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

注：このプロトコルのすべての動物の手順は、フロリダ大学の施設内動物管理使用委員会（IACUC）の承認を得て実施し、「実験動物の管理と使用に関する指針」に準拠しているされています。 1.基本実験は、断続的低酸素症の管理前に設定<ol><li>他の実験目的5,13,14のための大人の齧歯類とほぼ同じ方法で、全身マウスプレチスモグラフ室を使用して、異なるガス混合物への子犬12を公開します。チャンバは3-4仔マウスまたは1-2仔ラットを収容するために十分なサイズである4インチ径（容積= 450 ml）を、有しています。ここで、AIHプロトコルは、生後1-8（P1〜P8）歳の新生児に配信されます。 </li><li> 21％でチャンバーの酸素化を維持し、21％、「ベースライン」ガスを送達するために圧縮空気のガスタンクを使用してください。 「相対的な低酸素状態」を達成するために、10％酸素/ 90％窒素ガスのタンクを使用します。 NOTE：プラスチックチューブ（3/16 &quot;直径）は、流量計にガスタンクを接続しています。このように、流量計への入力は、ベースラインと低酸素気流のために一本のチューブそれぞれ構成されています。 </li><li>次のようにガス配管を構成します。 <ol><li>各チャンバーに1 L /分の流れを可能にするバイアス流調整器、流量計からのプラスチックチューブ（1/8 &quot;直径）を実行する（ すなわち 、4 L / minで4チャンバが使用される場合）。 </li><li>容積脈波室にバイアス流量調整からプラスチック管（直径1/8 &quot;）を実行します。各チャンバに1 L /分の空気流は、ガス交換は、生理学的および行動の観察に基づいて、動物に、非ストレス誘導されたものとみなさされる所定の流量です。 </li></ol></li><li>何のガス流が使用されていない、またはプロトコル中の脈波検査室のうち、他のチャンバに逃げるないようにキャップ付き室にバイアス流ユニットへとからすべての未使用の接続およびすべての未使用の開口部をシール。 </li><li> （室を置き、S）37℃に平衡化し、または体温を可能にするためにチャンバ内に動物を入れする前にインキュベーター。 </li></ol>断続的低酸素症のためのサイクルタイムの2キャリブレーション<ol><li> 、空気タンクと流量計との間で、すべてのチューブを検査し、適切な接続用計器とバイアス流ユニット、およびバイアス流ユニットとプレチスモグラフィーチャンバー（複数可）を流します。 例えば、使用されていない未使用のバイアス流ユニットのうちラインおよび任意のチャンバ開口部をチェック：すべての説明ラインと閉鎖の段階的チェックして、接続を確認し、手順1で説明したように接続が設定されています。 </li><li>手持ちO 2メートルで、周囲O 2センサを接続して、容積脈波室蓋にセンサーを取り付けます。 </li><li>両方のガスタンクを開き、ガスのオフサイクルの流れをクランプするプラスチック製のチューブで絶縁外科手術、長い取っ手止血のペアを1つ取り付けます。この方法では、一度に一つのガスタンク室あたり1リットル/分のガス流を供給しています。 「低酸素」ガス混合物をチャンバに供給される一方で、「ベースライン」のガス混合物をクランプ、およびその逆。 </li><li> 10％の目標レベルに到達し、21％に戻ることチャンバーO 2に必要な時間を記録します。その後のプロトコルの配信のための記録された時間を利用しています。 </li></ol>急性間欠性低酸素症の3管理<ol><li>ステップ2.1で説明したように、すべてのチューブとの接続を点検します。 </li><li>脈波検査室（複数可）に新生児を置き、チャンバーベースに容積脈波室の蓋を収容します。未使用室接続の適切なOリングシールにシールであるだけでなく、閉鎖を確認してください。これらの手順は、ガス混合物の適切な配送を確保するために重要です。 </li><li>子犬を保持室は37℃のインキュベーターに扱われることを置き、ドアを閉めます。室前のプロトコルを開始する37℃に平衡化することを許可します。 incubatで37℃で対照動物を維持または室内空気の酸素で。 </li><li>両方のガスタンクを開き、ガスのオフサイクルの流れをクランプするプラスチック製のチューブで絶縁外科手術、長い取っ手止血のペアを1つ使用します。この方法では、一度に一つのガスタンク室あたり1リットル/分のガス流を提供しています。 </li><li>正確に時間サイクルにハンドヘルドタイマーを使用し、したがって、チャンバへのガス流の交互に得られた、チューブ間の止血を切り替える時刻を示します。 </li><li> 2ステップ、20サイクルプロトコルのすべてのステップをオフにマークするために、図1に提供されるものなどの処理ログを使用して、各サイクルの完了を記録します。 </li><li>動物は指定範囲（37°Cになるインキュベーターの設定である33〜35°C）に維持されることを保証するために、サイクルごとの変更のインキュベーターの温度を監視します。 </li><li>密接にこのような発声、レベルの変化などの動物が見える苦痛なしでサイクルに耐えることを確認するために、プロトコル全体子犬の活動を監視四肢活性またはローリング。 </li></ol>脳室下帯から幹/前駆細胞の単離および培養4 注：コントロールに比べAIH次ニューロスフェア形成の変化は、組織や細胞レベルの変化を誘発するこのプロトコルの有効性を示すエンドポイントの例です。 <ol><li>セルを形成するニューロスフェアを単離するために、AIHが完了した直後にチャンバーからマウスまたはラット仔を削除します。プロトコル終端の30分以内にIACUCプロトコルに従って動物を生け贄に捧げます。 </li><li>以前の方法の章15,16と独立したビデオプロトコル8に発表されたように、SVZの組織を採取する神経球の文化に置き、標準の通路及びデリバティブニューロスフェアの展開を行っています。 </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...

