Beta-Chemokine CCL15 Affects the Adhesion and Migration of Hematopoietic Progenitor Cells.

BACKGROUND: Hematopoietic stem and progenitor cell (HPC) motility is essential for HPC transplantation. The chemokine CXCL12 is key for HPC motility. Further regulators are of interest to improve HPC transplantation and regenerative medicine. Here the impact of the human chemokine CCL15 on HPC motility was investigated. METHODS: CCL15 plasma concentrations were determined during HPC mobilization in humans. Activity of CCL15 on HPCs was investigated in murine assays, including chemotaxis, adhesion, and CFU-A assays, and competitive repopulation assays. RESULTS: During HPC mobilization with granulocyte colony-stimulating factor, blood plasma contains increased concentrations (1.1 ± 0.1 ng/ml) of activated CCL15(27-92) versus 0.4 ± 0.1 ng/ml in controls (p = 0.02). CCL15(27-92) significantly enhanced CXCL12-induced transwell migration of Lin-/Sca1+ HPCs and strengthened shear stress-dependent adhesion to vascular cell adhesion molecule-1 (VCAM-1). CCL15(27-92) dose-dependently reduced the colony size in CFU-A assays performed with murine bone marrow and Lin-/Sca1+ HPCs. CCL15(27-92) did not show a direct impact on cell cycle status of HPCs. In murine repopulation assays, pretreatment of bone marrow with CCL15(27-92) significantly increased competitive repopulation. CONCLUSION: Our results point to a regulation of HPCs by CCL15 by modulating migratory and adhesive properties of HPCs with the potency to improve HPC short-term engraftment in stem cell transplantation.

STAT activation status differentiates leukemogenic from non-leukemogenic stem cells in AML and is suppressed by arsenic in t(6;9)-positive AML.

Acute myeloid leukemia (AML) is characterized by an aberrant self-renewal of hematopoietic stem cells (HSC) and a block in differentiation. The major therapeutic challenge is the characterization of the leukemic stem cell as a target for the eradication of the disease. Until now the biology of AML-associated fusion proteins (AAFPs), such as the t(15;17)-PML/RARα, t(8;21)-RUNX1/RUNX1T1 and t(6;9)-DEK/NUP214, all able to induce AML in mice, was investigated in different models and genetic backgrounds, not directly comparable to each other. To avoid the bias of different techniques and models we expressed these three AML-inducing oncogenes in an identical genetic background and compared their influence on the HSC compartment in vitro and in vivo. These AAFPs exerted differential effects on HSCs and PML/RARα, similar to DEK/NUP214, induced a leukemic phenotype from a small subpopulation of HSCs with a surface marker pattern of long-term HSC and characterized by activated STAT3 and 5. In contrast the established AML occurred from mature populations in the bone marrow. The activation of STAT5 by PML/RARα and DEK/NUP214 was confirmed in t(15;17)(PML/RARα) and t(6;9)(DEK/NUP214)-positive patients as compared to normal CD34+ cells. The activation of STAT5 was reduced upon the exposure to Arsenic which was accompanied by apoptosis in both PML/RARα- and DEK/NUP214-positive leukemic cells. These findings indicate that in AML the activation of STATs plays a decisive role in the biology of the leukemic stem cell. Furthermore we establish exposure to arsenic as a novel concept for the treatment of this high risk t(6;9)-positive AML.

Platelet Precursor Cells Can Be Generated from Cultured Human CD34+ Progenitor Cells But Display Recirculation into Hematopoietic Tissue upon Transfusion in Mice.

