我们要感谢埃里克 Ullian 博士 (UCSF) 在设计这些程序方面的知识投入, 迈克尔. 沃德博士 (NIH) 就 iNeuron 分化和 Barlas 进行初步图像分析提供技术咨询。

1. 细胞培养和试剂制备
注: 本节中的协议按照它们在差异化协议中的显示顺序编写 (第2节)。请参阅材料和目录编号的材料表。
<ol><li>
为细胞培养制备涂层板材。
	<ol>
<li>稀释细胞外基质 (ECM) 涂层溶液与 DMEM/F12 介质, 以准备1毫克/毫升的库存解决方案。整除稀释的 ECM 库存溶液成30个3毫升的锥形管, 并立即存储在-20 °c。对于一个工作的解决方案, 并用重悬3毫升的 ECM 储存成33毫升的预冷 DMEM/F12 介质, 使总体积为36毫升的浓度为80µg/毫升。</li>
		<li>涂层1毫升每井的6井细胞培养板与 ECM 工作解决方案。增加一个额外的1毫升 DMEM/F12 每井 (如有必要), 以确保充分覆盖油井的表面。在使用前, 允许涂敷的6井细胞培养板在室温下坐至少1小时。 注: 镀膜板可贮存在孵化箱内, 或在4摄氏度内存放2周。</li>
	</ol>
</li>
	<li>
准备培养基配方, 生长因子和小分子。
	<ol>
<li>要准备500毫升的人类多潜能干细胞 (hPSC) 中27, 根据制造商的指示添加20x 和500x 补充剂。</li>
		<li>要准备500毫升的神经培养基 (NM), 添加肝素到最终浓度为2毫克/毫升, 1x 抗生素/防霉溶液, 1x B27 补充或 1x N2 补充, 500 毫升瓶 DMEM/F12 与 l-谷氨酰胺补充作为以前详细28。</li>
		<li>要准备一个10毫米 (1,000x) 溶液的 Y27632 激酶抑制剂 (Y), 添加10毫克的粉末到3毫升磷酸盐缓冲盐水 (PBS)。过滤消毒, 整除, 并存储在-20 摄氏度。在10µM 工作浓度的介质中使用。</li>
		<li>要准备一个20毫米 (10,000x) 的 SB431542 溶液, 添加10毫克的粉末到1.3 毫升的二甲基亚砜 (亚砜)。要准备一个2毫米 (1,000x) 的 DMH1 溶液, 添加10毫克的粉末到13.1 毫升的亚砜。过滤消毒, 整除, 并存储每个解决方案在-20 摄氏度。在介质中使用2µM 工作浓度 (NM + SB431542 + DMH1)。 注: 本议定书建议在 SB431542 和 DMH1 的2µM 工作浓度的基础上, 根据我们的观察和报告, 从其他29指出, 这种浓度有效地促进神经诱导从 hPSCs。更高浓度也报告了30。另外, 有了替代介质, 也证明了小分子不需要高神经转换31。因此, 工作浓度将因不同的细胞培养环境而异, 而最佳浓度应由个体研究人员来检验。</li>
		<li>为了准备单独的100µg/毫升 (10,000x) 的表皮生长因子 (EGF) 和成纤维细胞生长 factor-2 (FGF2) 的解决方案, 每增加1毫克10毫升的 PBS + 0.1% BSA。整除 EGF 和 FGF2 库存解决方案成50µL 整除数和存储在-80 摄氏度。在介质中使用每一个10毫升的工作浓度;例如, 添加5µL EGF 和5µL FGF2 到50毫升的 nm (nm + EGF + FGF2)。</li>
		<li>为制备盐酸强力霉素 (Dox) 的库存溶液, 首先将亚砜中的粉末溶解至100毫克/毫升, 贮存在-20 摄氏度。进一步稀释它使2毫克/毫升 (1,000x) 股票解决方案在 PBS 和存储在-20 °c。在介质中使用2µg/毫升工作浓度;例如, 添加50µL Dox 到50毫升的 nm (nm + Dox)。 注: 解冻后不要再冻结溶液。</li>
	</ol>
</li>
</ol>2. 人诱导多潜能细胞神经亚型的生成 (hPSCs)
注: 所有细胞培养应保持在一个孵化器与 5% CO2在37摄氏度。这些文化保持在房间的氧气水平, 虽然较低的水平可能被利用。
<ol><li>
从 hPSCs 中生成和维持星形胶质细胞祖细胞 (hAstros)。 注: 本节提供了一种简单、简化的差异化协议。有关详细说明 (具有胚体形成、莲座丛选择和区域图案步骤), 请参阅前面描述的协议28 (图 1A, 虚线绿色框)。<ol>
