我们感谢 Gerbren 各布斯和 Jurjen Broeke 的专家技术援助, 普里斯·坎贝尔 Leferink 为这两条控制 iPSC 线的产生和特点作出贡献, 并向 Lisa Gasparotto 展示我们的程序。

1. 准备工作
注意: 对于所有步骤, 请参阅特定物料的材料表。
<ol><li>
为 hPSC 文化准备500毫升定义的 hPSC 培养基 注: 步骤 2.1-2.6 使用介质。<ol>
<li>在4° c 夜间解冻 hPSC 培养基补充剂 (o/n)。从中瓶中取出12.5 毫升的 hPSC 基培养基, 然后加入10毫升的补充剂和2.5 毫升 (使 100 U/升) 青霉素/链霉素 (笔/链球菌) 的瓶子。</li>
		<li>贮存于4° c, 2 周内使用。 注: hPSC 培养基可通过添加10µM 岩石抑制剂 (RI) 和10% 亚砜来冻结细胞。</li>
	</ol>
</li>
	<li>
准备 1 L 神经维持培养基 (NMM) 进行分化培养 注: 步骤 3.1-4.4 使用介质。<ol>
<li>混合谷氨酰胺强化 DMEM/F12 和神经碱性培养基 (1:1 比) 在一个1升瓶, 然后补充 (1x) N2 补充剂, (1x) B27 补充, 5 µg/毫升胰岛素, 1.5 毫米 l-谷氨酰胺, 100 µM 非必需氨基酸 (NEAA), 100 U/l 笔/链球菌, 10 µMβ-基</li>
		<li>4° c 贮存介质, 3 周内使用。 注: 在将补充剂添加到混合基介质之前, 请删除适当的音量以根据库存浓度调整所需的添加元件体积。</li>
	</ol>
</li>
	<li>
为传代 hPSCs 准备500毫升0.5 毫米 EDTA 工作溶液 注: 步骤2.4 和2.5 使用介质。<ol>
<li>在流罩下, 将49毫升磷酸缓冲盐水 (pbs) 从500毫升无菌 pbs 瓶转移到50毫升管。在50毫升管中加入0.5 毫升的 0.5M EDTA 和0.9 克氯化钠。轻轻地混合溶解。</li>
		<li>过滤消毒解决方案使用0.22 µm 过滤器和转移到500毫升消毒 PBS 瓶。室温贮存 (RT)。</li>
	</ol>
</li>
	<li>
为 hPSC 文化准备 hPSC 文化板块 注: 使用板的步骤 2.1-2.6。<ol>
<li>制作 50x hPSC-适当的贴附涂层 (评定总署) 的工作解决方案: 解冻4° c 的评定总署的一瓶。稀释评定总署在1:1 的比例与 DMEM/F12 和转移为400µL 等分成1.5 毫升管。存储50x 工作解决方案评定总署在-80 ° c。 注: (重要) 评定总署在 RT 中快速固化, 因此所有组件 (DMEM、管、等) 都必须在冰上 (或在4° c) 上保存。</li>
		<li>50x 工作溶液的解冻管在4° c, 然后在冷 DMEM/F12 中稀释50x。添加750µL/井稀释评定总署为6WP。孵育板至少1小时在37° c。 注: 在4° c 下, 在 1 h 潜伏期后将钢板包裹起来, 可将该板材储存1周。预热盘至37° c 前使用。</li>
	</ol>
</li>
	<li>
为鉴别培养制备抗粘连 (AA) 板 注: 使用板的步骤 3.1-4.1。<ol>
<li>用95% 乙醇生产5毫克/毫升聚 (2-羟乙基甲基丙烯酸酯) 溶液。在37° c 时摇动 o/n 直到得到清晰的解。存储在 RT。 注: 通过0.22 µm 过滤器过滤可去除溶。</li>
		<li>在培养基中加入多聚, 使其覆盖每一个井的底部。在37° c 下孵育2天, 并检查板材以确保液面/均匀涂层的完全蒸发。AA 板可以包装和存储在 RT。</li>
	</ol>
</li>
	<li>
制备聚 l-鸟氨酸/层粘连蛋白 (巴解组织/林) 板的分化培养 注: 使用板的步骤 4.2-4.4。<ol>
<li>用20µg/毫升巴解组织溶解在无菌 PBS 中, 涂上井的表面积。在37° c 下孵育板 o/n。吸气, 用 PBS 冲洗3次。 注: (可选) 带巴解组织的孵育板可包装并贮存在4° c, 直至需要。</li>
		<li>用10µg/毫升的林在无菌 PBS 中溶解, 在巴解组织涂层的水井表面涂敷。孵育至少2小时在37° c 或 o/n 在4° c。用 PBS 将林和洗井 2-3x, 然后立即添加适当的培养基和/或细胞。 注: (可选) 去除的 LAM 溶液可贮存在4° c, 再使用2倍。(重要)不允许涂覆的表面晾干;为了防止这种情况立即添加 PBS 或适当的媒体。 
		 </li>
	</ol>
</li>
</ol>2. 议定书 1: 无馈线 hPSC 文化
注: 干细胞是从非商业组织 (行 H01, 请参阅材料表) 获得的。三 hiPSC 控制线 (hvs51, 60, 和 88) 是由三健康的人患者 (成纤维细胞从匿名, 非可识别的捐赠者获得, 因此免于 IRB 批准) 产生的成纤维细胞,10, 17。
<ol><li>保持 hPSCs 的无饲养文化
	<ol>
<li>在解冻和电镀 hPSCs 到评定总署的 hPSC 介质板 (见步骤 2.2), 维护 hPSCs 在37° c 与 5% CO2。刷新 hPSC 介质 (参见步骤 2.3), 除解冻或传代后的次日, 并检查显微镜下的细胞 (目标: 2.5x/0.06, 5 x/0.12 Ph0, 10 x/0.25 Ph1) 观察生长速率并识别分化的潜在区域 (图3, 左上面板显示差异的示例)。</li>
		<li>通过 hPSCs 每3-4 天, 或当文化达到 > 80% 汇合。如果少于5% 的细胞表现出分化, 使用正常的 hPSC 传代方法 (见步骤 2.4), 否则使用温和的方法 (见步骤 2.5)。当不再需要的文化, hPSCs 可能会冻结长期存储 (见步骤 2.6)。</li>
	</ol></li>
	<li>hPSC 介质中的解冻 hPSCs
	<ol>
<li>将所需的 hPSC 介质的体积转化为解冻过程的无菌管 (9 毫升/低温管) 和准备好的评定总署接收解冻的细胞。补充在两个管中的培养基与10µM RI。</li>
		<li>从2存储中检索 cryotube, 并将其直接放入水浴 (37 ° c) 中。当仅一个小冰晶遗骸的, 从水浴去除并且转移低温管子的内容到管子为解冻过程 (总容量10毫升)。离心管在 290 x g 为5分钟在 RT。</li>
		<li>使用血清学吸管从评定总署的油井中移除评定总署的溶液 (见步骤 1.4), 目的是接收细胞, 并添加 hPSC 培养基与10µM RI。 注意: (重要) 不要用吸针吸入评定总署的解决方案, 也可以将管线固化并堵塞到真空泵。</li>
		<li>用10µM RI 将 hPSC 培养基中的上清液和重细胞去除。将单元格分布到目标板, 比例为1低温管/井6WP。孵育在37° c 与 5% CO2, 并且不刷新媒介为1天。 注意: 在 5% O2处启动单元格可能会增加细胞存活率。</li>
	</ol></li>
	<li>刷新 hPSC 介质
	<ol>
<li>在 RT 或水浴中, 在无菌管中加热必要的 hPSC 培养基体积;建议2毫升/井6WP。 注: (可选): 通过添加额外的 hPSC 培养基, hPSCs 可以保持额外的一天没有刷新;但是, 不要让这一周超过一次。</li>
		<li>从含有 hPSCs 的水井中吸取培养基, 添加新鲜的 hPSC 培养基。</li>
