这项工作是与来自英国的再生医学平台奖（MRC MR / L022974 / 1和MR / K026666 / 1）与中国国家留学基金支持。

注:在本协议中使用的所有试剂供应商信息已经列于表1。当细胞是有一个与它们直接接触的所有媒体/板应该是无菌的并且至少在室温下。 1.传代人多能干细胞（hPSCs）到521层粘连蛋白注意:下面描述的细胞传代过程基于单个细胞，是理想的从hPSCs肝细胞样细胞的均匀群体的推导。菌落电镀也可以适用与先前已24所述。 <ol><li>根据需要准备层粘连蛋白包被的平板。 <ol><li>在4℃解冻重组521层粘连蛋白（LN-521）的100微克/毫升的股票。 </li><li>稀释解冻的LN-521在冰冷的1X DPBS（与钙 /镁离子 ），以使5微克/毫升的溶液。 </li><li>添加5微克的1毫升/毫升的LN-521溶液涂覆一个6孔板的一个孔中和岩石中的孔均匀分布的。 </li><li>孵育所述板在2 37℃/ 5％CO 2的细胞培养孵化- 4小时紧急使用或在4℃冰箱中过夜。 </li><li>根据需要存储层粘连蛋白包被的板在4℃冰箱中。保持在一个平面上的板和它们密封，以避免蒸发和污染。 注意:不要让涂井干涸;顶起来额外1X DPBS（有钙 /镁离子 ），如果需要的话。 2周内使用平板。 </li></ol></li><li>允许所需预涂覆板的编号，以在使用前，在37℃进行0.5达到室温或孵育所述板 - 1小时。 </li><li>小心吸出涂层的LN-521溶液，而不会损坏涂层表面。注意:这是至关重要的在其上播种细胞前不要损坏涂覆的表面。 </li><li>立即添加补充有10μM的Rho相关激酶（ROCK）抑制剂Y 2预热-mTeSR1介质的1毫升7632至一个6孔板的孔。离开板在细胞培养孵化器接收细胞。 注意:不要让层粘连蛋白涂井干。 </li><li>吸从维护良好的hPSCs培养基在约75％至85％的汇合。从6孔板的一个孔用1mL室温1X DPBS（无钙 /镁离子 ）洗细胞一次。 </li><li>添加0.5mL的1×的Accutase到细胞中并在37℃下6孵育 - 8分钟以解离细胞。 注:要检查消化是否足够或不长，轻轻拍打板，并检查细胞是否可以很容易地取下。如果是的话，那么现在是时候停止酶反应;如果没有，延长消化了额外的1 - 2分钟。 </li><li>加入2毫升新鲜mTeSR1培养基补充有10μM的Y27632到细胞中终止的解离。移液器上下使用P1000尖端几次，使单细胞悬浮液。 </li><li>算使用血球的活细胞。使用台盼蓝染色并排除死细胞14 </li><li>计算出所需的细胞的总数。对于常规HPSC传代，种子每一个6孔板的孔4×10 5至5×10 5个细胞（ 即，4.21×10 4〜5.26×10 4个 每平方厘米）。为传代hPSCs为肝细胞分化，种子6.5×10 5〜7.5×10 5（ 即，6.84×10 4〜7.89×10 4每厘米2）每一个6孔板的孔的细胞。 注意:对于每个细胞系的接种密度可能需要根据这里给出的肝分化的经验密度较小的优化。 </li><li>转移所需要的细胞悬浮到无菌的15毫升或50毫升的离心管中并离心，在115×g离心，在室温下搅拌3分钟。 </li><li>慢慢吸出上清液，然后重悬细胞沉淀在补充有10μM的Rho相关激酶（ROCK）inhibito新鲜，温暖mTeSR1介质- [R Y27632，使用适当的介质进行所需的细胞密度。 注意:使用ROCK抑制剂的强烈建议，以增强细胞附着和存活率。 </li><li>种子的细胞所制备的板和摇动它们来回和侧方向均匀地分布在细胞。 注:关键是要确保细胞均匀分布在井板是用于常规细胞培养或肝细胞分化的实验。 </li><li>放置在细胞培养箱板和保持在37℃/ 5％CO 2的细胞24小时，以使它们能够连接和恢复。 </li><li>检查第二天细胞和如果细胞 - 细胞接触已经建立撤回ROCK抑制剂。保持在mTeSR1培养基中的细胞的常规培养，或根据需要切换到分化培养基。 注意:如果将细胞用所提到的密度接种，在汇合应是理想的日常维护或肝细胞differentiatioñ。 </li></ol> 2.区分hPSCs对重组层粘连蛋白肝细胞样细胞<ol><li>准备分化培养基。 <ol><li>使人类激活素A股解决方案:溶解人激活素的粉末，使无菌0.2％牛血清白蛋白（BSA）/ DPBS 100微克/毫升原液。让小等份并将其存放在-20°C。使用1:1000。 </li><li>使小鼠的Wnt 3a中原液:溶于小鼠的Wnt 3a中粉末以使在无菌0.2％BSA / DPBS 10微克/毫升储液。让小等份并将其存放在-20°C。使用在1:200。 </li><li>使人类肝细胞生长因子（HGF）原液:溶解人类HGF粉末，使在无菌0.2％BSA / DPBS 10微克/毫升储液。让小等份并将其存放在-20°C。使用1:1000。 </li><li>使制瘤素M（OSM）原液:溶解制瘤素M（OSM）粉末，使在无菌0.2％BSA / DPBS 20微克/毫升储液。让小等份和s撕毁他们在-20°C。使用1:1000。 </li><li>使内胚层吸库存培养基500mL的2％B27补充物（50×， 减去维生素A）和1％青霉素/链霉素（最终浓度为100国际单位/毫升和100微克/毫升，分别）;顶起来使用罗斯威尔公园纪念研究所1640（RPMI 1640）培养基500毫升。注:存放库存在4℃和两周内使用。从库存等分试样中，（100毫微克/毫升和50毫微克/毫升，最终浓度）在各平台改变添加新鲜活化素A和Wnt 3a上。 </li><li>使500毫升的KSR / DMSO分化培养基:80％敲除DMEM（KO-DMEM），20％敲除血清替代品（KSR），0.5％Glutamax的，1％非必需氨基酸（NEAA），0.1毫摩尔β-巯基乙醇， 1％DMSO和1％青霉素/链霉素（最终浓度为100国际单位/毫升和100微克/毫升，分别）。真空过滤。储存在4°C和两周内使用。 </li><li>让500毫升HepatoZYME成熟培养基:1％胃肠utaMAX，10μM可的松21-半琥珀酸钠盐（HCC）和1％青霉素/链霉素（最终浓度为100国际单位/毫升和100微克/毫升，分别）;顶起来使用HepatoZYME培养基500毫升。注:存放库存在4℃和两周内使用。从库存等分试样中，（在10毫微克/毫升和20毫微克/毫升，终浓度分别）为每个介质变化补充新鲜HGF和OSM。 </li></ol></li><li>种子hPSCs对LN-521肝细胞分化，如如果LN-521 / LN-111被用作基底在部分1中所述，外套与LN-521和LN-111板（1:3的比例）在5微克/毫升的最终层粘连蛋白的浓度;其余治疗应该是相同的纯LN-521包被的平板。 注:LN-521 / LN-111是不理想的hPSCs的常规培养;它仅用于分化实验。 </li><li>检查细胞汇合接种后24小时。引发细胞分化，一旦细胞汇合达到约40％。 - [Remove废mTeSR1介质和添加补充有100纳克/毫升激活素A和50ng / mL的Wnt信号3a中新鲜胚层吸介质。把这种分化1天。 注:强烈建议开始分化一天接种细胞后。 </li><li>改变内胚层吸介质每24小时3天的人类胚胎干细胞（胚胎干细胞）。作为人类诱导多能干细胞（人iPS细胞），2天以上的细胞延长此阶段素对定形内胚层样的细胞，但仅使用用100ng补充内胚层吸介质/ mL的激活素A，这两天12 。 注:为了确保成功的内胚层规范，可以检查内胚层标记物，如FOXA2和SOX17的表达。根据免疫荧光染色，所述的细胞的80％以上是阳性的在我们的实验室两个标记。 </li><li>切换到第4天（为人类胚胎干细胞）KSR / DMSO分化培养基/ 6天（为iPS细胞）。更改介质D.aily为第3天，然后在此分化阶段的第五天。 注:需要在这个分化阶段的第四天没有喂养。使用未补充的KO-DMEM至中改变之前洗一次细胞，如果有许多死细胞。为了检查此阶段分化是否成功与否，人们可以检测肝祖细胞标记，诸如甲胎蛋白，CK19，和HNF4A的表达。细胞的近90％将利好根据我们的经验这些标记。 </li><li>后KSR / DMSO分化阶段的5天，切换到HepatoZYME成熟阶段。去除KSR / DMSO中后，用纯HepatoZYME基础培养基洗涤细胞一次。添加HepatoZYME成熟培养基补充有10毫微克/毫升HGF和20毫微克/毫升的OSM。 </li><li>改变介质每48小时7 - 10天，在该点的细胞准备用于标准表征或进一步使用。 注:肝细胞标志物表达的考试，代谢功能试验（如细胞色素P450活性），尿素和白蛋白分泌试验，糖原存储测试，和吲哚花青绿（ICG）摄取试验是典型表征方法。 注:该分化方案的时间表示于图5。在我们的实验室，我们例行检查派生HLCS"肝细胞特异性标志物的表达水平，白蛋白分泌，细胞色素P450（CYP）3A和1A2活动。 </li></ol>

