We thank Matt Raymond for editorial assistance. This work was supported by funds from the National Institute of Health (U01HL099773, R01HL116221, and P51 RR000167) and The Charlotte Geyer Foundation.

1.ウイルス調製物および転写因子の組み合わせ<ol><li> ETV2、GATA1、GATA2、TAL1およびLMO2のためのタンパク質をコードするDNA（ 表1）を含む PSIN-EF1αレンチウイルス発現プラスミドを含む溶液を準備します。 </li><li> 230ナノメートル、260ナノメートル、および280 nmで分光光度計を用いてUV吸収を記録することにより、レンチウイルス産生のために用いられたプラスミド調製物の濃度および純度を測定します。注：1.8よりも大きなDNA調製実証A260 / 280およびA260 / 230の値は、一般的に良い品質と考えられています。下部A260 / 230の値は、フェノールのような塩またはいくつかの溶媒を用いて不純物を示しながら、低いA260 / 280の値は、タンパク質汚染を示すことができます。推奨プラスミド濃度は、1〜3μgの/μlです。 </li><li>以前に発表されたプロトコル11で説明したように形質導入のためのレンチウイルスを生成します。汎骨髄可能性を秘めたHEの誘導はETV2およびGATA1、またはETV2およびGATA2の共発現を必要とします。赤血球および巨核球ポテンシャルのHEの誘導はGATA2およびTAL1が必要です。 LMO2の添加は大幅にGATA2とTAL1 10の存在下でhPSCsからエリスロ巨核球細胞の歩留まりを向上させることができます。 </li></ol> 2. hPSCs培養プロトコル<ol><li>継代培養前に70〜80％の密集度にシャープなエッジとの緊密なコロニーで多能性幹細胞を増殖させます。マークと削除は、自発的に細胞を継代する前にコロニーを分化しました。 </li><li>使用前に4℃で、または少なくとも1時間37℃で一晩、製造者の指示及び被覆6ウェルプレートによれば（ 表2参照 ）は、市販の細胞外マトリックスゲルを希釈します。細胞を継代する前に、行列を吸引、新鮮な商業完全culturemediumの2ミリリットルを追加します（ 表2を参照）、37でプレートを保ちます ℃、5％CO 2の細胞が播種のための準備が整うまで。 </li><li>パッセージhPSCs <ol><li> ASPIRAテhPSCsで6ウェルプレートの1コンフルエントウェルから培地および（0.22μmのフィルターで濾過滅菌したDMEM / F12中2 mg / mlの）予め温めておいたディスパーゼII溶液1.5mlを追加します。コロニーの縁部が表面から持ち上げ始めるまで37℃、5％CO 2で5~7分間インキュベートします。 </li><li>吸引しディスパーゼII溶液を、注意深く追加し、新鮮なDMEM / F12培地2mlを吸引することにより、二回コロニーを洗います。次いで、5 mlの血清ガラスピペットを使用して、細胞懸濁液を上下に数回ピペッティングすることにより（ 表2参照）新鮮な商業完全培地3mLでhPSCsを解離させます。 </li><li> （ 表2参照 ）は、市販の完全培地1.5 mlを含有する6ウェルプレートの各ウェルに細胞懸濁液を0.5mlを加えます。インキュベーターにプレートを位置づける際に均一な細胞分布を確保するために、前後および左右に数回にプレートを攪拌。 1 37℃、5％CO 2で細胞を維持します8-24時間。 </li><li>新鮮な商業完全培養培地への次の日変化媒体（ 表2を参照）、hPSCs形態を調べます。 HPSCの形態を保持し、小さな、よく付着したコロニーで成功継代結果。 hPSCsは、日常的によくミリリットルfreshmediumあたり2.5〜3で供給され、これ以上の70〜80％以下のコンフルエンスで4-5日毎に継代する必要があります。 注：hiPSCsは、マウス胚性線維芽細胞（MEF）で維持した場合は、無フィーダー条件に転送する必要があり、効率的な分化を確実にするために導入する前に2〜3回継代しました。 </li></ol></li></ol> hPSCsからHematoendothelial Pprecursorsの3誘導（0日目、hPSCsの形質導入） <ol><li>反応媒体は、市販の完全無血清培地の1.3ミリリットルを組み合わせる調製するためには、ウイルス、ポリブレン、およびY27632 ROCK阻害剤（ 表1）（表2参照 ）：最終concentraに1.3 mlの培地に10mMのROCK阻害剤の1.3μlを添加します10μMの化、および最終濃度6 / mlのポリブレンへ。 <ol><li>細胞当たり各ウイルスの0.5〜1.0 MOI（感染の多重度）の最終濃度でウイルスの濃縮物（複数可）の適切な量を加えます。単一細胞懸濁液の準備ができるまで氷上または4℃で反応媒体を保管してください。 注：GATA1 / ETV2、GATA2 / TAL1の各ウイルスにMOIの同じ量、およびGATA2 / TAL1 / LMO2反応混合物は、分化の誘導に最適です。しかし、ETV2に対する相対GATA2のためにMOIを倍増ETV2 / GATA2形質導入された培養液で、差別化を強化します。したがって、これらの培養のために、比1：ETV2のMOIとGATA2の1 MOIのための2を推奨します（例えば、ウイルス濃縮物は、約6.8×10 7粒子/ mlと推定されている場合にETV210μlのとGATA2ウイルス濃縮物の20μlを添加6ウェルプレートの35mmのウェル中の1 ETV2 / GATA2の反応当たり0.68×10 6細胞 ）。 </li></ol></li><li>単一細胞SUSPにhPSCsを準備ension。 <ol><li> 4日目のセクション2で説明したように最後の通路以下の無フィーダー条件でhPSCsを育て、倒立顕微鏡下で細胞密度および形態を観察します。 hPSCsは、自発的な分化とシャープなエッジとの緊密なコロニーに成長し、伝達の日に〜60〜70％の密集度に到達する必要があります。 </li><li>培地を吸引、ウェルあたり市販の細胞解離試薬（ 表1）の1.5〜2ミリリットルを追加し、5-7分間、5％CO 2で37℃でインキュベートします。 </li><li>商業完全培地の等容量に細胞を収集し、細胞をカウントし、生存率を測定、ROCK阻害剤の10μMと（ 表2参照）。その後、ペレット5分間200×gで遠心分離によって細胞とは、1mlあたり3.4から5×10 6細胞の濃度にROCK阻害剤の10μMと商業完全培地中で細胞を再懸濁します。 注：細胞の生存率は、成功したウイルス形質導入およびその後の分化に重要です。一般的に、導入前の細胞の生存率は90％を超えている必要があります。 </li></ol></li><li> 3.1で調製した反応媒体の1.3ミリリットルに、0.68から1×10 6個の細胞を含む、200μlの細胞懸濁液を追加します。 </li><li> 、2.2で調製した一晩コーティングした6ウェルプレートのマトリックス溶液を吸引し、6ウェルプレートの1ウェルに、反応混合物を転送し、細胞を均一に分配します。 24時間、5％CO 2で37℃でインキュベートします。 24時間後、細胞の少なくとも80％がマトリックスに付着する必要があります。 </li></ol> hPSCsからHematoendothelial前駆体の4誘導（日1-7） <ol><li> 24時間の形質導入後、ウイルス含有培地を除去し、商用の不完全な細胞培養培地で付着した細胞を洗浄した（表1を参照）、造血サイトカインを含有する市販の不完全な細胞培養培地のウェルあたり3 mlを加え：SCF 100ng / mlの、TPOを50 ng / mlで、bFGFを（以下、3F媒体と呼ぶ）20 ng / mlで。 </li><li>死細胞および破片を除去するために、日2、3、4日目に新鮮な3F-培地で培地全体を交換してください。浮動造血細胞が出現しているときに4 mlの総容量を維持しつつ、4日後、一日おきに3F-培地の半分を交換してください。 注：PSIN-EF1αレンチウイルス発現プラスミドにより形質導入された細胞の正の選択を可能にピューロマイシン耐性遺伝子を組み込んでいます。ピューロマイシンを1μg/ mlの任意の残留未分化hPSCsを除去するために、分化の最初の1~2日の間に培地に添加することができます。 </li><li>倒立顕微鏡下で4日目の細胞形態を守ってください。典型的な内皮形態を有する細胞のタイトなクラスタが現れ始める（ 図2A、3A）。一部の地域ではhPSCsの形態を保持することができる一方でラウンド血液細胞は、分化の7日目に、5日目から表示されます。以下に説明するように、彼と造血分化を分析します。 注：成功hematoendothelial異なりますiationは緩く、基礎となるフラット内皮細胞に付着したとして屈折丸い細胞を表示された血液細胞の産生をもたらします。これらの細胞は、積極的に小さな凝集体を形成し増殖し、最終的に切り離し、フロート（ 図2Aおよび3A）。生血液細胞は、光を反射し、培養条件に拡張する能力に基づいて、死んだと死細胞から視覚的に区別することができます。 </li></ol> 5.分化の造血内皮（HE）ステージの分析。 <ol><li>内皮形態を有する細胞の顕微鏡観察により分化の3日目と4の間にHEの形成を検出します。分化のこの段階での文化には、浮遊血球はありません。 HEの存在は、免疫染色によって確認し、VE-カドヘリン、CD73、CD226、およびCD43抗体を用いたフローサイトメトリー分析することができます。 <ol><li> 3日目にフローサイトメトリー分析用細胞によるHE形成を評価するために一つの実験から4日目。 37℃で5〜10分間（ 表1参照）の商業的細胞解離試薬と培養液をインキュベートすることによって細胞を集め、5％CO 2、次いで（に記載の染色手順フローサイトメトリー染色反応あたり1×10 5個の細胞まで使用12）。 注：VEカドヘリン+ HE細胞は初期造血マーカーCD226の発現を獲得するが、CD73とCD43 6の発現を欠きます。対照的に、非HE細胞は、CD73（ 図2B、3B）を発現します 。 </li></ol></li><li> HPSC由来のHEのための免疫蛍光染色。 <ol><li>リン酸緩衝生理食塩水（PBS）で細胞の付着した単層を洗浄した後、室温で30〜60分間の4％パラホルムアルデヒドで細胞を固定します。 </li><li> PBSで再び細胞を洗浄し、次いで、室温で20分間PBS中0.1％トリトンX-100を用いて透過性。 </li><li> 3回、PBS 2で細胞を洗浄し、その後でインキュベート30〜120分間、10％ウシ胎児血清（FBS）を含むPBSからなるブロッキング緩衝液。 </li><li>一次抗体の両方を含む、染色溶液を調製する（1：1,000希釈）、マウス抗ヒトCD43およびウサギ抗ヒトVE-カドヘリンをPBS中の5％FBS、（ 表1）を用いて。 </li><li>細胞からブロッキング緩衝液を吸引除去して、コメントを追加一次抗体で染色バッファーのウェルあたり1ミリリットルを追加します。室温で3時間、または一晩4℃でインキュベートします。 </li><li> 5％FBSを含む2mlのPBSを加えることによって、細胞を3回洗浄します。 </li><li> 5％FBSを含むPBS中で500倍に希釈：ロバ緑色蛍光色素と結合した抗ウサギ、および赤色蛍光色素（ 表1）と結合した抗マウス1で二次抗体を含有する、第二の染色緩衝液を準備します。二次染色バッファーウェルあたり1 mlを加え、室温で1時間インキュベートします。 </li><li>血清を含まないPBSで3回洗浄します。 dHで300 nmのDAPI作業溶液で染色細胞10〜15分間2 O。 </li><li> PBSで2回細胞を洗浄し、染色された細胞をウェルにPBSを2〜3mlのを追加します。緑、赤、青の顕微鏡フィルターで蛍光を観察します。 </li></ol></li></ol>誘導性造血前駆細胞の6分析<ol><li> 3.1で説明したように、浮遊細胞は、3F-培地の半分を変更し、10〜14日まで、大幅に拡大されるまで2日ごとに、分化培養を維持します。 </li><li> 37℃で5〜10分間（ 表1参照）解離および商業細胞解離試薬で細胞を処理することにより、細胞の単層を分化収集 °C、5％CO 2。 </li><li>文化12で造血を評価するために、抗VEカドヘリン、CD43およびCD45抗体を用いたフローサイトメトリーを実行します。アップETV2とGATA2形質導入した細胞の40％への細胞の大部分は、同時発現するが、VE-CADこととCD43に注意してください。以下の抗体は、E以下の特定の細胞系統を検出するために使用され得ますxpansionまたは誘導された血液前駆細胞の分化：CD235a（赤血球細胞）、CD41a（巨核球）、CD32（骨髄細胞のすべてのタイプ）、CD66b（好中球）およびCD163（マクロファージ）。 </li><li>製造業者の指示に従ってコマーシャルクローン原性培地（ 表1）を用いて、CFCアッセイを行います。 <ol><li>商業クローン原性培地3ml中に1〜2×10 4個の細胞を移すコロニー形成細胞及び造血コロニーの種類の数を評価する（ 表3参照）。最適な播種密度は、互いに別々に成長し、重複しない単一コロニーの同定および単離を可能にします。播種密度は、分化効率に応じて実験間で異なるが、商業クローン原性培地1mlあたり1×10 4個の細胞を超えてはなりません。 </li><li>穏やかにボルテックスにより商業クローン原性培地中の細胞を混合し、2つの35ミリメートルの超低接着ディッシュにサスペンションの3ミリリットルを転送CFCアッセイのため、各皿にサスペンションの1.5ミリリットル。均一媒体を配布し、14日間、37℃、5％CO 2でインキュベートします。料理を邪魔しないでください。 7日目、アッセイの10日目にコロニー形成を観察します。 </li><li> 14日目に、その形態に応じて、造血コロニーを同定し、カウントします。 </li></ol></li></ol>

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Cynataのための創立株主およびコンサルタントです。

TFの過剰発現によるhPSCsの造血分化のための上記の方法、それによってより最大30万血液細胞の産生を可能にする、ヒトES細胞やiPS細胞から彼と骨髄とerytho-magakaryocytic前駆細胞の生成のための迅速かつ効率的なアプローチを表し、 1個の百万多能性幹細胞10。この方法は、複数のhESCと性IPSCライン10で一貫性のある分化を示しました。 ETV2とGATA2、GATA1の要因だけでなく、GATA2およ?...

自己再生および血液を含む三胚葉の細胞に分化するために、造血開発の機構研究のためにそれらの貴重なツールを作る、血液疾患のモデリング、薬物スクリーニングにヒト多能性幹細胞（hPSCs）のユニークな能力、毒性試験、および細胞の治療法の開発。内皮造血移行1,2を通じて造血内皮（HE）から胚進行中の血液形成するので、培養液中で彼の世代は、造血移行および造血仕様に内皮を制御する分子メカニズムを研究するために不可欠であろう。 HEの研究のための現在の方法は、造血サイトカイン3-5、および造血支持する間質細胞6,7または細胞外の2次元文化のあるhPSCsの共培養を加えた集合体でhematoendothelial分化（したEB）の誘導に基づいています行列やCytokines 8,9。これらの古典的な分化方法は、細胞表面で作用し、最終的にhematoendothelial発展を導く転写プログラムの活性化につながる分子経路のカスケードを開始する外部信号の導入に基づいています。したがって、これらのシステムでhPSCsの分化の効率は、それらの信号の効果的な誘導、核へのシグナル伝達、および特定の転写調節因子の結果として生じる活性化に依存しています。また、従来の分化培養では、彼の研究は、細胞選別を使用して、HE細胞を単離する追加の工程を必要とします。ここでは、造血転写因子の過剰発現によるHEと血液の直接誘導するための単純なプロトコルを記述します。この方法は、面倒なセルソーティングの手順を使用して、HEを単離を必要とせずに、皿および造血への遷移内皮の直接観察では、彼を効率的に誘導することができます。ヒト多能性幹細胞からのHE及び血液の形成を効率的にわずか数の転写因子（TFS）を過剰発現させることによって誘導することができます。 hPSCsから堅牢な汎骨髄造血を誘導することができるTFの最適な組み合わせは、ETV2およびGATA1またはGATA2が含まれています。対照的に、GATA2およびTAL1の組み合わせがerythromegakaryocytopoiesis 10を誘導します 。 CD73 - -徐々に初期造血マーカーCD43 7の式で定義された造血表現型を獲得HE細胞これらの因子の過剰発現を通じてhPSCsのプログラミングは、インクルードは+ CD43-CAD VEに直接hPSCsを区別します。ヒト多能性幹細胞法の直接のプログラミングのためのこのレンチウイルスベースの方法は、機構研究、造血への遷移内皮の研究、および造血開発と仕様の転写調節のために、彼と血液細胞の生成に適用されます。 Cが、urrentプロトコルは、導入遺伝子の構成的発現を使用して、血液の生産を記載し、同様の結果が変更されたmRNAの10を使用して得ることができました。

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 1.&lt;/strong&gt; &lt;strong&gt;Induction of hPSCs differentiation with trancription factors and analysis of hemogenic endothelium and blood cells.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>pSIN4-EF1a-ETV2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61061</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-GATA2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61063</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-GATA1-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61062</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-TAL1-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61062</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-LMO2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61064</td><td>Lentiviral Vector</td></tr><tr><td>Hexadimethrine bromide (Polybrene)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>107689-10G</td><td>Cationic polymer used to increase the efficiency of infection</td></tr><tr><td>Y-27632 (Dihydrochloride) ROCK inhibitor</td><td>STEMCELL Technologies</td><td>72302</td><td>RHO/ROCK pathway inhibitor Inhibits ROCK</td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>A11105-01</td><td>Cell Dissociation Reagent</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Incomplete (growth factor- free) culture medium&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; mTeSR1 Custom formulation&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>MCF</td><td>&lt;a name=&quot;RANGE!D12&quot;&gt;Serum-free mTeSR1 medium without bFGF and TGFb&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>human SCF</td><td>Peprotech</td><td>300-07</td><td>Premium grade</td></tr><tr><td>human TPO</td><td>Peprotech</td><td>300-18</td><td>Research grade</td></tr><tr><td>human FGF-basic</td><td>Peprotech</td><td>100-18B</td><td>Premium grade</td></tr><tr><td>CD144 (VE-cad) FITC</td><td>BD Biosciences</td><td>560411</td><td>Endothelial marker (FACS)</td></tr><tr><td>CD226 PE</td><td>BD Biosciences</td><td>338305</td><td>Hematopoietic (FACS)</td></tr><tr><td>CD43 PE</td><td>BD Biosciences</td><td>560199</td><td>Hematopoietic (FACS)</td></tr><tr><td>CD73 APC</td><td>R&amp;amp;D Systems</td><td>FAB5795A</td><td>Endothelial marker (FACS)</td></tr><tr><td>CD45 APC</td><td>BD Biosciences</td><td>555485</td><td>Hematopoietic (FACS)</td></tr><tr><td>7AAD</td><td>Life Technologies</td><td>A1310</td><td>Live/Dead assay (FACS)</td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>Cell fixation&amp;nbsp;</td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T9284-500ML&amp;nbsp;</td><td>Permeabilization&amp;nbsp;</td></tr><tr><td>FBS</td><td>Fisher Scientific</td><td>SH3007003</td><td>Fetal bovine serum</td></tr><tr><td>Mouse anti-human CD43</td><td>BD Biosciences</td><td>551457</td><td>Pure, primary antibody for Immunofluorescence (IF) staining</td></tr><tr><td>Rabbit anti-human VE-cadherin</td><td>BenderMedSystem</td><td>BMS158</td><td>Primary (IF)</td></tr><tr><td>Anti-rabbit Alexa Fluor 488-conjugated</td><td>JacksonResearch</td><td>715-486-152</td><td>Secondary (IF)</td></tr><tr><td>Anti-mouse Alexa Fluor 594-conjugated</td><td>JacksonResearch</td><td>715-516-150&amp;nbsp;</td><td>Secondary (IF)</td></tr><tr><td>DAPI nucleic acid stain</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>D1306&amp;nbsp;</td><td>Live/Dead assay (IF)</td></tr><tr><td>Clonogenic medium MethoCult H4435 Enriched</td><td>STEMCELL Technologies</td><td>4435</td><td>CFC-assay</td></tr><tr><td>Wright Stain solution</td><td>Sigma -Aldrich</td><td>32857</td><td>Staining cytospins</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 2.&lt;/strong&gt; &lt;strong&gt;hPSCs culture.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Human Pluripotent Stem Cells (hPSCs)&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>hESCs (WA01, WA09) human Embryonic Stem cells; iPSCs (DF-19-9-7T, DF-4-3-7T) transgene-free induced Pluripotent Stem Cells</td><td>hPSCs are able to self-renew and to differentiate into cells of three germ layers</td></tr><tr><td>Complete serum-free medium for culture of hPSCs&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; mTeSR1 media&amp;nbsp;&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>M500&amp;nbsp;</td><td>Serum-free&amp;nbsp; medium with growth factors for feeder free culture of ESC/iPSCs</td></tr><tr><td>Matrigel</td><td>BD Biosciences/ Corning&amp;nbsp;</td><td>356234</td><td>Matrix for maintenance of human ESC/iPSCs</td></tr><tr><td>DMEM/F-12, powder&amp;nbsp;</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>12500-062</td><td>Basal Medium&amp;nbsp;</td></tr><tr><td>HyClone Dulbecco&#039;s PBS powder</td><td>Fisher Scientific</td><td>dSH30013.04</td><td>PBS</td></tr><tr><td>Dispase II, powder</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>17105-041</td><td>Neutral protease, Cell dissociation</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 3.&lt;/strong&gt; &lt;strong&gt;Primers for detection of virus genomic integration.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primer&#039;s Name&lt;/strong&gt;</td><td>&lt;strong&gt;Forward (Fwd)&amp;nbsp; 5&amp;rsquo; --&gt;3&amp;rsquo;&lt;/strong&gt;</td><td>&lt;strong&gt;Reverse (Rev) 5&amp;rsquo;--&gt;3&amp;rsquo;&lt;/strong&gt;</td><td>&lt;strong&gt;Discription&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>pSIN EF1a Fwd</td><td>TTC CAT TTC AGG TGT CGT GA</td><td>--</td><td>EF1a promoter sequence</td></tr><tr><td>GATA1 Rev</td><td>--</td><td>TCC CTG TAG TAG GCC AGT GC</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>GATA2 Rev</td><td>--</td><td>GGT TGG CAT AGT AGG GGT TG</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>TAL1 Rev</td><td>--</td><td>AGG CGG AGG ATC TCA TTC TT</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>LMO2 Rev</td><td>--</td><td>GGC CCA GTT TGT AGT AGA GGC</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>ETV2 Rev</td><td>--</td><td>GAA CTT CTG GGT GCA GTA AC&amp;nbsp;</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr></tbody></table>

direct induction of hemogenic endothelium and blood by overexpression of transcription factors in human pluripotent stem cells

開発中に、造血細胞は、内皮細胞、造血内皮（HE）の専門サブセットから生じます。 in vitroでのモデルHE開発は、内皮、造血転移の機構研究および造血仕様のために不可欠です。ここでは、転写因子の異なるセットの過剰発現を介して、ヒト多能性幹細胞（hPSCs）から彼を効率的に誘導するための方法を説明します。 GATA2およびTAL1転写因子の組み合わせは赤血球と巨核球電位とHEの製造を可能にしながらETV2 GATA1およびGATA2またはTFの組み合わせが、汎骨髄電位HEを誘導するために使用されます。 GATA2およびTAL1の組み合わせにLMO2の添加は、実質的に分化を促進し、赤血球および巨核球細胞の生産を増加させます。この方法では、hPSCsからHE誘導の効率的かつ迅速な手段を提供し、内皮hematopoietiの観察を可能にします培養皿のC推移。プロトコルはhPSCsの導入手順およびHEの形質導入後の分析や血液前駆細胞を含んでいます。

転写因子の過剰発現によってhPSCsからHE及び血液誘導の概略図は、GATA1またはGATA2組合せで、図1 ETV2に示されている、TAL1 +/- LMO2の組み合わせは主にエリスロ-巨核球造血を誘導GATA2ながら、汎骨髄造血を誘導します。どちらのTFの組み合わせは直接、その後造血分化の明確なスペクトルと血液前駆細胞に変換HE細胞を誘導しました。血液への多能性状態からhPSCsの分化は、彼は平均4日?...

Watch this Scientific Journal Video about Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells at JoVE.com

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

ヒト多能性幹細胞における転写因子の過剰発現によって、造血内皮と血液の直接誘導

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

direct-induction-hemogenic-endothelium-blood-overexpression

Research

JoVE Journal

Developmental Biology

7.5K ビュー.  University of Wisconsin-Madison. This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors. 

ビデオ: Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

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

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

転写因子との再プログラミングマウス胚線維芽細胞造血プログラムを誘導するために

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

センダイウイルスや遠心分離を用いて血液細胞から誘導多能性幹細胞の生成

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

エピソームベクトルを用いたヒト末梢血単核細胞からの統合フリー人工多能性幹細胞の生成

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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 ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

フィーダーとゼノフリーの定義条件におけるヒト多能性幹細胞からの血原性内皮分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

転写因子の強制発現によるヒト線維芽細胞のヘモジェニックリプログラミング

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

ヒト多能性幹細胞からの血原性内皮細胞の指向性分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

STEMCCAレンチウイルスベクターを用いた末梢血からのヒト人工多能性幹細胞の生成

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

生物学

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

多能性幹細胞からのヒト血管オルガノイドの作製

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...