<li>种子簇的 hPSCs (图 1B) 到 ECM 涂层6井板 (见步骤 1.1) 与2毫升的 hPSC 中 + Y 每井。每天给干细胞喂食, 直到它们有50% 的汇合, 并根据需要进行分裂。<ol>
<li>要分裂 hPSCs, 添加1毫升的传代试剂到水井和吸入1分钟后, 室温孵化细胞5分钟, 增加1毫升的 hPSC 培养基, 并分裂合计1:10 到新的 ECM 涂层的油井2毫升的 hPSC 中 + Y 每井。</li>
		</ol>
</li>
		<li>保持干细胞, 直到它们约50% 汇合, 然后改变培养基到 NM + SB431542 + DMH1 诱导神经分化 (0 天)。当细胞约为95% 汇合时, 将1:6 分裂成新的 ECM 涂层的井在同一培养基中。</li>
		<li>在14天, 分离的细胞与脱离溶液和转移到一个无涂层的瓶子与 Y, 以促进形成的骨料。 注: Y 的增加用于促进细胞存活和球体形成, 但不包括在每一个饲料。</li>
		<li>对于神经元祖细胞和神经元 (hNeurons) 的生成, 请按照下面的描述使用这些 day-14。或者, 在神经元成熟发生或 gliogenesis 开始之前, 利用这些细胞在以后的时间点从单层或球形的培养。 注意: 默认情况下, 此协议产生背皮层星形胶质细胞。然而, 星形胶质细胞子类型可以是区域指定的加法模式格局来说如果需要28,32,33。维甲酸 (RA) 可以添加到 caudalize 细胞成脊髓表型, 而声波刺猬 (嘘), smoothened 激动剂 (凹陷), 或 purmorphamine 将产生腹型。</li>
		<li>对于在自发形成的3D 聚合体 (hAstrospheres) 中的星形胶质细胞祖和星状胶质, 切换到 NM + EGF + FGF2 和饲料每周 (或根据需要, 以确保稳定的 pH 值) 作为以前的详细34。<ol>
<li>当暗中心出现并去除自发附着的球体时, 轻轻地将 hAstro 骨料与脱离溶液分开。每周轻轻地折断球体以保持球体的健康, 避免坏死的核心。 注: 不超过5分钟的治疗与脱离解决方案。</li>
			<li>经过4-6 月的扩张, 确认细胞的身份和冷冻保存培养基 (根据制造商的指示) 或替代贮存条件, 长期保存液氮, 或立即使用实验。</li>
		</ol>
</li>
		<li>解冻 hAstrospheres 的冷冻库存, 在室温下迅速解冻一小瓶, 将其内容转移到空的15毫升锥形管中, 离心机在 300 x g处为1分钟. 小心地吸入上清, 用 DMEM/F12 冲洗, 并重复两次洗涤步骤。添加到一个 T25 瓶, 总体积为6毫升的 NM + EGF + FGF2 + Y (或扩大根据需要)。</li>
	</ol>
</li>
	<li>
从 hPSCs 中生成和维持诱导神经元 (iNeurons)。
	<ol>
<li>种子转基因 hPSCs (以稳定的强力霉素诱导 neurogenin 2 转基因35) 在 ECM 涂覆6井板与2毫升的 hPSC 中 + Y 每井 (如上文所述非转基因线)。每天用2毫升的介质喂养, 直到它们准备在70% 融合。按步骤2.1.2 中所述拆分单元格。</li>
		<li>当细胞约35% 汇合, 添加 NM + Dox 诱导分化为 iNeurons。在继续步骤3.2 之前, 用 NM + Dox 维持2天的单层培养。 注意: 细胞将转变为神经元祖, 但仍不能扩展突起 (图 1C)。</li>
	</ol>
</li>
</ol>3. 3D 球形 Cocultures 的制备和维护
<ol><li>在悬浮液中从3D 聚合体中分离 hAstros (如步骤2.1.5.1 中所述)。</li>
	<li>
从2D 单层分离 hNeurons 或 iNeurons。
	<ol>
<li>将介质从6井板中取出, 并将分离液的500µL 添加到每个含单层培养的井中。孵育37°c 5 分钟, 轻轻地添加2-3 毫升 DMEM/F12, 以去除附着的细胞。</li>
