Los autores desean agradecer a Sanjiv Gambhir por el don del gen reportero fluc/mrfp/sr39tk. Este trabajo fue financiado por la Red de Células Madre (SCN) de Canadá, y la Fundación de Jesse Journey.

1. Mantenimiento y Propagación de mioblastos C2C12 <ol><li> Placa de mioblastos C2C12 en un matraz de 75 cm 2 y mantener las células en el suero de alta glucosa Dulbecco Modificado de Eagle (HG-DMEM) suplementado con suero fetal bovino (FBS) a una concentración final de 10%. No permitir que las células se hacen confluentes en cualquier momento ya que esto agotar la población myoblastic. Medio debe ser cambiado cada otro día Nota:. Medio siempre caliente a 37 ° C en un baño de agua antes de su uso. </li><li> Cuando mioblastos ser aproximadamente 80% confluentes, las células de paso a un nuevo matraz. Aspirar el medio de cultivo. Lavar las células con 4-5 ml de solución salina balanceada de Hanks (HBSS) para eliminar todos los restos de medio de cultivo que contiene tripsina inhibidor de suero. Brevemente enjuague la capa de células con 2-3 ml de 0,25% (w / v) de tripsina-EDTA solución para disociar los mioblastos adherentes del matraz. Aspirar tripsina y el matraz lugar en incubadora a 37 ° C durante 5 min. </li><li> Durante esta incubaciónción, preparar un nuevo matraz con 9 ml de medio de HG-DMEM/10% de FBS. Después de tripsinización, añadir 10 ml de medio completo a las células y los tiempos de pipeta 4-5 para garantizar la recogida de todas las células en el medio. Añadir 1 ml de suspensión de células en el matraz de nuevo y se incuban en 5% de CO 2 a 37 ° C. </li></ol> 2. La transfección celular C2C12 <ol><li> Una vez que las células han alcanzado el 50% de confluencia, transfectar de acuerdo con las instrucciones del fabricante de Invitrogen. En pocas palabras, se combinan 90 l Lipofectamine 2000 reactivo con 4,5 ml de OPTI-MEM medio. En otro tubo, se combinan 36 g CMV-trifusion ADN del gen reportero con 4,5 ml de OPTI-MEM. Flick cada tubo para combinar y esperar 5 min. Combinar el contenido de ambos tubos, mezclar suavemente y se incuba a temperatura ambiente durante exactamente treinta minutos Nota:. Un matraz de segunda también se debe configurar para que sólo las células no transfectadas recibir Lipofectamine y OPTI-MEM para servir como un control negativo. </li><li> Eliminar el medio de las células C2C12 y añadir 15,5ml de medio fresco HG-DMEM/10% de SFB. Añadir medio de transfección para llevar el volumen total a 20 ml. </li><li> Permitir que las células a transfectar durante la noche durante al menos 20 horas a 37 ° C. </li><li> Al día siguiente, se aspira el medio de transfección y se añaden 10 ml HG-DMEM/10% de FBS. </li><li> Ver las células bajo un microscopio de fluorescencia invertido. Permite capturar tanto de campo claro y rojo imágenes de fluorescencia (utilizando un cubo de filtro TRITC; mRFP ex / em: 584/607 nm). Contar el número de células que expresan RFP observe bajo fluorescencia dividido por el número total de células observar bajo campo claro para múltiples campos de visión para mayor eficacia de transfección. </li></ol> 3. Evaluación de la supervivencia celular / MTT ensayo <ol><li> Un día antes de la transfección, la placa de 1x10 5 células C2C12 en cada pocillo en una placa de 24 pocillos. Las células deben ser plateado en volúmenes de 500 l en FBS HG-DMEM/10%. </li><li> Al día siguiente, los mioblastos transfectar durante la noche como se ha descrito previamente. Siga las sugerencias del fabricante para la transfección reagenvolúmenes t para una placa de 24 pocillos. Incubar un conjunto de pocillos con Lipofectamine sólo (sin ADN) como control. Ver células bajo fluorescencia para asegurar que la transfección adecuada se ha producido. </li><li> Retirar el medio de transfección y se incuban con mioblastos 5 mg / ml de azul de tiazolilo tetrazolio (MTT) en FBS HG-DMEM/10%. Añadir D-luciferina a ocho de los pozos transfectadas, y se incuba a 37 ° C durante cuatro horas. </li><li> Retire medio de pozos. Solubilizar azul cristales de formazán mediante la adición de 180 l de isopropanol a cada