私たちは、IPSCの生成に関する専門知識とプロトコルを共有するため、それぞれにChR2とシナプシン-tdTomatoレンチウイルス構築物についてC.チャイをK. DeisserothとS.帝に感謝し、ワイオミング州レオン技術サポートのために、共有試薬および専門知識のためのゴーラボのメンバー。この作品は、ELGにその競争的研究プログラム（002から082 2008 NRF-CRP NRF）の下で国立研究財団シンガポールにより、アボット栄養とELGに優秀の学術センター（ACE）グラクソ·スミスクライン（GSK）から研究賞によってサポートされていましたし、 GJAへ：GJA、及び文部科学省によって資金を供給韓国国立研究財団（NRF）のワールドクラス研究所（WCI）プログラムで、韓国の科学技術（MEST）（WCI 2009-003 NRF認可番号）

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No conflicts of interest declared.

光遺伝学は、ニューロン13の定義された集団の活性化のための時間的·空間的な精度を提供します。我々の実験では、顕微鏡対物レンズの全視野は、光刺激のChR2を発現するだけ皮質ニューロンを30秒間照射した。これは、私たちは、シナプスの接続は、共培養内のニューロンの異なる集団の間に形成されたかどうかを判断することができました。我々の結果は、これらのニューロンがChR2をを表現するシナプス前皮質ニューロンからシナプス入力を受け取ることを示している、光刺激はIPSC由来の神経細胞でPSC周波数を増大させることを明らかにした。これは、順番に、IPSC由来の神経細胞が正常に皮質ニューロンを持つ回路に組み込まれていることを確立した。 この共培養系を生成するためのいくつかの重要なステップがある。これは、メッキを進める前に、最小限の細胞死に、マウスNPC由来の星状細胞培養が健康であることを確認することが重要です他の種類の細胞。皮質ニューロンと人間のNPCは健康的なアストロサイト上によく付着し、電気生理学的記録は、培養条件に大きく依存しています。このプロトコルの他の重要なステップは、星状細胞培養へのエレクトロポレーションおよび皮質ニューロンのメッキです。多数の細胞がエレクトロポレーション後に死滅したように、少なくとも600万皮質ニューロンを、24ウェルプレート中で星状細胞培養上にメッキする電気穿孔されることを保証することが重要である。私たちは、より少ない600万人以上の細胞をエレクトロポレーションした時、生き残るニューロンの数は、電気生理学的記録に影響を与えた密な神経回路網を形成していなかったことを観察した。エレクトロポレーション皮質ニューロンの数は、異なる研究間の比較を可能にするために、各共培養実験のための一貫している必要があります。酵素消化に関与するすべての手順については、それはあまりにもあれば細胞死につながる可能性があるために、消化液中で細胞を残さないことが不可欠である。 このアプローチは、他のニューロンとのシナプス接触を形成するために、患者固有の線維芽細胞から誘導された神経細胞の能力をスクリーニングすることができる。神経発達障害、レット症候群（RTT）に罹患している患者からの線維芽細胞から誘導されたニューロンは、シナプス伝達の欠陥を示す：RTTニューロンは、おそらく少ないシナプス接続14に、健康なニューロンに比べPSC周波数及び振幅の有意な減少を示す。