RESUMO
The role played by certain domestic species such as dogs as a translational model in comparative oncology shows great interest to develop new therapeutic strategies in brain tumors. Gliomas are a therapeutic challenge that represents the most common form of malignant primary brain tumors in humans and the second most common form in dogs. Gene-directed enzyme/prodrug therapy using adipose mesenchymal stem cells (Ad-MSCs) expressing the herpes simplex virus thymidine kinase (TK) has proven to be a promising alternative in glioblastoma therapy, through its capacity to migrate and home to the tumor and delivering local cytotoxicity avoiding other systemic administration. In this study, we demonstrate the possibility for canine Ad-MSCs (cAd-MSCs) to be genetically engineered efficiently with a lentiviral vector to express TK (TK-cAd-MSCs) and in combination with ganciclovir (GCV) prodrug demonstrated its potential antitumor efficacy in vitro and in vivo in a mice model with the human glioblastoma cell line U87. TK-cAd-MSCs maintained cell proliferation, karyotype stability, and MSCs phenotype. Genetic modification significantly affects its secretory profile, both the analyzed soluble factors and exosomes. TK-cAd-MSCs showed a high secretory profile of some active antitumor immune response cytokines and a threefold increase in the amount of secreted exosomes, with changes in their protein cargo. We also found that the prodrug protein is not released directly into the culture medium by TK-cAd-MSCs. We believe that our work provides new perspectives for glioblastoma gene therapy in dogs and a better understanding of this therapy in view of its possible implantation in humans.
Assuntos
Neoplasias Encefálicas/terapia , Ganciclovir/administração & dosagem , Glioblastoma/terapia , Herpes Simples/enzimologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Timidina Quinase/genética , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Cães , Ganciclovir/farmacologia , Genes Transgênicos Suicidas , Terapia Genética , Glioblastoma/genética , Herpes Simples/genética , Humanos , Lentivirus/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Timidina Quinase/metabolismo , Transdução Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Thymidine kinase expressing human adipose mesenchymal stem cells (TK-hAMSCs) in combination with ganciclovir (GCV) are an effective platform for antitumor bystander therapy in mice models. However, this strategy requires multiple TK-hAMSCs administrations and a substantial number of cells. Therefore, for clinical translation, it is necessary to find a biocompatible scaffold providing TK-hAMSCs retention in the implantation site against their rapid wash-out. We have developed a microtissue (MT) composed by TKhAMSCs and a scaffold made of polylactic acid microparticles and cell-derived extracellular matrix deposited by hAMSCs. The efficacy of these MTs as vehicles for TK-hAMSCs/GCV bystander therapy was evaluated in a rodent model of human prostate cancer. Subcutaneously implanted MTs were integrated in the surrounding tissue, allowing neovascularization and maintenance of TK-hAMSCs viability. Furthermore, MTs implanted beside tumors allowed TK-hAMSCs migration towards tumor cells and, after GCV administration, inhibited tumor growth. These results indicate that TK-hAMSCs-MTs are promising cell reservoirs for clinical use of therapeutic MSCs in bystander therapies.
Assuntos
Células-Tronco Mesenquimais , Neoplasias , Animais , Efeito Espectador , Linhagem Celular Tumoral , Ganciclovir/farmacologia , Camundongos , Neoplasias/terapia , Simplexvirus , Timidina QuinaseRESUMO
The existence of radio- and chemotherapy-surviving cancer stem cells is currently believed to explain the inefficacy of anti-glioblastoma (GBM) therapies. The aim of this study was to determine if a therapeutic strategy specifically targeting GBM stem cells (GSC) would completely eradicate a GBM tumor. In both the in vitro and the in vivo models, ganciclovir therapy targeting proliferating GSC promotes the survival of a quiescent, stem-like cell pool capable of reproducing the tumor upon release of the therapeutic pressure. Images of small niches of therapy-surviving tumor cells show organized networks of vascular-like structures formed by tumor cells expressing CD133 or OCT4/SOX2. These results prompted the investigation of tumor cells differentiated to endothelial and pericytic lineages as a potential reservoir of tumor-initiating capacity. Isolated tumor cells with pericyte and endothelial cell lineage characteristics, grown under tumorsphere forming conditions and were able to reproduce tumors after implantation in mice.
