وتكرس هذه المخطوطة لذكرى الدكتور Wenlan وانغ الذي وافته المنية يوم 26 مايو، 2011. نشكر الدكتور دوغلاس كير لتوفير بسخاء HBG3 الخلايا زارة التربية والعلم المستخدمة في هذه الدراسة. وقد تم تمويل هذا العمل من قبل Nemours، منحة (2 RR016472-10) في إطار برنامج INBRE من المركز الوطني للبحوث الموارد (NCRR)، وجائزة المنحة كوبر من NCRR (5 P20 RR020173-05) لدعم مركز طب الأطفال في مستشفى بحوث ألفريد دوبون الأول للأطفال، ويلمنجتون في ديلاوير، الولايات المتحدة الأمريكية.

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	<li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110, 385-397 (2002).</li><li>Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+motoneurons+derived+from+mouse+embryonic+stem+cells.">Functional properties of motoneurons derived from mouse embryonic stem cells.</a> J. Neurosci. 24, 7848-7858 (2004).</li><li>Wichterle, H., Peljto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+to+spinal+motor+neurons.">Differentiation of mouse embryonic stem cells to spinal motor neurons.</a> Curr. Protoc. Stem Cell. Biol. Chapter 1, Unit 1H.1.1-Unit 1H.1.9 (2008).</li><li>Kiris, E. <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-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem Cell Res. 6, 195-205 (2011).</li><li>Wu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+assessment+of+a+cell+model+of+spinal+muscular+atrophy.">Proteomic assessment of a cell model of spinal muscular atrophy.</a> BMC Neurosci. 12, 25 (2011).</li><li>McMahon, 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=Noggin-mediated+antagonism+of+BMP+signaling+is+required+for+growth+and+patterning+of+the+neural+tube+and+somite.">Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite.</a> Genes Dev. 12, 1438-1452 (1998).</li><li>Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+posterior+neural+tissue+is+induced+by+FGF+in+the+chick+embryo.">Early posterior neural tissue is induced by FGF in the chick embryo.</a> Development. 125, 473-484 (1998).</li><li>Launay, C., Fromentoux, V., Shi, D. L., Boucaut, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+truncated+FGF+receptor+blocks+neural+induction+by+endogenous+Xenopus+inducers.">A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.</a> Development. 122, 869-880 (1996).</li><li>Sinha, S., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purmorphamine+activates+the+Hedgehog+pathway+by+targeting+Smoothened.">Purmorphamine activates the Hedgehog pathway by targeting Smoothened.</a> Nat. Chem. Biol. 2, 29-30 (2006).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 14071-14076 (2002).</li><li>Lee, S. M., Danielian, P. S., Fritzsch, B., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+FGF8+signaling+from+the+midbrain-hindbrain+junction+regulates+growth+and+polarity+in+the+developing+midbrain.">Evidence that FGF8 signaling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.</a> Development. 124, 959-969 (1997).</li></ol>

الإعلان عن أي تضارب في المصالح.

