Name: expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting
Uploaded: 2017-06-30T14:00:00
Description: Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

Laboratori Contreras e Watt e Dr. William Raphael. Questi esperimenti sono stati supportati da NHLBI F31 HL128035-01 (standardizzazione del protocollo di digestione tissutale), NHLBI 5R01HL117847-02 e 2P01HL070687-11A1 (animali) e NHLBI 5R01HL117847-02 (isolamento e coltura cellulare).

Tutte le procedure descritte in questo documento seguono le linee guida stabilite dal Comitato istituzionale per la cura e l&#39;uso degli animali (IACUC) della Michigan State University. Tutti i buffer e i media devono essere protetti dalla luce. 1. Preparazione di buffer, supporti e strumenti <ol><li> Preparare la soluzione di Krebs Ringer Bicarbonato-Buffered (KRBB): 135 mM di cloruro di sodio, 5 mM di potassio, 1 mM di solfato di magnesio, 0,4 mM di fosfato di potassio fosfato, 5,5 mM ...

<ol>
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A., Thelen, K., Ayala-Lopez, N., Watts, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+and+adipogenic+potential+of+perivascular+adipose+tissue+adipocyte+progenitors+is+dependent+on+sexual+dimorphism+and+vessel+location.">The distribution and adipogenic potential of perivascular adipose tissue adipocyte progenitors is dependent on sexual dimorphism and vessel location.</a> Physiol Rep. 4 (19), (2016).</li><li>Lee, Y. H., Petkova, A. P., Granneman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+adipogenic+niche+for+adipose+tissue+remodeling+and+restoration.">Identification of an adipogenic niche for adipose tissue remodeling and restoration.</a> Cell Metab. 18 (3), 355-367 (2013).</li><li>Lee, Y. H., Petkova, A. P., Mottillo, E. P., Granneman, J. 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L&#39;obiettivo principale del presente esperimento è l&#39;isolamento, l&#39;espansione e l&#39;induzione adipogenica di APC dai depositi PVAT. Qui presentiamo un protocollo per l&#39;isolamento di APC basato sull&#39;identificazione delle cellule che esprimono i marker di superficie CD34 e PDGFRα. Queste proteine ​​superficiali sono state precedentemente identificate in APC con elevati tassi di proliferazione e il potenziale di differenziarsi in adipociti bianchi o marroni in vari depositi adiposi <sup class="xr...

Il tessuto adiposo perivascolare (PVAT), grazie alla sua prossimità ai vasi sanguigni, è una componente principale di segnalazione paracrina nella funzione vascolare 1 . L&#39;espansione di questo tessuto adiposo dipende dal fenotipo delle cellule progenitrici adipociti (APC) presente 2 , 3 . L&#39;isolamento delle cellule da tessuti adiposi è difficile, poiché gli adipociti primari sono fragili, fluttuanti e di dimensioni variabili. Alcune tecniche di isolamento possono anche alterare il fenotipo e la morfologia delle cellule aumentando la sintesi proteica infiammatoria e riducendo l&#39;espressione genica adipogenica 4 , sottolineando l&#39;importanza di un protocollo che mantiene l&#39;integrità delle cellule. La cultura delle cellule primarie e delle sottopopolazioni specifiche preadipocitiche fornisce un approccio riduzionista alla crescita in vivo e mantiene un equivalente equipaggiamento genetico cellulare 5 , anche se ilMe con queste cellule è limitata a causa del deterioramento con l&#39;invecchiamento, o senescenza 6 . I preadipociti provenienti da vari depositi adiposi, compresi i depositi sottocutanei e omentali, mostrano anche differenze nella proliferazione 7 , che sottolinea l&#39;importanza di raccogliere le cellule da siti anatomici specifici. Le cellule dei precursori dai depositi adiposi bianchi non PVAT sono stati caratterizzati negli studi precedenti 7 , 8 , 9 , ma meno conosciuti sui fenotipi PVAT APC. Le tecniche qui descritte consentono l&#39;analisi di popolazioni APC specifiche e definite con un impatto minimo sulla loro morfologia, sulla loro vitalità e sul potenziale di proliferare e differenziare. La selezione di celle magnetiche attivata (MCS) è suscettibile di applicazioni a valle, come la cultura, in quanto le perle si dissolvono senza alterare la cella. MCS è anche economico, e una volta che l&#39;anticorpo conLe centrazioni sono state standardizzate, la necessità di analisi della citometria a flusso è minima. Studi in vitro con precursori PVAT possono anche dare un&#39;occhiata al potenziale che queste cellule primarie possono avere.

