这项工作得到了首席科学家办公室 (TCS/16/37) 的奖项和一份湄公河博士奖学金的支持。

1. 将人类多潜能干细胞 (hPSCs) 播种成96个肝细胞分化的井板
注: 本议定书所用试剂和设备已列于表 1。用于此实验的介质必须是无菌的, 在室温下用于细胞培养。本协议这一部分中描述的所有试剂/解决方案卷都基于一个96井板的单井, 除非另有规定。
<ol><li>
准备层粘连涂层96井板。
	<ol>
<li>解冻一小瓶重组层粘连蛋白 521 (100 µg/毫升) (LN-521) 2 小时到一夜之间4摄氏度。</li>
		<li>通过稀释 LN-521 1x Dulbecco 的磷酸盐缓冲盐水 (DPBS) (含钙2 +/毫克2 +), 制备5µg/毫升溶液。</li>
		<li>使用一个自动液体处理分配器与标准管配药盒添加50µL 的 LN-521 解决方案每井96井板。 注: 除另有规定外, 所有容积均由自动液体处理分配器与标准管配药盒一起执行。先解决这个问题, 直到它到达磁带的配药端, 然后进行免除。在相同的实验中, 可以根据需要多次重复这个过程来准备多个96井板。</li>
		<li>孵化 LN521-coated 板在37°c/5% CO2 2 到 4 h。 注: LN521-coated 板可储存长达两周, 摄氏4摄氏度。将盘子密封在平坦的表面上, 避免蒸发和污染。</li>
	</ol>
</li>
	<li>预热所需的预涂板的数量在37°c 30 分钟到1小时。</li>
	<li>使用8通道吸尘器吸入涂层表面的剩余 LN-521 溶液。 注意: 要避免与涂层表面接触, 避免涂层降解, 这是至关重要的。</li>
	<li>立即添加50µL 每井 mTeSR1 培养基补充10µM 蛋白相关激酶 (岩石) 抑制剂 Y27632 和保持板在孵化, 直到细胞播种37摄氏度/5% CO2。 注: 请勿让层粘连蛋白涂层的水井干燥。</li>
	<li>
在 mTeSR1 培养基中制备 3 x 105细胞/毫升的细胞悬浮液, 辅以10µM 岩石抑制剂 Y27632。 注意: 每个单元格线的播种密度都需要优化。<ol>
<li>从培养皿中吸取培养基, 并将其与未分化的 hPSCs 培养在约75% 至85% 融合培养的 LN-521 上, 如前所述12。 注: hPSCs 在 mTSER1 的 LN-521 上培养, 每24小时改变培养基, 一旦细胞达到80% 融合, 如前所述12, 则定期传代。</li>
		<li>用5毫升室温 1x DPBS (不含钙2 +/毫克2 +) 冲洗细胞, 并吸入缓冲液。</li>
		<li>将5毫升的1x 温和细胞离解试剂添加到细胞中, 并在37摄氏度孵育6到8分钟, 用于细胞离解。 注意: 为了停止反应, 检查显微镜下的基质细胞脱离。细胞应部分脱离板块。如果没有, 将孵化期延长1到2分钟。</li>
		<li>通过吸气温和细胞离解试剂和添加5毫升新鲜 mTeSR1 培养基补充10µM 岩石抑制剂 Y27632 细胞, 终止反应。使用一个细胞刮板来解除细胞从基质和吸管向上和向下使用 P1000 提示几次混合。</li>
		<li>利用基于台盼蓝染色排除法的 hemocytometer 对活细胞进行计数, 并用所需浓度制备细胞悬浮液。</li>
		<li>将所需的细胞数注入无菌的15毫升或50毫升离心管, 并在室温下将细胞离心在 115 x g上以5分钟为单位旋转。 注: 对于一个96井板, H9 细胞线需要5毫升的 mTeSR1 培养基, 含有 3 x 105细胞/毫升。</li>
