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1。マトリゲル被覆プレートとメディアの準備<ol><li>マトリゲルは、EBSの差別化の取り付けのための好ましい基質であることが判明し、また、これらの10からニューロンの効率的な伸長を促進されたマトリゲル被覆プレートの作製。 <ol><li>氷の上で一晩マトリゲルの雪解け10ミリリットル。 </li><li>翌日、氷のように冷たいDMEM/F-12の2ボリュームとゼリー状のマトリゲルを希釈し、-20℃で凍結保存することができる予冷した15 mlコニカルチューブに1mlのアリコートを準備</li><li>神経分化にプレートフォーマットを決めて。例えば、出発点として、私たちは12ウェルフォーマットにおける分化の最初のラウンドを実行することをお勧めします。 4℃でプレクール4 12ウェルプレートすべて予め希釈マトリゲルは解凍し、溶解するまで上下にピペッティングし、続いて予冷DMEM/F-12 9mlを添加することにより凍結マトリゲルの1アリコートを溶かす。 </li><li>その後、氷（最終マトリゲル希釈：1:75）にDMEM/F-12の15ミリリットルを含む新しい50 mlチューブに内容を転送します。次に、予め冷却し、12ウェルプレートの各ウェルに希釈したマトリゲルの0.5 mlを加える。パラフィルムでプレートを包み、室温で一晩放置する。 </li><li>翌日、4℃にプレートを転送コー​​トされたプレートは、使用前に数週間のために保存することができます。 </li></ol></li><li>メディア</li></ol> EB形成培地（1 96ウェルプレートでの差別化を行うのに必要な〜15ミリリットル- 図2の表1及びスキームを参照してください ）： 0.4％（PVA）とDMEM/F-12 1X N2 1X B27 0.05％BSA 20 ng / mlでのFGF2 10μMのY-27632 1X PenStrepGln 分化培地（1 96ウェルプレートでの差別化を行うのに必要な〜30ミリリットルは、1マトリゲル被覆した12ウェルプレートにおけるニューロン形成に続いて、 表1を参照してください ）： DMEM/F-12 1X N2 <br/&gt; 1X B27 0.05％BSA 0.5μMのPD0325901 15μMのSB431542 0.5μMのDorsomorphin 1X PenStrepGln 2。強制EB形成<ol><li>お好みのフィーダを含まない条件下でhPSCsを栽培しています。我々は、マトリゲル被覆された6ウェルプレート15日 、MEF馴化培地を使用しています。しかし、このような自家製または市販の定義されたメディアなど、他の培養系でも動作するはずです。分化実験開始時に、hPSCsはサブコンフルエントと活発に成長しなければなりません。 </li><li>機械的にステレオ顕微鏡下で滅菌したプラスチック製のピペットチップを用いて自発的に分化したコロニーを削除します。 </li><li> 1X、Y-27632を含むAccutaseを用いて単一細胞にPBS、およびダイジェストを使用して残りの未分化コロニーを洗う。 2分間、200×gで遠心分離によってペレット単一細胞、EB形成の少量の培地で再懸濁し、血球計数器を用いて細胞力価を決定します。 </li><li>に対する細胞のアリコートを追加ミリリットル当たり40,000〜80,000細胞間の力価になり、残りのEB形成媒体。 </li><li>マルチチャンネルピペットを使用して、1ウェルあたり4,000〜8,000個で、その結果、96V底プレートにウェル当たり100μlを移す。 1分間400 xgでスイングアウトプレート遠心機でスピン96ウェルプレートはウェルの底部での細胞を収集し、細胞培養インキュベーターに転送する。 図1に示すように、EBは、一晩形成することになる。 </li></ol> 3。神経外胚葉誘導<ol><li>次の日（0日）、分化培地2mlを含む3.5センチメートル細菌皿に小さな音量でステレオ顕微鏡と転送で96VプレートからEBを収集します。優しくEBを洗うには、数秒のために皿を攪拌。 </li><li> 100分化培地のウェルあたりの超低付着U底96ウェルプレートを追加します。実体顕微鏡下では、96U-ウェルに洗浄皿から小さなボリュームで転送EBS（我あたり1 EBLL）。神経外胚葉の形成を可能にするために4日間細胞培養インキュベーターに96Uプレートを置きます。 </li></ol> 4。ニューロン形成<ol><li> 4日目に、ステップ3.1のように洗浄皿を準備し、さらに分化培地（ウェル当たり1ミリリットル）で予め温めマトリゲル被覆した12ウェルプレートにDMEM/F-12を交換してください。 </li><li> EBのメッ​​キ<ol><li> 96UプレートからEBを収集し、マトリゲル被覆した12ウェルプレート（ウェル当たり約8 EBS）のウェルに、これら上記のように、そして慎重にシードそれらを一つずつ洗っ：EBSは均等の平穏な成長を可能にするためにウェル内に分配されるべきである次回日間のニューロン（ 図2の&quot;5日目&quot;画像を参照してください）。 </li><li>バックインキュベーターに12ウェルプレートの譲渡の前に、EBを緩く室温で約10分間アタッチすることができます。その後、細胞培養インキュベーターに12ウェルプレートを慎重に移す。ニューロンは、笙のように、次の4日以内に放射状にめっきしたEBから外に成長するだろう図2のWN（ &quot;8日目&quot;右の画像）。 </li></ol></li></ol> 5。単層としてニューロン形成<ol><li>点4）の手順の代わりに、手順の4日目に、分化培地2mlを含む3.5センチメートル細菌皿にEBを集め、次いでPBS含有皿に少量のEBを転送して、にAccutaseを含む別の皿。 </li><li>分化培地の小さなボリュームに再懸濁し、単一細胞、2分間、200×gで遠心分離に消化し、血球計数器を用いて細胞力価を決定しましょう​​。分化培地を用いml当たり1-2X10 6細胞（Y-27632は除く）に力価を調整します。 </li><li>予め温めておいたマトリゲル被覆した12ウェルプレート（ウェル当たり1ミリリットル）、および細胞培養インキュベーターへの転送では、プレートの細胞外。単層としてニューロンの形成は日7から8（ 図3C）間で観察された。それは、しかし、注意すべきは、この単層バリアント手順は完全に細胞の生存および最も適した基板に関して最適化されていません。 </li></ol>

