RESUMO
BACKGROUND/AIMS: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice. METHODS: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections. RESULTS: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging. CONCLUSION: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Miocárdio/patologia , Miócitos Cardíacos/transplante , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Rastreamento de Células , Técnicas de Cocultura , Imunidade Inata , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Microscopia de Fluorescência , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miocárdio/imunologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Infiltração de Neutrófilos , Imagem Óptica , Células-Tronco Pluripotentes/citologia , Polímeros/químicaRESUMO
BACKGROUND/AIMS: Embryonic stem (ES) cells have got a broad range differentiation potential. The differentiation is initiated via aggregation of non-differentiated ES cells into embryoid body (EB) capable of multi-lineage development. However experimental variables present in standard differentiation techniques lead to high EB heterogeneity, affecting development into the cells of desired lineage, and do not support the process automatization and scalability. METHODS: Here we present a novel pipe based microbioreactor (PBM) setup based on segmented flow, designed for spatial maintenance of temperature, nutrition supply, gas supply and sterility. RESULTS: We verified PBM feasibility for continuous process generating cardiac cells starting from single ES cell suspension followed by EB formation for up to 10 days. The ES cells used in the study were genetically modified for cardiac-specific EGFP expression allowing optical monitoring of cardiomyocytes while EBs remained within PBM for up to 10 days. Efficiency of cardiac cells formation within PBM was similar compared to a standard hanging drop based protocol. CONCLUSION: Our findings ensure further development of microfluidic bioreactor technology to enable robust cardiomyocytes production for needs of drug screening, tissue engineering and other applications.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias Murinas/citologia , Miócitos Cardíacos/citologia , Animais , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Linhagem Celular , Citometria de Fluxo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Camundongos , Microfluídica/instrumentação , Microfluídica/métodos , Microscopia , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Engenharia TecidualRESUMO
SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.
Assuntos
Alternativas aos Testes com Animais/métodos , Testes de Toxicidade/métodos , Alternativas aos Testes com Animais/legislação & jurisprudência , Alternativas aos Testes com Animais/organização & administração , Animais , Biomarcadores/análise , Células Cultivadas , Qualidade de Produtos para o Consumidor , União Europeia , Regulamentação Governamental , Ensaios de Triagem em Larga Escala , Humanos , Técnicas In VitroRESUMO
A long-term goal of numerous research projects is to identify biomarkers for in vitro systems predicting toxicity in vivo. Often, transcriptomics data are used to identify candidates for further evaluation. However, a systematic directory summarizing key features of chemically influenced genes in human hepatocytes is not yet available. To bridge this gap, we used the Open TG-GATES database with Affymetrix files of cultivated human hepatocytes incubated with chemicals, further sets of gene array data with hepatocytes from human donors generated in this study, and publicly available genome-wide datasets of human liver tissue from patients with non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular cancer (HCC). After a curation procedure, expression data of 143 chemicals were included into a comprehensive biostatistical analysis. The results are summarized in the publicly available toxicotranscriptomics directory ( http://wiki.toxbank.net/toxicogenomics-map/ ) which provides information for all genes whether they are up- or downregulated by chemicals and, if yes, by which compounds. The directory also informs about the following key features of chemically influenced genes: (1) Stereotypical stress response. When chemicals induce strong expression alterations, this usually includes a complex but highly reproducible pattern named 'stereotypical response.' On the other hand, more specific expression responses exist that are induced only by individual compounds or small numbers of compounds. The directory differentiates if the gene is part of the stereotypical stress response or if it represents a more specific reaction. (2) Liver disease-associated genes. Approximately 20 % of the genes influenced by chemicals are up- or downregulated, also in liver disease. Liver disease genes deregulated in cirrhosis, HCC, and NASH that overlap with genes of the aforementioned stereotypical chemical stress response include CYP3A7, normally expressed in fetal liver; the phase II metabolizing enzyme SULT1C2; ALDH8A1, known to generate the ligand of RXR, one of the master regulators of gene expression in the liver; and several genes involved in normal liver functions: CPS1, PCK1, SLC2A2, CYP8B1, CYP4A11, ABCA8, and ADH4. (3) Unstable baseline genes. The process of isolating and the cultivation of hepatocytes was sufficient to induce some stress leading to alterations in the expression of genes, the so-called unstable baseline genes. (4) Biological function. Although more than 2,000 genes are transcriptionally influenced by chemicals, they can be assigned to a relatively small group of biological functions, including energy and lipid metabolism, inflammation and immune response, protein modification, endogenous and xenobiotic metabolism, cytoskeletal organization, stress response, and DNA repair. In conclusion, the introduced toxicotranscriptomics directory offers a basis for a rationale choice of candidate genes for biomarker evaluation studies and represents an easy to use source of background information on chemically influenced genes.
