Name: efficient generation and editing of feeder-free ipscs from human pancreatic cells using the crispr-cas9 system
Uploaded: 2017-11-08T13:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System at JoVE.com

实验室的工作得到了博士后奖学金的资助, Dr. 安贾莉 Nandal, 以及马里兰州干细胞研究基金对 BT (TEDCO) 的探索性赠款。

1. 重新编程协议 <ol> <li> 从主人类胰腺细胞中生成人类 iPSC <ol> <li> 用1.5 毫克/毫升的冷胶原蛋白涂上6个板, 并允许它在37和 #176 上凝胶; C 为 1 h. </li>
		<li> 在 Prigrow III 培养基 (~ 1-1.5 和 #215; 10 5 细胞) 上, 在天6上的胶原涂层-2-井板上的人原发性胰腺细胞, 达到约2.5 和 #215; 10 5 单元格或至少在当天的每井 60% 70-100转导 (0 天)。在第一项研究中, 在0天, 至少需要 2-3 口井以获得一个良好的70-100。至少使用一个井作为控件来计算单元格. </li>
		<li> 在转导日 (0 天), 温暖1毫升的 Prigrow III 培养基在水浴为每井转。在1毫升 10% FBS 培养基中, 用1毫升的胰蛋白酶/EDTA 为5分钟或直到细胞分离以执行细胞计数时, 从一个控制井中收获细胞。要制作 10% FBS 培养基, 加入 DMEM、10% FBS、1% l-谷氨酰胺、1% 丙酮酸钠和1% 非必需氨基酸. </li>
		<li> 使用单元计数器计数和检查单元格的可行性, 并根据细胞数和病毒滴度计算到达目标所需的每种病毒的体积。使用科斯的感染多样性 = 5, hc c-myc = 5 和 hKlf4 = 3 为胰腺细胞. </li>
		<li> 在-80 和 #176 中解冻一组仙台矢量管; C 存储在冰上, 并小心地将三仙台矢量管的计算量添加到1毫升的 Prigrow III 介质, 5ml 到37和 #176; c. 吸管轻轻地混合溶液。在6井板中的一个井, 足以换与仙台病毒载体产生重新编程的细胞. </li>
		<li> 从细胞中吸出 Prigrow III 培养基, 并慢慢地将重新编程的病毒混合物添加到含有这些细胞的井中。在一夜之间孵育细胞37和 #176; C 恒温气氛 5% CO 2 . </li>
		<li> 小心地将病毒混合物丢弃在细胞上, 并在第二天以新鲜的 Prigrow III 培养基取代, 24 小时后转导。培养5维的细胞, 每隔一天改变培养基. </li>
		<li> 在5天, 准备 MEFs 0.1% 明胶预 10 cm 培养皿 (1.3 x10 6 细胞/碟)。生长 MEFs 在 T175 烧瓶中, 直到天 4, 然后在天 5, 暴露细胞到6000拉德从 a 和 #947;-辐射源在播种单元格 13 或使用商业 MEF 源之前. </li>
		<li> 在6天, 使用1毫升的细胞剥离解决方案10分钟, 以分离转细胞, 板块所有在 Prigrow III 培养基与岩石抑制剂 (5 毫米股票, 10 和 #181; M 最后) 和孵化过夜, 在37和 #176; C 恒温详情重 5% CO 2 . </li>
		<li> 将第二天的 Prigrow III 介质更改为干细胞介质。为500毫升的干细胞培养基, 添加 DMEM/F-12, 20% (v/v) 淘汰赛血清置换 (KOSR), 5 ng/毫升 bFGF;1毫米 l-谷氨酰胺;100和 #181; 不必要的氨基酸和100和 #181; m 2 基。现在每天都要温和地改变媒介. </li>
		<li> 定期观察这些板块, 以发现细胞团簇或菌落的出现, 表明重新编程的细胞。转化后的细胞形成具有鹅卵石形态、大核和核仁的无性系团聚体。标记可能和 #39; iPSC 和 #39; 殖民地并定期检查它们的生长. </li>
		<li> 近四周后转导作为菌落准备采摘或转移, 准备 24-良好的 MEF 板通过电镀 1.3 x 10 6 细胞/明胶预板, 为转移的单一殖民地. 
		<ol> <li> 根据分析证书上的稀释因子, 准备 aliquoting 的矩阵膜 (如 如 , 基质)。添加一个分到25毫升的 DMEM/F-12, 以大衣四 6-井板 (1 毫升/井) 和孵化在室温 (RT) 至少1小时前使用。手动选取12-24 个菌落, 使用无菌吸管针尖将其吸入, 并在500和 #181 中转移到 MEF 板上; 干细胞介质的 L. </li>
			<li> 抽吸井中的介质, 以轻轻地扰乱或分离菌落。也挑选24-48 殖民地的矩阵膜涂层 24-井板。对于膜涂层板, 使用 mTeSR1 (补充10和 #181; M 岩石抑制剂) 为24小时, 并改变每天。在采摘殖民地的 6-10 d 之内, 他们准备好转移到新的24井和/或12井和 #160; 膜板用于克隆扩张. </li>
		</ol> </li> <li> 如果需要, 将 MEFs 上的菌落分离使用1毫克/毫升胶原酶20分钟, 用 hESC/mTeSR1 洗两次, 然后转移到 #160; 膜涂层12或24孔板。如果有足够的强大的殖民地生长在矩阵膜从步骤 1.1.12, 冻结在 MEFs 使用 iPSC 冷冻媒体, 根据制造商和 #39; s 的指导方针的殖民地. </li>
		<li> 1.1.12 使用500和 #181 将在膜上镀的鲁棒菌落分离; 中性20分钟, 再在基体膜上涂上 12-井板。手动刮去任何分化的细胞或污染 MEF 饲养细胞, 以丰富的多能克隆的 #160; 膜. </li>
		<li> 在 6-8 d 之后扩展克隆, #160; 用中性溶液游离膜涂层板, 并在新鲜 #160 上电镀小细胞团聚体; 膜涂层 6-或12孔板。每天更换 mTeSR1 介质。通过碱性磷酸酶染色、免疫多标记 (OCT3/4 和 NANOG)、多能面标记物的分析和鉴别检测来表征重编程克隆。种植和培养在膜涂层板上的克隆所有的化验, 包括 CRISPR 编辑如上所述, 除非其他涂层解决方案被明确提到. </li>
	</ol> </li> <li> 描述人类干细胞 <ol> 碱性磷酸酶染色 <li> 注: 此处使用的是商用套件。请参阅材料表。
		<ol> <li> 板假定人类的干细胞在 24-井板和文化为 4-5 d 与每日媒介变动. </li>
			<li> 启动染色协议, 吸入培养基, 用1毫升 1x PBS 洗涤细胞, 0.05% 吐温 20 (PBST). </li>
