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Adult mouse hematopoietic stem cells: purification and single-cell assays

Nature Protocols volume 1, pages 2979–2987 (2006)Cite this article

Abstract

Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34−/lowc-Kit+Sca-1+lineage marker− (CD34−KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34−KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.

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Figure 1: Sorting gates for CD34−KSL cells.
Figure 2: Clonal assays for CD34−KSL cells.
Figure 4: Immunostaining steps.
Figure 5: Long-term reconstitution with single CD34−KSL cells.
Figure 6: Immunostaining for CD34−KSL cells.
Figure 3: Single-cell liquid cultures.

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Acknowledgements

The authors thank A. S. Knisely for critically reviewing the manuscript. The work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Hideo Ema, Yohei Morita, Satoshi Yamazaki, Azusa Matsubara, Jun Seita, Yuko Tadokoro, Hiroyoshi Kondo & Hiromitsu Nakauchi

  2. ReproCell Inc., A3-12F Imperial Hotel Tower, 1-1-1 Uchisaiwai-cho, Chiyoda-ku, Tokyo, 100-0011, Japan

    Yohei Morita & Satoshi Yamazaki

  3. Department of Hematology and Oncology, Musashino Red Cross Hospital, 1-26-1 Kyonan, Musashino, Tokyo, 180-8610, Japan

    Hina Takano

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  1. Hideo Ema
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Correspondence to Hiromitsu Nakauchi.

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Ema, H., Morita, Y., Yamazaki, S. et al. Adult mouse hematopoietic stem cells: purification and single-cell assays. Nat Protoc 1, 2979–2987 (2006). https://doi.org/10.1038/nprot.2006.447

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