Name: generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions
Uploaded: 2020-11-09T15:00:00
Description: Watch this Scientific Journal Video about Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions at JoVE.com

تم دعم هذا العمل من خلال المنح المقدمة من المعاهد الوطنية للصحة (R00 AG054616 إلى YAH و T32 GM136566 إلى KC) ، وكلية الطب بجامعة ستانفورد وزمالة Siebel (الممنوحة ل SC). Y.A.H. هو أستاذ متعدية في GFL من مركز علم الأعصاب الانتقالي في معهد براون للعلوم الانتقالية.

1. تحريض الخلايا العصبية البشرية من الخلايا الجذعية البشرية متعددة القدرات
<ol><li>تحضير فيروس العدس (~ 5 أيام ، بروتوكول مفصل كما هو موضح سابقا16)<ol><li>لوحة ~ 1 مليون خلية HEK293T كل قارورة T75 ، ليكون لها ~ 40 ٪ متقاربة عند إجراء transfection. قم بنقلها باستخدام البلازميدات التي تعبر عن Ngn2 المحرض...

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H., Park, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+reprogramming+methods+for+generation+of+induced-oligodendrocyte+precursor+cells.">Comparison of reprogramming methods for generation of induced-oligodendrocyte precursor cells.</a> Biomolecules &amp; Therapeutics (Seoul). 25 (4), 362-366 (2017).</li><li>Ehrlich, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+generation+of+oligodendrocytes+from+human+induced+pluripotent+stem+cells+using+transcription+factors.">Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (11), 2243-2252 (2017).</li><li>Rodrigues, G. M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+and+scalable+differentiation+of+human+oligodendrocyte+precursors+from+pluripotent+stem+cells+in+a+3D+culture+system.">Defined and scalable differentiation of human oligodendrocyte precursors from pluripotent stem cells in a 3D culture system.</a> Stem Cell Reports. 8 (6), 1770-1783 (2017).</li><li>Hu, B. Y., Du, Z. W., Li, X. J., Ayala, M., Zhang, S. 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بالإضافة إلى الدعم الجسدي والأيضي لتحقيق الاستقرار في هياكل المشبك وتسهيل توصيل الإشارة الملحية عن طريق الميالين ، يمكن لخلايا سلالة oligodendrocyte تشكيل نمط النشاط العصبي عبر محادثات متقاطعة سريعة وديناميكية مع الخلايا العصبية5،6،7. بينما في...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61778/generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions</a>.&#160;The Representative Results section has been updated.
Figure 3 was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61778/61778fig03.jpg" /> 
Figure 3: Co-culture of iNs and iOPCs.&#160;(A) Representative bright field image of co-cultured iNs and iOPCs at Day 7, showing a proper density for further maturation. (B) Representative immunofluorescence image of iNs and iOPCs co-cultured for 28 days. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Right, a segment of iN axon ensheathed by iOL process (MBP+). (C) Synapse formation assayed in 4-week-old co-cultures. Cells were stained for Synapsin 1 (Syn1, green) and MAP2 (red), and synaptic puncta were quantified by confocal analysis of density along the dendritic segments as described17,18. (D) In our co-cultures of iNs and iOPCs (7 days of co-culturing), the expression of astrocyte markers, ALDHL1 and GFAP, is minimal (top), and the expression of microglia markers, TMEM119, TREM2, and CD33, is not detected (N.D.) by qPCR. The contamination from these two glial cell types is thus excluded. <a href="https://www.jove.com/files/ftp_upload/61778/61778fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61778/61778fig3v2.png" /> 
Figure 3: Co-culture of iNs and iOPCs.&#160;(A) Representative bright field image of co-cultured iNs and iOPCs at Day 7, showing a proper density for further maturation. (B) Representative immunofluorescence image of iNs and iOPCs co-cultured for 28 days. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Right, a segment of iN axon ensheathed by iOL process (MBP+). (C) Synapse formation assayed in 4-week-old co-cultures. Cells were stained for Synapsin 1 (Syn1, green) and MAP2 (red), and synaptic puncta were quantified by confocal analysis of density along the dendritic segments as described17,18. (D) In our co-cultures of iNs and iOPCs (7 days of co-culturing), the expression of astrocyte markers, ALDHL1 and GFAP, is minimal (top), and the expression of microglia markers, TMEM119, TREM2, and CD33, is not detected (N.D.) by qPCR. The contamination from these two glial cell types is thus excluded.&#160;(E) Coculturing iOPC with iN leads to the formation of neuron-OPC synapses. The fluorescence-tagged post-synaptic marker PSD95-mCherry is expressed only in OPCs, and display a diffuse pattern in single cultures (left) but aggregate to form puncta in cocultures (right, indicated by arrows; Tuj1, neuronal marker). (F) The expression of well-characterized oligodendroglial genes that can sense and respond to neuronal activities in the pure cultures of iOPCs at Day 14.&#160;<a href="https://www.jove.com/files/ftp_upload/61778/61778fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The fourth&#160;paragraph was updated from:
Co-culturing of iNs and iOPCs 
This protocol is optimized specifically for co-culturing iNs and iOPCs and allow our real-time monitoring of the inter-cellular communications between these two cell types along the course of neural development. The ideal plating densities for both cell types need to be decided with a series of cell number titration to achieve proper differentiation (Figure 3A). After 4 weeks in co-cultures, the iOPCs are expected to be adequately differentiated into OLs that are positive for specific markers such as MBP and extend processes to ensheath axons (Figure 3B). The co-culture system can robustly boost up the number of synapses, indicating that the iOPCs provide a neuronal support through physical contacts or release of trophic factors (Figure 3C). We can maintain the co-cultures in acceptable health condition for up to 6 weeks and observe that the synapse number and other neuronal attributes plateau around the fifth week. Of note, astrocytes and microglia are not present in our preparations and their absence can be documented by checking the expression of specific markers (Figure 3D).
to:
Co-culturing of iNs and iOPCs 
This protocol is optimized specifically for co-culturing iNs and iOPCs and allow our real-time monitoring of the inter-cellular communications between these two cell types along the course of neural development. The ideal plating densities for both cell types need to be decided with a series of cell number titration to achieve proper differentiation (Figure 3A). After 4 weeks in co-cultures, the iOPCs are expected to be adequately differentiated into OLs that are positive for specific markers such as MBP and extend processes to ensheath axons (Figure 3B). The co-culture system can robustly boost up the number of synapses, indicating that the iOPCs provide a neuronal support through physical contacts or release of trophic factors (Figure 3C). We can maintain the co-cultures in acceptable health condition for up to 6 weeks and observe that the synapse number and other neuronal attributes plateau around the fifth week. Of note, astrocytes and microglia are not present in our preparations and their absence can be documented by checking the expression of specific markers (Figure 3D).&#160;The iOPCs express a good number of well-characterized genes that can potentially respond to and mediate the activity-dependent signals from neighboring neurons, in a paracrine (e.g. neurotrophins and metabolites) and/or a synaptic manner (Figure&#160;3E and 3F).&#160;

