We previously show that protein-kinase C-β (PKC-β) orchestrates the tumor interaction with the surrounding microenvironment. Malignant B-cells in ALL and CLL induce PKC-β overexpression in bone marrow stroma cells (BMSCs), and in turn, this overexpression is required for the support provided by stromal cells to the tumor.

Fragestellung: Für die adulte Stammzellforschung ist das Fachgebiet der Gynaekologie und Geburtshilfe prädestiniert, da in diesem Fachgebiet die biologische Resource in Form von Nabelschnurblut und –stroma reichlich vorhanden ist. Die Migration der HSZ innerhalb ihres Mikrokosmos ist massgeblich für den Erfolg z.B. einer Stammzell-Transplantation verantwortlich. Das Ziel der vorliegenden Arbeit ist die Darstellung der xenotransplantierten CD34+-Zellen mittels MRT und Korrelation der Signalergebnisse mit der tatsächlich im Tierpräparat darzustellenden Anzahl an transplantierten Zellen am Endostium. Methode: Humane CD34+-Zellen wurden nach Abnahme von NS-Blut postpatum mittels microbeads (Miltenyi Biotech) separiert. Nach einer Eisenmarkierung galt das Interesse der Darstellung des Migrations- und ´Homing´-Verhalten mittels Magnetresonanztomographie in Balc-c-Mäusen zu unterschiedlichen Zeitpunkten. Ergebnisse: Ca.0,3% der aus NS-Blut gewonnenen mononukleären Zellen expremieren. Es konnten pflastersteinartige Kolonien bis zu 12 Wochen nach Aussiedelung in die Zellkultur nachgewiesen werde und so die Vitalität der Zellen bewiesen werden. Nach Injektion von CD34+-Zellen in die Schwanzvene von Balb-c-Mäusen konnten wir eine deutliche Signalzunahme bei der MRT in Leber, Milz, Lunge, Femur und Knochenmark 2 und 24h post injectionem nachweisen. Die nötige Mindestdosis an markierten CD34+-Zellen lag hierfür bei 5×19´6-Zellen. Nach 48h wurden die Mäuse geopfert. Erste histologische Untersuchungen zeigen korrelierend zur Bildgebung in der Magnetresonanztomographie die tatsächliche Präsenz humaner CD34+-Zellen im Knochenmark. Schlussfolgerung: Erste Ergebnisse zeigen die tatsächliche Etablierung einer Methodik zur Markierung humaner CD34+-Zellen und Visualisierung dieser Zellen post injectionem in der MRT im Xenotransplantationsmodell.

Umbilical cord blood (UCB) is frequently collected for the purpose of hematopoietic stem cell therapy. However, the use of these cells is infrequent. To investigate other possible applications we recently showed that UCB is an excellent source of endothelial colony-forming cells which improve vessel density and heart function ofter myocardial infarction (Ott, Keller, et al., FASEB J , 2005; 19:992–4). We here investigated whether UCB or the umbilical cord would be suitable as a routine source of mesenchymal colony-forming cells (CFU-F). CD34+ and AC133+ cells were selected by MACS (Miltenyi) from mononuclear UCB cells. The resulting cells were cultured at limiting dilutions on fibronectin in medium with 2% FCS (BioWhittaker: EGM-2). CFU-F were isolated from 3 of 7 (42%) UCB donors investigated. However, the CFU-F frequency is extremely low: only 1.3 in 100 ml of UCB. Similraly to the endothelial cells we previously described, these few UCB-derived stromal cells (CBSC) could easily be expanded. In Parallel, we also investigated CFU-F from the human umbilical vein: human umbilical vein stromal cells (HuvSC). Here, CFU-F were isolated from all donors when veins were prepared within 6 hours of collection. The CFU-F frequency was estimated to be 3 to 5 per 10 cm of umblical vein. Like the UCB-derived CFU-F, HuvSC were easily expandable. To find out whether these stromal cells showed characteristics of immature cells, we investigated the presence of embryonic markers by RT- and realtime PCR. We confirmed the expression of POU5F1 (Oct4), SSEA-4 and Stella-related (DPPA3). Immature HuvSC were shown to express mRNA of many genes associated with mesenchymal lineages but did not express the hematopoietic markers CD45 or CD34. HuvSC were shown to differentiate into adipogenic, osteogenic and myogenic lineages, though their differentiation is not as pronounced as bone marrow derived MSC. HuvSC did also support long-term production of immature cobblestone area-forming cells week 6. In conclusion, UCB and the umbilical vein contain CFU-F with characteristics of immature mesenchymal stem cells. However, not all UCB collections contained CFU-F and the CFU-frequency is very low. Thus, UCB can, in our view, not be used as routine MSC source. On the other hand, CFU-F grew from small stretches of umbilical vein (HuvSC), making these cells excellent source of mesenchymal progenitors for tissue engineering purposes.

