نشكر براد Lackford لقراءة وتحرير مخطوطة. وأيد هذا البحث من قبل المعهد الوطني لعلوم الصحة البيئية، المعاهد الوطنية للصحة برنامج بحوث ضمن الجدران Z01ES102745 (إلى GH).

1. Oct4GiP خلية ES الفأر الصيانة تفضلت الخلايا Oct4GiP الدكتور أوستن سميث. كانت مشتقة من الفئران 129/Ola يحمل التحوير Oct4-GFPiresPac 4،5. يتم الاحتفاظ بها في الجيلاتين المغلفة لوحات زراعة الأنسجة في المتوسط ​​ESGRO درجة كاملة زائد نسيلي (ميليبور)، أو في المدى المتوسط ​​M15: DMEM (إينفيتروجن) تستكمل مع FBS 15٪، 1000 يو / مل ESGRO (ميليبور)، 1X غير الأحماض الأمينية الأساسية (إينفيتروجن)، 1X EmbryoMax Nucleasides (ميليبور)، و 10 ميكرومتر β-المركابتويثانول. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لوحات معطف مع الجيلاتين 0.1٪ (سيغما) في درجة حرارة الغرفة لمدة 30 دقيقة (0.1 مل الجيلاتين حل / سم 2). </li><li style=";text-align:right;direction:rtl"> خلايا Oct4GiP صفيحة الجيلاتين في مغلف بالمطاط في لوحات ~ 2 × 10 4 / سم 2. </li><li style=";text-align:right;direction:rtl"> انقسام الخلايا كل يومين مع التربسين 0.05٪. </li></ol> 2. ترنسفكأيشن سيرنا في خلايا Oct4GiP الفحص مراسل Oct4GiP هو الأكثر ملاءمةنفذت tly في الجيلاتين المغلفة مسطحة القاع 96 - 384 أو جيدا لوحات (كورنينج أو دينار بحريني) في المتوسط ​​M15. ويرد الإجراء شامل في الشكل 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> سيرنا، دهن المجمعات الجمعية العمومية: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لترنسفكأيشن في 96-جيدا لوحات، وتجمع الدهون المجمعات سيرنا، في U-96-أسفل جيدا لوحات. </li><li style=";text-align:right;direction:rtl"> في كل بئر، خلط 10 OptiMEM ميكروليتر (إينفيتروجن) و 0.3 Lipofectamine ميكرولتر 2000 (إينفيتروجن)، واحتضان في درجة حرارة الغرفة لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إضافة 5 سيرنا بمول إلى خليط للدهون، OptiMEM واحتضان لمدة 15 دقيقة. وسيرنا: نسبة الدهون هو 100 بمول: 6ul. </li><li style=";text-align:right;direction:rtl"> نقل المجمعات سيرنا-الدهون في OptiMEM من لوحة U-أسفل لوحة مسطحة القاع الجيلاتين المغلفة مع ماصة متعدد القنوات. </li><li style=";text-align:right;direction:rtl"> لترنسفكأيشن في 384 جيدا لوحات، وإعداد المجمعات سيرنا-الدهون باستخدام 0.1 ميكرو لتر Lipofectamine 2000 و 2 بمول سيرنا في 10 OptiMEM ميكرولتر. </li></ol></li></ol> ملاحظة: إن تركيز سيرنا في النهائيوtransfections هو 50 نانومتر. تنفيذ البيولوجية 3-4 مكررات لكل ترنسفكأيشن سيرنا. إعداد الخلائط ماجستير في المجمعات سيرنا-الدهون في لوحة تحت القاع وقسامة في لوحة الجيلاتين المغلفة مسطحة القاع. <ol start="2" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Oct4GiP طلي الخلية وترنسفكأيشن: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لترنسفكأيشن في 96-جيدا لوحات، وجمع الخلايا Oct4GiP وresuspend منهم في المتوسط ​​M15 جديدة ل9 × 10 4 خلية / مل. </li><li style=";text-align:right;direction:rtl"> أضف 100 ميكرولتر من التعليق على كل خلية جيدا باستخدام ماصة متعدد القنوات. </li><li style=";text-align:right;direction:rtl"> خلط تعليق خلية مع المجمعات سيرنا، الدهون قبل aliquoted في البئر بواسطة pipeting صعودا وهبوطا 3-5 مرات. نصائح لتغيير transfections سيرنا مختلفة. </li><li style=";text-align:right;direction:rtl"> لترنسفكأيشن في لوحات 384 بشكل جيد، وخلايا Oct4GiP resuspend إلى 6 × 4 10 خلية / مل وقسامة 30 تعليق خلية ميكروليتر إلى كل بئر. </li></ol></li></ol> ملاحظة: تصفيح الخلية الأمثل كثافة عادة ما يكون 3 × 10 5 خلية / سم 2، ولكن مAY تتطلب مزيدا من التحسين siRNAs أن تؤثر بشكل كبير في نمو الخلايا أو قدرتها على البقاء. ومنخفض الكثافة الطلاء يؤدي إلى بقاء الخلية الفقراء خلال ترنسفكأيشن. وارتفاع كثافة الطلاء يؤدي إلى خلفية عالية نظرا لارتفاع خلية التمايز بفعل التقاء. إذا كان ذلك ضروريا تصفيح اختبار، والكثافة بين 2-4 خلايا س 5 10 / سم 2. <ol start="3" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> متوسط ​​التغير: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة المتوسطة القديمة باستخدام متعدد القنوات الشافطة (كورنينج) أو فراغ عصا (VP-العلمي). يجب الحرص على عدم خدش الخلايا في الجزء السفلي من لوحات. </li><li style=";text-align:right;direction:rtl"> تغذية الخلايا مع الطازجة وفاق متوسط ​​خلية (100 ميكروليتر لوحة 96-جيدا وميكرولتر 30 ل384 لوحة جيد) باستخدام متعدد القنوات ماصة كل يوم. </li></ol></li></ol> 3. تحليل نظام مراقبة الأصول