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61778/generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions</a>.&#160;The Representative Results section has been updated.

Erratum: Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

تشكل خلايا سلالة الخلايا قليلة التغصن - بما في ذلك خلايا سلائف الخلايا قليلة التغصن (OPCs) ، والخلايا قليلة التغصن الميالينية ، والأنواع الانتقالية بينهما - مجموعة رئيسية من خلايا الدماغ البشرية1 التي تشارك بنشاط في العديد من الوظائف الحيوية من أجل التشغيل السليم وصيانة نظامنا العصبي المركزي طوال التطور العصبي والشيخوخة2،3،4 . في حين أن oligodendrocytes معروفة جيدا بإنتاج المايلين لتسهيل انتقال النشاط العصبي ودعم الصحة المحورية في المادة البيضاء ، فإن OPCs وفيرة (~ 5٪) في المادة الرمادية حيث يكون الميالين نادرا وتؤدي وظائف إشارات تعتمد على النشاط للتحكم في سلوك التعلم وتكوين الذاكرة5،6،7،8 . كيف تعمل الخلايا الدبقية قليلة التغصن والخلل الوظيفي في التسبب في مرض الزهايمر (AD) وغيرها من الحالات التنكسية العصبية المرتبطة بالعمر لم تتم دراستها9. وتتمثل أوجه القصور في نظام نموذجي مناسب وأوجه القصور في المعارف العامة لتوجيه مسار تجريبي إلى الأمام في الأسباب الرئيسية لهذه الفجوة.
في ضوء أحدث الاختراقات في اشتقاق خلايا الدماغ البشري من الخلايا الجذعية متعددة القدرات بما في ذلك الخلايا الجذعية الجنينية (ES) والخلايا الجذعية المستحثة متعددة القدرات (iPS) ، ظهرت هذه النماذج الخلوية جنبا إلى جنب مع أدوات تحرير الجينات الحديثة كأدوات قوية للتعامل مع العلاقة المعقدة للتفاعلات الخلوية في الدماغ ، وهي قادرة على إظهار مظاهر المرض الخاصة بالإنسان10 ، 11. بالنظر إلى أن أنواع خلايا الدماغ الفردية يمكن أن تظهر تأثيرات مميزة وحتى متضاربة في مواجهة نفس الظروف المعززة لمرض الزهايمر12,13 ، فإن منهجية الخلايا الجذعية هذه تقدم بشكل فريد معلومات خاصة بنوع الخلية تم تفويتها سابقا باستخدام نماذج ثابتة في الجسم الحي أو في المختبر توفر فقط قراءات مجمعة من مجموعات من أنواع خلايا الدماغ. في العقد الماضي ، تم تطوير عدد كبير من البروتوكولات الموثوقة لتوليد الخلايا العصبية البشرية من التمايز العابر للخلايا الجذعية الجنينية / الجذعية المستحثة متعددة القدرات أو التحويل المباشر من أنواع الخلايا الأخرى المتمايزة نهائيا (مثل الخلايا الليفية)14,15. على وجه الخصوص ، يمكن أن يؤدي تطبيق عوامل النسخ العصبية الرئيسية (على سبيل المثال ، neurogenin 2 ، Ngn2)16 على الخلايا الجذعية البشرية متعددة القدرات إلى توليد مجموعة متجانسة من أنواع الخلايا العصبية المميزة جيدا للمزارع النقية دون الحاجة إلى التثاقف مع الخلايا الدبقية12،17،18. بالنسبة للخلايا قليلة التغصن البشرية المستحثة ، هناك عدد قليل من البروتوكولات المنشورة التي يمكن أن تولد خلايا وظيفية تشبه إلى حد كبير نظيراتها الأولية ، مع مجموعة واسعة من الكفاءة والطلب في الوقت والموارد19،20،21،22،23،24،25،26،27،28 . حتى الآن ، لم يتم تطبيق أي من هذه البروتوكولات للتحقيق في كيفية استجابة الخلايا قليلة التغصن وتأثيرها على التسبب في مرض الزهايمر.
هنا ، نصف بروتوكولاتنا المحسنة للثقافات الفردية والمختلطة للخلايا العصبية المستحثة البشرية (iNs) و OPCs / oligodendrocytes (iOPCs / iOLs). يعتمد بروتوكول iN الموصوف هنا على نهج Ngn216 المستخدم على نطاق واسع ، وله ميزة إضافية تتمثل في كونه خاليا من الدبقية. تكون iNs الناتجة متجانسة وتشبه إلى حد كبير الخلايا العصبية المثيرة للطبقة القشرية 2/3 ، مع مورفولوجيا هرمية مميزة ونمط التعبير الجيني وميزات فيزيولوجية كهربية17,18 (الشكل 1). للتغلب على بعض الحواجز الأساسية في التمايز الموجه للخلايا الجذعية متعددة القدرات ، قمنا بتطوير طريقة بسيطة وفعالة للمعالجة المسبقة لجرعة منخفضة من ثنائي ميثيل سلفوكسيد (DMSO)29،30 ، وأبلغنا عن ميل معزز لخلايا ES / iPS البشرية للانتقال إلى iOPCs و iOLs31 ، بناء على بروتوكول تم تكييفه على نطاق واسع بواسطة Douvaras و Fossati 32 . لقد قمنا بتبسيط البروتوكول بشكل أكبر ودمجنا مركبا قويا يعزز التمايز ، كليماستين7،33،34 ، لتسريع عملية النضج الدبقي قليل التغصن. نتيجة لذلك (الشكل 2) ، يمكن إنشاء iOPCs في أسبوعين (~ 95٪ إيجابي للعلامة O4) و iOLs في أربعة أسابيع (معبرا عن علامات ناضجة MBP و PLP1). ومن المثير للاهتمام ، وجدنا أن iOPCs وحدها تفرز كمية ملحوظة من β الأميلويد (Aβ) ، بما يتفق مع البيانات النسخية المستقلة التي تظهر التعبير الوفير لبروتين السلائف الأميلويد (APP) ومعالجة البروتياز β-secretase (BACE1) في خلايا سلالة الخلايا قليلة التغصن35,36. علاوة على ذلك ، يعزز نظام الاستزراع المشترك iN-iOPC الخاص بنا تغليف المحاور من خلال عمليات iOL الإيجابية MBP ويوفر دعما كبيرا لتشكيل المشبك العصبي (الشكل 3). وبالتالي ، فإن البروتوكولات التي قمنا بتفصيلها أدناه لها مزايا تقنية وبيولوجية على طرق الاستزراع المشترك للخلايا العصبية قليلة التغصن المفهرسة سابقا ، وتبشر بنمذجة أفضل للتنكس العصبي في مرض الزهايمر.

