نشكر لورا كلارك لتقديم المساعدة لها لا تقدر بثمن. ويدعم هذا العمل NCE : الجذعية شبكة الخليوي ، والمعاهد الوطنية للصحة CIHR.

1. إعداد حلول تشريح وحلول أنزيم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل السائل الشوكي الدماغي الاصطناعي (ACSF) وسائل الاعلام المصل خالية في وقت سابق والثلاجة. </li><li style=";text-align:right;direction:rtl"> تزن خارج حمض Kynurenic (0.2 ملغ / مل) ، وتذوب في 10 مل من ACSF هيلو في بالحمام المائي 37 درجة مئوية في وقت مبكر لأنها لا تذوب بسهولة. </li><li style=";text-align:right;direction:rtl"> تزن خارج التربسين (1.33 ملغ / مل) وهيالورونيداز (0.67 ملغ / مل) ووضعه في أنبوب واحد 15 مليلتر وابقائه في -20 درجة مئوية لحين الحاجة إليها. إضافة محلول حمض Kynurenic لهذا الأنبوب والتصفية باستخدام حقنة 22 ميكرون فلتر فقط قبل استخدامه. </li><li style=";text-align:right;direction:rtl"> تزن خارج التربسين المانع (Ovamucoid : 1 ملغ / مل) ، وتذوب في وسائل الإعلام المصل خالية من الدفء والتصفية باستخدام محقنة 22μm التصفية. </li><li style=";text-align:right;direction:rtl"> جعل وسائل الإعلام الطلاء : مصل خالية تحتوي على وسائل الإعلام FGF2 (10 نانوغرام / مل) والهيبارين (2 ميكروغرام / مل). </li></ol> 2. عزل الخلايا الجذعية من شبكية العين بالعين ماوس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم تنفيذ عزل الخلايا الجذعية الشبكية في غرفة معقمة محددة مكرسة لثقافة التجارب الأولية. قبل البدء في هذا الإجراء ، وإعداد مجهر تشريح ومصدر ضوء الباردة ، ومن ثم وضع الأدوات المعقمة تشريح. كل غطاء لديه معقم الساخنة حبة لتعقيم الأدوات بين الخطوات. </li><li style=";text-align:right;direction:rtl"> سنقوم عزل الخلايا الجذعية من أعين الشبكية التي تم وضعها على الفور في طبق بتري معقمة تحتوي على السائل الشوكي الدماغي الاصطناعي (ACSF) بعد إزالتها من الفئران التضحية الحيوانية وافق وفقا لأخلاقيات البروتوكولات. </li><li style=";text-align:right;direction:rtl"> تشريح تحت المجهر ، وتنظيف العين مع ملقط : تخلص من الشعر والنسيج الضام الذي تعلق على القرنية / الحدود الصلبة. ثم نقل العين إلى صحن جديد مع ACSF. </li><li style=";text-align:right;direction:rtl"> بينما يمسك بلطف على العين ثابت مع ملقط مسنن ، واستخدام الزاوية الصغيرة تشريح المقص لإزالة عضلات العين. إزالة العصب البصري وكذلك إذا كان لا تزال تعلق على العين. ليس محاولة لإسحق العين أثناء هذه العملية. نقل عينيه على طبق جديد مع ACSF. </li><li style=";text-align:right;direction:rtl"> استخدام المقبل منحني الدقيقة تشريح مقص لقطع في نصف العين : تبدأ في العصب البصري وقطع طريق وسط القرنية ، الاجتماع مرة أخرى في العصب البصري حيث بدأ الخفض. وهو مثالي إذا قطعتين من العين هي ما يعادل تقريبا في الحجم لأن ذلك سوف يجعل الخطوة التالية أسهل. </li><li style=";text-align:right;direction:rtl"> عقد القرنيات باستخدام اثنين من الملقط ، بلطف قشر العين نصفين منفصلين. إزالة والتخلص من العدسة ، والأحشاء ، والعصبية للشبكية العين من القذائف. نقل القذائف على طبق جديد مع ACSF. </li><li style=";text-align:right;direction:rtl"> الآن نحن على استعداد لعزل ظهارة الهدبية. لتبدأ ، توجيه قذيفة العين بحيث القرنية على يمينك والشبكية الصباغي الطلائية (RPE) هو على اليسار. دبوس برفق قذيفة أسفل العين مع ملقط على التوالي على الجانب RPE. </li><li style=";text-align:right;direction:rtl"> المقبل ، واستخدام مشرط لقطع القرنية والقزحية بعيدا عن ظهارة الهدبية للعين عن طريق دفع برفق لأسفل على مشرط بدلا من استخدام الاقتراحات النشر. </li><li style=";text-align:right;direction:rtl"> بعد ذلك ، قص