We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.

1. Obtención de embriones de ratón con CPC de ratón CES <ol><li> Preparar fibroblastos embrionarios de ratón (MEFs) capa alimentadora. <ol><li> Medio MEF caliente (10% FBS en DMEM) a 37 ° C. </li><li> Preparar placas recubiertos con gelatina. <ol><li> Añadir 1 ml de 0,1% de gelatina en agua en un pocillo de una placa de 6 pocillos o 5 ml en una placa de 10 cm. </li><li> Deja placas o platos a 37 ° C o temperatura ambiente durante al menos 30 minutos. Aspirar la gelatina antes de su uso. </li></ol></li><li> Descongele irradiado MEFs rápidamente en un baño de agua a 37 ° C, con agitación suave. </li><li> Transferencia de las células suavemente a un tubo cónico de 15 ml o 50 ml. Añadir 5 ml de medio MEF pre-calentado. Agitar lentamente las células y centrifugar a 200 xg durante 5 min. </li><li> Resuspender las células en medio MEF y contar las células viables. Utilice los siguientes volúmenes de medio MEF: 2 ml de células por pocillo de una placa de 6 pocillos y 10 ml para las células de una placa de 10 cm. </li><li> Las células de la placa en placas de 6 pocillos o placas de 10 cm a 2.1x 10 6 por placa / placa. </li><li> Incubar las placas MEF / platos a 37 ° C, 5% de CO 2 al menos 24 horas antes de su uso. NOTA: Deseche las placas MEF / platos de más de una semana. </li></ol></li><li> Preparar ratón CES <ol><li> Cultura del ISL1 cre-; Rosa26YFP / MEF2C-cre; Rosa26YFP ratón CES en MEFs hasta 90% de confluencia. Medio de células ES Uso compuesta de 410 ml de DMEM, 75 ml de FBS, 0,1 mM MEM NEAA, 2 mM L-glutamina, 0,1 mM de piruvato, 100 U Pen / Strep, y 0,1 mM β-mercaptoetanol. </li><li> Preparar placas de 6 pocillos recubiertos con gelatina como se describe en 1.1.2. </li><li> Aspirar medio de placas y enjuague células con DPBS. <ol><li> Aspirar DPBS y añadir 0,4 ml de 0,25% de tripsina en un pocillo de placa de 6 pocillos. Se incuban las células a 37 ° C, 5% de CO 2 durante 5 min. Añadir 1 ml de medio ES pre-calentado a cesar reacción tripsina. </li></ol></li><li> Células Cosecha y centrifugar las células a 200 xg y aspirar medio. Resuspender las células en 1 ml de medio de células ES y contar las células. &lt;/ Li&gt; <li> CES placa en placas recubiertas de gelatina en densidad de 3 x 10 4 / cm 2. Incubar a 37 ° C, 5% de CO 2 durante aproximadamente 2 días en que los CES alcanzan aproximadamente 90% de confluencia. Cambie el medio cotidiano. </li><li> Cuando los CES son 90% de confluencia, repita los pasos 1.2.2-1.2.5 para eliminar por completo los alimentadores del MEF. </li></ol></li><li> Inducir CPC diferenciación por la formación EB <ol><li> Preparar medio de diferenciación como sigue: 36 ml de IMDM, 12 ml F12 de Ham, 2 mM L-glutamina, 0,34 ml de BSA (7,5%), 0,25 ml N2, 0,5 ml B27, 50 mg / ml AA, y 0,45 mM 1-tioglicerol. </li><li> Aspirar medio de las células y enjuague con DPBS. Aspirar DPBS y añadir 0,4 ml de 0,25% de tripsina en un pocillo de placa de 6 pocillos. Incubar a 37 ° C durante 5 min. </li><li> Añadir 1 ml de medio de diferenciación que cese la reacción y la pipeta hacia arriba y abajo para dispersar a grupos de células en células individuales. </li><li> Centrifugar los CES a 200 xg durante 5 minutos y utilizar el medio. <ol><li> Resuspender 1 x 10 6 &lt;/ Sup&gt; CES de 1 ml de medio de diferenciación. Contar las células, y cultivar las células en condiciones de