Este estudo foi financiado pelo National Institutes of Health Research concessão R01-DE021683-01 e National Institutes of Health Research concessão R01-DE019434 para MTL; Howard Hughes Medical Institute Research Fellowship para MTCDCW foi apoiado pela ACS Franklin Martin Faculdade Research Fellowship, O Laboratório Hagey for Pediatric Regenerative Medicine, e do Instituto de Pesquisa de Saúde Infantil da Universidade de Stanford Faculdade Scholar Award.

NOTA: Todas as amostras dos pacientes foram obtidos com o consentimento informado, e os protocolos experimentais foram revistos e aprovados pela Stanford University Institutional Review Board (Protocolo nº 2188 e # 9999). 1. Isolamento celular e Cultura: <ol><li> Obter tecido adiposo subcutâneo humano de pacientes saudáveis ​​do sexo feminino submetidos a lipoaspiração eletiva do abdômen, flanco, e / ou região de coxa sob anestesia local / geral. Certifique-se de que Institutional Review Board (IRB) a aprovação foi obtida para o protocolo de isolar ASC de tecidos humanos, e siga as precauções de segurança institucionais ao trabalhar com esses materiais. </li><li> Para obter o SVF a partir do tecido adiposo lipoaspirated, lava-se a primeira lipoaspirado três vezes com volumes iguais de solução salina tamponada 1x com fosfato estéril (PBS). Aspirar cuidadosamente e descartar a camada aquosa inferior. </li><li> Preparar o tampão de digestão de colagenase: 0,075% de colagenase do Tipo I em HankBalanced Salt Solution (HBSS). Preparar tampão de FACS: 2% de FBS, 1% P188 e 1% de Pen-Strep em PBS. Filtrar ambas as soluções através de um tamanho de poro de 0,22 um fluxo rápido filtro polietersulfona disponível comercialmente. </li><li> Para digerir o tecido adiposo lavada, adicionar um volume igual de tampão de digestão com colagenase e colocar vaso de digestão de forma segura em um banho de água com agitação durante 60 min a 37 ° C (cerca de 180 agitações / min). NOTA: É melhor utilizar um vaso de digestão volume maior do que o necessário, pois isso permite uma digestão durante a agitação máxima (isto é, 250 ml de tampão de digestão com colagenase, de 250 ml de tecido adiposo lavado em um frasco estéril 1L). </li><li> Neutraliza-se a actividade enzimática por adição de um volume igual de tampão de FACS e permitindo a sentar-se à TA durante 5 min. Em seguida, centrifugar a 233 xg durante 20 min a 4 ° C. </li><li> Aspirar cuidadosamente e desprezar o sobrenadante, tomando cuidado para não perturbar o SVF pellet de alta densidade. </li><li> Ressuspender o sedimento em 5-10 ml de RT vermelhocélulas de tampão de lise, com base no tamanho da pelota. Deixar a solução sentar à temperatura ambiente durante 5 min e centrifugar a 233 xg durante 5 min à temperatura ambiente. </li><li> Aspirar o sobrenadante e ressuspender o sedimento, adicionando 5-10 ml de meio de crescimento tradicional, com base no tamanho da pelota (Dulbecco Modified Eagle Medium [DMEM] / FBS a 10% / 1% de solução de penicilina-estreptomicina [pen-strep]). </li><li> Filtra-se a suspensão através de um filtro de células de 100 mm de nylon para remover os detritos celulares. </li><li> Centrifuga-se a 233 xg durante 5 min a 4 ° C, e desprezar o sobrenadante sem perturbar o sedimento. </li><li> Ressuspender o sedimento em 5-10 ml de meio de crescimento tradicional, com base no tamanho do sedimento. </li><li> Coloque 2 x 10 6 células em uma placa de cultura de 15 centímetros padrão e estabelecer culturas primárias O / N a 37 ° C / 21% de O2, 5% de CO 2. Mantém-se a níveis sub-confluente para impedir a diferenciação espontânea a 37 ° C / 21% de O2, 5% de CO 2 em meio de crescimento. A densidade celular em um plenamente: NOTAconfluente 15 centímetros da placa é de aproximadamente 4-6 x 10 6 células. </li></ol> 2. Coloração <ol><li> ASC Cultura em meio de crescimento tradicional, a 37 ° C / 21% de O2, 5% de CO 2, por 36 horas antes da coloração / classificação. </li><li> ASC carona de placas de cultura utilizando Accutase (de acordo com o protocolo do fabricante). </li><li> Lave as células uma vez com tampão de FACS (PBS com 2% de FBS e 1% de pen-strep). </li><li> Centrifuga-se a 233 xg durante 5 min a 4 ° C. </li><li> Desprezar o sobrenadante sem perturbar o sedimento. Ressuspender as células em 0,5-1 mL de tampão de FACS (dependendo do número de células e a quantidade de anticorpo desejado). NOTA: O