אנו מודים אוון Dysart ומארק לאונרד לסיוע מומחה טכני הפקת וידאו ועריכה, מאט קוק לסיוע באיסוף הנתונים, ושרה גלברד לסיוע מערכת מומחה. העבודה מומנה על ידי מענקים מהמכון ההפעלה הקנדי לבריאות מחקר (CIHR, MOP 62,626) כדי SALB, יוזמה אימון אסטרטגי ומחקר בריאות התוכנית להעניק SALB ו SF (CIHR תוכנית הכשרה Lipidomics ניווניות, TGF 9121), תשתיות תמיכה של הקרן הקנדית אונטריו חדשנות חדשנות אמון על המדע הבדיוני. ת&quot;י קיבל מלגת בוגר אונטריו במדע וטכנולוגיה עם תרומות של הקונסורציום לחקר מחלת הפרקינסון. NV מקבל המכון הזדקנות CIHR תוכנית הכשרה Lipidomics ניווניות שלאחר מקצועי המילגה. אנו בתודה להכיר תמיכה חינוכית תוכנה טכני המסופקים על ידי Autodesk המחקר.

<ol>
	<li>Kempermann, G., Jessberger, S., Steiner, B., Kronenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milestones+of+neuronal+development+in+the+adult+hippocampus.">Milestones of neuronal development in the adult hippocampus.</a> Trends Neurosci. 27, 447-452 (2004).</li><li>Campos, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurospheres:+insights+into+neural+stem+cell+biology.">Neurospheres: insights into neural stem cell biology.</a> J Neurosci Res. 78, 761-769 (2004).</li><li>Imbeault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+controls+gap+junction+protein+expression+and+function+in+postnatal+hippocampal+neural+progenitor+cells.">The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells.</a> BMC Neurosci. 10, 13-13 (2009).</li></ol>

כל הניסויים בבעלי חיים בוצעו בהתאם להנחיות והתקנות שנקבעו על ידי אוניברסיטת אוטווה Animal Care הוועדה והמועצה הקנדית על טיפול בבעלי חיים.

פרוטוקול זה מתאר הליך לתרבות סדרתי שלאחר הלידה cryosection NPCs בהיפוקמפוס, בתרגום ביטוי החלבון על ידי immunodetection, ולבסוף לשחזר ולנתח את המיקום הטופוגרפי של תאים immunopositive בתוך neurosphere 3D כולו. על ידי שילוב של ביולוגיה של התא תרבות וטכניקות עיבוד, הדמיה מיקרוסקופית (OpenLab, אימפרוביזציה ועוד תוכנות ניתוח התמונה), תוכנת עריכת גרפיקה יכולות (Adobe Photoshop), ו 3D אנימציה והתוכנה יכולות compositing (Autodesk Maya), אנו מציגים מתודולוגיה לשחזר את הרכב הסלולר כולו של תרבויות 3D מ 2 ד תמונות סידורי המאפשר שחזור נאמן של עמדת הסלולר ביטוי חלבון בכל תרבות neurosphere. פרוטוקול זה יכול להיות בשילוב עם יעילות שווה ההרשמה ושיקום epifluorescent סעיפים סדרתי דק או confocal Z-ערימות דרך קטעים סדרתי עבה. בגלל התרחבות המשובטים של NPCs אחת משחזר תרבות neurosphere רבים בשלבים נצפתה במהלך הנוירוגנזה gliogenesis במוח שלאחר הלידה, את ההשפעה של מבנה 3D שלה מייצג NPC חשוב גורל הקובע צאצאים שנחשפו בסביבה ניסויית אותו 1. סטייה בשושלת וביטוי חלבון הליבה והפריפריה סעיפים neurosphere סדרתי מציע כי גורמים פיזיים מרובים לרבות העמדה התא, לחץ מכני, וכוח גזירה תפקיד חשוב בוויסות NPC ביולוגיה 2. יתר על כן, הביטוי connexin חלבון, ניתחו כאן, הוכח לשנות בהתאם NPCs כאשר הם מתורבתים כמו neurospheres 3D של השעיה או מצופה על מצע laminin עם תקשורת connexin בתיווך פונקציונלי שונה אם התאים בתרבית על מצע פלסטיק או 3D בחינם צף תרבויות 3. ההשפעה של עמדת הסלולר על גורל NPC עדיין לא ברור. זה מאתגר מבחינה טכנית כדי לנתח את ההשפעה של מיקום מרחבי בתוך התרבות 3D על הביולוגיה NPC בהתחשב בכך הערכה antigenic השושלת ו איתות חלבונים דורש שיבוש של ארכיטקטורת 3D כדי לאפשר permeabilization התא נוגדנים גישה לכל התאים. כאן, אנו מראים כי מתודולוגיה להדמיה היברידית מספק אמצעי לקבוע אם חלבונים מסוימים התערוכה לגרסאות המגוירות מיקומית ייחודי בתרבות 3D, ניתן להשתמש בהם כדי להסיק ולבדוק תפקוד החלבון ופיקוח ביחס לוקליזציה אזוריים בתוך neurosphere, וגם, ואולי הכי חשוב , מספק את הנתונים מיקומית נדרש ללמוד את ההשפעה של הטופוגרפיה 3D ומיצוב הסלולר על גורל NPC בתרבות 3D.