		<li>收集细胞和介质在一个15毫升圆锥管和离心机在 300 x g 1 分钟, 吸入上清, 添加1毫升的新鲜培养基, 和吸管上下轻轻与1000µL 微, 以实现一个单一的细胞悬浮。</li>
	</ol>
</li>
	<li>
使用 microwell 培养板形成3D 球体。
	<ol>
<li>在 microwell 板的每个井中加入0.5 毫升的防粘漂洗液, 准备板材。离心板在 2000 x g的5分钟吸入冲洗液, 添加1毫升的 DMEM/F12 到每个井洗, 并再次吸入。 注: 始终确保离心机在使用过程中平衡。</li>
		<li>使用 hemocytometer 或自动单元格计数器对每个单元格类型进行计数。将所需的分离 hAstros 和 hNeurons 或 iNeurons 的比例添加到每个井中, 总体积为2毫升的 NM (包括 Dox, 如果使用 iNeurons)。离心板在 100 x g 3 分钟, 并返回到孵化器。 注:24-井 microwell 板 (见材料表) 包含 300 microwells 每井。增加至少 6 x 105单元格 (对于每一个 2 x 10 3个单元格的球体) 和最大 6 x 106个单元格 (对于 2 x 10 4 个单元格的球体), 每井都有2毫升的介质。对于此范围内特定密度的可行球体, 请使用已定义的单元格数量并除以300以计算每个球体的单元格数。如果需要, 还可以使用其他球形成形方法和工具。按照制造商的指示, 可接受的细胞密度范围。</li>
		<li>允许细胞在未来2天内在 microwell 板块内自组装成密集的球体 (图 2A)。如果文化变黄, 请更换50% 媒体。 注: 在移除和添加介质时, 仅交换半介质和吸管, 以免干扰球体。球体可以从 microwells 中抬起, 并与更高的力量融合在一起。</li>
		<li>2天后, 用1000µL 微轻轻地从 microwells 中移除球体 (图 1D)。用介质轻轻地向 microwells 底部施加力, 以除去任何附加的附着球体。让球体在15毫升锥形管中沉淀, 吸入旧介质, 加入新鲜的介质。</li>
	</ol>
</li>
	<li>
建立了3D 球体的微调瓶生物反应器系统。
	<ol>
<li>用70% 乙醇清洁磁性搅拌板表面, 将其放入细胞培养孵化器中。高压釜单独的微调瓶消毒。</li>
		<li>向每个微调瓶添加最小 50–60 mL 介质的球体 (图 1D, 嵌入)。将烧瓶放在 60 rpm 的磁力搅拌板上。在微调烧瓶中培养球体, 直到它们准备好进行数据收集。 注: 突触形成需要至少3周的培养。如果不需要微调瓶生物反应器系统, 可以在细胞培养瓶或菜肴的固定条件下培养球体。球体也可以嵌入到 ECM 或水凝胶中。</li>
	</ol>
</li>
</ol>4. 用多电极阵列测量活突触生理学 (多边环境协定)
<ol><li>
为细胞培养准备多边环境协定。
	<ol>
<li>清洁表面的每一个多边环境协定与1毫升洗涤剂为1小时冲洗与无菌去离子 (DI) 水, 冲洗与70% 乙醇消毒, 然后空气干燥的生物安全柜在紫外线下。 注: 可在无菌条件下贮存在4摄氏度的 DI 水中, 直至使用。</li>
		<li>将50毫克的巴解组织溶入100毫升硼酸缓冲液中, 制备0.5 毫克/毫升的聚鸟氨酸溶液。将每个多边环境协定的表面涂上1毫升的巴解组织, 使表面亲水性, 并在37摄氏度孵育至少4小时. 去除巴解组织, 用 DI 水洗涤表面, 加入1毫升 ECM (见步骤 1.1.1), 并在37摄氏度孵化至少4小时。</li>
		<li>移除 ECM 并将球体置于多边环境协定表面的1.5 毫升介质中, 确保球体位于电极阵列的顶端 (图3A、3A)。允许坚持2天。每2–3天更改一半的媒体 (或者更经常需要), 以确保这些球体不会受到干扰。 注: 增加生长因子可改善文化生存和成熟。</li>
	</ol>
</li>
	<li>
测量神经球体的电活动。
	<ol>
<li>设置具有温度控制 headstage 的多电极阵列 (多边环境协定) 系统。创建一个具有5赫兹高通滤波器和 200 hz 低通滤波器的软件程序, 以及5x 标准偏差 (SD) 的峰值阈值。</li>
