Nadia Quirici

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.

Expression of integrins in human bone marrow

Expression of integrins, a superfamily of glycoprotein α/β 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 β1/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 longterm bone marrow cultures (LTBMC), VLA‐1 and 3 were present respectively on 35% and 40% of the adherent cells which included flbroblasts and endothelial cells, as shown by double‐labelling experiments; VLA‐2 was expressed only on a subpopulation of flbroblasts. β2/LeuCAM molecules were absent from platelets, megakaryocytes and HLA‐DR+/myelo‐peroxidase− early myeloid precursors, and appeared progressively during maturation in both lymphoid and myeloid cells. Expression of β3/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.

Anti-L-NGFR and -CD34 Monoclonal Antibodies Identify Multipotent Mesenchymal Stem Cells in Human Adipose Tissue

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.

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.

Antiendothelial cell antibodies induce apoptosis of bone marrow endothelial progenitors in systemic sclerosis.

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.

Recombinant human erythropoietin stimulates vasculogenesis and wound healing in a patient with systemic sclerosis complicated by severe skin ulcers

Systemic sclerosis (SSc) is often complicated by severe skin ulcers that are unresponsive to traditional treatments. Vascular alterations are responsible for the ischaemic features of the disease in both the skin and visceral organs. Defective neoangiogenesis correlates with an abnormally reduced quantity of circulating endothelial progenitor cells (EPCs) caused by impaired maturation potential and proliferative capacity of bone‐marrow endothelial stem cells. We report a patient with nonhealing cutaneous ulcers successfully treated with recombinant human erythropoietin (rHuEPO). The possible biological effects of this drug were also investigated. Before rHuEPO treatment, the bone‐marrow sample contained reduced numbers of EPCs, which were functionally impaired. After a 6‐month rHuEPO cycle, a marked increase in endothelial progenitor markers was seen, along with a significant reduction in their apoptotic rates. The clinical and laboratory data variations before and after rHuEPO treatment give new insights into the pathogenetic role of impaired endothelial stem‐cell maturation and defective neoangiogenesis in patients with SSc.

EXPRESSION AND QUANTIFICATION OF IGG AND IGM MOLECULES ON THE SURFACE OF LYMPHOCYTES OF CATTLE INFECTED WITH BOVINE LEUKAEMIA VIRUS

Immunogold-labelled antibodies were used with scanning electron microscopy (SEM) and fluorescent-labelled antibodies were used with flow cytometry (FACS) to evaluate the expression and quantity of IgG and IgM molecules on the surface of the lymphocytes of cattle infected with bovine leukaemia virus (BLV). The BLV-infected animals were divided serologically and haematologically into groups with (BLV+PL+) and without (BLV+PL-) persistent lymphocytosis (PL). The percentage of IgM-bearing cells was significantly higher in the BLV+PL+ group than in the BLV-PL- and BLV+PL- groups by FACS. There was a significantly higher percentage of IgG-bearing cells in the BLV+PL+ group than in the BLV-PL- and BLV+PL- groups by SEM, but no differences were found by FACS. A significantly higher intensity of IgM expression was observed in the BLV+PL+ group by SEM. A higher intensity of IgG expression in some animals was detected only by SEM. An increase in the number of larger IgM cells were observed in the BLV+PL- and BLV+PL+ groups by SEM. The SEM analysis was more sensitive than FACS in this experiment.

The Surface Phenotype of Lymphatic Leukemia, Cells: An Immunoscanning Electron Microscopy Study.

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.

Immunoanalysis of IgG and IgM molecules on the surface of BLV infected cattle lymphocytes

The BLV-infected animals were divided into lymphocytotic (9 animals: BLV+PL+) and non lymphocytotic (11 animals: BLV+PL -). BLV-free animals formed a control group (7 animals: BLV-PL-). Flow cytometry immunofluorescence (FACS) and scanning electron microscopy (SEM) were used to evaluate the expression of IgG and IgM molecules on the surface of the lymphocytes of cattle infected with the bovine leukaemia virus (BLV). The percentage of IgM-bearing cells was significantly higher in the BLV+PL+ (75% and 74%) than in BLV+PL (43% and 50%) and BLV-PL (39% and 44%) groups by FACS and SEM respectively. The percentage of IgG-bearing cells was higher in BLV+PL+ (6%) and BLV+PL (8%) compared to BLV-PL (0.7%) determined by FACS and significantly higher in BLV+PL+ (62%) in relation to BLV+PL (17%) and BLV-PL (8%) groups ascertained by SEM. A significantly higher intensity of IgM expression was observed in the BLV+PL+ group by SEM and FACS; a higher intensity of IgG expression in BLV+PL+ group was detected only by SEM. The results suggest that FACS and SEM are complementary. The finding of significant differences in the expression of IgG molecules in BLV+ groups compared with BLV - animals confirms the sensitivity of the SEM method.