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

NOTA: Tutte le procedure animali in questo protocollo sono condotte con l&#39;approvazione della University of Florida Istituzionale Animal Care and Use Committee (IACUC) e sono in conformità con la &#39;Guida per la cura e l&#39;uso di animali da laboratorio&#39;. 1. Impostare sperimentale di base in funzione Prima Amministrazione intermittente ipossia <ol><li> Esporre cuccioli 12 alle diverse miscele di gas che utilizzano un corpo intero camere del mouse pletismografo più o meno allo stesso modo di roditori adulti per altri scopi sperimentali 5,13,14. Le camere hanno un diametro di 4 pollici (volume = 450 ml), che è di ampie dimensioni per ospitare 3-4 cuccioli del mouse o 1-2 cuccioli di ratto. Qui, i protocolli di AIH sono consegnati a neonati di età compresa tra 1-8 giorni post-natale (P1-P8). </li><li> Utilizzare serbatoi di gas compresso l&#39;aria di consegnare il gas 21% &quot;di base&quot;, che mantiene l&#39;ossigenazione camera al 21%. Per raggiungere &quot;ipossia relativa&quot;, utilizzare un / 90% serbatoio di gas di azoto 10% di ossigeno. NONE: Tubi flessibili (&quot;diametro 3/16) collega i serbatoi di gas a un flussometro. Pertanto, l&#39;ingresso del flussometro è costituito da un tubo per ciascuna linea di base e ipossia flusso d&#39;aria. </li><li> Configurare il tubo del gas come segue. <ol><li> Eseguire tubazione di plastica (&quot;diametro 1/8) dal flussometro di un regolatore di flusso di polarizzazione, che consente flussi di 1 L / min per ciascuna camera (cioè, 4 L / min se si utilizzano 4 alloggiamenti). </li><li> Eseguire tubazione di plastica (&quot;diametro 1/8) dal regolatore di flusso polarizzazione alle camere pletismografia. Flusso d&#39;aria a 1 L / min per ciascuna camera è una portata predeterminata in cui lo scambio di gas è considerata non-stressanti agli animali basate su osservazioni fisiologiche e comportamentali. </li></ol></li><li> Chiudere tutte le connessioni inutilizzate da e per l&#39;unità di flusso pregiudizi e tutte le aperture non utilizzate alle camere con tappi in modo che nessun flusso di gas sfugge ad altre stanze non in uso, o fuori delle camere pletismografia durante il protocollo. </li><li> Posizionare la camera (s) In un incubatore prima di mettere animali nella camera per raggiungere l&#39;equilibrio a 37 ° C, o della temperatura corporea. </li></ol> 2. Taratura di tempi di ciclo per ipossia intermittente <ol><li> Controllare tutti i tubi, tra bombole e flussometro, flussometro e unità di flusso pregiudizi, e il flusso di pregiudizi unità e la camera pletismografia (s) per le connessioni appropriate. I collegamenti sono impostati come descritto al punto 1. Verificare la connessione da un controllo graduale di tutte le linee descritte e chiusure: ad esempio, il controllo linee usate fuori dalla unità di flusso di polarizzazione e le aperture di camera non è in uso. </li><li> Collegare un sensore ambientale O 2 con una O 2 metri portatile e quindi collegare il sensore al coperchio della camera pletismografia. </li><li> Aprire entrambi i serbatoi del gas e collegare un paio di chirurgia, emostatici manico lungo isolati con tubi di plastica per bloccare il flusso di fuori-ciclo del gas. In questo modo, un solo serbatoio alla volta è fornire il / flusso del gas min 1 L per ogni camera. Bloccare la miscela di gas &quot;di base&quot;, mentre la miscela di gas &quot;ipossia&quot;, è consegnato alla camera, e viceversa. </li><li> Registrare il tempo necessario per la camera 2 O per raggiungere i livelli obiettivo di 10% e tornare al 21%. Utilizzare i tempi di consegna per il successivo protocollo. </li></ol> 3. La somministrazione di ipossia acuta intermittente <ol><li> Controllare tutti i tubi e le connessioni come descritto al punto 2.1. </li><li> Posizionare neonati nella camera pletismografia (s) e ospitare le palpebre camera pletismografia nella base della camera. Confermare una corretta tenuta della guarnizione O-ring è così come la chiusura di tutti i collegamenti da camera inutilizzate. Questi passaggi sono fondamentali per assicurare la consegna adeguata di miscele di gas. </li><li> Posizionare le camere che tengono i cuccioli da trattare nel 37 ° C incubatore e chiudono la porta. Lasciare camera di equilibrare a 37 ° C prima di iniziare il protocollo. Mantenere animali di controllo a 37 ° C nel incubato in camera d&#39;aria ossigenazione. </li><li> Aprire entrambi i serbatoi di gas e utilizzare una coppia di chirurgia, emostatici manico lungo isolati con tubi di plastica per bloccare il flusso di fuori-ciclo del gas. In questo modo, un solo serbatoio alla volta è fornire il / flusso del gas min 1 L per ogni camera. </li><li> Utilizzare un timer manuale a proprio cicli di tempo e per indicare il momento di cambiare le hemostats tra tubi, con conseguente l&#39;alternanza del flusso di gas verso le camere. </li><li> Registrare il completamento di ogni ciclo utilizzando un registro trattamento come quello fornito in Figura 1 per delimitare tutte le fasi del 2-step, 20 protocollo ciclo. </li><li> Monitorare la temperatura dell&#39;incubatrice ad ogni cambio ciclo per assicurare gli animali sono mantenuti al raggio designato (33-35 ° C, che è l&#39;impostazione sull&#39;incubatrice che si traduce in 37 ° C). </li><li> Seguire da vicino l&#39;attività cucciolo tutto il protocollo per garantire che gli animali tollerano cicli senza angoscia visibile come vocalizzi, alterato livello diattività arto o di rotolamento. </li></ol> 4. Isolamento e la coltura di cellule staminali / progenitrici dalla zona subventricolare NOTA: Il cambiamento di formazione neurosfere seguito EAI rispetto ai controlli è un esempio di un endpoint che dimostra l&#39;efficacia di questo protocollo per suscitare cambiamenti tessutale e a livello cellulare. <ol><li> Per isolare neurosfere formano cellule, rimuovere topo o ratto cuccioli dalla camera subito dopo EAI è completata. Sacrificare gli animali secondo protocolli IACUC entro 30 minuti dal termine del protocollo. </li><li> Raccolto il tessuto SVZ, inserire nella cultura neurosfere e condurre iter standard e l&#39;espansione delle neurosfere derivate, come precedentemente pubblicato su metodi capitoli 15,16 e un protocollo video separata 8. </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invited+review:+Intermittent+hypoxia+and+respiratory+plasticity.">Invited review: Intermittent hypoxia and respiratory plasticity.</a> J Appl Physiol. 90 (6), 2466-2475 (2001).</li><li>Fuller, D. D., Zabka, A. G., Baker, T. L., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phrenic+long-term+facilitation+requires+5-HT+receptor+activation+during+but+not+following+episodic+hypoxia.">Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.</a> J Appl Physiol. 90 (5), 2001-2006 (2001).</li><li>Fuller, D. D., Johnson, S. M., Olson, E. B., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+pathways+to+phrenic+motoneurons+are+enhanced+by+chronic+intermittent+hypoxia+after+cervical+spinal+cord+injury.">Synaptic pathways to phrenic motoneurons are enhanced by chronic intermittent hypoxia after cervical spinal cord injury.</a> J Neurosci. 23 (7), 2993-3000 (2003).</li><li>Baker-Herman, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+is+necessary+and+sufficient+for+spinal+respiratory+plasticity+following+intermittent+hypoxia.">BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia.</a> Nat Neurosci. 7 (1), 48-55 (2004).</li><li>Zhu, L. 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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...

