;博士ルーシーセンター、博士ブリジットウィルフォードとMSU ALAC認定マウスの施設での研修や動物のケアと支援のためのマイクBasett、博士ドウェインワイズ博士ニールまず、原稿編集のためKourtneyウィルキンソン：著者はの非常に貴重な助けに感謝セザールモンロイ、ハンナスウォープ、およびMSUのビデオ制作とその支援のためのボビーハドルストン、顕微鏡と画像キャプチャと彼の支援のための。この研究は、インスティテューショナルリサーチとミシシッピ州立大学で生物科学科のオフィスによって資金を供給された。

パート1：妊娠中のマウスの開腹<ol><li>頸椎脱臼を使用して、10.5から11.5 DPCで妊娠C57BL6J雌マウスを安楽死させる。 </li><li>その後抗菌石鹸で腹部をきれいに、そして、それを剃る。 </li><li>シェービング後は、生理食塩水で腹部を洗う。 </li><li>その後、滅菌ガーゼで腹部を乾かします。 </li><li>新しい滅菌フィールドでマウスをカバー。 </li><li>鉗子と解剖鋏を使用して腹側切開を行います。 </li><li>腹腔からの全体の子宮を識別し、削除。マウスの胚は子宮内部に表示されます。 </li><li> D - PBSで満たしたシャーレに子宮を転送し、それを氷上で保管してください。 </li><li>滅菌メスや鉗子を用いて、胎盤及び各胚の余分な胚組織を削除します。 </li><li> D - PBSで満たされた新鮮なシャーレに胚を移す。 </li><li>マウス胚の長さを測定します。我々は、サイズが胚の年齢に応じて異なることがわかった。例8.5のDPCは、16mmの平均で測定さ11mmの12.5 DPCの平均で測定さ6mmの10.5 DPCの平均で測定。 </li><li>その後、DNA抽出のための彼らの尾を取り除く。 </li></ol>パート2：生殖隆起の解剖実体顕微鏡と光源ショットFostec下<ol><li>シャーレにろ紙を置きます。その後、胚を乾燥させ、解剖のために固定するために、ろ紙の上にマウス胚を置きます。 </li><li>滅菌メスを用いて、臍帯の起源上記のマウス胚の横カットを行います。 </li><li>実体顕微鏡下で、無菌細かい鉗子と滅菌細かいからかいの針を使用して、泌尿器系が表示されるように腸や肝臓を取り除く。 腎臓は、腹腔内の横方向に配置され、そして膀胱は腎臓に比べて内側と尾側に位置しています。生殖腺の隆起が膀胱の両側に配置されているので、胚が雄として識別することができます。女性では、生殖腺の尾根はしっかりと腎臓の尾外側端に接続されている。 </li><li>その背後にある針をスライドさせて生殖隆起ピール。生殖隆起は、それを支える組織を離れて切断することによって除去される。 </li><li>新鮮なD - PBSで満たされた新しいペトリ皿に生殖腺隆起を転送する。 顕微鏡的に、男性の生殖隆起は、形状が大きく、楕円形、ストリップする必要があります。対照的に、女性の生殖隆起を発見する必要がある、細長い形状を有し、雄の生殖隆起と比較して小さくなる。 </li></ol>パート3：生殖隆起の消化実体顕微鏡の下で： <ol><li>中腎の解剖：中腎と罰金とシャープな針を使用して尾根から生殖腺を区切ります。 <ul><li>それはPGCの導出過程で生い茂るれる体細胞組織であるため、生殖隆起から中腎を削除することが重要です。 </li></ul></li><li>生殖隆起の消化：新鮮なD - PBSの0.5ulドロップできれいな生殖腺隆起を収集。コラゲナーゼ/ディスパーゼ溶液20ml（1mg/ml時の最終濃度）を追加します。 <ul><li>胚は、個別に処理されるべきです。それぞれの胚は、独自の個別およびラベル付けペトリ皿を持っている必要があります。 </li></ul></li><li>生殖腺のリッジは、ミンシング：滅菌針および滅菌細かい鉗子4号を使用して小片に生殖隆起をカット。 </li><li>インキュベーション：ペトリ皿を37℃インキュベーターに転送されます℃で15分間。 <ul><li>インキュベーション時間は、組織間で異なる可能性があります。 </li></ul></li><li>ピペッティング：15分後、組織は、ピペッティングにより解離させることができる。 40〜100 mmの間で直径と引っ張らガラスキャピラリーピペットを使用すると上下にピペッティングでピースを分割。これは、小さな塊を形成します。最後の洗浄のための事前暖めD - PBSのドロップの0.5ミリリットルに塊を転送する。 </li><li>洗浄後、エッペンドルフチュー​​ブに塊を転送する。 <ul><li>上のコラゲナーゼに組織を公開しないでください。 </li><li>単一の細胞懸濁液を作成しないでください。これはコロニー形成のために重要です。それは単一の細胞懸濁液の場合は、始原生殖細胞は分化し、移行する傾向がある。 </li><li>このプロセスは20分以内にする必要があります、またはあなたの細胞は、生存能力を失うことになる。 </li></ul></li><li>室温で5分間2000rpmで遠心する。 </li><li>遠心後、上清を除去し、メディアから補足ノック​​（37℃で予め温めておいた）0.5 mlにペレットを再懸濁します。 </li></ol>パート4：始原胚細胞の培養 &quot;あなたは分離し、導出のため始原生殖細胞の生存を維持するためにすぐに次の手順を実行する必要があります。 <ol><li> 24時間このプロセスの前に、張J.ら、2008年からプロトコルを以下の有糸分裂的不活化MEF - CF1フィーダー層を準備する必要があります。 <ul><li> oneプレグナで20皿を準備するNT雌性マウス（6-8胚）。 </li><li> MEFには5-6継代後（成長を促進し、差別化を防ぐ）フィーダー層となる能力を失う。 </li><li> 48時間以上経過したMEFを使用しないでください。 </li></ul></li><li>すぐにメッキの始原生殖細胞の前に、MEFの培地は、徐々にMEFフィーダー層から削除する必要があります。 </li><li>その後、MEFフィーダー層上に予め温めておいた補充ノックアウト培地0.5ml /ウェルを追加。 <ul><li> MEFのメディアは、始原生殖細胞の分化を誘導するウシ胎児血清が含まれています。 </li><li>グロー因子の活性を確保するために使用する前にノックアウト媒体は、直ちに正貨特定の成長因子を補充する必要があります。 </li></ul></li><li> MEFフィーダー層上に対称的な分布、プレートの始原生殖細胞を使用する。 <ul><li>これらの作品が近すぎる場合は、集約する傾向があり、不十分な添付または分化を開始高密度コロニーを作る。 </li><li>培養皿は、胚胚細胞株名、継代数、および日付で標識されている必要があります。 </li></ul></li><li>慎重に、37℃、5％CO 2インキュベーターに培養皿を転送する。 </li><li>細胞を24時間ごとに監視します。 </li><li> 48時間後、コロニーの付着を妨げることを避けるために文化の非常に上からメディア250mlのを削除します。その後、新鮮なノックアウト添加培地（37℃で予め温めておいた℃）250 mlを加える。 </li><li>手順7の後に、別の48時間待機してから、完全にすべてのメディアを取り出し、新鮮なノックアウト添加培地（37℃で予め温めておいた）に交換してください。 PGCコロニー形成されるまで8-10日のためにこの2日おきください。 </li></ol>パート5：胚胚細胞<ol><li> PGCの分離の10日後、胚性生殖細胞のコロニーが形成されています。 </li><li>胚性生殖細胞系は、マニュアルの文章ごとに8-10​​日で生き続けています。彼らは未分化な形態を提示し、そのようなalkalyneホスファターゼ、OCT - 4、SSEA - 1、およびSOX - 2のような多分化能マーカーを発現し続ける</li></ol>ノート<ul><li>無菌/滅菌条件は、培養室で不可欠です。 </li><li>導出と文化のために、始原生殖細胞は、浄水器クラスIIバイオセーフティキャビネット内で処理されます。 </li><li>インキュベーションは、加湿37℃、5％CO 2インキュベーターです。 </li><li>すべての培地および試薬は4℃で保存されている0.2ミクロンフィルター、° Cで使用する前に濾過し、使用前に37℃で事前に暖めています。 </li><li>解剖の手順中に、すべての胚や組織は氷上で保存する必要があります。 </li><li>すべての試薬のフラスコは、キャビネット内に配置する前にエタノールで除染されています。 </li><li>手袋、白衣、看護師のキャップの使用は必須です。 </li></ul>結論私達は分離する方法を示していますビデオ、派生、および10.5から11.5 DPCのマウス胚の生殖腺尾根から文化胚性生殖細胞を提供している。再現性の分離および胚生殖細胞株の長期的な文化は、男性と女性の胚で配偶子形成の発達過程で、遺伝的およびエピジェネティックな胚のパターン、生殖腺形成、周囲の臓器の環境への影響を初期胚発生の研究のための重要な基盤を提供します。とがんと不妊につながる経路の決定。 <img src="/files/ftp_upload/1635/1635figure1.jpg" alt="図1" />

