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 border="0" cellpadding="0" cellspacing="0" style="width:719px;" width="717">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">DPBS with Calcium and Magnesium</td>
			<td style="width:171px;">ThermoFisher</td>
			<td style="width:119px;">14040133</td>
			<td style="width:107px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Gentle cell dissociation reagent</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342 Ready Flow Reagent</td>
			<td style="width:171px;">thermofisher</td>
			<td>R37165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Recombinant Laminin 521</td>
			<td>BioLamina</td>
			<td>LN521-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Serum Albumin ELISA</td>
			<td>Alpha Diagnostics</td>
			<td>1190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Serum Alpha Fetoprotein ELISA</td>
			<td>Alpha Diagnostics</td>
			<td>500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 medium</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Operetta High-Content Imaging System</td>
			<td>PerkinElmer</td>
			<td>HH12000000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">PBS, no calcium, no magnesium</td>
			<td style="width:171px;">ThermoFisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:171px;">Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rho-associated kinase (ROCK)inhibitor Y27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503-1MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STEMdiff Definitive Endoderm Supplement CJ</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STEMdiff Definitive Endoderm Supplement MR</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STEMdiff Endoderm Basal Medium</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STEMdiff Hepatic Progenitor Medium</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:323px;">TWEEN 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Antibodies</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A6684</td>
			<td style="width:107px;">1:200 (mouse)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Alpha-fetoprotein</td>
			<td>Sigma-Aldrich</td>
			<td>A8452</td>
			<td style="width:107px;">1:400 (mouse)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HNF-4&#945;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td>sc-8987</td>
			<td>1:400 (rabbit)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">IgG</td>
			<td style="width:171px;">DAKO</td>
			<td style="width:119px;"></td>
			<td>1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sox17</td>
			<td>R&#38;D Systems, Inc.</td>
			<td>AF1924</td>
			<td>1:200 (Goat)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Software</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:323px;">Columbus Image Data Storage and Analysis system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td style="width:107px;"></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

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

hepatic-progenitor-specification-from-pluripotent-stem-cells-using

Research

JoVE Journal

Biology

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

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

科研

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

推导与表征的转基因无人类诱导多能干细胞系，并转换成定义的临床级条件

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

使用三维共培养系统NICO-1评估卵巢癌干细胞样细胞的血管遗传特性

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

表征 hiPSC 驱动 MSC 中的表面抗原和增殖能力

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

作者聚焦：iPSC 和遗传病研究的进展 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

人诱导多能干细胞分化为间充质基质细胞的两种代表性方法比较

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...