		<li>培养 hPSCs 在一个孵化器在37° c 和 5% CO2。</li>
	</ol></li>
	<li>hPSC 介质中的通道 hPSCs
	<ol>
<li>将所需的 hPSC 介质的体积转移到无菌管, 用于传代过程, 并用于制备评定总署的板材以接收传代细胞。以10µM RI 为目的板补充培养基。在 RT 或水浴中加热介质管。 注: 如果使用比材料表中列出的替代涂层材料, 则准备和处理将有所不同。</li>
		<li>使用血清学吸管从评定总署的油井中移除评定总署的溶液 (见步骤 1.4), 目的是接收细胞, 并添加 hPSC 培养基与10µM RI。 注意: (重要) 不要用吸针吸入评定总署的溶液, 因为它可能会凝固并堵塞真空泵的管线。</li>
		<li>从井中抽出 hPSCs 传代, 用0.5 毫米 edta 冲洗两次细胞, 再加入0.5 毫米 edta, 在37° c 孵育2-5 分钟。 注: 1 毫升/井6WP 是一个足够的体积 EDTA 的洗涤和孵化。</li>
		<li>检查显微镜下的水井 (目标: 2.5x/0.06, 5 x/0.12 Ph0, 10 x/0.25 Ph1)。如果细胞开始分离, 用 hPSC 培养基吸出 EDTA 溶液并释放细胞。 注: (重要) 注意不要在吸 EDTA 时移除整个 hPSC 菌落 (不要等到整个菌落分离)。不要冲洗细胞超过5次, 因为这可能伤害 hPSCs 和影响多。此外, 不要让细胞站在 hPSC 介质与 RI 前冲洗细胞从水井, 因为他们可能会重新坚持板。</li>
		<li>根据经验确定 (通常与汇合, 殖民地大小和增长率), 转移 hPSCs 到目标板块的井用分裂比率 1:4-1:16 (即, 1 井从原始的板材到4井目标板)。孵育在37° c 与 5% CO2, 并且不刷新媒介为1天。 注: 分裂率高达 1:16-1:20 是可能的, 以防止拥挤和改善外观的殖民地。</li>
	</ol></li>
	<li>用温和法 (g-法) hPSCs 通道
	<ol>
<li>将所需的 hPSC 介质的体积转移到无菌管, 用于传代过程, 并用于制备评定总署的板材以接收传代细胞。以10µM RI 为目的板补充培养基。热媒管在 RT, 或在水浴在37° c。</li>
		<li>使用血清学吸管从评定总署的油井中移除评定总署的溶液 (见步骤 1.4), 用于接收细胞, 并添加 hPSC 培养基与10µM RI。 注: (重要) 不要用吸针吸入评定总署, 也可以将管线固化并堵塞到真空泵。</li>
		<li>从 hPSCs 的井中抽出培养基传代, 用0.5 毫米 EDTA 冲洗两次细胞。在第二次洗涤, 等待三十年代之前, 吸 EDTA, 然后添加1毫升 PBS 和孵化4-9 分钟在37° c。等待时, 用4毫升 PBS 制备无菌管。 注: 1 毫升/井6WP 是足够的 EDTA 的洗涤量。</li>
		<li>检查显微镜下的水井 (目标: 2.5x/0.06, 5 x/0.12 Ph0, 10 x/0.25 Ph1)。如果细胞开始分离, 小心地敲击盘子的两侧来帮助自由的殖民地。当 > 50% 的殖民地是自由漂浮, 使用5毫升血清吸管转移到1毫升 pbs 的菌落到含有4毫升 pbs 的管 (不研制)。 注意: (重要) 因为目的是清理 hPSC 的区域性, 请检查以确定区分的单元格是否仍然附着在板上。此外, 这是没有必要通过所有的殖民地在一个良好的使用这个过程, 所以仍然依附的殖民地可能会留下。</li>
		<li>在 RT 中等待5-10 分钟, 使细胞在试管中沉淀 (不要离心)。从试管中抽吸 PBS, 注意不要移除已沉淀的 hPSCs。仔细重 hPSC 培养基中的细胞 (不研制), 并将细胞转移到目标板上, 使用分裂率为 1:4-1:16。孵育在37° c 与 5% CO2, 并且不刷新媒介为1天。</li>
	</ol></li>
	<li>冻结 hPSCs
	<ol>
<li>根据合流, 使用1井的 hPSCs, 在2-3 天 (最大) 的文化, 以填补1-2 冷冻保存瓶储存在 LN 在2。</li>
		<li>在传代 (步骤 2.5) 的末端, 使用500µL 或1毫升的 hPSC 培养基将细胞从1井的6WP 到1或2冷冻小瓶, 分别 (500 µL/瓶)。每管, 添加500µL 2x 冷冻培养基含有 hPSC 培养基, 20 µM RI, 20% 亚砜。 注: (可选) 细胞可直接在1毫升/瓶的1x 冷冻介质中传输。</li>
		<li>将低温管置于低温容器中 (含异丙醇) 预冷却至4° c, 并立即贮存在-80 ° c。</li>
		<li>第二天, 将低温管传输至 LN2罐中进行长期贮存。</li>
	</ol></li>
</ol>3. 议定书 2: 3D "化" 的区别
<ol><li>
hPSCs 传代的改性 G 化方法的建立
	<ol>
<li>将所需的 NMM 量转移到无菌管的目标井数。补充 4 ng/毫升 FGF2 和10µM RI。在 RT 处加热介质管, 或在37° c 的水浴中预热。 注: 根据起始井的汇合, hPSCs 集中在分配到目的板材在2:1 或3:1 比率 (即, 2 口井从原始的板材到1井目标板材), 以末端容量2.5 毫升/井6WP。</li>
		<li>将含有 hPSCs 的井中的培养基抽吸, 用0.5 毫米 EDTA 清洗两次细胞。在第二次洗涤, 等待三十年代之前, 吸 EDTA, 然后添加1毫升 PBS 和孵化4-9 分钟在37° c。等待时, 用4毫升 PBS 制备无菌管。 注: 1 毫升/井6WP 是足够的 EDTA 的洗涤量。(重要)最好使用在最后一段时间内不超过3天的 hPSCs, 在解冻后至少1-2 通道。</li>
		<li>检查显微镜下的水井 (目标: 2.5x/0.06, 5 x/0.12 Ph0, 10 x/0.25 Ph1)。如果细胞开始分离, 通过轻轻敲击板的两侧释放细胞。将细胞转移到含有5毫升吸管的4毫升 PBS 的试管中。 注意: (重要) 光冲洗和研磨被允许分解菌落和收集松散的细胞, 但忽略仍然附着在板上的细胞。</li>
		<li>允许管在 RT 上坐10分钟, 用于重力分离。(可选) 如果需要, 可以将细胞轻轻地离心 (不大于 200 x g, 在 RT, 5 分钟)。</li>
		<li>从试管中吸取 PBS, 注意不要移除固定的 hPSCs, 重 NMM 的细胞, 用 4 ng/毫升 FGF2 和10µM RI, 然后以2:1 或3:1 的比例分布到 AA 涂层板上。孵育在37° c 与 5% CO2, 并且不刷新媒介为3天, 除非需要 (参见步骤 3.2.1)。 注: (可选) 为了方便转移细胞, 在分布前将培养基的一部分添加到目标板的井中, 并重在较小的体积内。此外, hPSCs 可能被染色和计数, 以确定确切的起始细胞密度。然而, 最终的体积在目标板块应该是2.5 毫升/井的6WP。</li>
	</ol>
</li>
	<li>
在37° c (5% CO2) 上保持自由浮动差异化培养
	<ol>
<li>每天检查盘子中的颜色变化, 死细胞堆积, 结块, 并坚持到井底。<ol>
