This study was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant (371697-2011, AT). The authors want to acknowledge the help of bariatric surgeons Drs S. Biron, F-S. Hould, S. Lebel, O. Lescelleur, P. Marceau as well as Christine Racine and Caroline Gagnon from the IUCPQ Tissue Bank. We thank Mr Jacques Cadorette from the IUCPQ&#8217;s audiovisual services for video shooting and editing.

Dichiarazione etica: Il progetto è stato approvato dal Comitato Etico della Research IUCPQ prima del reclutamento dei pazienti. Ai fini del presente articolo / video, abbiamo ottenuto i tessuti da 2 pazienti: 1) A 62 anni, paziente di sesso maschile con un BMI di 50,7 kg / m 2 e 2) 35 anni paziente di sesso femminile con un BMI di 57 kg / m 2. Esperimenti può essere fatto con due scomparti grasso, ma sono limitati a un compartimento grasso ai fini di questo video. Aspetti tecnici del video sono state effettuate con il paziente e 1 FABP4 immunofluorescenza è stata eseguita con le cellule dedifferenziate da paziente 2. Elaborazione 1. Campione <ol><li> Chiedi chirurghi a raccogliere il tessuto adiposo dal comparto grasso omentale e sottocutaneo, al momento della chirurgia bariatrica laparoscopica. </li><li> Portare rapidamente campioni adipose al laboratorio a temperatura ambiente ed elaborare immediatamente. </li><li> Eseguire la digestione in laboratorio, in un ambiente non sterile. Ilcellule saranno eventualmente trasferiti nel locale cultura e coltivati ​​in condizioni sterili. Per evitare la contaminazione, preparare tampone KRH con acqua distillata e filtrata e seguire da una filtrazione (filtro 0.22μM) prima della digestione. Tubi accuratamente puliti con etanolo prima cessione nel cofano coltura cellulare per pallone e preparazione piastra. </li><li> Collocare il tessuto adiposo su un piatto pre-pesate e il peso record. Fissare una piccola parte di ogni campione di tessuto (meno di 1 cm 2) in 10% formalina tampone a temperatura ambiente per almeno 24 ore prima della paraffina. Utilizzare questo esempio incorporato per esperimenti di immunoistochimica (tecnica non mostrato). </li><li> Inserire un altro pezzo in una provetta da 50 ml e flash-congelamento in azoto liquido prima della conservazione a -80 ° C per ulteriori studi sui tessuti adiposi interi (ad esempio, l&#39;espressione genica - tecnica non mostrati). </li></ol> 2. Collagenase Digestione <ol><li> Posizionare il restante pezzo di tessuto adiposo in un ml tu 50essere per la digestione. </li><li> Aggiungere 4 ml di KRH-WB integrati con collagenasi (350 U / ml) per grammo di campione nel tubo di digestione. </li><li> Tritate tessuto adiposo con le forbici. </li><li> Inserire sospensione tessuto adiposo macinate in un agitatore, 37 ° C, 90 rpm massima, per una incubazione di 45 minuti (massimo 1 ora). </li></ol> 3. Purificazione di adipociti e preadipociti <ol><li> Versare la soluzione traslucido con pochi pezzi di grasso attraverso un nylon 400 micron maglia in un bicchiere di plastica. </li><li> Con pinzette, strofinare la preparazione cellulare sulla rete di nylon e lavare con 5 ml di KRH-WB. </li><li> Delicatamente trasferire la sospensione cellulare filtrato