Adhesion Of Hematopoietic Cells Research Articles

BCR-ABL1 Disrupts SDF-1-Dependent Hematopoietic Cell Migration and Adhesion through the LFA-1 Integrin-Mediated Mechanism.

Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, are essential for normal stem/progenitor cell movement, adherence, and retention within the bone marrow environment. Two mechanisms through which BCR-ABL1 are thought to disrupt CXCR4-mediated chemotactic responses have been described in leukemia:1.the inhibition of CXCR4 expression (Geay et al. 2005, Cancer Res.) and2.intra-cellular signaling defects without modification of CXCR4 expression (Salgia et al. 1999, Blood; Ptasznik et al. 2002, J. Exp. Med.).These opposing mechanisms suggest that the actual situation is more complex and that new signaling paradigms are needed. To address this, we studied the effects of BCR-ABL1 on SDF-1-dependent, integrin-mediated, migration and adhesion of hematopoietic precursors. Stimulation of BCR-ABL1(−) hematopoietic cells with SDF-1 showed reduced cell adherence to surfaces coated with ICAM-1 (a ligand for the LFA-1 integrin), which was associated with down-regulated expression of activation-dependent epitopes of the β2 integrin, LFA-1, on hematopoietic cells. Inhibition of Lyn expression with siRNA prevented the SDF-1-triggered down-regulation of LFA-1 and cell adherence, indicating that CXCR4 inhibited the function of LFA-1 through Lyn. Expression of BCR-ABL1 in these cells resulted in increased expression of activation-dependent epitopes of LFA-1 and prevented SDF-1-dependent regulatory effects on both LFA-1 affinity and ICAM-1 adherence. Also, expression of BCR-ABL1 prevented Lyn-mediated regulation of cell adhesion to ICAM-1 as well as Lyn-mediated regulation of LFA-1 affinity. These results indicate that BCR-ABL1 constitutively increases the affinity of the LFA-1 integrin to its ligand ICAM-1, locking the integrin into an “active” conformation. The net result is the loss of responsiveness of LFA-1 to SDF-1-induced ‘inside-out' signaling involving CXCR4 and Lyn kinase. Because in our experiments BCR-ABL1 had no significant effect on the expression of CXCR4 in Mo7e cells, transfected with low and high amounts of p210-BCR-ABL, or in primary BCR-ABL(+) cells from CML blast crisis patients (n=3), we conclude that BCR-ABL1 inhibits CXCR4-triggered ‘inside-out' integrin signaling rather than CXCR4 expression. Taken together, we propose that BCR-ABL1 disrupts the signaling link between the chemokine receptor, CXCR4 and the β2 integrin LFA-1 so as to inhibit normal SDF-1-mediated chemotaxis and adhesion in hematopoietic cells.

Tie-2-Expression Controls the Radio-Sensitivity of Hematopoietic Stem/Progenitor Cells.

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie-2) and its ligand, angiopoietin-1 (Ang-1) play an important role in the remodeling and maturation the vessels. However, Tie-2 expressed in hematopoietic stem cells has been reported to interact with Ang-1 on stromal osteoblasts in the bone marrow niche. This interaction leads to tight adhesion of hematopoietic stem cells to stromal cells, resulting in the maintenance of long-term repopulating activity of hematopoietic stem cells. Although the high radio-sensitivity of hematopoietic stem/progenitor cells is a serious cause of radiation damage in accidentally irradiated victims, the relationship of Tie-2/Ang-1-signals relate to radio-sensitivity is unclear. In addition, a diagnosis of specific radio-sensitivity in patients with malignant diseases allows radio- and/or chemo-therapy to be performed more effectively. Therefore, the radio-protection activity and the possible association of to radio-sensitivity in hematopoietic stem/progenitor of Tie-2/Ang-1 signaling was evaluated. This study was approved by the Committee of Medical Ethics of Hirosaki University School of Medicine. CD34+ hematopoietic stem/progenitor cells were isolated using a magnet sorting kit from human placental and umbilical cord blood units (n = 33) at the end of full-term deliveries after obtaining informed consent from the mothers. Each purified CD34+ fraction individually (81.3 ± 13.7%) was analyzed for the expression of Tie-2 using a flow cytometer. The range of Tie-2-expression was 1.6 ∼ 12.4% (mean = 5.13%). Based on the Tie-2-positive rate, all 33 fractions were classified into CD34+/Tie-2low fraction (Tie-2 positive rate; < 5%, n = 17) or CD34+/Tie-2high fraction (Tie-2 positive rate; ≥ 5%, n = 16). The CD34+ cells were exposed to X-rays at 2 Gy by an X-rays generator (150 kVp, 20 mA, 0.8 ∼ 0.95 Gy/min). The radio-sensitivity of the hematopoietic stem/progenitor cells of each specimen was measured using clonogenic assays. Specifically, the BFU-E, CFU-GM and CFU-GEMM of each sample were assessed in triplicate using a 14-day culture assay in a methylcellulose medium with 5 growth factors (EPO; 4 U/ml, SCF and IL-3; 100 ng/ml, G-CSF and GM-CSF; 10 ng/ml). In addition, CFU-Meg was assessed in triplicate using a 14-day plasma clot technique with 2 growth factors (TPO; 50 ng/ml, SCF; 100 ng/ml). Unexpectedly, the total CFC number decreased with the expression of Tie-2 in CD34+ cells and the total CFC number in CD34+/Tie-2high fractions was lower than that of CD34+/Tie-2low fractions. Moreover, the survival of total CFC at 2 Gy and Tie-2-expression were positively correlated in the CD34+ cells and the total CFC number in CD34+/Tie-2high fractions was more resistant to X-rays than that of CD34+/Tie-2low fractions. Furthermore, to clarify the interaction radio-sensitivity and Tie-2-expression, the effect of Ang-1 on the radio-protective activity in X-irradiated CD34+ cells was examined. However, no protective effect was observed. Although previous reports have showed that CD34+/Tie-2+ cells were more primitive than CD34+/Tie-2− cells, these results suggested that Tie-2-expression appeared to enhance the radio-resistant ability of CD34+ cells and that it was a marker for a radio-sensitivity. Since the direct activation by Ang-1 was not associated with the survival of X-irradiated CD34+ cells, another signaling mechanism may thus be involved in the radio-resistance activity of Tie-2.

Thrombopoietin promotes β1-integrin–mediated adhesion in hematopoietic cells via the small GTPase Rap1

The interactions between cells and extracellular matrices in the bone marrow microenvironment are critical for normal hematopoiesis, controlling cell survival, proliferation, differentiation, and motility. A number of hematopoietic growth factors and cytokines can mediate these interactions by changing expression and/or activity of specific integrins, or by changing cell shape. Thrombopoietin (TPO) has previously been shown to stimulate adhesion. in certain hematopoietic cell types, although the exact mechanisms by which adhesion is promoted remain elusive. The role of TPO in hematopoietic cell adhesion was determined with fibronectin adhesion and binding assays, flow cytometry, and immunocytochemistry using the hematopoietic cell line UT-7/TPO and bone marrow-derived primary mouse megakaryocytes. The role of Rapl in TPO-mediated adhesion was determined using a RaplGAP overexpressing UT-7/TPO cell line, in which Rapl could not be activated. We found that TPO promoted hematopoietic cell adhesion by causing cytoskeletal reorganization and not by increasing integrin expression, localization, or affinity, as previously hypothesized. Through studies using the UT-7/TPO-RaplGAP cell line, we found that TPO-mediated cell shape change occurred via activation of Rapl. These data demonstrate an important role for TPO in mediating interactions in the bone marrow microenvironment and make a significant contribution to our understanding of how TPO may affect hematopoiesis.

Adhesion of hematopoietic progenitor cells to human mesenchymal stem cells as a model for cell−cell interaction

The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining "stemness" has been demonstrated. Human mesenchymal stem cell (MSC) feeder layers represent a surrogate model for this interaction. Specific adhesion molecules are responsible for this cell-cell contact. To define cell-cell contact between HPC and MSC, we have studied adhesive interaction of various fractions of HPC by using a novel assay based on gravitational force upon inversion. Adherent and nonadherent cells were separated and further analyzed with regard to gene expression and long-term hematopoietic culture initiating cell (LTC-IC) frequency. HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to human MSC (CD34(+) vs CD34(-), CD34(+)/CD38(-) vs CD34(+)/CD38(+), slow dividing fraction vs fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the nonadherent fraction. Furthermore, genes coding for adhesion proteins and extracellular matrix were higher expressed in the adherent subsets of CD34(+) cells (fibronectin 1, cadherin 11, vascular cell adhesion molecule-1, connexin 43, integrin beta-like 1, and TGFBI). In this study we have demonstrated that primitive subsets of HPC have higher affinity to human MSC. The essential role of specific junction proteins for stabilization of cell-cell contact is indicated by their significant higher expression.

