This research was supported by the NIH Common Fund and Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases.

1.准备基底膜基质和塑料制品的涂装<ol><li> -20℃放置冷冻基底膜基质的库存在冰上解冻过夜，在4℃。 </li><li>解冻后，基底膜基质的吸移管2毫克等分入预冷的微量离心管中。直到需要℃，-20存储这些。 </li><li>为了准备基底膜基质涂层钢板，解冻一个等份冰上直到最后一块冰，在离心管消失（通常在〜2小时）。 </li><li>解冻后，基底膜基质添加至12毫升冷（4℃）的DMEM / F12，使基底膜基质涂布液。 </li><li>加入基底膜基质上解决了相应的培养容器。为一个6孔板，分配每孔1ml。摇动板以确保基底膜基质溶液完全覆盖每个孔中。 </li><li>封口膜密封基底膜基质涂层板/皿，孵育袋鼠米的温度在使用前1小时。备选地，2周涂层的内店铺基底膜基质包被的平板/菜在4℃下并使用。 注：添加额外的DMEM / F12向基底膜基质涂覆后的板/培养皿防止干燥。用4℃储存基底膜基质涂覆后的板/培养皿之前，将其放置在生物安全柜，并允许它达到室温至少30分钟。 </li><li>完全加入培养基和细胞的前抽吸基底膜基质。 </li></ol> 2.准备E8介质<ol><li>通过解冻E8补充过夜，在4℃下制备E8培养基。 </li><li>从500毫升的库存除去10毫升E8基础培养基并丢弃。 </li><li>吸取了整个10毫升小瓶E8补充直接进入490毫升E8基础培养基。不热情完全E8媒体在37℃水浴，因为温度反复波动会降低的bFGF在完井TE E8中。 </li><li> 2周另外补充的范围内使用完整的E8媒体。 </li></ol> 3.解冻iPS细​​胞的<ol><li>取下液氮和地点干冰冷冻iPS细​​胞的小瓶。 </li><li>迅速解冻在37℃的水浴的小瓶;漩涡在水浴小瓶，直到冰遗体只有一小片段。 </li><li>喷用70％乙醇和转移小瓶至生物安全柜中。 </li><li>加入1毫升室温E8介质滴加直接向小瓶中。 </li><li>使用2毫升吸管，转移细胞悬液滴加到9ml E8培养基在15ml锥形管中。摇动管经常以确保细胞和培养基迅速拌匀。 </li><li>离心细胞，在200×g离心5分钟。 </li><li>吸出上清液，和重悬细胞沉淀在E8的适当体积补充有10μM的Y-27632。 </li><li>添加细胞地下室适当数量MEMBRANê基质包被的孔，并将其放置在37℃，5％CO 2的培养箱中过夜。建议板至少0.2×10 6的iPSC每一个6孔板的井，使解冻后快速恢复。 </li></ol> 4.维护和iPS细胞的常规传代<ol><li>每天刷新E8媒体。 </li><li>监视形态和细胞的汇合与倒置显微镜。高品质的iPS细胞在生长具有明显的边界平整的殖民地;单个菌落拥有“铺路石样”外观。 </li><li>通道iPSCs的细胞，当他们到达〜70％汇合。 </li><li>通过加入0.9克NaCl和500μl的0.5M的EDTA至500ml的DPBS制备的EDTA传代方案。拌匀溶解NaCl和真空过滤消毒。暖在之前传代37℃水浴中传代溶液的等分试样。 </li><li>向通道，抽吸掉废培养基并用温水PAS等体积一次洗涤细胞saging解决方案。吸出物，以及移液管足够的EDTA传代溶液涂覆的细胞（每一个6孔板的孔1毫升）中。 </li><li>放置细胞的倒置显微镜下，观察的iPSC集落。孔殖民地和提高境内的外观应在2至5分钟变得明显。 </li><li>小心吸了EDTA传代的解决方案。 </li><li>用10毫升吸管，分配4毫升E8介质（如果使用6孔板）在高压下直接进入每个孔进行传代。 </li><li>收集的iPSC团块，并分裂成根据从1分流比阱的适当数量：8至1:12不要过度吸管，因为细胞团块解体将导致生存能力差。 </li><li>将板在培养箱中，并摇动板背面的往复和侧到另一侧的几次，以分散细胞。 </li></ol> 5.准备的MEF和iPS细胞的Tansfection的<ol><li> 48小时前transfectioN，通道的iPSC在〜1：6成四个或更多个井基底膜基质包被的6孔板的，使得它们将是70％汇合2天因此。 </li><li>第二天，解冻DR4的MEF成由DMEM（高葡萄糖），补充有10％FBS和1x MEM-NEAA的MEF培养基。 </li><li>板DR4的MEF成两个10厘米菜肴〜2×10 4个细胞/ cm 2孵育过夜，在37℃。 </li><li>在转染的当天，在iPS细胞进行转染前30分钟改变MEF培养基至E8补充有10μM的Y-27632。 </li><li>可选：如果iPSCs的转染后的流式细胞术分析是需要的，除去从4℃的基底膜基质涂覆后的板和放置在室温下。 </li><li>可选：4小时在转染之前，补充预染的iPSC培养用Y-27632以10μM的最终浓度。 </li></ol> 6.温和，细胞分离试剂治疗iPS细胞的转染次使用电穿孔系统<ol><li>从4℃除去P3的初级细胞的转染溶液，并允许它来室温〜30分钟。整个加入100μl补充添加到使用前的转染溶液。 </li><li>温暖柔和的细胞分离试剂在37℃水浴。 </li><li>得到AAVS1 TALENS（PZT-AAVS1-L1和PZT-AAVS1-R 1）和AAVS1-CAG-EGFP -20℃供体。 </li><li>从培养箱中取出的iPSC和用DPBS洗涤一次。 </li><li>加入1毫升平缓细胞解离试剂以每孔，并孵育iPSCs的在37℃下5分钟，或直至细胞大于50％的从培养容器中解离。 </li><li>吸取细胞上下用P1000吸管解离从培养容器中的任何剩余的细胞，并打散的iPSC团块几次。 </li><li>加2ml E8培养基到每个孔中，并且吸管上下数次用10毫升吸管给菲尔特呃分解细胞团块成单个细胞 注：转染效率显著下降，如果细胞团块不够分类。 </li><li>收集的iPSC在15ml锥形管中，并离心，在100×g离心3分钟。 </li><li>吸去上清液，并在E8介质的最小量重悬细胞。 </li><li>算使用的应用的一个重要污点诸如0.4％台盼蓝后血球细胞。确保细胞被充分分解，同时计数（1-3单元格每个“丛”）。 </li><li>分配3×10 6细胞到每个2的15毫升锥形管中，并再次降速，在100×g离心3分钟。 注：低速离心降低了细胞应激，并允许电前容易再悬浮iPS细胞的。 </li><li>电穿孔系统设置为对人胚胎干细胞系H9（程序的CB-150）的细胞类型特异性的程序。 </li><li>离心后，从CEL吸出上清液升颗粒。一个小球，加10微克的HR捐助作为对照样本。的其他粒料加10微克的人力资源供体，连同5微克每TALEN（PZT-AAVS1-L1和PZT-AAVS1-R 1）作为实验样品。 </li><li>重悬在100μl的P3的初级细胞的转染溶液各细胞沉淀，并转移到一个试管中。 </li><li>完成转染，并立即加入500μl室温E8培养基到每个试管中。 </li><li>转移的iPS细胞转染滴含步骤5.4准备DR4 MEF中一个10厘米的菜。 </li><li>可选：从每个实验添加数滴成基底膜基质包被的6孔板的一个孔中，如果流式细胞转染效率anaylsis是期望的。 </li><li>重复步骤6.15-6.17完成两个样本。次日，洗涤细胞用DPBS两次并切换培养基NutriStem，这似乎支持的iPSC培养上馈线优于E8。 </li><li> TRansient EGFP表达的峰在48到72小时后转染;根据需要评估转染效率。 </li></ol> 7.嘌呤霉素选择针对性的iPS细胞的<ol><li>开始嘌呤霉素选择转染后72小时，当iPS细胞达到70％汇合。 </li><li>对于NCRM5 iPS细胞，开始在0.5微克/毫升嘌呤霉素（1/2全剂量）稀释成NutriStem培养基嘌呤霉素选择。最优嘌呤浓度可能会发生变化，从0.25至1微克/ MLL一些iPSC系。 </li><li>培养iPS细胞在NutriStem + 0.5微克/毫升嘌呤霉素3天，每日刷新介质。 </li><li> 3天之后，增加嘌呤霉素的浓度为1微克/毫升。 </li><li>在1微克/毫升嘌呤选择培养iPSCs的另一个7至8天，或直到明显的菌落出现菌落采摘足够大。 </li></ol> 8.殖民地采摘和扩大目标iPS细胞的<ol><li>利用基底膜基质涂布一个96孔板通过分配50μl的基底膜基质涂层溶液（2毫克基底膜基质稀释到12毫升的DMEM / F12）到每个孔中。存储板在4℃下过夜后使用。 </li><li>使基底膜基质涂覆后的板来室温之后，吸出基底膜基质和分配100μl的E8补充有10μM的Y-27632到每个孔中。 </li><li>放置在倒置显微镜成生物安全柜，并用70％乙醇喷雾轻轻消毒。 </li><li>拉玻璃巴斯德吸管过本生灯，以获得理想的殖民地采摘工具，有一个微小的“钩子”的提示。消毒用70％乙醇，并放置在通风橱中干燥。 </li><li>删除包含从孵化器的目标IPSC的殖民地的菜，并放入显微镜下的引擎盖。 </li><li>通过跟踪菌落周围边框圈与菌落采摘工具挑选iPS细胞。</li><li>使用集落拾取工具的“钩”，以轻轻刮去并从板的表面去除的iPSC集落。对于较大的殖民地，与绘图殖民地采摘工具季度殖民地将有助于重新电镀后的细胞生长的X。 </li><li>一旦细胞团块被分离和自由浮动，使用P200移液器设置为30〜微升收集的iPS细胞。板单元直接放入96孔板中。 </li><li>可选：使用P20移液管设置为〜5微升收集的iPSC成的eppendorf管中，再加入50微升平缓细胞解离试剂，在37℃消化5分钟。消化后，加入500μlE8介质进入eppendorf管以稀释平缓细胞解离试剂和在标准的台式微量离心机降速细胞在200×g离心5分钟。然后吸大部分的培养基中，并离开〜30微升重新悬浮的细胞转移到96孔板中。 注：此做法将促进细胞团块，拿起殖民地的快速扩张的解离。 </li><li>继续菌落采摘直到的iPSC集落的足够数量的已被收集（大约建议每个实验20-30集落）。 </li><li>菌落采摘后，将96孔板在培养箱过夜。刷新次日完整的E8媒体，文化，直到〜70％汇合。 </li><li>传代细胞从96孔到24孔，然后进入6孔，按照步骤4.5-4.10描述。 注：EDTA溶液和E8介质的卷应该使用井比6孔板较小时，可以按比例减少。 </li><li>评估由结PCR和Southern印迹分析靶向成功确认针对性盒一体化随机插入 ​​载体16和不存在。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

