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1。馈线维护人类iPS细胞系和传<ol><li>准备基质胶涂层的菜肴。解冻一小时人类ESC合格的冰矩阵的一小瓶，在25毫升的DMEM-F12培养基中稀释。加入1 ml各35毫米的钢板（或等值金额，每面面积，如果使用其他的菜肴），冰和藏在心里，确保所有的板材及管材预冷。铝箔盖板。 </li></ol> 注:的基底膜股票浓度不同的批次。稀释指令在数据表上显示。 <ol start="2"><li>保持板置于冰上过夜，第二天从冰中删除。板可存放在冰箱里长达一个星期后方可使用。 </li><li>准备mTESR1完全培养基（或解冻一小瓶的Nutristem培养基），和H-ESM（人ESC培养基）根据下表。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td> ħESM </td><td> 终浓度 </td><td> FOR 500毫升 </td></tr><tr><td>淘汰赛血清替代（KSR） </td><td> 20％的</td><td>百毫升</td></tr><tr><td> 100X的GlutaMAX </td><td> 2毫米</td><td> 5毫升</td></tr><tr><td> MEM非必需氨基酸</td><td> 0.1毫米</td><td> 5毫升</td></tr><tr><td>青霉素，链霉素</td><td> 100 U / ml的 - 0.1毫克/毫升</td><td> 5毫升</td></tr><tr><td> 2 - 巯基乙醇</td><td> 0.1毫米</td><td> 900微升</td></tr><tr><td> B27补充剂 - 没有维他命A </td><td> 1％ </td><td> 10毫升</td></tr><tr><td> N2补充</td><td> 1％ </td><td> 5毫升</td></tr><tr><td>淘汰赛的DMEM </td><td> - </td><td> 370毫升</td></tr></tbody></table> 免介质可以被存储在4℃下不超过2周。要准备完全培养基，加入碱性FGF只是江前终浓度为20 ng / ml的ë键馈送。 </li></ol> 注意:空调，小鼠胚胎成纤维细胞的完整的H-ESM可以用来代替的mTESR1或Nutristem的iPS细胞。 <ol start="4"><li>将涂层板的培养箱中在37℃下为30分钟至2小时，传代前。 </li><li>传代细胞大约每5天，即使他们还没有达到汇合。殖民地不应接触。 </li><li>更换生长培养基中，用1毫升分散酶（1毫克/毫升，溶解10毫克/毫升在PBS中的库存）。 </li><li>拆下区分拉玻璃巴斯德移液器领域。应被删除的区域包括火山口状或囊状结构的菌落的中心，从菌落中分离的细胞已成为任何地方和扁平似乎已被向外迁移。 </li><li>在37°C下孵育文化罩，直到殖民地的边界变得更亮，并开始分离（通常5-7分钟左右）。抛弃它ð中性蛋白酶。用1毫升H-ESM清洗并丢弃。 </li><li>在1毫升H-ESM，使用单元格升降器（刮刀刮刀）到收获的殖民地，并收集在15毫升的Eppendorf管中。用1毫升H-ESM冲洗，并加至管。转速为1,000 RCF 4分钟，去除上清。 </li><li>悬浮颗粒在1毫升预热的生长培养基（无论是mTESR1或Nutristem）。避免分手团块太多 - 吸管向上和向下小于6-8X。确定镀细胞应该是多少，并删除不必要的细胞（通常为1:4或1:5的比例进行稀释，1:5）， 例如 ，2板，取出600微升，然后加入1.6毫升新鲜培养基。 </li><li>板删除基底膜。广场细胞一滴一滴，盘子上均匀分布。避免过度摇晃管，因为这会导致细胞走向中心。 </li><li>清洗管1毫升培养基和板之间的鸿沟。最终在每个盘量应该是1.5-2毫升。 </li><li>传代后的一天，除了每天更换介质。虽然这是理想介质日常生活，这可能会偶尔每两天一次的频率改变最后一段已经过去了，如果不超过2-3天，不影响多能性和分化潜能。 </li><li>如果单元格行必须冻结，再暂停，而不是在1-2毫升mFreSR的冷冻介质使用2毫升吸管1毫升/离心管。将小瓶在cryobox中在-80°C和转移到液氮3-4天。 </li></ol> 注:必须进行手动显微切割iPS细胞在体视显微镜下。 体外受精 （IVF）工作站（ 如试管婴儿工作站的来自KSystem），用立体显微镜集成，让细胞在无菌条件下操作，同时它们保持在加热表面积。如果不能做到这一点，可以移动在立体显微镜下定期层流罩。 2。胚体聚合和心脏电磁炉Ñ <ol><li>根据下表，准备EBM-20培养基。