poca desprendimiento placa Petri a una concentración de 1 x 10 5 células / ml en medio de diferenciación a 37 ° C para la primera agregación EB. </li></ol></li><li> Dos días después de la formación de EB, EB transferir desde platos de tubos de 50 ml. Haz girar a 200 xg durante 1 min. Retire medio y enjuague EBS con 10 ml de DPBS sin Ca2 + y Mg2 +. </li><li> Aspirar DPBS y añadir 1 ml de 0,25% de tripsina. Incubar a 37 ° C durante 5 min. Añadir 4,5 ml de medio de diferenciación para detener la reacción. Pipetear hacia arriba y hacia abajo para disociar EBS en células individuales. </li><li> Centrifugar las células a 200 xg durante 5 min. Células medianas y volver a suspender las Aspirar en 1 ml de medio de diferenciación. </li><li> Contar las células, y se diluye 2 x 10 6 células a 1 x 10 5 células / ml en medio de diferenciación. Añadir VEGF, Bmp4 y activina A al medio a una concentración final de 5 ng / ml, 0,8 ng / ml, y 5 ng / ml, respectivamente. Culture las células en una placa de Petri para la segunda formación de EB a 37 ° C. </li><li> 40 horas después de la segunda formación de EB, EB transferir a tubos de 50 ml. Enjuague con DPBS una vez, disociar EBS con 1 ml de tripsina 0,25% a 37 ° C durante 5 min. Pipetear hacia arriba y hacia abajo para disociar EBS en células individuales. </li><li> Resuspender las células en StemPro-34 medio con 5 ng / ml de VEGF, 10 ng / ml de bFGF y 12,5 ng FGF10 / ml. Placa de las células a 2 x 10 5 / cm 2 en placas recubiertas con gelatina. Cultura en incubadora a 37ºC durante aproximadamente 32 horas antes del aislamiento del PCCh. </li></ol></li><li> Aislar mCPCs <ol><li> Aspirar medio y enjuague con DPBS. Añadir 0,5 ml 0,25% de tripsina a las placas de cultivo a 37 ° C durante 5 min. Añadir 4,5 ml de medio de diferenciación para detener la reacción. Pipetear hacia arriba y hacia abajo para disociar EBS en células individuales. Recoger las células por centrifugar las células y volver a suspender en el 2% de FBS / PBS para FACS-purificación. </li><li> Ejecutar CES indiferenciadas en clasificador como control negativo. Cambio de tensión para ajustar el scat adelanteter (FSC) y la dispersión lateral (SSC) para seleccionar la población deseada. Utilice adelante dispersar frente a dispersión lateral y altura de dispersión hacia adelante frente a la anchura de excluir escombros y doblete. En la sub-población seleccionada, de acuerdo con la distribución de los controles de la ESC, dibuja una puerta de YFP positivo en histograma para eliminar la autofluorescencia celular. Recoger YFP + CPC de YFP + gating. </li><li> Placa de los CPC purificados (células YFP +) en placas de 6 pocillos con medio de diferenciación (1.3.1). Cultura adicionales 3-5 días a 37 ° C para la diferenciación de los CPC en cardiomiocitos y SMC. </li></ol></li></ol> 2. Derivación de Human Embryonic CPC de los CES Humanos <ol><li> Preparar Alimentadores <ol><li> Preparar fibroblastos embrionarios de ratón (MEFs) capa de alimentación como se describe anteriormente a una densidad de 2 x 10 4 / cm 2. </li></ol></li><li> Mantener humanos CES <ol><li> Mantener ISL1 humana: cre; pCAG-flox-STOP-flox-GFP o RFP CES rutinariamente en MEF utilizando medio hESC regular (Knockout DMEM / F-12 385 ml, Knockout SR 100 ml, 2 mM L-glutamina, 0,1 mM NEAA, 0,1 mM β-mercaptoetanol, 10 ng / ml de FGF básico). Antes de la diferenciación cardiaca, transferir los