我々は博士Arieh Shalhav率いる泌尿器シカゴセクションの大学、泌尿器研究博士キャリーRinker-シェーファーのディレクターの支持を認めるたい。また、博士ミシェルルボー率いるシカゴ総合がんセンター（UCCCC）大学の支援に感謝します。また、博士マークリンゲン率いるヒト組織リソースセンター中核施設、レスリーマーティンとメアリージョーフェケテの支援を専門的技術支援に感謝するとともに。またテリー李が運営免疫中核施設に感謝します。米国癌協会制度研究助成（ACS-IRG、＃IRG-58-004）;;がんセンターサポートグラント（P30 CA14599）;ブリンソンこの作品は外科シカゴ科、泌尿器科のセクション大学によって資金を供給された財団;アルビンバウムファミリー基金、シカゴ大学のがん研究財団女子会; S.クレーゲルはHHMIでサポートされています：マックに - 卒業生フェローヒップ（56006772）とがん生物学トレーニンググラント（T32-CA09594）。最後に、私たちは原稿の彼らの重要な評価のためにロバート·クラーク博士VenkateshKrishnan、とネイサンStadickに感謝したいと思います。

この研究は米国国立衛生研究所の実験動物のケアと使用に関する指針の勧告に厳密に従って行った。プロトコルはシカゴインスティテュー動物実験委員会の大学（IACUC、プロトコル番号72066および72231）によって承認された。すべての手術麻酔下で行い、すべての努力を最小限に抑えるために苦しんで行われた。ヒト胚性幹細胞株WA01（H1、NIH登録＃0043）がmTeSR1メディアを使用してフィーダーに依存しないプロトコルを使用してWiCell（マディソン、WI）、培養から取得しました（電池技術を幹、バンクーバー、BC）。 ES細胞を解凍通路10内で使用された。 1。マウスまたはラット胚から尿生殖洞の分離<ol><li> 5分間のCO 2吸入によりタイムアウト妊娠マウス（日17.5）またはラット（日18.5）を安楽死させる。死亡は、ラットにおけるバイタルサインの停止によって確認される。その後、人道的にその首を破る。 Rにおいては、この時点で安楽死させている。腹部皮膚及び脇腹を殺菌するために70％エタノールを散布。腹部の皮膚や筋肉の層をカットした後、子宮を切った羊膜嚢からラット胚を分離し、へその緒を切った。氷冷ハンクス平衡塩類溶液（HBSS）を含む50ミリリットルファルコンチューブに胚を移し、氷上に保つ。胎児は、外科はさみで断頭により安楽死させる。 </li><li> 100ミリメートルペトリ皿に胚を置きます。臍（ 図1A）のレベルでハサミで胚を二等分する。胃や小腸を取り外します。雄の胚（ 図1B及び図1C）の女性胚と精巣で卵巣を明らかにする。 （卵巣は腎臓の尾極に配置されています。精巣、膀胱のレベルで約です）。この発達段階では、両方の男性と女性の胚から単離さUGSMは、ホストの存在下で前立腺上皮系統を誘導することができるテストステロン。4この発達時点の後、しかし、前立腺芽がUGSMに形成し始め、純粋UGSMの分離は非常に困難である。4 </li><li>解剖顕微鏡下で、膀胱と腹部の後壁を公開するVannasはさみで真ん中のラインで腹壁をカット。添付ファイルから無料アッパー生殖（オス胚における、尿管、ウォルフ管とミュラー管をカット、女性胚において、尿管とミュラー管をカット）。臍帯から膀胱を解放。恥骨とデュモン細かい先端の鉗子との泌尿生殖洞の前壁からぶっきらぼうに別をカットします。最後に、直腸から泌尿生殖洞の後壁を分離。下端（尿道から）で、膀胱と尿生殖洞をカットし、氷冷HBSSで泌尿生殖洞を一括します（膀胱）を格納する。 </li></ol> 2。尿生殖洞間充織の分離<ol><li&gt;解剖顕微鏡（ 図1D）の下尿生殖洞から膀胱を削除します。 </li><li> 1％のカルシウムでトリプシン（のCa 2 +）とマグネシウム（Mg 2 +）自由HBSSを含むファルコンチューブに尿生殖洞を転送します。 75分間、4℃の冷蔵庫にチューブを置きます。 </li><li>トリプシンを取り外し、DMEM/F12培地中で、10％胎児Bovin血清（FBS）をトリプシンを中和。 </li><li>慎重に針またはデュモン細かい先端鉗子（上皮管をそのまま維持すると上皮チューブから粘着性の間葉スリーブをいじめることは間葉の上皮細胞の混入の可能性を減少させ、これはあなたの幹細胞由来のヒトと一緒に形成する齧歯類腺になることが腺上皮）（ 図1D）。12 </li><li> + 1％非必須アミノ酸（NEAA）プラス1nM​​のR1881; DMEM/F12培地+が10％FBS + 1％ペニシリン/ストレプトマイシン（0.5 mg / mlのペニシリン/ストレプトマイシン）を含む新たなファルコンチューブにUGSMを移す。 PLエース37℃のインキュベーター内チューブ5％CO 2で一晩。 </li><li> 200 xgで組織を遠心分離し、上清を捨てる。リン酸緩衝生理食塩水（PBS）（組織ペレットを乱さないでください）​​で洗ってください。 </li><li>再懸濁する組織に0.2％コラゲナーゼ（DMEM/F12で）を追加します。 1時間穏やかに揺れて37℃でインキュベートする。 </li><li>精力的に渦消化組織三回、それは均質な単一細胞混合物となり、コラゲナーゼを中和するDMEM/F12培地+ 10％FBS + 1％P / S + 1％NEAA + 1nMのR1881を追加するまで。 </li><li> 200×gで遠心分離した後、HBSSでUGSM細胞を再懸濁することにより洗浄し、完全にUGSMを洗浄を3回繰り返します。最後DMEM/F12に間葉細胞を中断し、血球や細胞計数装置を用いて細胞を数える。 </li><li> Accutase消化（;ビルリカ、MAミリポア）を使用して; mTeSR1メディア（バンクーバー、BC電池技術を幹）とのフィーダーフリーのプロトコルを使用して培養ヒトES細胞を解離する。ヒトES細胞を洗浄暖かいDMEM/F12メディアで彼らの成長のログインフェーズ中に、その後暖かい1X Accutaseソリューションを追加して、単一の細胞がコロニーから一様に解離されるまで15〜20分間37℃でインキュベートする。 Accutaseを中和し、穏やかにピペッティング上下細胞塊を混合し、分割するために、1xの定義されたトリプシンインヒビター（グランドアイランド、NYインビトロジェン）の等量を追加します。ピペット200×gで3分間遠心チューブと遠心分離機への細胞、およびDMEM/F12メディアで再懸濁細胞ペレット。 200が再びxgで3分間、チューブを遠心し、上清を除去し、次いで、所望の音量で冷たいHBSSまたはDMEM/F12で再懸濁する。 </li><li>ヒトES /間葉系細胞の1:2.5比hES細胞を追加します。3これは、10ミリリットルの注射あたり4E 4ヒトES細胞と1E 5 UGSM細胞（シングルラットUGS 2E 6間葉細胞について通常利回り）の細胞組成になります。 5分間200×gで遠心分離した後、上清を捨てる。 75％マトリゲル（BD Biosciences社、と再懸濁サンノゼ、カリフォルニア、扱いやすくするために25％の冷たいHBSSと混合）とサブ腎カプセル注入のため氷の中にチューブを置きます。 </li></ol> 3。腎被膜の下に組換え組織の移植<ol><li>無菌手術領域を準備します。 </li><li>ケタミン/キシラジンIPを持つ男性の無胸腺ヌードマウスを麻酔、ケタミンの1時01分08秒の比率を含む新鮮なミックスを使用：キシラジン：生理食塩水。投与は100 mg / kgでケタミンおよび2.5 mg / kgをキシラジンなります。つま先ピンチに反応しない動物は、完全に20〜30分間の下でなければなりません。 </li><li>外科ベタジン溶液及び外科用ドレープを適用することにより、続いて70％のアルコールの3交互ワイプでクレンジング、手術部位を（ヌードマウスを使用していない場合）シェービングでマウスを準備します。 </li><li>麻酔中の乾燥を防ぐために、マウスの目に目水分軟膏（Altalube眼軟膏）を置きます。 </li><li>手術中に、マウスの呼吸速度および中核体温が監視される。呼吸速度笙40-90回/分以内に安定して維持される自民。中核体温は、粘膜と足の裏ピンクの色を観察して監視される。 </li><li>背面に1.0センチメートル長い縦皮膚切開を行います。 0.5センチメートル縦切開で腹壁をカットし続けています。 </li><li>優しく腹部の腎臓を絞り出す、滅菌生理食塩水の滴を使って腎臓の表面を湿った状態に保つ。 </li><li>そっと穿刺空の27ゲージ注射針を使用して腎カプセル。非常に浅い穿刺で十分です。あなたの携帯電話、混合物を注入するためにあなたの鈍針を挿入してここは次のようになります。 </li><li>あなたの細胞混合液10μlと28ゲージの鈍注射針を埋める。ゆっくり穿刺部位を挿入し、腎被膜下の領域に到達するために腎臓実質貫通している。腎被膜下に腎臓にボーラスを形成する細胞の混合液10μlを注入します。撤退針。 </li><li> ABDOに腎臓を返す端末空洞。 4-0ナイロン縫合糸で腹膜の筋層を固定します。 </li><li>縫合糸またはステープルのどちらかで皮膚を閉じます。 </li><li>滅菌生理食塩水を使用して皮膚から血液をきれいにします。 </li></ol> 4。術後鎮痛とモニタリング<ol><li>術後の痛み、37 1mlのを管理するためにブプレノルフィン（0.1 mg / kgを12時間毎）、又はケトプロフェン（生理食塩水で3〜5 mg / kgを12時間毎）で動物を注入℃の生理食塩水を腹腔内に動物を暖かく保つために脱水を防ぐ。 </li><li>清潔なケージに動物を入れて、マイルドな加熱パッドの上に置きます。 </li><li>彼らは意識を取り戻し、ケージ内で移動された後、動物の部屋に戻って動物を送信します。 </li><li>次の3日間、病気や感染の予想外の兆候を毎日動物を観察します。マウスは、食料や水術後1日のアクセスを含むケージでの活動の観察と監視されています。マウスが少ない活動を表示すると、ケトプロフェンは腹腔内広告です一日一回再び仕え。 </li><li>皮膚ステープルまたは縫合糸2週間後に手術を取り外します。 </li><li>異種移植組織は8週間後の手術は早くも収穫することができる。異種移植片は、小動物超音波（ 図2A）と、固定組織を用いたin vivoで画像化することができる免疫組織化学（ 図3）を用いて種々のタンパク質のための切片で染色することができる。 </li></ol>

