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Chapter 11 Hematopoietic Microenvironment 123

ensure that they remain quiescent in the absence of antigen stimula- discovery of SCID mice led to development of two powerful models.
tion. The BM also contains a large proportion of regulatory T cells, The first, known as SCID-hu mouse, was generated by engrafting
which have recently been found to exclusively protect HSPCs and human thymus and fetal liver. This model was most informative for
early progenitors from rejection after allogeneic transplantation, the study of human lymphoid development and is still used for
arguing that the endosteal surface acts as an immune privileged site. testing novel HIV drugs. The second model, hu-SRC (for SCID-
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Surprisingly, BM harbors specific niche cells (osteocalcin OLCs) repopulating cell), through the pioneering work of John Dick and
which provide instructive cues to T-competent progenitors that colleagues, enabled investigation of human HSPC engraftment and
migrate from the BM to the thymus, since deletion of the osteocalcin- differentiation. Further modifications of the SCID model led to
expressing cells leads to decreased intrathymic T-cell precursors and generation of NOD/SCID–IL2 receptor γ chain knock-out (NSG)
impaired generation of mature T-cells in the presence of normal strain, which supports robust normal human multilineage (myeloid
thymic function. Specific OLCs therefore appear to be important in and lymphoid) hematopoietic engraftment, as well as engraftment of
T-lymphopoiesis. Their damage in the settings of conditioning for acute myeloid leukemia (AML) and acute lymphoblastic leukemia
transplantation or graft-versus-host disease (GVHD) may compro- (ALL) cells from patients. The sensitivity of the transplant assay is
mise the ability of BM to provide the early T-lineage cells for thymo- further increased by direct intrafemoral injection into the BM cavity;
cyte and ultimately, T-cell production. 23 remarkably, in NSG recipients, human hematopoietic engraftment
can be detected after intrafemoral transplantation of a single highly
purified human HSPC. Further advance in the field of xenotrans-
ERYTHROID NICHES plantation is the development of mouse strains that express several
human hematopoietic cytokines, thereby enabling support of innate
Erythroblastic islands were first described by a French hematologist immune cells (myeloid and NK cells).
Marcel Bessis more than 50 years ago and consist of developing Similar to mouse HSPCs, human HSPCs transplanted into mouse
erythroblasts surrounding a central macrophage. They are present in recipients preferentially traffic to the trabecular bone and home next
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the BM, fetal liver, and the spleen and in in vitro long-term BM to the endosteal surface. They are guided to their niches by the
cultures. The number of erythroblasts per island ranges from 10 cells CXCL12–CXCR4 pathway and cell adhesion molecules such as very
observed in sections of rat femur to 5 to 30 erythroblasts seen in late antigen-4 (VLA-4), very late antigen-5 (VLA-5), and lymphocyte
human BM. Some islands are located adjacent to the BM sinusoids, function-associated antigen-1 (LFA-1); of note, CXCL12 is expressed
and the others are scattered throughout the BM cavity. Within ery- by human OLCs, mesenchymal stromal, Nestin-positive, and endo-
throid islands, the macrophage functions as a “nurse cell” providing thelial cells. CD44 and hyaluronic acid cooperate with CXCL12 in
iron to the developing erythroblasts and phagocytosing the extruded human HSPC homing. Recent experiments identified α6 integrin
nuclei at the end of erythroid differentiation. CD49f as a novel marker for human HSPCs, alluding to functional
Adhesion between maturing erythroblasts and central macrophage importance of HSPC anchorage within BM microenvironment. 25,26
is mediated by several molecules, including erythroblast macrophage The cellular components of the human HSPC niche are yet to be
protein (Emp via homophilic binding), α4β1 integrin (VCAM-1), identified. One potential candidate is a population of mesenchymal
and αv integrin (ICAM-4); antibody-mediated blockade of each of subendothelial cells expressing CD146, which can be prospectively
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these molecule results in disruption of the islands. The most striking isolated from human BM. These perivascular cells were able to
effect is seen with the blockade of Emp, which causes significant establish both bone and hematopoietic microenvironment upon
increase in proliferation, maturation, and apoptosis of maturing subcutaneous transplantation; had a documented self-renewal capac-
erythroblasts in vitro. Of note, Emp-null fetuses die in utero from ity; and produced angiopoietin 1, a cytokine known to induce HSPC
severe anemia. quiescence. CD271 has been suggested as another marker for human
In addition to interaction within macrophages, maturing erythro- hematopoiesis-supporting mesenchymal population; in addition to
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blasts adhere to ECM proteins, fibronectin, and laminin for the CD146 perivascular cells, it labels CD146-endosteal population,
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maturation to proceed. Fibronectin protects erythroblasts from which colocalizes with hematopoietic CD34 cells in human BM.
apoptosis, partly through antiapoptotic bcl-xL, and laminin is The limitations of our knowledge of human hematopoietic
thought to localize reticulocytes to sinusoids as the initial step before microenvironment restrict our ability to accomplish in vitro stem cell
their release into circulation. expansion. The potential benefits of doing so are especially evident
in the context of cord blood transplantation when the number of
donor cells is small and in the setting of gene modified stem cells
MEGAKARYOCYTIC NICHES where gene transduction efficiencies are low. Several molecules have
been tested. One of them is Sonic hedgehog protein, which has been
Megakaryocytes localize to BM endothelial cells in vivo and release shown to induce proliferation of primitive human hematopoietic cells
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platelets into the marrow intravascular–sinusoidal space or the lung when added to highly purified CD34 CD38 lineage human cells;
capillaries. Although CXCL12 induces platelet production by mega- this effect translated into increased level of progenitor expansion in
karyocytes if preceded by migration through endothelial cells, this is NOD/SCID mice. The aryl hydrocarbon receptor antagonist, SR-1
not observed in the absence of endothelial cells, suggesting that also appears to increase HSPC. An engineered Notch ligand Delta 1
megakaryocyte interaction with specific molecules present on the and pleiotrophin have both been shown to increase HSPC. Prosta-
endothelial cells is necessary for thrombopoiesis. FGF4 and CXCL12 glandin E 2 enhances murine HSPC localization in the BM after brief
enhance the interaction of megakaryocytes with endothelial cells and in vitro exposure. This resulted in a two- to threefold increase in the
restore thrombopoiesis in mice deficient in thrombopoietin or its number of HSPCs compared with control (vehicle-exposed) cells. A
receptor c-mpl. Thus, chemokine-mediated localization of mega- phase 1 study in transplant recipients concurrently receiving PGE 2 -
karyocytes within a specific vascular microenvironment is necessary treated and vehicle-treated cord blood units showed that PGE 2 -treated
for their maturation and platelet production. cells generated durable multilineage engraftment and displayed
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greater efficacy, as evidenced by accelerated neutrophil recovery.
These results are encouraging but require validation in larger studies.
HUMAN BONE MARROW MICROENVIRONMENT
HEMATOPOIETIC MICROENVIRONMENT IN ACUTE
Because direct mechanistic studies of human BM microenvironment
cannot be undertaken, the bulk of our knowledge comes from experi- LEUKEMIA AND MYELODYSPLASIA
ments in xenotransplantation models. These initially involved fetal
sheep and heavily irradiated or nude mice as recipients, but only very Given a critical role of hematopoietic microenvironment in safeguard-
low level of human hematopoietic engraftment was observed. The ing cellular homeostasis in the BM, it is not surprising that alterations