この作品は、再生医療（RN2-00921から1）、およびNIHの資金国立研究賞（F32-HL104924）カリフォルニア工科大学から新しい教員賞IIによって賄われていた

1。マウス胚性フィーダーの準備<ol><li>ヒトES細胞の共培養に使用されるMEFには、以前に分裂を受けてから細胞を阻害するための放射線照射またはマイトマイシン-Cで扱われるべきである。 </li><li> MEFの播種前に二時間、コートは各60mmのプラズマは、0.05％ゼラチン2mlの培養皿を治療した。 </li><li> 〜20,000細胞/ cm 2で治療を受けたMEFとプレートのバイアルを解凍します。 </li><li>細胞が一晩付着することができます。 </li></ol> 2。 MEFの上に共同播種hESCの<ol><li>新しい植民地は、以下のように、新しいMEFコートディッシュ上に凍結保存培養または継代培養から現在開始されてもよい。 </li><li>温めておいた1×PBS 3mlで1回継代する大型のhESCコロニーを含むプレートを洗浄してください。 </li><li>コラゲナーゼIV / PBS溶液（1 mg / ml）を3 mlを添加し、37℃で5〜10分間インキュベート℃、 （注：細胞がアップトリプシンで見られるように異なるか、ボールになることはありません）。 </li><li>細胞は、まだですがコラゲナーゼ、5 mlのピ​​ペットチップのエッジを使用して、小さな細胞塊に分割するか、コロニーのグリッドパターンを作成します。 </li><li>表面からのすべてのセルを外れ、培養プレートの底をこすり取る際にピペットチップの先端を（培養プレートに垂直方法で保持）を使用します。 </li><li> 5分間（1000 rpm）でファルコンチューブとスピンに細胞を回収する。 </li><li>遠心分離後、ファルコンチューブに細胞ペレットを残して、培地を吸引除去する。 </li><li>メッキされたMEFに前日からのMEF培地を吸引する。 </li><li>再プレート原版から1:3の割合で細胞、hESCの培地3mlを持つフィード</li></ol> 3。共培養からMEFを枯渇させる注：ヒトESCの培養は、通常の文化の10と14日の間で、高密度でなければなりません。 <ol><li>プレートの端で吸引ピペットの先端を入れ、吸引優しくMEFのシートの端をプルアップすることができます。 </li><li> R皿からメディアを残したまま削除し、先端が細胞シートをキャッチして、徐々にプレートの上円弧形で先端を移動することができます。注：シートは、ピペットの先端に詰まらせることがありますが、それは手動で培養皿の表面からシートを引っ張ってからユーザーを防ぐべきではありませんので、これは問題ではありません。細胞はアスピレーターの最後に立ち往生している場合は、完全な吸引のための細胞シートを分割する培養皿の上部を使用することができます。 </li><li>直ちにhESCの培地を交換し、インキュベーターにバックプレートを置く。 </li></ol> 4。代表的な結果 MEFの除去プロセスの最終結果は、小さな平穏なhESCの多能メーカー、SSEA-4の発現を維持しながら、非常に大規模なコロニー（ 図2）に大幅な拡大を受けることができるコロニー（ 図1D）、10月¾、およびTRA 1から81（ 図を生成3）。 図1。 ESCのコロニー形態MEFの除去前と後の培養皿上に播種後）1日目とB）2日目でのヒト胚性幹細胞のコロニー。C）の高密度 MEFに囲まれたヒト胚性幹細胞のコロニー（H7）。 2 hESCのコロニーが赤くダッシュで概説されています。D）が記載され吸引法を用いてのMEFを除去した後、我々は、無傷のコロニーを取り巻く非常に少数のMEFを持つ絶縁hESCの植民地が残されています。 <img alt="図2" src="/files/ftp_upload/3951/3951fig2.jpg" /> 図2。 10日のMEF除去後ESCコロニー拡張。直ちにMEFの枯渇した後、元のhESCコロニー（左）は、非常に大きなコロニー（右）が適切に展開するために許可されているximately800μmで、10倍。元植民地が複合コロニーと同じ倍率（10倍）に結像されることに注意してください。 <img alt="図3" src="/files/ftp_upload/3951/3951fig3.jpg" /> 図3。 MEFの欠乏のhESCコロニーの免疫染色。アップコロニーの免疫同定が10日にMEFの枯渇はコロニーが10月¾、SSEA-4、TRA-1-81染色により明らかなように多能性マーカーの発現を維持することを示した後。さらに、これらのhESCコロニーはFLK-1の不在によって示されるように、中胚葉の運命に向かって分化を示さない。A、E、I、およびM）immunoflouscentlyステンドコロニーの透過光画像、20、10、10と2倍であり、それぞれA、B、F、J、およびN）の核を示す- 。免疫蛍光染色されたコロニーのDAPI染色された細胞を、20、10、10、2xを、それぞれCD）の発現を示すヒト幹細胞マーカーのみ ​​、SSEA-4、コンポジット映像、20倍になったGH）のみ¾ヒト幹細胞マーカー10月の発現を示し、合成画像、10倍。KL）ヒト幹細胞マーカーの発現を示すトラ1から81のみ、コンポジット映像、10倍。MP）最後の行は、イメージがこの倍率、2x、およびこれらのコロニーで見られる典型的な滑らかな境界線を描いたコロニーを描いている場合ではない急行O）Flk-1を、中胚葉分化の初期マーカー、またやったんでしたDDR2の染色の欠如によって示されるように、彼らはP）は 、任意の線維芽細胞が含まれています。 補足ビデオ。シート吸引によって線維芽細胞を削除しています。 <a href="http://www.jove.com/files/ftp_upload/3951/3951movie1.mp4" target="_blank">ムービーを見るにはここをクリック</a> 。

