Riconosciamo la Mayo Clinic Microscopy Cell Analysis Core Flow Cytometry Facility per l'assistenza allo smistamento FACS.

Tutte le procedure sperimentali sugli animali sono state eseguite sotto l'approvazione del Comitato istituzionale per la cura e l'uso degli animali della Mayo Clinic.
1. Preparazione della soluzione 
<ol><li>Preparare la soluzione di collagenasi al 2% (w/v) sciogliendo la collagenasi II 2 g nella soluzione salina bilanciata di Hanks da 100 mL (HBSS). Aliquota 200 μL ciascuno per ogni utilizzo.</li>
	<li>Preparare il mezzo di neutralizzazione mescolando 84 mL F-12 medium, 15 mL di siero di cavallo e 1 mL penicillina/streptomicina.</li>
	<li>Preparare il tampone di citometria a flusso sciogliendo 500 mg di albumina di siero bovino (BSA) e 400 μL di 0,5 M EDTA in 100 mL della salina tamponata con fosfato (DPBS) di Dulbecco.</li>
</ol>2. Dissezione del cuscinetto adiposo epididimale e della dissociazione tissutale
<ol><li>Eutanasia umana (isoflurane/lussazione cervicale) di un topo maschio. In questo protocollo è stato utilizzato un mouse FVB di quattro mesi.</li>
	<li>Applicare/spruzzare il 70% di etanolo sull'addome ed esporre la cavità addominale inferiore utilizzando forbici e forcep pulite.</li>
	<li>Individuare il cuscinetto grasso epididimico (prossimale/attaccato ai teste). Tenere la giunzione tra teste e cuscinetto grasso epididimale con forcette smussate e tirare delicatamente per liberare il cuscinetto grasso epididimale.</li>
	<li>Rimuovere i teste tagliando con forbici e incubare il cuscinetto di grasso epididimale in 5 ml di HBSS integrato con 3% di BSA in un tubo conico da 50 ml a temperatura ambiente per 15 minuti.</li>
	<li>Centrifuga a 150 x g per 7 min a 4 °C.</li>
	<li>Prendere il cuscinetto di grasso epididimale galleggiante dal tubo conico da 50 ml e tritare finemente il tessuto usando forbici pulite.</li>
	<li>Aggiungere 200 μL di collagenasi soluzione al 2% in 3,8 ml di HBSS in un tubo di coltura da 13 ml. Aggiungere il tessuto tritato e incubare in un'incubatrice rotante a 5 giri/min per 1 h a 37 °C. Invece di un incubatore rotante, l'incubatore di coltura cellulare a 37 °C può essere usato alternativamente con agitazioni occasionali.</li>
	<li>Trasferire l'intero contenuto in un tubo conico da 50 ml e aggiungere 10 ml di mezzo di neutralizzazione. Mescolare delicatamente invertendo il tubo 2-3 volte.</li>
	<li>Preparare un altro tubo conico da 50 ml dotato di un filtro a celle da 70 μm. Filtrare il tessuto digerito nel nuovo tubo utilizzando il filtro cellulare.</li>
	<li>Trasferire il flusso fino a un tubo conico da 15 ml e centrifugare a 350 x g per 10 min a 4 °C.</li>
	<li>Rimuovere con cura i pellet di cellule supernatanti e resuspend con 5 mL di DPBS. Centrifuga a 350 x g per 10 min a 4 °C.</li>
	<li>Rimuovere con cura il supernatante e aggiungere 50 μL di tampone di citometria di flusso. Mescolare bene tubazione delicatamente e mantenere le cellule sul ghiaccio. A causa del volume del pellet cellulare e della piccola quantità di supernatante residuo, il volume totale di cellule nel buffer di citometria del flusso sarà maggiore di 50 μL (circa 100 μL).</li>
</ol>3. Etichettatura degli anticorpi e smistamento delle cellule attivate a fluorescenza (FACS)
<ol><li>Dopo aver mescolato bene le cellule con una tubazione delicata, trasferire 54 μL della sospensione cellulare in un tubo di microcentrifugo da 1,7 ml. Aggiungere 6 μL di reagente bloccante FcR e mescolare bene tubazione delicatamente. Incubare a 4 °C per 10 min. Salvare le cellule rimanenti dopo aver preso 54 μL e tenerle nel ghiaccio da utilizzare come controllo non macchiato nel passaggio 3.5 e nel passaggio 3.6.</li>
