Este trabalho foi financiado pelo NIH NINDS conceder R01 NS074388 581 (CL Limoli), Instituto da Califórnia de Medicina Regenerativa (CIRM) Grant RS1-00413 (CLL), Formação CIRM Grant TG2-0115 (MMA), e um Grant CIRM para JOVE em apoio da documentação de vídeo.

<ol>
	<li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ionizing+radiation-induced+damage+in+human+neural+stem+cells.">Consequences of ionizing radiation-induced damage in human neural stem cells.</a> Free. Radic. Biol. Med. , (2010).</li><li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+radiation-induced+cognitive+impairment+through+cranial+transplantation+of+human+embryonic+stem+cells.">Rescue of radiation-induced cognitive impairment through cranial transplantation of human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 19150-19155 (2009).</li><li>Boyer, A., Khan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+11.">Ch. 11.</a> Treatment planning in Radiation Oncology. , (2007).</li><li>Lawson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+application+of+IMRT+based+radiosurgery+to+treat+multiple+or+large+irregular+lesions+and+verification+of+infra-red+frameless+localization+system.">Intracranial application of IMRT based radiosurgery to treat multiple or large irregular lesions and verification of infra-red frameless localization system.</a> J. Neurooncol. 97, 59-66 (2010).</li><li>Ling, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissioning+and+quality+assurance+of+RapidArc+radiotherapy+delivery+system.">Commissioning and quality assurance of RapidArc radiotherapy delivery system.</a> Int. J. Radiat. Oncol. Biol. Phys. 72, 575-581 (2008).</li><li>Otto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+modulated+arc+therapy:+IMRT+in+a+single+gantry+arc.">Volumetric modulated arc therapy: IMRT in a single gantry arc.</a> Med. Phys. 35, 310-317 (2008).</li><li>Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in stereotaxic coordinates. , (2007).</li><li>Hess, D. C., Borlongan, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapy+in+ischemic+stroke.">Cell-based therapy in ischemic stroke.</a> Expert. Rev. Neurother. 8, 1193-1201 (2008).</li><li>Hefferan, M. P., Johe, K., Feldman, E. L., Lunn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+immunosuppressive+therapy+for+spinal+grafting+of+human+spinal+stem+cells+in+a+rat+model+of+ALS.">Optimization of immunosuppressive therapy for spinal grafting of human spinal stem cells in a rat model of ALS.</a> Cell. Transplant. , (2011).</li></ol>

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Considerável pesquisa está em andamento a explorar a miríade de formas que as células-tronco pode ser usada clinicamente para restaurar as funções normais de tecidos danificados, com idades entre 8 e doentes. A eventual realização desses esforços exigirá uma compreensão detalhada do comportamento das células enxertadas dentro microambientes únicos que são distintos de tecido normal intacta. Nosso trabalho demonstrou que dentro do leito tecido irradiado, NSCs cranialmente enxertada pode restaurar funcionalmente cognição, onde sobrevivem, migram e se diferenciam ao longo de linhagens neuronais e gliais 2. Precisamente como estas células mediar a recuperação da cognição é incerto no momento, mas não depende de realização de uma série de procedimentos experimentais cuidadosamente controladas de maneira reprodutível. Temos detalhada desses procedimentos crítica aqui em esforços para acelerar o potencial de translação de terapias com células-tronco para amenizar efeitos adversos cognitivos associados com amanejo clínico do cérebro e outras formas de câncer. Considerações adicionais que são susceptíveis de ter um impacto significativo da qualidade dos dados são destacadas a seguir. Terapias de transplante com base dependem de células-tronco como o reagente crítico, e, consequentemente, o cuidado deve ser exercido de forma adequada caracterizar culturas, manter a esterilidade e usar números passagem pareados por fiabilidade dos resultados. Transplantadas células-tronco humanas foram marcadas com BrdU antes da cirurgia, para fornecer um meio para segui-los in vivo. Nas nossas condições experimentais, NSCs transplantadas não foram submetidos a proliferação extensiva, de modo que a diluição do rótulo BrdU não era problemático. Alternativamente, transplantaram células-tronco humanas podem ser distinguidas das células do hospedeiro por imunomarcação para marcadores específicos humanos tais como a proteína da matriz nuclear (h-NUC ou hNUMA) 9 ou humana antígeno específico nuclear (HuNu) 2. Células-tronco humanas também podem ser rotulados comuma variedade de marcadores fluorescentes para facilitar a sua identificação dentro do cérebro host. Atenção a parâmetros de irradiação definir técnicas de entrega precisos dose, e aquelas aqui descritas modelo atual prática clínica em oncologia de radiação. O transplante de células-tronco reprodutível no cérebro também é crítica, e é realizado através de um instrumento digital estereotáxico. A capacidade de micromanipulate precisamente de uma seringa para a implantação de micro-células-tronco em pequenas estruturas cerebrais como o hipocampo reduz o erro humano. Com este aparelho, NSCs foram transplantadas em quatro locais distintos abrangendo o anterior para regiões posteriores do hipocampo de ratos. Dorso-ventral (DV) coordenadas foram determinadas com base na experiência com atímicos nude (ATN) ratos de tal forma que os procedimentos cirúrgicos não causaria danos à formação do hipocampo 2. A secção coronal através do cérebro do rato revela estruturas fundamentais da formação hipocampal including giro denteado (DG), o hilo denteado (DH) e CA1 e CA3 subcampos (Fig. 8). NSCs transplantados, introduzida dorsalmente a partir do trato agulha visível (Nt, redline), são visualizadas como coloração escura (marrom) células depositadas no site de transplante de liberação (Tr), logo abaixo do corpo caloso (CC), que, posteriormente, migrar todo o septo -temporal eixo do hipocampo.