Mobilization and recruitment of endothelial progenitor cells (EPC) contributes to vasculogenesis in vivo. So far, applications for cell therapy are limited by the number of available cells. Expansion of EPC or their progeny may, therefore, facilitate its therapeutic use in ischemic disease. The aim of this study was to expand CD34+ EPC-derived progeny from different sources, characterize them, and investigate their potential for use in therapeutic vasculogenesis. CD34+ cells from G-CSF-mobilized peripheral blood (PB) and cord blood (CB) were isolated using immunomagnetic beads and cultured in endothelial cell medium. Cells were expanded up to 16 (PB) and up to 46 (CB) population doublings, respectively. Immunophenotypic and mRNA expression analyses showed a high degree of similarity between the cultured cells and human umbilical vein endothelial cells (HUVEC). By day 14 after transplantation, transplanted human CD31-positive EPC-derived cells were detected. These cells expressed the proliferation marker Ki67 and formed vessel-like structures in ischemic myocardium. Most strikingly, transplantation of EPC-derived cells improved left ventricular function after experimental ischemia, as shown by echocardiography. In conclusion, cells cultured from CD34+ EPC can be expanded in vitro to clinically relevant numbers. In vivo, these cells proliferate, form vascular structures, and improve left ventricular function after experimental myocardial infarction. Therefore, in vitro expanded EPC-derived endothelial cells may be beneficial in the treatment of ischemic disease.

We here investigated whether receptor tyrosine kinase signaling (ckit, flt3) was sufficient to maintain hematopoietic stem cells (HSC) in culture, or whether, like in embryonic stem cells, signaling through gp130 is also required. Sorted CD34+ AC133+ (CD33/ CD38/ CD71)- cells from human umbilical cord blood (CB) were cultured in the presence of combinations of ckit-ligand stem cell factor (SCF), flt3-ligand (FL) and different stimulators of gp130 (IL-6, IL-11, LIF, OSM, CT1 and CNTF). Of these cytokines, SCF stimulated cell division by itself. Gp130-stimulating cytokines did not give any additional cell division activity. When SCF was included in the cytokine cocktail, progenitor levels (CFC, CAFCw2, CAFCw6) was very similar for all combinations of cytokines tested after this initital 6-day culture. To study more immature progenitors, cells were harvested after 6 days of serum-free culture and cultured for another 6 weeks on FBMD-1 stromal cells (LTC-CAFC). These experiments showed that in initial 6 day cultures with FL or gp130-stimulators alone, very little hematopoietic activity remained. SCF maintained CFC, and LTC-CAFCw2 and LTC-CAFCw6 at about half the original level. However, combinations of SCF with OSM consistently increased the number of LTC-CAFCw2 and w6 about two-fold. This finding suggests that very immature hematopoietic progenitors were at least maintained in serum-free cultures. To study the most immatore cells, we performed in transplantations into sublethally irradiated NOD/SCID mice. When CB cells were cultured with SCF alone, NOD/SCID-repopulating cell (CRU) frequency (1 in 900) dropped to about 20% in 6 days (1 in 5500). Similarly, cultures without growth factors or with FL or OSM showed a decrease to less than 10% of the original CRU number. In contrast, the combination of SCF and OSM maintained CRU at about half the original level (1 in 1900). This study shows that signals through ckit and gp130 synergize to maintain CRU level. However, to achieve expansion of human NOD/SCID-repopulating cell numbers, additional signaling pathways need to be stimulated.

