私たちの仕事は、スイス国立科学財団（SNF）、システム生物学（SystemsX.ch）、筋ジストロフィー協会アメリカ（MDA）、協会·フランセーズcontreレミオパシー（AFM）、米国ミトコンドリア病財団、スイスのイニシアティブによってサポートされています（UMDF）、ゲーベルト-RUF財団まれな疾患プログラム（GRF）、筋疾患（SSEM / FSRMM）、スイスライフ &quot;JubiläumsstiftungエリーゼVolksgesundheitウントmedizinische Forschung&quot;、ロシュ研究財団と大学の研究のためのスイス社会バーゼルの。

1。初代ヒト筋細胞培養の準備<ol><li>植-RE-植技術を4に記載のヒト筋細胞の培養を確立します。最初に、生検から非筋肉組織を除去する。その後、線維芽細胞は外植片から出てくることができ、プラズマの凝塊で1 mm 3で 、筋肉の外植片を、埋め込 ​​むことができます。 </li><li>埋め込まれた外植片は、プラズマゼラチンコートしたディッシュに移し、それらを通常の成長培地（MEM、25％の培地199、10％ウシ胎児血清[FBS]、1％ペニシリン/ストレプトマイシン、10μg/ mlのインスリンを補足に成長させてい単分子層では10 ng / mlのヒト上皮成長因子[EGF]、12.5 ng / mlのヒト線維芽細胞成長因子[FGF]）。これらの条件の下で外植片から出てくる細胞は筋肉細胞である。これらの細胞は、外植片を除去した後、単層で成長していきます。 </li><li>文化aneurally通常の成長培地に0.1％ゼラチンコートプレート上で細胞。細胞はすることができますaneurally培養し、必要に応じて増幅した。これは、共培養を行うために3と8の間の通路で筋細胞を使用することをお勧めします。それは若い筋管を支配することが重要であり、このポイントの後、共培養は失敗のリスクが高い。さらに、筋管が大きすぎると厚すぎてはいけません。 </li><li>神経支配が予定されている2日前、プレート、通常の増殖培地中皿当たり500,000個の細胞の密度で35mmの組織培養皿（または6ウェルプレート）の筋細胞。その密度で、細胞は次の日に核融合を開始するための適切なコンフルエントに、その後の神経支配でその翌日である。これらの条件を使用して、筋管では、最適な支配、したがって、その後の繊維の収縮のために右の段階にある。 </li><li>次の日に、25％の培地199、5％FBS、10μg/ mlのインスリンおよびペニシリン/ストレプトマイシン、1％を添加したMEMで構成される融合培地で通常の成長培地を交換してください。 </li></ol> 2。 Isolati<html>ラット胚脊髄外植片の上に脊髄外植片を分離するために全体の手順は、10％ウシ胎児血清（FBS）を含むハンクス平衡塩溶液（HBSS、GIBCO / Invitrogen社REF 14170）で、双眼顕微鏡下で実行されます。 <ol><li>さまざまな開発段階では全体の手順を危うくするとして、それは、ラットの胚はED 13と14の間にあることが重要です。 CO 2を妊娠女性を犠牲にしてHBSS培地中の全胚チェーンを収集します。それぞれの胚を分離し、安楽死のためにそれらを首をはねる。 </li><li>ワンピースの身体の残りの部分から胚脊髄の解剖とその周辺の筋肉や結合組織を除去します。機能的な神経支配を確保するため、脊髄に接続された後根神経節（DRGS）を保つために非常に注意してください。 </li><li>各植は、少なくとも2 DRG（1 mm 3の外植片）それに接続が含まれている方法で、横方向に各脊髄のスライス。 </li></ol><html>ラット胚脊髄外植片を持つ人間の筋肉細胞のove_title &quot;&gt; 3。神経分布<ol><li>単層上に媒体の微細な層を残して、筋肉の細胞培養に融合培地を除去します。 </li><li> 、筋細胞層の上に慎重に35mm皿あたり5つ等間隔に植片を外植片を配置します。 </li><li>非常にゆっくりと追加し、各植に賢明な、融合培地をドロップします。それは植が最初に付着し、細胞と接触するのを許容する生き残るために筋細胞用培地を追加する必要がありますが、あまりにも多くはありません。媒体が（あまりにも強く、またはあまりにも多くのメディア）が正しく追加されていない場合、外植片をフロートされ、筋細胞を支配するために失敗します。 </li><li>外植片が付着し始めたら、35 mmディッシュあたり2 mlに融合培地を上回ると、インキュベーターに戻す非常に慎重に皿を置く。 </li></ol> 4。異種神経筋共培養のメンテナンス<ol><li>週二回の融合培地を変更します。 </li></ol>_title &quot;&gt; 5。代表的な結果できるだけ早く二日として支配した後は、各外植片から新興国やボタンのような構造（ 図1）で表される筋細胞との接触を確立する突起を見ることができます。 早くも支配した後4-6日として、いくつかの個々の繊維は、契約を開始します。 収縮は週二回培地を変更するだけで維持され、何ヶ月も持続する。 約2週間後、脊髄片自体が退化したが、神経支配が保存されている。これは正常です。神経成分の微細な層は、筋細胞の層の表面にとどまります。この神経ネットワークは、モータユニットの機能を維持するのに十分である。 外植片は、神経支配の翌日培地に浮遊した場合、それらが付着することはありません。筋細胞の層がある場合にこの現象が発生した十分にコンフルエント場合、またはメディアがあまりにも大まかに追加されません。時には、植付着がない突起が出現します。これはおそらくそれに接続されているDRGの欠如によるものである。その場合においても、神経支配は確立されません。外植片は、まだ簡単に切り離すことができますように、最初の週は非常に慎重に料理を操作します。融合メディアを交換するために、細胞の剥離を避けるために最小限の速度で液滴を追加します。 <img alt="図1" src="/files/ftp_upload/3616/3616fig1.jpg" /> 図1脊髄外植片筋細胞共培養1日目から15日まで。

