Name: using human induced pluripotent stem cell-derived hepatocyte-like cells for drug discovery
Uploaded: 2018-05-19T12:30:00.000+00:00
Duration: 12 min 40 s
Description: Watch this Scientific Journal Video about Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery at JoVE.com

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
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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。
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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>
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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>
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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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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illustration+of+SSMD,+z+score,+SSMD*,+z*+score,+and+t+statistic+for+hit+selection+in+RNAi+high-throughput+screens.">Illustration of SSMD, z score, SSMD*, z* score, and t statistic for hit selection in RNAi high-throughput screens.</a> J Biomol Screen. 16 (7), 775-785 (2011).</li><li>Lorenz, C., 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=Human+iPSC-Derived+Neural+Progenitors+Are+an+Effective+Drug+Discovery+Model+for+Neurological+mtDNA+Disorders.">Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.</a> Cell Stem Cell. 20 (5), 659-674 (2017).</li><li>Rashid, S. 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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=Hepatic+progenitor+cells+of+biliary+origin+with+liver+repopulation+capacity.">Hepatic progenitor cells of biliary origin with liver repopulation capacity.</a> Nat Cell Biol. 17 (8), 971-983 (2015).</li><li>Ogawa, M., 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+cholangiocytes+from+human+pluripotent+stem+cells.">Directed differentiation of cholangiocytes from human pluripotent stem cells.</a> Nat Biotechnol. 33 (8), 853-861 (2015).</li><li>Sampaziotis, F., 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=Cholangiocytes+derived+from+human+induced+pluripotent+stem+cells+for+disease+modeling+and+drug+validation.">Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.</a> Nat Biotechnol. 33 (8), 845-852 (2015).</li><li>Mallanna, S. 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. E., 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=Diverse+Population+Cohorts+of+hiPSCs+and+Derived+Hepatocyte-like+Cells+Reveal+Functional+Genetic+Variation+at+Blood+Lipid-Associated+Loci.">Diverse Population Cohorts of hiPSCs and Derived Hepatocyte-like Cells Reveal Functional Genetic Variation at Blood Lipid-Associated Loci.</a> Cell Stem Cell. 20 (4), 558-570 (2017).</li><li>Davidson, M. D., Ware, B. R., Khetani, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+liver+cells+for+drug+testing+and+disease+modeling.">Stem cell-derived liver cells for drug testing and disease modeling.</a> Discov Med. 19 (106), 349-358 (2015).</li><li>Choi, S. M., 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+drug+screening+and+gene+correction+for+treating+liver+disease+using+patient-specific+stem+cells.">Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells.</a> Hepatology. 57 (6), 2458-2468 (2013).</li><li>Tafaleng, E. N., 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=Induced+pluripotent+stem+cells+model+personalized+variations+in+liver+disease+resulting+from+alpha1-antitrypsin+deficiency.">Induced pluripotent stem cells model personalized variations in liver disease resulting from alpha1-antitrypsin deficiency.</a> Hepatology. 62 (1), 147-157 (2015).</li><li>Jing, R., Duncan, C. B., Duncan, S. 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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?...

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

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

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

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 ...

科研

发育生物学

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

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

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

多能干细胞半自动化生产肝样细胞的研究

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

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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

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

人类多能干细胞有效分化为肝细胞

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

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

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

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

从多能干细胞中无血清自由生产三维人类肝球

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

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

生物学

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

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

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

用于生成 iPSC 衍生的 3D 人肝类器官的综合方案

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

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

用于肝病研究的外周血人肝球

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

生物工程

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

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

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

从人类胚胎干细胞中引导肝细胞样细胞诱导的有效方法

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

免疫与感染

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

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

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

使用定义的分化系统从多能干细胞的肝祖原体规范

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

通过线粒体-ALCAM 分选纯化人 iPSC 来源的肝细胞

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% &plusmn; 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

作者聚焦:推进从人诱导多能干细胞中纯化肝细胞用于再生医学

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.

活线粒体染色在细胞分选中纯化人诱导多能干细胞肝细胞的应用

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...