volume da suspensão de células, de acordo com as recomendações do fabricante do anticorpo FACS, determina a concentração de anticorpos. No entanto, a titulação da concentração de anticorpo, utilizando uma concentração de partida de 1: 100, é normalmente aconselhada se é a primeira vez que a utilização de um anticorpo. </li><li> Contar o número total decélulas utilizando um hemocitómetro. </li><li> Reserva alíquotas de 100 ul em tubos de 1,5 ml de centrífuga a ser utilizado para uma amostra não corado e controlos de cor única (de acordo com o número de anticorpos fluorescentes utilizados). </li><li> Adicionar os anticorpos monoclonais marcados com fluorescência adequados (em concentrações pré-determinadas, segundo as instruções do fabricante) e incubar durante 20-30 minutos em gelo, protegida da luz. <ol><li> Para etiquetar subpopulação osteogénico: Diluir anti-CD90 (APC CD90 anti-humano (Thy1)) ou anti-CD105 (FITC anti-CD105 humano (endoglina)) em tampão de SCAF até uma concentração de 1:50. </li><li> Para identificar as outras populações de células (por exemplo, hematopoiética e células endoteliais): Diluir anti-CD45 (anti-CD45 humano do Pacífico azul), anti-CD34 (anti-CD34 humano APC), e / ou anti-CD31 (anti-humano CD31 PE ) em tampão de FACS para uma concentração de 1:50. </li><li> Se não utilizando anticorpos directamente conjugados, utilizar um anticorpo primário biotinilado com um segundo conjugado de estreptavidina-apropriadaanticorpo ária, tal como estreptavidina PE-Cy7, a uma diluição de 1: 100. </li></ol></li><li> Após a incubação, lavar as células duas vezes: encher os tubos com tampão de SCAF e centrifugar a 233 xg durante 5 min a 4 ° C, o sobrenadante por aspiração. </li><li> Ressuspender as células em 400-500 amostras ul FACS tampão e filtro através de um 70 um filtro de células nylon para remover aglomerados de células. </li><li> Transferir células marcadas em tubos de FACS com topo do filtro e manter as amostras em gelo durante a duração da análise. </li></ol> 3. activadas por fluorescência celular NOTA: Os passos seguintes mandato conhecimento prévio em células activadas por fluorescência (FACS) ou a assistência de um técnico especializado. <ol><li> Usando o software FACS adequadas, parcelas de análise de projeto para examinar dispersão para a frente (FSC), dispersão lateral (SSC), ficoeritrina (PE) -Texas Red, Blue Pacific, Fluorescein Isothiocyanate (ITCF), aloficocianina (APC) e qualquer outro uso fluorophored para marcar as células. </li><li> Antes de carregar no separador de células, brevemente vórtice cada amostra para voltar a suspender as células. </li><li> Começar com a análise da amostra não corado, de modo a definir portas para a população total de células, para excluir os detritos celulares, e para determinar a linha de base da autofluorescência. </li><li> Adiciona-se iodeto de propídio (PI) para a amostra não corado e analisá-lo, a fim de visualizar a população de células vivas na população total de células. Construir um portão em torno desta população. </li><li> Analisar uma amostra de controlo de cor única para cada fluorocromo, a fim de definir a compensação de fluorescência. </li><li> Analisar a amostra corada para definir a posição de portas para a classificação. </li><li> Adicionar PI para a amostra corada de forma a corar as células mortas. </li><li> Organize as populações de células marcadas. Separar por quer tubos de 1,5 ml de centrífuga ou tubos cónicos (15 ml) contendo meio de cultura. </li><li> Cessar a triagem manual, uma vez a quantidade necessária de células foi obtido. </li><li> Nósing FACS, executar uma verificação de pureza por análise de uma pequena fracção (por exemplo., células 500) da população de células obtidas. NOTA: Este passo confirma a pureza das populações de células classificadas. Idealmente, a pureza deverá ser superior a 90-95%. </li><li> Centrifugar células a 233 xg durante 5 min a 4 ° C imediatamente após a conclusão de triagem e ressuspender em 1 ml de meio fresco contendo 10% de FBS. NOTA: A quantidade de material utilizado pode ser aumentado com base no tamanho do sedimento celular. </li><li> Placa em placas revestidas com gelatina para melhorar a viabilidade celular. Dependendo do rendimento celular final, a placa de 300.000 células por cavidade em uma placa de 6 poços, 100000 células por poço numa placa de 12 poços. </li></ol>

