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264 Part IV: Molecular and Cellular Hematology Chapter 18: Hematopoietic Stem Cells, Progenitors, and Cytokines 265

transmembrane domains, and by extension, the two intracellular kinase M-CSF and SCF. Like the other two cytokines, FL displays a 4α-helix
domains. Once SCF binds to c-Kit the affinity for dimer formation bundle tertiary structure and exists in both membrane-bound and
overcomes the D4 electrostatic repulsion and the two kinase domains soluble states, the result of alternate splicing of the primary transcript
are brought together to initiate signaling. The intracellular mediators that does or does not include a cleavage site for its release from the cell
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activated by SCF binding to c-Kit include phosphoinositol 3′-kinase membrane. 188
(PI3K), mitogen-activated protein kinases (MAPKs), phospholipase C Unlike SCF levels that remain relatively static regardless of blood
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gamma (PLCγ), and c-Src (Chap. 17; reviewed in Ref. 174). cell counts, blood concentrations of FL can rise more than 25-fold in
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SCF is synthesized by marrow fibroblasts and other cell types. Sol- response to pancytopenia. Interestingly, only pancytopenia, and not
uble SCF is a highly glycosylated 36-kDa protein released from its initial individual lineage deficiencies cause an increase in blood FL concen-
site on the cell membrane by proteolytic processing. An alternatively trations, suggesting that the cytokine is a bona fide regulator of stem
spliced form of SCF messenger RNA (mRNA), that does not encode the or primitive hematopoietic cells. Consistent with this conclusion,
cleavage site, remains on the cell membrane, and is a more potent stim- transplantation data indicate that HSCs from Flt3-deficient mice do
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ulus of c-Kit-receptor-bearing cells. The ratio of soluble to membrane not effectively reconstitute the hematopoietic system, being three- to
encoding SCF mRNA varies widely in different tissues, ranging from eightfold less efficient in repopulation as wild-type cells, a conclusion
10:1 in the brain, to 4:1 in the marrow, to 0.4:1 in the testis. reinforced by its genetic combination with c-Kit mutant mice. 190
The importance of SCF to hematopoiesis is easily demonstrated; Like SCF, FL appears to act on HSCs only in synergy with other
although nullizygous mice (Sl/Sl) are embryonic lethal because of a hematopoietic cytokines, 191,192 a finding particularly true for its combi-
number of developmental defects, the presence of a partially functional nation with TPO. 193,194 In addition, FL is a potent stimulus of B lymp-
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allele (Sl ) allows compound heterozygotes (Sl/Sl ) to survive into adult- hopoiesis and granulocyte-macrophage proliferation and development,
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hood, albeit with severe anemia because of diminished numbers/quality particularly of the latter toward the dendritic cell lineage. 195,196
of HSCs. In addition to its critical role in the development of embryonic Thrombopoietin TPO is a 45- to 70-kDa hormone that was
and fetal hematopoiesis, treatment of adult mice with an antibody that cloned by both traditional biochemical purification and expression
neutralizes the SCF receptor, c-Kit, also results in severe pancytopenia, cloning strategies based on the use of a then orphan class I cytokine
indicating an important hematopoietic role for the receptor/ligand pair receptor, first identified as the cellular homologue of the murine-trans-
throughout life. forming oncogene v-mp1. TPO bears extensive sequence homology to
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When present in culture SCF alone can maintain the long-term erythropoietin (EPO), sharing 20 percent identity and an additional 25
repopulating ability of murine Sca-1+/Rh /Lin− hematopoietic cells, percent similarity. The hormone is produced in several organs, includ-
lo
suggesting that the cytokine can promote the survival of HSCs in vitro. ing the liver, kidney, skeletal muscle, and the marrow stroma. Based on
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However, alone, SCF is only a weak stimulator of cell proliferation, pri- murine liver transplantation studies about half of steady-state TPO pro-
marily inducing the development of mast cells both in vitro and in vivo. duction occurs in that organ, but in states of thrombocytopenia the
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Nevertheless, in the additional presence of IL-3, IL-6, IL-11, G-CSF, or marrow stroma increases production substantially. 160,163 The hormone
