注：無菌培養条件を維持するためには、このプロトコルの手順のすべては、無菌室の安全基準を用いて実施し、層流フードの下に行われている。 開始する前に、すべてのメディアは℃、適切にガス処刑37に平衡であることを確認してください。 Geltrexコーティングした培養皿の準備注：CELLstartコート培養皿を使用するための付録を参照してください。 <ol><li> 2〜8雪解けGeltrexの一管（1mL）をゆっくり° CとノックアウトD-MEM/F-12の99 mLので1:100にそれを希釈する。穏やかに解決策を混ぜる。 注：一部のiPS細胞株は、最適な成長のための異なるGeltrexの希釈が必要な場合があります。代替希釈については、付録を参照してください。 </li><li> Geltrex溶液（35 - mmディッシュに1 mLを、60 mmディッシュでは1.5 mL）でそれぞれ培養皿の表面全体をカバーしています。 </li><li>乾燥を防ぎ、37℃で1時間のための皿をインキュベートするためにパラフィルムで各皿をシール℃に 注：最大1ヶ月間この時点では、4℃でGeltrexコーティングした培養皿が格納されることがあります。乾燥からGeltrexを防ぐためパラフィルムで各皿をシール。 </li><li>使用する前に、層流フードへGeltrex -コートディッシュを転送し、それらが室温（約1時間）に平衡化することができます。 </li></ol>完全ノックアウトSRフィーダーを含まない培地を準備<ol><li> 10μg/ mLの基本的なFGFの溶液1 mLを準備するには、無菌的に以下のコンポーネントを混在させること。 -20℃で分注しソリューションと店舗までの6ヶ月間。 <table border="0"><tbody><tr><td>塩基性FGF </td><td> 10μgの</td></tr><tr><td> D - PBS </td><td> 990μL </td></tr><tr><td> 10％BSA </td><td> 10μL </td></tr></tbody></table> 注：BSAは、同じ濃度でHSAまたはノックアウトSRで置換することができます。 </li><li> 2 mg / mLのディスパーゼの溶液50mlを準備するには、無菌的に以下のコンポーネントを混在させること。 4の解と店舗℃で2週間までを滅菌フィルタします。 <table border="0"><tbody><tr><td>ディスパーゼ</td><td> 100mgの</td></tr><tr><td> D - PBS </td><td> 50 mLの</td></tr></tbody></table></li><li>完全なノックアウトSRフィーダーフリー（KSR - FF）100mLを準備するには無菌的に以下の表に記載されているコンポーネントを組み合わせる。 <table border="1"><tbody><tr><td> コンポーネント </td><td> ストック濃度 </td><td> 最終濃度 </td><td> ボリューム </td></tr><tr><td>ノックアウトDMEM/F12（カタログ番号。12660〜012） </td><td> - </td><td> 1X </td><td> 76.8 mLの</td></tr><tr><td>グルタミン- I（製品番号35050〜061） </td><td> 200mMの</td><td> 2mMの</td><td> 1 mLの</td></tr><tr><td>ノックアウトSR（カタログ番号。10828〜028） </td><td> - </td><td> 20パーセント</td><td> 20 mLの</td></tr><tr><td>ノックアウトSR - GFC（カタログ番号。A10580 - 01） </td><td> 50X </td><td> 1X </td><td> 2 mLの</td></tr><tr><td> bFGFの（カタログ番号。PHG0024）。 </td><td> 10μg/ mLの</td><td> 20 ng / mLの</td><td> 200μL </td></tr></tbody></table> 最大1週間までには2-8 ° CでKSR - FF媒体を格納することがあります。 </li><li>温度とガスに完全培地を事前に平衡化する直前に、無菌的に0.1mMの最終濃度は2 -メルカプトエタノールの必要量 （55 mMストック濃度）を追加します 。例えば、KSR - F 100mLを準備するFの培地は55 mM 2 - メルカプトエタノール（1:550希釈）182μLを加える代わりに、2 - メルカプトエタノールは、1X完了培地に添加し、2〜8℃で1週間までのCで保存することができます。 </li></ol>凍結/凍結保存培地の準備凍結保存培地は2つのメディアから構成され、彼らは別々のタイミングで細胞に添加され、分離されたままにしておく必要があります凍結培地及び凍結培地のB.。彼らは、それぞれの凍結保存媒体の総容積の50％が必要とされています。必要に応じて凍結保存媒体の総体積は、フリーズしたように各バイアルに1 mLです。ヒトES細胞の90％コンフルエント60ミリメートル皿は凍結保存媒体でヒトES細胞のバンク2バイアルに使用することができます。 超過を確実にするために付加的な2 - 5mLを使用して各凍結培地の十分なボリュームを準備します。 <table border="1"><tbody><tr><td>凍結培地（最終容量の50％）： </td><td> 50パーセントDMEM/F12 </td><td> 50％のKSR </td></tr><tr><td>凍結培地B（最終容量の50％）： </td><td> 80パーセントDMEM/F12 </td><td> 20％DMSO </td></tr></tbody></table>例： あなたが細胞の20バイアルを凍結している場合は、凍結保存培地の20mlのが必要になります。 24mL凍結保存培地の合計のために超過分を4mlのを追加。それはあなたが凍結培地（24mLの50％）および凍結培地Bの12mL（24mLの50％）の12mL必要があることを意味します。 <table border="1"><tbody><tr><td>凍結培地（12 mL）中： </td><td> 6 mLのDMEM/F12 </td><td> 6 mLのKSR </td></tr><tr><td>凍結培地B（12 mL）中： </td><td> 9.6 mLのDMEM/F12 </td><td> 2.4mLのDMSO </td></tr></tbody></table>凍結保存培地の最終組成は65％DMEM/F12、25％KSR、および10％DMSOである。 Cryopreserving性IPSC <ol><li> 37℃の水浴中でディスパーゼのプレ暖かい必要なボリューム。必要なボリュームの詳細については、下記の表1を参照してください。 </li><li> 37℃の水浴で15分間でKSR - FFの必要量を事前に平衡化します。必要なボリュームの詳細については、表の1belowを参照してください。 </li><li>ピペットを用いて培養容器から使用済み培地を吸引し、そしてD - PBSで細胞を2回すすいでください。 </li><li>ゆっくりと培養容器（例えば、60 mmの培養皿あたりのディスパーゼ溶液1 mL）に予め温めておいたディスパーゼのソリューションを追加します。コー​​ト全体の細胞表面をにスワール培養容器を。 </li><li> 3分間37℃で培養容器をインキュベートする。 </li><li>インキュベーターから容器を削除、ディスパーゼソリューションを吸引し、そして穏やかに、D - PBSで細胞を洗浄する。 </li><li>優しくセルスクレイパーを用いて培養皿の表面から細胞をこすり、および滅菌15mlの遠心チューブに細胞を移す。 </li><li>静かに切り離されていない任意のセルを&quot;オフ噴霧&quot;、KSR - FFで二回培養皿をすすぎます。プールは、15mlのチューブで細胞と培地を洗い流してください。 </li><li>ペレットに室温で5分間、細胞を200 xgでチューブを遠心分離します。 </li><li>慎重に細胞ペレットを乱すことなく上清を吸引し、それを捨てる。 </li><li> cryovialsの十分な量を準備します。細胞はDMSOと接触していると、彼らは2〜3分以内に分注し、凍結して下さい。 </li><li>ゆっくりと完全に[穏やかに5 mLの血清学的ピペットを使用して上下にピペッティングして]を凍結培地Aにチューブの底からの細胞ペレットと再サスペンド細胞を取り除くために、チューブをフリック。穏やかに混合して細胞懸濁液を渦巻くながら、塊の均一な懸濁液に続いて、、滴下方法で、それに凍結培地Bの等量を追加します。 注：この時点で、細胞はDMSOと接触している、と作業が効率的に実行する必要があります。いったん細胞はDMSOと接触している、それらは2〜3分以内に分注し、凍結して下さい。 </li><li>各クライオバイアルに細胞懸濁液のアリコートを1ミリ​​リットル。 </li><li> ℃で一晩迅速ミスターフロスティでバイアルを配置[急速に凍結する]、-80に転送します。 </li><li> -80℃で一晩保管° Cの後、長期保管のための液体窒素タンクにセルを転送する。 </li></ol> IPSCバイアルの雪解けと細胞の回復注：KSR - FFは、KSR含有MEF -馴化培地、またはフィーダで使用するためのKSRの培地で置き換えられるかもしれません。メディアを準備するための指示については、付録を参照してください。 50 mlチューブにKSR - FF媒体のプレ暖かい10mLを。 <ol><li>ちょうどあなたのめっき前に室温にGeltrex - コートディッシュの適切な量を平衡化し、皿から液体を吸引するR細胞。 </li><li>慎重にヒトES細胞（またはIPSC）液体窒素貯蔵タンクからバイアルを取り外してください。 </li><li>ちょうど小さな冷凍チャンクがバイアルになるまで急速に、37 ° Cの水浴中で細胞の凍結バイアルを解凍。 </li><li>それを除染し、無菌組織培養フードにそれを転送するためにイソプロパノール70％スプレーバイアル。 </li><li>無菌的に5 mLのピペットを用いて15 mLコニカルチューブにバイアルの内容全体を転送する。 </li><li> KSR - FF 1 mlのバイアルをリンスし、15 mlの遠心チューブに以下を追加。 </li><li>ゆっくりと、KSR - FFの4 mLの15mlのコニカルチューブに細胞に滴下して加える。培地を添加しながら、穏やかに細胞を混合する前後にチューブを移動します。 （これは細胞に浸透圧ショックを減らす）。 </li><li>室温で2分間1000rpm（200 × g）で細胞懸濁液を15 mLの試験管を遠心します。 </li><li>慎重に吸引し、細胞ペレットを乱すことなく、上清を捨てます。 </li><li> 5 mLのピペットを用いて予め温めておいたKSR - FF（例えば4mL/60 mmディッシュ）で細胞ペレットを再サスペンドし、細胞ペレットが完全に分散されるまで静かに上下のセルをピペッティング。生存率が70％未満の場合は70％を超えるの生存率が期待されている、しかし、それは、コンフルエントに達するために、細胞のために時間がかかることがあります。 </li><li>同じピペットを使用して、細胞懸濁液を室温に平衡化Geltrexコーティングした培養皿に滴下して転送する。 </li><li>空気中の4〜6％CO 2の加湿雰囲気下で、37℃インキュベーターで培養皿を置きます。均等に細胞を分散させる西側パターンに南、東、北に慎重にスワール容器。 </li><li>静かに培養皿24時間の解凍後、細胞破片を除去すると料理が約70〜80％コンフルエントになるまで、新鮮な栄養素を提供するために、その後毎日流体変更。あなたのiPS細胞の継続的な文化と継代については、&quot;完全ノックアウト血清代替フィーダーを含まない培地を用いてヒトiPS細胞のフィーダーフリー培養と継代&quot;というタイトルの我々のプロトコルを参照してください。 </li></ol>予想される結果細胞は、前の凍結保存にコンフルエントおよそ70から80パーセントにする必要があります。アップカーリングとコロニーの縁に沿ってコロニーの折り畳みのディスパーゼの消化は結果。細胞を収穫した後、彼らは、凍結保存メディアの小さな塊として凍結されています。バイアルを融解されると、細胞が回復し、小さな塊としてプレコートしたディッシュに付着する。彼らは急速に5-6日でコンフルエント皿につながる成長する。 表1 - 推奨ボリューム<table border="1"><tbody><tr><td> コンポーネント </td><td> 35mmのディッシュ </td><td> 60ミリメートルディッシュ </td><td> 100mmディッシュ </td></tr><tr><td>完全にノックアウトSR培地</td><td> 2 mLの</td><td> 4 mLの</td><td> 10mLの</td></tr><tr><td> Geltrexソリューション</td><td> 1 mLの</td><td> 1.5 mLの</td><td> 4〜5 mLの</td></tr><tr><td>ディスパーゼ</td><td> 0.5mLの</td><td> 1 mLの</td><td> 3〜4 mLの</td></tr><tr><td>すすぎのためのD - PBS </td><td> 2 mLの</td><td> 4 mLの</td><td> 10mLの</td></tr></tbody></table>してください<a href="https://www.jove.com/files/ftp_upload/2237/2237_appendix.pdf">付録を見るにはここをクリック</a> 。

