RESUMEN
Cell therapy using induced pluripotent stem cell (iPSC) derivatives may result in abnormal tissue generation because the cells undergo numerous cycles of mitosis before clinical application, potentially increasing the accumulation of genetic abnormalities. Therefore, genetic tests may predict abnormal tissue formation after transplantation. Here, we administered iPSC derivatives with or without single-nucleotide variants (SNVs) and deletions in cancer-related genes with various genomic copy number variant (CNV) profiles into immunodeficient mice and examined the relationships between mutations and abnormal tissue formation after transplantation. No positive correlations were found between the presence of SNVs/deletions and the formation of abnormal tissues; the overall predictivity was 29%. However, a copy number higher than 3 was correlated, with an overall predictivity of 86%. Furthermore, we found CNV hotspots at 14q32.33 and 17q12 loci. Thus, CNV analysis may predict abnormal tissue formation after transplantation of iPSC derivatives and reduce the number of tumorigenicity tests.
Asunto(s)
Células Madre Pluripotentes Inducidas , Animales , Pruebas de Carcinogenicidad , Reprogramación Celular , Variaciones en el Número de Copia de ADN , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Mutación , Polimorfismo de Nucleótido SimpleRESUMEN
Kynurenine, which is generated from tryptophan by indoleamine 2,3-dioxygenase 1 (IDO1), binds to the aryl hydrocarbon receptor (AhR). Here, we report that kynurenine was produced by undifferentiated human embryonic stem cells (hESCs) and by induced pluripotent stem cells (iPSCs). In undifferentiated hESCs, kynurenine stimulated the AhR to promote the expression of self-renewal genes. The kynurenine-AhR complex also stimulated the expression of IDO1 and AHR, activating a positive feedback loop. Inhibition of IDO1 activity reduced the proliferation of undifferentiated ESCs but did not stimulate their differentiation. Substantial amounts of free kynurenine were present in the culture medium, providing a paracrine signal for maintenance of the undifferentiated state. Kynurenine was not present in the medium of differentiated ESCs or iPSCs. When ESCs were induced to undergo ectodermal differentiation, the abundance of kynurenine in the medium was reduced through activation of the main kynurenine catabolic pathway mediated by kynurenine aminotransferase 2 (KAT2, also known as AADAT), resulting in the secretion of 2-aminoadipic acid (2-AAA) into the culture medium. Inhibition of KAT2 activity blocked ectodermal differentiation. Thus, kynurenine metabolism plays an important role in the maintenance of the undifferentiated state and in ectodermal differentiation. Furthermore, kynurenine in the culture medium is a biomarker for the undifferentiated state, whereas the presence of 2-AAA in the culture medium is a biomarker of ESCs and iPSCs that have committed to differentiate along the ectoderm lineage.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Quinurenina/metabolismo , Receptores de Hidrocarburo de Aril/biosíntesis , Transducción de Señal , Diferenciación Celular , Línea Celular , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
Embryonic Stem Cells (ESC) possesses two distinct features; self-renewal and the potential to differentiate. Here we show the differentiation potential and growth rate of ESC correlates positively with the expression level of the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). When ESCs are maintained in feeder-free conditions and single cell seeding, ESC KhES-1 having 4520 copies or more of CHD7 in 5 ng total RNA show differentiation potential, but this is lost when the CHD7 copy number is reduced in KhES-1 to less than 696 by alternative culture conditions. Introduction of siCHD7 reduced differentiation potential and growth rate of KhES-1. Interestingly, KhES-1 underwent spontaneous differentiation when mCHD7 was introduced and we could not obtain CHD7-overexpressing ESC in culture. These data suggest that CHD7 drives differentiation, and there is a lower limit for CHD7 to initiate differentiation and an upper limit for CHD7 if maintained in undifferentiated state, and such upper limit varies depending on culture condition. As CHD7 drives cell growth, ESC with the highest permissible CHD7 level in the given culture become dominant in a couple of passages. Thus, we can select differentiation resistance-free cell clones by optimizing the culture system using CHD7 as an index.
Asunto(s)
Diferenciación Celular/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Proliferación Celular , Células Cultivadas , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed "patterned culture"), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed "non-patterned cultures"). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.
