Name: generation of standardized and reproducible forebrain-type cerebral organoids from human induced pluripotent stem cells
Uploaded: 2018-01-23T14:00:00.000+00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells at JoVE.com

这项工作得到了北莱茵-威斯特伐利亚 (初级研究小组) 创新科学和研究部的支持, 并受到了时代网神经元 JTC 2015 神经发育紊乱的支助。

1. iPSC 集料的产生
<ol><li>iPSC 殖民地单细胞单细胞培养的研究<ol>
<li>准备一个基底膜萃取物 (BME) 涂层 6-井板。解冻 BME 在冰在4° c 为 2-3 h, 稀释它与冷的 Dulbecco 的修改过的老鹰中等 F12 (DMEM-F12; 1:50 稀释), 盖板材与1毫升或稀释的 BME 解答的井, 并且在4° c 隔夜存放板材。</li>
		<li>在 pbs 中以0.5 毫米乙酸酸 (edta) 为4分钟, 在室温 (RT) 中, 在磷酸缓冲盐 (pbs) 中, 用0.5 毫米 edta 将6个井板中的至少2口井的培养基和洗涤完好的 iPSC 菌落吸进两次。抽出 EDTA 溶液, 用5毫升的 DMEM-F12 培养基将其从盘底冲洗出来, 轻轻地将其分离。收集他们在一个15毫升的管和颗粒他们的离心 (4 分钟在 1200 x g在 RT)。 注意:从商业可用的成纤维细胞重新编程干细胞已成功使用10。</li>
		<li>用500µL 的细胞离解试剂在37° c 下吸出上清液, 孵育 iPSC 菌落6分钟。</li>
		<li>吸管细胞悬液几次轻轻向上和向下的1毫升吸管, 打破剩余的细胞簇成单细胞。</li>
		<li>在细胞悬浮液中加入4毫升的 DMEM-F12, 以稀释细胞离解试剂。</li>
		<li>在 RT 处将单元格向下旋转 1200 x g , 以4分钟为单位。</li>
		<li>重在2毫升的 iPSC 培养基中补充5µM Y-27632 和种子细胞成 BME 涂层 6-井板的一个井。 注意:使用在材料表中指定的 iPSC 介质, 以单细胞单层 iPSC 培养。</li>
		<li>第二天, 用新鲜的 iPSC 培养基取代培养基, 缺乏 Y-27632。从这一点上, 继续培养细胞, 每天改变培养基, 直到干细胞100% 汇合。根据起始井的细胞线和 70-100, 这将需要2-4 天。</li>
		<li>一旦汇合, 通过干细胞。吸气培养基, 并在细胞上应用500µL 的细胞离解试剂。</li>
		<li>孵育细胞5-10 分钟在37° c。轻轻摇动盘子以分离细胞。</li>
		<li>用2毫升的 DMEM-F12 从井中清洗细胞, 然后在15毫升的管子中收集。添加 DMEM-F12 为5毫升的总体积。</li>
		<li>在 RT 中向下旋转 1200 x g处的单元格, 并吸入上清液4分钟。</li>
		<li>种子的细胞在 iPSC 培养基补充5µM Y-27632 在一个1:2 到1:4 的比例在一个 BME 涂层 6-井板 (2 毫升/井中)。</li>
		<li>继续培养细胞2-5 天, 并改变 iPSC 培养基缺乏 Y-27632 每天。一旦汇合, 段落干细胞 (步骤 1.1. 4-1. 1.8)。 注意:培养干细胞至少2通道作为单层在材料表中指定的 iPSC 介质中, 然后使用它们生成 iPSC 聚合以允许细胞适应培养条件。</li>
	</ol>
</li>
	<li>使用单层 iPSC 培养时, 他们是70-90% 汇合的世代 iPSC 聚合体。 注意: iPSC 适应了培养条件, 因为单个细胞在分离和聚集过程中不易受到压力, 导致细胞死亡。单层 iPSC 培养需要显示典型的多能形态学, 没有分化的证据。定期测试培养物对支原体的污染。仅工作与无支原体 iPSC 文化。<ol>
<li>从一个6孔板的井中吸取培养基, 并在细胞上应用500µL 的细胞离解试剂。</li>
		<li>孵育细胞5-10 分钟在37° c。轻轻摇动盘子以分离细胞。</li>
		<li>使用2毫升的 DMEM-F12 从井中清洗细胞, 并将其收集在15毫升的管中。添加 DMEM-F12 为10毫升的总体积。</li>
		<li>对于细胞计数, 采取25µL 从细胞悬浮和混合与25µL 的台盼蓝标记死细胞。使用计数室计数活细胞。</li>
		<li>从15毫升管中的细胞悬浮液中收集足够的细胞 (每 iPSC 聚集4500细胞)。</li>
		<li>在 RT 中向下旋转 1200 x g处的单元格, 并吸入上清液4分钟。</li>
		<li>重细胞在适当的容量 iPSC 培养基补充与50µM Y-27632 获得4500活细胞每150µL。 注意:使用高浓度的 Y-27632 (50 µM) 对干细胞的生存至关重要。</li>
		<li>板150µL 在低附着96井 U 底板的每个井中, 并将其放置在37° c 和 5% CO2的孵化器中。 注意:在生成 iPSC 聚合时, 应考虑到20天的质量控制需要至少 6 organoids (参见5节)。</li>
	</ol>
</li>
</ol>2. 前外胚层的诱导
<ol><li>每天在组织培养显微镜下使用4X 或10X 的功率透镜密切监测 iPSC 聚集体的形态学变化。观察在1天, 细胞聚集与清楚的边界。在37° c 和 5% CO2的孵化器中继续培养 iPSC 聚合体。 注意:一定数量的死细胞/井是正常的, 不会影响化的世代。</li>
	<li>从2天开始喂养 iPSC 聚合体, 并每隔一天通过轻轻地吸气大约2/3 的培养基, 而不干扰底部的细胞聚集。添加额外的100µL iPSC 培养基缺乏 Y-27632。 注意:在4-6 天内, 细胞团聚体将是350-450 µm 直径, 并表现出平滑的边缘。</li>
