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.

모든 동물 실험 절차는 요코하마 시립 대학의 동물 보호 지침에 따라 수행 하였다. 급성 간 손상 마우스 모델 1 세대 <ol><li> 1 ㎎ / ㎖ DT 원액을 1 mg을 디프테리아 독소 (DT) 내지 (0.85 % NaCl 용액 100 ㎖에 0.6 g의 페놀) phenolized 0.85 % NaCl 용액 1 ㎖를 추가한다. 주 : 1 밀리그램의 농도 DT는 / ㎖ 3 ° C ~ 8 ° C에서 약 2 년 동안 저장 될 수있다. </li><li> 직렬 phenolized 0.85 % NaCl 용액에서 0.3 μg의 / ㎖ DT에 1 ㎎ / ㎖ DT 원액을 희석 작업 솔루션으로 0.3 μg의 / ㎖의 DT 용액의 분취 량을 준비합니다. 참고 :이 작업 솔루션은 새로 제조해야한다. </li><li> 치사 용량 (~ 50 % 치사)를 사용하여 실험을 수행, 8 주령 생쥐에게 DT의 1.5 μg의 / kg 투여 량을 관리. <ol><li> 지느러미 드러 누움에 마우스를 잡고 DT의 복강 내 주사를 수행하고 needl를 삽입무릎의 굽힘 아래 전자는 왼쪽이나 정중선의 오른쪽. 방광의 침투를 방지하기 위해 중간 선을 피하십시오. 각도 몸에 바늘이 약 45 °. </li><li> 인슐린 주사기를 이용하여 마우스의 체중 20g 당 0.3 μg의 새로 제조 / ㎖의 100 ㎕ DT와 마우스를 주입. 예를 들어, 18 g의 마우스는 0.3 μg의 / ㎖의 DT 솔루션의 9​​0 μl를 받아야합니다. </li></ol></li><li> 48 시간 후 DT 주입에서는, 혈관을 팽창하기 위해 약 10 분 동안 37 ℃의 수욕 마우스 꼬리 제한 수단에 마우스를 위치시키고 가온하여 꼬리 정맥으로부터 혈액을 수집한다. 그런 다음, 날카로운 메스 블레이드와 함께 끝에서 꼬리 2cm에 1 mm 닉을하고 마이크로 캐 필러 튜브에 혈액을 수집합니다. <ol><li> 5 분 1,500 XG에서 혈액을 포함하는 마이크로 캐 필러 튜브를 원심 분리기와 뜨는 및 세포 펠렛을 분리하여 혈청을 수집합니다. </li></ol></li><li> 1시 20분 희석 마우스 혈청의 피펫 50 μL GOT / AST- 상PIII는 슬라이드. 제조사의 프로토콜에 따라 다목적 건조 화학 자동 분석기를 사용하여 650 nm에서의 반응 생성물 (청색 색소)의 흡광도를 측정한다. 참고 : 아스 파르 테이트 아미노 전이 효소 (AST)의 판독은 / 글루타민산 옥 살로 트랜스 아미나 제 (GOT) 활동이 자동으로 표시됩니다. 16,000 IU / L 급성 간 손상을 앓고있는 것으로 간주 하였다 - 이일은 DT가 주입, 쥐의 약 60 % 12,000 사이 AST 값을 나타내 게시합니다. 이러한 마우스는 새로운 케이지 전달 및 세포 이식을 사용 하였다. </li></ol> 인간의 간 줄기 세포의 2. 준비 <ol><li> CDCP1 + CD90 + CD66- 아군를 얻을 CDCP1, CD90 및 CD66의 세포 항원을 이용한 셀 소터 인간 일차 태아 간세포에서 인간 간 줄기 세포를 분리 한 다음, 콜라겐 IV 코팅 된 배양 접시에 절연 세포군 시드, 앞서 15 일 보도했다. 배아의 인간의 기본 태아 간 세포를 사용하여주 14과 18 사이의 나이. </li><li> 배양액을 흡인 100-mm 배양 접시에 90 % 합류 10 ml의 PBS로 세포를 세척 한 후 PBS를 제거 - 80 % 배양 된 간 줄기 세포를 수집한다. 37 ° C에서 5 분 동안 2 ml의 0.05 % 트립신 / EDTA 용액으로 세포를 Trypsinize. 주 : 세포 분화 쥐 그들의 증식 능력에 영향을 미칠 수 있기 때문에, 90 % 이상의 세포가 합류 세포 이식에 적합하지 않다. </li><li> 현미경 하에서 세포의 분산액의 상태를 모니터한다. 세포가 떠하거나 자유롭게 유동 할 때 나타나는 10 % FBS를 함유하는 DMEM / F12 8 ml를 첨가하여 효소 소화를 멈춘다. </li><li> 천천히 10 ㎖의 혈청 피펫을 사용하여 세포를 정지하고, 15 mL의 원뿔형 튜브에 세포를 옮긴다. 100 XG에 5 분, 4 ° C를위한 세포를 원심 분리기. </li><li> 조심스럽게 10 % FBS를 함유하는 10 ㎖의 DMEM / F12에서 세포 펠렛을 재현 탁하고 현미경 카운팅 챔버를 이용하여 세포 수를 결정한다. </li><li>각 마우스 PBS 씩 50 μL 당 1.0 × 10 6 세포로 세포를 나누어 이식까지 얼음에 저장합니다. </li></ol> 인간의 간 줄기 세포의 3 Intrasplenic 이식 <ol><li> 37 ° C 전기 가열 패드에 깨끗한 케이지를 놓습니다. </li><li> 노즐 아래에 배치하여 - (분당 산소의 2 L에서 1.5 % (부피 / 부피) 1) 마우스 사용 이소 플루 란 흡입 마취. 