<li>可选: 无论介质更改时间表如何, 刷新 (包括前3天不刷新) 介质 (如果已变成黄色), 并按照中的更改/刷新说明 (步骤 3.3)。如果大多数细胞出现死亡, 按照指示进行介质变化/刷新与重力分离 (步骤 3.4)。 注意: 预期形成的 EBs 和成长为大细胞聚集物, 但细胞和细胞聚集可以聚集成大质量不是由于个人的生长/增殖。如果这被观察, 轻的研磨打破大量是允许的。如果细胞开始黏附在 AA 板表面, 剩余的漂浮的内容可能直接地转移到新的井或转移在过程中中度变化或刷新。不要试图转移附着在板上的细胞。</li>
		</ol>
</li>
		<li>3天, 将培养基改成 NMM, 4 ng/毫升 FGF2。每隔一天刷新一次介质。</li>
		<li>7天, 用1µM 维甲酸 (RA)、100 ng/毫升 FGF8B 和 4 ng/毫升 FGF2 将培养基改 NMM。每隔一天刷新一次介质。 注意: (重要) RA 是光敏的。保护镭文化样品免受光照。</li>
		<li>14天, 用 100 ng/毫升 FGF8B、100 ng/毫升 FGF4 和 20 ng/ml FGF2 将培养基改成 NMM。每隔一天刷新一次介质。</li>
		<li>17天, 将培养基改成 NMM, 100 ng/毫升 FGF8B。每隔一天刷新一次介质。</li>
		<li>在21天, 改变培养基到 NMM 与 100 ng/毫升脑源性神经营养因子 (BDNF) 和 10 ng/毫升胶质来源神经营养因子 (GDNF)。每隔一天刷新一次介质。</li>
		<li>在28天, 改变培养基到 NMM 与 100 ng/毫升 BDNF 和 10 ng/毫升 GDNF, 3 ng/毫升, 100 ng/毫升神经营养因子 3 (NT3), 和25毫米氯化钾. 每隔一天刷新一次介质。</li>
		<li>35天, 收集 3D organoids 进行分析。</li>
	</ol>
</li>
	<li>
更改/刷新3D 文化的差异媒体
	<ol>
<li>将所需的 NMM 量与相应的组件进行传输 (请参阅步骤 3.2. 2-3. 2.7 的组件时间表) 到无菌管。温水浴在37° c。</li>
		<li>把盘子轻轻摇动, 直到细胞沉淀到井底边缘。用血清吸管小心去除2毫升的旧培养基, 避免移除细胞, 然后加入2毫升的新鲜培养基。孵育在37° c 与 5% CO2。 注: (重要) 最终音量应为2.5 毫升/6WP。如果蒸发发生, 不要去除2毫升的旧培养基, 因为这将进一步干燥细胞培养;相反, 添加额外的媒体。</li>
	</ol>
</li>
	<li>
利用重力分离改变/刷新微分介质
	<ol>
<li>将所需的 NMM 量与适当的元件转移到无菌管。温水浴在37° c。</li>
		<li>将井内的内容转移至无菌管, 并允许该管在 RT 处坐10分钟, 用于重力分离。</li>
		<li>使用吸管去除管中的旧介质, 注意不要移除已沉淀的细胞, 在 NMM 中用适当的成分重, 然后分配给新的 AA 涂层井。孵育在37° c 与 5% CO2。 注: (可选) 为了方便起见, 在分配前可以在目标井中添加一部分培养基, 将细胞悬浮在较低的体积内。最终体积应为2.5 毫升/6WP。</li>
	</ol>
</li>
</ol>4. 议定书 3: 备选2D 差异文化
<ol><li>
按照3节中的步骤启动和维护区域性, 以便在12天后使用3D 协议
	<ol>
<li>按照步骤3.3 和3.4 执行步骤 3.1-3.2. 3 和更改/刷新介质。</li>
	</ol>
</li>
	<li>
切换到并保持为2D 单层培养
	<ol>
<li>在13天的差异化, 按照指示的变化/刷新介质与重力分离 (步骤 3.4), 只分配细胞/骨料到巴解组织/LAM 涂层板 (见步骤 1.6) 的结束量2.5 毫升/井的6WP。 注: 在初始电镀期间, 培养基可辅以10µM RI, 以帮助细胞的黏附和存活。(重要)最好是均匀地在井中传播细胞, 以避免低密度或拥挤的板块, 并通过 (见步骤 4.4) 必要的。必须根据细胞系的增殖率和产品的用途, 从经验上确定巴解组织/LAM 涂层板 (即、6WP、12WP、等) 的首选尺寸。指令将给6WP 的体积, 并可以转换为每加倍的井号 (即, 2 毫升/良好的 6WP, 1 毫升/良好的 12WP,等)</li>
		<li>14天, 用 100 ng/毫升 FGF8B、100 ng/毫升 FGF4 和 20 ng/ml FGF2 将培养基改成 NMM。如步骤4.3 所述, 每隔一天刷新一次介质。</li>
		<li>17天, 将培养基改成 NMM, 100 ng/毫升 FGF8B。如步骤4.3 所述, 每隔一天刷新一次介质。</li>
		<li>在21天, 改变培养基到 NMM 与 100 ng/毫升 BDNF 和 10 ng/毫升 GDNF。如步骤4.3 所述, 每隔一天刷新一次介质。</li>
		<li>在28天, 改变培养基到 NMM 与 100 ng/毫升 BDNF 和 10 ng/毫升 GDNF, 3 ng/毫升凹陷, 100 ng/毫升 NT3, 和25毫米氯化钾. 每隔一天刷新一次介质, 如步骤4.3 所述。</li>
		<li>在35天, 收集用于分析的单元格, 或与扩展区域性的步骤4.2.5 保持相同的介质 (未测试潜在限制)。</li>
	</ol>
</li>
	<li>
更改/刷新2D 文化的差异媒体
	<ol>
<li>将所需的 NMM 量与适当的组件进行传输 (请参阅步骤 4.2. 2-4. 2.5 的组件时间表) 到无菌管。温水浴在37° c。</li>
		<li>从水井中吸取培养基, 然后加入2毫升新培养基。孵育在37° c 与 5% CO2。 注: (可选) 端部容积可保持在2.5 毫升/6WP, 使用吸管去除2毫升的旧培养基和增加2毫升的新鲜培养基。在井中保留部分旧培养基, 防止细胞与空气接触, 可在改变步骤时减少对细胞的冲击。</li>
	</ol>
</li>
	<li>
通道2D 分化培养
	<ol>
<li>将所需的 NMM 量与相应的组件 (参见步骤 4.2. 2-4. 2.5) 转到无菌管、传代过程, 以及分别准备用于接收传代细胞的巴解组织/LAM 涂层板。用10µM RI 补充目的板的培养基。在 RT 或在37° c 的水浴中加热介质管。为了节省组件, 可以单独使用 NMM 在传代过程中清洗细胞。 注: (重要) 如果产品将用于钙显像实验中, 确保通过细胞在2-6 天前分化结束。</li>
		<li>从水井中吸取传代。添加300µL/井的胰蛋白酶为基础的离解剂 (见材料表), 旋流板覆盖井, 然后立即删除离解剂。</li>
		<li>允许板在 RT 上坐2分钟, 然后通过敲击板的两侧松开电池。添加600µL/良好定义的胰蛋白酶抑制剂 (dti) 和转移的细胞 dti 到一个无菌管与5毫升 NMM。</li>
		<li>离心管在 290 x g 为15分钟, 在 RT. 吸气培养基, 再加5毫升 NMM 到试管。</li>
		<li>离心管在 290 x g 为15分钟, 在 RT. 吸气培养基, 并在含有 RI 的适当培养基中重细胞。</li>
		<li>根据初始汇流、扩散速率和原点与目标板的大小差异, 将巴解组织/LAM 板上的单元分布在 1:1-1:12 的分裂比例上, 并在37° c 与 5% CO2之间保持。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