* כל ריאגנטים כימיים אחרים סטנדרטיים מופיע בפרוטוקול הם מן סיגמא אולדריץ. כל שאר תקן תרבות ריאגנטים רקמה מן Invitrogen.

localizing protein in 3d neural stem cell culture: a hybrid visualization methodology

חשיבותה של טופוגרפיה (3D) 3 מימדי להשפיע גזע עצביים תא אב (NPC) הוא נרחב פנוטיפ הודה עדיין מאתגר ללמוד. כאשר ניתק מן המוח העוברי או שלאחר הלידה, NPCs יחיד יתרבו ההשעיה כדי ליצור neurospheres. לתאי הבת בתוך תרבויות אלה באופן ספונטני לאמץ שושלות התפתחותיים שונים (נוירונים, oligodendrocytes, האסטרוציטים ו) במהלך התרחבות למרות היותו חשוף הסביבה תאיים אותו. זה משחזר את התקדמות בשלבים רבים של שנצפה במהלך הנוירוגנזה gliogenesis במוח שלאחר הלידה והוא משמש לעתים קרובות ללמוד ביולוגיה NPC בתוך בסיסי בסביבה מבוקרת. הערכת ההשפעה המלאה של הטופוגרפיה 3D ומיצוב הסלולר בתוך תרבויות אלה על גורל NPC עם זאת, קשה. כדי למקם חלבונים היעד ולזהות שושלות NPC ידי immunocytochemistry, ריחוף ללא neurospheres חייב להיות מצופה על מצע או מחולק סדרתי. עיבוד זה נדרש כדי להבטיח permeabilization תא המקבילה וגישה נוגדן בכל תחום. כתוצאה מכך, תמונות של cryosections epifluorescent 2D או 3D שחזורים confocal של Z-ערימות יכול רק לספק מידע מרחבי על המיקום בתוך תא נפרד פרוסות 3D פיזי או דיגיטלי ולא לדמיין עמדה בתחום הסלולר ללא פגע. כאן, לחזור על הטופוגרפיה של התרבות neurosphere היתר ניתוח מרחבי של ביטוי חלבון לאורך התרבות כולה, אנו מציגים פרוטוקול לבידוד, הרחבה, חתך הסידורי של שלאחר הלידה neurospheres בהיפוקמפוס מתאים immunodetection epifluorescent או confocal של חלבונים היעד. Connexin29 (Cx29) הוא ניתח כדוגמה. בשלב הבא, באמצעות שילוב של עריכה גרפית ו מודלים 3D תוכנות ליישם בקפדנות כדי לשמור על פרט ביולוגיים, אנו מתארים כיצד להרכיב מחדש את המיקום המבני 3D של דימויים אלה מפה דיגיטלית תאים שכותרתו בתוך neurosphere המלא. מתודולוגיה זו מאפשרת הדמיה וניתוח של העמדה של חלבונים היעד תאים ברחבי הטופוגרפיה התרבות כולה 3D ותאפשר ניתוח מפורט יותר של יחסים מרחביים בין תאים במהלך הנוירוגנזה gliogenesis במבחנה. שני Imbeault ו Valenzuela תרמו באותה מידה יש ​​לשקול המחברים first משותף.

Watch this Scientific Journal Video about Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology at JoVE.com

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

כאן אנו מתארים כיצד לייצר, להתרחב, immunolabel תאים בהיפוקמפוס לאחר הלידה אבי עצביים (NPCs) בתרבות (3D) תלת ממדי. בשלב הבא, תוך שימוש בטכנולוגיות הדמיה היברידית, אנו מדגימים כיצד תמונות דיגיטליות של cryosections immunolabelled יכול לשמש כדי לשחזר ולמפות את המיקום המרחבי של תאים immunopositive ברחבי neurosphere 3D כולו.

חלבון לוקליזציה ב 3D תרבות גזע עצביים נייד: מתודולוגיה ויזואליזציה היברידי

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

localizing-protein-3d-neural-stem-cell-culture-hybrid-visualization

Research

JoVE Journal

Neuroscience

12.6K Views.  University of Ottawa. כאן אנו מתארים כיצד לייצר, להתרחב, immunolabel תאים בהיפוקמפוס לאחר הלידה אבי עצביים (NPCs) בתרבות (3D) תלת ממדי. בשלב הבא, תוך שימוש בטכנולוגיות הדמיה היברידית, אנו מדגימים כיצד תמונות דיגיטליות של cryosections immunolabelled יכול לשמש כדי לשחזר ולמפות את המיקום המרחבי של תאים immunopositiv...

Video: Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection והתרבות של נוירונים Commissural מחוט השדרה עובריים

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

גדילה והתמיינות של מבוגרים בהיפוקמפוס סנאי הארקטי קרקע תאים גזע עצביים

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

הקמת עכבר עובריים גזע עצביים תרבית תאים באמצעות Assay Neurosphere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

עצבית, המושבה יצירת Assay נייד: Assay להפלות בונה תאים בתום גזע עצביים מן ובתאים עצבית

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Assay Neuroblast: Assay לדור והעשרה של ובתאים העצבית הבחנה בין צאצאיהם גזע עצביים Cell שימוש cytometry זרימה

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

בידוד והתרבות של תאים עצביים לפסגה מהצינור עובריים עצבית Murine

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

ספירת בתאי גזע עצביים באמצעות מבחני המשובטים

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

פיתוחו של פרוטוקול מעודן עבור השתלת Subretinal טרנס-scleral של תאי אפיתל הפיגמנט ברשתית האנושי בעיניים עכברוש

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

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

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

דור פשוטה של תרבות תשואה גבוהה של נוירונים המושרה מ Fibroblasts עור מבוגר

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

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.