ظهارة الهدبية بعيدا عن RPE. عدم استخدام ملقط مسنن لنقل شريط من ظهارة الهدبية إلى صحن 35 ملم جديدة تحتوي على ACSF. </li><li style=";text-align:right;direction:rtl"> في نفس الطريق ، وعزل ظهارة الهدبية من قذيفة العين الأخرى. </li><li style=";text-align:right;direction:rtl"> مرة واحدة جمعت كل من شرائط الهدبية الظهارية ، ونقل الشرائط الى صحن 35 ملم تحتوي على 2 مل من Dispase. مكان الطبق مع شرائح في الحاضنة 37 درجة مئوية لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> بعد 10 دقيقة ، ونقل شرائح من Dispase في صحن يحتوي على 2 مل من التربسين تصفيتها ، هيالورونيداز وحمض Kynurenic. ضع طبق في 37 لمدة 10 دقائق درجة مئوية. </li><li style=";text-align:right;direction:rtl"> الآن نعود إلى مجهر تشريح. في حين عقد الصلبة إلى أسفل مع ملقط على التوالي ، واستخدام الجزء السفلي من ملقط مسنن غير منحنية لكشط البشرة بلطف الهدبية بعيدا عن الصلبة. </li><li style=";text-align:right;direction:rtl"> إزالة الصلبة من الطبق. كل ذلك ينبغي أن يترك في الطبق الآن هي الخلايا من الاهتمام والحل الانزيم. </li><li style=";text-align:right;direction:rtl"> باستخدام ماصة القطن توصيل النار مصقول ، ونقل هذا الحل في أنبوب 14 مل. متمزج هذا الحل 30 مرات لتحطيم الخلايا الظهارية من خلال إجبار برفق في حل والخروج من ماصة. </li><li style=";text-align:right;direction:rtl"> أنبوب الطرد المركزي لمدة 5 دقائق عند 1500 دورة في الدقيقة. عندما يتم الطرد المركزي ، وإزالة أنبوب من أجهزة الطرد المركزي منذ بعناية الخلايا المعرضة للنزوله أسفل الأنبوب في هذه المرحلة. </li><li style=";text-align:right;direction:rtl"> نضح بلطف غالبية طاف به ماصة مصقول النار ، ثم إضافة 1 مل من مثبط التربسين في المصل خالية من وسائل الإعلام إلى الخلايا. استخدام القطن بئر صغيرة ماصة لتوصيل متمزج العينة ما يقرب من 50 مرات حتى يتم تعليق وحيد الخلية. </li><li style=";text-align:right;direction:rtl"> أنبوب الطرد المركزي مرة أخرى لمدة 5 دقائق عند 1500 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة طاف واستبدالها مع 1 مل من المتوسط ​​الطلاء الخاص. متمزج بلطف باستخدام ماصة الزجاج المصقول إلى النار resuspend الخلايا. </li><li style=";text-align:right;direction:rtl"> بعد العدجي الخلايا ، لوحة لهم في الكثافة المطلوبة في صفيحة ال 24 أيضا. نحن لوحة عادة 10 خلية / ميكرولتر من خلال تعبئة كل أول بئر مع المتوسط ​​ثم إضافة تعليق خلية للحصول على الحجم النهائي من 500 ميكرولتر من وسائل الإعلام. </li><li style=";text-align:right;direction:rtl"> وضع لوحة في الحاضنة 37 2 درجة مئوية ثاني حيث أنه لن يتم نقل حتى كنت تعول المجالات التي تنشأ بعد 7 أيام. </li></ol> 3. نتائج عزل الخلايا الجذعية الشبكية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عندما يتم تنفيذ هذا الإجراء بنجاح ، يجب أن تشريح الخلايا الظهارية الهدبية تبدو هذه بعد فصلها ومطلي في كثافة منخفضة (الشكل 1). </li><li style=";text-align:right;direction:rtl"> بعد 7 أيام في الثقافة ، ويتم الاعتماد على الخلايا الجذعية الشبكية المجالات التي تنشأ. وينبغي أن يكون المجال أكثر من 75 ميكرومتر في القطر والتعويم الحر لتحسب بوصفها مجال الخلايا الجذعية المشتقة. (الشكل 2). ومع ذلك ، فإن بعض الخلايا محدودة الانتشار وشكل الأجسام الشبه الكروية التي لا تستوفي معيار حجم وسوف لا تكون محسوبة على مجالات الخلايا الجذعية المشتقة ؛ يظهر مثال كروي من هذا القبيل هنا (الشكل 2). </li></ol> 4. ممثل النتائج <img src="/files/ftp_upload/2209/2209fig1.jpg" alt="الشكل 1" /> الشكل 1. <img src="/files/ftp_upload/2209/2209fig2.jpg" alt="الشكل 2" /> الشكل 2.

