パートプレート結合フィーダー細胞として使用されるNIH/3T3細胞を発現するCD40リガンドの準備を示しています：PBMCからヒトCD40活性化B細胞を生成するためのプロトコルは2つの部分に分かれています。パートBは、実際のCD40 - Bの文化を説明します。 フィーダー細胞のA.準備（tCD40L NIH/3T3） tCD40L NIH/3T3は完全にコンフルエントになることはありません接着マウス繊維芽細胞株、です。細胞は、そのため1週間に2回分かれています。以上の6週間以上の培養が推奨されていません。 <ol><li>滅菌ピペットを用いて初代培養から古い培地を除去し、1 × PBS 10 mLで細胞を洗浄。洗浄後PBSを吸引除去する。 </li><li> 37℃で5-10分は75 cm 2のフラスコに4 mLのトリプシン/ EDTAを追加℃に細胞を分離するために穏やかなタッピングを使用してください。 </li><li>野生型の培地10mLを加え、穏やかに回転さ。 </li><li>滅菌ピペットで50mLのチューブに細胞懸濁液を移し、5分間、225 × gで細胞をスピンダウン。 </li><li>上清を除去し、野生型の培地10mLにペレットを再懸濁します。細胞懸濁液のアリコートの細胞数をカウントし、適切な数の細胞を3つの50 mLの試験管を準備します。 <ol><li>継代のための1.5 × 10 6細胞</li><li> 0.2 × 10 6細胞/ウェルCD40 - B細胞の培養に用いる照射</li><li>フリーズして残り（必要に応じて）。 </li></ol></li><li> 5分で225 × gで細胞をスピンダウン。 </li><li>上清を取り除く。 <ol><li>継代培養のために：再懸濁し、1.5 × 10 6細胞を75センチメートル2細胞培養フラスコ（細胞密度1.5 × 10 5細胞/ mL）10 mLの野生型媒体において、[0.7 mg / mLの] G - 418を追加し、で細胞をインキュベート37℃、5％CO 2とC。週2回のセルを分割する。 </li><li> CD40 - B細胞の培養には：1つの6ウェルプレート用1.2 × 10 6細胞を必要とする。 0.1 × 10 6細胞/ mLの密度で野生型の培地で細胞を再懸濁し、78 Gyの時にそれらを照射する。よくそれぞれ、37でそれらをインキュベート° C、5％のCO 2に細胞懸濁液のプレートを2mL。 tCD40L NIH/3T3細胞が接着のときB細胞の刺激のために、この準備のプレートを使用する（少なくとも4時間：顕微鏡による遵守を確認するには、B細胞の刺激を開始するために24時間以上待機しません）。 （bに進みます。） </li></ol></li></ol> B. CD 40 - Bの細胞培養 CD40刺激（0日）のための末梢血単核細胞のI.の準備： ご注意：あなたは、フィーダー細胞が接着していることを確かめる続行する前に。常に使用直前に増殖培地にインターロイキン-4およびシクロスポリンの新鮮なソリューションを追加します。 <ol><li>新鮮あるいは適切に融解したPBMCを、取る。再懸濁し、それらを洗浄し、他の細胞を除去するために7分間で190 × gで7分と二度目の256 × gで初めてスピンダウンするの1X PBS 50mLのに二回末梢血単核細胞。上清を捨て、PBS 20mLに細胞を懸濁します。細胞懸濁液の一定量の細胞数を決定します。 </li><li> 5分で225 xgで細胞の必要量をスピンダウン。 6ウェルプレート4の場合は× 10 6細胞/ウェルので、プレート当たり24 × 10 6細胞、必要とされています。 </li><li>上清を除去し、新鮮なT -細胞の伸長を防止するための成長因子としてのインターロイキン-4および0.63μg/ mLのシクロスポリンの50 U / mLで補ったCD40 - B培地で1 × 10 6細胞/ mLでPBMCを再懸濁します。 （与えられた濃度は1枚のMLの培地を参照してください！）。 </li><li> 6ウェルプレートtCD40L NIH/3T3細胞をプレインキュベートから上清を取り除く。 </li><li>ウェルあたり最初とCD40 - B洗浄培地2mlで2番目のステップで2mLのPBSで接着tCD40L NIH/3T3細胞を洗浄。 </li><li>穏やかに6ウェルプレートの各ウェルにPBMC懸濁液を4mL（1 × 10 6細胞/ mL）を追加します。 </li><li> 37細胞をインキュベート° C、5％のCO 2。 </li><li> 7日目に、細胞を（3.2に進みます。）reculture。 </li></ol> II。 CD40 - B細胞（7日目、その後3〜4日ごと）のRecultivati​​on： <ol><li> 50 mlチューブに10 mLのピペットとプール一緒に再懸濁することにより6 - ウェルプレートからCD40 - B細胞の収穫クラスター。 </li><li> 7分で225 × gでスピンダウンし、完全にCD40 - Bの洗浄媒体を使用して上清を交換してください。アリコートの細胞の量をカウントしながら、5分間、225 xgで細胞をスピンダウン。 1 × 10 6細胞/ mLの濃度でCD40 - B培地で再懸濁し、CD40 - B細胞。 </li><li>培地に50 U / mLおよび0.63μg/ mLのシクロスポリンの濃度のインターロイキン-4の新鮮なソリューションを追加。 </li><li> 6ウェルプレートtCD40L NIH/3T3細胞をプレインキュベートから上清を取り除く。 </li><li>よく最初とCD40 - B洗浄培地2mlで2番目のステップのあたり2mLのPBSで付着細胞を洗浄する。 </li><l私は&gt;静かに6ウェルプレートの各ウェルにCD40 - B懸濁液を4mL（1 × 10 6細胞/ mL）を追加します。 </li><li> 37プレートをインキュベート℃、5％のCO 2。 </li><li>継代培養細胞は、再び3〜4日ごとに14日間の合計の後に高純度のヒトCD40活性化B細胞で終わるために。 </li></ol> C.トラブルシューティング - 何かのCD40 - B細胞は増殖しないのですか？ <ol><li>あなたは、フィーダー細胞のCD40リガンド発現について確認しましたか？ </li><li>プレートに結合刺激のために使用されるフ​​ィーダー細胞は、24時間以上経過すべきではない？ </li><li>マイコプラズマ可能で汚染されていますか？ </li><li>補充のために使用されるインターロイキン- 4ソリューションは、新鮮に解凍し、適切な生物学的活性を有していた？ </li><li>シクロスポリン正しい濃度で添加されたのはいつですか？ </li></ol><img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="図1" /> 図1：してください<a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">こちらをクリックして</a>図1の拡大版のために。 <img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="図2" /> 図2：してください<a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">こちらをクリックして</a>図2の拡大バージョンのために。

