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.

כל הפרוצדורות בבעלי חיים בוצעו בהתאם להנחיות צער בעלי חיים של אוניברסיטת העיר יוקוהמה. 1. דור של במודל העכבר הפגיע בכבד החריף <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 1 מ&quot;ל של תמיסת 0.85% NaCl phenolized (פנול 0.6 גרם ב 100 מ&quot;ל של תמיסת 0.85% NaCl) עד 1 רעלן דיפתריה מ&quot;ג (DT) כדי להפוך פתרון מניות 1 מ&quot;ג / מ&quot;ל ​​DT. הערה: DT בריכוז של 1 מ&quot;ג / מ&quot;ל ​​ניתן לאחסן כ 2 שנים 3 מעלות צלזיוס עד 8 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> סדרתי לדלל את פתרון מניות 1 מ&quot;ג / מיליליטר DT 0.3 מיקרוגרם / מיליליטר DT בתמיסת phenolized 0.85% NaCl ולהכין aliquot של פתרון DT 0.3 מיקרוגרם / מיליליטר כפתרון עבודה. הערה: הפתרון עובד זה אמור להיות מוכן טרי. </li><li style=";text-align:right;direction:rtl"> כדי לערוך ניסויים שימוש במינון sublethal (~ 50 הקטלניות%), לנהל עכברים 8 שבועות בן מנה 1.5 מיקרוגרם / ק&quot;ג של DT. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בצע זריקה intraperitoneal של DT ידי החזקת העכבר שכיבה על הגב והכנס את needlה להלן העיקול של הברכיים, שמאלה או ימינה של קו האמצע. הימנע קו האמצע כדי למנוע חדירה של שלפוחית ​​השתן. זווית את המחט על כ 45 מעלות לגוף. </li><li style=";text-align:right;direction:rtl"> באמצעות מזרק אינסולין, להזריק עכברים עם 100 μl של המוכן טרי 0.3 מיקרוגרם / מ&quot;ל ​​DT לכל 20 גרם של משקל העכבר. לדוגמה, עכבר 18 גרם צריך לקבל 90 μl של פתרון DT 0.3 מיקרוגרם / מ&quot;ל. </li></ol></li><li style=";text-align:right;direction:rtl"> בשעה 48 הזרקת שעות שלאחר DT, לאסוף דם מווריד הזנב על ידי הצבת העכבר בתוך עוצר ומחמם הזנב העכבר באמבט המים 37 מעלות צלזיוס למשך כ 10 דקות כדי להרחיב את כלי הדם. לאחר מכן, לבצע ניק 1-מ&quot;מ ס&quot;מ הזנב 2 מהקצה עם להב אזמל חד לאסוף את הדם עם צינור microcapillary. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צנטריפוגה צינור microcapillary המכיל את הדם ב 1500 XG במשך 5 דקות לאסוף את הסרום על ידי הפרדת גלולת supernatant ותא. </li></ol></li><li style=";text-align:right;direction:rtl"> פיפטה 50 μl של 1:20 סרום עכבר מדולל על GOT / AST-PIII מחליק. מדוד את הספיגה של מוצר התגובה (צבע כחול) ב 650 ננומטר באמצעות נתח רב תכליתי אוטומטית יבשה-כימיה על פי הפרוטוקול של היצרן. הערה: מתוך לקריאה של aminotransferase aspartate (AST) / transaminase oxaloacetic גלוטמית (GOT) פעילות מוצגת באופן אוטומטי. 2 ימים שלאחר DT זריקה, כ -60% מהעכברים הציג ערכים AST בין 12,000 - 16,000 IU / L ונחשבו סובל מנזק כבד חריפה. עכברים אלו הועברו לכלוב חדש ומשומש כמקבל השתלה תאית. </li></ol> 2. הכנה בתאי גזע אנושית כבדיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבודד תאי גזע בכבד אדם מתאי כבד עוברי עיקרי אדם עם סדרן תא באמצעות CDCP1, CD90, ו אנטיגנים תא CD66 להשיג subpopulation CDCP1 + CD90 + CD66-, אז זרע אוכלוסיית התאים המבודדת על קולגן תרבות מנות IV-מצופות, כפי שדווח בעבר 15. השתמש בתאים בכבד עובר עיקריים אדם של עובריגיל בין שבועות 14 ו -18. </li><li style=";text-align:right;direction:rtl"> אסוף בתאי גזע כבד אנושיים תרבותיים עד 80% - מפגש 90% על תרבות מנות 100 מ&quot;מימ, לשאוב את מדיום התרבות, לשטוף את התאים עם 10 מיליליטר PBS, ולאחר מכן להסיר את PBS. Trypsinize את התאים עם פתרון 0.05% טריפסין / EDTA 2 מ&quot;ל במשך 5 דקות ב 37 מעלות צלזיוס. הערה: תאים בבית יותר מ 90 מפגש% אינם מתאימים השתלת תאים מאז התמיינות תאים עשויה להשפיע יכולת השגשוג שלהם בעכברים. </li><li style=";text-align:right;direction:rtl"> לפקח על מצב של פיזור התאים תחת מיקרוסקופ. כאשר התאים מופיעים בריחוף או לזרום בחופשיות, לעצור את העיכול אנזימטי על ידי הוספת 8 מיליליטר של DMEM / F12 המכיל 10% FBS. </li><li style=";text-align:right;direction:rtl"> להשעות את התאים לאט בעזרת פיפטה סרולוגיות 10 מ&quot;ל ולהעביר את התאים לתוך צינור חרוטי 15 מ&quot;ל. צנטריפוגה התאים למשך 5 דקות ב 100 XG ו -4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> בזהירות resuspend התא גלולה ב 10 מ&quot;ל DMEM / F12 המכיל 10% FBS ולקבוע מספרים סלולריים באמצעות תא ספירה מיקרוסקופ. </li><li style=";text-align:right;direction:rtl">מחלקים את התאים לתוך 1.0 x 10 6 תאים לכל 50 μl של aliquots PBS עבור כל עכבר הפרט ולאחסן על הקרח עד להשתלה. </li></ol> 3. השתלת Intrasplenic של תאי גזע אנושי כבדיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים לכלוב נקי על כרית חימום חשמלי 37 ° C. </li><li style=";text-align:right;direction:rtl"> להרדים את שאיפת isoflurane באמצעות העכבר (1 - 1.5% (כרך / כרך) ב 2 ליטר של חמצן לדקה) על ידי הצבתו מתחת זרבובית. לחלופין, השתמש צינורות המכילים גזה ספוגה isoflurane. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא כי העכבר הוא מורדם על ידי צובט את השודד האחורי בקלילות. אם אידיוט ברך מתקבל, למקם את העכבר בחזרה לתא. השתמש במשחת וטרינר על העיניים כדי למנוע יובש לאחר הרדמה. </li></ol></li><li style=";text-align:right;direction:rtl"> לגלח את האתר כירורגית באמצעות קוצץ חשמלי ולהחיל 70% (כרך / כרך) אתנול povidone יוד לעקר. ואז לעשות חתך בעור 1-ס&quot;מ בכנף שמאל ממש מתחת לגבול costal של הצלעות, ואחריו חתך עלדופן הבטן, כמו גם את הצפק. </li><li style=";text-align:right;direction:rtl"> בזהירות לחשוף את הטחול. השתמש במזרק microinjection 100 μl עם מחט 32-G 1/2-inch להזריק ישירות 1.0 x 10 6 בתאי גזע בכבד האדם 50 μl PBS לתוך הטחול של כל עכבר. הטה את המחט בזווית 5 מעלות להזרקה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא כי ועומק הזרקה הוא פחות ממחצית עובי של הטחול. המחט צריכה להיכנס הטחול בקצה אחד (ראש) וכן להפקיד את התאים בקצה השני (הזנב). </li></ol></li><li style=";text-align:right;direction:rtl"> כדי למנוע דליפה של התאים לאחר ההזרקה, להפעיל לחץ בעדינות בעזרת האצבע למשך 2 דקות ולאחר מכן להסיר את המחט. </li><li style=";text-align:right;direction:rtl"> מניח את הטחול בחזרה לתוך גוף העכבר ולסגור את החלל עם תפר ריצה נורמלי השרירים והעור. </li><li style=";text-align:right;direction:rtl"> מעבירים את העכברים לכלוב מחומם מראש 37 ° C מיד לאחר ההשתלה. כדי להבטיח את העכבר הוא מסוגל לנשום בנוחות, הצב את העכבר על צדו בכלוב,רקת קשר בין מצעי כלוב באתר של ניתוח. </li><li style=";text-align:right;direction:rtl"> צג העכברים עד ההרדמה פגה כדי להבטיח את התפרים נשארים סגורות העכברים לחזור טרום ניתוח תנאים. </li><li style=";text-align:right;direction:rtl"> ודא העכברים מסופקים עם מים ומזון שתייה רגילים. אחרי השעה 1, להחזיר את הכלוב לחדר העכבר של מרכז בעלי החיים לפקח על העכברים יומיים. </li></ol> 4. איתור של מושתלי האדם כבדית גזע hepatocytes נגזר-Cell בכבד העכבר הערה: ההליכים הבאים, להרדים כל בעלי החיים באמצעות מנת יתר של קטמין ו xylazine ואחריו נקע בצוואר הרחם. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ב 4 - 6 שבועות לפרסם ההשתלה, להרדים את העכברים ולפתוח את חלל הבטן על ידי חיתוך cutis בו זמנית fascia באמצעות מספריים כירורגיות. </li><li style=";text-align:right;direction:rtl"> לנתח את רקמת החיבור מעל הצפק, באמצעות מספריים כמו מפזר, וחתך את הצפק לאורך alba Linea כדי לפתוח את הצפקחָלָל. </li><li style=";text-align:right;direction:rtl"> הרם את החזה עם מלקחיים, לנקב את הסרעפת, ולחתוך בכל צד של עצם החזה עד דרך אבנט צוואר הרחם. </li><li style=";text-align:right;direction:rtl"> לנתק את הווריד הנבוב בצד החזה של הכבד ולמשוך הוושט דרך הכבד לכיוון הקדמי. הסר את הסרעפת על מנת להסיר את הכבד. הערה: הקפד לשמור את קצות מספריים הצביע כלפי מעלה על מנת למנוע כל נזק לאיברים החזה מתחת. התימוס יש נטייה להיצמד מדי פעם בצד הגבי של החזה וזה יש להימנע בזהירות. </li><li style=";text-align:right;direction:rtl"> באמצעות הסרעפת כידית, מתחיל ללחוץ על הכבד מתוך חלל הבטן. הווריד הנבוב הפנים יקיים כבד במקום. חותכים את הווריד הנבוב פנים; להיזהר שלא לשחרר את הכליה התקינה בטרם עת. הערה: יש עכברים כבדים ארבעה אונות מורכב אונת חציון, באונה שמאלית, אונה ימנית, ואונת caudate. את כיס המרה מושעה ב הסתעפות קטנה שלאונת החציון. </li><li style=";text-align:right;direction:rtl"> הפרד את האונות אחד מהשני בצומת והטמעת אותם בטמפרטורת חיתוך אופטימלי מתחמת (אוקטובר) על פי הפרוטוקול של היצרן. Freeze ב- OCT המכיל את רקמת הכבד של רשתות מתכת על cryostat. </li><li style=";text-align:right;direction:rtl"> חותכים חלקים מדגם בעובי של 5 מיקרומטר באמצעות cryostat ב C -18 ° ולעגן אותם בשקופיות מיקרוסקופ RT. אחסן את המניה ב -80 ° C. הכן את השקופיות עבור hematoxylin ו eosin (H &amp; E) מכתים immunofluorescence פי נהלים שדווח בעבר 16. </li></ol> 5. איתור של הפרשת אלבומין אדם חישוב שיעור Chimeric <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי למדוד כינון מחדש כבד אנושי, לבצע אלבומין האנושי ELISA שימוש בסרום וערכת ELISA אדם אלבומין פי הוראות היצרן. </li><li style=";text-align:right;direction:rtl"> חשב את שיעור כימרי הכבד האנושי על ידי בזמן אמת ניתוח PCR של רמות ביטוי בכבד השלם-אנושי. קבע את האקס יחסיתpression יחס רמה תקטין אדם ספציפי כדי יקטין צלב אדם ועכבר (1 יחס) והיחס של אקטין ספציפי בעכבר כדי יקטין צלב אדם ועכבר (יחס 2). שיעור כימרי מחושב כיחס 1 / (יחס 1 + יחס 2). </li></ol>

