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.

所有的动物实验过程是按照横滨市立大学的动物保护准则进行的。 1.代急性肝损伤小鼠模型<ol><li>加入1 ml phenolized 0.85％NaCl溶液（0.6克苯酚在100毫升0.85％NaCl溶液），以1毫克白喉毒素（DT），制成1毫克/毫升DT原液。注:DT以1mg / ml的浓度可在3℃至8℃储存约2年。 </li><li>连续稀释phenolized 0.85％的NaCl溶液中的1mg / ml的DT原液至0.3微克/毫升DT和制备0.3微克/毫升DT溶液作为工作溶液的等分试样。注:该工作液应新鲜配制。 </li><li>进行使用亚致死剂量（〜50％致死）实验，辖8周龄小鼠DT的1.5微克/千克的剂量。 <ol><li>通过按住鼠标背卧执行DT腹腔注射，并插入业务承包商Ë膝盖的弯曲下面，左面或中线的权利。避免中线以防止膀胱的渗透。角针在约45°到主体。 </li><li>使用胰岛素注射器，注射的老鼠用100微升每个20 G鼠标重量的新鲜0.3微克/毫升的DT。例如，18-G鼠标应该接受90微升0.3微克/毫升DT解决方案。 </li></ol></li><li>在48小时后DT注射，通过将鼠标在一个限制器和温热小鼠尾部在37℃水浴中约10分钟，以扩张血管收集从尾静脉血液。然后，制成1毫米切口，从用锋利的手术刀刀片尖端的尾2厘米并用微毛细管收集血液。 <ol><li>离心含有血液的微毛细管在1500×g离心5分钟，并通过分离上清液和细胞沉淀收集血清。 </li></ol></li><li> 1:20稀释的小鼠血清中吸取50微升到GOT / AST-PIII幻灯片。使用根据制造商的协议的多功能自动干化学分析仪在650nm测定反应产物（蓝色染料）的吸光度。 注:读出谷草转氨酶（AST）的/自动显示谷草转氨酶（GOT）的活性。后2天DT注射，将小鼠的约60％12,000表现AST值 - 16000 IU / L，被认为是从急性肝损伤痛苦。这些小鼠被转移至新笼子并用作细胞移植。 </li></ol> 2.人力肝干细胞的制备<ol><li>隔离从人初级胎儿肝细胞与使用CDCP1，CD90和CD66细胞抗原以获得CDCP1 + CD90 + CD66-亚群细胞分选仪人肝干细胞，然后接种于涂覆IV型胶原的培养皿的分离的细胞群，如先前报道15。使用胚胎的人类首要胎肝细胞周14至18岁之间的年龄。 </li><li>收集培养至80％的人类肝干细胞 - 90％汇合于100毫米的培养皿，吸出培养基，用10毫升的PBS洗涤细胞，然后取出PBS。 Trypsinize用2ml的0.05％胰蛋白酶/ EDTA溶液中的细胞5分钟，在37℃下。注意:在大于90％汇合的细胞不适合于细胞移植自细胞分化可能在小鼠中影响其增殖能力。 </li><li>监测细胞分散液的显微镜下的状态。当细胞似乎漂浮或自由流动，通过加入8毫升含有10％FBS的DMEM / F12的停止酶消化。 </li><li>悬浮细胞慢慢使用10毫升血清吸管将细胞转移到15mL的锥形管中。离心细胞，在100 xg离心5分钟，4℃。 </li><li>小心重悬细胞沉淀在含有10％FBS的10的10ml DMEM / F12，并确定使用显微镜计数室的细胞数。 </li><li>划分细胞向每50μl的PBS等份为每个单独的小鼠的1.0×10 6个细胞，并在冰上储存直至移植。 </li></ol> 3.人肝脏干细胞内移植<ol><li>将干净的笼子里37℃的电热垫。 </li><li>通过将它放置在喷嘴的下面 - （在2L每分钟的氧气的1.5％（体积/体积）1）麻醉小鼠用异氟醚吸入。另外，使用含有用纱布浸湿异氟醚管。 <ol><li>确保鼠标轻轻捏后足垫麻醉。如果膝反射是引出，将鼠标回到室内。使用对眼睛兽医药膏，以防止干燥时的麻醉下。 </li></ol></li><li>使用电动推剪剃手术部位，并应用70％（体积/体积）乙醇和聚维酮碘消毒。然后使在左侧面只是肋的肋缘下方1厘米的皮肤切口，接着在切口腹壁，以及腹膜。 </li><li>小心露出脾脏。用100微升注射注射器带32 G1 / 2英寸的针直接注入1.0×10 6人肝干细胞在50μlPBS中进入各小鼠的脾脏。倾斜以5°角的注射针头。 <ol><li>确保注入的深度为脾脏不到一半的厚度。针应在一端（头部）进入脾脏，并在另一端（尾）沉积的细胞。 </li></ol></li><li>为了防止注射后的细胞泄漏，用手指2分钟轻轻地施加压力，然后取出针。 </li><li>放置脾放回鼠标主体和与正常运行缝合的肌肉和皮肤关闭腔。 </li><li>转移的小鼠成37℃预热的笼立即移植以下。为了保证鼠标能够舒适地呼吸，将鼠标在其一侧的笼子里，一排尿笼床上用品和手术部位之间的接触。 </li><li>监测小鼠直到麻醉消退确保缝线保持关闭并且小鼠返回到手术前的条件。 </li><li>确保将小鼠具有正常的饮用水和食品。 1小时后，返回笼动物中心的鼠标间，每日监测小鼠。 </li></ol> 4.移植的检测人肝干在小鼠肝细胞衍生肝细胞注:对于以下步骤，使用安乐死颈椎脱位氯胺酮和甲苯噻嗪过量的所有动物。 <ol><li>在4 - 第6周后移植，安乐死的小鼠和通过同时切割表皮和使用手术剪筋膜打开腹腔。 </li><li>解剖结缔组织腹膜以上，使用剪刀作为吊具，并切断腹膜沿白线，打开腹腔腔。 </li><li>解除胸骨用镊子，穿刺隔膜，并通过胸骨的每一侧穿过宫颈腰带切开。 </li><li>断绝对肝脏的胸侧的腔静脉，并通过在向前方向肝脏拉食道。为了除去肝脏去除隔膜。 注意:请小心保持剪刀提示，以防止下胸器官的损害向上指出。胸腺有偶尔紧贴胸骨背侧的趋势，这一点必须小心避免。 </li><li>使用隔膜作为手柄，启动拉动肝脏出腹腔。内部腔静脉将举行到位肝脏。切内部腔静脉;要小心，不要过早解放右肾上腺。 注:老鼠有四裂肝脏包括中叶，左叶，右叶和尾状叶。胆悬浮在小分叉中叶。 </li><li>在路口彼此分开的瓣，并根据制造商的方案在最佳切割温度（OCT）化合物嵌入其中。冻结包含在低温恒温器的金属网格肝组织的十月</li><li>用切低温恒温器5微米厚的切片样本在-18°C和安装它们RT显微镜载玻片。在-80℃保存的股票。制备用于苏木精和曙红（H＆E）和根据先前报道的方法16免疫荧光染色的幻灯片。 </li></ol> 5.人力分泌白蛋白的检测和嵌合率的计算<ol><li>来测量人体肝重构，执行人白蛋白ELISA使用血清，并根据制造商的说明一个人白蛋白ELISA试剂盒。 </li><li>通过实时计算人源化肝脏的嵌合率表达水平的PCR分析在整个-人源化肝脏。确定相对前人特异性肌动蛋白的人类 - 小鼠横动蛋白PRESSION水平比率（比率1）和小鼠 - 特异性肌动蛋白的人类 - 小鼠横肌动蛋白的比率（比例为2）。嵌合率计算为比率1 /（比1+比率2）。 </li></ol>