RATIONALE AND OBJECTIVES:To compare and optimize ferumoxides labeling of human hematopoietic progenitor cells from umbilical cord blood and from peripheral blood for subsequent in vivo tracking with a clinical 1.5 T MR scanner.MATERIALS AND METHODS:Human hematopoietic progenitor cells, derived from umbilical cord blood or peripheral blood, were labeled with Ferumoxides by simple incubation or lipofection. Cellular iron uptake was quantified with spectrometry. Then, 3 x 10(7)-labeled cells were injected into the tail vein of 12 female nude Balb/c mice. The mice underwent magnetic resonance imaging before and 24 hours after injection. Precontrast and postcontrast signal intensities of liver, spleen, and bone marrow were measured and tested for significant differences with the t-test. Immunostains served as a histopathologic standard of reference.RESULTS:After labeling by simple incubation, only umbilical cord blood cells, but not peripheral blood cells, showed a significant iron uptake and could be tracked in vivo with magnetic resonance imaging. Using lipofection, both cell types could be tracked in vivo. A significant decline in signal intensity was observed in liver, spleen, and bone marrow at 24 hours after injection of efficiently labeled ferumoxides cells (P < .05). Histopathology proved the distribution of iron oxide-labeled cells to these organs.CONCLUSION:Hematopoietic progenitor cells from umbilical cord blood can be labeled by simple incubation with an Food and Drug Administration-approved magnetic resonance contrast agent with sufficient efficiency to provide an in vivo cell tracking at 1.5 T. Progenitor cells from peripheral blood need to be labeled with adjunctive transfection techniques to be depicted in vivo at 1.5 T.

We recently established that two midgestation-derived stromal clones-UG26-1B6, urogenital ridge-derived, and EL08-1D2, embryonic liver-derived-support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC-stroma contact is required for this stem cell maintenance. Adult bone marrow ckit(+) Ly-6C(-) side population (K6-SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which factors might be involved in HSC maintenance, we compared the gene expression profile of EL08-1D2 and UG26-1B6 with four HSC-nonsupportive clones. We found that EL08-1D2 and UG26-1B6 both expressed 21 genes at a higher level, including the putative secreted factors fibroblast growth factor-7, insulin-like growth factor-binding proteins 3 and 4, pleiotrophin, pentaxin-related, and thrombospondin 2, whereas 11 genes, including GPX-3 and HSP27, were expressed at a lower level. In summary, we show for the first time long-term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support.

Apoptosis is an essential process in embryonic tissue remodeling and adult tissue homeostasis. Within the adult hematopoietic system, it allows for tight regulation of hematopoietic cell subsets. Previously, it was shown that B-cell leukemia 2 (Bcl-2) overexpression in the adult increases the viability and activity of hematopoietic cells under normal and/or stressful conditions. However, a role for apoptosis in the embryonic hematopoietic system has not yet been established. Since the first hematopoietic stem cells (HSCs) are generated within the aortagonad-mesonephros (AGM; an actively remodeling tissue) region beginning at embryonic day 10.5, we examined this tissue for expression of apoptosis-related genes and ongoing apoptosis. Here, we show expression of several proapoptotic and antiapoptotic genes in the AGM. We also generated transgenic mice overexpressing Bcl-2 under the control of the transcriptional regulatory elements of the HSC marker stem cell antigen-1 (Sca-1), to test for the role of cell survival in the regulation of AGM HSCs. We provide evidence for increased numbers and viability of Sca-1(+) cells in the AGM and subdissected midgestation aortas, the site where HSCs are localized. Most important, our in vivo transplantation data show that Bcl-2 overexpression increases AGM and fetal liver HSC activity, strongly suggesting that apoptosis plays a role in HSC development.

