Federica Servida

Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies

OBJECTIVE Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into multiple mesodermal tissues. We previously reported that monoclonal antibodies to the low-affinity nerve growth factor receptor (alpha-LNGFR) stain bone marrow (BM) mesenchymal cells. We now show that LNGFR antibodies label primitive MSCs with high specificity and purity in adult BM, and compare these cells to those isolated by plastic adherence (PA) and CD45(-)anti-glycophorin A(-) selection. MATERIALS AND METHODS Low-density mononuclear cells (LD-MNCs) from normal BM were separated by PA or immunomagnetic selection for NGFR(+) or CD45(-)alpha-glycophorin A(-) cells. The three fractions were grown in Iscoves modified Dulbecco medium + 20% fetal bovine serum +/- basic fibroblast growth factor (bFGF) in order to assess their proliferative capacity and evaluate their phenotype during culture. The clonogenic potential of the MSCs was assessed using a colony-forming unit fibroblast (CFU-F) assay, whereas multipotential differentiation was determined after culture in adipocytic and osteoblastic conditioned media. RESULTS The NGFR(+) mesenchymal cells grown without growth factors showed persistent NGFR expression (rapidly down-regulated after the addition of bFGF) and persistent CFU-F activity. The NGFR(+) fractions were rich in clonogenic precursors: CFU-F median frequency was 1584/1 x 10(6) cells (range 325-13,793) in the NGFR(+) cells and 35/1 x 10(6) cells (range 27-112) in the LD-MNCs. The NGFR(-) fraction never showed any residual CFU-F activity. Compared with the other two fractions, the NGFR(+) cells (+/- bFGF) showed a 1 to 3 log greater expansion in the number of fibroblastic cells and a greater capacity to give rise to adipocyte colonies and induce osteoblastic differentiation, and they had similar effects in supporting the growth of hematopoietic precursors. CONCLUSION The data suggest that positive selection using low-affinity NGFR antibodies makes it possible to obtain homogeneous multipotent MSCs.

Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells

We report a method of purifying, characterizing and expanding endothelial cells (ECs) derived from CD133+ bone marrow cells, a subset of CD34+ haematopoietic progenitors. Isolated using immunomagnetic sorting (mean purity 90 ± 5%), the CD133+ bone marrow cells were grown on fibronectin‐coated flasks in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin growth factor (IGF‐1). The CD133+ fraction contained 95 ± 4% CD34+ cells, 3 ± 2% cells expressing VEGF receptor (VEGFR‐2/KDR), but did not express von Willebrand factor (VWF), VE‐cadherin, P1H12 or TE‐7. After 3 weeks of culture, the cells formed a monolayer with a typical EC morphology and expanded 11 ± 5 times. The cells were further purified using Ulex europaeus agglutinin‐1 (UEA‐1)‐fluorescein isothiocyanate (FITC) and anti‐FITC microbeads, and expanded with VEGF for a further 3 weeks. All of the cells were CD45− and CD14−, and expressed several endothelial markers (UEA‐1, VWF, P1H12, CD105, E‐selectin, VCAM‐1 and VE‐cadherin) and typical Weibel–Palade bodies. They had a high proliferative potential (up to a 2400‐fold increase in cell number after 3 weeks of culture) and the capacity to modulate cell surface antigens upon stimulation with inflammatory cytokines. Purified ECs were also co‐cultivated with CD34+ cells, in parallel with a purified fibroblastic cell monolayer. CD34+ cells (10 × 105) gave rise to 17 951 ± 2422 CFU‐GM colonies when grown on endothelial cells, and to 12 928 ± 4415 CFU‐GM colonies on fibroblast monolayers. The ECs also supported erythroid blast‐forming unit (BFU‐E) colonies better. These results suggest that bone marrow CD133+ progenitor cells can give rise to highly purified ECs, which have a high proliferative capacity, can be activated by inflammatory cytokines and are superior to fibroblasts in supporting haematopoiesis. Our data support the hypothesis that endothelial cell progenitors are present in adult bone marrow and may contribute to neo‐angiogenesis.

Myeloid microvesicles are a marker and therapeutic target for neuroinflammation.

Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS).

The apoptogenic response of human myeloid leukaemia cell lines and of normal and malignant haematopoietic progenitor cells to the proteasome inhibitor PSI.

Degradation of several intracellular proteins involved in cell cycle control and tumour growth is regulated by the ubiquitin‐dependent multicatalytic protease complex (proteasome). We report that proteasome inhibitor Z‐Ile‐Glu(OtBu)‐Ala‐Leucinal (PSI) was cytotoxic on most human myeloid leukaemia cell lines at IC50 doses ranging from 5 to 25 nmol/l. Additionally, PSI pre‐treatment enhanced cytotoxicity by taxol and cisplatinum. PSI was more active on leukaemic than on normal CD34+ bone marrow progenitors because the 50% growth inhibition of colony‐forming unit granulocyte macrophage (CFU‐GM) from cases of chronic myelogenous leukaemia (CML) and normal subjects was achieved by 15 nmol/l and 50 nmol/l PSI respectively. PSI killed cells by apoptosis as revealed by ultrastructural changes, nuclear DNA fragmentation, cleavage of poly (ADP‐ribose) polymerase (PARP) and of β‐catenin, and was antagonized by ectopic expression of Bcl‐2 but not by inactivating mutations of p53. This event was associated with a slight accumulation of Bcl‐2, a decrease of Bax but no changes in Bcl‐XL protein expression at any time point. In Ph+ cell lines BCR‐ABL protein was only down‐regulated after 48 h of treatment with 10 nmol/l PSI. Altogether, these results indicate that PSI, alone or in association with other cytotoxic agents, has anti‐tumour activity against myeloid malignancies and is more effective on leukaemic than on normal haematopoietic progenitor cells.