Assuntos
Bases de Dados Genéticas , Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatopatias/genética , Bibliotecas de Moléculas Pequenas/toxicidade , Toxicogenética/métodos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Análise de Componente Principal , Bibliotecas de Moléculas Pequenas/química , Toxicogenética/estatística & dados numéricosRESUMO
The umbilical cord blood derived endothelial progenitor cells (EPCs) contribute to vascular regeneration in experimental models of ischaemia. However, their ability to participate in cardiovascular tissue restoration has not been elucidated yet. We employed a novel coculture system to investigate whether human EPCs have the capacity to integrate into living and ischaemic cardiac tissue, and participate to neovascularization. EPCs were cocultured with either living or ischaemic murine embryonic ventricular slices, in the presence or absence of a pro-angiogenic growth factor cocktail consisting of VEGF, IGF-1, EGF and bFGF. Tracking of EPCs within the cocultures was performed by cell transfection with green fluorescent protein or by immunostaining performed with anti-human vWF, CD31, nuclei and mitochondria antibodies. EPCs generated vascular tube-like structures in direct contact with the living ventricular slices. Furthermore, the pro-angiogenic growth factor cocktail reduced significantly tubes formation. Coculture of EPCs with the living ventricular slices in a transwell system did not lead to vascular tube-like structures formation, demonstrating that the direct contact is necessary and that the soluble factors secreted by the living slices were not sufficient for their induction. No vascular tubes were formed when EPCs were cocultured with ischaemic ventricular slices, even in the presence of the pro-angiogenic cocktail. In conclusion, EPCs form vascular tube-like structures in contact with living cardiac tissue and the direct cell-to-cell interaction is a prerequisite for their induction. Understanding the cardiac niche and micro-environmental interactions that regulate EPCs integration and neovascularization are essential for applying these cells to cardiovascular regeneration.
Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Comunicação Celular , Células Endoteliais/citologia , Sangue Fetal/citologia , Coração/fisiologia , Neovascularização Fisiológica , Células-Tronco/citologia , Animais , Técnicas de Cocultura , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura , Sobrevivência de Tecidos , Transfecção , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Cell transplantation offers the promise in the restoration of ventricular function after an extensive myocardial infarction, but the optimal cell type remains controversial. Human unrestricted somatic stem cells (USSCs) isolated from umbilical cord blood have great potential to differentiate into myogenic cells and induce angiogenesis. The present study evaluated the effect of USSCs on myocardial regeneration and improvement of heart function after myocardial infarction in a porcine model. METHOD AND RESULTS: The distal left anterior descending artery of Yorkshire pigs (30 to 35 kg) was occluded by endovascular implantation of a coil. Four weeks after infarction, single-photon emission computed tomography technetium 99m sestamibi scans (MIBI) and echocardiography were performed. USSCs (100 x 10(6)) or culture media were then directly injected into the infarcted region (n=8 per group). Pigs were immunosuppressed by daily administration of cyclosporin A. At 4 weeks after transplantation, MIBI and echocardiography were repeated and heart function was also assessed with a pressure-volume catheter. The infarcted myocardium and implanted cells were studied histologically. MIBI showed improved regional perfusion (P<0.05) and wall motion (P<0.05) of the infarct region in the transplant group compared with the control. Ejection fraction evaluated by both MIBI and echocardiography decreased in the control group but increased in the transplant group (P<0.01). Scar thickness of the transplant group was higher than the control. The grafted cells were detected 4 weeks after transplantation by both immunohistochemistry and in situ hybridization. CONCLUSIONS: Engrafted USSCs were detected in the infarct region 4 weeks after cell transplantation, and the implanted cells improved regional and global function of the porcine heart after a myocardial infarction. This study suggests that the USSC implantation will be efficacious for cellular cardiomyoplasty.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Infarto do Miocárdio/cirurgia , Células-Tronco Pluripotentes/transplante , Animais , Cateterismo Cardíaco , Circulação Coronária , Feminino , Sangue Fetal/citologia , Coração/fisiologia , Humanos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Regeneração , Volume Sistólico , Sus scrofa , Tomografia Computadorizada de Emissão de Fóton Único , Transplante Heterólogo , UltrassonografiaRESUMO
During development of the heart, mitochondria proliferate within cardiomyocytes. It is unclear whether this is a response to the increasing energy demand or whether it is part of the developmental program. To investigate the role of the electron transport chain (ETC) in this process, we used transgenic murine embryonic stem (ES) cells in which the green fluorescent protein gene is under control of the alpha-myosin heavy chain promoter (alpha-MHC), allowing easy monitoring of cardiomyocyte differentiation. Spontaneous contraction of these cells within embryoid bodies (EBs) was not affected by inhibition of the ETC, suggesting that early heart cell function is sufficiently supported by anaerobic ATP production. However, heart cell development was completely blocked when adding antimycin A, an inhibitor of ETC complex III, before initiation of differentiation, whereas KCN did not block differentiation, strongly suggesting that specifically complex III function rather than mitochondrial ATP production is necessary for early heart cell development. When the underlying mechanism was examined, we noticed that antimycin A but not KCN lead to inhibition of spontaneous intracellular Ca++ oscillations, whereas both substances decreased mitochondrial membrane potential, as expected. We postulate that mitochondrial complex III activity is necessary for these Ca++ oscillations, which in turn are a prerequisite for cardiomyocyte differentiation.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/fisiologia , Transporte de Elétrons/fisiologia , Coração Fetal/citologia , Mitocôndrias Cardíacas/fisiologia , Miocárdio/citologia , Miócitos Cardíacos/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Antimicina A/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Genes Reporter , Idade Gestacional , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Cadeias Pesadas de Miosina/genética , Cianeto de Potássio/farmacologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Miosinas Ventriculares/genéticaRESUMO
