Name: pluripotent stem cell derived cardiac cells for myocardial repair
Uploaded: 2017-02-03T15:00:00.000+00:00
Duration: 6 min 37 s
Description: Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).

全ての実験手順は、アラバマ大学バーミンガム校の動物ガイドラインに従って行われています。 1. hiPSC-のCMにhiPSCsの区別<ol><li>一晩4℃で予め冷却した成長因子低減ゼラチン状タンパク質混合物でコート6ウェルプレートのウェル。使用前にゲル状のタンパク質混合物を吸引。 10μMのROCK阻害剤とのmTeSR1培地補充で5％CO 2、37℃で細胞（ウェル当たり1×10 5細胞）プレコーティングしたプレート上にhiPSCsをシードし、文化。 </li><li>細胞が90％コンフルエンスに達するまで毎日培地をリフレッシュ。その後、5％CO 2、37℃2日分以上の細胞成長因子低減ゼラチン状タンパク質培地への混合物（6ウェルプレート当たり0.5 mgのゼラチン状のタンパク質混合物、ウェルあたり2 mlの培地）、および文化を追加します。 <ol><li>培地を交換するには、静かにして真空を経由してペトリ皿から培地を吸い出します細胞をタッチアウト、および転送ピペットを使用して、新しいメディアを追加します。 </li></ol></li><li>成長因子低減ゼラチン状タンパク質混合物、インスリンなしB27および100ng / mlのアクチビンAを補ったRPMI1640培地で培地を交換し、分化を開始します。 24時間の培養を5％CO 2、37℃で細胞。 </li><li>インスリンなしB27を補充されたRPMI1640培地、10ng / mLの骨形成タンパク質4（BMP-4）、および10ng / mLの塩基性線維芽細胞成長因子（bFGF）を含む培地を交換し、 96時間培養を5％CO 2、37℃で細胞。 </li><li> B27を補充したRPMI1640培地で培地を交換し、培養を5％CO 2、37℃で細胞を続けます。 3日ごとに培地を更新します。 </li><li>分化を開始した後3日〜、顕微鏡下で細胞を収縮のクラスターを観察します。最初の収縮を観察した後〜7日のクラスタを収集し、ハンクス平衡塩溶液でそれらを洗います。 <ol><li>穏やかに振盪しながら37ºCで10分間100 U / mlのコラゲナーゼIVを含むハンクスバッファーでそれらをインキュベートすることによってプレートにクラスタ化された細胞を解離させます。その後、5分間、エチレンジアミン四酢酸（EDTA）中の0.25％トリプシンを加えます。 </li><li> RPMI / B27培地中で、10％ウシ胎児血清を酵素溶液を中和した後、RPMI / B27培地中で細胞を再懸濁。 </li><li>血球計数器により細胞数をカウントします。培養非心筋細胞は、培養皿の表面に付着することを可能にする、少なくとも3時間、5％CO 2、37℃で10cmの皿に細胞。 </li></ol></li><li> hiPSC-のCMが含まれている細胞懸濁液を、収集し、ゲル状のタンパク質混合物でコーティングされた表面上でそれらを培養することによって細胞（10cmディッシュあたり1×10の周りに6個の細胞）を維持します。 </li></ol> 2. hiPSC-ECのにhiPSCsの区別<ol><li>目をインキュベートすることにより単一細胞にhiPSC集団を解離5分間、5％CO 2、37℃でのキレート剤とEM。新鮮なmTeSR1培地を含む15ミリリットルの遠心チューブに細胞を移します。 </li><li> 5分間、200×gで細胞をスピンダウン。 10μMのROCK阻害剤を補充したmTeSR1培地で細胞を再懸濁します。血球計数器を用いて細胞密度を決定します。 </li><li> 24ウェルプレートの1ウェルに20 NIH単位/ mlのトロンビン溶液の250μlのを追加します。 </li><li> 12.5 mg / mlでフィブリノゲン溶液の250μlに1×10 6 hiPSCsを追加します。そして、トロンビンロードウェルに細胞含有フィブリノーゲン溶液の250μlを添加します。混合物を分以内hiPSC含有フィブリン足場を形成するために固化観察します。 </li><li>カバーガラスをピンセットで6ウェルプレートのウェルに凝固、細胞を含む足場を転送し、インスリンなしで2％のB27を補充した2 mlのEBM2培地で足場を培養することによって分化を開始し、アクチビンA（50ngの/ミリリットル）、及びBMP-4（25 ngの/ ml）を、5％CO 2で24時間<sUB&gt; 2、37℃です。 <ol><li>細胞を穏やかに触れることなく真空を介して、古い培地を吸引して培地を交換してください。インスリンなしB27を補充した新しいEBM2培地、血管内皮増殖因子（VEGF、50ngの/ mlで）、エリスロポエチン（EPO、100ng / mlの）、およびトランスフォーミング増殖因子β1（TGFβ1、を10ng / ml）を、培養に追加48時間、5％CO 2、37℃で足場。 </li><li>さらに48時間、5％CO 2、37℃で培地および培養足場を更新します。その後、培地に200 UのコラゲナーゼIV（100U / ml）を添加することによって、足場から細胞を放出します。 </li><li>彼らがリリースされた後に細胞をスピンダウン。 B27、VEGF-A（50 ngの/ mlの）、およびSB-431542（10μM）の2％を補充した2ミリリットルEGM2-MV培地で培地を交換してください。 2日毎に培地を更新します。 </li><li>約10日後（ すなわち 、分化が開始された後に14〜日に）、Hを分離、100 U / mlのコラゲナーゼIVで細胞を解離フローサイトメトリーを介した分化細胞の集団からIPSC-のECは、EC特異的マーカータンパク質の発現について解析する（ 例えば 、CD31、CD144）3。 </li><li>確立されたプロトコル3を介して、単離されたhiPSC-のECを展開します。 </li></ol></li></ol> 3. hiPSC-のSMCにhiPSCsの区別<ol><li>コンフルエント（〜2日）まで、毎日培地交換で、5％CO 2、37℃でゲル状のタンパク質の混合物でコーティングされたプレート、および文化へのhiPSCs mTeSR1培地中で細胞をシード。 </li><li> 3日間RPMI1640培地と2％のB27プラスインスリンでCHIR99021（5μM）およびBMP-4（10ng / ml）で細胞を培養することにより、中胚葉系細胞への分化を開始します。 </li><li> 主に合成表現型とhiPSC-たSMCを生成するには： <ol><li>培養RPMI1640私におけるVEGF-A（25ngの/ ml）を、線維芽細胞成長因子β（FGFβ、5 ngの/ mlで）を有する細胞<html> dium、4日間、5％CO 2、37℃で2％のB27マイナスインスリン。 </li><li> 5％CO 2、37℃で培養VEGF-A（25ngの/ ml）でのセル、FGFβ（5 ngの/ ml）をRPMI1640培地中で、2％のB27プラスインスリン 2日間Cを°。 </li><li>培養血小板由来成長因子β（PDGFβ、を10ng / ml）を、TGFβ、5％CO 2、37℃でRPMI1640（3 NG / ml）を、2％のB27を加えたインスリンのセル 4日間Cを°。 </li></ol></li><li> 主に収縮表現型とhiPSC-たSMCを生成するには： <ol><li> RPMI1640および2％のB27のマイナスインスリンにおけるVEGF-A（25 ngの/ ml）及びFGFβ（5 ngの/ ml）で培養4日間細胞を。 </li><li> RPMI1640および2％B27プラスインスリンにおけるPDGFβ（5ng / ml）およびTGFβ（2.5 ngの/ ml）で7日間培養細胞を。 </li></ol></li><li> 〜6日間RPMI1640代謝培地を含有する乳酸（4ミリモル）で細胞を培養することにより、最終的なhiPSC-SMC集団を精製します。 </li></ol>jove_title &quot;&gt; 4。IGF-含むマイクロスフェアを作成します<ol><li>水浴中で45℃にオリーブオイルを加熱します。 </li><li>加熱を50℃に10％ゼラチン溶液5ml。 </li><li>オリーブオイル、攪拌にゼラチンを追加し、水浴に氷を添加することにより、5℃に、その後急速に冷却します。 </li><li> 25分後に、マイクロスフェアの形成を誘導するためにオリーブオイルを冷蔵（4℃）アセトンを追加します。 </li><li> 5の温度を維持します 1時間°C;その後、オリーブオイルを除去するために、予め冷却したアセトンでそれらを5回洗浄し、マイクロスフェアを収集し、4℃で空気乾燥し、それらを可能にします。 </li><li>架橋を誘導するために、一晩4℃で70％エタノールおよび0.25％グルタルアルデヒド溶液でマイクロスフェアを再懸濁し、次いで、100mMグリシンとの混合物を中和します。 </li><li> 5μgのIGFの30分間、0.1％ウシ血清アルブミンを含有するH 2 Oを蒸留15μlの5 mgの微小球を混合してマイクロスフェアに負荷IGF。 </li></ol> 5.損傷部位の上にパッチを作成し、細胞を注入<ol><li> 1ミリリットル最小必須培地（MEM）で一緒にhiPSC由来のSMC（100万）のCM（2百万円）と、電気部品（100万）を一時停止します。 </li><li> 1ミリリットルフィブリノゲン溶液（25 mg / mlで）中の微小球の5ミリグラムを一時停止します。 </li><li>細胞含有液、フィブリノーゲン/マイクロスフェア溶液と別の注射器、およびトロンビン溶液1mlを有する第3シリンジで1注射器を記入（80 NIH単位は/ミリリットル、2μlの400 mMの塩化カルシウムおよび200mMのεアミノカプロンを補っ酸）。 </li><li>外科2の前に説明したように左冠動脈前下行枝の結紮を介し豚の心に心筋梗塞を誘発します。 注：簡単な、女性ヨークシャーブタ（〜13キロ、年齢の45日）でテラゾール/キシラジン（4.4ミリグラム/キログラム）の筋肉内注射で鎮静され、およびイソフルラン（0.5から5パーセント）で全身麻酔下挿管。 4 番目の intercostal開胸を行い、左前下降冠状動脈を露出しています。冠状動脈を60分間結紮によって閉塞され、次いで、結紮を除去します。即時の電気的除細動は、心室細動の場合に行われます。 </li><li>豚の心臓の梗塞領域の心外膜上の位置滅菌プラスチックリング（〜2.5センチメートル）、その後、同時にリングにマイクロスフェア/フィブリノゲンとトロンビンのソリューションを注入します。 </li><li>混合物は、（〜30秒）固化することを許可し、その後、梗塞心筋に固化した混合物を介して細胞含有注射器の針を挿入し、細胞を注入します。動物の大きさに応じて、3-0または2-0モノクリル縫合糸で筋肉層、皮下組織、および胸壁を閉じます。ブプレノルフィン0.01〜0.02ミリグラム/ kgの筋肉内注射の8時間ごとに、手術後の最初の3日間、疼痛を制御するために使用されます。 </li><li>心臓磁気共鳴を用いた心機能を評価します外科手術、一週間、手術手順2、3、4後4週間前にイメージング（MRI）。最終的なMRI研究の後、動物を犠牲にし、組織学および分子解析2、3、4のための彼らの心を処理します。 </li></ol>