<ol>
	<li>Bongso, A., Fong, C. Y., Ng, S. C., Ratnam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+inner+cell+mass+cells+from+human+blastocysts.">Isolation and culture of inner cell mass cells from human blastocysts.</a> Hum. Reprod. 9, 2110-2117 (1994).</li><li>Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., Bongso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat. Biotechnol. 18, 399-404 (2000).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+scalable+culture+of+human+embryonic+stem+cells+in+feeder-free+conditions.">Automated, scalable culture of human embryonic stem cells in feeder-free conditions.</a> Biotechnol Bioeng. 102, 1636-1644 (2009).</li><li>Kolhar, P., Kotamraju, V. R., Hikita, S. T., Clegg, D. O., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+surfaces+for+human+embryonic+stem+cell+culture.">Synthetic surfaces for human embryonic stem cell culture.</a> J. Biotechnol. 146, 143-146 (2010).</li><li>Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+system+using+human+foreskin+fibroblasts+as+feeder+cells+allows+production+of+human+embryonic+stem+cells.">A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.</a> Hum. Reprod. 18, 1404-1409 (2003).</li><li>McKay, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+cell+line+for+derivation+and+culture+of+hESc/hiPSc.">Human feeder cell line for derivation and culture of hESc/hiPSc.</a> Stem Cell Res. 7, 154-162 (2011).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Stein, G. S., Shi, M. J., Stencel, K., Borowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thawing, Seeding, and Changing Medium of Human Embryonic Stem Cells. , (2010).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol. Biol. 767, 107-123 .</li></ol>

特別な利害関係は宣言されません。

本稿で提示した方法は、ヒト胚性細胞培養物から線維芽細胞フィーダー細胞を除去するための迅速かつ安価な代替手段を提供しています線維芽細胞の除去に成功し、より長い幹細胞培養に発症、これらの細胞のコンフルエントな単層しっかりの存在に依存しています。 7-10日後に、成長のhESCコロニーはコロニー間のますます密線維芽細胞単層を生成し、外方向にフィーダー線維芽細胞をプッ?...

