This research was supported in part by Program for Creating Start-ups from Advanced Research and Technology (START Program) from the Japan Society for the Promotion of Science (ST261006IP, TM) and by Program for the Strategic Research Foundation at Private Universities (2014-2019) (S1411018, TM) from the Ministry of Education, Sports, Science and Technology.

I campioni di grasso sottocutaneo umano sono stati ottenuti da pazienti sottoposti a chirurgia nei dipartimenti di Chirurgia Plastica, Urologia, Chirurgia Pediatrica e Chirurgia Ortopedica dell&#39;Ospedale Nihon dell&#39;Università Itabashi (Tokyo, Giappone). I pazienti hanno dato consenso informato scritto, e il Comitato Etico della Nihon University School of Medicine ha approvato lo studio. 1. Preparazione del tessuto <ol><li> Portare il campione di tessuto dalla sala operatoria al laboratorio. </li><li> Lavare 1-2 g di tessuto adiposo con 5 ml di PBS (-) a temperatura ambiente (RT), una volta, in un tubo da 50 ml e quindi posizionare il campione in un piatto di plastica 10 cm. </li></ol> 2. Collagenasi Digestione <ol><li> Rimuovere il campione dalla PBS (-), poi aggiungere 10 ml di soluzione sterile 0,1% (w / v) soluzione di collagenasi (collagenasi di tipo II) del recipiente contenente il campione di tessuto. </li><li> Macinare il tessuto con le forbici chirurgiche per circa 15 min, fino a raggiungere un composto frullatarenza. </li><li> Successivamente, digerire il tessuto tritato nel 0,1% (w / v) di collagenasi a 37 ° C per 30 min (60 rpm) in un tubo da 50 ml con leggera agitazione su un agitatore. </li><li> Filtrare il campione digerito attraverso un filtro 100 micron in un tubo da 50 ml. Avanti, passare 10 ml di DCM (vedi Tabella dei Materiali) integrato con il 2% FBS attraverso il filtro. </li></ol> Isolamento 3. cellulare <ol><li> Centrifugare le cellule filtrate a 100 xg per 1 min a RT. </li><li> A questo punto, preparare 5 ml di DCM integrato con il 2% FBS in un tubo da 50 ml. </li><li> Successivamente, elaborare le cellule di grasso galleggianti utilizzando una pipetta ml 1.000 e aggiungere le cellule di un terreno contenente 50 ml di tubo. </li><li> Successivamente, scuotere il tubo da 50 ml delicatamente per miscelare il medium DCM con le cellule. </li><li> Poi, centrifugare la provetta a 100 xg per 1 min a RT. </li><li> Eliminare il mezzo sul fondo del tubo con un ago 18 G e una siringa da 20 ml. </li><li> Successivamente, aggiungere 10 ml di DCM supplemented con 2% FBS alle cellule raccolte. </li><li> Mescolare il DCM con le cellule agitando il tubo da 50 ml con delicatezza. </li><li> Centrifugare la provetta a 100 xg per 1 min a RT. </li></ol> 4. Cellule placcatura <ol><li> Aggiungere 41 ml di DCM o DMEM supplementato con 20% FBS in un pallone 12,5 centimetri. </li><li> Successivamente, utilizzando una pipetta 200 ml aggiungere 40 ml di cellule di grasso al pallone. </li><li> Poi, riempire il pallone con DCM o DMEM supplementato con il 20% FBS. PUNTO CRITICO: A questo punto, in modo che le cellule sono diffuse in tutto il mezzo. Controllare visivamente le cellule staccate e poi tenere il pallone sul coperchio di una capsula di Petri giro per mantenerla piatta. </li><li> Infine, il recipiente all&#39;interno dell&#39;incubatrice e lasciato indisturbato in un incubatore CO 2 5% a 37 ° C per 7 giorni. </li><li> Dopo una settimana di incubazione, invertire il pallone e verificare la presenza di cellule DFAT. Successivamente, valutare DFAT generazione delle cellule di competenza, come descritto da 10 a questo punto fibroblcellule AST-simile (cellule DFAT) si trovano in regioni distinte dalle cellule grasse. </li><li> Aggiungere 5 ml di DCM o DMEM supplementato con il 20% FBS al pallone dopo la rimozione del precedente mezzo di coltura cellulare. Permettono alle cellule di crescere DFAT per un&#39;altra settimana dopo aver cambiato il mezzo. </li><li> Utilizzando un contatore di cellule, contare il numero di cellule DFAT ottenuti in diverse condizioni di coltura cellulare (DCM vs. DMEM). </li></ol>

