Cette étude a été soutenue par les Instituts nationaux de recherche en santé subvention R01-DE021683-01 et National Institutes of Health Research subvention R01-DE019434 à MTL; Howard Hughes Medical Institute bourse de recherche à MTCDCW a été soutenu par l&#39;ACS Franklin Martin Faculté de bourses de recherche, le laboratoire Hagey for Pediatric médecine régénérative, et de la Faculté Scholar Award Child Health Research Institute de l&#39;Université de Stanford.

REMARQUE: Tous les échantillons de patients ont été obtenus avec le consentement éclairé, et les protocoles expérimentaux ont été examinés et approuvés par le Conseil d&#39;examen Stanford University institutionnel (Protocole N ° 2188 et # 9999). 1. Isolement et culture cellulaire: <ol><li> Obtenir le tissu adipeux sous-cutané humain provenant de patients sains de sexe féminin subissant lipoaspiration élective de l&#39;abdomen, du flanc, et / région ou de la cuisse sous anesthésie locale / générale. Assurez-vous que l&#39;Institutional Review Board (IRB) l&#39;approbation a été obtenue pour le protocole d&#39;isoler ASC de tissus humains, et suivre les précautions de sécurité institutionnels tout en travaillant avec de tels matériaux. </li><li> Pour obtenir le SVF à partir du tissu adipeux lipoaspirated, le lipoaspirat premier laver trois fois avec des volumes égaux de 1x solution saline tamponnée au phosphate stérile (PBS). Aspirer soigneusement et jeter la couche aqueuse inférieure. </li><li> Préparer le tampon collagénase de digestion: 0,075% collagénase de type I dans HankBalanced Salt Solution (HBSS). Préparer tampon FACS: 2% de FBS, 1% et 1% P188 Pen-Strep dans du PBS. Filtrez les deux solutions en utilisant un 0,22 um taille des pores débit rapide filtre polyéthersulfone disponible dans le commerce. </li><li> Pour digérer le tissu adipeux lavé, ajouter un volume égal de tampon de digestion à la collagénase et le placer récipient de digestion en toute sécurité dans un bain-marie à agitation pendant 60 min à 37 ° C (environ 180 secousses / min). REMARQUE: Il est préférable d&#39;utiliser un récipient de digestion de volume plus important que nécessaire, car cela permet à la digestion maximale pendant l&#39;agitation (c.-250 ml de tampon de digestion collagénase, 250 ml de tissu adipeux lavé dans un flacon stérile 1L). </li><li> Neutraliser l&#39;activité enzymatique par addition d&#39;un volume égal de tampon pour FACS et permettant de se asseoir à la température ambiante pendant 5 min. Ensuite, centrifuger à 233 g pendant 20 min à 4 ° C. </li><li> Aspirer soigneusement et jeter le surnageant, en prenant soin de ne pas perturber le culot SVF-haute densité. </li><li> Reprendre le culot dans 5 à 10 ml de RT rougeTampon de lyse cellulaire, selon la taille du culot. Laissez la solution de se asseoir à la température ambiante pendant 5 min et centrifuger à 233 g pendant 5 min à température ambiante. </li><li> Aspirer le surnageant et remettre le culot en ajoutant 5-10 ml de milieu de croissance traditionnelle, basée sur la taille de pellets (milieu de Eagle modifié Dulbecco [DMEM] / 10% de FBS / 1% de pénicilline-streptomycine solution [pen-strep]). </li><li> On filtre la suspension à travers un filtre cellulaire en nylon de 100 um pour éliminer les débris cellulaires. </li><li> Centrifuger à 233 g pendant 5 min à 4 ° C, et jeter le surnageant sans perturber le culot. </li><li> Reprendre le culot dans 5 à 10 ml de milieu de croissance traditionnelle, basée sur la taille du culot. </li><li> Placez 2 x 10 6 cellules dans une boîte de culture de 15 cm standard et établir des cultures primaires O / N à 37 ° C / 21% d&#39;O 2, 5% de CO 2. Maintenir au niveau sous-confluentes à prévenir une différenciation spontanée à 37 ° C / 21% O 2, 5% CO 2 dans des milieux de croissance. La densité cellulaire dans un cadre entièrement: NOTEconfluentes 15 cm plaque est d&#39;environ 4-6 x 10 6 cellules. </li></ol> 2. coloration <ol><li> CSA de la culture dans un milieu de croissance classique à 37 ° C / 21% O 2, 5% de CO 2, pendant 36 heures avant la coloration / tri. </li><li> ASC Ascenseur à partir de plaques de culture à l&#39;aide Accutase (selon le protocole du fabricant). </li><li> Laver les cellules une fois avec du tampon FACS (PBS avec 2% de FBS et 1% de Pen-strep). </li><li> Centrifuger à 233 g pendant 5 min à 4 ° C. </li><li> Jeter le surnageant sans perturber le culot. Remettre en suspension les cellules dans 0,5 à 1 ml de tampon FACS (selon le nombre de cellules et la quantité souhaitée de l&#39;anticorps). NOTE: Le volume de la suspension cellulaire, selon