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<li>Martinez-Hernandez, A., Delgado, F. M., Amenta, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+extracellular+matrix+in+hepatic+regeneration.+Localization+of+collagen+types+I%2C+III%2C+IV%2C+laminin%2C+and+fibronectin%5BTitle%5D)+AND+%22Lab+Invest%22%5BJournal%5D)">The extracellular matrix in hepatic regeneration. Localization of collagen types I, III, IV, laminin, and fibronectin.</a> Lab Invest. 64 (2), 157-166 (1991).</li>
<li>Lorenzini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterisation+of+a+stereotypical+cellular+and+extracellular+adult+liver+progenitor+cell+niche+in+rodents+and+diseased+human+liver%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver.</a> Gut. 59 (5), 645-654 (2010).</li>
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C.大卫博士干草是Stemnovate有限公司的联合创始人和董事。

为了推进人类多能干细胞研究和转化医学，无异物系统，与所需电流的良好生产规范准则的规定。键任何分化过程是细胞外基质（ECM）。不仅ECM支持细胞附着，而且还提供了访问关键信号因子，影响细胞的决心和表现型25,26。 层粘连蛋白在体内的多功能细胞外基质蛋白。在肝脏中，层粘连蛋白分泌的局部切除27之后是用于肝脏再生的关键和所需的肝祖细胞维持28。在肝脏维持和再生层粘连蛋白的重要性是在我们的肝细胞分化的系统测试市售的重组人层粘连蛋白的基础。 他出众patocyte分化相比，基质胶时LN-521和LN-521 / LN-111基板实现。衍生HLCS显然极化和在盘组织，并与他们的凝胶状蛋白质混合物的对应时，他们的细胞功能被显著改善。标的这些改进是污染冒号的下调，fibroblast-和茎在层粘连蛋白细胞相关的基因，以及在细胞增殖和迁移相关基因表达10下降。 总之，这里描述的方案产生肝细胞样细胞是在自然界接近成人肝细胞。该工艺重现性好，易于自动化，并能经济高效地缩放应用程序可以。重要的是，批与批变异已经显著相比下降到使用基质胶，导致此领域内的研究人员的改进的分化系统的技术。 </p&gt;