		<li>将多边环境协定放在 headstage 上, 记录自发电活动 (图 3B, B)。保存原始数据, 包括穗频和振幅进行统计分析。 注: 可与多边环境协定一起使用灌注系统添加药理剂或增加新鲜培养基的长期记录。额外的峰值后处理可以按需要执行36,37。</li>
	</ol>
</li>
</ol>5. 免疫细胞化学检测突触密度
<ol><li>
准备试剂。
	<ol>
<li>要制造500毫升的4% 多聚甲醛 (粉煤灰) 的溶液, 在玻璃烧杯或通风罩中加入400毫升 1x PBS。搅拌时加入搅拌棒和加热至60摄氏度;不要煮沸。将20克粉煤灰粉加入加热 PBS 溶液中, 通过缓慢添加 1 N 氢氧化钠, 将 pH 值提高到6.9。 注: 4% 粉煤灰溶液可 aliquoted, 储存在4摄氏度, 长达一个月。</li>
		<li>添加20克或30克蔗糖到100毫升 PBS, 分别制作20% 和30% 溶液的蔗糖。</li>
		<li>添加1毫升洗涤剂到10毫升 PBS, 以准备10% 库存解决方案。反转几次到融汇。</li>
		<li>添加250µL 10% 洗涤剂库存解决方案 (最终浓度 0.25%) 和500µL 每个山羊和驴血清 (最终浓度5% 每) 到10毫升 PBS 准备主阻塞缓冲区。</li>
		<li>添加100µL 山羊和驴血清 (最终浓度为 1%) 到10毫升 PBS, 以准备二级阻塞缓冲器。</li>
	</ol>
</li>
	<li>
准备样品。
	<ol>
<li>将球体转换成15毫升锥形管, 让它们在管子的底部沉淀, 并吸入旧的介质。冲洗球体与500µL pbs, 让解决, 并吸入 pbs。添加500µL 4% 粉煤灰 (或足够覆盖球体) 和孵化4°c 30 分钟。</li>
		<li>吸入粉煤灰, 并轻轻地洗两次 PBS。加入20% 蔗糖溶液, 在4摄氏度孵育几个小时或一夜。小心吸入, 加入30% 蔗糖溶液, 并在4摄氏度孵育几个小时或一夜。 注: 在进行下一步之前, 样品可在 PBS 或蔗糖中储存4摄氏度至1周。如果不切片, 保持为3D 球体 (图 4A-C) 在1.5 毫升管, 并进行步骤5.3。</li>
		<li>如果切片球体, 小心地转移球体到一个嵌入的低温模子在干冰之上。吸入30% 蔗糖溶液, 慢慢将组织嵌入溶液倒入模具中, 直到完全覆盖样品, 避免气泡。等到解决方案冻结并存储在-80 °c, 直到恒温器切片。</li>
		<li>使用恒温器-20 摄氏度, 将嵌入块切片成30个µm 部分 (或根据需要) 并转移到玻璃幻灯片。储存在-80 摄氏度, 直到准备着色。</li>
	</ol>
</li>
	<li>
Immunostain 的球体。
	<ol>
<li>用疏水性的 PAP 笔勾勒出幻灯片的边缘, 以防止液体溢出。用抗体制备主、二次阻断溶液 (见材料表)。在每张幻灯片或管中添加500µL 的主阻塞缓冲器, 室温孵育30分钟。</li>
		<li>取出液体, 添加500µL 的新鲜主阻断缓冲液稀释的主要抗体, 每一个幻灯片或管, 并孵化过夜在4摄氏度。去除主抗体溶液, 用 PBS 洗涤 3x, 每10分钟。</li>
		<li>添加500µL 的二级阻断缓冲器, 稀释二次抗体的每一个幻灯片或管和孵化在4°c 1 小时. 去除二次抗体解决方案和洗涤3x 与 PBS 为10分钟每。 注: 推荐的抗体列表在材料表中提供。其他抗体或染料可按需要使用。不要将荧光样品暴露在光线下 (步5.3.3 向前)。</li>
		<li>添加50µL 的安装溶液滴状, 以覆盖表面, 并小心地放置在幻灯片上的盖玻片, 避免气泡。允许幻灯片在室温下干燥24小时, 并存储在4摄氏度。</li>
	</ol>
</li>
	<li>
图像的幻灯片。
	<ol>
<li>使用共焦或双光子荧光显微镜与63X 油浸泡目的是图像前和后突触蛋白丰度和定位在球形切片 (图 4D)。使用20X 或40X 目标来可视化 hAstros 的形态学特征。</li>
		<li>使用 ImageJ 软件打开荧光图像文件。使用单元格计数器插件手动计数前突触后 puncta, 并采用前述38或其他方法的无偏计数方法。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

