Selective lodgement or homing of transplanted hemopoietic stem cells in the recipient's bone marrow (BM) is a critical step in the establishment of long-term hemopoiesis after BM transplantation. However, despite its biologic and clinical significance, little is understood about the process of homing. In the present study, we have concentrated on the initial stages of homing and explored the functional role in vivo of some of the adhesion pathways previously found to mediate in vitro adhesion of hemopoietic cells to cultured BM stroma. We have found that homing of murine hemopoietic progenitors of the BM of lethally irradiated recipients at 3 h after transplant was significantly reduced after pretreatment of the donor cells with an antibody to the integrin very late antigen 4 (VLA4). This inhibition of marrow homing was accompanied by an increase in hemopoietic progenitors circulating in the blood and an increased uptake of these progenitors by the spleen. Similar results were obtained by treatment of the recipients with an antibody to vascular cell adhesion molecule 1 (VCAM-1), a ligand for VLA4. Furthermore, we showed that administration of the same antibodies (anti-VLA4 or anti-VCAM-1) to normal animals causes mobilization of hemopoietic progenitors into blood. These data suggest that hemopoietic cell lodgement in the BM is a regulatable process and can be influenced by VLA4/VCAM-1 adhesion pathway. Although additional molecular pathways are not excluded and may be likely, our data establish VCAM-1 as a BM endothelial addressin, analogous to the role that mucosal addressin cell adhesion molecule (MAdCAM) plays in lymphocyte homing. Whether splenic uptake of hemopoietic progenitors is passive or controlled through different mechanisms remains to be clarified. In addition, we provide experimental evidence that homing and mobilization are related phenomena involving, at least partly, similar molecular pathways.

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic 1,2 . We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.Low neutrophil numbers lead to opportunistic infections. There are two hereditary human neutropenia syndromes: cyclic hematopoiesis 4 , comprising three-week oscillations of blood cells, and SCN 3 , consisting of statically low neutrophil counts progressing to leukemia. Heterozygous mutations of ELA2 cause cyclic hematopoiesis and about two-thirds of SCN cases. Mutations in WAS (different from those that cause Wiskott-Aldrich thrombocytopenia) also cause SCN 5 . Owing to its severity, SCN usually arises from new mutations, and additional genes associated with neutropenia have not yet been identified.

Interaction of hemopoietic cells with the elements of the underlying bone marrow stroma, the unique site of their "homing" in adult individuals, is essential for sustained normal hemopoiesis. However, the specific molecules responsible for homing and for the continuing interaction of hemopoietic cells with the bone marrow stromal cells in vivo, or those involved in progenitor/stem cell trafficking through the bloodstream, have not been defined. A large repertoire of adhesion receptors, especially of the integrin family, appear to play a prominent role in promoting adhesion of hemopoietic stem cells to cultured marrow stromal cells in vitro. To test the functional role of cytoadhesion molecules in vivo, we treated primates systemically with either anti-a4-or anti-P2-integrin antibodies, whose antigens are found in the majority of hemopoietic progenitors and in many differentiated cells. We found that anti-a4 (anti-VLA4, anti-CD49d) but not anti-P2

It was previously reported that treatment with the sulfated polysaccharide fucoidan or the structurally similar dextran sulfate increased circulating mature white blood cells and hematopoietic progenitor/stem cells (HPCs) in mice and nonhuman primates; however, the mechanism mediating these effects was unclear. It is reported here that plasma concentrations of the highly potent chemoattractant stromal-derived factor 1 (SDF-1) increase rapidly and dramatically after treatment with fucoidan in monkeys and in mice, coinciding with decreased levels in bone marrow. In vitro and in vivo data suggest that the SDF-1 increase is due to its competitive displacement from heparan sulfate proteoglycans that sequester the chemokine on endothelial cell surfaces or extracellular matrix in bone marrow and other tissues. Although moderately increased levels of interleukin-8, MCP1, or MMP9 were also present after fucoidan treatment, studies in gene-ablated mice (GCSFR ؊/؊ , MCP1 ؊/؊ , or MMP9 ؊/؊ ) and the use of metalloprotease inhibitors do not support their involvement in the concurrent mobilization. Instead, SDF-1 increases, uniquely associated with sulfated glycan-mobilizing treatments and not with several other mobilizing agents tested, are likely responsible. To the authors' knowledge, this is the first published report of disrupting the SDF-1 gradient between bone marrow and peripheral blood through a physiologically relevant mechanism, resulting in mobilization with kinetics similar to other mobilizing CXC chemokines. The study further underscores the importance of the biological roles of carbohydrates. IntroductionStromal-derived factor 1 (SDF-1) is a highly potent chemoattractant both in vitro and in vivo for mature leukocytes and hematopoietic progenitor/stem cells (HPCs), which carry its receptor CXCR4. [1][2][3][4][5][6][7] This highly conserved chemokine is constitutively expressed by virtually all tissues, 8 including bone marrow (BM). 3 It is expressed as 2 alternatively spliced isoforms, the predominant ␣ form and the ␤ form containing 4 additional amino acids at the C terminus, each possessing a heparin-binding domain. 9,10 The SDF-1-CXCR4 interaction plays a dominant role in hematopoiesis, and mice deficient in either gene die in utero exhibiting defects in B-cell lymphopoiesis and BM myelopoiesis. 4,5 Additionally, a critical role for CXCR4 on human cells in engraftment to the BM of nonobese diabetic/severe combined immunodeficiency mice 6,7 has been shown. Although involvement of SDF-1 in mobilization-the egress of HPCs from the BM to the peripheral blood (PB)-has also been speculated, direct evidence has only recently been obtained in mice using a synthetic SDF-1 analog 11 or following injection with an adenovirus expressing human SDF-1. 12 Previously, we reported that the sulfated polysaccharide fucoidan (FucS) and the structurally similar dextran sulfate (DexS) can elevate circulating white blood cells (WBCs) and mobilize HPCs within hours in a selectin-independent manner in mice and nonhuman primates. 13...

