Questo studio è stato sostenuto dal National Institutes of Health Research concessione R01-DE021683-01 e National Institutes of Health Research concessione R01-DE019434 a MTL; Howard Hughes Medical Institute Research Fellowship per MTCDCW è stata sostenuta dalla ACS Franklin Martin Faculty Research Fellowship, Il Laboratorio Hagey Pediatrica Medicina Rigenerativa, e la Stanford University Child Health Research Institute Faculty Scholar Award.

NOTA: Tutti i campioni dei pazienti sono stati ottenuti con il consenso informato, e protocolli sperimentali sono stati esaminati e approvati da Stanford University Institutional Review Board (protocollo # 2188 e # 9999). 1. Isolamento delle cellule e Cultura: <ol><li> Ottenere tessuto adiposo sottocutaneo umano da pazienti di sesso femminile sani sottoposti a lipoaspirazione elettiva del ventre, fianchi, e / o regione della coscia in anestesia locale / generale. Assicurarsi che Institutional Review Board (IRB) di approvazione è stata ottenuta per il protocollo di isolare ASC da tessuti umani, e seguire le precauzioni di sicurezza istituzionali mentre si lavora con questi materiali. </li><li> Per ottenere il SVF dal tessuto adiposo lipoaspirated, prima lavare il lipoaspirato tre volte con volumi uguali di 1x tampone fosfato salino (PBS) sterile. Con attenzione aspirare ed eliminare lo strato acquoso inferiore. </li><li> Preparare il buffer collagenasi digestione: 0,075% Tipo I collagenasi a Hank diSoluzione salina bilanciata (HBSS). Preparare tampone FACS: 2% FBS, 1% P188 e l&#39;1% Pen-Strep in PBS. Filtro entrambe le soluzioni che utilizzano una dimensione dei pori 0,22 micron filtro polietersulfone flusso veloce disponibile in commercio. </li><li> Per digerire il tessuto adiposo lavato, aggiungere un volume uguale di tampone di digestione collagenasi e posizionare vaso di digestione saldamente in un bagno d&#39;acqua agitazione per 60 min a 37 ° C (circa 180 frullati / min). NOTA: E &#39;meglio utilizzare un volume di digestione nave più grande del necessario, in quanto ciò permette di digestione massima durante l&#39;agitazione (cioè, 250 ml di tampone collagenasi digestione, 250 ml di tessuto adiposo lavati in un pallone sterile 1L). </li><li> Neutralizzare attività enzimatica aggiungendo un volume uguale di tampone FACS e consentendo di sedersi a temperatura ambiente per 5 min. Successivamente, centrifugare a 233 xg per 20 min a 4 ° C. </li><li> Aspirare accuratamente e scartare il surnatante, facendo attenzione a non disturbare il pellet SVF ad alta densità. </li><li> Risospendere il pellet in 5-10 ml di RT rossotampone di lisi cellulare, in base alle dimensioni del pellet. Lasciare soluzione sedersi a temperatura ambiente per 5 min e centrifugare a 233 xg per 5 minuti a temperatura ambiente. </li><li> Aspirare il surnatante e risospendere il pellet aggiungendo 5-10 ml di mezzi di crescita tradizionali, in base alle dimensioni del pellet (Dulbecco Modified Eagle Medium [DMEM] / 10% FBS / 1% soluzione di penicillina-streptomicina [pen-strep]). </li><li> Filtrare la sospensione attraverso un 100 micron filtro cella nylon per rimuovere i detriti cellulari. </li><li> Centrifugare a 233 xg per 5 minuti a 4 ° C e scartare il supernatante senza disturbare il pellet. </li><li> Risospendere il pellet in 5-10 ml di mezzi di crescita tradizionali, in base alle dimensioni del pellet. </li><li> Inserire 2 x 10 6 cellule in un piatto di coltura standard di 15 cm, stabilire colture primarie O / N a 37 ° C / 21% O 2, 5% CO 2. Mantenere a livelli sub-confluenti per impedire differenziazione spontanea a 37 ° C / 21% O 2, 5% CO 2 nei substrati. Densità cellulare in uno completamente: NOTAconfluenti 15 centimetri piatto è di circa 4-6 x 10 6 cellule. </li></ol> 2. Colorazione <ol><li> ASC culturali in mezzo di crescita tradizionale a 37 ° C / 21% O 2, 5% di CO 2, per 36 ore prima della colorazione / smistamento. </li><li> ASC Ascensore da piastre di coltura utilizzando Accutase (come da protocollo del produttore). </li><li> Lavare le cellule una volta con tampone FACS (PBS con il 2% e l&#39;1% FBS pen-strep). </li><li> Centrifugare a 233 xg per 5 minuti a 4 ° C. </li><li> Scartare il surnatante senza disturbare il pellet. Risospendere le cellule in 0,5-1 ml di tampone FACS (a seconda del numero di cellule e la quantità di anticorpo desiderato). NOTA: Il