Protocol abstract
Nature Protocols 3, - 1932 - 1940 (2008)
Published online: 20 November 2008 | doi:10.1038/nprot.2008.194
Subject Categories: Cell and tissue culture | Model organisms
In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood
Christopher Y Park1,2, Ravindra Majeti1,3 & Irving L Weissman1,2
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.
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 1050 Arastradero Road, Palo Alto, California 94304, USA.
- Department of Pathology, Stanford University, Palo Alto, California 94305, USA.
- Department of Medicine, Stanford University, Palo Alto, California 94305, USA.
Correspondence to: Christopher Y Park1,2 e-mail: cypark@stanford.edu
nature-products
A-Z product listing
- 1,000
propidium iodide(Molecular Probes/Invitrogen) - 15-ml conical tubes(Falcon)
- 2 mM glutamax(GIBCO)
- 2% wt/vol Dextran 500(GE Healthcare)
- 5-ml polystyrene round-bottomed tubes(BD)
- Alexa 488 or PE-Cy7-conjugated CD45.1, clone A20.1.7(eBiosciences)