pocillo. Agitar a 37 ° C durante 15 min. </li><li> Evitando cualquier precipitado, la solución de la pipeta en una placa de 96 pocillos y se leyó la absorbancia a 575 nm. </li></ol> 4. Preparación de mioblastos para Trasplante <ol><li> Retire medio, lavar con HBSS mioblastos y células Tripsinizar como se indica en el punto 1.1. </li><li> Volver a suspender las células en 4 ml de medio completo. </li><li> El uso de un hemocitómetro, contar las células para generar volúmenes que contienen 10 6 mioblastos. Pipetear volmen en microtubos estériles 1,5 ml. Con una eficacia de transfección de ~ 10%, estos valores indican que 100.000 células que expresan luciferasa son detectables en el escáner GE ExploreOptix después del trasplante. </li><li> Alcanzar un volumen de inyección final de 15 l, que contiene 10 6 células C2C12. Si es necesario, microtubos de centrífuga a 2.000 rpm durante un minuto. Aspirar cuidadosamente el sobrenadante con una pipeta. Vuelva a suspender las células en 15 l de DMEM carente HG-FBS. </li></ol> 5. Implantación Celular <ol><li> Anestesie ratón con un 2% de isoflurano / 2% O 2. Pluck pelo de la zona extremidad posterior dorsal. Mantenga la anestesia con isoflurano al 1,5% / 2% O 2. </li><li> Asegurar C2C12 mioblastos son bien suspendido. Con el ratón en una posición de decúbito prono, extender la extremidad posterior y el uso de una jeringa de insulina a inyectar células directamente en el lateral de la cabeza del músculo gastrocnemio en un ángulo de 30 °. Inyectar mioblastos transfectados en la extremidad posterior derecha y int mioblastos sin transfectaro el contralateral (izquierda) miembro posterior. </li><li> Realizar un análisis de bioluminiscencia línea de base: Transfiera rápidamente ratón a la etapa en el escáner óptico, por el que se el ratón en una posición boca abajo. Conecte la línea de anestésico. Para asegurar el ratón permanece anestesiado, una segunda persona debe estar presente para conectar la línea de anestésico mientras que los otros lugares que el animal en el escáner. </li><li> Suavemente extender la extremidad posterior de modo que el área de inyección es visible. Cinta traseras piernas en posición con un adhesivo suave, como la cinta médica. </li><li> Cerrar la cámara y asegurarse de que la luz no puede tener acceso al interior del escáner, ya que esto aumentará la señal de fondo detectada. El escáner debe ajustarse a los parámetros incluidos en las instrucciones de su fabricante para obtener imágenes de bioluminiscencia. En concreto, asegúrese de &quot;no láser&quot; está seleccionado. </li><li> Dibuje una región de interés (ROI) en la zona de desplumado y exploración inicial. </li></ol> 6. La inyección de sustrato Fluc, D-luciferina, en ratón mdx &lt;ol&gt; <li> Mientras que el ratón es anestesiaron intraperitonealmente inyectar 150 mg / kg de sustrato de luciferasa de luciérnaga, D-luciferina (de una 40 mg / ml de solución madre, compuesto de acuerdo con las instrucciones del fabricante). </li><li> Recuperar ratón y permitir un período de 15 minutos antes de preparar la captación de ratón para el siguiente ciclo. </li></ol> 7. BLI a Target Luc-expresando PSM después del implante en modelos de ratón de DMD <ol><li> Después de que el período de absorción, anestesiar ratón de nuevo, como se describe en 5,1. </li><li> Transferir el ratón de nuevo al dispositivo de lectura óptica y realice otro escaneo de bioluminiscencia de la misma manera como la exploración de fondo. </li><li> Aunque un período de 20 min la absorción debe proporcionar una intensidad de señal máxima, un posterior análisis puede ser realizado. </li><li> Una vez completada la adquisición de imágenes, sacrifica el ratón de acuerdo con las directrices establecidas por el Comité de Ética Institucional Animal y el Consejo Canadiense de Protección de los Animales (CCPA). Aislar el músculo extremidades posteriores y el lugar inmediatamente in 10% de formalina para arreglar para empotramiento parafina. Realizar tinción inmunohistoquímica para la luciferasa para confirmar inyección intramuscular de mioblastos. </li></ol>