<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> コメント（オプション） </td></tr><tr><td> DDR2メモリ</td><td>サンタクルス</td><td> 7555 </td><td>線維芽細胞マーカー</td></tr><tr><td> DAPI </td><td>カルビオケム</td><td> 268298 </td><td>細胞核マーカー</td></tr><tr><td> SSEA-4 </td><td>ミリポア</td><td> MAB4304 </td><td>ヒトESCマーカー</td></tr><tr><td> Oct 3月4日</td><td>サンタクルス</td><td> 9081 </td><td>ヒトESCマーカー</td></tr><tr><td> Flk-1の</td><td> BDファーマ</td><td> 555307 </td><td>初期の分化マーカー</td></tr><tr><td> TRA-1-81 </td><td> Acris </td><td> AM20377AF4-S </td><td>ヒトESCマーカー</td></tr></tbody></table> 表1特定の理由が表紳士はhESCの免疫染色に使用されます。

rapid fibroblast removal from high density human embryonic stem cell cultures

マウス胚線維芽細胞（MEF）は胚盤胞の分離1の後に、ヒト胚性幹細胞（hESC）文化を確立するために使用されていました。このフィーダシステムは、細胞膨張時に自発的分化を受けてからのヒトES細胞を維持しています。しかし、この共培養法は、労働集約的で高度に訓練された人材を必要とし、利回りが低いのhESC純度4。多くの研究室では、hESCの培養におけるフィーダー細胞の数（ すなわち、マトリックスコートディッシュや他のフィーダー細胞タイプ5-8を組み込む）を最小化しようとしました。これらの改変された培養系では、いくつかの約束を示しているが、hESCの文化の不要な自発的分化を遅らせるために、マイトマイシンC処理したマウスembyronic線維芽細胞とヒトES細胞を培養するための標準的な方法を取って代わられていない。したがって、hESCの拡張に使用されるフ​​ィーダー細胞は、分化実験中に除去されるべきである。いくつかの手法がhESCのCOを浄化するために利用可能であるがフィーダからlonies（FACS、MACS、または薬剤耐性ベクターの使用）は、これらの技術は、集中的な高価な、そして/またはhESCのに破壊的労働である。このプロジェクトの目的は、ヒトES細胞の純粋な人口の収穫を可能に精製する方法を考案しました。我々は、コンフルエントhESCの文化では、MEFの人口はMEFのシートの簡便かつ迅速な吸引を用いて除去することができることを観察した。この除去は、 &quot;横細胞から細胞へのスチレン培養皿に低い結合親和性を持っているMEFの結合し、独自の生成中に線維芽細胞が外側にプッシュする幹細胞コロニーの能力など、いくつかの要因に依存していますニッチ &quot;。 hESCの多能性は、その後の維持を確保するため、MEFを除去した後の10日に、SSEA-4、Oct3 / 4の発現およびTRA 1から81までを調べた。また、hESCのコロニーはhESCの拡張の追加レベルを提供し、MEFを除去した後に大規模な地層中への成長を続けることができました。

Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

遷移文化にフィーダーフリー条件への継続的な努力にもかかわらず、ヒト胚性幹細胞（hESCの）の導出と文化がマウス胚性フィーダー細胞（MEF）と共培養に大きく依存して残っている。ここでは、急速に、実験開始までにhESCの文化からフィーダを除去するための新規な方法を示しています。

高密度ヒト胚性幹細胞の培養線維芽細胞からの迅速な除去

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

rapid-fibroblast-removal-from-high-density-human-embryonic-stem-cell

Research

JoVE Journal

Biology

11.7K ビュー.  University of California, Merced. 遷移文化にフィーダーフリー条件への継続的な努力にもかかわらず、ヒト胚性幹細胞（hESCの）の導出と文化がマウス胚性フィーダー細胞（MEF）と共培養に大きく依存して残っている。ここでは、急速に、実験開始までにhESCの文化からフィーダを除去するための新規な方法を示しています。

ビデオ: Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

トリプシンで継代色合いヒト胚性幹細胞、線

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

マウス表皮神経堤幹細胞（EPI - NCSC）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

動物の無血清ヒト幹細胞培養のための効率的なサプリメントとしてプールしたヒト血小板可溶化物（pHPL）の調製

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

ヒト胚性幹細胞のコロニーの成長のビデオバイオインフォマティクス解析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

オリゴデンドロサイト前駆体に胚性幹細胞の分化

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

げっ歯類からプライマリ大人の線維芽細胞培養の確立

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.