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私たちの以前に公表されている手順10を含むhPSCs、、からのEB形成を伴う最も分化プロトコルは、手動または必然的にEBのサイズで異質につながる集約などhESCの酵素アシスト収穫に基づいています。この事実は、それによって培地スループットスケールで特に困難な体系的な分析を、レンダリング、いくつかの細胞株を分化効率の変動につながる。さらに、マニュアル解剖自体はスケ?...

胚由来のヒト胚性幹細胞（hESC）や人工多能性幹細胞（hiPSCs）を含むhPSCsは、それらがin vitroで1-2の様々な細胞系統を生じさせることができる多能意味です。多数の分化した細胞の種類は、これらの細胞は、科学的研究と細胞ベースのスクリーニングのアプローチのための貴重なツールを紹介し、細胞ベースの治療のための約束を保持するという考えを支持し、現在までにヒトES細胞に由来している。 ヒトES細胞の神経細胞分化プロトコルは、彼らはしばしば神経前駆細胞の分離マニュアル、および利息3-6の与えられたニューロンタイプへのその後の最終分化に続いて、第1誘導全体の神経運命に基づいているため、通常、複雑で時間のかかるものです。いくつかの点では、この複雑さは、このような最先端の指示分化プロトコルを段階的に早い人間開発、whicを模倣する傾向があることを考えれば驚くべき、不可避ではないhはそれ自体が非常に複雑である。その一方で、それは部分的にもはるかに完全に最適化されてからまだ分化プロトコルで、その結果、根本的な分子過程の理解の我々の欠如を反映しているかもしれない。 神経外胚葉への分化hPSCsの変換につきましては、ペラら BMP / SMAD1/5/8シグナル伝達の抑制はhESCの7の神経誘導を増強することを発見した。また、SMAD2 / 3シグナル伝達の不活性化は神経外胚葉の形成8を促進することが示されている。したがって、これら2つの経路の不活性化が組み合わさhPSCs 4,9のより効率的な神経誘導につながる傾向。さらに最近では、我々は第三シグナル伝達経路は、FGF / ERKは、ヒトES細胞における神経外胚葉コミットメントの最も早い段階の強力なリプレッサーとして機能することが示されている。逆に、これらのすべての3つの経路の同時抑圧はと神経外胚葉にhESCの近傍同種の変換につながるわずか4日10インチ続いて、機能的な神経細胞への高い効率的な終末分化は8日以内に観察された。得られたニューロンは、部分的に10彼らの急速な導出を説明するかもしれない末梢神経系の感覚神経細胞である可能性が高かった。感覚ニューロンのメンテナンスの欠陥は、家族性自律神経失調症11などの特定のヒトの疾患の原因を考えられている。ニューロンの特定のタイプを必要としない適用の目的のためだけでなく、さらなる機能解析のために、hiPSC技術12に基づいて、そのような疾患をモデル化するため、この分化の手順では、有用な基礎を提示することができる。 しかし、差別化戦略は、我々は当初のhESCコロニー10の塊から胚体の関与形成（EBS）を採用し、これはEBのサイズに関する異質性のかなり度をもたらし、また、いくつかのCEのニューロン形成効率を損なうように見えたLLライン。また、EBのサイズの異質性に起因して、体系的にニューロン形成中および後に追加の成長因子または小分子の効果をテストする能力がやや混乱しているように見えた。 また、他のコンテキスト13に示すように、本 ​​報告では、我々は非常にサイズ制御された方法でEBを生成するための培地スループット互換、強制集約ベースの技術に我々の前の手順を適応した。続いて、96ウェルプレートのV字型のウェル中に生成されたEBを懸濁液中の神経外胚葉の形成を開始するために、U字型、超低付着の96ウェルプレートに移した。 4日後、EBは、高効率と均一性で分化した神経細胞を生じさせるプレーティングすることができます。あるいは、日-4はEBは、単一細胞に解離し、そのように播種し、人間の神経細胞の低密度の単層で生じた。首尾一貫した結果は、これまで独立して2で得られたデrivedのhESCライン、HuES6とNCL3。 前提条件として、成功したEB形成は一緒hESCの生存13-14を促進することが知られているROCK阻害剤Y-27632で、合成ポリマー、ポリビニルアルコール（PVA）の使用に厳密に依存していた。その結果、96-Vプレートで形成されたEBの均質性は実質的にランダムな分割手法に基づいて大量培養としてEBの形成に比べて強化されました。このシステムは、このように付着した条件の下で高度に制御されたニューロンの形成に続いて懸濁培養における神経外胚葉形成、有意に改善されたプラットフォームを提供します。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>マトリゲルHC </td><td>ベクトン·ディッキンソン</td><td> 354263 </td><td>取り扱いに関する詳細については本文を参照</td></tr><tr><td> DMEM/F-12 </td><td>ライフテクノロジーズ</td><td> 21331-020 </td><td></td></tr><tr><td>ポリ（ビニルアルコール） </td><td>シグマ</td><td> 363170 </td><td>超音波ウォーターバスを使用してDMEM/F-12で0.4％で溶解/過熱を避ける/ 4℃で数週間保存することができる</td></tr><tr><td> N2サプリメント</td><td>ライフテクノロジーズ</td><td> 17502-048 </td><td> 100X、凍結アリコートとして保存</td></tr><tr><td> B27サプリメント</td><td>ライフテクノロジーズ</td><td> 12587-010 </td><td> 50X、凍結アリコートとして店舗</td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> A1595 </td><td>凍結アリコートとして10％= 500X、店舗</td></tr><tr><td>ペニシリン/ストレプトマイシンを含むL-グルタミン</td><td> PAA </td><td> P11-013 </td><td>または同等の</td></tr><tr><td> FGF2は</td><td> Peprotech社</td><td> 100-18B </td><td>凍結アリコートとして0.1％BSA / PBS = 1000X、ストア内の10μg/ mlで溶解</td></tr><tr><td> ROCK阻害剤Y-27632 </td><td> abcamBiochemicals </td><td> ASC-129 </td><td>凍結アリコートとしてDMSO = 1000X、店中の10mMで溶解</td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td>または同等の</td></tr><tr><td> Accutase </td><td> PAA </td><td> L11-007 </td><td></td></tr><tr><td> 96ウェルV底プレート</td><td>ヌンク</td><td> 277143 </td><td></td></tr><tr><td> PD0325901 </td><td>軸索Medchem </td><td>アクソン1408 </td><td>凍結アリコートとしてDMSO = 1000X、店舗が0.5mmで溶解</td></tr><tr><td> SB431542 </td><td> abcamBiochemicals </td><td> ASC-163 </td><td>凍結アリコートとしてDMSO = 1000X、ストア内の15mMで溶解</td></tr><tr><td> Dorsomorphin </td><td>サンタクルス</td><td> SC-200689 </td><td>凍結アリコートとしてDMSO = 1000X、店舗が0.5mmで溶解</td></tr><tr><td> 96ウェルU底超低アタッチメントプレート</td><td>コー​​ニング</td><td> 7007 </td><td></td></tr><tr><td> β-IIIチューブリン抗体（マウス） </td><td>シグマ</td><td> T8660 </td><td> 1:1000で使用</td></tr><tr><td> β-IIIチューブリン抗体（ウサギ） </td><td> Covance </td><td> PRB-435P </td><td> 1:2000で使用</td></tr><tr><td> BRN3A（POU4F1）抗体</td><td>サンタクルス</td><td> SC-8429 </td><td> 1:500で使用</td></tr><tr><td></td><td></td><td></td><td> 表1 &lt;/ strong&gt;の具体的な試薬や機器。 </td></tr></tbody></table>

