AEC هو زميل في التابوت جين الصندوق التذكاري تشايلدز للأبحاث الطبية ومختبرات ميرك للبحوث. نشكر دال Egli وكوان جيم التجريبية للحصول على المشورة والمناقشة.

<html> وقد تم التبرع تجميد الأجنة البشرية المستخدمة لاشتقاق الجذعية الجنينية البشرية خلية (HES) للموافقة المسبقة عن البحوث التالية المناسبة بموجب بروتوكولات البحوث التي تم مراجعتها والموافقة عليها من قبل لجنة على حد سواء على استخدام المواد البشرية وابحاث الخلايا الجذعية الجنينية لجنة الرقابة في جامعة هارفارد . ويستند بروتوكول مكتوب أدناه على نشر المعلمات التي أبلغ عنها كوان وآخرون. عام 2004 ، مع تعديلات طفيفة. جنين الثقافة أذاب أجنة بشرية باستخدام أدوات كوين ذوبان مزايا والثقافة في قطرات من المتوسط ​​العالمي تستكمل مع Plasmanate 15 ٪ حتى التنمية إلى مرحلة الكيسة الموسعة. Immunosurgery القائم على عزل كتلة الخلايا الداخلية إعداد : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد لوحات لإزالة الشفافة وزونا immunosurgery (أنظر أدناه) عن طريق إقامة قطرات من كل حل في لوحات 10cm. تراكب مع الزيوت المعدنية لمنع التبخر. إعداد صف واحد في الجنين المراد الاشتقاق. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> EmbryoMax Tyrodes الحمضية ، استخدم كما هو. </li><li style=";text-align:right;direction:rtl"> وسائل الاعلام HES اشتقاق الخلية : 75 ٪ KO - DMEM ، و 10 ٪ KO - مصل استبدال ، Plasmanate 10 ٪ ، 2.5 ٪ وفاق الخلية اختبار مصل بقري جنيني (FBS) ، 2mm وGlutamax - I ، 1 ٪ غير الأحماض الأمينية الأساسية ، و 50 وحدات / مل البنسلين ، 50μg/ml ستربتوميسين 0.055mM ب - المركابتويثانول ، 5ng/ml bFGF. </li><li style=";text-align:right;direction:rtl"> أرنب المضادة للجسم الإنسان خلايا الدم الحمراء الابتدائية ، وفقا لاقتراح إعادة التصنيع ، مخففة في 1:10 مع وسائل الاعلام HES اشتقاق الخلية. </li><li style=";text-align:right;direction:rtl"> غينيا الخنازير تكملة مصل ، وفقا لاقتراح إعادة التصنيع ، مخففة في 1:10 مع وسائل الاعلام HES اشتقاق الخلية. <img src="/files/ftp_upload/old/574_2008_9_18_6/574_Figure-01.png" alt="574_Figure - 01" width="600" height="166" /> </li></ul></li><li style=";text-align:right;direction:rtl"> يوازن جميع اللوحات الى 37 درجة مئوية في حاضنة زراعة الأنسجة قبل الاستخدام. </li><li style=";text-align:right;direction:rtl"> سحب 50μl - 100μl ماصات الشعرية (VWR) لاستخدامها في نقل الأجنة. قطع الماس ينتهي مع القلم واستخدام مع أنابيب ماصة فم مجهزة تصفية 0.2μm. </li></ol> Immunosurgery الداخلي : ملاحظات : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في هذا الإجراء ، يتم تدمير خلايا الأديم الظاهر الغاذي عن التعرض قصيرة إلى الأجسام المضادة الموجهة ضد الخلايا البشرية جنبا إلى جنب مع النشاط مكملا. ولذلك ، والبروتوكول يعمل بشكل أفضل مع أجنة عالية الجودة مع الأديم الظاهر الغاذي سليمة ، وإلا السلامة الهيكلية للالكيسة يمنع الحركة من كونها أيضا عرضة لردود الفعل المناعية. </li><li style=";text-align:right;direction:rtl"> تنفيذ كافة الإجراءات باستخدام نطاق تشريح مجهزة مرحلة ساخنة. </li></ul> الخطوات التالية : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شطف الفم ماصة لاستخدامها لنقل الأجنة مع FBS لمنع الأجنة من الالتصاق. </li><li style=";text-align:right;direction:rtl"> نقل الكيسة طبق من ثقافة إلى قطرة من عقد HES AT الطبق. </li><li style=";text-align:right;direction:rtl"> يبدأ الإجراء عن طريق نقل الكيسة من انخفاض HES من خلال سلسلة من قطرات AT. في الانخفاض الثالث ، ومشاهدة لفسخ الشفافة زونا (50-30 ثانية عادة). قد يكون حريصا على عدم الإفراط في النزاهة علاج أو الجنين للخطر. <img src="/files/ftp_upload/old/574_2008_7_21_2/574_Figure-02.png" alt="574_Figure - 02" width="551" height="273" /> </li><li style=";text-align:right;direction:rtl"> نقل الكيسة من خلال