Sensitivity of human multiple myelomas and myeloid leukemias to the proteasome inhibitor I

The proteasome inhibitor PSI is potently cytotoxic in vitro against human chronic myeloid leukemia (CML) and acute myeloid leukemias (AML). Here, we have tested proteasome inhibitor I (PSI) in a panel of 11 human multiple myeloma (MM) cell lines and found that it has antiproliferative activity, with an IC50 between 4.5 and 557 nM at 48 h. PSI potentiated the toxicity of a number of chemotherapeutic agents in myeloid leukemia but not in MM cell lines, while in combination with therapeutic proteasome inhibitor PS-341 (Bortezomib) it had a synergistic effect. PSI suppressed the growth of AML cell lines more effectively than PS-341. CFU-GM colony assays revealed that CD34+ bone marrow progenitors from CML and AML patients were more sensitive to PSI than those from normal subjects (IC50: 5, 15 and 50 nM for AML, CML and normal, respectively). Moreover, the growth of normal primitive progenitors (LTC-IC) was unaffected by 15 nM PSI (P=0.576). PSI-induced cell death required RNA transcription and protein synthesis, but not DNA replication, was accompanied by the upregulation of Bcl-2 and modest reduction of Bax and Bcl-XL proteins, and involved the activation of caspases 2, 3, 7 and 8. These findings lend additional support to preclinical investigations with PSI.

Structural Basis for Bivalent Smac-Mimetics Recognition in the IAP Protein Family

XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.

Rational design, synthesis and characterization of potent, non-peptidic Smac mimics/XIAP inhibitors as proapoptotic agents for cancer therapy

Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2-BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.

Indeterminate cell histiocytosis in association with later occurrence of acute myeloblastic leukaemia

Indeterminate cell histiocytosis (ICH) is a proliferation of indeterminate CD1a+, CD68+, S100+ and CD207− dermal dendritic cells. We describe a 39‐year‐old man who developed diffuse ICH and, 6 years later, acute myeloblastic leukaemia (AML). He was treated with cyclophosphamide, etoposide and vinblastine until 2003. In August 2004, he presented dyspnoea, hyperpyrexia and infiltration of the lung parenchyma, compatible with an AML invasion, and died after a course of induction chemotherapy. Cytomorphology and immunophenotype analyses suggested an ICH clonal evolution. The leukaemogenic role of etoposide is discussed. ICH has previously been reported in association with B‐cell malignancy, but only one case has shown systemic progression.

Effect of inositol hexaphosphate (IP 6 ) on human normal and leukaemic haematopoietic cells

Summary. Inositol hexaphosphate (IP6), a naturally polyphosphorylated carbohydrate, has been reported to have significant in vivo and in vitro anticancer activity against numerous tumours, such as colon, prostate, breast, liver and rhabdomyosarcomas. To confirm this activity in haematological malignancies and to characterize some of the mechanisms of IP6 action, we analysed its effects on human leukaemic cell lines and fresh chronic myelogenous leukaemia (CML) progenitor cells using a combined cellular and molecular approach. IP6 had a dose‐dependent cytotoxic effect on all of the evaluated cell lines, with accumulation in the G2M phase in two out of five cell lines tested. At the molecular level, cDNA microarray analysis after IP6 exposure showed an extensive downmodulation of genes involved in transcription and cell cycle regulation and a coherent upregulation of cell cycle inhibitors. Furthermore, IP6 treatment of fresh leukaemic samples of bone marrow CD34+ CML progenitor cells significantly inhibited granulocyte–macrophage colony‐forming unit (CFU‐GM) formation (P = 0·0062) in comparison to normal bone marrow specimens, which were not affected. No differentiating effect on HL60 cells was observed. Taken together, our results confirm the antiproliferative activity of IP6 and suggest that it may have a specific antitumour effect also in chronic myeloid leukaemias, via active gene modulation.

Haematopoietic abnormalities after autologous stem cell transplantation in lymphoma patients.

Haematopoietic reconstitution after autologous stem cell transplantation (ASCT) was evaluated at different times in 26 lymphoma patients. All of the patients showed a significant decrease in the number of both committed (CFU-C) and more primitive progenitor cells (LTC-IC). The expansion of bone marrow progenitor cells in a ‘stroma-free’ long-term liquid culture system supplemented with SCF, IL-3, IL-6 and GM-CSF from 19 transplanted patients was significantly reduced compared to normal controls. The stromal cell compartment, evaluated by means of a CFU-F assay, was also greatly reduced. The number of haematopoietic and stromal cell progenitors was, nevertheless, very similar to their pre-transplant values. Bone marrow histology, which was evaluated at different times after transplant, showed an increase in reticulin fibres, the dilatation of parenchymal sinusoids and some morphological evidence of trilineage dysplasia in 11 patients; however, the same abnormalities were seen in the majority of pre-transplant samples. No cytogenetic abnormalities were observed in 15 patients before transplant, but four subsequently developed persistent clonal karyotypic alterations and five showed non-clonal abnormalities that generally disappeared over time. Our data suggest that both the stromal and the haematopoietic compartments are somehow damaged after ASCT for lymphoma; however, these defects generally pre-exist the transplant conditioning regimen and seem to become less pronounced over time.