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

NOT: Bu protokol tüm hayvan prosedürleri Florida Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) Üniversitesi onayı ile gerçekleştirilen ve &#39;Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu&#39; ile uyumlu vardır. 1. Temel Deney Seti yukarı Aralıklı Hipoksi İdaresi Öncesi <ol><li> Diğer deneysel amaçlar 5,13,14 yetişkin kemirgenler kadar aynı şekilde bir tüm vücut fare Pletismograf odaları kullanarak farklı gaz karışımları için yavrular 12 Açığa. Odaları 3-4 fare yavrular ya da 1-2 sıçan yavrular karşılamak için yeterli büyüklükte bir 4 inç çapı (hacim = 450 ml) var. Burada, AIH protokolleri doğum sonrası gün 1-8 (P1-P8) yaş yenidoğanlarda teslim edilir. </li><li> % 21 kamara oksijenasyonu tutar% 21 &quot;taban&quot; gaz teslim basınçlı hava gaz tankları kullanın. &quot;Göreli hipoksi&quot; elde etmek için,% 10 oksijen /% 90 azot gazı tankı kullanın. DEĞİLE: Plastik boru (3/16 &quot;çapında) bir akış ölçere gaz tankları bağlanır. Bu nedenle, akış ölçere girişi bir tüp taban çizgisi ile hipoksi hava akışı için, her oluşur. </li><li> Aşağıdaki gibi gaz boru yapılandırın. <ol><li> Her odaya 1 L / dk akışını sağlayan bir önyargı akış regülatörü, akış ölçer plastik boru (1/8 &quot;çap) Run (yani 4 L / dk 4 odacıklı kullanılırsa). </li><li> Pletismografi odalarına önyargı akış düzenleyici plastik boru (1/8 &quot;çap) çalıştırın. Hava akımı, 1 L / saat, her bölmeye dakika gaz alışverişi olmayan stres yapıcı fizyolojik ve davranış gözlemlere dayanarak hayvanlara olduğu kabul edildiği bir önceden tespit edilmiş bir akış hızıdır. </li></ol></li><li> Hiçbir gaz akış protokolü sırasında kullanılmadığı, ya da pletismografi odaları dışında diğer odalarına kaçar ve böylece kapakları odalarına ve önyargı akış biriminden kullanılmayan tüm bağlantıları ve tüm kullanılmayan açıklıkları kapatılmalıdır. </li><li> (Odasına yerleştirin ler) 37 ° C&#39;ye kadar dengeye veya vücut ısısı sağlamak için bölme hayvanların koyarak önce bir inkübatör içine. </li></ol> Aralıklı Hipoksi için Döngüsü Times 2. Kalibrasyon <ol><li> Hava tankları ve akış ölçer arasındaki tüm hortumunu kontrol edin doğru bağlantıları için sayaç ve önyargı akış ünitesi ve önyargı akış ünitesi ve pletismografisi odasını (ler) akar. Örneğin, kullanımda olmayan kullanılmayan önyargı akış biriminin dışına hatları ve herhangi bir odacık açıklıkları kontrol: Tüm açıklanan hatları ve kapanışları bir adım adım çekle bağlantısını kontrol edin Adım 1&#39;de açıklandığı gibi bağlantı kurulmaktadır. </li><li> Bir el O 2 metre ile ortam O 2 sensörünü bağlayın ve sonra pletismografisi odası kapak sensörü takın. </li><li> Her iki gaz tankları açın ve gaz off-döngüsü akışını kelepçe plastik boru ile izole cerrahi, uzun saplı hemostat bir çift takın. Bu şekilde, bir seferde sadece bir gaz tankeri odasının başına 1 L / dk gaz akışını iletiyor. &quot;Hipoksik&quot; gaz karışımı tersi odasına teslim ve ise &quot;taban&quot; gaz karışımı Kelepçe. </li><li> % 10 hedef düzeylere ulaşması ve% 21 geri dönmek için hazne O 2 için gerekli süreyi kaydedin. Sonraki protokol teslimat için kaydedilen süreleri yararlanın. </li></ol> Akut Aralıklı Hipoksi 3. İdare <ol><li> Adım 2.1 anlatıldığı gibi tüm boru ve bağlantıları kontrol edin. </li><li> Pletismografisi odasının (ler) yenidoğan yerleştirin ve oda üssü haline pletismografisi odası kapaklarını ev. Kullanılmayan odası bağlantılarının uygun bir O-ring conta ile mühür olduğu gibi kapatma onaylayın. Bu adımlar, gaz karışımları uygun teslimat sağlanması için kritik öneme sahiptir. </li><li> Yavrular tutan odaları 37 ° C inkübatör içine tedavi edilmesi yerleştirin ve kapağını kapatın. Kamara önce protokolü başlamadan 37 ° C gelmesini sağlayınız. Incubat 37 ° C &#39;de kontrol hayvanları korumakveya oda havası oksijenasyonu de. </li><li> Her iki gaz tankları açın ve gaz off-döngüsü akışını kelepçe plastik boru ile izole cerrahi, uzun saplı hemostat bir çift kullanın. Bu şekilde, bir seferde sadece bir gaz tankeri odasının başına 1 L / dk gaz akışının sağlamaktadır. </li><li> Hassas bir zaman döngü elde tutulan bir zamanlayıcı kullanarak ve böylece odalarına gaz akışının münavebe ile sonuçlanan, borular arasındaki hemostat geçiş zamanı gösterir. </li><li> Örneğin 2 aşamalı, 20 çevrim protokolünde tüm adımları kapalı işaretlemek için Şekil 1&#39;de verilen biri olarak tedavi günlüğünü kullanarak her döngünün tamamlanmasını kaydedin. </li><li> Hayvanlar belirlenen aralığın (37 ° C ile sonuçlanan inkübatör ayarı 33-35 ° C) muhafaza edilir sağlamak için her döngü değişikliğinde inkübatör sıcaklığını izleyin. </li><li> Yakından hayvanlar gibi sesler çıkarma, değişmiş seviyesi gibi görünen sıkıntı olmadan çevrimleri tahammül emin olmak için protokol boyunca yavru etkinliğini izlemekuzuv faaliyet veya yuvarlanma. </li></ol> Subventricular Zone Kök / Progenitör Hücreler 4. İzolasyon ve Kültür NOT: kontrollere göre AIH aşağıdaki neurosphere oluşumunda değişiklik doku ve hücresel düzey değişiklikleri ortaya çıkarma, bu protokolün etkinliğini gösteren bir bitiş noktası bir örnektir. <ol><li> Hücreleri oluşturan neurospheres izole etmek için, AIH tamamlandıktan hemen sonra odadan fare veya sıçan yavrular kaldırın. Protokol fesih 30 dk içinde IACUC protokollerine göre hayvanların Kurban. </li><li> Daha önce yöntemler bölümlerde 15,16 ve ayrı bir video protokolü 8 yayımlanan, SVZ doku hasat neurosphere kültürü yerleştirmek ve standart geçit ve türev neurospheres genişlemesini yürütmek. </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...

