Name: generation of a humanized mouse liver using human hepatic stem cells
Uploaded: 2016-08-29T15:00:00.000+00:00
Duration: 11 min 44 s
Description: Watch this Scientific Journal Video about Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells at JoVE.com

We wish to thank the Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, for providing the mice. We also thank S. Aoyama and Y. Adachi of the ADME (Absorption, Distribution, Metabolism, Excretion) &amp; Toxicology Research Institute, Sekisui Medical Company Ltd., Japan, and K. Kozakai and Y. Yamada for assistance with LC-MS/MS analysis. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan to Y-W.Z. (18591421, 20591531, and 23591872); by the Jiangsu innovative and entrepreneurial project for the introduction of high-level talent and the Jiangsu science and technology planning project (BE2015669); and by grants to H.T. for Strategic Promotion of Innovative Research and Development (S-innovation, 62890004) from the Japan Science and Technology Agency.

全ての動物実験手順は、横浜市立大学の動物保護ガイドラインに従って行いました。 急性肝障害モデルマウスの1世代<ol><li> 1mg / mlのDTストック溶液を作製するために1mgのジフテリア毒素（DT）から（0.85％のNaCl溶液100 mlの0.6グラムフェノール）フェノール化0.85％のNaCl溶液1mlを加えます。注：1ミリグラムの濃度のDT / mlで8℃に3℃で約2年間保存することができます。 </li><li>シリアルフェノール化0.85％NaCl溶液中0.3 / mlのDTに1mg / mlのDTストック溶液を希釈し、作業溶液として0.3 / mlのDT溶液のアリコートを準備します。注：この作業溶液を新たに準備する必要があります。 </li><li>致死未満量（〜50％の致死率）を使用して実験を行うために、8週齢のマウスにDT1.5μgの/ kgの用量を投与します。 <ol><li>背側横臥位でマウスを保持することによって、DTの腹腔内注射を行い、needlを挿入膝の曲げ下のeは、左または正中線の右側。膀胱の浸透を防止するために、正中線を避けてください。角度身体への針は約45°です。 </li><li>インスリン注射器を用いて、マウス体重20gのあたり新たに調製した0.3 / mlのDTを100μlをマウスに注射します。例えば、18-グラムのマウスには、0.3 / mlのDTの溶液を90μLを受けるべきです。 </li></ol></li><li> 48時間後DT注射で、血管を拡張するために、約10分間37℃の水浴中でマウスの尾の拘束にマウスを置き、温めて尾静脈から血液を収集します。そして、鋭いメスの刃で先端から尾2センチメートルに1ミリニックを行い、マイクロキャピラリーチューブに血液を収集します。 <ol><li> 5分間1500×gで血液を含むマイクロキャピラリーチューブを遠心し、上清および細胞ペレットを分離することにより血清を収集します。 </li></ol></li><li> GOT / AST-上に1：20希釈したマウス血清のピペットを50μlPIIIはスライドします。製造業者のプロトコルに従って多目的自動乾式化学分析装置を用いて650nmで反応生成物（青色色素）の吸光度を測定します。 注：アスパラギン酸アミノトランスフェラーゼ（AST）の読み出しは、/グルタミン酸オキサロトランスアミナーゼ（GOT）活性が自動的に表示されます。 2日は、DTの注射後、マウスの約60％は12,000の間でAST値を示した - 16,000 IU / Lと急性肝障害を患っていると考えられました。これらのマウスは、新しいケージに移し、細胞移植のレシピエントとして使用しました。 </li></ol>ヒト肝幹細胞の調製<ol><li> CDCP1 + CD90 + CD66-の亜集団を得ることがCDCP1、CD90、およびCD66細胞抗原を使用して、セルソーターでヒト初代胎児肝細胞からのヒト肝幹細胞を分離し、その後、コラーゲンIVでコーティングした培養皿上で単離された細胞集団をシード、以前に15を報告しました 。胚性のヒト初代胎児肝細胞を使用してください週14と18の間の年齢。 </li><li> 、100 mmの培養皿上で90％コンフルエンス培地を吸引、10mlのPBSで細胞を洗浄し、その後PBSを削除 - 80％まで培養したヒト肝幹細胞を収集します。 37℃で5分間2mlの0.05％トリプシン/ EDTA溶液で細胞をトリプシン処理。