Los autores desean agradecer la ayuda inestimable de: Dr. Neal First y Kourtney Wilkinson manuscrito de edición, Dr. Lucy Senter, Dr. Brigit Willeford y Mike Basett de ayuda con el cuidado y entrenamiento de los animales en las instalaciones de MSU ALAC ratón acreditados, el Dr. Dwayne Sabio por su ayuda con la microscopía y la captura de imágenes, César Monroy, Swoope Hannah, y Bobbie Huddleston por su ayuda en la producción de vídeo en la MSU. Esta investigación fue financiada por la Oficina de Investigación Institucional y el Departamento de Ciencias Biológicas de la Universidad Estatal de Mississippi.

Parte 1: La laparotomía ratón embarazadas <ol><li> Por dislocación cervical, la eutanasia a una mujer embarazada ratón C57BL6J femenina en 10,5-11,5 DPC. </li><li> Limpia el abdomen con jabón antiséptico, y luego, se le exige. </li><li> Después de afeitarse, lavar el abdomen con una solución salina. </li><li> A continuación, seque el abdomen con una gasa estéril. </li><li> Cubrir el ratón con un campo estéril. </li><li> Haga una incisión ventral con las pinzas y tijeras de disección. </li><li> Identificar y eliminar todo el útero de la cavidad abdominal. Embriones de ratón será visible en el interior del útero. </li><li> Transferencia al útero en una placa Petri llena con D-PBS, y mantener en hielo. </li><li> Con un bisturí estéril y pinzas, extraer la placenta y extra tejidos embrionarios de cada embrión. </li><li> La transferencia de embriones en una nueva placa de Petri llena de D-PBS. </li><li> Medir la longitud de los embriones de ratón. Encontramos que el tamaño difiere según la edad del embrión. Por ejemplo 8,5 dpc medido en un promedio de 6 mm, 10,5 dpc medido en un promedio de 11 mm, y el 12,5 dpc medido en un promedio de 16 mm. </li><li> Luego, quite la cola para la extracción de ADN. </li></ol> Parte 2: Disección cresta gonadal Bajo microscopio estereoscópico y Fuente de luz Schott Fostec <ol><li> Coloque un papel de filtro en una placa de Petri. Luego, coloque el embrión de ratón en la parte superior del papel de filtro para secar el embrión e inmovilizar a la disección. </li><li> Con un bisturí estéril, hacer un corte transversal del embrión de ratón por encima del origen del cordón umbilical. </li><li> Bajo un microscopio estereoscópico, utilizando unas pinzas finas estéril y una aguja estéril burla fina, quitar los intestinos y el hígado para que el aparato urinario esté visible. Los riñones están situados lateralmente en la cavidad abdominal, y la vejiga se encuentra medial y caudal en comparación con los riñones. El embrión puede ser identificado como masculino porque las crestas gonadales se encuentran a ambos lados de la vejiga. En la mujer, las crestas gonadales están firmemente sujeto al extremo caudal lateral de los riñones. </li><li> Pelar la cresta gonadal a cabo por el deslizamiento de una aguja detrás de él. La cresta gonadal se elimina cortando los tejidos que la sostienen. </li><li> Transferencia de las crestas gonadales a una nueva placa de Petri llena de frescos D-PBS. Microscópicamente, la cresta gonadal masculino debe ser despojado, grandes y ovalados. Por el contrario, la cresta gonadal femenino debe ser visto, tienen forma alargada y ser más pequeño en comparación con la cresta gonadal masculino. </li></ol> Parte 3: La digestión cresta gonadal Bajo microscopio estereoscópico: <ol><li> Disección mesonefros: Separar la gónada del mesonefros y el canto con una aguja fina y afilada. <ul><li> Es importante eliminar el mesonefros de la cresta gonadal, ya que es un tejido somático, que crecen en exceso en el proceso de derivación PGC. </li></ul></li><li> La digestión cresta gonadal: Recoger limpia crestas gonadales en una gota de agua dulce 0.5ul D-PBS. Añadir 20 ml de colagenasa / Dispasa solución (concentración final en 1mg/ml). <ul><li> Los embriones deben ser tratados individualmente. Cada embrión debe tener su propio plato de petri separados y etiquetados. </li></ul></li><li> Gonadal canto Picado: Corte la cresta gonadal en trozos pequeños con una aguja estéril y estéril pinzas finas N º 4. </li><li> Incubación: placas de Petri se transfieren a una incubadora a 37 ° C durante 15 minutos. <ul><li> El tiempo de incubación puede variar entre los tejidos. </li></ul></li><li> Pipeteo: Después de 15 minutos, el tejido se puede disociar con pipeta. Utilizando sacó pipetas capilares de vidrio con un diámetro entre 40 mm -100 Romper las piezas pipeteando arriba y hacia abajo. Esto formará pequeños grupos. La transferencia de los macizos de 0,5 ml de una pre calentado D-PBS caída de un lavado final. </li><li> Después del lavado, la transferencia de los grupos a los tubos eppendorf. <ul><li> No más de exponer el tejido de la colagenasa. </li><li> No