This work was supported by the Brain Research Program through the National Research Foundation (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (Grants NRF-2015M3C7A1028790); and by a grant from the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (HI14C3347).

1. הכנת חומרים והתרבות בינונית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עבור לנתיחה ו דיסוציאציה של SCz, לעטוף את מטריקס המוח, סכין גילוח פיפיות, מלקחיים עם רדיד אלומיניום, ולאחר מכן לעקר אותם על ידי מעוקר. </li><li style=";text-align:right;direction:rtl"> כן 50 מיליליטר של חיץ קר PBS לשטוף את מוח העכבר כולו. </li><li style=";text-align:right;direction:rtl"> הגדרת מיקרוסקופ לנתיחה ולהכין את כלי הניתוח הנדרש דיסקציה של (מספריים autoclaved מלקחיים) המוח ואת הבידוד של (מזרק 1 מ&quot;ל, 30 מחט G, מטריקס המוח, מלקחיים בסדר) SCz. </li><li style=";text-align:right;direction:rtl"> הכנת N2 בינוני: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן F-12 / DMEM (+ L-glutamin, + סודיום ביקרבונט) בינוני עם 2% B27, 1% תוספי N2, ו -1% פניצילין, סטרפטומיצין. הערה: מדיום הגידול מורכב גורמים בינוניים וצמיחת N2 (20 ng / mL גורם גדילה באפידרמיס המטוהר (EGF) ו -20 ng / mL גורם גדילה פיברובלסטים בסיסית (bFGF2)). </li></ol></li><li style=";text-align:right;direction:rtl"> הכן חיץ העיכול (40 יחידה / מ&quot;ל ​​פפאין, 2.4 יחידת / dispas מ&quot;להדואר השני, ו -2% פניצילין, סטרפטומיצין ב PBS) לעיכול רקמה. </li><li style=";text-align:right;direction:rtl"> הכנת צלחת ציפוי Coverslip: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן-פולי-L ornithine (אש&quot;ף; 0.01%) ו laminin (10 מיקרוגרם / מ&quot;ל מומס O DH 2). כדי 6 צלחות גם מעיל לשמירה על SCz-aNSCs כמו בשכבה או 18 מ&quot;מ coverslips עבור immunostaining, דגירה אותם עם אש&quot;ף לילה ב 4 מעלות צלזיוס. לאחר מכן לשטוף אותם 3 פעמים עם DH 2 O. אפשר הצלחות ו coverslips להתייבש אחרי הכביסה האחרונה. </li><li style=";text-align:right;direction:rtl"> לאחר מכן, דגירה צלחות עם laminin לילה בשעה 4 ° C. לאחר מכן, לשטוף אותם 3 פעמים עם DH 2 O. זהירות: אל לייבש את laminin, אשר ישפיע מצורף התא. הערה: פתרונות הציפוי ניתן לעשות שימוש חוזר 3 פעמים. </li></ol></li></ol> בידוד דיסוציאציה 2. למבוגרים SCz <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני הכנת תרבות, למקם את מטריקס המוח גילוח פיפיות על הקרח. הערה: אין להקפיא מטריצת המוח, משום brAin יכול לצרף אליו. </li><li style=";text-align:right;direction:rtl"> הקורבן עכבר (8 שבועות) בחנק CO 2 או נקע בצוואר הרחם. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותך את הראש במספריים חדים לאחר ריסוס 70% אתנול. כדי לשתק את הראש, להחזיק בשני צידי הראש בחוזקה. חותכים את העור עם מספריים על קו האמצע לכיוון הזנב-מקורי. זה מקדם את הסרה מלאה של העור מהגולגולת. </li><li style=";text-align:right;direction:rtl"> הסר את חלק הזנב של גולגולת (אחורי למבדה) ראשונה, ולאחר מכן הכנס את המלקחיים בין הגולגולת והמוח במיקום קו אמצע הגבה. תפוס שמאל (או ימין) עצם הקודקוד ובזהירות לקלף אותו. חזור על הליך זה עבור הסרת עצם הקודקוד האחר. הערה: ניתוק של עצב הראייה והסרת קרומי המוח מהמוח להקל לנתק את המוח מהגולגולת. </li><li style=";text-align:right;direction:rtl"> מעבירים את המוח למאגר קר PBS (25 מ&quot;ל) ולשטוף אותו פעמיים כדי להסיר דם עודף. </li></ol></li><li style=";text-align:right;direction:rtl"> מניח את המוח לתוך מטריצת המוח על קרח make חתכי עטרה להשיג 1 מ&quot;מ פרוסות עבות. מעבירים את פרוסות המוח (1 מ&quot;מ) הכוללים את האזורים SCz הממוקמים בחלק האחורי של המוח על PBS קר בצלחת פטרי פלסטיק 