New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X.

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Intravenous Grafts of Human Amniotic Fluid-Derived Stem Cells Reduce Behavioral Deficits in Experimental Ischemic Stroke.

Amniotic fluid has been investigated as new cell source for stem cells in the development of future cell-based transplantation. This study reports isolation of viable human amniotic fluid-derived stem cells, labeled with multimodal iron oxide nanoparticles, and its effect on focal cerebral ischemia-reperfusion injury in Wistar rats. Middle cerebral artery occlusion of 60 min followed by reperfusion for 1 h, 6 h, and 24 h was employed in the present study to produce ischemia and reperfusion-induced cerebral injury in rats. Tests were employed to assess the functional outcome of the sensorimotor center activity in the brain, through a set of modified neurological severity scores used to assess motor and exploratory capacity 24 h, 14, and 28 days after receiving cellular therapy via tail vein. In our animal model of stroke, transplanted cells migrated to the ischemic focus, infarct volume decreased, and motor deficits improved. Therefore, we concluded that these cells appear to have beneficial effects on the ischemic brain, possibly based on their ability to enhance endogenous repair mechanisms.

Intravenous grafts of human amniotic fluid-derived stem cells reduce behavioral deficits in experimental ischemic stroke

Amniotic fluid has been investigated as new cell source for stem cells in the development of future cell-based transplantation. This study reports isolation of viable human amniotic fluid-derived stem cells, labeled with multimodal iron oxide nanoparticles, and its effect on focal cerebral ischemia–reperfusion injury in Wistar rats. Middle cerebral artery occlusion of 60 min followed by reperfusion for 1 h, 6 h, and 24 h was employed in the present study to produce ischemia and reperfusion-induced cerebral injury in rats. Tests were employed to assess the functional outcome of the sensorimotor center activity in the brain, through a set of modified neurological severity scores used to assess motor and exploratory capacity 24 h, 14, and 28 days after receiving cellular therapy via tail vein. In our animal model of stroke, transplanted cells migrated to the ischemic focus, infarct volume decreased, and motor deficits improved. Therefore, we concluded that these cells appear to have beneficial effects on the ischemic brain, possibly based on their ability to enhance endogenous repair mechanisms.

New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Intravenous grafts of human amniotic fluid-derived stem cells reduce behavioral deficits in experimental ischemic stroke

Amniotic fluid has been investigated as new cell source for stem cells in the development of future cell-based transplantation. This study reports isolation of viable human amniotic fluid-derived stem cells, labeled with multimodal iron oxide nanoparticles, and its effect on focal cerebral ischemia–reperfusion injury in Wistar rats. Middle cerebral artery occlusion of 60 min followed by reperfusion for 1 h, 6 h, and 24 h was employed in the present study to produce ischemia and reperfusion-induced cerebral injury in rats. Tests were employed to assess the functional outcome of the sensorimotor center activity in the brain, through a set of modified neurological severity scores used to assess motor and exploratory capacity 24 h, 14, and 28 days after receiving cellular therapy via tail vein. In our animal model of stroke, transplanted cells migrated to the ischemic focus, infarct volume decreased, and motor deficits improved. Therefore, we concluded that these cells appear to have beneficial effects on the ischemic brain, possibly based on their ability to enhance endogenous repair mechanisms.

New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo.

BACKGROUND: Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. METHODS/RESULTS: We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. CONCLUSIONS: Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Lipid nanoemulsion passive tumor accumulation dependence on tumor stage and anatomical location: a new mathematical model for in vivo imaging biodistribution studies.

Nanoparticle delivery to tumor tissue is one of the most important applications of nanomedicine. However, the literature shows that this pharmacological event is highly-affected by several tumor biology characteristics, including tumor size and maturation. Thus, the objective of the present study is to report on the investigation of the biodistribution of a lipid nanoemulsion (NE) in a breast cancer tumor model using in vivo imaging techniques. As highlights of this study, we can indicate that the biodistribution was measured in different tumor sites (primary and metastatic tumors) and in the same experimental mice for four subsequent weeks. With this approach it is possible to observe that the NE tumor delivery is significantly altered during tumor growth and metastasis progression. Furthermore, in the present report we introduce a phenomenological mathematical model that successfully explains the delivery behavior of a hydrophobic infrared fluorescent NE marker to both primary tumor and metastatic lesions. We believe that these data, in addition to the phenomenological mathematical model, are relevant to understanding how the stage of tumor development can alter macromolecule and/or nanoparticle delivery to tumor tissues, thus improving the efficacy of the passive delivery features promoted by tumor biology.

Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo

Background Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. Methods/results We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. Conclusions Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology

Background: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo

Background Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. Methods/results We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. Conclusions Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology

Background: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

Acid-sensitive lipidated doxorubicin prodrug entrapped in nanoemulsion impairs lung tumor metastasis in a breast cancer model.

