نقدم الشكر الخاص لكيم لينو، وستيف جوزمان ساتيارثي Ujit للمساعدة التقنية خلال تطوير هذا الأسلوب. تشكيله مايرون هوثورن، Spengel ريتشارد، وروبرتو روخاس الدوائر المستخدمة هنا وقدمت الكثير في أمس الحاجة إليها المساعدة التقنية. والجدير بالذكر أن إنتاج روبرتو روخاس الشكل 4. كما نشكر للمشورة سكوت فريزر لا تقدر بثمن قبل وضع الفحص الكيميائي من أعلاه. وأيد هذا العمل من قبل جزئيا على درجة 5S06GM048680-13-NIH-MBRS لMEdB وجائزة من، CSU برنامج الدراسات العليا نورثريدج دعم الرسالة إلى CW.

<ol>
	<li>Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avian+embryo+as+a+model+to+study+the+development+of+the+neural+crest:+a+long+and+still+ongoing+story.">The avian embryo as a model to study the development of the neural crest: a long and still ongoing story.</a> Mechanisms of Development. 121, 1089-1102 (2004).</li><li>Baker, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neural Crest and Cranial Ectodermal Placodes. , (2005).</li><li>Gammill, L. S., Roffers-Agarwal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+labor+during+trunk+neural+crest+development.">Division of labor during trunk neural crest development.</a> Dev. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Eph-related+receptors+and+ligands+confer+rostrocaudal+pattern+to+trunk+neural+crest+migration.">Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration.</a> Curr. Biol. 7, 571-580 (1997).</li><li>Gammill, L. S., Gonzalez, C., Gu, C., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+trunk+neural+crest+migration+requires+neuropilin+2/semaphorin+3F+signaling.">Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling.</a> Development, Cambridge, England. , 133-199 (2006).</li><li>De Bellard, M. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+polymorphonuclear+leukocytes+to+orient+in+gradients+of+chemotactic+factors.">Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.</a> The Journal of Cell Biology. 75, 606-616 (1977).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chicken+embryo.">A series of normal stages in the development of the chicken embryo.</a> J. Morph. 88, 49-52 (1951).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> The Journal of Experimental Medicine. 115, 453-466 (1962).</li><li>Davis, E. M., Trinkaus, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+cell-to+cell+contacts+for+the+directional+movement+of+neural+crest+cells+within+a+hydrated+collagen+lattice.">Significance of cell-to cell contacts for the directional movement of neural crest cells within a hydrated collagen lattice.</a> Journal of Embryology and Experimental Morphology. 63, 29-51 (1981).</li><li>Zicha, D., Dunn, G. A., Brown, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> Journal of Cell Science. 99, 769-775 (1991).</li></ol>

الإعلان عن أي تضارب في المصالح.