			<li> 在单元格中添加0.5 毫升的固定解决方案, 并在 RT 中孵育2到 5 min. 抽出固定溶液, 然后用 PBST 洗净细胞。不要让油井干涸. </li>
			<li> 通过混合溶液 A、B 和 c 将每个井的新制备的 AP 衬底溶液加0.5 毫升, 在室温下孵育5至15分钟, 在黑暗中孵化细胞 (用箔或深色容器包裹). 
			注意: 密切监测颜色变化, 并停止反应时, 颜色变亮, 以避免非特异性染色. </li>
			<li> 通过将 AP 衬底溶液吸进并用2毫升 1x PBS 冲洗两次井来停止反应. </li>
			<li> 观察在显微镜下的菌落, 使用任何明亮的野外显微镜和相机捕捉4X 或10X 放大图像. </li>
		</ol> </li> <li> 免疫 <ol> <li> 干细胞 24-井板和文化中的4-5 维, 每日媒体更改. </li>
			<li> 用 pbs 将细胞一次性冲洗一次, 在 pbs 中用4% 甲醛 (粉煤灰) 修复20分钟. </li>
			<li> 在 RT 上使用 PBS 清洗三次, 10 分钟 (3x, 10 分钟). </li>
			<li> 在 PBS 中使用10% 正常的山羊或驴血清 (NS, 取决于动物的二级抗体), 在 RT 中饱和40分钟。对于只有细胞表位, 包括 0.3% TX-100 在 pbs (TX/pbs) 到 permeabilize. </li>
			<li> 在 RT 中使用 PBS 清洗3x5 分钟. </li>
			<li> 将 OCT4、NANOG、TUJ1、NKX2-5 和 SOX17 抗体稀释在 PBS 中 5% NS 的新鲜溶液中, 在 RT 或隔夜在4和 #176 孵育2小时; C. </li>
			<li> 在 RT 中使用 PBS 清洗 3x 5 分钟. </li>
			<li> 在 RT 中稀释适当的次要 Alexa 氟488或568抗体, 在 5% NS/PBS 和孵育细胞为1小时. </li>
			<li> 在 RT 中为5分钟清洗 3x. </li>
			<li> 在 PBS 中孵育 DAPI 10分钟和洗涤2x 在 RT 5 分钟. 使用荧光显微镜拍照. </li>
		</ol> </li> <li> 荧光活化细胞分类 (干细胞) <ol> <li> 在6井板块和文化中, 直到菌落成为80% 汇合 (至少 10 5 细胞/样本) 与每日 mTeSR1 介质变化. </li>
			<li> 在实验的当天, 将单元格隔离并与单个单元格中断分离, 如协议中所述。用 10% FBS 培养基冲洗. </li>
			<li> 对于表面标记, 重在 0.5-1 毫升的 10% FBS 介质中, 并保持在冰上. </li>
			<li> 细胞内标记, 重在1毫升4% 的粉煤灰/PBS 和孵化10分钟室温。用 10% FBS 培养基冲洗。重在 0.1% TX/PBS 和孵化15分钟冰。再用 10% FBS 培养基冲洗。重在 0.5-1 毫升的 10% FBS 培养基和保持冰. </li>
			<li> 添加50和 #181; l 细胞悬浮到50和 #181; l 10% FBS 培养基中含有推荐量的 TRA-1-60、TRA-1-81 或 SSEA-4 抗体, 孵育30分钟至 1 h. </li>
			<li> 用 10% FBS 清洗两次. </li>
			<li> 重100和 #181; 10% FBS 培养基中含有荧光二次抗体, 孵育30分钟, 10% FBS 和重在适当容积 10% FBS。活细胞可以区别于死细胞由碘碘化染料添加在和 #60; 1 和 #181; g/mL 在过程中估计细胞凋亡. </li>
		</ol> </li> <li> 三沿袭差异 <ol> <li> 种子两个6井板, 1.5-2 x 10 6 干细胞/mTeSR1 介质与10和 #181; M 岩石抑制剂. </li>
			<li> 允许细胞生长 2-3 d, 每天 mTeSR1 介质变化, 直到油井80-90% 汇合. </li>
			<li> 对于外胚层感应, 根据制造商和 #39 的指示, 在6井板的2-3 口井中添加神经感应介质 (尼姆). </li>
			<li> 将培养基改 RPMI 含 2% B27 补充剂减去胰岛素和12和 #181; M CHIR99021 用于胚层2-3 口井的感应) 14 。只添加 RPMI 含有 2% B27 补充减去胰岛素的下一个24小时. 添加5和 #181; M IWP4 到 RPMI 培养基与 2% B27 补充减去胰岛素的下一个 24 h。72小时后, 用含有 2% B27 补充剂的 RPMI 取代培养基, 从而在 2-3 d. </li> 中产生跳动的心肌细胞。
			<li> 对于内胚层感应, 将6孔板的井中介质改成 MCDB 131, 加1.5 克/升碳酸氢钠, 1x Glutamax, 10 毫米葡萄糖, 0.5% BSA, 100 ng/毫升 GDF8, 5 和 #181; M CHIR99021 为 24 h. 在同一介质中没有 CHIR99021对于 2-3 d 15 . </li>
			<li> 根据步骤2中的所有三沿袭免疫协议, 并检查相关标记的表达式. </li>
		</ol> </li> </ol> </li> </ol> 2。人 iPSC 的基因组编辑使用 CRISPR-Cas9 <ol> <li> 在无菌条件下 Cas9 蛋白和 sgRNA <ol> <li> 分 Cas9 蛋白的制备并将离心冷冻在-80 和 #176 的管子上; C. </li>
		<li> 根据麻省理工学院 (http://www.genome-engineering.org/crispr/) 或任何其他 crispr 指南设计 webtool 的软件设计两个针对每个基因的目标导向 rna。目标的第一或第二外显子的基因 (基于开放阅读框架) 产生挖空。选择两个参考线, 使它们紧密地切割在一起 (〜 30-100 bp 分开), 我们可以很容易地可视化的删除使用同一组筛选底漆。根据较高的质量分数和较低的不相干站点数量, 从软件输出中选择参考线。设计筛选底漆使用程序底漆爆炸 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)。筛选底漆应至少 100 bp 远离 Cas9 切割地点和 pcr 产品的大小最好是在 400-700 bp 之间, 以方便扩增 pcr. </li>