原代人类组织和衍生物细胞类型是经常使用，都为基于细胞的筛选和在诊所。然而，进入这些细胞被严重限制，由于器官捐献和细胞表型后的隔离1的损失不足。 hPSCs代表一个有前途的替代主组织和促进遗传限定的和可再生的人类体细胞的产生。从hPSCs源性肝样细胞（HLCS）已显示出在这一领域的承诺。 HLCS像在各个方面的原代人肝细胞，包括细胞形态，肝细胞的基因表达，代谢功能，和敏感性药物和病毒2，3，4，5，6，7，8。此外，无限增殖和既research-和GMP级hPSCs的自我更新能力有助于其应用9,10。 以上研究的十年已经产生了一些有效的肝细胞分化程序2，3，5，11，12，13，14，15，16，17，18，19，20。然而，大多数这些系统的使用不确定的成分和/或病毒转导来驱动肝细​​胞癌规范。为了改善在尺度该技术的可靠性，开发出强健肝细胞分化是重要这是真正定义的系统，无异物，以及GMP兼容。 层粘连蛋白（逻辑节点）是可以影响细胞粘附，增殖，迁移和分化的重要的细胞外基质蛋白。层粘连蛋白是一种α一β和一个γ链组成的异源糖蛋白。最近，重组人层粘连蛋白已经产生并在细胞生物学中使用。 LN-511已显示，以支持hPSCs 21的维护，而LN-521和E-cadherin的混合物允许克隆衍生和人胚胎干细胞22的扩张。 LN-111，在另一方面，支持从hPSCs 23衍生的肝细胞样细胞的维持。然而，我们的报告之前，层粘连蛋白521和111没有被用来从hPSCs 10产生与成熟特性HLCS。 在这里，我们对LN-521培养hPSCs详细过程和上区分他们要么LN-521或LN-521和LN-111（LN-521 / LN-111）的共混物。我们通过优化单细胞接种到产生HLCS的多种格式的14高度重复性和同质单层分化协议。我们相信，我们定义的分化系统是一个简单，经济有效的方法来制造HLCS积极申请，代表在该领域迈出了一步显著。

<table><tbody><tr><td>Human Recombinant Laminin 521</td><td>BioLamina</td><td>LN521-02</td><td></td></tr><tr><td>Human Recombinant Laminin 111</td><td>BioLamina</td><td>LN111-02</td><td></td></tr><tr><td>Recombinant mouse Wnt3a</td><td>R&amp;amp;D Systems</td><td>1324-WN-500/CF</td><td></td></tr><tr><td>Human Activin A</td><td>Peprotech</td><td>120-14E</td><td></td></tr><tr><td>Human Hepatocyte Growth Factor</td><td>Peprotech</td><td>100-39</td><td></td></tr><tr><td>Human Oncostatin M</td><td>Peprotech</td><td>300-10</td><td></td></tr><tr><td>Rho-associated kinase (ROCK) inhibitor Y27632&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Y0503-1MG</td><td></td></tr><tr><td>Hydrocortisone 21-hemisuccinate sodium salt</td><td>Sigma-Aldrich</td><td>H4881</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>mTeSR1 medium</td><td>STEMCELL Technologies</td><td>05850</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>21875</td><td></td></tr><tr><td>Knockout DMEM</td><td>Life Technologies</td><td>10829</td><td></td></tr><tr><td>HepatoZYME</td><td>Life Technologies</td><td>17705</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>12587-010</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Life Technologies</td><td>10828</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Life Technologies</td><td>11140</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Life Technologies</td><td>31350</td><td></td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>DPBS with Calcium and Magnesium</td><td>ThermoFisher</td><td>14040133</td><td></td></tr></tbody></table>

defined and scalable generation of hepatocyte-like cells from human pluripotent stem cells

