We thank Mrs. Matsumoto for the technical assistance.

1.細胞培養<ol><li>四倍を誘導するために細胞を得ます。現在までに、この技術は、ヒト線維芽細胞株TIG-1、BJ、IMR-90およびテロメラーゼ不死化TIG-1（TIG-HT）に適用できることが確認されました。 </li><li> α改変または/ 5％（Vでインキュベートすることにより、10％（v / v）の熱不活性化ウシ胎児血清（FBS）を補充した研究する細胞型に適した任意の他の細胞培養培地で最小必須培地中で細胞を増殖させます37℃でのv）のCO 2雰囲気。継代細胞ごとに3〜4日には、サブコンフルエント密度を超えません。 注：集団倍加レベル（PDL）細胞増殖と細胞齢を評価するために、継代時に各時間を計算しなければなりません。 PDLは、細胞数から計算することができ、以下の式（X初期PDLである）を用いてディッシュ（N1）と次の継代（N2）で採取された細胞数に播種。 PDL = X +（N1を記録 - N2をログ）/ログ2 </li></ol> 2.誘導とエスタ二倍体線維芽細胞から四倍細胞のblishment <ol><li>振り切りなしで四倍誘導します。 注：次のようにいくつかの細胞株（ 例えば TIG-1）がデメコルチン（DC）で連続的に処理することにより、ほぼ完全に四倍になることができます。 <ol><li>治療前の日に1または2 100mm皿に継代細胞は、四倍誘導しました。通常、四倍体細胞の誘導のために皿当たり5×10 5×10 6〜1細胞を調製。 </li><li> 4日間は0.1μg/ mLのDCを含む培地で指数関数的に増殖する細胞を扱います。 </li><li>薬物を含まない培地でのDCを含む培地を交換し、37℃で5％（v / v）のCO 2雰囲気中で細胞をインキュベートします。細胞は通常、1週間以内に増殖を再開します。 </li></ol></li><li>振り切りで四倍誘導します。 注：ほとんどの細胞はDCとの単純な処理の結果として二倍体と四倍体集団の混合物は、上記（2.1）説明になります。 Pのため、いくつかの変更（ 図1）を次のように二倍体の集団を除去するrocedureが必要です。 <ol><li>いくつかのT75への継代細胞は、四倍を誘導するために、治療前の日をフラスコ。通常、四倍体細胞の誘導のために少なくとも5×10 6細胞（フラスコ当たり1×10 6細胞）を準備します。 </li><li>有糸分裂中の細胞を逮捕するために18時間 - 16のためには0.1μg/ mLのDCを含む培地で指数関数的に増殖する細胞を扱います。有糸分裂中の細胞を阻止するのに必要な時間は、標的細胞に依存してもよいし、可能な限り多くの有糸分裂細胞を得るために、予備実験によって決定されるべきです。例えば、遅い成長している細胞が速く増殖する細胞よりも長い時間DCで治療すべきです。 注：細胞が長すぎる、この段階での治療を受けている場合、彼らは分裂滑りを受ける可能性があり、それができなく次のステップで振り切りにより有糸分裂細胞を収集すること、皿表面に付着します。 </li><li> WHで、振り切り法によりDC-逮捕有糸分裂細胞を収集ICH緩く付着した有糸分裂細胞を遠心分離し、フラスコと培地での洗浄の穏やかに振盪（300×gで、5分間）によって収集されます。この段階での適切な方法により、細胞数をカウントします。 </li><li> 60mm培養皿に収集した有糸分裂細胞を再シードし、さらに3日間は0.1μg/ mLのDCで細胞を扱います。細胞の10％未満が、この処理に耐えることができるので、皿当たり以上、1×10 6個の有糸分裂細胞を播種。 </li><li>薬物を含まない培地でのDCを含む培地を交換し、細胞は通常、1週間以内に発生した増殖を再開するのを待ちます。 </li></ol></li><li>同様に、元の二倍体細胞に継代細胞。 </li></ol> DNA倍数性の3.検討注：これは、十分に確立された手順で、技術的なマニュアルは、詳細な手順と技術的なヒントについては、を参照されたいです。 <ol><li>ソリューション詐欺で細胞をインキュベートすることにより-収穫細胞（1×10 6、5×10 5）37℃で10分間、0.05％トリプシンおよび0.02％EDTAをtaining、10％FBSを含む培地によりトリプシンを中和します。 </li><li>培地中の遠心細胞及び遠心分離（300×gで、5分間）、続いてリン酸緩衝生理食塩水（PBS）5mLに再懸濁することにより、それらを洗浄します。 </li><li>次の2つの方法のいずれかによってDNA分析のための細胞を調製します<ol><li>未固定細胞10を分析するための方法 注：この方法は、細胞周期分析のために市販のキットを用いて行うことができます。