私たちは、手続きのフィギュアデザインとデモの支援のためにRoxannエテュ-アーバーに感謝しています。ラボでの研究が発見はありません付与、コール財団からの移行賞によってサポートされていました。自然科学とカナダの工学研究評議会（NSERC）とイノベーションのためのカナダ基金（CFIリーダーファンドはありません。31377を付与）419226から2012。サンテ（FRQS） - KMHは、フォンデRECHERCHEケベックためChercheur-Boursierジュニアです。

このプロトコルで説明されているすべての手順は、制度上の動物の倫理委員会によって承認され、動物実験ガイドラインにカナダの評議会に従ってきました。 注意：滅菌/特定病原体フリーの住宅事情を維持するために、生物学的安全キャビネットまたは層流フードの内側に生きたマウスのダイレクトなハンドリングに関連するすべての手続きを行っています。適切に動物に提供される装置、住宅資材、飼料と水を抑制し、クリーンまたはケージを滅菌します。注射のための血液採取のためにのみ、滅菌、使い捨ての針を使用してください。無菌技術は、移植の準備中に重要です。 レシピエントマウスの作製<ol><li>時間をかけて末梢血および骨髄再構成の個々のフォローアップを有効にするには、耳のノッチ（またはローカルの動物倫理委員会8によって承認された他の方法）とレシピエントマウスを識別します。移植後のフォローアップのためのマウスを計量。米国（25g未満）7〜12週齢での電子のレシピエントマウス。 注：提供された例では、CD45.1 + CD45.2 +レシピエントマウスは、コンジェニックB6.SJLマウスとC57BL / 6マウスを交配することによって家の中で飼育しました。 </li><li>造血活性を破壊するために450ラドの2用量でレシピエントマウスに照射。移植前、移植の前に最初の投与日及び第二の1〜2時間を与えます。 注：X線またはガンマ線は、両方の適切な設備の利用可能性に応じて使用することができます。 </li></ol>ドナーと競合骨髄細胞の調製<ol><li>ドナー（試験および対照を、CD45.2 +）を安楽死させると、競合他社（CD45.1 +; B6.SJL）頸椎脱臼や地元の動物倫理委員会によって承認された方法を用いて、続いてCO 2窒息によりマウス。 </li><li>空のペトリ皿中または滅菌ガーゼ上で、オープン広い背中、足にマウスを置き、私は（清潔な作業面を維持します）生物学的安全キャビネットをnside。 70％エタノール/ 30％Hと皮膚と毛を湿ら2 O（v / v）です。 </li><li>手術用ブレードまたは鋭いハサミで足を切断してください。脚部に沿って皮膚を開き、鋸歯状の鉗子を用いて皮膚を引き離すカット。余分な筋肉を切り取ります。 </li><li>足の骨を引っ張ると反力として骨盤に対してはさみの刃を使用して大腿骨を脱臼。膝頭から脛骨と大腿骨を外し、筋肉や腱の残りのビットを削除します。 6ウェル組織培養プレート2-3ミリリットル中に骨を滅菌リン酸緩衝生理食塩水（PBS）を配置。 </li><li> 5ミリリットル滅菌PBSで骨を洗い流すことによって骨髄細胞を収集します。 <ol><li>ピンセットで軽く骨を持ち、骨の一端に満たした1mlシリンジに取り付けた25 Gの針を挿入します。プランジ​​ャーを押して、15ミリリットルコニカルチューブに細胞を収集します。骨の中心が白になるまで、必要に応じて繰り返します。 </li></ol></li><li>繰り返しに針を通すことにより細胞を解離単一細胞懸濁液を得ます。気泡や過剰な力を避けてください。注：この段階で、細胞生存率を改善するために、わずかに大きい針（22 G）を使用します。 </li><li>均一な細胞懸濁液を確実にし、破片や凝集塊を除去するために70μmのナイロンストレーナを介して細胞を渡します。 </li><li>血球計数器を用いて細胞計数のためにアリコートを削除します。 4℃で5分間、200×gで、残りの細胞懸濁液を遠心。滅菌PBS（10 8細胞/ ml）に、必要に応じて細胞密度を再調整します。細胞を氷上に保管してください。 </li></ol> 3.