Este trabajo está financiado por el NIH / NCI subvención R01CA-118916

1. Extracto de las células epiteliales madre de piel humana <ol><li> Antes de iniciar el procedimiento de aislamiento de las células epiteliales del tallo que se necesita para preparar los medios respectivos y los reactivos (ver tabla 1). </li><li> Piel fresca humano adulto cuero cabelludo de los procedimientos de cirugía estética o biopsia por punción se recoge, a continuación, se incuban en FBS DMEM / 10% / Dispasa (4 mg / mL) durante la noche a 4 ° C. Incubación de 2-4 horas a 37 ° C también es efectiva. Trozos de piel debe tener un ancho máximo de 1 cm para permitir la enzima de penetrar. </li><li> Transferencia de la piel en una placa de Petri esterilizadas, saque cada pelo de la piel, sujetando el tallo del pelo cerca de la superficie de la piel y tirando con firmeza y sin problemas. Seleccione los folículos en la fase de telogen en función de su morfología bajo un microscopio de disección, corte de la región bulto (Figura 1) la transferencia de los folículos en un tubo de 15 ml esterilizados. Folículos anágena también se puede utilizar si se corta en 1 / 3 superior del folículo para obtener folículo piloso &quot;bulto&quot; región (fig. 1B). </li><li> Incubar los fragmentos aislados del folículo en una mezcla de 0,05% de tripsina-EDTA (GIBCO) y Versene (0,53 mM EDTA 4NA, GIBCO) (1:1) durante 15-20 minutos a temperatura ambiente con agitación periódica, añadir 4 ml de DMEM + 10 % de SFB para detener la reacción, la desaceleración durante 5 minutos a 800 rpm. Por otra parte, el folículo explantes fragmento de tejido se pueden colocar en la cultura sin la tripsina digerir para cultivar células de los folículos individuales. </li><li> Desechar el sobrenadante con cuidado (~ 0.2-0.5 mL ahorrar para evitar la pérdida de las células) y vuelva a suspender en 1 ml de KCM (medio de queratinocitos), sin FEAG. </li></ol> 2. La cultura primaria de las células madre epiteliales <ol><li> Antes de que el cultivo de células madre epiteliales, la mitomicina C tratados con células 3T3-J2 (células alimentadoras) se deben preparar de la siguiente manera. 3T3-J2 células se cultivaron en DMEM con FBS al 10% antes del tratamiento. Retire el medio de cultivo de células y lavar dos veces con PBS, el tratamiento de células con 15μg / ml de mitomicina C en DMEM sin ​​suero durante 2 horas a 37 ° C, 5% de CO 2. Retire la mitomicina C que contienen DMEM y lavar dos veces con PBS, las células están listas para usar. (Mitomicina C tratados con células 3T3-J2 también se puede preparar previamente y almacenadas a -80 ° C. Descongelar y semillas de las células de un día antes de cultivo de células madre epiteliales primarias, se incuba a 37 ° C, 5% de CO 2. 150.000-200.000 3T3-J2 células por pocillo de la placa de 6 pocillos se recomienda. </li><li> Semilla del folículo del pelo aislado células madre a partir del paso de 1,5 sobre la mitomicina C tratados con células 3T3-J2 en KCM sin ​​noche a la mañana a 37 del FEAG ° C, 5% de CO 2, cambió a EGF contienen KCM al día siguiente. Las células se cultivan a 37 ° C en una atmósfera húmeda con 5% de CO 2. Todos los queratinocitos son alimentados con KCM contiene FEAG cada 2 días y se cultivan durante 14-20 días. Compruebe celular todos los días bajo el microscopio. Células madre del folículo del pelo se forman colonias (Figura 2A). Explantes del folículo del pelo se forman crecimientos después de 10-14 días (Figura 2B). </li></ol> 3. Las células madre epiteliales paso <ol><li> Lavar las células con PBS una vez. Quitar 3T3-J2 células alimentadoras por Versene (pre-calentado a RT) de tratamiento. Incubar la cultura servida en Versene durante 5 minutos, luego agite suavemente y aspirar de células alimentadoras. </li><li> Lave el cabello de las células madre del folículo, ya sea con o Versene tiempo PBS uno. </li><li> Añadir precalentado (a 37 ° C, crítico) de tripsina-EDTA (2 veces) y se incuba a 37 ° C durante 7 minutos o más si es necesario para que la célula madre de los folículos del pelo se puede desprender. No más de 15 minutos es lo mejor. </li><li> Añadir KCM w / o FEAG para detener la tripsina, suavemente la pipeta hacia arriba y abajo para dispersar las células, la desaceleración en 200 g, 5 minutos. </li><li> Vuelva a suspender las células con KCM sin EGF, Conde y re-placa de mitomicina C tratados con 3T3-J2 capa de células. </li></ol> 4. Inmortalizar las células epiteliales madre Las células primarias, células madre adultas, incluso, llegar a la senectud, después del paso de serie y el mes en la cultura. Inmortalización