	<li>Durante l'incubazione nella fase 3.1, preparare un cocktail di anticorpi combinando 11 μL ciascuno di anticorpi anti-Sca1-APC, anti-Ter119-FITC, anti-CD31-FITC e anti-CD45-FITC in un tubo di microcentrifugo. Mescolare bene con una pipettazione delicata e mantenere il ghiaccio protetto dalla luce.</li>
	<li>Dopo l'incubazione con reagente bloccante FcR per 10 min, aggiungere 40 μL del cocktail anticorpale e mescolare bene con pipettaggio delicato. Incubare a 4 °C per 10 min.</li>
	<li>Aggiungere 500 μL di tampone di citometria di flusso integrato con 1 μg/mL di DAPI nelle celle macchiate. Mescolare bene con tubazioni delicate e celle filtranti utilizzando una provetta da 5 ml con tappo a scatto del filtro a celle. Tieni le cellule sul ghiaccio.</li>
	<li>Aggiungere 500 μL di tampone di citometria di flusso alle cellule rimanenti nel passaggio 3.1 e mescolare bene con una pipettazione delicata. Filtrare le celle utilizzando una provetta da 5 ml con tappo a scatto del filtro cellulare. Tenere queste cellule sul ghiaccio e utilizzarle come controllo non macchiato nel passaggio seguente.</li>
	<li>Porta le celle macchiate e il controllo non macchiato allo strumento di analisi o ordinamento FACS.<ol>
<li>Identificare e isolare la popolazione APC+/FITC-/DAPI- con strategie di gating mostrate nella figura 1. Queste celle rappresentano cellule Sca1+/Ter119-/CD31-/CD45. I detriti e gli aggregati cellulari sono esauriti utilizzando grafici a dispersione in avanti (FSC) e a dispersione laterale (SSC). Quindi, DAPI- la popolazione è gated, seguita da gating di APC+/ FITC- popolazione. I controlli non macchiati devono essere utilizzati per aiutare a impostare i parametri di gating.</li>
		<li>Raccogliere le cellule in 500 μL di tampone di citometria di flusso in un tubo di microcentrifugo da 1,5 ml. Andando avanti, tutte le procedure devono essere eseguite in condizioni sterili per ridurre al minimo una contaminazione. Circa 50.000 - 100.000 cellule vengono raccolte da un topo.</li>
	</ol>
</li>
	<li>Contare le cellule usando un emocitometro per valutare la vitalità cellulare. Poiché gli aggregati cellulari possono interferire con ulteriori analisi a valle, viene valutata anche la presenza di aggregati cellulari per garantire una presenza minima (<5% del numero di cellule vitali) di questi aggregati.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+obesity+in+man:+cellular+character+of+the+adipose+tissue.">Experimental obesity in man: cellular character of the adipose tissue.</a> The Journal of Clinical Investigation. 50, 1005-1011 (1971).</li><li>Trayhurn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+adipose+tissue+function+and+dysfunction+in+obesity.">Hypoxia and adipose tissue function and dysfunction in obesity.</a> Physiological Reviews. 93, 1-21 (2013).</li><li>Halberg, N., 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=Hypoxia-inducible+factor+1alpha+induces+fibrosis+and+insulin+resistance+in+white+adipose+tissue.">Hypoxia-inducible factor 1alpha induces fibrosis and insulin resistance in white adipose tissue.</a> Molecular and Cellular Biology. 29, 4467-4483 (2009).</li><li>Upadhyay, J., Farr, O., Perakakis, N., Ghaly, W., Mantzoros, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+as+a+Disease.">Obesity as a Disease.</a> Medical Clinics of North America. 102, 13-33 (2018).</li><li>Cawthorn, W. P., Scheller, E. L., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+stem+cells+meet+preadipocyte+commitment:+going+back+to+the+future.">Adipose tissue stem cells meet preadipocyte commitment: going back to the future.