We thank Dr. Tadashi Koyama for technical help. This work was supported in part by JSPS KAKENHI Grant Numbers 23249045, 15K09211, 15K19172; a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan; a grant for research on allergic disease and immunology, Japan.

1. Preparazioni e coltura di cellule epiteliali del polmone <ol><li> cellule epiteliali del polmone umano A549 Cultura (linea cellulare aderente) a Dulbecco Modified Eagle Medium (DMEM) supplementato con siero fetale bovino al 10% (FBS), 100 UI / ml di penicillina, e 100 mg / ml di streptomicina. </li><li> Rimuovere e scartare il terreno di coltura di cellule dal piatto di cultura, e lavare una volta con 5 - 10 ml di tampone fosfato salino (PBS). Dopo il lavaggio, aspirare immediatamente la PBS. </li><li> Aggiungere 2 ml Tripsina / acido etilendiamminotetraacetico (EDTA) (0,05%) e incubare a 37 ° C e 5% CO 2 per 3 min. </li><li> Raccogliere le cellule staccate in provette da centrifuga contenenti terreno di coltura cellulare, centrifugare le provette a temperatura ambiente per 4 min a 150 × g. </li><li> Risospendere le cellule pellet in 2 ml di mezzo di coltura cellulare e rimuovere un campione per il conteggio delle cellule. Contare il numero di cellule utilizzando un emocitometro con Trypan blu macchia di verificare la vitalità delle cellule. </li><li> cellule Seed A549 su10 piatti cm polistirolo con una densità di 0,5 - 1,0 × 10 6 cellule / piatto con 10 ml di terreno per test gel contrazione. Seme le celle 6 pozzetti di polistirene ad una densità di 0,5 - 1,0 × 10 5 cellule / pozzetto con 2 ml di mezzo per la conferma EMT. Incubare le cellule a 37 ° C e 5% CO 2 per 24 ore. </li></ol> 2. Procedura EMT <ol><li> Aggiungere 10 ml di TGF-β1 (5 mg / ml) e 10 ml di TNF-α (10 ug / ml) alla piastra per test gel contrazione seminate in fase 1.6. Aggiungere 2 ml di TGF-β1 (5 mg / ml) e 2 ml di TNF-α (10 ug / ml) alla piastra di conferma EMT seminate in fase 1.6. Incubare a 37 ° C e 5% CO 2 per 48 ore. </li></ol> 3. Conferma di procedura EMT mediante PCR e Western Blotting <ol><li> Confermare il cambiamento nella morfologia (da ciottoli simile al mandrino forma) di cellule trattate usando la microscopia a contrasto di fase. Nota: Normcellule di Al A549 hanno cobble aspetto lapideo e di forma triangolare che è una caratteristica delle cellule epiteliali, ma dopo stimolazione con TGF-β1 e TNF-α, le cellule appaiono lungo e mandrino forma che è simile alle cellule mesenchimali 14,15. </li><li> Valutare l&#39;espressione di un marcatore epiteliale, come ad esempio E-caderina, e marcatori mesenchimali quali la N-caderina, vimentina, e α-actina del muscolo liscio utilizzando PCR o Western blotting. <ol><li> Estratto RNA totale dalle cellule utilizzando kit di estrazione di RNA 16 e sintetizzare cDNA utilizzando trascrittasi inversa 17 secondo il protocollo del produttore. </li><li> Misurare l&#39;espressione dei livelli di mRNA utilizzando in tempo reale del sistema PCR 18 e colorante cianinico monomerico kit per la PCR in base alle istruzioni del fabbricante. I primers specifici per GAPDH (gliceraldeide 3-fosfato deidrogenasi), ACTA2 (alpha-actina-2), CDH1 (caderina-1; E-caderina), e VIM (vimentina) sono mostrati in Tabella 1 </strong&gt;. </li><li> Lisare le cellule utilizzando un tampone di lisi (Tabella 2) soluzione contenente 1% di cocktail di inibitori delle proteasi. Misurare tutte le concentrazioni di proteine ​​del campione utilizzando un kit di analisi delle proteine ​​19,20, e applicare le stesse quantità di proteine ​​ad un gel di poliacrilammide. <ol><li> Eseguire SDS gel elettroforesi e trasferimento semi-dry delle proteine ​​di membrana PVDF 21. Incubare la membrana con anticorpi primari in tampone di bloccaggio per 1 - 2 ore. Dopo l&#39;incubazione, lavare due volte la membrana con tampone di lavaggio e incubare la membrana hr 1 secondo anticorpi. Nota: vedere la tabella Materiali / Attrezzature per gli anticorpi e le diluizioni utilizzati in questi studi. Vedere la Tabella 2 per i componenti del tampone di