	<li>在这个阶段, 池的细胞聚合与切割100µL 吸管尖端到低附件 6 cm 碟 (最多20集/碟) 在皮质诱导介质含有 DMEM-F12 与 N2 补充 (1:200), B27 补充 (1:100), 葡萄糖 (0.2 毫克/毫升), 循环腺苷单磷酸 (cAMP; 0.15 µg/毫升), 0.5% 非必需氨基酸 (NEAA), 1% l-丙-l-谷氨酰胺, 肝素 (10 µg/毫升), 和化合物 LDN-193189 (180 nm), A83-01 (500 nm), 和抑制剂 Wnt response-1 (IWR-1) (10 µg/毫升)。通过改变皮质诱导培养基3天后将其转移到6厘米的盘中来喂养细胞团聚体。</li>
	<li>在组织培养显微镜下使用4X 功率透镜密切监测皮层诱导的形态学变化。 注意:在皮质诱导培养基4-5 天后, 细胞团聚体的边缘应开始在表面亮起, 表明神经分化。在这个阶段, 假上皮细胞的放射组织出现。进入3节, 将单元聚合嵌入到 BME 矩阵中。</li>
</ol>3. 神经骨料在基质支架中的嵌入
<ol><li>解冻 BME 在冰在4° c 为 2-3 h. 分未稀释的 BME 在充足的数额。</li>
	<li>为嵌入程序准备一个塑料石蜡薄膜片。用无菌剪刀在4厘米 x 4 厘米大块的塑料石蜡薄膜, 把一块塑料石蜡膜在一个空的100µL 小费托盘100µL 提示, 并按戴手套的手指, 使小酒窝在塑料石蜡薄膜片创建 (1 dimple/细胞聚集需要)。用70% 乙醇清洁塑料石蜡膜, 用 UV 光照射 (功率:15 瓦特, 波长: 435 nm) 在闭合的无菌工作台下30分钟。</li>
	<li>将每个电池集料转移到塑料石蜡薄膜片的一个凹坑中, 使用100µL 的切口尖端, 直径为 1.5-2 mm 开口。在两个细胞聚合体被熔化的情况下, 不要将它们分开, 而是将它们一起转移到一个酒窝。</li>
	<li>使用未切割的100µL 吸管尖端, 轻轻地吸气周围的细胞聚合体。 注意:小心不要把细胞聚合体吸进尖端, 因为这会损坏聚合体。</li>
	<li>添加40µL 未稀释的 BME 到每个细胞聚合体。</li>
	<li>使用未切割的100µL 吸管尖端, 将每个细胞聚合体放置在 BME 滴中间。 注意:要非常小心, 不要伤害发展神经细胞与吸管尖端。</li>
	<li>用无菌钳将塑料石蜡薄膜片小心地转移到10厘米的培养皿中 (或另一种足够的细胞培养基), 并将盘放在孵化器中15-20 分钟, 使 BME 凝固。</li>
	<li>同时, 准备一个低附着的6厘米的盘含有5毫升皮质诱导培养基。</li>
	<li>BME 聚合后, 从塑料石蜡薄膜片中除去含有细胞集料的液滴。使用无菌钳将塑料石蜡膜翻转, 然后轻轻地将细胞聚合体挤压到略微倾斜的 (约30°) 低附着6厘米的盘子中, 直到水滴从塑料石蜡薄膜片上脱落。将最大16细胞聚合体转化为6厘米的盘。</li>
	<li>在37° c 下继续孵育细胞聚合体。</li>
</ol>4. 从神经集料中产生前脑型 Organoids
<ol><li>一天后, 嵌入一个 BME 矩阵, 放置化文化菜肴的摇摆细胞培养振动筛与倾斜角5°和 14 rpm, 安装在一个细胞培养孵化器。</li>
	<li>每天监控 organoids在 BME 矩阵中嵌入后, 均质上皮环状结构逐渐发展。</li>
	<li>当上皮环结构是可见的, 改变培养基到化分化培养基含有 DMEM-F12 与 N2 补充 (1:200), B27 补充 (1:100), 葡萄糖 (0.2 毫克/毫升), 阵营 (0.15 µg/毫升), 0.5% NEAA, 1% l-丙-l-谷氨酰胺,胰岛素 (2.5 µg/毫升)</li>
	<li>每3-4 天更换化分化培养基, 直至达到预期的分化时间点。然后修复 organoids (参见5节)。 注意:有时, 组织可能会表现出不带环形结构的光学半透明组织的芽。虽然这并不理想, 但这并不影响皮质结构的发展。organoids 可在化分化培养基中培养40天。对于延长培养期 (40-100 天), 化分化培养基可辅以 1:50 BME, 以增加组织复杂性, 并与 BDNF 和 GDNF, 以允许神经元存活和成熟14,15。</li>
</ol>5. 前脑型 Organoids 的固定和验证
<ol><li>为验证, 收集 6 organoids 在20天。用 3 organoids 进行 mRNA 分离和 PCR 分析。将额外的 3 organoids 转移到含有 PBS 的24井板上, 使用1毫升的吸管尖端, 开口 3-3. 5 毫米用于固定和顺序免疫细胞化学分析。</li>
	<li>洗 organoids 两次, 仔细地吸取 pbs 和更换新鲜 pbs 使用5毫升吸管。在冷4% 甲醛 (粉煤灰) 中固定 organoids 15 分钟 (pH 7.4)。 警告:当心, 粉煤灰是一种已知的人类致癌物。所有工作必须在一个化学油烟罩戴着腈手套做。建议佩戴安全眼镜。甲醛会对角膜造成不可逆转的损害。</li>
	<li>仔细吸气, 用室温 PBS 10 分钟洗涤三次。</li>
	<li>用30% 的蔗糖溶液 (重/卷, pbs 基) 取代 pbs, 将样品储存在4° c, 使 organoids 脱水。Organoids 可以存储多达7天, 直到进一步处理。</li>
	<li>在固定后的一天, 预染色的脱水 organoids 通过添加台盼蓝在约 1:50 10 分钟, 让 organoids 在 cryosectioning 程序的可视化。</li>
	<li>在 PBS 中制备含有10% 蔗糖、7.5% 明胶/卷的嵌入培养基, 并将其加热至75° c, 直至液态为止。</li>
	<li>用嵌入介质取代30% 蔗糖溶液, 将24个井板转移到加热板 (60 ° c) 上15分钟, 平衡 organoids。</li>
	<li>将嵌入模具的底部盖上一层嵌入介质, 并将其放在冰上聚合。</li>
	<li>将 organoids 从24层板转移到包含聚合嵌入介质的嵌入模具中, 在顶部添加附加的嵌入介质, 使 organoids 覆盖, 并在100% 乙醇/干冰冷冻浴中快速放置模具 (温度应在-30 至-50 ° c 之间), 至少1分钟至休克冻结。</li>
	<li>暂时将模具与钳子放在干冰上, 或者将其储存在-80 ° c 或直接进行 cryosectioning。</li>
	<li>Cryosection organoids 在20µm 厚度, 并收集在显微镜幻灯片的部分, 保持对幻灯片上的部分顺序的跟踪 (顺序摄取)。在-80 ° c 或直接进行免疫细胞化学染色之前, 允许切片在幻灯片上晾干数小时。</li>
</ol>