نوعية الخلايا زارة التربية والعلم هو المعلمة الأكثر أهمية بالنسبة لجيل من كفاءة الخلايا العصبية الحركية. ويجب أن زراعة الخلايا زارة التربية والعلم في PMEF لمنع التمييز عفوية. بالإضافة إلى ذلك من LIF يساعد في الحفاظ على خلايا زارة التربية والعلم في حالة غير متمايزة. كما ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> نهائي تركيز </td></tr><tr><td> PMEF </td><td> ميليبور </td><td> PMEF-H </td><td> NA </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> PMEF المتوسطة </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> إينفيتروجن </td><td> 11965-118 </td><td> NA </td></tr><tr><td> FBS </td><td> إينفيتروجن </td><td> 16140-071 </td><td> 10٪ </td></tr><tr><td> L-الجلوتامين </td><td> إينفيتروجن </td><td> 25030-081 </td><td> 1٪ </td></tr><tr><td> البنسلين / الستربتومايسين </td><td> إينفيتروجن </td><td> 15240-063 </td><td> 1٪ </td></tr><tr><td></td><td></TD&gt; <td></td><td></td></tr><tr><td> MES المتوسطة </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell تقنيات </td><td> 36250 </td><td> NA </td></tr><tr><td> وفاق خلية المؤهلين FBS </td><td> إينفيتروجن </td><td> 16141-079 </td><td> 15٪ </td></tr><tr><td> GlutaMax-I </td><td> إينفيتروجن </td><td> 35050-061 </td><td> 1٪ </td></tr><tr><td> غير الاساسيين من الأحماض الأمينية </td><td> إينفيتروجن </td><td> 11140-050 </td><td> 1٪ </td></tr><tr><td> النيوكليوسيدات </td><td> ميليبور </td><td> ES-008-D </td><td> 1٪ </td></tr><tr><td> β-المركابتويثانول </td><td> ميليبور </td><td> ES-007-E </td><td> 0.1 ملي </td></tr><tr><td> البنسلين / الستربتومايسين </td><td> ميليبور </td><td> TMS-AB2-C </td><td> 1٪ </td></tr><tr><td> LIF </td><td> ميليبور </td><td&gt; LIF2010 </td><td> 10 نانوغرام / مل </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> العصبية التفاضل المتوسطة </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell تقنيات </td><td> 36250 </td><td> NA </td></tr><tr><td> وفاق عبادة FBS </td><td> StemCell تقنيات </td><td> 06905 </td><td> 15٪ </td></tr><tr><td> غير الاساسيين من الأحماض الأمينية </td><td> إينفيتروجن </td><td> 11140-050 </td><td> 1٪ </td></tr><tr><td> أحادية ثيو الجلسرين </td><td> سيغما الدريخ </td><td> M-6145 </td><td> 1MM </td></tr><tr><td> رأس </td><td> إينفيتروجن </td><td> PHC1506 </td><td> 50 نانوغرام / مل </td></tr><tr><td> FGF-8 </td><td> إينفيتروجن </td><td> PHG0274 </td><td> 20 نانوغرام / مل </td></tr><tr><td> bFGF </td><td> إينفيتروجن </td><td> PHG0024 </td><td> 20 نانوغرام / مل </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> الفرقة المتعددة الجنسيات المتوسطة (التمايز) </td><td></td><td></td><td></td></tr><tr><td> ES-عبادة المتوسطة وبصل </td><td> StemCell تقنيات </td><td> 5801 </td><td> NA </td></tr><tr><td> استبدال خروج المغلوب المصل </td><td> إينفيتروجن </td><td> 10828-028 </td><td> 10٪ </td></tr><tr><td> N-2 الملحق </td><td> إينفيتروجن </td><td> 17502-048 </td><td> 1٪ </td></tr><tr><td> ملحقها-B </td><td> StemCell تقنيات </td><td> 07155 </td><td> 1٪ </td></tr><tr><td> حمض الأسكوربيك </td><td> StemCell تقنيات </td><td> 07157 </td><td> 1٪ </td></tr><tr><td> البنسلين / الستربتومايسين </td><td> ميليبور </td><td> TMS-AB2-C </td><td> 