עבודה זו מומנה ברצף על ידי גבולות NCCR בפרס טייס גנטיקה, Juvenile Diabetes Research Foundation גרנט 41-2009-775 וגרנט 12-126875 מDet Frie Forskningsråd / עוג Sundhed Sygdom. המחברים מודים המעבדה Spagnoli לאירוח וידאו הירי.

פרוטוקול זה נועד לגדול organoids לבלב שמקורם E10.5 העכברי ניתק תאי לבלב אפיתל. הפרוטוקול דורש אישור אתי לניסויים בבעלי חיים. 1. Dissection של גב הלבלב באד מE10.5 עוברי עכברים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מדיום חיוני להקריב את העכברים בעיתוי בהריון ביום עוברי (E) 10.5, פתח את הבטן עם זוג מספריים, להסיר את שתי קרנות הרחם ומניח אותם בצלחת פטרי 10 סנטימטר מלא מלוח פוספט הקר חיץ (PBS) או שונה Dulbecco (DMEM) שמר על קרח. הניסוי הכולל מההקרבה לזריעת תאים נעשה ב60-90 דקות על מנת למנוע נזק לתאים. </li><li style=";text-align:right;direction:rtl"> הפרד את הרחם למקטעי עובר בודדים באמצעות מספריים קטנים. העבר את העובר אחד לצלחת 35 מ&quot;מ פטרי עם PBS הקר ולדמיין אותו תחת מיקרוסקופ לנתח עם תאורה מלמעלה. עם מלקחיים נתיחה להסיר את sa שרירים, decidua וחלמון מסביבג ולחשוף את העובר. מניחים את העובר חזרה למדיום DMEM קר. בקלות ניתן להעביר עוברים באמצעות פיפטה / טפטפת העברת פלסטיק 3 מיליליטר. לבודד את כל עובר בודד בצורה דומה לפני שתמשיך. </li><li style=";text-align:right;direction:rtl"> הנח לעובר 35 מ&quot;מ צלחת פטרי נקי ב-PBS. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש במלקחיים דקים (0.05 רוחב מ&quot;מ) כדי להסיר את forelimb. הכניסו בעדינות את המלקחיים בפתיחה לנתק את מערכת עיכול מאזור חוט השדרה (איור 1). אופציונלי: כדי לראות בצורה נוחה יותר בבטן, להסיר את החלק העליון של גופו של העובר, עד ללב ובאזור הזנב מתחת לגבעול החלמון. </li><li style=";text-align:right;direction:rtl"> אתר את הקיבה, הכבד ומעי. באמצעות המלקחיים, לבודד את מערכת העיכול מהקיבה למעי ולמקם אותו בDMEM הקר על קרח (איור 1). ניצן הלבלב הגבי מצורף dorsally, אחורי אל הקיבה (איור 1). </li><li style=";text-align:right;direction:rtl"> לבודד כל אדם מערכת עיכול בסימיאופן lar לפני שתמשיך. אם העוברים צריכים להיות genotyped, לאסוף את הזנב בעת הנתיחה ולשמור על כל מערכת עיכול בודדת בבאר שונה בצלחת 24 גם עם DMEM הקר על קרח. </li></ol></li><li style=";text-align:right;direction:rtl"> הנח בדרכי עיכול באחד 35 מ&quot;מ צלחת פטרי נקייה בקור PBS. עכשיו להשתמש בתאורה מלמטה בשדה בהיר כדי לחזות ולנתח את ניצן הלבלב הגבי תחת מיקרוסקופ לנתיחה. תנאים אלה יהיו לייעל את ההדמיה של כרכים. באמצעות מחטי טונגסטן חדדו אלקטרוליטים או 20 מחטי מזרק G, לבודד את ניצן הלבלב עם mesenchyme מעט ככל האפשר סביב אפיתל (איור 1). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העבר את הניצן המבודד לתוך צלחת פטרי שמכילה תמיסה קרה dispase (1.25 מ&quot;ג / מיליליטר) ל2-3 דקות. מנקודה זו, העברה יכולה ביותר נוחות להיעשות עם 50 נימי זכוכית μl-משך להבה מחוברת לצינור גמיש שבשליטת פה. לחלופין, אבל עם יותר ריsk לאבד הניצן, השתמש פיפטה אוטומטית 10 μl (Pipetman) עם טיפים פלסטיק מתאימים. </li><li style=";text-align:right;direction:rtl"> בצע שאיפת ניצן לבלב ופליטה תחת שליטה מיקרוסקופית. שים ניצן הלבלב בחזרה ב-PBS. בהמשך לנקות את ניצן הלבלב המבודד מmesenchyme עם המחטים ושאיפה עדינה באמצעות נימי הזכוכית (איור 1). </li><li style=";text-align:right;direction:rtl"> כאשר כל mesenchyme מוסר, יש לשטוף את ניצן הלבלב בקור PBS; להעביר את כל ניצן לDMEM הקר בבארות בודדות (60 היטב מיני מגשים מלא ב10 μl של DMEM הקר). חשוב שלא להסיר את mesenchyme בdispase, מה שהופך את הרקמה דביקה מאוד. </li></ol></li></ol> 2. ציפוי ותרבות של תאים מפוזרים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העבר האפיטל גזור מכל העוברים עם נימי זכוכית משכה להבה לתוך בארות חרוטי של 60 היטב מיני מגשים מלא ב10 μl PBS לשטיפה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העבר את הניצן ל10 μl של 0.05% טריפסיןnd לתת לו לדגור על 37 מעלות צלזיוס למשך 4 דקות. להשבית טריפסין על ידי העברת הניצן להיטב עם 10 DMEM μl 10% עוברי עגל בסרום + (FCS). </li><li style=";text-align:right;direction:rtl"> לנתק את ההשעיה התא על ידי שאיפה דרך נימים דקות משכו עם חולץ פיפטה. זה חשוב כדי למנוע בועות בשלב זה תוך pipetting למעלה ולמטה כדי לנתק את התאים. organoids הלבלב להתחיל בצורה אופטימלית מקבוצות קטנות של 5-15 תאים ולכן ניתוק חלקי מומלץ (איור 1). </li></ol></li><li style=";text-align:right;direction:rtl"> בריכת תאים מכמה עוברים לתוך צינור Eppendorf על מנת למזער את ההבדלים בשל עיבוד פרט. לדלל את ההשעיה התא בMatrigel המצונן ביחס 1:3. Aliquot את התערובת לצלחת 96 היטב, 8 μl / טוב או בצלחת מותאמת להדמיה (ראה להלן). </li><li style=";text-align:right;direction:rtl"> דגירה את הצלחת ב-C ° 37 למשך 5 דקות, ומאפשרת Matrigel להתעבות. למלא את הבארות עם 70 μl של מדיום של בחירה (organoid או כדור, ראה לשוניתles 1 ו -2) ולהשאיר בסביבת humidified המכילה 5% CO 2 ו95% אוויר ב37 ° C. </li><li style=";text-align:right;direction:rtl"> החלף את המדיום בכל יום 4 ה. לפקח על מושבות לבלב גדלה מדי יום ולתעד את התהליך על ידי הדמיה. </li><li style=";text-align:right;direction:rtl"> ניתן להוסיף מולקולות או חלבונים קטנים של עניין למדיום בשלב זה לניסויי הפרעה, כפי שדווחו בעבר 28. </li></ol> טבלה 1: בינוני Organoid. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> שמו של חומר </td><td> ריכוז מניות </td><td> ריכוז במדיום סופי </td><td> נפח של המניה </td></tr><tr><td> פניצילין, סטרפטומיצין </td><td> 100% </td><td> 1% </td><td> 50 μl </td></tr><tr><td> החלפת סרום נוקאאוט (תוסף) </td><td> 100% </td><td> 10% </td><td> 500 μl </td></tr><tr><td> 2-mercaptoethanol </td><td> 14.3 M </td&gt; <td> 0.1 מ&quot;מ </td><td> 1 μl </td></tr><tr><td> Phorbol Myristate אצטט (PMA) </td><td> 16 מיקרומטר </td><td> 16 ננומטר </td><td> 5 μl </td></tr><tr><td> Y-27632 (מעכב ROCK) </td><td> 50 מ&quot;מ </td><td> 10 מיקרומטר </td><td> 1 μl </td></tr><tr><td> EGF </td><td> 50 מיקרוגרם / מיליליטר </td><td> 25 ng / ml </td><td> 2.5 μl </td></tr><tr><td> האנושי רקומביננטי R-spondin 1 </td><td> 250 מיקרוגרם / מיליליטר </td><td> 500 ng / ml </td><td> 10 μl </td></tr><tr><td> - או - </td><td></td><td></td><td></td></tr><tr><td> רקומביננטי עכבר 1 R-spondin </td><td> 250 מיקרוגרם / מיליליטר </td><td> 500 ng / ml </td><td> 10 μl </td></tr><tr><td> FGF1 האנושי רקומביננטי (aFGF) </td><td> 100 מיקרוגרם / מיליליטר </td><td> 25 מיקרוגרם / מיליליטר </td><td> 1.25 μl </td></tr><tr><td> הפרין (Liquemin) </td><td> 2500 U / ml </td><td> 2.5 U / ml </td><td> 2 μl </td></tr><tr><td> מלFGF10 אדם binant </td><td> 100 מיקרוגרם / מיליליטר </td><td> 100 ng / ml </td><td> 5 μl </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 μl </td></tr><tr><td> סך הכל </td><td></td><td></td><td> 5,000 μl </td></tr></tbody></table> טבלה 2: בינוני כדור. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> שמו של חומר </td><td> ריכוז מניות </td><td> ריכוז במדיום סופי </td><td> נפח של המניה </td></tr><tr><td> פניצילין, סטרפטומיצין </td><td> 100% </td><td> 1% </td><td> 50 μl </td></tr><tr><td> x50 B27 (תוסף) </td><td> 100% </td><td> 10% </td><td> 100 μl </td></tr><tr><td> FGF2 האנושי רקומביננטי (bFGF) </td><td> 100 מיקרוגרם / מיליליטר </td><td> 64 ng / ml </td><td> 3.2 μl </td></tr><tr><td> Y-27632 (מעכב ROCK) </td><td> 50 מ&quot;מ </td><td> 10 מיקרומטר </td><td> 1 μl </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845.8 μl </td></tr><tr><td> סך הכל </td><td></td><td></td><td> 5000 μl </td></tr></tbody></table> 3. הדמיה של ההתקדמות של פיתוח Organoid <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> organoids תמונה אחת ביום, או על ידי מיקרוסקופ הזמן לשגות באמצעות מיקרוסקופ הזמן לשגות ניאון. עבור זמן לשגות הדמיה, השתמש XY מיקרוסקופ פלואורסצנטי ההפוך אוטומטי (Z). </li><li style=";text-align:right;direction:rtl"> טיפות קטנות פיקדון של 3 μl בצלחות 4 היטב או צלחות זכוכית תחתונה מלאות ב2-5 בינוני מיליליטר. תמונה עם מטרה למרחקים ארוכים 10x. הערה: ניתן להשתמש בעכברים מהונדסים מבטא קליעים נותבים ניאון. סרט 1 מופעים