CES humanos en condición libre de alimentador de por lo menos 2 pasajes. </li></ol></li><li> Preparar los CES humanos en condición libre alimentador <ol><li> Preparar placas recubiertas para la cultura humanos CES. <ol><li> Descongele matrigel a 4 ° C durante la noche. Poner un tubo de 50 ml en hielo y añadir 30 ml de medio DMEM / F12 frío en el tubo. </li><li> Añadir 1 ml de matrigel en el medio fría y mezclar bien. Añadir 1 ml de frío matrigel-DMEM 12 media / en un pocillo de una placa de 6 pocillos o 5 ml en una placa de 10 cm. </li><li> Deja placas / platos a 4 ° C durante la noche o la temperatura ambiente durante 2 horas. Aspirar medio antes de su uso. </li></ol></li><li> Dividir humanos CES sobre las placas: Aspirar los medios de placa MEF y enjuague humanos CES con DPBS. A continuación, añadir 1 ml de dispasa (1 mg / ml) a un pocillo de placa de 6 pocillos. Se incuban las células en 37 ° Cincubadora hasta que los bordes de las colonias empiezan a separar. </li><li> Retire la solución dispasa. Enjuague con DPBS dos veces, a continuación, añadir 2,5 ml / por medio bien mTeSR o medio MEF acondicionado a la placa. </li><li> Raspe las células utilizando un rascador de células y cuidadosamente pipetear arriba y abajo para romper CES colonias humanas en pequeños grupos. </li><li> La transferencia de los macizos del CES y diluir 1: 3 en las placas recubiertas. </li><li> Añadir 2 ml / pocillo medio mTeSR o medio MEF acondicionado y cambiar a medio día. </li><li> Cultura CES humanos en medio mTeSR o medio MEF acondicionado en placas recubiertas durante al menos 2 pasajes. </li></ol></li><li> Inducir CPC diferenciación de los CES humanos <ol><li> Cuando las células son ~ 90% de confluencia, aspirar el medio y enjuague con DPBS. Entonces se incuban las hESCs en 0,5 mg / ml Dispasa a 37 ° C durante 20 min. </li><li> Enjuagar los hESCs con DPBS dos veces, eliminar las células a partir de placas con un levantador de célula, a continuación, volver a suspender las abrazaderas de células en medio de diferenciación (DMEM / F12 que contiene 18% de FBS, 0,1 mM NEAA, 2 mML-glutamina, 0,1 mM β-mercaptoetanol y 50 mg / ml de ácido ascórbico). </li><li> Transferir las células en placas de fijación ultra-bajas de 6 pocillos para la formación de EB. </li><li> Cambie medio de diferenciación día siguiente. </li><li> Reemplace medio cada 3 días y mantener EBS en cultivo en suspensión. </li></ol></li><li> Aislar HCPCS <ol><li> Recoger EBS a más tardar el día 9 de diferenciación para el aislamiento CPC humano. </li><li> Aspirar medio y enjuague EBS con DPBS dos veces. Añadir 0,5 ml 0,25% de tripsina a cada pocillo de placas de cultivo de 6 pocillos a 37 ° C durante 5 min. </li><li> Añadir un volumen igual de medio de diferenciación para detener la reacción. Pipetear hacia arriba y hacia abajo para disociar EBS en células individuales. Recoger las células por centrifugación a 200 xg durante 5 min y resuspender las células en 2% FBS / DPBS. Las células de filtro con filtro celular 40 micras y purificado FACS el PP + células CPC como se describe en 1.4.3. </li><li> Plate ordenadas HCPCS sobre las placas geltin-revestido. HCPCS Cultura en medio de diferenciación para 7-9 Días para diferenciarse en células de cardiomiocitos y el músculo liso. 7 días después de la siembra, cultivo de células con 5% Knockout SR en medio DMEM / F12 diferenciado. </li></ol></li></ol>