在本协议中, 我们描述了一个系统的方法来生产3D 球体的神经 cocultures。球体由星形胶质细胞和神经元组成, 独立于 hPSCs。虽然不是本协议的重点, 但从 hPSCs28中生成的纯星形胶质细胞是一个关键步骤, 如果没有事先的经验, 它在技术上可能具有挑战性。这些突触集成电路的第一步应该是细致的定时和注意细节。hPSC 星形胶质细胞的使用限制是长的分化过程;然而, 生产大量的细胞, 可?...

星形胶质细胞是中枢神经系统 (CNS) 内一种高度丰富的神经细胞类型, 其功能职责超出结构支持范围。通过可溶性 synaptogenic 因子和细胞外基质 (ECM) 组分的分泌, 星形胶质细胞在发育过程中帮助成熟突触的建立和聚集1。通过胞外信号2、3、4、5, 它们在保持突触的健康和可塑性方面发挥着关键作用, 并有助于维持稳定的长期稳定性。环境通过调节胞外钾和谷氨酸, 以及能量基质的分泌和 ATP6,7,8。最后, 它们可以通过影响 extrasynaptic 电流9来促进神经传递, 并且可以通过其他细胞类型 (如促进髓鞘形成10) 间接地影响活动。重要的是, 由于星形胶质细胞的异常或功能障碍会导致许多神经发育综合征和成人神经病理学, 显然需要将星形胶质细胞与神经网络中的神经元包括在一起, 以改善内源性脑环境模型。星形胶质细胞的一个整体特征是它们能够形成与神经元突触1,11,12的动态相互作用。在没有胶质细胞的情况下, 神经元形成数量有限的突触, 一般也缺乏功能成熟度13。
人星形胶质细胞显示形态学, 转录和功能特征-如增加的大小和复杂性的分支, 以及物种特异基因-不概括在啮齿目动物12,14, 15。因此, 利用人类多潜能干细胞 (hPSC) 衍生的神经细胞的研究已被广泛接受为一种检测中枢神经系统相关疾病的方法, 同时开发新的疗法, 伤害模型和文化范例16 ,17。此外, hPSCs 允许研究人类突触的形成和功能, 而不需要初级组织18,19。
我们理解各种细胞类型和信号对突触电路功能的贡献的障碍是缺乏相关的人脑模型。需要一个适当的平台来重述其突触网络的高保真度和重现性。最近, 3D 文化系统 (广为人知的 "organoids"、"球体" 或 "迷你大脑") 的生产中出现了兴趣,在细胞和宏观层面上对复杂的三维 (3D) 结构建模。3D. 在典型的2D 共培养范式21、22中, 文化系统保留 ECM 和细胞间的相互作用, 通常不存在或受到限制。为培养3D 神经球体23、24、25, 存在大量的技术;然而, 许多人需要长时间的文化周期 (几个月到几年) 来进行自发的开发和层的保存, 用户对输出的控制很少。
在这里, 我们说明了一个系统的方法, 迅速和一贯生物工程的神经相互作用之间的多种细胞类型 (预分化神经元和星形胶质细胞) 从 hPSCs 的组装细胞成球形 cocultures (' 小行星 ')26在3D 中概括了人类特有的形态学复杂性。这种高密度的神经系统产生均匀分散的神经亚型, 随着时间的推移而逐渐成熟, 可以用高通量的方式进行筛选或测定。我们首次证明, 人类星形胶质细胞诱发突触网络爆裂活动在这些 3D cocultures。此外, 该协议很容易适应产生不同大小的球体, 利用指定给中枢神经系统不同区域特性的细胞, 并根据需要研究多种其他细胞类型的相互作用。