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hiPSC-のCMの歩留まりの向上/純度 CMSにヒト幹細胞を分化するための従来のプロトコルは、多くの場合、低い収率および純度によって制限されます。例えば、ヒトES細胞-CMSのわずか35から66まで%は遅いミオシン重鎖またはcTnTの6を表明パーコール分離および心臓体形成を介して得られます。分化hiPSC-CM集団の純度は、実質的にCM特異的タンパク質<s...

ヒトの人工多能性幹細胞（hiPSCs）は、患者の免疫系によって拒絶されていない細胞の潜在的に無限の範囲と量に分化させることができるので、再生細胞療法のための最も有望な薬剤の一つです。 （平滑筋細胞を但し、自己複製および分化のための能力はまた、腫瘍形成につながる可能性があり、その結果、hiPSCsは、完全にこのような心筋細胞（CMS）、内皮細胞（EC）、および平滑筋細胞などの特定の細胞型に分化することが必要）、投与前。細胞投与の最も簡単で最も一般的な方法の1つは、直接心筋内注射であるが、本来の心筋組織により生着された移植細胞の数が非常に低いです。このスレの多くは、虚血組織の細胞毒性環境に起因することができます。しかし、マウスの胚性幹細胞（ESC）は、O、無傷心臓の心筋に直接注入しました。NLY〜配信500万細胞の40％は、それらが中に製造された高圧力によって針トラックを通って絞り出されたせいか、投与された細胞のかなりの割合は、投与部位を終了したことを示唆している3-5時間1、のために保持しました心筋収縮。 ここでは、（SMCの）4 hiPSC由来の心筋細胞（hiPSC-CMS）2、内皮細胞（hiPSC-ECS）3、および平滑筋細胞を生成するための新規かつ、実質的に、より効率的な方法を提示します。注目すべきは、このhiPSC-SMCプロトコルは、主に合成または収縮SMC表現型に向かって細胞を向けることによって、体細胞のSMC 5で観察された形態学的および機能的特性の広い範囲を模倣する最初のものです。我々はまた、サイトカインを含むフィブリンPを作成することにより、注入された細胞の生着率を改善細胞送達の方法を提供します注射部位の上にATCH。パッチは、少なくとも3日間の期間にわたってインスリン様成長因子（IGF）を放出することにより、心筋から出るセルを防止する針トラック、及び細胞生存を封止することにより、両方の細胞保持を改善するように見えます。