سلسلة من وسائل الإعلام HES اشتقاق الخلية ينخفض ​​إلى شطف AT. ينصح ماصة تحميلها مسبقا مع وسائل الاعلام لتحييد على الفور في رد الفعل. ويمكن عقد Blastocysts هنا حتى تم كل AT - المجهزة. </li><li style=";text-align:right;direction:rtl"> نقل الكيسة من خلال سلسلة من قطرات الضد الابتدائية ، وترك في الانخفاض الثالث لمدة 30 دقيقة من الحضانة عند 37 درجة مئوية في نسيج حاضنة الثقافة. </li><li style=";text-align:right;direction:rtl"> نقل الكيسة من خلال سلسلة من وسائل الإعلام HES اشتقاق الخلية ينخفض ​​إلى شطف الابتدائي. </li><li style=";text-align:right;direction:rtl"> نقل الكيسة من خلال سلسلة من قطرات تكمل ، وترك في الانخفاض الثالث لمشاهدة لتحلل خلايا الأديم الظاهر الغاذي. مراقبة الكيسة لأول 30 ثانية على خشبة المسرح ل&quot;فقاعات&quot; من خلايا الأديم الظاهر الغاذي كما ليز. إذا لوحظ تحلل ضمن هذا الإطار الزمني ، والحفاظ على طبق على خشبة المسرح المجهر لمراقبة رد فعل حتى الاكتمال. إذا لوحظ أي محتدما خلال أول 30 ثانية ، والعودة إلى الحاضنة ، والتحقق من كل 5 دقائق حتى أكثر من الأديم الظاهر الغاذي وقد lysed. وهذا يمكن أن يستغرق 5-15 دقائق. مراقبة عن كثب رد فعل على تقليل وقت رد الفعل والضرر المحتمل للكتلة الخلية الداخلية. <img src="/files/ftp_upload/old/574_2008_7_21_3/574_Figure-03.png" alt="574_Figure - 03" width="571" height="273" /> </li><li style=";text-align:right;direction:rtl"> نقل الكيسة من خلال سلسلة من وسائل الإعلام HES اشتقاق الخلية قطرات ليغسل تكمل النشاط. </li><li style=";text-align:right;direction:rtl"> باستخدام ماصة الزجاج مع قطره أصغر قليلا من الكيسة (سحبت من أنابيب VWR 10μl الشعرية) ، يوجه الكيسة صعودا وهبوطا لتجريد بعيدا أكثر من الأديم الظاهر الغاذي lysed. ز &quot;بديل =&quot; 574_Figure - 0 &quot;العرض =&quot; 577 &quot;ذروة =&quot; 270 &quot;/&gt; </li><li style=";text-align:right;direction:rtl"> لوحة ICM عزل الخلايا المغذية على المعطل mitotically الليفية من جنين فأر (MEFs). وينبغي إعداد MEFs قبل يوم والاشتقاق ، وتحولت إلى خلية HES اشتقاق وسائل الاعلام يوم. 4 - جيدا الأطباق نونك تعمل بشكل جيد لخطوات أولية من الاشتقاق. </li><li style=";text-align:right;direction:rtl"> لا تخل لوحة لأيام 1-2 الأولى. بعد ذلك ، والتحقق اليومي للثمرة مستعمرات الخلايا الجذعية الجنينية (عادة ما يبدو في غضون 1-2 أسابيع). يجب تغيير وسائل الإعلام في حجم نصف كل يوم وبداية أخرى في اليوم 3. </li></ol> وفاق نمو الخلية والركض عندما ثمرة الخلية HES وصلت حوالي 0.5mm أو أكثر ، وانها مستعدة للتوسع عن طريق التقاط الميكانيكية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تفريق ميكانيكيا نصف ثمرة ونقل لوحات جديدة تحتوي على مغذيات جديدة. وينبغي أن الخلايا الجذعية الجنينية كتل تكون حوالي 3-50 خلايا لكل منهما. ترك النصف الآخر من ثمرة باعتبارها احتياطية لمرور الأول (P1). يمكن الحصول على ثمرة (P0) من عدة مرات لأنها لا تزال في التوسع. <img src="/files/ftp_upload/old/574_2008_7_21_5/574_Figure-05.png" alt="574_Figure" width="639" height="219" /> </li><li style=";text-align:right;direction:rtl"> يمكن المستعمرات الثانوية موزعة على نحو مماثل ، وتوسعت عبر الأطباق اكبر الثقافة حتى الممرات 3-5 ، في الوقت الذي يمكن تكييفها للخطوط الخلايا الجديدة إلى الركض مع الأنزيمية التربسين. وينبغي أيضا على محتوى وسائل الاعلام المصل الثقافة سيخفض تدريجيا الى 1 ٪ و 0 ٪ خلال هذه المقاطع الأولية للحد من وفاق تمايز الخلايا. </li><li style=";text-align:right;direction:rtl"> وينبغي وضع راهن جميع الخلايا الجذعية الجنينية من خلال تجميد جزء من أسفل الممرات المبكر للتوسعات المستقبلية. </li><li style=";text-align:right;direction:rtl"> بالإضافة ، يجب أن تتسم كل سطر الخلية عن طريق تحليل النمط النووي والتحقق من الدراسات تعدد القدرات وتمايز في المختبر والمجراة. </li></ol>

