Este estudio fue apoyado por los Institutos Nacionales de Salud de Investigación de subvención R01-DE021683-01 y los Institutos Nacionales de Salud de Investigación de subvención R01-DE019434 a MTL; Instituto Médico Howard Hughes de Becas de Investigación para MTCDCW fue apoyado por la AEC Franklin Martin Facultad de Becas de Investigación, El Laboratorio Hagey Pediátrica Medicina Regenerativa, y el Premio Académico Facultad Instituto de Investigación de Salud Infantil de la Universidad de Stanford.

NOTA: Todas las muestras de los pacientes se obtuvieron con consentimiento informado, y los protocolos experimentales fueron revisados ​​y aprobados por la Universidad de la Junta de Revisión Institucional de Stanford (Protocolo # 2188 y # 9999). 1. Aislamiento de células y Cultura: <ol><li> Obtener tejido adiposo subcutáneo humano a partir de pacientes de sexo femenino sanos sometidos a lipoaspiración electiva del abdomen, flanco, y / o la región del muslo bajo anestesia local / general. Asegúrese de que la Junta de Revisión Institucional (IRB) haya obtenido la aprobación para el protocolo de aislamiento de ASC a partir de tejidos humanos, y siga las precauciones de seguridad institucionales mientras se trabaja con este tipo de materiales. </li><li> Para obtener el SVF del tejido adiposo lipoaspirated, primero lavar el lipoaspirado tres veces con volúmenes iguales de 1x solución salina tamponada con fosfato estéril (PBS). Aspirar con cuidado y desechar la fase acuosa inferior. </li><li> Preparar el tampón de digestión colagenasa: 0,075% colagenasa tipo I en HankBalanced Salt Solution (HBSS). Preparar tampón FACS: 2% de SFB, el 1% y el 1% P188 Pen-Strep en PBS. Filtra ambas soluciones utilizando un tamaño de poro de flujo rápido del filtro de polietersulfona 0,22 micras disponible en el mercado. </li><li> Para digerir el tejido adiposo lavado, añadir un volumen igual de tampón de digestión con colagenasa y colocar recipiente de digestión de forma segura en un baño de agua en agitación durante 60 min a 37 ° C (aproximadamente 180 batidos / min). NOTA: Lo mejor es usar un recipiente de digestión volumen más grande de lo necesario, ya que esto permite la digestión máxima durante la agitación (es decir, 250 ml de tampón de digestión con colagenasa, 250 ml de tejido adiposo lavado en un matraz estéril 1L). </li><li> Neutralizar la actividad enzimática mediante la adición de un volumen igual de tampón de FACS y permitir sentarse a TA durante 5 min. A continuación, se centrifuga a 233 xg durante 20 min a 4 ° C. </li><li> Aspirar con cuidado y desechar el sobrenadante, teniendo cuidado de no alterar el pellet SVF de alta densidad. </li><li> Resuspender el precipitado en 5 a 10 ml de RT rojoTampón de Lisis Celular, basado en el tamaño del pellet. Deje la solución para sentarse a TA durante 5 min y se centrifuga a 233 xg durante 5 min a TA. </li><li> Aspirar el sobrenadante y resuspender el precipitado mediante la adición de 5-10 ml de medio de crecimiento tradicional, basado en el tamaño de pellet (medio Eagle modificado de Dulbecco [DMEM] / 10% FBS / 1% de penicilina-estreptomicina solución de [pen-strep]). </li><li> Se filtra la suspensión a través de un filtro de células de 100 micras de nylon para eliminar los desechos celulares. </li><li> Centrifugar a 233 xg durante 5 min a 4 ° C, y descartar el sobrenadante sin perturbar el sedimento. </li><li> Resuspender el precipitado en 5 a 10 ml de medio de cultivo tradicionales, basados ​​en el tamaño de pellets. </li><li> Coloque 2 x 10 6 células en una placa de cultivo de 15 cm estándar y establecer cultivos primarios O / N a 37 ° C / 21% O 2, 5% de CO 2. Mantener a niveles sub-confluente para prevenir diferenciación espontánea a 37 ° C / 21% O 2, 5% de CO2 en medio de crecimiento. La densidad celular en un totalmente: NOTAconfluente placa de 15 cm es de aproximadamente 6.4 x 10 6 células. </li></ol> 2. La tinción <ol><li> ASC Cultura en medio de cultivo tradicional a 37 ° C / 21% de O 2, 5% de CO2, durante 36 horas antes de la tinción / clasificación. </li><li> ASC Ascensor de placas de cultivo utilizando Accutase (según el protocolo del fabricante). </li><li> Lave las células una vez con tampón FACS (PBS con FBS al 2% y 1% pen-strep). </li><li> Centrifugar a 233 xg durante 5 min a 4 ° C. </li><li> Descartar el sobrenadante sin perturbar el sedimento. Resuspender las células en 0,5-1 ml de tampón FACS (dependiendo del número de células y la cantidad de anticuerpo deseado). NOTA: El volumen de la suspensión de células, de acuerdo con las recomendaciones del fabricante anticuerpo FACS, determina la concentración de anticuerpo. Sin embargo, la valoración