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

HINWEIS: Alle tierischen Verfahren in diesem Protokoll sind mit Zustimmung der University of Florida Institutional Animal Care und Verwenden Committee (IACUC) durchgeführt und sind in Übereinstimmung mit dem Leitfaden für die Pflege und Verwendung von Labortieren. 1. Grundversuchsaufbau Bevor intermittierende Hypoxie Verwaltung <ol><li> Expose Welpen 12 zu den verschiedenen Gasmischungen unter Verwendung eines Ganzkörper-Maus Plethysmographen Kammern in der gleichen Weise wie erwachsenen Nagetieren für andere experimentelle Zwecke 5,13,14. Die Kammern haben einen Durchmesser von 4 Inch (Volumen = 450 ml), die eine ausreichende Größe ist, um 3-4 Maus Welpen oder 1-2 Rattenjungen zubringen. Hier werden AIH Protokolle Neugeborenen im Alter von postnatalen Tag 1-8 (P1-P8) geliefert. </li><li> Verwenden Sie Druckluft Gastanks, die 21% &quot;Baseline&quot; Gas, das Sauerstoffkammer mit 21% unterhält zu liefern. Um &quot;relative Hypoxie&quot; zu erzielen, verwenden Sie ein% Sauerstoff 10/90% Stickstoffgastank. NICHTE: Kunststoffschlauch (3/16 &quot;Durchmesser) verbindet die Gastanks mit einem Durchflussmesser. Somit wird der Eingang zu dem Durchflussmesser besteht aus jeweils zur Grundlinie und Hypoxie Luftstrom einen Röhre. </li><li> Konfigurieren Sie den Gasschlauch wie folgt. <ol><li> Laufen Kunststoffschlauch (1/8 &quot;Durchmesser) von dem Durchflußmesser zu einem Spannungsströmungsregler, der Ströme von 1 l / min ermöglicht, jede Kammer (dh 4 L / min bei 4 Kammern verwendet werden). </li><li> Führen Kunststoffschlauch (1/8 &quot;Durchmesser) von der Bias-Stromregler auf die Plethysmographie Kammern. Luftstrom bei 1 l / min zu jeder Kammer ist eine vorbestimmte Strömungsrate, mit der der Gasaustausch als Nicht verspannungsinduzierenden der Tiere basierend auf physiologische und Verhaltensbeobachtungen können. </li></ol></li><li> Verschließen Sie alle nicht verwendeten Verbindungen zu und von der Bias-Flow-Einheit und alle nicht verwendeten Öffnungen zu den Kammern mit Kappen, damit kein Gasstrom entweicht, um anderen Kammern nicht in Gebrauch ist, oder aus den Plethysmographie Kammern während des Protokolls. </li><li> Setzen Sie die Kammer (n) In einem Inkubator vor der Inbetriebnahme Tiere in der Kammer, um Äquilibrierung auf 37 ° C, oder Körpertemperatur zu ermöglichen. </li></ol> 2. Kalibrierung der Zykluszeiten für intermittierende Hypoxie <ol><li> Prüfen Sie die Verschlauchung zwischen Luftbehälter und Durchflussmesser, Durchflussmesser und Bias-Flow-Einheit und Bias-Flow-Einheit und Plethysmographie Kammer (n) für die richtigen Verbindungen. ZB ungenutzte Leitungen aus dem Bias-Flow-Einheit und keine Raumöffnungen Überprüfung nicht in Gebrauch: Die Anschlüsse sind wie in Schritt 1 beschrieben, Überprüfen Sie die Verbindung durch eine schrittweise Überprüfung aller beschriebenen Linien und Schließungen gesetzt. </li><li> Schließen Sie eine Umgebungs O 2 -Sensor mit einem Handheld-O 2 Meter und bringen Sie dann den Sensor mit dem Plethysmographie Kammerdeckel. </li><li> Öffnen Sie beide Gastanks und bringen ein Paar chirurgische, langstieligen hemostats mit