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作者没有什么可透露的。

脑 organoids 代表了一个强大的工具, 研究人脑的发展在体外, 因为它们提供了相关的物种背景和复杂的3D 排列细胞在一个组织的背景。因此, 他们弥合非人类动物模型和简化人的二维单层细胞培养技术之间的差距。但是, 它们的应用程序由于缺乏重复性9而受阻。我们已经开发出一种前脑型化协议, 它通过将 iPSC 的自组织能力与它们的可结合起来, 从而克服了大样本到样本的变异性。具体地说, 干细胞被聚合以促进自组织, 并随后抑制 TGF-？/SMAD 信号, 通过向 BMP (LDN-193189) 和 TGF β型受体抑制剂 (A83-01) 暴露培养促进背皮质分化。此外, 还采用了一种抑制 Wnt 通路 (IWR) 的化合物来防止 posteriorization。与 "内在的" 脑化协议19不同, 它们是基于无外部控制的自组装而产生的非均匀的脑 organoids, 并表现出大量的间歇性变化 (以极化的效率来衡量神经外胚层形成15), 这里描述的协议性产生同质的前脑特定的 organoids 从人类干细胞。
这些前脑型 organoids 可用于各种应用, 如神经发育研究, 进化研究, 包括基因功能研究, 疾病建模和, 潜在的, 药物检测和治疗的目的。然而, 该议定书最适合于研究人类皮质发育的早期方面。例如, 我们用前脑型 organoids 来检查 vRGC 行为的人的特定方面。更具体地说, 病理生理学的变化与严重形式的 lissencephaly, 人类皮质畸形的特点是近没有皮质折叠, 是解决。只有某些方面的这种疾病可以模仿老鼠的大脑是自然 lissencephalic。当将化系统应用于 lissencephaly 患者的干细胞时, 我们可以可靠地重述该疾病的人的特定方面, 并确定潜在的机制。更具体地说, 我们可以证明, 病人获得的 organoids 显示, 由一个开关从对称到不对称的 vRGCs 细胞分裂造成的大小显著减少。这个开关与 vRGCs 的微管网络组织的改变有关, 破坏了 VZ 生态位的结构, 改变了细胞黏附分子的表达, 导致了 n-钙黏蛋白/β-蛋白的活化作用受损。信号轴10。注意: β-蛋白依赖性的 vRGC 分裂模式的调节被认为是人类特有的, 因为β-蛋白的过度表达导致了小鼠的切皮质扩张, 随后皮质折叠20。因此, 我们的数据强调, 前脑型化系统代表了一个有希望的工具, 以量化的方式研究早期皮质发育的人类特定方面的体外。
未来的一个主要挑战是保持 organoids 的均匀性, 以达到更成熟的神经元表型。这可能由以下一种或多种实现: 在生物反应器系统中培养 organoids14, 应用漂浮支架的15, 补充分化培养基与神经生长, 或神经元生存因子。最后, 通过将前脑型 organoids 与不同区域身份21,22的脑 organoids 融合, 可以实现大脑复杂性的控制增加。
两者结合在一起, 前脑型化协议提供了一个容易适用和可靠的工具, 为早期皮质结构的生成体外。该协议产生了高度均匀的早期皮质组织横跨多个 iPSC 线, 并可用于可靠地生成个别特定的皮质组织。因此, 该系统特别适用于需要高度的同质性和重现度 (如疾病建模) 的应用。