<table border="0" cellpadding="0" cellspacing="0" width="757">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:325px;">Tissue Dissection</td>
			<td style="width:168px;"></td>
			<td style="width:108px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Dishes</td>
			<td>Handmade with Silicone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture Petri Dish</td>
			<td>Pyrex</td>
			<td></td>
			<td>7740 Glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td>Sylgard 170</td>
			<td>Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Braided Silk Suture</td>
			<td>Harvard Apparatus</td>
			<td>51-7615</td>
			<td>SP104</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope MZ6</td>
			<td>Leica</td>
			<td></td>
			<td>10447254</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomaster Microscope Fiber-Optic Light Source</td>
			<td>Fisher Scientific</td>
			<td>12562-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splinter Forceps</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Scissors</td>
			<td>George Tiemann &#38; Co</td>
			<td>105-400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KRBB Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>7487-88-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Phosphate Dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>7758-114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic/Antimicotic</td>
			<td>Corning</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Digestion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase Type 1</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Red Blood Cell Lysis Buffer</td>
			<td>BioLegend</td>
			<td>420301</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td>Thermo-Fisher Scientific</td>
			<td>2876</td>
			<td>Reciprocal Shaking Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Cabinet</td>
			<td>Thermo-Fisher Scientific</td>
			<td>1385</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotisserie Incubator</td>
			<td>Daigger</td>
			<td>EF4894C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-549</td>
			<td>Yellow</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 &#181;m Cell Strainer</td>
			<td>Thermo-Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Blue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td>Cole-Parmer</td>
			<td>UX-79001-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OctoMACS Kit</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(DMEM):F12 Medium</td>
			<td>Corning</td>
			<td>90-090</td>
			<td>Medium Base</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35016CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Donkey Serum</td>
			<td>AbCam</td>
			<td>AB7475</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD34</td>
			<td>Santa Cruz</td>
			<td>SC-7324</td>
			<td>FITC conjugated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PDGFR&#945;</td>
			<td>Thermo-Fisher Scientific</td>
			<td>PA5-17623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>712-007-003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS 10X</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>1X Working Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CO2 Cell Incubator</td>
			<td>Thermo-Fisher Scientific</td>
			<td>51030285</td>
			<td>Heracell 160i&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48- Well Plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-Well Plates, Black Wall</td>
			<td>Corning</td>
			<td>353376</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>144-55-8</td>
			<td>TC-Treated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Calf Serum</td>
			<td>Corning</td>
			<td>35011CV</td>
			<td>USA Origins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>50-81-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>58-85-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pantothenate</td>
			<td>Sigma-Aldrich</td>
			<td>137-08-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Corning</td>
			<td>61-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bone Morphogenic Protein 4</td>
			<td>Prospec Bio</td>
			<td>CYT-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal Growth Factor</td>
			<td>PeproTech</td>
			<td>400-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia Inhibitory Factor</td>
			<td>PeproTech</td>
			<td>250-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet-derived Growth Factor BB</td>
			<td>Prospec Bio</td>
			<td>CYT-740</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic Fibroblast Growth Factor</td>
			<td>PeproTech</td>
			<td>450-33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin</td>
			<td>Corning</td>
			<td>25-800-CR</td>
			<td>ITS Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IBMX</td>
			<td>Sigma-Aldrich</td>
			<td>28822-58-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>50-02-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T3 (Triiodothyronine)</td>
			<td>Sigma-Aldrich</td>
			<td>6893-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CyQUANT Proliferation Assay</td>
			<td>Thermo-Fisher Scientific</td>
			<td>C7026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AdipoRed Fluorescence Assay Reagent</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oil Red O Lipid Dye Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>O1391</td>
			<td>In Solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M1000 Microplate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse Inverted Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Sight DS-Qil Camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting

L&#39;espansione del tessuto adiposo perivascolare (PVAT), un importante regolatore della funzione vascolare attraverso la segnalazione paracrina, è direttamente correlata allo sviluppo dell&#39;ipertensione durante l&#39;obesità. La portata dell&#39;ipertrofia e dell&#39;iperplasia dipende dalla localizzazione del deposito, dal sesso e dal tipo di fenotipi di APC (Adipocyte Progenitor Cell) presenti. Le tecniche utilizzate per l&#39;APC e l&#39;isolamento dei preadipociti negli ultimi 10 anni hanno drasticamente migliorato l&#39;esattezza in cui le singole cellule possono essere identificate in base a determinati marcatori di superficie cellulare. Tuttavia, l&#39;isolamento di APC e di adipociti può essere una sfida a causa della fragilità della cellula, soprattutto se la cellula intatta deve essere mantenuta per le applicazioni di coltura cellulare. La selezione delle cellule magnetiche ( MCS) fornisce un metodo per isolare un maggior numero di APC vitali per unità di peso del tessuto adiposo. L&#39;APC raccolto da MCS può essere utilizzato per i protocolli in vitro per espandere la preaDipociti e li differenzia in adipociti attraverso l&#39;uso di cocktail di fattori di crescita che permettono l&#39;analisi del potenziale prolifico e adipogenico conservato dalle cellule. Questo esperimento si è concentrato sui depositi PVAT aortici e mesenterici, che svolgono ruoli chiave nello sviluppo della malattia cardiovascolare durante l&#39;espansione. Questi protocolli descrivono metodi per isolare, espandere e differenziare una popolazione definita di APC. Questo protocollo MCS consente l&#39;utilizzo dell&#39;isolamento in qualsiasi esperimento in cui è necessaria la classificazione delle celle con attrezzature minime o addestramento. Queste tecniche possono aiutare ulteriori esperimenti per determinare la funzionalità di popolazioni cellulari specifiche in base alla presenza di marcatori della superficie cellulare.

La capacità proliferativa dei preadipociti e il potenziale adipogenico dei precursori adipociti sono caratteristiche che vengono mantenute in vitro 11 . La proliferazione in vitro di SVF e APC isolati da aPVAT, mPVAT e GON di ratti maschi è stata valutata a 8, 24, 48 e 96 h dopo la placcatura usando un saggio quantitativo del DNA. Nessuna differenza di sito nel tasso di espansione di SVF è stata osservata in qualsiasi momento tranne c...

Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Qui riportiamo un metodo per l&#39;isolamento delle popolazioni di cellule progenitrici di Adipocyte (APC) da tessuto adiposo perivascolare (PVAT) utilizzando la selezione delle cellule magnetiche (MCS). Questo metodo consente un aumento dell&#39;isolamento di APC per grammo di tessuto adiposo se confrontato con la selezione delle cellule attivate con fluorescenza (FACS).

L&#39;espansione e l&#39;adipogenesi dell&#39;induzione di progenitori adipociti da tessuto adiposo perivascolare isolati da cellule magnetiche attivate

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

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Developmental Biology

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

Isolamento e differenziazione delle cellule staminali derivate da tessuto adiposo sottocutaneo da Suini tessuti adiposi

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

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

Isolamento e l&#39;espansione delle staminali mesenchimali stromali derivate / cellule da placenta umana 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 ...

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Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

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Induzione di acuta muscolo scheletrico Rigenerazione da cardiotossina Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

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

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Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

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Generazione di insulina senza impalcatura, tridimensionale esprimendo Pancreatoids dal Mouse progenitori pancreatico In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

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Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

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An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...