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Chapter 26 Biology of Erythropoiesis, Erythroid Differentiation, and Maturation 309

Philadelphia-negative MPN are heterogeneous have raised concerns cells and include collagens (types I, III, IV, and V), glycoproteins
whether JAK2 mutations represent the primary transformation event (fibronectin, laminins, thrombospondins, hemonectin, and tenascin),
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in these diseases. Mutations involving Mpl, LNK, TET2, ASXL1, and glycosaminoglycans (hyaluronic acid, chondroitin, dermatan,
EZH2, IDH1/2, CBL, and IKZF1 have been observed in 3% to 20% and heparan sulfate). 352,353 The production of extracellular matrix
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of MPN patients, and more recently Klampfl et al and Nangalia proteoglycans by mesenchymal stromal cells may be regulated by the
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et al have identified novel mutations in exon 9 of the calreticulin Wnt pathway. 354
(CALR) gene in the majority of ET and PMF patients who did not Besides providing structure to the marrow space and a surface
2+
harbor JAK2 or MPL mutations. CALR is a Ca binding protein for cell adhesion, the microenvironment is important for hema-
2+
mostly localized in the endoplasmic reticulum that regulates Ca topoietic cell homing, engraftment, migration, and the response
homeostasis and chaperones other proteins to the nucleus and other to physiologic stress and homeostasis. Although the functional
cellular compartments. Unexpectedly, the results of a large clinical consequences of the microenvironment ultimately must be defined
trial have recently identified that the JAK2 inhibitor ruxolitinib by in vivo studies in mice, dissection of the cellular components
effectively reduced spleen size and disease manifestation both in of the microenvironment, definition of the cytokines that are
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JAK2-mutated and JAK2-nonmutated patients, whereas the JAK2 produced by individual cells, and the nature of cell–cell interac-
inhibitor fedratinib reduced splenomegaly in two CALR-mutated tions have been aided by in vitro models. Long-term bone marrow
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patients. These results suggest that mutations leading to develop- cultures provide an experimental approach for such studies. 355,356
ment of MPN may occur along a unifying pathogenetic pathway Under these in vitro conditions, murine hematopoiesis can be
including JAK2 that may be, therefore, targeted by inhibitors of this maintained for 8 to 10 months and human hematopoiesis for 2 to
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enzyme. 343 3 months. An adherent layer consisting of fibroblasts, adipocytes,
and macrophages is a crucial component of the culture system. Pro-
genitor cells adherent to stroma are generally quiescent (dormant),
HEMATOPOIETIC MICROENVIRONMENT whereas those in the nonadherent cell compartment are in active
cell cycle. 356,357
In invertebrates such as worms and sessile marine creatures, eryth- In vitro studies have demonstrated that stromal cells, including
ropoiesis occurs adjacent to peritoneal and endothelial cells. In pre- endothelial cells and fibroblasts, elaborate cytokines such as GM-CSF,
mammalian species, the spleen is the primary site of erythropoiesis. G-CSF, IL-1, IL-3, IL-6, IL-11, KL, Flt-3 ligand, activin A, and basic
With evolutionary advancement, the function gradually shifts to the fibroblast growth factor, which influence, alone or in combination, the
9
liver and the sinusoidal cavities of bones. These observations suggest growth of adjacent marrow progenitors. 355–358 In addition to positive
that sufficient oxygen, a stagnated flow of blood to avoid dispersion regulators of replication and differentiation, stromal cells elaborate
of factors produced locally, and extensive and redundant surfaces for factors such as TGF-β, TPO, CXCL12L, IFN-γ, and TNF-α, which
cell–cell interactions are essential to supporting red cell production. exert a negative influence on proliferation and may help maintain
Similar sites support erythropoiesis during human development (see a dormant (noncycling) state. 356,357,359–361 Because some regulators
Ontogeny of Erythropoiesis). During both phylogeny and ontogeny, inhibit differentiation along certain lineages but not others, there is
the liver and spleen are primarily erythropoietic organs; granulocytic an intriguing possibility that lineage-specific regulation within the
9
cells dominate in the bone marrow. Within the bone marrow, microenvironment can be achieved through negative, rather than
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hematopoiesis is restricted to the extravascular space, where compact positive, factors. Several cytokines are expressed in a transmembrane
collections of cells are interspersed among venous sinuses. These form as well as a soluble (secreted) product. Others bind extracellular
sinuses originate adjacent to the endosteal bone surface and empty matrix, a mechanism that not only allows for high concentrations of
into a central longitudinal vein. Studies in mice demonstrate that a factor within the microenvironment that metabolically stabilizes
BFU-E follows a bimodal distribution with peaks adjacent to the these factors but also keeps them adjacent to developing progenitors.