de la concentración de anticuerpo, utilizando una concentración de partida de 1: 100, se aconseja por lo general si es la primera vez que utiliza un anticuerpo. </li><li> Cuente el número total decélulas utilizando un hemocitómetro. </li><li> Reserva alícuotas de 100 ul de 1,5 ml tubos de centrifugación que se utilizarán para una muestra sin mancha y los controles de un solo color (según el número de anticuerpos fluorescentes usados). </li><li> Añadir los anticuerpos monoclonales marcados con fluorescencia-apropiados (en concentraciones predeterminadas, según las instrucciones del fabricante) y se incuba durante 20 a 30 minutos en hielo, protegida de la luz. <ol><li> Para etiquetar subpoblación osteogénico: Diluir anti-CD90 (APC CD90 anti-humano (Thy1)) o anti-CD105 (FITC anti-humana CD105 (endoglina)) en tampón FACS a una concentración de 1:50. </li><li> Para etiquetar otras poblaciones de células (por ejemplo, hematopoyéticas y células endoteliales): Diluir anti-CD45 (Anti-CD45 humano Pacific Blue), anti-CD34 (Anti-CD34 humano APC), y / o anti-CD31 (Anti-Human CD31 PE ) en tampón FACS a una concentración de 1:50. </li><li> Si no está usando anticuerpos conjugados directamente, utilizar un anticuerpo primario biotinilado con un segundo conjugada con estreptavidina apropiadoary anticuerpo, tal como estreptavidina PE-Cy7, a una dilución de 1: 100. </li></ol></li><li> Después de la incubación, se lavan las células dos veces: llenar tubos con tampón FACS y se centrifuga a 233 xg durante 5 min a 4 ° C, aspirar el sobrenadante. </li><li> Resuspender las células en 400 a 500 muestras l FACS de amortiguación y filtrar a través de un 70 micras filtro de células de nylon para eliminar grupos de células. </li><li> Transferencia de células marcadas en tubos FACS con la parte superior del filtro y mantener las muestras en hielo durante la duración de análisis. </li></ol> 3. Clasificación células activadas por fluorescencia NOTA: Los siguientes pasos exigen conocimientos previos en células activadas por fluorescencia (FACS) o la ayuda de un técnico especializado. <ol><li> Utilizando el software FACS apropiadas, las parcelas de análisis de diseño para examinar dispersión frontal (FSC), la dispersión lateral (SSC), ficoeritrina (PE) -Texas Rojo, Azul Pacífico, isotiocianato de fluoresceína (FITC), Allophycocyanin (APC) y cualquier otro uso fluoróforod para etiquetar células. </li><li> Antes de cargar en el clasificador de células, vórtice brevemente cada muestra para resuspender las células. </li><li> Comenzar con el análisis de la muestra sin teñir para fijar puertas para la población total de células, para excluir los desechos celulares, y para determinar la autofluorescencia de línea de base. </li><li> Añadir yoduro de propidio (PI) a la muestra sin teñir y analizar con el fin de visualizar la población de células vivas dentro de la población total de células. Construir una puerta alrededor de esta población. </li><li> Analizar una muestra de control de un solo color para cada fluorocromo con el fin de ajustar la compensación de fluorescencia. </li><li> Analizar la muestra teñida para establecer la posición de las puertas para la clasificación. </li><li> Añadir PI a la muestra teñida con el fin de teñir las células muertas. </li><li> Ordena las poblaciones de células marcadas. Ordenar en cualquiera de 1,5 ml tubos de centrífuga o tubos cónicos (15 ml) que contenía medio de cultivo. </li><li> Cesar manualmente la clasificación una vez se ha obtenido la cantidad necesaria de células. </li><li> Nosotrosing FACS, realizar una comprobación de la pureza mediante el análisis de una pequeña fracción (por ejemplo., 500 células) de la población de células adquirida. NOTA: Este paso confirma la pureza de las poblaciones de células ordenados. Idealmente, la pureza debe ser mayor que el 90-95%. </li><li> Centrifugar las células a 233 xg durante 5 min a 4 ° C inmediatamente después de la finalización de la clasificación y resuspender en 1 ml de medio fresco que contiene 10% de SFB. NOTA: La cantidad de los medios utilizados se puede aumentar en función del tamaño de la sedimento celular. </li><li> Placa en placas recubiertas con gelatina para mejorar la viabilidad celular. Dependiendo del rendimiento celular final, placa 300000 células por pocillo en una placa de 6 pocillos, 100.000 células por pocillo en una placa de 12 pocillos. </li></ol>

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Ninguno de los autores tiene un interés financiero en cualquiera de los productos, dispositivos o medicamentos mencionados en este manuscrito. Ninguno de los autores tiene ningún interés financiero en competencia reportar.

