Increased Mortality Rate and Not Impaired Ribosomal Biogenesis is Responsible for Proliferative Defect in Dyskeratosis Congenita Cell Lines

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Human cadaver multipotent stromal/stem cells isolated from arteries stored in liquid nitrogen for 5 years

Introduction: Regenerative medicine challenges researchers to find noncontroversial, safe and abundant stem cell sources. In this context, harvesting from asystolic donors could represent an innovative and unlimited reservoir of different stem cells. In this study, cadaveric vascular tissues were established as an alternative source of human cadaver mesenchymal stromal/stem cells (hC-MSCs). We reported the successful cell isolation from postmortem arterial segments stored in a tissue-banking facility for at least 5 years. Methods: After thawing, hC-MSCs were isolated with a high efficiency (12 × 106) and characterized with flow cytometry, immunofluorescence, molecular and ultrastructural approaches. Results: In early passages, hC-MSCs were clonogenic, highly proliferative and expressed mesenchymal (CD44, CD73, CD90, CD105, HLA-G), stemness (Stro-1, Oct-4, Notch-1), pericyte (CD146, PDGFR-β, NG2) and neuronal (Nestin) markers; hematopoietic and vascular markers were negative. These cells had colony and spheroid-forming abilities, multipotency for their potential to differentiate in multiple mesengenic lineages and immunosuppressive activity to counteract proliferation of phytohemagglutinin-stimulated blood mononuclear cells. Conclusions: The efficient procurement of stem cells from cadaveric sources, as postmortem vascular tissues, demonstrates that such cells can survive to prolonged ischemic insult, anoxia, freezing and dehydration injuries, thus paving the way for a scientific revolution where cadaver stromal/stem cells could effectively treat patients demanding cell therapies

Proapoptotic Activity and Chemosensitizing Effect of the Novel Akt Inhibitor (2S)-1-(1H-Indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-amine (A443654) in T-Cell Acute Lymphoblastic Leukemia

Constitutively activated AKT kinase is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). Here, we report that the novel AKT inhibitor (2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2Hindazol-5-yl)pyridin-3-yl]oxypropan2-amine (A443654) leads to rapid cell death of T-ALL lines and patient samples. Treatment of CEM, Jurkat, and MOLT-4 cells with nanomolar doses of the inhibitor led to AKT phosphorylation accompanied by dephosphorylation and activation of the downstream target, glycogen synthase kinase-3â. Effects were time- and dose-dependent, resulting in apoptotic cell death. Treatment of Jurkat cells with A443654 resulted in activation of caspase-2, -3, -6, -8, and -9. Apoptotic cell death was mostly dependent on caspase-2 activation, as demonstrated by preincubation with a selective pharmacological inhibitor. It is remarkable that A443654 was highly effective against the drug-resistant cell line CEMVBL100, which expresses 170-kDa P-glycoprotein. Moreover, A443654 synergized with the DNA-damaging agent etoposide in both drug-sensitive and drug-resistant cell lines when coadministered [combination index (CI) = 0.39] or when pretreated with etoposide followed by A443654 (CI = 0.689). The efficacy of A443654 was confirmed using blasts from six patients with T-ALL, all of whom displayed low levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and constitutive phosphorylation of Akt on Ser473. At 1 ìM, the inhibitor was able to induce apoptotic cell death of T-ALL blast cells, as indicated by flow cytometric analysis of samples immunostained for active (cleaved) caspase-3. Because activated AKT is seen in a large percentage of patients with T-ALL, A443654, either alone or in combination with existing drugs, may be a useful therapy for primary and drug-resistant T-ALL. Originally published Molecular Pharmacology, Vol. 74, No. 3, Sep 200

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 TNF-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 IC50, dephosphorylated Akt and increased TRAIL-R2 levels, as demonstrated by western blot, RT-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. Upregulation of TRAIL-R2 expression was dependent on a protein kinase Cα/ c-Jun-NH2-kinase 2/c-Jun signaling pathway activated by perifosine through reactive oxygen species production. Perifosine synergized with TRAIL also in primary AML cells displaying constitutive activation of the Akt pathway, by inducing apoptosis, Akt dephosphorylation, TRAIL-R2 upregulation, cFLIP-L and XIAP downregulation, and c-Jun phosphorylation. The combined treatment negatively affected the clonogenic activity of CD34+ cells from AML patients. In contrast, CD34+ cells from healthy donors were resistant to perifosine and TRAIL treatment. Our findings suggest that the combination perifosine and TRAIL might offer a novel therapeutic strategy for AML. Originally published Cancer Research, Vol. 68, No. 22, Nov 200