Active Rap1, a small GTPase that induces malignant transformation of hematopoietic progenitors, localizes in the nucleus and regulates protein expression

Rap1, a member of the Ras superfamily, regulates cytoskeletal changes in lower eukaryots and integrin-mediated adhesion in hematopoietic cells. Sustained activation of Rap1 in mouse hematopoietic stem cells causes expansion of hematopoietic progenitors, followed by a myeloproliferative disorder mimicking chronic myeloid leukemia. Moreover, these mice develop a B-cell lymphoproliferative disorder resembling chronic lymphocytic leukemia. Here, we used HEK 293 cells as a tool to examine the molecular effects of Rap1. We observed that a constitutively active Rap1 mutant localized predominantly in the nucleus. Nuclear localization of endogenous Rap1-GTP was also detected upon physiologic activation. A potential consequence of nuclear localization of Rap1-GTP is the regulation of gene expression. We used a high throughput proteomic approach to identify gene products potentially modulated by Rap1-GTP. Out of 1000 proteins examined, 64 proteins were upregulated and 66 proteins were downregulated. The differentially expressed gene products belong to cytoskeletal regulator proteins, signaling molecules, transcription factors, viability regulators, and protein transporters. This analysis provides the first fingerprint of gene product expression regulated by Rap1 and may contribute to our understanding of malignant transformation mechanisms regulated by this small GTPase.

β1 integrin mediated adhesion increases Bim protein degradation and contributes to drug resistance in leukaemia cells

It has been shown that the tumour microenvironment confers resistance to chemotherapy. Specifically, it was previously reported that adhesion of haematopoietic tumour cells to fibronectin (FN) via beta1 integrins confers a multi-drug resistance phenotype commonly referred to as cell adhesion mediated drug resistance. The present study showed that the pro-apoptotic BCL-2 family member Bim was reduced when leukaemia cells were adherent to FN via beta1 integrins. beta1 integrin-mediated regulation of Bim in K562 cells was demonstrated to be partly a result of increased proteasomal-mediated degradation of Bim protein levels, and proteasome inhibitors prevent Bim degradation. Increased degradation of Bim was not related to activation of the mitogen-activated protein kinase pathway, as adhesion of K562 cells caused a reduction in phospho-extracellular signal-related kinase (ERK)1/2 levels. In addition, pharmacological inhibition of MAP/ERK (MEK) with PD98059 did not increase Bim levels. Reducing Bim levels by short hairpin RNA targeting inhibited imatinib and mitoxantrone-induced cell death. These results showed that beta1 integrin-mediated adhesion regulates Bim degradation and may contribute to the minimal residual disease associated with many haematopoietic malignancies. Together our data indicate that disrupting beta1 integrin-mediated regulation of Bim degradation may increase the efficacy of drugs, including imatinib, used to treat haematopoietic malignancies.

Thrombopoietin Promotes b1 Integrin-Mediated Adhesion in Hematopoietic Cells Via the Small GTPase Rap1.