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The authors have no competing financial interests to disclose.

最近的研究已经表明，在小鼠肝脏可以与人肝细胞，包括成人肝细胞和增殖性肝干细胞17被重新填充。这些重新填充肝脏已被用作临床前试验模型的药物代谢试验和药物发现和开发18;此外，他们提供了用于细胞的成熟和分化19 的体内环境。本研究的主要目的是要产生，允许人未成熟肝细胞增殖，成熟和分化，对采集的药物代谢活动的新颖性肝病的小鼠模型。 <p cla...

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技术/ SCID小鼠的肝细胞表达的白蛋白启动子的控制下的人类DT受体HB-EGF的基因并显示出下列DT给药12细胞毒性作用。为了评估DT处理对肝损伤的影响，DT剂量为1.5微克/公斤，分别注入8周龄ALB-Treck技术/ SCID小鼠和在肝脏中48小时后DT给药的病理改变进行组织学评估。与对照组（不与DT处理）相比，DT-治疗的小鼠表现出与血清AST活性升高拥堵修正杂乱无章的肝架构?...

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.

利用人体肝干细胞人源化小鼠肝脏生成

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

Research

JoVE Journal

Medicine

10.8K 次观看.  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.

视频: 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.

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

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

科研

免疫与感染

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.

在免疫缺陷小鼠中生成肝脏正交人子宫黑色素瘤异种移植平台

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

癌症研究

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.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

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.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

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

发育生物学

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.

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

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.

干细胞基工程免疫抗HIV感染的人性化小鼠模型

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.

建立与艾滋病毒相关的肝脏发病机制的双人性化TK-NOG小鼠模型

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.

用于肝病研究的外周血人肝球

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

生物工程

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.

生成双人源化 BLT（骨髓、肝脏、胸腺）小鼠模型

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