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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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 border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
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			<td style="width:119px;">11965-092</td>
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			<td>1:3000 dilution</td>
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			<td>1:3000 dilution</td>
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			<td height="21" style="height:21px;width:216px;">Anti-&#945;-Tubulin antibody</td>
			<td style="width:96px;">sigma aldrich</td>
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			<td>1:10000 dilution</td>
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			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-Actin, &#945;-Smooth Muscle antibody&#160;</td>
			<td style="width:96px;">sigma aldrich</td>
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			<td>1:10000 dilution</td>
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			<td>1:1000 dilution</td>
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			<td style="width:96px;">GE Healthcare</td>
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			<td>1:20000 dilution</td>
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			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934-100UL</td>
			<td>1:20000 dilution</td>
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			<td height="42" style="height:42px;width:216px;">blocking reagent</td>
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			<td style="width:119px;">RPN418</td>
			<td>2% in TBS-T</td>
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		<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>
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		<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>
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		<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>
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			<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:216px;">24 Well Plates, Non-Treated</td>
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			<td height="42" style="height:42px;width:216px;">Trypan Blue Solution, 0.4%</td>
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			<td style="width:119px;">15250-061</td>
			<td></td>
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			<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>
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			<td height="42" style="height:42px;width:216px;">reverse transcriptase</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">real time PCR system</td>
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			<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>
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		<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>
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		<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>
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		<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>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylamide gel</td>
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			<td style="width:119px;">2331810</td>
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			<td height="42" style="height:42px;width:216px;">western blotting detection reagent</td>
			<td style="width:96px;">GE Healthcare</td>
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			<td height="42" style="height:42px;width:216px;">cold CCD camera</td>
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			<td height="21" style="height:21px;width:216px;">Trypsin inhibitor</td>
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			<td style="width:119px;">163-11512</td>
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			<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>
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</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 ...

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

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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.

שיטה אנזימתי להציל תאי גזע mesenchymal מדוגמאות קרשו מח העצם

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

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.

במבחנה מודלים Vivo ללמוד קרנית האנדותל-mesenchymal מעבר

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.

בידוד של עכבר רירית הרחם אפיתל סטרומה תאים עבור<em&gt; במבחנה</em&gt; Decidualization

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.

במבחנה צמיחה של העכבר זקיקים Preantral תחת Microgravity מדומה

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.

במבחנה הבידול של גזע Mesenchymal אנושי לתוך תאים דמויי Cardiomyocyte תפקודית

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.

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 ...

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.

התפתחות במבחנה הנועד Quantitate גזע Hematopoietic, קדמון תאים (HSPCs) בפיתוח עוברי דג זברה

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.

ניתוח חישובית של Germline Caenorhabditis elegans ללמוד הפצה של גרעינים, חלבונים ו את שלד התא

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.

בדור חוץ גופית של Somite נגזרים מן האדם המושרה בתאי גזע Pluripotent

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.

הערכה של נזק חמצוני בעכבר הראשי פני השטח העינית/תאים גזע בתגובה אולטרה סגול (UV-C) נזק

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

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