对于成功的一代AAVS1安全港的最关键的步骤有针对性的人类iPSCs的是：（1）有效地提供TALEN和捐助质粒成iPS细胞转染; （2）优化的iPSC成单个细胞在转染后的转染和铺板密度之前的解离; （3）优化剂量和基于iPSC系的生长的药物选择的时间; （4）认真剖析，并有针对性的挑选殖民地，转移到新的板/孔。相比于Hockemeyer的纸张10用类似的方法，该协议中使用的一对开源AAVS1-TALENS的，不同的iPSC解离...

重新编程人类体细胞分化成胚胎干细胞样诱导多能干细胞（iPS细胞）的能力，首次发现了高桥等人，于2007年1。转导的逆转录病毒表达4转录因子人真皮成纤维细胞（将这样配成山因子的Oct3 / 4，SOX2，c-Myc的和Klf4）被证明是高度相似基于形态学，增殖，基因表达，和表观遗传状态的人类胚胎干细胞（胚胎干细胞）;关键的是，iPS细胞也能分化成所有三种胚层1的细胞。 iPS细胞的增殖潜力和分化的能力，使他们非常有吸引力的工具;通过重新编程从患有特定疾病的患者的细胞，iPS细胞可被用来作为体外疾病模型系统和作为潜在的治疗剂。 对于后者的目的，有几个问题必须的iPSCs的全部潜力之前得到解决在临床环境，可以实现; 在体外培养的人类胚胎干细胞和iPS细胞，重新编程和细胞维持期间使用异种衍生物的致瘤潜力，并且需要在体内跟踪移植细胞都是至关重要的障碍的临床应用的多能干细胞的（来自Hentze 等人 。 2）。一个理想的解决方案的需要跟踪分化的细胞移植后会涉及目视可检测的标记物抵抗沉默和花斑不管应用程序的。综合转基因稳健和持续的表达是最容易实现的，当外源DNA导入安全港位点;即，基因组位点，使综合向量的足够的转录，而在同一时间减轻表达扰动邻近基因3。一个这样的网站已经非常好特点，因为它的发现是腺相关病毒INTEGRATION站点1（AAVS1），在蛋白磷酸酶1调节亚基12C（PPP1R12C）基因的第一内含子。该位点已被证明不仅以允许持续和通过延长时间鲁棒表达的整合的转基因在培养和体外分化3，但也保护周围的基因从转录扰动4;两个特征被认为是由于内源染色质绝缘体元件侧翼AAVS1部位5的存在。 在刚刚过去的十年进展基因工程工具，极大地促进了易用性和效率与遗传操作的任何细胞类型可以实现的。虽然早期的成功实验，依靠内源性同源重组（HR）的极低水平与引进捐助来实现基因在胚胎干细胞6,7目标，采用特定地点核酸酶，如锌指核酸酶（ZFN），即显通过双链DNA断裂的产生ficantly诱导同源重组大大增加了这样的实验8,9的效率。这两个转录激活因子样效应植物病原黄单胞属（故事）和原核聚集定期相互间隔短回文重复（CRISPR）/ Cas9系统进入高效的站点特定的设计师核酸酶的再利用已经取得了基因打靶的多能干细胞的访问，可行的方法10-13。 最近的一篇论文描述了用于稳定整合绿色荧光报道基因盒的成在使用TALE核酸（TALENS）14人类iPSC的AAVS1安全港轨迹的有效方法。这些有针对性的iPS细胞保持其荧光即使在定向分化为心肌细胞，并移植到心肌梗死（MI）的小鼠模型，提供了强有力的证据，这样的效用稳定地荧光多能干细胞14。以获得目标菌落，基因陷阱方法用于其中一个拼接-受体（SA），2A自我裂解肽序列置于内源性PPP1R12C启动子的控制下的嘌呤霉素N-乙酰基转移酶（PAC）的基因;因此，只有那些掺入该DNA供体的AAVS1轨迹的iPSCs表达PAC，使它们可选择基于嘌呤霉素抗性; （ 图1，15）。该协议的细节产生AAVS1-GFP iPSCs的报道，在最近的一篇文章14，包括iPS细胞转染与TALENS的过程和9.8 kb的捐助为4.2kb DNA片段插入AAVS1安全港轨迹整合，选择iPS细胞的基础上，程序嘌呤霉素抗性，和用于克隆扩增拾取菌落。本文描述的技术可以应用到许多基因组工程实验。