1٪ </td></tr><tr><td> GlutaMax-I </td><td>إينفيتروجن </td><td> 35050-061 </td><td> 1٪ </td></tr><tr><td> D-جلوكوز </td><td> سيغما الدريخ </td><td> G-8270 </td><td> 0.15٪ في د 2 H 2 O </td></tr><tr><td> فبرونيكتين </td><td> StemCell تقنيات </td><td> 07159 </td><td> 5 ميكروغرام / مل </td></tr><tr><td> الهيبارين </td><td> سيغما الدريخ </td><td> H3149 </td><td> 20 ميكروغرام / مل في د 2 H 2 O </td></tr><tr><td> β-المركابتويثانول </td><td> ميليبور </td><td> ES-007-E </td><td> 0.1 ملي </td></tr><tr><td> حمض الريتينويك </td><td> سيغما الدريخ </td><td> R-2625 </td><td> 1 ميكرومتر </td></tr><tr><td> ش م خ </td><td> EMD كيماويات </td><td> 566660 </td><td> 1 ميكرومتر </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> الفرقة المتعددة الجنسيات المتوسطة (العصبون الحركي للثقافة) </td><td></td><td></td><td></td></tr><tr><td> ES-عبادة المتوسطة وبصل </td><td> StemCell تقنيات </td><td> 5801 </td><td> NA </td></tr><tr><td> استبدال خروج المغلوب المصل </td><td> إينفيتروجن </td><td> 10828-028 </td><td> 10٪ </td></tr><tr><td> N-2 الملحق </td><td> إينفيتروجن </td><td> 17502-048 </td><td> 1٪ </td></tr><tr><td> ملحقها-B </td><td> StemCell تقنيات </td><td> 07155 </td><td> 1٪ </td></tr><tr><td> حمض الأسكوربيك </td><td> StemCell تقنيات </td><td> 07157 </td><td> 1٪ </td></tr><tr><td> البنسلين / الستربتومايسين </td><td> ميليبور </td><td> TMS-AB2-C </td><td> 1٪ </td></tr><tr><td> GlutaMax-I </td><td> إينفيتروجن </td><td> 35050-061 </td><td> 1٪ </td></tr><tr><td> D-جلوكوز </td><td> سيغما الدريخ </td><td> G-8270 </td><td> 0.15٪ في د 2 H 2 O </td></tr><tr><td> فبرونيكتين </td><td> StemCELL تقنيات </td><td> 07159 </td><td> 5 ميكروغرام / مل </td></tr><tr><td> الهيبارين </td><td> سيغما الدريخ </td><td> H3149 </td><td> 20 ميكروغرام / مل في د 2 H 2 O </td></tr><tr><td> β-المركابتويثانول </td><td> ميليبور </td><td> ES-007-E </td><td> 0.1 ملي </td></tr><tr><td> BDNF </td><td> R &amp; D أنظمة </td><td> 248-BD-005/CF </td><td> 10 نانوغرام / مل </td></tr><tr><td> CNTF </td><td> R &amp; D أنظمة </td><td> 257-NY-010/CF </td><td> 10 نانوغرام / مل </td></tr><tr><td> GDNF </td><td> R &amp; D أنظمة </td><td> 212-GD-010/CF </td><td> 10 نانوغرام / مل </td></tr><tr><td> NT-3 </td><td> R &amp; D أنظمة </td><td> 267-N3-005/CF </td><td> 10 نانوغرام / مل </td></tr></tbody></table> NA = غير قابلة للتطبيق. الجدول رقم 1. PMEF وسائل الإعلام للثقافة الخلية زارة التربية والعلم أو تمييز. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> &lt;قوي اسم&gt; من كاشف </td><td> شركة </td><td> فهرس العدد </td><td> نهائي تركيز </td></tr><tr><td> 0.1٪ الجيلاتين </td><td> StemCell تقنيات </td><td> 07903 </td><td> NA </td></tr><tr><td> بولي DL-الأورنيثين </td><td> سيغما الدريخ </td><td> P0421 </td><td> 0.1 ملغ / مل في د 2 H 2 O </td></tr><tr><td> فأر laminin </td><td> ميليبور </td><td> CC095 </td><td> 2 ميكروغرام / مل في برنامج تلفزيوني </td></tr><tr><td> 0.25٪ التربسين / EDTA </td><td> StemCell تقنيات </td><td> 07901 </td><td> NA </td></tr><tr><td> Accumax </td><td> ميليبور </td><td> SCR006 </td><td> NA </td></tr></tbody></table> NA = غير قابلة للتطبيق. الجدول 2. الكواشف لطلاء والتفكك.