		<li>吸入上清液并轻轻并用重悬细胞颗粒, 直到在 mTeSR1 培养基中获得37摄氏度的均匀溶液, 辅以10µM 岩石抑制剂 Y27632。 注: 建议使用岩石抑制剂, 以改善细胞生存后再电镀。</li>
	</ol>
</li>
	<li>将细胞悬浮液的50µL 添加到96井板上, 含50µL mTeSR1, 10 µM 岩石抑制剂 Y27632。</li>
	<li>播种后, 返回到细胞孵化器在37摄氏度/5% CO2 24 小时, 以允许细胞附加。</li>
	<li>第二天检查细胞和提取岩石抑制剂一旦细胞与细胞接触已经建立。在40% 融合, 切换到分化介质 (图 1B)。 注: 如果细胞融合高于 40%, 则该板块不适合 HLC 分化 H9 细胞系。</li>
</ol>2. 96 井板重组 Laminins 中肝样细胞 hPSCs 的鉴别
<ol><li>
准备分化培养基和生长因子。
	<ol>
<li>通过将人体激活µg 在 PBS 中的不育0.2% 牛血清白蛋白 (BSA) 中的粉末, 制备人激活。整除的库存和存储在-20 摄氏度。使用在 1:1, 000。</li>
		<li>通过将小鼠 Wnt3a 粉溶于无菌 0.2% BSA, 制备小鼠 Wnt3a 库存溶液 (10 µg/毫升)。整除的库存和存储在-20 摄氏度。使用1:200。</li>
		<li>在无菌 0.2% BSA 中溶解人肝细胞生长因子 (10 µg/毫升), 制备人肝癌 (HGF) 库存液。整除的库存和存储在-20 摄氏度。使用1:1000。</li>
		<li>通过在无菌 0.2% BSA 中溶解粉末, 制备 Oncostatin M (OSM) 库存溶液 (20 µg/毫升)。整除的库存和存储在-20 摄氏度。使用在 1:1, 000。</li>
		<li>确定的内胚层: 准备内胚层分化培养基, 包括 2% B27 补充 (50x, 不含胰岛素), 1% 青霉素/链霉素 (最终浓度: 100 毫升/ml 和100µg/毫升, 分别) 添加到罗斯威尔公园纪念研究所 1640 (RPMI 1640) 基部培养基。贮存在4摄氏度, 并在两周内使用。在每个培养基变化, 补充所需的体积与激活 A 和 Wnt3a 的最终浓度为 100 ng/毫升和 50 ng/毫升, 分别。</li>
		<li>Hepatoblast 分化: 制备 Hepatoblast 分化培养基, 包括20% 的剔除血清置换 (KSR), 0.5% 补充 l-谷氨酰胺, 1% 非必需氨基酸 (NEAA), 0.1 毫米β巯基乙醇, 1% 亚砜, 1%青霉素/链霉素 (最终浓度在100毫升/ml 和100µg/毫升, 分别) 添加到淘汰赛 DMEM (高 DMEM)。过滤器在真空之下并且存放在4°c。两周内使用。</li>
		<li>肝细胞分化: 制备肝细胞分化培养基, 由1% 替代补充 l-谷氨酰胺, 10 µM 氢化物, 21-琥珀酸酯钠盐 (HCC), 1% 青霉素/链霉素 (最终浓度在100毫升/mL 和100µg/毫升, 分别) 添加到 HepatoZYME 培养基。将库存储存在4摄氏度, 并在两周内使用。对于每个培养基的变化, 补充所需的体积与 HGF 和 OSM (最终浓度在 10 ng/毫升和 20 ng/毫升, 分别)。</li>
	</ol>
</li>
	<li>24 h 后播种, 使用自动手持电子通道吸管系统, 小心去除介质, 并添加100µL 的新鲜内胚层吸介质, 补充 100 ng/毫升激活 A 和 50 ng/毫升 Wnt3a。 注意: 一旦添加了最终的内胚层培养基, 分化过程就开始了。一旦细胞播种优化, 细胞分化通常在24小时内启动。所有介质的变化是通过删除介质与自动手持电子通道吸管系统和配药100µL 的新鲜介质使用自动液体处理分配器与标准管配药盒, 除非另有指定。一个自动手持电子通道吸管系统使用96井头, 使用300µL 提示。简而言之, 90 µL 被吸入从井避免任何接触与细胞, 并且重要的是不干燥细胞在媒介变化期间减少细胞压力。</li>
