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.

תכשירי 1. והתרבות של תאי אפיתל הריאות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תאי אפיתל ריאה אנושית A549 תרבות (שורת תאים חסיד) ב בינוני הנשר שונה של Dulbecco (DMEM) בתוספת 10% בסרום שור העובר (FBS), 100 IU / פניצילין מ&quot;ל, 100 מיקרוגרם / מ&quot;ל ​​סטרפטומיצין. </li><li style=";text-align:right;direction:rtl"> סר וזורק תקשורת תרבית תאים מן מנת התרבות, ולשטוף פעם עם 5 - 10 מיליליטר של בופר פוספט (PBS). לאחר הכביסה, מיד לשאוב PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 2 מ&quot;ל טריפסין / חומצה ethylenediaminetetraacetic (EDTA) (0.05%) ו לדגור על 37 מעלות צלזיוס, 5% CO 2 דקות 3. </li><li style=";text-align:right;direction:rtl"> אוסף את התא המנותק צינורות צנטריפוגה המכילים בינוני תרבית תאים, צנטריפוגה הצינורות ב RT במשך 4 דקות ב g 150 ×. </li><li style=";text-align:right;direction:rtl"> Resuspend התאים גלולה ב 2 מ&quot;ל של מדיום תרבית תאים ולהסיר מדגם עבור ספירת תאים. ספירת התאים באמצעות hemocytometer עם כתם כחול Trypan לבדוק כדאיות התא. </li><li style=";text-align:right;direction:rtl"> תאי A549 זרע על10 ס&quot;מ קלקר צלחות בצפיפות של 0.5 - 1.0 × 10 6 תאים / תבשיל עם 10 מ&quot;ל של מדיום עבור assay התכווצות ג&#39;ל. זרעים התאים על 6 צלחות קלקר גם בצפיפות של 0.5 - 1.0 × 10 5 תאים / היטב עם 2 מ&quot;ל של מדיום לאישור EMT. דגירת התאים ב 37 מעלות צלזיוס, 5% CO 2 למשך 24 שעות. </li></ol> נוהל EMT 2. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 10 μl של-β1 TGF (5 מיקרוגרם / מ&quot;ל) ו -10 μl של-α TNF (10 מיקרוגרם / מ&quot;ל) לצלחת עבור assay התכווצות ג&#39;ל זורעים בשלב 1.6. הוסף 2 μl של-β1 TGF (5 מיקרוגרם / מ&quot;ל) ו -2 μl של-α TNF (10 מיקרוגרם / מ&quot;ל) לצלחת לאישור EMT זורעים בשלב 1.6. לדגור על 37 מעלות צלזיוס, 5% CO 2 למשך 48 שעות. </li></ol> 3. אישור של נוהל EMT ידי PCR ו המערבי סופג <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אשר שינוי במורפולוגיה (מאבן דמוי חלוק כדי ציר צורה) של תאים שטופלו באמצעות מיקרוסקופ לעומת שלב. הערה: נורםיש תאי A549 אל חלוק אבן דמוית ומראה בצורה משולש היא מאפיין של תאי אפיתל, אך לאחר גירוי עם-β1 TGF ו-α TNF, התאים להופיע ארוכים ציר בצורה דומה תאי mesenchymal 14,15. </li><li style=";text-align:right;direction:rtl"> חישוב הביטוי של סמן אפיתל, כגון E-cadherin, וסמנים mesenchymal כגון N-cadherin, vimentin, אקטין שריר α-חלקה באמצעות PCR או המערבי סופג. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חלץ RNA הכולל את התאים באמצעות ערכת חילוץ RNA 16 לסנתז cDNA באמצעות transcriptase הפוכה 17 על פי פרוטוקול של היצרן. </li><li style=";text-align:right;direction:rtl"> למדוד ביטוי של רמות ה- mRNA באמצעות מערכת בזמן אמת PCR 18 וערכת PCR לצבוע cyanine monomeric פי הוראות היצרנים. פריימרים ספציפיים עבור GAPDH (glyceraldehyde dehydrogenase 3-פוספט), ACTA2 (אלפא-אקטין 2), CDH1 (cadherin-1; E-cadherin), ו VIM (vimentin) מוצגים בלוח 1 </stרונג&gt;. </li><li style=";text-align:right;direction:rtl"> Lyse את התאים באמצעות חיץ תמוגה (טבלה 2) תמיסה המכילה קוקטייל מעכב 1% פרוטאז. למדוד את כל ריכוזי חלבון מדגם באמצעות ערכת assay חלבון 19,20, ולהחיל אותן כמויות של חלבון ג&#39;ל polyacrylamide. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בצע-ג&#39;ל אלקטרופורזה SDS והעביר חצי יבש של חלבוני קרום PVDF 21. דגירה הממברנה עם נוגדנים ראשוניים בחסימת המאגר עבור 1 - שעות 2. לאחר דגירה, לשטוף פעמים הממברנה עם חיץ לשטוף דגירת hr קרום 1 עם נוגדנים שניים. הערה: עיין חומרים / ציוד שולחן עבור נוגדנים דילולים שימוש במחקרים אלה. ראה טבלה 2 עבור רכיבי חסימת חיץ. </li><li style=";text-align:right;direction:rtl"> לשטוף את הממברנה עם חיץ לשטוף פעמיים. צלם תמונות של הקרום עם ערכת זיהוי המערבית סופגת 22 באמצעות 11,23 מצלמת CCD קר. </li></ol></li></ol></li></ol> 4. Assay התכווצות ג&#39;ל EMT הערכה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשאוב התקשורת מותנה מספינת תרבית תאים, ולשטוף היטב עם 5 - 10 מ&quot;ל של PBS להסיר תאים מתים. לאחר הכביסה, מיד לשאוב PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 2 מ&quot;ל של 0.05% טריפסין / EDTA ו לדגור על 37 מעלות צלזיוס, 5% CO 2 דקות 3. </li><li style=";text-align:right;direction:rtl"> אוספים את התאים מנותקת צינורות צנטריפוגה המכיל DMEM בתוספת מעכב טריפסין (1 מ&quot;ג / מ&quot;ל), צנטריפוגה צינורות ב RT במשך 4 דקות ב 150 × גרם. </li><li style=";text-align:right;direction:rtl"> מערבבים סוג 1 ג&#39;ל קולגן עם מים מזוקקים, ו -4 × מרוכזים DMEM להתאים את הכרכים כדי להשיג ריכוז הקולגן של 1.75 מ&quot;ג / מ&quot;ל, 1 × ריכוז DMEM. הקפד לשמור המדיום ג&#39;ל על הקרח במהלך שלב זה. הערה: כדי להפוך 6 מ&quot;ל של מדיום ג&#39;ל, ומערבבים היטב 3.5 מ&quot;ל של ג&#39;ל קולגן מסוג 1 (3 מ&quot;ג / מ&quot;ל), 1.5 מ&quot;ל של 4 × מרוכזים DMEM, ו 1ml של מים מזוקקים. </li><li style=";text-align:right;direction:rtl"> Resuspend התא גלולה ב 500 μl של PBS ולהסיר מדגם עבור ספירת תאים. לספורמספר התאים באמצעות hemocytometer עם כתם כחול trypan לבדוק כדאיות התא. </li><li style=";text-align:right;direction:rtl"> הוסף בינוני ג&#39;ל להתאים את הכרכים להשיג צפיפות התאים של 3.0 × 10 5 תאים / טוב (6.0 × 10 5 תאים / מ&quot;ל) בעדינות אך במהירות לערבב אותו על ידי pipetting ללא gelation. </li><li style=";text-align:right;direction:rtl"> לוותר 0.5 מיליליטר של התערובת לבאר כל צלחת 24 גם שאינם מטופלים במהירות ובזהירות לעשות בצורה גלילית מסודרת. היזהר שלא לאפשר כל בועות אוויר לזהם את הג&#39;לים (איור 1 א). הערה: בינוני ג&#39;ל המכיל את התאים הוא צמיג ויכול ג&#39;ל טופס סהר צורה בקלות הבאר. </li><li style=";text-align:right;direction:rtl"> דגירה את הצלחת במשך 15 דקות בתוך חממת תא ב 37 מעלות צלזיוס, 5% CO 2 ולחות 95% להגליד לחלוטין. </li><li style=";text-align:right;direction:rtl"> ניתוק ג&#39;לים מהצלחת בלי לשבור על ידי הזזת מרית מעוקרת באופן לצייר היקף בכיוון אחד. בעזרת מרית, להעביר את הג&#39;לים בעדינות תרבות מנות 60 מ&quot;מ רקמההמכיל 5 מ&quot;ל של FBS% DMEM / 1 עם או בלי-β1 TGF (5 ng / ml) ו-α TNF (10 ng / ml). </li><li style=";text-align:right;direction:rtl"> נער בעדינות את הכלים כדי להבטיח ג&#39;לים צף על המדיום. מדגירים את תא חממה על 37 מעלות צלזיוס, 5% CO 2 ולחות 95%. </li></ol> מדידת 5. ג&#39;ל גודל <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מדוד את גודל ג&#39;ל קולגן לאחר 0, 24, 48, ו -72 שעות באמצעות מערכת ניתוח תמונה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפעל את מערכת תיעוד ג&#39;ל (איור 2 א), ולשים מנות לתוך ארון מגן אור. ואז מסירים את המכסה של הצלחות בארון הכלים. </li><li style=";text-align:right;direction:rtl"> פתח את התוכנה לניתוח ג&#39;ל קשורה. לחץ על &quot;כפתור רכישת תמונה&quot; בבר-התפריט כדי להציג את התמונה של הג&#39;לים בקבינט. לאחר מכן, לחץ &quot;לרכוש&quot; בשורת התפריטים כדי לצלם את ג&#39;לים (איור 2 ב). </li><li style=";text-align:right;direction:rtl"> לחץ על &quot;כפתור זיהוי&quot; בשורת התפריטים (איור 2 ג) ולהתאים באזור המדידה (i עיגול צהובn איור 2C) על ידי גרירת העכבר. לאחר מכן, לחץ על &quot;איתור אוטומטי כפתור&quot; בשורת התפריטים (ראה כפתור באיור 2D). התוכנה מזהה אוטומטית את הג&#39;לים מראים מפת חום (איור 2 ד). </li><li style=";text-align:right;direction:rtl"> לחץ &quot;אישור&quot; כדי לחלץ את קווי המתאר של ג&#39;ל על ידי עיבוד תמונה ולחשב אזורים המוקפים המתאר (האיור 2E). אחרי השטח חושב, באזור המחושב מוצג חלון מוקפץ נפרד. הערה: אם את הג&#39;לים חופפים זה לזה במהלך ההדמיה, להעביר ג&#39;לים בעדינות בעזרת קצה פיפטה סטרילית. </li></ol></li></ol>