<ol>
	<li>Emery, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+muscular+dystrophies.">The muscular dystrophies.</a> Lancet. 359, 687-695 (2002).</li><li>Blake, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+and+genetics+of+dystrophin+and+dystrophin-related+proteins+in+muscle.">Function and genetics of dystrophin and dystrophin-related proteins in muscle.</a> Physiol. Rev. 82, 291-329 (2002).</li><li>Goldring, K., Partridge, T., Watt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> J. Pathol. 197, 457-467 (2002).</li><li>Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+that+participate+in+regeneration+of+skeletal+muscle.">Cells that participate in regeneration of skeletal muscle.</a> Gene Ther. 9, 752-753 (2002).</li><li>Moss, F. P., Leblond, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+as+the+source+of+nuclei+in+muscles+of+growing+rats.">Satellite cells as the source of nuclei in muscles of growing rats.</a> Anat. Rec. 170, 421-435 (1971).</li><li>Snow, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autoradiographic+study+of+satellite+cell+differentiation+into+regenerating+myotubes+following+transplantation+of+muscles+in+young+rats.">An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats.</a> Cell Tissue Res. 186, 535-540 (1978).</li><li>Kuang, S., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+biology+of+satellite+cells+and+their+therapeutic+potential.">The emerging biology of satellite cells and their therapeutic potential.</a> Trends Mol. Med. 14 (2), 82 (2008).</li><li>Hoffman, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+dystrophin+in+muscle-biopsy+specimens+from+patients+with+Duchenne's+or+Becker's+muscular+dystrophy.">Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy.</a> N. Engl. J. Med. 318, 1363-1368 (1988).</li><li>Ray, P., Tsien, R., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+validation+of+improved+triple+fusion+reporter+gene+vectors+for+molecular+imaging+of+living+subjects.">Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects.</a> Cancer Res. 67 (7), 3085 (2007).</li><li>Ray, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+lights.">Molecular imaging in living subjects: seeing fundamental biological processes in a new lights.</a> Genes Dev. 17, 545-580 (2003).</li><li>Sicinski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+muscular+dystrophy+in+the+mdx+mouse:+a+point+mutation.">The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.</a> Science. 244, 1578-1580 (1989).</li><li>Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathogenesis of neurodegenerative disorders. In: Contemporary neuroscience. X, 294 (2001).</li><li>Andersson, J. E., Bressler, B. H., Ovalle, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+regeneration+in+hind+limb+skeletal+muscle+of+the+mdx+mouse.">Functional regeneration in hind limb skeletal muscle of the mdx mouse.</a> J. Muscle Res. Cell Motil. 9, 499-515 (1988).</li></ol>

Los autores no tienen nada que revelar.

En este estudio, hemos descrito un rápido y fiable de imagen molecular enfoque, gen indicador de mioblastos diana de manera no invasiva / PSM tras el trasplante. Aunque este estudio demuestra la localización a corto plazo de los PSM trasplantadas a través de bioluminescense formación de imágenes (BLI), la manera en que las células están dirigidos puede, de hecho, ser fácilmente aplicada a una evaluación longitudinal de un injerto de células, a través de la implantación de células que expre...