الميدانية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خلية تفكك: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد أربعة أيام من ترنسفكأيشن، وإزالة المتوسطة باستخدام متعدد القنوات الشافطة (كورنينج) أو فراغ عصا (VP-العلمي). </li><li style=";text-align:right;direction:rtl"> ل96-جيدا لوحات، وشطف الخلايا مرة واحدة مع 100μل PBS. </li><li style=";text-align:right;direction:rtl"> إضافة 25 ميكرولتر التربسين 0.25٪ على كل جيدا باستخدام ماصة متعدد القنوات. </li><li style=";text-align:right;direction:rtl"> احتضان في درجة حرارة الغرفة ل5minutes مع الإثارة في بعض الأحيان، وفحص البصر لضمان حياد تام للخلايا. </li><li style=";text-align:right;direction:rtl"> إضافة 90 ميكرولتر من برنامج تلفزيوني مع FBS 10٪ لإبطال نشاط التربسين وفصل الخلايا في تعليق خلية واحدة بواسطة pipetting المتكررة. </li><li style=";text-align:right;direction:rtl"> ل384 جيد لوحات، وتنأى الخلايا مع 10 ميكرولتر التربسين وإخماد مع 30 PBS ميكرولتر مع FBS 10٪. </li></ol></li><li style=";text-align:right;direction:rtl"> تحليل نظام مراقبة الأصول الميدانية: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحليل مضان GFP على محلل دينار بحريني FACS LSRII مجهزة وحدة HTS أو غيرها من محلل نظام مراقبة الأصول الميدانية مجهزة على نحو مماثل (مثل Accuri أو Intellicyt). </li><li style=";text-align:right;direction:rtl"> استخدام وضع الإنتاجية العالية على HTS وتحليل 10 ميكرولتر من التعليق الخلية. تعيين العتبة عدد الخلايا إلى 1.0 X 4 10. </li><li style=";text-align:right;direction:rtl"> ضبط الجهد PMT وعتبة للقبض على خلايا في المؤامرة إلى الأمام في مقابل الجانب مبعثر. بوابة للسكان الخلية الحية في forwارض مقابل الجانبية مبعثر مؤامرة. </li><li style=";text-align:right;direction:rtl"> خلق مؤامرة الرسم البياني للقناة GFP. تعيين البوابة في قناة GFP بحيث ~ 10٪ من الخلايا على ما يبدو GFP سلبية في الخلايا transfected وهمية أو سيطرة سيرنا. </li><li style=";text-align:right;direction:rtl"> تحديد٪ GFP سلبية الخلايا من كل معاملة: الخلايا المتمايزة٪ =٪ GFP سلبية الخلايا. مقارنة بين الخلايا٪ فرق بين التجريبية وtransfections ضبط سيرنا باستخدام 2 الذيل اختبار t، ويسجل مشاهدات ايجابية مع القيم ف &lt;0.01. </li></ol></li></ol> 4. ممثل النتائج Oct4، نانوغ (NANOG) وSox2 ثلاثة الجينات التي تلعب دورا حاسما في الحفاظ على خلايا ES الذاتي تجديد 6،7،9-11. الشكل 2 يبين أن مقايسة مراسل Oct4GiP يمكن الكشف بسهولة التفريق بسبب إسكات هذه العوامل في Oct4GiP خلايا ES. رقم 2A يبين خلايا ES Oct4GiP GFP هي إيجابية عندما حافظت على وفاق<html> الخلايا. الشكل 2B يبين مؤامرة مبعثر إلى الأمام في مقابل الجانب والرسم البياني للقناة GFP من الخلايا Oct4GiP transfected مع ضبط أو Oct4 siRNAs. فمن الضروري بوابة للخلايا الحية في مؤامرة مبعثر إلى الأمام في مقابل الجانب، حيث أن الخلايا الميتة والبقايا هي GFP سلبية وستزيد من الخلفية. لا من ناحية أخرى، صدرة ضيقة التمييز لاستبعاد كتل الخلية ممكن حاجة دائما. في الخلايا ضبط سيرنا transfected، ينبغي أن الغالبية العظمى من الخلايا تكون GFP إيجابية. إذا واضح GFP سلبية السكان موجودة في آبار ضبط سيرنا transfected، قد يكون تم الخلايا Oct4GiP بدء أو إجراء ترنسفكأيشن للخطر والنتيجة قد لا تكون للتفسير. الشكل 2C يظهر في الرسم البياني شريط من الخلايا المتمايزة٪ (الخلايا المتمايزة٪ =٪ GFP سلبية خلايا) من التحكم، Oct4، Nanog، وSox2 سيرنا خلايا transfected. <html> d/3987/3987fig1.jpg &quot;/&gt; الشكل 1. الخطوط العريضة للOct4GiP مقايسة مراسل. <img alt="الشكل 2" src="/files/ftp_upload/3987/3987fig2.jpg" /> الشكل 2. يمكن Oct4GiP فحص كشف مراسل ES تمايز الخلايا التي تسببها نانوغ (NANOG) Oct4، أو إسكات Sox2. أ) تم تحليل E14Tg2a (من النوع البري، خط أسود) والخلايا Oct4GiP (الخط الأخضر) من قبل نظام مراقبة الأصول الميدانية. رسم بياني للقناة GFP يدل على أن الخلايا Oct4GiP هي GFP إيجابية. ب) تم transfected الخلايا Oct4GiP في 96-جيدا لوحات مع التحكم أو Oct4 سيرنا، ونظام مراقبة الأصول الميدانية تحليلها بعد 4 ايام ترنسفكأيشن. وtransfected إلى الأمام مقابل المؤامرات مبعثر جانب ورسوم بيانية للقناة GFP من الخلايا transfected ترد. C) الخلايا Oct4GiP مع التحكم، Nanog، Oct4، أو Sox2-سيرنا. تم تحديد الخلايا المتمايزة٪ من٪ GFP سلبية الخلايا بعد 4 ايام ترنسفكأيشن، وتآمر كما يعني + / - الخطأ المعياري للمتوسط ​​(ن = 4).و = &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot; الهدف = &quot;_blank&quot;&gt; اضغط هنا لعرض أكبر شخصية.