periosteum and midcavity, whereas CFU-E and later erythroid cells Among the factors elaborated by stromal cells, KL has the most
have a broad distribution with highest incidence toward the axis profound effect on erythropoiesis. Mice unable to synthesize KL die
d
of the femur, adjacent to the central vein, 9,344 thus suggesting that in utero because of severe anemia. Steel-Dickie (Sl ) mice that are
the local anatomy (specialized niches?) influences the maturation of unable to make the membrane-restricted form of KL are viable but
erythroid cells. severely anemic, whereas other lineages in these animals are marginally
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The bone marrow microenvironment consists of three broad affected or not affected by this defect. The fact that erythropoiesis
components: stromal cells (e.g., fibroblasts, endothelial cells, mesen- is abnormal, despite high levels of circulating EPO and the presence
chymal stem cells and their diverse descendant progeny), accessory of soluble KL, suggests that normal erythroid differentiation and
cells (monocytes, macrophages, megakaryocytes, T cells), and extra- maturation require both a functional membrane-restricted KL/KIT
cellular matrix (a protein–carbohydrate scaffold). In the bone marrow, and an EPO signaling pathway. Cross-phosphorylation of EPOR by
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although early studies described two distinct hematopoietic niches for KL may provide a basis for the predominantly erythroid effect.
hematopoietic stem cells/progenitor cells (i.e., the “endosteal” niche Furthermore, data suggest that tyrosine cross-phosphorylation of
and the “endothelial or vascular niche” within the medulla), more EPOR is sustained longer when cells are cultured on steel stromal
recent studies suggest that this distinction may be artificial, because cells engineered to express the membrane-restricted form of KL than
both cellular structures can be intimately associated in trabecular cells expressing the soluble form. 364
bone. Thus mesenchymal stem cell–derived osteoprogenitor cells and The soluble form of KL is produced by proteolytic cleavage
stromal reticular cells (nestin+ or leptinR+) are intimately associated of membrane KL and is released in the circulation. Soluble KL
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with sinusoidal endothelium and seem to be pivotal organizers of the effectively supports erythroid maturation in vitro but is dispens-
bone marrow niche. 345–347 Reciprocal communication of hematopoi- able for steady-state erythropoiesis, because targeted mutant mice
etic stem cells with cells/matrix in their bone marrow niche ensures expressing exclusively the more stable membrane isoform of KL, KL2,
both their quiescent state and self-renewal dynamics. lacking the major proteolytic cleavage site, have normal hematocrit
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EPO, in addition to promoting erythropoiesis directly, enhances values. However, these mice recover poorly from radiation-induced
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erythropoiesis indirectly by decreasing the interaction of hematopoi- anemia. In wild-type mice, sublethal radiation induced a transient
etic stem cells with their niches, reducing the amount of trabecular fourfold increase in KL in the serum (from <0.5 up to >2 ng/mL),
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bone and downregulating CXCR4 expression, the receptor for reaching a peak after 7 days. In contrast, the proteolytic-cleavage
CXCR12L/SDF1, on hematopoietic stem cells. 349 mutant KL KL2/KL2 mice did not release soluble KL into the serum after
Accessory cells are progeny of hematopoietic stem cells; hence sublethal radiation, and survival was significantly diminished because
after marrow transplantation these cells are of donor origin, whereas of anemia. This phenotype is remarkably similar to that of mice
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stromal cells remain mostly host derived. 350,351 Extracellular matrix lacking the dimerization domain of GR. Recent data indicate that
molecules are synthesized and secreted by microenvironmental soluble KL specifically induce expression of GRα in human erythoid