Ce travail a été soutenu en partie par le National Institutes of Health: HD054347 et NS063997-01 à VLS. Ce travail a également été soutenu en partie par le Fonds Empire State de cellules souches par le ministère de l&#39;État de New York contrat Santé # C024324 au VLS. Les opinions exprimées ici sont uniquement celles de l&#39;auteur et ne reflètent pas nécessairement celles du Conseil du Empire State de cellules souches, le New York State Department of Health, ou l&#39;Etat de New York. VLS est un Doris Duke Clinical Scientist Lauréat du Prix du développement. Nous remercions également le professeur Timothy Vartanian pour son don de l&#39;anti-O1, anti-O4 anticorps.

1. Préparation des solutions et matériaux pour la dissection et l&#39;entretien de la culture de cellules souches neurales <ol><li> Préparer 100ml moyennes dissection (KNOCKOUT DMEM/F12, Invitogen) à l&#39;avance et réfrigérer. </li><li> Prepare100 milieu de culture ml (Stem Pro NSC GDF, Invitrogen) et conserver à 37 ° C dans un bain d&#39;eau. </li><li> Préparer la cellule de congélation moyenne (10% KNOCKOUT DMEM/F12 FBS + 5% DMSO) pour la cryoconservation des cellules à long terme. </li><li> Si désiré, préparer paraformaldéhyde à 4% (PFA) pour la fixation des tissus. </li><li> Stérile, pinces et les lames de bistouri autoclave avec des poignées sont utilisés pour la dissection. </li><li> Mettez de côté un pistolet pipette, 10 ml pipettes de transfert, et 40 tamis cellulaire uM (BD Falcon 352 340) pour la dissociation. </li><li> Mettez de côté quelques 10 cm de boîtes de culture (BD) pour la dissection, 50 tubes à centrifuger ml (BD) pour la dissociation, des tubes de 1,5 ml pour centrifugeuse de stockage des tissus et des coupes congelées (BD) pour les cellules de congélation. </li></ol> 2. Isoler les cellules souches neurales du cerveau fœtal humain <ol><li> La récolte des tissus viables immédiatement après la résiliation du fœtus est essentielle à la réussite de la procédure. Pour des interventions électives, le calendrier d&#39;obtenir des échantillons peuvent être organisées à l&#39;avance afin de minimiser le temps après la mort du fœtus. Les produits de conception sont récoltés dans les 2 heures après la procédure, mais, idéalement, peut être réalisé sur les procédures électives dans des minutes. Les tissus fœtaux sont souvent fragmentées. Cependant, en général une partie importante du cerveau reste intact pour une identification visuelle. Limites d&#39;âge gestationnel (AG) sont déterminés par la loi statutaire, mais ont été effectuées en utilisant ce protocole entre 18-22 semaines AG. </li><li> Cerveau du fœtus est placé dans une boîte de 10 ml de Petri contenant glacée KNOCKOUT DMEM/F12 solution. Identifier les différentes parties du cortex par des repères anatomiques. Limites pour les cortex frontal et pariéto-occipitale sont orientés par l&#39;intersection de l&#39;extrapolation sillon central et la scissure de Sylvius. Disséquer les tissus du cortex frontal antérieur à le sillon central et le long de la frontière de la fissure sylvienne avec les lames chirurgicales, en s&#39;assurant de garder le ventricule intact et en bon état. </li><li> Retirez tout le sang résiduel et les méninges du bloc séparé du cortex frontal. Si l&#39;échantillon est de qualité suffisante, il est idéal pour disséquer le bloc en plusieurs petits échantillons à des fins diverses: articles (fixés dans du paraformaldéhyde 4%, PFA) et protéine / ARNm dosages (rapide gelés dans -80 ° C). </li><li> Transfert du bloc du cerveau sélectionnés dans un tube à centrifuger de 50 ml, et ajouter glacée KNOCKOUT DMEM/F12 solution à environ 3 fois le volume du tissu. Doucement dissocient le tissu par pipetage mécanique avec une pipette de 10 ml, transférer jusqu&#39;à ce que tous les tissus est fragmenté (en général 20-30 fois), puis filtrer les cellules à travers une cellule de 40 uM crépine (BD Falcon 352 340) pour obtenir seule ou presque seule cellule suspension. </li><li> Centrifuger la suspension de cellules à 2000 rpm et la température ambiante pendant 5 min, resuspendre le culot cellulaire dans 10 ml du milieu de culture frais chaud (Stem Pro NSC AFD), et compter le nombre de cellules avec un hémocytomètre. </li><li> Ajouter 5 ml de milieu de culture chaud dans tous les 25 cm de flacons de culture 2, et le transfert 2X10 6 cellules pour chaque ballon. Les cultures sont maintenues dans un 37 ° C / 5% de CO 2 incubateur pour 1 semaine avant l&#39;analyse. Changer la moitié de la moyenne une fois par semaine pour des cultures plus ou expériences. </li></ol> 3. Manipulaton des cellules souches neurales pour une caractérisation plus poussée ou d&#39;expérimentations <ol><li> Neurosphères forment habituellement dans les 1 à 2 semaines selon les conditions de culture recommandée avec un diamètre allant NSC entre 200 et 400 um. Neurosphères à ce stade peut être dissociée de 0,2 g / L d&#39;EDTA en calcium et en magnésium libre milieu de Hanks (Hanks) à 37 ° C pendant 15 min afin d&#39;obtenir des cellules individuelles. Suspension de cellules sont filées à 2000 rpm, rincés dans l&#39;eau douce Hanks, et réétalées en milieu de culture chaud pour subculture. </li><li> Pour initier la différenciation, les cellules dissociées sont étalées sur des lamelles de 1 poly-D-lysine/laminin revêtement à