Pier Luigi Tazzari

Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro

BackgroundTerm Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC).ResultsThe recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical profile, i.e., positive for CD105, CD73, CD29, CD44, CD166 and negative for CD14, CD34, CD45, consistent with that reported for bone marrow-derived MSCs. In addition, amniotic membrane-isolated cells underwent in vitro osteogenic (von Kossa stain), adipogenic (Oil Red-O stain), chondrogenic (collagen type II immunohistochemichal detection) and myogenic (RT-PCR MyoD and Myogenin expression as well as desmin immunohistochemical detection) differentiation. In angiogenic experiments, a spontaneous differentiation into endothelial cells was detected by in vitro matrigel assay and this behaviour has been enhanced through Vascular Endothelial Growth Factor (VEGF) induction. According to these findings, VEGF receptor 1 and 2 (FLT-1 and KDR) were basally expressed in AM-hMSCs and the expression of endothelial-specific markers like FLT-1 KDR, ICAM-1 increased after exposure to VEGF together with the occurrence of CD34 and von Willebrand Factor positive cells.ConclusionThe current study suggests that AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues. AM-hMSCs may potentially assist both bone and cartilage repair, nevertheless, due to their angiogenic potential, they may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.

Activity of the Novel Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 against T-Cell Acute Lymphoblastic Leukemia

Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival, and drug resistance. These observations lend compelling weight to the application of PI3K/Akt/mTOR inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. NVP-BEZ235 was cytotoxic to a panel of T-ALL cell lines as determined by MTT assays. NVP-BEZ235 treatment resulted in cell cycle arrest and apoptosis. Western blots showed a dose- and time-dependent dephosphorylation of Akt and mTORC1 downstream targets in response to NVP-BEZ235. Remarkably, NVP-BEZ235 targeted the side population of both T-ALL cell lines and patient lymphoblasts, which might correspond to leukemia-initiating cells, and synergized with chemotherapeutic agents (cyclophosphamide, cytarabine, dexamethasone) currently used for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic to T-ALL patient lymphoblasts displaying pathway activation, where the drug dephosphorylated eukaryotic initiation factor 4E-binding protein 1, at variance with rapamycin. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment of those T-ALLs that have aberrant upregulation of this signaling pathway for their proliferation and survival.

CTLA‐4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction

CTLA‐4 (CD152) is a cell surface receptor that behaves as a negative regulator of the proliferation and the effector function of T cells. We have previously shown that CTLA‐4 is also expressed on neoplastic lymphoid and myeloid cells, and it can be targeted to induce apoptosis. In our study, we have extended our analysis and have discovered that surface expression of CTLA‐4 is detectable by flow cytometry on 30 of 34 (88%) cell lines derived from a variety of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (but not in primary osteoblast‐like cultures). However, by reverse transcriptase‐PCR, CTLA‐4 expression was detected in all cell lines. We have also found, by immunohistochemistry, cytoplasmic and surface expression of CTLA‐4 in the tumor cells of all 6 osteosarcoma specimens examined and in the tumour cells of all 5 cases (but only weakly or no positivity at all in neighbouring nontumor cells) of ductal breast carcinomas. Treatment of cells from CTLA‐4‐expressing tumor lines with recombinant forms of the CTLA‐4‐ligands CD80 and CD86 induced apoptosis associated with sequential activation of caspase‐8 and caspase‐3. The level of apoptosis was reduced by soluble CTLA‐4 and by anti‐CTLA‐4 scFvs antibodies. The novel finding that CTLA‐4 molecule is expressed and functional on human tumor cells opens up the possibility of antitumor therapeutic intervention based on targeting this molecule.

Nuclear apoptotic changes: An overview

Apoptosis is a form of active cell death essential for morphogenesis, development, differentiation, and homeostasis of multicellular organisms. The activation of genetically controlled specific pathways that are highly conserved during evolution results in the characteristic morphological features of apoptosis that are mainly evident in the nucleus. These include chromatin condensation, nuclear shrinkage, and the formation of apoptotic bodies. The morphological changes are the result of molecular alterations, such as DNA and RNA cleavage, post‐translational modifications of nuclear proteins, and proteolysis of several polypeptides residing in the nucleus. During the last five years our understanding of the process of apoptosis has dramatically increased. However, the mechanisms that lead to apoptotic changes in the nucleus have been only partially clarified. Here, we shall review the most recent findings that may explain why the nucleus displays these striking modifications. Moreover, we shall take into consideration the emerging evidence about apoptotic events as a trigger for the generation of autoantibodies to nuclear components. J. Cell. Biochem. 82: 634–646, 2001.