<table><tbody><tr><td>&lt;strong&gt;Protocol Section 1&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>mTeSR1 medium</td><td>Stem cell technologies</td><td>5850</td><td></td></tr><tr><td>Growth-factor-reduced matrigel</td><td>Corning lifescience</td><td>356231</td><td></td></tr><tr><td>Y-27632</td><td>Stem cell technologies</td><td>72304</td><td></td></tr><tr><td>B27 supplement, serum free</td><td>Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>RPMI1640</td><td>Fisher Scientific</td><td>11875-119</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC-010</td><td></td></tr><tr><td>BMP-4</td><td>R&amp;amp;D</td><td>314-BP-010</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>232-FA-025</td><td></td></tr><tr><td>Collagenase IV</td><td>Fisher Scientific</td><td>NC0217889</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (Dextrose, KCl, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, NaHCO&lt;sub&gt;3&lt;/sub&gt;, NaCl, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; anhydrous)</td><td>Fisher Scientific</td><td>14175079</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10438018</td><td></td></tr><tr><td>6-well plate</td><td>Corning Lifescience</td><td>356721</td><td></td></tr><tr><td>10 cm dish</td><td>Corning Lifescience</td><td>354732</td><td></td></tr><tr><td>Cell incubator</td><td>Panasonic</td><td>MCO-18AC</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Versene</td><td>Fisher Scientific</td><td>15040066</td><td></td></tr><tr><td>Fibrinogen</td><td>Sigma-Aldrich</td><td>F8630-5g</td><td></td></tr><tr><td>Thrombin</td><td>Sigma-Aldrich</td><td>T7009-1KU</td><td></td></tr><tr><td>EMB2 medium</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>VEGF</td><td>ProSpec-Tany</td><td>CYT-241</td><td></td></tr><tr><td>EPO</td><td>Life Technologies</td><td>PHC9431</td><td></td></tr><tr><td>TGF-&amp;#946;</td><td>Peprotech</td><td>100-21C</td><td></td></tr><tr><td>EGM2-MV medium</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>SB-431542</td><td>Selleckchem</td><td>S1067</td><td></td></tr><tr><td>CD31</td><td>BD Bioscience</td><td>BDB555445</td><td></td></tr><tr><td>CD144</td><td>BD Bioscience</td><td>560411</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>Fisher Scientific</td><td>12565269</td><td></td></tr><tr><td>Eppendorff Centrifuge</td><td>Eppendorf</td><td>5702R</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CHIR99021</td><td>Stem cell technologies</td><td>720542</td><td></td></tr><tr><td>PDGF-&amp;#946;</td><td>Prospec</td><td>CYT-501-10ug</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 4&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Olive oil</td><td>Sigma-Aldrich</td><td>O1514</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818100</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898</td><td></td></tr><tr><td>IGF</td><td>R&amp;amp;D</td><td>291-G1-01M</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Fisher Scientific</td><td>15561020</td><td></td></tr><tr><td>Heating plate</td><td>Fisher Scientific</td><td>SP88850200</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>15-462-10Q</td><td></td></tr><tr><td>&lt;strong&gt;Protocol Section 5&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>&amp;#949;-aminocaproic acid</td><td>Sigma-Aldrich</td><td>A0420000</td><td></td></tr><tr><td>MEM medium</td><td>Fisher Scientific</td><td>12561-056</td><td></td></tr><tr><td>Syringe</td><td>Fisher Scientific</td><td>1482748</td><td></td></tr><tr><td>Anesthesia ventilator</td><td>Datex-Ohmeda</td><td>47810</td><td></td></tr><tr><td>Anesthesia ventilator</td><td>Ohio Medical</td><td>V5A</td><td></td></tr><tr><td>Defibrillator</td><td>Physiol Control</td><td>LIFEPAK 15</td><td></td></tr><tr><td>1.5 T MRI</td><td>General Electric</td><td>Signa Horizon LX</td><td></td></tr><tr><td>7 T MRI</td><td>Siemens</td><td>10018532</td><td></td></tr><tr><td>Gadolinium Contrast Medium (Magnevist)</td><td>Berlex</td><td>50419-188-02</td><td></td></tr><tr><td>2-0 silk suture</td><td>Ethilon</td><td>685H</td><td></td></tr><tr><td>3-0 silk suture</td><td>Ethilon</td><td>622H</td><td></td></tr><tr><td>3-0 monofilament suture</td><td>Ethilon</td><td>627H</td><td></td></tr></tbody></table>