<ol>
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S., Akkina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+normal+macrophages+from+human+embryonic+stem+(hES)+cells+for+applications+in+HIV+gene+therapy.">Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.</a> Retrovirology. 3, 24 (2006).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-efficiency+system+for+the+generation+and+study+of+human+induced+pluripotent+stem+cells.">A high-efficiency system for the generation and study of human induced pluripotent stem cells.</a> Cell Stem Cell. 3, 340-345 (2008).</li></ol>

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UGSMを用いて組織の組換えは、前立腺および前立腺癌の開始につながる分子事象の開発を調査するために非常に便利なテクニックです。 UGSMの誘導電位が前立腺研究の多くの用途に用いられてきたが、これらは増強腫瘍間質上皮相互作用を研究し、異種間前立腺組換え体を形成し、前立腺細胞株および腫瘍を取る含むUGSMの7,17-20適切な製剤。しかし、げっ歯類上皮は腺形成になり、結?...

前立腺癌のより良い人間モデル系のための多大な必要性が存在する。具体的には、遺伝的に直接前立腺癌の開始において特定の遺伝子の役割を識別するために操作可能な通常の、非悪性前立腺組織の関連するヒトモデル系は非常に有益であろう。ゲノム時代の到来は、癌の形成において役割を有することができる多数の遺伝子を同定している。実験的な人間のモデルシステムの欠如は、しかし、深刻な機能的にテストし、候補前立腺がん感受性遺伝子を特徴づけるために我々の能力を損なう。理想的なモデルシステムは、適切なトランスジェニックげっ歯類モデル系と組み合わせて、癌感受性遺伝子の迅速かつより迅速な機能解析を容易にするであろう。さらに、このような人間のモデル系はへ新しい治療法の発見と検証に向けて発癌前立腺のシグナル伝達機構の分子特性解析を可能にする前立腺癌の形成を防止する。 ヒト胚性幹細胞（ES細胞）異種移植片のようなヒト前立腺組織を形成することができる。 2006年にテイラーらは 、ヒトES細胞は、8〜12週の期間内に齧歯類泌尿生殖洞間葉（UGSM）で再結合されたとき、生体内で前立腺上皮を形成するように誘導することができることを報告した。1これらの研究はで以前の研究に基づいていたその齧歯胚UGSMを示すクーニャラボは、幹細胞とin vivoでの胚上皮細胞集団の前立腺分化を促進することができます。2,3前立腺は、尿生殖洞（UGS）と呼ばれ、前胚17日（にマウスE17胚原基から開発;ラットにおける日E18）UGSを除去することができ、物理的に上皮（UGSE）と間葉（UGSM）に分割4この組織換えアプローチはかなり特定の前立腺の開発と発がんの我々の理解を、強化しています。LYの成長因子、ホルモンシグナル伝達経路と前立腺間質と上皮の間の分子の関係。5-8この方法は、幹同一または異なる種からの上皮細胞とUGSMのex vivoでの組み合わせを含み、これらの細胞/組織組換え体を移植し、栽培されているとマウスのホスト内の異種移植。4,9 生体成長の期間の後、インプラントは、間質組織に埋め込 ​​まれている前立腺上皮腺構造が含まれています。さらに、染色は、そのような構造が本当に前立腺とヒト由来のかどうかを判断するために実施することができます。10,11

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Hank's Balanced Salt Solution (HBSS)</td>
 <td>GIBCO</td>
 <td>14170</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>GIBCO</td>
 <td>11330</td>
 <td></td>
 </tr>
 <tr>
 <td>R1881</td>
 <td>Sigma</td>
 <td>965-93-5</td>
 <td>Mix to 1 ug/ml in Ethanol (1,000x stock)</td>
 </tr>
 <tr>
 <td>NEAA</td>
 <td>GIBCO</td>
 <td>11140</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen-Strep Solution</td>
 <td>GIBCO</td>
 <td>15070</td>
 <td>100x stock</td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>BD Biosciences</td>