The mitochondrial electron transport chain (ETC) is the most important source of reactive oxygen species (ROS) in mammalian cells. To assess its relevance to the endogenous generation of oxidative DNA damage in the nucleus, we have compared the background (steady-state) levels of oxidative DNA base modifications sensitive to the repair glycosylase Fpg (mostly 7,8-dihydro-8-oxoguanine) in wild-type HeLa cells and HeLa rho0 cells. The latter are depleted of mitochondrial DNA and therefore are unable to produce ROS in the ETC. Although the levels of ROS measured by flow cytometry and redox-sensitive probes in rho0 cells were only 10-15% those of wild-type cells, steady-state levels of oxidative DNA base modifications were the same as in wild-type cells. Mitochondrial generation of ROS was then stimulated in HeLa wild-type cells using inhibitors interfering with the ETC. Although mitochondrial ROS production was raised up to 6-fold, none of the substances nor their combinations induced additional oxidative base modifications in the nuclear DNA. This was also true for glutathione-depleted cells. The results indicate that the contribution of mitochondria to the endogenously generated background levels of oxidative damage in the nuclear DNA is negligible.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , DNA/metabolismo , Guanosina/análogos & derivados , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células/química , Células/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA-Formamidopirimidina Glicosilase/metabolismo , Transporte de Elétrons , Proteínas de Escherichia coli/metabolismo , Citometria de Fluxo , Guanosina/química , Guanosina/metabolismo , Células HeLa , Humanos , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Nucleotídeos/química , Nucleotídeos/metabolismo , OxirreduçãoRESUMO
BACKGROUND: Cervical carcinomas are second most frequent type of women cancer. Success in diagnostics of this disease is due to the use of Pap-test (cytological smear analysis). However Pap-test gives significant portion of both false-positive and false-negative conclusions. Amendments of the diagnostic procedure are desirable. Aetiological role of papillomaviruses in cervical cancer is established while the role of cellular gene alterations in the course of tumor progression is less clear. Several research groups including us have recently named the protein p16INK4a as a possible diagnostic marker of cervical cancer. To evaluate whether the specificity of p16INK4a expression in dysplastic and neoplastic cervical epithelium is sufficient for such application we undertook a broader immunochistochemical registration of this protein with a highly p16INK4a-specific monoclonal antibody. METHODS: Paraffin-embedded samples of diagnostic biopsies and surgical materials were used. Control group included vaginal smears of healthy women and biopsy samples from patients with cervical ectopia. We examined 197 samples in total. Monoclonal antibody E6H4 (MTM Laboratories, Germany) was used. RESULTS: In control samples we did not find any p16INK4a-positive cells. Overexpression of p16INK4a was detected in samples of cervical dysplasia (CINs) and carcinomas. The portion of p16INK4a-positive samples increased in the row: CIN I - CIN II - CIN III - invasive carcinoma. For all stages the samples were found to be heterogeneous with respect to p16INK4a-expression. Every third of CINs III and one invasive squamous cell carcinoma (out of 21 analyzed) were negative. CONCLUSIONS: Overexpression of the protein p16INK4a is typical for dysplastic and neoplastic epithelium of cervix uteri. However p16INK4a-negative CINs and carcinomas do exist. All stages of CINs and carcinomas analyzed are heterogeneous with respect to p16INK4a expression. So p16INK4a-negativity is not a sufficient reason to exclude a patient from the high risk group. As far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages application of immunohisto-/cytochemical test for p16INK4a may be regarded as a supplementary test for early diagnostics of cervical cancer.
Assuntos
Biomarcadores Tumorais/análise , Inibidor p16 de Quinase Dependente de Ciclina/análise , Neoplasias do Colo do Útero/patologia , Adenocarcinoma/química , Adenocarcinoma/patologia , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Citoplasma/patologia , Progressão da Doença , Epitélio/patologia , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Humanos , Miométrio/citologia , Miométrio/patologia , Invasividade Neoplásica , Estadiamento de Neoplasias , Valores de Referência , Sensibilidade e Especificidade , Displasia do Colo do Útero/patologia , Neoplasias do Colo do Útero/química , Vagina/citologia , Esfregaço VaginalRESUMO
Pluripotent stem cells have great potential for regenerative medicine; however, their clinical use is associated with a risk of tumor formation. We utilized pluripotent cells expressing green fluorescent protein and puromycin resistance under control of the Oct4 promoter to study the persistence of potential pluripotent cells under embryoid body (EB) culture conditions, which are commonly used to obtain organotypic cells. We found that i.) OCT4-expressing cells dramatically decrease during the first week of differentiation, ii.) the number of OCT4-expressing cells recovers from day 7 on, iii.) the OCT4-expressing cells are similar to embryonic stem cells grown in the presence of leukemia inhibitory factor LIF but express several markers associated with germ cell formation, such as DAZL and STRA-8 and iv.) the persistence of potentially pluripotent cells is independent of supportive cells in EBs. Finally, OCT4-expressing cells, isolated from EBs after 2-month of culture, were further maintained under feeder-free conditions in absence of LIF and continued to express OCT4 in 95 % of the population for at least 36 days. These findings point to an alternative state of stable OCT4 expression. In the frame of the landscape model of differentiation two attractors of pluripotency might be defined based on their different characteristics.