إجراء البحوث الكيميائي على البلدان المساهمة الصافية الجذع وقد ثبت تحديا لسلسلة من الأسباب. البلدان المساهمة الصافية الجذع تشكل الخلايا الجذعية غير المتجانسة التي من شأنها أن تفرق السكان إذا مثقف على المدى الطويل، وبالتالي، يجب الحصول على الجذع من البلدان المساهمة ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> التعليقات (اختياري) </td></tr><tr><td> DMEM </td><td> أوميغا العلمية </td><td> DM-22 </td><td></td></tr><tr><td> البنسلين الستربتوميسين الحل </td><td> أوميغا العلمية </td><td> PS-20 </td><td> 100X الأسهم تركيز </td></tr><tr><td> الجلوتامين L- </td><td> أوميغا العلمية </td><td> GS-60 </td><td> 100X الأسهم تركيز </td></tr><tr><td> المصل البقري الجنين </td><td> أوميغا العلمية </td><td> FB-11 </td><td> # 105247 الكثير (أو آخر يمكن مقارنتها) </td></tr><tr><td> تعديل زيجموند غرفة </td><td> المنزل الذي أدلى </td><td> N / A </td><td> حجم الخزان: ~ 160 ميكرولتر عصام؛ لمواصفات إضافية، انظر الشكل. (4) وبروتوكول إضافي تلفيق </td></tr><tr><td> خلية ثقافة طبق </td><tد&gt; Denville </td><td> T6040 </td><td> 40 × 10 مم </td></tr><tr><td> فبرونيكتين </td><td> BD </td><td> 354008 </td><td> هيأهم للأوراق 10X من تمييع 1 ملغ FN في 1 مل O 2 H و 9 مل DMEM </td></tr><tr><td> Coverslips </td><td> الصياد </td><td> 12-548-B </td><td> Precleaned؛ 22 × 22 مم </td></tr><tr><td> L15 المتوسطة </td><td> الحرارية العلمية </td><td> SH30525.02 </td><td></td></tr><tr><td> الفازلين </td><td> وسائل الراحة </td><td> 011110794642 </td><td> 100٪ </td></tr><tr><td> الطرد المركزي أنبوب </td><td> Biologix </td><td> 10-9152 </td><td> 15 مل </td></tr><tr><td> Dispase </td><td> نظم الخلايا </td><td> 4Z0-850 </td><td> 10X الأسهم تركيز </td></tr><tr><td> محقنة </td><td> BD </td><td> 309602 </td><td> 1 مل </td></tr><tr><td> إبرة </td><td> BD </td><td> 305127 </td><td> 25 غ × 1.5 بوصة </td></tr><tr><td> الكسا فلور 488-الغلوبولين المناعي </td><td> أناnvitrogen </td><td> A21042 </td><td> الأسهم هو 2 ملغ / مل و 7 الشامات صبغ / خلد الغلوبولين المناعي </td></tr><tr><td> تشريح الملقط </td><td> FST </td><td> منوعات. </td><td> دومون # 5 أو 55؛ التوالي الرؤوس؛ الفولاذ المقاوم للصدأ أو التيتانيوم </td></tr><tr><td> التنغستن الإبرة </td><td> N / A </td><td> N / A </td><td> المنزل الذي أدلى؛ ضعت في حامل دبوس </td></tr><tr><td> الملقط حادة </td><td> تيمان </td><td> 160-18 </td><td> تستخدم لنقل الأجنة إلى وقارع الأجراس من صفار البيض </td></tr></tbody></table> بروتوكول الإضافي: التصنيع لإحدى دوائر المحكمة زيجموند معدلة يرجى الرجوع إلى الشكل 4 كمرجع لبروتوكول أدناه: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شراء ورقة سميكة من الاكريليك 3/16 &quot;مصقول (4.45 مم سماكة الفعلي). </li><li style=";text-align:right;direction:rtl"> باستخدام جدول رأى، قطع الفراغات غرفة كبيرة الحجم للأبعاد الخام من 33،25 مم × 64،57 ملم. هذا يسمح 3،175 مم للمواد اضافية بالقطع. </li><li style=";text-align:right;direction:rtl"> تعيين فارغة على غرفة السادسحد ذاتها. مع آلة الطحن ومم 6.35 (1/4 &quot;) نهاية بت طاحونة، والانتهاء من الآلات الجانبين من الغرفة لأبعادها بالضبط: 30.07 مم × 61،39 ملم. </li><li style=";text-align:right;direction:rtl"> وضع فارغة الغرفة على آلة طحن وتحديد موقع مركز فارغة على طول X على حد سواء ومحاور ذ مع مكتشف حافة، ثم الصفر موقع المركز. </li><li style=";text-align:right;direction:rtl"> الحصول على غرفة ارتفاع (Z-المحور) من خلال لمس بت طاحونة حد للالسطح العلوي وصفر في الارتفاع. </li><li style=";text-align:right;direction:rtl"> باستخدام مم 3.91 (0.154 &quot;) نهاية بت طاحونة، ويقابل بت 3،03 مم على طول محور س (اتجاه إيجابي) لخزان الأولى. بيغن بالقطع في غرفة على عمق 2،84 ملم بينما تتحرك على طول المحور ص ( الاتجاه الإيجابي) إلى 7.62 مم (0.300 &quot;)، ثم تعبر إلى 7.62 ملم (0.300&quot;) في الاتجاه (سلبي) مقابل طول الخزان كاملة من 15.24 ملم (0.600 &quot;). تعويض قليلا إلى 3.03 مم (0.119 &quot;) على طول محور س (اتجاه سلبي) وكرر نفس العملية للخزان الثاني. </li><li style=";text-align:right;direction:rtl"> وضع دائرة على حافتهوحفر حفرة باستخدام مم 1.09 (0.043 بوصة) على قمة الحفر نهاية كل خزان (4 الكل) الذي يربط نهاية الخزان إلى جانب قاعة لتحميل المتوسطة خلال التجريب. </li><li style=";text-align:right;direction:rtl"> نقع في غرفة البئر بماء دافئ وصابون للمساعدة على إزالة أي ملوثات كيميائية. </li><li style=";text-align:right;direction:rtl"> نقع وشطف جيدا في غرفة مزدوجة الماء المقطر لإزالة أي الصابون. الدوائر هي الآن جاهزة للاستخدام كما هو موضح أعلاه. </li></ol>

