We wish to thank the following individuals: Drs. Tim Krohne and Eyal Banin (along with Dr. Mandy Lehmann and David Friedlander) for generous help developing the differentiation protocols. Dr. Felicitas Bucher provided assistance differentiating the RPE cells used in this study. We also acknowledge the National Eye Institute (NEI grants EY11254 and EY021416), California Institute for Regenerative Medicine (CIRM grant TR1-01219), and the Lowy Medical Research Institute (LMRI) for very generous funding for this project.

1. إخراج تمايز الخلايا الجذعية المشتقة من خلية RPE ملاحظة: يتم تنفيذ جميع الخطوات حضانة بها في 37 درجة مئوية في 5٪ CO 2 <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحفاظ على حس أو الوركين خطوط (إطعام يوميا) في وسائل الإعلام الصيانة (MM، الجدول 1). مرة واحدة وقد وصلت خطوط معايير كافية من مراقبة الجودة، والمحافظة عليها على طبقة من الفأر الجنينية الخلايا المغذية (MEFS) المصنف في مناطق ذات كثافة من 250،000 / بئر من لوحة جيدا ستة. ويمكن أيضا أن تتولد الثقافات خالية من كره بعد بروتوكول التي وضعتها تاكر وآخرون. 40. </li><li style=";text-align:right;direction:rtl"> السماح للمستعمرات الخلايا الجذعية للوصول إلى confluency. </li><li style=";text-align:right;direction:rtl"> التحول إلى وسائل الإعلام التمايز مع نيكوتيناميد (DM / NIC، الجدول 1)؛ تغذية يوميا. </li><li style=";text-align:right;direction:rtl"> بعد ثلاثة أسابيع في الثقافة، والتحول إلى وسائل الإعلام التمايز مع نيكوتيناميد وإما Activin-A أو IDE-1 (DM / NIC / AA أو DM / NIC / IDE1، الجدول 1) لتعزيز RPE التمايز. </lI&gt; <li style=";text-align:right;direction:rtl"> بعد خمسة أسابيع في الثقافة، والتبديل إلى DM / NIC. الوجبات اليومية لم تعد ضرورية مرة واحدة المؤشرات درجة الحموضة في توقف وسائل الإعلام تحول الأصفر اليوم بعد الرضاعة. </li><li style=";text-align:right;direction:rtl"> انتظر الجزر الخلايا الصباغية للمثول التي تكون كبيرة بما يكفي لخفض وإزالة (انظر الشكلين 2D-F و3A). </li></ol> 2. عزل الصباغي الفشوت <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> معطف الآبار من 24 لوحة جيدا مع مصفوفة الذي يحاكي البيئة الطبيعية خلية (خالية من كره هو الأفضل). ملاحظة: شفرات القرنية وحادة، ملقط غرامة ذات الرؤوس هي مفيدة جدا للاختيار. ورقة كاملة من خلايا متمايزة يمكن فصل بينما نقطف. تجنب ذلك من خلال العمل بعناية والبداية اختيار المستعمرات في وسط الطبق. </li><li style=";text-align:right;direction:rtl"> شغل العديد من الآبار من لوحة 24-جيدا مع وسائل الإعلام التمايز (DM، الجدول 1) حسب الحاجة. هذا القرار سيعتمد على عدد من المستعمرات المصطبغةالتي تم جمعها. </li><li style=";text-align:right;direction:rtl"> في غطاء ثقافة الخلية مع نطاق تشريح، وقطع يدويا من الجزر المصطبغة. تقطيع كل جزيرة في الجزء السفلي من لوحة أصلية باستخدام مشرط في حوالي 2-6 قطع والاستيلاء على أجزاء مصطبغة مع ملقط حادة. </li><li style=";text-align:right;direction:rtl"> نقل 1-2 قطعة المصطبغة في كل 24 عاما أيضا. </li><li style=";text-align:right;direction:rtl"> لا تغيير وسائل الاعلام لمدة 3-5 أيام حتى خلايا تلتزم، ثم البدء في تغيير وسائل الإعلام ثلاث مرات في الأسبوع. (اذا لم تلتزم بعد هذا الوقت، واحتمال جيد بأنهم سوف أبدا). </li><li style=";text-align:right;direction:rtl"> توسيع الخلايا لمدة 3-4 أسابيع في وسائل الإعلام التمايز (DM، الجدول 1) - يتم عرض صورة للثقافة نموذجية في الشكل 3B. الخلايا قد لا يزال غير ملء البئر كله ولكن الانتظار لفترة أطول لا يبدو للمساعدة. تغذية خلايا ثلاث مرات في الأسبوع. </li><li style=";text-align:right;direction:rtl"> بعد حوالي 3 أسابيع، يدويا إزالة مجموعات من الخلايا البيضاء في كتل إذا لزم الأمر مع ملقط حادة. لا تفعل هذه الخطوة إلا عند الضرورة - فمن السهل أن إدخال الملوثات. وهو مثالي لمحاولة للحصول على أنقى سكان RPE من المستعمرات المصطبغة الأصلية في الخطوة 1.6. </li></ol> 3. الركض الخلايا الجذعية