bloccaggio. </li><li> Lavare la membrana con tampone di lavaggio due volte. Scattare foto della membrana con il kit di rilevamento occidentale blotting 22 con una camera CCD 11,23 freddo. </li></ol></li></ol></li></ol> 4. Gel Contrazione Assay per EMT valutazione <ol><li> Aspirare il supporto condizionata dalla nave coltura cellulare, e lavare bene con 5 - 10 ml di PBS per rimuovere le cellule morte. Dopo il lavaggio, aspirare immediatamente la PBS. </li><li> Aggiungere 2 ml di 0,05% tripsina / EDTA e incubare a 37 ° C e 5% CO 2 per 3 min. </li><li> Raccogliere le cellule staccate in provette da centrifuga contenenti DMEM integrato con inibitore della tripsina (1 mg / ml), centrifugare le provette a temperatura ambiente per 4 minuti a 150 × g. </li><li> Mescolare tipo gel 1 collagene con acqua distillata, e 4 × DMEM concentrati per regolare i volumi per ottenere una concentrazione di collagene di 1,75 mg / ml e 1 x concentrazione DMEM. Essere sicuri di mantenere il supporto gel sul ghiaccio durante questa fase. Nota: Per fare 6 ml di mezzo di gel, mescolare bene 3,5 ml di tipo 1 collagene gel (3 mg / ml), 1,5 ml di 4 × DMEM concentrato e 1 ml di acqua distillata. </li><li> Risospendere il pellet di cellule in 500 microlitri di PBS e rimuovere un campione per il conteggio delle cellule. Contareil numero delle cellule utilizzando un emocitometro con trypan blue macchia per verificare la vitalità cellulare. </li><li> Aggiungere il mezzo gel per regolare i volumi di raggiungere una densità cellulare di 3,0 x 10 5 cellule / pozzetto (6,0 × 10 5 cellule / ml) e delicatamente ma rapidamente mescolare pipettando senza gelificazione. </li><li> Versare 0,5 ml della miscela in ciascun pozzetto di una piastra non trattata 24 pozzetti rapidamente e con attenzione per rendere forma cilindrica ordinata. Fare attenzione a non consentire eventuali bolle d&#39;aria per contaminare i gel (Figura 1A). Nota: Il mezzo gel contenente le cellule è viscoso e può facilmente gel e la forma forma di mezzaluna nel pozzo. </li><li> Incubare la piastra per 15 minuti in un incubatore cellule a 37 ° C, 5% di CO 2 e il 95% di umidità al gel completamente. </li><li> Staccare gel dalla piastra senza rompere spostando una spatola sterilizzato in modo per disegnare una circonferenza in una direzione. Utilizzando una spatola, trasferire delicatamente i gel a 60 mm piatti di coltura dei tessuticontenente 5 ml di DMEM / FBS 1% con o senza TGF-β1 (5 ng / ml) e TNF-α (10 ng / ml). </li><li> Agitare delicatamente i piatti per garantire gel stanno galleggiando sul supporto. Incubare in un incubatore cellule a 37 ° C, 5% di CO 2 e il 95% di umidità. </li></ol> 5. Misura di Gel Size <ol><li> Misurare le dimensioni gel di collagene dopo 0, 24, 48, e 72 ore utilizzando un sistema di analisi dell&#39;immagine. <ol><li> Accendere il sistema di documentazione gel (Figura 2A), e mettere i piatti nel mobile luce di schermatura. Poi togliere il coperchio di piatti nel mobile. </li><li> Aprire il relativo software di analisi di gel. Fare clic sul pulsante &quot;acquisizione delle immagini&quot; nella barra dei menu per visualizzare l&#39;immagine del gel nel cabinet. Quindi, fai clic su &quot;acquisire&quot; nella barra dei menu per scattare foto dei gel (Figura 2b). </li><li> Fare clic sul pulsante &quot;rilevamento&quot; nella barra dei menu (Figura 2C) e regolare la regione di misura (cerchio giallo in Figura 2C) trascinando il mouse. Quindi, fare clic sul pulsante &quot;auto-detect&quot; nella barra dei menu (vedi pulsante nella Figura 2D). Il software rileva automaticamente i gel che mostra una mappa termica (Figura 2D). </li><li> Fai clic su &quot;OK&quot; per estrarre il contorno dei gel di elaborazione delle immagini e per calcolare le aree circondate da contorno (Figura 2E). Dopo che l&#39;area viene calcolata, l&#39;area calcolata viene visualizzata nella finestra pop-up separata. Nota: se il gel si sovrappongono l&#39;un l&#39;altro durante l&#39;imaging, spostare i gel delicatamente con un puntale sterile. </li></ol></li></ol>