人类多能干细胞（hPSCs）具有对生物医学研究具有重要价值。 hPSCs可以缩放和分化为在人体中发现的所有类型的细胞。 hPSCs人类肝细胞样细胞（HLCS）的分化已被广泛研究，并有效的分化方法已经确立。细胞外基质和生物刺激，包括生长因子，细胞因子和小分子的组合使得有可能生成类似于原代人肝HLCS。然而，大多数的程序仍然使用不确定的成分，从而产生批与批变异。这作为一个显著障碍的技术的应用。为了解决这个问题，我们开发了一个定义系统，肝细胞分化用重组人层粘连蛋白的结合细胞外基质与无血清分化过程。高效的肝细胞规范是实现的，与去monstrated两个HLC功能和表型的改进。重要的是，这个系统易于扩展使用的研究和GMP级HPSC线基于细胞的造型和治疗有希望的进展。

从hPSCs分化肝细胞一种人胚胎干细胞系，H9，以及一个人类诱导多能干细胞系，33D6，被用于肝细胞分化。在图1-3中的结果来自H9细胞，而在图4中是从33D6细胞。接种到层粘连单细胞24小时后已建立的细胞 - 细胞接触。将细胞在40％汇合达到后，分化进程启动（ 图1A和图4A）。关于层粘连蛋白（二者LN-521和LN-521 / LN-111），这些细胞经历了连续的形态变化并引起偏振光HLCS（ 图1和图4A）。 肝细胞样细胞特征达ý18 HLCS收集和评估的代表肝细胞标志物，HNF4A和ALB（ 图2A）的表达。 18天HLCS的免疫染色表明，近90％的细胞表达HNF4α（ 图2B）。这些细胞极化的层粘连蛋白和展出了多边形的外观，标志着E-cadherin和F-肌动蛋白的表达（ 图2B）。 细胞色素P450（CYP）活性也进行评估。所述CYP450s进行肝细胞的重要代谢功能。衍生于凝胶状蛋白质混合物18天HLCS，如基质胶，LN-521，或LN-521 / LN-111，为CYP3A活性进行了测试。 HLCS表现出对比基质胶层粘连蛋白底物（ 图3）显著更高CYP3A活性。重要的是，相比于商业的人原代肝细胞时（HU1339）在这些基板上再镀，HLCS有近10倍更高的水平CYP3A的活动10。 iPS细胞的分化相似，人类胚胎干细胞。的细胞表现出在外观上（ 图4A）的顺序变化。衍生HLCS表示一个关键肝细胞转录因子，HNF4α（ 图4B），且具CYP3A活性和分泌白蛋白（ 图4C和D）。值得注意的是，从33D6衍生HLCS相比于衍生HLCS（ 图3）的H9细胞显示的减小CYP3A，但它仍然是相当于人原代肝细胞10。然而，这些HLCS的白蛋白分泌较原代肝细胞10低得多。 <img alt="图1" src="/files/ftp_upload/55355/55355fig1.jpg" /> 图1:肝分化过程中的顺序形态学变化。 &lt;/ STRONG&gt;（一）未分化的胚胎干细胞作为种子细胞单约40％汇合达到播种后24小时。 （B）的吸后，将细胞表现出对4（C）的一天的典型的内胚层的形态在到达肝细胞样阶段，它们显示对第9天（D）的一个明确的多边形形状的成熟阶段之后，偏振HLCS准备为在一天18比例尺= 200微米这里显示进一步表征。 <a href="http://ecsource.jove.com/files/ftp_upload/55355/55355fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55355/55355fig2.jpg" /> 图2:HLCS表征。 （A）的肝细胞特异性标志物基因表达，HNF4A（左）和ALB（右）。使用衍生一天HLCS 18表达水平进行了分析从两个LN-521和LN-521 / LN-111人类胚胎干细胞，并将其标准化为管家基因GAPDH和表示相对于胚胎干细胞。结果代表三次生物学重复，且误差棒代表标准偏差（SD）。 * P &lt;0.05，** P &lt;0.01;非配对t检验。一个关键的肝标记，HNF4α和偏振标记，E-cadherin和F-肌动蛋白（二）蛋白的表达。第18天对LN-521和LN-521 / LN-111进行染色用于上述标记和用Hoechst 33342的阴性对照复染HLCS用相应的免疫球蛋白G（IgG）的执行。 HNF4α阳性细胞的百分比和SD被示出。这是从图4个随机字段计算。图像是在20X放大倍数下拍摄。比例尺= 100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55355/55355fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:HLCS的代谢功能鉴定。基质胶（MG），LN-521，或LN-521 / LN-111培养细胞CYP3A的细胞色素P450活性进行了测试。数据代表三次生物学重复，且误差棒代表SD。 ** P &lt;0.01;单向ANOVA和Tukey的事后检验。 <a href="http://ecsource.jove.com/files/ftp_upload/55355/55355fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55355/55355fig4.jpg" /> 图4:HLCS标准表征。在LN-521培养iPS细胞分化成HLCS。标准特性测试是在17日进行的HLCS。 （A）的肝分化过程中的细胞的连续形态;所显示的时间点 - [R<html> epresent细胞上天1，4,9，和HNF4α表达17.（B）的免疫染色。阳性细胞的百分比和SD是基于图4个随机字段示出。图像是在20X放大倍数下拍摄。比例尺= 100微米。在17日HLCS（C）CYP3A的活动。数据代表6生物学重复和误差条表示标准差。衍生HLCS（四）白蛋白分泌过文化24小时。数据代表4个生物学重复和误差条表示标准差。 <a href="http://ecsource.jove.com/files/ftp_upload/55355/55355fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55355/55355fig5.jpg" /> 图5:分化方案的示意图时间轴。T ="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells at JoVE.com