Kunststoffschlauch isoliert, um die Off-Cycle-Gasstrom zu klemmen. Auf diese Weise wird nur ein Gastank zu einer Zeit liefert die 1 l / min Gasfluss pro Kammer. Klemmen Sie den &quot;Baseline&quot; Gasgemisch, während die &quot;Hypoxie&quot; Gasgemisch in die Kammer geliefert wird, und umgekehrt. </li><li> Notieren Sie sich die Zeit für die Kammer O 2 erforderlich, um die Zielwerte von 10% zu erreichen und zurück zu 21%. Nutzen Sie die aufgezeichneten Zeiten für nachfolgende Protokoll Lieferung. </li></ol> 3. Verwaltung von akuter intermittierender Hypoxie <ol><li> Überprüfen Sie alle Schläuche und Anschlüsse, wie in Schritt 2.1 beschrieben. </li><li> Zeigen Neugeborenen in der Plethysmographie Kammer (n) und das Haus des Plethysmographie Kammerdeckel in die Kammer Basis. Bestätigen Sie eine ordnungsgemäße Abdichtung zu der O-Ringdichtung ist, sowie Schließung von nicht genutzten Kammer Verbindungen. Diese Schritte sind entscheidend für die Gewährleistung der entsprechenden Lieferung von Gasgemischen. </li><li> Zeigen die Kammern halten die Welpen in die 37 ° C-Inkubator behandelt werden und die Tür schließen. Ermöglichen Kammer auf 37 ° C vor dem Beginn des Protokolls äquilibrieren. Aufrechtzuerhalten Kontrolltieren bei 37 ° C im INCUBAToder bei Raumluftsauerstoffversorgung. </li><li> Öffnen Sie beide Gastanks und verwenden Sie ein Paar chirurgische, langstieligen hemostats mit Kunststoffschlauch isoliert, um die Off-Cycle-Gasstrom zu klemmen. Auf diese Weise wird nur ein Gastank zu einer Zeit liefert die 1 l / min Gasstrom pro Kammer. </li><li> Verwenden eines Handauslöser auf präzise Zeitzyklen und die Zeit, um die blutstillende Mittel zwischen den Rohren Schalters anzuzeigen, wodurch der Wechsel der Gasströmung zu den Kammern. </li><li> Nehmen Sie den Abschluss eines jeden Zyklus mit einem Behandlungsprotokoll, wie in Abbildung 1 zur Verfügung gestellt, um zu kennzeichnen Sie alle Schritte in der 2-Stufen, 20-Zyklus-Protokoll. </li><li> Überwachen Sie den Inkubator Temperatur in jedem Zyklus ändern, um sicherzustellen Tiere werden an der vorgesehenen Bereich (33 bis 35 ° C, was die Einstellung auf dem Inkubator, in 37 ° C ergibt, ist) gehalten. </li><li> Pup Tätigkeit während des Protokolls genau überwachen, um sicherzustellen, dass die Tiere vertragen Zyklen ohne sichtbare Not wie Laute, veränderten NiveauGliedmaßentätigkeit oder Walzen. </li></ol> 4. Isolierung und Kultur der Stamm- / Vorläuferzellen aus dem Subventrikularzone HINWEIS: Die Veränderung der Neurosphäre Bildung nach AIH Vergleich zu Kontrollen ist ein Beispiel für einen Endpunkt, die die Wirksamkeit des Protokolls zeigt den Gewebe- und Zellebene Veränderungen hervorzurufen. <ol><li> Um Neurosphären bildenden Zellen zu isolieren, zu entfernen Maus oder Ratte Welpen aus der Kammer unmittelbar nach AIH ist abgeschlossen. Opfern die Tiere nach IACUC Protokolle innerhalb von 30 Minuten von Protokoll-Terminierung. </li><li> Ernten Sie die SVZ Gewebe, legen Sie in die Neurosphärenkultur und Verhalten Standarddurchgang und Expansion von derivativen Neurosphären, wie zuvor im Verfahren Kapiteln 15,16 und einem separaten Video-Protokoll 8 veröffentlicht. </li></ol>