Bu yöntemin amacı, yeniden üretilebilir ve etkili bir yenidoğan kemirgenlere sistemik alçaltılmış havada bulunan oksijen aralıklı nöbetleri sunmaktır. Kök hücre biyolojisi işlemek için kesintili hipoksi (İH) kullanımı için gerekçe büyüme ortamı O 2 içeriği değiştirilmiş olduğu in vitro hücre kültürü deneylerinde kaynaklanır. % 20 &quot;standart&quot; koşullar O 2, artan çoğalmasında% 3 O 2 sonuçlarında kök / progenitör hücre popülasyonları hücrelerin uzatılmış kültürü ile karşılaştırıldığında Özellikle, apoptozu azalmış ve nöronal verim 1,2 arttı. Bu grup, sistemik IH yönetimi ile önemli bir deneyime sahiptir ve solunum plastisitede 3-7 IH rolü üzerine kapsamlı çalışmalar yürütmüştür. Bu çalışma, kronik IH kemirgen CNS 8-10 nörogenezisi arttığı son bulgu in vivo akut keşif için temelini oluştururBir önkoşullanma uyarıcı olarak hipoksi (yani., doku hasat öncesinde) nöral kök / progenitör hücrelerin (NPC) 11 sonraki kültürüne. Fare yavrular akut aralıklı hipoksi (AIH) kısa (&lt;1 saat) Dönem maruz kaldılar Dikkate değer, subventriküler bölge (SVZ) hasat edildi hücreler önemli ölçüde neurospheres veya yapışık tek tabaka hücreleri gibi genişleme kapasitesi artmıştır. AİH protokol aynı zamanda bir &quot;nöron kaderi&quot; transkripsiyon faktörü (Pax6) artmış ekspresyonu ile ilişkili bulunmuştur. Buna göre, in vivo AIH protokolleri için &quot;prime&quot; NPC&#39;ler önce kültürüne bir araç sağlayabilir. Örneğin, bu yaklaşım için uygulamalar yaralı merkezi sinir sistemi içine transplantasyondan önce hücre popülasyonlarının genişletilmesi, ya da sadece önceden in vitro deneylere kültürlenmiş hücrelerin nöronal farklılaşma artan içerebilir. Bundan başka, bu sistemik olarak uygulanması, çünkü herhangi birorgan doku veya hücre içindeki çalışma için bir adaydır. Bu nedenle, yazılı olarak protokol küçük memeliler aralıklı oksijen manipülasyon çalışmalarının geniş bir yelpazede potansiyel uygulanabilir. Bu yaklaşımın bazı avantajları vardır. Diğer yayınlanan çalışmada, yenidoğanlar tedaviden önce, kronik dozlama için daha az işleme izin veren hipobarik odacıklarda baraj, bir çöp muamelesi ve tedavisi 9 süresince anne teması muhafaza edildi. Geçerli yaklaşım üreme kadın, ya da her bir deney için farklı bir baraj kullanımına tekrarlanan tedaviler atlar. Bu protokol ayrıca hassas çöp eşleştirilmiş ve aynı yaştaki yenidoğanların çalışma sağlar. Temsili veriler bu protokolün bir başka önemli gücü, AIH, teslim olarak, nöral kök hücre biyolojisinde güçlü ve tutarlı bir biyolojik tepkiyi ortaya çıkaran hangi ile yani Çabukluğu göstermektedir. Bu doku ve hücre düzeyinde biyolojik ka ortaya çıkarmak için bu protokol için bir emsal oluştururHücre biyolojisi değiştiren cal değişir. Bu rapor AIH için kemirgen yavrular yanı sıra neurospheres olarak yetiştirilen SVZ hücrelerin nüfus analizi sergilemek için kullanılan ayrıntılı prosedürler açıklayacağım.