复杂性和成本是干细胞研究人员在选择或发展差异性协议时的相关因素。这是特别真实的, 因为它是一个公开的问题, 需要多少外部控制, 以产生所需的细胞类型, 或-以不同的姿势-如何胜任 hPSCs 是在生产自己的发展环境, 如果留给自己足够的营养.外部因素的介绍在体外可能很好地产生所需的细胞产品, 但它们也可能干扰内在的发育能力细胞将显示在体内。这种考虑是很重要的, 特别是如果目标是使用病人衍生的干细胞疾病建模。广泛使用的模式和/或生长因子可以掩盖疾病表型。本报告中详述的协议遵循先前研究的趋势, 以降低复杂性、成本和/或使用外部模式因素8,9。
根据 Muguruma et al.)报告的结果和我们自己最近的研究, 似乎没有一致的努力去重现体内条件, 就可以实现对小脑命运的分化, 就像以前的研究所做的那样1,2,3,4,8,10. 有趣的是, 这两项研究使用了不同的生长因子, 这表明两者都不是必需的, 尽管两者都使用了 FGF2。我们运行了额外的测试, 其中 FGFs 被选择性地排除在协议之外, 并显示出单元格能够生成相同的产品, 而不需要外部 FGFs10。我们的研究之间的差异是合格的事实, 我们使用不同的 hPSC 线和培养方法, 诱导神经分化与 RA, 并包括成分, 以支持颗粒细胞的生存和成熟 (BDNF, GDNF, 下垂, 和氯化钾)11 -14。此外, 与 Muguruma et al.相比, 使用了一种不太复杂的启动方法。他们的协议始于在96WPs 中生成统一的 EBs, 这使得它们彼此之间的物理和化学隔离。在 EB 形成期间, 这里的公司在6WPs 的时候都比较拥挤, 这使得它们可以自由地相互作用。这可能会对 EBs 和后来的 organoids (包括信号化合物的内在生成) 的物理和化学环境产生差异性的影响, 这是未知的, 可以探索。此外, 当我们显示与小脑起源相关的基因的表达, 在形态学上与 Muguruma et al.报告的结构相似, 我们不能排除神经元样结构的生成,是非小脑的身份。未来的研究, 使用 Muguruma et al.报告的大型抗体组(即、ATOH1、CALB、等) 将进行此类分配, 并比较两个协议的产品, 更具决定性。
在3D 协议中, 重要的是开始并保持足够数量的细胞在文化中, 以确保足够数量的最终产品进行分析。在协议的早期, 由于有重大的死亡, 我们建议在文化的前3天 (图 1) 中开始使用超过 500 EBs/井。这不应该是很难实现给定的殖民地大小的 hPSCs 在无饲养的文化, 但可能不是那么容易对那些仍然使用馈线依赖的方法。考虑到大量的细胞, 重要的是要注意在培养基中的颜色变化 (表明 pH 值的变化), 并积累死细胞。必须纠正这两种情况, 以防止文化崩溃。也可能有细胞和聚集成块状的结构。虽然它可能仍然导致骨料, 可以分析, 产品数量将大大减少, 所以把它们分解成较小的聚合与温和的研磨可以是有用的。但是, 避免干扰正常的聚合, 它们本身可以增长到大的大小 (图 4)。如果集料变得太稀, 建议结合水井, 使骨料不完全孤立。产品的可变性 (数量、大小和形态) 是3D 细胞培养中的一个众所周知的问题, 包括那些从孤立的、统一的 EB 形成步骤开始的协议, 建议一个不太复杂的启动过程 (如此处所述的协议) 可能更实用8,15。尽管这种异质性是研究人员需要牢记的东西, 特别是在分析过程中, 报告的协议生成的产品与其他3D 协议8、9、15中发现的一致。根据大小和形态学, 它们属于神经丛的范围内的大脑化, 如最近的审查所描述的克拉瓦和兰开斯特15, 最适合的分类的椭球。特别值得注意的是, 存在的3D 结构暗示神经花环与流明, (分) 心室区, 和菱形唇状特征 (图 5和 图 6) 由其他组标识8,15,16,17. 由于每个实验都至少产生了一个具有假定 VZ/SVZs 和小脑相关标记 (ZIC1, KIRREL2) 的聚合体, 这些都是确定使用我们的协议的3D 微分的成功的有用标准, 与 RL 一样提供额外支持的功能。延长过去35天的文化长度没有被测试, 但可以被追求, 以确定最大程度的增长, 复杂性和成熟度允许的这种技术。
2D 协议使用相同的非附着的 EB 形成和神经感应过程作为3D 协议, 因此上述评论也适用。一旦电镀, 应考虑不同的考虑因素。EBs 应该迅速的坚持, 使细胞向外扩散到盘子里。如果有黏附的问题, 增加 RI (如果未使用), 减少的媒介的容量或实验性变动在巴解组织或 LAM 集中可能适用。重要的是要保持细胞的生长过于密集或稀疏 (最好在 20-80% 70-100) 在井中;每日监测和及时传代是重要的, 以避免过度70-100 或漂浮的细胞。与3D 协议不同的是, 在文化中不应该有重大的死亡, 尽管可能会有一些地区的增长缓慢, 或者是扩散率的减慢。传代影响细胞的成熟状态 (例如, 去除细胞过程和开发细胞之间的网络), 并且应该记住当接近点, 细胞将被收集或分析以某种方式。例如, 对于钙显像, 这是非常重要的通道细胞之间的2-6 天之前, 分析。传代太接近分析可能意味着细胞没有时间连接和/或成熟, 太远可能导致细胞过度拥挤, 使成像困难。虽然实验之间的差异可能存在, 结果是一致的那些报告在最初的2D 小脑协议1,2。ICC 染色和基因表达分析证实了颗粒细胞标记 ZIC1 细胞的存在, 同时也识别与其他神经和小脑特征相关的标记 (图 7和图 8)。钙显像, 其中包括电刺激细胞孵育 fluor5 染料, 表明功能性神经元活动 (图 9,补充图 1, 和补充图 2), 虽然这是不确定的, 如果这些是颗粒细胞。值得商榷的是, 通过延长35天的培养时间, 使细胞有更多的成熟, 功能性神经元活动的数量应该增加。这一潜力可以在未来探索。
除了上面建议的研究线之外, 还有兴趣确定2D 和3D 协议之间的产品标识 (数量和质量) 的差异。外部 FGFs 的重要性没有在2D 协议中进行测试, 如果知道在电镀后缺少3D 结构, 以及相关的信号通路, 会使2D 文化或多或少地依赖于那些早期的模式化合物, 这将是很有用的。更多的剥离协议 (例如, 没有 RA, BDNF, 凹陷) 是同样可行的进一步调查的线。最后, 未来的研究可能会受益于新的研究工具, 更好地表征 (和评估) 人特有的小脑神经元亚型的生成效率。
考虑到给定的注意事项, 两个报告的协议可以用于小脑分化, 产品适合不同的目的。它们可以作为研究人员进行试验研究的实际出发点, 测试细胞系对这种分化的可行性, 或者作为其他类型靶向神经分化的基本模型。