</a> Journal of Lipid Research. 53, 227-246 (2012).</li><li>Cho, D. S., Lee, B., Doles, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refining+the+adipose+progenitor+cell+landscape+in+healthy+and+obese+visceral+adipose+tissue+using+single-cell+gene+expression+profiling.">Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.</a> Life Science Alliance. 2, 201900561 (2019).</li><li>Burl, R. B., 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=Deconstructing+Adipogenesis+Induced+by+beta3-Adrenergic+Receptor+Activation+with+Single-Cell+Expression+Profiling.">Deconstructing Adipogenesis Induced by beta3-Adrenergic Receptor Activation with Single-Cell Expression Profiling.</a> Cell Metabolism. 28, 300-309 (2018).</li><li>Hepler, C., 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=Identification+of+functionally+distinct+fibro-inflammatory+and+adipogenic+stromal+subpopulations+in+visceral+adipose+tissue+of+adult+mice.">Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.</a> eLife. 7, 39636 (2018).</li><li>Merrick, D., 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=Identification+of+a+mesenchymal+progenitor+cell+hierarchy+in+adipose+tissue.">Identification of a mesenchymal progenitor cell hierarchy in adipose tissue.</a> Science. 364, 2501 (2019).</li><li>Schwalie, P. C., 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=A+stromal+cell+population+that+inhibits+adipogenesis+in+mammalian+fat+depots.">A stromal cell population that inhibits adipogenesis in mammalian fat depots.</a> Nature. 559, 103-108 (2018).</li><li>Raajendiran, A., 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=Identification+of+Metabolically+Distinct+Adipocyte+Progenitor+Cells+in+Human+Adipose+Tissues.">Identification of Metabolically Distinct Adipocyte Progenitor Cells in Human Adipose Tissues.</a> Cell Reports. 27, 1528-1540 (2019).</li><li>De Matteis, R., 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=In+vivo+physiological+transdifferentiation+of+adult+adipose+cells.">In vivo physiological transdifferentiation of adult adipose cells.</a> Stem Cells. 27, 2761-2768 (2009).</li><li>Hanamsagar, R., 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=An+optimized+workflow+for+single-cell+transcriptomics+and+repertoire+profiling+of+purified+lymphocytes+from+clinical+samples.">An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples.</a> Scientific Reports. 10, 2219 (2020).</li><li>Marinovic, M. P., 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=Crotamine+induces+browning+of+adipose+tissue+and+increases+energy+expenditure+in+mice.">Crotamine induces browning of adipose tissue and increases energy expenditure in mice.</a> Scientific Reports. 8, 5057 (2018).</li><li>Takahashi, H., 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=Biological+and+clinical+availability+of+adipose-derived+stem+cells+for+pelvic+dead+space+repair.">Biological and clinical availability of adipose-derived stem cells for pelvic dead space repair.</a> Stem Cells Translational Medicine. 1, 803-810 (2012).</li><li>Cowan, C. M., 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=Adipose-derived+adult+stromal+cells+heal+critical-size+mouse+calvarial+defects.">Adipose-derived adult stromal cells heal critical-size mouse calvarial defects.</a> Nature Biotechnology. 22 (5), 560-567 (2004).</li><li>Hagberg, C. E., 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=Flow+Cytometry+of+Mouse+and+Human+Adipocytes+for+the+Analysis+of+Browning+and+Cellular+Heterogeneity.">Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity.</a> Cell Reports. 24, 2746-2756 (2018).</li></ol>