<table border="1"><tbody><tr><td> Nombre de reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> C2C12 mioblastos </td><td> ATCC </td><td></td><td></td></tr><tr><td> Dulbecco modificada de Eagle </td><td> Life Technologies </td><td> 12800-017 </td><td></td></tr><tr><td> suero bovino fetal </td><td> Life Technologies </td><td> 12483020 </td><td></td></tr><tr><td> 0,25% de tripsina-EDTA </td><td> Life Technologies </td><td> 25200-72 </td><td></td></tr><tr><td> Hanks Balanced Salt Solution </td><td> Sigma Aldrich </td><td> H6648 </td><td></td></tr><tr><td> Lipofectamine 2000 </td><td> Life Technologies </td><td> 11668-019 </td><td></td></tr><tr><td> Nikon Eclipse TE2000-5 </td><td> Nikon Instruments Inc. </td><td></td><td></td></tr><tr><td> Xenolight D-luciferina </td><td> PerkinElmer </td><td> 122796 </td><td> 40 mg / ml en PBS </td></tr></tbody></table>

molecular imaging to target transplanted muscle progenitor cells

La distrofia muscular de Duchenne (DMD) es una grave enfermedad genética neuromuscular que afecta a 1 de cada 3.500 varones, y se caracteriza por la degeneración progresiva de los músculos 1, 2. En los pacientes, la capacidad de los residentes células musculares satélite (SCS) para regenerar las miofibras dañadas vuelve cada vez más ineficaz 4. Por lo tanto, el trasplante de células progenitoras musculares (PSM) / mioblastos de sujetos sanos es un enfoque terapéutico prometedor para DMD. Una limitación importante para el uso de la terapia con células madre, sin embargo, es la falta de tecnologías de imágenes fiables de seguimiento a largo plazo de las células implantadas, y para evaluar su eficacia. Aquí, describimos una manera no invasiva, en tiempo real enfoque para evaluar el éxito del transplante de mioblastos. Este método tiene la ventaja de un gen de fusión informador unificada compuesta de genes (luciferasa de luciérnaga [fluc], monomérico proteína fluorescente roja [mRFP] SR39 y la timidina quinasa [sr39tk]) cuya Expression se pueden obtener imágenes con diferentes modalidades de imágenes 9, 10. Una variedad de técnicas de imagen, como la tomografía por emisión de positrones (PET), emisión de fotón único tomografía computarizada (SPECT), resonancia magnética (MRI), formación de imágenes ópticas y de alta frecuencia de ultrasonido en 3D están disponibles, cada uno con sus ventajas y limitaciones 11. Bioluminiscencia de imágenes (BLI) estudios, por ejemplo, tienen la ventaja de ser un coste relativamente bajo y de alto rendimiento. Es por esta razón que, en este estudio, se hace uso de la luciferasa de luciérnaga (fluctuaciones) secuencia de gen reportero contenido dentro del gen de fusión y formación de imágenes de bioluminiscencia (BLI) para la localización a corto plazo de mioblastos C2C12 viables después de la implantación en un ratón modelo de DMD (distrofia muscular en el cromosoma X [mdx] ratón) 12-14. Es importante destacar que, BLI nos proporciona un medio para examinar la cinética de los PSM etiqueta post-implantación, y será útil para rastrear las células repetidamente en time y la migración siguiente. Nuestro enfoque de genes reportero además nos permite fusionar múltiples modalidades de imagen en un sujeto vivo solo, dada la naturaleza tomográfico, buena resolución espacial y la capacidad de escalar a grandes animales y seres humanos 10,11, PET será la base del trabajo futuro que sugieren que puede facilitar la traducción rápida de los métodos desarrollados en las células a modelos preclínicos y para aplicaciones clínicas.

Tras la confluencia 50-60%, mioblastos C2C12 fueron transfectadas transitoriamente con el gen de fusión antes mencionada construcción reportera compuesto de la luciferasa de luciérnaga [fluc], proteína monomérica fluorescente de color rojo [mRFP] y SR39 timidina quinasa [sr39tk] (Figura 1A). La eficiencia de transfección se calculó a través de microscopía de fluorescencia (Figuras 1B, C), haciendo uso de la secuencia de mRFP en nuestro constr...

Watch this Scientific Journal Video about Molecular Imaging to Target Transplanted Muscle Progenitor Cells at JoVE.com

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Un medio no invasivo para evaluar el éxito de trasplante de mioblastos se describe. El método se aprovecha de un gen de fusión informador unificada compuesta de genes cuya expresión se pueden obtener imágenes con diferentes modalidades de imágenes. Aquí, se hace uso de un<em&gt; Fluc</emReportero&gt; secuencia de genes a las células diana a través de imágenes de bioluminiscencia.

