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Stem Cells Handbook pp 155–161Cite as

Hematopoietic Stem Cells

Identification, Characterization, and Assays

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Abstract

The small population of pluripotent hematopoietic stem cells (PHSCs) in the bone marrow consists of short-term reconstituting cells (STRCs) and long-term reconstituting cells (LTRCs), based on how quickly the transplanted cells can produce progeny in an irradiated recipient. They can be “purified” using a combination of cell size; density; fluorescent dye uptake; resistance to cytotoxic chemicals; and cell-surface markers including Thy 1.1 (T), Sca-1 (S), c-kit (K), lineage (L), CD38 (38), and CD34 (34). Using five-color fluorescence-activated cell sorting the long term, very primitive mouse LTRCs are L-/lo, S+, K+, 38+, 34-, and appear to mature to L-/lo, S+, K+, 38+, 34+ cells and then to L-/lo, S+, K+, 38-, 34+; thus, STRCs acquire CD34 and lose CD38 on maturation from CD34- LTRCs. CD34 has been used to isolate PHSCs for human transplantation studies; therefore, the LTRC may be lost during this procedure. Experimental transplantation studies indicate that the best reconstitution occurs when both cell populations are present, the more mature cells activating the immature cells after myeloablation, whereas the mature cells provide negative control in normal animals. Functionally the type of assay that has been most widely used for the quantitation of mouse stem cells is the in vivo repopulating assay. Different numbers of donor cells are combined with a standard number of normal bone marrow cells. The normal cells protect against the immediate effects of myeloablation and compete with the donor stem cells. The proportions of mature cells derived from the donor stem cells are determined by the detection of a donor-specific marker, such as an isoenzyme, Y-chromosome, or congenic antigen. Similarly, using limiting dilution transplant of a donor test population of cells and a standard number of stem cell—compromised serially transplanted cells, the relative contribution of the donor cells is measured as a competitive repopulating unit. Finally, the repopulating stem cell unit assay using complete myeloablation and busulfan-treated bone marrow radioprotective support cells provides comparatively rapid and sensitive detection of the very small numbers of LTRCs present in limiting dilution transplants. This procedure utilizes busulfan because it appears preferentially stem cell toxic, and it provides radioprotective support cells that are unable to compete effectively with normal donor stem cells in the population under investigation. Stem cells are selected based on their ability to produce both lymphoid and myeloid repopulation in severely ablated mice, rather than competitive ability.

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Authors
  1. Ian Ponting PhD
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  2. Yi Zhao MD
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  3. W. French Anderson MD
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Editors and Affiliations

  1. Wadsworth Center and Ordway Research Institute, Empire State Plaza, Albany, NY, USA

    Stewart Sell MD

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Ponting, I., Zhao, Y., Anderson, W.F. (2004). Hematopoietic Stem Cells. In: Sell, S. (eds) Stem Cells Handbook. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59259-411-5_14

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  • DOI: https://doi.org/10.1007/978-1-59259-411-5_14

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61737-367-1

  • Online ISBN: 978-1-59259-411-5

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