pluripotent stem cell derived cardiac cells for myocardial repair

ヒト人工多能性幹細胞（hiPSCs）が完全に投与する前に、特定の細胞型に分化されなければならないが、心筋細胞（hiPSC-CMS）にhiPSCsを区別するための従来のプロトコル、内皮細胞（hiPSC-ECS）、および平滑筋細胞（平滑筋細胞）は、しばしば低収量、純度、および/または貧弱な表現型の安定性によって制限されます。ここで、我々は、新規hiPSC-のCMを生成するためのプロトコル、-ECs、実質的に従来の方法よりも効率的です-SMCs、ならびに投与のサイト上で作成されたサイトカインを含むパッチで細胞注入を組み合わせるための方法を提示します。パッチは、長期間にわたってインスリン様成長因子（IGF）を放出することにより、心筋から絞り出されてから細胞を防ぐために、針の軌道、及び細胞生存を封止することにより、注入された細胞の保持の両方を改善します。心筋虚血再灌流傷害のブタモデルにおいて、生着率が時より2倍大きかったです細胞は、細胞とパッチの両方を有するサイトカイン含有貼付せずに細胞に比べ、パッチ、および治療を投与したが、一人ではない細胞を用いて、心機能および梗塞サイズの大幅な改善と関連していました。

差別hiPSC-CMを、-ECs、および-SMCsのキャラクタリゼーション hiPSCsの微分容量は2、3、4を評価しました。フローサイトメトリー心臓トロポニンT（cTnTの）の発現の分析は、最終的なhiPSC-CM集団の純度は90％（ 図1A、1B、パネルB1）を超え...

Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

我々は、心筋細胞、内皮細胞、および平滑筋細胞およびパッチ媒介性サイトカインの送達と細胞注射を組み合わせることにより、移植細胞の生着を改善する配送方法に、ヒト人工多能性幹細胞を分化するための3つの新規かつより効率的なプロトコルを提示します。

多能性幹細胞は、心筋の修復のための心臓細胞を派生します

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

pluripotent-stem-cell-derived-cardiac-cells-for-myocardial-repair

Research

JoVE Journal

Developmental Biology

8.2K ビュー.  University of Alabama at Birmingham. 我々は、心筋細胞、内皮細胞、および平滑筋細胞およびパッチ媒介性サイトカインの送達と細胞注射を組み合わせることにより、移植細胞の生着を改善する配送方法に、ヒト人工多能性幹細胞を分化するための3つの新規かつより効率的なプロトコルを提示します。 

ビデオ: Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

胚性幹細胞から心臓前駆細胞の誘導

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

発生生物学

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

マイクロコンタクトプリントスルーアライン機能心筋組織の生成

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

生物工学

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

心臓の細胞治療幹細胞 DNA の完全性を評価します。

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

遺伝学

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

体外ヒト間葉系幹細胞の心筋細胞のような細胞への分化

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

プロ線維性シグナル伝達の抑制を促進する誘導心筋細胞マウス胚性線維芽細胞のリプログラミング因子を介した

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

心筋内細胞送達：マウスハーツの観測

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

医学

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下で心臓前駆細胞の内皮への分化の誘導

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

患者末梢血単核細胞からのヒト心筋細胞の発生と拡大

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

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

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

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

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

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

生物学

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

フローサイトメトリーによる高効率心筋細胞へのヒト多能性幹細胞の分化と特性

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...