<ol>
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F., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+transplanted+into+models+of+late+stages+of+retinitis+pigmentosa+preferentially+adopt+a+glial+or+a+retinal+ganglion+cell+fate.">Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate.</a> Invest Ophthalmol Vis Sci. 48 (1), 446-4454 (2007).</li><li>Canola, K., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cells+committed+towards+the+photoreceptor+fate+for+retinal+transplantation.">Generation of cells committed towards the photoreceptor fate for retinal transplantation.</a> Neuroreport. 18 (9), 851-855 (2007).</li><li>Djojosubroto, M. 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F., Kooy, D. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+neural+stem+cells+proliferate+in+response+to+EGF+and+FGF+in+the+developing+mouse+telencephalon.+Dev+Biol.">Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol.</a> , 208-201 (1999).</li><li>Coles-Takabe, B. L., Brain, I., Purpura, K. A., Karpowicz, P., Zandstra, P. W., Morshead, C. M., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+look:+growing+clonal+versus+nonclonal+neural+stem+cell+colonies.">Don't look: growing clonal versus nonclonal neural stem cell colonies.</a> Stem Cells. 26 (11), 2938-2944 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

هذا البروتوكول توضح كيفية عزل الخلايا الجذعية من الظهارة الشبكية الهدبية من الفأرة 1-3 العين. يمكن للمنهجية لعزل شرائط من ظهارة الهدبية تختلف ، ومع ذلك فقد تم تحسين الانزيمات ومنهجية لتحقيق تعليق وحيد الخلية في هذا البروتوكول. من المهم أيضا أن يجرد من ظهارة الهدبية التي تم عزلها لا تحتوي على كميات كبيرة من RPE أو القرنية منذ هذه تظهر أن يكون لها أثر سلبي على إجمالي عدد من المجالات التي يمكن أن تكون معزولة عن كل عين. بالإضافة إلى ذلك ، وسائل الاعلام المصل الحرة التي نتخذها التي صيغت في الأصل لneurospheres الدماغ 13 ، مثالية لزراعة الخلايا الجذعية الشبكية المجالات. ينبغي للمرء أيضا التأكد من أن ليست منزعجة من الخلايا بعد أن تم مطلي انهم وضعت في الحاضنة بحيث مجالات النمو لا التجميعية معا 14. مرة واحدة في الخلايا الجذعية في شبكية العين قد شكلت نسيلي المجالات ، ويمكن عدهم على الحصول على عدد من الخلايا الجذعية المحتملين في العين. المجالات ومن ثم يمكن بعد ذلك فصل واحد والى خلايا passaged لقدرات ذاتية التحقق تجديد أو متمايزة الى أنواع مختلفة من الخلايا العصبية للشبكية وRPE باستخدام تركيبات مختلفة من عوامل النمو و / أو البروتينات.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stemi 2000 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cold Light Source</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>KL1500 </td>
<td>dual arm optic light</td>
</tr>
<tr>
<td>Curved Mini Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-10</td>
<td>&nbsp;</td>
<tr>
<td>Angled Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15005-08</td>
<td>&nbsp;</td>
<tr>
<td>#7 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11272-30</td>
<td>non-serrated</td>
</tr>
<tr>
<td>#5 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11251-10</td>
<td>straight</td>
</tr>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11051-10</td>
<td>serrated curved</td>
</tr>
<tr>
<td>#3 Scalpel handle</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2586-M36-10</td>
<td>&nbsp;</td>
<tr>
<td>#10 Scalpel blades</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2580-M90-10</td>
<td>&nbsp;</td>
<tr>
<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>FST</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>VWR/BD </td>
<td>CACB354235</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1005</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6254</td>
<td>&nbsp;</td>
<tr>
<td>Kynurenic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>K3375</td>
<td>1000 IU</td>
</tr>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10109878001</td>
<td>&nbsp;</td>
<tr>
<td>35 mm dishes</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA354235</td>
<td>&nbsp;</td>
<tr>
<td>24-well plate</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA73521-004</td>
<td>&nbsp;</td>
<tr>
<td>Cotton-plugged pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14672-400</td>
<td>fire-polish</td>
</tr>
<tr>
<td>14 mL Tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352057</td>
<td>Polystyrene</td>
</tr>
<tr>
<td>Serum-free Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref# 13 for formulation</td>
</tr>
<tr>
<td>FGF2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3149</td>
<td>100 IU</td>
</tr>		</tbody>
		</table>
		</div>