AIM: To develop an acid-sensitive lipidated, doxorubicin (Dox) prodrug (C16-Dox) to be entrapped in lipid nanoemulsion (NE-C16-Dox) as a nanocarrier to treat breast cancer models (in vitro and in vivo). RESULTS: We report the efficacy of NE-C16-Dox in in vitro experiments, as well as the improved chemotherapeutic index and tumor-control efficacy compared with treatment with free Dox in an in vivo murine 4T1 breast cancer model. In addition, NE-C16-Dox allowed the use of a higher dose of Dox, acceptable biocompatibility and a significant reduction in lung metastasis. CONCLUSION: Taken together, these results indicate that NE-C16-Dox is promising for breast cancer treatment, thus creating possibilities to translate these nanotechnology concepts to clinical applications.

Mesenchymal stem cell-like properties of CD133+ glioblastoma initiating cells.

Glioblastoma is composed of dividing tumor cells, stromal cells and tumor initiating CD133+ cells. Recent reports have discussed the origin of the glioblastoma CD133+ cells and their function in the tumor microenvironment. The present work sought to investigate the multipotent and mesenchymal properties of primary highly purified human CD133+ glioblastoma-initiating cells. To accomplish this aim, we used the following approaches: i) generation of tumor subspheres of CD133+ selected cells from primary cell cultures of glioblastoma; ii) analysis of the expression of pluripotency stem cell markers and mesenchymal stem cell (MSC) markers in the CD133+ glioblastoma-initiating cells; iii) side-by-side ultrastructural characterization of the CD133+ glioblastoma cells, MSC and CD133+ hematopoietic stem cells isolated from human umbilical cord blood (UCB); iv) assessment of adipogenic differentiation of CD133+ glioblastoma cells to test their MSC-like in vitro differentiation ability; and v) use of an orthotopic glioblastoma xenograft model in the absence of immune suppression. We found that the CD133+ glioblastoma cells expressed both the pluripotency stem cell markers (Nanog, Mush-1 and SSEA-3) and MSC markers. In addition, the CD133+ cells were able to differentiate into adipocyte-like cells. Transmission electron microscopy (TEM) demonstrated that the CD133+ glioblastoma-initiating cells had ultrastructural features similar to those of undifferentiated MSCs. In addition, when administered in vivo to non-immunocompromised animals, the CD133+ cells were also able to mimic the phenotype of the original patient's tumor. In summary, we showed that the CD133+ glioblastoma cells express molecular signatures of MSCs, neural stem cells and pluripotent stem cells, thus possibly enabling differentiation into both neural and mesodermal cell types.

Evaluation of umbilical cord mesenchymal stem cell labeling with superparamagnetic iron oxide nanoparticles coated with dextran and complexed with Poly-L-lysine.

OBJECTIVE: The objective of this study was to evaluate the effect of the labeling of umbilical cord vein derived mesenchymal stem cells with superparamagnetic iron oxide nanoparticles coated with dextran and complexed to a non-viral transfector agent transfector poly-L-lysine. METHODS: The labeling of mesenchymal stem cells was performed using the superparamagnetic iron oxide nanoparticles/dextran complexed and not complexed to poly-L-lysine. Superparamagnetic iron oxide nanoparticles/dextran was incubated with poly-L-lysine in an ultrasonic sonicator at 37°C for 10 minutes for complex formation superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine by electrostatic interaction. Then, the mesenchymal stem cells were incubated overnight with the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine and superparamagnetic iron oxide nanoparticles/dextran. After the incubation period the mesenchymal stem cells were evaluated by internalization of the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine and superparamagnetic iron oxide nanoparticles/dextran by Prussian Blue stain. Cellular viability of labeled mesenchymal stem cells was evaluated by cellular proliferation assay using 5,6-carboxy-fluorescein-succinimidyl ester method and apoptosis detection by Annexin V- Propidium Iodide assay. RESULTS: mesenchymal stem cells labeled with superparamagnetic iron oxide nanoparticles/dextran without poly-L-lysine not internalized efficiently the superparamagnetic iron oxide nanoparticles due to its low presence detected within cells. Mesenchymal stem cells labeled with the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine efficiently internalized the superparamagnetic iron oxide nanoparticles due to greater presence in the cells interior. The viability and apoptosis assays demonstrated that the mesenchymal stem cells labeled and not labeled respectively with the superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine continue to proliferate over seven days and the percentage of cells in early or late apoptosis is low compared to the percentage of live cells over the three days. CONCLUSION: Our results showed that the use of poly-L-lysine complexed with superparamagnetic iron oxide nanoparticles/dextran provides better internalization of these superparamagnetic iron oxide nanoparticles in mesenchymal stem cells Thus, we demonstrated that this type of labeling is not cytotoxic to the mesenchymal stem cells, since the viability and apoptosis assays showed that the cells remain alive and proliferating. The efficiency of this type of labeling in mesenchymal stem cells can provide non-invasive methods for monitoring these cells in vivo.

