The authors acknowledge funding sources responsible for this work: 5K12HD055929 (HHR), 5R01NS080180-02 (DDF).

NOTE: Toutes les procédures d&#39;animaux dans ce protocole sont menées avec l&#39;approbation de l&#39;Université de Floride institutionnel de protection des animaux et l&#39;utilisation Comité (IACUC) et sont en conformité avec le «Guide pour le soin et l&#39;utilisation des animaux de laboratoire». 1. expérimentale de base mis en place avant l&#39;administration intermittente hypoxie <ol><li> Exposer chiots 12 aux différents mélanges de gaz en utilisant un corps entier chambres de pléthysmographe souris de la même manière que les rongeurs adultes à d&#39;autres fins expérimentales 5,13,14. Les chambres ont un diamètre de 4 pouces (volume = 450 ml), ce qui est de taille suffisante pour accueillir les souriceaux 3-4 ou 1-2 de jeunes rats. Ici, protocoles HAI sont livrés à des nouveau-nés âgés de jour postnatal 1-8 (P1-P8). </li><li> Utilisez des réservoirs de gaz de l&#39;air comprimé pour livrer le gaz de 21% &quot;de base&quot;, qui maintient la chambre d&#39;oxygénation à 21%. Pour atteindre &quot;hypoxie relative&quot;, utiliser un / 90% réservoir de gaz d&#39;azote de 10% d&#39;oxygène. NE PASE: tube en plastique (3/16 &quot;de diamètre) relie les réservoirs de gaz à un débitmètre. Ainsi, l&#39;entrée du compteur de débit est constitué d&#39;un tube pour chaque ligne de base et le flux d&#39;air hypoxie. </li><li> Configurer le tuyau de gaz comme suit. <ol><li> Exécuter un tube en plastique (1/8 &quot;de diamètre) à partir du débitmètre à un régulateur de débit de polarisation, ce qui permet de flux 1 L / min à chaque chambre (à savoir, 4 L / min si les chambres 4 sont utilisés). </li><li> Exécuter un tube en plastique (1/8 &quot;de diamètre) à partir du régulateur d&#39;écoulement de polarisation aux chambres de pléthysmographie. Airflow à 1 L / min à chaque chambre est un débit prédéterminé au cours de laquelle l&#39;échange de gaz est réputé être non-stress induisant des animaux basés sur des observations physiologiques et comportementaux. </li></ol></li><li> Sceller toutes les connexions inutilisées depuis et vers l&#39;unité de débit de polarisation et toutes les ouvertures inutilisées dans les chambres avec des bouchons de sorte qu&#39;aucun écoulement de gaz échappe à d&#39;autres chambres ne sont pas utilisés, ou sur des chambres d&#39;Plethysmographie pendant le protocole. </li><li> Placer la chambre (s) Dans un incubateur avant de mettre les animaux dans la chambre pour permettre l&#39;équilibrage à 37 ° C, ou la température du corps. </li></ol> 2. Calibrage des temps de cycle pour l&#39;hypoxie intermittente <ol><li> Inspectez tous les tuyaux, entre les réservoirs d&#39;air et débitmètre, débitmètre et l&#39;unité de flux de polarisation, et le flux de polarisation unité et la chambre (s) de pléthysmographie pour les connexions appropriées. Les connexions sont mis en place comme décrit à l&#39;étape 1. Vérifiez la connexion d&#39;un chèque par étapes de toutes les lignes et les fermetures décrites: par exemple, la vérification de lignes non utilisées hors de l&#39;unité de débit de polarisation et des ouvertures de chambre ne sont pas utilisés. </li><li> Connectez un capteur de température ambiante O 2 O 2 avec un compteur à main, puis fixez le capteur sur le couvercle de la chambre de pléthysmographie. </li><li> Ouvrez les deux réservoirs de gaz et fixez une paire de pinces hémostatiques chirurgicaux, à long manche isolés avec des tubes en plastique pour bloquer le flux hors cycle du gaz. De cette façon, un seul réservoir de gaz à la fois met en œuvre le 1 L / min débit de gaz pour chambre. Fixer le &quot;niveau de référence&quot; mélange de gaz tandis que le &quot;hypoxique&quot; mélange de gaz est livré à la chambre, et vice versa. </li><li> Noter le temps nécessaire pour que la chambre de O 2 pour atteindre les niveaux de 10% de cible et revenir à 21%. Utiliser les temps enregistrés pour la livraison de protocole ultérieur. </li></ol> 3. Administration de l&#39;hypoxie aiguë intermittente <ol><li> Inspectez tous les tuyaux et les connexions comme décrit dans l&#39;étape 2.1. </li><li> Placez les nouveau-nés dans la chambre (s) de pléthysmographie et abriter les couvercles des chambres de pléthysmographie dans la base de chambre. Confirmer une bonne étanchéité à l&#39;joint torique est ainsi que la fermeture de toutes les connexions de chambre inutilisés. Ces étapes sont essentielles pour assurer la bonne exécution des mélanges de gaz. </li><li> Placez les chambres de retenue les chiots à être traités dans le 37 ° C incubateur et fermer la porte. Laisser chambre à équilibrer à 37 ° C avant le début du protocole. Maintenir les animaux témoins à 37 ° C dans le Incubatou à l&#39;air ambiant oxygénation. </li><li> Ouvrez les deux réservoirs de gaz et d&#39;utiliser une paire de pinces hémostatiques chirurgicaux, à long manche isolés avec des tubes en plastique pour bloquer le flux hors cycle du gaz. De cette façon, un seul réservoir de gaz à un moment administre le 1 L / flux de gaz min par chambre. </li><li> Utilisez une minuterie à main pour précisément cycles de temps et d&#39;indiquer le temps de passer les pinces hémostatiques entre tubes, entraînant ainsi l&#39;alternance du flux de gaz dans les chambres. </li><li> Constater la réalisation de chaque cycle à l&#39;aide d&#39;un journal de traitement tel que celui fourni dans la Figure 1 pour marquer toutes les étapes du 2-step, 20 protocole de cycle. </li><li> Surveiller la température de l&#39;incubateur à chaque changement de cycle pour assurer que les animaux sont maintenus à la gamme désigné (33-35 ° C, ce qui est le réglage de l&#39;incubateur qui se traduit par 37 ° C). </li><li> Surveiller de près l&#39;activité des petits tout au long du protocole afin d&#39;assurer que les animaux tolèrent cycles sans détresse visibles tels que les vocalisations, altération de l&#39;étatactivité de membre ou de roulement. </li></ol> 4. Isolement et culture de cellules souches / progénitrices de la zone sous-ventriculaire REMARQUE: Le changement dans la formation de neurosphère suivante AIH rapport aux témoins est un exemple d&#39;un point de terminaison qui démontre l&#39;efficacité de ce protocole pour obtenir des changements Tissue et au niveau cellulaire. <ol><li> Pour isoler neurosphères formant des cellules, retirez rat ou de souris chiots de la chambre immédiatement après AIH est terminée. Sacrifier les animaux selon des protocoles IACUC dans les 30 minutes de la fin du protocole. </li><li> Récolter le tissu SVZ, placer dans la culture de neurosphère et mener passage standard et expansion de neurosphères dérivés, comme précédemment publié dans les méthodes chapitres 15,16 et un protocole de vidéo séparée 8. </li></ol>