haga una suspensión de células individuales. Esto es importante para la formación de colonias. Si se trata de una suspensión de células individuales, PGCs tienden a diferenciar y migrar. </li><li> Este proceso no debe ser superior a 20 minutos, o sus células pierden su viabilidad. </li></ul></li><li> Centrifugar a 2000 rpm durante 5 minutos a temperatura ambiente. </li><li> Después de centrifugar, separar el sobrenadante y resuspender el precipitado en 0,5 ml de golpear complementado con los medios de comunicación (pre-calentado a 37 ° C). </li></ol> Parte 4: Cultura células germinales primordiales &quot;Usted debe realizar los siguientes pasos rápidamente para preservar la viabilidad de PGCs para el aislamiento y la derivación. <ol><li> 24 horas antes de este proceso debe preparar una mitóticamente inactivadas MEF-CF1 alimentador de capa, siguiendo el protocolo de Zhang J. et al 2008. <ul><li> Preparar 20 platos de un pregnant hembra del ratón (6-8 embriones). </li><li> MEFs perder la capacidad de ser una capa de alimentación (promover el crecimiento y evitar la diferenciación) después de 5-6 pasajes. </li><li> No use más de 48 horas MEFs. </li></ul></li><li> Inmediatamente antes de PGCs recubrimiento, el medio MEF debe retiran lentamente de la capa de alimentación MEF. </li><li> A continuación, añadir 0,5 ml / pocillo de pre-calentado medio suplementado por nocaut en la capa de alimentación MEF. <ul><li> MEF medios de comunicación contiene suero bovino fetal al que induce la diferenciación de PGCs. </li><li> Medio de octavos de final debe ser complementado con factores de crecimiento específico de especie inmediatamente antes de su uso para asegurar la actividad del factor de crecimiento. </li></ul></li><li> Mediante la distribución simétrica, PGCs placa sobre la capa de alimentación MEF. <ul><li> Si estas piezas están muy cerca, que tienden a agruparse y hacer densas colonias que se adhieren mal o comienzan a diferenciarse. </li><li> Placas de cultivo deben ser etiquetados con el nombre de embrión línea celular germinal, el número de pases, y la fecha. </li></ul></li><li> Con cuidado, la transferencia de las placas de cultivo en una incubadora a 37 ° C y 5% CO 2. </li><li> Monitor de las células cada 24 horas. </li><li> Después de 48 horas, eliminar 250 ml de los medios de comunicación de la parte superior de la cultura para no molestar a la adhesión de la colonia. A continuación, añadir 250 ml de agua dulce complementado knock-out medios de comunicación (pre-calentado a 37 ° C). </li><li> Después del paso 7, esperar otras 48 horas, y luego, eliminar por completo todos los medios de comunicación y sustituirlo por nuevos medios de comunicación complementado knock-out (pre-calentado a 37 ° C). Haga esto cada dos días durante 8-10 días hasta el PGC formar colonias. </li></ol> Parte 5: embrionario las células germinales <ol><li> Después de 10 días de aislamiento PGC, colonias de células germinales embrionarias se forman. </li><li> Líneas de células germinales embrionarias se mantiene vivo gracias a pasajes manual de cada 8-10 días. Siguen presentando una morfología indiferenciada y expresan marcadores de pluripotencialidad como alkalyne fosfatasa, Oct-4, SSEA-1 y Sox-2 </li></ol> Notas <ul><li> Condiciones de esterilidad / asepsia son esenciales en la sala de la cultura. </li><li> Para la derivación y la cultura, PGCs se procesan en un gabinete de seguridad Clase II purificador Bio. </li><li> Incubaciones se encuentran en una húmeda 37 ° C y 5% de CO 2 incubadora. </li><li> Todos los medios y los reactivos son filtrados antes de su uso en un filtro de 0,2 um, se almacenan a 4 ° C, y pre-calentado a 37 ° C antes de su uso. </li><li> Durante los pasos de la disección, todos los embriones y los tejidos se debe mantener en hielo. </li><li> Todos los frascos de reactivos deben descontaminarse con etanol antes de colocar en un armario. </li><li> El uso de guantes, bata de laboratorio, y las tapas de la enfermera son obligatorios. </li></ul> Conclusión Hemos proporcionado un video que muestra cómo aislar, se derivan, y la cultura de las células germinales embrionarias a partir de las crestas gonadales de 10.5-11.5 embriones de ratón DPC. El aislamiento reproducibles y la cultura a largo plazo de las líneas de células germinales embrionarias proporciona una base fundamental para el estudio del desarrollo embrionario temprano, los patrones germinal genética y epigenética, la formación de las gónadas, los efectos ambientales de los órganos circundantes durante el proceso de desarrollo de la gametogénesis en los embriones masculinos y femeninos, y la determinación de las vías que conduce al cáncer e infertilidad. <img src="/files/ftp_upload/1635/1635figure1.jpg" alt="Figura 1" />

