Martin Bonamino

Altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient.

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions, and emotions, usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.

An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits. Aims We herein report a convenient and affordable method based on in house made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes. Results This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41–59%), the hard to transfect murine T cells (mean 38%, 36–42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as in vitro expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis. Conclusions We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs.

Retinoic Acid-Treated Pluripotent Stem Cells Undergoing Neurogenesis Present Increased Aneuploidy and Micronuclei Formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.

Human Mesenchymal Cells from Adipose Tissue Deposit Laminin and Promote Regeneration of Injured Spinal Cord in Rats

Cell therapy is a promising strategy to pursue the unmet need for treatment of spinal cord injury (SCI). Although several studies have shown that adult mesenchymal cells contribute to improve the outcomes of SCI, a descripton of the pro-regenerative events triggered by these cells is still lacking. Here we investigated the regenerative properties of human adipose tissue derived stromal cells (hADSCs) in a rat model of spinal cord compression. Cells were delivered directly into the spinal parenchyma immediately after injury. Human ADSCs promoted functional recovery, tissue preservation, and axonal regeneration. Analysis of the cord tissue showed an abundant deposition of laminin of human origin at the lesion site and spinal midline; the appearance of cell clusters composed of neural precursors in the areas of laminin deposition, and the appearance of blood vessels with separated basement membranes along the spinal axis. These effects were also observed after injection of hADSCs into non-injured spinal cord. Considering that laminin is a well-known inducer of axonal growth, as well a component of the extracellular matrix associated to neural progenitors, we propose that it can be the paracrine factor mediating the pro-regenerative effects of hADSCs in spinal cord injury.

Agathisflavone enhances retinoic acid-induced neurogenesis and its receptors α and β in pluripotent stem cells.

Flavonoids have key functions in the regulation of multiple cellular processes; however, their effects have been poorly examined in pluripotent stem cells. Here, we tested the hypothesis that neurogenesis induced by all-trans retinoic acid (RA) is enhanced by agathisflavone (FAB, Caesalpinia pyramidalis Tull). Mouse embryonic stem (mES) cells and induced pluripotent stem (miPS) cells growing as embryoid bodies (EBs) for 4 days were treated with FAB (60 μM) and/or RA (2 μM) for additional 4 days. FAB did not interfere with the EB mitotic rate of mES cells, as evidenced by similar percentages of mitotic figures labeled by phospho-histone H3 in control (3.4% ± 0.4%) and FAB-treated groups (3.5% ± 1.1%). Nevertheless, the biflavonoid reduced cell death in both control and RA-treated EBs from mES cells by almost 2-fold compared with untreated EBs. FAB was unable, by itself, to induce neuronal differentiation in EBs after 4 days of treatment. On the other hand, FAB enhanced neuronal differentiation induced by RA in both EBs of mES and miPS. FAB increased the percentage of nestin-labeled cells by 2.7-fold (mES) and 2.4 (miPS) and β-tubulin III-positive cells by 2-fold (mES) and 2.7 (miPS) in comparison to RA-treated EBs only. FAB increased the expression of RA receptors α and β in mES EBs, suggesting that the availability of RA receptors is limiting RA-induced neurogenesis in pluripotent stem cells. This is the first report to describe that naturally occurring biflavonoids regulate apoptosis and neuronal differentiation in pluripotent stem cells.

Retroviral vectors and transposons for stable gene therapy: advances, current challenges and perspectives

Gene therapy protocols require robust and long-term gene expression. For two decades, retrovirus family vectors have offered several attractive properties as stable gene-delivery vehicles. These vectors represent a technology with widespread use in basic biology and translational studies that require persistent gene expression for treatment of several monogenic diseases. Immunogenicity and insertional mutagenesis represent the main obstacles to a wider clinical use of these vectors. Efficient and safe non-viral vectors are emerging as a promising alternative and facilitate clinical gene therapy studies. Here, we present an updated review for beginners and expert readers on retro and lentiviruses and the latest generation of transposon vectors (sleeping beauty and piggyBac) used in stable gene transfer and gene therapy clinical trials. We discuss the potential advantages and disadvantages of these systems such as cellular responses (immunogenicity or genome modification of the target cell) following exogenous DNA integration. Additionally, we discuss potential implications of these genome modification tools in gene therapy and other basic and applied science contexts.

Chimeric Antigen Receptors in Cancer Immuno-Gene Therapy: Current Status and Future Directions

The concept of chimeric antigen receptors (CARs) as molecules able to redirect T lymphocytes toward tumor cells is currently being exploited in the field of cancer immunotherapy. Despite promising preliminary results, some clinical trials evidenced limitations for this technology that must be overcome for more extensive application of CARs in tumor immunotherapy. We describe here the fundaments of these molecules in terms of structure, function, possible targets and pre-clinical and clinical applications. We also discuss strategies that can potentially overcome the limitations seen so far, paving the road to a wider application of this exciting new technology.

Hydroxyurea dose impacts hematologic parameters in polycythemia vera and essential thrombocythemia but does not appreciably affect JAK2-V617F allele burden

A recent publication by Antonioli et al. shows that the continuous use of hydroxyurea (HU) does not appreciably reduce JAK2 V617F allele burden in patients with polycythemia vera (PV) or essential thrombocythemia (ET).[1][1] Our results confirm and extend these data. To assess the effects of a

Membrane cholesterol depletion reduces breast tumor cell migration by a mechanism that involves non-canonical Wnt signaling and IL-10 secretion

BackgroundMigration and invasion are hallmarks of cancer cells that allow their dissemination to other tissues. Studying the cellular and molecular basis of cancer cell migration can help us to understand and control cancer metastasis. Many membrane molecules have been described as being involved in tumor cell migration, and among them is cholesterol.MethodIn the present study we investigated the role of membrane cholesterol in the migration of breast tumor cells. The human mammary gland/breast epithelial cell line MDA-MB 231 was used and membrane cholesterol was depleted with methyl-β-cyclodextrin (MbCD). Cell migration was measured in a cell-based scratch assay and the involvement of the Wnt signaling was tested using Lef-1/TCF reporter activation in permanently transfected MDA-MB 231 cells. Cell morphology was analyzed using fluorescent phalloidin to label F-actin structures.ResultsOur results show that membrane cholesterol depletion reduces cell migration, induces changes in cell morphology and in membrane protrusions. Cholesterol depletion also induces an increase in the secretion of IL-10, a cytokine involved in the control of cell migration. In addition, we show that these effects do not involve the activation of the canonical Wnt/beta-catenin pathway. Interestingly, Wnt5a inhibited cell migration in similar levels as MbCD, pointing to a role for the non-canonical Wnt pathway and IL-10 in this process.ConclusionTaken together, our results suggest a correlation between membrane cholesterol, non-canonical Wnt signaling, IL-10 secretion and breast tumor cell migration. These results could contribute to the understanding of breast tumor cell migration and may be used in the development of new therapeutic strategies targeted to breast cancer metastasis.

PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.