		<li> 设计两个互补的 sgRNA 寡核苷酸 (根据引导序列的长度为19-22 核苷), 并在每一端 Bsa1 剪切位置。寡核苷酸可以通过商业和退火的方式合成, 形成双 DNA。对于退火, 添加1和 #181; l 每个100和 #181; M 指南, 25 和 #181; 退火缓冲器 (10 毫米三, pH7.5-8.0, 50 毫米氯化钠, 1 毫米 EDTA) 和23和 #181; 水的 l。在 thermocycler 95 和 #176 开始孵化; c 为2分钟, 然后逐渐冷却到25和 #176; c 超过 45 min. </li>
		<li> 克隆2和 #181; 将产生的片段转化为 Bsa1 限制酶, 消化1和 #181; in-house T7 启动子 pCR2.1 向量, Cas9 结合使用 T4 DNA 连接, 根据制造商和 #39; s 协议. </li>
		<li> 序列的克隆 DNA 片段和当保真度建立, 在体外 转录的克隆使用 T7 试剂盒生成单导 rna。用商用套件和洗在核糖核酸水中净化 sgRNAs。检查 RNA 浓度. </li>
	如上所述, </ol> </li> <li> 转染 hiPSCs <ol> <li> 区域性 hiPSCs 在 mTeSR1 介质中, 直到单元格为40-50% 汇合. </li>
		<li> nucleofection 前两个小时, 用2毫升的 prewarmed mTeSR1 培养基取代含有10和 #181 的培养基; M 岩石抑制剂. </li>
		<li> 一小时后, 通过吸气和 #160 为 nucleofected 细胞准备目标井; 膜, 如上图所示, 从12孔板 prewarmed 1 毫升的 mTeSR1 培养基, 用10和 #181; M 岩石抑制剂。保持在37和 #176; C 用于孵育. </li>
		<li> 为每个样品准备 nucleofection 的主组合 (根据样品适当缩放), 添加16.4 和 #181; P3 原细胞补充剂; 3.6 和 #181; 1 nucleofector 试剂盒; 0.5 和 #181; g Cas9 蛋白和0.5 和 #181; 每 sgRNA 的 g在22和 #181; L 每反应容量。pMAX GFP 载体也 nucleofected 根据制造商和 #39; 在细胞中的建议, 以粗略估计 iPSC 转染的效率. </li>
		<li> 在从 iPSC 井中吸出含有岩石抑制剂的介质后, 用2毫升的 RT PBS 清洗每一个井。然后抽吸 PBS, 加入1毫升的细胞剥离液, 并孵育37和 #176; C 为 10 min. </li>
		<li> 重3毫升的 mTeSR1 培养基中的细胞, 轻轻吸管向上和向下产生一个单细胞悬浮。将离解的细胞转移到15毫升离心管中, 其中含有5毫升 mTeSR1 介质. </li>
		<li> 计数单元格计数器并计算 0.5 x 10 6 细胞/转染所需的总体积。在15毫升离心管中放置所需的细胞数量, 在 200 x g 上进行离心, 在 RT 和吸气上清5分钟. </li>
		<li> 重每单元 0.5 x 10 6 22 和 #181 中的单元格; 在步骤4中准备的转染主混合的 L。快速将细胞转移到 nucleocuvette 带的一个井的中央室。使用程序 CB150 将条带放入 nucleofector 设备和 nucleofect 单元格中. </li>
		<li> nucleofection 后, 快速添加80和 #181; prewarmed mTESR1 培养基中含有10和 #181; M 岩石抑制剂对 nucleofected 细胞的每一个井。轻轻地混合移上下. </li>
		<li> 轻轻地将电池从条带移到 #160 的井中; 在步骤3中制备含 mTeSR1 介质的膜预 12-井板. </li>
		<li> 1 d 后, 更换为无岩石抑制剂的新鲜 mTeSR1 培养基。收获细胞 2-3 d 后 nucleofection 为单细胞排序. </li>
	</ol> </li> <li> 在排序前的某一天对目标 hiPSCs <ol> <li> 进行单单元隔离, 在 10% FBS 介质中播种 2 x 10 6 单元/明胶涂层板, 准备96良好的 MEF 板。大约70-80% 的克隆存活后, nucleofection 和 2-3 MEF 板可以为每个编辑实验准备. </li>
		<li> 在夜间孵化后, 将培养基改为干细胞培养基 (如前所述) 辅以 100 ng/ml bFGF, 1x SMC4 (抑制剂添加到媒体, 以提高单细胞的生存能力), 和5毫克/毫升纤维连接蛋白促进粘连. </li>
		<li> 在单单元格排序前, 将12孔板上的 hiPSCs 中的介质从 mTeSR1 替换为 mTeSR1 培养基, 并辅以 1x SMC4. </li>
		<li> 从 hiPSCs 中吸取培养基, 用 PBS 轻轻冲洗细胞。在各井中添加500和 #181; 在37和 #176 孵育, 在吸出 PBS 后10分钟;通过添加1毫升的 mTeSR1 (unsupplemented) 到每一个井和移上下轻轻几次, 产生单细胞悬浮. </li>
		<li> 将细胞悬浮在15毫升锥管和离心机5分钟, 在 200 x g at RT. 吸气上清和重1毫升的 mTeSR1 细胞. </li>
		<li> 在步骤1中准备的96孔板的单个井中, 使用 100 mm 喷嘴在无菌条件下的单元格分类器对单个单元格中的单元进行排序. </li>
		<li> 分类后四天, 菌落形成应明显; 此时, 用干细胞培养基补充 1x SMC4, 以取代培养基. </li>
		<li> 排序后八天, 用干细胞介质和培养基取代培养基 2 d. </li>
		<li> 分离的殖民地使用1毫克/毫升胶原酶20分钟, 并转移到 24-井膜涂层板 mTeSR1 与岩石抑制剂。在复制或扩展它们之后, 让单细胞克隆生长并提取他们的基因组 DNA。用基因特异引物通过 PCR 对靶 DNA 进行放大. </li>
		<li> 使用片段分析器试剂盒对所有克隆的 PCR 扩增目标基因组区域进行初始筛选, 方法是按照制造商和 #39 的说明, 通过凝胶/染料混合运行它们。通过与示例一起运行的相应梯子进行比较, 估计软件 PROSize 中产生的片段大小。序列的预期和 #39; 编辑和 #39; 克隆和分析顺序数据以确认编辑或删除. </li>
		<li> 用于区分 monoallelic 和 biallelic 克隆, 用 PCR 和克隆在 pJET1.2 向量中放大编辑后的序列。发送至少8-10 克隆以进行排序, 并检查序列以确认一个或两个等位基因中的编辑. </li>
		<li> 一旦通过基因分型确定了成功的目标 iPSC 克隆, 检查以确认它们没有丢失多或通过该过程获得染色体异常. </li>
	</ol>
	</li>
</ol>

