Name: efficient derivation of retinal pigment epithelium cells from stem cells
Uploaded: 2015-03-08T14:00:00.000+00:00
Duration: 7 min 7 s
Description: Watch this Scientific Journal Video about Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells at JoVE.com

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. בימוי התמיינות של הרשתית המופק מתאי גזע הערה: כל שלבי הדגירה מתבצעות על 37 מעלות צלזיוס ב 5% CO 2 <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשמור על קווי הס או ירכיים (להאכיל יומי) בתקשורת תחזוקה (MM; טבלת 1). ברגע שהקווים הגיעו סטנדרטים מספקים של בקרת איכות, לשמור אותם בשכבה של תאים מזינים עכבר עוברי (MEFs) זורעים בצפיפות של 250,000 / גם צלחת גם שש. יכולות גם להיות שנוצרו תרבויות ללא קסנו הבא הפרוטוקול שהוקם על ידי et al טאקר. 40. </li><li style=";text-align:right;direction:rtl"> לאפשר למושבות של תאי גזע להגיע confluency. </li><li style=";text-align:right;direction:rtl"> לעבור לתקשורת בידול עם Nicotinamide (DM / NIC; טבלת 1); להאכיל יומי. </li><li style=";text-align:right;direction:rtl"> אחרי שלושה שבועות בתרבות, לעבור לתקשורת בידול עם nicotinamide וגם Activin-או IDE-1 (DM / NIC / AA או DM / NIC / IDE1; טבלת 1) כדי לשפר את בידול הרשתית. </li&gt; <li style=";text-align:right;direction:rtl"> אחרי חמישה שבועות בתרבות, לחזור לDM / NIC. האכלות יומיות אינן נחוצות עוד פעם אינדיקטורים pH בתחנת התקשורת הופכת צהוב ביום שלאחר ההאכלה. </li><li style=";text-align:right;direction:rtl"> חכה לאיים של תאי פיגמנט להופיע כי הם גדולים מספיק כדי לחתוך ולהסיר (ראו איורים 2D-F ו3A). </li></ol> 2. איי פיגמנט בידוד <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מעיל הבארות של צלחת 24 גם עם מטריצה ​​המחקה את סביבת התא הטבעית (ללא קסנו עדיף). הערה: להבי קרנית ומלקחיים חדים, קנס שקצו הם מאוד שימושיים לקטיף. גיליון השלם של תאים מובחנים יכול לנתק תוך קטיף. למנוע זאת על ידי עבודה בזהירות ובהתחלת קטיף מושבות במרכז הצלחת. </li><li style=";text-align:right;direction:rtl"> מלא בארות רבות של הצלחת 24 גם עם תקשורת בידול (DM; טבלת 1) במידת צורך. החלטה זו תהיה תלויה במספר מושבות פיגמנטשנאסף. </li><li style=";text-align:right;direction:rtl"> במנדף תרבית תאים עם היקף לנתח, לחתוך באופן ידני את איי פיגמנט. לקצוץ את כל איון בתחתית הצלחת המקורית באמצעות אזמל בכ 2-6 חתיכות ולתפוס את חלקי פיגמנט עם מלקחיים חדים. </li><li style=";text-align:right;direction:rtl"> העבר 1-2 חתיכות פיגמנט לכל 24 גם. </li><li