in un tubo da 50 ml con il tubo di plastica in esso e una siringa 60cc attaccata all&#39;estremità tubo. </li><li> Sia la sospensione con adipociti maturi riposare per circa 10 min, permettendo alle cellule di raggiungere la parte superiore del buffer galleggiamento. </li><li> Lentamente aspirare il tampone nella parte inferiore del tubo utilizzando 60cc Syrinaspirazione ge. </li><li> Aggiungere 20 ml di KRH-WB per lavare. Ripetere la procedura dal punto 3.4 per 2 lavaggi aggiuntivi. </li><li> Raccogliere il buffer di portare la sospensione adipociti ad un volume finale di 5 o 10 ml, a seconda della quantità di cellule. Perseguire con passaggi nella sezione 5. </li><li> Recupero della frazione stromale-vascolare dal buffer raccolti con la siringa 60cc mediante centrifugazione (3000 rpm, RT, 5 min) per ulteriore coltura cellulare primaria se desiderato (tecnica non mostrato). </li></ol> Cell Count 4. adipociti maturi <ol><li> Carico 10 ml di sospensione degli adipociti scosso delicatamente in una camera di conteggio (emocitometro). Eseguire conta delle cellule in quadruplicato. </li><li> Calcolare il numero di cellule mature isolate. </li></ol> 5. Coppia degli adipociti dedifferenziazione in T-25 Flask <ol><li> Riempire a 25 cm 2 tessuto pallone di coltura a ¾ del volume con il siero di vitello DMEM / F12-20%. </li><li> Secondo il conteggio delle cellule, versare 500.000 cellule mature nel pallone. </li&gt; <li> Riempire il pallone completamente utilizzando un tubo da 50 ml con il mezzo ed eliminare come bolle che possibile. </li><li> Avvitare il tappo non ventilate sul pallone. </li><li> Pulire il pallone con etanolo prima dell&#39;incubazione per evitare la contaminazione. </li><li> Incubare la beuta capovolto per una settimana senza toccarlo per evitare movimenti nella cultura che potrebbero disturbare l&#39;adesione cellulare. </li><li> Prima invertendo il matraccio a 7 giorni di coltura invertito, manipolare delicatamente il matraccio e rimuovere tutta medie nel pallone per aspirazione, evitando movimenti bruschi. </li><li> Aggiungere 12 ml di siero di vitello% DMEM-F12-20 e coltivare cellule con tecniche standard. Un filtrato, tappo ventilato può essere aggiunto al pallone. </li></ol> 6. maturo degli adipociti dedifferenziazione in un 6-pozzetti <ol><li> Inserire un coprioggetto sul fondo di ciascun pozzetto di una piastra da 6 pozzetti </li><li> Aggiungi un ½ &quot;boccola di plastica sulla parte superiore di ogni coprioggetto. </li><li> Riempire pozzetti con 8 ml di 20% di siero di vitello-DMEM. </li><li> Metti un coprioggetto su ogni boccola in plastica. </li><li> Inserire puntale tra la slitta e il tubo di iniettare le cellule sotto il vetrino (50.000 cellule per pozzetto). </li><li> Incubare le piastre in un incubatore standard di coltura cellulare a 37 ° C con 5% di CO 2 per una settimana. </li><li> Coprioggetto Reverse con le cellule attaccate in ogni pozzetto contenente 2 ml di media integrato con siero di vitello del 20% e perseguire la cultura. </li><li> Utilizzare coprioggetto con cellule in fase di de-differenziazione per diversi scopi, tra cui immunofluorescenza (tecnica non mostrato). </li></ol>

