这项研究得到了MRC博士培训伙伴关系（MR/K501293/1）、英国再生医学平台（MRC MR/L022974/1和M/K026666/1）、首席科学家办公室（TCS/16/37）的奖项的支持。

1. 层压素-521上的人类多能干细胞（hPSC）维护
<ol><li>将人类多能细胞 （hPSCs） 保持在 37 °C 和 5% CO2 的 6 井板层压素-521 （LN-521）中。每天用2兆升的干细胞维持介质（即mTeSR1介质）喂养细胞，每口6井板的井，直至选定的播种日（第0天）。</li>
	<li>确保在细胞采集之前实现所需的 70%至 80% 的细胞汇流。</li>
</ol>2. 拉米宁-521多井制备和hPSC播种的差异化
注:对于未在 LN-521（例如母体或纤维素）上维护的 hPSC，在传递前将 hPSC 拆分为 LN-521 和文化 1 周，并引起差异化，以提高流程 15、17、18 的效率。
<ol><li>拉米宁涂层板制备<ol>
<li>在 4 °C 下解冻一小瓶重组 LN-521（100 μg/mL），2 小时或过夜。</li>
		<li>通过稀释冰冷的 1x DPBS 中的解冻 LN-521，使用 Ca2+/Mg2+来准备 8 μg/mL 解决方案。</li>
		<li>在 24 井板的每口井中加入 0.25 mL 的 8 μg/mL LN-521 解决方案，或在 96 井板的每口井中加入 0.05 mL 溶液。将盘子轻轻地从一边摇到另一边，均匀地涂上LN-521溶液的油井。 注:对于 96 井板格式，可以使用半自动管道执行体积分配、细胞播种和介质更改。有关详细信息，请参阅梅塞格尔-里波莱斯等人。</li>
		<li>在使用前将 LN-521 涂层板密封，并采用半透明、灵活的薄膜，并在 4 °C 下过夜。 注:LN-521 涂层板在 4 °C 下存储时最多可使用 2 周。 避免层压涂层油井的任何干燥。</li>
	</ol></li>
	<li>在细胞播种当天，在 37 °C 的细胞培养培养器中加热预涂板，30~60 分钟。</li>
	<li>吸气 LN-521 解决方案。 注意:避免吸气器与井底直接接触，以防止 LN-521 涂层损坏。</li>
	<li>将 0.5 mL 的干细胞维护介质与新鲜补充的 10 μM Rho 相关激酶 （ROCK） 抑制剂 Y27632 分配到 24 井板的每口井或 96 井板的每口井 0.05 mL。将盘子放在孵化器中，直到准备好细胞播种。</li>
	<li>在预定的播种日（第 0 天），hPSC 汇流在 70°80% 之间，标记 6 井板底部任何自发分化区域。 注:自发分化可以通过细胞大小和/或不同细胞形态的存在，使用相位对比显微镜可视化。</li>
	<li>吸气并丢弃标记的分化区域和井中的废介质。在室温下用 1 mL 的 DPBS 清洗每口井，而无需 Ca2+/Mg2+ 。</li>
	<li>在每口井中加入1mL无酶分离试剂（见 材料表），在37°C下孵育8~10分钟，直到细胞明显脱离板。</li>
	<li>使用细胞刮刀轻轻地将细胞从井中分离出来。派珀特每个井上下2×4倍的内容与P1000移液器产生一个单细胞悬架。对于每个细胞线，池细胞从所有维护井进入无菌50mL管。</li>
	<li>用1兆L的干细胞维持介质清洗每个清空良好的。将洗涤添加到相应的管中，其中包含来自适当细胞线的聚合细胞。</li>
	<li>对于每个细胞系，在汇集的样本上执行三个可行的细胞计数。计算每个细胞系的平均活细胞计数（活细胞/mL）。</li>
	<li>在 RT 以 250 x g 为集中样品离心 5 分钟，在 RT. 吸气超高分子，然后在 1×3 mL 的 RT 干细胞维护介质中重新使用细胞颗粒，新鲜补充 10 μM ROCK 抑制剂 Y27632。</li>
	<li>对于每个细胞系，计算步骤 2.4 时准备的井数所需的细胞数（参见 表 1）。重新使用干细胞维持介质重新使用所需的细胞编号，新补充了 10 μM ROCK 抑制剂 Y27632。</li>
	<li>将计算的体积添加到第 2.4 步的准备板和预涂板的井中，而不删除之前添加的体积。每口井的总容积为 1 mL，24 井板为 0.1 mL，96 井板总容量为 0.1 mL。轻轻摇动板从一边到另一边，来回，以确保均匀的细胞分散整个井。 注:在井中均匀的细胞分布是确保同质细胞播种和成功分化的关键。</li>
	<li>将种子板放入孵化器中，并立即轻轻地来回摇动种子板，从一边到另一边均匀地分配细胞，并在37°C和5%CO2下保持培养。</li>
</ol>3. 将 hpscs 区分为层压素 - 521 上的肝祖体
