私たちは、NIHの助成金R01 DE021683、DE019434 R01、U01 HL099776、オーク財団と小児再生医療のためのHagey研究所の支援を認める。

注：倫理声明：脊椎動物に関わるすべての研究は実験動物管理（APLAC）にスタンフォード紛争処理パネルにより承認に従ってプロトコルで実施した。 1.準備<ol><li> （1.077グラム/ mlの密度に調整したポリスクロースおよびジアトリゾ酸ナトリウムを含んでいる）市販の密度勾配細胞分離培地10mlを50mlコニカルチューブにRTに来ることを可能にする。 </li><li>フローサイトメトリー（FACS）は、1×リン酸緩衝生理食塩水（PBS）、2％ウシ胎児血清（FCS）を用いて緩衝液を準備。の50mlアリコートを取り、密度勾配細胞分離培地工程の間に使用するために、RTで、このアリコートを維持する。 </li><li>単離手順の間に単離された組織や細胞を維持する準備ができた氷のバケツを持っている。 </li></ol> 2.文化メディアを準備します<ol><li>基礎培地を準備します。MEM（最小必須培地）、フェノールレッド（500ミリリットル）、グルタミン酸（1×）、FCS（10倍）、10,000単位/ mlのpenicilliなしn個（1×）。 </li><li> 0.22μmのフィルターで基本培地をフィルタリングします。 </li><li>骨髄マクロファージ誘導培地を準備します。 <ol><li> ステップ2.1で調製した基礎培地の50ミリリットルを取る。 </li><li> 10 -7 Mの最終濃度で、プロスタグランジンE2（PGE2、ミスター352.465グラム/モル）を追加します。 </li><li>を10ng / mlでマクロファージコロニー刺激因子（M-CSF）を追加します。 </li><li>メディアを刺激骨髄マクロファージの最終的な解決をフィルタリングしないでください。 </li></ol></li><li>破骨細胞誘導培地を準備します。 <ol><li> ステップ2.1で調製した基礎培地の50ミリリットルを取る。 </li><li>追加10 -7 Mの最終濃度で、プロスタグランジンE2（PGE2氏352.465グラム/モル） </li><li>を10ng / mlでマクロファージコロニー刺激因子（M-CSF）を追加します。 </li><li>を10ng / mlでRANKLを追加します。 </li><li>破骨細胞誘導培地の最終的な解決をフィルタリングしないでください。 </li></ol></li></ol> 3.骨髄の単離<ol><li> 1 C57BL / 6マウスを安楽死させる頸椎脱臼に続く二酸化炭素吸入法を用いて。 注：げっ歯類は、適切な技術、機器や薬剤を使用訓練を受けた者が安楽死させなければなりません。 </li><li>位置及び解剖ボード上でマウスを固定し、70％アルコールで噴霧する。 </li><li>大腿骨、脛骨、上腕骨と背骨を解剖する。 <ol><li>仰臥位でマウスで始まる。縦に下肢に沿って切開を行い、軟部組織の添付ファイルを分割することによって大腿骨と脛骨の長さに沿って切開することから始めます。最後に、スケルトンから完全に自由な骨に膝と股関節から骨を脱臼。 </li><li>上腕骨を分離するために上肢の長さに沿って切開を作るために進んでください。軟組織の莢添付ファイルを解剖することによって、肩と肘の関節での上腕骨を脱臼。 </li><li>マウスが発生しやすい位置するように最後に、マウスを裏返します。正中線で、背骨の長さに沿って皮膚切開を行います。傍脊椎ソフトを観察脊椎の骨縁に沿って脊椎および切開の各側の組織、傍脊椎軟組織塊を通って解剖する。はさみを使って、首の付け根に背骨全体に水平カットを行い、背骨の終わりに、テール挿入のすぐ近位、軟組織の添付ファイルから背骨を解放する。 </li></ol></li><li>骨が収穫された後、氷上でFACS緩衝液中にそれらを保つ。 </li><li>筋肉と骨から軟部組織をきれいにティッシュペーパーを使用してください。 </li><li> FACS緩衝液3mLで乳棒と乳鉢にきれいに骨を置きます。 </li><li>乳棒と乳鉢で静かに骨をつぶす。 </li><li>吸引除去血まみれ流体オフと70μmのセルストレーナーに溶液を通すことにより、50ミリリットルコニカルチューブに移す。 </li><li> 5ミリリットルのFACSを粉砕骨にバッファに追加し、さらにつぶす。再び、ピペットを用いて流体を除去し、70μmのストレーナーを50ミリリットルコニカルチューブに移す。 </li><li>一般的に骨のTWIをつぶす新鮮なFACS緩衝液中で3回、流体が赤に染色しなくなるまで、乳鉢と乳棒、新鮮なFACS緩衝液をさらに5ミリリットル追加するたびに使用するCE。 </li><li>細胞ペレットを得るために、4℃で5分間、200×gでの骨髄液を集める。 </li></ol>骨髄細胞の4勾配分離<ol><li> FACS緩衝液10ml中に再懸濁上清を吸引（RTで維持される）。 </li><li>市販の密度勾配細胞分離培地（RT）10 mlを含む50mlコニカルチューブを取る。 </li><li>密度勾配細胞分離媒体に骨髄液レイヤ。ゆっくり電動ピペットを用いてこれを行います。角度ピペット円錐管の上部付近内面に先端部とは約30°にコニカルチューブを傾け。ピペット上の低速設定を用いて、密度勾配細胞分離媒体を妨害しないように静かに骨髄液を排出する。 注：最終的にはそこに意志二つの溶液（細胞溶液および密度勾配細胞分離培地溶液）との間の明確な定義である。 </li><li> RT遠心機を用いて、15​​分間、200×gでバランスのとれた遠心分離器で密度勾配細胞分離培地および細胞溶液を遠心する。また、密度勾配細胞分離媒体勾配を用いて、適切な相分離を確実にするために遠心分離機に加減速を除去。 </li><li>遠心分離後、関心の骨髄細胞を含む濁った中間層をオフに吸引除去する。 </li><li>新しいコニカルチューブにこれらの細胞を移す。細胞を洗浄するために、（氷上で保持）追加のFACS緩衝液20ミリリットルを追加します。 </li><li> 4℃で5分間、200×gで遠心分離して細胞ペレットを得た。 </li><li>血球計数器を用いて細胞計数を可能にするためにメディアを刺激するマクロファージを1mlの最終細胞ペレットを再懸濁する。 </li></ol> 5.細胞の計数<ol><li>細胞液10μlを取り、混ぜるパンブルー10μlのと。負荷られたミックス10μlの血球計算板の上に、溶液の1ml当たり細胞数を得る。 </li></ol> 6.細胞培養<ol><li> 24ウェルプレートの各ウェルにマクロファージ刺激培地2mlを置く。 </li><li>各ウェルに200,000細胞を追加します。 注：我々の経験では、細胞プレーティング密度は破骨細胞の体外培養において十分なために最も重要な要件の一つである。 </li><li>静かに37℃での定期的なインキュベーター内でプレートと場所を攪拌する。 </li><li> 3日間のメディアを変更しないでください。 </li><li>マクロファージ刺激媒質中で3日後、破骨細胞のメディアにメディアを変更します。 </li><li>以下、大、多核破骨細胞は、培養プレート上で表示されている必要があり、その時点で、5-7日間、毎日メディアを変更。 </li></ol> 7.染色と酒石酸抵抗性酸性ホスファターゼ（TRAP） <ol><li> インビトロ CULの5-7日後トゥーレは、培養ウェルから培地を吸引除去する。 </li><li> 1×PBSで3回優しく井戸を洗ってください。 </li><li> 4％パラホルムアルデヒド溶液を添加することによって細胞を固定し、4℃で1時間放置する。 </li><li>この間、37℃に予備温暖化（市販されている）、TRAP染色を準備します。 </li><li>ホルムアルデヒド中で1時間後、パラホルムアルデヒドをオフに吸引し、1×PBSで穏やかに3回洗浄する。 </li><li>染色されるプレートの各ウェルに予め温めTRAP染色の1ミリリットルを追加します。 </li><li>遮光し1時間37℃のインキュベーターでプレートを置きます。 </li><li> 1時間後、TRAP染色をオフに吸引除去する。 </li><li>予め温めておいた脱イオン水でウェルを3回洗浄する。 </li><li> 1-2分間ギルのヘマトキシリン​​（注意）とよく対比。 </li><li>明視野顕微鏡を用いた画像破骨細胞。 </li><li>核はブルーレイを出るときに汚れの十分な色強度は、一般的に、達成されるまで脱イオン水でウェルを洗ってください電子メール。 </li></ol> 8.破骨細胞再吸収アッセイ<ol><li>骨基質または無機結晶リン酸カルシウムコーティングのいずれかでプレコートされている市販の24ウェルのポリスチレン培養皿を使用する。 </li><li>各ウェルにマクロファージ刺激メディアの2ミリリットルを置きます。 </li><li>各ウェルに、ステップ2の終了時に得られた20万新たに単離した骨髄細胞を加える。 </li><li>穏やかに3日間37℃のインキュベーター内に配置する前にプレートを攪拌する。 </li><li> 3日目に、破骨細胞分化メディアにメディアを変更。 </li><li> 5〜7日間破骨細胞分化培地毎日メディアの変更を行います。 </li></ol>破骨細胞再吸収活動9.定量<ol><li>以下に説明するように鉱化基板上に破骨細胞分化培地中でインビトロ培養の5~7日後、resorpti形成する破骨細胞の能力を評価するために、石灰化した表面を分析ピット上、破骨細胞活性の指標。 </li><li>吸収アッセイプレートから培地を吸引し、各ウェルに10％の漂白剤溶液100μlを加える。 </li><li> RTで15分間、10％の漂白剤溶液で細胞をインキュベートする。 </li><li>蒸留水でウェルを3回洗浄する。 </li><li>残りの水を吸引し、プレートから余分な水分を振り払うと、3-5時間、空気乾燥させ。 </li><li>非再吸収基板のより容易な可視化を可能にするために、市販のフォン·コッサ染色キットでプレート対比。 </li><li>再吸収面の代表部分の画像を取るために明視野顕微鏡を使用してください。 </li><li>画像解析ソフトウェアを使用して、破骨細胞の吸収活性の定量化を可能にするために再吸収表面の割合を計算する。 </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+and+targeting+osteoclastic+activity+in+prostate+cancer+bone+metastases.">Understanding and targeting osteoclastic activity in prostate cancer bone metastases.</a> Current molecular medicine. 13, 626-639 (2013).</li></ol>