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25, 659-664 (2004).</li><li>Bergwelt-Baildon, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107, 2786-2789 (2006).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> Blood. 85, 1903-1912 (1995).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63, 2836-2843 (2003).</li><li>von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99, 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155, 249-256 (2009).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15, 955-965 (2008).</li></ol>

メディアのA.の準備： <table border="1"cellspacing="0"><tbody><tr><td colspan="2"> フィーダー細胞の野生型媒体 </td><td colspan="2"> フィーダー細胞を選択培地 </td></tr><tr><td> 試薬 </td><td> 濃度 </td><td> 試薬 </td><td> 濃度 </td></tr><tr><td> DMEM -ハムの/ F12 </td><td></td><td> DMEM -ハムの/ F12 </td><td></td></tr><tr><td> L -グルタミン</td><td> 365μg/ mLの</td><td> L -グルタミン</td><td> 365μg/ mLの</td></tr><tr><td> FBS </td><td> 10パーセント</td><td> FBS </td><td> 10パーセント</td></tr><tr><td> HEPES </td><td> 10 mMの</td><td> HEPES </td><td> 10 mMの</td></tr><tr><td>ゲンタマイシン</td><td> 15μg/ mLの</td><td>ゲンタマイシン</td><td> 15μg/ mLの</td></tr><tr><td></td><td></td><td> G - 418 </td><td> 0.7 mg / mLの</td></tr></table><table cellspacing="0" border="1"><tbody><tr><td colspan="2"> CD40 - Bの洗浄媒体 </td><td colspan="2"> CD40 - B培地 </td></tr><tr><td> 試薬 </td><td> 濃度 </td><td> 試薬 </td><td> 濃度 </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td> </td><td> IMDM </td><td> </td></tr></tbody><tbody><tr><td> L -グルタミン</td><td> 584μg/ mLの</td><td> L -グルタミン</td><td> 584μg/ mLの</td></tr></tbody><tbody><tr><td> HEPES </td><td> 25 mMの</td><td> HEPES </td><td> 25 mMの</td></tr></tbody><tbody><tr><td>ゲンタマイシン</td><td> 15μg/ mLの</td><td>ゲンタマイシン</td><td> 15μg/ mLの</td></tr></tbody><tbody><tr><td> </td><td> </td><td> RHトランスフェリン</td><td> 50μg/ mlの</td></tr></tbody><tbody><tr><td> </td><td> </td><td> RHインスリン</td><td> 5μg/ mLの</td></tr></tbody><tbody><tr><td> </td><td> </td><td> AB - Humanserum </td><td> 10パーセント</td></tr></tbody></table> B.その他の試薬： <table cellspacing="0" border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> オーダーNo </td></tr><tr></tr><tr><td> DMEM / HamのF12 </td><td> PAA </td><td>猫番号：E15 - 813 </td></tr><tr><td> IMDM </td><td>インビトロジェンGIBCO ® </td><td> REF 21980-032 </td></tr><tr><td>ダルベッコPBS（10倍） </td><td> PAA </td><td>猫なしH15 - 011 </td></tr><tr><td> AB -ヒト血清</td><td>インビトロジェン</td><td>猫なし34005100 </td></tr><tr><td>ホロトランスフェリン人間</td><td>シグマ</td><td>猫なしT0665 </td></tr><tr><td>ヒトインスリン</td><td>シグマ</td><td>猫なしI2643 </td></tr><tr><td> Gencin ® </td><td>デルタの選択</td><td>アートなし7395800 </td></tr><tr><td>組み換えヒトインターロイキン-4 </td><td> Immunotools </td><td>猫なし11130045 </td></tr><tr><td>シクロスポリン</td><td>ノバルティス</td><td>猫なしNDC 0078-0109-01 </td></tr><tr><td> FBS </td><td>ロンザ</td><td>猫なしDE14 - 802C </td></tr><tr><td> HEPESバッファー</td><td> PAA </td><td>猫なしS11 - 001 </td></tr><tr><td> G - 418硫酸</td><td> PAA </td><td>猫なしP02 - 012 </td></tr><tr><td>トリプシン/ EDTA（10倍） </td><td>インビトロジェンGIBCO ® </td><td> REF 15400-054 </td></tr></tbody></table> C.その他の用品： <table cellspacing="0" border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> オーダーNo </td></tr><tr></tr><tr><td>滅菌ピペットチップ</td><td>ザルスタット</td><td></td></tr><tr><td> 6ウェルプレート</td><td> NUNC </td><td>猫なし140675 </td></tr><tr><td> 50mLのコニカルチューブ</td><td> BDファルコン™ </td><td>猫なし352070 </td></tr><tr><td>組織培養フラスコ</td><td>ザルスタット</td><td>猫なし83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