我々の同時培養系は発達障害を表示するニューロンに対する薬物効果の検査を可能にする。これは、罹患した人間のNPCの播種の間、並びに神経分化の時間にわたって化合物への連続暴露中に目的の化合物の添加によって行うことができる。 この共培養系はまた、薬物試験のためのモデルとして使用することができるが、このアプローチの限界は、各候補化合物の効果を調査するプロセスは長くとして面倒であることであるそれは、ハイスループットスクリーニング法ではない。候補化合物で処理した後、IPSC由来のニューロンは、個別のPSCの各化合物の効果を決定するためにパッチを適用する必要があります。また、患者から多くの利用可能な線維芽細胞およびiPS細胞は、現在、存在しない。したがって、より多くの患者の細胞を有し、また、ハイスループットスクリーニングのためにこのプロトコルを適応させるために、近い将来において重要であろう。例えば、このようなイオンまたは膜電位の遺伝的にコードセンサーとして動作インジケータとIPSC由来のニューロンを標識することにより、サイドステッピング時間のかかる電気生理学的手順をすることにより、実質的にスループットレートを増大させることが可能であろう。電気生理学的記録を実行する線維芽細胞を再プログラミングから、この共培養系を生成するプロセス全体としては、90日以上、それはそれぞれのリプログラミングや神経誘導ステップの後、人間性IPSCまたはNPCはどちらが展開することをお勧めします必要があり、凍結保存将来の実験のために必要な時間を短縮する。 我々の実験では、我々はシナプシンプロモーター下皮質ニューロンにChR2を発現した。このプロモーターはパン神経であるため、我々は、おそらく抑制性と興奮性皮質ニューロンの両方を光刺激された。将来の実験の目的は、特に、阻害または刺激性のいずれかの回路を調べるためである場合、より特異的プロモーターを使用することができる。代替的に、皮質ニューロンは、トランスジェニック（またはウイルスを注射したマウス）にChR2の発現が特異的にニューロンの遺伝的に定義された亜集団を標的とするから得ることができる。例えば、Creリコンビナーゼは、フロックスChR2を持つマウスと交配されているパルブアルブミン含有介在ニューロン（PVの介在ニューロン）に発現しているマウスから収穫皮質組織は、ChR2をを表現する唯一の皮質ニューロンは、PVの介在ニューロン15になります状況が得られます。私たちの共同Cu中などChR2を発現介在ニューロンの使用ltureシステムは、パッチを適用したIPSC由来のニューロンがPVの介在ニューロンとの回路に組み込むことができるかどうかの決定を可能にします。 以前の研究9に基づき、皮質ニューロンは、in vitroで 14日後に樹状突起が重複する成熟している。光刺激は、パッチを適用したIPSC由来の神経細胞からの応答を誘発しない場合にはこのように、それはニューロンは、皮質ニューロンとシナプス結合を行う能力を持っていないことを示す必要があります。 IPSC由来のニューロンのいずれか他のIPSC由来の神経細胞または皮質ニューロンからシナプス入力を受信することができる。従来のパッチクランプ記録を用いた場合には、シナプス入力がどこから来ているのかを区別することは不可能である。私たちのシステムの利点は、皮質のシナプス前入力からシナプス後応答が選択的に誘発し、識別することができるということです。ここで概説した手順を統合するIPSC由来の神経細胞の能力を評価するために、簡単なアプローチを提供定義された神経回路網に。

