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

to other cells. In such cultures, disruption of adherence causes pro- As HSCs mature into committed progenitors, they migrate toward
grammed cell death; for example, endothelial cells undergo apoptosis the marrow vasculature, and into a higher oxygen tension atmosphere.
upon forced detachment in vitro, as a result of disruption of multiple This would appear to be required to gain the additional metabolic rate
integrins. Integrins also influence the proliferation of cells by affecting afforded by oxidative phosphorylation. As one component of this “met-
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the G to S phase transition of the cell cycle. These effects also operate abolic switch,” marrow hematopoietic progenitor cells express lower
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in vivo; α integrin (a component of the α β collagen receptor) null mice levels of PDKs, reducing PDK-mediated suppression of oxidative phos-
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have a hypoplastic dermis, and the growth of α −/− fibroblasts on col- phorylation seen in HSCs. 102
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lagen is substantially reduced. 92
Hematopoietic stem and progenitor cells express multiple inte- CELL-CYCLE CHARACTERISTICS
grins, including α β (also termed very-late antigen [VLA] 4), which
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binds to either vascular cell adhesion molecule (VCAM) 1 or fibronec- Adult hematopoietic cells display altered engraftment capacity depen-
tin, and α β (VLA5), which binds to a region of fibronectin distinct dent on their phase in the cell cycle. Using primitive hematopoietic cell
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from the β β binding domain. Moreover, primitive hematopoietic cells populations several investigators have demonstrated that only quiescent
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are thought to express integrin αIIbβ , the platelet fibrinogen receptor, G /G phase cells engraft into lethally irradiated recipient animals; cells
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based on the death of multiple hematopoietic lineages in mice express- in the S and early G phase display minimal engraftment capacity,
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ing a suicide transgene under control of the integrin αIIb promoter. a situation that can be experimentally manipulated; elimination of p21,
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However, the physiologic significance of this finding is uncertain at a key cell-cycle progression gene, enhances stem cell expansion. This
present. finding correlates well with findings that the profile of expressed genes
The avidity of progenitor cell–integrin interactions can be altered in a highly selected population of primitive hematopoietic cells shifts
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by external effectors; numerous cytokines and chemokines, including when they are induced from G phase into the cell cycle. However,
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cytokines critical for stem cell function (stem cell factor [SCF], TPO, even though this cell-cycle dependence of engraftment of stem cells is
and CXCL12), enhance integrin-mediated binding. 94–96 Counterre- true for adult cells, the corresponding cell populations derived from
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ceptors for both integrins, such as VCAM1 and fibronectin (FN), are umbilical cord blood or fetal liver is not cell-cycle dependent. A bet-
highly expressed in the marrow matrix and on marrow stromal cells ter understanding of these findings is very likely to shed important new
(see “Matrix Proteins” in “The Hematopoietic Microenvironment” later). insights into the genes that regulate engraftment.
Integrin-based interactions with the stroma are responsible for hom-
ing and retention of stem and primitive progenitor cells in the marrow, GENE EXPRESSION PROFILE
as antibodies that interfere with the interaction can mobilize stem and It can be argued that the most critical feature of the HSC is its abil-
progenitor cells into the blood. However, it is uncertain whether inte- ity to quantitatively balance its three fates—apoptosis, self-renewal,
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grins can influence the survival or growth of HSCs, or affect their ulti- and differentiation—into the mature elements of the blood. Moreover,
mate developmental fate. the undifferentiated cell must express (at the least) the initiating genes
responsible for all possible developmental lineages. A useful conceptual
METABOLIC CHARACTERISTICS framework for this process can be constructed by considering the gene
expression profiles of stem and committed hematopoietic progenitors
One of the hallmarks of HSCs is their resistance to chemotherapy-in- that develop into the multiple hematopoietic differentiation pathways.
duced cytotoxicity. A primary reason for this property is high-level At each developmental step genes associated with the adopted pathway
expression of drug efflux pumps of the multidrug resistance class of should remain expressed or be upregulated, while the genes that specify
proteins. The presence of these verapamil-sensitive efflux pumps has the alternate lineage(s) are likely silenced. A thorough understanding
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enabled the separation of HSCs based on their low-level retention of of these gene expression profiles should help to explain the circuitry
various fluorescent markers such as rhodamine 123 and Hoechst 33342, of specific aspects of hematopoiesis, and of developmental biology in
the “Rh /Ho ” population of murine cells and the side population (SP) general.
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of cells in human marrow. However, before such maneuvers can be Initial studies using immortalized multipotent hematopoietic cell
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used for clinical stem cell enrichment procedures, the lack of toxicity lines reinforced this conceptual framework; pluripotency is character-
of the fluorescent dyes must be confirmed. Nevertheless, such experi- ized by the expression of multiple genes associated with multiple cell
mental strategies continue to shed important insights into HSC biology. fates. Studies of purified HSCs and lineage-committed progenitors
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As a consequence of residing physically removed from the marrow have also strengthened this hypothesis, revealing coexpression of sev-
vasculature, the stem cell microenvironment is hypoxic (see “Anatomy” eral different lineage-affiliated gene sets in single primitive hemato-
in “Hematopoietic Microenvironment”). The metabolic consequence is poietic cells. In contrast, the downstream progenitors of HSCs were
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that HSCs display a low metabolic state, with most of its ATP generation found to express only lineage-appropriate transcripts, such as for the
derived from glycolysis. As the HSC rarely undergoes cell division, it granulocyte colony-stimulating factor receptor (G-CSF-R) in granu-
can “afford” the low metabolic state of cells dependent on glycolysis. locyte-macrophage progenitors (GMPs), or β-globin and the erythro-
One of the transcription factors critical for HSC physiology, MEIS1, poietin receptor (EPOR) in committed erythroid progenitors. Similar
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appears responsible for this metabolic profile, by driving expression findings were reported for lymphoid committed cells, although some
of hypoxia-inducible factor (HIF)-1α, that upregulates expression of promiscuity was detected in B-cell progenitors. 109
the glycolytic enzyme machinery. In addition, HIF-1α also induces With these principles established, more ambitious efforts to cata-
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pyruvate dehydrogenase kinases (PDK1-4) that prevent mitochondrial logue all the genes expressed by each stage of hematopoietic develop-
pyruvate oxidation by suppressing pyruvate dehydrogenase complex, ment have been made possible by advances in microarray and whole
the first step that fuels the Krebs cycle. Stem cell down-modulation of transcriptome sequencing approaches to gene expression. On an even
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mitochondrial oxidative phosphorylation acts to reduce the generation broader scale, and as might be expected, comparisons of different types
of reactive oxygen species (ROS), which is highly toxic to HSCs. It has of stem cells (e.g., liver, skin, neural) reveals an overlap in the expressed
been suggested that this property, the avoidance of ROS generation, genes, supporting the hypothesis that the mechanisms responsible for
contributes to the longevity of the stem cell pool in any individual. critical stem cell properties, such as self-renewal, are shared among the
Kaushansky_chapter 18_p0257-0278.indd 261 9/19/15 12:05 AM