<table>
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>ThermoFisher</td>
			<td>PHG 0266</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>MilliporeSigma</td>
			<td>A9501</td>
			<td></td>
		</tr>
		<tr>
			<td>Clemastine</td>
			<td>MilliporeSigma</td>
			<td>SML0445</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ThermoFisher</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>MilliporeSigma</td>
			<td>D3072</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ScienCell</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>H1 human ES cells</td>
			<td>WiCell</td>
			<td>WA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus</td>
			<td>STEMCELL Technologies</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>ThermoFisher</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A medium</td>
			<td>ThermoFisher</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>ThermoFisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-AA</td>
			<td>R&#38;D Systems</td>
			<td>221-AA-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>VWR</td>
			<td>71002-812</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-REV</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>ThermoFisher</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Tocris</td>
			<td>4366</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Progenitor Freezing Media</td>
			<td>STEMCELL Technologies</td>
			<td>5838</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff SMADi Neural Induction Kit</td>
			<td>STEMCELL Technologies</td>
			<td>8581</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 triiodothyronine</td>
			<td>MilliporeSigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Tempo-iOlogo: Human iPSC-derived OPCs</td>
			<td>Tempo BioScience</td>
			<td>SKU102</td>
			<td></td>
		</tr>
		<tr>
			<td>TetO-Ng2-Puro</td>
			<td>Addgene</td>
			<td>52047</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions

في مرض الزهايمر (AD) والاضطرابات التنكسية العصبية الأخرى ، يعد الفشل الدبقي قليل التغصن سمة مرضية مبكرة شائعة ، ولكن كيف يساهم في تطور المرض وتطوره ، خاصة في المادة الرمادية للدماغ ، لا يزال غير معروف إلى حد كبير. يتميز الخلل الوظيفي لخلايا سلالة الخلايا قليلة التغصن بأوجه القصور في الميالين وضعف التجديد الذاتي لخلايا سلائف الخلايا قليلة التغصن (OPCs). يحدث هذان العيبان جزئيا على الأقل بسبب اضطراب التفاعلات بين الخلايا العصبية والخلايا قليلة التغصن على طول تراكم علم الأمراض. تؤدي OPCs إلى ظهور الخلايا قليلة التغصن الميالينية أثناء تطور الجهاز العصبي المركزي. في قشرة الدماغ الناضجة ، OPCs هي الخلايا التكاثرية الرئيسية (التي تضم ~ 5 ٪ من إجمالي خلايا الدماغ) وتتحكم في تكوين المايلين الجديد بطريقة تعتمد على النشاط العصبي. لم تتم دراسة مثل هذه الاتصالات من الخلايا العصبية إلى الخلايا العصبية قليلة التغصن بشكل كبير ، خاصة في سياق الحالات التنكسية العصبية مثل مرض الزهايمر ، بسبب نقص الأدوات المناسبة. في السنوات الأخيرة ، أحرزت مجموعتنا وغيرها تقدما كبيرا لتحسين البروتوكولات المتاحة حاليا لتوليد الخلايا العصبية الوظيفية والخلايا قليلة التغصن بشكل فردي من الخلايا الجذعية البشرية متعددة القدرات. في هذه المخطوطة ، نصف إجراءاتنا المحسنة ، بما في ذلك إنشاء نظام زراعة مشتركة لنمذجة اتصالات الخلايا العصبية قليلة التغصن. تشير نتائجنا التوضيحية إلى مساهمة غير متوقعة من OPCs / oligodendrocytes في الداء النشواني في الدماغ وسلامة المشبك العصبي وتسليط الضوء على فائدة هذه المنهجية لأبحاث مرض الزهايمر. هذا النهج الاختزالي هو أداة قوية لتشريح التفاعلات الخلوية غير المتجانسة المحددة من التعقيد المتأصل داخل الدماغ. من المتوقع أن تسهل البروتوكولات التي نصفها هنا الدراسات المستقبلية حول عيوب قلة التغصن في التسبب في التنكس العصبي.

