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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Gage, F.H. Mammalian neural stem cells. Science 287, 1433–1438 (2000).
Weissman, I.L. & Shizuru, J.A. The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases. Blood 112, 3543–3553 (2008).
Kitada, M. & Dezawa, M. Induction system of neural and muscle lineage cells from bone marrow stromal cells; a new strategy for tissue reconstruction in degenerative diseases. Histol. Histopathol. 24, 631–642 (2009).
Kuroda, Y., Kitada, M., Wakao, S. & Dezawa, M. Bone marrow mesenchymal cells: how do they contribute to tissue repair and are they really stem cells? Arch. Immunol. Ther. Exp. (Warsz) 59, 369–378 (2011).
Pittenger, M.F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999).
Prockop, D.J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71–74 (1997).
Dezawa, M. et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 309, 314–317 (2005).
Mizuno, H. et al. Myogenic differentiation by human processed lipoaspirate cells. Plast. Reconstr. Surg. 109, 199–209, Discussion 210–211 (2002).
Wakitani, S., Saito, T. & Caplan, A.I. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 18, 1417–1426 (1995).
Makino, S. et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J. Clin. Invest. 103, 697–705 (1999).
Rangappa, S., Entwistle, J.W., Wechsler, A.S. & Kresh, J.Y. Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J. Thorac. Cardiovasc. Surg. 126, 124–132 (2003).
Oswald, J. et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 22, 377–384 (2004).
Cao, Y. et al. Human adipose tissue-derived stem cells differentiate into endothelial cells in vitro and improve postnatal neovascularization in vivo. Biochem. Biophys. Res. Commun. 332, 370–379 (2005).
Dezawa, M. et al. Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. J. Clin. Invest. 113, 1701–1710 (2004).
Woodbury, D., Schwarz, E.J., Prockop, D.J. & Black, I.B. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364–370 (2000).
Mimura, T., Dezawa, M., Kanno, H., Sawada, H. & Yamamoto, I. Peripheral nerve regeneration by transplantation of bone marrow stromal cell-derived Schwann cells in adult rats. J. Neurosurg. 101, 806–812 (2004).
Dezawa, M., Takahashi, I., Esaki, M., Takano, M. & Sawada, H. Sciatic nerve regeneration in rats induced by transplantation of in vitro–differentiated bone-marrow stromal cells. Eur. J. Neurosci. 14, 1771–1776 (2001).
Schwartz, R.E. et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J. Clin. Invest. 109, 1291–1302 (2002).
Miyazaki, M. et al. Improved conditions to induce hepatocytes from rat bone marrow cells in culture. Biochem. Biophys. Res. Commun. 298, 24–30 (2002).
Tang, D.Q. et al. In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 53, 1721–1732 (2004).
Timper, K. et al. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochem. Biophys. Res. Commun. 341, 1135–1140 (2006).
Ferrari, G. et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, 1528–1530 (1998).
Orlic, D. et al. Bone marrow cells regenerate infarcted myocardium. Nature 410, 701–705 (2001).
Lee, J. et al. Migration and differentiation of nuclear fluorescence-labeled bone marrow stromal cells after transplantation into cerebral infarct and spinal cord injury in mice. Neuropathology 23, 169–180 (2003).
Tomita, M. et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells 20, 279–283 (2002).
Tamai, K. et al. PDGFRα-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia. Proc. Natl. Acad. Sci. USA 108, 6609–6614 (2011).
Sakaida, I. et al. Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice. Hepatology 40, 1304–1311 (2004).
Zuk, P.A. et al. Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell 13, 4279–4295 (2002).
De Bari, C., Dell'Accio, F., Tylzanowski, P. & Luyten, F.P. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum. 44, 1928–1942 (2001).
Ishizeki, K., Nawa, T. & Sugawara, M. Calcification capacity of dental papilla mesenchymal cells transplanted in the isogenic mouse spleen. Anat. Rec. 226, 279–287 (1990).
Kim, J.W. et al. Mesenchymal progenitor cells in the human umbilical cord. Ann. Hematol. 83, 733–738 (2004).
Crigler, L. et al. Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages. FASEB J. 21, 2050–2063 (2007).
Fuchs, E. & Segre, J.A. Stem cells: a new lease on life. Cell 100, 143–155 (2000).
Hirata, T.M. et al. Expression of multiple stem cell markers in dental pulp cells cultured in serum-free media. J. Endod. 36, 1139–1144 (2010).
Huang, H.I. et al. Multilineage differentiation potential of fibroblast-like stromal cells derived from human skin. Tissue Eng. Part A 16, 1491–1501 (2010).
De Bari, C. et al. Mesenchymal multipotency of adult human periosteal cells demonstrated by single-cell lineage analysis. Arthritis Rheum. 54, 1209–1221 (2006).
Sarugaser, R., Hanoun, L., Keating, A., Stanford, W.L. & Davies, J.E. Human mesenchymal stem cells self-renew and differentiate according to a deterministic hierarchy. PLoS ONE 4, e6498 (2009).
Kitada, M. Mesenchymal cell populations: development of the induction systems for Schwann cells and neuronal cells and finding the unique stem cell population. Anat. Sci. Int. 87, 24–44 (2012).
Kuroda, Y. et al. Unique multipotent cells in adult human mesenchymal cell populations. Proc. Natl. Acad. Sci. USA 107, 8639–8643 (2010).
Wakao, S. et al. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proc. Natl. Acad. Sci. USA 108, 9875–9880 (2011).
Kucia, M. et al. A population of very small embryonic-like (VSEL) CXCR4+SSEA-1+Oct-4+ stem cells identified in adult bone marrow. Leukemia 20, 857–869 (2006).
Kucia, M., Wysoczynski, M., Ratajczak, J. & Ratajczak, M.Z. Identification of very small embryonic like (VSEL) stem cells in bone marrow. Cell Tissue Res. 331, 125–134 (2008).
D'Ippolito, G. et al. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J. Cell Sci. 117, 2971–2981 (2004).
Toma, J.G., McKenzie, I.A., Bagli, D. & Miller, F.D. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells 23, 727–737 (2005).
De Kock, J., Vanhaecke, T., Biernaskie, J., Rogiers, V. & Snykers, S. Characterization and hepatic differentiation of skin-derived precursors from adult foreskin by sequential exposure to hepatogenic cytokines and growth factors reflecting liver development. Toxicol. In Vitro 23, 1522–1527 (2009).
Bianco, P., Sacchetti, B. & Riminucci, M. Osteoprogenitors and the hematopoietic microenvironment. Best Pract. Res. Clin. Haematol. 24, 37–47 (2011).
Sacchetti, B. et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131, 324–336 (2007).
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).
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.
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)
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)
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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