efficient differentiation of mouse embryonic stem cells into motor neurons

تمايز الخلايا الجنينية مباشرة من (ES) الجذعية إلى الخلايا العصبية الحركية وظيفي يمثل مورد واعدة لدراسة آليات المرض، لفحص مركبات عقار جديد، وتطوير علاجات جديدة لأمراض الخلايا العصبية الحركية مثل ضمور العضلات الشوكي (SMA) والتصلب الجانبي ( ALS). البروتوكولات الحالية العديد من استخدام مزيج من حمض الريتينويك (RA)، والقنفذ الصوتي (SHH) لتمييز الماوس الجذعية الجنينية (MES) الخلايا في الخلايا العصبية الحركية 1-4. ومع ذلك، فقد اجتمع كفاءة تمايز الخلايا زارة التربية والعلم في الخلايا العصبية الحركية فقط مع نجاح معتدل. قمنا بتطوير بروتوكول تمايز على مرحلتين (5) التي تحسن كبير في كفاءة التمايز مقارنة مع البروتوكولات المعمول بها حاليا. فإن الخطوة الأولى هي لتعزيز عملية neuralization بإضافة رأس وعوامل نمو الخلايا الليفية (FGFs). رأس هو بروتين مخلق العظام (BMP) وخصم متورطة في تحريض العصبية 1ccording إلى النموذج الافتراضي لتكوين الخلايا العصبية والنتائج في تشكيل الزخرفة العصبية الأمامية 6. جمعية جيل المستقبل مما يشير يعمل بالتآزر مع رأس في حفز تشكيل الأنسجة العصبية من خلال تعزيز الهوية الخلفي العصبية 7-9. في هذه الخطوة، ومعبي الخلايا زارة التربية والعلم مع رأس، bFGF، وجمعية جيل المستقبل 8 لمدة يومين لتعزيز التمايز نحو الأنساب العصبية. والخطوة الثانية هي للحث على العصبون الحركي المواصفات. وحضنت رأس / FGFs تتعرض الخلايا زارة التربية والعلم من التهاب المفاصل الروماتويدي، وناهض SHH، ناهض Smoothened (SAG)، لمدة 5 أيام لتسهيل العصبون الحركي جيل. لرصد التمييز بين فوضى في الخلايا العصبية الحركية، وكنا على خط خلية ES مستمدة من eGFP الماوس المعدلة وراثيا التعبير تحت سيطرة المروج الخلايا العصبية الحركية محددة Hb9 1. باستخدام هذا البروتوكول قوي، حققنا 51 ± 0.8٪ من كفاءة التمايز (ن = 3؛ ف &lt;0.01، الطالب في اختبار t) 5. نتائج من مناعيوأظهرت أن تلطيخ GFP + الخلايا التعبير عن الخلايا العصبية الحركية علامات محددة، وجزيرة 1 والكولين أسيتيل (الدردشة). بروتوكول لدينا تمايز على مرحلتين ويوفر وسيلة فعالة للتمييز بين الخلايا زارة التربية والعلم في الخلايا العصبية الحركية في العمود الفقري.

Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

قمنا بتطوير بروتوكول جديد لتحسين كفاءة المختبر في تمايز الخلايا الجذعية الجنينية في الخلايا العصبية الحركية. حصلت على خلايا ES متباينة الخلايا العصبية الحركية يتميز كما يتضح من التعبير عن علامات الخلايا العصبية الحركية والخلايا العصبية باستخدام تقنيات المناعى.

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 ...

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22.2K Views.  Alfred I. duPont Hospital for Children. قمنا بتطوير بروتوكول جديد لتحسين كفاءة المختبر في تمايز الخلايا الجذعية الجنينية في الخلايا العصبية الحركية. حصلت على خلايا ES متباينة الخلايا العصبية الحركية يتميز كما يتضح من التعبير عن علامات الخلايا العصبية الحركية والخلايا العصبية باستخدام تقنيات المناع?...

Video: Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

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 ...

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 ...

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 ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

إعداد خلايا الفأر الجنينية الخلايا الليفية مناسبة للالإنسان التثقيف الجنينية والتي يسببها الخلايا الجذعية المحفزة

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

بسرعة وكفاءة توليد الخلايا العصبية من الخلايا الجذعية المحفزة الإنسان في تنسيق اللوحة Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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 ...

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

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

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

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

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