This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1. مضغي الشكل الجسم (EB) تشكيل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الجيلاتين التكيف مع المجالس الاقتصادية والاجتماعية التي حافظت على الخلايا الليفية الماوس الجنين (MEFs). المجالس الاقتصادية والاجتماعية مرور 3 مرات في 6 جيدا وحة زراعة الأنسجة المغلفة مع 0.1٪ الجيلاتين لإزالة MEFs. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو الخلايا في وسائل الإعلام ESC الصيانة مع LIF للحفاظ على خلايا غير متمايزة (الجدول 1). جعل خلايا المؤكد لم تتجاوز 80٪ التقاء. استخدام مرور منخفضة (10 أو أقل بعد إزالة مقاطع من MEFs) الخلايا للتمايز. </li></ol></li><li style=";text-align:right;direction:rtl"> في اليوم قبل المجالس الاقتصادية والاجتماعية والثقافية وللEB التمايز، ومرور الخلايا بحيث أنها ستكون حوالي 50-70٪ متكدسة في اليوم التالي. الاستمرار في زراعة الخلايا ESC صيانة المتوسطة للاحتفاظ تعدد القدرات الخلية. </li><li style=";text-align:right;direction:rtl"> في يوم تمايز والإعلام الصيانة نضح ESC وتغسل مع 1 مل الفوسفات مخزنة المالحة (PBS: 137 مم كلوريد الصوديوم، 2.7 ملي بوكل، و 11.9 ملي العازلة الفوسفات، ودرجة الحموضة 7.4) لكل بئر من لوحة 6 جيدا. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 200 ميكرولتر من 0.25٪ trypsفي / EDTA إلى كل بئر واحتضان عند 37 درجة مئوية لمدة 3 دقائق. </li><li style=";text-align:right;direction:rtl"> إبطال نشاط التربسين / EDTA مع 800 ميكرولتر من وسائل الإعلام التمايز. الماصة صعودا وهبوطا في 2 مل الماصة المصلية مع طرف P200 إلى تفكك الخلايا في تعليق خلية واحدة. تأكد من أن طرف P200 يلائم بشكل مريح على نهاية حتى وسائل الإعلام لا ترتفع بين الماصة واجهة طرف. </li><li style=";text-align:right;direction:rtl"> الماصة صعودا وهبوطا بحوالي 5-10 مرات. والحرص على عدم خلق فقاعات خلال pipetting ل. </li></ol></li><li style=";text-align:right;direction:rtl"> نقل الخلايا إلى أنبوب مخروطي 15 مل. طرح حجم 10 مل مع برنامج تلفزيوني. أجهزة الطرد المركزي في 315 x ج لمدة 5 دقائق في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> إزالة طاف. غسل مرتين مع 5 مل PBS لإزالة ما تبقى وسائل الإعلام التي تحتوي على LIF. أجهزة الطرد المركزي كما في الخطوة 1.4 لكل غسل. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> resuspend الكرية خلية النهائي في 2 مل من وسائل الإعلام التمايز (الجدول 2). </li></ol></li><li style=";text-align:right;direction:rtl"> عد الخلايا مع عدادة الكريات أو خلية مكافحة وطبق 6،000-10،000 خلية / مل في 10 سم لوحة بيتري(ثقافة غير الأنسجة المعالجة). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحديد بالضبط خلية / مل تجريبيا لكل خط ESC. استخدام لوحات بتري معقمة تستخدم عادة للعمل بكتيرية أو لوحات التقيد منخفضة. ملاحظة: للحصول على أفضل النتائج التمايز، ينبغي أن تشكل EBS المجالات التي لا تزال معلقة دون ربط إلى اللوحة. اختبار العديد من العلامات التجارية لوحات منها لإيجاد أقل الالتزام. </li><li style=";text-align:right;direction:rtl"> احتضان المجالس الاقتصادية والاجتماعية التفريق في 37 ° C الأنسجة ثقافة حاضنة مع 5٪ CO 2. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><colgroup><col /><col span="2" /><col /><col /><col /></colgroup><tbody><tr><td> كاشف </td><td> الأسهم </td><td> تركيز النهائي </td><td> حجم </td><td> شركة </td><td> عدد كتالوج </td></tr><tr><td> DMEM </td><td> 1X </td><td></td><td> 410 مل </td><td> S<html> IGMA / الدريتش </td><td> D5796 </td></tr><tr><td> FBS (ES المفحوص) </td><td> 100٪ </td><td> 15٪ </td><td> 75 مل </td><td> Hyclone </td><td> SH30070.03E </td></tr><tr><td> الأحماض الأمينية غير الأساسية </td><td> 100X </td><td> 1X </td><td> 5 مل </td><td> تقنيات الحياة </td><td> 11140 </td></tr><tr><td> L-الجلوتامين </td><td> 100X </td><td> 1X </td><td> 5 مل </td><td> تقنيات الحياة </td><td> 35050 </td></tr><tr><td> Penncillin / الستبرتوميسين </td><td> 100X </td><td> 1X </td><td> 5 مل </td><td> تقنيات الحياة </td><td> 15070 </td></tr><tr><td> β-المركابتويثانول </td><td> 14.3 M </td><td> 114 ميكرومتر </td><td> 4 ميكرولتر </td><td> سيغما / الدريتش </td><td> M3148 </td></tr><tr><td> سرطان الدم التثبيطي عامل (LIF) </td><td> 10 7 وحدة / مل </td><td> 1،000 وحدة / مل </td><td> 50 ميكرولتر </td><td> ميليبور </td><td> ESG1107 </td></tr></tbody></table>ontent &quot;&gt; الجدول 1: ESC صيانة وسائل الإعلام. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td> كاشف </td><td> الأسهم </td><td> تركيز النهائي </td><td> حجم </td><td> شركة </td><td> عدد كتالوج </td></tr><tr><td> DMEM </td><td> 1X </td><td></td><td> 410 مل </td><td> سيغما / الدريتش </td><td> D5796 </td></tr><tr><td> FBS (العضو فحص لتمايز الأمثل) </td><td> 100٪ </td><td> 15٪ </td><td> 75 مل </td><td> Hyclone </td><td> SH30070.03E </td></tr><tr><td> الأحماض الأمينية غير الأساسية </td><td> 100X </td><td> 1X </td><td> 5 مل </td><td> تقنيات الحياة </td><td> 11140 </td></tr><tr><td> L-الجلوتامين </td><td> 100X </td><td> 1X </td><td> 5 مل </td><td> الحياة تقانةخيس </td><td> 35050 </td></tr><tr><td> Penncillin / الستبرتوميسين </td><td> 100X </td><td> 1X </td><td> 5 مل </td><td> تقنيات الحياة </td><td> 15070 </td></tr><tr><td> β-المركابتويثانول </td><td> 14.3 M </td><td> 114 ميكرومتر </td><td> 4 ميكرولتر </td><td> سيغما / الدريتش </td><td> M3148 </td></tr></tbody></table> الجدول 2: التمايز وسائل الإعلام. 2. إعداد الخلايا لعدوى فيروس EB <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في المرحلة المطلوبة للتنمية (بين يوم 2 ويوم 3)، ونقل EBS من طبق بتري 10 سم إلى 15 مل أنبوب مخروطي. غسل لوحة مع 5 مل PBS وإضافة إلى غسل أنبوب مخروطي. </li><li style=";text-align:right;direction:rtl"> بيليه EBS في 315 XG في درجة حرارة الغرفة لمدة 5 دقائق. غسل الخلايا EB مع 5 مل PBS. </li><li style=";text-align:right;direction:rtl"> إضافة 1 مل من محلول مذاب مفرزة الخلية (يحتوي على بروتين الكولاجين والانزيمات) ووضع الأنابيب في 37 ° C حمام الماء (أو حاضنة) لمدة 30 دقيقة مع أورينتالتحريض asional (من vortexing لعبها أو الضوء). </li><li style=";text-align:right;direction:rtl"> إضافة 1 مل من وسائل الإعلام التمايز. الماصة صعودا ونزولا مع الماصة 2 مل مع طرف الماصة P200. وضع أنبوب في 37 ° C حمام الماء مرة أخرى لمدة 30-60 دقيقة. </li><li style=";text-align:right;direction:rtl"> تدور أسفل الخلايا في 315 x ج لمدة 5 دقائق وغسل الخلايا مع برنامج تلفزيوني. </li></ol> عدوى فيروس 3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> resuspend الخلايا في 1.5 مل من وسائل الإعلام التمايز. نقل تعليق خلية إلى 6 جيدا غير الأنسجة الثقافة وحة المعالجة. </li><li style=";text-align:right;direction:rtl"> إعداد الارتجاعي من التقنيات القياسية في وقت مبكر. إضافة 5-100 ميكرولتر من طاف الفيروسي لخلايا اعتمادا على عيار الفيروسية. لا تضيف polybrene لتعزيز العدوى لأن هذا يحول دون إصلاح لاحق من EBS. </li><li style=";text-align:right;direction:rtl"> Spinoculate الخلايا مع الفيروس عن طريق الطرد المركزي في 2،120 x ج لمدة 90 دقيقة في درجة حرارة الغرفة. </li></ol> 4. التفريق الفيروسية الخلايا المصابة EB في قطرات شنقا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 2 مل من وسائل الإعلام التمايز إلى كل بئر من طnfected EB التعليق الخلية. </li><li style=";text-align:right;direction:rtl"> الماصة 3.5 مل من المصابين EB تعليق خلية في خزان معقم كاشف للmultipipettors. استخدام الماصة 15-20 multipipettor إلى صفوف من ثمانية 20 ميكرولتر قطرات على الغطاء المقلوب من 15 سم لوحة بيتري. </li><li style=";text-align:right;direction:rtl"> إضافة 10 مل من برنامج تلفزيوني العقيمة أو المياه إلى النصف السفلي من لوحة بيتري. ثم قلب الغطاء مع قطرات ومكان على طبق بيتري. تأكد من أن قطرات معلقة رأسا على عقب من غطاء مع برنامج تلفزيوني أو الماء أقل للحفاظ على ترطيب الغرفة. </li><li style=";text-align:right;direction:rtl"> ضع الأطباق في 37 ° C الأنسجة ثقافة حاضنة مع 5.0٪ CO 2. </li><li style=";text-align:right;direction:rtl"> بعد 2 أيام، جمع EBS شكلت في قطرات شنقا بواسطة قلب الغطاء والغسيل مع 4.5 مل من وسائل الإعلام التمايز. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وسائل الاعلام نقل وEBS للثقافة 10 سم غير الأنسجة بيتري لوحة جديدة. غسل غطاء مرة أخرى مع 3 مل من وسائل الإعلام التمايز ونقل إلى 10 سم لوحة. </li></ol></li><li style=";text-align:right;direction:rtl"> حصاد الخلايا لتحليلها من قبل التدفق الخلوي بعد ما مجموعه 8أيام الثقافة بدءا من نقل الخلايا الأولية إلى وسائل الإعلام التمايز (-LIF). </li></ol> 5. إعداد الخلايا لتحليل تدفق عداد الكريات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نقل الخلايا إلى أنبوب مخروطي 15 مل مع 10 مل الماصة. غسل لوحة مع 5 مل PBS ونقل إلى نفس أنبوب مخروطي. </li><li style=";text-align:right;direction:rtl"> خلايا بيليه في 315 ز س. غسل الكريات مع 10 مل PBS. </li><li style=";text-align:right;direction:rtl"> إضافة 1 مل من محلول مذاب مفرزة الخلية ووضع الأنابيب في 37 ° C حمام الماء لمدة 30 دقيقة مع الإثارة في بعض الأحيان. للكشف عن بعض مستضدات سطح الخلية، التربسين يمكن أن تستخدم بدلا من ذلك. </li><li style=";text-align:right;direction:rtl"> إضافة 1 مل سائل الإعلام التمايز والماصة صعودا وهبوطا مع 2 مل الماصة. استخدام غيض P200 إلى نهاية الماصة للمساعدة في كسر المتابعة هضمها EB في تعليق خلية واحدة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع أنبوب في 37 ° C حمام الماء مرة أخرى لمدة 60 دقيقة حتى يتسنى للبروتينات الغشاء متكاملة لإعادة تدوير إلى سطح الخلية. </li></ol></li><li style=";text-align:right;direction:rtl"> بيليه الخلايا في 315 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> غسل واي خلاياال 0.1٪ مصل بقري الزلال جزء V (BSA) التي أعدت في برنامج تلفزيوني (PBS / BSA)، واحتضان الخلايا مع محددة الموسومة fluorescently الأجسام المضادة. </li></ol> 6. تحليل التدفق الخلوي للدم الأسلاف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع 10 6 خلايا EB المستمدة في الجولة 5 مل 12 × 75 مم أنبوب أسفل. لضبط إعدادات تدفق عداد الكريات التعويض، استخدام الخرز التعويض وفقا لتعليمات الشركة المصنعة. </li><li style=";text-align:right;direction:rtl"> خلايا بيليه في 315 ز س. تخلصي من طاف و resuspend بيليه في برنامج تلفزيوني المتبقية الحل / BSA في الأنبوب. </li><li style=";text-align:right;direction:rtl"> للكشف عن ESC HPCS، احتضان الخلايا مع الأجسام المضادة الفلورسنت المسمى: مكافحة فأر CD41-PE (R-Phycoerytherin) ومكافحة فأر CD117 (cKit) -APC / CY7 (Allophycocyanin Cyanine 7). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تمييع الأجسام المضادة 1: 100 في برنامج تلفزيوني / BSA. إضافة 100 ميكرولتر من الأجسام المضادة المخفف لكل عينة. تحديد الصحيح التخفيف من الأجسام المضادة لكل بائع، استنساخ، والكثير أو الأجسام المضادة. تحديد التخفيف الأمثل لوصفها الخلايا بشكل فردي. </لى&gt; </ol></li><li style=";text-align:right;direction:rtl"> احتضان العينات على الجليد في الظلام لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> إضافة 2 مل PBS / BSA وبيليه الخلايا في 315 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> نضح وطاف بيليه resuspend في 300 ميكرولتر من برنامج تلفزيوني أو متساوي التوتر عازلة أخرى. </li><li style=";text-align:right;direction:rtl"> مرشح معلق الخلايا من خلال مصفاة غطاء الخلية (0.35 ميكرومتر) لإزالة الركام الخلية الكبيرة. </li><li style=";text-align:right;direction:rtl"> تحليل الخلايا على وعداد الكريات التدفق. </li></ol>