Asunto(s)
Técnicas de Cultivo de Célula , Células Madre Pluripotentes/citología , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Línea Celular , Proliferación Celular , Autorrenovación de las Células , Criopreservación , Medios de Cultivo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Matriz Extracelular/metabolismo , Células Nutrientes , Inestabilidad Genómica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipificación , Células Madre Pluripotentes/metabolismoRESUMEN
Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional -80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application.
Asunto(s)
Criopreservación/métodos , Crioprotectores/química , Dimetilsulfóxido/química , Glicol de Etileno/química , Derivados de Hidroxietil Almidón/química , Células Madre Pluripotentes/citología , Fosfatasa Alcalina/química , Diferenciación Celular , Ácido Edético/química , Citometría de Flujo , Congelación , Humanos , Cariotipificación , Temperatura , VitrificaciónRESUMEN
CD34+ cord blood cells can be reprogrammed effectively on dishes coated with a synthetic RGD motif polymer (PronectinF®) using a temperature sensitive Sendai virus vector (SeV TS7) carrying reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC. Dish-shaped human ES cell-like colonies emerged in serum-free primate ES cell medium (supplemented with bFGF) in 20% O2 culture conditions. The copy numbers of SeV TS7 vectors in the cytoplasm were drastically reduced by a temperature shift at 38°C for three days. Then, single cells from colonies were seeded on PronectinF®-coated 96-well plates and cultured under naïve culture conditions (N2B27-based medium supplemented with LIF, forskolin, a MAPK inhibitor, and a GSK inhibitor in 5% O2) for cloning purpose. Dome-shaped mouse ES cell-like colonies from single cells emerged on PronectinF®-coated dishes. These cells were collected and cultured again in primate ES cell medium supplemented with bFGF in 20% O2 and maintained on PronectinF®-coated dishes. Cells were assessed for reprogramming, including the absence of residual SeV and their potential for three germ layer differentiation. Generation of virus-free induced pluripotent stem cell (iPSC) clones from single cells under feeder-free conditions will solve some of the safety concerns related to use of xeno- or allogeneic-material in culture, and contribute to the characterization and the standardization of iPS cells intended for use in a clinical setting.
Asunto(s)
Células Clonales/citología , Células Madre Pluripotentes/citología , Animales , Células Cultivadas , Células Clonales/inmunología , Medios de Cultivo , Vectores Genéticos , Humanos , Cariotipificación , Factor 4 Similar a Kruppel , Ratones , Células Madre Pluripotentes/inmunología , Primates , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus Sendai/genéticaRESUMEN
Human leukemia inhibitory factor (LIF) was immobilized into insect virus-derived microcrystals (polyhedra) to generate LIF polyhedra (LIF-PH) that can slowly release LIF into embryonic stem (ES) cell culture media and thus maintain ES cells in an undifferentiated state. Assays of the biological activities of LIF-PH indicated that a single addition of LIF-PH to the ES cell culture medium can support the proliferation of mouse ES and induced pluripotent stem (iPS) cells continuously for 14 days, and suggest that LIF-PH can be successfully used in the place of a periodic addition of recombinant LIF to the media every 2-3 days. The release of LIF protein from LIF-PH was determined by enzyme-linked immunosorbent assay (ELISA). Maintenance of undifferentiated state of mouse ES and iPS cells cultured with LIF-PH was determined by the detection of pluripotency-related biomarkers Oct3/4 and stage-specific embryonic antigen-1 (SSEA-1) through immunostaining and measurement of alkaline phosphatase activity. In this paper, we propose a closed culture system for mass production of ES and iPS cells that utilize a slow-releasing agent of LIF.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Factor Inhibidor de Leucemia/farmacología , Animales , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Factor Inhibidor de Leucemia/química , Ratones , Factor de Transcripción STAT3/metabolismoRESUMEN
We report that iPS cells can be safely and effectively generated from fresh human cord blood (CB) cells with Sendai virus (SeV) vector carrying reprogramming factors OCT3/4, SOX2, KLF4, and c-MYC. The SeV vector is a single strand RNA virus having no DNA phase, and selectively infects the freshly isolated CD34+ CD45low+ fraction of CB cells corresponding to hematopoietic progenitors. Approximately twenty ES cell-like colonies emerged from 1 x 104 freshly isolated CD34+ CB cells around 18 days after SeV infection and were selected for passage to reduce the frequency of the remaining SeV-infected cells. The complete elimination of viral constructs was confirmed after several passages by immunostaining with monoclonal antibody against hemagglutinin-neuraminidase (HN) and by RT-PCR analysis. Five ES cell-like clones were selected to examine their in vitro potential for three germ layer differentiation and their capacity for teratoma formation. Generation of non-integrating Sendai virus (SeV) iPS cells from CB cells may be an important step to provide allogeneic iPS cell-derived therapy in the future.