또한, 이소 플루 란 적신 거즈를 포함하는 튜브를 사용합니다. <ol><li> 마우스를 가볍게 뒤쪽 발바닥을 곤란하게하여 마취되어 있는지 확인합니다. 무릎 경련이 유발되는 경우, 다시 실에서 마우스를 놓습니다. 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. </li></ol></li><li> 전기 면도기를 사용하여 수술 부위를 면도하고 (부피 / 부피) 에탄올 및 포비돈 요오드 소독 할을 70 %를 적용합니다. 그런 다음 절개에 이어 바로 리브의 늑골 테두리 아래 왼쪽 측면에서 1 cm의 피부 절개를 만들복벽뿐만 아니라 복막. </li><li> 조심스럽게 비장 노출됩니다. 직접 각 마우스의 비장에 50 ㎕의 PBS에 1.0 × 106 인간 간 줄기 세포를 주입하기 위해 32-G / 2 인치 바늘 100 μL 미세 주입 주사기를 사용한다. 주입을위한 5 ° 각도로 바늘을 기울입니다. <ol><li> 주사의 깊이가 비장의 절반 이하 두께 있는지 확인하십시오. 바늘을 한쪽 끝 (헤드)에서 비장을 입력하고, 다른 단부 (꼬리)의 셀을 증착한다. </li></ol></li><li> 주입 따라 셀의 누출을 방지하기 위해, 2 분간 부드럽게 손가락을 이용하여 압력을인가하고 바늘을 제거. </li><li> 다시 마우스 본체에 비장을 놓고 근육과 피부를위한 정상 주행 봉합와 공동을 닫습니다. </li><li> 즉시 이식 다음 37 ° C 미리 예열 케이지에 쥐를 전송합니다. 마우스를 보장하기는 편안하게 호흡 케이지에 옆에 마우스를 배치 할 수있다케이지 침구 및 수술 부위 사이의 접촉을 무효화. </li><li> 마취가 봉합 폐쇄 유지하기 위해 오프 착용하고 마우스 조건 수술 전으로 돌아갈 때까지 마우스를 모니터링합니다. </li><li> 마우스를 확인하는 것은 정상적인 마시는 물과 음식이 제공됩니다. 1 시간 후, 동물 센터의 마우스 방에 케이지를 반환하고 매일 쥐를 모니터링 할 수 있습니다. </li></ol> 4. 감지 이식 인간의 간 마우스 간에서 세포 유래 간세포 줄기 주 : 다음 절차는 자궁 경부 전위 다음 케타민과 자일 라진의 과다 복용을 사용하여 모든 동물을 안락사. <ol><li> 4시 - 육주, 이식을 게시 쥐를 안락사 동시에 커티스 절단 및 밴드 수술 가위를 사용하여 복강을 엽니 다. </li><li> , 복막 위의 결합 조직을 해부 확산기로 가위를 사용하고 복막을 열 수있는 원격 교육 알바 함께 복막을 잘라공동. </li><li> , 집게로 흉골을 들어 올려 다이어프램에 구멍 및 자궁 거들을 통해 흉골의 각면을 잘라. </li><li> 간 흉부 측의 대정 맥을 절단하고, 앞쪽 방향으로 간을 통해 식도를 당깁니다. 간을 제거하기 위하여 진동판을 제거한다. 참고 : 위의 팁은 아래의 흉부 장기의 손상을 방지하기 위해 위쪽으로 지적 유지하기 위해주의해야합니다. 흉선은 때때로 흉골의 지느러미 측면에 집착하는 경향이있다이 조심스럽게 피해야한다. </li><li> 핸들로 진동판을 사용하여, 복강 내에서 간 당기는 시작한다. 내부 대정맥은 장소에 간을 개최합니다. 내부 대정 맥을 잘라; 중간에 오른쪽 부신을 해방 않도록주의하십시오. 참고 : 마우스가 중간 엽으로 구성된 네 잎 모양 간, 왼쪽 엽, 우엽과 미상 로브 있습니다. 담낭은 작은 분기에 현탁중간 엽. </li><li> 접합에서 서로의 로브를 분리하고 제조 업체의 프로토콜에 따라 최적의 절삭 온도 OCT () 화합물을 포함. 그라 이오 스탯의 금속 격자의 간 조직을 포함 OCT를 고정. </li><li> -18 ° C에서 그라 이오 스탯을 사용하여 5 μm의 두께 샘플 부분을 잘라 RT 현미경 슬라이드에 그들을 탑재합니다. -80 ° C에서 주식을 저장합니다. 헤 마톡 실린 및 에오신 (H &amp; E)과 이전에보고 절차 (16)에 따라 면역 형광 염색법에 대한 슬라이드를 준비합니다. </li></ol> 5. 인간 알부민 분비의 탐지 및 키메라 비율​​의 계산 <ol><li> 인간 간 재구성을 측정하기 위해, 혈청 및 제조자의 지시에 따라 인간 알부민 ELISA 키트를 사용하여 인간 알부민 ELISA 수행한다. </li><li> 전체 - 인간화 간에서 실시간으로 발현의 PCR 분석을 인간형 키메라 간 비율을 계산한다. 상대 예를 결정인간의 마우스 크로스 굴지 인간 고유의 굴지의 Pression의 수준 비율 (비율 1) 인간 - 마우스 크로스 굴지에 마우스 특정 굴지의 비율 (비율 2). 키메라 비율​​은 비율이 1 /로 (비율 1 + 비율 2)를 계산한다. </li></ol>