Assuntos
Antígeno AC133/genética , Glioma/genética , Glioma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Antígeno AC133/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismoRESUMO
A preclinical model of glioblastoma (GB) bystander cell therapy using human adipose mesenchymal stromal cells (hAMSCs) is used to address the issues of cell availability, quality, and feasibility of tumor cure. We show that a fast proliferating variety of hAMSCs expressing thymidine kinase (TK) has therapeutic capacity equivalent to that of TK-expressing hAMSCs and can be used in a multiple-inoculation procedure to reduce GB tumors to a chronically inhibited state. We also show that up to 25% of unmodified hAMSCs can be tolerated in the therapeutic procedure without reducing efficacy. Moreover, mimicking a clinical situation, tumor debulking previous to cell therapy inhibits GB tumor growth. To understand these striking results at a cellular level, we used a bioluminescence imaging strategy and showed that tumor-implanted therapeutic cells do not proliferate, are unaffected by GCV, and spontaneously decrease to a stable level. Moreover, using the CLARITY procedure for tridimensional visualization of fluorescent cells in transparent brains, we find therapeutic cells forming vascular-like structures that often associate with tumor cells. In vitro experiments show that therapeutic cells exposed to GCV produce cytotoxic extracellular vesicles and suggest that a similar mechanism may be responsible for the in vivo therapeutic effectiveness of TK-expressing hAMSCs.
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The encapsulation of mRNA in nanosystems as gene vaccines for immunotherapy purposes has experienced an exponential increase in recent years. Despite the many advantages envisaged within these approaches, their application in clinical treatments is still limited due to safety issues. These issues can be attributed, in part, to liver accumulation of most of the designed nanosystems and to the inability to transfect immune cells after an intravenous administration. In this context, this study takes advantage of the known versatile properties of the oligopeptide end-modified poly (ß-amino esters) (OM-PBAEs) to complex mRNA and form discrete nanoparticles. Importantly, it is demonstrated that the selection of the appropriate end-oligopeptide modifications enables the specific targeting and major transfection of antigen-presenting cells (APC) in vivo, after intravenous administration, thus enabling their use for immunotherapy strategies. Therefore, with this study, it can be confirmed that OM-PBAE are appropriate systems for the design of mRNA-based immunotherapy approaches aimed to in vivo transfect APCs and trigger immune responses to fight either tumors or infectious diseases.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Portadores de Fármacos/química , Células HeLa , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Polímeros/química , Células RAW 264.7RESUMO
Glioblastoma multiforme (GBM) is the most devastating primary brain tumor due to its infiltrating and diffuse growth characteristics, a situation compounded by the lack of effective treatments. Currently, many efforts are being devoted to find novel formulations to treat this disease, specifically in the nanomedicine field. However, due to the lack of comprehensive characterization that leads to insufficient data on reproducibility, only a reduced number of nanomedicines have reached clinical phases. In this context, the aim of the present study was to use a cascade of assays that evaluate from physical-chemical and structural properties to biological characteristics, both in vitro and in vivo, and also to check the performance of nanoparticles for glioma therapy. An amphiphilic block copolymer, composed of polyester and poly(ethylene glycol; PEG) blocks, has been synthesized. Using a mixture of this copolymer and a polymer containing an active targeting moiety to the Blood Brain Barrier (BBB; Seq12 peptide), biocompatible and biodegradable polymeric nanoparticles have been prepared and extensively characterized. In vitro studies demonstrated that nanoparticles are safe for normal cells but cytotoxic for cancer cells. In vivo studies in mice demonstrated the ability of the Seq12 peptide to cross the BBB. Finally, in vivo efficacy studies using a human tumor model in SCID mice resulted in a significant 50% life-span increase, as compared with non-treated animals. Altogether, this assay cascade provided extensive pre-clinical characterization of our polymeric nanoparticles, now ready for clinical evaluation.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Nanopartículas/administração & dosagem , Polímeros/administração & dosagem , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Bovinos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Glioma/metabolismo , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos SCID , Nanopartículas/metabolismo , Polímeros/metabolismo , Ratos , Ratos Wistar , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
MicroRNAs are critical regulators of gene networks in normal and abnormal biological processes. Focusing on invasive ductal breast cancer (IDC), we have found dysregulated expression in tumor samples of several microRNAs, including the miR-200 family, along progression from primary tumors to distant metastases, further reflected in higher blood levels of miR-200b and miR-7 in IDC patients with regional or distant metastases relative to patients with primary node-negative tumors. Forced expression of miR-200s in MCF10CA1h mammary cells induced an enhanced epithelial program, aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization in vivo. MiR-200s also induced the constitutive activation of the PI3K-Akt signaling through downregulation of PTEN, and the enhanced mammosphere growth and ALDH activity induced in MCF10CA1h cells by miR-200s required the activation of this signaling pathway. Interestingly, the morphology of tumors formed in vivo by cells expressing miR-200s was reminiscent of metaplastic breast cancer (MBC). Indeed, the epithelial components of MBC samples expressed significantly higher levels of miR-200s than their mesenchymal components and displayed a marker profile compatible with luminal progenitor cells. We propose that microRNAs of the miR-200 family promote traits of highly proliferative breast luminal progenitor cells, thereby exacerbating the growth and metastatic properties of transformed mammary epithelial cells.