volume della sospensione cellulare, in conformità con le raccomandazioni del costruttore l&#39;anticorpo FACS, determina la concentrazione di anticorpi. Tuttavia, la titolazione di concentrazione di anticorpi, utilizzando una concentrazione iniziale di 1: 100, è di solito consigliata se è la prima volta di utilizzare un anticorpo. </li><li> Contare il numero totale dicellule utilizzando un emocitometro. </li><li> Riserva 100 aliquote ul a 1,5 ml provette da utilizzare per un campione senza macchia e controlli solo colore (in base al numero di anticorpi fluorescenti utilizzate). </li><li> Aggiungere gli anticorpi monoclonali fluorescenza marcata adeguati (a concentrazioni predeterminate, secondo le istruzioni del produttore) e incubare per 20-30 minuti sul ghiaccio, al riparo dalla luce. <ol><li> Per etichettare sottopopolazione osteogenic: Diluire anti-CD90 (APC CD90 anti-umano (Thy1)) o anti-CD105 (FITC anti-umano CD105 (endoglin)) in tampone FACS ad una concentrazione di 1:50. </li><li> Per etichettare altre popolazioni cellulari (ad esempio, ematopoietiche e cellule endoteliali): Diluire anti-CD45 (Anti-Human CD45 Pacific Blue), anti-CD34 (Anti-Human CD34 APC), e / o anti-CD31 (Anti-Human CD31 PE ) in FACS tampone a una concentrazione di 1:50. </li><li> Se non si usa anticorpi coniugati direttamente, utilizzare un anticorpo primario biotinilato con un secondo streptavidina coniugato appropriatoanticorpi ary, quali streptavidina PE-Cy7, a una diluizione di 1: 100. </li></ol></li><li> Dopo l&#39;incubazione, lavare le cellule due volte: riempire tubi con tampone FACS e centrifugare a 233 xg per 5 minuti a 4 ° C, aspirando surnatante. </li><li> Risospendere le cellule in 400-500 campioni microlitri FACS tampone e filtro attraverso un colino 70 micron cella nylon per rimuovere grumi di cellule. </li><li> Trasferire cellule marcate in tubi FACS con piano filtro e mantenere i campioni in ghiaccio per la durata dell&#39;analisi. </li></ol> 3. Fluorescence-activated cell sorting NOTA: Le seguenti operazioni impongono conoscenza nella cella fluorescenza attivate (FACS) o l&#39;assistenza di un tecnico specializzato. <ol><li> Utilizzando il software FACS appropriate, trame di analisi progettuale esaminare forward scatter (FSC), side scatter (SSC), ficoeritrina (PE) -Texas Red, Pacific Blue, fluoresceina isotiocianato (FITC), Allophycocyanin (APC) e qualsiasi altro uso fluoroforod per etichettare le cellule. </li><li> Prima di essere caricato nel cell sorter, brevemente vortice ogni campione per sospendere nuovamente le cellule. </li><li> Iniziare con analisi del campione non colorato per impostare porte per la popolazione totale di cellule, di escludere detriti cellulari, e determinare autofluorescenza basale. </li><li> Aggiungere ioduro di propidio (PI) al campione non colorato e analizzarlo per visualizzare la popolazione di cellule vive all&#39;interno della popolazione cellulare totale. Costruire un cancello intorno a questa popolazione. </li><li> Analizzare un campione di controllo unico colore per ogni fluorocromo per impostare la compensazione della fluorescenza. </li><li> Analizzare il campione colorato per impostare la posizione di porte per l&#39;ordinamento. </li><li> Aggiungere PI al campione macchiato per macchiare cellule morte. </li><li> Ordina le popolazioni di cellule etichettate. Ordina in entrambi 1,5 ml provette o tubi conici (15 ml) contenenti terreno di coltura. </li><li> Cessare manualmente classificare una volta ottenuta la quantità necessaria di cellule. </li><li> Noiing FACS, eseguire un controllo della purezza analizzando una piccola frazione (ad es., 500 cellule) della popolazione cellulare acquisita. NOTA: Questo passaggio conferma la purezza delle popolazioni cellulari ordinati. Idealmente, la purezza dovrebbe essere maggiore di 90-95%. </li><li> Cellule centrifugare a 233 xg per 5 min a 4 ° C immediatamente dopo il completamento della cernita e risospendere in 1 ml di mezzo fresco contenente 10% FBS. NOTA: La quantità di materiale utilizzato può essere aumentato in base alle dimensioni del pellet di cellule. </li><li> Piatto su piastre rivestite di gelatina per migliorare la vitalità cellulare. A seconda del rendimento cellulare finale, piastra 300.000 cellule per bene in un 6-pozzetti, 100.000 cellule per pozzetto in una piastra di 12 pozzetti. </li></ol>

<ol>
	<li>Narsinh, K. 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=Single+cell+transcriptional+profiling+reveals+heterogeneity+of+human+induced+pluripotent+stem+cells.">Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.</a> J Clin Invest. 121, 1217-1221 (2011).</li><li>Thorsen, T., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+large-scale+integration.">Microfluidic large-scale integration.</a> Science. 298, 580-584 (2002).</li><li>Warren, L., Bryder, D., Weissman, I. L., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+profiling+in+individual+hematopoietic+progenitors+by+digital+RT-PCR.">Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR.</a> Proc Natl Acad Sci U S A. 103, 17807-17812 (2006).</li><li>Warren, L. 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=Transcriptional+instability+is+not+a+universal+attribute+of+aging.">Transcriptional instability is not a universal attribute of aging.</a> Aging Cell. 6, 775-782 (2007).</li><li>Aalami, O. O., 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=Applications+of+a+mouse+model+of+calvarial+healing:+differences+in+regenerative+abilities+of+juveniles+and+adults.">Applications of a mouse model of calvarial healing: differences in regenerative abilities of juveniles and adults.</a> Plast Reconstr Surg. 114, 713-720 (2004).</li><li>Levi, 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=CD105+protein+depletion+enhances+human+adipose-derived+stromal+cell+osteogenesis+through+reduction+of+transforming+growth+factor+beta1+(TGF-beta1)+signaling.">CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor beta1 (TGF-beta1) signaling.</a> The Journal of biological chemistry. 286, 39497-39509 (2011).</li><li>Chen, X. D., Qian, H. Y., Neff, L., Satomura, K., Horowitz, 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=Thy-1+antigen+expression+by+cells+in+the+osteoblast+lineage.">Thy-1 antigen expression by cells in the osteoblast lineage.</a> J Bone Miner Res. 14, 362-375 (1999).</li><li>Malladi, P., Xu, Y., Yang, G. P., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+vitamin+D,+retinoic+acid,+and+dexamethasone+in+mouse+adipose-derived+mesenchymal+cells.">Functions of vitamin D, retinoic acid, and dexamethasone in mouse adipose-derived mesenchymal cells.</a> Tissue engineering. 12, 2031-2040 (2006).</li><li>Levi, 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=Depot-specific+variation+in+the+osteogenic+and+adipogenic+potential+of+human+adipose-derived+stromal+cells.">Depot-specific variation in the osteogenic and adipogenic potential of human adipose-derived stromal cells.</a> Plastic and reconstructive surgery. 126, 822-834 (2010).</li><li>James, A. W., 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=Estrogen/estrogen+receptor+alpha+signaling+in+mouse+posterofrontal+cranial+suture+fusion.">Estrogen/estrogen receptor alpha signaling in mouse posterofrontal cranial suture fusion.</a> PloS one. 4, e1720 (2009).</li><li>Locke, M., Feisst, V., Dunbar, P. R. <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:+human+adipose-derived+stem+cells:+separating+promise+from+clinical+need.">Concise review: human adipose-derived stem cells: separating promise from clinical need.</a> Stem Cells. 29, 404-411 (2011).</li><li>Glotzbach, J. 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=An+Information+Theoretic,+Microfluidic-Based+Single+Cell+Analysis+Permits+Identification+of+Subpopulations+among+Putatively+Homogeneous+Stem+Cells.">An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells.</a> PLoS One. 6, e21211 (2011).</li><li>Zhu, M., Heydarkhan-Hagvall, S., Hedrick, M., Benhaim, P., Zuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+isolation+of+adipose-derived+stem+cells+from+human+lipoaspirates.">Manual isolation of adipose-derived stem cells from human lipoaspirates.</a> J. Vis. Exp. , e50585 (2013).</li><li>Mitchell, J. 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=Immunophenotype+of+human+adipose-derived+cells:+temporal+changes+in+stromal-associated+and+stem+cell-associated+markers.">Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.</a> Stem Cells. 24, 376-385 (2006).</li><li>McMurray, R. 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=Nanoscale+surfaces+for+the+long-term+maintenance+of+mesenchymal+stem+cell+phenotype+and+multipotency.">Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency.</a> Nat Mater. 10, 637-644 (2011).</li><li>Katz, A. J., Tholpady, A., Tholpady, S. S., Shang, H., Ogle, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+and+transcriptional+characterization+of+human+adipose-derived+adherent+stromal+(hADAS)+cells.">Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.</a> Stem Cells. 23, 412-423 (2005).</li><li>McIntosh, K., 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=The+immunogenicity+of+human+adipose-derived+cells:+temporal+changes+in+vitro.">The immunogenicity of human adipose-derived cells: temporal changes in vitro.</a> Stem Cells. 24, 1246-1253 (2006).</li><li>Choudhery, M. S., Badowski, M., Muise, A., Pierce, J., Harris, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+age+negatively+impacts+adipose+tissue-derived+mesenchymal+stem+cell+expansion+and+differentiation.">Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation.</a> Journal of translational medicine. 12, 8 (2014).</li><li>Yoshimura, K., Suga, H., Eto, H. <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+stem/progenitor+cells:+roles+in+adipose+tissue+remodeling+and+potential+use+for+soft+tissue+augmentation.">Adipose-derived stem/progenitor cells: roles in adipose tissue remodeling and potential use for soft tissue augmentation.</a> Regenerative medicine. 4, 265-273 (2009).</li><li>Chung, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD90+(Thy-1)+Positive+Selection+Enhances+Osteogenic+Capacity+of+Human+Adipose-Derived+Stromal+Cells.">CD90 (Thy-1) Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells.</a> Tissue Eng Part A. , (2012).</li></ol>

