המחברים מודים Xiu-פינג וואנג לעזרה ראשונית עם תרבויות DESC. המחברים ממומנים בחלקו על ידי מלגות ומענקים מהמוסד הלאומי לבריאות (K99-DE022059 לAHJ, K12-GM081266 לMGC, K08-DE022377-02 לאה, וR01-DE021420 לODK).

1. לנתח תחתונה חמי-לסתות מעכבר למבוגרים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קודם לביצוע פרוטוקול זה, לקבל את אישור מוסדי דרוש ולהיות בטוח כדי לעמוד בכל הנחיות הטיפול בבעלי החיים. להרדים בעלי חיים באמצעות IACUC הסטנדרטי אושר נהלים. עבור עבודה זו CO מחנק 2 היה בשימוש ואחריו נקע בצוואר הרחם. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אופציונלי: ראש נפרד משאר הגוף באמצעות סכין גילוח. </li></ol></li><li style=";text-align:right;direction:rtl"> הסר את העור מן השפה התחתונה למחשוף. </li><li style=";text-align:right;direction:rtl"> באמצעות אזמל, עושה חתך בין שתי החותכות התחתונות, אשר מחוברות באופן רופף. לאחר הפעלת לחץ, הלסת התחתונה תהיה מחולקת לשני חצאים. </li><li style=";text-align:right;direction:rtl"> טריז האזמל בין condyle של הלסת ומפרקי לסת התחתונים, הזמן ולחתוך לצד השרירים הלסת לנתק את הלסת. </li><li style=";text-align:right;direction:rtl"> הסר את כל השריר, גיד, רצועה ועד שרק העצם נשאר. </li></ol> 2. לבודד את הארקטיק <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות אזמל # 15, לגלח בזהירות את העצם בזווית של ° 45 מהקצה הדיסטלי לקצה הפרוקסימלי של האזור הקרומי. השתמש בקצה של האזמל כדי לאסוף משם כל עצם שנותר ביניהם שברי עצמות בקצה. לאט לאט מתחיל לאסוף משם בצלחת קליפת המוח הלשונית של הלסת התחתונה, מתחיל ממש מתחת לשן טוחנת 3 rd. </li><li style=";text-align:right;direction:rtl"> המשך עד השן החותכת היא נקייה, עובדת בזהירות לכיוון השטח שבו CL. </li><li style=";text-align:right;direction:rtl"> הסר את צרור עצבים מכתשיים נחותים. </li><li style=";text-align:right;direction:rtl"> לעשות חתך נקי כדי להסיר תחילה את העצם ורקמות הפרוקסימלי לCL. </li><li style=";text-align:right;direction:rtl"> לעשות חתך שני distally עם האזמל, בערך מתחת לשן טוחנת 2 nd. אזור השן החותך הפרוקסימלי מכן גזור החוצה על ידי החדרת האזמל בין העצם לבין המשטח המדיאלי של השן החותכת הפרוקסימלי. זה חייב להיעשות בזהירות, כדי לא לקרוע את הרקמה. תנועת החתך היא הפרוקסימלי-דיסטלי. שלב חלופי: להסיר כמה שיותר עצם ככל האפשר לאורך האזור שמעלייncisor. ברגע שהשטח נוקתה, להסיר בזהירות את כל שן חותכת באמצעות גודל 5 מלקחיים כדי לטפל בחלק האמייל הקרוב ביותר לאזור CL. </li><li style=";text-align:right;direction:rtl"> הנח רקמה גזור (או CL המבודד כמו באיור 3E, או כל שן חותכת כמו בשלב חלופי 2.5) בcollagenase 2% ב 1X PBS עבור 3-4 שעות ב 4 ° C בצלחת היענות נמוכה. ל1-10 חותכות, השתמש בכל טוב 100 μl ב12 גם צלחת. לגדול יותר מ10 חותכות, השתמש 500 μl לכל טוב ב6 היטב צלחת נמוכה דבקות. </li></ol> 3. Microdissect שפתני צוואר הרחם Loop <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר את אזור CL מcollagenase והמקום בתקשורת DMEM/F12 הקרה. </li><li style=";text-align:right;direction:rtl"> משוך בעדינות בקצה התחתון של CL או באמצעות גודל 5 מלקחיים או מזרק אינסולין (1 סמ&quot;ק, 28 G ½); mesenchyme יתפרק תוך האפיתל נותר בשלמותה. CL ואפיתל הסמוך המשתרע רוחבי כמבנה &quot;כמו כנף&quot; יהיה גלויים. </li><li style=";text-align:right;direction:rtl"> להסירניצן apical מן האפיתל הסמוך על ידי ביצוע חתך בצורת V בכל צד, ומניח מייד בDMEM/F12 הקר על קרח. </li><li style=";text-align:right;direction:rtl"> חזור על פעולה עבור כל CLS, לאסוף ב1.5 צינור microfuge מיליליטר. </li><li style=";text-align:right;direction:rtl"> ספין למטה צינור microfuge עם דגימות, להסיר תקשורת, להחליף עם 100 פתרון ניתוק תא μl. </li><li style=";text-align:right;direction:rtl"> דגירה רקמות בפתרון ניתוק תא ל30 דקות ב 37 ° C. </li><li style=";text-align:right;direction:rtl"> לנתק את הרקמות לייצר השעיה תא בודדת על ידי pipetting בעדינות מעלה ומטה באמצעות פיפטה קצה נמוכה הידבקות 1,000 μl. יכולים גם להיות ממוקמים תאים דרך מסננת תא סטרילי כדי להשיג דיסוציאציה. </li><li style=";text-align:right;direction:rtl"> ספירת תאים עם hemocytometer, צלחת פלסטיק בתרבית רקמה או חומר רצוי אחר. </li></ol> 4. תרבות העיקרית של DESCs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פלייט תאים בצלחת 6 היטב ב1-6 x 10 4 תאים / מיליליטר בתקשורת DESC, אשר מורכב מDMEM/F12 בתוספת חלבון רקומביננטי EGF העכבר בריכוז של 20 nגר &#39;/ מיליליטר, חלבון רקומביננטי FGF בריכוז של 25 ng / ml, תוסף B27 1X, ופתרון 1% אנטיביוטיקה (פניצילין, 100 U / ml, סטרפטומיצין, 50 מיקרוגרם / מיליליטר). </li><li style=";text-align:right;direction:rtl"> לגדל תאים מצופים על צלחת 6 היטב על 37 מעלות צלזיוס ב 5% CO 2 ב 1 מיליליטר של תקשורת DESC. נא לא להפריע תרבויות ראשוניות במשך 5 ימים לאחר ציפוי לאפשר הידבקות תא והקמת מושבה. </li><li style=";text-align:right;direction:rtl"> לשינוי התקשורתי הראשון, החלף חצי מהנפח (500 μl) של המדיה ישנה עם חצי נפח (500 μl) של המדיה חדשה. בדקו מושבות תחת מיקרוסקופ כאשר תקשורת משתנות. </li><li style=";text-align:right;direction:rtl"> לאחר השינוי בתקשורת הראשון, ממשיך לשנות תקשורת (1-2 מיליליטר) כל 2 ימים. בדקו מושבות תחת מיקרוסקופ כאשר תקשורת משתנות. </li></ol> 5. DESCs המעבר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תקשורת לשאוב ולשטוף תאי 3x עם PBS חימם מראש. </li><li style=";text-align:right;direction:rtl"> להוסיף מראש חימם 0.25% טריפסין-EDTA בתמיסת מלח ולדגור על 37 מעלות צלזיוס למשך 10-15 דקות לDESCs לניתוק. </li><li style=";text-align:right;direction:rtl"> הוסף מדיה DESC לנטרל את טריפסין, בעדינות פיפטה לאורך צלחת תרבית רקמה כדי לאסוף את התאים, ומקום בצינור חרוטי 15 מיליליטר. </li><li style=";text-align:right;direction:rtl"> ספין למטה תאים ב XG 800 למשך 2 דקות. </li></ol>