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The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells.
RESUMO
Bioreactor systems allow safe and reproducible production of tissue constructs and functional analysis of cell behavior in biomaterials. However, current procedures for the analysis of tissue generated in biomaterials are destructive. We describe a transparent perfusion system that allows real-time bioluminescence imaging of luciferase expressing cells seeded in scaffolds for the study of cell-biomaterial interactions and bioreactor performance. A prototype provided with a poly(lactic) acid scaffold was used for "proof of principle" studies to monitor cell survival in the scaffold (up to 22 days). Moreover, using cells expressing a luciferase reporter under the control of inducible tissue-specific promoters, it was possible to monitor changes in gene expression resulting from hypoxic state and endothelial cell differentiation. This system should be useful in numerous tissue engineering applications, the optimization of bioreactor operation conditions, and the analysis of cell behavior in three-dimensional scaffolds.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Proliferação de Células , Processamento de Imagem Assistida por Computador/métodos , Medições Luminescentes , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Tecido Adiposo/metabolismo , Técnicas de Cultura de Células , Humanos , Células-Tronco Mesenquimais/metabolismo , Perfusão , Alicerces TeciduaisRESUMO
Conventional photodynamic therapy has shown to be beneficial in the treatment of a variety of tumors. However, one of its major limitations is the inadequate penetration depth of visible light. In order to overcome this constraint, we developed 80nm poly-methylmethacrylate core-shell fluorescent nanoparticles (FNP) loaded with the photosensitizer tetrasulfonated aluminum phthalocyanine (Ptl). To demonstrate the efficacy of our Ptl@FNP we performed in vitro and in vivo studies using a human prostate tumor model. Our data reveal that Ptl@FNP are internalized by tumor cells, favour Ptl intracellular accumulation, and efficiently trigger cell death through the generation of ROS upon irradiation with 680nm light. When directly injected into tumors intramuscularly induced in SCID mice, Ptl@FNP upon irradiation significantly reduce tumor growth with higher efficiency than the bare Ptl. Collectively, these results demonstrate that the newly developed nanoparticles may be utilized as a delivery system for antitumor phototherapy in solid cancers.
Assuntos
Indóis/administração & dosagem , Nanopartículas , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Humanos , Isoindóis , Masculino , Camundongos , Camundongos SCIDRESUMO
We synthesized novel tetraphenylethene (TPE) conjugates, which undergo unique self-assembly to form spherical nanoparticles that exhibited aggregation induced emission (AIE) in the near-infrared region. These nanoparticles showed significant singlet oxygen generation efficiency, negligible dark toxicity, rapid cellular uptake, efficient localization in cytoplasm, and high in vitro photocytotoxicity as well as in vivo photodynamic activity against a human prostate tumor animal model. This study demonstrates, for the first time, the power of the self-assembled AIE active tetraphenylethene conjugates in aqueous media as a nanoplatform for future therapeutic applications.