35 מ&quot;מ. זהירות: כדי להשיג מטוס מקביל סעיפי עטרה, להבטיח כי בקע המוח יוצב קו האמצע של מטריקס המוח; זה קריטי כדי למנוע וריאציות מיותר בסעיפים. הערה: קבל פרוסות מוח אחורי 2-3 מ&quot;מ עד גבחת; זה מאפשר הפרדה בין SCz מן SVZ. </li><li style=";text-align:right;direction:rtl"> תחת המיקרוסקופ לנתח עם בהגדלה נמוכה, מיקרו-לנתח את SCz מאזור בחומר הלבן של קליפת המוח ואת ההיפוקמפוס עם 6,9 מחט 30G מכופף. לאחר מכן, להסיר את האזורים בקליפה מעל SCz. מניח את אזור SCz גזור מן הפרוסות לתוך צלחת פטרי פלסטיק 35 מ&quot;מ על קרח בלי קר PBS. הערה: זיהום של אזורים נוספים המכילים נוירונים בוגרים יכול להשפיע על הכדאיות של aNSCs ו- B היווצרות neurosphereecause שהם עוברים מוות של תאים בתנאי תרבות aNSC. </li><li style=";text-align:right;direction:rtl"> באמצעות מחט כפוף 30 G, מייד לקצוץ הרקמות הגזורות לחתיכות קטנות. הערה: אם זמן רב יותר נדרש לנתח את רקמת SCz, לטבול את הרקמות הגזורות בקור PBS לפני החיתוך. </li><li style=";text-align:right;direction:rtl"> Re- להשעות את רקמת קצוץ עם 1 מ&quot;ל של חיץ העיכול, להעביר את רקמת לצינור 15 מ&quot;ל המכיל 2 מ&quot;ל של חיץ העיכול, דגירה אותו במשך 30 דקות באמבט מים 37 מעלות צלזיוס. הערה: יש לנער את הצינור כל 10 דקות לערבב היטב. </li></ol> 3. Subcallosal אזור נגזרות תרבית תאי גזע בוגרים העצביים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחץ על הצינור במתינות כדי לנתק את הרקמה מתעכלת, ולאחר מכן צנטריפוגות הצינור ב 145 XG במשך 5 דקות. בטל supernatant, מחדש תלויה הרקמה מתעכל עם 1 מ&quot;ל של מדיום מראש חימם N2 לשטוף את החיץ העיכול, בעדינות pipet הפתרון מדגם מקסימום 5 פעמים בעזרת פיפטה P1000. הערה: Over-triturating עם צרטיפ pipet יכול להפחית כדאיויות תא הצמיחה הבאה. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה הצינור ב 145 XG במשך 5 דקות. לאחר השלכת supernatant, מחדש להשעות את התא גלולה ב 1 מ&quot;ל של מדיום N2. </li><li style=";text-align:right;direction:rtl"> הכן 1 מ&quot;ל של מדיום N2 בצלחת 6-היטב שאינו מצופה ולהוסיף 1 מ&quot;ל של תאים מושעה לעשות נפח סופי של 2 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> להוסיף EGF (20 ng / ml) ו bFGF (20 ng / ml) לתוך כל טוב. הנער בעדינות את מנת תרבות 6 היטב ביד לערבב את גורמי הגדילה הוסיפו עם התאים המצופים. שמור את צלחת 6-היטב באינקובטור 37 מעלות צלזיוס, 5% CO 2. </li><li style=";text-align:right;direction:rtl"> להוסיף EGF (20 ng / ml) ו bFGF (20 ng / ml) זה טוב כל יום במשך 8 ימים. כל יום שלישי, להוסיף 200 μl של התקשורת N2 כדי לשמור על נפח 2 מ&quot;ל המשוער של המדיום. </li></ol> 4. Passaging של NSCs כמו Neurospheres וכדי תרבויות חד שכבתי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אסוף את neurospheres ולהעבירם צינור חרוטי חדש 15 מ&quot;ל. הערה: מספר neurospheres (&gt; 50 מיקרומטר קוטר) PEr היטב מן SCz של מוח עכבר אחת היה 64.3 ± 7.31, אשר היה פחות מזה של SVZ (190.5 ± 6.33) 9. </li><li style=";text-align:right;direction:rtl"> דגירה neurospheres עם 0.5 מ&quot;ל של חיץ ניתוק למשך 5 דקות באמבט מים 37 ° C כדי לנתק את neurospheres לתוך תאים בודדים. ניתן ניתק neurospheres הראשי לתוך תאים בודדים ומתוחזק על קטעים מספר תרבויות neurosphere או בשכבה. הערה: הרחבת SCz-aNSCs כמו בשכבה עדיפה על neurospheres כי SCz-aNSCs ניתן passaged&gt; 10 פעמים במתכונת תרבות בשכבה, אבל &lt;5 פעמים במתכונת תרבות neurosphere. זהירות: SCz-aNSCs להפגין צבירה חזקה, וקשה לנתק אותם לתוך תאים בודדים. לכן, פורמט תרבות neurosphere אינו מומלץ הרחבת תא שיגרתית. </li><li style=";text-align:right;direction:rtl"> בעדינות pipet הפתרון המדגם מעלה ומטה עם טפטפת P1000 פחות מ -5 