人脑显然是最复杂的器官之一, 并对所有人类的智力能力负责。因此, 深入了解人类特定的大脑发育是理解人类认知能力的重要前提。传统上, 转基因动物充当模型生物体来研究大脑发育。这些模型为大脑发育的原理提供了基本的洞察力。我们现在知道, 所有哺乳动物大脑发育的一个共同特征是祖细胞增殖、神经形成和神经元迁移的精确编排。然而, 在模型生物体的大脑, 如啮齿动物和人类, 特别是在大脑皮层, 存在着显著的结构差异。已提出的主要机制, 以促进灵长类皮层的进化是增加增殖的茎和祖细胞, 以及生成外桡神经胶质细胞 (oRGCs), 这只是很少发现在啮齿目动物1 ,2,3。
人类大脑皮层发育模型的方法包括: PSC 衍生的 telencephalic 祖细胞和大脑皮层投射神经元作为单层培养物的生成。这些标准化的差异性协议重放了人类皮层发育的某些方面, 如皮层神经再生的常规时间顺序4。然而, 当涉及到器官发育过程的重述时, 如空间模式和形态发生, 它们就会出现短缺。干细胞生物学的最新发展导致了从公司的3D 化文化的建立, 这是革命性的研究的体外人类器官。利用公司的能力, 自我组织成器官结构, 各种 organoids, 反映器官的关键结构和功能的性质, 包括肾脏, 肠道, 眼睛和大脑已经建立5。此类 organoids 包含多个器官特异的细胞亚型, 在体内组织与发育器官非常相似的在活体中5,6。此外, 细胞组成, 血统关系, 和基因网络研究使用单细胞 RNA 测序显示, 人脑 organoids 忠实重述人类胎儿大脑皮层的发展, 如基因表达程序的主要方面7,8. 然而, 目前阻碍其广泛应用的一个主要缺点是, 批量变更和化化异构性9。
在这里, 我们提供了一个简单的和标准化的前脑型化文化系统的详细协议。该系统的关键特点是它有效地和性生成了 PSC 派生的 organoids 几乎独占的背 telencephalic 身份。该协议基于我们最近的单元报告文件10中使用的方法。它结合了干细胞的自组织能力与选择性诱导皮质神经细胞, 并能在3周内有力地生成早期背 telencephalic 组织的同源培养物.该协议建立在先前报告的 SMAD 信号和 Wnt 抑制策略上, 它引导 PSC 向前神经谱系的分化11,12与矩阵嵌入相结合, 这促进大型连续上皮结构的形成13。我们已经成功地在不同的 iPSC 线上使用了描述的方法, 每个个体有几个克隆。结果表明, 该系统适用于可重复性和均匀性都具有重要意义的下游应用, 如疾病建模等。当将该协议应用于患有严重皮质畸形患者的干细胞时, 我们能够重述该病的病理特征体外, 并确定新的分子机制导致表型更改10。我们建议, 所描述的化协议可以用来弥合简化 PSC 的皮层单层培养和体内研究之间的差距, 并表示它代表一个可靠和稳定的细胞为基础的模型系统, 以模拟早期人体皮质发育在健康和疾病之外的人体。

<table><tbody><tr><td>A83-01</td><td>StemGent</td><td>130-106-274</td><td>500 nM</td></tr><tr><td>B27 Supplement</td><td>Gibco</td><td>17504-044</td><td>1 to 100</td></tr><tr><td>Basement membrane extract (e.g. Geltrex)</td><td>Gibco</td><td>A14132-02</td><td></td></tr><tr><td>Cyclic adenosine monophosphate (cAMP)</td><td>Sigma-Aldrich</td><td>A9501</td><td>0.15 &amp;micro;g/mL</td></tr><tr><td>Cell-dissociation reagent (TrypLE Express)</td><td>Gibco</td><td>12605028</td><td></td></tr><tr><td>Counting chamber e.g. Fuchs-Rosenthal</td><td>Karl Hecht</td><td>40449001</td><td></td></tr><tr><td>D-Glucose</td><td>Carl Roth</td><td>HN06.3</td><td>0.2 mg/mL</td></tr><tr><td>DMEM-F12 L-Glutamin</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>Embedding molds (Tissue-Tek Cryomold)</td><td>Sakura Finetek</td><td>4565</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>E6511</td><td>0.5 mM&amp;nbsp;</td></tr><tr><td>Gelantin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3149-25KU</td><td>10 ug/mL</td></tr><tr><td>Inhibitor of WNT response (IWR-1)</td><td>Enzo Life Science</td><td>BML-WN103-0005</td><td>10 ug/mL</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>91077C</td><td>2.5 &amp;micro;g/mL</td></tr><tr><td>IPSC medium for monolayer cultures (Pluripro)</td><td>Cell Guidance Systems</td><td>MK01</td><td></td></tr><tr><td>L-alanyl-L-glutamine (GlutaMax)</td><td>Gibco</td><td>35050038</td><td>1%</td></tr><tr><td>Low-adhesion 6 cm plates</td><td>Labomedic</td><td>2081646L</td><td></td></tr><tr><td>Low-adhesion 10 cm plates</td><td>Labomedic</td><td>2081646O</td><td></td></tr><tr><td>LDN-193189</td><td>Miltenyi Biotec</td><td>130-104-171</td><td>180 nM</td></tr><tr><td>N2 Supplement</td><td>Gibco</td><td>17502-048</td><td>1 to 200</td></tr><tr><td>Non-essential amino acids</td><td>Gibco</td><td>11140035</td><td>0.50%</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>4.00%</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Plastic paraffin film (Parafilm)</td><td>BRAND GMBH + CO KG</td><td>701606</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>Cell Guidance Systems</td><td>SM02-100</td><td>5 &amp;micro;M or 50 &amp;micro;M</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>Tubes 15 mL</td><td>Corning Life Sciences</td><td>734-0451&amp;nbsp;</td><td></td></tr><tr><td>Microscope Slides e.g. Superfrost Plus Microscope Slides</td><td>Thermo Scientific</td><td>4951PLUS4</td><td></td></tr><tr><td>Tissue culture 6 well plate</td><td>Falcon</td><td>734-0019</td><td></td></tr><tr><td>Tissue culture 24 well plate</td><td>Falcon</td><td>734-0949</td><td></td></tr><tr><td>Trypan blue stain</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Ultra-low-binding 96 well lipidure-coat plate A-U96</td><td>Amsbio</td><td>AMS.51011610</td><td></td></tr><tr><td>Antibodies</td><td></td><td></td><td></td></tr><tr><td>Sox2</td><td>R&amp;amp;D Systems</td><td>MAB2018</td><td></td></tr><tr><td>Pax6</td><td>Covance</td><td>PRB-278P-100</td><td></td></tr><tr><td>Otx2</td><td>R&amp;amp;D Systems</td><td>ab9566</td><td></td></tr><tr><td>Emx1</td><td>Sigma</td><td>HPA006421</td><td></td></tr><tr><td>N-cadherin</td><td>BD</td><td>610921</td><td></td></tr><tr><td>ZO-1</td><td>Life Tech</td><td>61-7300</td><td></td></tr><tr><td>P-Vimentin</td><td>Biozol</td><td>D076-3</td><td></td></tr><tr><td>Tpx2</td><td>Novus Biologicals</td><td>NB500-179</td><td></td></tr><tr><td>Acetylated &amp;alpha;-tubulin&amp;nbsp;</td><td>NEB/CS</td><td>5335</td><td></td></tr><tr><td>Alexa488 anti ms</td><td>Invitrogen</td><td>A11001</td><td></td></tr><tr><td>Alexa488 anti rb</td><td>Invitrogen</td><td>A11008</td><td></td></tr><tr><td>Alexa555 anti ms</td><td>Invitrogen</td><td>A21424</td><td></td></tr><tr><td>Alexa555 anti rb</td><td>Invitrogen</td><td>A21429</td><td></td></tr></tbody></table>

generation of standardized and reproducible forebrain-type cerebral organoids from human induced pluripotent stem cells

人类皮质高度膨胀, 呈现出具有特定功能区域的复杂结构, 提供更高的脑功能, 如认知。研究人类大脑皮层发育的努力受到了模型系统的可用性的限制。从啮齿类动物研究到人类系统的翻译结果受到物种差异的限制, 而人类原发性组织的研究由于缺乏组织的可用性以及伦理问题而受到阻碍。人类多能干细胞 (PSC) 技术的最新发展包括三维 (3D) 自组织器官培养系统的生成, 它在一定程度上模拟了人类特定的大脑发育体外。目前, 各种协议可用于整个大脑或脑区特定 organoids 的生成。从诱导 psc (iPSC) 中产生的同源和可重现前脑型 organoids 的方法, 我们在此之前建立和描述, 结合了 psc 对自组织的内在能力, 并将其引导分化为前神经谱系和基质嵌入支持连续神经细胞的形成。更具体地说, 本议定书涉及: (1) iPSC 聚合体的生成, 包括 iPSC 菌落向融合单层培养的转化;(2) 前外胚层的诱导;(3) 神经骨料在基质支架中的嵌入;(4) 从神经集料中产生前脑型 organoids;(5) 前脑型 organoids 的固定和验证。因此, 该协议提供了一种易于适用的系统, 用于生成标准化和可重现性的 iPSC 皮层组织结构体外。