de las células madre primaria es una forma efectiva para aliviar este problema. <ol><li> Plato principal las células madre epiteliales (~ 350,000 células / pocillo de 6 pocillos) en el paso 1 en la capa de alimentación (3T3-J2) y la cultura durante 2 días. </li><li> Cultura PA317 LXSN 16E6E7 línea celular un día después de la siembra de las células epiteliales primarias madre con DMEM con 10% de SFB. (Esta línea produce los LXSN16E6E7 anfotrópico retrovirus que codifica para el VPH 16 E6 y E7 marcos de lectura abierta, y que puede ser usado para infectar de forma estable e inmortalizar muchos tipos de células). </li><li> Tratar PA317 LXSN 16E6E7 línea celular con mitomicina C durante 2 horas (el mismo protocolo con 3T3-J2 células). Añadir mitomicina C tratados PA317 LXSN 16E6E7 línea celular de las células madre epiteliales primarias, co-cultivo durante 6 días en KCM con EGF. Renovar KCM fresco con EGF los medios de comunicación cada 2 días. </li><li> Quitar 3T3-J2 y PA317 LXSN 16E6E7 celda con Versene. Añadir mitomicina-C tratados 3T3-J2 células NHP (neomicina, higromicina, puromicina resistentes, ~ 200.000 células por pocillo) a las células madre epiteliales. Las células se seleccionan menos de 0,2 mg / ml de G418 (Gibco) para añadiritional 6 días. Renovar fresco KCM contiene G418 con EGF los medios de comunicación cada 2 días. Sobrevivir a las células madre epiteliales se inmortalizó células madre. Un pozo de primaria de las células madre epiteliales que no son co-cultivadas con PA317 LXSN 16E6E7 celular se debe establecer como un control para la selección G418, todas las células de este bien debe ser asesinado por G418 después de 6 días de la selección. </li></ol> 5. Resultados representante Pronta aprobación de las células madre de la piel epiteliales e inmortalizado células madre epiteliales forman colonias apretado que consiste en queratinocitos pequeñas (Figura 2A), rodeada de células alimentadoras. Si se cultivan en medio libre de suero con los suplementos definido, sin una capa de alimentación, las células madre se dispersan y forman colonias no ajustados (que crecen como células individuales y pequeños grupos). Inmortalizado células madre epiteliales mantienen un fenotipo estable durante&gt; 12 meses de paso continuo, pero tienden a formar colonias más estrictas que las células primarias. No forman colonias de anclaje independiente en los ensayos de agar blando, lo que indica que estas células inmortalizadas no poseen las características de las células cancerosas. Primaria y inmortalizado células epiteliales expresan madre del folículo piloso marcador de célula madre citoqueratina 15 (Figura 3), y son capaces de diferenciarse en la epidermis del folículo del pelo, y los linajes sebáceas (Figura 3B-D). <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Figura 1" /> Figura 1. Desplumados pelos. Después de arrancar desde dispasa tratados con la piel, folículos pilosos aparecer como telogen pelos club (A) con una bola de células que rodean la parte inferior del pelo, o pelos anágena (B) con una manga de epitelio que rodea la longitud del pelo. La región del bulbo telogen forma una región distinta a la morfología micro-disección, mientras que el bulto anágena es menos clara. El bulbo anágena contiene queratinocitos de la matriz, lo que representa el tránsito de amplificación de las células que también pueden ser aisladas y cultivadas. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Figura 2" /> Figura 2. Células madre culturas. (A) Las células epiteliales se derivan de la piel forma apretada colonias epiteliales (designado por E) cuando se cultivan en KCM con una capa de alimentación de las células (designado por F). (B) explantes del folículo piloso dar lugar a crecimientos epiteliales. La región del bulbo telogen (designado por T) está conectado a la fuente y rodeada de colonias de células epiteliales con células alimentadoras. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Figura 3" /> Figura 3. Diferenciación de las células madre. (A) Las células madre colonias mantener la expresión de marcadores epiteliales, como citoqueratina 15. (B) Las células cutáneas epiteliales madre pueden ser diferenciadas a lo largo del linaje folículo del pelo según lo determinado por la expresión K6Hf. (C) Las células pueden ser cultivadas en de-epidermalized dermis u otras matrices en la interfase aire-líquido y se forma una epidermis estratificada con capa córnea, la capa granular y la capa espinosa. (D) Las células madre pueden ser inducidas a lo largo del linaje sebáceas con aceite positiva glóbulos rojos.