体外鉴别 hPSCs 向小脑谱系的协议最初是在模仿活体小脑发育123,4的原理上操作的。因此, 他们需要一系列的因素, 在特定的时间, 以推动亲小脑模式和成熟。其中主要有 WNT、骨形态发生蛋白 (bmp) 和成纤维细胞生长因子 (FGFs), 在中后脑发育中具有已知的作用, 并形成峡组织者5,6,7。当然, 每一个额外的步骤和因素意味着增加劳动密集型的操作和更大的费用的研究员, 因此, 开发更简单的协议, 能够达到平等的结果是有趣的。这个实际问题与假设的问题很好地吻合, 即细胞是否需要对其发展进行如此严密的外部控制体外。
对于小脑分化, 2015年发表的一项协议讨论了使用大量生长因子的必要性, 方法是仅使用 FGF2、FGF19 和基质细胞衍生因子 1 (SDF1), 用于模式用途8。这项研究也不同于以前的小脑协议, 使用自由浮动的3D 文化系统。除了为小脑标记产生阳性细胞外, 他们的技术所产生的大脑 "organoids" 显示了相关的形态学, 在传统的2D 单层培养中, 如菱形唇状结构中不可用。虽然在生长因子方面不那么复杂和昂贵, 但在96井板 (96WPs) 中形成均匀的胚状体 (EBs) 和培养物等其他特征使其在初始步骤过程中变得复杂。同年发布的另一3D 协议, 报告了使用普通和廉价的细胞培养技术成功分化为神经谱系的方法9。虽然这组是研究皮质而不是小脑分化, 他们的概念的应用, 小脑分化不能打折扣。
我们最近报告了一个3D 的小脑差异化协议使用减少的模式因素 (即 FGF2, 4, 和 8), 以及一个简化的设置通过保持在6井板 (6WPs) 中的细胞, 以尽量减少中等要求10。为了辅助颗粒细胞的生产, 在最后的成熟阶段使用了快速激动剂 (凹陷)。凹陷是一种较便宜的化学替代声波刺猬 (嘘), 这已被用于早期的小脑协议, 由于它的作用促进颗粒细胞前体的生长 (控制点)在体内1,2, 11,12,13。差异化产品与其他3D 协议的一致性, 包括在形态学相关的结构中存在小脑相关的标记8,9。这样的结果强化了先前的信息, 即对于复杂的 3D体外差异化协议, 对体内环境的详细模拟可能不是必需的。
除了3D 协议之外, 本报告还描述了一个2D 协议, 它采用相同的快速设置、基本材料和减少增长因子的数量来设计。它能够从人类胚胎干细胞 (干细胞) 或诱导多能干细胞 (hiPSCs) 中产生细胞, 对与早期神经、小脑和颗粒细胞特征相关的标记物是阳性的。此外, 钙显像表明存在的功能性人神经元。在协议之间进行选择的能力, 为研究人员增加了一定的灵活性: (1) 生成特定的细胞类型, (2) 人脑发育和相关结构的建模, (3) 单层优化分析设置 (例如, 膜片钳录音), 或 (4) 混合神经文化中的细胞-细胞相互作用。他们的简单和低成本的性质使他们可以访问的研究人员谁是新的 hPSC 领域, 或需要的基础 hPSC 程序, 以探索进一步分化的选择。