פעמים צנטריפוגות את הצינור ב 145 XG במשך 5 דקות. בטל supernatant מחדש suלבלות את neurospheres עם 1 מ&quot;ל של מדיום N2. הערה: מעל triturating עם קצה פיפטה צר יכול להפחית כדאיויות תא הצמיחה הבאה. </li><li style=";text-align:right;direction:rtl"> כדי לספור את התאים, לעשות 1: תערובת 1 של השעית התא (10 μl) והפתרון הכחול 0.4% trypan, ולאחר מכן לספור את מספר תאי חיים על hematocytometer. לאחר ציפוי צלחת 6-היטב עם אש&quot;ף / laminin, צלחת התאים ב 2.5 x 10 מ&quot;ל 5 תאים / עם 2 מ&quot;ל של מדיום N2 עבור כל טוב. הערה: שינויי צפיפות תאים יכולים להשפיע על המצב ופוטנציאל בידול שלהם. </li><li style=";text-align:right;direction:rtl"> שמירה על SCz-aNSCs עם טיפול יומי של גורמי גדילה (2 מ&quot;ל, 20 ng / ml) במשך 5 ימים, ואת אותם מעבר להם. </li></ol> בידול 5. Subcallosal אזור שמקורם בתאי גזע בוגרים עצביים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ANSCs פלייט על coverslip מ&quot;מ 18 מצופה Laminin אש&quot;ף / עם 1 x 10 מ&quot;ל 5 תאים / מ&quot;ל 1 N2 עם גורמי גדילה (20 ng / ml) עבור בידול של-aNSCs SCz. </li><li style=";text-align:right;direction:rtl"> היום שאחרי,כשהתאים מחוברים היטב coverslip, להחליף את מדיום הגידול עם N2 להסיר את גורמי הגדילה. </li><li style=";text-align:right;direction:rtl"> לאחר 6 ימים, לשטוף את התאים הבדיל עם 1 מ&quot;ל של PBS להסיר פסולת התא ולתקן אותם עבור immunostaining. הערה: BrdU ניתן לשלב את ה- DNA החדש המסונתז של תאים מתרבים. לכן, אם רוצים, BrdU (10 מיקרוגרם / מ&quot;ל) ניתן להוסיף לחיות התאים לפני קיבוע של תאים. </li></ol> 6. בתאי גזע Immunostaining למבוגרים עצביים בדיל אבות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עבור immunostaining, לשטוף את התאים עם PBS ולתקן אותן עם PFA 4% למשך 20 דקות בטמפרטורת החדר. זהירות: PFA היא רעילה ביותר; להימנע ממגע עם העור והעיניים. </li><li style=";text-align:right;direction:rtl"> הסר את 4% PFA, לשטוף את התאים קבוע עם PBS 3 פעמים, ולאחר מכן לאחסן אותם ב 4 ° Cuntil הם נדרשים עבור immunostaining. </li><li style=";text-align:right;direction:rtl"> דגירה התאים על coverslip עם פתרון חסימה (אלבומין בסרום שור 3% ו -0.1% Triton X-100 ב 1x PBS) למשך 30 דקות לפחות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הכן את הנוגדנים העיקריים בפתרון חסימה טרי דגירת דגימות הלילה ב 4 מעלות צלזיוס. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Immunostaining aNSCs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכתם aNSCs באמצעות אנטי Nestin ונוגדנים נגד BrdU. לפני קיבעון, להוסיף 20 מיקרוגרם של BrdU אל aNSCs דגירה אותם עבור 2 שעות. בצע את הצעד denaturing באמצעות 2 N HCl למשך 20 דקות ב 37 מעלות Cbefore שתבצע את הפעולה חוסם. </li></ol></li><li style=";text-align:right;direction:rtl"> Immunostaining אבות הבדיל: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש אנטי-O4, אנטי bIII טובולין, וחלבון חומצי fibrillary אנטי גליה (GFAP) נוגדנים לתייג את האבות המובחנים. </li></ol></li></ol></li><li style=";text-align:right;direction:rtl"> שטפו את דגימות עם 3 פעמים PBS דגירה אותם עם נוגדנים משני מצומדות כדי צבעי ניאון (1: 500) בחסימת פתרון במשך 30 דקות בטמפרטורת החדר. לאחר מכן, לשטוף את דגימות 3 פעמים עם PBS. הערה: נוגדנים משני השתמש התואמים את המארחים של בפריימריזנוגדנים. Hoechest33343 (1: 2,000) משמש מכתים גרעיני. </li><li style=";text-align:right;direction:rtl"> הוסף פתרון גובר זכוכית שקופית והמשך עם הרכבה. שימו ותמונה המדגם על מיקרוסקופ confocal באורכי גל מרובים: FITC (488 ננומטר), Cy3 (543 ננומטר), Cy5 (647 ננומטר), ו Hoechest33343 (405 ננומטר). </li></ol>