למשל ההתרחבות הראשונית של organoids מPdx1-Ngn3-ER עכברי TM-ires-nGFP + 4. GFP הגרעיני מאפשר למשתמש לעקוב אחר תאים כיכולים להיות מיושמים אובייקטים בודדים אבל עקרונות דומים כדי לעקוב אחר תאים עם הקרינה קרום כזהכמו עכברי MT / מיליגאוס 29. </li><li style=";text-align:right;direction:rtl"> להתחיל זמן לשגות הדמיה שעה 3 לאחר זריעת התאים, כדי למנוע סחף להתמקד ולהגדיר האוטומציה שליטה בתוכנה כדי לצלם את 1 / שעה למשך 3 ימים או יותר בעמדות שהוגדרו באופן ידני. לכל תפקיד, לרכוש התערבות ההפרש לעומת זאת תמונה (DIC), כמו גם את אות ה-GFP, דיווח ביטוי סמן פלואורסצנטי. </li></ol> 4. שחזור של Organoids להיסטולוגיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את צלחת 96 היטב על קרח ולהסיר את המדיום, והחלפתו עם PBS הקר כקרח. זה depolymerizes חלקית Matrigel. </li><li style=";text-align:right;direction:rtl"> לשאוב בעדינות כל organoid פרטני, הסרת Matrigel מקיף באמצעות קצה 1,000 μl על מנת שלא לשבש את הארכיטקטורה הכוללת. העבר כל organoid להיטב עם קרח הקר PBS. שמור את הצלחת על קרח. קיבעון ישיר בMatrigel הוא גם אפשרי. </li><li style=";text-align:right;direction:rtl"> תקן organoid במשך 15 דקות paraformaldehyde 4% (PFA), cryopreserve זה בסוכרוז ולשבץ אותה בג&#39;לטין. לעבד כלorganoid לcryosectioning והיסטולוגיה כמתואר (ג&#39;והנסון et al. 2007) 4 בעבר. </li></ol> 5. שחזור של Organoids לPCR וביוכימיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את צלחת 96 היטב על קרח ולהסיר את המדיום. הוסף 60 μl של RNAlater בכל טוב על מנת לייצב ולהגן על RNA התאי. </li><li style=";text-align:right;direction:rtl"> לשבש את הג&#39;ל בכל אחד גם באופן מכאני על ידי באופן חלקי depolymerizing אותו על קרח. כך או לשחזר organoids הבודד באמצעות טיפ 1,000 μl על מנת שלא לשבש את הארכיטקטורה הכוללת או לשחזר גם כולו (עם Matrigel) ע&quot;י שיבוש ג&#39;ל מכאני עם טיפ μl 200. השתמש קצה 1,000 μl להעביר את התוכן גם לתוך צינור Eppendorf חופשי לא דביק RNAse שמר על קרח. </li><li style=";text-align:right;direction:rtl"> לשטוף את הבארות עם 60 RNAlater μl ולהוסיף תוכן שנותר לאותו הצינור Eppendorf. </li><li style=";text-align:right;direction:rtl"> ספין הצינורות במשך 5 דקות ב500-1,000 XG ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> הסר את supernatant, רק עוזב 20-30 μlשל RNAlater בצינור יחד עם גלולה. לביוכימיה, לאחסן דגימות יבשות ככל האפשר. </li><li style=";text-align:right;direction:rtl"> אין להקפיא דגימות בRNAlater באופן מיידי; חנות ב 4 ° CO / N (כדי לאפשר RNAlater לחדור את הרקמה ביסודיות). הרקמה יכולה להיות מאוחסנת ב-20 ° C לאחסון לטווח ארוך ובהמשך יכולה להיות מעובד לשיבוש והפקה של כמויות קטנות של RNA ברקמות. </li></ol>

<ol>
	<li>Pagliuca, F. W., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+functional+beta-cell.">How to make a functional beta-cell.</a> Development. 140, 2472-2483 (2013).</li><li>Percival, A. C., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+pancreatic+development+using+a+cell+lineage+label.">Analysis of pancreatic development using a cell lineage label.</a> Exp Cell Res. 247, 123-132 (1999).</li><li>Attali, 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=Control+of+beta-cell+differentiation+by+the+pancreatic+mesenchyme.">Control of beta-cell differentiation by the pancreatic mesenchyme.</a> Diabetes. 56, 1248-1258 (2007).</li><li>Johansson, K. 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=Temporal+control+of+neurogenin3+activity+in+pancreas+progenitors+reveals+competence+windows+for+the+generation+of+different+endocrine+cell+types.">Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types.</a> Dev Cell. 12, 457-465 (2007).</li><li>Speier, S., Rupnik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+in+situ+characterization+of+pancreatic+beta-cells.">A novel approach to in situ characterization of pancreatic beta-cells.</a> Pflugers Arch. 446, 553-558 (2003).</li><li>Golosow, N., Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitheliomesenchymal+interaction+in+pancreatic+morphogenesis.">Epitheliomesenchymal interaction in pancreatic morphogenesis.</a> Dev Biol. 4, 242-255 (1962).</li><li>Miralles, F., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follistatin+regulates+the+relative+proportions+of+endocrine+versus+exocrine+tissue+during+pancreatic+development.">Follistatin regulates the relative proportions of endocrine versus exocrine tissue during pancreatic development.</a> Development. 125, 1017-1024 (1998).</li><li>Petzold, K. M., Spagnoli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+ex+vivo+culturing+of+embryonic+pancreas.">A system for ex vivo culturing of embryonic pancreas.</a> J. Vis. Exp. , 3979 (2012).</li><li>Miralles, F., Battelino, T., Czernichow, P., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+plays+a+key+role+in+morphogenesis+of+the+pancreatic+islets+of+Langerhans+by+controlling+the+activity+of+the+matrix+metalloproteinase+MMP-2.">TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2.</a> J Cell Biol. 143, 827-836 (1998).</li><li>Hope, K., Bhatia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+interrogation+of+stem+cells.">Clonal interrogation of stem cells.</a> Nat Methods. 8, 36-40 (2011).</li><li>Sugiyama, T., Rodriguez, R. T., McLean, G. W., Kim, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+markers+of+fetal+pancreatic+epithelium+permit+prospective+isolation+of+islet+progenitor+cells+by+FACS.">Conserved markers of fetal pancreatic epithelium permit prospective isolation of islet progenitor cells by FACS.</a> Proc Natl Acad Sci U S A. 104, 175-180 (2007).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Dontu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes Dev. 17, 1253-1270 (2003).</li><li>Smukler, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+mouse+and+human+pancreas+contain+rare+multipotent+stem+cells+that+express+insulin.">The adult mouse and human pancreas contain rare multipotent stem cells that express insulin.</a> Cell Stem Cell. 8, 281-293 (2011).</li><li>Seaberg, R. 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=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nat Biotechnol. 22, 1115-1124 (2004).</li><li>Jin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony-forming+cells+in+the+adult+mouse+pancreas+are+expandable+in+Matrigel+and+form+endocrine/acinar+colonies+in+laminin+hydrogel.">Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.</a> Proc Natl Acad Sci U S A. 110, 3907-3912 (2013).</li><li>Sugiyama, 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=Reconstituting+pancreas+development+from+purified+progenitor+cells+reveals+genes+essential+for+islet+differentiation.">Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation.</a> Proc Natl Acad Sci U S A. 110, 12691-12696 (2013).</li><li>Huch, 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=Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5/R-spondin+axis.">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> Embo J. , (2013).</li><li>Sato, 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. , (2009).</li><li>Ootani, 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=Sustained+in+vitro+intestinal+epithelial+culture+within+a+Wnt-dependent+stem+cell+niche.">Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.</a> Nat Med. 15, 701-706 (2009).</li><li>Barker, N., 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=Lgr5(+ve)+stem+cells+drive+self-renewal+in+the+stomach+and+build+long-lived+gastric+units+in+vitro.">Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.</a> Cell Stem Cell. 6, 25-36 (2010).</li><li>Huch, 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=In+vitro+expansion+of+single+Lgr5++liver+stem+cells+induced+by+Wnt-driven+regeneration.">In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.</a> Nature. 494, 247-250 (2013).</li><li>Lukacs, R. U., Goldstein, A. S., Lawson, D. A., Cheng, D., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation+and+characterization+of+adult+murine+prostate+stem+cells.">Isolation, cultivation and characterization of adult murine prostate stem cells.</a> Nat Protoc. 5, 702-713 (2010).</li><li>Rock, J. R., 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=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proc Natl Acad Sci U S A. 106, 12771-12775 (2009).</li><li>Eiraku, 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Spence, J. R., 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=Directed+differentiation+of+human+pluripotent+stem+cells+into+intestinal+tissue+in+vitro.">Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.</a> Nature. 470, 105-109 (2011).</li><li>Lancaster, M. 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 10, (2013).</li><li>Greggio, 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=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