The interactions between hematopoietic stem cells (HSCs), non-hematopoietic cells and extracellular matrices in the surrounding microenvironment are critical for hematopoietic survival, differentiation and function. Integrins play many essential roles in mediating these interactions, including supporting HSC homing to hematopoietic tissues and maintaining stem cell properties at the osteoblastic and vascular niches in the bone marrow. We and others have previously demonstrated that adhesion of HSCs and mature hematopoietic cells to α4β1 and α5β1 integrin-specific matrices promotes cell survival and enhances differentiation of specific cell lineages. The hematopoietic cytokine thrombopoietin (TPO) is critically important in supporting HSCs both in vitro and in vivo. In vitro, TPO synergizes with other hematopoietic factors to promote proliferation, whilst mice lacking TPO or its receptor, c-Mpl, display greatly reduced numbers of long-term repopulating HSCs. We report on studies designed to elucidate the mechanisms by which TPO mediates hematopoietic progenitor adhesion. Using the cell line UT7-TPO as a model of c-Mpl-expressing hematopoietic progenitors, we found that TPO potently increased cell adhesion to fibronectin (FN) in a dose-dependent manner, with 50ng/ml human (h)TPO resulting in a 6-fold increase in adhesion after 3 hours. These results were confirmed in primary murine megakaryocytes where 50ng/ml hTPO doubled adhesion to FN. Adhesion was prevented by pre-treatment with specific pharmacological inhibitors to Janus kinase (JAK) and phosphoinositide-3 kinase (PI3K), both of which function downstream of TPO. TPO treatment did not increase levels of α4, α5 or β1 integrin at the cell surface and biotinylated-FN binding assays also confirmed that TPO-induced adhesion was not due to changes in integrin affinity. Further investigation revealed that TPO induced dramatic cytoskeletal changes in UT7-TPO cells, with TPO (50ng/ml) treatment resulting in a 3-fold increase in the number of spread cells on FN. These results suggest that TPO-induced adhesion is mediated via rearrangements in cytoskeleton and possibly integrin avidity. Recently the small GTPase Rap1 was shown to stimulate αIIbβ3 integrin-mediated adhesion to fibrinogen in megakaryocytic cell lines and platelets and can modulate the actin cytoskeleton in a number of cell lineages. Therefore, we investigated the role of Rap1 in TPO-induced adhesion. We have found that TPO activates Rap1 in UT7-TPO cells via PI3K and causes a rapid transient recruitment of Rap1 to the plasma membrane. This translocation of Rap1 has previously been attributed to the action of modulators of the actin cytoskeleton, such as profillin and Ena/VASP (enabled/vasodilator-stimulated phosphoprotein), which require Rap1-GTP for their activation. Further, over-expression of Rap1GAP in UT7-TPO cells ablated TPO-mediated adhesion and spreading on FN. These results suggest a mechanism through which Rap1 can influence cytoskeletal changes in these cells and mediate the favorable effects of TPO on hematopoietic cell adhesion. Taken together, our findings suggest that TPO increases cell adhesion to FN not by integrin activation, but via Rap1-mediated cytoskeletal reorganization, providing a further novel role for TPO in the bone marrow microenvironment.

Adhesion of Hematopoietic Progenitor Cells to Human Mesenchymal Stromal Cells as a Model for Interaction between Stem Cells and Their Niche.

Objective: The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining “stemness” has been demonstrated. Human mesenchymal stromal cell (MSC) feeder layers represent a surrogate model for this interaction. The molecular composition of this heterotypic cell-cell contact is yet unknown.Methods: To define this cell-cell contact between HPC and MSC, we have studied adhesion of various fractions of HPC with different preparations of MSC by using a novel assay based on gravitational force upon inversion. Adherent and non-adherent cells were then separated. Gene expression analysis by microarray (GeneChip Human Genome U133_Plus_2.0, Affymetrix) of the two populations was performed and the relationship to long-term hematopoietic culture initiating cell (LTC-IC) frequency examined.Results: HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to MSC from human bone marrow (CD34+vs. CD34−, CD34+/CD38−vs. CD34+/CD38+, slow dividing fraction vs. fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the non-adherent CD34+ cells, thus providing evidence for specific adhesive interaction of primitive HPC with MSC. Genes coding for adhesion proteins and extracellular matrix were highly expressed in the adherent fraction compared to non-adherent CD34+ cells. These genes included fibronectin1 (FN1), cadherin11, VCAM1, connexin43 and ITGBL1. Furthermore, affinity of CD34+ cells was analyzed on human MSC isolated from bone marrow (BM), adipose tissue (AT) and cord blood (CB). Affinity to BM-MSC was significantly higher compared to AT-MSC and CB-MSC. Gene expression in different MSC preparations (BM-MSC, AT-MSC and CB-MSC) correlated in various adhesion proteins with the differences observed in affinity of HPC (including cadherin11, VCAM1, N-cadherin, ITGB1, ITGA1, ITGA5, SDF-1 and osteopontin). Western blot analysis also confirmed higher protein expression of FN1, cadherin11, N-cadherin and ITGB1 in BM-MSC compared to AT-MSC and CB-MSC.Conclusion: MSC represent a model for the human hematopoietic niche. Primitive subsets of HPC have significantly higher affinity to BM-MSC. The essential role of specific junction proteins (cadherin11, VCAM1, N-cadherin) for stabilization of cell-cell contact is indicated by their significant higher expression on both sides of the heterotypic interaction.