CD34 is a sialomucin expressed on hematopoietic cells, endothelial cells and muscle satellite cells. Within the hematopoietic system, CD34 expression has been associated with very immature progenitor cells as well as hematopoietic stem cells (HSC), and it is widely used to assess stem cell activity in clinical protocols. In the past, HSC activity was thought to be retained exclusively in the subset of cells expressing CD34. This view has been challenged by recent observations in mice in which HSC activity was also found in the CD34-negative fraction. These findings have since been reproduced using human marrow and cord blood cells. However, the exact relationship between CD34+ and CD34− stem cells remains unclear. We investigated the regulation of CD34 expression as dependent on cell division history. To follow cell division, human cord blood cells were labeled with the fluorescent dye CFSE. Lin-CD34−CD133+CFSE+ (CD34−) and CD34+ populations were almost indistinguishable in their ability to produce CAFCweek6 content. After three days of serum-free culture with stem cell factor, Flt3 ligand and thrombopoietin, almost all initially CD34− cells had acquired expression of CD34, including all undivided cells. We found that, in cultures initiated from CD34− cells, virtually all CAFCweek6 were produced from the divided, now CD34+ cells, indicating these cells had self-renewed. In contrast, similar cultures from initially CD34+ cells demonstrated that hematopoietic activity associated with the undivided cell fraction. We did not find any hematopoietic activity in the cell fraction that remained CD34− or the fraction that lost CD34 after division. Analysis of mRNA expression showed that CD34− and CD34+ cells expressed almost equal levels of CD34, AC133, Flt1, Flk1 and Flt4, while CD34− cells expressed significantly lower levels of Tie1 and Tie2 than CD34+ cells. The expression of CD34 message in CD34− cells was explained by our observation that these cells contained intracellular CD34, indicating that they are “primed” to express the antigen on their cell surface. In conclusion, Lin−CD34−CD133+ cells acquire expression of CD34, even in the absence of cell divisions. These CD34− cells self-renew more rapidly in vitro than cells initially expressing CD34, and self-renewal is preceded by acquisition of CD34 antigen.

PURPOSETo label human hematopoietic progenitor cells with various magnetic resonance (MR) imaging contrast agents and to obtain 1.5-T MR images of them.MATERIALS AND METHODSHematopoietic progenitor cells, labeled with ferumoxides, ferumoxtran, magnetic polysaccharide nanoparticles-transferrin, P7228 liposomes, and gadopentetate dimeglumine liposomes underwent MR imaging with T1- and T2-weighted spin-echo and fast field-echo sequences. Data were analyzed by measuring MR signal intensities and R1 and R2* relaxation rates of labeled cells and nonlabeled control cells. Mean quantitative data for the various contrast agent groups were assessed for significant differences compared with control cells by means of the Scheffe test. As a standard of reference, MR imaging data were compared with electron microscopic and spectrometric data.RESULTSFor all contrast agents, intracellular cytoplasm uptake was demonstrated with electron microscopy and was quantified with spectrometry. When compared with nonlabeled control cells, progenitor cells labeled with iron oxides showed significantly (P <.05) increased R2*. Cells labeled with gadopentetate dimeglumine liposomes showed significantly increased R1. Detection thresholds were 5 x 10(5) cells for gadopentetate dimeglumine liposomes and ferumoxtran, 2.5 x 10(5) cells for ferumoxides and P7228 liposomes, and 1 x 10(5) cells for magnetic polysaccharide nanoparticles-transferrin.CONCLUSIONHematopoietic progenitor cells can be labeled with MR contrast agents and can be depicted with a standard 1.5-T MR imager.

