1. La eliminación del embrión y la placenta disección <ol><li> Para empezar, los embriones debe ser aislado de las presas embarazadas. </li><li> Los animales son sacrificados de acuerdo con los procedimientos aprobados - en nuestro caso vamos a usar una dosis letal del anestésico isoflurano </li><li> En primer lugar, rocíe el vientre con etanol y hacer una pequeña incisión con un par de tijeras. </li><li> Arranca la piel con las dos manos para descubrir el abdomen y se abre el peritoneo. </li><li> Con unas pinzas, recoja los cuernos uterinos y recogerlos con unas tijeras en cada extremo distal. Colocar los cuernos uterinos en una placa de Petri llena de PBS y el lugar en el hielo. También, recuerde lavar varias veces con PBS antes de aislar a la placenta. Ahora podemos aislar de la placenta. </li><li> Con dos pinzas, comience cuidadosamente pelar el tejido endometrial que rodea el embrión conceptuses. Hay que tener cuidado de no perforar o no causar daños estructurales a los embriones, ya que esto va a complicar la disección de los pasos posteriores. </li><li> Coloque el conceptuses aislado en una nueva placa de Petri con PBS en hielo y continuará hasta que todos conceptuses han sido liberados a partir del endometrio. </li><li> La transferencia de un embrión de una nueva placa de Petri con PBS colocado bajo el microscopio y con dos pinzas de pelar la decidua lejos de la placenta. Eliminar la mayor cantidad de la decidua posible, ya que las células de la decidua puede interferir con una sola suspensión celular y citometría de flujo. Un suave movimiento continuo peeling se recomienda para lograr los mejores resultados. </li><li> Corte el saco vitelino de distancia de la placenta en el cruce entre los dos órganos y tire del saco vitelino y los vasos vitelino fuera de la placenta. Se debe tener cuidado de no alterar la placa coriónica de la placenta, especialmente con los embriones tempranos. </li><li> Cuidadosamente la celebración de la placa coriónica de la placenta con un par de pinzas y el cordón umbilical con otro, tirar el cordón umbilical y el embrión conectado fuera de la placenta. </li><li> Retire el exceso de tejido de células gigantes en los bordes de la placenta con el fin de minimizar la célula aglutinación durante la preparación de una sola célula de la suspensión. </li><li> Recoger la placenta en un recipiente adecuado, como por ejemplo un tubo Falcon de 15 ml, con PBS 5% de FCS y sobre hielo. Hacer esto para todas las placentas disecados. </li><li> En este punto, usted puede proceder con la preparación de una suspensión de una sola célula de la citometría de flujo, ensayos funcionales, tales como el cultivo in vitro o el trasplante, o preparar la placenta entera para la fijación del tejido y de bloque fijo congelados incorporación eventual de inmunohistoquímica. </li></ol> 2. Preparación de una sola célula de suspensión de tejido de la placenta <ol><li> A continuación se muestra cómo generar una única suspensión de células de la placenta para el análisis de FACS. </li><li> Para preparar una suspensión de células individuales de la placenta, mecánica y la disociación enzimática es necesario. En primer lugar preparar una solución de colagenasa 0,1% en PBS con 10% de FCS y 1% de solución de penicilina / estreptomicina. </li><li> Añadir un volumen adecuado de solución de colagenasa a las placentas, dependiendo de la cantidad que usted tiene. Un volumen que es el doble del volumen de la placenta y entre 2 y 5 ml se recomienda. </li><li> Use una aguja de 16 G montado en una jeringa de 5 ml para romper mecánicamente el tejido de pases de la solución de colagenasa y la placenta a través de la aguja de 3 veces. Repita el procedimiento con una aguja 18-G. </li><li> Colocar la muestra en un 37 ° C, 5% de CO 2 incubadora durante 45 minutos. </li><li> Después de la incubación de 45 minutos en la colagenasa, la aprobación de la solución de células a través de una aguja de 20 G, y se incuba durante 45 minutos, en 37 ° C. </li><li> Después de la incubación total de 1,5 horas en la colagenasa, la aprobación de la solución de células a través de 22 G y las agujas 25-G 3 veces con cada aguja. </li><li> Filtrar la muestra a través de un filtro de 50 micras, unido a una nueva de 15 ml tubo Falcon, lavar con PBS 5% de FCS y se centrifuga a 4 ° C durante 5 minutos a 300 x g. </li><li> En este punto, tenemos una suspensión de células individuales para el análisis de FACS o ensayos funcionales. </li></ol> 3. La fijación del tejido de las secciones congeladas <ol><li> Inmediatamente después de aislar a la placenta de cada embrión, que se colocan en frío PBS 1X. Por lo general, una placa de 96 pozos que funciona mejor para placentas jóvenes (E8.5-11.5), pero con mayores placentas, E12.5 y mayores, una placa de 24 y puede ser más conveniente. </li><li> Cada placenta se transfiere a un nuevo pozo lleno de recién descongelado 4% paraformaldahyde o PFA. Para el E12.5 placentas, el tejido se trasladó a un nuevo pozo a mano, pero para el E10.5 puede quitar la PBS cuidadosamente con una pipeta y se añade la PFA. Es importante que la placenta está totalmente sumergida durante 2-4 horas a 4 ° C, dependiendo de la edad y el tamaño de los tejidos. </li><li> La transferencia de los tejidos en 30% de sacarosa, o si los tejidos son muy pequeños y frágiles, aspirar la PFA y añadir la sacarosa directamente. En esta etapa, las placentas estarán flotando en la solución. Deje que los tejidos pasar la noche a 4 ° C.</li><li> Después del paso de la noche en el 30% de sacarosa, en última instancia, los tejidos deben hundirse hasta el fondo del pozo, lo que indica la criopreservación adecuada. Eliminar la mitad de la solución de sacarosa y reemplazar el volumen de octubre y el lugar del tejido de vuelta en 4 ° C durante 1-2 horas. Esto facilita la penetración de octubre </li><li> La transferencia de los tejidos de 100% octubre durante 1 hora, después de lo cual puede ser embebido. </li></ol> 4. Incorporación de los tejidos fijados <ol><li> Etiqueta de moldes de plástico con la identificación de tejido apropiado (molde mostrar ejemplo). </li><li> Tire el tejido de la solución de octubre y reducir a la mitad de la placenta mediante la orientación de la placenta en forma de disco con el lado del cordón umbilical hacia abajo y cortar cuidadosamente con una cuchilla de afeitar limpia, asegurándose de mover la hoja de ida y vuelta antes de liberar la presión para asegurar un corte parejo. La placenta se reduce a la mitad para conseguir el mejor prientation para visualizar más tarde, el HSCs la placenta. </li><li> Coloque una placenta media en el fondo del molde con el borde del corte en contacto con la superficie del fondo del molde. Repita el procedimiento con un nuevo molde para la otra mitad. Por E10.5 placentas, las dos mitades se pueden colocar en el mismo molde con la misma técnica. </li><li> Cuando se vierte en el molde de octubre, puede ser difícil mantener la placenta en la orientación correcta. Asegurar la placenta en su lugar mediante la celebración de la segunda mitad del tejido con una pinza. Verter lentamente la solución octubre en el molde y evitar la producción de las burbujas. Por E10.5 placentas, mantenga la mitad del tejido con las pinzas y el resto de la mitad de otro tejido contra el exterior de la pinza. Verter lentamente la solución octubre como se describe para E12.5 placentas. </li><li> Una vez que el molde está lleno de octubre, rápidamente coloque el molde sobre el hielo seco, la OCT a su vez, un flash para congelar el tejido </li><li> Coloque el molde en una pequeña bolsa de plástico que contiene Drierite e inmediatamente guarde en el congelador de -80 º C. </li></ol>

