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258 Part IV: Molecular and Cellular Hematology Chapter 18: Hematopoietic Stem Cells, Progenitors, and Cytokines 259

hepatocytes replace hematopoietic cells and the latter shift to the mar- increases with age in some but not all strains of mice. 42,43 Also, HSC dif-
row, prior to birth. ferentiation in aged animals is skewed toward the myeloid rather than
The final shift in the site of hematopoiesis occurs before birth; lymphoid lineage. The molecular basis for these changes are undergo-
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although the marrow begins to populate with liver derived hematopoi- ing intense study. 45–48
etic cells at day 16 in the mouse and at 8 weeks gestation in the human, Another measure of stem cell kinetics is the time it takes for trans-
it is mostly myeloid in nature and contributes little to the circulating planted marrow cells to repopulate a lethally irradiated animal. Studies
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blood until just before birth. Hematopoietic stem and progenitor cells using retroviral markers suggest that HSCs can be divided into short-
circulate in large numbers during fetal life, as clinically witnessed by the term and long-term repopulating cells, based on the timing of their
use of umbilical cord blood as a rich source of HSCs for transplanta- appearance in the blood following intravenous transplantation (fewer
tion. However, shortly after birth neonatal blood has very few primitive than or more than 3 months following transplantation in mice). How-
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hematopoietic cells, as they begin to home to and lodge in the marrow. ever, a rapidly repopulating stem cell has been identified using a direct
Genetic studies reveal that marrow localization of HSCs is dependent on marrow injection strategy, a cell capable of generating large numbers
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the chemokine CXCL12 (previously known as stromal cell-derived fac- of erythroid and myeloid cells within 2 weeks of injection. Moreover,
tor [SDF]-1) as elimination of the chemokine or its receptor (CXCR4) by transplanting luciferase-labeled single stem cells, a strategy that
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leads to marrow hypoplasia. The shifts in localization of hematopoiesis allows the serial tracking of the cells during life, initially detected foci
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during mammalian development are likely the result of changes both were found to expand locally, seed other sites in the marrow or spleen,
in the cell surface adhesion molecules on hematopoietic stem and pro- and then recede with different kinetics. From these experimental
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genitors that occur during ontogeny, and the characteristics of stromal approaches it is clear that HSCs are heterogeneous.
cells of the yolk sac, AGM, fetal liver, and adult marrow that provide the
microenvironmental support of HSC survival, homing and lodgment,
self-renewal, proliferative expansion, and differentiation (Chap. 7). STEM CELL ASSAYS
Transplantation Assays
THE HEMATOPOIETIC STEM CELL Assays of Murine Stem Cells Experimental transplantation in ani-
mals affords the clearest estimation of HSC properties as the capacity
FUNCTIONAL DEFINITION to durably regenerate all of hematopoiesis in an otherwise lethally irra-
Although the concept of a common “mother cell” of all blood elements diated animal remains the gold standard for the field; moreover, the
technique can be made quantitative. Typically, either 2 × 10 genetically
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in the adult dates to Maximov in 1909, and its potential for participation marked, whole murine marrow cells, or reduced numbers of variably
in disease as proposed by Danchakoff in 1916, the basic concepts of a purified cells are infused intravenously into recipient animals who had
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hierarchical organization of stem and progenitor cells leading to mature previously received 90 to 110 cGy of whole-body irradiation. Blood cells
blood cell production were coalesced by Till and McCulloch using a and marrow are monitored for hematopoietic recovery in the following
spleen colony-forming assay, experimentally establishing the existence weeks and months, and the success of the transplant is measured by
of multipotential hematopoietic cells. The capacity to transplant mar- survival, and long-range contribution to hematopoiesis in the recipient.
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row cells and reconstitute all aspects of hematopoiesis in myeloablated The contribution of donor cells to recovery is established by analysis of
recipients provided an in vivo assay for the HSC, but it was not until the the posttransplant blood or marrow cells; the most common method of
development of clonal in vitro assays of lineage-committed progenitors distinguishing donor from residual recipient blood and marrow cells is
that a coherent model of blood cell production began to emerge. The the use of flow cytometry against isoforms of the cell membrane-bound
pioneering work of Pluznik and Sachs and of Bradley and Metcalf phosphatase CD45, present on virtually all hematopoietic cells. In a
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provided methods to enumerate and characterize marrow cells com- more quantitative embodiment of the strategy, limiting numbers of the
mitted to the hematopoietic lineage. These investigators independently genetically distinct cells (e.g., CD45.1+) are mixed with a “just adequate”
developed culture conditions that allowed colonies of leukocytes to (for full recovery) number of alternately marked cells (e.g., CD45.2+)
develop from single progenitors. However, as a result of the more fas- and the proportion of CD45.1 to total CD45.1+ plus CD45.2+ cells is
tidious conditions required for erythropoiesis and megakaryopoiesis in assessed following transplantation, yielding a calculation of the number
vitro, the description of methods to culture these progenitors did not of stem cells in the initial inoculum, an approach termed competitive
occur for another decade or more. 27–31 Work using density fractionation, repopulation. Because there exist both “short-term” and “long-term”
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cell sorting, and fluorescent dye exclusion methods has yielded purified repopulating cells, the degree of donor cell chimerism is tested 3 or
populations of stem cells, 32–36 common myeloid and lymphoid pro- more months following transplantation, to be certain that only the lat-
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genitors, and lineage-restricted hematopoietic progenitors, 39,40 methods ter are evaluated. For example, transplantation of megakaryocyte-ery-
that have greatly advanced our understanding of the cell and molecu- throid progenitor (MEP) cells allows for survival in a lethally irradiated
lar biology of blood cell development. Figure 18–1 depicts a working mouse, as these cells allow sufficient time for endogenous recovery of
model of this process.
the small number of relatively radio-resistant HSCs in the recipient
mouse. Consequently, survival alone following cell transplantation is
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STEM CELL KINETICS not a sufficient measure of the presence of stem cells in a given popula-
Based on transplantation data indicating that there are a remarkably tion. Thus, with the appropriate caveats, this approach allows an assess-
similar total-body number of HSCs in mice and cats, it has been esti- ment of the numbers or “quality” of HSCs in the test population (i.e.,
mated that all mammals, including humans, possess 2 × 10 stem cells, some genetically altered stem cell populations repopulate less robustly
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and because only a small fraction of these are cycling (and therefore than wild-type cells as a consequence of defects in cytokine receptors
contributing to blood cell production) at any given time, it is also clear or other genes that affect the self-renewal, survival, or proliferation of
that daily blood cell development from the few cycling stem cells to pro- stem cells). Based on the use of these experimental tools, we know most
duce the approximately 4 × 10 mature blood cells represents a mas- about murine HSCs. Obviously, this approach is not available to assess
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sive amplification process. However, the capacity of HSCs to contribute human HSCs. Instead, a number of alternate experimental approaches
to hematopoiesis changes with age (Chap. 9). The number of HSCs have been developed.

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