The authors wish to thank Carl Hobbs and Lindsey Majoram for their technical assistance, and Professor Pat Doherty for use of microscopy facilities. Dr. Agley was supported by a studentship from King&#8217;s College London. Funding from the Spurrell Trust is also acknowledged.

<ol>
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The authors declare that they have no competing financial interests.

Hemos descrito un procedimiento de clasificación immunomagentic para el enriquecimiento selectivo de precursores derivadas de músculo humanos a partir de pequeñas muestras de material de biopsia muscular. Esta técnica ha sido muy valiosa en nuestro laboratorio para superar la pérdida de cultivos de músculo humanos derivados de los fibroblastos, sino también para la comprensión del comportamiento único de poblaciones distintas de células progenitoras derivadas de músculo. Células miogénicas Una vez purificad...

La reparación y la regeneración del músculo esquelético requiere la acción de las células satélite 1, las células madre miogénicas 2,3. In vivo estas células existen en un estado quiescente reversible situado entre el sarcolema y la lámina basal de cada miofibrillas, pero se activan para proliferar, fusibles y diferenciarse como el tejido muscular están dañados, reparar y regenerar 3. Las células satélite se pueden aislar a partir de muestras de biopsia de músculo humano joven y de edad avanzada utilizando digestión enzimática 4 y sus propiedades miogénicos posteriormente se pueden estudiar en la cultura primaria 5. La eficiencia de este proceso de aislamiento con respecto a tanto el rendimiento y la pureza de la población de células depende de los métodos utilizados y puede variar de muestra a muestra. Los dos principales tipos de células adherentes obtenidas a partir de la digestión enzimática son las células satélite (células miogénicas ahora denominados o células precursoras de músculo), identificados inicialmente como células CD56 + / desmina, y mufibroblastos SCLE derivados, identificadas como CD56 - y TE7 + 5 células. Los fibroblastos tienen una tasa de proliferación rápida y no se someten a la detención del crecimiento y la diferenciación terminal irreversible al contacto célula-célula, como las células miogénicas; por lo tanto en poblaciones mixtas, los fibroblastos pueden invadir células miogénicas a dominar la cultura. Los fibroblastos a menudo se han visto como una irritación para los biólogos musculares, sin embargo, ahora existe un creciente interés en los fibroblastos como células dignos de estudio por derecho propio, en particular, ya que han demostrado tener un papel cooperativo con células miogénicas durante la reparación del músculo 6 . El aislamiento y purificación de diferentes tipos de células de músculo humano es por lo tanto una consideración metodológica importante cuando se trata de investigar el comportamiento innato de ambos tipos de células en cultivo. Activada por fluorescencia de células (FACS) es un método por el cual las células pueden ser ordenados para el estudio adicional y / o se contaron y seanalizada. FACS se ha demostrado para enriquecer de forma fiable células miogénicas humanos, pero el rendimiento de las células para el cultivo posterior hasta el momento no ha sido alto 7. Dado el potencial de replicación limitado de células somáticas, tales como células derivadas de células satélite miogénicas y muy pobre la proliferación y diferenciación asociados con la senescencia 4, se requieren métodos más suaves. Culturas individuales de fibras musculares ofrecen otra, menos agresivo, los medios de obtención de células satélite murinos todavía residente en su nicho sublaminal y después de su activación en la cultura 8,9. Sin embargo, esto a menudo no es posible a partir del material de biopsia de músculo humano (porque las fibras rara vez se pueden obtener de tendón a tendón) lo que significa que esta técnica puede no ser accesible a muchos laboratorios de investigación interesados ​​en el estudio de células derivadas de músculo humanos. Por otra parte, la técnica de la fibra individual sólo proporciona el número de células muy limitadas. Aquí se describe un sistema de clasificación basada en el gendigestión enzimática TLE de células utilizando colagenasa y dispasa seguido por dos rondas sucesivas de clasificación magnética de células activadas (MACS), que da a la vez una alta pureza (&gt; 95% de células miogénicas) y rendimiento (~ 2,8 x 10 6 ± 8,87 x 10 5 células / g de tejido) para experimentos en la cultura. CD56 se considera el marcador de superficie estándar de oro para la identificación de las células satélite humanos in situ e in vitro 10 11 y proporciona el candidato ideal marcador de superficie para la fijación del talón. En este enfoque anticuerpos CD56 conjugados con óxido de hierro y perlas superparamagnéticas que contiene polisacárido se une a las células y pasa a través de una columna de separación celular magnética de alto gradiente colocado en un fuerte campo magnético 12,13. Las columnas de separación se llenan con una matriz de lana de acero o de hierro esferas ferromagnéticas que sirven para enfocar líneas de campo magnético hacia su superficie generar fuertes gradientes de campo magnético (~ 4tesla) 14. En estas columnas incluso células ligeramente magnéticos son atraídos y absorbidos a su superficie 14. Sin consolidar (CD56 -) las células pasan a través de la columna mientras