<ol>
	<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=Clonal+and+population+analyses+demonstrate+that+an+EGF-responsive+mammalian+embryonic+CNS+precursor+is+a+stem+cell.">Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell.</a> Developmental biology. 175, 1-3 (1996).</li><li>Babu, H., Cheung, G., Kettenmann, H., Palmer, T. D., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enriched+monolayer+precursor+cell+cultures+from+micro-dissected+adult+mouse+dentate+gyrus+yield+functional+granule+cell-like+neurons.">Enriched monolayer precursor cell cultures from micro-dissected adult mouse dentate gyrus yield functional granule cell-like neurons.</a> PloS one. 2, e388 (2007).</li><li>Alvarez-Buylla, A., Garcia-Verdugo, 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=Neurogenesis+in+adult+subventricular+zone.">Neurogenesis in adult subventricular zone.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 22, 629-634 (2002).</li><li>Kempermann, G., Song, H., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+Adult+Hippocampus.">Neurogenesis in the Adult Hippocampus.</a> Cold Spring Harbor perspectives in biology. 7, a018812 (2015).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+neural+stem+cells.">Mammalian neural stem cells.</a> Science. 287, 1433-1438 (2000).</li><li>Seri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+organization+of+the+SCZ:+a+large+germinal+layer+containing+neural+stem+cells+in+the+adult+mammalian+brain.">Composition and organization of the SCZ: a large germinal layer containing neural stem cells in the adult mammalian brain.</a> Cereb Cortex. 16 Suppl 1, i103-i111 (2006).</li><li>Kim, W. 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=Evidence+for+the+spontaneous+production+but+massive+programmed+cell+death+of+new+neurons+in+the+subcallosal+zone+of+the+postnatal+mouse+brain.">Evidence for the spontaneous production but massive programmed cell death of new neurons in the subcallosal zone of the postnatal mouse brain.</a> The European journal of neuroscience. 33, 599-611 (2011).</li><li>Kim, H. J., Kim, J. Y., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+changes+in+the+subcallosal+zone+neurogenesis+of+mice.">Age-dependent changes in the subcallosal zone neurogenesis of mice.</a> Neurochemistry international. 61, 879-884 (2012).</li><li>Kim, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+Cortical+Neurogenesis+from+the+Neural+Stem+Cells+in+the+Adult+Mouse+Subcallosal+Zone.">Promotion of Cortical Neurogenesis from the Neural Stem Cells in the Adult Mouse Subcallosal Zone.</a> Stem Cells. , (2015).</li><li>Nunes, M. 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=Identification+and+isolation+of+multipotential+neural+progenitor+cells+from+the+subcortical+white+matter+of+the+adult+human+brain.">Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain.</a> Nature medicine. 9, 439-447 (2003).</li><li>Gould, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+widespread+is+adult+neurogenesis+in+mammals?.">How widespread is adult neurogenesis in mammals?.</a> Nature reviews. Neuroscience. 8, 481-488 (2007).</li><li>Gage, F. H., Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells:+generating+and+regenerating+the+brain.">Neural stem cells: generating and regenerating the brain.</a> Neuron. 80, 588-601 (2013).</li><li>Shaker, M. R., Kim, J. Y., Kim, H., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+secondary+neural+tube-derived+embryonic+neural+stem+cells+in+vitro.">Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.</a> Stem cells and development. 24, 1171-1181 (2015).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Molecular neurobiology. 34, 153-161 (2006).</li><li>Suslov, O. N., Kukekov, V. G., Ignatova, T. N., Steindler, 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=Neural+stem+cell+heterogeneity+demonstrated+by+molecular+phenotyping+of+clonal+neurospheres.">Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 14506-14511 (2002).</li><li>Richards, L. J., Kilpatrick, T. J., Bartlett, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+generation+of+neuronal+cells+from+the+adult+mouse+brain.">De novo generation of neuronal cells from the adult mouse brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 89, 8591-8595 (1992).</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>Gritti, 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=Epidermal+and+fibroblast+growth+factors+behave+as+mitogenic+regulators+for+a+single+multipotent+stem+cell-like+population+from+the+subventricular+region+of+the+adult+mouse+forebrain.">Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 19, 3287-3297 (1999).</li><li>Wachs, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficacy+of+clonal+growth+and+expansion+of+adult+neural+stem+cells.">High efficacy of clonal growth and expansion of adult neural stem cells.</a> Laboratory investigation; a journal of technical methods and pathology. 83, 949-962 (2003).</li><li>Xiong, F., 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=Optimal+time+for+passaging+neurospheres+based+on+primary+neural+stem+cell+cultures.">Optimal time for passaging neurospheres based on primary neural stem cell cultures.</a> Cytotechnology. 63, 621-631 (2011).</li><li>Lee, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Matrigel-based+culture+for+expansion+of+neural+stem+cells.">Optimization of Matrigel-based culture for expansion of neural stem cells.</a> Animal Cells and Systems. 19, 175-180 (2015).</li><li>Guo, W., Patzlaff, N. E., Jobe, E. M., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+multipotent+neural+stem+or+progenitor+cells+from+both+the+dentate+gyrus+and+subventricular+zone+of+a+single+adult+mouse.">Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse.</a> Nature protocols. 7, 2005-2012 (2012).</li><li>Walker, T. L., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mouse,+two+cultures:+isolation+and+culture+of+adult+neural+stem+cells+from+the+two+neurogenic+zones+of+individual+mice.">One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice.</a> JoVE (Journal of Visualized Experiments). , e51225 (2014).</li></ol>