这里描述的标准化前脑型化协议通常产生高度均匀的化文化几乎完全从人类干细胞的背部皮层的身份在20天的耕种 (协议概述在图 1A). 建议在协议的时间过程中执行几个质量控制步骤, 此处定义为: "go" (继续差异化过程) 和 "不 go" (建议终止批处理的区域性) (图1).通过拍摄图像和注释, 还可以很好地记录每个质量控制步骤。
产生前脑型 organoids 的第一个关键步骤是从高质量的 iPSC 文化开始。它是重要的干细胞不包含被区分的细胞的更大的组分。只使用 iPSC 的培养物, 在起始种群中呈现为未分化细胞的均匀单层 (图 1B, C)。此外, 从给定的单元格数开始 iPSC 聚合是至关重要的。对 iPSC 集料的第一次详细检查应在2天进行。在这个阶段, 聚集体应该形成紧凑的细胞芽与光滑的边缘 ("go"), 而不规则出现的骨料或与空洞的集合应丢弃 (' 不去 ') (图 1D, E)。下一个质量控制步骤应在协议的10天执行。在这个时间点, 细胞团聚体应显示光滑和光学半透明的组织外表面代表诱导的外胚层 (' go '), 而缺乏这种组织表明次优神经诱导 (' 不去 ') (图 1F, G).只有那些呈现半透明表面的聚合体 (图 1F) 才应嵌入到 BME 矩阵中。一旦植入, 皮质 organoids 将发展连续的上皮循环状结构, 这将迅速扩展。通过调查 organoids 是否已开发出极化神经外胚层, 分析15天和20日皮质诱导的效率, 如图 1H、J ("go") 所示。如果 organoids 没有开发出这样的上皮芽 (如图 1I, K中所示的 "无 go"), 请严格地修改所执行的质量控制步骤以进行故障排除。当严格遵循该协议时, 将生成高度标准化的化批次 (图2A、B), 它将在多个批次中和跨批 (图 2C) 中显示在极化神经外胚层形成中的≥90% 均匀性。导致极化神经外胚层形成的一个常见错误是增加 iPSC 聚合的起始细胞数, 或者从低质量的 iPSC 培养物, 如支原体污染的文化或含有分化细胞。
应在20天对生成的 organoids 的背 telencephalic 特性进行详细的验证。为此, 3 organoids 应固定, 并用于免疫荧光分析。分层上皮循环 (图 3A) 表示神经干细胞标记 Sox2 (图 3B, D), 前脑标记 Pax6 和 Otx2 (数字 3C, E), 和背部皮层标记 Emx1 (图 3F). 这些皮质环进一步特征为 n-钙黏蛋白和 ZO-1 (图 3G, H) 的顶端定位, 心室区桡胶质细胞 (vRGC) 衍生的微管, 从顶端到结构的基侧的跨度 (图 3I), 和顶端位于划分细胞, 染色阳性的磷酸化波形 (p-波形,图 3J)。细胞死亡可能存在于化结构中。中心细胞凋亡是正常的, 不影响皮层组织的发育。此外, 还应使用 3 organoids, 通过基因表达分析来评估该协议的同质性。前脑型 organoids 表现的背前脑标记 (FoxG1, Otx2, Emx1), 而中脑的表达 (FoxA2, Pax5) 和后脑 (HoxB2, HoxA4, HoxB4, HoxB6) 标记是不可检测的 (图 3K)。
批次的质量控制 organoids 可用于各种应用, 如分析的顶端径向胶质细胞的分裂平面。为此, 我们建议使用对 p-波形和 Tpx2 的抗体进行双染色 (图 3J)。在有丝分裂过程中, p-波形磷酸化 CDK1, 位于细胞核内, 从而在有丝分裂期16中标记所有细胞核。Tpx2 是一种微管相关蛋白, 可以在 interkinetic 核迁移过程中可视化有丝分裂纺锤和顶端过程17,18。使用这些标记, vRGC 分裂的三个方面在原则上可以被分析: (I) 细胞分裂是否发生在顶端边, (II) 分裂平面是否垂直对齐 (指示对称细胞分裂), 水平, 或斜 (指示不对称细胞分裂) 到顶端表面, 和 (III) 微管组织中心是否正常形成。
Organoids 可以也进一步被区分成更加复杂组织和分层的皮层组织结构。35± 2 organoids 内的皮质结构由心室区 (VZ)、内外脑室区 (SVZ) 以及皮质板 (CP) 样区组成。在 VZ 和内外部 SVZ, vRGCs, 中间祖 (IPs), 和细胞让人联想到 oRGCs 可以被识别。此外, 一个层状皮质的初步形成可以在 CP 样区观察到, 深皮层神经元表达 Tbr1 和 Ctip2 内和上皮质神经元表达 Satb2, 以及 Reelin 表达细胞在外部区域10。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56768/56768fig1.jpg"> 图 1: 化协议的示意图概述和 "go" 和 "不 go" 条件的说明.(A) 协议的示意图概述。CI 培养基: 皮质诱导培养基;CD: 皮质分化培养基。(B-C)一个最佳的90% 汇合 iPSC 单层培养 (B) 的图像和非合适的 iPSC 文化展示分化 (C)。(D-E)在大小、单元密度和表面外观 (D) 和两个 "无 go" 单元格聚合中的 iPSC 聚合最佳表现为单元备用腔 (e、上部聚合体) 或不规则边 (e, 下合计) 两天以下单元格聚合。(F-G)具有半透明和平滑边缘 (F) 和缺少光清除 (G) 的单元聚合的单元聚合。黄线正在可视化感兴趣的区域。(H-K)一个最佳的化与连续的上皮循环 (H, J) 和一个化, 未能开发径向组织的外胚层 (I, K), 分别在15天和当天20拍摄。刻度条, B-c 500 µm;d-K 200 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56768/56768fig1large.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/56768/56768fig2.jpg"> 图 2: 前脑型化协议的同质性和可重复.(A-B)在15天 (A) 和26天 (B) 中, 从一个批次中 organoids 的具有代表性的明亮域图像。(C) 20 天 organoids 的定量分析。Organoids 显示在外面表面 a 神经细胞, 可认识在明亮领域作为光学上清楚的表面组织以清楚的边界和放射状细胞建筑学的证据是定量的 (n = 3 每 iPSC 线以至少 16 Organoids 每实验)。刻度条, a-B: 500 µm. 错误栏± SD.