Assuntos
Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
Human embryonic stem cells (hESCs) can be propagated indefinitely in vitro in an undifferentiated pluripotent state, can differentiate into derivatives of all three germ layers and are of considerable interest for applications in regenerative medicine. Clinical application of hESCs, however, requires reliable protocols for cryopreservation. Current protocols for cryopreservation of hESCs suffer from low recovery rates of hESCs and loss of pluripotency after thawing. We therefore studied the effects of cryopreservation on the viability, proliferation potential, and the pluripotency status of hESCs by combining cellular readouts and transcriptomics. We identified biological processes and pathways affected by cryopreservation in order to understand the limited survival rate of hESCs by comparing transcriptomes of hESCs at different time points after thawing with cells that did not undergo cryopreservation. While the transcriptomes of cells post thawing were very similar to those of control non-frozen hESCs for the early time points, we observed increased expression of genes involved in apoptosis, embryonic morphogenesis, ossification, tissue morphogenesis, regeneration, vasculature development and cell death at later time points. Our data suggest that inhibition of anoikis apoptosis and the stress-induced differentiation pathways are promising targets for improving the survival rate and maintaining pluripotency of hESCs after cryopreservation.
Assuntos
Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica , Biomarcadores/metabolismo , Morte Celular , Diferenciação Celular/fisiologia , Proliferação de Células , Sobrevivência Celular , Células-Tronco Embrionárias/citologia , Humanos , Análise em Microsséries , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologiaRESUMO
Transplantation of purified pluripotent stem cell-derived cardiomyocytes into damaged myocardium might become a therapy to improve contractile function after myocardial infarction. However, engraftment remains problematic. Aim of this study was to investigate whether murine embryonic fibroblasts (MEFs) support the functional integration of purified embryonic stem cell-derived cardiomyocytes (ES-CMs). Neonatal murine ventricular tissue slices were subjected to oxygen and glucose deprivation to simulate irreversible ischemia. Vital tissue slices served as control. Vital and avital tissue slices were cultured with or without MEFs before coculturing with clusters of puromycin-selected ES-CMs. Integration of ES-CM clusters was assessed morphologically, motility by long-term microscopy, and functional integration by isometric force measurements. We observed a good morphological integration into vital but a poor integration into avital slices. Adding MEFs improved morphological integration into irreversibly damaged slices and enabled purified ES-CMs to migrate and to confer force. We conclude that noncardiomyocytes like MEFs support morphological integration and force transmission of purified ES-CMs by enabling adhesion and migration.
Assuntos
Fibroblastos/citologia , Ventrículos do Coração/patologia , Isquemia Miocárdica/patologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Engenharia Tecidual/métodos , Animais , Animais Recém-Nascidos , Adesão Celular , Diferenciação Celular , Movimento Celular , Técnicas de Cocultura/métodos , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Camundongos , Microtomia , Infarto do Miocárdio/patologia , Medicina RegenerativaRESUMO
We have established an in vitro Cre/loxP-based assay for monitoring cell fusion events that specifically traces the transport of cytoplasm from one cell to its fusion partner. Cells with a double fluorescence vector indicate fusion with cells expressing Cre recombinase by switching expression from red to green fluorescent protein through a Cre-mediated recombination event that simultaneously activates puromycin-acetyltransferase expression. This strategy allows for both the observation and puromycin selection of indicator cells that have undergone fusion with a Cre recombinase-expressing partner. A fusion protein of Cre with estrogen receptor (ER) can be used to control Cre recombinase activity through the tamoxifen-induced translocation of the Cre-ER fusion protein to the nucleus. Here we have established a new methodology that not only allows the monitoring of the transport of cellular contents, but also enables the purification of fused cells using puromycin.