<table border="1"><tbody><tr><td> Nome do material </td><td> Tipo </td><td> Companhia </td><td> Número Catálogo </td><td> Comentário </td></tr><tr><td> Digital Instrumento estereotáxica w/45 ° e 18 ° Earbars </td><td> Configuração craniana transplante cirúrgico </td><td> Leica Microsystems (anteriormente: MyNeuroLab) </td><td> 39463501 </td><td> Acessórios úteis: Palco com aquecedor e fonte de corrente variável (para manter a temperatura corporal durante a cirurgia) </td></tr></tbody></table>

stem cell transplantation strategies for the restoration of cognitive dysfunction caused by cranial radiotherapy

Radioterapia geralmente fornece o único recurso clínico para os que sofrem com tumores cerebrais primários ou metastáticos. Apesar de benéfico, irradiação craniana pode induzir um declínio progressivo na cognição e debilitante que pode, em parte, ser causada pela depleção de células-tronco neurais. Dado o aumento da sobrevida de pacientes diagnosticados com câncer no cérebro, qualidade de vida em termos de saúde cognitiva tornou-se uma preocupação crescente, especialmente na ausência de qualquer satisfatória tratamentos de longo prazo. Para resolver este grave problema de saúde que temos utilizado a substituição de células-tronco como uma estratégia para combater a radiação induzida por declínio cognitivo. Nosso modelo utiliza ratos atímicos nu submetidos a irradiação craniana. A radiação ionizante é entregue como quer a totalidade do cérebro ou como um feixe altamente focalizado para o hipocampo através de lin orelha de um ccelerator (LINAC) radiocirurgia estereotáxica base. Dois dias após a irradiação, hucélulas-tronco neurais homem (hNSCs) foram transplantadas para o stereotaxically hipocampo. Os ratos foram então avaliados para alterações na cognição sobrevivência da célula, enxertadas e para a expressão de marcadores de diferenciação específicos 1 e 4 meses após a irradiação. Nossos paradigmas testes cognitivos têm demonstrado que os animais enxertados com hNSCs apresentam melhoras significativas na função cognitiva. Estereologia imparcial revela sobrevida significativa (10-40%) das células enxertadas em 1 e 4 meses após o transplante, depende da quantidade e do tipo de células enxertadas. Enxertada células migram extensivamente, ao longo diferenciar linhagens glial e neuronal, e expressar uma gama de imaturos e maduros marcadores fenotípicos. Nossos dados demonstram direto benefícios cognitivos derivados de células-tronco humanas em vós enxertada, sugerindo que este procedimento poderá um dia pagar uma estratégia promissora para a restauração de longo prazo funcionais da cognição em indivíduos submetidos a radiot cranialherapy. Promover a divulgação dos procedimentos de crítica necessária para replicar e estender os nossos estudos, nós fornecemos a documentação escrita e visual dos vários passos-chave em nosso plano experimental, com ênfase em estereotáxica radiosurgey e transplante.

Watch this Scientific Journal Video about Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy at JoVE.com

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Pacientes com tumor cerebral rotineiramente submetidos à radioterapia cranial, e ao mesmo tempo benéfico, este tratamento muitas vezes resulta em disfunção cognitiva debilitante. Este grave problema por resolver tem no momento, nenhum recurso clínico, e tem impulsionado os nossos esforços para desenvolver terapias com células-tronco com base para a recuperação de induzida por radiação decrementa cognitiva.

Transplante de Células-tronco Estratégias para a Restauração da disfunção cognitiva causada pela radioterapia craniana

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

stem-cell-transplantation-strategies-for-restoration-cognitive

Research

JoVE Journal

Medicine

14.3K visualizações.  University of California Irvine. Pacientes com tumor cerebral rotineiramente submetidos à radioterapia cranial, e ao mesmo tempo benéfico, este tratamento muitas vezes resulta em disfunção cognitiva debilitante. Este grave problema por resolver tem no momento, nenhum recurso clínico, e tem impulsionado os nossos esforços para desenvolver terapias com células-tronco com base para a recuperação de induzida por radiação decrementa cognitiva.

Vídeo: Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Transplante ortotópico de aorta: um modelo de rato para estudar o desenvolvimento de vasculopatia crônica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Medicina

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Um modelo do rato de No Utero Transplante

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

Diminui quantificar cognitivos causados ​​pela radioterapia craniana

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Transplante de Células-tronco em um In vitro Modelo de isquemia / reperfusão Simulated

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Neural Stem Cell Transplantation em Experimental contusão Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

High Throughput caracterização de células-tronco adultas modificadas para liberação de fatores terapêuticos como estratégia de neuroproteção

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Normotérmica<em&gt; Ex Vivo</em&gt; Perfusão rim para a Preservação do rim Os enxertos antes do transplante

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Criação e transplante de uma folha de células-tronco adiposo-derivado (ASC) em um modelo de cicatrização de feridas diabética

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...