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td> EBM-20 </td><td> 终浓度 </td><td> FOR 500毫升 </td></tr><tr><td>胎牛血清（产地南美） </td><td> 20％的</td><td>百毫升</td></tr><tr><td> MEM非必需氨基酸</td><td> 0.1毫米</td><td> 5毫升</td></tr><tr><td>青霉素 - 链霉素</td><td> 100U/ml - 0.1毫克/毫升</td><td> 5毫升</td></tr><tr><td> 100X的GlutaMAX </td><td> 0.1毫米</td><td> 5毫升</td></tr><tr><td> 2 - 巯基乙醇</td><td> 50UM </td><td> 450微升</td></tr><tr><td> DMEM/F12 </td><td> - </td><td> 385毫升</td></tr></tbody></table> FBS原产南美洲的使用确定的分化效率是至关重要的，其他类型的血清可能影响就业的分化过程。为了制备完整的EBM-20，添加抗坏血酸（AA）至终浓度为50微克/毫升。完全生长培养基可以存储在4℃下不超过一个星期。 </li><li>收获的iPS菌落如上述（步骤1.6到1.9）。 </li><li>轻轻重悬在1毫升EBM-20与2毫升吸管。避免分手团块太多 - 吸管向上，上下不超过2〜3倍，体视显微镜下检查大小的菌落。超低附件板在1:1的比例（ 例如 35毫米的菜盘上，使用1 6孔板）。冲洗管，并添加1毫升EBM-20板。 </li></ol> 注:菌落的大小影响的分化过程，控制心脏感应的效率和时序。聚合胚大小均匀已经被证明可以减少分化过程中观察到的变异。 <ol start="4"><li>第3天，EBM-20一次改变，以避免重新使用显微镜胚MOVAL。保持吸液管靠近板边，并移除介质。 </li><li>在第7天，开关胚体板涂有0.1％明胶和以前在37℃下放置30分钟。如有必要，明胶可以取出细胞培养罩下板离开O / N。添加35-40 EBS每35毫米的菜。 </li><li>检查EBS每周两次击败区和变化EBM-20的。马克跳动区上的菜盖的顶部位于在体视显微镜下，以促进它们的本地化。各地区应出现后约10-20天。 </li><li>当出现自发性收缩的区域，切换到中EBM-2（准备，用含2％FBS的EBM-20代替）和隔离，如下文所述在第3条。 </li><li>继续检查其他地区殴打和改变介质的每周两次，直到跳动的领域都需要进一步的实验。 </li></ol> 注:效率的分化过程是高度依赖于若干因素，其中最严重的是特定的细胞系，并用于诱导心脏分化血清批次。为了获得最高的效率，测试细胞株血清心肌潜力和各批次。 3。打浆区的分离和培养<ol><li>外套12孔板（4 cm 2的区域），或用5微克/ cm 2的纤连蛋白的板，在PBS中稀释，以足够的音量完全覆盖整个表面（在12孔板中，每孔加入300μl总）。保持在细胞培养罩发现并晾干。板可以包装和储存在4°CO / N。 </li><li>删除跳动的区域用玻璃拉巴斯德移液管和手术刀。 1毫升吸管转移到纤​​维连接蛋白涂层钢板。 </li><li>加入1 ml EBM-2。 </li><li>十字板指示的地方除去细胞，并改变它的介质。板可保持长达1个月，其他地区可能开始玩b吃了以后。 </li></ol> 注 :如果需要更小的跳动团块，吸液管植面积和体视显微镜下数次，直到它打破成小块。这将导致在几个跳动细胞（电影3）集群。 <ol start="5"><li>每周两次，直到更改介质准备分析。 </li></ol>注意:从这个协议中获得的心肌细胞具有胎儿状的形态和功能特征，对一个成年人的样表型的成熟培养时间增加，进一步区分这些细胞可以通过以下方式获得。 4。单跳动细胞分离与分析<ol><li>外套2玻片（4 cm 2的区域）或其他适当的支持，用5微克/厘米2纤连蛋白在足够的PBS稀释，以覆盖整个培养表面。如果使用玻璃支持，大衣与层粘连蛋白和纤维连接蛋白（5微克/平方厘米&lt;SUP&gt;各2个）。 </li><li>删除板从第3跳动的区域用巴斯德吸管/手术刀。 </li><li>使用1毫升吸管，传递细胞含有500微升Ⅱ型胶原酶（480 U / ml的PBS与镁和钙）和孵化15分钟，在37℃，一次在孵化过程中晃动管15毫升管。 </li><li>移液器向上和向下与200μl的吸管。如果任何团块，转移到新鲜Ⅱ型胶原酶，并重复步骤4.3。 </li><li>重复步骤4.4直到没有留下大的团块。 </li><li>灭活胶原酶用3毫升EBM-2（AA）。管，然后可以留引擎盖下在加热机架，直到其他管准备。离心机在1000 RCF 4分钟。 </li><li>重悬浮颗粒在1毫升0.25％胰蛋白酶/ EDTA（从0.5％的股票在1X PBS稀释），在37°C孵育5分钟。合并馏分从相同的初始样品的消化。 </li><li>胰蛋白酶失活与4毫升EBM-2（AA）。通过在2.5毫升注射器没有更多的T细胞通过18G的针头汉7X。离心机在1000 RCF 4分钟。 </li><li>重悬细胞沉淀在1毫升EBM-2每口井和板。细胞可用于电镀后2-3天的功能和形态分析。 </li></ol>