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

这里介绍的方法描述了一个可扩展性和良好生产规范（GMP）-ready分化系统生成的多能干细胞的人类肝细胞样细胞。它作为一个具有成本效益的和标准化的系统以产生用于基础和应用的人肝研究人肝细胞样细胞。

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

defined-scalable-generation-hepatocyte-like-cells-from-human

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.5K 次观看.  University of Edinburgh. 这里介绍的方法描述了一个可扩展性和良好生产规范（GMP）-ready分化系统生成的多能干细胞的人类肝细胞样细胞。它作为一个具有成本效益的和标准化的系统以产生用于基础和应用的人肝研究人肝细胞样细胞。 

视频: Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

科研

发育生物学

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

多能干细胞半自动化生产肝样细胞的研究

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

从多能干细胞中无血清自由生产三维人类肝球

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

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

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

用于生成 iPSC 衍生的 3D 人肝类器官的综合方案

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

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

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

从人类胚胎干细胞中引导肝细胞样细胞诱导的有效方法

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...

免疫与感染

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

使用定义的分化系统从多能干细胞的肝祖原体规范

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

<meta charset="utf8"></head><body>通过基于图像的 ML 策略监测多能干细胞分化

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available PSC-to-functional cell differentiation systems are impeded by problems of severe line-to-line and batch-to-batch variability. Precise control of cell differentiation in real time is therefore important. In this protocol, we describe a non-invasive and intelligent strategy that overcomes the variability in cell differentiation by using bright-field image-based machine learning. Taking PSC-to-cardiomyocyte differentiation as an example, this methodology provides detailed information for control of the initial PSC state, early assessment and intervention in differentiation conditions, and elimination of the misdifferentiated cell contamination, together realizing consistently high-quality differentiation from PSCs to functional cells. In principle, this strategy can be extended to other cell differentiation or reprogramming systems with multiple steps to support cell manufacturing, as well as to further our understanding of the mechanisms during cell fate conversion.

<meta charset="utf8"></head><body>作者聚焦:使用基于活细胞明场成像的 ML 模型增强 PSC 到功能性细胞的分化

Available pluripotent stem cell (PSC)-to-functional cell differentiation systems are currently impeded by problems of severe line-to-line and batch-to-batch variability. Here, using cardiac differentiation as the main example, we present a protocol to intelligently monitor and modulate the process of PSC differentiation based on image-based machine learning.

一种基于活细胞图像的机器学习策略，用于监测多能干细胞分化

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available ...