这项工作得到国家卫生研究院 (DK55743、DK087377、DK102716 和 HG006398) 的支持。我们要感谢 Behshad Pournasr 博士, 詹姆斯 Heslop 博士, 并为他们的贡献而奔跑。

1. 人类诱导多潜能干细胞的培养
<ol><li>
涂层重组人 e-钙黏蛋白 fc 融合蛋白质 (e cad fc) 或其他适合 hPSC 文化的矩阵6<ol>
<li>将 Dulbecco 的磷酸盐缓冲盐水 (DPBS (+)) 稀释为15微克/毫升。</li>
		<li>涂层100毫米悬浮组织培养皿与5毫升稀释的 E cad Fc 和孵育在37˚C 至少 1 h. 移除基板, 并用5毫升的介质 (例如, mTeSR1 称为 M 介质从今以后)7。 注意:此协议中使用的区域性介质是在发布的协议7之后准备的。然而, 一些其他媒体的准备工作已经被描述, 或者是可以在商业上获得的, 这可能与程序兼容。</li>
		<li>从液氮中提取一小瓶冷冻 iPSCs。解冻在37˚C 直到一个小冰晶遗骸。轻轻地将细胞注入15毫升不育的锥形管, 其中含有4毫升的 M 培养基。</li>
		<li>离心机在 300 x g 的5分钟内移除上清, 并轻轻地重新悬浮细胞与5毫升培养基辅以10µM 的 Y-27632, 一种选择性抑制剂的与蛋白激酶相关的, 卷状线圈含有的蛋白酶 (岩石)。</li>
		<li>从步骤1.1.2 中移除 M 介质, 并将5毫升的细胞从步骤1.1.4 转移到 E cad-Fc 涂层的100毫米悬浮组织培养皿中。</li>
	</ol>
</li>
	<li>
在文化中维护人的 iPSCs
	<ol>
<li>一旦 iPSCs 到达大约80% 融合, 去除培养基并且洗涤一次与钙和镁自由 DPBS (-)。吸入 DPBS (-), 并添加足够的 0.02% EDTA 溶液覆盖板, 然后在室温下孵育高达3分钟。 注意:在80% 汇合处, 该板块应包含大约 2 x 107单元格。重要的是不要 overgrow 细胞, 并确保细胞保持不分化的形态学。细胞的干细胞可以通过染色与干细胞标记 Tra-1-60 或等效的8来确定。</li>
		<li>一旦细胞开始释放, 去除 0.02% EDTA 溶液和洪水的盘子与10毫升的 M 培养基释放细胞。用轻柔的吹打帮助 iPSCs 的支队。 注意:细胞分离的平均潜伏期是3分钟左右, 但这需要根据经验确定每 iPSC 线。细胞应该被释放作为小簇包含大约 5-10 iPSCs 每群。</li>
		<li>1/10 的悬浮细胞每新鲜 E cad-Fc 涂层100毫米悬浮组织培养皿含有5毫升的 M 培养基。培养在37˚C 以下 4% O2/5% CO2和改变媒介每日。 注意:虽然 iPSCs 在生理氧条件下例行培养, 以促进干细胞, iPSCs 也可以使用周围的氧气生长。</li>
	</ol>
</li>
</ol>2. 96 井板上人 iPSCs 与肝细胞样细胞分化的研究
注意:本节介绍与筛选兼容的格式 iPSCs 的区别。利用这种方法, 可以诱导人类 iPSCs 在20天内分化形成肝样细胞。虽然这是适合大多数化验, 文化的长度可以延长, 以改善成熟的细胞。
<ol><li>
低生长因子基底膜基质包衣96井组织培养板
	<ol>
<li>用冰冷无菌 DMEM/F12 组织培养培养基将基质稀释至2毫克/毫升, 制备库存。将矩阵分为 250 ul 整除数, 并存储在-80 ˚C, 直到需要。在冰上冷却10毫升的 DMEM/F12 30 分钟. 检索 250 ul 整除的2毫克/毫升矩阵股票和解冻冰。</li>
		<li>将基质与10毫升冷 DMEM/F12 混合使用预冷的吸管轻轻搅拌, 使最终浓度为0.05 毫克/毫升。</li>
		<li>将稀释基质的 100 ul 转移到96井组织培养板的每一个井中。在37˚C、4% O2/5% CO2中孵育至少 1 h. 丢弃该矩阵并替换为 M 介质的 50 ul 每井。存储在37°c, 4% O2/5% CO2直到需要为止。</li>
	</ol>
</li>
	<li>
平板细胞分化
	<ol>
<li>培养的 iPSCs 100 毫米板, 直到细胞接近80% 汇合。确保该板块包含大约 2 x 107单元格。通过细胞流式分析仪或 hemocytometer 确定细胞数。 注意:通过经验, iPSCs 细胞的质量可以通过显微镜来判断;然而, 接近98% 的细胞应该表达干细胞标记, 如 TRA-1-60 表位时, 测量由外地资产管制或染色。重要的是, 细胞保持积极的分裂, 并没有杂草丛生。</li>