<ol>
	<li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+fibrosis.">Cellular and molecular mechanisms of fibrosis.</a> The Journal of pathology. 214, 199-214 (2008).</li><li>Hardie, W. D., Glasser, S. W., Hagood, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+concepts+in+the+pathogenesis+of+lung+fibrosis.">Emerging concepts in the pathogenesis of lung fibrosis.</a> The American journal of pathology. 175, 3-16 (2009).</li><li>Kovacic, J. C., Mercader, N., Torres, M., Boehm, M., Fuster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+and+endothelial-to-mesenchymal+transition:+from+cardiovascular+development+to+disease.">Epithelial-to-mesenchymal and endothelial-to-mesenchymal transition: from cardiovascular development to disease.</a> Circulation. 125, 1795-1808 (2012).</li><li>Thiery, J. P., Acloque, H., Huang, R. Y., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions+in+development+and+disease.">Epithelial-mesenchymal transitions in development and disease.</a> Cell. 139, 871-890 (2009).</li><li>Kalluri, R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> The Journal of clinical investigation. 119, 1420-1428 (2009).</li><li>Forino, 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=TGFbeta1+induces+epithelial-mesenchymal+transition,+but+not+myofibroblast+transdifferentiation+of+human+kidney+tubular+epithelial+cells+in+primary+culture.">TGFbeta1 induces epithelial-mesenchymal transition, but not myofibroblast transdifferentiation of human kidney tubular epithelial cells in primary culture.</a> International journal of experimental pathology. 87, 197-208 (2006).</li><li>Yang, 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=Participation+of+miR-200+in+pulmonary+fibrosis.">Participation of miR-200 in pulmonary fibrosis.</a> The American journal of pathology. 180, 484-493 (2012).</li><li>Reynolds, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+inflammation+and+fibrosis:+an+alveolar+macrophage-centered+perspective+from+the+1970s+to+1980s.">Lung inflammation and fibrosis: an alveolar macrophage-centered perspective from the 1970s to 1980s.</a> American journal of respiratory and critical care medicine. 171, 98-102 (2005).</li><li>Camara, J., Jarai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition+in+primary+human+bronchial+epithelial+cells+is+Smad-dependent+and+enhanced+by+fibronectin+and+TNF-alpha.">Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-alpha.</a> Fibrogenesis &amp; tissue repair. 3, 2 (2010).</li><li>Kamitani, 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=Simultaneous+stimulation+with+TGF-beta1+and+TNF-alpha+induces+epithelial+mesenchymal+transition+in+bronchial+epithelial+cells.">Simultaneous stimulation with TGF-beta1 and TNF-alpha induces epithelial mesenchymal transition in bronchial epithelial cells.</a> International archives of allergy and immunology. 155, 119-128 (2011).</li><li>Mikami, 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=Lymphotoxin+beta+receptor+signaling+induces+IL-8+production+in+human+bronchial+epithelial+cells.">Lymphotoxin beta receptor signaling induces IL-8 production in human bronchial epithelial cells.</a> PloS one. 9, e114791 (2014).</li><li>Yamauchi, 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=Tumor+necrosis+factor-alpha+enhances+both+epithelial-mesenchymal+transition+and+cell+contraction+induced+in+A549+human+alveolar+epithelial+cells+by+transforming+growth+factor-beta1.">Tumor necrosis factor-alpha enhances both epithelial-mesenchymal transition and cell contraction induced in A549 human alveolar epithelial cells by transforming growth factor-beta1.</a> Experimental lung research. 36, 12-24 (2010).</li><li>Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts,+myofibroblasts,+and+wound+contraction.">Fibroblasts, myofibroblasts, and wound contraction.</a> The Journal of cell biology. 124, 401-404 (1994).</li><li>Ramos, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-1+reverts+epithelial-mesenchymal+transition+induced+by+TGF-{beta}1+through+MAPK/ERK+kinase+pathway+.">FGF-1 reverts epithelial-mesenchymal transition induced by TGF-{beta}1 through MAPK/ERK kinase pathway .</a> American journal of physiology. Lung cellular and molecular physiology. 299, L222-L231 (2010).</li><li>Ren, Z. X., Yu, H. B., Li, J. S., Shen, J. L., Du, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitable+parameter+choice+on+quantitative+morphology+of+A549+cell+in+epithelial-mesenchymal+transition.">Suitable parameter choice on quantitative morphology of A549 cell in epithelial-mesenchymal transition.</a> Bioscience reports. 35, (2015).</li><li>Brinkmann, V., Kinzel, B., Kristofic, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR-independent+activation+of+human+CD4++45RO-+T+cells+by+anti-CD28+plus+IL-2:+Induction+of+clonal+expansion+and+priming+for+a+Th2+phenotype.">TCR-independent activation of human CD4+ 45RO- T cells by anti-CD28 plus IL-2: Induction of clonal expansion and priming for a Th2 phenotype.</a> Journal of immunology. 156, 4100-4106 (1996).</li><li>Krug, M. S., Berger, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-strand+cDNA+synthesis+primed+with+oligo(dT).">First-strand cDNA synthesis primed with oligo(dT).</a> Methods in enzymology. 152, 316-325 (1987).