Increased hematopoietic stem cell mobilization in aged mice

AbstractHematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow in close association with a highly organized 3-dimensional structure formed by stroma cells, referred to as the niche. Mobilization of HSPCs from bone marrow to peripheral blood in response to granulocyte colony-stimulating factor (G-CSF) requires de-adhesion of HSPCs from the niche. The influence of aging of HSPCs on cell-stroma interactions has not been determined in detail. Using a mouse model of G-CSF–induced mobilization, we demonstrated that the ability to mobilize hematopoietic stem cells is approximately 5-fold greater in aged mice. Competitive mobilization experiments confirmed that enhanced mobilization ability was intrinsic to the stem cell. Enhanced mobilization efficiency of primitive hematopoietic cells from aged mice correlated with reduced adhesion of hematopoietic progenitor cells to stroma and with elevated levels of GTP-bound Cdc42. These results might indicate that stroma–stem cell interactions are dynamic over a lifetime and result in physiologically relevant changes in the biology of primitive hematopoietic cells with age.

Inhibition of RhoA GTPase activity enhances hematopoietic stem and progenitor cell proliferation and engraftment

AbstractRas-related Rho GTPases regulate actin cytoskeletal organization, adhesion, gene transcription, and cell-cycle progression. The Rac subfamily of Rho GTPases and Cdc42 has been shown to play essential roles in hematopoietic stem cell (HSC) engraftment and mobilization. Here, we study the role of RhoA, a related Rho GTPase, in HSC functions. Using retrovirus-mediated gene transfer of a dominant-negative (DN) mutant of RhoA (RhoAN19), we demonstrate that down-regulation of RhoA activity resulted in increased HSC engraftment and self-renewal as measured by competitive repopulation and serial transplantation assays. However, overexpression of RhoAN19 resulted in decreased migration toward SDF-1α and α4β1- and α5β2-integrin–mediated adhesion of hematopoietic progenitor cells in vitro. Low RhoA activity was associated with higher proliferation rate of hematopoietic progenitor cells and increased cells in active phases of cell cycle, most likely via decreasing p21Cip/Waf expression and increasing cyclin D1 levels. Thus, reducing RhoA activity by optimizing the balance between adhesion/migration and proliferation/self-renewal results in a net increase in HSC engraftment. This mechanism could provide a novel therapeutic target to enhance HSC therapies.

Na+/H+ Exchanger Regulatory Factor-1 Is a Hematopoietic Ligand for a Subset of the CD34 Family of Stem Cell Surface Proteins

CD34 and its relatives, podocalyxin and endoglycan, comprise a family of surface sialomucins expressed by hematopoietic stem/progenitor cells and vascular endothelia. Recent data suggest that they serve as either pro- or antiadhesion molecules depending on their cellular context and their post-translational modifications. In addition, their ability to function as blockers of adhesion may be further regulated by their subcellular localization in membrane microdomains via activation-dependent linkage with the actin cytoskeleton. To gain further insights into the function and regulation of CD34-type molecules, we sought to identify the intracellular ligands that govern their localization. Using both genetic and biochemical approaches, we have identified the Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1) as a selective ligand for podocalyxin and endoglycan but not for the closely related CD34. Furthermore, we show that NHERF-1 is expressed by all c-kit(+) /lineage marker(-)/Sca-1(+) cells, which are known to express podocalyxin and have long-term repopulating abilities. Finally, we show that these proteins relocalize and colocalize in response to cytokine signaling. The results suggest that this cytosolic adaptor protein may be important for mobilization of CD34-type proteins in the plasma membrane and may thereby regulate their ability to block or enhance hematopoietic cell adhesion.