細胞が手順で固定されていないと染色された核は時間をかけて破損しますので、この方法で調製した試料は、直ちに分析する必要があります。 <ol><li> 250mMのスクロースを含む40 mMのクエン酸緩衝液で細胞を洗浄。その後室温で10分間、250mMのスクロースを含有する200μLの40 mMのクエン酸緩衝液中の0.1％（v / v）のノニデットP40の30 / mlのトリプシンの250μLで細胞を治療します。 </li><li> 0.5μgの/ mLのトリプシンINHの200μLを追加ibitor及び250mMのスクロースを含む40mMのクエン酸緩衝液中の0.1 / mlのRNアーゼA、室温で10分間細胞をインキュベートします。 </li><li> 250mMのスクロースを含む40mMのクエン酸緩衝液中の0.4mg / mLのヨウ化プロピジウム（PI）の200μLを加え、細胞核を染色し、室温で10分間細胞をインキュベートします。 </li></ol></li><li>固定した細胞を分析するための方法<ol><li>細胞懸濁液を混合し、室温で30分間そのままの100％EtOH滴下を4mLを加えて1mlのPBSに細胞を懸濁し、80％エタノールで細胞を固定。この固定液中の細胞は数週間-20℃で保存することができます。 </li><li> PBS 5mLにそれらを再懸濁することにより洗浄細胞を遠心分離（300×gで、5分間）しました。 0.5ミリグラム/ mLのRNase Aで細胞を処理し、室温で30分間、50μg/ mlのヨウ化プロピジウム（PI）（0.5ミリグラム/ mlのRNase Aおよび1mg / mLのPIの25μLの475μL）でそれらを染色します。 </li></ol></li></ol></li><li>トンのDNA含有量を分析彼488 nmのレーザー、赤チャンネル発光フィルタ（ロングパス&gt; 610 nm）を用いてフローサイトメーターを用いて細胞。標的細胞の倍数性を評価するために、同時に本物の二倍体細胞から調製された基準試料を分析します。 <ol><li>代替的に、二倍体細胞対ビーズの蛍光強度比を評価するために、蛍光標準ビーズを加えます。二倍体細胞11の単四倍体細胞と凝集塊とを区別するために、フローサイトメーターのオプション&#39;パルス幅対パルス高さ&#39;を使用します。 </li></ol></li></ol>染色体の数および核型分析4.審査注：これらはすべてが順調に確立された手法であり、技術マニュアルか、製造元のプロトコルが詳細な手順と技術的なヒントについては、を参照されたいです。 <ol><li>中期で細胞を停止するために4時間を0.1μg/ mLのDCで、指数関数的に増殖する細胞を扱います。 </li><li>染色体スライドを準備します。 <ol><li&gt;をトリプシン処理により細胞を回収し（少なくとも5×10 5細胞）およびFBSを含む培地中で中断。 </li><li>培地中の遠心細胞（300×gで、5分間）し、10分間37℃で低張液（0.075 MのKCl 5 mL）で治療します。 </li><li>修正し、カルノア固定液で細胞を懸濁（メタノール：酢酸= 3：1）。 </li><li>細胞懸濁液の - （35μL25）、少量をドロップすることにより、スライドガラス上に細胞を広げました。このような高温の蒸気への暴露などの追加の手順が広がり、染色体を改善する必要があるかもしれません。 </li></ol></li><li> in situハイブリダイゼーション染色体数や核型分析や多色蛍光染色する細胞（mFISH）。 <ol><li>染色体数。 <ol><li> 20分 - 5％ギムザ溶液（又は蛍光顕微鏡検査のために0.5mg / mlのRNase Aを含むに5μg/ mLのPI）15を有する染色細胞。 </li><li>デジタル少なくとも50中期細胞を撮影。 </li><li>画像解析softwaを用いて細胞あたりの染色体数を数えます写真編集ソフトウェアを使用して染色体をタッチし、重複を手動で分離した後の再。染色体数を手動で採点することができますが、コンピュータ化されたスコアリングが推奨されます。 </li></ol></li><li>核型分析 注：この分析は、訓練を受けた技術者または専門の会社が行ってください。 <ol><li>標準G-バンディング技術12に従って染色細胞。 </li><li>標準karyograms 12を作るために染色体を分析します。 </li></ol></li><li> mFISH 注：この手順はオプションであり、必要に応じて行われるべきです。 <ol><li>製造業者のプロトコル13に記載のヒト染色体のための多色FISHプローブを使用して染色細胞。 </li><li>適切なフィルターとmFISH分析13については、ソフトウェアに蛍光顕微鏡を用いて染色体を分析します。 </li></ol></li></ol></li></ol>