L&#39;obiettivo di questo metodo è quello di fornire riproducibile ed efficace periodi intermittenti di sistemica di ossigeno ambiente abbassata a roditori neonatali. Il razionale per l&#39;utilizzo di ipossia intermittente (IH) per manipolare biologia delle cellule staminali in vitro proviene da esperimenti di coltura cellulare in cui tenore di O 2 i mezzi di crescita è alterata. In particolare, rispetto alle condizioni &quot;standard&quot; del 20% O 2, cultura estesa staminali / progenitrici delle cellule popolazioni cellulari in 3% O 2 risultati in un aumento della proliferazione, diminuita apoptosi neuronale e una maggiore resa 1,2. Questo gruppo ha una significativa esperienza con la somministrazione di IH sistemica, e ha condotto studi approfonditi sul ruolo della IH nella plasticità respiratoria 3-7. Questo lavoro, e la recente scoperta che IH cronica aumentata neurogenesi nei roditori CNS 8-10, costituisce la base per l&#39;esplorazione di acuta in vivoipossia come stimolo precondizionamento (es., prima del raccolto tessuti) sulla successiva coltura di cellule neurali staminali / progenitrici (NPC) 11. Sorprendentemente, quando cuccioli di topo sono stati esposti a un breve periodo di acuta ipossia intermittente (IAC) (&lt;1 ora), cellule che sono state raccolte dalla zona subventricolare (SVZ) era significativo aumento di capacità di espansione come neurosfere o cellule monostrato aderenti. Il protocollo AIH è stato anche associato con una maggiore espressione di un &quot;destino neuronale&quot; fattore di trascrizione (Pax6). Di conseguenza, in vivo protocolli AIH possono fornire un mezzo di &quot;prime&quot; NPC prima cultura. Ad esempio, applicazioni di questo approccio possono includere espansione popolazioni di cellule prima del trapianto nel sistema nervoso centrale feriti, o semplicemente aumentando la differenziazione neuronale di cellule coltivate prima esperimenti in vitro. Inoltre, perché questa è una consegna sistemica, qualsiasiorgano, tessuto o cellula è un candidato per studio simile. Pertanto, il protocollo come scritto è potenzialmente applicabile ad una vasta gamma di studi sulla manipolazione ossigeno intermittente in piccoli mammiferi. Ci sono alcuni vantaggi di questo approccio. In un altro lavoro pubblicato, i neonati sono stati trattati come una cucciolata con la diga in camere ipobariche, che consente la somministrazione cronica, meno manipolazione prima del trattamento, e mantenuto il contatto materno durante il trattamento 9. L&#39;attuale approccio bypassa trattamenti ripetuti ad una femmina di allevamento, o l&#39;uso di una diga differente per ciascun esperimento. Questo protocollo permette anche lo studio di precisi neonati lettiere corrispondenza e di pari età. Dati rappresentativi dimostrano un altro punto di forza di questo protocollo, vale a dire la rapidità con cui epatite autoimmune, come consegnato, provoca una risposta biologica potente e costante in biologia delle cellule staminali neurali. Questo stabilisce un precedente per questo protocollo di suscitare biologi tessutale e-livello cellularemodifiche CAL che alterano biologia cellulare. La relazione illustrerà le procedure dettagliate utilizzate per esporre cuccioli roditori per EAI, nonché l&#39;analisi della popolazione di cellule SVZ cresciuto come neurosfere.