ドナー細胞HSC同等の確立<ol><li>フローサイトメトリー染色のために5ミリリットル丸底ポリスチレンチューブに3×10 6個の細胞（30μL）と同等のものを転送します。 （2.5 / mlの最終濃度（PBS、0.1％ウシ血清アルブミン（BSA）および1mM EDTAを補充した）、染色緩衝液で希釈）、非特異的染色をブロックするためにCD16 / CD32に対する非標識抗体の等量を加えます。 RTで5分間インキュベート。 </li><li> 、ビオチン化リネージュカクテル（B220、CD3e、のCD11b、GR1、TER119）のSca1、CD117、CD135、CD150：染色緩衝液中で調製90μlの蛍光標識抗体のマスターミックスを追加します。光から保護して30分間、氷上でインキュベートします。 注：適切な希釈は、抗体のロットごとに決定されるべきです。本研究で用いた希釈液のための材料の表を参照してください。 </li><li>未結合抗体を除去するために4℃で5分間、200×gで細胞、ボルテックスおよび遠心分離に2ミリリットルの染色バッファーを追加します。フリックにより上清と再懸濁ペレットをデカント。 </li><li>染色緩衝液で希釈した10μlの蛍光色素結合ストレプトアビジンは（ 材料表を参照）を追加します。光から保護し、20分間、氷上でインキュベートします。未結合ストレプトアビジンを除去するためのステップ3.3のように洗ってください。 </li><li>少なくとも6つの検出器9とフローサイトメーターを用いて細胞サンプルを取得します。林の周波数を決定する-のSca1 + CD117 + CD135 - CD150 <su解析テンプレートを使用して、各試料中のp&gt; +細胞（HSCを推定） 図2に示すように、以前に10,11を報告しました 。 注意：その集団内の機能HSCの頻度は1/3と1/5 2,12の間に推定することができます。 </li><li>以下および図2で説明するように、ベースラインのような1つのサンプル（典型的には、競合他社、この例ではB6.SJL CD45.1 +）を使用して、すべてのサンプル11の推定HSC当量を確立します。 <ol><li>以下の式を使用して、必要な細胞の数を計算します。 HSCを#transplanted /受信者= 5×10 5細胞のxは（ベースラインサンプルのために決定されるように、この数は、野生型C57BL / 6マウスを、75と125の間に通常ある）HSC頻度を推定10-12。 注： 図2では、試料Aのために、結果は139のHSCです。 （他のサンプルのため、 図2の例のサンプルB）の合計＃移植された細胞=＃トランスHSC / HSC頻度を植えました。 注： 図2では、試料Bの場合、結果は197826（または約2×10 5）総骨髄細胞です。 </li></ol></li><li>上記で得られた結果を用いて、各サンプルのため移植片受容者ごとに必要な細胞の総数を計算します。 注意：この数は、ベースラインサンプル（競合他社）のために5×10 5であるべきです。 <ol><li>レシピエントマウスの数でステップ3.7から得られた数を乗算し、シリンジ内のデッドボリュームを補うために、少なくとも2つのさらなるマウスについての十分な細胞を追加します。 </li></ol></li><li>ステップ3.6で計算され、滅菌PBSを使用して、注射当たり200μlのための最終的な音量を調整すると1 HSC当量比：（ 例えば 、CD45.1 +）競合他社と1の試験細胞（CD45.2 +）を混ぜます。 注：5レシピエントマウスのグループが提案ボリュームは、このように3.5×10 6競技者の骨髄細胞を含有する1.4以上mlである（またはであろうために973 図2のサンプルA）の推定のHSC、およびテストセルの等価数は（ 図2に、サンプルBの等価）は197826×7 = 1384782骨髄細胞であろう。 </li></ol> 4.骨髄移植<ol><li>血管の拡張を確実にするために、横尾静脈をより見やすくするために、赤加熱ランプとステップ1.2で照射レシピエントマウスをウォームアップ。 </li><li> 27 G針（または小さい）を装備した1ミリリットルツベルクリン注射器を準備します。吸引し（3レシピエントマウス用）調製した細胞懸濁液の約750μlの。