الآراء الواردة في هذه الوثيقة أو التأكيدات هي آراء خاصة للكتاب ولا يمكن تفسيره بأنه مسؤول أو كما تعكس آراء زارة الجيش أو وزارة الدفاع. تم إجراء هذا البحث في حين أن الكتاب يتني A. غرين، ألبرتو مونيز وراميش ر Kaini عقد المجلس الوطني للبحوث ما بعد الدكتوراه البحوث الزمالة في USAISR. أجريت فحوصات ميكروأري من قبل Greehey طفولة مرفق معهد أبحاث السرطان ميكروأري الأساسية وإدارة المعلوماتية الحيوية في مركز العلوم الصحية UT سان انطونيو. وأيد هذا العمل من قبل الجيش الأمريكي برنامج السريرية التأهيلية الطب بحوث (CRMRP) وبرنامج بحوث الطب التشغيلية العسكرية (MOMRP).

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والكتاب ليس لديهم ما يكشف.

في الختام، ويصف هذا التقرير والأساليب المستخدمة في الثقافة خلايا الجذع، IPS-RPE، وRPE الجنين. RPE المستمدة من الجذع هي شكليا ووظيفيا مماثلة لRPE الجنين. يعبر عن IPS-RPE أيضا الجينات RPE مميزة بما في ذلك RPE65، CRALBP، PEDF، والانتقال بعيد المدى المحددة 16. لمزيد من تميز هذه الخلايا،...