المستمدة RPE <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فصل الخلايا مع 200 ميكرولتر مع الحل تفارق الخلية (ويفضل أن لا التربسين-كاشف التي يمكن المعطل من خلال التخفيف هو الأفضل) لمدة 5-8 دقائق عند 37 درجة مئوية، وتعطيل من قبل تمييع مع DM. استخدام ماصة 200 ميكرولتر ليغسل كل الخلايا ول resuspend لهم (إذا كان اختيار جيد يحتوي سوى عدد قليل من الخلايا، والنظر في تجميع الخلايا من اثنين من 24 بئرا). </li><li style=";text-align:right;direction:rtl"> الطرد المركزي (800 x ج لمدة 5 دقائق)، ونبذ طاف، و resuspend في 3 مل سائل الإعلام DM. </li><li style=";text-align:right;direction:rtl"> خلايا إعادة لوحة من واحد (أو اثنين) 24 آبار في مصفوفة واحدة المغلفة 6 جيدا في 3 مل من DM لكل بئر (1: 5 التوسع). </li><li style=";text-align:right;direction:rtl"> توسيع لمدة 1-2 أسابيع حتى كانت الخلايا ~ 90٪ متموجة. ويظهر صورة لثقافة نقية متباينة تماما في الشكل 3C. </li><li style=";text-align:right;direction:rtl"> فصل الخلايا مع 1 مل خلية دحل issociation لمدة 5-8 دقائق عند 37 درجة مئوية، على تعطيل من قبل التخفيف مع DM، استخدم 1،000 ماصة ميكرولتر ليغسل كل الخلايا، و resuspend لهم. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار (800 x ج لمدة 5 دقائق)، ونبذ طاف، و resuspend في 18 مل سائل الإعلام DM. </li><li style=";text-align:right;direction:rtl"> خلايا إعادة لوحة واحدة من 6 جيدا كل في ستة مصفوفة المغلفة 6 آبار في 3 مل من DM لكل بئر (1: 6 التوسع) أو المغلفة واحد 75CM 2 قارورة (1: 7.5 التوسع). </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 3،4-3،8 حسب الضرورة حتى يتم الحصول على ما يكفي من الخلايا. ملاحظة: في محاولة لجمع أكبر عدد ممكن من المستعمرات ممكن للتوسع بدلا من التخطيط لتضخيم الثقافات عبر الركض لأن كل مرور يدفع RPE فقد التمايز. </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+directed+differentiation+of+human+pluripotent+stem+cells+into+retinal+pigmented+epithelium.">Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium.</a> Stem Cells Transl Med. 2 (5), 384-393 (2013).</li><li>Fuhrmann, S., Levine, E. M., Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraocular+mesenchyme+patterns+the+optic+vesicle+during+early+eye+development+in+the+embryonic+chick.">Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick.</a> Development (Cambridge, England). 127 (21), 4599-4609 (2000).</li><li>Borowiak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+efficiently+direct+endodermal+differentiation+of+mouse+and+human+embryonic+stem+cells.">Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells.</a> Cell Stem Cell. 4 (4), 348-358 (2009).</li><li>Tucker, B. A., Anfinson, K. R., Mullins, R. F., Stone, E. M., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+synthetic+xeno-free+culture+substrate+for+induced+pluripotent+stem+cell+induction+and+retinal+differentiation.">Use of a synthetic xeno-free culture substrate for induced pluripotent stem cell induction and retinal differentiation.</a> Stem Cells Transl Med. 2 (1), 16-24 (2013).</li><li>Ramsden, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+retinal+regeneration:+past,+present+and+future.">Stem cells in retinal regeneration: past, present and future.</a> Development (Cambridge, England). 140 (12), 2576-2585 (2013).</li><li>Zhao, T., Zhang, Z. -. N., Rong, Z., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+induced+pluripotent+stem+cells.">Immunogenicity of induced pluripotent stem cells.</a> Nature. 474 (7350), 212-215 (2011).</li><li>Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+human+fibroblasts+into+retinal+pigment+epithelium-like+cells+by+defined+factors.">Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors.</a> Protei., &amp; Cell. 5 (1), 48-58 (2013).</li></ol>

