وقد تم دعم XHP بواسطة المعهد الوطني للمنح (NIH) الصحة من المعهد الوطني للشيخوخة (NIHK01AG024496) يونيس كينيدي وشرايفر المعهد القومي لصحة الطفل والتنمية البشرية (NIHR21HD056530).

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الكتاب تعلن المصالح المتنافسة. XHP هو مؤسس Xcelthera. XHP EYS ولها خصائص الفكرية المتصلة hESCs.

كان واحدا من التحديات الكبرى على حد سواء دراسة تنموية والترجمة السريرية كيفية توجيه إمكانات واسعة من تمايز الخلايا الجذعية المحفزة الإنسان إلى النمط الظاهري المطلوب بكفاءة ويمكن التنبؤ به. على الرغم من أن مثل هذه الخلايا يمكن التفريق بصورة عفوية إلى خلايا في المختبر من كل الطبقات الجرثومية التي تمر في مرحلة تجميع متعددة النسب ، في hESC المستمدة متعددة النسب المجاميع (الهيئات مضغي الشكل) ، سوى جزء صغير جدا من الخلايا (&lt;2 ٪) بشكل عفوي تفرق في العضلية 13-15 (الشكل 1A). انتقاء مناعي التالية ، كانت من أثرى السكان العضلية قادرة على العمل وأجهزة ضبط نبضات القلب البيولوجي لانقاذ ظيفة عضلة القلب التالفة ميكانيكيا والكترونيا بعد حقنها في قلب النماذج الحيوانية 13. في النماذج احتشاء القوارض ، تم engrafted hESC المستمدة السلالات القلب قادرة على البقاء وناضجة تصل إلى 12 أسبوعا ، وremuscular جزئياإيزي قلب المصابين وتحسين وظيفة مقلص 14،15. ومع ذلك ، فقد أعاق انعدام الكفاءة في توليد الإنسان التي ارتكبت في القلب ، من خلال خلايا متعددة النسب تمايز الخلايا المحفزة ومخاطر عالية من زرع مزيد من الترجمة التالية tumorigenicity السريرية. وضع استراتيجيات لتوجيه إمكانات واسعة من تمايز hESCs المحفزة وحدها ، ويمكن التنبؤ بها إلى النمط الظاهري القلب هو أمر حيوي لتسخير قوة البيولوجيا hESC في مجال القلب. استخدمنا من أجل تحقيق تحويل موحد للخلايا الجذعية المحفزة لنسب معينة ، وهو نظام قادر على تعريف الثقافة تأمين انتشار hESCs غير متمايزة لتحديد الظروف التي تسيطر عليها بشكل جيد للتحريض كفاءة hESCs المحفزة حصرا على وجه الخصوص النسب ذات الصلة سريريا عن طريق توفير بسيط من الجزيئات الصغيرة (الشكل 1B). وجدنا أن يسببها نيكوتيناميد القلب ، نسب التزام مباشر من عluripotent hESCs تحت ثقافة المعرفة التي تقدم للضرب مزيد من العضلية ذات كفاءة عالية (الشكل 2). Nkx2.5 هو النسخ المحفوظة homeobox evolutionally عاملا لا غنى عنه لتحقيق التنمية القلب الطبيعي والطفرات في الجينات البشرية المقترنة مع أمراض القلب الخلقية (CHD) ، وأكثرها شيوعا عيب خلقي الإنسان 16. التعبير عن Nkx2.5 هو اقرب علامة للحصول على خلايا القلب السلائف في جميع الفقاريات حتى الآن فحص وضروري لتحوجز القلب السليم وتكوين / نضوج نظام التوصيل الكهربائي 16. في الفقاريات ، وظهور نمط من التعبير Nkx2.5 المبكر تتزامن تقريبا مع توقيت ومجال المواصفات القلب في مرحلة التطور الجنيني المبكر ، وNkx2.5 الجين لا يزال يعبر عنها من خلال التنمية في القلب 16. في نموذج hESC لدينا ، وبدا حركة عدم الانحياز لتحريك القلب تحريض مباشر من hESCs المحفزة من خلال تشجيع التعبير عن القلب محددة transcription Nkx2.5 عاملا في العملية التي قد تحاكي مواصفات cardiomesoderm من الأديم الظاهر في المحفزة تخلق القلب الجنينية البشرية. وسوف تكشف الدراسات المستقبلية مراقبة الجزيئات الجينية وجينية في مجال التنمية البشرية كبدائل القلب ، مما قد يمهد الطريق