Assuntos
Benzotiazóis/química , Citoplasma , Nanopartículas/química , Imagem Óptica , Estilbenos/química , Animais , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos SCID , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Nanopartículas/metabolismo , Nanopartículas/toxicidade , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/toxicidade , Neoplasias da Próstata/terapiaRESUMO
Considerable research has been dedicated to restoring myocardial cell slippage and limiting ventricular remodeling after myocardial infarction (MI). We examined the ability of a three-dimensional (3D) engineered fibrin patch filled with human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) to induce recovery of cardiac function after MI. The UCBMSCs were modified to coexpress luciferase and fluorescent protein reporters, mixed with fibrin, and applied as an adhesive, viable construct (fibrin-cell patch) over the infarcted myocardium in mice (MI-UCBMSC group). The patch adhered well to the heart. Noninvasive bioluminescence imaging demonstrated early proliferation and differentiation of UCBMSCs within the construct in the postinfarct mice in the MI-UCBMSC group. The implanted cells also participated in the formation of new, functional microvasculature that connected the fibrin-cell patch to both the subjacent myocardial tissue and the host circulatory system. As revealed by echocardiography, the left ventricular ejection fraction and fractional shortening at sacrifice were improved in MI-UCBMSC mice and were markedly reduced in mice treated with fibrin alone and untreated postinfarction controls. In conclusion, a 3D engineered fibrin patch composed of UCBMSCs attenuated infarct-derived cardiac dysfunction when transplanted locally over a myocardial wound.
Assuntos
Fibrina/uso terapêutico , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Infarto do Miocárdio/terapia , Animais , Diferenciação Celular/genética , Sangue Fetal/citologia , Humanos , Camundongos , Infarto do Miocárdio/patologiaRESUMO
The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft) for posterolateral lumbar fusion (PLF) in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM) mesenchymal stem cells (MSCs) have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group), hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct). During the last three days of culture, dexamethasone (dex) and beta-glycerophosphate (ß-GP) were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4-L5). Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT), histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70%) than for mineral scaffold alone (22%) and hybrid constructs (35%). The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft). Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored by bioluminescence imaging (BLI). Although the cultured MSCs had osteogenic potential, their contribution to spinal fusion when seeded in mineral scaffolds, in the conditions disclosed here, remains uncertain probably due to callus interference with the scaffolds. At present, bone autografts are better than hybrid constructs for posterolateral lumbar fusion, but we should continue to seek better conditions for efficient tissue engineering.
Assuntos
Células da Medula Óssea/citologia , Fusão Vertebral/métodos , Alicerces Teciduais , Animais , Diferenciação Celular , Feminino , Medições Luminescentes , Camundongos , Camundongos Nus , Minerais/química , Imagem Molecular , Osteogênese , Ovinos , Alicerces Teciduais/química , Tomografia Computadorizada por Raios XRESUMO
Human mesenchymal stromal cells, whether from the bone marrow or adipose tissue (hASCs), are promising cell therapy agents. However, generation of abundant cells for therapy remains to be a challenge, due to the need of lengthy expansion and the risk of accumulating genomic defects during the process. We show that hASCs can be easily induced to a reversible fast-proliferating phenotype (FP-ASCs) that allows rapid generation of a clinically useful quantity of cells in <2 weeks of culture. Expanded FP-ASCs retain their finite expansion capacity and pluripotent properties. Despite the high proliferation rate, FP-ASCs show genomic stability by array-comparative genomic hybridization, and did not generate tumors when implanted for a long time in an SCID mouse model. Comparative analysis of gene expression patterns revealed a set of genes that can be used to characterize FP-ASCs and distinguish them from hASCs. As potential candidate therapeutic agents, FP-ASCs displayed high vasculogenic capacity in Matrigel assays. Moreover, application of hASCs and FP-ASCs in a fibrin scaffold over a myocardium infarct model in SCID mice showed that both cell types can differentiate to endothelial and myocardium lineages, although FP-ASCs were more potent angiogenesis inducers than hASCs, at promoting myocardium revascularization.
