We thank Kwon laboratory members for helpful discussions. This work was supported by grants from NHLBI/NIH (R01HL111198) and Maryland Stem Cell Research Fund (MSCRF).

すべてのマウスは、ジョンズ·ホプキンス大学の動物施設を-accredited実験動物管理の認定のためのアメリカ協会（AAALAC）に維持し、実験動物の管理と使用に関するガイドに概説された手順に従って飼育しました。制度動物実験委員会（IACUC）は、すべての実験プロトコールを承認しました。 1。実験の準備<ol><li>リン酸緩衝液中の10％ウシ胎児血清（FBS）（PBS）を用いてコート12ウェルプレートで60分間。 </li><li>コー​​ティング溶液を除去し、無血清培地（SFM）の200μlを添加します。メディアのフィルムは、表面領域全体を覆っていることを確認するために、プレートを振り回します。 </li></ol> 2.外科的処置<ol><li>完全な安楽死を確実にするために、頸椎脱臼に続いて、金融機関の動物管理委員会が承認したプロトコルを使用して妊娠マウスを安楽死させます。 </li><li>スプレーし、70％のマウスの腹の領域をきれいにエタノール。汚染を避けるために、厳密な無菌技術を維持しています。 </li><li>へその領域で0.5 cm 2の切開を作るために鉗子やハサミを使用してください。 </li><li> /ピンチ切開の上および下の皮膚をつかみ、ゆっくりと反対方向（ヘッドとテール）に皮膚を引っ張って指を使用してください。 </li><li>へその領域における膜内の最初の切開を作るために鉗子やハサミを使用してください。ゆっくりそれによって子宮を明らかに、それぞれの側の卵巣にへそからV字状に（2×1.5 cm 2で）膜をカット。 </li><li>このように卵巣から子宮を解放し、卵巣側の卵管を挟ま卵巣およびはさみに子宮を接続卵管を挟ま鉗子を使用してください。 </li><li>優しく鉗子で卵管によって子宮をプルアップし、はさみで膀胱領域のない子宮を切りました。 </li><li>ピンセットで卵管によってさらにアップ子宮を引き出し、それによってMから子宮を解放する、反対側にハサミで卵管をカットウーズ。 </li><li> 50mlのチューブに子宮を転送およびCa 2+およびMg 2+との無菌冷PBS 20mlのを追加します。 PBSは、胚の解剖中のCa 2+およびMg 2+を含まれている必要があります。 </li><li>ゆっくり血を子宮を洗浄するために10秒間チューブを振ります。 </li><li>鉗子で管から子宮を移し、10mlの皿にそれを置き、子宮の上に冷PBSを5ml加えます。 </li><li>実体顕微鏡下での子宮で皿を置きます。 注：2.13から2.19には実体顕微鏡を必要とする手順。 </li><li>静かに子宮を開き、子宮一つ一つからの胚で羊膜嚢を解剖するために鉗子の二組を使用してください。 <ol><li>慎重に、胚を囲む羊膜を開く1鉗子で嚢をつまんで優しく胚から削除し、胚から羊膜を切断し、解放するために他の鉗子を使用するために鉗子の二組を使用してください。特定の遺伝子型は、必要に応じて、遺伝子型決定のための羊膜を維持します。 </li></ol></li><li>側に胚を配置し、 第 1および第 2の切断に鉗子を使用NDアーチと咽頭嚢（ 図1A &#39;&#39;）との間の心に後方アーチ。 </li><li>静かに反対側に胚を反転し、アーチや咽頭嚢の間心に後方に1番目と2番目のアーチをカットする鉗子を使用しています。 </li><li>まだ胚に1 番目と2 番目の咽頭弓を接続している心臓の流出路を切断するために鉗子を使用してください。胚から、まだ蝶（ 図1B）の形で一緒に取り付けられているアーチを、削除します。 </li><li>ピンチする鉗子を使用し、残りの心臓の流出路と前腸内胚葉から1 番目の咽頭弓を解放します（ 図1（b） &#39;&#39; *破線で示すと）。 </li><li>ピンチする鉗子を使用し、残りの心臓の流出路から2 番目の咽頭弓をリリースと前腸内胚葉（ 図1B &#39;&#39;破線で示し、**）。 </li><li>ピンセットを用いて別々のアーチの各ペアを移し、よく指定の途中で両方のアーチを配置します。別々のウェルでプレート1 番目と2 番目の咽頭弓。アーチは、ステップ1.2で追加されたメディアのフィルムに接触していることを確認してください。彼らは、この期間中に取り付けるためのウェルの表面と接触する必要があるように、それは、アーチが媒体によって覆われていないことが重要です。 </li></ol> 3.インキュベーションとイメージング<ol><li>ウェル表面領域に取り付けるためのアーチのために2時間、37℃/ 5％CO 2で12ウェルプレートにプレーティングアーチをインキュベートします。 </li><li>ゆっくりウェルの側面の下、37℃SFMの200μlを添加します。アーチが取り付けられたままとO / Nをインキュベートすることを確認します。 </li><li>翌日、視覚的に咽頭弓が接続されていることを確認し、穏やか側下37°C SFMの200μlのを追加よく。アーチが取り付けられていないとフローティング残っている場合、メディアの膜のみが残っているまで、静かにアーチを除去せずにピペットでメディアを削除します。よく繰り返しステップ3.1の中央にアーチを配置するピペットチップを使用してください。 </li><li>毎日の監視;任意の蒸発したメディアを交換するごとに2日目のメディアの〜100〜200μlを添加します。 注：細胞を移行した後24〜48時間は、アーチの周りに表示され、増殖し、次の5-8日にわたって弓から移行されます。拍動心筋細胞の36から72時間の形成は、2 番目の咽頭弓（ 図2B）から観察することができます後。筋管形成は、3-7日付着（ 図2A）した後、 第 1 の咽頭弓から観察することができます。 </li></ol>