التوليد المباشر للخلايا العصبية التي يسببها الإنسان من الخلايا الجذعية البشرية متعددة القدرات من المهم جدا أن تظهر الخلايا الجذعية البشرية متعددة القدرات درجة عالية من تعدد القدرات للتوليد الناجح ل iNs أو iOPCs / iOLs. لذلك ، يجب تلطيخ الخلايا لعلامات محددة ، مثل Oct4 و SOX2 ، قبل البدء ف?...

Watch this Scientific Journal Video about Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions at JoVE.com

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

التفاعلات العصبية الدبقية في التنكس العصبي ليست مفهومة جيدا بسبب عدم كفاية الأدوات والأساليب. هنا ، نصف البروتوكولات المثلى للحصول على الخلايا العصبية المستحثة ، وخلايا سلائف الخلايا قليلة التغصن ، والخلايا قليلة التغصن من الخلايا الجذعية البشرية متعددة القدرات ونقدم أمثلة على قيم هذه الطرق في فهم المساهمات الخاصة بنوع الخلية في مرض الزهايمر.

توليد الخلايا العصبية البشرية والخلايا قليلة التغصن من الخلايا الجذعية متعددة القدرات لنمذجة تفاعلات الخلايا العصبية قليلة التغصن

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

The protocols described here, are expected to facilitate future studies on oligodendroglial defects, in the pathogenesis of neuro degeneration. This is an improved method that can be used to generate functional human oligodendrocyte lineage cells in only three to four weeks, and establish a co-culture system to model the neuron oligodendrocyte connections. The protocol can be used to study not only Alzheimer's disease, but any other disease associated aspect of neuron oligodendrocyte interactions.To begin, plate approximately one million HEK293T cells in each T75 flask to achieve 40%confluency, then transfect them with plasmids expressing tetracycline inducible NGN2 and puromycin resistant tetracycline transactivator gene, along with the three helper plasmids. Use PEI for transfection. Change the media after 16 hours.Harvest released viral particles by collecting culture media every day and replacing it with fresh media for three days. Pool the collected media for purification, then filter the virus through a 0.22 micrometer filter and centrifuge at 49, 000 times G for 90 minutes. After centrifugation, re-suspend the pellet in the appropriate volume of PBS glucose.Use commercially available H1 human ES cells to perform neuron induction. Culture the cells on extracellular matrix solution coated 6-well plates with ESL maintenance medium and incubate the plates at 37 degrees Celsius with 5%carbon dioxide. Two days before induction of differentiation, detach ES cells by adding one milliliter of cell detachment solution and incubating at room temperature for 10 minutes.Transfer the cells to a tube, then wash the well with two milliliters of media and add it to the same tube. Centrifuge the cells at 300 times G for five minutes. Then re-suspend the pellet in media and plate the cells onto matrix coated 6-well plates at the seating density of one times 10 to the fifth cells per well.On the next day, add lentiviruses, expressing NGN2 plus puro. R, and tetracycline transactivator together with polybrene in fresh ES cell maintenance medium. On day zero, add two micrograms per milliliter doxycycline in DMEM/F-12 medium supplemented with N2 and without morphogens.On day one, add puromycin in fresh DMEM/F-12, plus N2 and doxycycline to the final concentration of one microgram per milliliter. Select the transduced cells in puromycin for at least 24 hours. On day two, detach differentiating neurons with cell detachment solution and re-plate them on 24-well plates coated with matrix solution.Maintain them in MBA B27 medium without doxycycline. Culture pure induced neurons on the plates, coated with extracellular matrix-based solutions. To perform neural progenitor cell generation, culture H1 human ES cells and transdifferentiate them into neural progenitor cells, as described in the text manuscript.On day negative one, seed 0.5 to one million cells in ES cell maintenance medium into each well of a 6-well plate coated with growth factor reduced matrix solution. On day zero, treat