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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.

מחקרים שנעשו לאחרונה הראו כי הכבד העכבר ניתן אוכלס מחדש עם hepatocytes אדם, כולל hepatocytes מבוגר בתאי גזע בכבד שגשוג 17. כבדי אוכלס מחדש אלה שימשו דגמים ניסיוניים פרה-קליניים עבור בדיקות סמים מטבוליזם גילוי ופיתוח תרופות 18; בנוסף, סיפקו להם בסביבה vivo עבור התבגר...

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-TRECK / hepatocytes עכברי SCID המבטא את גן HB-EGF קולטן DT אדם בשליטת אמרגן אלבומין ולהציג השפעות ציטוטוקסיות הבאים DT ממשל 12. כדי להעריך את ההשפעות של טיפול DT על פגיעה בכבד, מינונים DT של 1.5 מיקרוגרם / ק&quot;ג הוזרקו בן 8 שבועות Alb-TRECK / SCID עכברים ואת שינויים פתולו...

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.

דור של כבד עכבר אנושי באמצעות תאי גזע אנושי כבדיה

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

Research

JoVE Journal

Medicine

10.8K Views.  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.

Video: 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.

שימוש בעכבר האנושי של BLT כמודל לתאי גזע מבוסס Gene Therapy גידול

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

Immunology and Infection

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.

הדור של מלנומה האדם Uveal האנושי פלטפורמת Xהשתל בעכברים חיסוני

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

Cancer Research

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.

דור של תת עורי וintrahepatic אדם xenografts קרצינומה hepatocellular בעכברים עם מערכת חיסון חלשים

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

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.

מודל אנושי העכבר Chimeric הכבד היפרכולסטרולמיה משפחתית באמצעות Hepatocytes נגזר תאי גזע Pluripotent המושרה

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

Developmental Biology

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.

הדור יציבה של hepatocyte דמויי תאים של אדם אוכלוסיות תאים גזע עובריים

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

Biology

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.

תאי גזע מהונדסים חסינות בהתבסס נגד HIV זיהום במודל העכבר האנושי

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.

הקמתה של הומניזציה Dual-TK-חעוגה מודל העכבר עבור הנגיף הקשור HIV פתוגנזה

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.

ספרואידים של כבד אנושי מדם היקפי למחקרי מחלות כבד

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

Bioengineering

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://app.jove.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.

יצירת מודל עכבר BLT (מח עצם, כבד, תימוס) כפול אנושי

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