analysis of trunk neural crest cell migration using a modified zigmond chamber assay

الخلايا العصبية قمة (البلدان المساهمة الصافية) هي عابرة السكان من الخلايا موجودة في الفقاريات التي التنمية الهجرة من الأنبوب العصبي الظهري (NT) بعد خضوعه ل1،2 الانتقال الظهارية-الوسيطة. بعد EMT، البلدان المساهمة الصافية الهجرة مسافات كبيرة على طول مسارات النمطية حتى وصولها الى اهدافها. البلدان المساهمة الصافية تفرق في مجموعة واسعة من أنواع الخلايا بما في ذلك الخلايا العصبية، الخلايا الدبقية، الخلايا الصباغية، والخلايا أليفة الكروم 1-3. قدرة البلدان المساهمة الصافية لتصل إلى مواقعها والتعرف على الهدف الصحيح هو التأسيسية لتشكيل المناسب لجميع الهياكل التي تحتوي على الجذع NCC المستمدة من المكونات 3. توضيح آليات التوجيه لجذع NCC الهجرة كان ذلك مسألة ذات أهمية كبيرة. وقد أظهرت العديد من الجزيئات لتوجيه NCC الهجرة 4. على سبيل المثال، من المعروف أن البلدان المساهمة الصافية الجذع صده العظة التوجيه السلبية مثل Semaphorin، Ephrin، والشق يغاندس 5-8. ومع ذلك، لاحتى مؤخرا أي من البلدان المساهمة الصافية chemoattractants الجذع تم تحديد 9. التقليدية في النهج المختبر لدراسة السلوك الكيميائي للخلايا ملتصقة تعمل بشكل أفضل مع الخلايا متجانس خلد، توزيع، ولكنها أكثر صعوبة لتطبيق بعض الثقافات الخلايا الجذعية الأولية التي تفتقر في البداية توزيع متجانس وتفرق بسرعة (مثل البلدان المساهمة الصافية). نهج واحد لتحقيق التجانس توزيع البلدان المساهمة الصافية للدراسات الجذع الكيميائي هو عزل البلدان المساهمة الصافية الجذع من المرحلة الابتدائية الثقافات يزدرع NT، ثم ارفع وreplate لها أن تكون ما يقرب من 100٪ متموجة. ومع ذلك، فإن هذا النهج يتطلب الطلاء كميات كبيرة من الوقت والجهد ليزدرع الخلايا بما فيه الكفاية، قاس، وتوزيع البلدان المساهمة الصافية الجذع بطريقة تختلف عن تلك الموجودة في الجسم الحي في ظروف. هنا، ونحن التقرير نهجا في المختبر قادر على تقييم ردود المهاجرة الكيميائي وغيرها من البلدان المساهمة الصافية دون الجذع requirinالجا متجانسة التوزيع الخلية. هذه التقنية تستخدم الوقت الفاصل بين التصوير الأولية، رابط الجأش البلدان المساهمة الصافية الجذع داخل غرفة زيجموند المعدلة (يوصف غرفة زيجموند القياسية في أماكن أخرى 10). من خلال تعريض البلدان المساهمة الصافية في محيط الجذع من الثقافة إلى أن الانحدار chemotactant هو عمودي على اتجاهها الطبيعي توقع، يمكن الكشف عن تغييرات في الاستقطاب الناجم عن الهجرة التدرج chemotactant التطبيقية. هذه التقنية غير مكلفة، تتطلب زراعة من إإكسبلنتس NT اثنين فقط في علاج تكرار، يتجنب قاسية رفع الخلية (مثل trypsinization)، ويترك البلدان المساهمة الصافية في الجذع توزيع أكثر مماثلة لفي ظروف الجسم الحي، التخفيضات مقدار الوقت بين explantation والتجريب (مما يقلل من خطر المحتمل تمايز)، ويسمح الوقت الفاصل بين تقييم الخصائص المهاجرة عديدة.