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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HoxB4+confers+definitive+lymphoid-myeloid+engraftment+potential+on+embryonic+stem+cell+and+yolk+sac+hematopoietic+progenitors.">HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.</a> Cell. 109, 29-37 (2002).</li><li>Galvin, K. E., Travis, E. D., Yee, D., Magnuson, T., Vivian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+signaling+regulates+the+bone+morphogenic+protein+pluripotency+pathway+in+mouse+embryonic+stem+cells.">Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells.</a> J Biol Chem. 285, 19747-19756 (2010).</li><li>Ismailoglu, I., Yeamans, G., Daley, G. Q., Perlingeiro, R. C., Kyba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+patterning+activity+of+SCL.">Mesodermal patterning activity of SCL.</a> Exp Hematol. 36, 1593-1603 (2008).</li><li>Kyba, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+hematopoietic+differentiation+of+embryonic+stem+cells+conditionally+expressing+Stat5.">Enhanced hematopoietic differentiation of embryonic stem cells conditionally expressing Stat5.</a> Proc Natl Acad Sci U S A. 100, 11904-11910 (2003).</li><li>Cao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-23a+regulates+TGF-beta-induced+epithelial-mesenchymal+transition+by+targeting+E-cadherin+in+lung+cancer+cells.">MiR-23a regulates TGF-beta-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells.</a> Int J Oncol. 41, 869-875 (2012).</li><li>Chan, 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=Molecular+basis+for+antagonism+between+PDGF+and+the+TGFbeta+family+of+signalling+pathways+by+control+of+miR-24+expression.">Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression.</a> EMBO J. 29, 559-573 (2010).</li><li>Huang, 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=Upregulation+of+miR-23a+approximately+27a+approximately+24+decreases+transforming+growth+factor-beta-induced+tumor-suppressive+activities+in+human+hepatocellular+carcinoma+cells.">Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells.</a> Int J Cancer. 123, (2008).</li><li>Min, 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=TGF-beta-associated+miR-27a+inhibits+dendritic+cell-mediated+differentiation+of+Th1+and+Th17+cells+by+TAB3,+p38+MAPK,+MAP2K4+and+MAP2K7.">TGF-beta-associated miR-27a inhibits dendritic cell-mediated differentiation of Th1 and Th17 cells by TAB3, p38 MAPK, MAP2K4 and MAP2K7.</a> Genes Immun. 13, 621-631 (2012).</li><li>Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D., Orkin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD41+marks+the+initiation+of+definitive+hematopoiesis+in+the+mouse+embryo.">Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.</a> Blood. 101, 508-516 (2003).</li><li>Mitjavila-Garcia, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD41+on+hematopoietic+progenitors+derived+from+embryonic+hematopoietic+cells.">Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells.</a> Development. 129, 2003-2013 (2002).</li><li>Warr, M. R., Pietras, E. M., Passegue, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+controlling+hematopoietic+stem+cell+functions+during+normal+hematopoiesis+and+hematological+malignancies.+Wiley+interdisciplinary+reviews.">Mechanisms controlling hematopoietic stem cell functions during normal hematopoiesis and hematological malignancies. Wiley interdisciplinary reviews.</a> Systems biology and medicine. 3, 681-701 (2011).</li><li>Dahl, R., Ramirez-Bergeron, D. L., Rao, S., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spi-B+can+functionally+replace+PU.1+in+myeloid+but+not+lymphoid+development.">Spi-B can functionally replace PU.1 in myeloid but not lymphoid development.</a> Embo J. 21, 2220-2230 (2002).</li><li>Dahl, 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=Regulation+of+macrophage+and+neutrophil+cell+fates+by+the+PU.1:C/EBPalpha+ratio+and+granulocyte+colony-stimulating+factor.">Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor.</a> Nat Immunol. 4, 1029-1036 (2003).</li></ol>