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لا يمكن للمقايسة مراسل Oct4GiP وصفنا أعلاه قياس كميا مدى التجديد الذاتي مقابل التمايز. بالمقارنة مع الطرق الأخرى المتاحة، مثل ال 12 مورفولوجيا القاعدة والانتشار / البقاء القائم على المقايسات، فإنه يوفر أعلى حساسية والإنتاجية، فضلا عن قياس أكثر مباشرة للدولة وفاق ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> ESGRO متوسط ​​درجة كاملة زائد نسيلي </td><td> ميليبور </td><td> SF001-500P </td></tr><tr><td> DMEM (الجلوكوز السامية 1X) </td><td> إينفيتروجن </td><td> 11965 </td></tr><tr><td> 0.25٪ التربسين-EDTA </td><td> إينفيتروجن </td><td> 25200 </td></tr><tr><td> Lipofectamine 2000 </td><td> إينفيتروجن </td><td> 1001817 </td></tr><tr><td> OPTI-MEM (خفض متوسط ​​المصل) </td><td> إينفيتروجن </td><td> 31985 </td></tr><tr><td> ESGRO mLIF (10 7 units/1ml) </td><td> ميليبور </td><td> DAM1776540 </td></tr><tr><td> MEM NEAA (غير الضرورية الأحماض الأمينية) </td><td> إينفيتروجن </td><td> 11140 </td></tr><tr><td> 100X النيوكليوسيدات لخلية ES </td><td> ميليبور </td><td> 10620-1 </td&gt; </tr><tr><td> 2-المركابتويثانول </td><td> سيغما </td><td> M7522-100ML </td></tr><tr><td> ES-المؤهلين مصل بقري جنيني </td><td> إينفيتروجن </td><td> 10437 </td></tr><tr><td> Nanog سيرنا </td><td> إينفيتروجن </td><td> MSS231181 </td></tr><tr><td> Oct4 سيرنا </td><td> Dharmacon </td><td> D-046256-02 </td></tr><tr><td> Sox2 سيرنا </td><td> Dharmacon </td><td> M-058489-01 </td></tr></tbody></table> سيرنا السيطرة: مزدوج سيرنا استهداف يراعة luciferase المراسل (5&#39;-CGTACGCGGAATACTTCGA) توليفها من قبل Dharamcon.