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没有财务权益披露。

多能干细胞有潜力分化成CMS自发，虽然与线之间的低效率和高变异。因此，发展新型感应方法集中在提高效率的过程和走向更明确的协议。然而，大多数这些新的分化方法是相当复杂的，需要精心设计的培养条件下，定时的精细控制，试剂浓度，与昂贵的细胞因子和生长因子的治疗。此外，各种iPS细胞株中的内源性的细胞因子信号电平的差异使其难以标准化，这往往必须调整到适当的水平，每种?...

从历史上看，推动人类发展和疾病的遗传和分子机制的调查已基于上一代转基因动物模型。然而，众多的人类表型未能成功复制老鼠，主要是因为现有两个物种之间的生物差异。另一方面，获得人体组织可能会受到限制的，通常不允许足够的材料来获得深入的实验研究。心血管生物学领域遭受来自这两个限制:人体心脏的生理是显着的不同，鼠标和心脏组织是一个重大的数量只有验尸报告或在心脏手术期间访问。因此，找到一个最佳来源的分化功能的人类CMS成为心血管生物学中的中心话题，多已作出努力，来解决这个问题。已经提出了各种细胞类型，我ncluding骨骼肌成肌细胞，本地心脏干细胞，骨髓单核细胞，血管内皮前体细胞，间充质干细胞。然而，获得的数据，使用这些细胞并不一致4。 人类胚胎干细胞（ESC）的推导第5和Yamanaka和汤姆森6,7诱导多能干细胞（iPS）的开创性发现后似乎提供解决方案:这些细胞具有无限增长的能力和潜力给予上升到所有三个胚层的细胞衍生工具，包括CMS。 iPS细胞的使用提供了进一步的优点:来自自体成体细胞，它们携带的个人或患者衍生它们的相同的基因组。因此，他们允许在体外人类遗传性疾病的调查和机制的发展模式，推动发展的。凭借这一特性，iPS细胞也可以Øvercome问题相关的免疫排斥和伦理问题8。出于这些原因，尽管胚胎干细胞仍然是干细胞生物学领域的黄金标准，世界各地的研究人员现在正在向iPS细胞技术。 iPS细胞在体外人类疾病模型的各种条件，包括先天性心血管疾病9,10。近日，应用iPS细胞疾病模型已进行单基因心脏疾病（ 如长QT综合征，儿茶酚胺敏感性多形性室性心动过速）和病理学心脏缺陷是一个复杂的表型（ 即 Leopard和蒂莫西·综合征，扩张型心肌病）的一部分。这些报告证实， 在体内的疾病患者特异性的iPS细胞分化成CMS显示类似的表型和功能特性。 然而，有仍有许多挑战，提高疗效诱导的iPS细胞转化为心脏谱系。通过聚集形成从人类胚胎干细胞自发产生的CMS称为胚状体（EB）已被证明成功的17。由于这一发现，许多其他的方法已经被提出。许多研究小组已经大大提高了工作效率的分化协议，并已走向化学定义和动物产品的培养试剂（见马默里，C. 等，流通研究 （2012年）进行全面检讨现有的所有方法3 ）。 然而，传统的方法基于EB聚集的仍然是最常用的执行功能研究和调查疾病机制。我们建议的协议是基于iPS细胞的聚集成胚体培养在血清和抗坏血酸的存在下，这已被证明，以提高心脏的分化过程，与positively影响这些细胞的成熟18,19。在这篇文章中，我们将通过这种方法，在细节和将展示如何使用无饲养层的iPS细胞系产生特定病人的iPS衍生CMS。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Media and Reagents</td>
		</tr>
		<tr>
			<td>Human ESC-qualified Matrix</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Thaw the 5 ml bottle at 4 &deg;C on ice O/N, aliquot in pre-chilled cryo-tubes on ice, according to the data sheet dilution instructions and store at -20 &deg;C .</td>
		</tr>
		<tr>
			<td>Fibronectin, from human plasma, 0.1% solution</td>
			<td>Sigma</td>
			<td>F0896-2MG</td>
			<td>Working concentration: 5 &mu;g/cm2</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td>Working concentration: 5 &mu;g/cm2</td>