الخلايا الجذعية لديها القدرة على تكرار بلا حدود والقدرة على التمايز إلى أي نوع من الخلايا الجسدية. وقد أثارت تطوير تقنيات لإعادة برمجة الخلايا الجسدية إلى خلايا الجذعية المحفزة الإثارة كبيرة في الأوساط البحثية وبين الأطباء، وظهور شخصية تجديد الأنسجة تلوح في الأفق 1. الجذعية المحفزة التي يسببها (آي بي إس) الخلايا يحمل نفس الميزات من إمكانات غير محدودة وتعدد القدرات تنسخي كما الجذعية الجنينية (ES) الخلايا في حين التحايل على المعضلات الأخلاقية المرتبطة المجالس الاقتصادية والاجتماعية. بالإضافة إلى ذلك، سوف الخلايا الجذعية المشتقة من المريض لا تحفز الاستجابة المناعية، مما يزيد كثيرا من احتمال التطبيقات العلاجية الناجحة 2-3. في ظل ظروف ثقافة محددة، وقد ثبت أن خلايا الجذع على التمايز إلى عدة أنواع مختلفة من الخلايا في المختبر، بما في ذلك العضلية، الخلايا العصبية، خلايا بيتا في البنكرياس، الكبد، وظهارة الصباغية الشبكية (RPE) 4-12. وRPE هو عبارة عن طبقة من الخلايا الظهارية المتخصصة المصطبغة تقع في الجزء الخلفي من شبكية العين التي تنفذ العديد من الوظائف التي تعتبر أساسية للصحة البصرية وظيفة مثل امتصاص الضوء الشارد، البلعمة من شرائح مبصرة الخارجي، وتجهيز الرتينوئيدات لإنتاج من حامل اللون البصرية. خلل في RPE بسبب الضرر أو مرض يؤثر تأثيرا عميقا الصحية مبصرة وظيفة البصرية كما يتضح من الأمراض المسببة للعمى التي هي نتيجة لالكامنة علم الأمراض RPE مثل الضمور البقعي المرتبط بالعمر (AMD)، ومرض ستارغاردت، والتهاب الشبكية الصباغي (RP) 13. لم تتحقق علاجات فعالة حقا أن يمكن استعادة الرؤية، واستبدال RPE المريضة مع RPE صحية قد يكون الخيار الأفضل لمنع فقدان البصر 14-15. RPE المستمدة من الجذع (IPS-RPE) هو مصدر ممكن من الخلايا لتحل محل RPE التالفة. IPS-RPE عن characterisالبروتينات RPE التشنج الانتقال بعيد المدى المحددة، CRALBP، PEDF، وRPE65؛ يعرض الكلاسيكية المصطبغة للغاية التشكل RPE سداسية؛ ويؤدي وظائف RPE مثل البلعمة، وتجهيز ريتينويد، وإفراز 11 - 5،16 الشبكية رابطة الدول المستقلة. ولكن، قبل IPS-RPE يمكن استخدامها علاجيا، وIPS-RPE يجب أن تتسم بدقة. فهم العوامل التي تحكم RPE التمايز ضروري لتحسين المحصول والنقاء من الخلايا لاستخدامها في التطبيقات السريرية. تمايز الخلايا هي نتيجة لدرجة عالية من التنظيم التعبير الجيني. ويلزم إعادة جينية من الجينوم وتنسيق عوامل النسخ لقرارات مصير الخلية التي تحدث أثناء تمايز والتنمية 17. تنظيم رسالة الحمض النووي الريبي (مرنا) الترجمة من قبل الرنا الميكروي (ميرنا) يقدم حتى الآن مستوى آخر من التنظيم الذي يؤثر على الخلايا مصير 1. MiRNAs قصيرة، ~ 22 الإقليم الشمالي، وأطوال من النيوكليوتيدات التي إما ترجمة قمع من قبلالربط إلى 3 &#39;UTR مرنا أو استهداف مرنا للتدهور. تم الكشف عن MiRNAs في جميع الأنسجة تقريبا وحتى الآن تم تسجيل أكثر من 2،000 microRNAs الإنسان فريدة من نوعها في قاعدة البيانات miRBase. منذ miRNAs تتطلب التكامل الجزئي فقط لربط مرنا الهدف، يمكن ميرنا واحدة يحتمل أن ربط عشرات أو مئات من الأهداف، والعكس بالعكس، أي ومرنا واحدة يمكن أن تكون مستهدفة من قبل العديد من miRNAs مختلفة. هذه الخاصية ملزمة منحل يزيد بشكل كبير من مستوى تعقيد التنظيم وكذلك مستوى صعوبة تحديد وظائف لل miRNAs الفردية ودور كل المسرحيات في الوظائف الخلوية 18-21. ومع ذلك، فقد أظهرت الدراسات أن تهذب ميرنا التعبير الجيني أثناء تمايز عن طريق التأثير في الوضع الحامض النووي 17. في دراسة أكثر تحديدا إلى RPE، وقد تبين miR-204/211 لتعزيز النمط الظاهري الظهارية من RPE 22. مجموعة أخرى بتحليل البيانات الشخصية ميرنامن RPE خلال تمايز الخلايا الجذعية الجنينية من وكشفت مجموعات متميزة من ميرنا وأعرب خلال عملية التمايز 23. في الواقع، يمكن تمييز ملامح ميرنا دون لبس أنواع الخلايا، بما في ذلك خلايا ES، خلايا السلائف، وخلايا متباينة عضال 24،25. وأعرب أكثر من 250 miRNAs في شبكية العين. استنادا إلى هذه الدراسات، ونحن نفترض أن miRNAs تلعب دورا هاما خلال تمايز RPE من الجذع. الهدف من هذا التقرير هو لوصف بروتوكولات للتمايز RPE من IMR90-4 خلايا الجذع، وجمع الحمض النووي الريبي لتحليلها من قبل ميكروأري، وتحليل البيانات ميكروأري لتحديد miRNAs التي أعرب تفاضلي بين ثلاثة أنواع الخلايا، وخلايا الجذع ، IPS-RPE وRPE الجنين. تم استخراج الحمض النووي الريبي مجموع من الثقافات من كل نوع من الخلايا والمهجنة لميكروأري ميرنا، التي تحتوي على تحقيقات محددة ل1،205 miRNAs الإنسان و144 miRNAs الفيروسية. تم مقارنة النتائج ميكروأريد لتحديد التي تم miRNAs وأعرب تفاضلي بين أنواع مختلفة من الخلايا. وقد تم اختيار miRNAs مع 2 أضعاف أضعاف أو أكبر تغيير في التعبير لمزيد من التحليل. تم استخدام برنامج حاسوبي لتحليل ميرنا تحديد أهداف محتملة للmiRNAs وأعرب تفاضلي لتوليد الشبكات الخلوية التي ينظمها miRNAs المحدد.