	<li>对于人类胚胎干细胞 (干细胞), 每天改变内胚层分化培养基三天。如果对人类诱导的多潜能干细胞 (hiPSCs) 进行分化, 则使用激活 A 延长内胚层期2天。</li>
	<li>在确定的内胚层感应后, 切换到 KSR/亚砜介质 hepatoblast 规格, 并改变培养基每2天5天。 注: 如果井中有许多非附着细胞, 请使用非补充 DMEM 在下一次培养基改变前清洗细胞。由于协议的第二阶段 (5 天) 的持续时间, 在 hepatoblast 规范的最后一天, 将有3个中等变化, 最后一期。</li>
	<li>hepatoblast 分化后, 改用 HepatoZYME 培养基进行肝细胞成熟。在去除 KSR/亚砜培养基后, 用 HepatoZYME 清洗细胞, 并添加100µL HepatoZYME 成熟培养基, 补充 10 ng/毫升 HGF 和 20 ng/毫升 OSM。</li>
	<li>每48小时更换一次培养基7到10天。在18天的分化, 通过分析基因表达和 CYP P450 代谢活动的 HLCs 特征。</li>
</ol>3. 重组 Laminins 分化的肝细胞样细胞的特征
<ol><li>用染色检测肝细胞特异标记物和偏振标记的表达。</li>
	<li>用 CYP P450 测定法测试肝细胞代谢功能, 如前所述12。</li>
	<li>确定板块变化, 计算每个板块每井单元的总数量。</li>
</ol>4. 高通量免疫细胞化学和图像采集
<ol><li>在18天的分化, 洗井三次与 1x DPBS, 修复 HLCs 通过配药50µL 4% 多聚甲醛 (粉煤灰), 并孵化在室温下15至30分钟。固定后, 使用自动洗板机清洗三次与 PBST (0.1% 补间) (不含钙2 +/毫克2 +)。 注: 除另有规定外, 本节中的所有洗涤均使用自动洗板机进行。所有洗涤都是按照相同的协议进行的。</li>
	<li>Permeabilize 100 µL 的 PBST (不含钙2 +/毫克2 +), 在室温下孵化20分钟。接下来, 在 PBST 中用100µL 10% BSA 进行蛋白阻断, 用平板振动筛在柔和晃动中孵化1小时。在蛋白阻断后, 添加50µL 1% BSA 在 PBST 含有的主要抗体, 孵化在4°c 过夜与温和晃动使用平板振动筛。 注意: 不要在蛋白质块和抗体添加之间进行清洗。</li>
	<li>24小时后, 用 1x DPBS (不含钙2 +/毫克2 +) 清洗三次, 并在 PBST 中添加50µL 1% 的血清二次抗体。在室温下在黑暗中孵化1小时。</li>
	<li>继发抗体孵化后, 用 1x DPBS 冲洗3次。添加50µL 的 DPBS 与 DAPI (10 µg/毫升) 和孵化5–10分钟在室温下在黑暗中。</li>
	<li>最后, 用 DPBS 洗3次, 在井里留下100µL 1x DPBS。车牌已准备好进行成像。 注意: 印版应在黑暗中储存4摄氏度, 直到成像。</li>
	<li>采用高含量成像系统对板材进行图像处理, 并在井中获得多个领域, 以获得良好的表现。使用哥伦布软件 (高含量成像分析系统软件) 量化 HLCs 中不同标记的表达式。 注意: 哥伦布是一个高含量分析软件, 它提供细胞分型的细胞分割分析。同样的结果可以通过使用 CellProfiler, 一个开放源码的生物图像定量分析22的软件来实现。</li>
</ol>