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The authors have nothing to disclose.

This work reports the development of a protocol to expose neonatal rodents to AIH. The parameters described here effectively alter in situ neural stem cell biology, which is observable over several rounds of cell passage. Specifically, AIH increases the number of non-adherent neurospheres, the expansion of cells within each neurosphere (refected by sphere diameter), the expansion of adherent NPC populations, and the presence of neuroblasts in both non-adherent and adherent populations. It should be emph...

Le but de ce procédé est de fournir de manière efficace et reproductible des matchs intermittente systémique oxygène ambiant réduit de rongeurs nouveau-nés. La justification de l&#39;utilisation hypoxie intermittente (IH) pour manipuler la biologie des cellules souches in vitro provient d&#39;expériences de culture cellulaire dans laquelle la teneur en O 2 du milieu de croissance est modifiée. Plus précisément, par rapport à des conditions «standard» de 20% de O 2, de la culture prolongée de souches / progénitrices des cellules de populations de cellules dans 3% d&#39;O 2 résultats à une prolifération accrue, une diminution de l&#39;apoptose neuronale et un rendement accru de 1,2. Ce groupe a une grande expérience de l&#39;administration de IH systémique, et a mené des études approfondies sur le rôle de l&#39;IH dans la plasticité respiratoire 3-7. Ce travail, et la découverte récente que IH chronique augmente la neurogénèse chez le rongeur CNS 8-10, constitue la base pour l&#39;exploration de aiguë in vivohypoxie comme un stimulus de préconditionnement (ie., avant la récolte de tissu) sur la culture subséquente de cellules souches / progénitrices neurales (PNJ) 11. Remarquablement, lorsque les souriceaux ont été exposés à une brève période (&lt;1 h) de l&#39;hypoxie aiguë intermittente (AIH), les cellules qui ont été récoltées dans la zone sous-ventriculaire (SVZ) avaient augmenté de façon significative la capacité d&#39;expansion que neurosphères ou monocouches de cellules adhérentes. Le protocole AIH était également associée à une expression accrue d&#39;un &quot;destinée neuronale&quot; facteur de transcription (Pax6). En conséquence, in vivo protocoles HAI peuvent fournir un moyen de «prime» PNJ avant la culture. Par exemple, les demandes de cette approche peuvent inclure l&#39;expansion des populations de cellules avant la transplantation dans le système nerveux central blessé, ou augmentant simplement la différenciation neuronale de cellules en culture avant des expériences in vitro. En outre, parce que cela est une administration systémique, touteorgane, le tissu ou la cellule est un candidat pour étude similaire. Par conséquent, le protocole écrit est potentiellement applicable à une large gamme d&#39;études sur la manipulation de l&#39;oxygène intermittente chez les petits mammifères. Il ya certains avantages à cette approche. Dans d&#39;autres travaux publiés, les nouveau-nés ont été traités comme une litière avec le barrage en chambre hypobare, qui permet le dosage chronique, moins de manipulation avant le traitement, et maintenu le contact maternelle pendant le traitement 9. L&#39;approche actuelle contourne traitements répétés à une femelle reproductrice, ou l&#39;utilisation d&#39;un barrage différent pour chaque expérience. Ce protocole permet également l&#39;étude des nouveau-nés de la litière appariés et appariés pour l&#39;âge précis. Les données représentatives démontrent un autre atout majeur de ce protocole, à savoir la rapidité avec laquelle AIH, tel que livré, provoque une réponse biologique puissante et cohérente dans neuronal la biologie des cellules souches. Cela établit un précédent pour ce protocole de susciter biologi tissulaire et cellulaire niveauCAL changements qui modifient la biologie cellulaire. Le présent rapport décrit les procédures détaillées utilisées pour exposer rongeurs chiots à IAC ainsi que l&#39;analyse de la population de cellules SVZ grandi comme neurosphères.