BACKGROUND: Whereas ex vivo expanded megakaryocytic progenitor cells have been investigated for their ability to support platelet regeneration, the question whether more mature platelet-like particles expanded from hematopoietic progenitor cells may be useful for transfusion purposes remains largely elusive. METHODS: Human peripheral blood progenitor cells (PBPCs) were enriched using surface expression of CD34 by immunoselection. CD34+ enriched PBPCs were expanded ex vivo in serum-free medium supplemented with cytokines. As a proof-of-principle, distribution of expanded CD61+ particles was analyzed after transfusion into Non-Obese Diabetic/ Severe Combined Immunodeficiency (NOD/SCID) mice. RESULTS: Highest ex vivo expansion for CD41+/CD61 + cells was achieved when medium was supplemented with SCF, TPO and IL-3. During expansion culture, CD34 marker expression decreased from 85 to 2-8%, while megakaryocytic cells appeared and CD41 and CD61 expression increased from 3 to about 30%. After transfusion of the expanded cells in NOD/SCID mice, CD61 + cells located mainly to bone marrow and to a lesser degree to spleen, but also circulated in blood. CONCLUSIONS: Platelet-like particles using cytokine-substituted serumfree medium can be generated efficiently from CD34+ expansion cultures, but mainly home to hematopoietic tissue.

The AF4.MLL fusion protein is capable of inducing ALL in mice without requirement of MLL.AF4.

The chromosomal translocation t(4;11)(q21;q23) is the most frequent genetic aberration of the human MLL gene, resulting in high-risk acute lymphoblastic leukemia (ALL). To elucidate the leukemogenic potential of the fusion proteins MLL.AF4 and AF4.MLL, Lin(-)/Sca1(+) purified cells (LSPCs) were retrovirally transduced with either both fusion genes or with MLL.AF4 or AF4.MLL alone. Recipients of AF4.MLL- or double-transduced LSPCs developed pro-B ALL, B/T biphenotypic acute leukemia, or mixed lineage leukemia. Transplantation of MLL.AF4- or mock-transduced LSPCs did not result in disease development during an observation period of 13 months. These findings indicate that the expression of the AF4.MLL fusion protein is capable of inducing acute lymphoblastic leukemia even in the absence of the MLL.AF4 fusion protein. In view of recent findings, these results may imply that t(4;11) leukemia is based on 2 oncoproteins, providing an explanation for the very early onset of disease in humans.

Suspension medium influences interaction of mesenchymal stromal cells with endothelium and pulmonary toxicity after transplantation in mice.

Intravenous (i.v.) transplantation and subsequent homing of Mesenchymal Stromal Cells (MSC) may be adversely influenced by their relatively high adhesion capacity and their tendency to aggregate, leading to clogging of capillaries especially in the lungs. We evaluated the ability of murine MSC suspended in EDTA or heparin in buffered saline solution on their spontaneous adhesion to endothelial cells in vitro, under shear stress and their in vivo tolerability after i.v. injection. We show that suspension of MSC in heparin was highly beneficial, avoiding clinical symptoms in 95% of mice, whereas application of MSC suspended in PBS/EDTA or control buffer caused severe pulmonary reactions and partly, death. In vitro studies using parallel plate flow chambers revealed increased adhesion of MSC suspended in PBS/EDTA to endothelial cells compared with MSC in PBS/heparin. These data provide a means to predict and to interfere with toxicity of i.v. transplanted MSC.

Bone marrow derived cells in the tumour microenvironment contain cells with primitive haematopoietic phenotype.

Infiltration of bone marrow derived cells is part of the angiogenic switch required for uncontrolled tumour growth. However, the nature of the tumour-infiltrating cells from bone marrow has not been fully elucidated. To investigate the phenotype of bone marrow derived cells within a tumour, we employed the Lewis lung carcinoma (LLC) murine tumour model. We followed bone marrow derivation of tumour-infiltrating cells through transplantation of CD45.2 bone marrow cells into pre-irradiated CD45.1 mice. We found robust CD45.2 donor type chimerism in bone marrow and blood of CD45.1 recipient tumour-bearing mice. Flow cytometric analysis of LLC tumours showed, in addition to previously described pro-angiogenic CD45(+)VEGFR2(+)'endothelial progenitor cells' (EPC), or CD45(+)Tie2(+)'Tie2-expressing monocytes' (TEM), incorporation of donor type lineage marker negative (Lin(-)) and Lin(-)Sca1(+) undifferentiated haematopoietic cell types. Immunohistochemical analysis confirmed the extravasal location of the primitive haematopoietic cells. Flow-cytometric sorting of bone marrow cells and subsequent analysis in haematopoietic colony-forming assays revealed that cells with a Lin(-)Sca1(+) phenotype, which were initially negative for VEGFR2 and Tie2, gave rise to VEGFR2(+) and/or Tie2(+) cells. Moreover, Lin(-) bone marrow cells pre-labelled with the membrane dye PKH26 (a red fluorochrome) and transplanted i.v. into tumour-bearing mice were found to extravasate and incorporate into LLC tumours within 24 hrs. Thus, primitive haematopoietic precursors which are thought to be precursors of EPC and TEMs, constitute a part of the tumour microenvironment. This makes them an attractive target cell population for tumour-directed cellular therapies.