<ol><li>使用内皮基础介质补充正确的添加剂，为确定性内皮诱导（第 1 阶段）做好准备。<ol>
<li>第 0 天，在 4 °C 下隔夜解冻内德姆巴沙尔中等的瓶子。</li>
		<li>根据需要准备阶段 1 中等 1（用于第 1 天）。</li>
		<li>解冻补充 MR 和补充 CJ 在冰上。</li>
		<li>稀释补充MM和补充CJ 1:100在内皮基础介质。</li>
		<li>根据需要准备第 1 阶段中 2，以便在第 2/4 天使用。</li>
		<li>稀释补充CJ 1:100在内皮基础介质。</li>
	</ol>
</li>
	<li>使用肝祖体介质为随后的肝祖细胞规范（第 2 阶段）分化做好准备。<ol>
<li>第 4 天，在 4 °C 下隔夜解冻肝祖体介质瓶。 注:本实验使用1%青霉素/链霉素（最终浓度分别为100 IU/mL和100微克/mL）。不需要抗生素;抗生素的使用由用户自行决定。</li>
	</ol>
</li>
	<li>在分化的第一天，从井中取出10μM ROCKi Y-27632介质的废干细胞维护介质，代之以24井板每井0.5 mL的完整阶段介质1和96井板每井0.1mL。</li>
	<li>在第2天、第3天和第4天，取出乏中型井，每口井每口0.5mL的阶段中等2，为24口井板或每口96井板0.1ML。</li>
	<li>第5天，通过免疫细胞化学修复油井，进行明确的内皮分化分析。对于剩余的油井，取出废油井，每口井用0.5兆升的肝祖遗传分化介质喂每口井，每口井24口井板或每口96口井0.1兆升。在第 6 天、第 7 天和第 9 天再次刷新介质。</li>
	<li>第10天，收获井进行肝祖分化分析，或进行进一步肝细胞样细胞分化。 注:此时，样品要么用4%的副甲醛（PFA）进行免疫细胞化学分析，要么为ELISA采集超高分子，并采集细胞进行蛋白质定量。</li>
</ol>4. 层压素-521上hPSC产生的肝祖代分化培养物的特征
<ol><li>第 5 天，使用免疫染色检测确定性内皮特异性标记的表达。</li>
	<li>第10天，使用免疫染色检测肝祖特异性标记的表达。</li>
	<li>第10天，根据制造商的指示，使用试剂盒测量AFP和ALB分泌物，并根据双辛酸（BCA）蛋白质检测确定，使每毫克蛋白质正常化。</li>
	<li>通过量化每口井 HNF4® 阳性细胞的百分比来评估 96 井板的肝祖变异性。</li>
</ol>5. 免疫细胞化学和图像采集
<ol><li>在分化的第 5 天和第 10 天，用 1 倍 DPBS 清洗细胞 3 倍，每口 24 井板的 0.5 mL 和 96 井板每井 0.1 mL。在 RT 用柔和的摇晃孵育盘子 2 至 5 分钟。 注:使用没有Ca2+/Mg2+ 的 DPSB 进行免疫细胞化学。</li>
	<li>通过为 24 井板每井增加 0.3 mL 的 PFA 和 96 井板每井增加 0.1 mL，在 RT 中用 4% 的副甲醛 （PFA） 修复细胞 15 至 30 分钟。</li>
	<li>按步骤 5.1 中描述的 1 倍 DPBS 清洗 3 倍。</li>
	<li>使用 0.1% Tween、1x DPBS 和在 RT 孵育 20 分钟，通过每口 24 井板的 0.3 mL 和 96 井板每井 0.1 mL 加入 0.3 mST 来测量膜。</li>
	<li>通过在 PBST 中用 10% BSA 孵育细胞 1 小时，在 24 井板每井添加 0.3 mL 的 BSA，在 96 井板每井中加入 0.1 mL 的 BSA，使用板摇床轻轻摇晃，从而执行蛋白质块。</li>
	<li>蛋白质阻断后，用PBST中1%BSA稀释的原发性抗体代替阻断溶液，在4°C下孵育，一夜之间轻轻摇晃。 注意:不要在蛋白质块和抗体添加之间清洗。</li>
	<li>24 小时后，用 PBST 清洗井 3 倍。</li>
	<li>在 PBST 中将二级抗体添加到 1% BSA 中。在黑暗中用温柔的摇晃在 Rt 孵育 1 小时。</li>
	<li>二次抗体孵化后，用1x DPBS清洗井3倍，根据制造商的说明在黑暗中在RT下用轻柔的摇晃分配霍奇斯特污渍10分钟。</li>
	<li>用 1 倍 DPBS 清洗 3 倍。这些板现在已准备好成像。 注意:在黑暗中将板材存放在 4 °C 处，直到成像。</li>
	<li>免疫造血术后使用高含量成像显微镜对多井板进行成像。建议对多个视图领域进行图像采集，以获得井的真正表示。使用商业软件（见材料表）对不同标记的表达进行评估（图1）。 注:细胞分割也可以使用图像分析开源软件，如细胞程序或斐济20，21。</li>
</ol>