注：細胞分化マウスにおけるそれらの増殖能に影響を与えることができるので、90％以上のコンフルエンスの細胞を、細胞移植に適していません。 </li><li>顕微鏡下で細胞分散の状態を監視します。細胞が浮遊または自由に流れるように見える場合には、10％FBSを含むDMEM / F12を8mlを添加することにより酵素消化を停止します。 </li><li>ゆっくりと10-mlの血清学的ピペットを用いて細胞を懸濁し、15 mLコニカルチューブに細胞を移します。 100×gで5分間、4℃のための細胞を遠心します。 </li><li>慎重に10％FBSを含む10 mlのDMEM / F12中で細胞ペレットを再懸濁し、顕微鏡計数チャンバーを用いて細胞数を決定します。 </li><li>各個々のマウスのためのPBSのアリコート50μlのあたり1.0×10 6細胞に細胞を分割し、移植まで氷上で保存します。 </li></ol>ヒト肝幹細胞の3脾臓内移植<ol><li> 37°C電気加熱パッドの上にきれいなケージを置きます。 </li><li>ノズルの下に置くことによって - （分当たりの酸素の2 Lで1.5％（体積/体積）1）イソフルラン吸入を用いてマウスを麻酔。また、イソフルランに浸したガーゼを含むチューブを使用します。 <ol><li>マウスは軽くリアフットパッドをつまんで麻酔されていることを確認してください。膝ジャークが誘発されている場合は、バックチャンバー内にマウスを置きます。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 </li></ol></li><li>電気バリカンを使用して手術部位を剃るし、滅菌するために70％（体積/体積）エタノール及びポビドンヨードを適用します。その後、切開に続いてちょうど肋骨の肋骨境界線下の左脇腹に1 cmの皮膚切開を作ります腹壁、ならびに腹膜。 </li><li>慎重に脾臓を露出させます。直接各マウスの脾臓に50μlのPBS中に1.0×10 6個のヒト肝幹細胞を注入するために32-G、1/2インチの針で100μlのマイクロインジェクションのシリンジを使用してください。注射のための5°の角度で針を傾けます。 <ol><li>注入の深さは脾臓の半分以下の厚さであることを確認してください。針は、一方の端部（ヘッド）で脾臓を入力し、もう一方の端（テール）で細胞を付着させる必要があります。 </li></ol></li><li>注射後の細胞の漏れを防止するために、穏やかに2分間の指を使って圧力を適用し、針を取り除きます。 </li><li>バックマウス本体に脾臓を置き、筋肉組織や皮膚のための通常走行縫合糸で空洞を閉じます。 </li><li>すぐに移植後37℃で予熱したケージにマウスを転送します。マウスを確保するために、快適に息をケージにその側にマウスを配置することができ、ケージ寝具、手術の部位との間の接触が無効になります。 </li><li>麻酔は、縫合糸は閉じたままとマウスは、手術前の状態に戻り確実にするために切れるまでマウスを監視します。 </li><li>マウスを確認し、通常の飲料水と食料が設けられています。 1時間後、動物中央のマウスの部屋にケージを返し、毎日マウスを監視します。 </li></ol>移植されたヒト肝4.検出は、マウス肝臓における細胞由来肝細胞を幹注：以下の手順については、頸椎脱臼が続くケタミンとキシラジンの過剰摂取を使用して、すべての動物を安楽死させます。 <ol><li> 4時 - 6週間、移植後、マウスを安楽死させると同時に、手術用はさみを使用して真皮と筋膜を切断して腹腔を開きます。 </li><li>スプレッダーとしてハサミを使用して、腹膜上記の結合組織を解剖し、腹膜を開くには、白線に沿って腹膜をカット空洞。 </li><li>鉗子で胸骨を持ち上げ、横隔膜に穴を開け、最大子宮頸ガードルを介して胸骨の両側を切断。 </li><li>肝臓の胸部側の大静脈を切断し、前方方向に肝臓を通って食道を引っ張ります。肝臓を除去するために、振動板を取り外します。 注：はさみのヒントを出さないように注意してください下に胸部臓器の損傷を防止するために、上向きに指摘しました。胸腺は時折胸骨の背側にしがみつく傾向があり、これは慎重に避けなければなりません。 </li><li>ハンドルとしてダイヤフラムを使用して、腹腔から肝臓を引っ張って開始します。インテリア大静脈は、所定の位置に肝臓を開催します。インテリア大静脈をカット。途中で右副腎を解放しないように注意してください。 注：マウスは中葉、左葉、右葉、および尾状​​葉からなる4ローブ肝臓を持っています。胆嚢はの小さな分岐に懸濁されます中葉。 </li><li>接合部で互いにローブを分離し、製造業者のプロトコルに応じて最適な切削温度（OCT）化合物でそれらを埋め込みます。クライオスタット上の金属グリッドでの肝臓組織を含む10月をフリーズします。 </li><li> -18℃でクライオスタットを用いて5μmの厚さのサンプルのセクションをカットし、RT顕微鏡スライド上にマウントします。 -80℃での在庫を保管してください。ヘマトキシリンおよびエオシン（H＆E）と以前に報告された手順16に従って、免疫蛍光染色のためのスライドを準備します。 </li></ol> 5.ヒトアルブミンの分泌の検出とキメラ率の計算<ol><li>ヒト肝臓の再構成を測定するために、製造業者の指示に従って血清およびヒトアルブミンELISAキットを用いてヒトアルブミンELISAを行います。 </li><li>全ヒト肝臓における発現レベルのリアルタイムPCR分析によりヒト肝キメラ率を計算します。相対元を決定しますヒト - マウスクロスアクチンに対するヒト特異的アクチンのPRESSIONレベル比（比1）、ヒト - マウスクロスアクチンに対するマウス特異的アクチンの比率（比率2）。キメラ率は、比1 /ように（比1 +比2）で算出されます。 </li></ol>