Lipidomics study of mesenchymal stromal cells derived from human placenta

The interest for lipid metabolism in the stem cell field has increased in the last few years (1,2,3). Membrane lipidomics embraces many aspects of cell metabolism and the role of lipids is now considered more than merely inert and structural in delimitating the extra- and intra-cellular compartments (4,5). Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. The aim of our work was to study membrane lipidomics of mesenchymal stromal cells derived from human placenta and correlate it to specific biological properties, by using chemically- defined tailored lipid supplements (Refeed®). In the experimental study, the cell membranes of freshly isolated mesenchymal stromal cells obtained from human fetal membranes (FM-MSCs) were characterized for fatty acid composition. Then, we investigated cell morphology, viability, proliferation, differentiation and immunomodulation after in-vitro exposure to Refeed® supplements. Control MSCs were cultured without lipid supplementation. Our results showed a significant reduction of membrane fluidity for in-vitro primary cells, with cell membrane fatty acid composition greatly differing from the in-vivo one. By tailoring lipid supplementation, the fatty acid composition and biophysical properties of in-vitro cell membranes resulted more similar to the in-vivo counterparts, with higher omega-6 fatty acid content and increased membrane fluidity. These modifications of membrane composition and properties had no effect on cell morphology and viability, whereas ameliorated cell proliferation rate, diffentiation ability and immunomodulatory properties. In particular, supplemented FMMSCs showed an increased expression of cell membrane molecules like Vascular Endothelial Growth Factor Receptors 1 (VEGFR-1 or Flt-1) and 2 (VEGFR-2 or KDR), that correlated with a more efficient response to angiogenic commitment. Moreover, regarding immunomodulation, supplemented FM-MSCs displayed an increased expression of the tolerogenic cell surface protein HLA-G, that positively influenced the in-vitro cell immunomodulatory ability. Finally, these data suggest that specific lipid supplementation have functional consequences on in-vitro MSC behavior and may influence cell-based therapeutic approaches

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

BACKGROUND: Term 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). RESULTS: The 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. CONCLUSION: The 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

A combination of temsirolimus, an allosteric mTOR inhibitor, with clofarabine as a new therapeutic option for patients with acute myeloid leukemia

Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiating cells (LICs) function. In this study, we assessed the therapeutic potential of a combination consisting of temsirolimus [an allosteric mTOR complex 1 (mTORC1) inhibitor] with clofarabine, a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. The drug combination (CLO-TOR) displayed synergistic cytotoxic effects against a panel of AML cell lines and primary cells from AML patients. Treatment with CLO-TOR induced a G0/G1-phase cell cycle arrest, apoptosis, and autophagy. CLO-TOR was pro-apoptotic in an AML patient blast subset (CD34+/CD38−/CD123+), which is enriched in putative leukemia initiating cells (LICs). In summary, the CLO-TOR combination could represent a novel valuable treatment for AML patients, also in light of its efficacy against LICs

Pre-transplant CD69+ extracellular vesicles are negatively correlated with active ATLG serum levels and associate with the onset of GVHD in allogeneic HSCT patients

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Rabbit anti-T lymphocyte globulin (ATLG) in addition to calcineurin inhibitors and antimetabolites is a suitable strategy to prevent GVHD in several transplant settings. Randomized studies already demonstrated its efficacy in terms of GVHD prevention, although the effect on relapse remains the major concern for a wider use. Tailoring of ATLG dose on host characteristics is expected to minimize its side effects (immunological reconstitution, relapse, and infections). Here, day -6 to day +15 pharmacokinetics of active ATLG serum level was first assayed in an explorative cohort of 23 patients by testing the ability of the polyclonal serum to bind antigens on human leukocytes. Significantly lower levels of serum active ATLG were found in the patients who developed GVHD (ATLG_AUC(CD45): 241.52 +/- 152.16 vs. 766.63 +/- 283.52 (mu g*day)/ml, p = 1.46e(-5)). Consistent results were obtained when the ATLG binding capacity was assessed on CD3+ and CD3+/CD4+ T lymphocytes (ATLG_AUC(CD3): 335.83 +/- 208.15 vs. 903.54 +/- 378.78 (mu g*day)/ml, p = 1.92e(-4); ATLG_AUC(CD4): 317.75 +/- 170.70 vs. 910.54 +/- 353.35 (mu g*day)/ml, p = 3.78e(-5). Concomitantly, at pre-infusion time points, increased concentrations of CD69+ extracellular vesicles (EVs) were found in patients who developed GVHD (mean fold 9.01 +/- 1.33; p = 2.12e(-5)). Consistent results were obtained in a validation cohort of 12 additional ATLG-treated HSCT patients. Serum CD69+ EVs were mainly represented in the nano (i.e. 100 nm in diameter) EV compartment and expressed the leukocyte marker CD45, the EV markers CD9 and CD63, and CD103, a marker of tissue-resident memory T cells. The latter are expected to set up a host pro-inflammatory cell compartment that can survive in the recipient for years after conditioning regimen and contribute to GVHD pathogenesis. In summary, high levels of CD69+ EVs are significantly correlated with an increased risk of GVHD, and they may be proposed as a tool to tailor ATLG dose for personalized GVHD prevention