近年では、ニューロン及び神経回路機能の我々の理解は、神経細胞の興奮性を制御するいくつかの光遺伝学的ツールで革命をもたらしてきた。そのようなツールが光活性陽イオンチャネルであり、チャネルロドプシン2（ChR2を）：光刺激1-3に対応してChR2を消防活動電位を発現するニューロン。神経細胞が光に通常は区別されませんので、この驚くべき能力は、ニューロン4の遺伝的に定義された集団の活動の正確な時間的および空間的制御のための新たな道を切り開くと大幅に脳機能の研究を加速しています。 ChR2をニューラルネットワーク6の活性を調節する神経発達5の監視が 、異なる目的のために幹細胞研究するために適用されている。 ここでは、神経細胞の共培養系の有用性を高めるためにChR2を用いる。共培養システムは、異なる細胞型の間の相互作用の評価を可能にする。ニューロンおよびグリアの共培養は、神経系の主要な細胞型は、一般的に、これらの異なる細胞型の間のシグナリングを調査するために、ならびに生理学的反応のためにこのシグナリングの重要性を評価するために、損傷の伝播及び検査するために使用されている潜在的にこのシグナル伝達7に関与する分子機構。以前の研究は、ヒトiPS細胞がラット皮質初代培養含有ニューロン、アストロサイト、オリゴデンドロサイト、およびミクログリア8の存在下でニューロンに非常に迅速に分化することを明らかにした。 このプロトコルでは、我々は、ヒト人工多能性幹細胞（iPS細胞）に由来するラット皮質ニューロン及びニューロン間のシナプス結合を調べるために、共培養系でのChR2を利用する方法を実証する。我々は、アストロサイトに解剖するステップと収穫脳組織9と同様に、マウス神経前駆細胞を維持し、分化する（NPCの）を記述し、人間のNPC値intOニューロンは、共培養系を作成する。この共培養系を確立するために、我々は、ヒトiPS細胞を生成するために、 文献 10沖田らによって記載統合のない再プログラミング法を用いた。 Li らによって記載されているように、これらのiPS細胞は、その後、効率的小分子阻害剤を用いた化学的に定義された条件でのNPCに分化した。11 共培養では、ニューロンの異なる集団は、トマト（tdTomato）二量体蛍光タンパク質、タンデム経由IPSC由来のニューロンの皮質ニューロンにおけるChR2を発現の検出とタグ付けを介して同定することができます。皮質ニューロンの光遺伝学的光刺激とIPSC由来のニューロンからの電気生理学的記録を組み合わせると、互いに回路を形成するニューロンのこれら2つのタイプの能力の評価を可能にする。具体的には、ChR2を発現皮質神経細胞の光刺激は、シナプス回路を形成しているIPSC由来のニューロンにおけるシナプス応答を呼び起こす輝皮質ニューロンネットワークと。我々は、増加したシナプス後電流が実際にそのような条件下でIPSC由来のニューロンで検出されたことを示している。このプロトコルは、疾患患者からの線維芽細胞から派生iPS細胞を使って、別の神経疾患モデルの要約を含む実験条件、種々の条件下でシナプス回路の評価を可能にする。