Asunto(s)
Sangre Fetal/citología , Células Madre Hematopoyéticas/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Proliferación Celular , Separación Celular , Células Cultivadas , Reprogramación Celular , Técnicas de Cocultivo , Células Nutrientes , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Vectores Genéticos , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipificación , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Virus Sendai/genética , Factores de Tiempo , TransfecciónRESUMEN
Expression of the Wnt modulator secreted frizzled related protein 4 (Sfrp4) is upregulated after heart ischemic injury. We show that intramuscular administration of recombinant Sfrp4 to rat heart ischemic injury and recanalization models prevents further deterioration of cardiac function after the ischemic injury. The effect of Sfrp4 persisted for at least 20 weeks when Sfrp4 was administered in a slow release system (Sfrp4-polyhedra) to both acute and subacute ischemic models. The histology of the dissected heart showed that the cardiac wall was thicker and the area of acellular scarring was smaller in Sfrp4-treated hearts than in controls. Increased amounts of both the inactive serine 9-phosphorylated form of glycogen synthase kinase (GSK)-3ß and the active form of ß-catenin were observed by immunohistology 3 days after lateral anterior descendant ligation in control, but not in Sfrp4-treated hearts. All together, we show that administration of Sfrp4 interferes with canonical Wnt signaling that could mediate the formation of acellular scar and consequently contributes to the prevention of aggravation of cardiac function.
Asunto(s)
Pruebas de Función Cardíaca , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/fisiopatología , Proteínas Proto-Oncogénicas/uso terapéutico , Animales , Materiales Biocompatibles/farmacología , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Fibrosis , Humanos , Inyecciones Intramusculares , Masculino , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Proteínas Proto-Oncogénicas/administración & dosificación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
OBJECTIVE: Cord blood banks provide fully human leukocyte antigen-typed cells, from which a set of standard induced pluripotent stem (iPS) cells for use in allogenic transplantation can be derived. Hence, the ability to generate iPS cells from cord blood cells has the potential to provide a suitable source for clinical transplantation. The aim of this work is to determine the reprogramming methods, culture conditions, and cell fractions that can be used to generate iPS cells from cord blood cells effectively. MATERIALS AND METHODS: CD34(+), mononucleated, and derived adherent cells from cord blood were cultured in hematopoietic medium (X-vivo10 containing 50 ng/mL interleukin-6, 50 ng/mL soluble interleukin-6 receptor, 50 ng/mL stem cell factor, 10 ng/mL thrombopoietin, and 20 ng/mL Flit3/4 ligand) 3 days prior to viral infection. Cells were then infected with retroviral constructs driving the expression of OCT3/4, SOX2, Krüppel-like factor 4, c-MYC, and enhanced green fluorescent protein together with or without the p53 knockdown lentiviral construct Shp53 pLKO.1-puro. Infected cells were then cultured for an additional 4 days in hematopoietic culture medium before being transferred onto mouse embryonic fibroblast (MEF) or SNL76/7 feeder cells in human embryonic stem cell medium (Dulbecco's modified Eagle medium/F-12 containing 20% knockout serum replacement, 200 mM L-glutamine, 1% non-essential amino acids (NEAA), 0.1 mM 2-mercaptoethanol, and 4 ng/mL basic fibroblast growth factor). Subsequently, the number of embryonic stem cell-like colonies that emerged in the following 4 weeks was scored. Expression of a number of pluripotency makers were examined by immunochemistry and reverse transcriptase polymerase chain reaction. Finally, the differentiation potential of selected colonies was determined by teratoma formation in severe combined immunodeficient mice and in vitro culture. RESULTS: Repression of p53 expression by the addition of a lentiviral p53 short-hairpin RNA expression vector increased the frequency of formation of iPS-like colonies from 1 (on average) to around 100 per 2 x 10(4) cells when infected cells were grown on SNL feeder cells. CONCLUSIONS: iPS cells can be generated easily from CD34(+) cord blood cells through the addition of p53 inhibition to standard reprogramming conditions.