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Il sequenziamento dell'RNA a singola cellula (scRNA-seq) sta rapidamente guadagnando trazione come un potente strumento per studiare contemporaneamente diverse popolazioni cellulari a livello di singola cellula. A causa degli elevati costi associati alla preparazione del campione e all'elevato sequenziamento della produttività, è imperativo ottimizzare gli input cellulari (alta vitalità e purezza) per aumentare la probabilità di successo sperimentale. Alcuni protocolli di preparazione cellulare si basano sulla rimozi...

Il tessuto adiposo gioca un ruolo chiave nel metabolismo energetico. L'energia in eccesso viene immagazzinata sotto forma di lipidi e il tessuto adiposo è in grado di espansione o retrazione significativa a seconda dello stato nutrizionale e della domanda energetica. L'espansione del tessuto adiposo può derivare da un aumento delle dimensioni degli adipocite (ipertrofia) e/o da un aumento del numero di adipocite (iperplasia); quest'ultimo processo strettamente regolato dalla proliferazione e differenziazione delle cellule progenitrici adipose1,2. Durante l'obesità, il tessuto adiposo si espande eccessivamente e la disfunzione tissutale - tra cui ipossia, infiammazione e resistenza all'insulina -si sviluppa spesso 3,4. Queste complicanze sono fattori di rischio per molte malattie croniche tra cui ipertensione, diabete, malattie cardiovascolari, ictus e cancro5. Pertanto, limitare l'espansione incontrollata dei tessuti adiposi e mitigare le patologie tissutali adipose sono le principali priorità di ricerca biomedica. Durante l'espansione del tessuto adiposo, le cellule staminali derivate dai tessuti adiposi residenti (ASC) proliferano e si differenziano in sequenza in preadipociti (cellule progenitrici impegnate) e quindi in adipociti maturi6. Recenti studi di sequenziamento dell'RNA a singola cellula (scRNA-seq) mostrano che queste popolazioni di cellule progenitrici adipose (APC) (ASC e preadipociti) mostrano una sostanziale eterogeneità molecolare e funzionale7,8,9,10,11,12. Ad esempio, gli ASC mostrano una ridotta capacità di differenziazione adipogenica, ma presentano anche maggiori capacità di proliferazione ed espansione, rispetto ai preadipociti7. Ulteriori differenze molecolari sono riportate all'interno delle popolazioni ASC e preadipocite, anche se la rilevanza funzionale di queste differenze rimane poco chiara7. Insieme, questi dati evidenziano la complessità del pool di cellule progenitrici adipose e sottolineano la necessità di sviluppare e standardizzare strumenti per comprendere e manipolare meglio queste popolazioni di cellule critiche.
Questo protocollo descrive in dettaglio l'isolamento delle popolazioni di cellule progenitrici ad alta vitalità Sca1+ adipose dalle pastiglie di grasso epididimomale del topo adatte per analisi sensibili a valle, tra cui studi a cella singola (sequenziamento scRNA) e coltura cellulare. L'isolamento e la dissociazione dei cuscinetti di grasso epididimali è statoeseguito come descritto in precedenza 7,13 con lievi modifiche che migliorano la vitalità delle APC isolate. In breve, le cellule dissociate dai cuscinetti adiposi epididimali sono macchiate di anticorpi contro Sca1, un marcatore sia per gli ASC che per i preadipociti6,7e altri marcatori di lignaggio (Lin): Ter119 (cellule erythroid), CD31 (cellule endoteliali) e CD45 (leucociti). Sca1+/Ter119-/CD31-/CD45-/DAPI- le celle vengono quindi ordinate in base all'ordinamento cellulare attivato a fluorescenza (FACS). È importante sottolineare che questo protocollo è stato convalidato da un riuscito isolamento e analisi di cellule progenitrici sca1+/lin- adipose praticabili riportate in un recente studio di sequenziamento dell'RNA a singola cellula che ha identificato sottopopolazioni funzionalmente eterogenee all'interno di ASC e preadipociti7.

<table><tbody><tr><td>1.7 mL microcentrifuge tube</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>13 mL culture tube</td><td>Thermo Fisher Scientific</td><td>50-809-216</td><td></td></tr><tr><td>15 mL conical tube</td><td>Greiner Bio-one</td><td>188 271</td><td></td></tr><tr><td>5 mL test tube with cell strainer snap cap</td><td>Thermo Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>50 mL conical tube</td><td>Greiner Bio-one</td><td>227 261</td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Thermo Fisher Scientific</td><td>22-363-548</td><td></td></tr><tr><td>Anti-CD31-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-519</td><td></td></tr><tr><td>Anti-CD45-FITC antibody</td><td>Miltenyi Biotec</td><td>130-102-491</td><td></td></tr><tr><td>Anti-Sca1-APC antibody</td><td>Miltenyi Biotec</td><td>130-102-833</td><td></td></tr><tr><td>Anti-Ter119-FITC antibody</td><td>Miltenyi Biotec</td><td>130-112-908</td><td></td></tr><tr><td>BSA</td><td>Gold Biotechnology</td><td>A-420-500</td><td></td></tr><tr><td>Collagenase type II</td><td>Thermo Fisher Scientific</td><td>17101-015</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190-144</td><td></td></tr><tr><td>F-12 medium</td><td>Thermo Fisher Scientific</td><td>11765-054</td><td></td></tr><tr><td>FcR blocking reagent</td><td>Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>Hanks&#039; balanced salt solution (HBSS)</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>16050-122</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Propidium iodide solution</td><td>Miltenyi Biotec</td><td>130-093-233</td><td></td></tr></tbody></table>