<ol>
	<li>Kelly, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+heart+field.">The second heart field.</a> Curr Top Dev Biol. 100, 33-65 (2012).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Tzahor, E., Evans, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharyngeal+mesoderm+development+during+embryogenesis:+implications+for+both+heart+and+head+myogenesis.">Pharyngeal mesoderm development during embryogenesis: implications for both heart and head myogenesis.</a> Cardiovasc Res. 91, 196-202 (2011).</li><li>Uosaki, H., 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=Direct+Contact+with+Endoderm-like+Cells+Efficiently+Induces+Cardiac+Progenitors+from+Mouse+and+Human+Pluripotent+Stem+Cells.">Direct Contact with Endoderm-like Cells Efficiently Induces Cardiac Progenitors from Mouse and Human Pluripotent Stem Cells.</a> PLoS One. 7 (10), e46413 (2012).</li><li>Cheng, P., 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=Fibronectin+mediates+mesendodermal+cell+fate+decisions.">Fibronectin mediates mesendodermal cell fate decisions.</a> Development. 140, 2587-2596 (2013).</li><li>Kwon, C., 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=A+regulatory+pathway+involving+Notch1/beta-catenin/Isl1+determines+cardiac+progenitor+cell+fate.">A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate.</a> Nature cell biology. 11, 951-957 (2009).</li><li>Shenje, L. T., 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=Precardiac+deletion+of+Numb+and+Numblike+reveals+renewal+of+cardiac+progenitors.">Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.</a> Elife. 3, e02164 (2014).</li><li>Grevellec, A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharyngeal+pouches+and+clefts:+Development,+evolution,+structure+and+derivatives.">The pharyngeal pouches and clefts: Development, evolution, structure and derivatives.</a> Semin Cell Dev Biol. 21, 325-332 (2010).</li><li>Kwon, C., Cordes, K. R., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+acts+at+multiple+developmental+stages+to+promote+mammalian+cardiogenesis.">Wnt/beta-catenin signaling acts at multiple developmental stages to promote mammalian cardiogenesis.</a> Cell Cycle. 7, 3815-3818 (2008).</li><li>Cho, G. S., Fernandez, L., Kwon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+heart:+perspectives+on+stem-cell+therapy.">Regenerative medicine for the heart: perspectives on stem-cell therapy.</a> Antioxid Redox Signal. 21, 2018-2031 (2014).</li></ol>

The authors have nothing to disclose.

このビデオでは、我々は9.5日の咽頭弓古いマウス胚第一および第二の単離および培養する方法を示します。咽頭のアーチは、最初のアーチ8を第二のアーチとヘッド筋前駆細胞-in胚10,11の間に心臓をするために、マルチ強力な心臓前駆細胞ビルディングブロックを含む発生中の胚9のcraniolateral側に表示されるバルジを過渡セグメント化されています。 <p class="jove_conten...