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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.

최근의 연구는 마우스 간 성인 간세포 증식 및 간 줄기 세포 (17)를 포함하여 인간 간세포 증식을 할 수 있음을 보여 주었다. 이 다시 채워 간 약물 대사 시험 및 신약 개발 18 전임상 실험 모델로 사용되고있다; 뿐만 아니라, 그들은 세포 성숙과 분화 (19)에 대한 생체 내 환경을 제공하고 있습니다. 본 연구의 주된 목적은 약물 대사 활동의 획득을 위해, 인간 미성숙 간...

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.

장백의-트렉 / SCID 쥐 간세포는 알부민 프로모터의 조절 하에서 인간 DT 수용체 HB-EGF 유전자를 발현하고 DT 정부 (12) 아래의 세포 독성 효과를 나타낸다. 간 손상에 DT 치료의 효과를 평가하기 위해, 1.5 μg의 / kg의 DT의 용량은 8 주령 장백의 - 트렉 / SCID 마우스에 주입하고 간 48 시간 포스트 DT 관리의 병리학 적 변화는 조직 학적으로 평가 하였다. (DT하지 처리) 대조군 ...

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.

인간 간 줄기 세포를 이용한 인간형 마우스 간 세대

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 조회수.  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.

비디오: 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.

줄기 세포 기반 유전자 치료 종양 모델로 BLT 인간화 마우스를 사용하여

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

연구

면역학 및 감염병학

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.

면역결핍 마우스에서 간 직교 인간 편도 흑색종 Xenograft 플랫폼의 생성

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

암 연구학

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.

면역 결핍 마우스의 피하 및 간내 인간의 간세포 암 이종 이식의 생성

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

의학

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.

가족성 콜레스테롤 혈 증 인간의 간 공상 마우스 모델을 사용 하 여 유도 만능 줄기 세포 유래 Hepatocytes

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

발생학

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.

인간 배아 줄기 세포 인구에서 Hepatocyte 같은 전지의 강력한 생성

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

생물학

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.

인간화 된 마우스 모델에 HIV 감염에 대 한 줄기 세포를 기반으로 설계된 내성

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 관련 간 병인을 위한 이중 인간화 TK-NOG 마우스 모델 구축

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.

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

생체공학

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.

이중 인간화 BLT(Bone-Marrow, Liver, Thymus) 마우스 모델 생성

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