</li><li>Morozumi, 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=Simultaneous+detection+of+pathogens+in+clinical+samples+from+patients+with+community-acquired+pneumonia+by+real-time+PCR+with+pathogen-specific+molecular+beacon+probes.">Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes.</a> Journal of clinical microbiology. 44, 1440-1446 (2006).</li><li>Smith, P. 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical biochemistry. 150, 76-85 (1985).</li><li>Wiechelman, K. J., Braun, R. D., Fitzpatrick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+bicinchoninic+acid+protein+assay:+identification+of+the+groups+responsible+for+color+formation.">Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation.</a> Analytical biochemistry. 175, 231-237 (1988).</li><li>Ursitti, J. A., Mozdzanowski, J., Speicher, D. 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=Electroblotting+from+polyacrylamide+gels.">Electroblotting from polyacrylamide gels.</a> Current protocols in protein science. Chapter 10, Unit 10.7 (2001).</li><li>Kricka, L. J., Voyta, J. C., Bronstein, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+methods+for+detecting+and+quantitating+enzyme+activity.">Chemiluminescent methods for detecting and quantitating enzyme activity.</a> Methods in enzymology. 305, 370-390 (2000).</li><li>Noguchi, 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=An+integrative+analysis+of+the+tumorigenic+role+of+TAZ+in+human+non-small+cell+lung+cancer.">An integrative analysis of the tumorigenic role of TAZ in human non-small cell lung cancer.</a> Clinical cancer research : an official journal of the American Association for Cancer Research. 20, 4660-4672 (2014).</li><li>Kasai, H., Allen, J. T., Mason, R. M., Kamimura, T., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta1+induces+human+alveolar+epithelial+to+mesenchymal+cell+transition+(EMT).">TGF-beta1 induces human alveolar epithelial to mesenchymal cell transition (EMT).</a> Respiratory research. 6, 56 (2005).</li><li>Chen, 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=Integrin-mediated+type+II+TGF-beta+receptor+tyrosine+dephosphorylation+controls+SMAD-dependent+profibrotic+signaling.">Integrin-mediated type II TGF-beta receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling.</a> The Journal of clinical investigation. 124, 3295-3310 (2014).</li><li>Saito, 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=An+integrated+expression+profiling+reveals+target+genes+of+TGF-beta+and+TNF-alpha+possibly+mediated+by+microRNAs+in+lung+cancer+cells.">An integrated expression profiling reveals target genes of TGF-beta and TNF-alpha possibly mediated by microRNAs in lung cancer cells.</a> PloS one. 8, e56587 (2013).</li><li>Dvashi, Z., 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=Protein+phosphatase+magnesium+dependent+1A+governs+the+wound+healing-inflammation-angiogenesis+cross+talk+on+injury.">Protein phosphatase magnesium dependent 1A governs the wound healing-inflammation-angiogenesis cross talk on injury.</a> The American journal of pathology. 184, 2936-2950 (2014).</li><li>Hallgren, 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=Enhanced+ROCK1+dependent+contractility+in+fibroblast+from+chronic+obstructive+pulmonary+disease+patients.">Enhanced ROCK1 dependent contractility in fibroblast from chronic obstructive pulmonary disease patients.</a> Journal of translational medicine. 10, 171 (2012).</li><li>Kobayashi, 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=Matrix+metalloproteinase-9+activates+TGF-beta+and+stimulates+fibroblast+contraction+of+collagen+gels.">Matrix metalloproteinase-9 activates TGF-beta and stimulates fibroblast contraction of collagen gels.</a> American journal of physiology. Lung cellular and molecular physiology. 306, L1006-L1015 (2014).</li><li>Horie, 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=Histamine+induces+human+lung+fibroblast-mediated+collagen+gel+contraction+via+histamine+H1+receptor.">Histamine induces human lung fibroblast-mediated collagen gel contraction via histamine H1 receptor.</a> Experimental lung research. 40, 222-236 (2014).</li><li>Kohyama, 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=PGD(2)+modulates+fibroblast-mediated+native+collagen+gel+contraction.">PGD(2) modulates fibroblast-mediated native collagen gel contraction.</a> American journal of respiratory cell and molecular biology. 27, 375-381 (2002).</li><li>Muir, A. 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=Esophageal+epithelial+cells+acquire+functional+characteristics+of+activated+myofibroblasts+after+undergoing+an+epithelial+to+mesenchymal+transition.">Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition.</a> Experimental cell research. 330, 102-110 (2015).</li><li>Zhong, Q., 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=Role+of+endoplasmic+reticulum+stress+in+epithelial-mesenchymal+transition+of+alveolar+epithelial+cells:+effects+of+misfolded+surfactant+protein.">Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells: effects of misfolded surfactant protein.</a> American journal of respiratory cell and molecular biology. 45, 498-509 (2011).</li><li>Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cytokines+augments+TGF-beta1-induced+epithelial-mesenchymal+transition+in+A549+cells+by+up-regulating+TbetaR-I.">Inflammatory cytokines augments TGF-beta1-induced epithelial-mesenchymal transition in A549 cells by up-regulating TbetaR-I.</a> Cell motility and the cytoskeleton. 65, 935-944 (2008).</li></ol>