<table>
	<tbody>
		<tr>
			<td>DMEM/F12+Glutamax</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td>glutamine fortified DMEM/F12</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>neural basic medium</td>
		</tr>
		<tr>
			<td>N2 supplement&#160;</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Imgen</td>
			<td>PT468-B</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids (NEAA)</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly (2-hydroxyethyl methacrylate)</td>
			<td>Sigma</td>
			<td>P3932</td>
			<td>aka Poly-Hema</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>E8 medium and supplement</td>
			<td>Gibco</td>
			<td>A1517001</td>
			<td>hPSC medium and supplement</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S-5886</td>
			<td></td>
		</tr>
		<tr>
			<td>y-27632 (ROCK inhibitor)</td>
			<td>SelleckChem</td>
			<td>S1049-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D-2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>Gibco</td>
			<td>A1413302</td>
			<td>hPSC-appropriate adherent coating (PAAC)</td>
		</tr>
		<tr>
			<td>0,5M EDTA</td>
			<td>Gibco</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 um filter</td>
			<td>VWR</td>
			<td>28145-77</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tube</td>
			<td>VWR</td>
			<td>525-0130</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>83804360</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>PM996</td>
			<td>wrap for culture plates</td>
		</tr>
		<tr>
			<td>cryotubes</td>
			<td>ThermoFisher</td>
			<td>368632</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12563-029</td>
			<td>trypsin-based dissociation agent&#160;</td>
		</tr>
		<tr>
			<td>Defined Trypsin Inhibitor (DTI)</td>
			<td>Gibco</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-2</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-4</td>
			<td>R&#38;D Systems</td>
			<td>100-31</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-8B</td>
			<td>Peprotech</td>
			<td>100-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Derived Neurotrophic Factor</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Glial Derived Neurotrophic Factor</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurotrophic Factor 3</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Smoothened Agonist (SAG)</td>
			<td>Cayman</td>
			<td>11914</td>
			<td>CAS 912545-86-9</td>
		</tr>
		<tr>
			<td>Axiovert 40C microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Brightfield imaging microscope</td>
		</tr>
		<tr>
			<td>Axiocam</td>
			<td>Zeiss</td>
			<td></td>
			<td>Brightfield imaging - image aquisition</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td>521-0996</td>
			<td>centrifuge for cell culture</td>
		</tr>
		<tr>
			<td>PBS (gebufferde natrium oplossing)</td>
			<td>Braun Medical</td>
			<td>3623140</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Serological pipets</td>
			<td>VWR</td>
			<td>612-4950</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Serological pipets</td>
			<td>VWR</td>
			<td>612-4951</td>
			<td></td>
		</tr>
		<tr>
			<td>6-wells culture plates</td>
			<td>VWR</td>
			<td>734-2323</td>
			<td></td>
		</tr>
		<tr>
			<td>12-wells culture plates</td>
			<td>VWR</td>
			<td>734-2324</td>
			<td></td>
		</tr>
		<tr>
			<td>hESCs</td>
			<td>WiCELL</td>
			<td></td>
			<td>line H01</td>
		</tr>
	</tbody>
</table>