Assuntos
Tecido Adiposo/metabolismo , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica , Transplante de Células-Tronco Mesenquimais , Infarto do Miocárdio/terapia , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Xenoenxertos , Humanos , Células-Tronco Mesenquimais , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Infarto do Miocárdio/metabolismoRESUMO
In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (µCT) to evaluate bone regeneration and high-resolution µCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3weeks after implantation. Results from µCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea , Diferenciação Celular , Fibrina/farmacologia , Medições Luminescentes , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Crânio , Microtomografia por Raio-X , Tecido Adiposo/metabolismo , Animais , Becaplermina , Células Endoteliais/metabolismo , Matriz Extracelular/química , Humanos , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos SCID , Osteoblastos/metabolismo , Crânio/lesões , Crânio/metabolismo , Crânio/patologiaRESUMO
BACKGROUND: Adipose tissue-derived progenitor cells (ATDPCs) isolated from human cardiac adipose tissue are useful for cardiac regeneration in rodent models. These cells do not express cardiac troponin I (cTnI) and only express low levels of PECAM-1 when cultured under standard conditions. The purpose of the present study was to evaluate changes in cTnI and PECAM-1 gene expression in cardiac ATDPCs following their delivery through a fibrin patch to a murine model of myocardial infarction using a non-invasive bioluminescence imaging procedure. METHODS AND RESULTS: Cardiac and subcutaneous ATDPCs were doubly transduced with lentiviral vectors for the expression of chimerical bioluminescent-fluorescent reporters driven by constitutively active and tissue-specific promoters (cardiac and endothelial for cTnI and PECAM-1, respectively). Labeled cells mixed with fibrin were applied as a 3-D fibrin patch over the infarcted tissue. Both cell types exhibited de novo expression of cTnI, though the levels were remarkably higher in cardiac ATDPCs. Endothelial differentiation was similar in both ATDPCs, though cardiac cells induced vascularization more effectively. The imaging results were corroborated by standard techniques, validating the use of bioluminescence imaging for in vivo analysis of tissue repair strategies. Accordingly, ATDPC treatment translated into detectable functional and morphological improvements in heart function. CONCLUSIONS: Both ATDPCs differentiate to the endothelial lineage at a similar level, cardiac ATDPCs differentiated more readily to the cardiomyogenic lineage than subcutaneous ATDPCs. Non-invasive bioluminescence imaging was a useful tool for real time monitoring of gene expression changes in implanted ATDPCs that could facilitate the development of procedures for tissue repair.
Assuntos
Modelos Animais de Doenças , Fibrina/administração & dosagem , Medições Luminescentes/métodos , Infarto do Miocárdio/terapia , Transplante de Células-Tronco/métodos , Gordura Subcutânea/transplante , Animais , Diferenciação Celular/fisiologia , Transplante de Células/métodos , Células Cultivadas , Endotélio Vascular/química , Endotélio Vascular/patologia , Feminino , Humanos , Camundongos , Camundongos SCID , Infarto do Miocárdio/patologia , Miocárdio/química , Miocárdio/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/administração & dosagem , Células-Tronco/química , Células-Tronco/fisiologia , Gordura Subcutânea/químicaRESUMO
Multipotent human adipose tissue mesenchymal stromal cells (hAMSCs) are promising therapy vehicles with tumor-homing capacity that can be easily modified to deliver cytotoxicity activating systems in the proximity of tumors. In a previous work, we observed that hAMSCs are very effective delivering cytotoxicity to glioma tumors. However, these results were difficult to reconcile with the relatively few hAMSCs surviving implantation. We use a bioluminescence imaging (BLI) platform to analyze the behavior of bioluminescent hAMSCs expressing HSV-tTK in a U87 glioma model and gain insight into the therapeutic mechanisms. Tumor-implanted hAMSCs express the endothelial marker PECAM1(CD31), integrate in tumor vessels and associate with CD133-expressing glioma stem cells (GSC). Inhibition of endothelial lineage differentiation in hAMSCs by Notch1 shRNA had no effect on their tumor homing and growth-promoting capacity but abolished the association of hAMSCs with tumor vessels and CD133+ tumor cells and significantly reduced their tumor-killing capacity. The current strategy allowed the study of tumor/stroma interactions, showed that tumor promotion and tumor-killing capacities of hAMSCs are based on different mechanisms. Our data strongly suggest that the therapeutic effectiveness of hAMSCs results from their association with special tumor vascular structures that also contain GSCs.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Endotélio Vascular/citologia , Glioma/patologia , Células-Tronco Mesenquimais/citologia , Efeito Espectador , Linhagem Celular Tumoral , Linhagem da Célula , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Glioma/irrigação sanguínea , Glioma/terapia , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Neoplásicas/fisiologia , RNA Interferente Pequeno/genética , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMO
Acid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine-1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic, and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the nonmetastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confirm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer and suggest that our new potent and specific AC inhibitors could act by counteracting critical growth properties of these highly aggressive tumor cells.