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BT 是更新生物科学 Inc. 的创始成员

重组人体体细胞干细胞, 为基础生物学研究、个性化医学、疾病建模、药物开发和再生医学等领域提供了重要的推动力16。许多当前和广泛使用的人类 iPSC 世代的方法要求使用病毒与集成入宿主基因组或 episomal 载体的风险, 以低重编程效率。在这里, 我们提出了一种有效的方法, 产生无饲养干细胞的人胰腺细胞与仙台病毒, 这导致 "无足迹" 和有效的重新编程。由此产生的人类干细胞无病毒转基因, 维持多, 避免使用未知的外源因素。在无馈线条件下, 干细胞的生成效率较低, 因此我们将原代胰腺细胞重新编程到饲养细胞上, 然后将菌落迁移到无饲养膜, 用于扩张、培养和表征。这些技术的融合提供了稳定、可靠和高效的 iPSC 编程。
我们还从人成纤维细胞和其他主要细胞产生干细胞, 这表明, 仙台病毒可以用来重新编程的各种细胞。重要的考虑因素是: 对原细胞的最佳70-100 需要重新编程, 因为太少或太多的细胞影响重编程效率;当细胞仍处于最佳分割状态时, 原细胞的早期通路;仔细滴定的病毒载体, 导致最小细胞凋亡和高效率导致容易回升的强大的殖民地;通过 PCR 法对10-15 通道的仙台病媒转基因表达的损失进行验证, 以确保干细胞的 "无足迹";和严格的无菌条件下的 iPSC 在膜上的培养。
我们采用了一种方便有效的 CRISPR 基因组修饰方法来编辑 iPSC 基因组。该技术结合 Cas9 蛋白和 sgRNA、RNP 配合物来识别和切割互补 DNA 序列。它可以很容易地适应目标的基因组序列, 只需改变 20 bp 指南 RNA。我们的方法提供了一个简单的协议, 有效和可重复的编辑人类干细胞, 因为他们是更 labor-intensive, 难以传感器, 和昂贵的维护比其他细胞。我们为每个目标基因设计至少两个相邻但不重叠的参考线, 以便一组筛选底漆可用于筛选已编辑的克隆。此外, 使用两个指南〜 30-100 bp 分离产生一个大的删除, 这可以很容易地可视化在初步筛选和确保蛋白质消耗。这些指南可以在 HEK-293 细胞中进行测试, 最初是为了估计效率, 然后在 iPSC 中开始试验。此外, 建立常规试剂转染 iPSC 的参数是至关重要的。采用 pMAXGFP 质粒、转染效率和 #62; 80%、基因打靶/干扰效率为干细胞3-10%。在我们的协议中, 直接将 Cas9 RNP 配合物转化为细胞, 提供快速的行动和快速的周转, 并保持高的目标修改率。我们的方法采用单细胞分类后 nucleofection 的指导和 Cas9 蛋白, 克服了一个重大障碍的基因打靶在混合 iPSC 人口和确保克隆的细胞。这种 ' 新颖 ' 的整体方法是直接的, 易于复用, 只需要几个星期, 不需要任何抗生素选择。我们没有观察到任何效率的损失, 通过依次引入 CRISPRs 到细胞系或干细胞的删除, 虽然需要在这种情况下进行核型分析, 以确保基因组的稳定性。还应检查克隆是否有多标记的表达式。虽然不是本出版物的重点, 但该协议可以通过将修复模板包括在转染鸡尾酒1718中, 来诱导精确的基因修改。
最后, 根据我们的经验, iPSC 编辑协议的重要考虑因素是: 对指南进行合理的设计, 以尽量减少或完全避免目标突变, 特别是在 "种子区" 或接近 PAM 主题的修改;优化干细胞的 nucleofection 效率, 确保 RNPs 的交付;验证准备好的指南的质量; 指南和 Cas9 蛋白浓度的滴定, 并通过体外验证它们的活性生物化学的裂解法或如本文所描述的人类 cellssuch 中的 HEK 细胞, 更易于培养和染;使用早期通道干细胞, 在日志阶段的增长和达到〜 50% 70-100;在单细胞分类后, 小心地处理细胞, 以确保细胞的存活。
我们认为, 上述概述的协议详细描述, 将使读者有一个路线图, 以复制的方式生成和编辑 iPSC。