<ol>
	<li>Sharpe, P. T., Young, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test-tube+teeth..">Test-tube teeth..</a> Sci Am. 293, 34-41 (2005).</li><li>D'Souza, R. N., Klein, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+molecular+mechanisms+that+lead+to+supernumerary+teeth+in+mice+and+men:+Current+concepts+and+novel+approaches.">Unraveling the molecular mechanisms that lead to supernumerary teeth in mice and men: Current concepts and novel approaches.</a> Cells Tissues Organs. 186, 60-69 (2007).</li><li>Jernvall, J., Thesleff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+shape+formation+and+tooth+renewal:+evolving+with+the+same+signals.">Tooth shape formation and tooth renewal: evolving with the same signals.</a> Development. 139, 3487-3497 (2012).</li><li>Yen, A. H., Yelick, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+Tissue+Regeneration+A+Mini-Review.">Dental Tissue Regeneration A Mini-Review.</a> Gerontology. 57, 85-94 (2011).</li><li>Jheon, A. H., Seidel, K., Biehs, B., Klein, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+molecules+to+mastication:+the+development+and+evolution+of+tooth+development.">From molecules to mastication: the development and evolution of tooth development.</a> WIREs Dev Biol. 2, 165-182 (2013).</li><li>Wang, X. 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=An+integrated+gene+regulatory+network+controls+stem+cell+proliferation+in+teeth.">An integrated gene regulatory network controls stem cell proliferation in teeth.</a> PLoS Biol. 5, (2007).</li><li>Harada, H., 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=Localization+of+putative+stem+cells+in+dental+epithelium+and+their+association+with+Notch+and+FGF+signaling.">Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling.</a> J Cell Biol. 147, 105-120 (1999).</li><li>Juuri, E., 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=Sox2++stem+cells+contribute+to+all+epithelial+lineages+of+the+tooth+via+Sfrp5++progenitors.">Sox2+ stem cells contribute to all epithelial lineages of the tooth via Sfrp5+ progenitors.</a> Dev Cell. 23, 317-328 (2012).</li><li>Seidel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+regulates+the+generation+of+ameloblast+progenitors+in+the+continuously+growing+mouse+incisor.">Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor.</a> Development. 137, 3753-3761 (2010).</li><li>Smith, C. E., Warshawsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+renewal+in+the+enamel+organ+and+the+odontoblast+layer+of+the+rat+incisor+as+followed+by+radioautography+using+3H-thymidine.">Cellular renewal in the enamel organ and the odontoblast layer of the rat incisor as followed by radioautography using 3H-thymidine.</a> Anat Rec. 183, 523-561 (1975).</li><li>Smith, C. E., Warshawsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+cell+turnover+in+the+enamel+organ+of+the+rat+incisor.+Evidence+for+ameloblast+death+immediately+after+enamel+matrix+secretion.">Quantitative analysis of cell turnover in the enamel organ of the rat incisor. Evidence for ameloblast death immediately after enamel matrix secretion.</a> Anat Rec. 187, 63-98 (1977).</li><li>Parsa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+by+FGFR2b+controls+the+regenerative+capacity+of+adult+mouse+incisors.">Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors.</a> Development. 137, 3743-3752 (2010).</li><li>Chang, 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=Self-renewal+and+multilineage+differentiation+of+mouse+dental+epithelial+stem+cells.">Self-renewal and multilineage differentiation of mouse dental epithelial stem cells.</a> Stem Cell Res. 11, 990-1002 (2013).</li><li>Biehs, 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=Bmi1+represses+Ink4a/Arf+and+Hox+genes+to+regulate+stem+cells+in+the+rodent+incisor.">Bmi1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor.</a> Nat Cell Biol. 15, 846-852 (2013).</li><li>Hwang, W. S., Tonna, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+Analysis+of+Labeling+Indices+and+Migration+Rates+of+Cellular+Component+of+Mouse+Incisors+Using+Tritiated+Thymidine+(H3tdr).">Autoradiographic Analysis of Labeling Indices and Migration Rates of Cellular Component of Mouse Incisors Using Tritiated Thymidine (H3tdr).</a> J Dent Res. 44, 42-53 (1965).</li><li>Li, C. 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=E-cadherin+regulates+the+behavior+and+fate+of+epithelial+stem+cells+and+their+progeny+in+the+mouse+incisor.">E-cadherin regulates the behavior and fate of epithelial stem cells and their progeny in the mouse incisor.</a> Dev Biol. 366, 357-366 (2012).</li><li>Smith, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+chemical+events+during+enamel+maturation.">Cellular and chemical events during enamel maturation.