Dual Inhibition of Class IA Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia

Recent investigations have documented that constitutively activated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it strongly influences growth and survival. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. However, our knowledge of PI3K/Akt/mTOR signaling in T-ALL is limited and it is not clear whether it could be an effective target for innovative therapeutic strategies. Here, we have analyzed the therapeutic potential of the dual PI3K/mTOR inhibitor PI-103, a small synthetic molecule of the pyridofuropyrimidine class, on both T-ALL cell lines and patient samples, which displayed constitutive activation of PI3K/Akt/mTOR signaling. PI-103 inhibited the growth of T-ALL cells, including 170-kDa P-glycoprotein overexpressing cells. PI-103 cytotoxicity was independent of p53 gene status. PI-103 was more potent than inhibitors that are selective only for PI3K (Wortmannin, LY294002) or for mTOR (rapamycin). PI-103 induced G(0)-G(1) phase cell cycle arrest and apoptosis, which was characterized by activation of caspase-3 and caspase-9. PI-103 caused Akt dephosphorylation, accompanied by dephosphorylation of the Akt downstream target, glycogen synthase kinase-3beta. Also, mTOR downstream targets were dephosphorylated in response to PI-103, including p70S6 kinase, ribosomal S6 protein, and 4E-BP1. PI-103 strongly synergized with vincristine. These findings indicate that multitargeted therapy toward PI3K and mTOR alone or with existing drugs may serve as an efficient treatment toward T-ALL cells, which require up-regulation of PI3K/Akt/mTOR signaling for their survival and growth.

Thoracic aortas from multiorgan donors are suitable for obtaining resident angiogenic mesenchymal stromal cells.

The clinical use of endothelial progenitor cells is hampered by difficulties in obtaining an adequate number of functional progenitors. This study aimed to establish whether human thoracic aortas harvested from healthy multiorgan donors can be a valuable source of angiogenic progenitors. Immunohistochemical tissue studies showed that two distinct cell populations with putative stem cell capabilities, one composed of CD34+ cells and the other of c‐kit+ cells, are present in between the media and adventitia of human thoracic aortas. Ki‐67+ cells with high growth potential were located in an area corresponding to the site of CD34+ and c‐kit+ cell residence. We thus isolated cells (0.5 ∼ 2.0 × 104 aortic progenitors per 25 cm2) which, upon culturing, coexpressed molecules of mesenchymal stromal cells (i.e., CD44+, CD90+, CD105+) and showed a transcript expression of stem cell markers (e.g., OCT4, c‐kit, BCRP‐1, Interleukin‐6) and BMI‐1. Cell expansion was adequate for use in a clinical setting. A subset of cultured cells acquired the phenotype of endothelial cells in the presence of vascular endothelial growth factor (e.g., increased expression of KDR and von Willebrand factor positivity), as documented by flow cytometry, immunofluorescence, electron microscopy, and reverse transcription‐polymerase chain reaction assays. An in vitro angiogenesis test kit revealed that cells were able to form capillary‐like structures within 6 hours of seeding. This study demonstrates that thoracic aortas from multiorgan donors yield mesenchymal stromal cells with the ability to differentiate in vitro into endothelial cells. These cells can be used for the creation of an allogenic bank of angiogenic progenitors, thus providing new options for restoring vascularization at ischemic sites.

Synergistic proapoptotic activity of recombinant TRAIL plus the Akt inhibitor Perifosine in acute myelogenous leukemia cells.