		<li>从每100毫米的盘子中收获 iPSCs, 吸入培养基, 用5毫升的 DPBS (-) 冲洗。用3毫升的细胞脱离溶液 (例如、accutase) 代替冲洗, 在37摄氏度、4% O2/5% CO2上孵育约2分钟。 注意:通过显微镜观察细胞脱离是很重要的。不要过度消化与 accutase。其目的是将细胞作为小簇释放, 治疗最少。</li>
		<li>一旦细胞开始分离, 收获通过增加7毫升的 M 培养基。吸管多次将细胞菌落分离成大约 3-6 细胞的小簇 (图 1A)。</li>
		<li>将 iPSCs 收集成15毫升无菌离心机管, 离心机在 300 x g 处进行5分钟切除, 并在5毫升 M 介质中重新悬浮细胞颗粒。</li>
		<li>通过将悬浮细胞的 50 ul 吹打到每个井中均匀地将细胞分布到96井基体涂层板上。要设置每个96井板, 请使用一个100毫米的 iPSCs 板, 在大约80% 融合, 其中包含约 5 x 106单元格以设置差异。使用多通道吸管加速这个过程。将单元格在37˚C、4% O2/5% CO2中过夜。 注意:重要的是, 将相等数量的细胞存入每个井中, 以确保可重现的分化。</li>
	</ol>
</li>
	<li>
诱导细胞分化
	<ol>
<li>通过显微镜检查96井板, 以确保细胞至少覆盖每个个体表面的70%。如果覆盖率小于 70%, 请用新鲜培养基替换培养基, 并在37˚C、4% O2/5% CO2上孵化额外的24小时。 注意:在不诱导分化的情况下, 孵化细胞超过48小时, 通常会降低分化效率。使用环境氧也会产生差异, 尽管效率可能受到影响.
</li>
		<li>1天到2日分化, 取代培养基与 RPMI 补充 2% B27 (没有胰岛素), 100 ng/毫升激活 A, 10 ng/毫升 BMP4, 20 ng/毫升 FGF2。在37˚C, 在环境 O2/5% CO2中添加100个中等的 ul 和孵育, 每日中等变化2天。 注意:处理大量的96井板需要使用辅助吸管或机器人。12通道和96通道吸管是日常介质变化的理想选择。</li>
		<li>3天到5日的分化, 取代培养基与 RPMI 补充 2% B27 (没有胰岛素) 和 100 ng/毫升激活 a 文化细胞在37˚C, 在环境 O2/5% CO2 , 每日中等变化3天。</li>
		<li>在分化日 5, 在用新鲜培养基替换之前, 用 100 ul 0.02% EDTA 溶液在三十年代处理好每一个井, 然后用新鲜培养基去除和替换。 注意:如果分化为内胚层是有效的, 区分细胞的单层很容易从板块剥离。0.02% EDTA 溶液的简单治疗有助于缓解细胞脱离。这种治疗不会影响分化为肝细胞。</li>
		<li>为区分天6到 10, 替换培养基与 RPMI/2% B27 (与胰岛素) 补充 20 ng/毫升 BMP4 和 10 ng/毫升 FGF2 和孵化在37˚C, 4% O2/5% CO2为5天以每日媒介变动。这一阶段诱导细胞分化为肝祖细胞。</li>
		<li>为区分在天11到15之间, 替换培养基与 RPMI 或 2% B27 (与胰岛素) 补充与 20 ng/毫升 HGF。孵育在37˚C, 4% O2/5% CO2为5天以每日媒介变动。</li>
		<li>为区分在天16到20之间, 替换培养基与肝细胞培养培养基 (HCM) 补充与 20 ng/毫升的 Oncostatin (OSM)。将单元格孵化为37˚C, 在环境 O2/5% CO2中, 每日中等变化为5天。 注意:通过 ELISA 测定白蛋白、AFP 或 APOB 在培养基中的相对水平, 可以有效地测定单个井间分化的重现性 (见 3.2)。</li>
	</ol>
</li>
</ol>3. 小分子筛查
注意:下面描述了用于识别可用于降低家族性高胆固醇血症 iPSCs 中肝样细胞培养基中 APOB 水平的化合物的过程。对该模型的描述及其在降胆固醇药物鉴别中的应用已经详细描述了以前的5,9。在本例中, 对南卡罗来纳州复合集合 (SC3) 库中的1000个化合物进行了筛选。每种化合物的浓度为2微克/毫升。
<ol><li>
小分子 iPSC 肝细胞的治疗
	<ol>
<li>稀释主复合库, 以产生20微克/毫升浓度为2% 亚砜的化合物的工作储存板, 并贮存在-20 ˚C。</li>
		<li>准备足够的 96 iPSC 的肝细胞, 允许每个化合物进行测试。 注意:要筛选的化合物数量将决定所需的96个井板的数量。一般情况下, 每个化合物都应准备一个井, 再加上任何控制样品的额外水井。通常避免板块最外层的井, 以减少可能的边缘效应, 可以破坏解释。可以使用 z 评分方法识别命中, 而无需使用复制。如果包含复制, 则更适合使用 t 测试。</li>
		<li>在分化的完成 (天 20, 看见 2.3.7), 替换文化媒介与 100 ul 新的 HCM 补充与 20 ng/毫升 Oncostatin m.孵育在37˚C, 4% O2/5% CO2为确切地 24 h. 将培养基提取到新鲜的96井板中, 并贮存在-20 ˚C。使用此示例计算在添加药物前培养基中 APOB 的水平。</li>
		<li>添加 90 ul 新的 HCM 补充与 20 Oncostatin/毫升的每一个井。在吹打混合后, 将每种化合物的 10 ul 添加到工作库存中。将亚砜添加到0.2% 以控制水井, 使其与所处理样品的最终浓度相匹配, 并在37˚C、环境 O2/5% CO2中孵化, 精确 24 h. 收集培养基并贮存在-20 ˚C。使用此示例可以确定在使用化合物治疗后 APOB 的水平。</li>
		<li>可选-其余的细胞可以处理染色, 细胞的生存能力, 或分析的 mRNA 水平使用标准的方法。 注意:这个屏幕被设计用来识别对 APOB 水平有快速影响的药物。然而, 根据疾病模型, 下游化验, 或药物作用的机制, 这可能是有益的治疗样品多天。</li>
	</ol>
</li>
	<li>
通过 ELISA 确定 APOB 水平
	<ol>
<li>根据制造商的协议, 使用人类 APOB ELISA 开发套件, 从药物前和后处理样品中确定载脂蛋白 B (APOB) 浓度。</li>
		<li>
准备以下解决方案
		<ol>
<li>洗涤缓冲器, 由0.05% 吐温20在 PBS, pH 7.4 组成。存储在4˚C, 直到使用。</li>
			<li>孵化缓冲, 由0.05% 吐温20和0.1% 牛血清白蛋白 (BSA) 在 PBS, pH 7.4。存储在4˚C, 直到使用。</li>
		</ol>
</li>
		<li>APOB (单克隆 20/17) 抗体稀释到2µg/毫升在 PBS, pH 7.4。添加 100 ul 的稀释抗体每井和孵化在4˚C 过夜。</li>
		<li>从化验盘中取出抗体, 用 PBS 的 200 ul, pH 7.4, 分别冲洗两次。</li>
		<li>添加 200 ul/井0.05% 吐温 20, 0.1% 牛血清白蛋白 (BSA) 在 PBS, pH 7.4 和孵化在一个震动平台1小时室温。</li>
		<li>在 PBS 中使用0.05% 个补间20和 0.1% BSA 准备一个8点标准曲线, pH 值7.4 具有以下浓度的 APOB: 300 ng/毫升, 200 ng/毫升, 175 ng/毫升, 150 ng/毫升, 125 ng/毫升, 100 ng/毫升, 75 ng/毫升, 50 ng/毫升。</li>
		<li>稀释测试样品1:4 与0.05% 吐温20和 0.1% BSA 在 PBS, pH 7.4。</li>
		<li>1 h 的阻塞与孵化缓冲, 丢弃的缓冲和洗涤化验板五倍 200 ul 0.05% 吐温20在 PBS, pH 7.4。</li>
		<li>完全删除任何残留洗涤缓冲和转移 90 ul 的稀释样品和 APOB 标准的化验板。在室温下孵育1小时。</li>
		<li>丢弃样品, 并与 200 ul 0.05% 吐温20在 PBS, pH 7.4 每井清洗。重复洗涤总共5次。增加 100 ul 每井的单克隆 LDL 11-生物素稀释 1:1, 000 在0.05% 吐温20和 0.1% BSA 在 PBS, pH 7.4。在1小时的震动平台上孵育室温。</li>
		<li>丢弃试剂并用 200 ul 洗涤缓冲液洗涤5次。增加 100 ul 每井链亲和素抗体 (1:1, 000 稀释使用孵化缓冲), 然后在室温下孵育1小时的震动平台。</li>
		<li>丢弃试剂, 用 200 ul 0.05% 吐温20在 PBS 中清洗, pH 值为7.4。重复洗涤总共5次。从检测板上完全去除残留洗涤缓冲器, 并在 Tetramethylbenzidin (TMB) 底物溶液中添加 100 ul, 并在室温下孵育8分钟。</li>
		<li>不去除 TMB 衬底, 直接增加 100 ul 每井的停止解决方案 (1N HCl)。</li>
		<li>用平板阅读器测定 450 nm 的吸光度。</li>
		<li>可以建立一个标准曲线, 使用四参数逻辑回归方法, 然后用于定义每个示例10中 APOB 的浓度。</li>
		<li>检查药物前: APOB 在每个样本中的药物后比, 以确定有可能降低培养基中 APOB 水平的化合物。</li>
	</ol>
</li>
</ol>

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K., Duncan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+hepatocytes+from+pluripotent+stem+cells.">Differentiation of hepatocytes from pluripotent stem cells.</a> Curr Protoc Stem Cell Biol. 26 (Unit 1G 4), (2013).</li><li>Song, Z., 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=Efficient+generation+of+hepatocyte-like+cells+from+human+induced+pluripotent+stem+cells.">Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.</a> Cell Res. 19 (11), 1233-1242 (2009).</li><li>Cai, J., 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=Directed+differentiation+of+human+embryonic+stem+cells+into+functional+hepatic+cells.">Directed differentiation of human embryonic stem cells into functional hepatic cells.</a> Hepatology. 45 (5), 1229-1239 (2007).</li><li>Si-Tayeb, K., 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=Highly+efficient+generation+of+human+hepatocyte-like+cells+from+induced+pluripotent+stem+cells.">Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.</a> Hepatology. 51 (1), 297-305 (2010).</li><li>Pashos, E. 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作者声明他们没有竞争的财政利益。

基于目标的药物发现, 小分子被发现影响特定蛋白质的活性, 已成为许多现有筛查工作的重点。尽管这种方法提供了大量的药物, 但基于反转表型的屏幕, 经典药理学, 在识别临床上有效的1的第一类化合物方面更成功。表型药物发现的一个缺点是它依赖于适当的疾病模型的可用性。这可能是一个挑战, 当细胞模型的疾病是不可用的, 或当共同的研究动物物种未能概括的临床症状。然而, 从病人体细胞中产生 iPSCs 的能力为培养3型人类疾病提供了新的机会。对于一些疾病, 如罕见的遗传性疾病, 患者数量很少, 难以获得和同意。然而, 随着基因组工程学的出现, 将致病突变引入 iPSC 基因组是相对简单的, 这使得甚至可以对稀有疾病进行模型化。
一旦 iPSCs 在手里, 就有必要产生一种适当的细胞类型, 在这种情况下, 疾病的症状就会显现出来。例如, 影响神经功能的疾病可能需要产生神经元或胶质细胞17, 而影响肝脏的可能需要肝细胞9,18或胆道上皮干细胞研究19,20,21. 此要求在考虑使用 iPSCs 进行药物发现时引入了一些警告。首先, 了解疾病的细胞基础是很重要的。这可能具有挑战性, 特别是当细胞采用基于位置或环境的特征时。这对于在基因表达谱和酶功能上有差异的肝细胞来说是正确的, 这取决于它们在肝小叶中的位置。第二, 与任何孤立的细胞一起工作可以防止疾病的研究, 反映了组织结构的中断, 如肝硬化, 细胞通讯, 如神经肌肉疾病, 或那些受炎症影响, 如炎症肠道疾病。
为了研究肝病, 一些研究小组描述了从 iPSCs13,22,23,24,25中生成肝细胞样细胞的协议。虽然大多数这些协议是有效的, 但技术的局限性是细胞不能完全重述新鲜肝细胞的功能。这意味着确定从 iPSCs 生成的单元格是否显示评估给定突变的影响所需的功能是很重要的。在新鲜肝细胞中, 表达不完全匹配的基因是那些编码 CYP450 蛋白25,26。其中一些蛋白质在药物代谢和 xenobiotic 反应中起着作用。在考虑屏幕设计时, 这一点很重要, 因为许多药物需要产生活性代谢物, 这可能会影响到 iPSC 的肝细胞。正在积极寻求提高肝细胞质量的努力27。尽管有这些告诫, 但有几项研究表明, 在肝代谢中先天错误的患者中 iPSCs, 可以成功地用于产生像肝细胞一样, 在文化中反映疾病的病理生理学9,18,28,29。
要使筛网成功, 必须保证肝细胞与 iPSCs 的均匀分化。当96井板块的分化未能产生高质量的肝细胞时, 最常见的原因是在亚优条件下, 父母 iPSCs 或 iPSCs 的培养质量较差。干细胞的 iPSCs 必须谨慎地维持在高质量的媒体, 每天变化如1.2 号议定书所述。培养皿中多能细胞的密度应仔细监测, 因为如果细胞变得过度汇合, 它们就会失去干细胞, 自发地分化。我们还发现, 培养细胞的生理氧浓度促进干细胞, 是有益的, 在某些阶段的分化。然而, 如果没有适当的设备, 仍然可以使用环境氧两种培养多潜能细胞和诱导分化。我们建议通过测量包括 OCT-3/4、北美和 TRA-1-60 在内的多个干细胞标记的表达, 并在不考虑文化条件的情况下进行广泛的分化之前, 经常定义股票 iPSCs 的干细胞。我们还观察到, 当细胞形成内胚层的高效率, 他们有一种倾向, 从表面上的板块作为细胞板。我们发现, 用 0.02% EDTA 溶液对 iPSC 衍生的内胚层细胞进行简单的治疗有助于减少细胞脱离。最后, 有效分化所需细胞的最佳浓度在 iPSC 线之间有差异, 并可影响细胞脱离。因此, 重要的是要在经验上确定有效分化所需的细胞浓度, 尤其是在使用新的 iPSC 文化的时候。
总之, 我们已经描述了一种步进式的分化协议, 它从 iPSCs 中产生肝样细胞, 与化学筛相兼容。肝样细胞在96井板中作为单分子膜生成, 过程有效, 重现性好。我们展示了筛选化合物的可行性, 以降低培养基中 APOB 水平的能力。鉴别格式与多种检测方法兼容, 包括 ELISA、qRT PCR 和高含量成像。我们相信, 该领域将找到有用的方法, 以确定潜在的疗法和药理鉴定的途径影响肝细胞分化和功能在未来的应用5,30。

成功地确定可用于治疗罕见疾病的药物依赖于可用于筛查的化验方法的发展。假设或基于目标的屏幕 (反向药理学) 是有用的, 但需要详细了解该疾病的分子基础。表型筛 (经典药理学) 避免了对生物化学途径的详细理解, 而是依赖于精确地反映疾病病理生理学的模型的发展。尽管对以目标为基础的方法的热情, FDA 批准的一流药物的分析显示, 表型屏幕已远远超过成功的1。该方法的总体目标是建立一个高通量筛选平台, 可用于鉴别代谢性肝病的小分子。一些体外模型被描述包括原发肝细胞、肝癌干细胞和肝祖细胞2。然而, 这些模型大多有局限性, 需要新的模型, 可以准确地重述代谢性肝脏缺陷的病理生理学的文化。最近, 人类多潜能干细胞结合基因编辑提供了一个机会, 以模型甚至罕见的稀有疾病的文化, 而不需要直接访问病人的3。虽然使用病人特定的 iPSCs 作为一种工具来发现小分子治疗罕见肝病的概念是合理的, 但只有少数报告表明这种方法的可行性4。然而, 我们最近建立了一个平台, 使用 iPSC 的肝细胞成功地识别药物, 可用于治疗肝代谢缺陷5。
该协议解释了将人类 iPSCs 与96井板中的肝细胞样细胞进行鉴别的过程, 并利用它们来筛选小分子库。并以高胆固醇血症为例, 描述了代谢性肝病的终点分析。这种方法应有助于研究小分子在感染性肝病、代谢性肝病、药物毒性和其他肝病中的作用和应用。

<table border="0" cellpadding="0" cellspacing="0" width="959">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:588px;">100 mm x 20 mm sterile tissue culture dishes&#160;</td>
			<td style="width:164px;">Corning</td>
			<td style="width:119px;">430167</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm x 20 mm sterile suspension culture dishes</td>
			<td>Corning</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-wells tissue culture plate&#160;</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human Albumin</td>
			<td>Dako</td>
			<td>A 0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human FOXA2(6C12)</td>
			<td>Novus Biological</td>
			<td>H00003170-M12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human HNF4 alpha</td>
			<td>Santa Cruz</td>
			<td>SC-6556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human Oct-3/4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-9081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human SOX17</td>
			<td>R&#38;D</td>
			<td>AF1924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-human TRA-1-60 FITC conjugated</td>
			<td>Millipore</td>
			<td>FCMAB115F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Activin A Recombinant Human Protein&#160;</td>
			<td>Invitrogen</td>
			<td>PHC9563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement, minus insulin&#160;</td>
			<td>Invitrogen</td>
			<td>0050129SA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement, serum free&#160;</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BMP4 Recombinant Human Protein&#160;</td>
			<td>Invitrogen</td>
			<td>PHC9533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Dissociation Reagent StemPro&#160; Accutase&#160;</td>
			<td>Invitrogen</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTiter-Glo Luminescent Cell Viability Assay&#160;</td>
			<td>Promega</td>
			<td>7572</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS+(calcium, magnesium)</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS-(no calcium, no magnesium)</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12, HEPES&#160;</td>
			<td>Invitrogen</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ELISA human APOB ELISA development kit</td>
			<td>Mabtech</td>
			<td>3715-1H-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibroblast Growth Factor 2 (FGF2)</td>
			<td>Invitrogen</td>
			<td>PHG0023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepatocyte Culture Medium (HCM Bullet Kit)&#160;</td>
			<td>Lonza</td>
			<td>CC-3198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepatocyte Growth Factor&#160; (HGF)</td>
			<td>Invitrogen</td>
			<td>PHC0321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine&#160;</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution</td>
			<td>Invitrogen</td>
			<td>11140076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oncostatin M (OSM) Recombinant Human Protein&#160;</td>
			<td>Invitrogen</td>
			<td>PHC5015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin&#160;</td>
			<td>Invitrogen</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Feeder free pluripotent stem cell medium: mTesR1&#160;</td>
			<td>STEMCELL technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reduced Growth Factor Basement Membrane Matrix&#160;</td>
			<td>Invitrogen</td>
			<td>A1413301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium, HEPES&#160;</td>
			<td>Invitrogen</td>
			<td>22400105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemAdhere Defined Matrix for hPSC (E-cad-Fc)</td>
			<td>Primorigen Biosciences</td>
			<td>S2071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMB-ELISA Substrate Solution</td>
			<td>Thermo Scientific&#160;</td>
			<td>34022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TRA-1-60 FITC conjugated</td>
			<td>Millipore</td>
			<td>FCMAB115F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versene (EDTA) 0.02%&#160;</td>
			<td>Lonza</td>
			<td>17-711E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Y-27632 ROCK inhibitor</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
	</tbody>
</table>

using human induced pluripotent stem cell-derived hepatocyte-like cells for drug discovery

将人类诱导的多能干细胞 (iPSCs) 与肝细胞样细胞 (HLCs) 进行鉴别的能力为研究肝代谢中先天错误提供了新的机会。然而, 为了提供一个平台, 支持识别可能用于治疗肝病的小分子, 该程序需要一种与筛选数以千计的化合物相兼容的区域性格式。在这里, 我们描述了一个协议使用完全定义的文化条件, 使人类 iPSCs 的可再生分化为肝样细胞在96井组织培养板。我们还提供了一个例子, 使用该平台, 以筛选化合物的能力, 以降低载脂蛋白 B (APOB) 产生的 iPSC 肝细胞产生的家庭高胆固醇血症患者。与药物发现相兼容的平台的可用性应该允许研究人员确定新的治疗方法, 用于影响肝脏的疾病。

生成肝细胞-类似于单元格:图 1描述了在将人类 iPSCs 分化为类似肝细胞时发生的变化的时间范围。iPSCs 的文化在 E Cad Fc 提供大约2毫米直径殖民地表达多能标记 OCT4 (图 1A-B)。在 e-钙黏蛋白基体上生长的细胞的形态学与其他表面培养的不同。他们倾向于有一个扁平的形态学与更大的细胞质表面区域 (图 1A)。虽然人类多潜能干细胞可以在许多不同的基质上有效地培养11, 但在 e-钙黏蛋白基质上 iPSCs 的培养被认为能提高多功能细胞的同质性, 并允许无酶在通道12中对单元格的操作。然而, 一些替代矩阵, 包括基底膜基质, laminins, 及其与受精和小鼠胚胎成纤维细胞 (MEF), 也可以用来维护人类的 iPSCs, 所有应该与描述的过程13 兼容。在所有情况下, 单元格干细胞都可以通过 Tra-1-60 或等效的8来确认。
要初始化差异, 应将 iPSCs 收集为小簇 (图 1A)。在分化开始时, 被镀的细胞应该覆盖大约80% 的板材表面。经过5天的治疗与生长因子, 细胞表达的蛋白质, 是典型的表达在明确的内胚层, 如 SOX17 和 FOXA2 (图 1B)。在这个阶段, 超过90% 的细胞通常会表达这些标记。经过5天的分化, 内胚层转化为肝的命运, 除了 FOXA2, 细胞表达 HNF4a, 这可以识别整个分化过程的其余部分 (图 1B)。由于细胞采用肝的命运, 他们应该覆盖的表面的板块作为单层。在添加了 HGF 后, 细胞开始表达在胎儿肝细胞中发现的蛋白质, 包括 AFP (图 1B)。脂质滴可以在细胞的细胞质内观察到。在分化完成后, 细胞采用立方形态, 细胞核中含有突出的核仁, 有多个脂滴的大细胞质。肝样细胞的一个子集将是多核的。大多数细胞表达了大量的肝细胞蛋白, 包括白蛋白 (图 1B)14。
使用高胆固醇血症作为疾病模型的高效高通量屏幕:高胆固醇血症反映血清中低密度脂蛋白胆固醇 (LDL C) 浓度过高。在家族性高胆固醇血症 (FH) 中, 血清 ldl c 水平的增高主要是由于低密度脂蛋白受体 (LDLR) 中的突变, 它介导吸收 LDL C 对肝细胞进行清除。我们以前描述的 iPSCs 从一个病人的纯合家族性高胆固醇血症和他们的使用, 以产生肝细胞, 反映病人的肝病在文化中9。结果表明, 这些细胞可以被成功地用作药物发现的平台。在筛选出2500种药物的图书馆时, 发现心脏苷类蛋白在 iPSC 肝细胞、原发性人肝细胞和具有人性化肝脏的小鼠中均有降低低密度脂蛋白 C 的能力。此外, 在检查病人病历时, 我们发现, 被规定为心脏病治疗的心脏苷的患者胆固醇水平下降。这些研究证实, iPSC 的肝细胞可以成功地用于屏幕识别治疗。
为了提供一个与筛查相适应的平台, 重要的是可重现性, 并尽量减少良好的变异。图 2显示了一个96井板上每个井的染色结果, 用于检测20天的 HNF4a 和白蛋白的分化。如图所示, 这些肝细胞蛋白在所有水井中的分布都是相似的。
低密度脂蛋白 C 的中心蛋白成分为 APOB。在这里, 我们使用 iPSCs 筛选 APOB 降低化合物, 以提供一个例子, 使用 iPSC 衍生肝细胞的小分子筛查 (图 3A)。APOB 可通过 ELISA 方法在溶液中容易测定。由于 ELISA 可以在大量的样品上进行, 所以可以作为屏幕的基础。通过使用称为 z 因子 (或 z ')15的测量, 最好确定任何检测对高通量筛选的适用性。这一统计参数计算的有效性的检测, 以区分阳性和阴性对照样品。带有 Z 因子 > 0.5 的检测被认为与屏幕15兼容。如图 3B中所示, APOB 检测的 Z ' = 0.73, 它确认了使用该平台进行药物发现的适用性。
在iPSC 培养基中有能力降低 APOB 水平的化合物的标识-派生肝细胞: 为了确定该平台是否可用于识别影响 APOB 水平的新化合物, 我们从南卡罗来纳州的复合收集 (SC3) 代表集 (RSL) 中筛选了1000个小分子。MUSC 是由南卡罗来纳州的治疗发现中心产生的, 包括结构多样并反映父库的化合物。
后药物: APOB 的药物前比为每个化合物 (图 3C), 并通过 z 评分分析 (图 3D)16实现命中识别。在这种情况下, 小分子减少 APOB 以 z 分数≤-2.0 作为潜在命中被考虑了。当筛查1000小分子时, z 分数为2.0 的潜在命中数通常比较容易控制。然而, 在进行更大的屏幕时, 我们将依靠更保守的分数-3.0, 基于3西格玛规则, 以增加对潜在目标的信心。在此示例中, 我们确定了24个可能的命中, 其 APOB 比率从0.86 到 0.44 (图 4A) 不等。对四份样品 (n = 4 生物复制) 进行了二次检测, 以排除对细胞活力有负面影响的化合物, 并确定哪些化合物能重现性降低培养基中 APOB 的含量。在原来的24候选化合物中, 四被发现是可重现的 (p ≤0.05) (图 4B-C)。在进一步的研究中, 2 的这些化合物被发现对细胞的生存能力产生负面影响, 这表明 APOB 中观察到的减少可能是细胞丧失的结果。其余两种化合物将被认为是后续研究的良好候选者。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57194/57194fig1.jpg"> 图 1: 人 iPSCs 的步进肝分化.(A)在收获前 (左面板) 和 iPSCs 后 iPSC 菌落的形态学 (右面板)-在 accutase 治疗后, 细胞应分离成 3-6 细胞/簇. 