<ol>
	<li>Muruganandan, S., Sinal, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+as+clinically+relevant+models+for+the+study+of+cytochrome+P450-dependent+metabolism.">Mice as clinically relevant models for the study of cytochrome P450-dependent metabolism.</a> Clin. Pharmacol. Ther. 83, 818-828 (2008).</li><li>Nishimura, T., 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=Using+chimeric+mice+with+humanized+livers+to+predict+human+drug+metabolism+and+a+drug-drug+interaction.">Using chimeric mice with humanized livers to predict human drug metabolism and a drug-drug interaction.</a> J. Pharmacol. Exp. Ther. 344, 388-396 (2013).</li><li>Suemizu, H., 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=Establishment+of+a+humanized+model+of+liver+using+NOD/Shi-scid+IL2Rgnull+mice.">Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice.</a> Biochem. Biophys. Res. Commun. 377, 248-252 (2008).</li><li>Tateno, C., 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=Near+completely+humanized+liver+in+mice+shows+human-type+metabolic+responses+to+drugs.">Near completely humanized liver in mice shows human-type metabolic responses to drugs.</a> Am. J. Pathol. 165, 901-912 (2004).</li><li>Hasegawa, M., 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=The+reconstituted+'humanized+liver'in+TK-NOG+mice+is+mature+and+functional.">The reconstituted 'humanized liver'in TK-NOG mice is mature and functional.</a> Biochem. Biophys. Res. Commun. 405, 405-410 (2011).</li><li>Azuma, H., 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=Robust+expansion+of+human+hepatocytes+in+Fah&#8722;/&#8722;/Rag2&#8722;/&#8722;/Il2rg&#8722;/&#8722;+mice.">Robust expansion of human hepatocytes in Fah&#8722;/&#8722;/Rag2&#8722;/&#8722;/Il2rg&#8722;/&#8722; mice.</a> Nat. Biotechnol. 25, 903-910 (2007).</li><li>Rhim, J. A., Sandgren, E. P., Degen, J. L., Palmiter, R. D., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+diseased+mouse+liver+by+hepatic+cell+transplantation.">Replacement of diseased mouse liver by hepatic cell transplantation.</a> Science. 263, 1149-1152 (1994).</li><li>Haridass, D., 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=Repopulation+efficiencies+of+adult+hepatocytes,+fetal+liver+progenitor+cells,+and+embryonic+stem+cell-derived+hepatic+cells+in+albumin-promoter-enhancer+urokinase-type+plasminogen+activator+mice.">Repopulation efficiencies of adult hepatocytes, fetal liver progenitor cells, and embryonic stem cell-derived hepatic cells in albumin-promoter-enhancer urokinase-type plasminogen activator mice.</a> Am. J. Pathol. 175, 1483-1492 (2009).</li><li>Sharma, A. D., 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=Murine+embryonic+stem+cell-derived+hepatic+progenitor+cells+engraft+in+recipient+livers+with+limited+capacity+of+liver+tissue+formation.">Murine embryonic stem cell-derived hepatic progenitor cells engraft in recipient livers with limited capacity of liver tissue formation.</a> Cell. Transplant. 17, 313-323 (2008).</li><li>Peltz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+'humanized'+mice+improve+drug+development+in+the+21st+century.">Can 'humanized' mice improve drug development in the 21st century.</a> Trends Pharmacol. Sci. 34, 255-260 (2013).</li><li>Zhu, S., 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=Mouse+liver+repopulation+with+hepatocytes+generated+from+human+fibroblasts.">Mouse liver repopulation with hepatocytes generated from human fibroblasts.</a> Nature. 508, 93-97 (2014).</li><li>Saito, M., 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=Diphtheria+toxin+receptor-mediated+conditional+and+targeted+cell+ablation+in+transgenic+mice.">Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice.</a> Nat. Biotechnol. 19, 746-750 (2001).</li><li>Ishii, T., 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=Transplantation+of+embryonic+stem+cell-derived+endodermal+cells+into+mice+with+induced+lethal+liver+damage.">Transplantation of embryonic stem cell-derived endodermal cells into mice with induced lethal liver damage.</a> Stem Cells. 25, 3252-3260 (2007).</li><li>Machimoto, T., 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=Improvement+of+the+survival+rate+by+fetal+liver+cell+transplantation+in+a+mice+lethal+liver+failure+model.">Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model.</a> Transplantation. 84, 1233-1239 (2007).</li><li>Taniguchi, H., Zheng, Y. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatic+stem+cell,+method+for+preparation+of+the+same,+method+for+induction+of+differentiation+of+the+same,+and+method+for+utilization+of+the+same.">Human hepatic stem cell, method for preparation of the same, method for induction of differentiation of the same, and method for utilization of the same.</a> Japan Patent. , (2015).</li><li>Zhang, R. -. R., 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=Human+hepatic+stem+cells+transplanted+into+a+fulminant+hepatic+failure+Alb-TRECK/SCID+mouse+model+exhibit+liver+reconstitution+and+drug+metabolism+capabilities.">Human hepatic stem cells transplanted into a fulminant hepatic failure Alb-TRECK/SCID mouse model exhibit liver reconstitution and drug metabolism capabilities.</a> Stem Cell Res. Ther. 6, 1-12 (2015).</li><li>Strom, S. C., Davila, J., Grompe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Mice+with+Humanized+Liver:+Tools+for+the+Study+of+Drug+Metabolism,+Excretion,+and+Toxicity.">Chimeric Mice with Humanized Liver: Tools for the Study of Drug Metabolism, Excretion, and Toxicity.</a> Hepatocytes. , 491-509 (2010).</li><li>Zhang, D., Luo, G., Ding, X., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+experimental+models+of+drug+metabolism+and+disposition+in+drug+discovery+and+development.">Preclinical experimental models of drug metabolism and disposition in drug discovery and development.</a> Acta Pharm. Sin. B. 2, 549-561 (2012).</li><li>Nowak, G., 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=Identification+of+expandable+human+hepatic+progenitors+which+differentiate+into+mature+hepatic+cells+in+vivo.">Identification of expandable human hepatic progenitors which differentiate into mature hepatic cells in vivo.</a> Gut. 54, 972-979 (2005).</li><li>Naglich, J. G., Metherall, J. E., Russell, D. W., Eidels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+a+diphtheria+toxin+receptor:+identity+with+a+heparin-binding+EGF-like+growth+factor+precursor.">Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.</a> Cell. 69, 1051-1061 (1992).</li><li>Mitamura, T., Higashiyama, S., Taniguchi, N., Klagsbrun, M., Mekada, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diphtheria+toxin+binds+to+the+epidermal+growth+factor+(EGF)-like+domain+of+human+heparin-binding+EGF-like+growth+factor/diphtheria+toxin+receptor+and+inhibits+specifically+its+mitogenic+activity.">Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity.</a> J. Biol. Chem. 270, 1015-1019 (1995).</li><li>Liu, Y., Yang, R., He, Z., Gao, W. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+organs+from+stem+cells.">Generation of functional organs from stem cells.</a> Cell Regener. 2, 1 (2013).</li></ol>