<table border="0" cellpadding="0" cellspacing="0" width="1437">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:588px;">NAME OF MATERIAL/EQUIPMENT</td>
			<td style="width:123px;">COMPANY</td>
			<td style="width:123px;">CATALOG #</td>
			<td style="width:603px;">COMMENTS/DESCRIPTION</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10mL</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar 6 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Essential 8 Medium</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td>Store basal medium at 4&#176;C. Store supplement at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Store at room temp. Once dissolved in H2O, store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS, no calcium, no magnesium</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DR4 MEF 2M IRR - Academic</td>
			<td>GlobalStem</td>
			<td>GSC-6204G</td>
			<td>Store in liquid Nitrogen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life Technologies</td>
			<td>11995-040</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined Fetal Bovine Serum, US Origin</td>
			<td>HyClone</td>
			<td>SH30070.03</td>
			<td>Store at -20&#176;C. Thaw at 4&#176;C overnight and aliquot. Store aliquots at -20&#176;C until needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector Core unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001B</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector X unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001X</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P3 Primary Cell 4D-Nucleofector X Kit L (24 RCT)</td>
			<td>Lonza</td>
			<td>V4XP-3024</td>
			<td>Upon arrival, remove Primary Cell Solution and supplement and store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C and warm an aliquot in a 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NutriStem XF/FF Culture Medium</td>
			<td>Stemgent</td>
			<td>01-0005</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1)</td>
			<td>Addgene</td>
			<td>52637 and 52638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1-CAG-EGFP Homologous Recombination donor</td>
			<td>Addgene</td>
			<td>22212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin Dihydrochloride</td>
			<td>Life Technologies</td>
			<td>A11138-03</td>
			<td>Store at -20&#176;C. Prepare working aliquots of 1 mg/mL in ddH2O.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend XTR (Refrigerated), 120V 60Hz</td>
			<td>Thermo Scientific</td>
			<td>75-004-521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TX-750 4 &#215; 750mL Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
	</tbody>
</table>

transfection, selection, and colony-picking of human induced pluripotent stem cells talen-targeted with a gfp gene into the aavs1 safe harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

该协议的可视化图2提供的，用句其间iPS细胞在不同介质中无论是绿色E8或蓝色的NutriStem突出培养。它转染仅高质量iPSCs的是很重要的;检查培养皿整个例行维护和验证的iPSC培养包含主要鲜明菌落轴承鹅卵石样形态（ 图3A）;分化的细胞不应该占据的培养物的10％以上。 iPS细胞的Transfectability是由于它的小尺寸的评估和优化使用小PMAX-GFP载体（ 图4A-B）中 ，如转染PM...

Watch this Scientific Journal Video about Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor at JoVE.com

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

transfection-selection-colony-picking-human-induced-pluripotent-stem

Research

JoVE Journal

Developmental Biology

19.3K 次观看.  National Institutes of Health. TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

视频: Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

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

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

从培养儿童原发性鼻腔上皮细胞和重新编程为诱导多能干细胞

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

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

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

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

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

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

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 ...

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

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

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

人诱导多潜能干细胞衍生肝细胞的药物发现方法

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

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

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 ...