<table>
	<tbody>
		<tr>
			<td>6 well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Detachment solution</td>
		</tr>
		<tr>
			<td>AggreWell plate</td>
			<td>Stemcell Technologies</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>Stemcell Technologies</td>
			<td>7010</td>
			<td>Prevent cell adhesion to microwell plates</td>
		</tr>
		<tr>
			<td>Anti/anti</td>
			<td>Thermofisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>BrainPhys neuronal medium</td>
			<td>Stemcell Technologies</td>
			<td>5790</td>
			<td>Neurophysiological basal medium alternative</td>
		</tr>
		<tr>
			<td>Circular glass coverslips</td>
			<td>Neuvitro</td>
			<td>GG-12-oz</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS10</td>
			<td>Stemcell Technologies</td>
			<td>7930</td>
			<td>Cryopreservation medium with 10% DMSO</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>10565-042</td>
			<td>With GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>DMH-1</td>
			<td>Stemcell Technologies</td>
			<td>73634</td>
			<td>HAZARD: Toxic if swallowed. Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332100</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride (Dox)</td>
			<td>Sigma</td>
			<td>D3072-1ml</td>
			<td>HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-2 (FGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting solution</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisherbrand</td>
			<td>22-037-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332500</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Hemacytometer or automatic cell counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149-50KU</td>
			<td>Working concentration: 2 mg/mL</td>
		</tr>
		<tr>
			<td>Magnetic plate</td>
			<td>DLAB</td>
			<td>8030170200</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.</td>
		</tr>
		<tr>
			<td>MEA 2100 System</td>
			<td>Multichannel Systems</td>
			<td>MEA2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Tissue embedding solution for cryosectioning</td>
		</tr>
		<tr>
			<td>Pap Pen (Aqua Hold)</td>
			<td>Scientific Device Laboratory</td>
			<td>9804-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Acros Organics</td>
			<td>169650025</td>
			<td>HAZARD: Toxic if inhaled. Working concentration: 4% in PBS</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Stemcell Technologies</td>
			<td>CA008-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-l-ornithine (PLO)</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>Working concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Rectangular glass cover slips</td>
			<td>Fisherfinest Premium Superslip</td>
			<td>12-545-88</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stemcell Technologies</td>
			<td>5872</td>
			<td>Detachment and passaging reagent</td>
		</tr>
		<tr>
			<td>Rho-Kinase Inhibitor Y27632- (Y)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Working concentration: 10 uM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemcell Technologies</td>
			<td>72234</td>
			<td>Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Spinner flasks</td>
			<td>Fisher Scientific</td>
			<td>4500-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Chemical</td>
			<td>S5-3</td>
			<td>Working concentration: 20% or 30% in PBS</td>
		</tr>
		<tr>
			<td>T25 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>T75 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-209</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>Terg-A-zyme</td>
			<td>Sigma</td>
			<td>Z273287-1EA</td>
			<td>Detergent. Working concentration: 1%</td>
		</tr>
		<tr>
			<td>TeSR-E8 basal medium</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Human pluripotent stem cell (hPSC) medium</td>
		</tr>
		<tr>
			<td>TeSR-E8 supplements</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Supplements for human pluripotent stem cell medium</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td>Detergent for cell permeabilization. Working concentration: 0.25% in blocking buffer</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488</td>
			<td>Thermofisher</td>
			<td>A-11029</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>AlexaFluor 594</td>
			<td>Thermofisher</td>
			<td>A-11037</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ezrin</td>
			<td>Thermofisher</td>
			<td>MA5-13862</td>
			<td>Primary antibody; astrocytes perisynaptic</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Chemicon</td>
			<td>MAB360</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Aves</td>
			<td>GFP-1020</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Glt1</td>
			<td>Gift from Dr. Jeffrey Rothstein</td>
			<td>n/a</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Homer</td>
			<td>Synaptic Systems</td>
			<td>160 011</td>
			<td>Primary antibody; neurons, post-synaptic</td>
		</tr>
		<tr>
			<td>MAP2</td>
			<td>Synaptic Systems</td>
			<td>188 004</td>
			<td>Primary antibody; neurons</td>
		</tr>
		<tr>
			<td>PSD95</td>
			<td>Abcam</td>
			<td>ab2723</td>
			<td>Primary antibody; neurons, post-synaptic</td>
		</tr>
		<tr>
			<td>S100</td>
			<td>Abcam</td>
			<td>ab868</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Synapsin 1</td>
			<td>Synaptic Systems</td>
			<td>106 103</td>
			<td>Primary antibody; neurons, pre-synaptic</td>
		</tr>
		<tr>
			<td>TuJ1/&#946;3-tubulin (TUBB3)</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>Primary antibody; neurons</td>
		</tr>
	</tbody>
</table>

