Davide Soligo

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.

Neuro-glial differentiation of human bone marrow stem cells in vitro

Bone marrow (BM) is a rich source of stem cells and may represent a valid alternative to neural or embryonic cells in replacing autologous damaged tissues for neurodegenerative diseases. The purpose of the present study is to identify human adult BM progenitor cells capable of neuro-glial differentiation and to develop effective protocols of trans-differentiation to surmount the hematopoietic commitment in vitro. Heterogeneous cell populations such as whole BM, low-density mononuclear and mesenchymal stem (MSCs), and several immunomagnetically separated cell populations were investigated. Among them, MSCs and CD90+ cells were demonstrated to express neuro-glial transcripts before any treatment. Several culture conditions with the addition of stem cell or astroblast conditioned media, different concentrations of serum, growth factors, and supplements, used alone or in combinations, were demonstrated to alter the cellular morphology in some cell subpopulations. In particular, MSCs and CD90+ cells acquired astrocytic and neuron-like morphologies in specific culture conditions. They expressed several neuro-glial specific markers by RT-PCR and glial fibrillary acid protein by immunocytochemistry after co-culture with astroblasts, both in the absence or presence of cell contact. In addition, floating neurosphere-like clones have been observed when CD90+ cells were grown in neural specific media. In conclusion, among the large variety of human adult BM cell populations analyzed, we demonstrated the in vitro neuro-glial potential of both the MSC and CD90+ subset of cells. Moreover, unidentified soluble factors provided by the conditioned media and cellular contacts in co-culture systems were effective in inducing the neuro-glial phenotype, further supporting the adult BM neural differentiative capability.

Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential.

The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.

Prognostic Factors in Primary Cutaneous B-Cell Lymphoma: The Italian Study Group for Cutaneous Lymphomas

PURPOSE Primary cutaneous B-cell lymphomas (PCBCLs) are a distinct group of primary cutaneous lymphomas with few and conflicting data on their prognostic factors. PATIENTS AND METHODS The study group included 467 patients with PCBCL who were referred, treated, and observed in 11 Italian centers (the Italian Study Group for Cutaneous Lymphomas) during a 24-year period (1980 to 2003). All of the patients were reclassified according to the WHO-European Organisation for Research and Treatment of Cancer (EORTC) classification. RESULTS Follicle center lymphoma (FCL) accounted for 56.7% of occurrences, followed by marginal-zone B-cell lymphoma (MZL; 31.4%); diffuse large B-cell lymphoma (DLBCL), leg type, was reported in 10.9% of patients. Radiotherapy was the first-line treatment in 52.5% of patients and chemotherapy was the first-line treatment in 24.8% of patients. The complete response rate was 91.9% and the relapse rate was 46.7%. The 5- and 10-year overall survival (OS) rates were 94% and 85%, respectively. Compared with FCL/MZL, DLBCL, leg type, was characterized by statistically significant lower complete response rates, higher incidence of multiple cutaneous relapses and extracutaneous spreading, shorter time to progression, and shorter OS rates. The only variable with independent prognostic significance on the OS was the clinicopathologic diagnosis according to the WHO-EORTC classification (DLBCL, leg-type, showed a significantly worse prognosis v FCL and MZL; P < .001), whereas the only variable with independent prognostic significance on disease-free survival was the presence of a single cutaneous lesion (P = .001). CONCLUSION Our study identifies a possible PCBCL subclassification and the extent of cutaneous involvement as the two most relevant prognostic factors in PCBCL. These data can be considered reasonably as the clinical background for an appropriate management strategy.

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.

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.

Myelodysplastic syndrome with increased marrow fibrosis: a distinct clinico-pathological entity

Summary Seventeen cases of myelodysplastic syndrome (10 primary and seven secondary to previous radio‐chemotherapy), characterized by trilineage dysplasia, severe bone marrow fibrosis and a high number of megakaryocytes, are described. All of these patients had similar clinical and prognostic features consisting of pancytopenia, modest or absent visceral enlargement and poor survival. The use of CD61 antibodies, which recognize megakaryocytic cells at all stages of maturation, confirmed that these patients had a higher number of these cells than either normal subjects or patients affected by myelodysplastic syndrome (MDS) without fibrosis. Furthermore, primary and secondary MDS with fibrosis, although clinically and histopathologically similar, differed in terms of the number of megakaryoblasts which were significantly higher in primary forms (P<0·02). We conclude that MDS with fibrosis may represent a clinico‐pathological entity which needs to be distinguished from other MDS subtypes as well as from idiopathic myelofibrosis or malignant myelosclerosis.

Modulated Generation of Neuronal Cells from Bone Marrow by Expansion and Mobilization of Circulating Stem Cells with in Vivo Cytokine Treatment

The aim of the present study is to determine whether the expansion and mobilization of circulating bone marrow (BM) stem cells by in vivo treatment with granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF) increase the amount of BM-derived neuronal cells in mouse brain. The presence of BM-derived cells in the brain was traced by transplanting into lethally irradiated adults and newborns adult BM from transgenic mice that ubiquitously expressed enhanced green fluorescent protein (GFP). GFP+ and Y-chromosome+ donor-derived cells were present in several brain areas of all treated mice (cortical and subcortical areas, cerebellum, olfactory bulb). The presence of GFP+ cells expressing nuclear neural specific antigen (NeuN), neurofilament, and beta-III tubulin in cortical forebrain and olfactory bulb (OB) was higher in G-CSF-SCF treated groups (P < 0.05, analysis of variance, Fisher post hoc). We observed that overall the amount of double positive cells was higher in animals treated at birth than in adults and in OB than in forebrain areas (P < 0.05). Temporal cortical areas of cytokine-treated adult animals revealed a mean threefold increase in the number of GFP+ cells expressing the nuclear neural specific antigen (211 +/- 86 GFP+NeuN+/mm(3) in G-CSF + SCF treated mice and 66 +/- 33 GFP+NeuN+/mm(3) in control animals). GFP+ cells coexpressing neuronal markers contain only one nucleus and have a DNA index (a measure of DNA ploidy) identical to that of surrounding neurons, thus excluding donor cell fusion with endogenous cells as a relevant phenomenon under these experimental conditions. Our results indicate that G-CSF and SCF administration modulates the availability of GFP+ cells in the brain and enhances their capacity to acquire neuronal characteristics. Cytokine stimulation of autologous stem cells might be seen as a new strategy for neuronal repair in neurodegenerative diseases.

Induction of neurotrophin expression via human adult mesenchymal stem cells: implication for cell therapy in neurodegenerative diseases.

In animal models of neurological disorders for cerebral ischemia, Parkinsons disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFRp75) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophins expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders.