Le soutien et les conseils de Markus et Robert Pajtler Bohrer ont été inestimables pour la visualisation de ce protocole.

Les auteurs indiquent que les expériences sur les animaux ont été effectuées en conformité avec les Communautés européennes la directive du Conseil (86/609/CEE), suivant les directives du NIH concernant les soins et l&#39;utilisation d&#39;animaux pour des procédures expérimentales et les règlements énoncés par le comité institutionnel soin et l&#39;utilisation comité (IACUC) à l&#39;Université de Duisburg-Essen (Allemagne). Partie 1: Configuration (non montré sur la vidéo) PRÉPARATION DES MATÉRIAUX ET SOLUTIONS (avant de prendre les oeufs de l&#39;incubateur) <ul><li> Les œufs ont à incuber dans un incubateur humidifié pendant 11 jours à 37 ° C (100 ° F). </li><li> De pulvérisation d&#39;éthanol à 70%, des assiettes propres, précis pince propre, 4 ° C (40 ° F) à froid stérile DMEM sur la glace et 60 ml seringues stériles </li><li> 50 ml stériles tubes Corning doivent être à moitié rempli avec 4 ° C à froid DMEM à mélanger les embryons macérés dans un rapport de 1:1 (en masse macérée: DMEM). Dès que les œufs sont sortis de l&#39;incubateur de la dissection des embryons de poussins doit être démarré. </li><li> Stériliser les outils de dissection à l&#39;autoclave ou le jour de la dissection, immerger les outils dans l&#39;éthanol 70% pendant 20 minutes. </li></ul> Partie 2: Dissection de l&#39;embryons de poulet (montré sur la vidéo) <ol><li> Mouiller les oeufs incubés avec un spray d&#39;éthanol à 70% pendant au moins 30 secondes et les sécher soigneusement avec du papier absorbant propre molle tout en ne serrant les oeufs de façon excessive. Note: Nous recommandons de ne pas disséquer plus de 60 embryons de poulet simultanément. </li><li> Ouvrez les oeufs avec soin par s&#39;écrasant sur un bord tranchant et sortir l&#39;embryon avec prudence ne pas détruire le sac jaune ou l&#39;embryon de poulet lui-même. </li><li> Recherche pour le début du cordon ombilical et rapidement ouvert l&#39;emballage claires sans détruire le sac vitellin ou tout petits vaisseaux. Puis décortiquer le cordon ombilical avec une pince précise propre. </li><li> Prenez l&#39;embryon hors du sac jaune. Remarque: Les embryons doivent être décapité rapidement (pas montré dans la vidéo). </li><li> Recueillir les embryons dans un milieu essentiel minimal à 4 ° C. </li></ol> Partie 3: Macération des embryons de poulet <ol><li> Prenez le piston d&#39;une seringue stérile de 60 ml. </li><li> Transférer environ 10 embryons dans un 60 mL. </li><li> Remettre le piston attentivement dans la seringue afin de ne pas risquer de perdre les produits de macération. </li><li> Laisser macérer en appuyant sur le piston. </li><li> Recueillir la masse macérée dans les tubes Corning préparés à moitié rempli avec du DMEM à un ratio de 1:1 (en masse macérée: DMEM). En utilisant 10 embryons un volume d&#39;environ 25 ml est produite. </li><li> Placer le mélange sur un agitateur pendant 45 min à 4 ° C. </li></ol> Partie 4: La centrifugation de l&#39;embryon de poulet - Suspension du DMEM <ol><li> Transfert l&#39;embryon de poulet - la suspension du DMEM dans les tubes de centrifugation. </li><li> Ajouter hyaluronidase stérile à une concentration de 1 mg par 25 mg d&#39;embryon. </li><li> Tare des tubes de centrifugation très précisément. Note: Cette étape doit être effectuée avec prudence que la centrifugation est géré au G-Force très élevée et des dommages matériels ou centrifugeuse pourrait se produire si l&#39;aide de tubes déséquilibré. Avec une précision de pesage de haute selon la machine sur les trois positions après la virgule pour tous les tubes doivent être reçus en ajustant le montant manquant avec du DMEM. </li><li> Réduire la température de la centrifugeuse à 4 ° C. Remarque: outre le rotor de centrifugation doivent être stockés dans une chambre de refroidissement ou d&#39;un réfrigérateur pendant la nuit. </li><li> Centrifugeuse 6 heures au moins pour 180.000 XGX h. Note: Pour de meilleurs résultats concernant la séparation des phases à usage unique 266,000 XGX h. Si possible, utilisez le réglage vide. </li></ol> Partie 5: filtration de l&#39;extrait d&#39;embryon de poulet Après la centrifugation, trois phases peuvent être remarqué: La phase supérieure miteux liquide avec des fragments de gras, la phase liquide clair au milieu et le culot au fond du tube de centrifugation. Remarque: Secouer ou le déplacement rapide du mélange doit être centrifugé strictement évités, car cela détruirait le résultat de l&#39;étape de centrifugation en perdant la phase liquide clair central. Note: Toutes les étapes suivantes doivent être effectuées en conditions stériles sous un banc de flux d&#39;air laminaire: <ol><li> Pipeter la phase liquide clair dans un système de filtre avec des pores de 0,45 um. Puis passer à la pompe à vide connectée. Note: Ne pas toucher le culot au fond. Cela conduirait à une contamination de l&#39;extrait et un blocage du système de filtration. </li><li> Transvaser l&#39;extrait pré-dans un système de filtre avec des pores de 0,22 um et l&#39;interrupteur sur la pompe à vide reliée à nouveau. </li><li> Aliquoter l&#39;extrait d&#39;embryon de poulet atteintdans les stériles de 15 mL tubes Corning. </li><li> Stocker rapidement les aliquotes à -80 ° C (-62 ° F). Note: La dernière pour aller jusqu&#39;à deux ans PECO s&#39;il est conservé à -80 ° C. </li></ol>

<ol>
	<li>Bronner-Fraser, M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+reveals+multipotency+of+some+avian+neural+crest+cells.">Cell lineage analysis reveals multipotency of some avian neural crest cells.</a> Nature. 335, 161-164 (1988).</li><li>Stemple, D. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Rao, M. S., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+controlled+in+vitro+differentiation+of+murine+multipotent+neural+crest+stem+cells.">Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.</a> J Neurobiol. 32, 722-746 (1997).</li><li>Paratore, C., Goerich, D. E., Suter, U., Wegner, M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+glial+fate+acquisition+of+neural+crest+cells+are+regulated+by+an+interplay+between+the+transcription+factor+Sox10+and+extrinsic+combinatorial+signaling.">Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling.</a> Cell. 128, 3949-3961 (2001).</li><li>Kim, J., Lo, L., Dormand, E., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX10+maintains+multipotency+and+inhibits+neuronal+differentiation+of+neural+crest+stem+cells.">SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells.</a> Neuron. 38, 17-31 (2003).</li><li>Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao, M., Shah, N. M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+determination+and+the+control+of+neuronal+identity+in+the+neural+crest.">Cell lineage determination and the control of neuronal identity in the neural crest.</a> Review Cold Spring Harb Symp Quant Biol. 62, 493-504 (1997).</li><li>Dorsky, R. I., Moon, R. T., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+and+cell+fate+specification+in+premigratory+neural+crest.">Environmental signals and cell fate specification in premigratory neural crest.</a> Review Bioessays. 22, 708-716 (2000).</li><li>Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+lineage+specification+in+the+neural+crest.">Factors controlling lineage specification in the neural crest.</a> Int Rev Cytol. 197, 1-33 (2000).</li><li>Dupin, E., Real, C., Ledouarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+crest+stem+cells:+control+of+neural+crest+cell+fate+and+plasticity+by+endothelin-3.">The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3.</a> Int Rev Cytol. 73, 533-545 (2001).</li><li>Maurer, J., Fuchs, S., J&#228;ger, R., Kurz, B., Sommer, L., Schorle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+controlled+differentiation+of+neural+crest+stem+cell+lines+using+conditional+transgenesis.">Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis.</a> Differentiation. 75, 890-891 (2007).</li><li>Wu, Y. Y., Mujtaba, T., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stem+and+precursor+cells+from+fetal+tissue.">Isolation of stem and precursor cells from fetal tissue.</a> Methods Mol Biol. 198, 29-40 (2002).</li></ol>

Ce protocole est basé sur les modifications de procédures qui ont été décrites dans le passé, 10,11. En plus des publications anciennes, nous montrent ici les différentes étapes différentes du processus plus étendu. Cela soutient l&#39;expérimentateur avec des détails importants assurant une procédure correcte et des résultats fiables. En outre, comme le processus de dissection est une partie essentielle de l&#39;extraction de l&#39;ECO, nous décrivons la procédure de prise des embryons hors d...

