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260 Part IV: Molecular and Cellular Hematology Chapter 18: Hematopoietic Stem Cells, Progenitors, and Cytokines 261

Assays of Human Hematopoietic Stem Cells Severely immuno- cells; most successful stem cell purification strategies employ several
compromised mice can be engrafted by human HSCs, provided their such techniques.
survival can be supported in a strictly controlled animal care environ- The antigenic proteins and glycoproteins that exclusively or pre-
ment and that the experiments take place prior to the development of dominantly present on HSCs include (1) CD34, a 90- to 110-kDa type I
other untoward effects in such animals (e.g., tumor formation). The glycoprotein that is postulated to mediate cell adhesion and/or cell-cycle
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first assay employing this strategy relies on the combined immuno- arrest 70–72 ; (2) CD90 (Thy1), a heavily glycosylated glycophosphoinos-
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deficiency created by the severe combined immunodeficiency (SCID) itol-linked protein that participates in T-cell adhesion to stromal cells ;
and nonobese diabetic (NOD) genetic mutations. Subsequently, these (3) CD117 (the c-Kit receptor), which supports primitive hematopoi-
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mice were found to bear some ability to reject or alter the developmen- etic cell survival and proliferation 76,77 ; (4) AA4, a murine molecule
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tal characteristics of human cell repopulation, leading other investiga- homologous to the human phagocyte C1q complement receptor ; (5)
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tors to add genetic defects to the NOD-SCID background that improve Sca1, a murine surface molecule shown by knockout studies to be
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the engraftment of normal and pathologic human marrow cells, such necessary for normal stem cell development ; (6) CD133, a 115-kDa
as NOD-SCID/β -microglobulin null, or the most commonly used pentaspan cell-surface glycoprotein expressed on the apical surface of
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NOD-SCID/γc null, or by crossing with mice that also express human neuroepithelial and HSCs that has been proposed to function in estab-
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hematopoietic cytokines. Such animal models have allowed the assess- lishing or maintaining plasma membrane protrusions ; (7) CD164, a
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ment of (1) stem cell numbers in human CD34+ cells from mobilized cell-surface sialomucin that is present in several alternately spliced iso-
blood or umbilical cord blood, (2) assessment of the effects of gene forms and that enhances blood cell homing and inhibits CD34+/CD38−
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therapy vectors, 59,60 cell-cycle inhibitors, or cytokine cocktails designed cell proliferation, and CD150, a member of the signaling lymphocyte
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to expand stem cell numbers 58–64 on the retention of repopulating capac- activation molecule (SLAM) family of lymphocyte proliferation recep-
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ity, or (3) the study of fundamental biologic properties of human HSCs tors ; and (8) CD110 (the thrombopoietin [TPO] receptor c-Mpl)
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in vivo, such as the cell-cycle restriction of repopulating cells. 65 present on virtually all repopulating HSCs, and established to be vital
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for human HSC physiology as genetic elimination of the receptor leads
Surrogate In Vitro Stem Cell Assays to congenital amegakaryocytic thrombocytopenia at birth and aplastic
Although in vivo assays remain the gold standard, NOD-SCID and anemia shortly thereafter (Chap. 117).
more severely immunocompromised mice are difficult to maintain and Many or most of the surface membrane proteins found on HSCs
remain expensive and quite cumbersome methods to assess human are also present on cells that have begun to differentiate toward spe-
HSC quality and quantity. As a result, a number of culture-based meth- cific lineages, precluding the exclusive use of positive selection alone
ods were developed to more quickly and quantitatively evaluate human for stem cell purification. Thus, a number of stem cell purification strat-
HSC function. Generally, each relies on long-term cell growth in culture egies include negative selection, based on cell surface markers absent
and other special features to establish their validity as a model of the on HSCs but present on mature blood cells and their corresponding
human HSC. unilineage-committed progenitors. Typically, cocktails of negatively
The ability to grow marrow cells in culture for extended periods of selecting antibodies include CD38, HLA-DR, CD3, CD4, CD5, or CD8
time provided an important tool to explore HSC biology. In long-term for T lymphocytes; CD11b, CD14, or Gr-1 to exclude macrophages and
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cultures human or murine marrow is incubated in serum-containing granulocytes; CD10, CD19, CD20, or B220 to eliminate B lymphocytes;
medium under defined conditions, and after several weeks the stromal and glycophorin A or Ter119 to remove erythroid cells. The products
layer that has developed is recharged with fresh marrow cells, which that result from the use of such combinations of negative-selecting anti-
then produce mature blood cells and their progenitors for many months. bodies are termed Lin− cells.
Cell fractionation studies show that the HSC resides adherent to the A particularly difficult problem is presented by separating true
stromal cell layer in such cultures, and that enzymatic disruption of HSCs from their progeny committed to the lymphoid or myeloid lin-
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the stromal layer will allow one to reseed a secondary stromal cell layer eage, but not differentiated beyond that stage. Several studies clarify the
with the capacity to produce hematopoietic cells for a period of weeks cell surface profile of the common lymphoid progenitor (CLP) as Lin−/
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to months, thereby defining an in vitro assayable cell termed the long- interleukin (IL)-7R(receptor)α+/Thy1−/Sca-110 /c-kit low37 and the
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term culture-initiating cell (LTC-IC). A second assay that was devel- common myeloid progenitor (CMP) as Lin−/IL-7Rα−/c-Kit+/Sca-1−.
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oped based on similar principles is the cobblestone area-forming cell The cell surface phenotype of human HSCs includes CD34+/CD38−/
(CAFC), which, when evaluated by phase-contrast microscopy, gives KDR(VEGFR2)+/Thy1+/CD133+/Lin−, although most of these mark-
rise to complex colonies of multiple hematopoietic cell types under the ers require careful clinical assessment before their widespread use in
stromal cell layer of long-term cultures. Unfortunately, when careful patients can be considered. At present, at least for murine cells, that
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comparisons are made between these assays and transplantation stud- problem has been solved. The most effective cell sorting method for
ies, the true HSCs comprise only a fraction of the repopulating cells purifying murine HSC is the E-SLAM approach, negatively selecting for
found in marrow. Thus, conclusions about stem cell behavior from such CD48 and positively selecting for CD45, CD201, and CD150. It is esti-
in vitro assays cannot be considered rigorous. mated that such a cell population is at least 50 percent pure HSC based
on single-cell transplantation experiments in mice.
CELL SURFACE PHENOTYPE
Numerous investigators have used monoclonal antibodies to an increas- STEM CELL INTEGRINS
ing number of hematopoietic cell surface proteins to negatively and/ Integrins are a family of heterodimeric single-pass transmembrane
or positively enrich for stem and primitive hematopoietic progenitor proteins (18 α and 8 β subunits form at least 24 different cell surface
cells. Although the function of only a few of these stem cell markers adhesion receptors in humans) characterized by multiple immunoglo-
is known, it has not impeded their use for research and/or therapeu- bin (Ig)-like extracellular domains that allow two-way communica-
tic benefit. Others have taken advantage of the capacity of primitive tion between a cell and its environment. A large number of cell types
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hematopoietic cells to extrude fluorescent organic chemicals or on their require contact for survival; in vitro, this is usually manifest as integrin-
buoyant density to obtain purified populations of these scarce marrow dependent cell adhesion, either to extracellular matrix protein(s) or

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