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OBJECTIVE: Vascular abnormalities represent the main component of the pathobiology of systemic sclerosis (SSc), progressing from structural derangements of the microcirculation with abortive neoangiogenesis to final vessel loss. Since circulating endothelial progenitor cells (EPCs) are important in the vascular repair process, we undertook this study to examine their numbers in the peripheral blood (PB) of SSc patients and to evaluate whether their status is related to impaired quantitative and/or qualitative aspects of the bone marrow (BM) microenvironment. METHODS: Circulating EPCs from 62 SSc patients were evaluated by flow cytometry and characterized as CD45 negative and CD133 positive. BM EPCs, identified as CD133 positive, were isolated from 14 SSc patients and grown to induce endothelial differentiation. In addition, progenitor numbers and functional properties of hematopoietic and stromal compartments were analyzed by various assays. RESULTS: We found that EPCs were detectable in the PB of patients with SSc, and their number was significantly increased in patients with early-stage disease but not in those with late-stage disease. All of the examined BM samples contained reduced numbers of EPCs and stromal cells, both of which were functionally impaired. Both endothelial and stromal progenitors expressed vascular endothelial growth factor receptor, indicating that BM is strongly induced to differentiate into the endothelial lineage; furthermore, only BM EPCs from patients with early disease led to endothelial differentiation in vitro. CONCLUSION: This study provides the first demonstration that in SSc, there is a complex impairment in the BM microenvironment involving both the endothelial and mesenchymal stem cell compartments and that this impairment might play a role in defective vasculogenesis in scleroderma. [ABSTRACT FROM AUTHOR]

Background: Autologous fat grafting is a widely adopted surgical technique in both the reconstructive and aesthetic fields. This study aimed to compare centrifuged lipoaspirates harvested and refined by the Coleman technique with noncentrifuged lipoaspirates in terms of cell number and viability, phenotypic profile, and clonogenic and proliferative potential of adipose-derived stem cell (ADSC) populations., Methods: For each patient, both a centrifuged sample using the Coleman's technique and a noncentrifuged sample of adipose tissue were collected. Adipose-derived stem cells from both the centrifuged fraction (CF) and the noncentrifuged fraction (NCF) were isolated. The recovered ADSCs were used to set up flow cytometry analysis, colony-forming units-fibroblast (CFU-F) assays, and ADSC cultures., Results: The number of recovered cells was variable among the different donors but significantly higher in the CF donors. Cell viability, determined by the Trypan Blue dye assay, always exceeded 95 %, in both the CF and NCF fractions. Analysis of the putative ADSC subpopulations showed a significant enrichment of the mesenchymal and endothelial progenitors in CF compared with NCF. No differences in the clonogenic efficiency of the ADSC samples were observed when the same number of cells were plated from each fraction. On the contrary, when equal fat volumes were compared, the colony-forming ability of CF was always significantly higher than that of its NCF counterparts., Conclusions: This is the first study to comprehensively characterize the impact of Coleman's technique on the quality of lipoaspirates, showing that centrifugation is safe and feasible and does not impair cell viability, can augment the content in ADSC and the frequency of CFU-F, and reduces the number of proinflammatory blood cells.

Titanium and its alloys are the current materials to manufacture oral implants because of their excellent mechanical properties and biocompatibility. However the increasing needs of the patients to receive fast and reliable rehabilitation have forced materials scientists to modified the surface of the materials in order to increase the rate of osseointegration and minimize the times for healing. The presence of a reconstituted extracellular matrix (ECM), constituted of proteins and polysaccharides is a key factor for healing and regeneration of the tissues. The nano-fibrous feature of ECM improves cells proliferation and addresses their phenotype. Electrospinning technique is an efficient processing method to manufacture micro- and nano-sized fibrous structures mimicking the ECM. In this work we describe a method to obtain collagen coating made of nano-fibers onto titanium for oral implant manufacturing, using electrospinning. The obtained collagen coatings showed morphology, structure and chemical composition similar to that of ECM. Moreover the stem cells cultured onto titanium samples coated with electrospun collagen showed faster osteoblastic differentiation and more efficient deposition of mineralized matrix in comparison with those onto uncoated substrates. This effect was amplified using osteogenetic media.