<ol>
	<li>Lanzoni, G., 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=Concise+review:+clinical+programs+of+stem+cell+therapies+for+liver+and+pancreas.">Concise review: clinical programs of stem cell therapies for liver and pancreas.</a> Stem Cells. 31 (10), 2047-2060 (2013).</li><li>de Girolamo, L., 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=Mesenchymal+stem/stromal+cells:+a+new+"cells+as+drugs"+paradigm.+Efficacy+and+critical+aspects+in+cell+therapy.">Mesenchymal stem/stromal cells: a new "cells as drugs" paradigm. Efficacy and critical aspects in cell therapy.</a> Curr. Pharm. Des. 19 (13), 2459-2473 (2013).</li><li>Lindroos, B., Suuronen, R., Miettinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+adipose+stem+cells+in+regenerative+medicine.">The potential of adipose stem cells in regenerative medicine.</a> Stem Cell. Rev. 7 (2), 269-291 (2011).</li><li>Yan, J., Tie, G., Xu, T. Y., Cecchini, K., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+a+treatment+for+peripheral+arterial+disease:+current+status+and+potential+impact+of+type+II+diabetes+on+their+therapeutic+efficacy.">Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type II diabetes on their therapeutic efficacy.</a> Stem Cell. Rev. 9 (3), 360-372 (2013).</li><li>Ringden, O., Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+as+treatment+for+chronic+GVHD.">Mesenchymal stromal cells as treatment for chronic GVHD.</a> Bone Marrow Transplant. 46 (2), 163-164 (2011).</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>Lessard, J., 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=Generation+of+human+adipose+stem+cells+through+dedifferentiation+of+mature+adipocytes+in+ceiling+cultures.">Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.</a> J. Vis. Exp. (97), (2015).</li><li>Lessard, J., 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=Characterization+of+dedifferentiating+human+mature+adipocytes+from+the+visceral+and+subcutaneous+fat+compartments:+fibroblast-activation+protein+alpha+and+dipeptidyl+peptidase+4+as+major+components+of+matrix+remodeling.">Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.</a> PLoS One. 10 (3), 0122065 (2015).</li><li>Peng, X., 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=Phenotypic+and+Functional+Properties+of+Porcine+Dedifferentiated+Fat+Cells+during+the+Long-Term+Culture+In+Vitro.">Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro.</a> Biomed. Res. Int. 2015, 673651 (2015).</li><li>Kono, S., Kazama, T., Kano, K., Harada, K., Uechi, M., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+properties+of+feline+dedifferentiated+fat+cells+and+adipose-derived+stem+cells.">Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells.</a> Vet. J. 199 (1), 88-96 (2014).</li><li>Bellin, M., Marchetto, M. C., Gage, F. H., Mummery, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+the+new+patient.">Induced pluripotent stem cells: the new patient.</a> Nat. Rev. Mol. Cell Biol. 13 (11), 713-726 (2012).</li></ol>

The authors have nothing to disclose.