style=";text-align:right;direction:rtl"> אין לשנות תקשורת במשך 3-5 ימים, עד שתאים לדבוק, אז להתחיל לשנות מדיה שלוש פעמים בשבוע. (אם הם לא לדבוק בתום תקופה זו, הסבירות היא טובה כי הם לעולם לא). </li><li style=";text-align:right;direction:rtl"> להרחיב תאים במשך 3-4 שבועות בתקשורת בידול (DM; טבלת 1) - תמונה של תרבות טיפוסית מוצגת באיור 3. תאים עדיין לא יכולים למלא את כל טוב אבל מחכים כבר לא נראים כדי לעזור. להאכיל את תאים שלוש פעמים בשבוע. </li><li style=";text-align:right;direction:rtl"> לאחר כ 3 שבועות, להסיר באופן ידני אשכולות של תאים לבנים בגושים במידת צורך עם מלקחיים חדים. אל תעשו את הצעד הזה, אלא אם כן יש צורך - זה קל להציג את המזהמים. הוא אידיאלי לאנסה להשיג אוכלוסיות הטהורות ביותר של הרשתית ממושבות פיגמנט המקוריות בשלב 1.6. </li></ol> מקורן בתאי גזע 3. Passaging הרשתית <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ניתוק תאים עם 200 μl עם פתרון תא דיסוציאציה (רצוי לא טריפסין-מגיב כי יכול להיות מובטל בדילול עדיף) במשך 5-8 דקות על 37 מעלות צלזיוס, ולהשבית אותו על ידי דילול עם DM. השתמש פיפטה 200 μl כדי לשטוף את כל התאים וresuspend אותם (אם נבחר גם מכיל רק כמה תאים, שוקלים איגום התאים משני 24 בארות). </li><li style=";text-align:right;direction:rtl"> צנטריפוגה (800 XG במשך 5 דקות), להשליך supernatant, וגלול בתקשורת DM 3 מיליליטר. </li><li style=";text-align:right;direction:rtl"> תאים-צלחת מחדש מאחד (או שתיים) 24 בארות למטריצה ​​אחד 6-גם מצופה ב 3 מיליליטר של DM בכל טוב (1: 5 הרחבה). </li><li style=";text-align:right;direction:rtl"> להרחיב במשך 1-2 שבועות עד שהתאים הם ~ ומחוברות 90%; דמותה של התרבות הטהורה מובחנת באופן מלא מוצגת באיור 3 ג. </li><li style=";text-align:right;direction:rtl"> ניתוק תאים עם ד תא 1 מיליליטרפתרון issociation כ 5-8 דקות על 37 מעלות צלזיוס, להשבית אותו על ידי דילול עם DM, להשתמש 1,000 פיפטה μl כדי לשטוף את כל התאים, וresuspend אותם. </li><li style=";text-align:right;direction:rtl"> ספין למטה (800 XG במשך 5 דקות), להשליך supernatant, וגלול בתקשורת DM 18 מיליליטר. </li><li style=";text-align:right;direction:rtl"> תאים-צלחת מחדש מאחד 6-גם כל לשש מטריצה ​​מצופה 6-בארות ב 3 מיליליטר של DM בכל טוב (1: 6 הרחבה) או אחד מצופי 75cm 2 בקבוק (1: 7.5 הרחבה). </li><li style=";text-align:right;direction:rtl"> חזור על שלבים 3.4-3.8 צריכים, עד שמספיק תאים מתקבלים. הערה: נסה לאסוף מושבות רבות ככל האפשר להרחבה ולא מתכנן להגביר את התרבויות באמצעות passaging מאז כל קטע גורם dedifferentiation הרשתית. </li></ol>