Assuntos
Fusão Celular/métodos , Integrases/metabolismo , Microscopia de Fluorescência/métodos , Biologia Molecular/métodos , Animais , Linhagem Celular , Vetores Genéticos , Humanos , Imuno-Histoquímica , Integrases/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Puromicina , Receptores de Estrogênio , Reprodutibilidade dos Testes , Proteína Vermelha FluorescenteRESUMO
Here we describe the generation of a double-fluorescent Cre/loxP indicator system. This protocol involves (i) all cloning steps to generate the plasmid vector (3-5 months); (ii) a guide to prepare high-efficiency transformation competent E. coli; (iii) generation of double-fluorescent reporter cell lines (3-4 weeks); and (iv) the functional testing of the indicator cell lines by application of cell-permeable Cre recombinase. The indicator is designed to monitor recombination events by switching the fluorescence light from red to green. The red fluorescence, indicating the nonrecombined state, is accompanied by the expression of a resistance gene against the antibiotic blasticidin. Appearance of green fluorescence concomitantly with the activation of puromycin-acetyltransferase monitors the recombination of the indicator construct by the Cre recombinase. In summary, we have developed a plasmid vector allowing a fast, stable and straightforward generation of transgenic clones. The expression of red fluorescent protein enables the selection of positive clones upon transfection and significantly shortens the time for identification of stable clones. This feature and the option to select for recombined cells by puromycin application are advantages compared with other alternative methods. Moreover, we developed a method utilizing cell-permeable Cre protein to validate the transgenic clones. Ultimately, this powerful methodology facilitates Cre/loxP-based applications such as cell lineage tracking or monitoring of cell fusion.
Assuntos
Escherichia coli/genética , Vetores Genéticos/genética , Biologia Molecular/métodos , Transformação Bacteriana/genética , Fluorescência , Integrases/metabolismo , PuromicinaRESUMO
A variety of embryonic and adult stem cell lines require an initial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines.
Assuntos
Separação Celular/métodos , Técnicas de Cocultura/métodos , Células-Tronco/citologia , Animais , Sequência de Bases , Biotecnologia , Diferenciação Celular , Proliferação de Células , Primers do DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/citologia , Neurônios/metabolismo , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
UNLABELLED: In recent years, a large number of groups studied the fate of human stem cells in livers of immunodeficient animals. However, the interpretation of the results is quite controversial. We transplanted 4 different types of human extrahepatic precursor cells (derived from cord blood, monocytes, bone marrow, and pancreas) into livers of nonobese diabetic/severe combined immunodeficiency mice. Human hepatocytes were used as positive controls. Tracking of the transplanted human cells could be achieved by in situ hybridization with alu probes. Cells with alu-positive nuclei stained positive for human albumin and glycogen. Both markers were negative before transplantation. However, cells with alu-positive nuclei did not show a hepatocyte-like morphology and did not express cytochrome P450 3A4, and this suggests that these cells represent a mixed cell type possibly resulting from partial transdifferentiation. Using antibodies specific for human albumin, we also observed a second human albumin-positive cell type that could be clearly distinguished from the previously described cells by its hepatocyte-like morphology. Surprisingly, these cells had a mouse and not a human nucleus which is explained by transdifferentiation of human cells. Although it has not yet been formally proven, we suggest horizontal gene transfer as a likely mechanism, especially because we observed small fragments of human nuclei in mouse cells that originated from deteriorating transplanted cells. Qualitatively similar results were obtained with all 4 human precursor cell types through different routes of administration with and without the induction of liver damage. CONCLUSION: We observed evidence not for transdifferentiation but instead for a complex situation including partial differentiation and possibly horizontal gene transfer.