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Ceramidase Ácida/antagonistas & inibidores , Ceramidase Ácida/genética , Terapia de Alvo Molecular , Neoplasias da Próstata/genética , Ceramidase Ácida/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Ceramidas/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Lisofosfolipídeos/metabolismo , Masculino , Metástase Neoplásica , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
The angiogenic capacity of a new biomaterial composite of poly(lactic acid) and calcium phosphate glass (PLA/CaP) was analyzed by noninvasive bioluminescence imaging (BLI) and histological procedures. Human adipose tissue-derived mesenchymal stromal cells expressing cytomegalovirus (CMV) promoter regulated Photinus pyralis luciferase (hAMSC-PLuc) grew up to 30 times the initial cell load, in vitro, when seeded in PLA/CaP scaffolds, but suffered an initial growth crisis followed by recovery when the scaffolds were subcutaneously implanted in SCID mice. To analyze changes in gene expression, hAMSC-PLuc cells were double labeled with a CMV promoter regulated Renilla reniformis luciferase and a Photinus pyralis luciferase reporter regulated by either the PECAM promoter or a hypoxia response element (HRE) artificial promoter and seeded in PLA/CaP and PLA scaffolds implanted in SCID mice. Analysis by BLI showed that hAMSCs in scaffolds were induced to differentiate to the endothelial lineage and did this faster in PLA/CaP than in PLA scaffolds. Endothelial differentiation correlated with a decrease in the activity of HRE regulated luciferase expression, indicative of a reduction of hypoxia. Histological analysis showed that PLA/CaP scaffolds were colonized by a functional host vascular system. Moreover, colonization by isolectin B(4) positive host cells was more effective in PLA/CaP than in PLA scaffolds, corroborating BLI results.
Assuntos
Tecido Adiposo/metabolismo , Fosfatos de Cálcio , Diferenciação Celular/efeitos dos fármacos , Células Endoteliais/metabolismo , Vidro/química , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Alicerces Teciduais/química , Tecido Adiposo/citologia , Animais , Fosfatos de Cálcio/química , Fosfatos de Cálcio/farmacologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , Ácido Láctico/química , Ácido Láctico/farmacologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos SCID , Poliésteres , Polímeros/química , Polímeros/farmacologiaRESUMO
Stem cell therapies are promising strategies to regenerate human injured tissues, including ischemic myocardium. Here, we examined the acquisition of properties associated with vascular growth by human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs), and whether they promoted vascular growth in vivo. UCBMSCs were induced in endothelial cell-specific growth medium (EGM-2) acquiring new cell markers, increased Ac-LDL uptake, and migratory capacity as assessed by qRT-PCR, Western blotting, indirect immunofluorescence, and invasion assays. Angiogenic and vasculogenic potentials could be anticipated by in vitro experiments showing self organization into Matrigel-mediated cell networks, and activation of circulating angiogenic-supportive myeloid cells. In mice, following subcutaneous co-injection with Matrigel, UCBMSCs modified to co-express bioluminescent (luciferases) and fluorescent proteins were demonstrated to participate in the formation of new microvasculature connected with the host circulatory system. Response of UCBMSCs to ischemia was explored in a mouse model of acute myocardial infarction (MI). UCBMSCs transplanted using a fibrin patch survived 4 weeks post-implantation and organized into CD31(+)network structures above the infarcted myocardium. MI-treated animals showed a reduced infarct scar and a larger vessel-occupied area in comparison with MI-control animals. Taken together, the presented results show that UCBMSCs can be induced in vitro to acquire angiogenic and vasculogenic properties and contribute to vascular growth in vivo.