Actualmente, el aislamiento de las subpoblaciones homogéneos de ASC de la SVF de tejido adiposo humano sigue siendo un reto aunque objetivo deseable. El aislamiento de subpoblaciones ASC pro-osteogénicas es particularmente deseable, ya que tales células se pueden utilizar para estudiar la formación y la homeostasis de los tejidos esqueléticos. Sin embargo, el SVF del tejido adiposo alberga heterogeneidad significativa con respecto al vástago capacidad de la célula y el potencial de diferenciación. 11 ...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

<table border="0" cellpadding="0" cellspacing="0" width="618">
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			<td height="42" style="height:42px;width:275px;">Name of Reagent/Material</td>
			<td style="width:152px;">Company</td>
			<td style="width:112px;">Catalog Number</td>
			<td style="width:79px;">Comments</td>
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			<td height="21" style="height:21px;width:275px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">15140-122</td>
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			<td height="42" style="height:42px;width:275px;">DMEM, high glucose, GlutaMAX Supplement</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">10566-016</td>
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			<td height="21" style="height:21px;">PBS, pH 7.4</td>
			<td style="width:152px;">Gibco</td>
			<td>10010-023</td>
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			<td height="21" style="height:21px;">Betadine - Antiseptic Povidone/Iodine Solution</td>
			<td style="width:152px;">Purdue&#160;</td>
			<td>PFC-67618015017</td>
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			<td height="21" style="height:21px;">Hank&#39;s Balanced Salt Solution, 1X</td>
			<td style="width:152px;">Cellgro</td>
			<td>21-023-CV</td>
			<td style="width:79px;"></td>
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			<td height="21" style="height:21px;">Fetal Bovine Serum, Certified, US Origin</td>
			<td style="width:152px;">Gibco</td>
			<td>16000-044</td>
			<td style="width:79px;"></td>
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			<td height="21" style="height:21px;">Collagenase from&#160;Clostridium histolyticum</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td>C0130-5G</td>
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			<td height="42" style="height:42px;">ACCUTASE Cell Detachment Solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td>7920</td>
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			<td height="21" style="height:21px;width:275px;">APC Mouse Anti-Human CD90</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">559869</td>
			<td style="width:79px;"></td>
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			<td height="42" style="height:42px;width:275px;">FITC Mouse anti-Human CD105 (Endoglin)</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">561443</td>
			<td style="width:79px;"></td>
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			<td height="42" style="height:42px;width:275px;">Anti-Human CD45 eFluor 450 (Pacific Blue replacement)&#160;</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">48-9459-41</td>
			<td style="width:79px;"></td>
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			<td height="21" style="height:21px;width:275px;">Anti-Human CD34 APC</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">17-0349-41</td>
			<td style="width:79px;"></td>
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			<td height="21" style="height:21px;width:275px;">Anti-Human CD31 (PECAM-1) PE</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">12-0319-41</td>
			<td style="width:79px;"></td>
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			<td height="21" style="height:21px;width:275px;">Streptavidin PE-Cyanine7</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">25-4317-82</td>
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			<td height="21" style="height:21px;width:275px;">BD FACS Aria II instrument</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:112px;"></td>
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isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

El uso de CD90 como un marcador para las células con una mejora de los resultados de la osteogénesis en el aislamiento de una población altamente enriquecido de ASC humanos (Figura 1A, 1B). ASC se tiñeron con Pacific Blue-CD45 conjugado anti-humano, conjugado con FITC anti-CD105 humano, y CD90 anti-humano conjugado con APC. Después de la clasificación, el nivel de pureza era mayor que 98%, cuantificada por análisis post-especie. Definición de grupos de células en ba...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

El aislamiento y enriquecimiento de Human adiposo derivado de células estromales de la osteogénesis mejorada

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

Research

JoVE Journal

Developmental Biology

11.4K vistas.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Video: Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

El aislamiento y la diferenciación de células madre derivadas de tejido adiposo subcutáneo de la especie porcina tejidos adiposos

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Investigación

Biología del Desarrollo

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

La separación inmunomagnética de Sca1 depósitos de grasa específica<sup&gt; alta</sup&gt; Células Madre derivadas de tejido adiposo (ASC)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Aislamiento y expansión de células madre mesenquimales / estromales derivadas de tejido de placenta humana

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

La diferenciación condrogénica de inducción de las células madre derivadas de tejido adiposo por la gravedad centrífuga

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

El aislamiento rápido de BMPR-IB + derivadas de tejido adiposo células estromales de uso en un Defecto La curación Modelo de calota

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Aislamiento de ratón endometrial epiteliales y células del estroma de<em&gt; In Vitro</em&gt; decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

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 and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

El aislamiento y enriquecimiento de hígado progenitoras subconjuntos identificados por una combinación de marcadores de superficie Novel

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Aislamiento, cultivo y diferenciación de células estromales de médula ósea y progenitores de los osteoclastos de los ratones

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

Aislamiento y caracterización de células estromales mesenquimales cardiacas de Endomyocardial biópticos muestras de pacientes de miocardiopatía arritmogénica

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...