Zielsetzung: Adulte Stammzellen aus Nabelschnur und -blut haben vermutlich ähnliche pluripotenten Eigenschaften wie embryonale Stammzellen. Sie haben den Vorteil, dass eine Nutzung zu Forschungszwecken im Gegensatz zur Forschung an embryonalen Stammzellen ethisch unbedenklich ist und Nabelschnur und -blut als Abfallprodukte post partum ausreichend zur Verfügung stehen. In einem multi-zentrischen Forschungsprojekt, das vom Bayerischen Staatsministerium für Gesundheit, Ernährung und Verbraucherschutz (BayStMGEV) im Rahmen der Gesundheitsinitiative BayernAktiv gefördert wird, sollen die Eigenschaften von adulten Stammzellen näher untersucht werden.

The first definitive long-term repopulating hematopoietic stem cells (HSCs) emerge from and undergo rapid expansion in the embryonic aorta–gonad–mesonephros (AGM) region. To investigate the presumptive unique characteristics of the embryonic hematopoietic microenvironment and its surrounding tissues, we have generated stromal clones from subdissected day 10 and day 11 AGMs, embryonic livers (ELs) and gut mesentery. We here examine the ability of 19 of these clones to sustain extended long-term cultures (LTCs) of human CD34 + umbilical cord blood (UCB) cells in vitro . The presence of in vitro repopulating cells was assessed by sustained production of progenitor cells (extended LTC-CFC) and cobblestone area-forming cells (CAFC). The embryonic stromal clones differed greatly in their support for human HSCs. Out of eight clones tested in the absence of exogenous cytokines, only one (EL-derived) clone was able to provide maintenance of HSCs. Addition of either Tpo or Flt3-L + Tpo improved the long-term support of about 50% of the tested clones. Cultures on four out of 19 clones, ie the EL-derived clone mentioned, two urogenital-ridge (UG)-derived clones and one gastrointestinal (GI)-derived clone, allowed a continuous expansion of primitive CAFC and CFU-GM with over several hundred-fold more CAFC week6 produced in the 12th week of culture. This expansion was considerably higher than that found with the FBMD-1 cell line, which is appreciated by many investigators for its support of human HSCs, under comparable conditions. This stromal cell panel derived from the embryonic regions may be a powerful tool in dissecting the factors mediating stromal support for maintenance and expansion of HSCs.

The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSC-supportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs.

Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers,isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage.

In this study, we investigated the homing and initiation of division of fluorescently labelled adult mouse bone marrow cells after their intravenous injection into lethally irradiated congenic mice. After 2 h, only 3% of the transplanted cells remained in the blood, and ∼35% could be retrieved from the marrow, liver and spleen in approximately equal numbers. Subsequently, the proportion of injected cells found in blood, liver and spleen decreased further, but increased slightly (to ∼17%) in the marrow. Homing of progenitors followed a similar pattern. At 22 h post transplant, almost half of the injected cells in the blood, liver and spleen had completed a first mitosis; although these did not include progenitors with in vitro clonogenic ability. at the same time, >90% of the injected cells recovered from the marrow had not yet divided. Parallel studies with CD44−/− mice showed these to contain a numerically and functionally normal stem cell population whose homing and activation in either CD44+/+ or CD44−/− hosts appeared unaltered. These results indicate homing mechanisms that favor more stable retention of transplanted marrow cells in the marrow of the recipient, more rapid activation of some of those cells that home to other sites, and a lack of change in either of these responses when either the transplanted or the recipient cells do not express CD44. Bone Marrow Transplantation (2000) 26, 559–566.

The combined use of rigorous assays for quantitating transplantable stem cell numbers and precise cell labeling and tracking procedures have provided definitive evidence that stem cell self-renewal divisions can occur in vitro in the absence of stromal feeder layers. These findings set the stage for defining conditions that may alter the ability of these cells to maintain their primitive status when mitogenically activated.

