Etiquetado de manganeso de células estaminales embrionarias humanas <ol><li> Trypsinize los tallos de embriones humanos que se han cultivado en el alimentador sin condiciones durante 5 minutos. Neutralizar la tripsina mediante la adición de medios de cultivo. </li><li> Vuelta a las células por centrifugación a 800 rpm durante 5 minutos a 20 ° Celsius. Después de volver a suspender el pellet de células, contar el número de células por tinción con azul tripán. Con base en el recuento de células obtenidas, las células se dividen en cuatro alícuotas de 3 millones de células en tubos cónicos. Precipitar las células por centrifugación de nuevo. </li><li> Justo antes de comenzar el etiquetado de manganeso, cloruro de manganeso se disuelven fresco con una solución de cloruro de sodio al 0,9% para hacer una solución 0,1 mM de cloruro de manganeso. </li><li> Con el fin de observar la actividad de los canales de calcio, cuatro muestras se prepararon. La primera muestra es de nuestro control, que contiene las células incubadas en el 0,9% de cloruro sódico solo. La segunda muestra contiene células incubadas en el 0,1 mM MnCl2. Muestra 3 y 4 contienen las células se incubaron en mM MnCl 0,1 2, ya sea con 5 mM de Ca 2 +-canal agonista (s )-(-)- Bay K8644, o 250 M de la Ca 2 +-canal antagonista verapamilo. Incubar las cuatro muestras a 37 ° C con 5% de CO 2 durante 30 minutos. </li><li> Después de 30 minutos, centrifugar las células a 800 rpm durante 5 minutos a 20 ° Celsius. Y aspirar el sobrenadante y lavar las células marcadas dos veces con PBS. Después del lavado, vuelva a suspender cada pellet con 200 l de PBS y la transferencia en los tubos de PCR. Estas pastillas son generalmente de color blanco. </li></ol> Etiquetado de hierro de las células estaminales embrionarias <ol><li> Mix de grado clínico sulfato de protamina con agua destilada hasta obtener un 1 mg / ml de solución madre. </li><li> En un tubo madre de embriones humanos que contienen medio de cultivo celular, añadir ferumoxides a 100 mg / mL seguido por la adición de 12 mg / ml de sulfato de protamina. Mezcle la solución vigorosamente durante cinco minutos. </li><li> Añadir solución mixta a la misma cantidad de los medios de cultivo celular para lograr una concentración final de 50 mg / ml de ferumoxides y 6 mg / ml de sulfato de protamina a las células de la etiqueta. Se incuban las células en esta solución durante 12 horas. </li><li> Lavar las células dos veces con PBS, con el último lavado, que contiene heparina 10U/ml para disolver la matriz extracelular ferumoxides-protamina sulfato complejo. Después del lavado, trypsinize células para obtener una suspensión de células individuales. </li><li> Contar las células y la alícuota en el número requerido de muestras. Después del lavado, vuelva a suspender cada pellet con 200 l de PBS y la transferencia en los tubos de PCR. Estas pastillas deben ser de color marrón oscuro de color naranja. </li></ol> Realización de imágenes por resonancia magnética celular <ol><li> Hacer un fantasma con el fin de estabilizar los tubos que contienen los gránulos de las células y también para evitar los artefactos del aire circundante durante la exploración. Para ello, mezcla de 0,8% de agar y el 1% de sulfato de cobre en agua destilada y llevar a ebullición en el microondas durante 5-7 minutos. Enfriar esta mezcla por lo menos 1 hora antes de la exploración para obtener un gel. </li><li> Los tubos que contienen los gránulos de las células marcadas se coloca en el espectro para la exploración. Celular in vitro una resonancia magnética realizada en Signa HDx 3T MRI (GE Medical Systems) mediante el uso de la bobina de rodilla clínica. </li><li> Examinar las células de manganeso etiquetado para cada una de las tres muestras usando una secuencia de eco de espín: TR, el tiempo de repetición = 800 ms. TE: tiempo de eco = mínimo, FOV, campo de visión = 12 x 12 cm; matriz = 256x256, NEX 1. </li><li> Examinar las células de hierro etiquetados mediante el uso de una secuencia de eco de gradiente. Nosotros utilizamos los siguientes parámetros para esta imagen: TR = 100 ms, TE = 10 ms, FA, ángulo de inclinación = 30 °; FOV = 12 x 12 cm; matriz = 256 x 256, NEX 1. </li></ol> Análisis de los resultados de la RM <ol><li> Analizar las imágenes de resonancia magnética de 4 diferentes muestras de células de manganeso etiquetado: <ul><li> Muestra # 1 es de nuestro control, que contiene las células incubadas en el 0,9% de cloruro sódico solo. </li><li> Muestra N ° 2 contiene las células incubadas en el 0,1 mM MnCl2. </li><li> Muestra # 3 contiene las células incubadas en el 0,1 mM MnCl 2 con 5 M de Ca 2 +-canal agonista (s )-(-)- Bay K8644. </li><li> Muestra # 4 contiene las células incubadas en mM MnCl 0,1 2 con 250 M de la Ca 2 +-canal antagonista verapamilo. </li></ul></li><li> Como se predijo, las diferencias en la intensidad de señal de manganeso células marcadas son visibles. En comparación con las células marcadas únicamente con el manganeso, la intensidad de la señal con claridad el aumento de la presencia de los agonistas de los canales de calcio y disminuye en presencia de antagonistas de los canales de calcio. El área donde la intensidad de la señal se ha incrementado de manganeso células marcadas se miden mediante el uso de la imagen J (NIH, Bethesda, MD, EE.UU.). El mismo software se utiliza para analizar el área de la oscuridad fuera de resonancia de la señal del hierro células marcadas. El tamaño de la señal oscura de la plancha marcado células depende del número de células o el número de partículas de hierro contenido en las células. En este caso, Un millón de células muestra un área más pequeña de la señal oscura en comparación con la señal de los tres millones de células. </li></ol>