シリンジ内に気泡がないことを確認してください。 </li><li>拘束装置にマウスを挿入します。尾を調べ、尾の両側にはっきりと見えるはず外側尾静脈を探します。エタノールで注射部位を拭きます。 、アップベベル針を挿入し、皮膚に平行で、静かにプランジャーを押してください。注射は簡単にする必要があります。力再ある場合quiredは、針が静脈ではありません。 注意：古いマウスは厚い皮を持って、より困難な静脈を見つけることができます。針が静脈内にない場合は、最初の注射部位に注射針の近位を挿入します。 </li><li>針を外し、出血を止めるために数秒間滅菌ガーゼで注射部位を押してください。きれいなケージにマウスを転送します。 注意：針があまりにも鈍くなることがあり、その後3回の注射のために同じ注射器と針を使用してください。 </li><li>移植後のフォローアップのために、最初の5日間、マウスを再水和するために滅菌PBSを皮下1ミリリットルを注入。感染を防ぐために水を飲んで抗生物質を追加します（必要に応じて、オプション）。最初の3週間は2〜3日ごとにマウスを計量し、その移植前の体重（またはローカルの動物倫理委員会によって決定される）以上の15から20までパーセントを失ったマウスを安楽死させます。 </li></ol>末梢血の5.分析<ol><li>一滴の血液を収集する（Approx。 EDTAチューブに50-100μl）を。 <ol><li>下顎静脈から血液を採取するには、顎骨の下に位置するヘアレススポットに近い顔の皮膚を穿刺し、血液13の滴を受け取るために捕集管を配置するために、ランセットまたは22 G針を使用しています。多系列再構成に従うように、移植後4、8、12および16週目に血液を採取します。 </li></ol></li><li>ステップ5.1.1で採取した血液の低下に1ミリリットル新たに調製したRT赤血球溶解バッファー（9部0.16 M NH 4 Clで+ 1部0.17 Mトリス塩酸pHは7.65）を追加します。混合し、フローサイトメトリーに適した5ミリリットルの丸底ポリスチレンチューブにサンプルを移します。 RTで4分間放置してみましょう。 </li><li>氷冷PBSの4ボリュームを追加します。エンド・ツー・エンドを回して混合し、氷上ですぐに転送します。 4℃で200×gで遠心分離10分。 </li><li>フリックにより上清と再懸濁ペレットをデカント。上清を明確にし、赤でなければなりません。簡単にバッファと渦を染色2ミリリットルを追加します。 200 XGAで5分間遠心トン4°C。 </li><li>フリックにより上清と再懸濁ペレットをデカント。 </li><li> CD45.1、CD45.2、CD3e、CD19、GR1：ステップ3.3から3.1に詳述さ一般的なプロトコルを使用して染色、フローサイトメトリーと、次のマスターミックスのための抗体を続行します。 </li><li>少なくとも6つの検出器9とフローサイトメーターを用いて細胞サンプルを取得します。 図3に設けられた分析テンプレートを使用して、各試料中の各系統（CD19 + Bリンパ球、CD3e + Tリンパ球、GR1 明るい顆粒球）および10,11を公開としてドナー由来細胞の頻度を決定します。 </li></ol>骨髄再構成の6分析<ol><li>部2ステインにマスターミックスのためのステップ3.3から3.1に詳述さ一般的なプロトコルを使用してフローサイトメトリー分析のための細胞と以下の抗体を詳述したように骨髄細胞を収集：リネージュカクテル、のSca1、CD117、CD135、CD150、CD45.1 、CD45.2。 LINを検出ステップ3.4で説明したように蛍光色素結合ストレプトアビジンを使用してEAGEカクテル抗体。 </li><li>少なくとも8の検出器9とフローサイトメーターを用いて細胞サンプルを取得します。のみ6が利用可能な場合はCD135 +細胞が除外されるように、系統パネルと同じチャンネルにCD135を追加します。 図4に、10が公開され提供される分析テンプレートを使用して、各サンプル中のCD150 +のHSC -のSca1 + CD117 + CD135 -ドナー由来林の周波数を決定します。 </li></ol>