Asunto(s)
Antígenos CD34/análisis , Sangre Fetal/citología , Células Madre Pluripotentes/citología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Diferenciación Celular , Células Cultivadas , Medios de Cultivo , Embrión de Mamíferos , Células Madre Embrionarias/citología , Fibroblastos , Expresión Génica , Técnicas de Transferencia de Gen , Genes myc/genética , Genes p53/genética , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Humanos , Secuencias Invertidas Repetidas , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Lentivirus/genética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , ARN/genética , Retroviridae/genética , Factores de Transcripción SOXB1/genéticaRESUMEN
Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 alpha-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast growth factor-2 (FGF-2), FGF-7 and epidermal growth factor (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. Growth factors produced by both methods were functional, inducing the growth of fibroblast cells and keratinocytes. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Citocinas/metabolismo , Proteínas Inmovilizadas/metabolismo , Reoviridae/química , Proteínas Virales/química , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 7 de Crecimiento de Fibroblastos/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Confocal , Células 3T3 NIH , Estructura Secundaria de Proteína , Rayos Ultravioleta , Proteínas Virales/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. The cells have already been applied in various clinical situations because of their expansion and differentiation capabilities. The cells lose their capabilities after several passages, however. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 or Nanog gene into the cells. Sox2 and Nanog are not only essential for pluripotency and self-renewal of embryonic stem cells, but also expressed in somatic stem cells that have superior expansion and differentiation potentials. We found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. Significantly, in the presence of bFGF in culture media, most of the Sox2-expressing cells were small, whereas the control cells were elongated in shape. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. These results demonstrate not only an effective way to maintain proliferation and differentiation potentials of MSCs but also an important implication about the function of bFGF for self-renewal of stem cells including MSCs.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Células Madre Mesenquimatosas/citología , Médula Ósea , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas HMGB/genética , Proteínas HMGB/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Proteína Homeótica Nanog , Osteogénesis , Factores de Transcripción SOXB1 , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transducción GenéticaRESUMEN
Mesenchymal stromal cells (MSCs) derived from human bone marrow are expected to be utilized for the purpose of tissue engineering, because of their extensive self-renewal or proliferation capability. The capability decreases after several passages, however. Basic fibroblast growth factor (bFGF) is commonly used for culture of various cells including bone marrow-derived MSCs. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the bFGF gene into the passaged cells by retroviral system. The bFGF-expressing MSCs, even at 7 to 8 passages after the infection, showed consistent proliferation capability. The capability was not detected in control cells even in culture media containing the bFGF protein. Thus, we could not mimic the effect of forced expression of bFGF by exogenously adding the bFGF protein in culture media. Although we expressed the shortest isoform of bFGF, which was considered to be mostly cytosolic, we found the protein mostly in the nucleus. Our observations demonstrate not only an effective way to maintain proliferation potentials of MSCs, but also a possibility that there may be mechanistic and functional differences in the signal transduction events between endogenously expressed and exogenously added bFGF protein in MSCs.