preparation of adipose progenitor cells from mouse epididymal adipose tissues

L'obesità e i disturbi metabolici come diabete, malattie cardiache e cancro sono tutti associati a un drammatico rimodellamento dei tessuti adiposi. Le cellule progenitrici adipose residenti nei tessuti (APC) svolgono un ruolo chiave nell'omeostasi dei tessuti adiposi e possono contribuire alla patologia tissutale. L'uso crescente di tecnologie di analisi a singola cellula , tra cui il sequenziamento dell'RNA a singola cellula e la proteomica a singola cellula, sta trasformando il campo delle cellule staminali / progenitrici consentendo una risoluzione senza precedenti dei cambiamenti di espressione delle singole cellule nel contesto dei cambiamenti a livello di popolazione o tessuto. In questo articolo, forniamo protocolli dettagliati per sezionare il tessuto adiposo epididimale del topo, isolare le singole cellule derivate dai tessuti adiposi ed eseguire lo smistamento delle cellule attivate a fluorescenza (FACS) per arricchire per uno Sca1+/ CD31-/CD45-/Ter119- APC praticabile. Questi protocolli consentiranno agli investigatori di preparare APC di alta qualità adatti per analisi a valle come il sequenziamento dell'RNA a cella singola.

In questo esperimento sono stati utilizzati topi FVB maschi di quattro mesi. Dopo l'esclusione di detriti e doppietti utilizzando appezzamenti FSC/SSC, le cellule vitali (DAPI- popolazione) sono state gated, seguite dalla selezione di APC+/FITC- popolazione (Figura 1). I gate DAPI, APC e FITC sono stati disegnati in base al controllo non macchiato. Le strategie di gating sono mostrate nella figura 1.
D...

Watch this Scientific Journal Video about Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues at JoVE.com

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Presentiamo un metodo semplice per isolare le cellule progenitrici adipose altamente vitali dalle pastiglie di grasso epididimale del topo utilizzando lo smistamento delle cellule attivato a fluorescenza.

Preparazione di cellule progenitrici adipose dai tessuti adiposi epididimali del topo

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

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5.6K Views.  Mayo Clinic. Presentiamo un metodo semplice per isolare le cellule progenitrici adipose altamente vitali dalle pastiglie di grasso epididimale del topo utilizzando lo smistamento delle cellule attivato a fluorescenza.

Video: Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Generazione di cellule staminali adiposo umano attraverso dedifferenziazione di adipociti maturi in Culture soffitto

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

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Biologia dello sviluppo

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 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. ...

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

<meta charset="utf8"></head><body>Isolamento ed elaborazione di adipociti bianchi murini per analisi di trascrittoma ed epigenoma

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Isolamento ed elaborazione di adipociti bianchi murini per analisi di trascrittoma ed epigenoma

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Genetica

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Isolamento, espansione e adipogenico induzione di CD34 + CD31 + cellule endoteliali dal tessuto adiposo umano omentale e sottocutaneo

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Immunologia e infezioni

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

Isolamento di sottopopolazioni di cellule stromali adipogeniche e fibro-infiammatorie da depositi adiposi intra-addominali murini

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

Isolamento e differenziazione dei preadipociti bianchi e marroni primari dai topi appena nati

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Biologia

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Adipociti di topo differenziati in coltura primaria: un modello di insulino-resistenza

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

Coltura di preadipociti isolati da tessuto adiposo perivascolare murino

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Autore in primo piano: Il significato dell'isolamento, della coltura e dell'induzione adipogenica dei preadipociti derivati da SVF dal tessuto adiposo perivascolare di topo 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Preparazione di cellule progenitrici adipose dai tessuti adiposi epididimali del topo

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Isolamento ed elaborazione di adipociti bianchi murini per analisi di trascrittoma ed epigenoma

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