وكان في جزء منه دعم هذا العمل عن طريق منح لWD من المعاهد الوطنية للصحة (وRO1 NS059043 ES015988 RO1) ، جمعية التصلب المتعدد الوطنية ، شركة روش مؤسسة البحوث فقر الدم ، فيلدشتاين المؤسسة الطبية ، والمستشفيات Shriners للأطفال. نود أن نشكر الدكتور ديفيد متعة وجنيفر الطائرة للحصول على اقتراحات. لم نعلن المصالح المتنافسة المتعلقة بهذه المادة.

إجراءات تفصيلية لتوليد الخلايا السليفة oligodendrocyte (OPCs) من GFP - Olig2 الخلايا الجذعية الجنينية (mESCs) : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويتم شراء خط خلية فأر ES - GFP Olig2 (G - Olig2) ، من مجموعة نوع الثقافة الأميركية (آي تي ​​سي سي). يتم بشكل روتيني passaged mESCs كل 3 أيام على an المشع طبقة الخلايا الليفية الماوس الجنينية المغذية (MEF). ومطلي الخلايا MEF إلى 0.1 ٪ الجيلاتين المغلفة زراعة الأنسجة لمدة ستة جيدا لوحة واحدة على الأقل قبل يوم والركض والمجالس الاقتصادية والاجتماعية. في مستنبت MESC متوسطة Dulbecco لتعديل النسر (DMEM) (GIBCO) تستكمل مع 20 ٪ مصل بقري جنيني (GIBCO) ، 2 مم L - الجلوتامين ، 1 البيروفات الصوديوم ملم ، 0.1 ملم β - المركابتويثانول / ، 1 ٪ من الأحماض الأمينية غير الأساسية ( NEAA) ، وسرطان الدم عامل مثبط 1000 U / مل. </li><li style=";text-align:right;direction:rtl"> وزارة التربية والعلم المستعمرات trypsinized (TrypLE ، 5 دقائق ، و 37 درجة مئوية) في الخلايا واحدة وعلقت في المصل خروج المغلوب المتوسطة (KSR) الاستبدال ، ثم يتم نقل الخلايا إلى مرفق منخفضة جدا Costar ستة كذلك لوحة (كورنينج) في خلية كثافة 50000 خلية / سم 2. في ظل هذه الظروف ، mESCs شكل هيئات مضغي الشكل (EBS). KSR المتوسطة ، وتتألف من الحد الأدنى الضروري المتوسطة (- MEM) تستكمل مع KSR 20 ٪ ، 1 البيروفات الصوديوم مم ، NEAA 1 ٪ ، و 0.1 ملي β - المركابتويثانول /. </li><li style=";text-align:right;direction:rtl"> من يوم 4 الى يوم 7 ، يتم تضمين حمض الريتينويك (RA ، 0.2 M) وpurmorphamine (بور ، 1 م) كما هو مبين في الشكل. 1A في KSR أو متوسطة N2. المتوسط ​​هو N2 - MEM يحتوي 1X تكملة N2 ، 1 البيروفات الصوديوم مم ، NEAA 1 ٪ ، و 0.1 ملي β - المركابتويثانول /. يتم تغيير المتوسطة اليومية. </li><li style=";text-align:right;direction:rtl"> في يوم 8 ، ومصنفة EBS باستخدام TrypLE (5min و 37 درجة مئوية) ومطلي على أطباق polyornithine المغلفة 0.01 ٪ في المتوسط ​​OPC المتوسطة التي تتكون من N2 وعامل نمو الخلايا الليفية - 2 (FGF - 2 ، و 20 نانوغرام / مل). يتم تغيير المتوسطة كل يومين. </li><li style=";text-align:right;direction:rtl"> وtrypsinized الخلايا (TrypLE ، 3 دقائق و 37 درجة مئوية) وreplated تقريبا كل اسبوع عندما تصبح متموجة. في يوم 30 ، ما يزيد على 80 ٪ من الخلايا تظهر GFP/Olig2 التعبير وNG2 إيجابية ، سمة من OPCs. </li><li