Adhesive interactions between haemopoietic progenitor cells and stromal elements involve a number of different molecules, some of which may be progenitor‐lineage‐ and stage‐specific. CD44 is one such molecule, although little is known about the mechanism(s) by which it is involved. In this study, several anti‐CD44 monoclonal antibodies (mAb) increased the adherence of clonogenic cells, without affecting the total number of types of progenitors recoverable from the adhesion cultures. All of these mAb recognized epitopes on the globular head of CD44. In contrast, two mAb that recognized other regions of CD44 reduced progenitor adhesion to stroma. The mechanism by which one of the anti‐CD44 mAb (L178) enhanced progenitor adhesion did not involve CD44‐crosslinking, and was independent of VLA‐4‐, VLA‐5‐ or LFA‐1‐mediated interactions, Ca or Mg cations, or accessory cells. In addition, CD44 expression on both progenitors and stromal cells contributed to L178‐enhanced progenitor adhesion. Baseline adherence of erythroid progenitors to stroma required tyrosine kinase activity, whereas that of granulopoietic progenitors did not. However, the increase in adhesion did require tyrosine kinase activation. Additional experiments suggested that enhanced adhesion of CFU‐GM to stroma may also be adenylate cyclase‐dependent. Taken together, the present studies indicate both similarities and differences in the mechanisms of CD44‐mediated adhesion of erythroid and granulopoietic progenitors to stromal cells.

The alpha 4 beta 1 integrin very late activation antigen-4 (VLA-4) has been implicated to play a role in the adhesive interactions between hematopoietic progenitor cells (HPC) and bone marrow stromal cells which express the vascular cell adhesion molecule-1 (VCAM-1) or produce fibronectin (FN). Here, we summarize some of the recent advances made in the elucidation of the role of these particular adhesive interactions for the regulation of normal hematopoiesis. HPC bind to stroma mainly through VLA-4/VCAM-1 interactions. There is evidence which suggests that more primitive HPC constitutively express VLA-4 in a high-affinity state. In vitro studies in the mouse have shown that monoclonal antibodies (mAb) against VLA-4 partly block the development of lymphocytes, myelopoietic cells, and erythropoiesis, whereas in the human system outgrowth of TdT(+) B cells is severely retarded by such mAb. In vivo studies revealed that VLA-4 is involved in erythropoietic development, and is particularly important for homing and lodgement of HPC in the bone marrow. Hematopoiesis in mice with deficient expression of alpha 4 integrin or VLA-4's ligand VCAM-1 appears to develop normally. However, chimeras developed from wild-type blastocysts and beta(-/-) embryonic stem cells do not contain beta 1(-/-) hematopoietic cells, although these are present as blood islands in the yolk sac. These beta 1(-/-) hematopoietic cells are capable of forming colonies, indicating that beta 1-integrin is not involved in hematopoietic differentiation, but is primarily important for migration of hematopoietic cells into the fetal hematopoietic organs. In addition to the role of VLA-4 in migration, it may also have other regulatory functions. It has been demonstrated that ligation of VLA-4 induces phosphorylation of the protein tyrosine kinase (PTK) pp 125(FAK) as well as other proteins which may be involved in the regulation of ligand affinity. Indeed, it has been shown that tyrosine kinase-dependent stimulation of CD34(+) hematopoietic cell lines with c-kit ligand (KL), IL-3 or GM-CSF transiently activates the ability of VLA-4 to bind to VCAM-1 or FN. These events are most probably involved in the induction of quiescence in HPC which adhere to stromal cells. This claim was recently substantiated: when HPC were treated with Fab fragments of an anti-VLA-4 mAb, entry into S-phase of the cell cycle was prevented. Taken together, the present data point to a role for VLA-4 in HPC migration, cell cycle regulation, erythropoiesis and B-lymphopoiesis. Moreover, these insights may explain how defects in adhesive behaviour of leukemic HPC through VLA-4 contribute to their dysregulated growth and provide a rationale for therapeutically correcting those defects.