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">mTeSR1 media + 5X supplement</td>
			<td style="width:156px;">Stem Cell Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">M-7154</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:235px;">Non essential amino acids</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Knockout serum replacement</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Gentamicin&#160;</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">MEM media</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">10370-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">N1 supplement</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">N-6530-5ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Taurine</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T-8691-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hydrocortisone</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">H0888-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Fetal bovine serum</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>SH3008803HI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triiodo-l-thyronine sodium salt</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T6397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Sodium hydroxide</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal RPE media RTEGM kit</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">195406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Dispase</td>
			<td style="width:156px;">Life Technologies</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Matrigel</td>
			<td style="width:156px;">BD Biosciences</td>
			<td style="width:119px;">354277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Phosphate buffered saline</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Trypsin</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td style="width:119px;">25200-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec washing buffer</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-092-987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec rinsing buffer</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-092-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Anti-TRA-1-60 microbead kit</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-095-816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec cell sorter column</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-021-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RNeasy micro kit</td>
			<td style="width:156px;">Qiagen</td>
			<td style="width:119px;">74004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">QIA shredder</td>
			<td style="width:156px;">Qiagen</td>
			<td style="width:119px;">79654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RNA 6000 Pico LabChip kit</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">G2938-90046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Human miRNA microarray v16</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">G4471A</td>
			<td></td>
		</tr>
	</tbody>
</table>

microrna expression profiles of human ips cells, retinal pigment epithelium derived from ips, and fetal retinal pigment epithelium

الهدف من هذا التقرير هو لوصف بروتوكولات لمقارنة الرنا الميكروي (ميرنا) لمحات من الإنسان يسببها المحفزة الجذعية (آي بي إس) الخلايا، والظهارة الصبغية الشبكية (RPE) المشتقة من خلايا الجذع الإنسان (IPS-RPE)، وRPE الجنين. وتشمل البروتوكولات جمع الحمض النووي الريبي لتحليلها من قبل ميكروأري، وتحليل البيانات ميكروأري لتحديد miRNAs التي أعرب تفاضلي بين ثلاثة أنواع الخلايا. يتم شرح طرق لثقافة خلايا الجذع وRPE الجنين. كما يتم وصف بروتوكول يستخدم لتمايز RPE من الجذع الإنسان. وقد تم اختيار تقنية استخراج الحمض النووي الريبي وصفنا للسماح الانتعاش القصوى من الحمض النووي الريبي الصغيرة جدا للاستخدام في ميكروأري ميرنا. أخيرا، وأوضح مسار الخلوية وتحليل شبكة من البيانات ميكروأري. وهذه التقنيات تسهيل المقارنة بين ملامح ميرنا من ثلاثة أنواع مختلفة من الخلايا.

كانت تزرع خلايا الجذع (الشكل 1A) في ظروف التمايز للحث على التمايز إلى خلايا IPS-RPE. وIPS-RPE عرضت النمط الظاهري RPE الكلاسيكية من سداسية المصطبغة مورفولوجيا الخلايا (الشكل 1B) مماثلة لRPE الجنين (الشكل 1C). من أجل فه...

Watch this Scientific Journal Video about MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium at JoVE.com

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

والرنا الميكروي (ميرنا) لمحات من الجذعية المحفزة التي يسببها الإنسان (آي بي إس) الخلايا، والظهارة الصبغية الشبكية (RPE) المشتقة من الإنسان يسببها المحفزة الجذعية (آي بي إس) خلايا (IPS-RPE)، وRPE الجنين، وتمت مقارنة.

ملامح التعبير microRNA من خلايا الجذع الإنسان، الشبكية المشتقة من الظهارة الصبغية المحفزة، والجنين الظهارة الصبغية الشبكية

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

microrna-expression-profiles-human-ips-cells-retinal-pigment

Research

JoVE Journal

Biology

11.3K Views.  JBSA Fort Sam Houston.  والرنا الميكروي (ميرنا) لمحات من الجذعية المحفزة التي يسببها الإنسان (آي بي إس) الخلايا، والظهارة الصبغية الشبكية (RPE) المشتقة من الإنسان يسببها المحفزة الجذعية (آي بي إس) خلايا (IPS-RPE)، وRPE الجنين، وتمت مقارنة. 

Video: MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

سوبريتينال زرع الخلايا الظهارية الصبغية الشبكية المستمدة من الخلايا الجذعية الجنينية البشرية إلى نموذج كبير العينين من ضمور الجغرافية

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

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 ...

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 ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

الاستئصال بوساطة النيتروكتاز / ميترونيدازول ومنصة MATLAB (RpEGEN) لدراسة تجديد صبغة شبكية الزرد

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

الثقافة الأولية للخلايا الظهارية الصبغية الشبكية الخنازير

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

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 ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

عزل الخلايا الظهارية الصبغية الشبكية من عيون خنزير غينيا

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

نمذجة وتحليل تنكس الشبكية

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

تسليط الضوء على المؤلف: كشف التسبب في الضمور البقعي المرتبط بالعمر واكتشاف العلاجات المحتملة 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.