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(121), e55355 (2017).</li><li>Villarin, B. L., 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=Polymer+Supported+Directed+Differentiation+Reveals+a+Unique+Gene+Signature+Predicting+Stable+Hepatocyte+Performance.">Polymer Supported Directed Differentiation Reveals a Unique Gene Signature Predicting Stable Hepatocyte Performance.</a> Advanced Healthcare Materials. 4 (12), 1820-1825 (2015).</li><li>Wang, Y., 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=Multiomics+Analyses+of+HNF4&#945;+Protein+Domain+Function+during+Human+Pluripotent+Stem+Cell+Differentiation.">Multiomics Analyses of HNF4&#945; Protein Domain Function during Human Pluripotent Stem Cell Differentiation.</a> iScience. 16, 206-217 (2019).</li></ol>

大卫·海是斯坦诺瓦特有限公司的联合创始人和股东。其余作者证明他们在本文所讨论的主题或材料中不存在利益冲突。

大规模地从多能干细胞中产生人类肝祖细胞，可能是尸体衍生材料的有希望的替代品。协议标准化和可重复性是确保生物医学研究的技术翻译和影响的关键。为了解决这个问题，以前的工作重点是开发一个步骤分化协议从hESC和iPSC使用定义的添加剂和矩阵15，23，24，25，26，27，28。通过这样做，肝细胞表型和可重复性得到了改善，使分化过程的半自动化。所呈现的系统通过与现成的细胞培养介质和方便的肝细胞分化系统的结合而得到加强。
此前，多能细胞密度在分化方案开始前被强调为实现肝祖细胞同质种群的关键变量26。利用这个更精细的程序，可以使用一系列启动细胞密度（表1）逐步产生大量的干细胞衍生肝祖。在第5天，最终的内皮诱导被Six17染色（图3）验证。通过测试的ESC和iPSC线实现了对最终内皮的高效和强大的分化，超过80%的表达Six17（图3）。在第10天，肝祖系显示出一种均匀的鹅卵石状形态，肝干细胞标记对法新社和HNF4+（>86%，图4）都非常丰富。使用手动和半自动化技术的组合，可以执行多种板格式的区分19。
以目前的形式，细胞分化适用于 体外 实验。然而，在临床应用之前可能需要细胞浓缩，以确保肝祖体的同质种群为分娩做好准备。
最后，这里描述的协议为该领域提供了一个标准化的方法，以大规模地产生肝祖。未来的工作将侧重于生产用于后续 HLC 分化、成熟和维护的新介质。