<ol>
	<li>Zhang, H. H., Kumar, S., Barnett, A. H., Eggo, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ceiling+culture+of+mature+human+adipocytes:+use+in+studies+of+adipocyte+functions.">Ceiling culture of mature human adipocytes: use in studies of adipocyte functions.</a> J Endocrinol. 164 (2), 119-128 (2000).</li><li>Matsumoto, 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=Mature+adipocyte-derived+dedifferentiated+fat+cells+exhibit+multilineage+potential.">Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.</a> J Cell Physiol. 215 (1), 210-222 (2008).</li><li>Poloni, 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=Human+dedifferentiated+adipocytes+show+similar+properties+to+bone+marrow-derived+mesenchymal+stem+cells.">Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells.</a> Stem Cells. 30 (5), 965-974 (2012).</li><li>Perrini, S., 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=Differences+in+gene+expression+and+cytokine+release+profiles+highlight+the+heterogeneity+of+distinct+subsets+of+adipose+tissue-derived+stem+cells+in+the+subcutaneous+and+visceral+adipose+tissue+in+humans.">Differences in gene expression and cytokine release profiles highlight the heterogeneity of distinct subsets of adipose tissue-derived stem cells in the subcutaneous and visceral adipose tissue in humans.</a> PLoS One. 8 (3), e57892 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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+derived+from+mouse+and+human+white+mature+adipocytes.">Pluripotent stem cells derived from mouse and human white mature adipocytes.</a> Stem Cells Transl Med. 3 (2), 161-171 (2014).</li><li>Sugawara, A., Sato, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+dedifferentiated+fat+cells+for+periodontal+tissue+regeneration.">Application of dedifferentiated fat cells for periodontal tissue regeneration.</a> Hum Cell. 27 (1), 12-21 (2014).</li><li>Kikuta, S., 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=Osteogenic+effects+of+dedifferentiated+fat+cell+transplantation+in+rabbit+models+of+bone+defect+and+ovariectomy-induced+osteoporosis.">Osteogenic effects of dedifferentiated fat cell transplantation in rabbit models of bone defect and ovariectomy-induced osteoporosis.</a> Tissue Eng Part A. 19 (15-16), 1792-1802 (2013).</li><li>Obinata, 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=Transplantation+of+mature+adipocyte-derived+dedifferentiated+fat+(DFAT)+cells+improves+urethral+sphincter+contractility+in+a+rat+model.">Transplantation of mature adipocyte-derived dedifferentiated fat (DFAT) cells improves urethral sphincter contractility in a rat model.</a> Int J Urol. 18 (12), 827-834 (2011).</li><li>Jumabay, 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=Dedifferentiated+fat+cells+convert+to+cardiomyocyte+phenotype+and+repair+infarcted+cardiac+tissue+in+rats.">Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats.</a> J Mol Cell Cardiol. 47 (5), 565-575 (2009).</li><li>Ohta, Y., 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=Mature+adipocyte-derived+cells,+dedifferentiated+fat+cells+(DFAT),+promoted+functional+recovery+from+spinal+cord+injury-induced+motor+dysfunction+in+rats.">Mature adipocyte-derived cells, dedifferentiated fat cells (DFAT), promoted functional recovery from spinal cord injury-induced motor dysfunction in rats.</a> Cell Transplant. 17 (8), 877-886 (2008).</li><li>Moreno-Navarrete, J. M. F. -. r., Symonds, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Adipose Tissue Biology. , 17-38 (2012).</li><li>Poulos, S. P., Dodson, M. V., Hausman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+models+for+differentiation:+preadipocytes+and+adipocytes.">Cell line models for differentiation: preadipocytes and adipocytes.</a> Exp Biol Med (Maywood. 235 (10), 1185-1193 (2010).</li><li>Casteilla, L., Penicaud, L., Cousin, B., Calise, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+adipose+tissue+depot+for+sampling:+factors+in+selection+and+depot+specificity).">Choosing an adipose tissue depot for sampling: factors in selection and depot specificity).</a> Methods Mol Biol. 456, 23-38 (2008).</li><li>Tchernof, A., Despres, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+human+visceral+obesity:+an+update.">Pathophysiology of human visceral obesity: an update.</a> Physiol Rev. 93 (1), 359-404 (2013).</li><li>Rodbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+Isolated+Fat+Cells.+I.+Effects+of+Hormones+on+Glucose+Metabolism+and+Lipolysis.">Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis.</a> J Biol Chem. 239, 375-380 (1964).</li><li>Watson, J. 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=Comparison+of+Markers+and+Functional+Attributes+of+Human+Adipose-Derived+Stem+Cells+and+Dedifferentiated+Adipocyte+Cells+from+Subcutaneous+Fat+of+an+Obese+Diabetic+Donor.">Comparison of Markers and Functional Attributes of Human Adipose-Derived Stem Cells and Dedifferentiated Adipocyte Cells from Subcutaneous Fat of an Obese Diabetic Donor.</a> Adv Wound Care. 3 (3), 219-228 (2014).</li></ol>