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簡単にin vitroで破骨細胞の多数を隔離し、育成する能力は、骨の生物学および破骨細胞媒介性疾患の理解を進めるために支援を担当してきました。それが最近、破骨細胞の形成、分化および生存16-18の主要な調節因子として同定されたときには、これにつながるRANKLの同定だった。 それは、骨髄からの破骨細胞のin vitro栽培は播種密度に大きく依存し?...

骨リモデリングは動的であり、骨吸収の1骨形成のカップリングを含む。この緊密に調節されたプロセスは、通常の恒常性の間に、そして怪我や病気に応じて骨格を維持する責任がある。 破骨細胞は、骨の有機および無機マトリックスの両方を再吸収することができるユニークな多核細胞である。破骨細胞は、骨髄2-5の単球/マクロファージ系統に由来するものである。破骨細胞の機能または形成の異常は、骨粗しょう症のような一般的な条件を含む臨床病理、さまざまな可能性があります。 in vitroで破骨細胞を生成する能力は、骨の生物学6の我々の理解における大きな進歩を可能にした。その結果、新たな治療薬は、かなりの罹患率および死亡率の原因である破骨細胞関連疾患を治療するために出現している7 </s&gt;まで。骨量および強度の恒常性維持は、骨形成骨芽細胞と骨吸収破骨細胞8,9の協調行動が必要です。骨ホメオスタシス、骨質量および密度10の病原性の喪失をもたらす破骨細胞活性を増加させた閉経後骨粗鬆症を含む多くの疾患、変更される。ヒト疾患のトランスジェニックマウスモデルの利用の増加に伴い、人間の骨疾患11-13における破骨細胞の役割を解読するためのより多くの機会がある。 多くのバリエーションが9,12,14を記載して破骨細胞培養技術のための多数のプロトコルが、文献に現れる。興らは、マウスの骨髄細胞からの破骨細胞形成アッセイの彼らの説明では、以下に記載されるプロトコルに類似した方法論を説明します。しかし、長骨の収穫次骨髄細胞を解放するために、興ら α-MEM完全培地で骨髄腔をフラッシュ14。Catalfamoは破骨細胞機能における高血糖の影響を調べ、骨髄フラッシングによって動員され、すべての細胞は、非接着細胞を12に廃棄され、その時点で24時間、また、ボイルらによって使用される技術のために培養される方法が記載されている9これらの以前に公表されたプロトコルは、1つの骨の両端を切断しなければならないように、また、貴重な骨髄の針刺し損傷および損失の危険を誘発退屈練習を、骨髄をフラッシュするの練習を必要とする。我々が説明するプロトコルは、Weischenfeldt らによって記載マクロファージの単離方法と同様である破骨細胞を単離するための乳鉢と乳棒の使用を実装しています。15 我々の経験では、しかし、多くの場合、その結果、破骨細胞の産生の点で可変転帰における以前に公表された技法の結果を使用して、その破骨細胞の単離およびインビトロ培養物中に存在する破骨細胞を育てることができないことで。したがって、我々は、最初の存在下で、マクロファージ、続いて破骨細胞を形成するメッキされた細胞の70〜80％のおおよその収率で、in vitroで多核破骨細胞の多数を生成するために、マウス骨髄の一貫した分離を可能にするプロトコルを考案した破骨細胞誘導培地。