咽頭中胚葉細胞は、心臓の部品や咽頭の筋肉を生じさせます。胚発生の間に、第2の心臓フィールドから多能心臓前駆細胞は、咽頭中胚葉から移行し、心臓流出路および右心室を移入し、それらの異常な発達は密接先天性心疾患、先天性欠損症の主な原因と出生defect-に関連付けられていますヒト1-3関連死。最近の研究では、咽頭中胚葉が中胚葉心臓頭蓋顔面の開発4の重要な部分作る、心臓に加えて、筋肉を頭に寄与することを実証しました。このように、誘導、増殖、および咽頭中胚葉で頭と心前駆細胞の分化を含む発達プロセスは、活発な調査5-7中です。 最近まで、心臓前駆細胞は、拡張withoを受けるかどうかは不明のままで部分的には、それらの細胞環境に関する情報の欠如UT分化。我々の最近の研究では、咽頭のアーチは、心臓や筋肉前駆細胞の更新のための微小環境として機能し、数週間かけて8 ex vivoで培養することができることを示唆しています。この外植法は心臓頭蓋顔面前駆細胞ex vivoでの開発を研究するための新規かつユニークな機会を提供しています。

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS + Ca2 + Mg2</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10cm petri plates</td>
			<td>Fishersci</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Dissection forceps</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Serum Free Media:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Cellgro</td>
			<td>15-016-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F12</td>
			<td>Cellgro</td>
			<td>10-080-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>N2-SUPPLEMENT</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27-retinoic acid</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>10% BSA (in PBS) &#224; (Invitrogen Cat#. P2489)</td>
			<td>Life Sciences</td>
			<td>P2489</td>
			<td></td>
		</tr>
		<tr>
			<td>100X Glutamine (Gibco Cat.# 25030-081)</td>
			<td>Gibco</td>
			<td>35050-61</td>
			<td></td>
		</tr>
		<tr>
			<td>100X Pen/Strep (Gibco Cat#. 15070-063)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo culture of pharyngeal arches to study heart and muscle progenitors and their niche

発生中の胚の咽頭中胚葉は、頭と心筋組織の広範な地域に貢献しています。我々は、ex vivoで （も鰓のアーチと呼ばれる）咽頭弓で頭と心前駆細胞の発達を研究するための新規な方法を開発しました。この方法を使用して、我々は最近、第二の咽頭弓が自己複製心臓前駆細胞が含まれており、マウスの心臓の開発中に前駆細胞の拡大のための微小環境として機能することを記載しています。前駆細胞は、アーチの内側未分化と広大なままであるが、すぐに彼らはアーチの外に移動するような機能心筋細胞になります。また、最初の咽頭弓は弓を離れた後の筋管を生じさせる筋肉前駆細胞が含まれていることを報告しました。ここでは、解剖とマウス胚の開発からの第1および第2の咽頭弓のex vivo培養のための手順を示しています。この方法は、頭と心前駆/ devの筋肉を研究することを可能ex vivoで詳細に心筋細胞および筋管形成を含むelopment、。

彼らは増殖し、1 番目と2 番目の咽頭弓からの移行のように、開発時には、顔の筋肉や心臓前駆細胞は（ 図1A、A &#39;とA&#39; &#39;）は、それぞれ、頭と心の筋肉組織になるために、追跡することができます。咽頭弓を培養すると、ex vivoで詳細に心臓や筋肉の発達を研究するためのユニークな方法を提供しています。解剖と咽頭弓の付着した後、?...

Watch this Scientific Journal Video about Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche at JoVE.com

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽頭アーチのex vivo培養は、ハートと筋前駆細胞とそのニッチを研究します

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

ex-vivo-culture-pharyngeal-arches-to-study-heart-muscle-progenitors

Research

JoVE Journal

Developmental Biology

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

7.7K ビュー.  Johns Hopkins University School of Medicine. Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

ビデオ: Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

使い方<em&gt;エクスビボ</em&gt;全体の胎児器官の直立飛沫培養物をマウス器官中に発生過程を研究します

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

発生生物学

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

チック小脳スライスと顆粒細胞前駆体の空間的にターゲットを絞ったエレクトロポレーションの ex vivo培養

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜の伸長文化検定と<em&gt; ex vivoで</em&gt;心外膜由来の細胞移動の評価

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

フローサイトメトリーによるマウス胚における赤血球系前駆細胞およびその子孫の同定

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

細胞非自律影響を研究するためのキメラゼブラフィッシュ胚における個々の網膜前駆細胞におけるトランスジェニック遺伝子発現のin vivoイメージング

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

分離、カルチャ、および骨髄間質細胞とマウスから破骨細胞前駆細胞の分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性的な刺激の収縮活動に伴うラット骨格筋の表現型適応を研究への応用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

生体内でショウジョウバエの蛹の筋腱の形態形成の観察

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

最初心臓フィールドのような心臓前駆細胞およびひと多能性幹細胞からのような心室心筋細胞の生成

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

げっ歯類胎盤の Ex 生体灌流

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...