les recommandations du fabricant d&#39;anticorps FACS, détermine la concentration d&#39;anticorps. Cependant, le titrage de la concentration en anticorps, en utilisant une concentration de départ de 1: 100, est habituellement conseillé si ce est la première fois d&#39;utiliser un anticorps. </li><li> Comptez le nombre total decellules en utilisant un hémocytomètre. </li><li> Réserve aliquotes de 100 ul dans 1,5 ml tubes de centrifugeuse à utiliser pour un échantillon non colorées et les contrôles d&#39;une seule couleur (selon le nombre d&#39;anticorps fluorescents utilisés). </li><li> Ajouter les anticorps monoclonaux marqués par fluorescence appropriés (à des concentrations prédéterminées, selon les instructions du fabricant) et incuber pendant 20-30 min sur la glace, à l&#39;abri de la lumière. <ol><li> Pour étiqueter sous-population ostéogénique: Diluer anti-CD90 (CD90 APC anti-humain (Thy1)) ou anti-CD105 (FITC anti-CD105 humain (endogline)) dans un tampon FACS à une concentration de 1:50. </li><li> Pour marquer d&#39;autres populations de cellules (par exemple, hématopoïétiques et les cellules endothéliales): Diluer anti-CD45 (anti-humain CD45 Pacific Blue), anti-CD34 (anti-humain CD34 APC), et / ou anti-CD31 (anti-humain CD31 PE ) dans un tampon FACS à une concentration de 1:50. </li><li> Si vous ne utilisez anticorps directement conjugués, utiliser un anticorps primaire biotinylé avec une seconde de streptavidine conjugué appropriéeary anticorps, tels que la streptavidine PE-Cy7, à une dilution de 1: 100. </li></ol></li><li> Après incubation, laver les cellules deux fois: remplir les tubes avec du tampon FACS et centrifuger à 233 g pendant 5 min à 4 ° C, aspiration surnageant. </li><li> Remettre en suspension les cellules dans 400 à 500 échantillons ul FACS tampons et filtrer à travers une cellule de nylon filtre 70 um pour éliminer les grumeaux de cellules. </li><li> Transfert cellules marquées dans des tubes FACS avec le dessus du filtre et conserver des échantillons sur la glace pendant la durée de l&#39;analyse. </li></ol> 3. fluorescence tri cellulaire activé REMARQUE: Les étapes suivantes prescrivent des connaissances préalables dans la cellule activé par fluorescence (FACS) ou l&#39;assistance d&#39;un technicien qualifié. <ol><li> Utilisation du logiciel FACS appropriées, parcelles d&#39;analyse de conception pour examiner diffusion vers l&#39;avant (FSC), diffusion latérale (SSC), la phycoérythrine (PE) -Texas rouge, bleu Pacifique, isothiocyanate de fluorescéine (FITC), Allophycocyanin (APC) et toute autre utilisation de fluorophored pour marquer les cellules. </li><li> Avant de charger dans le trieur de cellules, vortex brièvement chaque échantillon pour remettre en suspension les cellules. </li><li> Commencer avec l&#39;analyse de l&#39;échantillon non coloré afin de mettre en portes pour la population totale de la cellule, pour exclure les débris cellulaires, et de déterminer autofluorescence base. </li><li> Ajouter l&#39;iodure de propidium (PI) à l&#39;échantillon non colorées et les analyser afin de visualiser la population de cellules vivantes dans la population totale de la cellule. Construire une porte autour de cette population. </li><li> Analyser un échantillon de contrôle d&#39;une seule couleur pour chaque fluorochrome pour régler la compensation de fluorescence. </li><li> Analyser l&#39;échantillon teinté pour régler la position des portes pour le tri. </li><li> Ajouter PI à l&#39;échantillon coloré pour colorer les cellules mortes. </li><li> Trier les populations cellulaires marqués. Trier soit 1,5 ml dans des tubes à centrifuger ou tubes coniques (15 ml) contenant du milieu de culture. </li><li> Cesser tri manuellement une fois que la quantité requise de cellules a été obtenue. </li><li> Nousing FACS, effectuer une vérification de la pureté en analysant une petite fraction (ex., 500 cellules) de la population de cellules acquise. NOTE: Cette étape confirme la pureté des populations de cellules triées. Idéalement, la pureté soit supérieure à 90-95%. </li><li> Centrifuger les cellules à 233 g pendant 5 min à 4 ° C immédiatement après la fin du tri et remettre en suspension dans 1 ml de milieu frais contenant 10% de FBS. REMARQUE: La quantité de support utilisé peut être augmentée en fonction de la taille du culot cellulaire. </li><li> Plate sur des plaques revêtues de gélatine pour améliorer la viabilité des cellules. Selon le rendement cellulaire finale, plaque 300000 cellules par puits dans une plaque à 6 puits, 100 000 cellules par puits dans une plaque de 12 puits. </li></ol>