une densité de 1x10 5 cellules par lamelle (24mmX24mm). Différenciation des oligodendrocytes est réalisé par le maintien des cellules en KNOCKOUT DMEM/F12 (Invitrogen, Main, MD) 2% B27 (50X, Invitrogen, Main, MD) 10 ng / ml de bFGF 100 ng / ml SHH + 10ng/ml PDGF-AA pour deux jours, puis passer dans le même milieu sans facteurs de croissance pour un autre 5 jours. Différenciation neuronale est réalisée par des cellules maintien en KNOCKOUT DMEM/F12 2% B27 (50X) pendant 7 jours. Différenciation astrocytaire est fait par culture de cellules de KNOCKOUT DMEM/F12 1% de FBS pendant une semaine. </li><li> La transfection de cellules souches neurales à des gènes peut être fait avec des cellules dissociées des neurosphères préformés. Ici, nous avons montré les cellules souches neurales transfectées avec EGFP-C1 par électroporation. L&#39;électroporation de EGFP-C1 construction a été faite en utilisant AMAXA Kits Nucleofector des cellules souches neurales de souris (VPG-1004) avec AMAXA Nucleofector périphérique (Lonza AAD-1001), suite à l&#39;instruction de l&#39;entreprise. En bref, 5 ug d&#39;ADN avec 1 x 10 6 cellules ont été mélangés avec 100 ul milieu de transfection, et après l&#39;électroporation d&#39;impulsion, les cellules ont été remises en suspension dans les cellules souches neurales maintenir moyennes pour la culture d&#39;autres. La différenciation des cellules transfectées ont été traitées avec la dissociation du jour neurosphères 3-4 après l&#39;électroporation, suivant les mêmes procédures décrites dans l&#39;étape 3.2. </li></ol> 4. La congélation des cellules souches neurales et les subcultures <ol><li> Suspensions cellulaires dissociées sont centrifugés et remis en suspension dans un milieu de congélation avec une concentration de 1x10 7 cellules / flacon / ml. Lentement geler les cellules de -20 ° C, -80 ° C puis transfert à l&#39;azote liquide pour stockage de longue durée. </li><li> Les cellules sont décongelées rapidement à 37 ° C bain-marie et remis en suspension dans réchauffé DMEM/F12 10% de sérum pour un lavage, centrifugé pour éliminer le milieu de congélation, et remis en suspension dans le milieu de culture réchauffé. </li></ol> 5. Les résultats représentatifs: Les cellules souches neurales à partir d&#39;un fœtus normal à 18 semaines d&#39;âge gestationnel ont été cultivés suivant les méthodes décrites et neurosphères peuvent être observés après une semaine avec, bordures lisses et de taille assez homogène (figure 2a). Ces neurosphères peuvent être transfectées avec EGFP-C1 ou d&#39;autres constructions et suivis en microscopie à fluorescence (Fig.2b,). Fondée neurosphères ont ensuite été dissociées avec EDTA et plaqués en cellules dispersées sur des lamelles de revêtement. Cellules différenciées selon les protocoles respectifs ont été fixés avec parafamaldehyde 4%, et colorés avec des marqueurs différents types cellulaires spécifiques. Multipotentiality est observée avec l&#39;expression des marqueurs indicatifs de neurones (Fig2C, D, la rhodamine) astrocytes (Fig2E, F, la rhodamine) et des oligodendrocytes (Fig2G, H, la rhodamine). Les cellules pas subi l&#39;électroporation de EGFP ont également été différenciées en types de cellules différentes et colorées avec des marqueurs de cellules spécifiques différentes. Multipotentiality est observée avec l&#39;expression des marqueurs indicatifs de neurones (Fig3A, B, fluorescéine) astrocytes (Fig3C, la rhodamine et Fig3D, fluorescéine) et des oligodendrocytes (Fig3E, F, la rhodamine). <img src="/files/ftp_upload/2681/2681fig1.jpg" alt="Figure 1"/> Figure 1. Schéma de la procédure expérimentale pour isoler des cellules souches neurales du cerveau du fœtus humains jetés <img src="/files/ftp_upload/2681/2681fig2.jpg" alt="Figure 2"/> Figure 2. Indifférenciées et différenciées des cellules neurales humaines propagés in vitro. (A) neurosphères sont présentés en microscopie à contraste de phase montrent lisses, rondes et des frontières croissance rapide après culture pendant plus de 1 semaine. (B) Introduction de différents plasmides et les constructions peuvent être atteints grâce à la transfection. Trois jours après l&#39;EGFP-C1 transfection, les cellules multiples montrent l&#39;expression de la protéine fluorescente verte comme on le voit dans immunomarquage fluorescéine et de microscopie à fluorescence. EGFP-C1 neurosphères transfectées sont dissociés et différencié dans des conditions différentes dans les neurones (C, D), les astrocytes (E, F) et des oligodendrocytes (G, H) et vu sous la fluorescence rhodamine. Parallèlement, des cellules transfectées EGFP positifs sont affichés sous la fluorescéine fluorescence. Les cellules transfectées (têtes de flèche blanche) sont indiscernables de cellules non transfectées (flèches blanches). Les noyaux cellulaires sont colorés avec Hoechst33342. Barres d&#39;échelle sont de 200 um pour A, 100 um pour B et 25 um pour CH. <img src="/files/ftp_upload/2681/2681fig3.jpg" alt="Figure 3"/> Figure 3. Neurosphères sans transfection sont dissociés et différencié dans des conditions différentes dans les neurones (A, B, fluorescéine), les astrocytes (C, la rhodamine, D, fluorescéine) et des oligodendrocytes (E, F, la rhodamine). Les noyaux cellulaires sont colorés avec Hoechst33342. Barres d&#39;échelle sont de 25 um pour AF.