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<td>11 days incubated chicken eggs</td>
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<td>S&ouml;rries-Trockels Vermehrungszucht GmbH &amp; Co.KG</td>
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<td>Alcoholic disinfection spray</td>
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<td>Sch&uuml;lke &amp; Mayr GmbH</td>
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<td>DMEMGlutamax</td>
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<td>GIBCO Invitrogen</td>
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<td>Syringe sterile (60 ml)</td>
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<td>BD Biosciences</td>
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<td>Tubes sterile (50; 15 ml)</td>
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<td>Greiner Bio-One GmbH</td>
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<td>Pipet tips sterile</td>
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<td>Starlab GmbH</td>
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<td>Hyaluronidase typeIV-S</td>
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<td>Sigma-Aldrich</td>
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<td>Milipore stericups sterile (0.45; 0.22 &mu;l)</td>
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<td>Fisher Scientific AG</td>
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<td>Pipettes sterile (25; 10; 5 ml)</td>
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<td>Greiner Bio-One GmbH</td>
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<td>Centrifuge L5-50</td>
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<td>Rotor type Ti-45</td>
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production of chick embryo extract for the cultivation of murine neural crest stem cells

La crête neurale provient de la neuro-ectoderme cours de l&#39;embryogenèse et persiste seulement temporairement. Les premières expériences déjà épreuve cellules progénitrices pluripotentes à être une partie intégrante de la crête neurale 1. Phénotypiquement, les cellules souches neurales de crête (NCSC) sont définis par exprimant simultanément p75 (bas-affine récepteur du facteur de croissance nerveuse, LNGFR) et SOX10 pendant leur migration de la crête neurale 2,3,4,5. Ces cellules progénitrices peuvent se différencier en cellules musculaires lisses, cellules chromaffines, neurones et cellules gliales, ainsi que les mélanocytes, les cartilages et les os 6,7,8,9. Pour cultiver NCSC in vitro, une moyenne spéciale cellules de la crête neurale tige (NCSCM) est requise 10. La partie la plus complexe de la NCSCM est la préparation d&#39;extrait d&#39;embryon de poulet (PECO) qui représente une source essentielle de facteurs de croissance pour le NCSC ainsi que pour d&#39;autres types d&#39;explants de neurones. Autres ingrédients NCSCM côté PECO sont disponibles commercialement. Produire du CCE de laboratoire utilisant un équipement standard, il est de grande importance pour connaître les détails difficiles que l&#39;isolement, la macération, la centrifugation et la filtration. Dans ce protocole, nous décrivons des techniques précises pour produire une quantité maximisée de qualité pure et de haute PECO.

Watch this Scientific Journal Video about Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells at JoVE.com

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

Pour cultiver des cellules souches neurales de crête (NCSC) in vitro, un milieu spécial (NCSCM) est nécessaire. Partie essentielle de NCSCM est extrait d&#39;embryon de poulet (PECO). Nous décrivons ici les techniques précises pour produire une quantité maximisée de qualité pure et de haute PECO, y compris des détails comme l&#39;isolement, la macération, la centrifugation et la filtration.

Production d&#39;extrait d&#39;embryon de poulet pour la culture des neurones murins Cellules souches Crest

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

production-chick-embryo-extract-for-cultivation-murine-neural-crest

Research

JoVE Journal

Neuroscience

12.6K vues.  University Children's Hospital Essen. Pour cultiver des cellules souches neurales de crête (NCSC) in vitro, un milieu spécial (NCSCM) est nécessaire. Partie essentielle de NCSCM est extrait d'embryon de poulet (PECO). Nous décrivons ici les techniques précises pour produire une quantité maximisée de qualité pure et de haute PECO, y compris des détails comme l'isolement, la macération, la centrifugation et la filtration.

Vidéo: Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Isolement et culture des cellules souches neurales Crête des follicules pileux humains

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Recherche

Neurosciences

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.

Isolement et culture de cellules de crêtes neurales de tube neural embryonnaire murin

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

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Dérivation sans chargeur-de cellules progénitrices de la crête neurale souches pluripotentes humaines cellules

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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.

Énumération des cellules souches neurales utilisant clonales Assays

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.

Élaboration d’un protocole raffiné pour Trans-sclérale Transplantation sous-rétinienne des cellules épithéliales humaines pigmentaire dans les yeux de Rat

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

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

La détection de 5-Hydroxymethylcytosine dans les cellules souches neurales et le cerveau des souris

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

﻿Releasing Murine Neural Stem Cells by Stereotaxic Intracerebroventricular Injection of Release Cocktail

Releasing Murine Neural Stem Cells by Stereotaxic Intracerebroventricular Injection of Release Cocktail   

Libération de cellules souches neurales murines par injection intracérébrale-ventriculaire stéréotaxique d’un cocktail de libération 

Dérivation de neurones entériques à partir de cellules souches pluripotentes humaines

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

Pleins feux sur l’auteur : Culture robuste de neurones entériques humains à partir de cellules souches pluripotentes humaines

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is presented.

Différenciation des lignées du système nerveux entérique à partir de cellules souches pluripotentes humaines

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls ...