Name: selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts
Uploaded: 2012-02-20T14:00:00
Description: Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

この作品は、フリードライヒ失調研究アライアンスとアーノルドファミリー財団とMDアンダーソンがんセンターの幹細胞と再生科学総合研究センターからのパイロット助成金によって支えられている。

1。ウイルスの生産と伝達<ol><li> DMEM培地（DMEM高グルコース、不活化10％FBS、熱、2 mM L-グルタミン、抗生物質なし）10ml中〜7-8×10 6％10 cmのプレートの密度でプレートフェニックス周りに細胞。 37一晩インキュベーターや文化の場所°C、5％CO 2。 </li><li>ベクトル符号化OCT3 / 4、Sox2の、KLF4、c-mycの、またはGFP遺伝子（プラスミドAddgene 17217、17218、17219、17220）、35μlのFuGENE 6トラン?...

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特に神経系と神経ヒトの疾患、勉強、特に十分なヒト細胞モデルの到達不能に起因する困難されています。多様な種類の細胞にそれらを区別するために誘導多能性幹細胞および潜在的に容易に得られる体細胞を再プログラムする能力は、遺伝性疾患の細胞モデルを作成する可能性を開いた。さらに、性IPSCは、再生医療の将来非常に有望なものである。したがって、多能性に体細胞を再プログ...

<table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> 11965 </td></tr><tr><td> DMEM/F12 </td><td>インビトロジェン</td><td> 11330 </td></tr><tr><td> KSR </td><td>インビトロジェン</td><td> 10828 </td></tr><tr><td>非必須アミノ酸</td><td>インビトロジェン</td><td> 11140 </td></tr><tr><td>酪酸ナトリウム</td><td>シグマ</td><td> B5887 </td></tr><tr><td> Y27632 </td><td> Stemgent </td><td> 04から0012 </td></tr><tr><td> bFGFの</td><td> Stemgent </td><td> 03から0002 </td></tr><tr><td> TRA-1-81抗体</td><td> Stemgent </td><td> 09から0069 </td></tr><tr><td> Oct3 / 4の抗体</td><td>サンタクルス</td><td> SC-8628 </td></tr><tr><td> NANOG抗体</td><td>に：Cell Signaling Technology </td><td> 4903S &lt;/ TD&gt; </tr><tr><td> TRA-1-60抗体</td><td>ミリポア</td><td> MAB4360 </td></tr><tr><td> Sox2の抗体</td><td>に：Cell Signaling Technology </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td>ミリポア</td><td> MAB4304 </td></tr><tr><td> CF1のMEF </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td>客観的マーカー</td><td>ニコン</td><td> MBW10010 </td></tr><tr><td>マトリゲル</td><td> BDバイオサイエンス</td><td> 354277 </td></tr><tr><td> mTeSR1 </td><td> StemCellテクノロジーズ</td><td> 05850 </td></tr><tr><td> β-メルカプトエタノール</td><td>シグマ</td><td> M7522 </td></tr><tr><td> FuGENE 6トランスフェ</td><td>ロシュ社</td><td> 11814443001 </td></tr><tr><td>ポリブレン</td><td>シグマ</td><td> H9268 </td></tr><tr><td>オブジェクトマーカー</td><td>ニコン</td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

ここに我々は1-3ナトリウム、酪酸の存在下でOCT3 / 4、Sox2の、KLF4およびc-myc（OSKM）をコードするレトロウイルスベクターを用いたヒト人工多能性幹細胞（hiPSC）にヒト成人線維芽細胞を再プログラミングのプロトコルを提示します。我々は、フリードライヒ失調症患者（GM03665、コーリエル医学リポジトリ）から派生した遅く通路（&gt; P10）ヒト成人線維芽細胞を再プログラムするためにこのメソッドを使用していました。プログラミングのアプローチは、ウイルス含有培地の存在下で線維芽細胞の反復的な遠心分離を用いた高効率伝達プロトコルが含まれています。書き換えhiPSCコロニーがTRA-1-81、多能性細胞の表面マーカーのライブ免疫染色を用いて同定した、非書き換え線維芽細胞から分離し、手動で4,5継代。これらのhiPSCその後、マトリゲルプレートに移し、直接プログラミングプレートから、フィーダーフリーの条件下で増殖させた。最初の通過から始めて、hiPSCのコロニーは、hES-L特性を実証IKE形態。このプロトコルを使用して、選択したコロニーの70％以上が正常に拡大し、細胞株に確立することができます。確立されhiPSC行は表面マーカーTRA-1-60及びSSEA-4と同様に、核のマーカーOCT3 / 4、Sox2、及びNanogの特徴を含む多能性マーカーを表示されます。ここで紹介するプロトコルが確立され、フリードライヒ失調症患者と対照個体6、人間の新生児線維芽細胞と同様に、ヒトケラチノサイトから得られた成人の繊維芽細胞を用いてテストされています。

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

我々はとレトロOCT3 / 4、Sox2の、KLF4およびc-myc（OSKM）をコードするベクターとライブ染色により正しく再プログラムhiPSCの識別を用いたヒト人工多能性幹細胞（hiPSC）TRA-にヒト体細胞の効率的なリプログラミングのためのプロトコルを提示1から81抗体。

大人の線維芽細胞から再プログラムヒト人工多能性幹細胞のコロニーを選択し、分離

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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Biology

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

StemgentヒトTFのレンチセットでヒト線維芽細胞を再プログラムすることによって誘導多能性幹細胞の生成

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Isolating Stem Cells from Soft Musculoskeletal Tissues

ソフト筋骨格組織から幹細胞を単離する

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

ヒト人工多能性幹細胞をトランスフェクトしNucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

培養ヒト胚および誘導多能性幹細胞に適したマウス胚性線維芽細胞の調製

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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

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, ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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

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 ...