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Actualmente, o isolamento de subpopulações homogéneas de ASC de SVF de tecido adiposo humano continua a ser um desafio embora objectivo desejável. Isolamento de subpopulações ASC pró-osteogénicas é particularmente desejável, uma vez que tais células podem ser utilizadas para estudar a formação e a homeostase do tecido esquelético. No entanto, o SVF de tecido adiposo abriga heterogeneidade significativa no que diz respeito a conter capacidade celular e potencial de diferenciação. 11 A base mole...

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 style="width:112px;">2602A43</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 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>
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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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			<td height="21" style="height:21px;width:275px;">BD FACSDiva Software</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.

Usando CD90 como um marcador para células com melhores resultados de osteogénese no isolamento de uma população altamente enriquecidas de ASC humanos (Figura 1A, 1B). ASC foram coradas com azul-Pacífico CD45 conjugado anti-humano, conjugado com FITC anti-CD105 humano, e CD90 anti-humano conjugado com APC. Após a separação, o nível de pureza foi superior a 98%, tal como quantificado por análise post-tipo. Definição de grupos de células com base em perfis de trans...

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.

Isolamento e Enriquecimento da Human adiposo-derivadas células estromais para Osteogenesis Aprimorado

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 visualizações.  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.

Vídeo: 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.

Isolamento e diferenciação de células tronco derivadas de tecido adiposo de Suínos Subcutânea tecidos adiposos

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

Biologia do Desenvolvimento

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.

A separação imunomagnética Sca1 de gordura Depot-específica<sup&gt; alta</sup&gt; células-tronco derivadas de tecido 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.

Isolamento e expansão de CTM / células estromais derivadas de placenta humana 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 ...

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.

Condrogênica Diferenciação indução de células-tronco derivadas de tecido adiposo por gravidade centrífugas

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.

Isolamento rápido de BMPR-IB + adiposo-derivados de células estromais para uso em um Defeito Modelo Cura calota craniana

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.

Isolamento de rato Endometrial epiteliais e estromais para células<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.

Isolamento e Caracterização de células mesenquimais do estroma a partir de cordão umbilical humano e 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.

Isolamento e Enriquecimento de fígado progenitoras Subconjuntos identificado por um marcador combinação de superfícies 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.

Isolamento, cultura e diferenciação de células do estroma da medula óssea e osteoclasto progenitores dos ratos

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.

Isolamento e caracterização de cardíacas células estromais mesenquimais de endomiocárdica Bioptic amostras de pacientes Arrhythmogenic cardiomiopatia

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