Erythrocytic precursor cells show potent shear stress resistant adhesion and home to hematopoietic tissue in vivo.

BACKGROUND: Transfusion of erythropoietic precursor cells has been suggested as an alternative to conventional red blood cells. However, little is known about the fate of transfused erythrocytic precursors after they enter the bloodstream. STUDY DESIGN AND METHODS: Erythrocytic precursors were classified by flow cytometry into different maturation stages. Precursors were enriched using cell surface expression of CD71 and Ter119 antigens and analyzed under shear stress in a parallel plate flow chamber and after fluorescence tagging with PKH and transfusion into anemic mice. RESULTS: We found that at all maturation stages, erythrocytic precursors expressed the adhesion receptor very late antigen (VLA)-4 with a frequency decreasing from 90% to approximately 60% during maturation. In contrast, expression of the beta(2)-integrins LFA-1 and Mac-1 and the rolling receptor P-selectin glycoprotein ligand-1 increased from 10% to 20% to approximately 50% during erythrocytic maturation. The chemokine receptor CXCR4 was expressed at low levels during differentiation stages. In vitro shear stress adhesion analysis showed that erythrocytic precursors can efficiently activate VLA-4 such that it binds its cognate ligand, vascular cell adhesion molecule (VCAM)-1. The coimmobilization of stromal cell-derived factor-1 alpha with VCAM-1 strengthened this adhesion. Transfusion of primitive (CD71+) or more mature (Ter119+) erythrocytic precursors into mice showed that both populations selectively and efficiently home to hematopoietic tissues. CONCLUSION: Our results demonstrate that erythrocytic precursor cells of different maturation stages are capable of homing to hematopoietic organs. This work has implications for the development of transfusion protocols that use ex vivo expanded, but not fully matured, erythrocytic precursors from cultured stem cell populations.

Shear stress-mediated adhesion of acute myeloid leukemia and KG-1 cells to endothelial cells involves functional P-selectin.

Acute myeloid leukemia (AML) shows malignant behavior through the ability of immature cells to circulate in blood and to invade peripheral tissues. Whereas binding of human AML cells to endothelial cells (ECs) through E-selectin has been shown to occur using classical adhesion assays, little is known about the ability of endothelial P-selectin to support this process. We therefore characterized the ability of AML blasts and KG-1 cells to bind to endothelial selectin type ligands. Flow cytometry revealed that, in addition to various integrin adhesion receptors, AML cells regularly express the P-selectin glycoprotein ligand (PSGL)-1, a ligand for P- and E-selectin on ECs. In parallel flow chambers, AML cells both rolled and adhered to TNF-alpha pretreated human umbilical vein endothelial cells (HUVECs). Pretreatment of HUVECs with anti-P- or anti-E-selectin function blocking antibodies significantly reduced both, rolling and subsequent arrest of primary AML cells. Intravital microscopy of i.v. injected fluorescence-labeled KG-1 cells into P-selectin deficient or wild type mice confirmed a significant role of endothelial P-selectin in the binding of human primary AML cells to ECs also in vivo. Thus, the currently available data suggest a role of P- and E-selectin in coordinated circulation of AML cells. Thus, P- or E-selectin mediated adhesion of AML cells may provide a target for the development anti-leukemic therapies.

In vitro differentiation of human mesenchymal stem cells to epithelial lineage.

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.

Arsenic but not all-trans retinoic acid overcomes the aberrant stem cell capacity of PML/RARalpha-positive leukemic stem cells.

BACKGROUND AND OBJECTIVES: Stem cells play an important role in the pathogenesis and maintenance of most malignant tumors. Acute myeloid leukemia (AML) is a stem cell disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARalpha fusion protein. Treatment of APL with all-trans retinoic acid (ATRA) induces CR, but not molecular remission (CMR), because the fusion transcript remains detectable, followed by relapse within a few months. Arsenic induces high rates of CR and CMR followed by a long relapse-free survival (RFS). Here we compared the effects of ATRA and arsenic on PML/RARalpha-positive stem cell compartments. DESIGN AND METHODS: As models for the PML/RARalpha-positive LSC we used: (i) Sca1+/lin- murine HSC retrovirally transduced with PML/RARalpha; (ii) LSC from mice with PML/RARalpha-positive leukemia; (iii) the side population of the APL cell line NB4. RESULTS: In contrast to ATRA, arsenic abolishes the aberrant stem cell capacity of PML/RARalpha-positive stem cells. Arsenic had no apparent influence on the proliferation of PML/RARalpha-positive stem cells, whereas ATRA greatly increased the proliferation of these cells. Furthermore ATRA induces proliferation of APL-derived stem cells, whereas arsenic inhibits their growth. INTERPRETATIONS AND CONCLUSIONS: Taken together our data suggest a relationship between the capacity of a compound to target the leukemia-initiating cell and its ability to induce long relapse-free survival. These data strongly support the importance of efficient LSC-targeting for the outcome of patients with leukemia.

Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells.

To explore the initial steps by which transplanted mesenchymal stem cells (MSCs) interact with the vessel wall in the course of extravasation, we studied binding of human MSCs to endothelial cells (ECs). In a parallel plate flow chamber, MSCs bound to human umbilical vein ECs (HUVECs) similar to peripheral-blood mononuclear cells (PBMCs) or CD34(+) hematopoietic progenitors at shear stresses of up to 2 dynes/cm(2). This involved rapid extension of podia, rolling, and subsequent firm adhesion that was increased when ECs were prestimulated with TNF-alpha. MSC binding was suppressed when ECs were pretreated with function-blocking anti-P-selectin antibody, and rolling of MSCs was induced on immobilized P-selectin, indicating that P-selectin was involved in this process. Preincubation of HUVECs with anti-VCAM-1 or of MSCs with anti-VLA-4 antibodies suppressed binding of MSCs to HUVECs but did not enhance inhibition by anti-P-selectin, indicating that both P-selectin and VCAM-1 are equally required for this process. Intravital microscopy demonstrated the capacity of MSCs to roll and adhere to postcapillary venules in vivo in a mouse model in a P-selectin-dependent manner. Thus, MSCs interact in a coordinated fashion with ECs under shear flow, engaging P-selectin and VCAM-1/VLA-4.

Role of the monomeric GTPase Rho in hematopoietic progenitor cell migration and transplantation.

To investigate the role of the monomeric guanosine triphosphatase (GTPase) Rho on migration of hematopoietic progenitor cells (HPC), we employed different clostridial toxins which inhibit the Rho family of GTPases. Pretreatment with C2I-C3, a cell-accessible C3 transferase fusion protein that targets Rho, increased chemokinetic migration of the factor-dependent multipotent cell line Factor Dependent Cell Paterson with mixed lineage differentiation potential (FDCP-mix) and of primary lineage marker-depleted HPC in vitro. In contrast, treatment with lethal toxin (LT) from Clostridium sordellii, which predominantly inactivates Rac, and with toxin B from C. difficile, which inactivates Rho, Rac and Cdc42, decreased in vitro migration. When HPC pretreated with LT or toxin B were transplanted into mice, homing to the bone marrow was impaired, whereas C2I-C3 treatment did not alter HPC homing. However, in a competitive hematopoietic repopulation experiment in C57BL/6 mice, pretreatment of bone marrow cells with any of the inhibitors, including the Rho inhibitor C2I-C3, resulted in suppressed donor-type hematopoiesis. Our data indicate that whereas Rac supports HPC cell cycling, migration, short-term homing and hematopoietic regeneration, Rho coordinates down-regulation of HPC migration and is required for hematopoietic regeneration.