isolation of retinal stem cells from the mouse eye

الكبار الخلايا الجذعية الشبكية (RSC) هي الخلية النادرة هادئة وجدت داخل ظهارة الهدبية (م) من العين الثدييات 1،2،3. وأدلى م حتى من غير ذوي البشرة الداخلية وطبقات الخلايا الصباغية الخارجي ، ومصنوعة المستعمرات نسيلي RSC التي تنشأ من خلية واحدة من المصطبغة CE يصل كل من الخلايا الصباغية وغير الصباغية ، والتي يمكن أن تكون متمايزة لتشكيل جميع أنواع الخلايا العصبية للشبكية وRPE لل. هناك بعض الجدل حول ما اذا كانت جميع الخلايا ضمن مجالات تحتوي على كل ما لا يقل عن بعض الصباغ 4 ، ولكن الخلايا لا تزال قادرة على تشكيل أنواع مختلفة من الخلايا الموجودة داخل الشبكية العصبية 1-3. في بعض الأنواع ، مثل البرمائيات والأسماك ، وعيونهم قادرة على التجدد بعد إصابة 5 ، ولكن ؛ العين الثدييات لا تظهر هذه الخصائص على التجدد. نحن نسعى للتعرف على الخلايا الجذعية في الجسم الحي وفهم الآليات التي تبقي الخلايا الجذعية الثديية هادئة شبكية العين 6-8 ، وحتى بعد الاصابة فضلا عن استخدامها كمصدر محتمل للخلايا للمساعدة في إصلاح نماذج الجسدية أو الوراثية للإصابة العين من خلال زرع 12/09. نحن هنا تصف كيفية عزل الخلايا الظهارية الهدبية من العين الفأر وتنمو بها في الثقافة من أجل تشكيل الخلايا الجذعية نسيلي الشبكية المجالات. حيث لا توجد علامات معروفة من الخلايا الجذعية في الجسم الحي ، وهذه المجالات هي الطريقة الوحيدة المعروفة لتحديد مستقبلي السكان الخلايا الجذعية داخل ظهارة الهدبي للعين.

Watch this Scientific Journal Video about Isolation of Retinal Stem Cells from the Mouse Eye at JoVE.com

Isolation of Retinal Stem Cells from the Mouse Eye

في هذا الفيديو ، سوف نقوم شرح كيفية عزل الخلايا الجذعية من الظهارة الشبكية الهدبية للعين الماوس وتنمو لهم في مجالات الثقافة لتشكيل نسيلي الشبكية. المجالات التي يتم عزل تمتلك خصائص أساسية للخلايا الجذعية : الذات وتجديد multipotentiality.

عزل الخلايا الجذعية من شبكية العين بالعين ماوس

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

isolation-of-retinal-stem-cells-from-the-mouse-eye

Research

JoVE Journal

Biology

24.7K Views.  University of Toronto. في هذا الفيديو ، سوف نقوم شرح كيفية عزل الخلايا الجذعية من الظهارة الشبكية الهدبية للعين الماوس وتنمو لهم في مجالات الثقافة لتشكيل نسيلي الشبكية. المجالات التي يتم عزل تمتلك خصائص أساسية للخلايا الجذعية : الذات وتجديد multipotentiality.

Video: Isolation of Retinal Stem Cells from the Mouse Eye

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

العزلة وتحليل الخلايا الجذعية المكونة للدم من المشيمة

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

عزل الخلايا الجذعية ماوس اللعابية الغدة

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

جيل من الفئران المستمدة من الخلايا الجذعية المحفزة المستحثة

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

العزلة وثقافة الأسنان طلائي الخلايا الجذعية من الكبار ماوس القاطعة

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

حقن الخلية الداخلية عبر كتلة من الخلايا الجذعية الجنينية يؤدي إلى ارتفاع في أسعار تشيميريسم

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

الثقافات الخلية الأولية من ظهارة صباغ الشبكية الماوس

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

عزل وتوصيف والتفريق في القلب من الخلايا الجذعية من قلب الماوس الكبار

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

تشريح فعال وثقافة الخلايا الظهارية صبغة الفأر الأولية

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

عزل الخلايا الظهارية المصطبغة بالشبكية الأولية للفأر

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

توليد فعال ومتسق لظهارة صبغية شبكية العين / المشيمية من العين البشرية للتحليل النسيجي

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...