The authors have no competing financial interests to disclose.

最近の研究は、マウスの肝臓は、成人肝細胞および増殖性肝幹細胞17を含むヒト肝細胞で再増殖することができることを示しています。これらの再増殖した肝臓は薬物代謝試験および薬物の発見と開発18のための前臨床実験モデルとして使用されています。加えて、それらは、細胞成熟および分化19のためのインビボ環境を提供しています。本研究の主要な目的?...

Mice are commonly used for pharmaceutical testing since biomedical research in humans is restricted1; however, these models are not always useful since they may inaccurately simulate the effects observed in humans. Most drugs in current medical use are metabolized primarily in the liver. However, the same drug can be metabolized into different metabolites in mouse and human livers because of inter-species differences. Thus, it is often difficult to determine during development whether a potential drug poses any risks for clinical applications2,3. 
To address this problem, "humanized" mouse livers have been developed by growing human liver cells inside mice4-6; these models exhibit drug responses similar to those observed in the human liver. The primary mouse models currently used for humanized liver generation include uroplasminogen activator (uPA+/+) mice4,7, fumarylacetoacetate hydrolase (Fah&#8722;/&#8722;) mice6, and the recently reported thymidine kinase (TK-NOG) mice. 
However, previous reports have shown that transplanted human immature cells or stem cells are less competitive than adult human hepatocytes in Alb-uPA tg(+/&#8722;)Rag2(&#8722;/&#8722;) mouse livers8-10. Moreover, Fah&#8722;/&#8722; mice provide a growth advantage only for differentiated hepatocytes and not for immature liver progenitor cells11. The transplantation of human hepatic stem cells (HpSCs) into TK-NOG mice in the lab has been unsuccessful. Hence, no useful mouse model for the efficient engraftment of human immature liver cells currently exists. 
Thus, we developed a novel Alb-TRECK/SCID mouse model that could be efficiently repopulated with human immature hepatocytes. This transgenic mouse model expresses human heparin-binding EGF-like growth factor (HB-EGF) receptors under the control of a liver cell-specific albumin promoter. Following the administration of diphtheria toxin (DT), these mice develop fulminant hepatitis due to conditional ablation of hepatocytes, enabling donor cell residency and proliferation12. Although mouse hepatocytes have been successfully transplanted into Alb-TRECK/SCID mice in previous studies13,14, the generation of a humanized liver using Alb-TRECK/SCID mice has yet to be reported. 
In this study, humanized livers were generated in Alb-TRECK/SCID mice via transplantation of HpSCs. This humanized liver provides an in vivo environment for universal stem cell differentiation and the ability to predict human drug metabolism patterns and drug-drug interactions.

<table><tbody><tr><td>Human albumin</td><td>Sigma</td><td>A6684</td><td>Mouse</td></tr><tr><td>Human CK19</td><td>Dako</td><td>M088801</td><td>Mouse</td></tr><tr><td>Human nuclei</td><td>Millipore</td><td>MAB1281</td><td>Mouse</td></tr><tr><td>Human CK8/18</td><td>Progen</td><td>GP11</td><td>Guinea pig</td></tr><tr><td>CDCP1</td><td>Biolegend</td><td>324006</td><td>Mouse</td></tr><tr><td>CD90</td><td>BD</td><td>559869</td><td>Mouse</td></tr><tr><td>CD66</td><td>BD</td><td>551479</td><td>Mouse</td></tr><tr><td>GOT/AST-PIII</td><td>Fujifilm</td><td>14A2X10004000009</td><td></td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>11320-033</td><td></td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520</td><td></td></tr><tr><td>0.05% Trypsin-EDTA&amp;nbsp;</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Diphtheria Toxin</td><td>Sigma</td><td>D0564-1MG</td><td></td></tr><tr><td>Human Albumin ELISA Kit</td><td>Bethyl Laboratories</td><td>E88-129</td><td></td></tr><tr><td>Syringe (1 ml)</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>32G 1/2&quot; needle</td><td>TSK</td><td>PRE-32013</td><td></td></tr><tr><td>O.C.T.Compound(118 ml)</td><td>Sakura Finetek Japan</td><td>4583</td><td></td></tr><tr><td>MoFlo high-speed cell sorter</td><td>Beckman Coulter</td><td>B25982</td><td></td></tr><tr><td>DRI-CHEM 7000</td><td>Fujifilm</td><td>14B2X10002000046</td><td></td></tr></tbody></table>

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.

