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Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells

Nature Protocols volume 8, pages 1391–1415 (2013)Cite this article

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Abstract

Multilineage-differentiating stress-enduring (Muse) cells are distinct stem cells in mesenchymal cell populations with the capacity to self-renew, to differentiate into cells representative of all three germ layers from a single cell, and to repair damaged tissues by spontaneous differentiation into tissue-specific cells without forming teratomas. We describe step-by-step procedures for isolating and evaluating these cells. Muse cells are also a practical cell source for human induced pluripotent stem (iPS) cells with markedly high generation efficiency. They can be collected as cells that are double positive for stage-specific embryonic antigen-3 (SSEA-3) and CD105 from commercially available mesenchymal cells, such as adult human bone marrow stromal cells and dermal fibroblasts, or from fresh adult human bone marrow samples. Under both spontaneous and induced differentiation conditions, they show triploblastic differentiation. It takes 4–6 h to collect and 2 weeks to confirm the differentiation and self-renewal capacity of Muse cells.

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Figure 1
Figure 2: Schematic diagram of the self-renewal of Muse cells.
Figure 3: Cell density and the confluency of mesenchymal cells.
Figure 4: Isolation of Muse cells by fluorescence-activated cell sorting (FACS) with anti–SSEA-3 antibody.
Figure 5: Characterization of M-clusters.
Figure 6: Expression of pluripotency markers in M-clusters.
Figure 7: Spontaneous differentiation of Muse cells on a gelatin-coated coverslip.
Figure 8: Induced differentiation of Muse cells into mesodermal-, endodermal- and ectodermal-lineage cells.

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Acknowledgements

This work was supported by the Japan New Energy and Industrial Technology Development Organization. Antibodies against Oct3/4 were a gift from H. Hamada (Osaka University).

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Author notes

  1. Yasumasa Kuroda, Shohei Wakao and Masaaki Kitada: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Anthropology, Tohoku University Graduate School of Medicine, Sendai, Japan

    Yasumasa Kuroda & Mari Dezawa

  2. Department of Stem Cell Biology and Histology, Tohoku University Graduate School of Medicine, Sendai, Japan

    Shohei Wakao, Masaaki Kitada, Toru Murakami, Makoto Nojima & Mari Dezawa

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  1. Yasumasa Kuroda
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Contributions

Y.K., S.W., M.K. and M.D. contributed to the development of the methodology, Y.K., S.W., M.K., M.N. and M.D. performed the experiments and all the authors prepared the figures. M.K. and M.D. contributed to the description of the protocols.

Corresponding authors

Correspondence to Yasumasa Kuroda, Masaaki Kitada or Mari Dezawa.

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Competing interests

S.W., M.K. and M.D. have filed a patent application (in Japan, the United States, Canada, Europe, China, Korea, Australia, India and Singapore) on some of the methods described in this article.

Supplementary information

Supplementary Figure 1

Telomerase activity in Muse cells. Telomerase activity in naive normal adult human dermal fibroblast (NHDF), Muse cells derived from NHDF (Muse (NHDF)), adult human bone marrow stromal cells (BMSCs), Muse cells derived from BMSCs (Muse (BDNF)), and Hela cells. The sample of Hela cells without the treatment for DNA amplification was used for the negative control (Polymerase (-)). Telomerase activity analysis was performed with TRAPEZE XL Telomerase Activity Detection kit (Millipore, Cat. No. S7707) and Ex Taq Polymerase (Takara, Cat. No. RR001A) under the protocol provided by the manufacturer, and 1.0 x 106 cells of each cell type were applied to this assay. The fluorescence intensity was measured by a microplate reader Infinite M1000 PRO (Tecan). A part of data in Supplementary Figure 1 is modified from Wakao et al. (2011). Reference Wakao et al. Proc Natl Acad Sci U S A 108, 9875-9880 (2011). (PDF 540 kb)

Supplementary Figure 2

The growth curve of Muse cells in M-cluster formation. Each M-cluster derived from normal adult human dermal fibroblast was treated with Trypsin-EDTA solution at 37 °C for 15 min followed by pipetting with a glass micropipette. The number of cells in each well was counted at days 2, 3, 5, 7, 8, 11, and 14. In M-cluster formation Muse cells proliferated constantly during day 2 to day 11, but they ceased cell division after day 11. The doubling time during this time span was calculated as 43.4 h (1.81 d). Supplementary Figure 2 is modified from Kuroda et al. (2010). Reference Kuroda, Y., et al. Proc Natl Acad Sci U S A 107, 8639-8643 (2010). (PDF 539 kb)

Supplementary Figure 3

Differentiation marker expression in naive Muse cells. (a-j) Immunocytochemistry for differentiation markers in Muse cells. Muse cells isolated from normal adult human dermal fibroblast are negative for the differentiation markers (red) neurofilament (NF) (a), smooth muscle actin (SMA) (b), cytokeratin-7 (CK7) (c), alpha-fetoprotein (a-FP) (d), Desmin (e), Osteocalcin (f), Nestin (g), Musashi-1 (h), NeuroD (i), and MAP-2 (j). (k) Oil red staining of Muse cells. Naive Muse cells are negative for Oil red. (l) The mRNA expression in Muse cells. Muse cells do not express differentiation markers alpha-fetoprotein (α-FP), GATA6, MAP-2, Nkx2.5, and albumin. Human fetal liver (Liver) was used as positive control for a-FP and albumin, and whole human embryo (Embryo) for GATA6, MAP-2, and Nkx2.5. Scale bar, 30 μm (a-k). Supplementary Figure 3l is modified from Wakao et al. (2011). Reference Wakao et al. Proc Natl Acad Sci U S A 108, 9875-9880 (2011). (PDF 1382 kb)

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Kuroda, Y., Wakao, S., Kitada, M. et al. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nat Protoc 8, 1391–1415 (2013). https://doi.org/10.1038/nprot.2013.076

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