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J., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+retinal+degenerative+diseases+with+human+iPS-derived+cells:+current+status+and+future+implications.">Modeling retinal degenerative diseases with human iPS-derived cells: current status and future implications.</a> Expert Review Of Ophthalmology. 8, 213-216 (2013).</li><li>Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cell+modeling+of+Best+disease:+insights+into+the+pathophysiology+of+an+inherited+macular+degeneration.">iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degeneration.</a> Hum Mol Genet. 22 (3), 593-607 (2013).</li><li>Buchholz, D. 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.

בכתב יד זה אנו מתארים את הצעדים כדי ליצור ביעילות מספר גדול של תרבויות iPS-הרשתית טהורות. הראינו בעבר שימוש בפרוטוקול זה בבימויו בידול עם Activin שאנחנו יכולים ליצור iPS-הרשתית שמאוד דומה לhRPE מבוסס על transcriptomics, פרוטאומיקה, metabolomics, ופונקציונליות, וכי הם לעכב ניוון רשתית כאשר ...

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.

<table><tbody><tr><td>Corneal knife&amp;nbsp;</td><td>Surgipro</td><td>SPOI-070</td><td>knife x 1</td></tr><tr><td>DMEM/F-12, HEPES</td><td>Life Technologies</td><td>11330-032</td><td>500 ml x 4&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, 1X w/out Ca or Mg</td><td>VWR</td><td>45000-434</td><td>500 ml x 6</td></tr><tr><td>Fetal Bovine Serum, Regular (Heat Inactivated)</td><td>VWR</td><td>45000-736</td><td>500 ml x 1</td></tr><tr><td>FGF-Basic (AA 10-155) Recombinant Human Protein</td><td>Life Technologies</td><td>PHG0021</td><td>100 &amp;micro;g x 1</td></tr><tr><td>IDE-1</td><td>Stemgent</td><td>04-0026</td><td>2 mg x 1</td></tr><tr><td>Knockout DMEM</td><td>Life Technologies</td><td>10829-018</td><td>500 ml x 1&amp;nbsp;</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>10828-028</td><td>500 ml x 1</td></tr><tr><td>L-Glutamine 200 mM&amp;nbsp;</td><td>Life Technologies</td><td>25030-081</td><td>100 ml x 1</td></tr><tr><td>MEM Non-Essential Amino Acids Solution 100X&amp;nbsp;</td><td>Life Technologies</td><td>11140-050</td><td>100 ml x 1</td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636-100G</td><td>100 g x 1</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-148</td><td>20 ml x 1</td></tr><tr><td>Recombinant Human/Murine/Rat Activin A&amp;nbsp;</td><td>PeproTech</td><td>120-14E</td><td>10 &amp;micro;g x 2</td></tr><tr><td>Synthemax-T Surface 6 Well Plates</td><td>Corning</td><td>3877</td><td>Case(12) x 1</td></tr><tr><td>TrypLE-Express Enzyme (1X), no phenol red&amp;nbsp;</td><td>Life Technologies</td><td>12604-021</td><td>500 ml x 1&amp;nbsp;</td></tr><tr><td>Vacuum Filter/Storage Bottle System, 0.1&amp;micro;m pore, 500ml&amp;nbsp;</td><td>Corning</td><td>431475</td><td>Case(12) x 1&amp;nbsp;</td></tr></tbody></table>

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.

הצעדים המפורטים בכתב היד הזה, כפי שהוא מתואר באיור 1, יכולים לשמש כדי ליצור בקלות הרשתית מתאי גזע כפי שדווחו בעבר 10,12. לאחר שמירת קווי iPS במשך כמה שבועות, מושבות פיגמנט יתחילו להופיע במושבות לאחר 5-7 שבועות (תרבויות בן 7 שבוע מוצגות באיור 2 א-ג). מוש?...

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.

גזירה יעילה של תאי הרשתית פיגמנט האפיתל מתאי גזע

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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

9.2K 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

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.

תאים (iPS) נובע רשתית פיגמנט האפיתל (RPE) מגזע המושרה pluripotent על ידי בגדלים שונים של גופי Embryoid

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

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.

הבידול מהירה, מכוונת של תאים אפיתל הפיגמנט ברשתית מתאי גזע Pluripotent האנושי עובריים או מושרה

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

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

קסנו נטולת מוגדר ותנאים מזין ללא תרבות לדגמי הדור של האדם נגזר iPSC רשתית תא

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

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.

הנדסת רקמות אפיתל הפיגמנט ברשתית השתלת-מתאים נגזר תאי גזע עובריים

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

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

ביצוע subretinal זריקות במכרסמים כדי לספק תאי הרשתית פיגמנט האפיתל בהשעיה

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Medicine

Isolation of Retinal Stem Cells from the Mouse Eye

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 non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

בידוד של גזע תאים רשתית העין של העכבר

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 ...

Biology

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

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

פרופילי הביטוי microRNA של תאי iPS האנושי, רשתית פיגמנט האפיתל נגזר משב&quot;ס, ועוברי הרשתית פיגמנט האפיתל

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

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

מערכת אינדוקציה אורגנויד רשתית להפקה של רקמות רשתית תלת-ממדיות מתאי גזע פלוריפוטנטיים אנושיים

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

השתלת אפיתל פיגמנט רשתית במודל פרימטים לא אנושי למחלות רשתית ניוונית

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

אפיון ודינמיקה מורפולוגית של RPE מורין ראשוני מתורבת

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

מחבר זרקור: בידוד יעיל של תאי אפיתל פיגמנט רשתית ראשוניים מעכברים בוגרים לחקר רטינופתיה

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

שיטה פשוטה לבידוד ותרבית של תאי אפיתל פיגמנט ברשתית מעכברים בוגרים

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...