<ol>
	<li>Cowan, C. A., Klimanskaya, I., McMahon, J., Atienza, J., Whitmyer, J., Zucker, J. P., Wang, S. P., Morton, C. C., McMahon, A. P., Powers, D., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+embryonic+stem-cell+lines+from+human+blastocysts.">Derivation of embryonic stem-cell lines from human blastocysts.</a> N ENGL J MED. 350 (13), 1353-1356 (2004).</li></ol>

The authors have nothing to disclose.

بروتوكول المعروضة هنا يقدم الباحثون مع موجزة وسهلة لمتابعة الخطوط العريضة لكيفية اشتقاق الخلايا الجذعية الجنينية البشرية عن طريق عزل خطوط immunosurgical من كتلة الخلايا الداخلية.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Quinn's Advantage Thaw Kit </td>
<td>&nbsp;</td>
<td>Sage</td>
<td>ART-8016</td>
<td>&nbsp;</td>
<tr>
<td>Global medium</td>
<td>&nbsp;</td>
<td>Life Global</td>
<td>GMGB-050</td>
<td>&nbsp;</td>
<tr>
<td>Plasmanate</td>
<td>&nbsp;</td>
<td>Talecris</td>
<td>61325</td>
<td>&nbsp;</td>
<tr>
<td>EmbryoMax Acidic Tyrodes</td>
<td>&nbsp;</td>
<td>Chemicon/Millipore</td>
<td>MR-004-D</td>
<td>&nbsp;</td>
<tr>
<td>KO-DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>10829-018</td>
<td>&nbsp;</td>
<tr>
<td>KO-Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>ES cell-tested fetal bovine serum, Defined</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30070.03</td>
<td>&nbsp;</td>
<tr>
<td>Glutamax-I</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>35050-079</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>11140-076</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>15070-063</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human basic fibroblast growth factor</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256-029</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol </td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-human red blood cell primary antibody</td>
<td>&nbsp;</td>
<td>Rockland</td>
<td>109-4139</td>
<td>&nbsp;</td>
<tr>
<td>Guinea-pig complement serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-1639</td>
<td>&nbsp;</td>
<tr>
<td>Capillary pipettes in 10 microL, 50 microL, 100 microL sizes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouth pipettes with tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>supplied with capillary pipettes from VWR</td>
</tr>
<tr>
<td>0.2 microm Acrodisc syringe filter </td>
<td>&nbsp;</td>
<td>Pall Life Sciences</td>
<td>PN4192</td>
<td>&nbsp;</td>
<tr>
<td>0.5% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>25300-120</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>NUNC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nikon SMZ1500 dissection scope equipped with Nikon Tokai Hit ThermoPlate warming stage </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouse embryonic fibroblasts (E12.5)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

derivation of human embryonic stem cells by immunosurgery

قدرة الخلايا الجذعية الجنينية البشرية لتجديد الذات وتميز في جميع أنواع الخلايا في الجسم يشير إلى أن لديهم وعدا كبيرا للتطبيقات الطبية والبحوث كأداة لمعالجة المسائل الأساسية في التنمية والمرض. هنا ، ونحن نقدم موجزة ، خطوة بخطوة بروتوكول لاشتقاق خلايا جذعية جنينية من أجنة من جراء العزلة immunosurgical من كتلة الخلايا الداخلية.

Watch this Scientific Journal Video about Derivation of Human Embryonic Stem Cells by Immunosurgery at JoVE.com

Derivation of Human Embryonic Stem Cells by Immunosurgery

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 ...

derivation-of-human-embryonic-stem-cells-by-immunosurgery

Research

JoVE Journal

Biology

33.8K Views.  Harvard. قدرة الخلايا الجذعية الجنينية البشرية لتجديد الذات وتميز في جميع أنواع الخلايا في الجسم يشير إلى أن لديهم وعدا كبيرا للتطبيقات الطبية والبحوث كأداة لمعالجة المسائل الأساسية في التنمية والمرض. هنا ، ونحن نقدم موجزة ، خطوة بخطوة بروتوكول لاشتقاق خلايا جذعية جني?...

Video: Derivation of Human Embryonic Stem Cells by Immunosurgery

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

انتشار الخلايا الجنينية البشرية (ES) الجذعية

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

سبر للنشاط مجمع الميتوكوندريا في خلايا المنشأ الجنينية البشرية

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

مناعي لونين من الخلايا الجذعية الجنينية البشرية

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

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 ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

الكروموسومات التحضير انتشار الخلايا الجذعية الجنينية البشرية لKaryotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

تجميد والذوبان الخلايا الجذعية الجنينية البشرية

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 ...

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

اشتقاق الخلايا الجذعية من الأرومة الغاذية Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...