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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. 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יש המחברים אין ניגודי אינטרסים לחשוף.

הפרוטוקול שפותח במחקר זה כולל שני שלבים. השלב הראשון מתבצע על מנת לגרום EMT, ואילו השלב השני הוא assay התכווצות ג&#39;ל. מאז חשוב לאשר תאים עברו EMT, שלב 2 מספק השלמה מצוינת שינויי ביטוי מורפולוגיים גן. מחקרים קודמים הראו כי EMT של תאי A549 הושר על ידי TGF-β1 רק 24; עם זאת, כפי שכב?...

פיברוזיס מעורב בפתוגנזה של מחלות פרוגרסיבי שונות כרוניות, כגון מחלת ריאות אינטרסטיציאלית, סיסטיק לב, שחמת כבד, אי ספיקת כליות סופנית, טרשת מערכתית, ומחלות אוטואימוניות מחלה 1. בין מחלות ריאה אינטרסטיציאלית פיברוזיס ריאתי אידיופתי (IPF) הוא מחלה מתקדמת כרונית ומראה פרוגנוזה גרועה. סימן היכר הפתולוגי של IPF הוא הפיתוח של מוקדי fibroblastic המורכבים פיברובלסטים מופעל myofibroblasts המשויכים הפרוגנוזה. מקורותיה של פיברובלסטים ריאתי כאלו מוצעות לנבוע מכמה תאים mesenchymal, כולל fibroblasts ריאות תושב במקור ומדחסים fibrocytes ממח העצם. לאחרונה, מעבר mesenchymal-אפיתל (EMT) הוצע להיות מזוהה עם ההיווצרות של תאי mesenchymal 2, ולתרום בפתוגנזה של פרעות fibrotic. הוא חשב כי EMT משחק תפקידים חשובים בתהליך של התפתחות עוברת, ריפוי פצעים, והתקדמות של סרטן, כולל חדירת גידול וגרור 3. בעקבות התהליך של EMT, תאי אפיתל להשיג את היכולת של תאי mesenchymal ידי אובדן של סמנים אפיתל, כגון E-cadherin, ועל ידי ביטוי של סמני mesenchymal, כגון vimentin, יקטין שריר α-חלקה (SMA) 4,5. מחקרים קודמים הראו הראיות כי תהליך EMT כבר קשור להתפתחות של סיסטיק רקמות הכליה 6 וריאות 7. בנוסף, דלקת כרונית מקדמת fibrotic מחלת 8; יתר על כן, ציטוקינים דלקתיים כגון חבר superfamily גורם גידול נמק 14 (TNFSF14; LIGHT), tumor necrosis factor (TNF) -α, ו- interleukin-1β, הוכחו לשפר EMT 9-12. assay התכווצות ג&#39;ל קולגן, assay התכווצות תא מבוסס קולגן, אשר פיברובלסטים מוטבעים מהסוג Iקולגן ג&#39;ל תלת ממדית, הוא אידיאל במודל חוץ גופית לצורך הערכה של התכווצות. התכווצות היא אחת הפונקציה האופיינית של פיברובלסטים ותורמת תיקון פצע נורמלי סיסטיק 13. ב assay זה, הוא חשב כי את הקובץ המצורף של פיברובלסטים Type I קולגן באמצעות מנגנוני integrin תלוי אמור לייצר מתח מכאני בתנאים מסוימים, וכתוצאה מכך להוביל להתכווצות רקמות. כאן, בפיתוח של assay התכווצות ג&#39;ל מדווח להיות מותאם על מנת להעריך את רכישת פונקצית התכווצות בתאים שעברו EMT. דו&quot;ח זה מדגים כי assay שונה זו מתאימה להערכת contractility בתאי mesenchymal שעברו EMT.

<table><tbody><tr><td>DMEM</td><td>sigma aldrich</td><td>11965-092</td><td>For A549 medium</td></tr><tr><td>FBS</td><td>GIBCO</td><td>10437</td><td></td></tr><tr><td>Transforming Growth Factor-&amp;beta;1, Human, recombinant</td><td>Wako Laboratory chemicals</td><td>209-16544</td><td></td></tr><tr><td>Recombinant Human TNF-&amp;alpha;</td><td>R&amp;amp;D systems</td><td>210-TA/CF</td><td></td></tr><tr><td>E-Cadherin (24E10) Rabbit mAb</td><td>Cell Signaling Technology</td><td>#3195</td><td>1:3,000 dilution</td></tr><tr><td>Vimentin (D21H3) Rabbit mAb</td><td>Cell Signaling