肝病是一个全球性的健康挑战，每年在全球造成约200万人死亡。虽然存在许多模型系统来研究肝病和临床干预，但细胞系统常规使用受到重大缺陷的限制（供审查见Szkolnicka等人）。先进的人类多能干细胞（hPSC）培养和体细胞分化方法代表了为临床3、4开发基础生物医学研究和分化细胞可再生能源的有前途的技术。
迄今为止，肝细胞分化的多个协议已经开发出5，6，7，8。这些协议试图利用小分子和生长因子9，10的组合来重建人类肝脏发育的各个方面。大多数协议包括一个循序渐进的分化过程，其中 hPSC 被准备为确定内皮，然后是肝祖体规范 11，12，13，并以 HLC 规范结束。这些协议产生的HLC显示胎儿和成人表型的混合物。这包括α胎儿蛋白（法新社）的表达，如肝细胞标记，如HNF4+和白蛋白（ALB），以及药物代谢能力14，15，16。在实验室之间，HLC 的差异可能有所不同:因此，制定标准化协议是必要的。这将使研究人员能够有效地生成和应用干细胞衍生的HLC大规模的基础和临床研究。
开发了一种肝祖分化系统，该系统可以使用易于遵循的指南应用于人类胚胎和诱导多能干细胞系。这个过程产生异质种群的肝祖体在不同的培养皿格式，从细胞培养瓶到96井板。提供以下协议，以24和96井的形式产生干细胞衍生肝祖。
下面介绍的协议中使用的细胞密度分别指定为 24 井和 96 井板的一口井（参见 表 1）。不同细胞培养板格式和细胞线需要优化起始单元格编号。建议启动细胞密度协议优化是2×105 细胞/厘米2。对于密度优化，可以通过一次添加 50，000 个单元格/cm2 ±来测试多个单元格密度。

<table><tbody><tr><td>DPBS with Calcium and Magnesium</td><td>ThermoFisher</td><td>14040133</td><td></td></tr><tr><td>Gentle cell dissociation reagent</td><td>STEMCELL Technologies</td><td>7174</td><td></td></tr><tr><td>Hoechst 33342 Ready Flow Reagent</td><td>thermofisher</td><td>R37165</td><td></td></tr><tr><td>Human Recombinant Laminin 521</td><td>BioLamina</td><td>LN521-02</td><td></td></tr><tr><td>Human Serum Albumin ELISA</td><td>Alpha Diagnostics</td><td>1190</td><td></td></tr><tr><td>Human Serum Alpha Fetoprotein ELISA</td><td>Alpha Diagnostics</td><td>500</td><td></td></tr><tr><td>mTeSR1 medium</td><td>STEMCELL Technologies</td><td>5850</td><td></td></tr><tr><td>Operetta High-Content Imaging System</td><td>PerkinElmer</td><td>HH12000000</td><td></td></tr><tr><td>PBS, no calcium, no magnesium</td><td>ThermoFisher</td><td>14190250</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Rho-associated kinase (ROCK)inhibitor Y27632</td><td>Sigma-Aldrich</td><td>Y0503-1MG</td><td></td></tr><tr><td>STEMdiff Definitive Endoderm Supplement CJ</td><td>STEMCELL Technologies</td><td></td><td></td></tr><tr><td>STEMdiff Definitive Endoderm Supplement MR</td><td>STEMCELL Technologies</td><td></td><td></td></tr><tr><td>STEMdiff Endoderm Basal Medium</td><td>STEMCELL Technologies</td><td></td><td></td></tr><tr><td>STEMdiff Hepatic Progenitor Medium</td><td>STEMCELL Technologies</td><td></td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Albumin</td><td>Sigma-Aldrich</td><td>A6684</td><td>1:200 (mouse)</td></tr><tr><td>Alpha-fetoprotein</td><td>Sigma-Aldrich</td><td>A8452</td><td>1:400 (mouse)</td></tr><tr><td>HNF-4&amp;alpha;</td><td>Santa Cruz</td><td>sc-8987</td><td>1:400 (rabbit)</td></tr><tr><td>IgG</td><td>DAKO</td><td></td><td>1:400</td></tr><tr><td>Sox17</td><td>R&amp;amp;D Systems, Inc.</td><td>AF1924</td><td>1:200 (Goat)</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Columbus Image Data Storage and Analysis system</td><td>PerkinElmer</td><td></td><td></td></tr></tbody></table>

hepatic progenitor specification from pluripotent stem cells using a defined differentiation system