style=";text-align:right;direction:rtl"> لتوليد OPCs ارتفاعه ، ونا BacMam + يستخدم عدة قنوات لتقديم نا ت 1.2 الوحيدات في هذه OPCs MESC المشتقة. خلط الكاشف BacMam (1 لتر ، مكون A) مع برنامج تلفزيوني على الحجم النهائي من 5 مل. ثم ، يتم إضافة كاشف BacMam مختلطة في الثقافة المستمدة من OPCs MESC في صحن الثقافة 60 ملم (50-70 ٪ في التقاء) قبل الشطف مع برنامج تلفزيوني. بعد حضانة 2 ساعة ، ويتم إزالة كاشف BacMam المختلطة واستبدالها المتوسطة OPC تستكمل مع محسن 1X. ومحسن هو مكون B تشكيلها في DMSO (العنصر جيم). المقبل ، بعد الاحتضان 2 ساعة إضافية يتم إزالة المتوسطة مع محسن واستبدالها المتوسطة OPC كاملة. ويعاير الخلايا لمدة 24 ساعة في وقت لاحق. </li></ol> ممثل النتائج : بعد بروتوكول التمايز هو مبين في الشكل. وعلقت في البداية 1A ، G - Olig2 mESCs في المتوسط ​​وKSR لمدة 4 أيام لتشكيل هيئات مضغي الشكل (EBS) (الشكل 1B). ثم ، ونحن على تعامل بالتسلسل EBS مع التهاب المفاصل الروماتويدي وبور. لم لا تظهر أي EBS مضان GFP في يوم (4) وأعرب قوية في مضان GFP D8 (الشكل 1B). عند هذه النقطة الوقت ، كانت لtrypsinized EBS مطلي والمتوسطة في N2 تستكمل مع عوامل النمو FGF - 2. بعد مزيد من الثقافة لمدة 22 يوما (D30) ، وصلت النسبة المئوية للخلية + GFP 80.3 ± 0.6 ٪ وفقا لنتائجنا التدفق الخلوي. كما هو مبين في الشكل. 1C ، GFP + الخلايا تتداخل مع NG2 تلطيخ باستمرار (96.4 ± 1.3 ٪ من الخلايا GFP كانت إيجابية NG2 إيجابية ، و94.8 ± 1.5 ٪ من الخلايا إيجابية NG2 GFP كانت إيجابية). وبالتالي ، فإن هذه الخلايا وأعرب عن GFP/Olig2 NG2 ، وتعريف عليها كما OPCs. فشل OPCs MESC المشتقة (76 خلايا) لاطلاق امكانات العمل على إزالة الاستقطاب (الشكل 2A). بعد transducing مع الفيروسة العصوية تحمل ت نا 1.2 الوحيدات ، يمكن تصنيف هذه OPCs MESC المستمدة من (94.1 ٪ ، و 16 من 17 خلايا) وارتفاعه (الشكل 2B). <img alt="الشكل 1" src="/files/ftp_upload/1960/1960fig1.jpg" /> الشكل 1. تمايز GOlig - MESC OPCs إلى (A) مخطط يبين بروتوكول الجسم مضغي الشكل (EB) والقائم على جزيء صغير يحركها التمايز. في D8 ، وكانت مصنفة في EBS ومطلي. وpassaged الخلايا مرة واحدة في الأسبوع عندما أصبحت متكدسة. (B) D8 ، ولكن ليس D4 EBS ، أظهرت GFP التعبير. (C) وكان من Immunostaining NG2 (الحمراء) بما يتفق مع GFP (الخضراء) في التعبير D30. وقد استخدم دابي (الأزرق) لتحديد النوى. شريط الحجم : 20 ملم. <img alt="الشكل 2" src="/files/ftp_upload/1960/1960fig2.jpg" /> الشكل 2. جيل من OPCs التشويك من nonspiking MESC المستمدة من فيروس OPCs بوساطة فشل نا التعبير القناة الخامس (أ) عقدت في غشاء المحتملة -60 بالسيارات وOPCs MESC المستمدة من امكانات العمل لاطلاق النار ، حتى عندما كانت الخلية استقطابها إلى 0 MV. (ب) مثال على مسك المستمدة OPCإطلاق إمكانات العمل (الضوء الاحمر) بعد تنبيغ الفيروسية.