The drosophila ortholog of the endolysosomal membrane protein, endolyn, regulates cell proliferation

Endolyn (CD164) is a sialomucin that regulates the proliferation, adhesion, and migration of human haematopoietic stem and progenitor cells. This molecule is predominately localized in endocytotic compartments, where it may contribute to endolysosomal biogenesis and trafficking. In order to more closely define the function of endolyn from an evolutionary view-point, we first analyzed endolyn orthologs in species ranging from insects, fish, and birds to mammals. The predicted molecular structures of the endolyn orthologs from these species are well conserved, particularly with respect to significant O-linked glycosylation of the extracellular domain, and the high degree of amino acid similarities within their transmembrane and cytoplasmic domains, with the latter possessing the lysosomal target signal, YXXphi. Focusing on Drosophila, our studies showed that the subcellular distribution of endolyn in non-polarized Drosophila S2 cells resembles that of its human counterpart in hematopoietic cells, with its predominant localization being within intracellular vesicles, while a small fraction occurs on the cell surface. Both Y --> A and L --> A mutations in the YHTL motif perturbed the normal subcellular distribution of Drosophila endolyn. Interestingly, embryonic and early larval development was often arrested in endolyn-deficient Drosophila mutants. This may partly be due to the role of endolyn in regulating cell proliferation, since knock-down of endolyn expression in S2 cells resulted in up to 50% inhibition of cell growth, with a proportion of cells undergoing apoptosis. Taken together, these results demonstrate that endolyn is an evolutionarily conserved sialomucin fundamentally involved in cell proliferation in both the human and Drosophila melanogaster.

Th-P16:305 Fenofibrate treatment reduces circulating conjugated diene level and increases glutathione peroxidase activity

Our recent studies have shown that annexin II, expressed on the cell surface of osteoblasts, plays an important role in the adhesion of hematopoietic stem cells (HSCs) to the endosteal niche. Similarly, prostate cancer (PCa) cells express the annexin II receptor and seem to use the stem cell niche for homing to the bone marrow. The role of the niche is thought to be the induction and sustenance of HSC dormancy. If metastatic PCa cells occupy a similar or the same ecological niche as HSCs, then it is likely that the initial role of the HSC niche will be to induce dormancy in metastatic cells. In this study, we demonstrate that the binding of PCa to annexin II induces the expression of the growth arrest-specific 6 (GAS6) receptors AXL, Sky, and Mer, which, in the hematopoietic system, induce dormancy. In addition, GAS6 produced by osteoblasts prevents PCa proliferation and protects PCa from chemotherapy-induced apoptosis. Our results suggest that the activation of GAS6 receptors on PCa in the bone marrow environment may play a critical role as a molecular switch, establishing metastatic tumor cell dormancy.

Marrow stromal cells from patients affected by MPS I differentially support haematopoietic progenitor cell development

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

CD34+ Marrow Cells from MDS Patients Express Homing-Associated Cell Surface Molecules but Impaired In Vitro Migration.

Homing and retention of hematopoietic stem cells in the marrow microenvironment are multi-step processes mainly controlled by interactions between chemokines, adhesion receptors and proteases. In the case of MDS, whether these interactions are perturbed and how they are implicated in the pathogenesis of the disease or progression to AML is not known. In this study we evaluated the cell surface expression of homing-associated adhesion molecules, CD11a, CD26, CD45, CD49d, CD49e and CXCR4 by flow cytometric analysis in BM mononuclear (MN) and CD34+ cells from 19 MDS patients [7 RA, 1RARS, 11RAEB(t)] and age-matched healthy individuals (n=9). CD34+ and MN cells from RAEB(t) samples expressed higher levels of CD11a and CD49e (2–3 fold increase) and 1,5–3 fold lower levels of CD45 compared with the cells from the healthy control group, as measured by the mean fluorescent intensity. None of the remaining molecules was found to have altered expression in the RAEB(t) subtype. A similar expression pattern of all homing-associated molecules was seen in the cells from the RA/RARS and normal control samples. However, when CD34+ cells from MDS patients were allowed to migrate in vitro toward a gradient of SDF-1α (125ng/ml), they displayed an impaired chemotactic response [mean values of % input: 9% (0,5–30%)], whereas those from healthy individuals actively migrated [mean values of % input: 20% (10–50%)]. Both migrated and non migrated CD34+ cells from MDS patients displayed little hematopoietic activity in vitro (CFC and LTC-IC). The addition of neutralizing monoclonal antibody anti-CXCR4 decreased in vitro chemotactic response of normal and MDS derived CD34+ cells by 90% and 80% respectively indicating that migration was dependent on SDF1/CXCR4 interactions. To further investigate the differences observed in response to SDF1a, immunofluorescence detection of intracellular expression of CXCR4 was performed. MDS derived CD34+ and MN cells from all the subtypes studied significantly expressed high levels of intracellular CXCR4 and were induced to cell surface CXCR4 expression after 48 hours of SCF (100ng/ml) and IL-6 (20ng/ml) stimulation, as was seen with normal cells.In conclusion marrow CD34+ cells from MDS patients showed reduced capacity to migrate in vitro, although no significant perturbations of adhesion molecules expression were detected. We are currently investigating the signaling events and pathways, which control homing and adhesion of hematopoietic cells in the marrow.