rapid and efficient generation of neurons from human pluripotent stem cells in a multititre plate format

ヒト多能性幹細胞（hPSCs）からニューロンを生成するための既存のプロトコルはしばしば彼らが前終末分化に神経前駆細胞の単離および拡張を伴う多段階の手続であるという点で、手間がかかります。これらの時間のかかるアプローチと比較して、我々は最近、3シグナル伝達経路、TGFβ/SMAD2、BMP/SMAD1、およびFGF / ERK、を合わせた抑制が機能して神経細胞への分化即時続いhPSCs、神経外胚葉からの迅速な誘導を促進することを見出した。ここでは、さらに、その再現性を向上させ、半ばスループットのアプリケーションと互換性を持たせるために、小説multititreプレートフォーマットに我々の手順を適応している。それが付着した条件下でのニューロンへの分化の一層の4日間続いフローティング球における外胚葉形成の4日後（胚様体）を含む。このプロトコルを用いて得られたほとんどの細胞は双極感覚ニューロンであるように見えます。また、手順は非常に効率的であり、特定の専門的なスキルを必要とせず、コスト効率の高い小分子との単純な化学的に定義されたメディアに基づいています。これらの特長により、プロシージャは、さらなる機能調査のための有用なプラットフォームとしてだけでなく、どのようなタイプの人間の感覚神経細胞や神経細胞を必要とする細胞ベースのスクリーニングアプローチのための役割を果たすことができる。

我々の以前の報告書では、多くの他の人と並んで、hPSCsからEBを単に低接着皿10へhPSCsのルーチンを分割する際に凝集体を再播種することによって生成することができます。その結果、EBのサイズ（ 図1C、上側）の広範な分布で、これは通常の結果。このことから生じたもう一つの問題は、懸濁液中に維持されたEBは、EBのサイズのさらに高い異質性につながる、互いに凝集?...

Watch this Scientific Journal Video about Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format at JoVE.com

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

多能性ヒト幹細胞の神経分化（hPSCs）のためのプロトコルは、多くの場合、時間がかかり、実質的な細胞培養のスキルを必要とします。ここでは、制御された方法で人間の神経細胞の、簡便、迅速、かつ効率的に生成できるように、multititreプレートフォーマットへの小分子ベースの分化手順を適応している。

Multititreプレートフォーマットでヒト多能性幹細胞から神経細胞の迅速かつ効率的な生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

rapid-efficient-generation-neurons-from-human-pluripotent-stem-cells

Research

JoVE Journal

Biology

43.5K ビュー.  Max Planck Institute for Molecular Biomedicine. 多能性ヒト幹細胞の神経分化（hPSCs）のためのプロトコルは、多くの場合、時間がかかり、実質的な細胞培養のスキルを必要とします。ここでは、制御された方法で人間の神経細胞の、簡便、迅速、かつ効率的に生成できるように、multititreプレートフォーマットへの小分子ベースの分化手順を適応している。

ビデオ: Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

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 ...

生物学

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 ...

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

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

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

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

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

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, ...

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 ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

高解像度メルト解析を用いたPCRを使用して、迅速かつ効率的なゼブラフィッシュ遺伝子型決定

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

の効率的な胞子形成<em&gt;サッカロマイセス・セレビシエ</em&gt; 96マルチウェルフォーマットで

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

組織学的分析のためのヒトの眼からの網膜色素上皮/脈絡膜フラットマウントの効率的かつ一貫した生成

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...