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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. 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=Neurogenesis+in+the+adult+rat+brain+after+intermittent+hypoxia.">Neurogenesis in the adult rat brain after intermittent hypoxia.</a> Brain Res. 1055, 1-6 (2005).</li><li>Zhang, J. X., Chen, X. Q., Du, J. Z., Chen, Q. M., Zhu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+exposure+to+intermittent+hypoxia+enhances+mice+performance+in+water+maze+and+8-arm+radial+maze+tasks.">Neonatal exposure to intermittent hypoxia enhances mice performance in water maze and 8-arm radial maze tasks.</a> Journal of neurobiology. 65 (1), 72-84 (2005).</li><li>Zhu, L. L., Wu, L. Y., Yew, D. 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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...

Das Ziel dieser Methode ist es, reproduzierbar und effizient zu liefern intermittierende Anfälle von systemischen abgesenkt Umgebungssauerstoff zu neonatalen Nagetiere. Der Grund für die Verwendung intermittierender Hypoxie (IH) Stammzellen biology manipulieren stammt von in vitro Zellkulturexperimente, in denen O 2 -Gehalt des Wachstumsmediums verändert wird. Insbesondere im Vergleich zu &quot;normalen&quot; Bedingungen von 20% O 2, verlängerte Kultur von Stammzellen / Vorläuferzellpopulationen Zellen in 3% O 2 führt zu einer erhöhten Proliferation, Apoptose verringert und erhöhte neuronale Ausbeute 1,2. Diese Gruppe hat umfangreiche Erfahrungen mit der Verabreichung von systemischen IH und hat umfangreiche Studien über die Rolle der IH in Atem Plastizität 3-7 durchgeführt. Diese Arbeit und die jüngste Entdeckung, dass chronische IH erhöhte Neurogenese im Nagetier CNS 8-10, bildet die Grundlage für die Erforschung der akuten in vivoHypoxie als Vorkonditionierung Stimulus (dh., vor der Gewebeernte) auf die anschließende Kultur von neuronalen Stammzellen / Vorläuferzellen (NPC) 11. Bemerkenswert ist, wenn die Maus Jungtiere wurden auf eine kurze (&lt;1 Stunde) Zeit der akute intermittierende Hypoxie (AIH) ausgesetzt sind, Zellen, die aus der Subventrikularzone (SVZ) geerntet wurden, hatten signifikant erhöhte Kapazität für die Expansion als Neurosphären oder anhaftenden Monolayer-Zellen. AIH Protokoll wurde auch mit einer erhöhten Expression eines &quot;neuronalen Schicksals&quot; Transkriptionsfaktor (Pax6) zugeordnet ist. Dementsprechend wird in vivo AIH Protokolle kann ein Mittel zur &quot;prime&quot; NPCs vor der Kultur zu schaffen. Beispielsweise könnten Anwendungen für diesen Ansatz sind die Erweiterung Zellpopulationen vor der Transplantation in das verletzte Zentralnervensystems, oder einfach die Erhöhung der neuronalen Differenzierung von kultivierten Zellen vor der in vitro-Experimente. Ferner kann, da dies eine systemische Verabreichung, jedeOrgan, Gewebe oder Zellen, ist ein Kandidat für ähnliche Studie. Daher ist das Protokoll geschrieben potenziell für eine breite Palette von Studien über intermittierende Sauerstoff Manipulation in kleinen Säugetieren. Es gibt bestimmte Vorteile für diese Vorgehensweise. In anderen veröffentlichten Arbeiten wurden Neugeborenen als ein Wurf mit der Staumauer in Unterdruckkammern, die für die Langzeitanwendung vor der Behandlung ermöglicht, weniger Handling behandelt und gepflegt mütterlichen Kontakt während der Behandlung 9. Der derzeitige Ansatz umgeht wiederholten Behandlungen zu einer Zuchthündin, oder die Verwendung einer anderen Damm für jedes Experiment. Dieses Protokoll erlaubt auch Studium der präzisen Wurf abgestimmt und altersangepassten Neugeborenen. Repräsentative Daten zeigen eine weitere wesentliche Stärke dieses Protokolls, nämlich die Schnelligkeit, mit der AIH, wie geliefert, löst eine leistungsstarke und einheitliche biologische Reaktion in neuronalen Stammzellbiologie. Dies schafft einen Präzedenzfall für dieses Protokoll, um Gewebe- und Zellebene biologi entlockencal Änderungen, die Zellbiologie zu ändern. Dieser Bericht wird die detaillierten Verfahren zur Belichtung von Nagetier Welpen zur AIH sowie die Populationsanalyse der SVZ-Zellen als Neurosphären verwendet gewachsen zu skizzieren.