Assuntos
Diferenciação Celular , Fígado/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Transplante Heterólogo , Albuminas/análise , Animais , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco/química , Células-Tronco/fisiologiaRESUMO
We have used HeLa cells without mitochondrial DNA (rho0-cells) and transient rho0-phenocopies, obtained from wild-type cells by short-term treatment with ethidium bromide, to analyze how the absence of a functional mitochondrial respiratory chain slows down proliferation. We ruled out an energetic problem (ATP/ADP content) as well as defective synthesis of pyrimidine, iron-sulfur clusters or heme as important causes for the proliferative defect. Flow cytometric analysis revealed that reactive oxygen species were reduced in rho0-cells and in rho0-phenocopies, and that, quite unusually, all stages of the cell cycle were slowed down. Specific quenching of mitochondrial ROS with the ubiquinone analog MitoQ also resulted in slower growth. Some important cell-cycle regulators were reduced in rho0-cells: cyclin D3, cdk6, p18INK4C, p27KIP1, and p21CIP1/WAF1. In the rho0-phenocopies, the expression pattern did not fully duplicate the complex response observed in rho0-cells, and mainly p21CIP1/WAF1 was downregulated. Activities of the growth regulatory PKB/Akt and MAPK/ERK-signaling pathways did not correlate with proliferation rates of rho0-cells and rho0-phenocopies. Our study demonstrates that loss of a functional mitochondrial electron transport chain inhibits cell-cycle progression, and we postulate that this occurs through the decreased concentration of reactive oxygen species, leading to downregulation of p21CIP1/WAF1.
Assuntos
Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Doenças Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Crescimento Celular , Proliferação de Células , Regulação para Baixo , Etídio/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Heme/biossíntese , Humanos , Doenças Mitocondriais/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Uridina/farmacologia , Uridina Trifosfato/metabolismoRESUMO
Infection with high-risk human papillomaviruses (HPV) can lead to the development of cervical carcinomas. This process critically depends on the virus-encoded E6 and E7 oncoproteins, which stimulate proliferation by manipulating the function of a variety of host key regulatory proteins. Here we show that both viral proteins dose-dependently interfere with the transcriptional activity of NF-kappaB. A variety of experimental approaches revealed that a fraction of the E7 proteins is found in association with the IkappaB kinase complex and attenuates induced kinase activity of IkappaB kinase alpha (IKKalpha) and IKKbeta, thus resulting in impaired IkappaBalpha phosphorylation and degradation. Indirect immunofluorescence shows that E7 impairs TNFalpha-induced nuclear translocation of NF-kappaB, thus preventing NF-kappaB from binding to its cognate DNA. While E7 obviates IKK activation in the cytoplasm, the E6 protein reduces NF-kappaB p65-dependent transcriptional activity within the nucleus. We suggest that HPV oncogene-mediated suppression of NF-kappaB activity contributes to HPV escape from the immune system.
Assuntos
NF-kappa B/metabolismo , Proteínas Oncogênicas Virais/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Quinase I-kappa B , NF-kappa B/fisiologia , Transporte Proteico , Transcrição Gênica/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Epstein-Barr virus (EBV) transforms primary B cells in vitro. Established cell lines adopt a lymphoblastoid phenotype (LCL). In contrast, EBV-positive Burkitt's lymphoma (BL) cells, in which the proto-oncogene c-myc is constitutively activated, do not express a lymphoblastoid phenotype in vivo. The two different phenotypes are paralleled by two distinct programmes of EBV latent gene expression termed latency type I in BL cells and type III in LCL. Human B cell lines were established from a conditional LCL (EREB2-5) by overexpression of c-myc and inactivation of EBV nuclear protein 2 (EBNA2). These cells (A1 and P493-6) adopted a BL phenotype in the absence of EBNA2. However, the EBV latency I promoter Qp was not activated. Instead, the latency III promoter Cp remained active. These data suggest that the induction of a BL phenotype by overexpression of c-myc in an LCL is not necessarily paralleled by an EBV latency III-to-I switch.