The authors have nothing to disclose.

ייצור בקנה מידה גדולה של תאי הבטא תפקודיים במבחנה הוא עדיין לא יעיל 1. בהקשר זה מאתגר, לימודי ביולוגיה התפתחותית עשויים לעזור בפענוח האותות המדויקים הנדרשים להתמיינות של תאי הבטא תפקודיים. פרוטוקול זה מאפשר לתחזוקה, ההתרחבות וההתמיינות של תאי אב עובריים ב?...

תרבות איבר מספקת מודל שימושי שמגשר על הפערים בין מורכב, אך רלוונטי מאוד בחקירות vivo וסימולציה הנוחה אבל משוערת של מודלים קו סלולרי. במקרה של הלבלב, אין שורת תאים שווה ערך לחלוטין לאבות לבלב למרות שיש הופכים שורות תאים לדמות תאים האנדוקרינית ואקסוקרינית. כל הלבלב המבוגר לא יכול להיות מתורבת; איונים האנדוקרינית מבודדים יכולים להישמר במשך מספר שבועות ללא התפשטות תאים ויכולים להישמר פרוסות רקמה במבחנה לכמה שעות 5. תרבות לבלב עוברי כבר בשימוש נרחב לא רק ללמוד הפיתוח שלה, אלא גם כדי לחקור אינטראקציות אפיתל-mesenchymal 4,6,7, לתמונה מעבדת 8 או להתערב באופן כימי עימם 9. שתי שיטות תרבות איבר משמשות בעיקר: הראשון מורכב בculturing ניצני לבלב על צלחות פיברונקטין מצופה 2, שהוא convenient למטרות הדמיה; האפשרות השנייה היא תרבות האיברים במסננים בממשק האוויר הנוזלי 3,4 השומר הכי טוב המורפוגנזה. למרות שמאוד שימושי, שיטות אלה יביאו למידה מסוימת של השטחה; ההתרחבות של אבות היא מוגבלת מאוד בהשוואה להתפתחות התקינה ולאוכלוסייה מתחילה מורכבת הכוללת את כל הסוגים של תאי לבלב ותאי mesenchymal. יכולת התרבות ולהרחיב את התאים ראשוניים מפוזרים היא בעל ערך ללמוד יחסי שושלת ולחשוף את התכונות הפנימיות של סוגי תאים מבודדים 10. סוגיאמה ואח&#39; 11. יכול לשמור על אבות לבלב ואת אבות האנדוקרינית שמרה על כמה דמויות פונקציונליות ל3-5 ימים בתרבות בשכבות מזין. Pancreatospheres, בדומה לneurospheres 12 וmammospheres 13, הורחב מאיונים למבוגרים ותאי ductal למרות שטבעו של האבות / תאי גזעשיוצרים תחומים אלה אינו ברור. בנוסף, בניגוד להתפתחות פיזיולוגית, pancreatospheres הכיל כמה נוירונים 14,15. התחומים גם הופקו לאחרונה מאבות עובריים לבלב 16,17 והתחדשות pancreata 18 עם התרחבות אב טובה והבחנה שלאחר מכן, אך לא הצליחו לשחזר המורפוגנזה. המודלים 3D מהתאים מפוזרים ולעתים קרובות מוגדרים שעצמי לארגן את האיברים מוקטנים פרחו לאחרונה ולדמות את מחזור הפיתוח או מבוגר של איברים מרובים כגון המעי 19,20, הקיבה 21, הכבד 22, הערמונית 23 וקנה הנשימה 24. במקרים מסוימים, המורפוגנזה והתמיינות התפתחותיים כבר סכמו ב3D מתאי גזע עובריים, כמו במקרה של כוסות אופטיות 25, מעי 26 או מוח 27. כאן, אנו describe שיטה להרחיב אבות לבלב multipotent ניתק בפיגום 3D Matrigel בו הם יכולים לבדל ולארגן את עצמו.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
			<td>M0815-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

אבות לבלב הגבי E10.5 ניתקו וזורעים ב3D Matrigel לסכם פיתוח לבלב. יכולים בקלות להיות בעקבות אבות עם כתבי ניאון. במקרה שלנו השתמשתי בעכבר מהונדס המבטא את חלבון ה-GFP הגרעינית הנשלט על ידי אמרגן Pdx1 (Pdx1-Ngn3-ER TM-nGFP) (סרט 1) בהעדר טמוקסיפן ובכך ללא הפעלת Neurog3 4 (איור 2)...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

שיטת התרבות תלת ממדים שתוארה בפרוטוקול זה משחזרת פיתוח לבלב מאבות מפוזרים עוברי לבלב של עכבר, כולל הרחבתן משמעותית, בידול והמורפוגנזה לתוך איבר מסועף. שיטה זו ניתנת להדמיה, התערבות ומניפולציה תפקודיות של הנישה.

במבחנה הלבלב Organogenesis ממפוזר אבות עכבר עובריים

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

in-vitro-pancreas-organogenesis-from-dispersed-mouse-embryonic

Research

JoVE Journal

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K Views.  Swiss Institute for Experimental Cancer Research.  שיטת התרבות תלת ממדים שתוארה בפרוטוקול זה משחזרת פיתוח לבלב מאבות מפוזרים עוברי לבלב של עכבר, כולל הרחבתן משמעותית, בידול והמורפוגנזה לתוך איבר מסועף. שיטה זו ניתנת להדמיה, התערבות ומניפולציה תפקודיות של הנישה. 

Video: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

מבחני מושבת Vitro לאפיון ובתאי Tri-עצמה מבודדת מלבלב Murine למבוגרים

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Developmental Biology

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

בידוד יעיל של איי פונקציונלית מן היילודים עכבר לבלב

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

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

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

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

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

בידול יעיל של pluripotent בתאי גזע כדי NKX6-1<sup&gt; +</sup&gt; אבות הלבלב

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

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

בידוד, תרבות והדמיה של צבירי תאים עוברי האנושי לבלב

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Medicine

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

דור של אינסולין נטולת לגרדום, תלת מימדי לבטא Pancreatoids העכבר אבות הלבלב במבחנה

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

יעיל הדור של הלבלב/תריסריון גנים Homeobox חלבון 1+ אבות הלבלב במעי הקדמי/אחורי של hPSCs בתרבויות אדהזיה

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

התמיינות של תאי גזע פלוריפוטנטיים אנושיים למבשרי תאי בטא בלבלב במערכת תרבית דו-ממדית

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Assay כמותי עבור אינסולין להביע המושבה יוצרי אבות

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

תא האנדותל שיתוף תרבות מתווכת ההבשלה של עובריים אנושיים לתאי גזע לאינסולין בתאי לבלב הפקת בגישה הבחנה בימוי

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

מערכת ל<em&gt; Vivo לשעבר</em&gt; Culturing של לבלב עוברי

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

במבחנה הלבלב Organogenesis ממפוזר אבות עכבר עובריים

Summary

Explore More Videos

Chapters in this video