synaptic microcircuit modeling with 3d cocultures of astrocytes and neurons from human pluripotent stem cells

我们理解各种细胞类型和信号对突触电路功能的作用的障碍是缺乏研究人脑的相关模型。解决这个问题的一项新兴技术是使用三维 (3D) 神经细胞培养, 称为 ' organoids ' 或 ' 球体 ', 长期保存细胞间相互作用, 包括细胞外黏附分子。然而, 这些文化系统是费时的, 没有系统地产生。在这里, 我们详细的方法, 以迅速和一贯地产生 3D cocultures 神经元和星形胶质细胞从人类多潜能干细胞。首先, 对预分化的星形胶质细胞和神经元祖先进行离解和计数。接下来, 细胞被结合在球形形成的菜肴与一个蛋白激酶抑制剂, 并在特定的比率, 以产生可再生的大小球体。经过几个星期的文化作为漂浮球体, cocultures (' 小行星 ') 最终被切片的染色或电镀后, 多电极阵列, 以测量突触密度和强度。一般情况下, 该协议将产生3D 神经球体, 显示成熟的细胞类型限制标记, 形成功能性突触, 并表现自发突触网络突发活动。与单层培养相比, 该系统允许对疾病机制进行药物筛选和调查。

当正确执行时, 此协议将产生由 hPSCs (图 1A-1C) 生成的星形胶质细胞28、33、34和神经元35的功能 cocultures 的定义种群, 如详细的前26和这里描述的步骤2.1–2.2。这个逐步的过程, 使用 microwell 板, 预计产生3D 的神经球体的大小和形状一致 ...

Watch this Scientific Journal Video about Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells at JoVE.com

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

在本协议中, 我们的目的是描述一种可重现的方法, 将分离的人类多能干细胞衍生神经元和星形胶质细胞组合成3D 球体 cocultures, 在自由漂浮条件下维持这些球体, 随后用 immunoanalysis 和多电极阵列记录测量球体的突触电路活动。

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 ...

synaptic-microcircuit-modeling-with-3d-cocultures-astrocytes-neurons

Research

JoVE Journal

Developmental Biology

12.1K 次观看.  Houston Methodist Research Institute. 在本协议中, 我们的目的是描述一种可重现的方法, 将分离的人类多能干细胞衍生神经元和星形胶质细胞组合成3D 球体 cocultures, 在自由漂浮条件下维持这些球体, 随后用 immunoanalysis 和多电极阵列记录测量球体的突触电路活动。

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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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 ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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 ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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

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

人类多能干细胞有效分化为肝细胞

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

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

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

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

从脐带血源性多能干细胞中提取三维皮肤有机细胞

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

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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 ...