通过重编程因子的过度表达, 将人体细胞重新编程为多能状态, 使干细胞研究在疾病建模、再生医学和药物开发等领域有了革命性的改变。一些病毒重新编程的方法可以提供重新编程的因素和生成干细胞, 但该过程是劳动密集型, 而不是非常有效的1。病毒方法虽然很有效, 但却与集成和瘤2、34的问题有关。在这篇手稿中, 我们报告了使用胞质仙台病毒提供重新编程的因素和建立无足迹 iPSC 线, 没有任何病毒载体序列集成到他们的基因组5。仙台病毒是一种 RNA 病毒, 被稀释出来的细胞细胞质〜10通道感染后, 产生了丰富的重编程因素, 导致快速和有效的重新编程6,7。建立的干细胞可以很容易地转换为无饲养介质, 以避免使用小鼠胚胎成纤维细胞 (MEFs) 作为饲养细胞8。
在本刊物中, 除了概述仙台病毒介导的重新编程, 我们还描述了一个改进的编辑干细胞的协议, 它有可能提供无限的人类细胞与期望的基因改造的研究。我们已经使用 CRISPR/Cas9 技术的修改干细胞, 这是现在被用于广泛的应用, 包括敲门和敲除, large-scale 基因组删除, 汇集图书馆筛选的基因发现, 基因工程许多模型有机体和基因疗法9,10,11。这项技术涉及形成的配合物的链球菌化脓衍生 Cas9 核酸和 20-滨海指南 rna, 实现目标识别通过基地配对与基因组目标序列相邻的 3 ' 核苷酸 protospacer 相邻主题 (PAM) 序列。Cas9 核酸诱发双链断裂〜3核苷酸从 PAM 网站, 这是随后修复主要由同源结束连接 (NHEJ) 通路导致插入或删除在开放阅读框架, 从而功能基因敲除12。
我们改进的协议包括了人类胰腺细胞培养的细节, 对分裂灭活小鼠胚胎成纤维细胞 (MEFs) 的重新编程, 以达到更高的重编程效率, 随后适应无饲养的文化在基质, 定性为建立的干细胞, CRISPR 引导 RNA 设计和准备, 交付到干细胞作为 RNP 复合体, 单细胞排序生成克隆线编辑干细胞, 容易筛选和识别编辑, 并表征单细胞克隆。本研究通过引入 Cas9 蛋白和两个 CRISPR sgRNA RNP 配合物来诱导双链断裂 (DSBs) 并删除干预段, 从而有效地生成了基因组缺失。这种方法利用了两个指南, 在开放阅读框架中产生删除, 高效率的 NHEJ 导致低数量的克隆需要的特点, 并容易初步筛选克隆的自动毛细管电泳装置, 碎片分析仪。这些有效的基因组编辑方法, 以生成人类干细胞疾病模型将很快成为一个标准和常规的方法在任何干细胞实验室。最后, 精确的基因组编辑将使其超越干细胞疾病模型, 并有可能帮助催化细胞疗法。