<ol>
	<li>Gekas, C., Dieterlen-Lievre, F., Orkin, S. H., Mikkola, H. K. 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+placenta+is+a+niche+for+hematopoietic+stem+cells.">The placenta is a niche for hematopoietic stem cells.</a> Dev Cell. 8, 365-375 (2005).</li><li>Rhodes, K. E., Gekas, C., Wang, Y., Lux, C. T., Francis, C. S., Chan, D. N., Conway, S., Orkin, S. H., Yoder, M. C., Mikkola, H. K. 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+Emergence+of+Hematopoietic+Stem+Cells+Is+Initiated+in+the+Placental+Vasculature+in+the+Absence+of+Circulation.">The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation.</a> Cell Stem Cell. 2, 252-263 (2008).</li></ol>

Los procedimientos experimentales descritos en este protocolo permitirá que para el aislamiento y la visualización de tejido de la placenta y la madre hematopoyéticas y las células progenitoras. Para un resumen completo sobre el rendimiento esperado y el número de celular de las CMH en la placenta y otros órganos hematopoyéticos fetal durante el desarrollo fetal que se refieren a Gekas, et al. 2005. Para la localización de las CMH en desarrollo y otras células hematopoyéticas de la placenta que se refieren a Rodas, et al. 2008.

Lista de Materiales: La glucosa 100 mM 10 mM NaN3 15 ml tubos cónicos (BD) 16-G, 18 G, 20 G, 22 G y 25-G agujas (BD) 30% de sacarosa solución 35 mm placas de Petri (BD) Paraformaldehído al 4% 50 - m filtros celular (Celltrics) Jeringas de 5 ml (BD) Fosfatasa alcalina sustrato Kit I (Vector Rojo, Vector) Fosfatasa alcalina sustrato Kit III (Blue Vector, Vector) Antibióticos (penicilina / estreptomicina, P / S) solución Colagenasa tipo I (Sigma) Cryomold Moldes desechables de vinilo de la muestra (10 mm x 10 mm x 5 mm, Tissue-Tek) Citoqueratina primaria de anticuerpos (DakoCytomation) Pinzas de disección, de acero inoxidable o de titanio, el número 5 o 55 Drierite De suero fetal bovino (FBS) Fisherbrand plástico portaobjetos del microscopio Mailer (Fisher) Glucosa oxidasa 1000u/ml Levamisol (Vector) Montaje de los medios de comunicación, de base acuosa medio de montaje (VectamountTM AQ) Suero normal de caballo (Vector) La temperatura óptima de corte (octubre, Tissue-Tek) Kit de sustrato de peroxidasa DAB (Vector) Tampón fosfato salino (PBS) con calcio / magnesio (w Ca2 + / Mg2 +) Tampón fosfato salino (PBS) sin calcio / magnesio (w / o Ca2 + / Mg2 +) Caja de diapositivas de plástico (capacidad para 100 diapositivas) Tubería de plástico (Nalgene 180 PVC no tóxico autoclave) Polipropileno de fondo redondo de tubo de ensayo, 5 ml (12x75 mm, BD Falcon) Proteinasa K 20mg/ml solución (Amresco) Investigación de Productos International Corp Súper Pluma de Papanicolaou HT * Marcadores de diapositivas (2,5 mm, Fisher) De cultivo de tejidos tratados en forma de U (de fondo redondo) placas de 96 pozos, o bien 24 (BD Falcon) Tween 20, SigmaUltra (Sigma) Kit de amplificación tiramida (Invitrogen) Vectastain ABC de fosfatasa alcalina Standard Kit (Vector) Vectastain ABC Kit estándar Elite (Vector)