To potentiate the response of acute myelogenous leukemia (AML) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity, we have examined the efficacy of a combination with perifosine, a novel phosphatidylinositol-3-kinase (PI3K)/Akt signaling inhibitor. The rationale for using such a combination is that perifosine was recently described to increase TRAIL-R2 receptor expression and decrease the cellular FLICE-inhibitory protein (cFLIP) in human lung cancer cell lines. Perifosine and TRAIL both induced cell death by apoptosis in the THP-1 AML cell line, which is characterized by constitutive PI3K/Akt activation, but lacks functional p53. Perifosine, at concentrations below IC(50), dephosphorylated Akt and increased TRAIL-R2 levels, as shown by Western blot, reverse transcription-PCR, and flow cytometric analysis. Perifosine also decreased the long isoform of cFLIP (cFLIP-L) and the X-linked inhibitor of apoptosis protein (XIAP) expression. Perifosine and TRAIL synergized to activate caspase-8 and induce apoptosis, which was blocked by a caspase-8-selective inhibitor. Up-regulation of TRAIL-R2 expression was dependent on a protein kinase Calpha/c-Jun-NH(2)-kinase 2/c-Jun signaling pathway activated by perifosine through reactive oxygen species production. Perifosine also synergized with TRAIL in primary AML cells displaying constitutive activation of the Akt pathway by inducing apoptosis, Akt dephosphorylation, TRAIL-R2 up-regulation, cFLIP-L and XIAP down-regulation, and c-Jun phosphorylation. The combined treatment negatively affected the clonogenic activity of CD34(+) cells from patients with AML. In contrast, CD34(+) cells from healthy donors were resistant to perifosine and TRAIL treatment. Our findings suggest that the combination of perifosine and TRAIL might offer a novel therapeutic strategy for AML.

Induction of apoptosis by ribosome‐inactivating proteins and related immunotoxins

Immunotoxins have been prepared with 3 ribosome‐inactivating proteins (RIPs), namely, momordin, pokeweed anti‐viral protein from seeds (PAP‐S) and saporin, linked to the Ber‐H2 monoclonal antibody directed against the CD30 antigen of human lymphocytes. Either the RIPs or the immunotoxins induced apoptosis in the CD30+ L540 cell line, as shown by the morphological aspects of the cells, by the DNA fragmentation visible at the electrophoresis, and by the formation of DNA breaks evidenced by 2 cytofluorometric techniques (propidium‐iodide staining and fluoresceine‐isothiocyanate conjugate dUTP incorporation). The AC50 (concentration causing apoptosis in 50% of the cells) is in the range 10‐8 to 10‐7 M in the case of RIPs, and 10‐11 to 10‐10 M in the case of the immunotoxins.

Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton's jelly in the horse

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Whartons Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2  h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 days) (P<0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.

In vitro anti-tumour activity of anti-CD80 and anti-CD86 immunotoxins containing type 1 ribosome-inactivating proteins

Immunotoxins specific for the CD80 and CD86 antigens were prepared by linking three type 1 ribosome‐inactivating proteins (RIPs), namely bouganin, gelonin and saporin‐S6, to the monoclonal antibodies M24 (anti‐CD80) and 1G10 (anti‐CD86). These immunotoxins showed a specific cytotoxicity for the CD80/CD86‐expressing cell lines Raji and L428. The immunotoxins inhibited protein synthesis by target cells with IC50s (concentration causing 50% inhibition) ranging from 0·25 to 192 pmol/l as RIPs. The anti‐CD80 immunotoxins appeared 1–2 log more toxic for target cells than the anti‐CD86 ones. Immunotoxins containing saporin and bouganin induced apoptosis of target cells. The toxicity for bone marrow haemopoietic progenitors of these conjugates was also evaluated. Bouganin and related immunotoxins at concentrations up to 100 nmol/l did not significantly affect the recovery of committed progenitors or of more primitive cells. The saporin‐containing immunotoxins at concentrations ≥ 1 nmol/l showed some toxicity on colony‐forming unit cells (CFU‐C). The expression of the CD80 and CD86 molecules is prevalently restricted to antigen‐presenting cells and is also strong on Hodgkin and Reed–Sternberg cells in Hodgkins disease. Present results suggest that immunotoxins targeting type 1 ribosome‐inactivating proteins to these antigens could be considered and further studied for the therapy of Hodgkins disease or other CD80/CD86‐expressing tumours.