</a> Crit Rev Oral Biol Med. 9, 128-161 (1998).</li><li>Lapthanasupkul, 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=Ring1a/b+polycomb+proteins+regulate+the+mesenchymal+stem+cell+niche+in+continuously+growing+incisors.">Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continuously growing incisors.</a> Dev Biol. 367, 140-153 (2012).</li><li>Chavez, M. 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=Characterization+of+dental+epithelial+stem+cells+from+the+mouse+incisor+with+two-dimensional+and+three-dimensional+platforms.">Characterization of dental epithelial stem cells from the mouse incisor with two-dimensional and three-dimensional platforms.</a> Tissue Eng Part C Methods. 19, 15-24 (2013).</li><li>Kawano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+dental+epithelial+cell+line+(HAT-7)+and+the+cell+differentiation+dependent+on+Notch+signaling+pathway.">Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway.</a> Connect Tissue Res. 43, 409-412 (2002).</li><li>Briggaman, R. A., Abele, D. C., Harris, S. R., Wheeler, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+a+viable+suspension+of+postembryonic+human+epidermal+cells.">Preparation and characterization of a viable suspension of postembryonic human epidermal cells.</a> J Invest Dermatol. 48, 159-168 (1967).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+epidermal+cells+from+embryonic+mouse+skin.">Isolation and cultivation of epidermal cells from embryonic mouse skin.</a> Naturwissenschaften. 58, 421 (1971).</li><li>Fusenig, N. E., Worst, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+epidermal+cell+cultures.+I.+Isolation+and+cultivation+of+epidermal+cells+from+adult+mouse+skin.">Mouse epidermal cell cultures. I. Isolation and cultivation of epidermal cells from adult mouse skin.</a> J Invest Dermatol. 63, 187-193 (1974).</li><li>Rheinwald, J. G., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+cultivation+of+strains+of+human+epidermal+keratinocytes:+the+formation+of+keratinizing+colonies+from+single+cells.">Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.</a> Cell. 6, 331-343 (1975).</li><li>Barrandon, Y., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+clonal+types+of+keratinocyte+with+different+capacities+for+multiplication.">Three clonal types of keratinocyte with different capacities for multiplication.</a> Proc Natl Acad Sci U S A. 84, 2302-2306 (1987).</li><li>Blanpain, 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=Self-renewal,+multipotency,+and+the+existence+of+two+cell+populations+within+an+epithelial+stem+cell+niche.">Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche.</a> Cell. 118, 635-648 (2004).</li><li>Toma, J. 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=Isolation+of+multipotent+adult+stem+cells+from+the+dermis+of+mammalian+skin.">Isolation of multipotent adult stem cells from the dermis of mammalian skin.</a> Nat Cell Biol. 3, 778-784 (2001).</li><li>Nowak, J. A., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+epithelial+stem+cells.">Isolation and culture of epithelial stem cells.</a> Methods Mol Biol. 482, 215-232 (2009).</li><li>Rheinwald, J. G., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+and+the+multiplication+of+cultured+human+epidermal+keratinocytes.">Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.</a> Nature. 265, 421-424 (1977).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35, 567-576 (1993).</li><li>Lesuisse, C., Martin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+mouse+cortical+neurons+as+a+model+for+neuronal+development,+aging,+and+death.">Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death.</a> J Neurobiol. 51, 9-23 (2002).</li><li>Felszeghy, S., Suomalainen, M., Thesleff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling+is+required+for+the+survival+of+epithelial+stem+cells+in+the+continuously+growing+mouse+incisor.">Notch signalling is required for the survival of epithelial stem cells in the continuously growing mouse incisor.</a> Differentiation. 80, 241-248 (2010).</li><li>Fujimori, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+in+the+dental+mesenchyme+regulates+incisor+development+by+regulating+Bmp4.">Wnt/beta-catenin signaling in the dental mesenchyme regulates incisor development by regulating Bmp4.</a> Dev Biol. 348, 97-106 (2010).</li><li>Klein, O. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+FGF+signaling+loop+sustains+the+generation+of+differentiated+progeny+from+stem+cells+in+mouse+incisors.">An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors.</a> Development. 135, 377-385 (2008).</li></ol>