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Los métodos de extracción de células y la cultura se describen son sorprendentemente fácil y reproducible. Hemos generado cultivos de células madre epiteliales de docenas de personas a través de un amplio rango de edades, incluidos los pacientes con defectos de la piel hereditaria 18. Lo mejor es comenzar el proceso en el día de la cosecha de tejidos, sin embargo, las células se mantienen viables en los medios de comunicación en hielo durante varios días, lo que facilita el envío de noche si es nec...

Los medios de comunicación de queratinocitos (KCM) [DMEM y Ham s F12 (GIBCO, 3:1), la adenina (Sigma, de 180 mm), el 10% de suero fetal bovino (GIBCO), la toxina del cólera (ICN, 0,1 nM), a la penicilina / estreptomicina (GIBCO, 100 U / ml y 100 mg / ml, respectivamente), hidrocortisona (Sigma, 0,4 mg / ml, 1,1 mM), T/T3 (transferrina, GIBCO, 5 mg / ml, 649 nM, y triyodo-l tironina, Sigma, 2 nM) , la insulina (Sigma, 5 mg / ml, 862 nm), y EGF (Sigma, 10 ng / ml, 1,6 nM), pH 7.2]

isolation and culture of adult epithelial stem cells from human skin

La homeostasis de los tejidos de auto-renovación depende de las células madre adultas. Como células madre indiferenciadas sufren divisiones asimétricas, que generan las células hijas que mantienen el fenotipo de células madre y la amplificación de tránsito de las células (células TA), que migran desde el nicho de células madre, se someten a la rápida proliferación y diferenciación terminal para repoblar el tejido. Las células epiteliales madre han sido identificados en la epidermis, folículos pilosos, y en el intestino como las células con un alto potencial de proliferación in vitro y como lento en bicicleta etiqueta de retención de las células in vivo 1-3. Adulto, el tejido de células madre específicas son responsables de la regeneración de los tejidos en los que residen durante el recambio fisiológico normal, así como en momentos de estrés 4-5. Por otra parte, las células madre son generalmente considerados como multi-potentes, que poseen la capacidad de dar lugar a múltiples tipos de células dentro del tejido 6. Por ejemplo, los roedores células madre del folículo del pelo puede generar epidermis, glándulas sebáceas y los folículos pilosos 7-9. Hemos demostrado que las células madre de la región de bulto del pelo humano folículo exhibición multi-potencialidad 10. Las células madre se han convertido en una herramienta valiosa en la investigación biomédica, debido a su utilidad como un sistema in vitro para el estudio de la biología del desarrollo, la diferenciación, la tumorogénesis y por su posible utilidad terapéutica. Es probable que las células madre adultas del epitelio será útil en el tratamiento de enfermedades como la displasia ectodérmica, monilethrix, el síndrome de Netherton, la enfermedad de Menkes, la epidermólisis bullosa hereditaria y alopecias 11-13. Además, otros problemas de piel como quemaduras, heridas crónicas y úlceras se beneficiarán de las terapias con células madre 14,15. Dado el potencial para la reprogramación de células adultas en un estado pluripotente (células iPS) 16,17, las células adultas de fácil acceso y ampliable madre en la piel humana puede proporcionar una valiosa fuente de células para la inducción y la terapia posterior para una amplia gama de enfermedades, incluyendo la diabetes y la enfermedad de Parkinson.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Una forma rápida y robusta de aislar células viables de adultos epiteliales madre de la piel humana se describe. El método utiliza la digestión enzimática de la matriz de colágeno la piel, seguido por el punteo de los folículos pilosos y el aislamiento de las suspensiones de células individuales o fragmentos de tejido para el cultivo celular.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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JoVE Journal

Biology

28.2K vistas.  University of Massachusetts Medical School. Una forma rápida y robusta de aislar células viables de adultos epiteliales madre de la piel humana se describe. El método utiliza la digestión enzimática de la matriz de colágeno la piel, seguido por el punteo de los folículos pilosos y el aislamiento de las suspensiones de células individuales o fragmentos de tejido para el cultivo celular.

Video: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Investigación

Biología

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 of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

El aislamiento de células madre de cáncer de páncreas xenoinjertos

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

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

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

Inducción y Análisis de epitelial a mesenquimal de transición

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

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Aislamiento Manual de células madre derivadas de tejido adiposo de Lipoaspirates Humanos

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Aislamiento, cultivo y trasplante de células musculares satélite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Aislamiento y cultivo de células madre epiteliales dental del adulto Ratón Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...