<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.

Die ersten Versuche, basierend auf historischen Daten, wurden unter Verwendung von 1 min Zykluslängen durchgeführt. Auf der Grundlage der nachfolgenden Kalibrierungen in Schritt 2 durchgeführt wird, wurde bestimmt, dass das O 2 in der Kammer, betrug 13% nach 1 min nach dem Spülen und Hypoxie, sie habe eine ähnliche Zeit, um die 21% Grundlinie zurück. Allerdings war eine 2 Minuten-Zyklus aus, um beides zu erreichen 10% Sauerstoff und eine Rückkehr zu 21% während des &quot;Baseline&quot; Zyklus. Anschli...

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

Dieser Artikel beschreibt die Methodik für die Verwaltung von kurzen Perioden intermittierender Hypoxie zu postnatalen Tag 1-8 Maus oder Ratte Welpen. Dieser Ansatz effektiv löst eine robuste Gewebeniveau &quot;Priming-Effekt&quot; an kultivierten neuralen Vorläuferzellen, die innerhalb von 30 Minuten von Hypoxie Exposition geerntet werden.

Lieferung von<em&gt; In-vivo-</em&gt; Akuter intermittierender Hypoxie in Neonatal Nagetiere in den Prime Subventrikularzone abgeleiteten neuronalen Vorläuferzellkulturen

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

delivery-vivo-acute-intermittent-hypoxia-neonatal-rodents-to-prime

Research

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 Aufrufe.  University of Florida.  Dieser Artikel beschreibt die Methodik für die Verwaltung von kurzen Perioden intermittierender Hypoxie zu postnatalen Tag 1-8 Maus oder Ratte Welpen. Dieser Ansatz effektiv löst eine robuste Gewebeniveau "Priming-Effekt" an kultivierten neuralen Vorläuferzellen, die innerhalb von 30 Minuten von Hypoxie Exposition geerntet werden. 

Serie: 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.

Direkte Induktion von humanen neuralen Stammzellen aus peripherem Blut hämatopoetischen Vorläuferzellen

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

Forschung

Entwicklungsbiologie

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.

Stammzell-ähnlichen<em&gt; Xenopus</em&gt; Embryonale Explantate bis Früh Neurale Entwicklungs- Eigenschaften Studieren<em&gt; In-vitro-</em&gt; Und<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.

Initiieren Differenzierung in Immortalisierte multipotenten Vorläuferzellen Otic

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.

Technik auf Zielmikroinjektions an die Entwicklungs<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.

Mit dem Hauptneurosphäre Kulturen Primäre Cilia zu studieren

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.

Induktion von Hypoxie im lebenden Frosch und Zebrafisch Embryonen

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.

Eine neuartige Mamma-Fat-Pad-Transplantationstechnik zur Visualisierung der Gefäßerzeugung von vaskulären endothelialen Stammzellen

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.

Quantitative ganze-Mount Immunfluoreszenz Analyse der kardialen Vorläuferzellen Populationen in Mausembryonen

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.

Echtzeit Live-Zelle Durchflusszytometrie, Calcium Zustrom, Porenbildung und Phagozytose durch P2X7-Rezeptoren im Erwachsenen neurale Vorläuferzellen zu untersuchen

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.

Kontrolliertes kortikales Wirkungsmodell der Maushirnverletzung mit therapeutischer Transplantation von humaninduzierten Pluripotenten Stammzell-abgeleiteten Neuralzellen

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