Evaluation of umbilical cord mesenchymal stem cells labeling with superparamagnetic iron oxide nanoparticles coated with dextran and complexed with Poly-L-Lysine / Avaliação da marcação de células-tronco mesenquimais de cordão umbilical com nanopartículas superparamagnéticas de óxido de ferro recobertas com Dextran e complexadas a Poli-L-Lisina

Objetivo: O objetivo deste estudo foi avaliar o efeito da marcação de células-tronco mesenquimais obtidas da parede da veia do cordão umbilical com nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran e complexadas a um agente transfector não viral denominado de Poli-L-Lisina. Métodos: A marcação das células-tronco mesenquimais foi realizada utilizando as nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran complexadas e não complexadas a Poli-L-Lisina. As nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran foram incubadas com o Poli-L-Lisina em um sonicador ultrassonico a 37ºC por 10 minutos, para a formação do complexo através de interação eletrostática. Em seguida, as células-tronco mesenquimais foram incubadas overnight com as nanopartículas de óxido de ferro superparamagnéticas complexadas e não com Poli-L-Lisina. Após o período de incubação as células-tronco mesenquimais foram avaliadas quanto à internalização do complexo nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina e nanopartícula de óxido de ferro superparamagnéticas /dextran através de ensaio citoquímico com azul de prússia. A viabilidade celular das célulastronco mesenquimais marcadas foi avaliada através do ensaio de proliferação celular utilizando o método de 5,6-carboxy-fluoresceinsuccinimidyl-ester e de morte celular através do método de anexinaiodeto de propídeo, ambos utilizando o recurso de citometria de fluxo. Resultados: Observamos nos ensaios citoquímicos que as célulastronco mesenquimais que foram marcadas com as nanopartícula de óxido de ferro superparamagnéticas /dextran sem a Poli-L-Lisina, não internalizaram com eficiência as nanopartículas devido pouca detecção de sua presença no interior das células. As células-tronco mesenquimais marcadas com o complexo nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina internalizaram com eficiência as nanopartículas devido à maior presença destas no interior das células. Os ensaios de viabilidade e morte celular demonstraram respectivamente que as células-tronco mesenquimais marcadas com as nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina continuam proliferando ao longo de sete dias e a porcentagem de células em apoptose inicial e tardia é baixa em relação à porcentagem de células vivas ao longo de três dias. Conclusão: Evidenciamos através de nossos resultados a necessidade da utilização da Poli-L-Lisina complexada com a nanopartícula de óxido de ferro superparamagnéticas /dextran para melhor internalização nas célulastronco mesenquimais. Paralelamente, demonstramos que este tipo de marcação não é citotóxico para as células-tronco mesenquimais já que os testes de morte e viabilidade celular mostraram que as células continuam vivas e proliferando.

Differential neuroprotection by A(1) receptor activation and A(2A) receptor inhibition following pilocarpine-induced status epilepticus.

Aiming at a better understanding of the role of A(2A) in temporal lobe epilepsy (TLE), we characterized the effects of the A(2A) antagonist SCH58261 (7-(2-phenylethyl)-5-amino-2(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) on seizures and neuroprotection in the pilocarpine model. The effects of SCH58261 were further analyzed in combination with the A(1) agonist R-Pia (R(-)-N(6)-(2)-phenylisopropyl adenosine). Eight groups were studied: pilocarpine (Pilo), SCH+Pilo, R-Pia+Pilo, R-Pia+SCH+Pilo, Saline, SCH+Saline, R-Pia+Saline, and R-Pia+SCH+Saline. The administration of SCH58261, R-Pia, and R-Pia+SCH58261 prior to pilocarpine increased the latency to SE, and decreased either the incidence of or rate of mortality from SE compared with controls. Administration of R-Pia and R-Pia+SCH58261 prior to pilocarpine reduced the number of Fluoro-Jade B-stained cells in the hippocampus and piriform cortex when compared with control. This study showed that pretreatment with R-Pia and SCH58261 reduces seizure occurrence, although only R-Pia has neuroprotective properties. Further studies are needed to clarify the neuroprotective role of A(2A) in TLE.

The ultrastructural study of tumorigenic cells using nanobiomarkers.