isolation and analysis of hematopoietic stem cells from the placenta

Las células madre hematopoyéticas (HSC) tienen la capacidad de auto-renovación y generar todos los tipos celulares de los linajes de sangre durante toda la vida de un individuo. Todos los HSCs surgir durante el desarrollo embrionario, después de que su tamaño de la agrupación se mantiene gracias a la auto-renovación de las divisiones celulares. Identificar el origen anatómico de las CMH y los acontecimientos de desarrollo fundamental que regula el proceso de desarrollo de HSC se ha complicado ya que muchos sitios anatómicos participar durante la hematopoyesis fetal. Recientemente, hemos identificado la placenta como un órgano principal hematopoyéticas en HSCs se generan y se amplió en exclusiva nichos microambientales (Gekas, et al 2005, Rodas, et al 2008). En consecuencia, la placenta es una fuente importante de HSC durante su surgimiento y la expansión inicial. En este artículo se muestran las técnicas de disección para el aislamiento de los murinos placenta de E10.5 E12.5 y embriones, correspondientes a las etapas de desarrollo de la iniciación de las CMH y el pico en el tamaño de la piscina HSC en la placenta, respectivamente. Además, presentamos un protocolo optimizado para la disociación enzimática y mecánica de tejido de la placenta en una sola célula de suspensión para su uso en citometría de flujo o ensayos funcionales. Hemos encontrado que el uso de colagenasa por una sola célula de suspensión de la placenta produce rendimientos suficientes de las CMH. Un factor importante que afecta el rendimiento HSC de la placenta es el grado de disociación mecánica antes, y la duración de, tratamiento enzimático. Disponemos también de un protocolo para la preparación de las secciones fijas congeladas de tejido placentario para la visualización del desarrollo de HSC por inmunohistoquímica en sus nichos celulares precisos. Como hematopoyéticas antígenos específicos no se conservan durante la preparación de las secciones incluidas en parafina, que utilizan habitualmente fija las secciones congeladas para la localización de la placenta y HSCs progenitores.

Watch this Scientific Journal Video about Isolation and Analysis of Hematopoietic Stem Cells from the Placenta at JoVE.com

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hemos identificado la placenta como un órgano principal hematopoyéticas durante el desarrollo. Encontramos que las células madre hematopoyéticas (HSC), son generados y ampliado en la placenta en el único nichos microambientales. A continuación, describimos las técnicas experimentales necesarias para el aislamiento y la visualización de la CMH en la placenta del ratón.

Aislamiento y análisis de las células madre hematopoyéticas de la placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

isolation-and-analysis-of-hematopoietic-stem-cells-from-the-placenta

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JoVE Journal

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Cell Biology

Biology

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

SCIENTISTS IN ACTION

20.3K vistas.  University of California, Los Angeles. Hemos identificado la placenta como un órgano principal hematopoyéticas durante el desarrollo. Encontramos que las células madre hematopoyéticas (HSC), son generados y ampliado en la placenta en el único nichos microambientales. A continuación, describimos las técnicas experimentales necesarias para el aislamiento y la visualización de la CMH en la placenta del ratón.

Video: Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

Aislamiento y trasplante de células madre hematopoyéticas (HSC)

Investigación

Biología

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

El aislamiento de las primeras células madre hematopoyéticas murinas saco vitelino y AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Aislamiento de células madre de la retina del ojo del ratón

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral mediada por derribo Durante Ex Vivo Eritropoyesis de las células estaminales hematopoyéticas

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

El análisis fenotípico y aislamiento de células madre hematopoyéticas murinas y progenitores comprometidos Lineage

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Infección retroviral murino de células madre embrionarias células derivadas embrioide cuerpo para el Análisis de la diferenciación hematopoyética

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identificación de los factores clave que regula la auto-renovación y diferenciación de las células precursoras hematopoyéticas EML por análisis de secuenciación de ARN-

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Aislamiento, caracterización y diferenciación de células cardiacas desde el corazón de ratón adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...