Objective: Patients with systemic sclerosis (SSc) have significantly fewer and functionally impaired endothelial progenitor cells (EPC) in peripheral blood and bone marrow; further, endothelial apoptosis seems to play a primary role in the pathogenesis of vascular damage. We investigated whether the failure of bone marrow EPC is related to their apoptotic phenotype and analyzed the possible mechanisms inducing apoptosis., Methods: The presence of apoptotic cells was investigated in bone marrow aspirates taken from patients with SSc; microvessel density (MVD) and the immunohistochemical expression of vascular endothelial growth factor (VEGF) were also measured in bone marrow biopsies. A correlation between EPC apoptosis and the presence of antiendothelial cell antibodies (AECA) was also investigated., Results: We confirmed the presence of bone marrow EPC dysfunction in SSc, while hematopoiesis was not impaired. Bone marrow studies showed a high percentage of apoptotic progenitors, no signs of fibrosis or an altered MVD, and an increased VEGF index. The patients' bone marrow plasma showed significant titers of AECA, and their presence correlated with that of apoptotic progenitors. These findings were further confirmed by an in vitro assay in which the apoptosis of normal progenitors was induced by the addition of AECA+ purified IgG., Conclusion: Our results showed that apoptosis in patients with SSc involves the source compartment of endothelial progenitors and correlates with AECA activity. These findings support the hypothesis that AECA may play a pathogenetic role by affecting the bone marrow EPC machinery that should repair the peripheral vascular lesions.

Stem cells hold great promise in tissue engineering for repairing tissues damaged by disease or injury. Mesenchymal stem cells (MSCs) are multipotent cells able to proliferate and differentiate into multiple mesodermal tissues such as bone, cartilage, muscle, tendon, and fat. We have previously reported that the low-affinity nerve growth factor receptor (L-NGFR or CD271) defines a subset of cells with high proliferative, clonogenic, and multipotential differentiation ability in adult bone marrow (BM). It has been recently shown that adipose tissue is an alternative source of adult multipotent stem cells and human adipose-derived stem cells, selected by plastic adherence (PA hASCs), have been extensively characterized for their functional potentials in vitro. In this study, immunoselected L-NGFR(+) and CD34(+) subpopulations have been analyzed and compared with the PA hASCs. Phenotypic profile of freshly purified subpopulations showed an enrichment in the expression of some stem cell markers; indeed, a great percentage of L-NGFR(+) cells co-expressed CD34 and CD117 antigens, whereas the endothelial-committed progenitor markers KDR and P1H12 were mainly expressed on CD34(+) cells. Differently from PA hASCs, the immunoseparated fractions showed high increments in cell proliferation, and the fibroblast colony-forming activity (CFU-F) was maintained throughout the time of culture. Furthermore, the immunoselected populations showed a greater differentiative potential toward adipocytes, osteoblasts, and chondrocyte-like cells, compared to PA hASCs. Our data suggest that both CD34(+) and L-NGFR(+) hASCs can be considered alternative candidates for tissue engineering and regenerative medicine applications.

Murine bone marrow (BM) cells have been shown to undergo myogenic differentiation and participate in muscle repair in different muscle regeneration models. In the present paper, we report on a subset of cells (CD45+/desmin+) with myogenic potential being present at very low frequencies in human adult BM. By a simple culture method, we were able to obtain in vitro multinucleated myotubes in up to 20% of the cultures. Myotubes were generated using both BM flushed from rib fragments obtained during thoracotomy and BM derived from iliac crest aspirates. Cells of the different adherent and non-adherent fractions expressed numerous muscle specific markers by immunocytochemistry, real-time RT-PCR, flow cytometry, and Western blot analyses. Moreover, direct injection of whole BM into the right tibialis anterior muscle of immunodeficient mice (NOD/RAG) that had previously been treated with cardiotoxin to induce muscle degeneration, showed a variable but significant level of human cell engraftment (from 0.06 to 0.26% Dys+/FISH+ fibers). These data suggest that cells with skeletal muscle differentiation potential are present in adult human BM can differentiate in vitro and give rise to myogenic cells in vivo in immunodeficient mice after muscle damage. Further improvements might allow new approaches to cell-mediated therapies for muscular diseases.

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 Iscove's 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.

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 x 10(5)) 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.

Background: The long-term prognosis after surgical therapy for esophageal carcinoma depends on tumor stage and completeness of resection. Similarly to other epithelial tumors, the presence of micro deposits of neoplastic cells in the bone marrow may indicate residual disease and the potential for recurrence. This study assesses the prevalence of bone marrow-disseminated tumor cells in patients undergoing surgical resection for esophageal carcinoma. In addition, we investigated the agreement between immunohistochemical and molecular techniques for the detection of micrometastases in a subgroup of patients., Methods: Between January 1998 and November 1999, forty-eight patients with adenocarcinoma of the esophagogastric junction (n = 29) or squamous cell carcinoma of the thoracic esophagus (n = 19) and no evidence of overt metastatic disease entered the study. An immunohistochemical assay (capable of detecting 1 carcinoma cell in 7 x 10(5) bone marrow cells) was used to test bone marrow obtained by flushing a resected rib or by needle aspiration either of the iliac crest or of a rib. A polymerase chain reaction (PCR) molecular technique was also used to identify bone marrow and peripheral blood epithelial cells., Results: Cytokeratin-positive cells were found in 79.1% of the bone marrow samples obtained from the rib, and in only 8% of the needle aspirates either from the iliac crest or from a contiguous rib: This difference is probably explained by the improved removal of metastatic cells with the flushing of the rib. Comparable results were obtained at a qualitative level by the PCR technique on bone marrow. In addition, PCR-positive results were found in 3 of 18 peripheral blood samples. There was no association with tumor type, neoadjuvant therapy, or lymph node status. Patients with a pT3 or pT4 tumor showed, at a borderline statistical level, a higher proportion of cytokeratin-positive cells in the flushed rib., Conclusions: Bone marrow-disseminated tumor cells are present in the resected rib of a high proportion of patients undergoing esophagectomy for carcinoma, and immunohistochemistry seems to be the method of choice for their quantitative assessment. However, the prognostic and therapeutic implications of this finding need further investigation.