Watch this Scientific Journal Video about Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay at JoVE.com

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

يوصف هذا النهج لتحليل الهجرة من الخلايا explanted (خلايا الجذع قمة العصبية). هذه الطريقة غير مكلفة، لطيف، وقادرة على التمييز الكيميائي من كلا تنشيط كيميائي والتأثيرات الأخرى على الاستقطاب المهاجرة مثل تلك المشتقة من خلية خلية التفاعلات داخل الجذع العصبية الأولية قمة الثقافة الخلية.

تحليل العصبية كريست الهجرة جذع الخلية باستخدام التعديل الفحص غرفة زيجموند

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

analysis-trunk-neural-crest-cell-migration-using-modified-zigmond

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Neuroscience

12.4K Views.  California State University, Northridge. يوصف هذا النهج لتحليل الهجرة من الخلايا explanted (خلايا الجذع قمة العصبية). هذه الطريقة غير مكلفة، لطيف، وقادرة على التمييز الكيميائي من كلا تنشيط كيميائي والتأثيرات الأخرى على الاستقطاب المهاجرة مثل تلك المشتقة من خلية خلية التفاعلات داخل الجذع العصبية الأولية ق?...

Video: Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

العزل والتوسع في الخلايا الجذعية العصبية الكبار باستخدام الفحص Neurosphere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

تأسيس الخلايا الجذعية الجنينية الماوس العصبية الثقافة باستخدام الفحص Neurosphere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

العصبية تشكيل مستعمرة الفحص الخلوي : إن الفحص تميز بحسن نية الخلايا الجذعية العصبية من الخلايا العصبية السلف

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

العزلة وثقافة العصبية الخلايا الجذعية من بصيلات الشعر كريست الإنسان

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

استنساخه سحق الفأر العصب الوركي وتقييم لاحق للتجديد من قبل الجامعة جبل تحليل العضلات

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

تحليل للهجرة كريست العصبية والتمايز بواسطة الصليب الأنواع زرع

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

الفحص Neuroblast: والفحص للانشاء وإثراء خلايا متعلق بالخلايا العصبية السلف من التفريق العصبية الجذعية آله الخلية باستخدام التدفق الخلوي

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

شارك في تحليل بنية الدماغ والدالة باستخدام الرنين المغناطيسي الوظيفي وانتشارها المرجحة التصوير

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

العزلة والثقافة من الخلايا العصبية كريست من الأنبوبة العصبية الجنينية الفئران

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

A الفحص انجذاب غلفاني لتحليل حركية العصبية السلائف الهجرة خلية في الحقل الحالي مباشرة التطبيقية خارجيا الكهربائية

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...