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Il existe différentes approches envers les tissus de culture frais et la production de lignées cellulaires humaines. Historiquement, les tissus frais a été récolté et mis en culture immédiatement pour générer divers types de cellules dans le système nerveux central. Cette approche est toutefois clairement limité par le nombre d&#39;échantillons qui peuvent être obtenus, qui dans le cas des échantillons humains, est généralement assez faible. Étant donné le degré minimal de la manipulation, fraîchemen...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> KNOCKOUT DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td> Milieu de dissociation </td></tr><tr><td> Tige Pro NSC GDF </td><td> Invitrogen </td><td> A10509-01 </td><td> Le milieu de culture </td></tr><tr><td> Sérum de veau fœtal </td><td> Invitrogen </td><td> 10091-148 </td><td> Moyennes congelés </td></tr><tr><td> Hanks solution (-Ca 2 +,-Mg 2 +) </td><td> Invitrogen </td><td> 14175-095 </td><td> Milieu de dissociation </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D2650 </td><td> Moyennes congelés </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> 431788 </td><td> Milieu de dissociation </td></tr><tr><td> Paraformaldéhyde </td><td> Sigma-Aldrich </td><td> 158127 </td><td> Solution de fixation </td></tr><tr><td> bFGF </td><td> R &amp; D </td><td> 234-FSE </td><td> Milieu de différenciation </td></tr><tr><td> SHH </td><td> R &amp; D </td><td> 1845-SH </td><td> Milieu de différenciation </td></tr><tr><td> PDGF-AA </td><td> R &amp; D </td><td> 221-AA </td><td> Milieu de différenciation </td></tr><tr><td> B27 </td><td> Invitrogen </td><td> 17504-044 </td><td> Milieu de différenciation </td></tr><tr><td> De souris anti-MAP2 </td><td> Sigma-Aldrich </td><td> M2320 </td><td> 1:200 </td></tr><tr><td> Lapin anti-DCX </td><td> Signalisation cellulaire </td><td> 4604s </td><td> 1:200 </td></tr><tr><td> Lapin anti-GFAP </td><td> DAKO </td><td> Z0334 </td><td> 1:200 </td></tr><tr><td> Lapin anti-S100B </td><td> DAKO </td><td> Z0311 </td><td> 1:200 </td></tr><tr><td> Lapin anti-O1 </td><td> dons de professeur Timothy Vartanian * </td><td></td><td> 01:50 </td></tr><tr><td> Lapin anti-O4 </td><td> Cadeaux du professeur Timothy Vartanian * </td><td></td><td> 01:50 </td></tr><tr><td> Tamis cellulaire 40μm </td><td> BD Falcon </td><td> 352340 </td><td></td></tr></tbody></table> * Timothy Vartanian, MD, Ph.D., Département de neurologie et de neuroscience, Weill Cornell Medical College, New York, USA

generation of neural stem cells from discarded human fetal cortical tissue

Les cellules souches neurales (NSC) résident le long de la zone ventriculaire neuroépithélium lors du développement de la plaque corticale. Ces premiers ancêtres de finalement donner naissance à des progéniteurs intermédiaires et plus tard, les divers sous-types de cellules neuronales et gliales qui forment le cortex cérébral. La capacité de générer et de développer NSCs humaine (ce qu&#39;on appelle neurosphères) de tissus fœtaux jetés normale fournit un moyen avec lequel étudier directement les aspects fonctionnels de la normale du développement humain NSC 1-5. Cette approche peut également être dirigée vers la génération de NSC à partir connu des troubles neurologiques, procurant ainsi l&#39;occasion d&#39;identifier les processus pathologiques qui altèrent la prolifération progénitrices, la migration et la différenciation 6-9. Nous nous sommes concentrés sur l&#39;identification des mécanismes pathologiques chez l&#39;homme NSCs syndrome de Down qui pourraient contribuer à l&#39;accélération de phénotype de la maladie d&#39;Alzheimer 10,11. Ni in vivo, ni dans les modèles murins in vitro peut reproduire le répertoire de gènes identiques situés sur le chromosome 21 humain. Ici, nous utilisons une méthode simple et fiable pour isoler le syndrome de Down NSCs d&#39;humains avortés cortex fœtal et les faire grandir dans la culture. La méthodologie fournit des aspects spécifiques de la récolte des tissus, la dissection des repères anatomiques limitées, tri cellulaire, de placage et repiquage du CNS humaine. Nous fournissons également des protocoles de base pour induire la différenciation des NSCs humains en sous-types de cellules plus sélective.

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Une méthode simple et fiable sur l&#39;isolement et la culture des cellules souches neurales à partir des tissus humains jetés corticale du foetus est décrite. Cultures dérivées de humain connu des troubles neurologiques peuvent être utilisés pour la caractérisation des processus cellulaires et pathologiques moléculaires, ainsi que de fournir une plateforme pour évaluer l&#39;efficacité pharmacologique.

Génération de cellules souches neurales à partir de tissus humains mis au rebut corticale fœtale

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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15.4K vues.  Beth Israel Deaconess Medical Center. Une méthode simple et fiable sur l'isolement et la culture des cellules souches neurales à partir des tissus humains jetés corticale du foetus est décrite. Cultures dérivées de humain connu des troubles neurologiques peuvent être utilisés pour la caractérisation des processus cellulaires et pathologiques moléculaires, ainsi que de fournir une plateforme pour évaluer l'efficacité pharmacologique.

Vidéo: Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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.

Neural-Formant Colonie dosage cellulaire: Un test pour distinguer Bona Fide Cellules souches neuronales à partir de cellules progénitrices neurales

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). ...

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Neurosciences

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 ...

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.

Le test neuroblaste: Un test pour la génération et l&#39;enrichissement des cellules souches neuronales à partir de différenciation des cellules souches neurales Progeny Par cytométrie en flux

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.

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 ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

Une méthode d&#39;extension cGMP applicable pour les agrégats de neurones humains des cellules souches et progénitrices dérivées de cellules souches pluripotentes ou fœtale tissu cérébral

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

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 ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modélisation astrocytome pathogenèse<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</em&gt; Utilisation corticale astrocytes ou cellules souches neurales à partir de, souris génétiquement modifiées conditionnelle

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

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 ...

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.

Génération simple d’une Culture de haut rendement de neurones induits des fibroblastes de la peau humaine adulte

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 ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Enregistrements simultanés des potentiels corticaux champ Local, électrocardiogramme, électromyogramme et respiration rythmique d’un Rat librement mobile

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...