Technology</td><td>#5741</td><td>1:3,000 dilution</td></tr><tr><td>Anti-&amp;alpha;-Tubulin antibody</td><td>sigma aldrich</td><td>T9026</td><td>1:10,000 dilution</td></tr><tr><td>Monoclonal Anti-Actin, &amp;alpha;-Smooth Muscle antibody&amp;nbsp;</td><td>sigma aldrich</td><td>A5228</td><td>1:10,000 dilution</td></tr><tr><td>Anti-N-cadherin antibody</td><td>BD Transduction Laboratories</td><td>#610920</td><td>1:1,000 dilution</td></tr><tr><td>Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)</td><td>GE Healthcare</td><td>NA931-100UL</td><td>1:20,000 dilution</td></tr><tr><td>Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)</td><td>GE Healthcare</td><td>NA934-100UL</td><td>1:20,000 dilution</td></tr><tr><td>Blocking reagent</td><td>GE Healthcare</td><td>RPN418</td><td>2% in TBS-T</td></tr><tr><td>6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid</td><td>corning</td><td>#353046</td><td></td></tr><tr><td>100 mm Cell culture dish</td><td>TPP</td><td>#93100</td><td></td></tr><tr><td>DMEM, powder</td><td>life technologies</td><td>12100-046</td><td>For 4&amp;times; DMEM</td></tr><tr><td>Type 1 collagen gel</td><td>Nitta gelatin</td><td>Cellmatrix type I-A</td><td></td></tr><tr><td>24 Well cell culture plate</td><td>AGC TECHNO GLASS</td><td>1820-024</td><td></td></tr><tr><td>Gel Documentation System&amp;nbsp;</td><td>ATTO</td><td>AE-6911FXN</td><td>Gel imager</td></tr><tr><td>Gel analyzing software</td><td>ATTO</td><td>Densitograph, ver. 3.00</td><td>analysing software bundled with AE-6911FXN</td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>life technologies</td><td>25300054</td><td></td></tr><tr><td>24 Well Plates, Non-Treated</td><td>IWAKI</td><td>1820-024</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>life technologies</td><td>15250-061</td><td></td></tr><tr><td>RNA extraction kit</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>Reverse transcriptase</td><td>life technologies</td><td>18080044</td><td></td></tr><tr><td>Real time PCR system</td><td>Stratagene</td><td>Mx-3000P</td><td></td></tr><tr><td>SYBR green PCR kit</td><td>Qiagen</td><td>204145</td><td></td></tr><tr><td>Protease Inhibitor Cocktail (100x)</td><td>life technologies</td><td>78429</td><td></td></tr><tr><td>PVDF membrane</td><td>ATTO</td><td>2392390</td><td></td></tr><tr><td>Protein assay kit</td><td>bio-rad</td><td>5000006JA&amp;nbsp;</td><td></td></tr><tr><td>Polyacrylamide gel</td><td>ATTO</td><td>2331810</td><td></td></tr><tr><td>Western blotting detection reagent</td><td>GE Healthcare</td><td>RPN2232</td><td></td></tr><tr><td>Cold CCD camera</td><td>ATTO</td><td>Ez-Capture MG/ST</td><td></td></tr><tr><td>Trypsin inhibitor</td><td>sigma aldrich</td><td>T9003-100MG</td><td></td></tr><tr><td>Polyoxyethylene (20)Sorbitan Monolaurate</td><td>Wako Laboratory chemicals</td><td>163-11512</td><td></td></tr><tr><td>Polyoxyethylene (9) octyiphenyl ether</td><td>Wako Laboratory chemicals</td><td>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.