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood , 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

Platelet-Derived Microparticels Stimulate Proliferation of Colony-Forming Unit Granulocyte-Macrophage of Umbilical Cord Blood Hematopoietic Stem Cells.

Umbilical Cord blood has become an important source of hematopoietic stem-progenitor cells for transplantation, however hematopoietic recovery after transplantation with umbilical cord blood is slower than with bone marrow or mobilized peripheral blood. Adhesion molecules on hematopoietic cells are involved in hematopoietic cells' homing, which is considered the most important step of hematological recovery. Some articles indicated that expressions of adhesion molecules on CD34+ cells could predict the time to hematopoietic recovery after transplantation with bone marrow and peripheral blood of many adhesion molecules (such as CD62, CXCR4) are significantly lower on umbilical cord blood than on bone marrow. It is a possible reason for the difficulty in hematopoietic recovery after umbilical cord blood transplant. Platelet -derived microparticles (PMPs) are submicroscopic (<1 μm) membrane vesicles released from platelet if they are stimulated with agonists such as thrombin, collagen, or calcium ionophore A23187 or if exposed to high-stress shear forces. PMPs express several platelet-endothelium attachment receptors on their surface, for example, glycoprotein IIb/IIIa (CD41), Ib and IaIIa, and P-selectin (CD62P) and several other platelet relevant receptors such as CXCR4 and PAR-1. Some articles indicate that PMPs can affect the function of hematopoietic stem cells by increasing the adhesion of hematopoietic cells to fibrinogen, which suggests that PMP-transferred CD41 antigen plays an important role in this process. PMPs can also increase the survival of human hematopoietic cells including human CD34+ clonogenic progenitors. In our research, we observe the function of PMP to affect the cloning efficiency of colony-forming unit granulocyte-macrophage (CFU-GM).We adopt different concentrations of Thrombin (2U/ml, 1.5U/ml, 1.0U/ml and 0.5U/ml) to activate the platelet and acquire PMPs. Then PMPs were evaluated by using flow cytometry. Based on the result that stimulation of platelets by Thrombin (1U/ml) can acquire the best efficiency of PMPs, we used this concentration in all subsequent experiments. Umbilical cord mononuclear cells (MNCs) were obtained from healthy donors and isolate the MNCs by Ficoll-Hypaque density gradient centrifugation. Briefly, MNCs incubated with or without PMPs cultured in 2.7% methylcellulose. CFU-GM growth was stimulated with 30% umbilical cord serum, rhIL-3 and rh GM-CSF. Cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% CO2. Colonies were counted under an inverted microscope after 7 or 10 days. The research was divided into four groups: 1. control group; 2. PMPs(10μg/ml); 3. PMPs(50μg/ml); 4. PMPs(100μg/ml). The colony formation was enhanced with PMPs and is dependently stimulated with PMPs. The number of colonies in the group of PMPs(100μg/ml) is more than that of other groups. The number of colonies in control group, PMPs(10μg/ml), PMPs(50μg/ml) and PMPs(100μg/ml) are 57.4±3.2, 65.6±5.6, 77.1±1.7 and 87.8±5.0 per 1×105 respectively. These increases in different groups were statistically significant when compared with control group(p<0.05). To sum up, PMPs can affect the cloning efficiency of CFU-GM of umbilical cord hematopoietic stem cells and the efficiencies are depended on the concentration of PMPs.