<table><tbody><tr><td>Sendai viral vectors - CytoTune-iPS 2.0 Kit</td><td>Invitrogen</td><td>A16517</td><td>Thaw on ice; S No: 1</td></tr><tr><td>Trypsin EDTA</td><td>Gibco Life Tech</td><td>25300-054</td><td>0.05%, 100 ml; S No: 2</td></tr><tr><td>Rock inhibitor (Y-27632)</td><td>Milipore</td><td>SCM075</td><td>Use 10 &amp;mu;M; S No: 3</td></tr><tr><td>DMEM/F-12 medium</td><td>Invitrogen</td><td>11330-032</td><td>S No: 4</td></tr><tr><td>Serum replacement (KSR)</td><td>Gibco</td><td>10828028</td><td>S No: 5</td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>11960069</td><td>1X; S No: 6</td></tr><tr><td>Fetal bovine serum</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Aliquot; S No: 7</td></tr><tr><td>L-glutamine (Glutamax, 100X), liquid</td><td>Thermo Scientific</td><td>35050061</td><td>1/100; S No: 8</td></tr><tr><td>Non-Essential Amino Acids</td><td>Gibco</td><td>11140-050</td><td>1/100; S No: 9</td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985023</td><td>55 mM, 1/1,000; S No: 10</td></tr><tr><td>Hausser Hemacytometers</td><td>Hausser Scientific</td><td>02-671-54</td><td>S No: 11</td></tr><tr><td>0.1% Gelatin Solution</td><td>STEMCELL Technologies</td><td>7903</td><td>Incubate at 37&amp;ordm; C for 1 hour; S No: 12</td></tr><tr><td>SSEA-4 antibody</td><td>Santacruz</td><td>sc-21704</td><td>1/100; S No: 13</td></tr><tr><td>TRA-1-81 antibody</td><td>Cell Signaling</td><td>4745S</td><td>1/200; S No: 14</td></tr><tr><td>OCT4 antibody</td><td>Santa Cruz</td><td>sc-5279</td><td>1/1,000; S No: 15</td></tr><tr><td>Collagen I, Rat Tail</td><td>Life Technologies</td><td>A10483-01</td><td>Keep cold; S No: 16</td></tr><tr><td>Alexa Fluor fluorescent 488/ 568 (secondary antibodies)</td><td>Invitrogen</td><td>A21202/A10042</td><td>1/2,000; S No: 17</td></tr><tr><td>DPBS</td><td>Hyclone</td><td>SH30028LS</td><td>1X; S No: 18</td></tr><tr><td>100-mm tissue culture dish</td><td>Falcon</td><td>353003</td><td>S No: 20</td></tr><tr><td>96-well tissue culture plate</td><td>Falcon</td><td>353078</td><td>S No: 21</td></tr><tr><td>6-well tissue culture plate</td><td>Falcon</td><td>353046</td><td>S No: 22</td></tr><tr><td>Dissecting scope&amp;nbsp;</td><td>Nikon</td><td>SMZ745</td><td>S No: 23</td></tr><tr><td>Picking hood</td><td>NuAire</td><td>NU-301</td><td>S No: 24</td></tr><tr><td>15&amp;nbsp;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>188271</td><td>S No: 25</td></tr><tr><td>50&amp;nbsp;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>227261</td><td>S No: 26</td></tr><tr><td>Sodium pyruvate</td><td>Invitrogen</td><td>11360</td><td>S No: 28</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td>S No: 29</td></tr><tr><td>Prigrow III medium</td><td>ABM</td><td>TM003</td><td>S No: 31</td></tr><tr><td>Countess&amp;trade; Cell Counter</td><td>Invitrogen</td><td>C10227</td><td>S No: 32</td></tr><tr><td>Faxitron X-ray system</td><td>Faxitron</td><td>CellRad</td><td>S No: 33</td></tr><tr><td>Accutase</td><td>Innovative cell Technologies</td><td>AT-104</td><td>S No: 34</td></tr><tr><td>Collagenase</td><td>Life Technologies</td><td>17104019</td><td>1mg/ml stock; S No: 35</td></tr><tr><td>Dispase</td><td>STEMCELL Technologies</td><td>7923</td><td>S No: 36</td></tr><tr><td>hESC qualified matrigel</td><td>BD Biosciences</td><td>354277</td><td>To dilute, use cold DMEM/F-12; S No: 37</td></tr><tr><td>bFGF</td><td>R &amp;amp; D</td><td>233-FB</td><td>Stock 10 ug/ml; S No: 38</td></tr><tr><td>Paraformaldehyde</td><td>EMS</td><td>15710</td><td>4% stock in PBS; S No: 39</td></tr><tr><td>TRA-1-60</td><td>Santa Cruz</td><td>sc-21705</td><td>1/100; S No: 40</td></tr><tr><td>NANOG</td><td>ReproCELL</td><td>RCAB0004P-F</td><td>1/100; S No: 41</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416-100ML</td><td>S No: 42</td></tr><tr><td>Alkaline Phosphatase kit</td><td>Stemgent</td><td>00-0055</td><td>S No: 43</td></tr><tr><td>Cas9 protein</td><td>PNA Bio</td><td>CP01-50</td><td>Thaw and aliquot; S No: 44</td></tr><tr><td>Goat or donkey serum</td><td>Sigma</td><td>D9663/G9023</td><td>S No: 45</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100-100ML</td><td>S No: 46</td></tr><tr><td>DAPI</td><td>Thermo Scientific</td><td>D1306</td><td>S No: 47</td></tr><tr><td>Tris</td><td>Sigma</td><td>9285-100ML</td><td>S No: 48</td></tr><tr><td>NaCL</td><td>Sigma</td><td>S7653-250G</td><td>S No: 49</td></tr><tr><td>EDTA</td><td>Sigma</td><td>BP2482-500</td><td>S No: 50</td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202T</td><td>S No: 51</td></tr><tr><td>Mega Shortscript T7 kit</td><td>Thermo Scientific</td><td>AM1354</td><td>S No: 52</td></tr><tr><td>Mega Clear kit</td><td>Thermo Scientific</td><td>AM1908</td><td>S No: 53</td></tr><tr><td>SMC4</td><td>BD Biosciences</td><td>354357</td><td>S No: 54</td></tr><tr><td>Fibronectin</td><td>STEMCELL Technologies</td><td>7159</td><td>S No: 55</td></tr><tr><td>CloneJET cloning kit</td><td>Thermo Scientific</td><td>K1232</td><td>S No: 56</td></tr><tr><td>Fragment analyzer&lt;sup&gt;TM&lt;/sup&gt;</td><td>Advanced Analytical</td><td></td><td>S No: 57</td></tr><tr><td>mTeSR1 medium kit</td><td>STEMCELL Technologies</td><td>5850</td><td>Warm to room temperature; S No: 58</td></tr><tr><td>Freezing medium mFreSR&amp;trade;</td><td>STEMCELL Technologies</td><td>5855</td><td>S No: 59</td></tr><tr><td>Freezing medium CryoStor&amp;reg;</td><td>STEMCELL Technologies</td><td>7930</td><td>S No: 60</td></tr><tr><td>MEFs</td><td>Globalstem</td><td>GSC-6301G</td><td>S No: 61</td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>25030081</td><td>S No: 62</td></tr><tr><td>Human pancreatic cells</td><td>ABM</td><td>T0159</td><td>S No: 63</td></tr><tr><td>STEMdiff&amp;trade; Neural Induction Medium</td><td>Stemcell Technologies</td><td>5835</td><td>S No: 64</td></tr><tr><td>RPMI</td><td>Thermofisher</td><td>11875-093</td><td>S No: 65</td></tr><tr><td>2% B27-insulin</td><td>Thermofisher</td><td>A1895601</td><td>S No: 66</td></tr><tr><td>CHIR99021</td><td>Stemcell Technologies</td><td>72052</td><td>S No: 67</td></tr><tr><td>IWP4</td><td>Stemcell Technologies</td><td>72552</td><td>S No: 68</td></tr><tr><td>2% B27</td><td>Thermofisher</td><td>17504044</td><td>S No: 69</td></tr><tr><td>MCDB 131</td><td>Life Technologies</td><td>10372019</td><td>S No: 70</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S8761-100ML</td><td>S No: 71</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td>S No: 72</td></tr><tr><td>BSA</td><td>Proliant</td><td>68700</td><td>S No: 73</td></tr><tr><td>GDF8</td><td>Pepro-Tech</td><td>120-00</td><td>S No: 74</td></tr><tr><td>TUJ1 antibody</td><td>EMD Milipore</td><td>AB9354</td><td>S No: 75</td></tr><tr><td>NKX2-5 antibody</td><td>Santa Cruz</td><td>Sc-14033</td><td>S No: 76</td></tr><tr><td>SOX17 antibody</td><td>R &amp;amp; D systems</td><td>AF1924</td><td>S No: 77</td></tr><tr><td>Propidium iodide</td><td>Thermo Scientific</td><td>P3566</td><td>S No: 78</td></tr><tr><td>Amaxa 4D-nucleofector&amp;trade;</td><td>Lonza</td><td>AAF-1002</td><td>S No: 79</td></tr><tr><td>FACSAria II (cell sorter)</td><td>BD biosciences</td><td>SORP UV</td><td>S No: 80</td></tr></tbody></table>