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A laboratory Handbook. 1, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, 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=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> Current Opinion in Genetics and Development. 13, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> Molecular embryology: methods and protocols. 97, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> Developmental Biology. 187, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> Development. 128, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> J Vis Exp. , (2008).</li></ol>

Esta investigación se llevó a cabo después de la revisión y el animal de Atención Institucional y el empleo Comisión (IACUC) la aprobación de nuestro protocolo de ratón # 08030 (el papel del oxígeno de especies reactivas-ROS en el desarrollo de la línea germinal y la formación de tumores: mantener y criar protocolo) en la Universidad Estatal de Mississippi.

Pinzas N º 14 y 15, 35 mm de bisturí, tijeras de disección, campos quirúrgicos, jabón antiséptico, solución salina al 0,9%, la gasa, el contenedor de hielo de espuma, cuchilla de afeitar, bisturí de 35 mm, pinzas finas N º 4 y 5, con aguja fina con bromas mango, hojas de papel filtro, y tiró de pipetas de vidrio.<br /<br /&gt; Equipo: microscopio estereoscópico de disección, la fuente de luz Schott Fostec, microscopio invertido, micropipeta 10 y 200ul, centrífuga, el gabinete de seguridad biológica.

isolation and derivation of mouse embryonic germinal cells

La capacidad de las células germinales embrionarias (EG) a diferenciarse en células germinales primordiales (PGC) y más tarde en los gametos durante las primeras etapas de desarrollo es un modelo perfecto para hacer frente a nuestra hipótesis sobre el cáncer y la infertilidad. Este protocolo se muestra la forma de aislar las células germinales primordiales de las gónadas en desarrollo en 10.5-11.5 días después de embriones de ratón coitum (dpc). El desarrollo de las crestas gonadales de embriones de ratón (C57BL6J) se disocian por rotura mecánica con colagenasa, está chapada en una capa de fibroblastos de embriones de ratón de alimentación (MEF-CF1) que antes era mitóticamente inactivados con mitomicina C en la presencia de los medios de octavos de final y que será completada con un inhibidor de la leucemia Factor (LIF), el factor de crecimiento de fibroblastos (bFGF), y factor de células madre (SCF). El uso de estos métodos optimizados para la identificación del PCG, el aislamiento, y el establecimiento de condiciones de cultivo permite que las culturas a largo plazo de las células EG de más de 40 días. Las líneas embrionarias germinales de células mostró fenotipo embrionario y la expresión de marcadores utilizados común del estado pluripotente. El aislamiento y la derivación de células germinales en la cultura una herramienta para entender su desarrollo in vitro y ofrece la oportunidad de controlar el daño acumulado en los niveles genéticos y epigenéticos que después de la exposición al estrés oxidativo.

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

La capacidad de las células germinales embrionarias a diferenciarse en células germinales primordiales durante etapas tempranas del desarrollo es un modelo perfecto para hacer frente a nuestra hipótesis sobre el cáncer y la infertilidad. Este protocolo se muestra la forma de aislar las células germinales primordiales de las gónadas en desarrollo de embriones de 10.5 a 11.5 días después de ratón coitum.

El aislamiento y la derivación de células embrionarias de ratón Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

isolation-and-derivation-of-mouse-embryonic-germinal-cells

Research

JoVE Journal

Biology

16.0K vistas.  Mississippi State University. La capacidad de las células germinales embrionarias a diferenciarse en células germinales primordiales durante etapas tempranas del desarrollo es un modelo perfecto para hacer frente a nuestra hipótesis sobre el cáncer y la infertilidad. Este protocolo se muestra la forma de aislar las células germinales primordiales de las gónadas en desarrollo de embriones de 10.5 a 11.5 días después de ratón coitum.

Video: Isolation and Derivation of Mouse Embryonic Germinal Cells

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 ...

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivación de células madre de ratón a partir de blastocistos Trofoblasto

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultas y embrionarias del músculo esquelético Cultura Microexplant y aislamiento de células madre de músculo esquelético

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Aislamiento y cultivo de células embrionarias progenitoras aviar valvular

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Aislamiento y cultivo de células de la zona nefrogénica del riñón de embriones de ratón

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparación de las células embrionarias de fibroblastos de ratón adecuados para el cultivo de células estaminales embrionarias humanas pluripotentes inducidas, y

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Aislamiento de células endoteliales embrionarias ventriculares

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...