الكتاب ليس لديهم المصالح المالية المتنافسة في الكشف عنها.

كما نوقش أعلاه، يمكن أن تتولد استنساخ ESC التي تعبر عن inducibly الجينات ذات الاهتمام باستخدام نظم الدوكسيسيكلين، ومع ذلك، جيل من هذه السطور هو وقتا طويلا وعمالة كثيفة. الموصوفة في هذا البروتوكول هو وسيلة للتعبير عن الجينات في المصالح في تعليق خلية واحدة أعدت من ESC المستمد...

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

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			<td height="21" style="height:21px;width:193px;">RW4 ESCs</td>
			<td style="width:144px;">ATCC</td>
			<td>CRL-12418</td>
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	</tbody>
</table>

retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

في هذه الدراسات مهيلم RW4 (مشتقة من 129X1 / SVJ سلالة الماوس) المجالس الاقتصادية والاجتماعية تم استخدامها. تم عزل EBS في 3 أيام من التمايز. للحصول على أفضل النتائج يجب أن يكون EBS كروية وغير ملتصقة (الشكل 1A، B). بعد spinoculation بفيروس المشارك تعبر عن GFP علامة الفلورسنت جنبا إل...

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

عدوى فيروسات الفئران الجنينية الجذعية المستمدة خلية خلايا الجسم مضغي لتحليل التمايز لدم

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

retroviral-infection-murine-embryonic-stem-cell-derived-embryoid-body

Research

JoVE Journal

Biology

8.4K Views.  Harper Cancer Research Institute. Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation. 

Video: Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

العزلة وزرع الخلايا الجذعية المكونة للدم (HSCs)

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

اشتقاق خلايا الجذعية المكونة للدم من الفئران خلايا الجذعية الجنينية

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

عزل الخلايا الجذعية المكونة للدم في وقت مبكر من الكيس المحي الفئران وAGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

الخلايا الجذعية الجنينية المستمدة من الخلايا البطانية لعلاج نقص التروية Hindlimb

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

الفيديو المعلوماتية الحيوية تحليل النمو البشري الخلية الجذعية الجنينية كولوني

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

تمايز الخلايا الجذعية الجنينية في السلائف Oligodendrocyte

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral بوساطة خلال ضربة قاضية السابقين فيفو تكون الكريات الحمر في الإنسان خلايا الجذعية المكونة للدم

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

المظهري التحليل وعزل الخلايا الجذعية المكونة للدم الفئران والأسلاف النسب، الملتزم

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

كفاءة تمايز الخلايا الجذعية الجنينية في الخلايا العصبية الحركية

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

بروتوكول الفيديو من عدوى فيروسات الرجعية في الابتدائية المعوية عضي الثقافة

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...