Induction and detection of human mesenchymal stem cell migration in the 48-well reusable transwell assay.

The induction and detection of chemotactic migration in mesenchymal stem cells (MSC) are complicated by their adherent nature. We describe here the experimental details for inducing the transmigration of MSC seeded in microchemotaxis chambers and reliably identifying the translocated cells. A combination of 8-microm pore-sized membranes and a two-step staining procedure resulted in the detection of MSC migration against a gradient of human plasma which plateaued after 4 h. Microscopic enumeration of the transmigrated cells clearly distinguished contaminating nonmigrated cells from transmigrated cells. Chemokinetic and chemotactic effects could be separated and were influenced by precoated fibronectin or vitronectin. Thus, the technique described allows rapid and reliable induction and determination of migration in adherent MSC.

NAT screening of blood donors for severe acute respiratory syndrome coronavirus can potentially prevent transfusion associated transmissions.

BACKGROUND: The severe acute respiratory syndrome (SARS) was first described in February 2003. Close contact with symptomatic patients appears to be the main route of transmission, whereas blood transfusion transmission could not be ruled out. STUDY DESIGN AND METHODS: A SARS coronavirus (SARS-CoV) detection kit developed by C. Drosten (Bernhard Nocht Institute, BNI) was used to amplify SARS-CoV sequences from blood donor samples. We tested 31,151 blood donor samples in minipools of up to 96 samples. To validate the sensitivity of the assay, routine donor minipools (88 +/- 8 samples per pool) were spiked with plasma of an imported case of SARS or of a subsequently infected contact person, respectively. Gamma-irradiated cell culture supernatants of Vero E6 cells, infected with SARS-CoV, were used as positive controls. RESULTS: None of 31,151 blood donors were positive for the presence of SARS. Two 96-member plasma pools that were each spiked with 100 microL of plasma of the German index patient or his wife, respectively, were positive. Overall, 0.85 percent of test results had to be considered invalid owing to negative internal controls. CONCLUSION: A real-time CoV PCR test is able to detect SARS-CoV in viremic blood donor samples even in the beginning of the disease when patients present minor clinical symptoms. Thus the assay could potentially help to prevent transfusion-associated SARS-CoV transmissions.

Comparison of TaqMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication.

In this study, TaqMan PCR was used to assess viral replication of HIV-1 infected cells in vitro. This PCR technique was compared with p24 ELISA as a standard method to monitor HIV-1 replication in cell culture. Hut78 T-lymphoblastoid cells were infected with different titres of HIV-1(IIIb) (MOI 0.05-0.0005). The course of HIV-1 replication was monitored by determination of p24 concentrations by ELISA in cell culture supernatants and by quantitation of HIV-1 gag RNA by TaqMan RT-PCR. Additionally, the number of HIV-1 proviral copies was assessed by TaqMan PCR. Monitoring of HIV-1 replication by p24 ELISA and TaqMan RT-PCR revealed comparable kinetics of infection. Both methods provided similar data on the exponential increase and on plateauing of HIV-1 replication. Furthermore, both methods were equally sensitive. However, a 7 log linearity of TaqMan HIV-1 gag PCR was demonstrated without dilution of the specimen, in contrast to p24 ELISA, where because of its narrow range of detectable p24 concentrations, sample dilution was necessary. Although determination of the number of proviral copies by TaqMan PCR does not measure HIV-1 replication, the kinetics of proviral copy number following in vitro inoculation of cells with HIV-1 was nearly the same as the kinetics of HIV-1 RNA copy numbers. In conclusion, TaqMan real-time RT-PCR was demonstrated as a reliable and sensitive tool to quantify and monitor HIV-1 replication in cell culture. It is suggested, therefore, that this technique be an alternative method to monitor HIV-1 replication in vitro.