que las células CD56 + etiquetados con microperlas magnéticas se conservan hasta la retirada del campo magnético 12,15. Las suspensiones celulares de cualquier etapa del proceso de clasificación se pueden colocaron en placas a la densidad deseada para la experimentación adicional. A raíz de una intervención dada los constituyentes celulares pueden identificarse utilizando inmunocitoquímica, utilizando imágenes de campo amplio o microscopía de fluorescencia confocal y analizados cuantitativamente usando un enfoque de análisis de imágenes que permite una rápida medición objetiva de todas las células marcadas en cualquier imagen dada. En nuestro laboratorio hemos utilizado este enfoque doble clasificación inmunomagnética seguido por análisis de imagen 16 para demostrar que CD56 - fibroblastos humanos transdifferentiate fácilmente en los adipocitos, mientras que las células myogenics de origen satélite son altamente resistentes a esta conversión adipogenic 5.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">Collagenase&#160; D</td>
			<td style="width:111px;">Roche&#160;</td>
			<td style="width:89px;">11088866001</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">Dispase II</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;">D4693-1G</td>
			<td style="width:140px;">Must be filter sterilized before use</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">Trypsin/EDTA</td>
			<td style="width:111px;">(Gibco) Invitrogen</td>
			<td style="width:89px;">15400-054</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">100 micron cell strainer</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:89px;">352360</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">Collagen solution ( 3 mg/ml in 0.1% acetic acid)</td>
			<td style="width:111px;">Sigma, Dorset, UK</td>
			<td style="width:89px;">C8919&#160;</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">Minisart SRP15 syringe filter (0.2 micron)</td>
			<td style="width:111px;">Sartorius</td>
			<td style="width:89px;">17573ACK</td>
			<td style="width:140px;">Polytetrafluorethylene (PTFE) membrane</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:169px;">CD56 human Microbeads</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-050-401</td>
			<td style="width:140px;">Be aware of the limited shelf life of microbeads</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">Anti- fibroblast Microbeads, human</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-050-601</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">40 &#956;m Pre-separation filters</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-041-407</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="129">
			<td height="129" style="height:129px;width:169px;">Large Cell Collumns</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-042-202</td>
			<td style="width:140px;">These columns come with a flow resistor. Use of the flow resistor is not necessary to obtain the high myogenic purities described here.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">LS columns&#160;</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-042-401</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:169px;">MiniMacs Seperator</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-042-102</td>
			<td style="width:140px;">This separator fits the large cell column but not the LS column.&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">MidiMACS</td>
			<td style="width:111px;">Miltenyi Biotech&#160;</td>
			<td style="width:89px;">130-042-302</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">MACS multistand</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-042-303</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">BSA</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;"></td>
			<td style="width:140px;">Must be filter sterilized before use</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">Oil Red O</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;">O0625</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">Triethyl phosphate</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;">538728</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="166">
			<td height="166" style="height:166px;width:169px;">Whatman Paper</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;">Z241121-1PAK</td>
			<td style="width:140px;">No. 42, Ashless. To prepare the filter fold the circular filter paper to make a semi circle, then fold the semi-circle in half again to form a cone shape. Fit the cone into a funnel for filtering.&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;"></td>
			<td style="width:111px;"></td>
			<td style="width:89px;"></td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:169px;">ProLong Gold Antifade Reagent</td>
			<td style="width:111px;">Molecular Probes, Invitrogen</td>
			<td style="width:89px;">P36930</td>
			<td style="width:140px;">This can be purchased with or without DAPI and does not quench initial fluorescence.</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:169px;">AxioVision</td>
			<td style="width:111px;">Carl Zeiss</td>
			<td style="width:89px;">Contact Zeiss</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:169px;">Adobe Photoshop CS5 Extended</td>
			<td style="width:111px;">Adobe (purchased from Pugh Computers)</td>
			<td>ADPH16982*&#160;</td>
			<td style="width:140px;"></td>
		</tr>
	</tbody>
</table>

isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle

La reparación y la regeneración del músculo esquelético requiere la acción de las células satélite, que son las células madre de músculo residente. Estos se pueden aislar a partir de muestras de biopsia de músculo humano usando digestión enzimática y sus propiedades miogénicas estudiados en cultivo. Cuantitativamente, los dos principales tipos de células adherentes obtenidas a partir de la digestión enzimática son: (i) las células satélite (denominadas células miogénicas o células precursoras del músculo), identificado inicialmente como CD56 + y más tarde como desmina + / CD56 + células y (ii) músculo- fibroblastos derivados, identificadas como CD56 - y TE-7 +. Los fibroblastos proliferan de manera muy eficiente en la cultura y en las poblaciones de células mixtas estas células pueden invadir células miogénicas de dominar la cultura. El aislamiento y purificación de diferentes tipos de células de músculo humano es por lo tanto una consideración metodológica importante cuando se trata de investigar el comportamiento innato de cualquiera de los tipos de células en cultivo. Aquí DescrIbe un sistema de clasificación basada en la digestión enzimática suave de las células utilizando colagenasa y dispasa seguido por clasificación magnética de células activadas (MACS), que da a la vez una alta pureza (&gt; 95% de células miogénicas) y buen rendimiento (~ 2,8 x 10 6 ± 8,87 x 10 5 células / g de tejido después de 7 días in vitro) de experimentación en la cultura. Este enfoque se basa en la incubación de la población de células derivadas de músculo mezclado con microperlas perlas magnéticas conjugadas con un anticuerpo contra CD56 y luego pasar las células a través de un campo magnético. Células CD56 + unidos a microperlas son retenidos por el campo mientras que CD56 - células pasan sin impedimentos a través de la columna. Las suspensiones celulares de cualquier etapa del proceso de clasificación pueden ser en placas y se cultivaron. A raíz de una intervención dada, la morfología celular, y la expresión y localización de proteínas, incluyendo factores de transcripción nuclear puede cuantificarse utilizando el etiquetado de inmunofluorescencia con anticuerpos específicos y una imagen Processing y el paquete de análisis.

Células miogénicas y fibroblastos purificadas pueden ser cultivadas en un medio de diferenciación adipogénica durante tres días seguidos por medio de la nutrición adipogenic desde cualquier lugar entre 7-30 días para evaluar su potencial para la adipogénesis. Uso de las poblaciones de células purificadas, la tinción con Oil Red O en combinación con inmunotinción para marcadores de linaje adipogénicos y miogénicas mostró que sólo la fracción de fibroblastos era capaz de diferenciación adipogénica <stro...

Watch this Scientific Journal Video about Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle at JoVE.com

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Aislamiento y Caracterización cuantitativa inmunocitoquímica de células miogénicas Primarias y fibroblastos de músculo esquelético humano

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

isolation-quantitative-immunocytochemical-characterization-primary

Research

JoVE Journal

Developmental Biology

15.8K vistas.  King's College London. The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells. 

Video: Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Aislamiento y caracterización de las células satélite de Músculos Rata Head Branchiomeric

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Investigación

Biología del Desarrollo

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Uso de mitocondrias aisladas a partir de cantidades mínimas de ratón músculo esquelético para alta microplacas rendimiento Mediciones respiratorias

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Aislamiento y caracterización de células individuales a partir de embriones de pez cebra

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Preparación y cultivo de células precursoras miogénica / mioblastos primarios de músculo esquelético de los seres humanos adultos y envejecidos

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Aislamiento y caracterización de las células mesenquimales del estroma de cordón umbilical humano y fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Aislamiento de los mioblastos humanos, evaluación de la diferenciación miogénica y medición de la entrada de calcio almacenada

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Identificación de células satélite del músculo esquelético por inmunofluorescencia con anticuerpos de laminina y Pax7

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

El aislamiento y cultivo de células epicárdicas principales derivan de ejemplares de corazón humano adulto y Fetal

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Basado en el ARN la reprogramación de fibroblastos primarios de humanos en células madre pluripotentes inducidas

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.