<table border="0" cellpadding="0" cellspacing="0" width="1017">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:503px;">Name of the Material/Equipment</td>
			<td style="width:165px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM, no glutamine, no phenol red</td>
			<td>Gibco</td>
			<td>51200-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-CSF, recombinant mouse</td>
			<td>Gibco</td>
			<td>PMC2044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Mouse TRANCE/RANK L/TNFSF11 (E. coli expressed)</td>
			<td>R&#38;D Systems</td>
			<td>462-TEC-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prostaglandin E2</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Phosphatase, Lekocyte (TRAP) kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osteoassay bone resorption plates, 24 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3987</td>
			<td></td>
		</tr>
	</tbody>
</table>

osteoclast derivation from mouse bone marrow

<html>破骨細胞は骨髄の単球/マクロファージ系統から誘導された高度に特殊化した細胞である。骨の有機および無機マトリックスの両方を再吸収する独自の能力は、それらが骨リモデリングの調節において重要な役割を果たしていることを意味する。一緒に、骨芽細胞および破骨細胞は骨吸収と健康と病気の間、正常な骨格を維持するために一緒に働く骨形成の両方を伴う動的結合プロセスに関与している。 体内の主要な骨吸収細胞としては、破骨細胞の分化または機能の変化は、体内で顕著な効果をもたらすことができる。改変された破骨細胞の機能に関連する疾患は、より一般的に過剰な破骨細胞骨吸収が形成を破砕するために罹りやすくする骨粗鬆症などの病態を観察することにより、造血のための骨髄空間を形成する失敗に致死性新生児疾患の重症度の範囲とすることができる。 ENT &quot;&gt; in vitroで高い数字で破骨細胞を単離する能力は、骨リモデリングサイクルを理解する上で大きな進歩を可能にしたこれらの疾患と闘う新たな治療戦略の発見のための道を開いてきた。 ここでは、隔離し、破骨細​​胞の多数が得られるマウス骨髄からの破骨細胞を育成するためのプロトコルを記述します。

<html>この方法の目的は、容易に、典型的には1週間で、in vitroで破骨細胞の多数を単離することであった。破骨細胞の多数の正常な分離は、酒石酸抵抗性酸性ホスファターゼ染色（ 図1A）を用いて確認した。大破骨細胞は、複数の核（通常は≥3核）を持つ大規模な紫色の細胞として可視化される。このプロトコルを使用して、破骨細胞あたり最大30核（ 図1B）で...

Watch this Scientific Journal Video about Osteoclast Derivation from Mouse Bone Marrow at JoVE.com

Osteoclast Derivation from Mouse Bone Marrow

破骨細胞は、体内の主要な骨吸収細胞である。大量に破骨細胞を単離する能力は、破骨細胞の生物学の理解における大きな進歩をもたらした。このプロトコルでは、培養し、インビトロで破骨細胞活性を定量化する、単離するための方法を記載する。

マウス骨髄からの破骨細胞の導出

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

osteoclast-derivation-from-mouse-bone-marrow

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Fibroblast Transformation and Muscle Stem Cells

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24.0K ビュー.  Stanford University School of Medicine. 破骨細胞は、体内の主要な骨吸収細胞である。大量に破骨細胞を単離する能力は、破骨細胞の生物学の理解における大きな進歩をもたらした。このプロトコルでは、培養し、インビトロで破骨細胞活性を定量化する、単離するための方法を記載する。 

ビデオ: Osteoclast Derivation from Mouse Bone Marrow

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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

骨髄前駆細胞から骨髄樹状細胞の培養

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

骨髄の造血細胞のホーミング

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

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 ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

ラット骨髄からの破骨細胞前駆体の分離・精製・分化

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

マウスの新鮮な骨髄のプロ血小板形成ダイナミクス

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

イン・ヴィヴォ マウスの頭蓋骨骨髄における巨核球とプロ血小板の2光子イメージング

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...