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The authors declare no competing financial interests.

This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability ...

A correction to the author list was made to: <a href="http://www.jove.com/video/52047/derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells">Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells</a>.
The following author is now listed as a co-corresponding author:
Lei Bu

A correction to the author list was made to: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells.

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Las enfermedades del corazón siguen siendo la causa principal en el mundo de hoy y las tasas de mortalidad se han mantenido prácticamente sin cambios en las últimas dos décadas (American Heart Association). Hay una necesidad crítica para el desarrollo de nuevas estrategias terapéuticas para prevenir o revertir la insuficiencia cardíaca con eficacia. Una estrategia prometedora es la terapia basada en células tras el rápido desarrollo de la biología de células madre 1. En este sentido, las CPC multipotentes podrían ser una fuente de células excelente para la terapia debido a su capacidad para proliferar, pero cometido sólo a la diferenciación de linaje cardiaco. Por lo tanto, el método eficiente y robusto para generar y aislar CPC es de gran importancia para los estudios de terapia celular cardiaca. Este protocolo se centra en CPC embrionarias identificados durante la embriogénesis temprana y cómo su generación a partir de los CES. Varios CPC han sido aislados de corazones embrionarias y adultas, incluso desde médulas óseas 2. Durante el desarrollo embrionario, morphoge huesoproteínas magnéticos (BMP), de tipo alas miembros de la familia del sitio de integración MMTV (Wnts) y nodal señales inducen el compromiso de la Mesp1 + mesodermo multipotentes 3. Células Mesp1 + luego se diferencian en los CPC embrionarias 4. Estos CPC están típicamente marcados por HCN4, NK2 homeobox 5 (Nkx2-5), Isl LIM homeobox 1 (ISL1), T-box 5 (Tbx5), y miocitos factor de promotor de 2C (MEF2C), forman campos cardíacos primarios y segundo, y contribuir a las partes principales de corazón durante cardiogénesis 5-10. CPC Tanto Nkx2-5 + y ISL1 + / + MEF2C son capaces de diferenciarse en cardiomiocitos, células musculares lisas (CML), y células endoteliales 5-8. Así, estas CPC dará lugar a la vasculatura cardiaca, así como tejidos cardíacos y son una fuente de células ideal para la terapia del corazón basado en células. En consecuencia, la generación de CPC in vitro ha sido un foco importante de investigación en estudios cardiovasculares. Desde CES tienen la expansión ilimitada capacidad de unnd representan las células ICM en la etapa de blastocisto, la diferenciación de los CES en CPC embrionarias después de la embriogénesis natural se considera un enfoque lógico y eficaz para obtener los CPC. Un enfoque ampliamente aplicada para obtener los CPC de los CES es agregar los CES en EBS 11. Para mejorar la eficiencia de diferenciación, se han utilizado factores químicos y de crecimiento definidos basados ​​en el conocimiento de desarrollo cardíaco 12-14. Sin embargo, no hay marcadores CPC definitivas, en particular no hay marcadores de la superficie celular, que son ampliamente aceptados en el campo. Para solucionar este problema, los CES están diseñados para marcar ISL1 + o MEF2C CPC + y sus derivados con los reporteros fluorescentes utilizando el sistema Cre / loxP. La recombinasa cre es eliminado en bajo el control de ISL1 / MEF2C promotor / potenciador. Modificado RFP proteína fluorescente o un gen YFP impulsado por un promotor constitutivo se pueden activar por escisión de codón de parada flox con recombinasa cre(ISL1: cre; pCAG-flox-STOP-flox-GFP o RFP / ISL1 cre-; Rosa26YFP / MEF2C-cre; Rosa26YFP) 5,6. Una vez que los CES se diferencian en CPC segundo campo corazón, ISL1 / MEF2C promotor / potenciador cre impulsado activará los reporteros fluorescentes y los CPC puede ser enriquecida por FACS-purificación. Brevemente, el método de agregación EB se utiliza para iniciar la diferenciación de ESC. Para mejorar la eficiencia de diferenciación, las células diferenciadas se tratan con ácido ascórbico (AA) y factores de crecimiento tales como Bmp4, activina A y VEGF 13,15. Este protocolo permite robusto y eficiente la diferenciación CPC usando ratón y humanos CES.

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derivation of cardiac progenitor cells from embryonic stem cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

El protocolo muestra derivación de CPC de varias líneas de células ES. CES se agregan para formar EBS para diferenciarse en los CPC. CES se mantienen rutinariamente en alimentadores MEF (Figura 1A, F) y alimentadores se eliminan antes de la diferenciación. Tras la agregación de los CES en EBS en medio de diferenciación (Figura 1B, g), la segunda EBS formados se tratan con Bmp4 y activina A para mejorar la diferenciación del mesodermo en líneas celulares de ratón (Figura...

Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Derivación de células cardiacas progenitoras a partir de células madre embrionarias

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells

Research

JoVE Journal

Developmental Biology

8.1K vistas.  University of Michigan. In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Video: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Derivación eficiente de células de la retina epitelio pigmentario de Células Madre

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Investigación

Biología del Desarrollo

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

La inducción directa de las células madre neurales humanas a partir de células de sangre periférica progenitoras hematopoyéticas

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Derivación sin alimentador de melanocitos partir de células madre pluripotentes

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

La regulación epigenética de Cardíaca diferenciación de células madre embrionarias y Tejidos

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Montaje entero horizontal: un nuevo protocolo de procesamiento e imágenes para tejidos gruesos y tridimensionales Secciones transversales de la piel

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Diferenciación rápida y dirigida de las células epiteliales del pigmento retiniano de humanos embrionarios o inducidas pluripotenciales células

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Sola célula Photoconversion en pez cebra intacto de la vida

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Análisis computacional de lo elegans de Caenorhabditis del Germline para estudiar la distribución de núcleos, proteínas y citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Derivación y diferenciación de células mesenquimales ovárico canino

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

En la generación de Vitro del Somite derivados humanos inducida por células madre pluripotentes

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...