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A laboratory Handbook. 1, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> Current Opinion in Genetics and Development. 13, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> Molecular embryology: methods and protocols. 97, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> Developmental Biology. 187, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> Development. 128, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> J Vis Exp. , (2008).</li></ol>

ミシシッピ州立大学：この研究は私たちのマウスのプロトコル＃08030（ホールディングおよびプロトコルを繁殖生殖系列の開発と腫瘍形成における活性酸素、ROSの役割）の見直しと動物実験と使用委員会（IACUC）承認後に実施された。

鉗子14号、15、メス35ミリメートル、解剖ハサミ、外科分野、抗菌石鹸、生理食塩水0.9％、ガーゼ、氷の泡コンテナ、カミソリの刃、メス35ミリメートル、微細な鉗子第4と5、罰金からかい針ハンドル、ろ紙シート、およびガラスピペットを引っ張った。<br /<br /&gt;設備：解剖顕微鏡、光源ショットFostec、倒立顕微鏡、マイクロピペット10、200ul、遠心分離、バイオセーフティキャビネット。

isolation and derivation of mouse embryonic germinal cells

初期の発達段階の間に後で配偶子への始原生殖細胞（始原生殖細胞）に分化し、胚性生殖細胞の能力（EG）は、がんと不妊についての我々の仮説に対応するために最適なモデルです。このプロトコルは、10.5から11.5日後に交尾（DPC）のマウス胚での開発生殖腺から始原生殖細胞を単離する方法を示しています。マウス胚から生殖腺隆起（C57BL6J）を開発してから以前に分裂ノックアウト媒体の存在下でマイトマイシンCで不活化し、白血病阻害剤を添加したマウス胚線維芽細胞のフィーダー層（MEF - CF1）に播種、コラゲナーゼと機械的な破砕により解離させた因子（LIF）、塩基性線維芽細胞増殖因子（bFGF）、および幹細胞因子（SCF）。培養条件のPCG識別、分離、および確立のためにこれらの最適化されたメソッドを使用すると、40日以上用EG細胞の長期培養を可能にします。胚性生殖細胞系は、多能性の状態の一般的な使用されるマーカーの胚の表現型と発現を示した。単離および培養中の胚細胞の派生は、in vitroでその開発を理解し、酸化ストレスへの曝露後の遺伝的およびエピジェネティックなレベルでの累積損傷を監視する機会を提供するツールを提供しています。

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

開発の初期段階で始原生殖細胞に分化する胚性生殖細胞の能力は、がんと不妊についての我々の仮説に対応するために最適なモデルです。このプロトコルは、10.5から11.5日後に交尾のマウス胚での開発生殖腺から始原生殖細胞を単離する方法を示しています。

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

isolation-and-derivation-of-mouse-embryonic-germinal-cells

Research

JoVE Journal

Biology

16.1K ビュー.  Mississippi State University. 開発の初期段階で始原生殖細胞に分化する胚性生殖細胞の能力は、がんと不妊についての我々の仮説に対応するために最適なモデルです。このプロトコルは、10.5から11.5日後に交尾のマウス胚での開発生殖腺から始原生殖細胞を単離する方法を示しています。

ビデオ: Isolation and Derivation of Mouse Embryonic Germinal Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

胚盤胞からマウス栄養膜幹細胞の導出

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

鳥類胚弁膜前駆細胞の単離と培養

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

マウス胎児の腎臓の腎性帯からの細胞の単離と培養

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

培養ヒト胚および誘導多能性幹細胞に適したマウス胚性線維芽細胞の調製

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚心室内皮細胞の単離

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

マウスプライマリ膵臓腺房細胞の単離と培養

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...