Adipociti maturi che subiscono in vitro de-differenziazione, un processo noto come la cultura del soffitto, può tornare ad un fenotipo più primitivo e acquisire capacità proliferative. Queste cellule sono indicati come le cellule di grasso dedifferenziato (DFAT). Il potenziale di differenziazione multilineare delle cellule DFAT è stata valutata. Citometria a flusso di analisi e analisi di espressione genica hanno rivelato che le cellule DFAT erano altamente omogenea rispetto ai ASC 6. Infatti il ...

Terapia cellulare e ingegneria dei tessuti sono temi caldi nel campo della medicina rigenerativa 1-5. Mentre queste modalità terapeutiche molto promettenti, diversi problemi tecnici attualmente ostacolano il loro uso clinico. A questo proposito, come nella generazione di cellule iPS, tutte le terapie ingegneria tissutale devono produrre celle libere di trasduzione del gene esterni al fine di mantenere la sicurezza del paziente. Di conseguenza, siamo stati il primo gruppo a produrre con successo cellule umane DFAT 6. Molti altri gruppi di ricerca hanno poi adottato il nostro metodo per generare cellule DFAT di origine mammiferi 7-9, evidenziando ulteriormente l&#39;utilità del nostro modello. Nel corso di diversi studi, abbiamo trovato che la qualità dell&#39;ambiente coltura cellulare può essere modificato regolando il contenuto del mezzo cellulare. Questa scoperta ha portato ad un aumento del tasso di successo della produzione di celle DFAT e migliore qualità delle cellule; entrambi i fattori criticigenerando in modo efficiente le cellule per i futuri studi clinici. A questo proposito, una migliore mezzo di coltura DFAT (DCM, un fluido simile al mesenchimali staminali medio cellule, che contiene insulina umana ricombinante, albumina sierica, acido L-glutammico, diversi acidi grassi e colesterolo) e un metodo per la generazione di cellule DFAT e la proliferazione è stato sviluppato (ulteriori informazioni sul contenuto di DCM è disponibile su richiesta). Utilizzando cellule di alta qualità DFAT questo metodo sono stati generati con la capacità di differenziarsi in diversi tipi di cellule, tra cui adipogenico, osteogenico, e le cellule condrogeniche. Complessivamente, questo convalidata protocollo coltura cellulare migliora la qualità delle cellule DFAT e può essere molto utile per migliorare applicazioni cliniche di terapia cellulare e ingegneria tessutale.

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			<td height="21" style="height:21px;width:243px;">CSTI303-MSC medium&#160;</td>
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			<td style="width:155px;">87-671</td>
			<td style="width:167px;">This medium is defined as DCM in the text</td>
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			<td height="40" style="height:41px;width:243px;">PBS(-)</td>
			<td style="width:143px;">Wako</td>
			<td style="width:155px;">166-23555</td>
			<td style="width:167px;">It does not contain Mg2+ and Ca2+</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEM medium</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:155px;">11965-092</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal Bovine Serum</td>
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			<td height="21" style="height:21px;width:243px;">Collagenase type II</td>
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			<td height="42" style="height:42px;width:243px;">Scissors</td>
			<td style="width:143px;">Takasago Medical Industry Co., Ltd</td>
			<td style="width:155px;">TKZ-F2194-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Shaker</td>
			<td style="width:143px;">TAITEC</td>
			<td style="width:155px;">Bioshaker V.BR-36</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Falcon Cell Strainer 100um Yellow</td>
			<td style="width:143px;">CORNING LIFE SCIENCES&#160;</td>
			<td style="width:155px;">DL 352360</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Falcon 12.5cm&#178; Rectangular Canted Neck Cell Culture Flask with Blue Vented Screw Cap</td>
			<td style="width:143px;">CORNING LIFE SCIENCES&#160;</td>
			<td style="width:155px;">353107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">18G needle</td>
			<td style="width:143px;">NIPRO</td>
			<td style="width:155px;">02-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">20ml Syringe&#160;</td>
			<td style="width:143px;">NIPRO</td>
			<td style="width:155px;">08-753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Z Series Coulter Counter</td>
			<td style="width:143px;">BECKMAN COULTER</td>
			<td style="width:155px;">383550</td>
			<td></td>
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</table>