oct4gip reporter assay to study genes that regulate mouse embryonic stem cell maintenance and self-renewal

تعدد القدرات الذاتية والتجديد هما الخصائص المميزة للخلايا الجذعية الجنينية (خلايا ES). وفهم الآلية الجزيئية الكامنة وراء يسهل إلى حد كبير على استخدام خلايا ES لدراسات البيولوجيا التطورية، والنمذجة المرض، واكتشاف المخدرات، والتجدد الطب (استعرضت في 1،2). إلى الإسراع في تحديد وتوصيف المنظمين رواية من خلية وفاق وصيانة النفس التجديد، قمنا بتطوير مضان مراسل القائم على فحص لقياس كمي لوضع الذات في تجديد خلايا ES فأر باستخدام خلايا Oct4GiP 3. الخلايا Oct4GiP تعبر عن ببروتين أخضر (GFP) في إطار السيطرة على المنطقة الجين المروج Oct4 4،5. مطلوب Oct4 لخلية ES الذاتي تجديد، ويعبر عنه بشكل كبير في الخلايا وفاق وسرعان ما انهارت التنظيم خلال التمايز 6،7. ونتيجة لذلك، GFP التعبير ومضان في الخلايا مراسل يرتبط بأمانةمع وفاق هوية الخلية 5، ومضان تنشيط الخلايا ويمكن فرز (FACS) يمكن استخدام التحليل أن تراقب عن كثب الوضع الذاتي تجديد الخلايا على مستوى خلية واحدة 3،8. مقرونا رني، يمكن استخدام الفحص مراسل Oct4GiP إلى التعرف بسرعة ودراسة المنظمين من صيانة الخلية وفاق والذاتي تجديد 3،8. بالمقارنة مع الطرق الأخرى لمعايرة الذاتي تجديد، وهو أكثر ملاءمة، الحساسة، والكمية، وأقل تكلفة. ويمكن القيام بها في 96 - أو 384 كذلك لوحات لدراسات واسعة النطاق مثل شاشات عالية الإنتاجية أو وراثية تحليل قشوة. أخيرا، وذلك باستخدام خطوط أخرى ES مراسل خلية نسب محددة، ويمكن أيضا للفحص وصفنا هنا يمكن تعديلها لدراسة مواصفات مصير خلال تمايز الخلايا ES.

Watch this Scientific Journal Video about Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal at JoVE.com

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

نحن وصفا لفحص مراسل مضان على التعرف بسرعة وتوصيف الجينات التي تنظم الماوس الخلايا الجذعية الجنينية وصيانة وتجديد النفس.

Oct4GiP الفحص مراسل لدراسة الجينات التي تنظم الفأر الخلايا الجذعية الجنينية الصيانة والتجديد الذاتي

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

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Research

JoVE Journal

Biology

10.4K Views.  National Institute of Environmental Health Sciences. نحن وصفا لفحص مراسل مضان على التعرف بسرعة وتوصيف الجينات التي تنظم الماوس الخلايا الجذعية الجنينية وصيانة وتجديد النفس.

Video: Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

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 ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

اشتقاق الخلايا الجذعية الجنينية البشرية التي Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

في المختبر والمجراة في ضوء بارد مراسل التصوير الجيني للخلايا منشأ جنينية بشرية

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

الثقافة والمحافظة على الخلايا الجذعية الجنينية البشرية

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

استخدام SC1 (Pluripotin) لدعم MESC التجديد الذاتي في غياب LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

العزلة واشتقاق خلايا الفأر الجنينية بيرشوت

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

كله يشن التحليل المناعى للالجنينية الاطراف الأوعية الدموية الجلدية : نظام نموذجي لدراسة الأوعية الدموية المتفرعة التشكل في الأجنة

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

تعديل الجذعية الجنينية الماوس الفحص خلية مقرها لقياس كفاءة قلبية التعريفي

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

مناعي لونين الفحص الجينات الأنسجة الخاصة باستخدام أجنة فئران في مرحلة مبكرة

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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.

جيل من الفئران المستمدة من الخلايا الجذعية المحفزة المستحثة

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