Takakura

  2. Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, USA

    Chang-Hyun Gil & Mervin C. Yoder

  3. Laboratory of Signal Transduction, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Nobuyuki Takakura

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  1. Hisamichi Naito
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Contributions

H.N designed the protocol, performed experiments, analyzed data and wrote the manuscript. T.W., M.I., T.I. and C.-H.G. performed experiments. M.I. prepared most of the cell suspensions. C.-H.G. independently replicated some of the results produced using this protocol in a different lab (in the United States). F.N.R. and S.S. helped with FACS data acquisition. M.C.Y. contributed to protocol design, supervised the project, and edited and reviewed the manuscript. N.T. contributed to protocol design, supervised the project, and reviewed the manuscript.

Corresponding authors

Correspondence to Hisamichi Naito, Mervin C. Yoder or Nobuyuki Takakura.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks David Smadja and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Naito, H., Kidoya, H., Sakimoto, S., Wakabayashi, T. & Takakura, N. EMBO J. 31, 842–855 (2012): https://doi.org/10.1038/emboj.2011.465

Wakabayashi, T. et al. Cell Stem Cell 22, 384–397 (2018): https://doi.org/10.1016/j.stem.2018.01.010

Naito, H. et al. Dev. Cell 48, 151–166.e7 (2019): https://doi.org/10.1016/j.devcel.2018.12.002

Integrated supplementary information

Supplementary Figure 1 FACS gating for isolation of CD157+ VESCs from lung, hind limb muscle, intestine and pancreas.

Representative FACS plots with the percentage of the parent gate for each population. All animal experiments in this figure were performed in accordance with the Institutional Animal Care and Use Committee of Osaka University and received approval from the Institutional Review Board.

Supplementary Figure 2 Fluorescence minus one (FMO) controls and FACS results of incomplete liver dissociation.

(a) Flow cytometric analysis of the liver showing the distribution of fluorescence of the FMO control and unstained control. (b) Flow cytometric analysis of the liver comparing successful (left) and incomplete (right) dissociation resulting in different percentages of cells in the CD31+CD45−fraction. All animal experiments in this figure were performed in accordance with the Institutional Animal Care and Use Committee of Osaka University and received approval from the Institutional Review Board.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

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Naito, H., Wakabayashi, T., Ishida, M. et al. Isolation of tissue-resident vascular endothelial stem cells from mouse liver. Nat Protoc 15, 1066–1081 (2020). https://doi.org/10.1038/s41596-019-0276-x

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