an optogenetic approach for assessing formation of neuronal connections in a co-culture system

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

ここで、共培養から星状細胞、皮質ニューロンおよびヒトの神経細胞の生成に関与する複数のステップを説明するプロトコルが示されている。ヒトiPS細胞は、エピソームベクター10と高密度（ 図1A）に維持されたNPCは11を作り、小分子阻害剤のカクテルで神経系統に指定を使用して線維芽細胞を再プログラムすることによって得た。 tdTomato（ 図1B）でタグ付けされた人間のNPCのアストロサイトへのマウスのNPCの分化、ChR2をを表現するラット皮質ニューロンのめっき、播種を：いくつかのステップが共培養を生成するのに必要とされる。分化の2日目に、グリア線維性酸性タンパク質（GFAP）+アストロサイトは容易に私たちの培養物（ 図1C）で観察することができる。マウスのNPCは、アストロサイトの単分子層（ 図1D）にChR2をを表現する皮質神経細胞をプレーティングする前に少なくとも1週間分化させた。 3の後人間のNPC分化の4週間、tdTomato +ニューロンは、培養物（ 図1E）で検出された。 この共培養系は、光刺激（ 図2A）に応答して、個々のIPSC由来のニューロンからシナプス後電流の一貫した検出を可能にする。光によって刺激されると、活動電位は、ChR2を（ 図2B）を発現する皮質ニューロンで生成された。 IPSC由来のニューロンは、現在の脱分極パルスの振幅が増加したように（ 図2D）増加し、活動電位発火を示す、また（ 図2C）励起可能である。したがって、これらの細胞は、成熟したニューロンの特性を示す。光がない場合には、IPSC由来のニューロンは、自発的なシナプス入力（ 図2E）を受けた。 GABA受容体を介して電流が外向きになるので、これらの内向きシナプス後電流は、主に、AMPA受容体によって媒介され、NMDA受容体は電流αを有していない-70 mVの保持電位トン。光刺激が大幅にシナプス後電流（ 図2E）の周波数を増加させているため、これらの入力のうちの少なくともいくつかは、ChR2を発現しているが、シナプス前皮質ニューロンからのものであった。シナプス入力の増加の時間経過を図2Fに示されている：皮質ニューロンの光刺激は、30秒の長い光のフラッシュを通して持続した。 ChR2を発現皮質神経細胞は（ 図2G）輝されたときのPSCの周波数が上昇したが、その振幅は（ 図2H）影響を受けなかった。要約すると、これらの結果は、IPSC由来のニューロンが成熟した神経細胞の性質を示し、シナプス前皮質ニューロンとシナプス結合を形成することができることを示している。 <img alt="図1" src="/files/ftp_upload/52408/52408fig1highres.jpg" /> 図1：共培養の生成のための模式図のNPCに性IPSCと神経誘導にヒト線維芽細胞を再プログラムするためのシステム。（A）のタイムライン。皮質ニューロンとアストロサイト培養物に対する成熟したニューロンへの人間のNPCの最終分化のために（B）タイムライン。（C）マウスNPCは急速に2の後GFAP +アストロサイトに分化するインビトロでの日数（D）1週間後にChR2を発現する皮質ニューロンのGFAP +星状細胞上にプレーティングした。（E）4〜5週目人間のNPCの分化後、電気生理学的記録は、tdTomato +ニューロンで行った。スケールバーは、100μmを表す。 <img alt="図2" src="/files/ftp_upload/52408/52408fig2highres.jpg" /> 図2：機能的なシナプスの接続性の光遺伝学的解析。 tdTomatoでタグ付けされた（A）IPSC由来細胞（赤色）は、皮質ニューロンが光活性を発現する共培養したチャンネル、ChR2を（緑）。 IPSC由来のニューロンからの記録は、皮質ニューロンまたは他のIPSC由来の神経細胞のいずれかから来ることができシナプス後電流（PSC）の応答を、明らかにした。（B）イルミネーション（青いバー）ChR2をを表現する皮質ニューロンにおける活動電位のシリーズを誘発。（ C）IPSC由来の細胞は、ChR2を発現皮質神経細胞の（E）光刺激（青いバー）。IPSC由来細胞の現在の関係を示す曲線D）焼成（。電流注入により誘発されるIPSC由来の神経細胞内のPSCの頻度が増加している光刺激によって生成PSC周波数の増加の。（F）時間のコース。青いバーが光刺激の時間を示している。 PSC周波数は光刺激（G）増加したが（G、H）、PSC振幅が（H）影響を受けなかった。 *はp &lt;0.05は、スチューデントのt検定を用いてコントロールと比較して示している。

Watch this Scientific Journal Video about An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System at JoVE.com

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

共培養系における神経結合の形成を評価するための光遺伝学的アプローチ

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

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JoVE Journal

Developmental Biology

13.3K ビュー.  Duke-NUS Graduate Medical School. A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2. 

ビデオ: An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化と共培養マウスの卵巣と卵胞

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

発生生物学

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

同軸ノズルを用いて多能性幹細胞のアルギン酸カプセル化

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

ゼブラフィッシュの発生における急性腎障害のモデル化のための正確な細胞除去法

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

機械的組織解離およびフローサイトメトリーによる哺乳動物雄胚細胞のマルチシード精製のための標準的アプローチ

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

大動脈リング共培養アッセイ:体外間葉系間質細胞の血管新生の可能性を評価するために便利なツール

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

移植組織の細菌付着を勉強する平行平板灌流システムのIn Vitroモデル

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

内皮コロニー形成細胞と間葉系幹細胞によって形成された共培養スフェロイドを用いた3次元血管新生アッセイ系

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

歯髄パラクリン信号に反応した神経突起の成長を研究する共培養法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

神経堤細胞の機械的信号を研究するための最適化されたO9-1/ヒドロゲルシステム

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

光遺伝学的TAEL/C120系を用いたゼブラフィッシュ胚における光誘導GFP発現

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...