Background and Objective: Clozapine is a diabenzodiazepine derivative characterized by a high therapeutic index in schizophrenic patients resistant to traditional neuroleptic drugs, because of the rarity of any extrapyramidal side effects, and its particular hematologic toxicity. According to the international literature, clozapine-induced neutropenia occurs mainly during the first 4-6 months of treatment, and its incidence decreases considerably over time. This neutropenic effect is not dose-dependent and normally clears up after drug discontinuation, although it may evolve into agranulocytosis. The aim of this study is to evaluate the in vitro toxic effect of clozapine and N-desmethylclozapine on both committed and immature human hematopoietic progenitor cells., Design and Methods: Cytotoxic assays were performed in vitro on normal human bone marrow samples treated with clozapine or with its metabolite N-desmethylclozapine. The clonogenic potential after treatment with both compounds was assessed on low density mononuclear cells (LD-MNC), purified CD34+ cells, cytokine driven liquid cultures and long term culture initiating cell (LTC-IC)., Results: Clozapine and N-desmethylclozapine had a dose-dependent inhibitory effect on in vitro growth of CFU-GM and BFU-E from normal bone marrow. The two drugs had toxic effects on purified CD34+ progenitor cells but no significant effect on LTI-IC., Interpretations and Conclusions: Our data indicate a cytotoxic effect, which is more pronounced with N-desmethylclozapine and at high doses, on the committed progenitor cell compartment but not on primitive hematopoietic cells. Furthermore, our data show that clozapine and N-desmethylclozapine have a direct effect on treated cells and do not induce apoptotic death.

Hemopoietic cells display a wide range of cell surface antigens which are either lineage specific or acquired during differentiation. Monoclonal antibodies can be used, in conjunction with colloidal gold markers, to identify under the scanning electron microscopy (SEM) at the single cell level, specific lineage or maturation stages in the hemopoietic bone marrow. Normal bone marrow cells, either gradient separated or purified by immuno-magnetic methods and leukemic cell samples, which can be considered as "frozen" stages of hemopoietic differentiation, have been studied with this method. Typical cell surface morphologies, which characterize immature progenitor cells and cells committed or differentiated towards the lymphoid, myeloid, erythroid and megakaryocytic lineage have been identified. Correlations between cell surface features and some hemopoietic cells functions have been attempted on the basis of these findings.

Antibovine leukocyte monoclonal antibodies were tested in order to use them for scanning electron microscopy in backscattered electron imaging mode. The tests revealed that numerous antibovine leukocyte monoclonal antibodies still recognize lightly glutaraldehyde prefixed antigens and can be used to identify various blood cell types. The results of these tests are presented and discussed. Clearcut differences in the surface morphology exist among bovine peripheral blood normal lymphocytes. Expression of IgM and IgG molecules was observed first of all on the surface of B lymphocyte microvilli.

Peripheral blood mononuclear cells (PBLs) from 14 patients with low grade non-Hodgkin's B-cell lymphomas with predominant splenomegaly were studied by means of scanning (SEM) and transmission electron microscopy (TEM). All patients had peripheral blood and bone marrow involvement, the absence of lymphoadenopathy, and, except in one case, immunophenotypic features of a malignant proliferation of mature spleen B-cells arising from outside the germinal center, but not consistent with CLL or HCL. Several distinctive cytological features were observed in PBLs of the different subgroups. The SEM surface features of PBLs in patients with intermediate differentiation lymphocytic lymphoma (IDL) (five cases), lymphoplasmacytoid immunocytoma (LP-IC) (two cases), and mixed small and large cells malignant lymphoma (one case) were characterized by the presence of numerous well-developed microvilli. Some distinctive TEM ultrastructural features were also seen in the different cases. In the two cases of splenic lymphoma with villous lymphocytes (SLVL), SEM revealed large and elongated surface microvilli generally arising from two or three poles of the cells. This surface morphology, confirmed by TEM analysis, may be pathognomonic of this disease. Four additional cases, tentatively classified as small lymphocytic lymphoma on the basis of immunophenotypic data, were extremely heterogeneous at both SEM and TEM analysis. The ultrastructural features revealed by SEM and TEM may be useful for the more precise characterization of this heterogeneous group of diseases, which is generally difficult to define even when immunophenotypic and molecular approaches are used.