<ol>
	<li>Xian, H., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+Olig2-expressing+progenitor+cells+derived+from+ES+cells.">Dividing Olig2-expressing progenitor cells derived from ES cells.</a> Glia. 47 (1), 88-101 (2004).</li><li>Clair, A., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+ES-cell-derived+neural+cells+marked+by+gene+targeting.">A subset of ES-cell-derived neural cells marked by gene targeting.</a> Stem Cells. 21 (1), 41-49 (2003).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Shin, S., Xue, H., Mattson, M. P., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+Olig2+expression+in+motor+neurons+and+oligodendrocytes+differentiated+from+embryonic+stem+cells.">Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells.</a> Stem Cells Dev. 16, 131-141 (2007).</li><li>Billon, N., Jolicoeur, C., Ying, Q. L., Smith, A., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+timing+of+oligodendrocyte+development+from+genetically+engineered,+lineage-selectable+mouse+ES+cells.">Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.</a> J Cell Sci. 115, 3657-3665 (2002).</li><li>Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-localization+of+NG2+proteoglycan+and+PDGF+alpha-receptor+on+O2A+progenitor+cells+in+the+developing+rat+brain.">Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.</a> J Neurosci Res. 43, 299-314 (1996).</li><li>Ligon, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+NG2+neural+progenitor+cells+requires+Olig+gene+function.">Development of NG2 neural progenitor cells requires Olig gene function.</a> Proc Natl Acad Sci U S A. 103, 7853-7858 (2006).</li><li>Dawson, M. R., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24, 476-488 (2003).</li><li>Chittajallu, R., Aguirre, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+in+the+mouse+white+and+grey+matter+display+distinct+physiological+properties.">NG2-positive cells in the mouse white and grey matter display distinct physiological properties.</a> J Physiol. 561, 109-122 (2004).</li><li>Karadottir, R., Hamilton, N. B., Bakiri, Y., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+and+nonspiking+classes+of+oligodendrocyte+precursor+glia+in+CNS+white+matter.">Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.</a> Nat Neurosci. 11, 450-456 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

يمكن أن الخلايا الجذعية الجنينية (المجالس) ، معزولة عن الجنين الكيسة ، تفرق في جميع الأنساب خلية من خلايا الكائن الحي 3 ، 4 ، وتوفير نظام نموذجي في المختبر لدراسة تطور الثدييات في وقت مبكر ، بما في ذلك مواصفات oligodendrocyte. وقد ثبت mESCs على التمايز إلى خلايا السلائف oligodendrocyte (OPCs) مع معاملة القنفذ سونيك (SHH) 5. وعلاوة على ذلك ، فإن التمايز OPC SHH التي يسببها من mESCs يحتفظ التوقيت الصحيح لوحظ في التطور الجنيني 6. وبالتالي ، تعتبر طبيعة التمايز في المختبر من OPC mESCs أن تكون متسقة مع ما تم تعلمه من التنمية في الجسم الحي. هنا ، وذلك باستخدام جزيئات صغيرة RA وبور ناهض SHH ، نحن متباينة بنجاح mESCs G - Olig2 في Olig2 + + + OPCs GFP NG2 ذات كفاءة عالية. OPCs ، والتي تتميز تعبير عن بروتيوغليكان NG2 (7) وعامل النسخ الحلزون ، الحلزون Olig2 حلقة 8 ، تولد oligodendrocytes في الجهاز العصبي المركزي النامية والناضجة ، حيث أنهم يشكلون نسبة كبيرة (~ 5 ٪) من مجموع الخلايا وهما نوع من الخلايا المتكاثرة الرئيسية 9. يتم التعبير عن ما يقرب من عقدين من الزمن الباحثين أظهروا قنوات الصوديوم في الخامس من subpopulation OPCs ويمكن تفعيلها عند الاستقطاب 10 و 11. علاوة على ذلك ، كانت الملاحظة الأخيرة التي ضرب 9 في الوضع الطبيعي في الجهاز العصبي المركزي الفئران المادة البيضاء ، OPCs (~ 50 ٪) ولدت إمكانات العمل عندما استقطابها تبعا للتعبير عن الجهد بوابات الصوديوم (نا V) قنوات ، وبالتالي يمكن تقسيمها الى التشويك وnonspiking المجموعات السكانية الفرعية. ومع ذلك ، فإن مهام هذه الخصائص التشويك لا تزال غير معروفة الى حد كبير. وجدنا أنه electrophysiologically ، ومسك OPCs المستمدة متباينة مع البروتوكول تعتمد SHH ، لم تكن في نفس الموقع OPCs الدماغ. بعد إدخال الوحيدات نا V 1.2 ، وكانت هذه الصامتة MESC المستمدة OPCs قادرة على ارتفاعه. وبالتالي ، قد التشويك / nonspiking MESC المستمدة OPCs وبروتوكول تمايز الموصوفة هنا يسهل (1) دراسة الفروق الوظيفية بين التشويك وnonspiking OPCs ، (2) عوامل الفرز الجديدة التي يمكن أن تعزز الصوديوم التعبير في قناة مسك OPCs المشتقة ، ( 3) تطوير وتحسين بروتوكول تمايز OPCs ESC من الإنسان أو الخلايا الجذعية المحفزة التي يسببها (iPSCs).