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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+protocol+for+total+body+irradiation+for+allogeneic+bone+marrow+transplantation+in+mice.">Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice.</a> Bone Marrow Transplant. 30 (12), 843-849 (2002).</li><li>Benz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+Stem+Cell+Subtypes+Expand+Differentially+during+Development+and+Display+Distinct+Lymphopoietic+Programs.">Hematopoietic Stem Cell Subtypes Expand Differentially during Development and Display Distinct Lymphopoietic Programs.</a> Cell Stem Cell. 10 (3), 273-283 (2012).</li><li>Eppert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+gene+expression+programs+influence+clinical+outcome+in+human+leukemia.">Stem cell gene expression programs influence clinical outcome in human leukemia.</a> Nat Med. 17 (9), 1086-1093 (2011).</li><li>McIntosh, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonirradiated+NOD,B6.SCID+Il2rgamma-/-+Kit(W41/W41)+(NBSGW)+mice+support+multilineage+engraftment+of+human+hematopoietic+cells.">Nonirradiated NOD,B6.SCID Il2rgamma-/- Kit(W41/W41) (NBSGW) mice support multilineage engraftment of human hematopoietic cells.</a> Stem Cell Reports. 4 (2), 171-180 (2015).</li></ol>

The authors declare that they have no competing financial interests.

ここで説明するプロトコルは、既知の競合他社のHSCに対するドナー（テスト）HSCの相対的な適合性を評価するために設計されています。競争の状況は（幹細胞のフィットネスの緩やかな減少を検出する可能性が高い）アッセイの相対感度を向上させ、照射し、注射の有効性のための内部の技術的な制御を提供します。しかし、それはHSCのフィットネスの絶対的な尺度として使用されるべきで?...

造血が損傷、放射線と細胞死によって失われた血液細胞の補充を確実に再生プロセスです。この処理は、主に成人の骨髄に存在する造血幹細胞（HSC）によって保証されます。また、造血幹細胞は、自己免疫疾患、血液悪性腫瘍および免疫不全1で治療目的のために使用することができます。よりよいそれらの増殖拡大と到達し、移植後のレシピエントの骨髄を生着する能力を含む、HSC機能を制御する機構を理解する必要性が存在します。最近の研究は、将来に向かって約50％の純度2-4に大人のHSCおよび胎児HSCを豊かにするSLAMファミリーメンバーCD150及びCD48を含むいくつかの細胞表面マーカーを、報告しているが、機能的な造血幹細胞のためのゴールドスタンダード尺度はへのin vivo再増殖アッセイのまま血液cを再確立する能力を決定します照射されたホスト5のエル生産。 in vivoでのクローン再増殖アッセイは、最初にティルとマカロック6が開発し、以来、洗練され、拡張されました。もともと定義されているように、HSCは自己複製と分化を通じて生涯の血液細胞の生産を確保します。異なる血液細胞系統（Tリンパ球、Bリンパ球、顆粒球、単球）、および連続移植を介して、自己再生能力の分析を介して分化する能力を：照射レシピエントへのHSCの転送は、このように我々が評価することを可能にします。アッセイは、通常、機能性および/ ​​またはHSCの2つの集団の量の比較を伴うだろう、 例えば 、細胞が造血幹細胞の維持や拡大に影響を与える可能性が別の因子で処理または未処理された異なる遺伝子型または細胞の2匹のマウスから来ます文化インチドナーキメラ、または転送されたドナーのHSCトンの貢献O血液細胞産生は、受信者、またはホストからのドナー細胞を区別する細胞表面マーカーまたは他の方法を用いて、末梢血および骨髄にフローサイトメトリー解析によって決定することができます。最も広く使用されているマーカーは確かに私たちは下記の実施例のために選択した遺伝子PtprcまたはCD45白血球抗原7のための2つの対立遺伝子です。 クローン再増殖アッセイは、競合的または非競合的のいずれかであることができます。非競合的設定では、対照および試験HSCは別個のレシピエントマウスに移し、各細胞型のための結果は、他の独立したであろう。競争力のある設定では、試験および対照両方HSCの機能は、競合他社HSCの集団に対して測定されます。ここで説明するプロトコルは、競争力のある設定を使用しても、非競合的状況に適合させることができます。どちらのアプローチは、その利点と限界を持っている、と私たちは中に詳細にそれらを比較します討論。我々はまた、移植されたHSCの数の株式を確保するための様々なアプローチを説明し、希釈アッセイ（LDA）を制限することにより、HSCの定量化のためのアッセイを適合させ、結果の解釈のための成功と失敗の移植の両方の例を提供する方法について説明します。