Imagen Molecular a Target trasplantó células progenitoras musculares

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

molecular-imaging-to-target-transplanted-muscle-progenitor-cells

Research

JoVE Journal

Medicine

9.1K vistas.  Lawson Health Research Institute. Un medio no invasivo para evaluar el éxito de trasplante de mioblastos se describe. El método se aprovecha de un gen de fusión informador unificada compuesta de genes cuya expresión se pueden obtener imágenes con diferentes modalidades de imágenes. Aquí, se hace uso de un Fluc secuencia de genes a las células diana a través de imágenes de bioluminiscencia.

Video: Molecular Imaging to Target Transplanted Muscle Progenitor Cells

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

En vivo en el infrarrojo cercano fluorescencia (NIRF) imagen intravascular molecular de placa inflamatoria, un enfoque multimodal de imágenes de la aterosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Investigación

Medicina

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Etiquetado de las células madre con Ferumoxytol, uno de nanopartículas de hierro aprobada por la FDA de óxido

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically inactive for prolonged periods of time, and begin with an unexpected bout of exercise1-4, but can also occur in athletes who exercise beyond their normal limits of training5. The symptoms associated with this painful phenomenon can range from slight muscle tenderness, to severe debilitating pain1,3,5. The intensity of these symptoms and the related discomfort increases within the first 24 hours following the termination of the exercise, and peaks between 24 to 72 hours post exercise1,3. For this reason, DOMS is one of the most common recurrent forms of sports injury that can affect an individual&#x2019;s performance, and become intimidating for many1,4.
For the last 3 decades, the DOMS phenomenon has gained a considerable amount of interest amongst researchers and specialists in exercise physiology, sports, and rehabilitation fields6. There has been a variety of published studies investigating this painful occurrence in regards to its underlying mechanisms, treatment interventions, and preventive strategies1-5,7-12. However, it is evident from the literature that DOMS is not an easy pathology to quantify, as there is a wide amount of variability between the measurement tools and methods used to quantify this condition6. It is obvious that no agreement has been made on one best evaluation measure for DOMS, which makes it difficult to verify whether a specific intervention really helps in decreasing the symptoms associated with this type of soreness or not. Thus, DOMS can be seen as somewhat ambiguous, because many studies depend on measuring soreness using a visual analog scale (VAS)10,13-15, which is a subjective rather than an objective measure. Even though needle biopsies of the muscle, and blood levels of myofibre proteins might be considered a gold standard to some6, large variations in some of these blood proteins have been documented 6,16, in addition to the high risks sometimes associated with invasive techniques. 
Therefore, in the current investigation, we tested a thermal infra-red (IR) imaging technique of the skin above the exercised muscle to detect the associated muscle soreness. Infra-red thermography has been used, and found to be successful in detecting different types of diseases and infections since the 1950&#x2019;s17. But surprisingly, near to nothing has been done on DOMS and changes in skin temperature. The main purpose of this investigation was to examine changes in DOMS using this safe and non-invasive technique.

The purpose of this investigation was to assess whether using an infra-red thermal camera is a valid tool for detecting and quantifying the muscle soreness after exercising.

El uso de la térmica de infrarrojos de imagen para detectar el envaramiento

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

MR Imagen Molecular del cáncer de próstata con un agente de contraste dirigidos Pequeño Molecular CLT1 Péptido

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Ensayo de alto rendimiento basado en células citometría de flujo para detectar anticuerpos a la N-metil-D-aspartato receptor o dopamina-2 del receptor en suero humano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Secuencial<em&gt; En vivo</em&gt; Imagen de células madre / progenitoras osteogénicas durante la reparación de la fractura

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Imágenes por resonancia magnética cuantitativa de la enfermedad del músculo esquelético

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

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.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

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

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Calcificación de células del músculo liso y de imagen de la calcificación aórtica y la inflamación

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

Mediciones de la contractilidad del musculo liso uterino humano desarrollo de fármacos de ayuda

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...