None of the authors have any commercial disclosures to declare.

في هذه المخطوطة نحن الخطوط العريضة الخطوات لتوليد بكفاءة أعداد كبيرة من الذهب الخالص الثقافات IPS-RPE. لقد أظهرنا سابقا باستخدام هذا البروتوكول التمايز الموجهة مع Activin A أننا يمكن أن تولد IPS-RPE التي تشبه بقوة hRPE على أساس transcriptomics، البروتينات، الايض، وظائف، وأنها تؤخر تنك?...

The various cell types that occupy the retina are organized in a functional architecture. The photoreceptors in the back of the retina are responsible for converting light into electrical impulses through phototransduction. However, phototransduction cannot occur without the coordinated efforts of other neighboring cell types including Mueller glia and retinal pigment epithelium (RPE) cells. A monolayer of RPE cells partitions the sensory retina from the choriocapillaris, the primary blood supply for photoreceptors, and are ideally situated to control multiple functions important for photoreceptor homeostasis. In fact, the RPE and photoreceptors are so co-dependent they are widely considered to be one single functional unit. (For a review of all the diverse functions of the RPE see Strauss, 20051.) Death or dysfunction of retinal pigment epithelium cells can induce age-related macular degeneration (AMD), the leading cause of permanent vision loss in industrialized countries2-4. 
AMD is a multifactorial disease of RPE, photoreceptors, and the choroidal vasculature; risk factors are diverse and include combinations of environmental and genetic influences5,6. Treatments for AMD are very limited, but one promising potential therapy is RPE cell replacement7,8. While the outcomes have been mixed, the transplantation of RPE cells in AMD patients (and in other patients with retinal degeneration) and also in rodent models of retinal degeneration, has the potential to transiently prevent significant photoreceptor atrophy9-23. (The animal model commonly used for these studies are Royal College of Surgeons (RCS) rats, which harbor a mutation in the MerTK gene. This mutation renders RPE cells incapable of phagocytosing photoreceptor outer segments and promotes retinal degeneration24.) While the reported survival rates of implanted RPE in the subretinal space of RCS rats and mice vary greatly, there is potential for them to survive for several months or years9,10,12,20. 
RPE cells can be obtained in sufficient numbers for transplantation by deriving them from pluripotent stem cells9-14,25-28. Several independent groups have demonstrated that these cells function in similar ways to their somatic counterparts, and long term studies suggest that they are safe upon implantation in rat and mouse disease models9,10,12,14,19,20,25,29-32. The use of induced pluripotent stem cells instead of embryonic stem cells may be advantageous since ethical issues and immunological challenges associated with allogeneic RPE may be obviated33,34. Another exciting application for iPS technology is disease modeling35. The ability to interrogate large numbers of RPE cells derived from patients with RPE diseases could be invaluable for understanding their molecular bases. This type of study has been performed recently with Best disease patient RPE and could pave the way for similar studies of inherited maculopathies36. 
The derivation of RPE from stem cells is a relatively simple process and can be done entirely in xeno-free conditions. The simplest strategy is to derive monolayers of RPE cells spontaneously, however, the yield can be significantly improved using directed differentiation techniques. But these techniques involve the use of exogenous protein factors that can be expensive and often generated in bacteria or other non-human sources10,12,37. In our studies we followed an established protocol10 that utilizes nicotinamide and Activin A, a signaling factor that has been shown to be sufficient for RPE specification38. Here we will demonstrate that the small molecule IDE-1 can adequately replace Activin A, thus reducing costs and alleviating concerns associated with the use of recombinant proteins39. Additionally, we utilize xeno-free serum replacement, and we culture the differentiating RPE cells on a synthetic xeno-free substrate. RPE cells have been shown previously to differentiate very effectively using this approach40.
When differentiated as a monolayer, we visualize pigmented colonies containing RPE cells after as early as five weeks12. Once they reach sufficient size, they can be manually excised and transferred to another dish for expansion. RPE cells are notorious for dedifferentiating with each passage, and the use of anything older than five passages should be avoided (we find that sufficient numbers of cells for characterization and transplantation in animal models can easily be generated after two or three passages). Once fully differentiated, we employ multiple techniques to characterize the cells anatomically and functionally to ensure that they will serve as adequate replacements for diseased RPE. The description of these techniques, and protocol for implanting the iPS-RPE in the subretinal space of rodents, are beyond the scope of this methods paper and have been previously published12,32,41. 
While developing standardized protocols for effective derivation of iPS-RPE is clearly important for the clinics, there is also significant preclinical work to still be done in animal models. There are concerns regarding immunogenicity of iPS-derived cells, and multiple different implantation techniques, including implanting cells on artificial substrates, are being explored42,43. For these reasons, we feel that the publication of standardized protocols is beneficial to facilitate both clinical and preclinical studies. Especially if direct comparisons will be done of iPS-RPE cells derived in different labs by different research groups.