cells for 24 hours with ES cell maintenance medium supplemented with 2%DMSO. On days one through six, change the full media with warm neural induction medium containing some add inhibitors from a commercial kit.On day seven, passage NPCs using cell detachment solution and plate them at a seating density of one to two times 10 to the fifth cells per well of a 24-well plate. Assay the differentiation efficiency by immunohistochemical staining for absence of pluripotentcy marker and presence of NPC markers. To generate the oligodendrocyte precursor cells, plate the NPCs in warm neural induction medium with some add inhibitors from a commercial kit.On day eight, prepare a solution of 1%DMSO in the OPC differentiation medium, and treat the plated NPCs for 24 hours. On day nine, replace the media with fresh OPC differentiation medium without DMSO, then feed the cells every other day until day 15. If the the cells reach confluence before day 15, passage them to the seating density of one to two times 10 to the fifth cells per well.On day 14, plate OPCs in OPC differentiation medium at a density of one to two times 10 to the fifth cells per well, in a 24-well plate. Test cells for the presence of OPC specific markers by IHC staining or qPCR, and for the absence of NPC markers. On day 15, replace media with OL maturation medium.Change the medium every other day or every day. When cells reach 90%confluence, split them at a one to three ratio for up to two passages or until cell division slows down substantially. If OPCs divide too fast and reach confluencey in less than three days, add Ara-C at a concentration of two to five micromolar for one to three days.On day 14, plate the IOPCs at a density of one times 10 to the fifth cells per well in a 24-well plate in OPC differentiation medium. On the next day, detach the induced human neurons with cell detachment solution. Add neurons onto the cultured OPCs, plating at the seating density of two times 10 to the fifth cells per well.Use co-culture medium containing neurobasal A medium, 2%B27 supplement, and 100 nanograms per milliliter T3 triiodothyronine. Change the medium on the next day and then every other day. If OPCs proliferate too fast and reach confluency in less than three days, add two to five micromolar Ara-C.It is important that the starting human pluripotent stem cells exhibit a high degree of pluripotentcy for the successful generation of induced neurons, or OPCs and oligodendrocytes. Therefore, cells should be stained for specific markers, such as OCT-4 and SOX2, before starting either of the induction protocols. Human H1 cells were used to obtain induced excitatory forebrain neurons.Pure neurons in culture started expressing synapse in one at day 14 to 16. IOPCs were generated in two weeks and mature oligodendrocytes in four to five weeks. DMSO treatment enriched the number of cells in the early G1 phase for better signaling Integration.Specific OPC markers were detected as early as two weeks after plating of ES H1 cells. The IOPC population at two weeks was fairly homogenous, with over 95%of cells positive for 04 staining and a high level of immunoreactivity for other markers. After initiation of oligodendrocyte maturation, specific oligodendrocyte markers were detected starting at day 28.The ideal plating densities for induced neurons, and IOPCs, need to be determined with a series of cell number titrations to achieve proper differentiation. After four weeks, in co-cultures, the IOPCs should be adequately differentiated into oligodendrocytes that are positive for specific markers such as MBP. This co-culture system can robustly boost the number of synapses, indicating that the IOPCs provide a neuronal support through physical contact or release of trophic factors.This described procedure can be further sophisticated by adding different brain cell types. For example, stem cells derived human astrocytes to model novel aspects of neuron glial interactions in health and disease.