The authors declare no conflict of interest.

Dedifferenziazione di adipociti maturi con la tecnica di coltura soffitto è un nuovo approccio per ottenere cellule staminali adipose da un piccolo campione di tessuto adiposo nativo. Sulla base della nostra esperienza e quella di altri 2, un grammo di tessuto è sufficiente al piatto un pallone da 25 cm 2 e per ottenere una popolazione di cellule DFAT per i quali l&#39;omogeneità è stata dimostrata da Poloni e collaboratori 3. Adipociti dedifferentiation sembra possibile con le cellule da qualsiasi donatore, indipendentemente dalla loro età, sesso e altre caratteristiche. Tra la popolazione risultante DFAT ottenuto, rimane qualche giro o in parte le cellule allungate che non dedifferentiate completamente. Queste cellule sono solitamente scartati attraverso il passaggio cultura galleggiano nel mix tripsina-media. Multipotenza di queste cellule è stabilita e supporta il loro uso per la terapia cellulare 2,3. La loro elevata capacità proliferativa è stato segnalato anche, che isa aspetto importante di coltura cellulare per le applicazioni delle cellule staminali 2. Gli studi con cellule DFAT umani hanno indicato che possono essere più efficiente di ASC dallo stesso donatore, in base alla loro capacità di replicazione e differenziazione 18. Un recente studio caso sostiene che le cellule DFAT erano più efficienti di differenziarsi in adipociti e osteoblasti, e avevano livelli di telomerasi elevati rispetto ASC dallo stesso individuo, un donatore con l&#39;obesità e il diabete 18. Pertanto, l&#39;uso della cultura massimale può fornire cellule staminali adipose più efficienti rispetto alla ASC già utilizzato. Tuttavia, sono necessari ulteriori esperimenti per valutare chiaramente questo punto. La 6-ben tecnica di coltura soffitto piastra consente lo studio del processo dedifferentiation stesso. Un numero minimo di cellule può essere placcato e consente lo studio di specifici punti temporali. Ad esempio, abbiamo raccolto il vetrino da un 6-pozzetti per eseguire immunofluorescenza da adipociti undergdedifferentiation oing (Figura 2). Esecuzione miscroscopy, con o senza di fluorescenza, è molto rilevante per valutare i vari aspetti della dedifferentiation. Oltre alle applicazioni di celle staminali, cellule DFAT possono rappresentare un interessante modello per gli studi fisiologici. Solo pochi studi hanno esaminato l&#39;espressione genica e le funzioni di entrambi i tipi di cellule. In breve, ASC e DFAT dallo stesso vano di grassi ha mostrato somiglianze nell&#39;espressione genica e la secrezione 4. Sono necessari altri confronti tra ASC e DFAT dallo stesso donatore. In conclusione, vi mostriamo in questa relazione tecnica come ottenere cellule DFAT dal tessuto adiposo umano utilizzando la tecnica consolidata di adiposo collagenasi tessuti digestione e la tecnica di coltura soffitto. Il nostro formato originale 6 pozzetti può contribuire ad aumentare la conoscenza del processo di de-differenziazione, mentre il metodo del pallone più comunemente utilizzato permette la generazione di grandi poporegola- di cellule DFAT. Il limite maggiore di questo protocollo è l&#39;accesso al tessuto adiposo umano che si basa sulla collaborazione con un team di chirurgia e di etica di gestione per ottenere paziente consenso informato per iscritto. Cellule mature sono molto sensibile, sono necessarie precauzioni nella manipolazione. Abbiamo ottimizzato il protocollo, in particolare il numero di adipociti da placcati in T-25flask e il formato 6 pozzetti, per ottenere risultati ottimali e potrebbe essere anticipato senza grandi modifiche aggiuntive o risoluzione di problemi.