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A., 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=A+dataset+of+images+and+morphological+profiles+of+30,000+small-molecule+treatments+using+the+Cell+Painting+assay.">A dataset of images and morphological profiles of 30,000 small-molecule treatments using the Cell Painting assay.</a> GigaScience. , (2017).</li><li>Pradip, A., 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=High+Content+Analysis+of+Human+Pluripotent+Stem+Cell+Derived+Hepatocytes+Reveals+Drug+Induced+Steatosis+and+Phospholipidosis.">High Content Analysis of Human Pluripotent Stem Cell Derived Hepatocytes Reveals Drug Induced Steatosis and Phospholipidosis.</a> Stem Cells Int. 2016, 2475631 (2016).</li><li>Donato, M. T., G&#243;mez-Lech&#243;n, M. J., Tolosa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+high-content+screening+technology+for+studying+drug-induced+hepatotoxicity+in+preclinical+studies.">Using high-content screening technology for studying drug-induced hepatotoxicity in preclinical studies.</a> Expert Opin Drug Discov. 12 (2), 201-211 (2017).</li><li>Carpenter, A. 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=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7, R100 (2006).</li></ol>

大卫. 干草是 Stemnovate 有限公司的共同创始人和董事。
大卫. 干草是 HigherSteaks 有限公司的董事。

我们的半自动程序是高效, 可靠和经济的, 允许生产的 HLCs 在规模 (图 1)。在平板上的水井间的细胞数量上没有任何显著的差异, 这使得这个平台成为细胞筛选的合适方法 (图 2)。此外, 半自动化工作流使用户能够一次生成大量的板, 这在使用手动过程5、12之前是不可能的。
重要的是, 自动化过程并没有影响分化产量, 大多数细胞表达 HNF4α (89.2%±2) 和白长袍 (图 3)。HLCs 还表达了 CYP P450 酶 CYP2D6 和 CYP3A4, 并显示 CYP1A2 和 CYP3A 代谢活动可比先前报告的实验 (图 3)5。尽管协议的标准化, 细胞融合在分化之前是关键的纯 HLC 分化。因此, 确保良好的细胞分布在井中是一个重要的考虑因素 (图 1)。这已经被证明是细胞系依赖, 因此细胞播种和密度优化是需要的每一个细胞线之前的放大。
在目前的形式, 这个平台不适合生产大量的 HLCs 用于临床应用, 这将最有可能通过使用细胞工厂和生物反应器实现。然而, 所制定的方法确实允许快速生成人类肝细胞进行疾病建模、药物筛选和/或药物再用途研究。此外, 该检测还可用于多路复用, 允许生成多参数数据集进行机械分析。展望未来, 我们还认为, 这种技术可以应用于更复杂的体外系统, 如3D 分化或作为一个平台, 以改善 HLC 表型的体外。
最后, 我们认为, 我们的简单和半自动系统将增加实验吞吐量, 减少实验性的变化, 从而提高生物数据集的质量和他们对人类生理学的推断。

原发性人肝细胞 (PHHs) 代表了目前肝脏生物医学研究的标准。尽管其优势, PHHs 代表一个有限的来源的成熟肝细胞与不稳定表型1。人类胚胎干细胞 (ESCs) 和人类诱导的多潜能干细胞 (iPSC) 被建议为生物医学研究的强大资源, 有望成为人类组织的可再生来源, 包括肝脏2、3、4.目前的肝细胞分化程序产生细胞, 表达肝细胞标记, 基因和功能类似 PHHs3,4,5,6,7, 8,9,10,11。最近, 使用定义的材料和矩阵, 如重组 laminins, 已改善细胞表型和实验再现性5,12,13,14。
传统的细胞培养技术严重依赖手工吹打, 既费时又容易出错。这限制了化验的吞吐量和你可以使用的平板格式。到目前为止, 大多数的研究描述 HLCs 的产生是劳动密集型的性质, 因此小规模在15,16,17。
现有的液体处理和吹打系统的进展, 结合了自动化显微镜和实验室分析仪, 使得有可能制定程序, 最大限度地减少人类干预的要求。我们利用这些技术, 开发了一种半自动分化系统, 用于为基础和应用生物医学研究生成大量的人体肝脏组织。这种方法可以使用人的 ESC 和 iPSC 线进行, 并且需要进行细微的调整, 具体取决于单元格线。将该系统与高含量分析相结合, 证明了 HLC 分型在18、19、20、21的规模上可以更少的耗时和执行。
总之, 这里描述的细胞培养技术的自动化导致了可靠性、实验时间管理和检测吞吐量的提高。