Research

JoVE Journal

Neuroscience

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

جيل من الخلايا الجذعية العصبية من الأنسجة الجنينية البشرية المهملة القشرية

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

كفاءة اشتقاق أسلاف الإنسان متعلق بالخلايا العصبية والخلايا العصبية من افرة القوة الخلايا الجذعية الجنينية البشرية التعريفي مع جزيء صغير

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

مواصفات من الخلايا العصبية الجلوتاميرجي الدماغ الانتهائي من خلايا الجذعية المحفزة

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

وهناك طريقة توسيع المركب-ينطبق على مجاميع من العصبية الجذعية البشرية والخلايا المشتقة من الخلايا السلف الجذعية المحفزة أو الجنين أنسجة المخ

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

خالية من التغذية اشتقاق الخلايا العصبية كريست السلف من افرة القوة البشرية الخلايا الجذعية

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

توجه الدوبامين العصبية التمايز من الخلايا الجذعية المحفزة الإنسان

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

جيل بسيطة من ثقافة عالية الغلة المستحث الخلايا العصبية من الخلايا الليفية الجلد الكبار البشرية

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

تحويل الخلايا الجذعية مستحث البشرية المستحثة (iPSCs) في العمود الفقري الوظيفي الشوكي والجمجمة الحركية باستخدام متجات البكالوريا

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

اشتقاق، توسعة، المحافظة على التبريد وتوصيف خلايا الدماغ البطانية الدقيقة من الخلايا الجذعية متعددة القدرات المستحثة من الإنسان

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

إنشاء منصة فيزيولوجية كهربية لنمذجة ALS مع الخلايا النجمية والخلايا العصبية المشتقة من الخلايا الجذعية البشرية متعددة القدرات الخاصة بالمنطقة

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...