an efficient method to obtain dedifferentiated fat cells

L&#39;ingegneria dei tessuti e terapia cellulare molto promettenti clinicamente. A questo proposito, cellule multipotenti, come le cellule staminali mesenchimali (MSC), possono essere utilizzati terapeuticamente, nel prossimo futuro, per ripristinare la funzione di organi danneggiati. Tuttavia, diversi problemi tecnici, compresa la procedura altamente invasiva di isolare cellule staminali mesenchimali e l&#39;inefficienza che circonda il loro amplificazione, attualmente ostacolano il potenziale utilizzo clinico di queste modalità terapeutiche. Qui, si introduce un metodo molto efficiente per la generazione di cellule adipose dedifferenziate (DFAT), cellule MSC simili. È interessante notare che le cellule DFAT possono essere differenziate in diversi tipi di cellule, tra cui adipogenico, osteogenico, e le cellule condrogeniche. Anche se altri gruppi hanno precedentemente presentato vari metodi per la generazione di cellule DFAT dal tessuto adiposo maturo, il nostro metodo ci permette di produrre cellule DFAT in modo più efficiente. A questo proposito, abbiamo dimostrato che terreno di coltura DFAT (DCM), integrato con il 20% FBS,è più efficace nel generare cellule DFAT di DMEM, integrati con il 20% FBS. Inoltre, le cellule DFAT prodotte dal nostro metodo di coltura cellulare può essere redifferentiated in diversi tipi di tessuto. Come tale, un modello molto interessante e utile per lo studio di dedifferentiation tessuto è presentato.

<html> In questo studio, il metodo e il kit di strumenti per la generazione di cellule DFAT stata migliorata (Figura 1). Il nostro metodo permette di generare cellule DFAT utilizzando sia DCM e medie DMEM contenente 20% FBS (Figura 2A). Come tale, abbiamo confrontato l&#39;efficienza di DCM e DMEM nella generazione di cellule DFAT. A questo proposito, DCM migliorata proliferazione cellulare DFAT di tre volte rispetto ai DMEM, indipendentemente dal numero ...

Watch this Scientific Journal Video about An Efficient Method to Obtain Dedifferentiated Fat Cells at JoVE.com

An Efficient Method to Obtain Dedifferentiated Fat Cells

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Un metodo efficiente per ottenere cellule di grasso dedifferenziato

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

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Developmental Biology

7.9K Views.  Nihon University. We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Video: An Efficient Method to Obtain Dedifferentiated Fat Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Derivazione efficiente di cellule retiniche epitelio pigmentato da cellule staminali

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Ricerca

Biologia dello sviluppo

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Un metodo enzimatico per salvare cellule staminali mesenchimali da coagulata midollo osseo da analizzare

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

Un metodo rapido ed efficace per la purificazione delle cellule endoderma generato da cellule staminali embrionali umane

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

La conversione epigenetico come un metodo semplice e sicuro per ottenere cellule che secernono insulina da adulti fibroblasti cutanei

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

Separazione immunomagnetica di Sca1 Fat Depot-specifica<sup&gt; alta</sup&gt; derivate da tessuto adiposo cellule staminali (ASC)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Differenziazione efficiente delle pluripotenti cellule staminali per NKX6-1<sup&gt; +</sup&gt; progenitori pancreatici

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Una tecnica novel di trapianto di grassi del grasso mammario per visualizzare la generazione di vascelli delle cellule staminali endoteliali vascolari

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Un metodo rapido ed efficiente per sezionare Pupal ali della drosofila adatto per Immunodetections o analisi di PCR

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Un metodo semplificato ed efficiente per isolare i Keratinocytes umani primari dal tessuto pelle adulta

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Un metodo efficiente per ottenere cellule di grasso dedifferenziato

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