Asunto(s)
Células de la Médula Ósea/citología , Factor 2 de Crecimiento de Fibroblastos/genética , Mesodermo/citología , Adulto , Anciano de 80 o más Años , Línea Celular , Proliferación Celular , Niño , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Humanos , Masculino , Retroviridae/genética , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , TransgenesRESUMEN
ABCA5 is a member of the ABC transporter A subfamily, and a mouse orthologue (mABCA5) in newborn mouse brain and neural cells was identified by reverse transcription-PCR. Full-length cDNA cloning revealed that mABCA5 consists of 1,642 amino acid residues and that its putative structure is that of a full-type ABC transporter having two sets of six transmembrane segments and a nucleotide binding domain. Immunohistochemical studies revealed that mABCA5 is expressed in brain, lung, heart, and thyroid gland. A subcellular localization analysis showed that mABCA5 is a resident of lysosomes and late endosomes. Abca5(-)(/)(-) mice exhibited symptoms similar to those of several lysosomal diseases in heart, although no prominent abnormalities were found in brain or lung. They developed a dilated cardiomyopathy-like heart after reaching adulthood and died due to depression of the cardiovascular system. In addition, Abca5(-)(/)(-) mice also exhibited exophthalmos and collapse of the thyroid gland. Therefore, ABCA5 is a protein related to a lysosomal disease and plays important roles, especially in cardiomyocytes and follicular cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Lisosomas/metabolismo , Lisosomas/patología , Transportadoras de Casetes de Unión a ATP/química , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Exoftalmia/genética , Exoftalmia/metabolismo , Exoftalmia/patología , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Miocardio/metabolismo , Miocardio/patología , Transporte de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándula Tiroides/metabolismo , Hormonas Tiroideas/metabolismoRESUMEN
PURPOSE: Telomerase activity (TA) has been extensively studied in tumors of many organs, but not the parathyroid gland. Therefore, we investigated TA in parathyroid tumors, and examined the mRNA expression of its catalytic subunits. METHODS: We examined 17 single adenomas, one hyperplastic parathyroid gland, and one metastatic parathyroid cancer and quantified TA by fluorescence-based TRAP analysis. We also studied the expression of telomerase catalytic subunits in nine adenomas and one cancer by reverse transcription - polymerase chain reaction (RT-PCR) analysis. RESULTS: Telomerase activity was not detected in any of the adenomas or the hyperplastic gland; however, the metastatic cancer was highly positive for TA. Both human telomerase RNA ( hTR) and human telomerase-associated pretein (hTEP-1) mRNA were detected universally in every specimen tested. Conversely, human telomerase reverse transcriptase (hTERT) mRNA was not detected by the conventional electrophoresis-based technique. Faint expression of hTERT mRNA was detected by real-time RT-PCR only in the sample of parathyroid cancer. DISCUSSION: We hypothesize that TA plays a role in the malignant transformation of parathyroid disease and suggest that hTERT mRNA expression could be the key step for TA as in other malignancies. Telomerase activity and hTERT may be useful molecular targets of parathyroid cancer.
Asunto(s)
Hiperparatiroidismo/enzimología , Neoplasias de las Paratiroides/enzimología , Telomerasa/biosíntesis , Telomerasa/metabolismo , Adenoma/enzimología , Adulto , Anciano , Dominio Catalítico , Proteínas de Unión al ADN , Femenino , Humanos , Hiperplasia/enzimología , Masculino , Persona de Mediana Edad , Glándulas Paratiroides/patología , ARN/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/genéticaRESUMEN
Anaplastic thyroid carcinoma is one of the most aggressive human malignancies. Outcomes of intensive multimodal therapy have been far from satisfactory. Furthermore, p53 gene dysfunction, often found in this type of cancer, is known to impair the efficacy of the therapeutic agents. Specific ligands for peroxisome proliferator activated receptor gamma (PPAR-gamma) induce growth suppression in some tumor cells. In this study, we investigated the role of PPAR-gamma in anaplastic thyroid cancer cell lines (OCUT-1, ACT-1). PPAR-gamma was expressed and functional in both cell lines. Activation of PPAR-gamma with its specific ligands, troglitazone and 15-deoxy-delta 12,14-prostaglandin J2, inhibited cell growth in a dose-dependent manner through inducing G1 cell cycle arrest. P53 protein expression differed in OCUT-1 and in ACT-1, though the levels stayed constant irrespective of ligand exposure in both cell lines. In contrast, p21 and p27 proteins were induced in a dose-dependent manner in both situations. This study showed that PPAR-gamma ligands were able to induce growth suppression in anaplastic thyroid cancer cells via a p53-independent, but p21- and p27-dependent cytostatic pathway. These tumor-suppressive effects of PPAR-gamma may provide a novel approach to the treatment of anaplastic thyroid cancer.