<a href="//cloudflare.jove.com/files/ftp_upload/56768/56768fig2large.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/56768/56768fig3.jpg"> 图 3:20 天前脑型 organoids 的验证(A-J)organoids 的免疫细胞化学特性。Organoids 在多个上皮循环中组织 (A, 1-4 与 DAPI)。上皮循环中的分层组织细胞表达神经干细胞标记 Sox2 (B, d), 前脑标记 Pax6 (C, d) 和 Otx2 (E), 以及背前脑标记 Emx1 (F)。皮质环结构表现出一个优良的附着的连接带, 在最顶端的一侧与积累的 n-钙黏蛋白 (G) 和透明拼装蛋白 1 (ZO-1;H). 心室视网膜的微管网络 (由乙酰α-蛋白、交流浴缸染色) 从顶端延伸到环路结构的基底侧 (I)。表达 p-波形 (p-Vim) 的增殖细胞位于顶端表面。有丝分裂纺锤形 Tpx2. 具有代表性的高放大图像垂直和水平分割平面显示在右侧 (J)。(K) 对2不同 iPSC 线的两个独立的 organoids 的20天的特定区域转录因子进行 rt-pcr 分析。胎儿脑部控制;AB: 成人大脑控制。鳞片棒, 200 µm;电子 I 10 µm.<a href="//cloudflare.jove.com/files/ftp_upload/56768/56768fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>表</td>
			<td>稀释</td>
		</tr>
<tr>
<td>Sox2</td>
			<td>1-300</td>
		</tr>
<tr>
<td>Pax6</td>
			<td>1-500</td>
		</tr>
<tr>
<td>Otx2</td>
			<td>1-500</td>
		</tr>
<tr>
<td>Emx1</td>
			<td>1-50</td>
		</tr>
<tr>
<td>n-钙黏蛋白</td>
			<td>1-500</td>
		</tr>
<tr>
<td>ZO-1</td>
			<td>1-100</td>
		</tr>
<tr>
<td>p-波形</td>
			<td>1-1000</td>
		</tr>
<tr>
<td>Tpx2</td>
			<td>1-500</td>
		</tr>
<tr>
<td>乙酰α-蛋白</td>
			<td>1-500</td>
		</tr>
<tr>
<td>Alexa488 抗 ms</td>
			<td>1-1000</td>
		</tr>
<tr>
<td>Alexa488 反 rb</td>
			<td>1-1000</td>
		</tr>
<tr>
<td>Alexa555 抗 ms</td>
			<td>1-1000</td>
		</tr>
<tr>
<td>Alexa555 反 rb</td>
			<td>1-1000</td>
		</tr>
</tbody></table>表 1:20 天 organoids 质量控制抗体。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>底漆</td>
			<td>序列</td>
		</tr>
<tr>
<td>Otx2 前锋</td>
			<td>tgcaggggttcttctgtgat</td>
		</tr>
<tr>
<td>Otx2 反向</td>
			<td>agggtcagagcaattgacca</td>
		</tr>
<tr>
<td>FoxG1 前锋</td>
			<td>ccctcccatttctgtacgttt</td>
		</tr>
<tr>
<td>FoxG1 反向</td>
			<td>ctggcggctcttagagat</td>
		</tr>
<tr>
<td>Emx1 前锋</td>
			<td>agacgcaggtgaaggtgtgg</td>
		</tr>
<tr>
<td>Emx1 反向</td>
			<td>caggcaggcaggctctcc</td>
		</tr>
<tr>
<td>FoxA2 前锋</td>
			<td>ccaccaccaaccccacaaaatg</td>
		</tr>
<tr>
<td>FoxA2 反向</td>
			<td>tgcaacaccgtctccccaaagt</td>
		</tr>
<tr>
<td>Pax5 前锋</td>
			<td>aggatgccgctgatggagtac</td>
		</tr>
<tr>
<td>Pax5 反向</td>
			<td>tggaggagtgaatcagcttgg</td>
		</tr>
<tr>
<td>HoxB2 前锋</td>
			<td>tttagccgttcgcttagagg</td>
		</tr>
<tr>
<td>HoxB2 反向</td>
			<td>cggatagctggagacaggag</td>
		</tr>
<tr>
<td>HoxA4 前锋</td>
			<td>ttcagcaaaatgccctctct</td>
		</tr>
<tr>
<td>HoxA4 反向</td>
			<td>taggccagctccacagttct</td>
		</tr>
<tr>
<td>HoxB4 前锋</td>
			<td>acacccgctaacaaatgagg</td>
		</tr>
<tr>
<td>HoxB4 反向</td>
			<td>gcacgaaagatgagggagag</td>
		</tr>
<tr>
<td>HoxB6 前锋</td>
			<td>gaactgaggagcggactcac</td>
		</tr>
<tr>
<td>HoxB6 反向</td>
			<td>ctgggatcagggagtcttca</td>
		</tr>
<tr>
<td>18s 向前</td>
			<td>ttccttggaccggcgcaag</td>
		</tr>
<tr>
<td>18s 反向</td>
			<td>gccgcatcgccggtcgg</td>
		</tr>
</tbody></table>表 2: 基因表达谱的引物和引物序列。