CD40活性化B細胞（CD40 - B細胞）癌免疫療法1-3免疫刺激抗原提示細胞（APC）の代替源として同定されている。樹状細胞（DC）、最も特徴的なAPCに比べて、CD40 - B細胞は、いくつかの異なる生物学的および技術的な性質を持っている。樹状細胞と同様に、B細胞はMHCと共刺激分子（図1b）の発現増加を示す、インターロイキン4およびCD40リガンド（CD40L）による刺激の後、強い遊走能と効率的にT細胞に存在する抗原提示を示す。しかし、未成熟または成熟DCとは対照的に、CD40 - B細胞はCD62L、CCR7/CXCR4から成る完全なリンパ節ホーミングトライアドを表現し、二次リンパ器官へのホーミングに必要な白血球機能抗原-1（LFA1、CD11a/CD18）、 （図1A）3。 CD40 - B細胞は、さらに高純度のCD40 - B健康なドナーからの細胞（患者あたり&gt; 10 9細胞）だけでなく、癌の非常に大量にin vitroで拡張することができる末梢血中の非常に少量から困難なしで生成することができます。患者（Fig.1c、D）1,4。 このプロトコルでは、ヒトPBMCから完全に活性化されたCD40 - B細胞を取得する方法を示します。細胞培養のための重要な分子は、CD40リガンド、インターロイキン-4（IL - 4）とシクロス​​ポリン（CSA）、3〜4日の文化のサイクルで補充されている。です。実験の目的のためにCD40刺激は、組換えヒトCD40リガンドを発現NIH/3T3細胞（tCD40L NIH/3T3）5によって提供されます。非トランスフェクト細胞の混入を避けるために、トランスフェクタントでヒトCD40リガンドの発現は、（図2）定期的にチェックする必要があります。 14日後にCD40 - B細胞の培養は、65日間にわたって95％以上の純粋なB細胞とCD40 - B細胞の増殖から構成されて関数1、4のいずれかを失うことなく頻繁に可能です。 CD40 - B細胞は、効率的に、プロセスを占有し、T細胞に存在する抗原6。彼らだけでなく、素数ナイーブを行うだけでなく、7,8メモリーT細胞を展開します。 CD40活性化B細胞はB細胞の活性化、分化と機能を研究するために使用することができます。また、彼らは腫瘍9に対する治療または予防接種のための有望なツールを表しています。

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells at JoVE.com

Generation of Human CD40-activated B cells

このビデオでは、提示<em&gt;生体外で</emヒトCD40活性化B細胞（CD40 - B）CD40リガンドとインターロイキン-4で刺激による末梢血単核細胞（PBMC）からの&gt;生成と展開。

ヒトCD40活性化B細胞の生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

generation-of-human-cd40-activated-b-cells

Research

JoVE Journal

Biology

15.1K ビュー.  University Hospital of Cologne, Department I of Internal Medicine. このビデオでは、提示生体外で生成と展開。

ビデオ: Generation of Human CD40-activated B cells

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

染色体分析のためのヒト胚性幹細胞の染色体スプレッドの準備

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

生物学

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 ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

ヒト胚性幹細胞集団から肝細胞様細胞の堅牢な生成

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

ヒト臍帯血由来血管内皮コロニー形成細胞の表現型および機能解析

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

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 Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

シングル細胞におけるゼブラフィッシュセグメンテーション時計のイメージングのための分散前体節中胚葉細胞培養物の世代

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

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 ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

ヒト末梢血からの血液伸長内皮細胞の生成と文化

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

トマト毛状根の発生によるDNAの修飾のハイスループットCRISPRベクター構築と特性

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Methods Article

ヒトCD40活性化B細胞の生成