efficient generation and editing of feeder-free ipscs from human pancreatic cells using the crispr-cas9 system

胚胎和诱导多能干细胞可以自我和分化成多个细胞类型的身体。因此, 多能细胞在再生医学研究中备受青睐, 目前正处于眼部疾病、糖尿病、心脏病和其他疾病的临床试验中。分化成专门的细胞类型的潜能, 加上基因组编辑技术的最新进展, 包括 CRISPR/Cas 系统, 为不同应用的 iPSC 基因组的剪裁提供了更多的机会包括疾病建模, 基因治疗, 和偏微分的途径, 命名为少数。在可用的编辑技术中, 来自链球菌化脓的 CRISPR/Cas9 已成为 site-specific 编辑真核基因组的首选工具。CRISPRs 是易于访问, 低廉, 高效的工程目标编辑。该系统需要一个 Cas9 核酸和一个引导序列 (20) 特定的基因组目标邻接一个3核苷 "NGG" protospacer 相邻主题 (PAM) 的目标 Cas9 到期望的基因组所在地, 旁边的通用 Cas9 结合示踪 RNA (一起被称为单一指南 RNA 或 sgRNA)。在这里, 我们提出了一个 step-by-步骤协议, 有效地生成馈线无关和无足迹 iPSC 和描述方法的基因组编辑 iPSC 使用 Cas9 核糖 (RNP) 配合物。基因组编辑协议是有效的, 可以很容易地复用 pre-complexing sgRNAs 为多个目标与 Cas9 蛋白, 同时传递到细胞。最后, 我们描述了一个简化的方法, 以识别和表征干细胞与所需的编辑。两者结合在一起, 预计将简化 iPSC 的生成和编辑, 以用于多种应用。

在本出版物中, 我们遵循了一个简单而有效的协议, 用于从人类胰腺细胞中利用集成或无足迹的仙台病毒载体生成 iPSC。图 1A显示了此重编程协议的示意图表示形式。人胰腺细胞的购买商业, 养殖在 Prigrow III 培养基和转与仙台病毒如上所述。转纺锤形的胰腺细胞没有显示任何形态学变化的〜5天后, 仙台病毒转导0天, 但随后成为圆形与更大的核和核仁。一些早期的殖民地在一个星期之后被看见了 post-transduction, 但他们没有被采摘, 象在我们的经验, 这些殖民地快速地离解并且通常是 ' 早期部分重新编程的细胞 ' 不建立健壮的殖民地。大约10-15 天 post-transduction, 小明亮的殖民地被观察了并且在成长以后被采摘了 (图 1B,右上)。人的 iPSC 殖民地是紧凑的, 严密地被包装以清楚的边缘 (被考虑作为 ' 完全地被重新编程的细胞) (图 1B,天23底部中间) 和 ' 部分重新编程的细胞 ' 是宽松的与空白在殖民地 (图 1B,23天右下角)。重组后的胰腺细胞菌落在18天 (图 1B, 左下角), 并在23天 (图 1B,底部中间) 手动拾取。殖民地被镀膜涂层板后采摘, 以获得无饲养 iPSC 的30-40 天 (图 2A,左)。其中一些 "健壮" 无馈线殖民地的扩展和特点, 以表达碱性磷酸酶, 多和表面标记的无饲养条件。所有的克隆试验都是阳性的碱性磷酸酶, 因为他们变成明亮的红色后的化验 (图 2A,右)。多标记 OCT4, NANOG, SSEA4, TRA-1-60 和 TRA-1-81 也被观察到高度表达在所有的克隆由免疫或外地资产管制局分析 (图 2B和图 3A)。
这些结果表明, 从胰腺细胞生成的人 iPSC 菌落在无饲养条件下表达了多标记。多还评估了人类 iPSC 的定向分化为三种细菌层: 外胚层, 胚层和内胚层。克隆膜能分化为 TUJ1-positive 神经元 (外胚层), NKX2-5-positive 跳动心肌细胞 (胚层) 和 SOX17-positive 胚细胞 (图 3B)。
经过深入的表征, 选择 3-稳健, 无饲养 iPSC 克隆线的基因组编辑研究。两端 BsaI 切口的两个参考线是为每个基因设计的, 以便将它们紧密地切割在一起 (即, n小于 ~ 100 bp)。协议示意图如图 4所示。这种用于目标基因的删除策略使我们能够很容易地删除一个基因的第一或第二外显子的一部分。这个策略非常有效, 我们可以在每次尝试中隔离编辑的克隆。指南被克隆成 BsaI 消化的载体和体外转录如上文所述。我们 nucleofected 指南 (rna) 和 Cas9 蛋白作为 RNP 配合物快速有效的编辑。我们还将细胞分类为 MEFs 的单细胞, 因为细胞的回收率比膜涂层板上单独排列的细胞要高。基因组 DNA 是从所有克隆分离出来的, 目标部分被 PCR 放大, 并在片段分析仪上进行分辨, 以筛选目标片段大小的变化与对照。将片段大小与示例中的梯形图进行比较, 以检测 "编辑" 的克隆 (图形 4,底部)。这使克隆的筛选容易, 因为我们可以通过和 #62; 90 克隆在一个板块和结果得到了准确的± 3 bp。然后对选定的克隆进行排序, 以确认野生类型和变异的克隆。野生克隆没有删除或添加基地, 而突变的克隆有添加或删除序列与野生。在序列中显示删除或添加的克隆被扩展并复制到 pJET1.2 向量中, 以通过每个克隆至少8-10 个菌落的顺序来识别 monoallelic 和 biallelic 克隆。Monoallelic 无性系在一个等位基因中被删除或添加的碱基, 另一种是不变的, 类似于野生基因。Biallelic 克隆在两个等位基因中都有缺失。大约300克隆被筛选为所有目标基因, 在每个案件辨认大约 9 monoallelic 删除克隆和 3 biallelic 删除克隆。与 monoallelic 克隆相比, 这大约相当于3倍的 biallelic 克隆频率的减少。在删除增的异质性反映了不完善和不一致的 NHEJ 修复。最后通过从靶细胞中提取 RNA, 并通过 rt-pcr 来证实该基因的表达。