In vitro dedifferentiation of mature adipocytes is achieved through a technique called ceiling culture1. Because of their natural tendency to float in aqueous solutions, isolated mature adipocytes adhere to the surface of an inverted flask fully filled with culture medium. Over a few days, cells modify their spherical morphology and become fibroblast-like cells. The resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent2. Research articles on adipocyte dedifferentiation, especially on human cells, are limited. However, they have already provided interesting information regarding multipotency, cell phenotype and replicative capacity of DFAT cells2. Mature adipocytes originating from various fat compartments have been successfully dedifferentiated including those originating from human visceral and subcutaneous adipose tissues2-4. In addition to these depots, Kishimoto and collaborators sampled adipose tissue from the buccal fat pads and dedifferentiated adipocytes into DFAT cells5. Matsumoto and collaborators successfully generated subcutaneous DFAT cells from patients covering a wide range of ages, and the majority of cells had a high proliferative rate and less than 6% of senescence even after 10 passages in culture2.
DFAT cells have been successfully re-differentiated into several lineages, including adipogenic, osteogenic, chondrogenic and neurogenic lineages2,3,6. These cells express several embryonic stem cell markers such as Nanog and the four identified pluripotent factors Oct4, c-myc, Klf4 and Sox23. They also express markers specific to each of the three germ layers7. In addition, DFAT cells are similar to Bone Marrow-derived Mesenchymal Stem Cells (BM-derived MSC) based on their epigenetic signature3. Exploiting the stem cell capacity of DFAT cells, many groups have investigated their potential to treat or improve various diseases8,9. Improvements of pathologic conditions, such as infracted cardiac tissue, spinal cord injury and urethral sphincter dysfunction, have been observed with DFAT cell injections in rat models of disease10-12. 
In addition to the stem cell properties of DFAT cells, they may represent a new cellular model for adipocyte physiology studies. The 3T3-L1 cell line is often used for this purpose as these cells differentiate into adherent, lipid-storing adipocytes under adipogenic stimulation13. However, these cells originate from mouse embryo tissue13. Also, depot-specificity cannot be investigated with this model and it may not fully reflect human adipocyte physiology14. Other laboratories work with isolated adipose cells from murine fat depots, but fat distribution is not dimorphic in mice and anatomical configuration of the rodent's abdominal cavity prevents from extrapolating directly to humans15. In order to study adipocytes in the context of the physiopathology of human obesity, consideration of body fat distribution and fat depot-specific differences has become essential16. Some limitations of primary preadipocyte cultures, including cell quantities obtained from adipose tissue biopsy samples and their senescence after a few passages in culture, created the need for alternate models. Perrini and collaborators investigated depot-specificity in gene expression of DFAT cells originating from visceral and subcutaneous fat and compared them to adipose-derived stem cells (ASC) from the same fat depot. They demonstrated that differences in gene expression and function where mainly found between depots than between cell types, suggesting that DFAT cells are physiologically close to ASC from the same depot. DFAT cells may represent an interesting alternative to available models for studies on fat distribution in the pathophysiology of human obesity. Moreover, ceiling culture is a promising method to obtain adult stem cells for tissue engineering purposes. 
Here, we describe collagenase digestion, a widely-used technique to isolate mature adipocytes from the subcutaneous and/or visceral fat samples17, and the subsequent steps to perform ceiling culture and dedifferentiate these cells into multipotent, fibroblast-like cells.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bovine serum albumine</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma</td>
			<td>A4036</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A0278</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Type I collagenase</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS-004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, no phenol red</td>
			<td>Gibco-Life Technologies</td>
			<td>11039-021</td>
			<td>Add to medium : 20% calf serum, gentamicin (50&#181;g/ml) and fungizone (2.5&#181;g/ml)</td>
		</tr>
		<tr>
			<td>Calf Serum, iron supplemented, from formula-fed calves</td>
			<td>Sigma</td>
			<td>C8056-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 In plastic bushing</td>
			<td>Iberville</td>
			<td>2704-CP</td>
			<td>SKU:1000120918 (Home Depot)</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Linde</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin soluton, neutral buffered, 10%</td>