この方法の目的は、再現性と効果的に新生児の齧歯類に全身の低下周囲の酸素の断続的な発作を提供することです。幹細胞生物学を操作するために、断続的低酸素症（IH）を使用するための理論的根拠は、増殖培地のO 2含有量が変更されたインビトロ細胞培養実験における由来します。具体的には、20％のO 2、3％O中の幹/前駆細胞集団の細胞増殖の増加は 2の結果の拡張された培養物の「標準的な」条件と比較した場合、アポトーシスの減少およびニューロン収量1,2を増加させました。 このグループは、全身IHの投与と豊富な経験を持っており、呼吸可塑3-7に IHの役割について鋭意検討しました。この作品、および慢性IHはげっ歯類CNS 8-10に神経新生を増加させることを最近の知見は、in vivoでの急性の探査のための基礎を形成プレコンディショニング刺激として低酸素状態（すなわち、組織の収穫に先立って）神経幹/前駆細胞（NPCの）11のその後の培養に。仔マウスは、急性間欠性低酸素症（AIH）の短い（&lt;1時間）の期間にさらされたときに驚くべき、脳室下帯（SVZ）から採取した細胞はかなりの神経球または接着単層細胞のような拡張のための容量を増加しました。 AIHプロトコルは、「ニューロンの運命」転写因子（Pax6の）の発現増加と関連していました。 従って、 インビボでの AIHプロトコルは、「プライム」のNPC培養前に手段を提供することができます。例えば、このアプローチのアプリケーションは、負傷した中枢神経系に移植前に細胞集団を拡大し、または単に以前のin vitro実験に培養細胞の神経分化を増加させる含めることができます。さらに、これは任意の、全身送達であるため、臓器、組織または細胞は、同様の研究のための候補です。したがって、記述されたようなプロトコルは、小型哺乳類で断続的な酸素操作に関する研究の広い範囲に適用できる可能性です。 このアプローチにはいくつかの利点があります。他の公開作品では、新生児を治療する前に、長期投与のために以下の取り扱いを可能にする低気圧チャンバ内のダム、とごみとして処理され、治療の9時の母体の接触を維持しました。現在のアプローチは、繁殖の女性、または各実験ごとに異なる​​ダムの使用に繰り返し治療をバイパスします。このプロトコルはまた、正確なゴミが一致したと年齢をマッチさせた新生児の研究を可能にします。代表的なデータは、このプロトコルの他のキーの強度、AIHとすなわち迅速性を実証し、配信として、神経幹細胞生物学における強力かつ一貫性のある生物学的応答を誘発します。これは、組織や細胞レベルのbiologiを引き出すために、このプロトコルのための先例を確立細胞生物学を変えるCAL変更。 このレポートではAIHにげっ歯類の子犬を露出するだけでなく、ニューロスフェアとして増殖SVZ細胞の集団の分析に使用する詳細な手順を概説します。

<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.

過去のデータに基づいて、初期の実験は、1分間のサイクル長を使用して行きました。上記のステップ2で実行される後続のキャリブレーションに基づいて、それは21％のベースラインに戻るには、同様の時間を要したこと、チャンバー内のO 2レベルは1分後に低酸素症のフラッシングで13％であったことを決定しました。しかし、2分間のサイクルを10％の酸素と、「ベースライン」サイ?...

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

この記事では、生後1-8マウスまたはラット仔に断続的低酸素症の短い期間を管理するための方法を説明します。このアプローチは、効果的に低酸素暴露の30分以内に収穫される培養神経前駆細胞の「影響をプライミング」強固な組織レベルを誘発します。

の配達<em&gt;インビボ</em&gt;急性間欠性低酸素症新生児げっ歯類で首相脳室下帯由来の神経前駆細胞の培養に

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

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Developmental Biology

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

7.2K ビュー.  University of Florida. この記事では、生後1-8マウスまたはラット仔に断続的低酸素症の短い期間を管理するための方法を説明します。このアプローチは、効果的に低酸素暴露の30分以内に収穫される培養神経前駆細胞の「影響をプライミング」強固な組織レベルを誘発します。 

ビデオ: 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.

末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

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

発生生物学

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.

幹細胞様<em&gt;アフリカツメガエル</em&gt;胚外植片を早期神経発達の特徴を研究するために<em&gt;インビトロ</em&gt;と<em&gt;インビボ</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.

不死化多能耳の前駆細胞に分化を開始

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.

開発にターゲットマイクロインジェクションのテクニック<em&gt;アフリカツメガエル</em&gt;腎臓

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.

一次繊毛を研究するために一次ニューロスフェア培養物を使用して

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.

生きた蛙およびゼブラフィッシュの胚における低酸素の誘導

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.

血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

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.

マウス胚における心臓前駆細胞集団の定量的ホール マウント免疫染色解析

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.

大人の神経前駆細胞における P2X7 受容体を貪食、細孔の形成、カルシウム流入を調査するためのリアルタイム生細胞フローサイトメトリー

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.

ヒト誘導多能性幹細胞由来神経細胞の治療移植によるマウス脳損傷の制御された皮質衝撃モデル

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