יש המחברים אין ניגודי אינטרסים לחשוף.

תאי אפיתל היו ראשון בהצלחה בתרבית לפני למעלה 40 שנות 21-24, ולאחרונה, בידוד מוצלח של תאי גזע אפיתל 25-27 יש הידע מתקדם של ביולוגיה האפיתל שלנו. אנו מדווחים על פרוטוקול לבידוד DESCs של השן החותכת העכבר המבוגר, אוכלוסייה יחסית understudied של תאי גזע שיש לו הפוטנציאל להניב...

אחד המאפיינים הייחודיים של בעלי חוליות היא האבולוציה של שיניים. השן הפכה מערכת מודל חשובה לתחומים רבים של מחקר, כמו המסלולים המולקולריים ומורפולוגיים התמחויות רלוונטיות לאיבר זה נחקרו מכמה נקודות מבט, ובכלל זה על ידי ביולוגים התפתחותיים והאבולוציונית 5. לאחרונה, תחום של רפואת רגנרטיבית החל לצבור תובנות רבות ערך באמצעות שן כמודל. בפרט, את הגילוי של תאי גזע אפיתל שיניים כבר מקדמה חשובה 6-13. כל המכרסמים יש שיניים חותכות הולכות ומתרחבות צמיחה שמונעת על ידי תאי גזע, מה שהופך את השיניים האלה מערכת מודל נגישה ללמוד ויסות תאי גזע בוגרים. ניסויי תיוג ב1970s 10,11, ואחרי ניסויי התחקות שושלת גנטית 8,9,12,14, הוכיחו כי DESCs מתגורר באזור הפרוקסימלי של השן החותכת. הגזעצאצאי תא בתא אפיתל בצד המהלך שפתני מהנישה המשוערת, המכונה לולאת שפתני צוואר הרחם (CL), ולתרום לאוכלוסייה של תאים הנקראת תאים (ת&quot;א) הגברת מעבר (איור 1). באופן ספציפי, DESCs מתגורר באפיתל החיצוני האמייל (OEE) וreticulum stellate (SR) 8,9,14, ואפיתל האמייל הפנימי (IEE) מעורר את תאי ת&quot;א שיתקדמו במספר מוגבל של מחזורי תא ולאחר מכן מהלך distally לאורכו של השן החותכת (איור 1). Ameloblasts ההבחנה בשן החותכת העכבר ימשיך לנוע distally לאורך השן החותכת בקצב מדהים של כ 350 מיקרומטר ביום אחד 15,16. כאשר הם עוברים, התאים להתמיין ameloblasts הבוגר וintermedium שכבת תאים (SI). לאחר ההפקדה בעובי המלא של מטריצת אמייל, רבים של ameloblasts עובר אפופטוזיס, והתאים שנותרו להתכווץ בגודל ולהסדיר התבגרות אמייל &lt;sup&gt; 17. השושלת של סוגי תאים האחרים בCL שפתני, כגון SR, היא פחות ברורה, ונתונים לגבי תאי הגזע בmesenchyme 18 ובCL הלשוני רק מתחילים לצוץ. שימוש במודל חותך העכבר, מספר הקבוצות עבד על מנת להבהיר את המסלולים הגנטיים ותהליכים ביולוגיים תא מעורב בחידוש איברים טבעי גזע מבוסס תאים. עם זאת, CL שפתני מכיל מספר קטן יחסית של תאים, מוערך בכ -5,000 לשן חותכת עכבר, מה שהופך את העבודה עם תאים ראשוניים מאתגרים. לכן, נעשו מאמצים לתרבות ולהרחיב את התאים האלה במבחנה 6,16,19,20 על מנת לפתוח דלתות חדשות לגישות ניסיוניות שאינן ברי השגה in vivo. המחקר שנערך לאחרונה רוב הוכיח כי תאים אלה יכולים גם עצמי לחדש ולהתמיין לamelogenin לבטא בתאים כאשר 13 בתרבית. כאן, אנו מתארים ומדגימים שיטה להדואר תרבות אמינה ועקבית של תאים משפתני העכבר CL.

<table border="0" cellpadding="0" cellspacing="0" width="584">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Forceps, size 5</td>
			<td style="width:83px;">Fine Science Tools</td>
			<td style="width:119px;">11251-30</td>
			<td style="width:167px;">FST by Dumont, Dumoxel,&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps Straight, fine, 3.5</td>
			<td style="width:83px;">Roboz</td>
			<td style="width:119px;">RS5070</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Razorblades</td>
			<td style="width:83px;">Electron Microscopy Services</td>
			<td style="width:119px;">#71960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Handle No.3</td>
			<td style="width:83px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Feather Blade No. 15</td>
			<td style="width:83px;">Electron Microscopy Services</td>
			<td style="width:119px;">#72044-15</td>
			<td>Surgical Stainless Steel</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Collagenase Type-1 filtered</td>
			<td style="width:83px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;">#4214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin syringe</td>
			<td style="width:83px;">BD</td>
			<td style="width:119px;">#329424</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accumax</td>
			<td style="width:83px;">EMD Millipore</td>
			<td style="width:119px;">#SCR006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:83px;">Gibco</td>
			<td style="width:119px;">11320-033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF</td>
			<td style="width:83px;">R&#38;D</td>
			<td style="width:119px;">2028-EG-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FGF2</td>
			<td style="width:83px;">R&#38;D</td>
			<td style="width:119px;">233-FB-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B27 Supplement</td>
			<td style="width:83px;">Gibco</td>
			<td style="width:119px;">10889-038</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of dental epithelial stem cells from the adult mouse incisor