<ol>
	<li>Bruvold, M., Nordhoy, W., Anthonsen, H. W., Brurok, H., Jynge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-calcium+interactions+with+contrast+media+for+cardiac+magnetic+resonance+imaging:+a+study+of+manganese+chloride+supplemented+with+calcium+gluconate+in+isolated+Guinea+pig+hearts.">Manganese-calcium interactions with contrast media for cardiac magnetic resonance imaging: a study of manganese chloride supplemented with calcium gluconate in isolated Guinea pig hearts.</a> Invest Radiol. 40, 117-125 (2005).</li><li>Aoki, I., 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=Cell+labeling+for+magnetic+resonance+imaging+with+the+T1+agent+manganese+chloride.">Cell labeling for magnetic resonance imaging with the T1 agent manganese chloride.</a> NMR Biomed. 19, 50-59 (2006).</li><li>Arbab, A. S., 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=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Arbab, A. S., Frank, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+MRI+and+its+role+in+stem+cell+therapy.">Cellular MRI and its role in stem cell therapy.</a> Regen Med. 3, 199-215 (2008).</li></ol>

Estos resultados indican que el manganeso entra a través de los canales de calcio voltaje-dependientes, y el manganeso puede ser utilizado como agente de contraste MRI, que también puede mostrar la viabilidad de las células. Por lo tanto, sugerimos que el manganeso guiada por resonancia magnética puede ser útil para controlar la viabilidad celular después del trasplante de células madre embrionarias humanas en el miocardio.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Manganese chloride tetrahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M8054</td>
<td>&nbsp;</td>
<tr>
<td>(S)-(-)-Bay K8644 </td>
<td>Reagent</td>
<td>Sigma</td>
<td>B133</td>
<td>&nbsp;</td>
<tr>
<td>(+)-Verapamil hydrochloride, minimum 99.0% titration </td>
<td>Reagent</td>
<td>Sigma</td>
<td>V4629</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride 0.85-0.90% W/V</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW 3257-1</td>
<td>&nbsp;</td>
<tr>
<td>Feridex I.V. </td>
<td>Reagent</td>
<td>Berlex Laboratories</td>
<td>NDC 59338-7035-5</td>
<td>ferumoxides injectable solution 11.2mg iron/mL</td>
</tr>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>American Pharmaceutical Partners</td>
<td>NDC 63323-229-05 </td>
<td>50 mg (10 mg/mL)</td>
</tr>
<tr>
<td>Knockout SR (Serum replacement for ES cells)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828</td>
<td>&nbsp;</td>
<tr>
<td>DMEM/F12 (1:1)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330</td>
<td>&nbsp;</td>
<tr>
<td>2- mercaptoethanol 98+%</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M 3148</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine 200mM 100x</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomyocin 10,000 units/ml</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>MEM Non-Essential Amino Acids 100x solution</td>
<td>Reagent</td>
<td>Gibco </td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>Signa HDx 3T MRI</td>
<td>Tool</td>
<td>GE Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>clinical Knee coil</td>
<td>Tool</td>
<td>GE Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>Reagent</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vitro labeling of human embryonic stem cells for magnetic resonance imaging