<table border="0" cellpadding="0" cellspacing="0" width="703">
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Microtainer tubes with K2EDTA</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">365974</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td>For blood sampling from the mandibular vein</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LabQuake Shaker rotisserie</td>
			<td style="width:89px;">Thermo&#160; Scientific</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">C415110</td>
			<td>Any other rotating mixer will work as well to prevent coagulation of blood samples</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Purified anti-mouse CD16/CD32 (clone 2.4G2, Fc Block)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">2.50</td>
			<td style="width:119px;">553142</td>
			<td>Alternatively use clone 93 from eBioscience (cat # 14-0161) or Biolegend (cat# 101310)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pe-Cy7-conjugated anti-mouse CD3e (clone 145-2C11)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">25-0031</td>
			<td>For most flow cytometry antibodies, the clone is important but the colours and companies can vary depending on the available equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-conjugated anti-mouse CD19 (clone 1D3)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">12-0193</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">APC-eFluor780 (APC-Cy7 equivalent)-conjugated anti-mouse GR1 (clone RB6-8C5)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">47-5931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FITC-conjugate anti-mouse CD45.1 (clone A20)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">2.50</td>
			<td style="width:119px;">11-0453</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">eFluor450-conjugated anti-mouse CD45.2 (clone 104)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.00</td>
			<td style="width:119px;">48-0454</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Biotinylated anti-human/mouse CD45R (B220) (clone RA3-6B2)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0452</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse CD3e (clone 145-2C11)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0031</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse CD11b (clone M1/70)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0112</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse GR1 (clone RB6-8C5)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-5931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse TER119 (clone TER119)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.63</td>
			<td style="width:119px;">13-5921</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">V500 streptavidin</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.50</td>
			<td style="width:119px;">561419</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-conjugated anti-mouse CD117 (clone 2B8)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">553355</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-Cy7-conjugated anti-mouse Ly6A/E (Sca1) (clone D7)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">558162</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PerCP-eFluor710-conjugated anti-mouse CD135 (clone A2F10)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.50</td>
			<td style="width:119px;">46-1351</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Alexa fluor 647-conjugated anti-mouse CD150 (clone TC15-12F12.2)</td>
			<td style="width:89px;">Biolegend</td>
			<td style="width:112px;">0.63</td>
			<td style="width:119px;">115918</td>
			<td>BD Biosciences and eBioscience do not carry the same clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1ml tuberculin syringe with 27G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">309623</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1ml tuberculin syringe with 25G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">309626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 um cell strainer</td>
			<td style="width:89px;">BD Falcon</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
	</tbody>
</table>

competitive transplants to evaluate hematopoietic stem cell fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

二次移植（さらに後述）を含む競争力の移植設定の一般的な説明は、 図1に見出すことができる。移植前の骨髄のHSCのための代表的な分析は、図2に記載されています。ダブレットの除外に関するより詳細な情報と死んだ細胞は、他の場所9見つけることができます。 図3及び<st...

Watch this Scientific Journal Video about Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness at JoVE.com

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

競争力の移植は、造血幹細胞のフィットネスを評価します

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

competitive-transplants-to-evaluate-hematopoietic-stem-cell-fitness

Research

JoVE Journal

Developmental Biology

15.1K ビュー.  Institut National de la Recherche Scientifique. This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting. 

ビデオ: Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

発生生物学

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血細胞の自動定量化 - 脱灰骨の組織学的画像における間質細胞の相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

失活ヒト真皮を用いて遺伝子組換え器官皮膚培養物の生成

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

大人の皮膚線維芽細胞からのインスリン分泌細胞を得るために安全かつ簡便な方法として、エピジェネティック変換

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

ひと多能性幹細胞からプリミティブかつ決定的な造血前駆細胞の分化を監督

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

赤血球の溶血症に対する造血幹細胞移植後の残留ドナー赤芽球系前駆細胞の検出

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

ゼブラフィッシュ初期胚細胞 (HSPCs) を造血幹・前駆細胞の量的にIn Vitroアッセイの開発

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

並べ替え内皮細胞ニッチ共培養と組み合わせて 1 つのセル インデックスを使用して胚性造血幹細胞前駆体のクローン解析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

骨髄異形成症候群の起源を評価するために造血幹細胞移植の使用

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.