ALB-トレック/ SCIDマウスの肝細胞は、アルブミンプロモーターの制御下で、ヒトDT受容体HB-EGF遺伝子を発現し、DT投与12以下の細胞毒性効果を発揮します。肝障害に対するDT処置の効果を評価するために、1.5μgの/キロのDT投与量は、8週齢のアルプ・トレック/ SCIDマウスに注射し、肝臓48時間後のDT投与の病理学的変化を組織学的に評価しました。 （いないDTで処?...

Watch this Scientific Journal Video about Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells at JoVE.com

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

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 ...

generation-of-a-humanized-mouse-liver-using-human-hepatic-stem-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.8K ビュー.  Yokohama City University. 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.

ビデオ: Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

幹細胞ベースの遺伝子治療腫瘍モデルとしてBLTヒト化マウスを用いた

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

免疫・感染学

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

免疫不全マウスにおける肝直交細胞性ヒトうveal黒色腫異種移植プラットフォームの生成

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

がん研究

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

免疫不全マウスの皮下および肝内ヒト肝細胞癌異種移植片の生成

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

医学

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウス モデル

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

発生生物学

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 ...

生物学

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

ヒト化マウスモデルにおけるHIV感染に対する幹細胞ベースの設計さイミュニティ

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

HIV関連肝病因に対する二重ヒト化TK-NOGマウスモデルの確立

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

肝疾患研究のための末梢血からのヒト肝スフェロイド

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

生物工学

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

Source: Daharsh, L. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/59773">A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the method for generating a double humanized bone BLT (marrow, liver, and thymus) mouse with a functional human immune system and a stable engrafted human-like gut microbiome.

二重ヒト化BLT(骨髄、肝臓、胸腺)マウスモデルの生成

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mice ...

ヒト肝幹細胞を用いたヒト化マウス肝臓の生成

要約

さらに動画を探す

この動画の章

幹細胞ベースの遺伝子治療腫瘍モデルとしてBLTヒト化マウスを用いた

免疫不全マウスにおける肝直交細胞性ヒトうveal黒色腫異種移植プラットフォームの生成

免疫不全マウスの皮下および肝内ヒト肝細胞癌異種移植片の生成

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウスモデル

ヒト胚性幹細胞集団から肝細胞様細胞の堅牢な生成

ヒト化マウスモデルにおけるHIV感染に対する幹細胞ベースの設計さイミュニティ

HIV関連肝病因に対する二重ヒト化TK-NOGマウスモデルの確立

肝疾患研究のための末梢血からのヒト肝スフェロイド

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34⁺ Cells Injection, and Cytokine Delivery

二重ヒト化BLT(骨髄、肝臓、胸腺)マウスモデルの生成

ヒト肝幹細胞を用いたヒト化マウス肝臓の生成

要約

さらに動画を探す

この動画の章

幹細胞ベースの遺伝子治療腫瘍モデルとしてBLTヒト化マウスを用いた

免疫不全マウスにおける肝直交細胞性ヒトうveal黒色腫異種移植プラットフォームの生成

免疫不全マウスの皮下および肝内ヒト肝細胞癌異種移植片の生成

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウス モデル

ヒト胚性幹細胞集団から肝細胞様細胞の堅牢な生成

ヒト化マウスモデルにおけるHIV感染に対する幹細胞ベースの設計さイミュニティ

HIV関連肝病因に対する二重ヒト化TK-NOGマウスモデルの確立

肝疾患研究のための末梢血からのヒト肝スフェロイド

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

二重ヒト化BLT(骨髄、肝臓、胸腺)マウスモデルの生成

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウスモデル

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34⁺ Cells Injection, and Cytokine Delivery