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我々は、開示することは何もありません。

筋細胞の培養は、通常、複数の細胞および細胞型接続の重要性を再現していないため、正常および病理学的文脈において筋細胞の機能を研究する生理学的なin vitroのツールは 、myologistsの最重要である。筋細胞に運動ニューロンの精製加えてシュワン細胞の存在は、5効率的に神経支配のために必要とされ、それに関連するプロトコルがここで説明する（3D +特殊なディス?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> HBSS </td><td> GIBCO / Invitrogen社</td><td> 14170 </td></tr><tr><td> MEM </td><td> GIBCO / Invitrogen社</td><td> 31095 </td></tr><tr><td>培地199 </td><td> GIBCO / Invitrogen社</td><td> 31153 </td></tr><tr><td>ウシ胎児血清</td><td>胎児のクローンPerbio </td><td> SH30066.03 </td></tr><tr><td> Insuline </td><td>シグマ</td><td> I9278 </td></tr><tr><td>ヒトEGF </td><td>シグマ</td><td> E9644 </td></tr><tr><td>ヒトFGF </td><td> SiのGMA </td><td> F0291 </td></tr><tr><td>ペニシリン/ストレプトマイシン溶液</td><td>ギブコ</td><td> 15140 </td></tr></tbody></table>

a functional motor unit in the culture dish: co-culture of spinal cord explants and muscle cells

神経成分の存在下で、細胞の分化が制限されており、運動ニューロンの刺激は、1が欠落している自発的に、ためにヒト初代筋細胞は単層でaneurallyまれに契約を培養した。これらの制限は、培養筋細胞の多くの神経疾患のin vitroでの研究を妨げる。重要なのは、単層、培養筋細胞の実験的な制約は、共培養における脊髄外植片と筋線維の機能的神経支配によって克服することができます。 ここでは、Askanas 2によって開発された方法に従って、差別化と繊維の収縮を完了するには、リード、ヒト初代筋細胞の効率的、適切な神経支配を達成するために必要な様々なステップを示しています。これを行うには、筋細胞はまだ脊髄スライスに接続された後根神経節で、ED 13.5でラット胎児の脊髄外植片と共培養されています。数日後、筋肉のfibers契約を開始し、最終的には筋細胞への接続という脊髄外植片から突出した機能的な神経によって支配によって横紋になる。この構造は、単に培養液の定期的な交換によって、多くのヶ月間維持することができます。 それは人間の筋肉の発達と神経支配の学際的な分析のための機能モデルを表して、この非常に貴重なツールのアプリケーションは、たくさんあり​​ます。実際には、完全なde novoの神経筋接合部のインストールは、基本的および生理学的文脈では、各ステップで多くのパラメータを簡単に測定できるように、培養皿で発生します。 ほんの一例、ゲノムおよび/またはプロテオミクス研究を引用する共培養上で直接実行することができます。両方のコンポーネントが別の種から来るので、さらに、プリ·ポストシナプスの効果が具体的かつ個別に、神経筋接合部で評価することができ、ラットとヒトであった。神経筋の共培養はまた、筋肉や神経筋疾患3から患っている患者から単離されたヒト筋細胞で行うことができるため、候補薬剤のスクリーニングツールとして使用することができます。最後に、特別な機器が、通常のBSL2施設は、培養皿の中で機能的なモータユニットを再現する必要はありません。このメソッドは、このように、正常および疾患のコンテキストで、筋肉だけでなく、神経筋機能の生理学的および機構の研究のための神経の研究コミュニティの両方の価値があります。

Watch this Scientific Journal Video about A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells at JoVE.com

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

培養筋細胞は、神経支配のある筋を再現するために不十分なモデルである<emin vivoでの&gt;</em&gt;。機能的なモータユニットを再現することができます<em&gt; in vitroで</em&gt;ラット胚脊髄の外植片を用いて分化したヒト初代筋細胞の神経支配によって。この資料では、脊髄の外植片と筋細胞の共培養が確立されている方法について説明します。

培養皿内の機能モーターユニット：脊髄外植片と筋細胞の共培養

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

a-functional-motor-unit-culture-dish-co-culture-spinal-cord-explants

Research

JoVE Journal

Neuroscience

15.2K ビュー.  University of Basel. 培養筋細胞は、神経支配のある筋を再現するために不十分なモデルである。機能的なモータユニットを再現することができます in vitroでラット胚脊髄の外植片を用いて分化したヒト初代筋細胞の神経支配によって。この資料では、脊髄の外植片と筋細胞の共培養が確立されている方法について説明します。

ビデオ: A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

神経突起伸長アッセイのためのコラーゲンゲルのチックStatoacousticガングリオンと脊髄の外植片の解剖と文化

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

ニワトリ胚脊髄スライス培養プロトコル

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

マイクロ流体共培養装置の使用歯胚神経支配の開発の分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

ゼブラフィッシュ<em&gt;その場での</em&gt;脊髄脊髄感覚運動ニューロンからの電気生理学的記録のための準備

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

マウスにおける線込みおよび脊髄窓移植

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

シアログリカンの代謝標識による原発神経幹および前駆細胞の細胞表面マーカーの同定

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

レンチ ウイルスのベクトルの直噴ハイライト ラット脊髄における複数の運動経路

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

PiggyBac ベクターを用いたヒト誘導多能性幹細胞 (iPSCs) の機能的脊髄および頭蓋運動ニューロンへの変換

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

ラットおよびマウス脳への神経幹細胞の動脈内デリバリー:脳虚血への応用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...