 <td>354230</td>
 <td></td>
 </tr>
 <tr>
 <td>KETASET (ketamine hydrochloride) </td>
 <td>Fort Dodge Animal Health</td>
 <td>NDC 0856-2013-01</td>
 <td>100 mg/ml; dilute 1:10 in sterile saline </td>
 </tr>
 <tr>
 <td>AnaSed (xylazine) </td>
 <td>VET-A-MIX, Inc.</td>
 <td>NADA 139-236</td>
 <td>20 mg/ml; dilute 1:10 in sterile saline</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>BD Biosciences</td>
 <td>215240</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase </td>
 <td>Sigma</td>
 <td>C2014</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketoprofen </td>
 <td>Fort Dodge</td>
 <td>NDC 0856-4396-01</td>
 <td>100 mg/ml; dilute 1:1,000 in sterile saline</td>
 </tr>
 <tr>
 <td>Altalube eye ointment </td>
 <td>Altaire Pharmaceuticals, Inc.</td>
 <td>NLC 56641-19850</td>
 <td></td>
 </tr>
 <tr>
 <td>Leica MZ16 F Stereomicroscope</td>
 <td>Leica</td>
 <td></td>
 <td>Any good dissecting scope can be used.</td>
 </tr>
 <tr>
 <td>Vannas spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15001-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Hamilton</td>
 <td>84855</td>
 <td></td>
 </tr>
 <tr>
 <td>Hamilton Needle, Small RN, 28 gauge, 0.5inches, Point Style #3 (Blunt)</td>
 <td>Hamilton</td>
 <td>7803-02 </td>
 <td>Custom Needle </td>
 </tr>
 <tr>
 <td>Ethanol Prep Pads</td>
 <td>Fisher Scientific</td>
 <td>06-669-62 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile Gauze Pads</td>
 <td>Fisher Scientific</td>
 <td>22-415-469 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethicon Vicryl Suture (4-0 FS-2)</td>
 <td>MedVet International</td>
 <td>J392H </td>
 <td>Needle-in, dissolvable suture </td>
 </tr>
 <tr>
 <td>Autoclip 9 mm Wound Clips</td>
 <td>Becton Dickenson</td>
 <td>427631</td>
 <td></td>
 </tr>
 <tr>
 <td>PVP Iodine Prep Pads</td>
 <td>Fisher Scientific</td>
 <td>06-669-98 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissector scissor with blunt end</td>
 <td>Fine Science Tools</td>
 <td>14072-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont fine tip forceps</td>
 <td>Fine Science Tools</td>
 <td>11252-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder with Scissor</td>
 <td>Fine Science Tools</td>
 <td>12002-14</td>
 <td></td>
 </tr>
 </tbody>
</table>

formation of human prostate epithelium using tissue recombination of rodent urogenital sinus mesenchyme and human stem cells