<table border="0" cellpadding="0" cellspacing="0" width="768">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:243px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Mouse plethysmography chambers</td>
			<td style="width:147px;">Buxco</td>
			<td style="width:136px;">PLY4211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Flow meter&#160;</td>
			<td style="width:147px;">Porter</td>
			<td style="width:136px;">F150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bias flow unit</td>
			<td style="width:147px;">AFPS</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Baseline Gas Mix</td>
			<td style="width:147px;">Airgas</td>
			<td style="width:136px;">AIZ300</td>
			<td>Compressed Air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hypoxic Gas Mix</td>
			<td style="width:147px;">Airgas</td>
			<td style="width:136px;">X03NI72C2000189</td>
			<td>10% Oxygen, balance nitrogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Oxygen Meter</td>
			<td style="width:147px;">Teledyne</td>
			<td style="width:136px;">AX-300</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of in vivo acute intermittent hypoxia in neonatal rodents to prime subventricular zone-derived neural progenitor cell cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Les premières expériences, basées sur des données historiques, ont été effectuées en utilisant des longueurs de cycle 1 min. Sur la base des calibrations ultérieures effectuées à l&#39;étape 2 ci-dessus, on a déterminé que le niveau d&#39;O 2 dans la chambre était de 13% à 1 min post-rinçage et l&#39;hypoxie, qu&#39;il a fallu un temps similaire pour revenir à la ligne de base de 21%. Cependant, un cycle de 2 min est suffisante pour atteindre à la fois 10% d&#39;oxygène et un retour à 21% ...

Watch this Scientific Journal Video about Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures at JoVE.com

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Cet article décrit la méthodologie de l&#39;administration de courtes périodes de hypoxie intermittente à jour 1-8 rat ou de souris chiots postnatales. Cette approche suscite effectivement un niveau de tissu robuste »d&#39;amorçage effet&quot; sur les cellules progénitrices neurales cultivées qui sont récoltées dans les 30 min d&#39;exposition à l&#39;hypoxie.

Livraison de<em&gt; In Vivo</em&gt; Aiguë intermittente hypoxie néonatale dans Rongeurs au Premier-ventriculaire Zone dérivés de cultures de cellules progénitrices neurales

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

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Developmental Biology

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

7.2K vues.  University of Florida.  Cet article décrit la méthodologie de l'administration de courtes périodes de hypoxie intermittente à jour 1-8 rat ou de souris chiots postnatales. Cette approche suscite effectivement un niveau de tissu robuste »d'amorçage effet" sur les cellules progénitrices neurales cultivées qui sont récoltées dans les 30 min d'exposition à l'hypoxie. 

Vidéo: Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Induction directe de neurales humaines cellules souches à partir de cellules progénitrices hématopoïétiques du sang périphérique

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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Biologie du développement

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Stem Cell-like<em&gt; Xenopus</em&gt; Embryonnaires explants à étudier les caractéristiques du développement précoce Neural<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Lancement de différenciation dans immortalisées cellules multipotentes progénitrices otique

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technique à la cible microinjection du Developing<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

En utilisant les cultures primaires Neurosphère à étudier primaire Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Induction de l&#39;hypoxie dans les écrevisses vivantes de grenouille et de poisson zèbre

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Une nouvelle technique de transplantation de graisse mammaire mammaire pour visualiser la génération de vaisseaux de cellules souches endothéliales vasculaires

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

Analyse quantitative Immunofluorescence entier-montent des Populations de progéniteurs cardiaques chez des embryons de souris

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Cytométrie en cellules vivantes en temps réel d’enquêter sur l’Influx calcique, la Formation de pores et phagocytose par les récepteurs P2X7 dans des cellules progénitrices neurales adultes

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Modèle d'impact cortical contrôlé des dommages de cerveau de souris avec la transplantation thérapeutique des cellules neurales pluripotentes induites par l'homme de cellules souches

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...