<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.

Gli esperimenti iniziali, basate su dati storici, sono stati condotti utilizzando durata del ciclo 1 min. Sulla base delle successive calibrazioni eseguite nella Fase 2, si è stabilito che il livello di O 2 nella camera era 13% a 1 min lavaggio post-ipossia e, che ha un tempo simile al ritorno alla linea di base 21%. Tuttavia, un ciclo di 2 minuti era sufficiente per realizzare sia il 10% di ossigeno e un ritorno al 21% durante il ciclo di &quot;base&quot;. Successivamente, sono stati utilizzati durata del c...

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

Questo articolo descrive la metodologia per la somministrazione di brevi periodi di ipossia intermittente per postnatali giorno 1-8 di topo o ratto cuccioli. Questo approccio suscita effettivamente un livello del tessuto robusto &quot;effetto priming&quot; su colture di cellule progenitrici neurali che vengono raccolte entro 30 minuti dall&#39;esposizione ipossia.

Consegna di<em&gt; In Vivo</em&gt; Acuta intermittente ipossia in Neonatale Roditore al Primo subventricolare Zone-derivati ​​colture cellulari progenitrici neurali

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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JoVE Journal

Developmental Biology

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

7.2K Views.  University of Florida.  Questo articolo descrive la metodologia per la somministrazione di brevi periodi di ipossia intermittente per postnatali giorno 1-8 di topo o ratto cuccioli. Questo approccio suscita effettivamente un livello del tessuto robusto "effetto priming" su colture di cellule progenitrici neurali che vengono raccolte entro 30 minuti dall'esposizione ipossia. 

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

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.

Cellule staminali simil-<em&gt; Xenopus</em&gt; Embrionali espianti di studiare Caratteristiche precoce dello sviluppo neurale<em&gt; In Vitro</em&gt; E<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, ...

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Biologia dello sviluppo

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.

Avvio di differenziazione in cellule multipotenti immortalizzate Otic progenitrici

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.

Tecnica a Target microiniezione per lo sviluppo<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 ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sviluppo di un modello di fegato Fibrotic etanolo-indotta in zebrafish per studiare progenitrici cellulo-mediata epatociti Rigenerazione

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

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.

Utilizzando colture di neurosfere primarie al primario dello studio 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.

Induzione di ipossia in rana vivente e embryos zebrafish

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.

Una tecnica novel di trapianto di grassi del grasso mammario per visualizzare la generazione di vascelli delle cellule staminali endoteliali vascolari

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.

Analisi quantitativa intero-monta immunofluorescenza delle popolazioni di cellule progenitrici cardiache in embrioni di topo

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.

In tempo reale Live-cella flusso Cytometry per indagare l'afflusso del calcio, la formazione del poro e fagocitosi dai recettori P2X7 in cellule progenitrici neurali adulto

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.

Modello di impatto corticale controllato di lesioni cerebrali del topo con trapianto terapeutico di cellule neurali derivate da cellule staminali indotte dall'uomo

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