The authors declare no conflicts of interest.

מאמר זה מתאר פרוטוקול מפורט ליצור NSCs מן SCz עכבר בוגר ולשמור אותם ליישומים שונים. ישנם שלושה שלבים קריטיים להקמת מערכת התרבות חוץ גופית צורך לטהר ולהרחיב SCz-NSCs. ראשית, חשוב להבטיח שאזור SCz הוא גזור החוצה בדיוק מאזורים נוירוגנית פוטנציאליים אחרים (איור 1 ב). ...

NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system1. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs2. These two well-developed culture systems have contributed to the characterization of aNSCs in vitro. 
NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury9. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain10. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing11. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>+L-glutamin, +Sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>BIOSOLUTION</td>
			<td>BP007a</td>
			<td>1X dilution</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociattion buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>04-942-078-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ICT</td>
			<td>AT-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>WPI</td>
			<td>555229F</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Storz</td>
			<td>E3321-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain matrix (1mm)</td>
			<td>RWD</td>
			<td>68707</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-edged razor</td>
			<td>DORCO</td>
			<td>ST-300</td>
			<td></td>
		</tr>
		<tr>
			<td>30 gage needle</td>
			<td>SUNGSHIM</td>
			<td>N1300</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>SPL</td>
			<td>10035</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>100mm dish</td>
			<td>SPL</td>
			<td>10090</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>Cover slip (18mm)</td>
			<td>Deckglaser</td>
			<td>111580</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well dish</td>
			<td>SPL</td>
			<td>32012</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>6 well dish</td>
			<td>SPL</td>
			<td>32006</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>Coating materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLO</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td>0.01%</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Primary antibodies&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td>mouse (1:1000)</td>
		</tr>
		<tr>
			<td>EGFR</td>
			<td>Abcam</td>
			<td>ab2430</td>
			<td>rabbit (1:1000)</td>
		</tr>
		<tr>
			<td>DCX</td>
			<td>Santa Cruz</td>
			<td>SC8066</td>
			<td>goat (1:500)</td>
		</tr>
		<tr>
			<td>Tuj1</td>
			<td>Sigma</td>
			<td>T2200</td>
			<td>rabbit (1:2000)</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Invitrogen</td>
			<td>13-0300</td>
			<td>rat (1:1000)</td>
		</tr>
		<tr>
			<td>O4</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td>mouse (1:500)</td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>Rat (1:500)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td rowspan="12">1:500</td>
		</tr>
		<tr>
			<td>anti-mouse cy3</td>
			<td>Jackson</td>
			<td>715-165-151</td>
		</tr>
		<tr>
			<td>anti-mouse 647</td>
			<td>Jackson</td>
			<td>715-606-150</td>
		</tr>
		<tr>
			<td>anti-rabbit 488</td>
			<td>Alexa</td>
			<td>A21206</td>