TPO, SCF exerts profound effects on the generation of hematopoietic acts on megakaryocyte (MK) progenitors to enhance their survival and
progenitor cells of all lineages, 176–178 pointing to primitive hematopoietic proliferation and on immature MKs to promote their differentiation,
cells as critical targets. The molecular mechanisms of such synergy are but surprisingly not on mature cells during platelet formation. Mul-
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beginning to emerge. A physical association of c-Kit and EPOR has tiple lines of evidence also indicate that TPO can exert profound effects
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been detected following SCF stimulation of cells bearing both receptors, on the HSC. The hormone also supports the survival of candidate HSC
an event that is essential for their functional synergy. 180 populations, and acts in synergy with IL-3 and SCF to induce these cells
Flt3 Ligand FL was cloned as the binding partner for the then into the cell cycle and increase their output of both primitive and com-
newly identified novel orphan receptor Flt3, a protein most closely mitted hematopoietic progenitor cells of all lineages. 200,201 These proper-
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related to the receptors for macrophage colony-stimulating factor ties are also seen in vivo. For example, administration of the hormone
(M-CSF) (hence the term flt = fms-like tyrosine kinase), and c-Kit. to myelosuppressed animals leads to more rapid recovery of all hemato-
FL is expressed by T lymphocytes and marrow stromal cells. 164,181 The poietic lineages, including primitive cells, 202–205 and genetic elimination
Flt3 receptor is a 160-kDa cell-surface molecule expressed primarily on of TPO or its receptor severely reduces the number of marrow stem
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primitive hematopoietic cells. Like c-Kit, activation of Flt-3 results and progenitor cells of all lineages to 15 to 25 percent of normal val-
in activation of several signaling mediators, including the p85 subunit ues. 87,206,207 In addition, as noted in “Flt3 Ligand” above, TPO acts in
of phosphatidylinositol 3-kinase, SHP, PLCγ, and a guanosine 5′- synergy with FL to expand primitive hematopoietic cells in suspension
triphosphatase (GTPase)-activating protein, activating Ras. Normal culture, and when used to supplement LTC, the hormone maintained
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Flt3 signaling also activates the MAPKs extracellular regulated kinase HSC numbers for up to 2 months, compared to standard LTCs in
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(ERK)-1 and ERK2 but leads to only weak phosphorylation of signal which repopulating HSCs are no longer detectable at this time.
transducer and activator of transcription (STAT)-5, in contrast to an The TPO receptor, the product of the cellular protooncogene
oncogenic form of the receptor, identified in 25 percent of patients with c-Mpl, is a member of the cytokine receptor family that includes EPO,
myelodysplastic syndromes or acute myelogenous leukemia. 184,185 In the G-CSF, growth hormone, leptin, and many others. Upon binding TPO,
leukemic cells of such patients Flt3 bears an internal tandem duplica- the homodimeric c-Mpl activates its tethered Jak2 kinases, leading to
tion of the kinase domain, resulting in the constitutive activation of the phosphorylation of three of the cytoplasmic domain tyrosine residues.
receptor. Clinically, this is associated with reduced likelihood of patient These phosphotyrosine residues then act as docking sites for several
survival; hence, this observation has led to an attempt to control the secondary signaling molecules, including STATs, MAPKs, and PI3K,
growth of such mutant-receptor-bearing cells with specific Flt3 kinase ultimately leading to the expression of a number of transcription factors
inhibitors, with some success (Chap. 88). (e.g., homeobox-containing proteins, HIFs) and cell-survival molecules
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FL was initially cloned using a soluble form of the receptor to iden- (e.g., BclXL). A more complete discussion of the molecular mechanisms
tify ligand-bearing cells. As their receptors bear a number of com- by which TPO affects the HSC is found in Chap. 17.
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mon structural features, it was not surprising to find that FL shares CXCL12 (Previously Termed Stromal Cell-Derived Factor 1)
significant structural homology, as well as biologic properties with both CXCL12 is produced by a number of the cells that occupy the

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