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Attualmente, l&#39;isolamento di sottopopolazioni omogenei di ASC dal SVF di tessuto adiposo umano rimane una sfida anche se obiettivo auspicabile. Isolamento di sottopopolazioni ASC pro-osteogeniche è particolarmente desiderabile, poiché tali cellule possono essere usati per studiare la formazione e l&#39;omeostasi dei tessuti scheletrici. Tuttavia, la SVF di tessuto adiposo ospita una significativa eterogeneità in materia di staminali la capacità delle cellule e potenziale di differenziazione. 11 Le bas...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

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		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Name of Reagent/Material</td>
			<td style="width:152px;">Company</td>
			<td style="width:112px;">Catalog Number</td>
			<td style="width:79px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Disposable 250 mL Conical Tubes</td>
			<td style="width:152px;">Corning (Thomas Scientific)</td>
			<td style="width:112px;">2602A43</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">DMEM, high glucose, GlutaMAX Supplement</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">10566-016</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS, pH 7.4</td>
			<td style="width:152px;">Gibco</td>
			<td>10010-023</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine - Antiseptic Povidone/Iodine Solution</td>
			<td style="width:152px;">Purdue&#160;</td>
			<td>PFC-67618015017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#39;s Balanced Salt Solution, 1X</td>
			<td style="width:152px;">Cellgro</td>
			<td>21-023-CV</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum, Certified, US Origin</td>
			<td style="width:152px;">Gibco</td>
			<td>16000-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from&#160;Clostridium histolyticum</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td>C0130-5G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ACCUTASE Cell Detachment Solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td>7920</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">APC Mouse Anti-Human CD90</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">559869</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">FITC Mouse anti-Human CD105 (Endoglin)</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">561443</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Anti-Human CD45 eFluor 450 (Pacific Blue replacement)&#160;</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">48-9459-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Anti-Human CD34 APC</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">17-0349-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Anti-Human CD31 (PECAM-1) PE</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">12-0319-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Streptavidin PE-Cyanine7</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">25-4317-82</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">BD FACS Aria II instrument</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">BD FACSDiva Software</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

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.

Utilizzando CD90 come marcatore per le cellule con risultati migliorati osteogenesi in isolamento di una popolazione altamente arricchito di ASC umani (Figura 1A, 1B). ASC sono state colorate con Pacific Blue coniugati CD45 anti-umane, coniugati con coniugato CD105 anti-umana, e APC-coniugato anti-CD90 umano. Dopo la cernita, il livello di purezza era superiore al 98%, come quantificato mediante analisi post-ordinamento. Gruppi di cellule in base ai profili trascrizionali co...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

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

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 ...

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

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JoVE Journal

Developmental Biology

11.4K Views.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Video: Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

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

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

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

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

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Biologia dello sviluppo

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 ...

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

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

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

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Condrogenico Differenziazione induzione di cellule staminali derivate da tessuto adiposo per gravità centrifuga

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

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Isolamento rapido di BMPR-IB + derivate da tessuto adiposo cellule stromali per l&#39;uso in un cranica difetto di guarigione Modello

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Isolamento di mouse endometriale epiteliali e stromali cellule per<em&gt; In Vitro</em&gt; decidualizzazione

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Isolamento e caratterizzazione di cellule mesenchimali stromali dal Cordone ombelicale e placenta fetale

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

L&#39;isolamento e arricchimento di fegato progenitrici sottoinsiemi identificati da un marcatore combinazione romanzo di superficie

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Isolamento, la cultura e la differenziazione delle cellule stromali del midollo osseo e dei progenitori degli osteoclasti dai topi

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

Isolamento e caratterizzazione di cellule Stromal Mesenchymal cardiache da Endomyocardial campioni bioptici di pazienti cardiomiopatia aritmogena

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