		</tr>
		<tr>
			<td>anti-rabbit cy3</td>
			<td>Jackson</td>
			<td>711-165-152</td>
		</tr>
		<tr>
			<td>anti-rabbit 647</td>
			<td>Jackson</td>
			<td>711-605-152</td>
		</tr>
		<tr>
			<td>anti-goat 488</td>
			<td>Alexa</td>
			<td>A11055</td>
		</tr>
		<tr>
			<td>anti-goat cy3</td>
			<td>Jackson</td>
			<td>705-165-147</td>
		</tr>
		<tr>
			<td>anti-goat 647</td>
			<td>Invitrogen</td>
			<td>A21447</td>
		</tr>
		<tr>
			<td>anti-rat 488</td>
			<td>Invitrogen</td>
			<td>A21208</td>
		</tr>
		<tr>
			<td>anti-rat cy3</td>
			<td>Jackson</td>
			<td>712-166-150</td>
		</tr>
		<tr>
			<td>anti-rat 647</td>
			<td>Jackson</td>
			<td>712-605-153</td>
		</tr>
		<tr>
			<td>Immunostaining materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Millipore</td>
			<td>82-100-6</td>
			<td>3% BSA with 0.1% Triton X-100 in PBS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>usb</td>
			<td>22686</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Biosesang</td>
			<td>P2031</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechest33342</td>
			<td>Life Technology</td>
			<td>H3570</td>
			<td>Dye for staining nuclei</td>
		</tr>
	</tbody>
</table>

isolation and culture of adult neural stem cells from the mouse subcallosal zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

הגדרת מערכת התרבות עבור aNSCs מאזור נוירוגנית ידוע חיונית להבנת התאים הללו לפיתוח פוטנציאל השימוש שלהם במוח תיקון 12. זה ידוע כי NSCs בשלבים התפתחותיים שונים או באזורים שונים מתנהגים בצורה שונה 3,4. לאחרונה, דווח כי נגזרות תאי SCz להפגין פוטנציא...

Watch this Scientific Journal Video about Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone at JoVE.com

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

בידוד והתרבות של תאי גזע בוגרים עצביים מאזור העכבר Subcallosal

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

isolation-culture-adult-neural-stem-cells-from-mouse-subcallosal

Research

JoVE Journal

Neuroscience

15.0K Views.  Korea University College of Medicine. Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described. 

Video: Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

בידוד הרחבה של תאים בוגרים עכבר גזע עצביים שימוש Assay Neurosphere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

הקמת עכבר עובריים גזע עצביים תרבית תאים באמצעות Assay Neurosphere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

בידוד ותרבות של תאי גזע עצביים קרסט מזקיקי שיער אנושיים

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

בידוד והתרבות של תאים עצביים לפסגה מהצינור עובריים עצבית Murine

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

גידול בתאי גזע עצבי מאזורים קונבנציונליים ולא קונבנציונליים של המוח מכרסם מבוגרים

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

בידוד והתרבות של תאי האנדותל מהמוח הקדמי עוברי

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

עכבר אחד, שתי תרבויות: בידוד והתרבות של תאי גזע עצביים למבוגרים משני האזורים העצביים של עכברים בודדים

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

גזירה ללא מזין של תאים עצביים קרסט אב קדמון מפלוריפוטנטיים אדם בתאי גזע

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

מיון תא של תאי גזע ועצביים ממבוגרי עכבר Subventricular האזור וחי-הדמיה של Dynamics תא המחזור שלהם

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.