Three-Dimensional Structure Prediction of the Interaction of CD34 with the SH3 Domain of Crk-L

The monomeric 115-kDa surface protein CD34, which is present on many stem cell populations, has been useful to enumerate the quality and viability of cell suspensions for engraftment. Although these studies assure the validity of CD34 as a stem cell marker, the functional role of this molecule has not been defined. CD34 has been demonstrated to regulate adhesion, differentiation, and proliferation of hematopoietic stem cells and other progenitors. The cytoplasmic domain of CD34 is known to be essential for its function. However, it is not clear how this domain's interactions with other molecules support the functional activity of CD34. Here we show that the cytoplasmic tail of CD34 is structurally similar to the carboxyl terminus of the gap junction protein Connexin 43 (Cx43). Because the activity of CD34 is mediated through its interaction with an SH3 domain of an intracellular protein, we attempted to define the SH3 binding region and amino acids involved in this interaction. We identified Glu325 to Ser334 as potential SH3 binding sites. Our results suggest that the interaction of the cytoplasmic tail of CD34 with the shallow proline-rich motif-binding groove of Crk-L is essential for the function of CD34 in stem cell development.

Mesenchymal Stem Cells for Myocardial Infarction

Recent studies indicate that cardiac transfer of adult stem cells can have a favorable impact on tissue perfusion and contractile performance of the infarcted heart. Several cell sources are being explored in an effort to regenerate infarcted myocardium, including hematopoietic stem cells, endothelial progenitor cells, cardiac resident stem cells, bone marrow–derived multipotent stem cells, and mesenchymal stem cells (MSCs). Each of these cell types may have its own profile of advantages, limitations, and practicability issues in specific settings. Studies comparing the regenerative capacity of distinct cell populations are scarce. Most clinical investigators have therefore chosen a pragmatic approach by using unselected bone marrow cells that contain different stem cell populations. Basic scientists, by contrast, are focusing more on specific cell populations in a quest to understand the biological foundations of cell therapy and to identify the most promising stem cells for cardiac regeneration.1 See p 214 MSCs are a rare population of self-renewing, multipotent cells present in adult bone marrow. Although MSCs represent <0.01% of all nucleated bone marrow cells, they can be readily expanded in vitro. In defined culture media, MSCs differentiate into several mesenchymal cell lineages, including cardiomyocytes.2,3 When injected into normal adult myocardium, MSCs differentiate into cardiomyocyte-like cells with sarcomeric organization.4 In an earlier study in pigs with myocardial infarction (MI), MSCs grafted into the infarcted area were shown to express muscle-specific markers and to improve regional wall motion.5 Ease of isolation, high expansion capability, and cardiomyogenic potential have led to the proposition that MSCs may be a good choice for cell-based therapies of MI.6 In a report published in this issue of Circulation , Dai et al7 have …

Delayed platelet engraftment in group O patients after autologous progenitor cell transplantation

Fucosylated glycans, including H-antigen, play critical roles in hematopoietic progenitor cell homing, adhesion, growth, and differentiation. H-active antigens are strongly expressed on CD34+ progenitor cells and committed megakaryocytic progenitors and may mediate adhesion to marrow stromal fibroblasts. We examined the possible influence of donor ABO type on platelet (PLT) engraftment after autologous peripheral blood progenitor cell transplant (PBPCT). A retrospective analysis of all patients who underwent a single autologous PBPCT between 1996 and 2000 were reviewed. Neutrophil and PLT engraftment were compared by patient ABO type and CD34+ cell dose by t test, chi-square test, analysis of variance, Kaplan-Meier probability, and log-rank test. Engraftment data was available in 195 patients. PLT engraftment was delayed in all patients, regardless of ABO type, at CD34+ PBPC doses of 2x10(6) to 3x10(6) per kg (p<0.001). When examined by ABO type, late PLT engraftment (PLT count>50x10(9)/L) was significantly delayed in group O patients relative to all non-group O patients (32.4 days vs. 19.6 days, p<0.001). Approximately 50 percent of group O patients required more than 40 days to achieve late PLT recovery (p<0.005). A group O phenotype may be associated with delayed PLT engraftment at lower CD34 doses.