במהלך EMT, תאי אפיתל לאבד סמנים אפיתל, כמו E-cadherin, ולהשיג ביטוי של סמנים mesenchymal, כגון vimentin ו α-שריר חלק אקטין 4,5. דגירה של תאי אפיתל ריאה האנושית A549 עם-β1 TGF ו- TNF-α גורמת EMT. הופעתו של תאי A549 נורמלים הם אבן חלוק כמו צורה וצורה משולשת היא מאפיין של תאי אפי...

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.

פיתוח של<em&gt; במבחנה</em&gt; Assay לאבחון תפקוד התכווצות של תאי mesenchymal שעבר אפיתל-mesenchymal מעבר

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

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Developmental Biology

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

12.2K 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

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Epicardial תולדת תרבות Assay ו<em&gt; Ex Vivo</em&gt; הערכה של נדידת תאים שמקורם Epicardial

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Enzyme- ואת הסרום ללא דגם תרבית תאי גזע עצבי לחקירות EMT המתאימים גילוי תרופות

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

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.

וזמינותו תרבות משותפת הטבעת אבי העורקים: כלי נוח כדי להעריך את הפוטנציאל אנגיוגנזה של תאי סטרומה Mesenchymal במבחנה

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

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

ניצול של זיזי סיליקון המיקרוסקופים כדי להעריך את התפקוד נייד התכווצות<em&gt; במבחנה</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Bioengineering

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

תפוקה גבוהה המתיחה כוח מיקרוסקופ באמצעות PDMS מגלה השפעות תלויי מינון של שינוי מקדם הצמיחה-β על המעבר אפיתל-to-מעבר משנה

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Assay Explant להערכת התנהגות ניידת של mesenchyme הגולגולת

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Biology

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

ניתוח פשוט בעל תפוקה גבוהה של התכווצות של תאים חד-תאיים באמצעות אלסטומרים מיקרו-מרופדים

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

להערכת ויטרו של תפקוד הלב באמצעות קרדיומיוציטים עור

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Medicine

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

בידוד של תאי שריר חלק אבי העורקים העיקריים הספציפיים למטופל ומדידות כיווץ חצי-קוונטיטיביות בזמן אמת במבחנה

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

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Epicardial תולדת תרבות Assay ו