للسيطرة على جزيء صغير بوساطة مباشرة والتشكيل مصير المحفزة hESC عند اشتقاق الأنساب سريريا ذات الصلة لعلاجات التجدد. حركة عدم الانحياز من دون علاج ، فإن &lt;hESCs 2 ٪ الخضوع التمايز عفوية إلى الضرب العضلية 12-15. مع العلاج حركة عدم الانحياز ، وكنا قادرين على توليد&gt; 95 ٪ السلائف القلب العضلية الجنينية والضرب&gt; 50 ٪ من hESCs حافظت ظل ثقافة تحدد في عملية التنمية البشرية قد تحاكي القلب الجنينية 12. في الآونة الأخيرة ، استخدمت يعرف مصير القلب ، لتحديد الجينات transdifferentiate الليفية الماوس إلى ما بعد الولادة العضلية الضرب مع انخفاض كفاءة 0.5 ٪ &lt;17 ، 18 &lt;/ سوب&gt;. ومع ذلك ، فقد تم إعادة برمجة الخلايا تاريخيا الجسدية المرتبطة الشاذ التعبير الجيني وتسارع الشيخوخة مع فائدة علاجية ضعاف 19-21. أخيرا ، تقتصر على البروتوكول وضعناها هنا لhESCs المحفزة المستمدة من الكتلة الخلوية الداخلية (ICM) أو الأديم الظاهر من 4،5 الكيسة الإنسان ، قد لا ينطبق على الخلايا المحفزة الأخرى ، بما فيها المجالس الاقتصادية والاجتماعية ، نشأت المجالس الاقتصادية والاجتماعية الحيوانية المستمدة من morula في وقت سابق (ثماني خلايا) في المرحلة 22 الأجنة والخلايا برمجتها بشكل مصطنع 23.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم الكاشف </th><th> شركة </th><th> فهرس العدد </th><th> تعليق (اختياري) </th></tr><tr><td> الجيلاتين </td><td> سيغما </td><td> G1890 </td><td></td></tr><tr><td> Matrigel </td><td> العلوم الحيوية دينار بحريني </td><td> 356231 </td><td> انخفاض عامل النمو </td></tr><tr><td> الإنسان laminin </td><td> سيغما </td><td> L6274 </td><td></td></tr><tr><td> نيكوتيناميد </td><td> سيغما </td><td> N0636 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 10565042 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 31053036 </td><td></td></tr><tr><td> DMEM - KO </td><td> Invitrogen </td><td> 10829018 </td><td></td></tr><tr><td> خروج المغلوب استبدال المصل </td><td> Invitrogen </td><td> 10828028 </td><td></td></tr><tr><td> MEM غير الضرورى حل الأحماض الأمينية (MNAA ، 100X) </td><td> Invitrogen </td><td> 11140050 </td><td></td></tr><tr><td> MEM حل الأحماض الأمينية (MEAA ، 100X) </td&gt; <td> Invitrogen </td><td> 11130050 </td><td></td></tr><tr><td> β - المركابتويثانول </td><td> Invitrogen </td><td> 21985023 </td><td></td></tr><tr><td> Albumax </td><td> Invitrogen </td><td> 11020021 </td><td></td></tr><tr><td> حمض الاسكوربيك </td><td> سيغما </td><td> A4403 </td><td></td></tr><tr><td> الإنسان ترانسفيرين </td><td> سيغما </td><td> T8158 </td><td></td></tr><tr><td> الإنسان bFGF </td><td> PeproTech </td><td> AF - 100 - 18B </td><td></td></tr><tr><td> الأنسولين البشري </td><td> Invitrogen </td><td> 12585014 </td><td></td></tr><tr><td> الإنسان activin A </td><td> PeproTech </td><td> 120 - 14E </td><td></td></tr><tr><td> تعريف FBS </td><td> Hyclone </td><td> SH30073 - 03 </td><td></td></tr><tr><td> 6 كذلك لوحة ultralow مرفق </td><td> كورنينج </td><td> 3471 </td><td></td></tr><tr><td> 6 كذلك لوحة </td><td> كورنينج </td><td> 3516 </td><td></td></tr></tbody></table>

efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction

حتى الآن ، كان عدم وجود مصدر الخلايا البشرية المناسبة لنكسة كبيرة في القلب في الإنسان تجديد عضلة القلب ، وإما عن طريق زرع خلية القاعدة أو أنسجة القلب 1-3 الهندسية. العضلية تصبح عضال ، متباينة بعد وقت قصير من الولادة ، وتفقد قدرتها على التكاثر. لا يوجد أي دليل على أن الجذعية / السلف الخلايا المشتقة من مصادر أخرى ، مثل نخاع العظم أو دم الحبل السري ، قادرة على أن تؤدي الى خلايا عضلة القلب مقلص التالية زرع في قلب 1-3. لم تكن الحاجة إلى تجديد أو إصلاح عضلة القلب التالفة التي اجتمع الكبار العلاج بالخلايا الجذعية ، سواء المحلية أو عبر تسليم خلية 1-3. ومستقرة وراثيا الخلايا الجذعية الجنينية البشرية (hESCs) وغير محدود القدرة على التوسع واللدونة غير مقيدة ، يقترحونها خزان المحفزة للاشتقاق في المختبر من كميات كبيرة من الخلايا الجسدية البشرية التي تقتصر على النسب في حاجةإصلاح وتجديد 4،5. نظرا لانتشار مرض القلب والأوعية الدموية في جميع أنحاء العالم والنقص الحاد في الأجهزة المانحة ، وهناك اهتمام كبير في العلاجات القائمة على hESC النامية كنهج بديل. ومع ذلك ، وكيفية التفريق قناة إمكانات واسعة من hESCs المحفزة بكفاءة ويمكن التنبؤ بها إلى النمط الظاهري المطلوب قد يشكل تحديا كبيرا لكلا دراسة تنموية والترجمة السريرية. المناهج التقليدية تعتمد على نسب متعددة من الخلايا المحفزة الميل التلقائي من خلال تمايز طبقة الجرثومية ، مما أدى إلى عدم كفاءة والتزام لا يمكن السيطرة عليها ، النسب التي يتم اتباعها في كثير من الأحيان عدم التجانس المظهري وعدم الاستقرار ، وبالتالي ارتفاع مخاطر tumorigenicity 6-8 (انظر التخطيطي في الشكل 1A). بالإضافة إلى ذلك ، قد غير معروف ملاحق البيولوجية الأجنبية / الحيوانية و / أو مغذيات التي عادة ما كانت تستخدم للعزل ، والتوسع ، والتفريق بين hESCs الاستفادة مباشرة من هذه الخلية specialiزيد الطعوم في المرضى الذين يعانون مشاكل 9-11. للتغلب على هذه العقبات ، ولقد عقدنا العزم على عناصر نظام ثقافة المعرفة الضرورية والكافية لإدامة الأديم الظاهر pluripotence من hESCs ، بمثابة منصة لاشتقاق نوفو دي hESCs سريريا مناسب وفعال توجيه مثل هذه hESCs موحد تجاه الأنساب سريريا ذات الصلة بواسطة الجزيئات الصغيرة 12 (انظر التخطيطي في الشكل 1B). بعد فحص مجموعة متنوعة من الجزيئات الصغيرة وعوامل النمو ، وجدنا أن هذه الشروط المحددة المقدمة نيكوتيناميد (NAM) كافية للحث على مواصفات cardiomesoderm مباشرة من hESCs المحفزة التي تقدم المزيد من cardioblasts التي ولدت مع الإنسان العضلية الضرب كفاءة عالية (الشكل 2 ). حددنا الشروط لتحريض مباشر من cardioblasts hESCs المحفزة دون التدخل المتعدد نسب مضغي الشكل مرحلة الجسم ، وكذلك تمكين لسيطرة فعالة على الاشتقاقكميات كبيرة من خلايا القلب البشرية عبر الطيف من المراحل التنموية لخلية مقرها المداواة.

Watch this Scientific Journal Video about Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction at JoVE.com

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

أقمنا على بروتوكول لتحريض مباشر من cardioblasts المحفزة الخلايا الجذعية الجنينية البشرية في ظل ظروف محددة المحافظة مع الجزيئات الصغيرة ، والتي تمكن من اشتقاق كميات كبيرة من أسلاف الإنسان والقلب العضلية وظيفية لإصلاح القلب والأوعية الدموية.

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 ...

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19.3K Views.  San Diego Regenerative Medicine Institute. أقمنا على بروتوكول لتحريض مباشر من cardioblasts المحفزة الخلايا الجذعية الجنينية البشرية في ظل ظروف محددة المحافظة مع الجزيئات الصغيرة ، والتي تمكن من اشتقاق كميات كبيرة من أسلاف الإنسان والقلب العضلية وظيفية لإصلاح القلب والأوعية الدموية.

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

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 ...

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

وبنقل العدوى إلى الإنسان Nucleofecting الجذعية المحفزة المستحثة خلايا

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

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 ...

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 ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

جيل من العضلية الإنسان: البروتوكول التمايز من خالية من الطاعم المستحثة الخلايا الجذعية المحفزة الإنسان

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...