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD90+(Thy-1)+Positive+Selection+Enhances+Osteogenic+Capacity+of+Human+Adipose-Derived+Stromal+Cells.">CD90 (Thy-1) Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells.</a> Tissue Eng Part A. , (2012).</li></ol>

Aucun des auteurs ont un intérêt financier dans l&#39;un des produits, dispositifs ou médicaments mentionnés dans ce manuscrit. Aucun des auteurs ne ont aucun intérêt financier concurrence à signaler.

Actuellement, l&#39;isolement des sous-populations homogènes de CSA de la SVF de tissu adipeux humain reste un défi si objectif souhaitable. L&#39;isolement de sous-populations de l&#39;ASC pro-ostéogéniques est particulièrement souhaitable, car ces cellules peuvent être utilisées pour étudier la formation et l&#39;homéostasie des tissus squelettiques. Toutefois, le SVF du tissu adipeux abrite hétérogénéité significative en ce qui concerne d&#39;endiguer la capacité des cellules et le potentiel de différ...

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">
	<tbody>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Disposable 250 mL Conical Tubes</td>
			<td style="width:152px;">Corning (Thomas Scientific)</td>
			<td style="width:112px;">2602A43</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<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>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<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>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS, pH 7.4</td>
			<td style="width:152px;">Gibco</td>
			<td>10010-023</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine - Antiseptic Povidone/Iodine Solution</td>
			<td style="width:152px;">Purdue&#160;</td>
			<td>PFC-67618015017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from&#160;Clostridium histolyticum</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td>C0130-5G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ACCUTASE Cell Detachment Solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td>7920</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<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>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">BD FACSDiva Software</td>
			<td style="width:152px;">BD Biosciences</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.

Utilisation de CD90 en tant que marqueur pour des cellules avec des résultats améliorés d&#39;ostéogenèse en isolement d&#39;une population hautement enrichi de CSA humains (Figure 1A, 1B). CSA ont été colorées avec Pacific Blue-CD45 anti-humain conjugué, conjugué à FITC anti-CD105 humain, et APC conjugué anti-CD90 humain. Après le tri, le niveau de pureté était supérieure à 98%, telle que quantifiée par une analyse post-tri. Définir les groupes de cellule...

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.

Isolement et enrichissement du tissu adipeux humain cellules stromales pour ostéogenèse accrue

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

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JoVE Journal

Developmental Biology

11.4K vues.  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.

Vidéo: 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.

Isolement et différenciation des cellules souches adipeuses de porcines sous-cutanée des tissus adipeux

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

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Biologie du développement

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.

Séparation immunomagnétique de Sca1 Fat Depot spécifique<sup&gt; haute</sup&gt; Cellules souches adipeuses (CSA)

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.

Isolement et expansion de mésenchymateuses cellules souches / stromales dérivées de placenta humain 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.

Chondrogène Différenciation Induction des cellules souches adipeuses par gravité centrifuge

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.

Isolement rapide de BMPR-IB + Adipose-Derived cellules stromales pour utilisation dans un calvarial Defect Modèle de guérison

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.

Isolement de souris endométriale épithéliales et stromales Cellules pour<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.

Isolement et caractérisation de cellules mésenchymateuses stromales du cordon ombilical humain et fœtale 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.

Isolement et enrichissement du foie progénitrices sous-ensembles identifiés par une combinaison de marqueurs de surface 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.

Isolement, Culture et différenciation des cellules stromales de la moelle osseuse et les ostéoclastes progéniteurs de souris

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.

Isolement et caractérisation de cardiaques cellules stromales mésenchymateuses d’endomyocardique Bioptic échantillons de Patients de la cardiomyopathie arythmogène

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