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56260/56260fig1.jpg"> 
图 1: 将人胰细胞重新编程为无饲养的多功能细胞 (iPSC).(a) 显示了从人类胰腺细胞生成 iPSC 的示意图。胰岛细胞生长在胶原涂层板, 然后转移到 MEFs 后与人仙台病毒载体转导。完全地被重新编程的殖民地然后转移了到干细胞合格的膜并且描绘了。(B) 仙台病毒转导后的形态学变化。在转导之前, 胰腺细胞表现出典型的纺锤样形态学 (左上、0天)。小, 紧密的殖民地出现在 MEFs 12 天, 类似于人类多潜能细胞的殖民地。通常, 完全重新编程的人类 iPSC 殖民地有非常明确的界限, 可以在23-30 天被拾起。在23天的一个离解的殖民地显示在右下角。所有的图像都是在100X 放大 (10X 物镜和10X 目镜) 拍摄的。缩放栏 = 100 µm.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56260/56260fig1large.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/56260/56260fig2.jpg"> 
图 2: 人类 iPSC 的特性.(a) 在无馈线条件 (40X、4X 物镜和10X 目镜) 采摘和培养后, iPSC 菌落的典型相衬图像. 仙台病毒重新编程后的胰腺细胞。碱性磷酸酶染色红 iPSC 菌落在右边。对于碱性磷酸酶染色, iPSC 的殖民地是固定和染色红色后, 添加的基质溶液从套件。照片是在40X 拍摄的。(B) 免疫为多标记 NANOG, OCT3/4 在无饲养人的胰腺细胞衍生的干细胞。DAPI 被用来染色核蓝色作为一个控制。所有的图像都是以100X 倍的倍率拍摄的。缩放条形图 = 100 µm 在所有图像中。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56260/56260fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
ontent "佛: 保持在一起. 在页内 =" 1 "><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56260/56260fig3.jpg"> 
图 3: iPSC 的多和分化电位的表征.(A) 对无馈线干细胞中的表面标记 SSEA4、TRA-1-60 和 TRA-1-81 的分析。细胞被分离到单细胞悬浮和染色为表面标记 SSEA-4, TRA-1-60 并且 TRA-1-81。紫峰含有 iPSC 阴性 (抗体控制) 细胞, 胰 iPSC 可想象为绿峰。(B) 荧光细菌层标记基因的成像。TUJ1 外胚层 (绿)、NKX2-5 胚层 (红色) 和 SOX17 内胚层 (红色) 在胰腺 iPSC 定向分化后的细胞中的表达。相应的控制核染色 DAPI 是蓝色的。所有的图像都是以100X 倍的倍率拍摄的。缩放栏 = 100 µm.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56260/56260fig3large.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/56260/56260fig4.jpg"> 
图 4: CRISPR/Cas9 删除策略示意图一 sgRNA 对被设计 (标记红色) 与 BsaI 切口站点在末端 (显示作为 F 和 R) 优选地瞄准第一个外显子, 退火, 被克隆在 BsaI 被消化的修改过的传染媒介 (pCR2.1, BsaI 站点突出显示灰色), 排序和在体外翻译。下划线的向量序列显示 BsaI 消化后的部分被删除。筛选底漆的位置用蓝色表示。Cas9 和指南 rna 是 nucleofected 在无饲养 iPSC 作为一个复杂的基因组缺失。干细胞是然后单细胞排序在96井 MEF 板材, 培养, 转移到膜, 扩大和筛选的片段分析仪。为了在分析仪上运行样品, 提取 Cas9 的基因组 dna, 在膜上引导转染的克隆, 通过在分析仪中的凝胶/染料混合, 选择潜在的 ' 编辑 ' 克隆 (底部中间)。相关的阶梯标记, 重量和潜在的编辑克隆标记。通过提取 RNA, 用 Q PCR 法进行分析, 证实了该基因的表达。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56260/56260fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System at JoVE.com

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

本协议详细描述了在无饲养条件下从人类胰腺细胞中生成无足迹诱导多能干细胞 (干细胞), 其次是使用 CRISPR/Cas9 均一进行编辑和修饰单细胞克隆。

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

efficient-generation-editing-feeder-free-ipscs-from-human-pancreatic

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.0K 次观看.  University of Maryland. 本协议详细描述了在无饲养条件下从人类胰腺细胞中生成无足迹诱导多能干细胞 (干细胞), 其次是使用 CRISPR/Cas9 均一进行编辑和修饰单细胞克隆。

视频: Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

科研

发育生物学

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

遗传学

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

建立基因组编辑的人多能干细胞系:从定位到隔离

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

CRISPR/Cas9在iPSC衍生肌肉预祖中恢复肌营养不良素表达的技术

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

生物学

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

将人多能干细胞分化为胰腺 β 细胞

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

作者聚焦：β细胞与多能干细胞分化的进展和挑战

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.