Monoclonal antibody QBEND10 is reactive with the CD34 antigen in aldehyde-fixed, decalcified, paraffin-embedded bone marrow biopsies. In normal bone marrow it stained endothelial cells lining arterioles and capillaries, sinusoidal (littoral) cells and 0.89% of all haemopoietic cells. QBEND10+ mononuclear cells were seen as isolated, randomly distributed mononuclear cells in normal and regenerating bone marrows. Conversely, QBEND10+ cells were increased and present in aggregates of three or more cells in 6/8 cases of acute leukemia; in two cases of CD34-negative leukemia and in two patients after complete remission no aggregates were seen. QBEND10 immunohistochemistry may therefore be useful for diagnosis and follow-up of myeloid leukemias. In addition, increased numbers of CD34+ cells arranged in clusters were seen in 4/9 cases of refractory anemia with excess blasts (RAEB), 1 case of chronic myelomonocytic leukemia, 3/3 cases of RAEB in transformation, and in 3/7 cases of chronic myelogenous leukemia: in all these cases, CD34 staining of the bone biopsy may have prognostic value. QBEND10+ endothelial cells were significantly increased in all the pathological conditions examined (1.43% of all nucleated cells versus 0.80% in normal bone marrow; p = 0.0063), but especially in myeloid leukemias and in two fibrotic syndromes examined.

Immunohistochemical studies were performed with monoclonal antibodies (MAbs) reactive on paraffin embedded bone marrow biopsies in 19 patients with myelodysplastic syndromes, 8 of them during r gamma-interferon treatment. CD15 MAbs stained mature myeloid cells predominantly located close to the bone marrow trabeculae. Anti-gpIIIa MAbs permitted precise identification of megakaryocytic cells including precursors and dysplastic megakaryocytes. Labelling with CD45 and CD68 MAbs, recognizing lymphocytes and macrophages respectively, was intense in patients in steady state, but progressively decreased during leukemic transformation. Increase in CD45+ and/or CD68+ cells was also observed in most bone marrow biopsies after 3 months of r gamma-interferon therapy.

Expression of integrins, a superfamily of glycoprotein alpha/beta heterodimers which integrate the cytoskeleton with the extracellular matrix and/or mediate cell-cell adhesive interactions, was examined on normal and leukaemic bone marrow cells by immunohistochemistry and immunotransmission electron microscopy (immuno-TEM). Among the beta 1/VLA molecules studied, VLA-2 and 6 were expressed on megakaryocytes and platelets, while VLA-4 was present on 40% of haemopoietic cells, including monocytes, erythroblasts and immature cells; this molecule was typically localized at sites of intercellular contact, as seen by immuno-TEM, suggesting it may be involved in interactions among haemopoietic cells during differentiation. In human long-term bone marrow cultures (LTBMC), VLA-1 and 3 were present respectively on 35% and 40% of the adherent cells which included fibroblasts and endothelial cells, as shown by double-labelling experiments; VLA-2 was expressed only on a subpopulation of fibroblasts. beta 2/LeuCAM molecules were absent from platelets, megakaryocytes and HLA-DR+/myeloperoxidase- early myeloid precursors, and appeared progressively during maturation in both lymphoid and myeloid cells. Expression of beta 3/cytoadhesin molecules was restricted to megakaryocytes and platelets and, in the adherent layer of LTBMC, to endothelial cells. The regulated expression and specific localization of integrins in the bone marrow suggest that these molecules may have a role in normal haemopoiesis.

Bone-marrow cells from 11 cases of T- and 18 cases of B-lymphatic leukemia, at different maturation stages, were examined by scanning electron microscope (SEMI. All cases were extensively studied for the expression of surface markers by immunofluorescence. In addition six cases of T- and 10 cases of B-cell leukemia were labeled with a panel of monoclon;tl antibodies (including CD3, CD5, CD7, CD10, CD4, CD8, CD19, CD20 and CD22) and, after incubation with a colloidal gold conjugate, observed with SEM in the back-scattered electron imaging mode. Early stages of leukemic lymphoid B- and T-cell differentiation are characterized by prevalently smooth cell surfaces. Short stub-like microvilli constantly appear on more mature T cells, while complex surface features like small ruffles and pleomorphic microvilli are present in well-differentiated B-cell proliferations. Surface microvilli can be interpreted als structural features of lymphoid cells, progressively expressed with maturation and differentiation of leukemic as well as normal cells.