缩放条 = 50 µm (B) 表格, 显示每个分化阶段 (上部) 的文化条件。染色 (下板) 在不同分化阶段进行标记表达。面板显示 OCT4 和 DAPI 染色核在 iPSCs, SOX17 和 FOXA2 在明确的内胚层, HNF4a 和 FOXA2 的肝祖细胞, HNF4a 和 AFP 在未成熟的肝细胞, 和 HNF4a 和白蛋白在相对成熟的肝样细胞。刻度条 = 100 µm (B)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57194/57194fig1large.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/57194/57194fig2.jpg"> 图 2:96-井板中人 iPSCs 的均匀肝分化.染色是在每一个96井板上进行的, 其中包含 iPSC 的肝细胞在20天的分化, 以检测 (a) 白蛋白和 (B) HNF4a。缩放条 = 100 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57194/57194fig2large.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/57194/57194fig3.jpg"> 图 3: 使用人 iPSC 源肝细胞进行小分子筛查.(A) 示意图概述了用于识别能降低 iPSC 肝细胞培养基中 ABOB 含量的化合物的方法。(B)图显示了 ELISA 检测的 APOB, 用于检测 iPSC 源肝细胞 (蓝色) 和/或未分化 iPSCs (橙色) 中87口井 (n = 87) 培养基中的含量。这些数据用于计算 z 因子 (z ' = 0.73)。( C) 图, 显示后药: iPSC 源肝细胞培养基中 APOB 浓度的前药比值, 与 SC3 24 小时的998种化合物进行治疗。(D) 图, 显示 (C) 中所示数据的 z 分数。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57194/57194fig3large.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/57194/57194fig4.jpg"> 图 4: 验证主命中.(A) 表, 显示在主屏幕中标识的化合物能够减少 APOB。(B、C)条形图, 显示了化合物对后药的影响: 药物前 APOB 浓度比 (B) 和 iPSC 肝细胞的细胞活力 (C) 的后续分析。误差线反射电子扫描电镜 (n = 4 生物复制); * p ≤0.05。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57194/57194fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery at JoVE.com

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

人诱导多潜能干细胞衍生肝细胞的药物发现方法

这里提出的协议描述了一个平台, 用于鉴别肝脏疾病的小分子。本文详细介绍了如何将 iPSCs 分化为96井板中具有肝细胞特征的细胞, 并利用细胞筛选出具有潜在治疗活性的小分子。

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

using-human-induced-pluripotent-stem-cell-derived-hepatocyte-like

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.2K 次观看.  Medical University of South Carolina. 这里提出的协议描述了一个平台, 用于鉴别肝脏疾病的小分子。本文详细介绍了如何将 iPSCs 分化为96井板中具有肝细胞特征的细胞, 并利用细胞筛选出具有潜在治疗活性的小分子。

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

科研

发育生物学

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

多能干细胞衍生的心肌修复心肌细胞

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

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

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 ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

微流控法评价人诱导的多能干细胞内皮细胞 (hipsc-ec) 对白细胞粘附的评价

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

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 ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...