前立腺癌の研究の進展が著しく、前立腺疾患の開始を根底に遺伝的および分子事象を理解するために我々の能力を制限するヒト由来とホルモンナイーブモデルシステムの可用性によって制限されます。発癌ヒト前立腺を研究するための優れたモデルシステムの開発に向けて、私たちと他の人が胎児の齧歯類前立腺間質のユニークなプロ前立腺誘導可能性を利用している、尿生殖洞間葉（UGSM）が呼ばれる。このような胚性幹細胞などの特定の多能性細胞集団と再結合するとき、UGSMは、テストステロン依存的に正常なヒト前立腺上皮の形成を誘導する。例えば、ヒトモデル系を調査し、実験的に、典型的なげっ歯類のトランスジェニックの研究と比較して加速した候補前立腺がん感受性遺伝子の能力を試験するために使用することができる。ヒト胚性幹細胞（ES細胞）遺伝的に培養ボイジャーで変更することができるのでグラム誘導性遺伝子発現やsiRNAは前に組織の組み換えにベクトルをノックダウン、そのようなモデルでは、機能遺伝子の結果、または促進するか、または発癌を防止すると考えられている遺伝子の組み合わせを、テストが容易になります。 UGSM細胞の純粋な集団を単離する技術は、しかし、挑戦されており、頻繁に学習すると、以前の専門知識を持つ誰かが個人的に教えることが必要です。また、免疫不全宿主の腎被膜下細胞混合の接種は、技術的に挑戦することができます。ここでは、アウトラインと齧歯類の​​胚及びヒト前立腺上皮を形成するために、組織の混合物の腎カプセル注入からUGSMの適切な分離を示しています。そのようなアプローチは、その現在の段階では、胚性幹細胞の異種移植生体内で必要とし、将来のアプリケーションは、潜在的にインビトロ腺形成または人工多能性幹細胞集団（iPS細胞）を使用して含むことができる。

、私たちの研究室では、一般的に使用されるH1（NIH-指定WA01、遺伝的に男性）は、ヒト胚性幹細胞株を用いて移植プロトコルを開発してきましたが、この行は厳密に品質管理のためにテストされています1。テイラーらの刺激的な報告書に構築および核型正常である13が適切に培養し、ヒトES細胞を維持拡大し、フィーダーフリー培養法（幹細胞技術を経由して、?...

Watch this Scientific Journal Video about Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells at JoVE.com

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

前立腺癌の開始を根底早い分子機構を解明するために、斬新かつ革新的な人間のモデル系とアプローチが必死に必要とされている。ヒト前立腺上皮を形成するために、多能性幹細胞集団を誘導するプリ前立腺尿生殖洞間葉（UGSM）の電位は、前立腺研究における強力な実験的なツールである。

齧歯尿生殖洞間充織とヒト幹細胞の組織の組換えを用いたヒト前立腺上皮の形成

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

formation-human-prostate-epithelium-using-tissue-recombination-rodent

Research

JoVE Journal

Medicine

13.6K ビュー.  University of Chicago. 前立腺癌の開始を根底早い分子機構を解明するために、斬新かつ革新的な人間のモデル系とアプローチが必死に必要とされている。ヒト前立腺上皮を形成するために、多能性幹細胞集団を誘導するプリ前立腺尿生殖洞間葉（UGSM）の電位は、前立腺研究における強力な実験的なツールである。

ビデオ: Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

における幹細胞移植 in vitroで模擬虚血/再灌流モデル

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

医学

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

マウスの眼の中に遺伝子治療ベクターと幹細胞の網膜下注入

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

ヒト前立腺癌転移の同所マウスモデル

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

ヒト前立腺癌サンプルからがん幹細胞の単離

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

マルチとシングルセルベース球アッセイを用いてヒト卵巣癌細胞における幹細胞特性の評価

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

RNA分析のためのヒト前立腺上皮のレーザーキャプチャーマイクロダイセクション

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

ヒト前立腺癌の前臨床同所性マウスモデル

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

ヒト肝幹細胞を用いたヒト化マウス肝臓の生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

顕微鏡を用いたひと脂肪組織血管機能評価

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...