The specific retention of intravenously administered hemopoietic cells within bone marrow is a complex multistep process. Despite recent insights, the molecular mechanics governing this process remain largely undefined. This study explored the influence of ␤ 2 -integrins on the homing to bone marrow and repopulation kinetics of progenitor cells. Both antifunctional antibodies and genetically deficient cells were used. In addition, triple selectin-deficient mice were used as recipients of either deficient (selectin or ␤ 2 ) or normal cells in homing experiments. The homing patterns of either ␤ 2 null or selectin null cells into normal or selectin-deficient recipients were similar to those of normal cells given to normal recipients. Furthermore, spleen colonyforming units and the early bone marrow repopulating activity for the first 2 weeks after transplantation were not significantly different from those of control cells. These data are in contrast to the importance of ␤ 2 -integrin and selectins in the adhesion/migration cascade of mature leukocytes. The special bone marrow flow hemodynamics may account for these differences. Although early deaths after transplantation can be seen in recipients deficient in CD18 and selectin, these are attributed to septic complications rather than homing defects. However, when ␤ 2 -or selectin-null donor cells are treated with anti-␣ 4 antibodies before their transplantation to normal or selectin-deficient recipients, a dramatic inhibition of homing (>90%) was found. The data suggest that the ␣ 4 ␤ 1 /vascular cell adhesion molecule-1 pathway alone is capable of providing effective capture of cells within the bone marrow, but if its function is compromised, the synergistic contribution of other pathways, that is, ␤ 2 -integrins or selectins, is uncovered. IntroductionRestriction of the developing hemopoietic cells to certain anatomic sites within the body, that is, the extravascular spaces of the bone marrow (BM), signifies a unique microenvironment capable of providing not only anchorage to hemopoietic cells, but of transmitting signals enabling their proliferation and differentiation. The relationship of hemopoietic cells with their microenvironment is a highly dynamic one allowing further expansion on demand and, depending on the stimuli, the movement of cells in and out of their microenvironment. The ability of intravenously administered cells to re-establish connections within the BM environment is a clinically exploited example of that flexibility. As the sinusoidal endothelial cells separate the extravascular space from the circulation, intravenously administered cells need first to tether to the sinusoidal endothelium, then to firmly anchor themselves within the extravascular space through reversible adhesion and transmigration steps. Although several insights regarding the molecular pathways that guide these processes have been obtained, our knowledge of this complex process remains incomplete. Early reports suggest that galactose-and mannose-specific lectins (present...

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Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45 ؉ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45 ؊ erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (⑀) and fetal (␥) globins, with little or no adult globin (␤). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms. IntroductionDuring human development, hematopoietic cells sequentially recruit new anatomic sites for their development, from the yolk sac, to the fetal liver, to the bone marrow (BM) in adults. Erythroid cells developing at these sites are distinguished morphologically, and they display distinct transcriptional factor and growth factor requirements, proliferative kinetics, and globin patterns. 1 Thus, erythroid cells maturing in yolk sac (primitive erythroid cells) have a characteristic morphology: they remain mostly nucleated at terminal maturation and synthesize mainly embryonic globins (⑀, , and ␣). Fetal cells have a macrocytic cell morphology and synthesize more than 80% fetal globins (␣2␥2), in contrast to adult cells that synthesize more than 90% adult globins (␣2␤2). Fetal and adult cells in circulation are enucleated, and both are considered of definitive type.Due to the transient nature of primitive erythropoiesis and because of ethical concerns in conducting experiments in human embryos, the regulation of primitive erythropoiesis has remained inadequately explored. Extensive research with murine embryonic stem (ES) cells differentiated through embryoid body (EB) formation and directed hematopoietic differentiation has shown that it recapitulates the earliest stages of murine hematopoietic development, as the appearance of primitive hematopoietic cells was followed by the emergence of definitive cells expressing the appropriate globin phenotypes. 2,3 Similar studies with human ES cells have been conducted only recently. [4][5][6][7][8][9][10][11] However, there are discrepancies among the studies published regarding the kinetics as well as the morphology and globin patterns of erythroid cell...

The mechanisms by which expression of the beta-like globin genes are developmentally regulated are under intense investigation. The temporal control of human embryonic (epsilon) globin expression was analyzed. A 3.7-kilobase (kb) fragment that contained the entire human epsilon-globin gene was linked to a 2.5-kb cassette of the locus control region (LCR), and the developmental time of expression of this construct was studied in transgenic mice. The human epsilon-globin transgene was expressed in yolk sac-derived primitive erythroid cells, but not in fetal liver or bone marrow-derived definitive erythroid cells. The absence of epsilon gene expression in definitive erythroid cells suggests that the developmental regulation of the epsilon-globin gene depends only on the presence of the LCR and the epsilon-globin gene itself (that is, an autonomous negative control mechanism). The autonomy of epsilon-globin gene developmental control distinguishes it from the competitive mechanism of regulation of gamma and beta-globin genes, and therefore, suggests that at least two distinct mechanisms function in human hemoglobin switching.