streamlined 3d cerebellar differentiation protocol with optional 2d modification

降低差异化协议的复杂性和成本对于研究人员来说是非常重要的。这种兴趣与外界对外部模式因素可能引入到人类多能干细胞 (hPSC) 脑发育或病理生理学模型 (如掩蔽疾病表型) 的可能意想不到的影响有关。在这里, 我们提出了两个小脑分化协议的 hPSCs, 设计了更简单的启动方法, 较少的模式因素, 和较少的物质要求比以前的协议。最近, 我们开发的文化程序, 产生自由漂浮的3维 (3D) 产品与其他大脑的 "化" 协议, 包括形态学相关的建模大脑发育, 如亚/室区-和菱形唇状结构。第二个使用附着的, 2D 单层的过程, 以完成分化, 这是可以产生功能小脑神经元, 因为产品是积极的小脑相关的标记, 并表现出神经元样钙的流入。这些协议为科学家提供了一个适合不同研究目的的选择, 以及一个测试其他类型的简化的神经分化的基本模型。

减少生长因子2D 和3D 小脑分化协议的可视化概述 图 1显示了2D 和3D 小脑差异化协议的总体时间线, 确定了外部因素和电镀时间。典型的进展为 hPSCs 接受3D 小脑分化被描绘在图 2: 以干细胞线 H01 开始作为殖民地在饲养自由文化在天 0 (图的上部左);在2天 (上中部) 接受 EB 形成;在14天 (右上), 在神经诱导与 RA 和 FGF8 后生长成更大的细胞聚集体, 有明显的流明;在28天 (左下) 形成不同大小和形状的骨料;在复杂的情况下发展, 在28天 (中下) 的单一聚合体的不同结构;并在35天 (右下) 对同一结构进行形态学更改。典型的进展为 hPSCs 接受2D 小脑分化被描述在图 3: 以干细胞线 H01 作为殖民地在饲养自由文化在天 0 (图左上部, 与圈子指示区分的细胞区域在干细胞殖民地之中);在2天 (上中部) 接受 EB 形成;在13天 (右上), 在神经诱导与 RA 和 FGF8 后生长成更大的细胞聚集体, 有明显的流明;在14天 (左下) 电镀后增殖为附着细胞;然后作为一个单层细胞, 在35天的低 (中下) 和高放大率 (右下) 更复杂/成熟的形态学。
3D 产品展示早期神经细胞的标志和结构 图 2(左下图) 和图 4显示了在整个培养过程中看到的3D 聚合形态的异质性, 这是由于不同的生长和/或成熟速率, 以及随机合并或分离聚合体。尽管异质性, 每一个分化产生的聚合显示早期神经和神经元标记, 包括小脑颗粒标记 ZIC1, 如免疫细胞化学 (ICC) 染色在图 5。更重要的是,图 5和图 6表明, 简单的3D 区域性 (具有减少的生长因子) 能够生成与大脑发育有关的复杂结构的聚合体, 如早期的神经细胞和菱形唇。
2D 产品展示小脑标记和功能性神经元活动 虽然2D 文化不能重现复杂的3D 结构, 但它们能够产生早期神经和神经元标记的细胞, 包括小脑颗粒细胞标记 ZIC1, 如图 7中的 ICC 染色所示。通过 rt-pcr 的基因表达分析, 如图 8所示, 支持 ICC 染色结果, 虽然早期颗粒细胞标记ATOH1的存在在实验和直线之间是可变的。钙显像更容易处理2D 文化。如图 9所示,补充视频 1和补充视频 2, 电刺激的细胞显示了钙的流入, 这是典型的神经元放电模式, 提示产生功能性神经元。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56888/56888fig1.jpg"> 图 1: 差异化协议的时间轴 (从0天开始差异化).实心线框表示当特定因素添加到培养基中时, 虚线框表示可选2D 修改的板涂层。对于 FGF2, 向下箭头指较低浓度 (4 ng/毫升), 向上箭头指较高浓度 (20 ng/毫升)。此图已从霍姆斯和 Heine10中修改。<a href="//cloudflare.jove.com/files/ftp_upload/56888/56888fig1large.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/56888/56888fig2.jpg"> 图 2: 3D 协议的代表性明图像.干细胞殖民地在 d0, EBs 在 d2, 聚合跟随归纳在 d14, 不同的大小和形态学集合 (编号 1-5) 在 d28, 一个单一的聚合具有唯一的, 可识别的特征 (由字母a-c) 在 d28, 和d35 中相同特征的可见变化。缩放条 = 100 µm。此图已从霍姆斯和 Heine10中修改。<a href="//cloudflare.jove.com/files/ftp_upload/56888/56888fig2large.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/56888/56888fig3.jpg"> 图 3: 2D 协议的代表性明图像.干细胞殖民地的 d0, EBs 在 d2, 归纳后, 在 d13, 在 d14 的电镀骨料后, 在 d35 的成熟后, 在5x 放大倍率, 20x 放大倍数。左上角的白色圆圈显示了干细胞菌落间分化细胞的面积。缩放栏 = 50 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56888/56888fig3large.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/56888/56888fig4.jpg"> 图 4: 明图像在3D 文化中显示出不同的大小和复杂性.聚合在 (A) 天 8 (干细胞) 和 (B) d35 (hiPSCs)。后一种图像由三独立的图像组成, 以显示整个聚合体。这两种聚合体都可能因合并较小的骨料或结构的损耗 (折断) 而受到影响。缩放条 = 200 µm。这个数字是从福尔摩斯和 Heine10重新发布的。<a href="//cloudflare.jove.com/files/ftp_upload/56888/56888fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56888/56888fig5.jpg"> 图 5: 3D 产品的 ICC 图像显示了相关的标记和结构.在文化 d35, 3D 产品陈列: PAX6 (绿色) 和 TBR2 (红色) 由神经莲座丛状形成的流明 (第一列);DCX (绿色) 和 NeuN (红色) 从外边缘心室区 (VZ) 状结构 (第二排) 传播;KIRREL2, 一个与小脑神经细胞相关的标记 (第三排, 左);ZIC1 与小脑颗粒细胞相关的标记物 (第三排, 右)。实验进行了多次使用四不同的 hPSC 线: 干细胞线 H01 (n = 5), 和 iPSC 线 hvs88 (n = 4), hvs60 (n = 3) 和 hvs51 (n = 1)。箭头指向菱形唇形 (RL) 状结构。缩放条 = 100 µm。这个数字是从福尔摩斯和 Heine10重新发布的。<a href="//cloudflare.jove.com/files/ftp_upload/56888/56888fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56888/56888fig6.jpg"> 图 6: 3D 产品中的大尺度心室区状结构.在文化 d35, 干细胞衍生的聚合是积极的 PAX6 (绿色) 和 TBR2 (红色) 通常与早期神经元发现的心室区 (VZs) 和脑室区 (SVZs),在体内。顶)星号 (*) 标记沿聚合体边缘运行的 VZ 状区域的顶端, 括号表示 VZ/SVZs 的深度/除法. (中) 合并信号显示 PAX6+/TBR2 细胞的散射部分在大小向右上端增加的 VZ。底部)在中间面板中由矩形指示的剖面的更高放大图像。缩放条 = 100 µm。此图已从霍姆斯和 Heine10中修改。<a href="//cloudflare.jove.com/files/ftp_upload/56888/56888fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56888/56888fig7.jpg"> 图 7: ICC 2D 产品的图像显示相关标记.在文化 d35, 2D 产品展览, 从干细胞 (顶排) 和 hiPSCs (下排), 细胞阳性: 颗粒细胞标记 ZIC1 和迁移小脑神经元标记 TAG1 (左栏);和神经元标记丝 (NF) 和 TAG1 (右栏)。缩放栏 = 50 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56888/56888fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56888/56888fig8.jpg"> 图 8: 2D 产品的 rt-pcr PCR.干细胞线 H01 (顶行) 和 hiPSC 线 hvs60 (下排) 的 mRNA 表达分析在2D 协议的结尾显示了产品与凝胶电泳为: 颗粒细胞标记 ZIC1, 颗粒细胞标记 ATOH1, 迁移小脑神经元标记 TAG1, 浦肯野细胞标记 Calbindin (CALB), 电压依赖性钙通道 CACNA1A (C-1A), CACNA1E (C-1E), 伽玛丁酸 (GABA) B 受体 1 (G-Br1), 和管家基因 EIF4G2)。
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56888/56888fig9.jpg"> 图 9: 2D 产品的钙成像/分析.在培养 d35, hPSC 分化产物被记录了2分钟显微镜下, 孵化后与 fluor5 染料。在三十年代, 细胞电刺激十年代在10赫兹. (A) 静止图像显示干细胞在0秒 (左) 和在开始电刺激在三十年代 (右)。箭头表示感兴趣的区域 (ROI), 用于分析钙的流入。(B) 图形分析显示相对荧光度与 ROI 时间的变化 (荧光 = (f-f0)/f0的变化, 其中 F0 = (∑F1-n)/n), 与神经元类似的峰值发生在之前、期间和之后刺激.黑条表示电刺激的长度。缩放栏 (白色) = 50 µm. 干细胞 (如图所示) 的完整录制和 hiPSC 线 (未显示) 分别在辅助视频 1和辅助视频 2中提供。录音以 AVI 格式, 以4x 速度。<a href="//cloudflare.jove.com/files/ftp_upload/56888/56888fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充视频 1: 从干细胞线 H01 的2D 产品的钙成像视频.在培养 d35, 干细胞线 H01 的分化产物在显微镜下记录了2分钟, 经 fluor5 染料孵化后。在三十年代, 细胞被电刺激十年代在10赫兹. 录音是在2帧/秒, 并处理成 AVI 视频在〜7帧/秒, 制作一个视频持久〜三十年代, 在〜 4 x 速度。<a href="http://cloudflare.jove.com/files/ftp_upload/56888/figS1_hESC_stim@10Hz.avi">请单击此处下载此文件.</a>
补充视频 2: 从 hiPSC 线 hvs51 的2D 产品的钙成像视频.在培养 d35, hiPSC 线 hvs51 的分化产物在显微镜下记录了2分钟, 经 fluor5 染料孵化后。在三十年代, 细胞被电刺激十年代在10赫兹. 录音是在2帧/秒, 并处理成 AVI 视频在〜7帧/秒, 制作一个视频持久〜三十年代, 在〜 4 x 速度。<a href="http://cloudflare.jove.com/files/ftp_upload/56888/figS2_hiPSC_stim@10Hz.avi">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification at JoVE.com