Despite recent advances, patients with malignant brain tumors still have a poor prognosis. Glioblastoma (WHO grade 4 astrocytoma), the most malignant brain tumor, represents 50% of all astrocytomas, with a median survival rate of <1 year. It is, therefore, extremely important to search for new diagnostic and therapeutic approaches for patients with glioblastoma. This study describes the application of superparamagnetic nanoparticles of iron oxide, as well as monoclonal antibodies, of immunophenotypic significance, conjoined to quantum dots for the ultrastructural assessment of glioblastoma cells. For this proposal, an immunophenotypic study by flow cytometry was carried out, followed by transmission electron microscopy analysis. The process of tumor cell labeling using nanoparticles can successfully contribute to the identification of tumorigenic cells and consequently for better understanding of glioblastoma genesis and recurrence. In addition, this method may help further studies in tumor imaging, diagnosis, and prognostic markers detection.

Labeling of human mesenchymal stem cells with quantum dots allows tracking of transplanted cells engrafted in infarcted pig hearts / A marcação de células-tronco mesenquimais humanas com quantum dots possibilita a identificação de células transplantadas enxertadas em corações de porcos após o infarto do miocárdio

Objective: The purpose of this paper was to validate fluorescent quantum dots QD as a cell marker for tracking human mesenchymal stem cells in vivo, using a pre-clinical model of acute myocardium infarction. Methods: Human umbilical cord mesenchymal stem cells were isolated and expanded in vitro. Mesenchymal stem cells were labeled with QD 655. Myocardium infarction induction in pigs was performed by occluding the left descending coronary artery for 60 minutes, with a balloon catheter. One day after the myocardium infarction, intracoronary injection of mesenchymal stem cells was performed. One week after cell transplantation, the animals were killed; their hearts were removed and underwent histological examination Results: All the mesenchymal stem cells were labeled with QD 655. The labeling process did not affect viability, proliferation, and osteogenic and adipogenic differentiation potential of the cells. Labeled mesenchymal stem cells were easily tracked in the histological sections of the infarcted area. Cells were observed with a frequency of two per section, while no cells were observed in the remote myocardium. Conclusion: These results indicate that QD 655 labeling is an efficient tool for tracking mesenchymal stem cells in vivo

Objetivo: Avaliar uma nova modalidade de marcação celular com quantum dots QD fluorescentes em células-tronco mesenquimais humanas (CTM), transplantadas em um modelo pré-clínico de infarto agudo do miocárdio. Métodos: CTM de cordão umbilical humano foram isoladas e expandidas in vitro. CTM foram marcadas passivamente com o nanocristal QD 655. A indução do infarto foi realizada pela cateterização e oclusão por 60 minutos da artéria coronária descendente anterior esquerda sob fluoroscopia. Um dia após o infarto, CTM foram transplantadas por via intracoronária. Uma semana após o transplante de CTM, os animais foram sacrificados e os corações removidos e analisados histologicamente. Resultados: Todas as CTM foram efetivamente marcadas com QD 655. A marcação não afetou a viabilidade, a taxa de proliferação e a capacidade de diferenciação em osteoblastos e adipócitos das células. CTM marcadas foram facilmente identificadas em cortes histológicos das áreas de infarto/borda de infarto com frequência média de duas células por corte histológico, enquanto que na região remota não foi detectada qualquer marcação. Conclusão: Os resultados indicam que a marcação de CTM com QD 655 é eficiente para rastreamento de células mesenquimais in vivo

Adult stem cells therapy for the heart: [review] / Uso terapêutico de células-tronco em Cardiologia: [revisão]

Acute myocardial infarction causes irreversible loss of cardiomyocytes and endothelial cells compromising the cardiac function of patients. For some patients, current therapeutic approaches are not sufficient to prevent myocardial remodeling, which aggravates the disease. The potential of many types of adult bone marrow-derived progenitor cells in replacing damaged myocardium with functional cells has been investigated in several clinical and experimental studies. It has been demonstrated that these cells have an overall beneficial effect on heart function although the mechanism or multiple mechanisms involved have not been elucidated yet. Here, it will be reviewed some aspects of this fast-growing and controversial of adult autologous progenitor/stem cells for cardiac repair.

O infarto agudo do miocárdio causa perda irreversível de cardiomiócitos e células endoteliais, que compromete a função cardíaca dos pacientes. Para alguns indivíduos, as abordagens terapêuticas atuais não são suficientes para evitar um remodelamento miocárdico, o que agrava a doença. O potencial de vários tipos de células progenitoras adultas derivadas da medula óssea para substituir o miocárdio lesado com células funcionais, tem sido investigado em vários estudos clínicos e experimentais. Já se demonstrou que tais células têm um efeito benéfico geral na função cardíaca, mas o mecanismo ou os diversos mecanismos envolvidos não foram ainda elucidados. Neste artigo revisamos alguns aspectos deste campo controverso e de rápida evolução – uso de células progenitoras/tronco autólogas de adultos para reparo cardíaco.