<table>
	<tbody>
		<tr>
			<td>405 LS Washer</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>31350</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS with Calcium and Magnesium</td>
			<td>ThermoFisher</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle cell dissociation reagent</td>
			<td>STEMCELL Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr>
			<td>GripTips for VIAFLO 96</td>
			<td>Integra</td>
			<td>6434</td>
			<td></td>
		</tr>
		<tr>
			<td>HepatoZYME-SFM</td>
			<td>Life Technologies</td>
			<td>&#160; 17705021</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Activin A</td>
			<td>Peprotech</td>
			<td>120-14E</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Hepatocyte Growth Factor</td>
			<td>Peprotech</td>
			<td>100-39</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Oncostatin M</td>
			<td>Peprotech</td>
			<td>300-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Recombinant Laminin 521</td>
			<td>BioLamina</td>
			<td>LN521-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone 21-hemisuccinate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H4881</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Life Technologies</td>
			<td>10828</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 medium</td>
			<td>STEMCELL Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr>
			<td>MultidropCombi Reagent Dispenser</td>
			<td>ThermoFisher</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Life Technologies</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>Operetta High-Content Imaging System</td>
			<td>PerkinElmer</td>
			<td>HH12000000</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse Wnt3a</td>
			<td>R&#38;D Systems</td>
			<td>1324-WN-500/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Rho-associated kinase (ROCK)inhibitor Y27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>21875</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard tube dispensing cassette</td>
			<td>ThermoFisher</td>
			<td>24072670</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>VIAFLO 96 Electronic 96-channel pipette</td>
			<td>Integra</td>
			<td>6001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, no calcium, no magnesium</td>
			<td>ThermoFisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution 4%, buffered, pH 6.9</td>
			<td>Sigma-Aldrich</td>
			<td>1.00496&#160;EMD MILLIPORE</td>
			<td></td>
		</tr>
		<tr>
			<td>P450-Glo CYP3A4 Assay and Screening System</td>
			<td>Promega</td>
			<td>V8801</td>
			<td></td>
		</tr>
		<tr>
			<td>P450-Glo CYP1A2 Assay and Screening System</td>
			<td>Promega</td>
			<td>V8771</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A6684</td>
			<td>1:200 (mouse)</td>
		</tr>
		<tr>
			<td>Alpha-fetoprotein</td>
			<td>Abcam</td>
			<td>ab3980</td>
			<td>1:500 (mouse)</td>
		</tr>
		<tr>
			<td>CYP2D6</td>
			<td>University of Dundee</td>
			<td>University of Dundee</td>
			<td>1:200 (sheep)</td>
		</tr>
		<tr>
			<td>CYP3A4</td>
			<td>University of Dundee</td>
			<td>University of Dundee</td>
			<td>1:200 (sheep)</td>
		</tr>
		<tr>
			<td>HNF-4&#945;</td>
			<td>Santa Cruz</td>
			<td>sc-8987</td>
			<td>1:400 (rabbit)</td>
		</tr>
		<tr>
			<td>E-cadherin</td>
			<td>Abcam</td>
			<td>ab76055</td>
			<td>1:200 (mouse)</td>
		</tr>
		<tr>
			<td>IgG</td>
			<td>DAKO</td>
			<td></td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Anti-Mouse 488 (secondary antibody)</td>
			<td>Life technologies</td>
			<td>A-11001</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Anti-sheep 488 (secondary antibody)</td>
			<td>Life technologies</td>
			<td>A-11015</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Anti-rabbit 488 (secondary antibody)</td>
			<td>Life technologies</td>
			<td>A-11008</td>
			<td>1:400</td>
		</tr>
	</tbody>
</table>

semi-automated production of hepatocyte like cells from pluripotent stem cells

人类多潜能干细胞代表着人类组织的可再生来源。我们的研究重点是从人类胚胎干细胞 (干细胞) 和诱导多潜能干细胞 (iPSCs) 中生成人肝组织。目前的分化过程产生的人肝细胞样细胞 (HLCs) 显示一个混合的胎儿和成人的特点。为了改善细胞的表型, 我们充分定义了我们的分化过程和细胞的利基, 导致细胞数量的生成, 显示改善的基因表达和功能。虽然这些研究标志着进展, 但能够产生大量的多井板进行筛查, 是有限的劳动密集型程序和批次的批次变化。为了解决这个问题, 我们开发了一个半自动的平台, 将多能干细胞分化为 HLCs. 采用液体处理和自动吹打系统进行96井板格式的干细胞播种和分化。在分化后, 采用全自动显微术和多井紫外线分析细胞表型。

