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In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood

Nature Protocols volume 3, pages 1932–1940 (2008)Cite this article

Abstract

Establishment of robust xenograft models is critical to studying human hematopoiesis in a physiologic setting. Using a recently developed immunodeficient mouse strain, we have established long-term multilineage human grafts and demonstrated their serially transplantability using limited numbers of purified human hematopoietic stem cells (HSCs). Herein, we describe our protocol for the isolation of human HSC (Lin-CD34+CD38−CD90+) from umbilical cord blood (CB) as well as the xenotransplantation system that allows stable engraftment of human hematopoietic cells with as few as ten HSCs. Isolation of CB mononuclear cells requires 2–3 h, and cells may be cryopreserved before transplantation. Isolation of HSC requires approximately 2–3 h, and transplantation requires 1 h. Short-term and long-term engraftment is assessed 4–6 weeks and 10–12 weeks post-transplantation, respectively, with preparation and analysis time requiring 4–8 h at each time point.

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Figure 1: Gating strategy for FACS-purification of human CB HSCs and MPP.
Figure 2: Engraftment of human CB HSCs in NSG mice.
Figure 3: BM engraftment of human CB HSC in primary and secondary recipients.
Figure 4: Flow chart for HSC transplantation and assessment of engraftment.

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Acknowledgements

We thank Libuse Jerabek and Theresa Storm for their lab management expertise and Adriane Mosley for animal husbandry. This research is supported by National Institutes of Health/National Cancer Institute grants R01CA86017 to I.L.W. and KO8CA1295470 to C.Y.P. R.M. is supported by the Walter and Idun Y. Berry Foundation.

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Authors and Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 1050 Arastradero Road, Palo Alto, 94304, California, USA

    Christopher Y Park, Ravindra Majeti & Irving L Weissman

  2. Department of Pathology, Stanford University, Palo Alto, 94305, California, USA

    Christopher Y Park & Irving L Weissman

  3. Department of Medicine, Stanford University, Palo Alto, 94305, California, USA

    Ravindra Majeti

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Contributions

C.Y.P. and R.M. contributed equally to this work.

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Correspondence to Christopher Y Park.

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Competing interests

C.Y.P and R.M. have no conflicts of interest to disclose. I.L.W. was a member of the scientific advisory board of Amgen and owns significant Amgen stock. I.L.W. cofounded and consulted for Systemix, is a cofounder and director of Stem Cells Inc., and cofounded Cellerant, Inc.

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Park, C., Majeti, R. & Weissman, I. In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood. Nat Protoc 3, 1932–1940 (2008). https://doi.org/10.1038/nprot.2008.194

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