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268 Part IV: Molecular and Cellular Hematology Chapter 18: Hematopoietic Stem Cells, Progenitors, and Cytokines 269

revealed the nonredundant role of these proteins in development of the to TPO. 235,236 Moreover, SCL, a transcription factor that when expressed
corresponding cell lineage. For example, genetic elimination of Pax5 in maturing hematopoietic cells inhibits cytokine-induced granulocytic
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eliminates B cells ; elimination of Ikaros leaves a mouse devoid of fetal and monocytic differentiation, maintaining them in an undifferentiated
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T cells, fetal and adult B cells, and their progenitors ; and loss of C/EBPα state, is enhanced by SCF and down-modulated by GM-CSF. And the
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leads to absolute neutropenia. Moreover, the exogenous expression of level of c-Myb, which determines whether a MEP develops an erythroid
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several transcription factors in lineage committed progenitor cells can or MK fate is affected by TPO, mediated by its induction of miR150.
redirect cell fate. For example, C/EBPα is expressed in myeloid progen- Thus, strong evidence supporting both extrinsic and intrinsic control
itor cells, and introduction of a regulatable C/EBPα gene into purified of lineage determination has been presented, and like the case for most
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erythroid progenitors causes their switch to the myeloid lineage. In conflicts in biology, it is most likely that elements of both mechanisms
further support of this hypothesis, several lines of evidence have been operate in hematopoiesis.
gathered, including the finding that forced expression of the antiapop-
totic gene bc1 in a growth factor-dependent multipotential hematopoi- STEM CELL SURVIVAL,
2
etic cell line resulted in growth factor independence and spontaneous
differentiation into all of the possible cell lineages that develop when the EXPANSION, SELF-RENEWAL,
corresponding growth factor(s) are added to the wild-type cells. 276
In addition to providing these and other arguments in favor of AND DIFFERENTIATION
a transcription factor–based intrinsic regulatory mechanism of stem Like nearly every other cell type, the HSC is subjected to a number of
cell fate, proponents of the intrinsic hypothesis point to feed-forward noxious stimuli, and must possess mechanism to survive such insults.
switch-like molecular mechanisms in which a stochastic increase in one Moreover, the regulation of HSC levels is carefully regulated, such as
of a binary set of such transcription factors reduces the level or activ- seen following transplantation, through both cell expansion and pro-
ity of those transcription factors responsible for alternate cell fates. An grammed cell death. The mechanisms that drive HSC survival are
example of this physiology is illustrated by the mutually antagonistic beginning to be determined.
effects of the erythroid transcription factor GATA1 and the myeloid A number of cytokines affect cell survival by regulating inhibitors
transcription factor PU.1; GATA1 acts to inhibit the myeloid activation of apoptosis, such as the Bcl proteins. In addition, in response to suble-
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potential of PU.1, and PU.1 blocks the binding of GATA1 to its genetic thal DNA damage, cytokines can also act to trigger DNA repair mecha-
target sites. Thus, when the level of GATA1 stochastically rises above nisms. For example, TPO acts to increase the efficiency of DNA-protein
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that of PU.1 in a CMP, the granulocyte-macrophage potential would be kinase-dependent nonhomologous end-joining in response to irradia-
extinguished and the MEP potential of the cell would march forward, tion or chemotherapy, helping to repair double-stranded DNA breaks. 282
unfettered. Alternately, CMPs in which PU.1 levels rise above that of The ability to divide symmetrically to generate identical daugh-
GATA1 would develop along the myeloid lineages, both through the ters is a feature of most cells, including HSCs. However, the multipo-
direct stimulation of myeloid gene expression by PU.1, and indirectly by tent stem cell possesses an added ability to undergo asymmetric cell
the blockade of GATA1-mediated erythroid and megakaryocytic gene divisions, yielding one committed progenitor daughter and one stem
expression programs. cell daughter, or two differentiating progeny; regulating the balance
between symmetric and asymmetric stem cell divisions becomes crit-
The Case for Humoral Mediators ical in maintaining proper HSC numbers and in meeting the demand
Although much evidence has been garnered in favor of an intrinsic for differentiated cells. The beginnings of the molecular origins of asym-
mechanism of stem and progenitor cell fate determination, propo- metric division in the HSC are under study. 283
nents of an extrinsic instructive hypothesis have also generated a large A question related to the previous discussion of whether intrinsic
amount of compelling evidence in favor of the importance of extrinsic or extrinsic factors determine HSC lineage fate, is whether intrinsic or
signals. One illustrative example of the capacity of certain extrinsic sig- extrinsic factors determine the possible outcomes for a dividing HSC
nals to impact specific patterns of differentiation is that the exogenous (two HSC progeny [stem cell expansion], one HSC and one differenti-
expression of an IL-2Rβ transgene in CLPs induces their differentiation ating cell [a self-renewal division], or two differentiating progeny). It is
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into myeloid cells. Subsequent studies revealed that the presence of clear that feedback mechanisms exist that govern the size of the stem cell
the exogenous receptor leads to upregulation of the GM-CSF-R in the pool, as following myeloablation and transplantation of a limited number
CLP, and that exogenous expression of GM-CSF-R could also lead a of HSCs, the pool expands toward that seen in a normal individual but
2
CLP toward monocyte/macrophage development. In separate studies, not beyond, even when subjected to forced overexpression of genes that
other cytokines were shown to direct myeloid lineage fate determina- enhance HSC expansion. HSCs do not appear to have a limit on their
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tion; compared to the differentiation profile seen when marrow cells capacity for expansion; experiments using serial transplantation of mar-
were cultured with SCF alone, an antiapoptotic stimulus, the addition of row cells revealed that even after four such maneuvers the transplantation
IL-5 greatly enhanced the number of marrow progenitor cells that gave of a limiting number of HSCs was associated with a 10-fold expansion in
rise to eosinophilic colonies, whereas the addition of TPO induced a the recipient, a level of expansion remarkably consistent from one serial
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predominance of megakaryocytic colonies, without significant changes transplant to the next. Thus, there does not appear to be an intrinsic limit
in the number of apoptotic cells in any of the three culture conditions. on HSC expansion that sets the size of the stem cell pool. Rather, evidence
These results were interpreted to indicate that while the SCF could from quantitative transplants suggests that there exist both intrinsic and
keep nearly all progenitor cells alive under the cell culture conditions extrinsic controls on the size of the stem cell pool.
employed, the second cytokine directed the multilineage progenitors Following transplantation the degree to which a limiting number
into specific cell fates. 280 of transplanted HSCs expand depends on the source of the cells; fetal
A number of external signaling events have been found to directly liver cells expand to a far greater degree than a similar number of adult
impact the transcriptional apparatus of the cell. For example, as previ- marrow-derived HSCs, indicating that an intrinsic mechanism governs
ously noted, two transcription factors that lead to the self-renewal and stem cell expansion divisions. However, evidence for an extrinsic mech-
expansion of HSCs, HOXB4, and HOXA9 are induced to higher levels anism that regulates stem cell expansion also exists, as the transplan-
of expression or to translocate into the nucleus of stem cells in response tation of a smaller number of either fetal liver or adult marrow HSCs

Kaushansky_chapter 18_p0257-0278.indd 268 9/19/15 12:06 AM