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著者は、彼らが競合する経済的利害関係を持っていないことを宣言します。

化学物質による、細胞質分裂阻害剤によるスピンドル阻害剤のいずれかによって二倍体細胞から四倍の誘導における主要な問題は、細胞がしばしば二倍体と四倍体集団の混合物となり、四倍体細胞は二倍体細胞から分離されなければならないということです。二倍体細胞の自由な四倍体の集団を単離するための最も一般的なアプローチは、希釈を制限することによって、FACSまたはクローニン...

倍数性は、哺乳動物種の特殊な組織でも、癌や変性疾患のような病的状態の様々なだけでなく、観察されました。倍数体（主に四倍体）細胞は、しばしば、頸部3,4のバレット食道1,2または扁平上皮内病変のようなヒト組織の前癌病変において観察され、そしてそれらの組織5における悪性異数性細胞の供給源であると考えられています、6。それは異数体細胞への四倍の変換は腫瘍形成の初期段階において重要な事象である可能性が示唆されていますが、このプロセスに関与するメカニズムは完全には理解されていません。非形質転換倍数体ヒト細胞を伝播することができる場所ないin vitroモデルが利用できなかったので、これは部分的です。 一部の研究者は、INHによって二核細胞の生成を介して非形質転換ヒト上皮細胞に四倍を誘発しました細胞質分裂7-9 ibiting。しかし、この方法では、不要な二倍体細胞は、蛍光活性化細胞選別（FACS）7,8によって除去されなければなりません または希釈9を制限することによってクローニング。これらの手順が面倒で実行することは容易ではないので、非形質転換四倍体細胞を確立するための簡単な方法は、この分野の研究のために望まれています。 本報告書では、比較的簡単な手順で、正常ヒト線維芽細胞またはテロメラーゼ不死化ヒト線維芽細胞から増殖性四倍体細胞を確立するためのプロトコルについて説明します。手順は、有糸分裂で二倍体細胞を逮捕するためにスピンドル毒デメコルチン（DC）を使用し、オフ振とうすることにより収集有糸分裂細胞は、更に、DCで処理されています。長時間DCで処理された二倍体有糸分裂細胞は、四倍体G1細胞に変換し、これらの細胞は、薬物除去後数日間増殖停止した後に四倍体細胞として増殖します。このプロトコルは、提供します染色体不安定性と倍数体ヒト細胞の発癌性との間の関係を研究するための有用なモデルを作成するための効率的な方法。

<table><tbody><tr><td>MEM-&amp;alpha;</td><td>Sigma-Aldrich</td><td>M8042-500ML</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>172012-500ML</td><td></td></tr><tr><td>Demecolcine solution (10 &amp;mu;g/mL in HBSS)</td><td>Sigma-Aldrich</td><td>D1925-10ML</td><td></td></tr><tr><td>BD CycleTES Plus DNA Reagent Kits</td><td>BD Biosciences</td><td>#340242</td><td>For examination of DNA ploidy by flow cytometry</td></tr><tr><td>Human chromosome multicolor FISH probe 24XCyte</td><td>MetaSystems</td><td>#D-0125-060-DI</td><td>Specialized filter set and software for mFISH analysis are necessary</td></tr><tr><td>Isis imaging system with mFISH&amp;nbsp; software&amp;nbsp;</td><td>MetaSystems</td><td></td><td>Specialized probe kit is necessary</td></tr></tbody></table>

establishment of proliferative tetraploid cells from nontransformed human fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

我々の経験では、TIG-1細胞を4日間は0.1μg/ mLのDC（ 図2A）との単純な連続処理によってほぼ完全に四倍にすることができます。これとは対照的に、このようなBJまたはIMR-90、およびTIG-HT細胞のような他の線維芽細胞株は、同じ治療後の二倍体と四倍体集団の混合物となり、振り切り法による有糸分裂細胞の単離はもちろん、中に必要ですDCの治療（通常は16?...

Watch this Scientific Journal Video about Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts at JoVE.com

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

非形質転換ヒト線維芽細胞から増殖性四倍体細胞の樹立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

establishment-proliferative-tetraploid-cells-from-nontransformed

Research

JoVE Journal

Developmental Biology

7.2K ビュー.  Saitama Medical University. Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

ビデオ: Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

免疫・感染学

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

ラミニン521マトリックスを用いたヒト誘導多能性幹細胞の統合された誘導

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

発生生物学

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

RNA に基づく誘導多能性幹細胞に主線維芽細胞のプログラムし直す

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

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

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

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 ...

生物学

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 ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人ヒト線維芽細胞の導出と拡張神経前駆細胞へのそれらの直接変換

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

神経科学

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

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

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 ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工学

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

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

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

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

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 ...

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

導入遺伝子のない人間の人工多能性の導出および特徴は、定義された臨床グレードの条件に細胞株および変換の幹

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...