Watch this Scientific Journal Video about Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells at JoVE.com

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

脑 organoids 代表了一个新的模型系统来研究早期人脑发育体外。本文提供了详细的方法, 以有效地产生同种背前脑型 organoids 的人诱导多能干细胞, 包括关键的表征和验证步骤。

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

generation-standardized-reproducible-forebrain-type-cerebral

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

21.2K 次观看.  University of Bonn. 脑 organoids 代表了一个新的模型系统来研究早期人脑发育体外。本文提供了详细的方法, 以有效地产生同种背前脑型 organoids 的人诱导多能干细胞, 包括关键的表征和验证步骤。

视频: Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

科研

发育生物学

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

一种从人类胚胎干细胞中生成脑器官的静态自导方法

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

从人诱导多能干细胞生成 3D 中脑类器官

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

强大且高度可重复的皮质脑类器官生成，用于在体外模拟大脑神经元衰老

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

神经科学

An In Vitro Method to Generate Human Brain Organoids

Source: Gabriel, E., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55372">Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating human brain organoids from induced pluripotent stem cells. It shows how the neurosphere forms a neuroectodermal layer, which suppresses the formation of other germ layers. The neurospheres are embedded in a 3D gel matrix and form neuroepithelial buds. When incubated with organoid media, these neuroepithelial buds differentiate into specific brain regions, resulting in the formation of mature brain organoids.

一种生成人脑类器官的体外方法

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Generation of Brain Organoids (23 ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Source: Koch, L. S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/64586">Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research.</a> J. Vis. Exp. (2023).
This video demonstrates an assay to generate cerebral organoids from induced pluripotent stem cells (iPSCs). Human iPSCs are induced to form embryoid bodies (EBs). The EBs are cultured over a polymer scaffold using an induction medium, promoting differentiation into neural stem cells, progenitor cells, and immature neurons. Finally, the EBs are embedded in a matrix and transferred to a maintenance medium for cerebral organoid formation.

产生诱导多能干细胞来源的大脑类器官

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Figure 1 illustrates a schematic ...

Generating Forebrain-Type Cerebral Organoids from Human-Induced Pluripotent Stem Cells

Source: Krefft, O. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56768">Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video outlines the creation of forebrain-type organoids from human induced pluripotent stem cells (iPSCs), demonstrating the progression from stem cell aggregates to complex structures that mimic the brain&#39;s cortex and differentiate into various neuron types.

从人诱导的多能干细胞生成前脑型大脑类器官

1. Generation of iPSC Aggregates

	Generation of single-cell monolayer cultures from iPSC colonies
	
		Prepare a basement membrane extract (BME) coated ...

Co-culture and Interaction Studies

Generation of Spheroids from Human Pluripotent Stem Cells

Source: Shaker, M. R., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63714">Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.</a> J. Vis. Exp. (2022).
This video demonstrates the transformation of human pluripotent stem cells (hPSCs) into spherical brain organoids. By using specific inhibitors, the hPSCs are guided to differentiate towards a neuronal lineage, forming neuroectodermal colonies. Special growth factors are then introduced, which encourage the self-organization and proliferation of the colonies, ultimately leading to the formation of spherical brain organoids.

从人多能干细胞生成球状体

1. Cortical brain organoid generation (Figure 1) 
NOTE: All the steps in this section of the protocol will occur in a Class 2 biosafety hood unless stated ...