Gli autori non hanno conflitti di interesse da dichiarare.

Il protocollo sviluppato in questo studio comprende due fasi. Il primo passo è eseguito per indurre EMT, mentre il secondo passo è il dosaggio gel contrazione. Poiché è importante confermare che le cellule sottoposti EMT, passaggio 2 fornisce un eccellente complemento ai cambiamenti di espressione morfologiche e geniche. Studi precedenti hanno mostrato che EMT delle cellule A549 è stata indotta da solo il 24 TGF-β1; tuttavia, come abbiamo riportato in precedenza 10, TNF-α aumenta EMT e l&#39...

La fibrosi è coinvolto nella patogenesi di diverse malattie croniche progressive, come la malattia polmonare interstiziale, fibrosi cardiaca, cirrosi epatica, insufficienza renale terminale, sclerosi sistemica, e malattie autoimmuni 1. Tra le malattie polmonari interstiziali, la fibrosi polmonare idiopatica (IPF) è una malattia cronica progressiva e mostra prognosi infausta. caratteristica patologica della IPF è lo sviluppo di focolai fibroblastici costituito da fibroblasti attivati ​​e miofibroblasti che sono associati con la prognosi. Le origini di tali fibroblasti polmonari sono proposti per essere derivato da varie cellule mesenchimali, tra cui fibroblasti polmonari residenti in origine e fibrociti circolanti dal midollo osseo. Recentemente, transizione epitelio-mesenchimale (EMT) è stata proposta per essere associato con la formazione di cellule mesenchimali 2, e contribuire alla patogenesi di malattie fibrotiche. Si pensa che EMT svolge un ruolo importante nel processo di sviluppo fetale, la guarigione della ferita, e la progressione del cancro, tra cui l&#39;invasione tumorale e metastasi 3. A seguito del processo di EMT, cellule epiteliali ottenere la capacità delle cellule mesenchimali da perdita di marcatori epiteliali, come la E-caderina, e l&#39;espressione di marcatori mesenchimali, quali vimentina, e α-actina del muscolo liscio (SMA) 4,5. Studi precedenti hanno mostrato l&#39;evidenza che processo EMT è stato associato con lo sviluppo della fibrosi tissutale nel rene e del polmone 6 7. Inoltre, l&#39;infiammazione cronica promuove fibrotica malattia 8; Inoltre, tali citochine infiammatorie come il fattore di necrosi tumorale membro superfamiglia 14 (TNFSF14, la luce), il fattore di necrosi tumorale (TNF) -α, e l&#39;interleuchina-1β, hanno dimostrato di migliorare EMT 9-12. assay contrazione di gel di collagene, un saggio contrazione delle cellule a base di collagene, in cui fibroblasti sono incorporati nel tipo Icollagene gel tridimensionale, è un ideale modello in vitro per la valutazione della contrattilità. Contrattilità è una delle funzioni caratteristiche di fibroblasti e contribuisce al normale ferita riparazione e fibrosi 13. In questo saggio, si ritiene che l&#39;attaccamento di fibroblasti di collagene di tipo I mediante meccanismi di integrine e dovrebbe produrre tensione meccanica in alcune condizioni, e di conseguenza portare alla contrazione del tessuto. Qui, lo sviluppo del test gel contrazione è segnalato per essere adattato per valutare l&#39;acquisizione della funzione contrattile nelle cellule che sottoposti EMT. Questo rapporto dimostra che questo saggio modificato è adatto per valutare la contrattilità delle cellule mesenchimali che hanno subito EMT.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:167px;">For A549 medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:96px;">GIBCO</td>
			<td style="width:119px;">10437</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Transforming Growth Factor-&#946;1, Human, recombinant</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">209-16544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinant Human TNF-&#945;</td>
			<td style="width:96px;">R&#38;D systems</td>
			<td style="width:119px;">210-TA/CF</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">E-Cadherin (24E10) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#3195</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vimentin (D21H3) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#5741</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-&#945;-Tubulin antibody</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">T9026</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-Actin, &#945;-Smooth Muscle antibody&#160;</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">A5228</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-N-cadherin antibody</td>
			<td style="width:96px;">BD Transduction Laboratories</td>
			<td style="width:119px;">#610920</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA931-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">blocking reagent</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN418</td>
			<td>2% in TBS-T</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid</td>
			<td style="width:96px;">corning</td>
			<td style="width:119px;">#353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mm cell culture dish</td>
			<td style="width:96px;">TPP</td>
			<td style="width:119px;">#93100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, powder</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">12100-046</td>
			<td>For 4&#215;DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">type 1 collagen gel</td>
			<td style="width:96px;">Nitta gelatin</td>
			<td style="width:119px;">Cellmatrix type I-A</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">24 well cell culture plate</td>
			<td style="width:96px;">AGC TECHNO GLASS</td>
			<td style="width:119px;">1820-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Documentation System&#160;</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">AE-6911FXN</td>
			<td>Gel imager</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">gel analyzing software</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">Densitograph, ver. 3.00</td>
			<td>analysing software bundled with AE-6911FXN</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 Well Plates, Non-Treated</td>
			<td style="width:96px;">IWAKI</td>
			<td style="width:119px;">1820-024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNA extraction kit</td>
			<td style="width:96px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">reverse transcriptase</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">18080044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">real time PCR system</td>
			<td style="width:96px;">Stratagene</td>
			<td style="width:119px;">Mx-3000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR green PCR kit</td>
			<td style="width:96px;">Qiagen</td>
			<td style="width:119px;">204145</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease Inhibitor Cocktail (100X)</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">78429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVDF membrane</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">2392390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">protein assay kit</td>
			<td style="width:96px;">bio-rad</td>
			<td style="width:119px;">5000006JA&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylamide gel</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">2331810</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">western blotting detection reagent</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN2232</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cold CCD camera</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">Ez-Capture MG/ST</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin inhibitor</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">T9003-100MG</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Polyoxyethylene (20)Sorbitan Monolaurate</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">163-11512</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">polyoxyethylene (9) octyiphenyl ether</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">141-08321</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an in vitro assay to evaluate contractile function of mesenchymal cells that underwent epithelial-mesenchymal transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Durante EMT, le cellule epiteliali perdono marcatori epiteliali, come E-caderina, e di ottenere l&#39;espressione di marcatori mesenchimali, quali vimentina e α-actina muscolo liscio 4,5. L&#39;incubazione delle cellule epiteliali umane A549 polmone con TGF-β1 e TNF-α induce EMT. L&#39;aspetto delle normali cellule A549 sono pietra ciottolo come forma e la forma triangolare che è una caratteristica delle cellule epiteliali (Figura 3A), ma dopo stimolate co...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition at JoVE.com

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Sviluppo di un<em&gt; In Vitro</em&gt; Test per valutare funzione contrattile del mesenchimali Le cellule che sono stati sottoposti a transizione epitelio-mesenchimali

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

development-an-vitro-assay-to-evaluate-contractile-function

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

12.1K Views.  The University of Tokyo Hospital. Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Video: Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

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 ...

Ricerca

Biologia dello sviluppo

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

In vitro e in modelli in vivo per studiare transizione corneale endoteliale-mesenchimale

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

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. ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

In Vitro Crescita dei follicoli preantrali Mouse sotto condizioni di microgravità simulata

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

In Vitro Differenziazione delle cellule staminali mesenchimali umane in funzionale del Cardiomyocyte-come le cellule

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

L'analisi di co-coltura di anello aortico: Un comodo strumento per valutare il potenziale angiogenico delle cellule stromali mesenchimali In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Sviluppo di un test In Vitro per quantificare progenitrici e staminali ematopoietiche cellule (HSPCs) nello sviluppo di embrioni di Zebrafish

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

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 ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

Generazione in Vitro di Somite derivati da umani indotta da cellule staminali pluripotenti indotte

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Valutazione dei danni ossidativi nelle cellule primarie della superficie oculare del topo/cellule staminali in risposta ai danni ultravioletti-C (UV-C)

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...