<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>
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		<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>
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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.

Tarihsel verilere dayalı ilk deneyler, 1 dk döngüsü uzunlukları kullanılarak yapılmıştır. Yukarıdaki 2. Adımda gerçekleştirilen sonraki kalibrasyon dayanarak, bunun% 21 başlangıca dönmek için benzer bir zaman aldı, odasında O 2 seviyesi 1 dk sonrası hipoksi flushing&#39;in 13% olduğu belirlenmiştir edildi ve. Bununla birlikte, 2 dakika döngüsü 10% elde oksijen ve &quot;taban&quot; döngüsü sırasında% 21 bir dönüş hem de yeterli olmuştur. Daha sonra, 2 dakika döngü uzunlukla...

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

Bu makale doğum sonrası gün 1-8 fare veya sıçan yavrular aralıklı hipoksi kısa süreli uygulanması için yöntem anlatılmaktadır. Bu yaklaşım etkili bir hipoksi maruz kalma 30 dakika içinde hasat edilir kültürlü nöral progenitör hücreler üzerindeki sağlam doku düzeyi &quot;etkisi priming&quot; ortaya çıkarır.

Teslimatı<em&gt; İn Vivo</em&gt; Akut Aralıklı hipoksi Yenidoğan Kemirgenler Başbakan subventricular bölgesi kökenli Sinir Progenitör Hücre Kültürleri için

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

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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 Görüntüleme.  University of Florida.  Bu makale doğum sonrası gün 1-8 fare veya sıçan yavrular aralıklı hipoksi kısa süreli uygulanması için yöntem anlatılmaktadır. Bu yaklaşım etkili bir hipoksi maruz kalma 30 dakika içinde hasat edilir kültürlü nöral progenitör hücreler üzerindeki sağlam doku düzeyi "etkisi priming" ortaya çıkarır. 

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.

Kök hücre benzeri<em&gt; Xenopus</em&gt; Embriyonik Eksplantlarındaki Erken Nöral Gelişim Özellikleri Okuyacak<em&gt; İn Vitro</em&gt; Ve<em&gt; İn Vivo</em

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

Gelişim Biyolojisi

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.

Ölümsüzleştirdi Multipotent Otic Progenitor Hücrelerde Farklılaşma başlatılıyor

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.

Gelişmekte olan mikroenjeksiyon Hedef Tekniği<em&gt; Xenopus</em&gt; Böbrek

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.

Zebra balığı bir Etanol kaynaklı Fibrotik Karaciğer Modelinin Geliştirilmesi progenitör hücre-aracılı Hepatosit Rejenerasyon Eğitim

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.

İlköğretim Cilia Eğitim için birincil Neurosphere Kültür Kullanımının

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.

Kurbağa ve Zebra balığı Embriyolarında Hipoksinin İndüklenmesi

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.

Vasküler Endotelyal Kök Hücrelerin Damar Oluşumunu Görselleştirmek İçin Yeni Bir Göğüs Göğsü Transplantasyon Tekniği

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.

Nicel bütün Dağı ayirt analiz fare embriyo kardiyak progenitör nüfusun

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.

Kalsiyum akını, gözenek oluşumu ve P2X7 reseptörleri yetişkin nöral progenitör hücreler tarafından fagositoz araştırmak için gerçek zamanlı canlı hücre Akış Sitometresi

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.

Insan kaynaklı pluripotent kök hücre türetilen nöral hücrelerin terapötik transplantasyonu ile fare beyin yaralanması kontrollü Kortiik darbe modeli

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