הבנת המנגנונים התאיים ומולקולריים העומדים בבסיס התחדשות שן וחידוש הפכה לנושא עניין רב 1-4, והשן החותכת העכבר מספקת מודל לתהליכים אלה. איבר מופלא זה גדל ברציפות לאורך כל חייו של בעל החיים ומייצר את כל סוגי התאים הדרושים מבריכות פעילה של תאי גזע בוגרים שוכנו בשפתני (לכיוון השפתיים) והלשוני (לכיוון הלשון) לולאת צוואר הרחם אזורים (CL). רק תאי גזע שיניים מCL שפתני להצמיח ameloblasts שיוצרים אמייל, כיסוי החיצוני של שיניים, על פני השטח שפתני. היווצרות אמייל סימטרית זה מאפשרת שחיקה בקצה השן החותכת, ואת האבות ותאי גזע בשן החותכת הפרוקסימלי להבטיח כי רקמות השיניים מתחדשת כל הזמן. היכולת לבודד ולגדל תאי גזע או אלה במבחנה מאפשרת הרחבה שלהם ופותחת דלתות למספר רבים של ניסויים לא השגה in vivo, כגון שעותבדיקת תפוקת igh של גורמי רגולציה בתאי גזע פוטנציאליים. כאן, אנו מתארים ומדגימים שיטה אמינה ועקבית לתאי תרבות מCL שפתני של השן החותכת העכבר.

החמים-הלסת התחתונה של העכבר כוללת אחד חותכת הולכים ומתרחב ושלוש טוחנות מושרשות (איור 1 א). כל השיניים מורכבות של דנטין ואמייל, שני הרכיבים mineralized של כתר השן (איורים 1 א ו 1 א &#39;). הבתים החותכים שתי גומחות תאי גזע שנקראים CL שפתני וCL הלשוני, ואמיי?...

Watch this Scientific Journal Video about Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor at JoVE.com

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

השן החותכת העכבר הולך ומתרחבת מספקת מודל ללימוד חידוש רקמות שיניים מתאי גזע שיניים אפיתל (DESCs). מערכת חזקה בעקביות ובאמינות קבלת תאים אלה מהשן החותכת והרחבתן במבחנה הוא דיווח כאן.

בידוד והתרבות של תאי גזע השיניים אפיתל ממבוגרי העכבר הארקטיק

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

isolation-culture-dental-epithelial-stem-cells-from-adult-mouse

Research

JoVE Journal

Biology

15.0K Views.  University of California, San Francisco.  השן החותכת העכבר הולך ומתרחבת מספקת מודל ללימוד חידוש רקמות שיניים מתאי גזע שיניים אפיתל (DESCs). מערכת חזקה בעקביות ובאמינות קבלת תאים אלה מהשן החותכת והרחבתן במבחנה הוא דיווח כאן. 

Video: Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

בידוד של גזע תאים רשתית העין של העכבר

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

בידוד תרבות למבוגרים גזע תאים אפיתל של העור האנושי

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

דור של עכברים שמקורם בתאי גזע pluripotent מושרים

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

בידוד ותרבות של Myofibers פרט ותאי הלווין שלהם משרירי שלד למבוגרים

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

בידוד תאי גזע בוגרים נאיבי מתרבויות פיברובלסטים אנושיות הראשוניות

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

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

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

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

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

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

בידוד ותרבות של עכברים ראשיים Keratinocytes מ neonatal ומבוגרים עור עכבר

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

בידוד, אפיון, התמיינות של תאי גזע הלב מהלב העכבר למבוגרים

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

בידוד ותרבות בו זמנית של מיוציטים פרך, מיוציטים חדריים, ולא מיוציטים מלב עכבר בוגר

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

בידוד ותרבות של קרטינוציטים אוראליים ראשוניים מחך העכבר למבוגרים

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...