细胞分化是用胚胎干细胞系 (H9) 进行的。半自动化平台用于区分和表征单元格 (图 1A)。采用自动液体处理分配器对基质和种子单细胞进行包衣。当细胞达到40% 融合 (0 天) 时, 细胞分化就开始了。媒体的变化是使用自动手持电子通道吸管系统, 以去除介质和液体处理分配器中添加 (图 1A)。采用全自动液体处理机与自动洗板机结合进行细胞固定和染色。使用高含量成像系统和哥伦布软件进行成像和定量。用已建立的测定方法测定细胞色素 P450 活性。继基质孵化后, 细胞上清液被收获, 生物发光记录使用多模微板块阅读器 (图 1A)。此外, 采用常规的相衬显微术测量肝细胞分化过程中体细胞形态学的变化 (图 1B)。
18天, 在 DAPI 染色后检查了井间细胞数的变异性 (图 2A)。七个视野被捕获每井和数量的原子核量化 (图 2A)。我们发现, 平均每井41662±3,366 细胞的水井之间没有统计学差异 (图 2B)。
在18天, HLCs 被固定和染色的典型肝细胞标记 (图 3A G) 和测试 CYP P450 活动。HLCs 表达了肝细胞标记物, 如 HNF4α (89.2% ±2阳性细胞)、白长袍 (92.8% ±6阳性细胞)、AFP (61.8% ±2阳性细胞)。HLCs 还表达了 CYP P450 蛋白、CYP2D6 和 CYP3A4, 并显示了一个明显的多边形外观, 如 e-钙黏蛋白表达所示。HLCs CYP P450 活动在18天进行了测量。HLCs 在295906±45,828 RLU/毫升/毫克的蛋白质和 CYP3A 在1066112±177,416 RLU/毫升/毫克的蛋白质 (图 3H) 展出 CYP1A2 活动。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57995/57995fig1v2.jpg"> 图 1:96 井格式的 PSCs 肝细胞分化.(A)用于半自动化细胞分化、固定、染色、成像和功能活动的设备示意图。(B) HLC 分化期间细胞形态学的代表性变化。简单地, PSCs 是向明确的内胚层, 其次是 hepatoblast 规范的特点是细胞显示鹅卵石样的形态, 并在肝细胞成熟之前, 以获得多角形。刻度条 = 100 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57995/57995fig1v2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57995/57995fig2v3.jpg"> 图 2: HLCs 井对井变异性的评估。(a)用 DAPI 染色的 HLCs 96 井板视图的表示法。刻度条 = 1 毫米(B)每井单元格数的量化。列 (顶部) 和行 (底部) 中每个井的平均单元数, 从七个视图字段中计算, 并使用哥伦布软件进行量化。整个板块的平均细胞数是每井 41662 3366 SEM 细胞。井间无统计学意义差异。采用单路方差分析与 Tukey 的后验统计试验。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57995/57995fig2v2large.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/57995/57995fig3v2.jpg"> 图 3:18 天 HLCs 的肝细胞标记表达和 CYP P450 活性测定.(A)肝细胞核因子4α (HNF4α) 表达的百分比是基于三十个井, 十个视场为一井。(B)白蛋白表达的百分比 +/-SEM, 是基于3个独立的井和十个视野每井。(C)甲胎蛋白 (AFP) 的百分比 +/-SEM 是根据3个独立的油井和十个视野的井。(D) e-钙黏蛋白染色。(E) CYP2D6 染色。(F) CYP3A4 染色。(g). 免疫球蛋白 G (IgG) 染色控制。刻度条 = 50 µm (H) CYP P450 1A2 和3A 代谢活动在 HLCs 18 天。数据代表六生物复制 +/-SEM. 活动被引述作为相对轻的单位 (RLUs)/毫升每1毫克蛋白质。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57995/57995fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells at JoVE.com

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

该协议描述了一种半自动化的方法, 从96井板格式的人类多潜能干细胞中产生肝样细胞。这一过程是快速和成本效益, 允许生产质量保证批次的肝细胞样干细胞的基础和应用人类研究。

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 ...

semi-automated-production-hepatocyte-like-cells-from-pluripotent-stem

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  University of Edinburgh. 该协议描述了一种半自动化的方法, 从96井板格式的人类多潜能干细胞中产生肝样细胞。这一过程是快速和成本效益, 允许生产质量保证批次的肝细胞样干细胞的基础和应用人类研究。

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

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

科研

发育生物学

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

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 ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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 ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

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

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

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

人诱导多潜能干细胞衍生肝细胞的药物发现方法

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

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

人多潜能干细胞第一心场样心脏祖细胞和心室样细胞的生成

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...