Las células madre embrionarias (células madre) han demostrado la capacidad de restaurar el miocardio lesionado. La resonancia magnética (MRI) se ha convertido en una de las modalidades de imagen predominante para evaluar la restauración del miocardio lesionado. Por otra parte, ex-vivo agentes etiquetado, como las nanopartículas de óxido de hierro, se han empleado para rastrear y localizar las células madre trasplantadas. Sin embargo, este método no hace un seguimiento de una propiedad fundamental de la biología celular sobre la viabilidad de las células trasplantadas. Se ha sabido que el cloruro de manganeso (MnCl2) entra en las células a través de voltaje de calcio (Ca 2 +) de canal cuando las células son biológicamente activos, y se acumula intracelularmente para generar un efecto de acortamiento T. Por lo tanto, sugerimos que el manganeso guiada por resonancia magnética puede ser útil para controlar la viabilidad celular tras el trasplante de células madre de en el miocardio. En este video, vamos a mostrar cómo etiquetar células madre con MnCl2 y cómo esas células pueden verse claramente mediante el uso de resonancia magnética in vitro. Al mismo tiempo, la actividad biológica de Ca 2 +-los canales serán moduladas utilizando tanto Ca 2 +-canal de agonistas y antagonistas para evaluar los cambios concomitantes de la señal.

Watch this Scientific Journal Video about In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging at JoVE.com

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

En este vídeo, se muestra como etiqueta de células madre embrionarias humanas (células madre) con cloruro de manganeso (MnCl<sub&gt; 2</sub&gt;) Que puede entrar en las células a través de los canales de calcio voltaje-dependientes cuando las células son biológicamente activos. Además, se muestra el uso de MnCl<sub&gt; 2</sub&gt; Como agente de cambio celular resonancia magnética para determinar la viabilidad de células madre in vitro de.

En el etiquetado in vitro de células estaminales embrionarias humanas de Resonancia Magnética

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

in-vitro-labeling-human-embryonic-stem-cells-for-magnetic-resonance

Research

JoVE Journal

Biology

10.1K vistas.  Stanford University. En este vídeo, se muestra como etiqueta de células madre embrionarias humanas (células madre) con cloruro de manganeso (MnCl 2) Que puede entrar en las células a través de los canales de calcio voltaje-dependientes cuando las células son biológicamente activos. Además, se muestra el uso de MnCl 2 Como agente de cambio celular resonancia magnética para determinar la viabilidad de células madre in vitro de.

Video: In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagación de la madre embrionarias humanas (ES) Células

Investigación

Biología

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivación de células estaminales embrionarias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etiquetado de las células madre con tintes fluorescentes para la detección no invasiva con imágenes ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondeo de la actividad mitocondrial complejo en células estaminales embrionarias humanas

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro e in vivo de imágenes de bioluminiscencia gen reportero de células estaminales embrionarias humanas

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

La cultura y el mantenimiento de células estaminales embrionarias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparación Extienda los cromosomas de las células estaminales embrionarias para cariotipo

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelación y descongelación células estaminales embrionarias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análisis de Crecimiento Humano Colonia células madre embrionarias

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...