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differentiation of embryonic stem cells into oligodendrocyte precursors

Oligodendrocytes هي الخلايا myelinating النظام العصبي المركزي. لعلاج الخلايا على التجدد في الأمراض المزيل ، هناك اهتمام كبير في اشتقاق النسب السكانية ، نقية من خلايا ملتزمة السلائف oligodendrocyte (OPCs) للزرع. تتميز OPCs عن هذا النشاط من النسخ Olig2 عامل والتعبير سطح NG2 بروتيوغليكان. باستخدام GFP - Olig2 (G - Olig2) الخلية الجذعية الجنينية الماوس (مسك) خط المراسل ، إلى أقصى حد ونحن الظروف لتمايز في mESCs Olig2 + + + OPCs GFP NG2. في بروتوكول لدينا ، ونحن تصف أول جيل من الهيئات مضغي الشكل (EBS) من mESCs. ثانيا ، نحن تصف العلاج من مسك المستمدة EBS مع جزيئات صغيرة : (1) حمض الريتينويك (RA) و (2) القنفذ سونيك purmorphamine ناهض (SHH) (بور) في ظل الظروف المحددة لتوجيه ثقافة التمايز EB في النسب oligodendroglial. هذا النهج ، يمكن الحصول على OPCs ذات كفاءة عالية (&gt; 80 ٪) في فترة زمنية مدتها 30 يوما. الخلايا المشتقة من mESCs في هذا البروتوكول مماثلة لphenotypically OPCs المستمدة من زراعة الأنسجة الابتدائية. ومسك OPCs المشتقة لا تظهر الخاصية التشويك وصف لsubpopulation OPCs من الدماغ في الموقع. لدراسة هذه الخاصية الكهربية ، ونحن تصف توليد MESC التشويك المستمدة OPCs بالإعراب عن ectopically نا<sub&gt; V</sub&gt; 1.2 الوحيدات. والخلايا والتشويك nonspiking تم الحصول عليها من هذا البروتوكول تساعد الدراسات الفنية المسبقة على المجموعات السكانية الفرعية اثنين من OPCs.

Watch this Scientific Journal Video about Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors at JoVE.com

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

وصفنا جزيء صغيرة مقرها بروتوكول للتمايز الخلايا الجذعية الجنينية لتصبح خلايا السلائف oligodendrocyte (OPCs). يولد هذا البروتوكول Olig2 + + NG2 OPCs ذات كفاءة عالية في موعد أقصاه 30 يوما من التمايز. نحن تصف أيضا طريقة لتوليد &quot;التشويك&quot; OPCs النار التي يمكن أن إمكانات العمل.

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 ...

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15.2K Views.  School of Medicine, University of California, Davis. وصفنا جزيء صغيرة مقرها بروتوكول للتمايز الخلايا الجذعية الجنينية لتصبح خلايا السلائف oligodendrocyte (OPCs). يولد هذا البروتوكول Olig2 + + NG2 OPCs ذات كفاءة عالية في موعد أقصاه 30 يوما من التمايز. نحن تصف أيضا طريقة لتوليد "التشويك" OPCs النار التي يمكن أن إمكانات العمل.

Video: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

انتشار الخلايا الجنينية البشرية (ES) الجذعية

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

ثقافة العصبية الماوس السلائف الخلايا الجذعية

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

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 ...

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 ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

التمايز في المختبر من خلايا الجنينية الماوس (MES) الجذعية باستخدام أسلوب إسقاط الشنق

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

كفاءة اشتقاق السلائف القلب البشري وافرة القوة العضلية من خلايا المنشأ الجنينية البشرية التعريفي مع جزيء صغير

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

التفريق بين الأطفال حديثي الولادة ماوس خلايا الجلد الجذعية المشتقة في مثل جرثومة خلايا<em&gt; في المختبر</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...