<ol>
	<li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Liao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cell+lines+from+adult+rat+cells.">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li><li>Dimos, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li><li>Park, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells.">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li></ol>

この記事の著者は、この記事で使用した試薬と器具を生産Life Technologies社によって採用されています。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>12660-012</td>
<td>Note: see appendix for the use of alternative DMEM products GlutaMAX-I (Cat. No. 35050-061)</td>
</tr>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PHG0024</td>
<td>Note: see appendix for alternative bFGF pack sizes.</td>
</tr>
<tr>
<td>2-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>17105-041</td>
<td>Note: see appendix for alternative passaging methods</td>
</tr>
<tr>
<td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A10480-01</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>DMSO, 500mL</td>
<td>&nbsp;</td>
<td>JT Baker</td>
<td>JT9224-1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Tissue Culture Hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette-Aid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile serological pipettes (5 mL, 10 mL)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60 mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Liquid nitrogen storage tank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol freezing containers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 mL Cryovials</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

cryopreserving and recovering of human ips cells using complete knockout serum replacement feeder-free medium

ヒトとマウスの線維芽細胞は、胚性幹細胞に非常に似て資質と1月3日 iPS細胞を生成するために再プログラムできることを2006年の発見は、創薬、細胞治療、および基礎研究のための多能性細胞の貴重な新しいソースを作成しました。 GIBCOの培地および試薬は、年間の多能性幹細胞研究の最前線にされている。 DMEMはノックアウト血清代替物を添加したノックアウトは胚性幹細胞の成長と現在のiPS細胞培養3-9最適なメディアです。この金の標準的なメディアシステムは、現在、ノックアウトSRの成長因子のカクテルを追加してフィーダーを含まない培養に使用することができます。 従来のヒトESやiPS細胞の培養法はマウスやヒトの線維芽細胞フィーダー層の使用、またはフィーダ-馴化培地を必要とする。これらの培養法は、スケールにハード、労働集約的であり、それが原因で未定義の条件に未分化腰の細胞を維持することは困難である。インビトロジェンでは、フィーダーフリーへの移行はお尻とhES細胞の培養を、まだノックアウトSRの使用を維持しながら簡単にできるように、ノックアウトSRの成長因子のカクテルを開発しました。

Watch this Scientific Journal Video about Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

このプロトコルはノックアウトSRの凍結保存培地でヒトiPS細胞をcryopreserving、完全ノックアウトSRフィーダーフリー（KSR - FF）培地やフィーダーベースのノックアウトSR培地でこれらの細胞を回収するための詳細な手順を説明します。

完全ノックアウト血清代替のフィーダーを含まない培地を用いてヒトiPS細胞のCryopreservingおよびリカバリ

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

cryopreserving-recovering-human-ips-cells-using-complete-knockout

Research

JoVE Journal

Biology

18.2K ビュー.  Life Technologies.  中文 日本語 

ビデオ: Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

数理モデルを使用して人口置換戦略の有効性を予測

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

生物学

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

ソックス- 2の強制発現によってMEFSからiPS細胞を生成し、OCT - 4、c - Mycの、とKLF4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

完全ノックアウト血清代替フィーダーを含まない培地を用いてヒトiPS細胞のフィーダーフリー適応、文化と継

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

ヒト心筋細胞の生成：フィーダーフリーヒト人工多能性幹細胞からの分化プロトコル

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

ヒト血清から小非コードRNAの分離

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

ヒトiPS細胞のマイクロRNA発現プロファイル、IPから派生網膜色素上皮、および胎児の網膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

エピソームおよびHDAC阻害剤を用いて血液からの効率的なiPS細胞の生成

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

骨馴化培地：調製およびバイオアッセイ

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

SPRi、ナノのSPRiおよびELISAアッセイを用いて血清中のヒト成長ホルモンの比較分析

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...