Asunto(s)
Proteínas Musculares , Receptores Citoplasmáticos y Nucleares/fisiología , Neoplasias de la Tiroides/patología , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/fisiología , Ciclo Celular , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/análisis , Humanos , Proteínas de Microfilamentos/análisis , Mutación , Receptores Citoplasmáticos y Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/análisis , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/análisisRESUMEN
Intraductal papillary-mucinous carcinoma (IPMC) of the pancreas, a new entity of pancreatic cancer with a favorable prognosis, has shown a gradual increase in the number of reported cases. Patients with high-frequency microsatellite instability (MSI-H) tumors have been shown to survive longer than those with low-frequency MSI (MSI-L) or microsatellite stable (MSS) tumors in colorectal and gastric cancer. We investigated whether MSI-H in patients with IPMC can contribute to a good prognosis. The formalin-fixed paraffin-embedded tumors and surrounding normal pancreatic tissues from 10 patients with IPMCs and 16 with intraductal papillary-mucinous adenomas (IPMAs) were provided for DNA extraction after microdissection. Polymerase chain reaction (PCR) was carried out using 8 microsatellite primer marker sets. The mixed PCR samples were analyzed using a genetic analyzer. MSI-H was determined by assessment of microsatellite variations in 3 or more of the 8 tested markers. Immunohistochemical staining of the MSI-responsible proteins hMLH1 and hMSH2 was conducted for both the IPMC and IPMA samples. Ten percent of IPMC harbored MSI-H tumors, whereas no MSI-H tumors were detected in the IPMAs. Thirty percent of IPMC tumors and 25% of IPMA tumors showed MSI-L. All IPMCs and IPMAs showed normal expression of both hMLH1 and hMSH2. MSI-H and loss of hMLH1 and hMSH2 are very rare events in both IPMCs and IPMAs. We conclude that a good prognosis for patients with IPMC is not associated with MSI-H.
Asunto(s)
Adenocarcinoma Mucinoso/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Papilar/genética , Repeticiones de Microsatélite , Neoplasias Pancreáticas/genética , Proteínas Adaptadoras Transductoras de Señales , Anciano , Proteínas Portadoras , Proteínas de Unión al ADN/genética , Femenino , Genes p16 , Genes p53 , Humanos , Inmunohistoquímica , Incidencia , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteína Smad4 , Transactivadores/genéticaRESUMEN
We investigated the adenoma-carcinoma sequence in intraductal papillary-mucinous neoplasm from the aspect of genetic changes. The formalin-fixed paraffin-embedded tumors and surrounding normal pancreatic tissues from patients with 16 intraductal papillary-mucinous adenoma of the pancreas (IPMA) and 10 intraductal papillary-mucinous carcinoma of the pancreas (IPMC) were provided for DNA extraction after microdissection. SSCP-DNA sequencing analysis demonstrated K-ras mutations at codon 12 in 75% of IPMA and 70% of IPMC, while those at codon 13 were observed neither in IPMA nor IPMC. There were no characteristic K-ras mutation types in IPMA and IPMC and no significant differences in incidence of K-ras mutations between the two categories. The frequencies of p53 mutations analyzed by SSCP-DNA sequencing were not high in IPMA (18.8%) and IPMC (30%), showing no significant difference between them. LOHs of APC in IPMA and IPMC were infrequent (6.3 and 20%, respectively) and showed no significant difference in incidence between the two categories. The LOH frequencies of DCC in IPMA and IPMC were 31.3 and 40%, respectively, and were not statistically different from each other. Taken together, genetic changes such as K-ras, p53, APC and DCC mutations may not be associated with adenoma-carcinoma sequence in intraductal papillary-mucinous neoplasm of pancreas.
Asunto(s)
Adenocarcinoma Mucinoso/genética , Adenoma/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Papilar/genética , Mutación , Neoplasias Pancreáticas/genética , Anciano , Femenino , Genes APC , Genes DCC , Genes p53 , Genes ras , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND & OBJECTIVES: Undifferentiated thyroid cancer is one of the most aggressive human malignancies, and the prognoses that have been reported are extremely poor. A number of studies have described the clinicopathologic features of this tumor and analyzed its biologic background to explain the extraordinarily aggressive nature of the tumor. Still the mechanism of cellular aggressiveness in undifferentiated thyroid cancer is not yet fully understood. METHODS: We established and characterized an undifferentiated thyroid cancer cell line, OCUT-1, derived from the surgical specimen obtained from a 74-year-old Japanese woman with advanced undifferentiated thyroid cancer. RESULTS: The cell line had already maintained over 100 passages and was stably cultured for more than a year. The cell line was observed to maintain not only its morphologic similarity to the primary cancer cells, but also its aggressive nature, including high proliferative activity, numerous genetic abnormalities, and cytokine production. Further, we have demonstrated the expression of telomerase activity in the cell, which could represent one of the responsible mechanisms for the cellular aggressiveness of this type of tumor. CONCLUSIONS: This cell line might be useful for further study concerning the correlation between telomerase activation and disease progression or anaplastic change in thyroid cancer.