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			<td height="21" style="height:21px;width:419px;">Recombinant Human/Murine/Rat Activin A&#160;</td>
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			<td height="21" style="height:21px;width:419px;">Synthemax-T Surface 6 Well Plates</td>
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			<td height="21" style="height:21px;width:419px;">TrypLE-Express Enzyme (1X), no phenol red&#160;</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">12604-021</td>
			<td>500 mL x 1&#160;</td>
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			<td height="21" style="height:21px;width:419px;">Vacuum Filter/Storage Bottle System, 0.1&#181;m pore, 500mL&#160;</td>
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			<td style="width:119px;">431475</td>
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efficient derivation of retinal pigment epithelium cells from stem cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

الخطوات الموضحة في هذه المخطوطة، كما هو مبين في الشكل رقم 1، ويمكن استخدامها لتوليد بسهولة RPE من الخلايا الجذعية كما ذكرت سابقا 10،12. بعد الحفاظ على خطوط المحفزة لعدة أسابيع، المستعمرات الصباغية تبدأ في الظهور في المستعمرات بعد 5-7 أسابيع (وترد الثقافات ?...

Watch this Scientific Journal Video about Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells at JoVE.com

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

اشتقاق كفاءة خلايا الظهارة الصبغية الشبكية من الخلايا الجذعية

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

efficient-derivation-retinal-pigment-epithelium-cells-from-stem

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JoVE Journal

Developmental Biology

9.1K Views.  The Scripps Research Institute. Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Video: Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

اشتقاق خلايا القلب السلف من الخلايا الجذعية الجنينية

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

المستمدة الشبكية الظهارة الصبغية (RPE) من المستحثة متعددة القدرات الجذعية (آي بي إس) الخلايا عن طريق أحجام مختلفة من الهيئات مضغي

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

اشتقاق خالية المغذية من الخلايا الصباغية من الخلايا الجذعية متعددة القدرات البشرية

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

إنتاج الخلايا الجذعية المحفزة من الفأر الأمنيوسي الخلايا السوائل عن طريق نظام ينقول

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

أفقي جبل كامل: A معالجة الرواية والتصوير بروتوكول للثدي، ثلاثة-- الأبعاد الأنسجة عبر-- أقسام الجلد

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

التفريق بين الخلايا الظهارية الصبغية الشبكية من الخلايا الجذعية Pluripotent المستحث أو الجنينية البشرية سريعة وموجهة

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

فوتوكونفيرسيون خلية واحدة في معيشة سليمة الزرد

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

التحليل الحسابي ل Germline ايليجانس كاينورهابديتيس دراسة توزيع الأنوية، والبروتينات، وسيتوسكيليتون

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

الاشتقاق وتمايز الخلايا الجذعية الوسيطة المبيض الكلاب

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

هندسة الأنسجة ظهارة صباغ الشبكية زرع مناسبة المستمدة من الخلايا الجذعية الجنينية البشرية

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...