肝病是一个不断升级的全球健康问题。虽然肝移植是一种有效的治疗方式，但由于供体器官供应不足，患者死亡率有所上升。器官短缺也影响人类肝细胞用于基础研究和临床的日常供应。因此，开发人类肝祖细胞的可再生来源是可取的，也是本研究的目标。为了能够大规模有效地生成和部署人类肝脏祖传，开发了可重复的肝祖分化系统。该协议有助于使用一系列细胞培养皿格式的用户之间的实验可重复性，并允许使用人类胚胎和诱导多能干细胞系进行区分。这些是现有分化系统的重要优势，将加强基础研究，并可能为临床产品开发铺平道路。

肝祖代区分从hESC （H9） 和 hiPSC （P106） 线是按照图 2中描述的步向协议进行的。在这里，多能干细胞作为单细胞在分化开始前被播种到LN-521涂层板中。细胞汇流是强健且可重复的分化的关键。一旦实现了正确的汇流（图2），就会开始分化。在第 5 天，通过 Sox17 表达评估了明确的内皮规范。在这两个细胞系中，Sox17分别以80%±0.5%和87.8%±H9和P106的SEM（图3）表示强烈。在第10天，肝祖宗展示了一个鹅卵石状的形态（图2）。此外，还评估了肝祖体规格为HNF4+、法新社、ALB和细胞克哈拉汀-19（CK19）表达，以及法新社和阿尔布蛋白分泌10、15、22（图4）。H9 和 P106 肝祖传培养都表示胎儿肝标记，如 HNF4+ （91% ± 0.5% 和 90% ± 0.2%）， 法新社（89.7%±1.8%和86%±1.2%），CK19（78.5%±3.2%和83.6±1.8%）（图4）。法新社分泌在第10天检测到两个细胞系（32.4±1.6和47.8±5.9 ng/mL/mg/mg/24h）（图5）。白蛋白合成在较低水平（30.7%±1.8%和27.2%±1.1%）（图4），未通过ELISA（图5）检测到。
该协议允许将肝祖体的标准化生产从24口井增加到96口井板。采用半自动管道从H9和P106细胞系生产96个肝祖板，如前所述17。细胞数变异性和肝祖分化效率通过 HNF4+ 表达的量化来评估。细胞分割是使用高含量成像仪器（图1）通过免疫荧光进行蛋白质定量的。在第10天，肝祖细胞在行间没有显著变异，H9和P106的HNF4+阳性细胞每井>94%为HNF4+阳性细胞（图6）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61256/61256fig1v2.jpg"> 图1:细胞分割管线概述。（A ） 使用原始图像，（B ）核染色用于核分割。（C） 根据形状和大小执行核分割质量控制步骤，仅量化清晰分割的核。（D） 在此之后，正HNF4+染色核被量化。（E） 最后，采用基于强度的阈值来识别 HNF4+ 表达细胞。在C和E中，绿色核代表选定的细胞，洋红色核表示废弃细胞。缩放栏 = 50 μm.<a href="https://www.jove.com/files/ftp_upload/61256/61256fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61256/61256fig2v2.jpg"> 图2:肝祖代分化与肝 祖分化协议的示意图表示。（B） 代表图像，突出分化过程中的形态变化。在第 0 天 （D0）， hpscs 提出了一个包装的单层细胞。在此之后，hPSC 在第 5 天 （D5） 被准备成最终的内皮。随后，第10天（D10）出现肝祖分化。肝祖器显示了一个鹅卵石状的细胞形态。 缩放栏 = 75 μm. <a href="https://www.jove.com/files/ftp_upload/61256/61256fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61256/61256fig3v2.jpg"> 图3:确定内皮规范的特征。在第5天，细胞被染色为Six17，一个明确的内皮标记。Sox17阳性细胞的百分比为80±H9为0.5%，P106为87.8±0.5%。百分比量化基于每口油井有6个视野的10口独立油井。数据显示为 Sem. 规模栏的平均± = 50 μm。<a href="https://www.jove.com/files/ftp_upload/61256/61256fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61256/61256fig4v3.jpg"> 图4:肝祖特征。 在第10天，肝祖传被染色肝标记（A） HNF4 +，（B） 法新社， 和（C） ALB.H9的正细胞比例分别为91%±0.4%、89.7%±1.8%和30.7%，±1.8%的HNF4+、法新社和阿尔布。对于P106，正细胞的百分比分别为90%±0.2%、86%+/-1.2%和27.2%，±1.1%的HNF4+、法新社和阿尔布。（D） 通过CK19表达评估胆囊细胞血统潜力:H9衍生肝祖细胞表示78.5%±3.2%CK19阳性细胞，而83.6%±1.8%的CK19阳性细胞被观察到P106肝祖细胞。免疫球蛋白G （IgG） 染色用作染色控制。百分比量化基于每口油井有6个视野的10口独立油井。数据显示为 Sem. 规模栏的平均± = 50 μm。 <a href="https://www.jove.com/files/ftp_upload/61256/61256fig4v3large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61256/61256fig5.jpg"> 图5:肝祖蛋白分泌分析。 在H9和P109的第10天，在肝祖文化中分析了α胎儿蛋白（法新社）和白蛋白（ALB）的分泌。这些数据代表三种生物复制品，误差条代表SD。 分泌的蛋白质从24h培养介质中量化为每毫克蛋白质每mL分泌蛋白质的纳米克，n = 3;ND = 未检测到。 <a href="https://www.jove.com/files/ftp_upload/61256/61256fig5large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61256/61256fig6v2.jpg"> 图6:96井板井对井变的评价。 （A） 可视化 96 井板视图的 H9 衍生肝祖体沾染 HNF4+ 。（B） HNF4+ 阳性细胞的量化。每排井的平均细胞数，从每口井的六个视图领域量化。整个板的平均细胞数为94.81%±每口井0.22个SEM HNF4+阳性细胞。油井之间没有发现统计学上显著的差异。（C） 可视化96井板视图的P106衍生肝祖体沾染HNF4+。（D） HNF4+ 阳性细胞的量化。每排井的平均细胞数，从每口油井的六个视图领域进行量化。整个板的平均细胞数为97.7%±每口井0.57个SEM HNF4+阳性细胞。行之间没有观察到统计学上显著的差异。H12 被用作免疫球蛋白 G （IgG） 染色控制。秤杆 = 1 毫米。采用了采用了采用图基事后统计测试的单向 ANOVA。 <a href="https://www.jove.com/files/ftp_upload/61256/61256fig6v2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1"><tbody>
<tr>
<td>板格式</td>
			<td>表面积 （cm2）</td>
			<td>每厘米细胞</td>
			<td>每口井的总细胞</td>
			<td>分配体积 （mL）</td>
			<td>细胞浓度（细胞/毫升）</td>
		</tr>
<tr>
<td>24 井板</td>
			<td>1.9</td>
			<td>210526</td>
			<td>400000</td>
			<td>0.5</td>
			<td>800000</td>
		</tr>
<tr>
<td>96井板</td>
			<td>0.32</td>
			<td>187500</td>
			<td>60000</td>
			<td>0.05</td>
			<td>1200000</td>
		</tr>
</tbody></table>表1:本协议中使用的 hPSC 电池线不同板格式的推荐细胞密度。

Watch this Scientific Journal Video about Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System at JoVE.com

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

本文的目标是提供一种标准化的方法，诱导人类肝祖细胞与多能干细胞的分化。这种程序的开发与即用媒体配方为用户提供了一个方便的系统，以产生人类肝细胞的生物医学研究和翻译。

使用定义的分化系统从多能干细胞的肝祖原体规范

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4.6K 次观看.  University of Edinburgh. 本文的目标是提供一种标准化的方法，诱导人类肝祖细胞与多能干细胞的分化。这种程序的开发与即用媒体配方为用户提供了一个方便的系统，以产生人类肝细胞的生物医学研究和翻译。

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

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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发育生物学

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

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

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

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 ...

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 ...

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 ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

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 ...