			<td>SIGMA</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60cc syringes</td>
			<td>BD Syringe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Ringer-Henseleit stock buffer (KRH)</td>
			<td></td>
			<td></td>
			<td>Prepare stock buffer as following: 25mM HEPES pH7.6, 125mM NaCl, 3.73mM KCl, 5mM CaCl2.2H2O, 2.5mM MgCl2.6H2O, 1mM K2HPO4. Adjust pH to 7.4.</td>
		</tr>
		<tr>
			<td>Krebs-Ringer-Henseleit-Working Buffer (KRH-WB)</td>
			<td></td>
			<td></td>
			<td>Add the following components freshly to KRH buffer: 4% bovine serum albumin, 5mM glucose, 0.1&#181;M adenosine, 560 &#181;M ascorbic acid</td>
		</tr>
		<tr>
			<td>KRH-WB supplemented with Type I collagenase</td>
			<td></td>
			<td></td>
			<td>Add 350U/ml of Type I collagenase</td>
		</tr>
		<tr>
			<td>T25 unvented cap tissue culture flask</td>
			<td>Sarsted or other brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>BD Falcon or other brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 22x22</td>
			<td>Fisherbrand</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile beakers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Importanti cambiamenti morfologici avvengono a maturare adipociti durante dedifferentiation (Figura 1). Come mostrato in Figura 2, le cellule in fase dedifferentiation state colorate con un anticorpo anti-FABP4 per analisi di fluorescenza. Le cellule con una morfologia rotonda espresso la proteina FABP4 mentre la maggior parte delle cellule di fibroblasti, come non ha fatto. Dopo dedifferentiation, cellule DFAT possono essere coltivati ​​con procedure standard per diversi passaggi. Siamo stati in grado di raggiungere più di 15 passaggi per omentale umana e sottocutanei linee cellulari DFAT (dati non riportati). <img alt="Figura 1" src="/files/ftp_upload/52485/52485fig1.jpg" /> Figura 1. Morfologia di dedifferentiating adipociti maturi nel tempo (A) a 4 giorni (B) a 7 giorni e (C) a 12 giorni di coltura. Foto sono state scattate in diversi punti temporali durante l&#39;incubazione con un Microscopio a contrasto di fasee. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52485/52485fig1large.jpg" target="_blank">Fare clic qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52485/52485fig2.jpg" /> Figura 2. Rilevamento di proteine ​​FABP4 in adipociti sottoposti dedifferenziazione. Le cellule sono state fissate dopo 13 giorni di de-differenziazione e colorati con anti-FABP4 anticorpi per immunofluorescenza. I nuclei sono stati visualizzati con colorazione DAPI. A sinistra: immagine Brightfield del corrispondente immunofluorescenza. A destra: L&#39;immagine unita viene mostrato (FABP4-rosso, Nuclei-blu-10X). Gli adipociti con morfologia rotonda FABP4 express, un marcatore adipociti maturi, mentre le cellule allungate esprimono più esso. Scala bar: 1 unità = 0,25 millimetri <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52485/52485fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures at JoVE.com

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

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 ...

generation-human-adipose-stem-cells-through-dedifferentiation-mature

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.6K Views.  IUCPQ Research Center. 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.

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

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.

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

Isolamento e arricchimento di Human derivate da tessuto adiposo cellule stromali di Osteogenesi avanzata

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

Ricerca

Biologia dello sviluppo

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Generazione di cellule staminali Integrazione-libero umano pluripotenti indotte Utilizzando capelli derivati ​​da cheratinociti

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Generazione di culture geneticamente modificate pelle organotipiche Uso devitalizzate derma umano

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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. ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Generazione di cellule staminali pluripotenti indotte da melanoma umano Linfociti infiltranti il ​​tumore

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generazione di integrazione-liberi cellule staminali pluripotenti indotte dalle cellule mononucleari di sangue periferico umano Utilizzando episomiale Vettori

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Regia di differenziazione dei progenitori ematopoietici primitivi e definitivo da cellule staminali pluripotenti umane

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Definito e Generazione scalabile di cellule epatociti-come da cellule staminali umane pluripotenti

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

Precondizionamento ipossico delle cellule progenitori derivate dal midollo osseo come fonte per la generazione di cellule Schwann mature

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Cella singola fotoconversione in Zebrafish intatto vivente

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...