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

我们描述了一个简化的 hPSCs 3D 差异化协议, 使用定义的培养基和减少生长因子, 能够产生早期的上皮结构和积极的小脑相关标记的细胞集料, 以及一个可选2D. 将细胞分化为单层, 产生功能性神经元。

简化的3D 小脑鉴别协议, 可选2D 修改

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

The overall goal of these differentiation cultures is to generate cerebellar cell lineages in 2D or 3D environments.These methods can help to answer basic questions regarding new developments or disease mechanisms underlying childhood brain disorders using patient derived stem cells.The main advantage of this technique is that it has a simple startup and maintenance with few factors and that it can be used as a base for testing more complex neural protocols.Demonstrating the procedure will be Lisa a technician from my laboratory.To set up the differentiation with modified method G-method of passing of hPSC's transfer the required volume of neural maintenance medium to a sterile tube and supplement with 4 nanograms per milliliter FGF2 and ten micromolar rock inhibitor.Then, keep the medium tube at room temperature or in water bath at 37

streamlined-3d-cerebellar-differentiation-protocol-with-optional-2d

Research

JoVE Journal

Developmental Biology

7.4K 次观看.  VU University Medical Center. The overall goal of these differentiation cultures is to generate cerebellar cell lineages in 2D or 3D environments.These methods can help to answer basic questions regarding new developments or disease mechanisms underlying childhood brain disorders using patient derived stem cells.The main advantage of this technique is that it has a simple startup and maintenance with few factors and that it can be used as a base for testing more complex neural protocols.Demonstrating the procedure will be...

视频: Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

在永生多能耳科内皮祖细胞分化启动

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

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 ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&amp;#239;ve-like State with Improved Multilineage Differentiation Potency

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

用DMSO对人多能干细胞的暂时性治疗促进分化

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...