		</tr>
		<tr>
			<td>PBS (no Mg and Ca), 10X</td>
			<td>Lonza</td>
			<td>BE17-515F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS (with Mg and Ca), 10X</td>
			<td>Bio Sera</td>
			<td>XC-S2067/500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dispase II, neutral protease, grade II</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td>Stock: 10 mg/ml in DMEM-F12 at -20 &deg;C, working solution: 1 mg/ml in PBS (no Mg/Ca)</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA, 0.5%</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase type 2</td>
			<td>Worthington</td>
			<td>4176</td>
			<td>Dilute to 480 U/ml (e.g. 1.6 mg/ml) in PBS with Mg and Ca</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Life Technologies</td>
			<td>21331-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KSR)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Thaw at 4 &deg;C, aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (origin South America)</td>
			<td>Invitrogen</td>
			<td>10270-106</td>
			<td>Batches should be tested for their cardiogenic potential. Thaw at 4 &deg;C, heat-inactivate at 56 &deg;C for 30 min, aliquot and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>PenStrep, penicillin-Streptomycin, 100X</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GlutaMAX, 100X</td>
			<td>Life Technologies</td>
			<td>35050-038</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM NEAA, 100X</td>
			<td>Invitrogen</td>
			<td>11140-135</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 supplement, 100X</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27 supplement, 50X</td>
			<td>Life Technologies</td>
			<td>12587-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>31350-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gelatin, from porcine skin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td>Make stock at 1% in water, store at -20 &deg;C. Dilute to 0.1% in PBS and keep at 4 &deg;C</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td>Combine Supplement 5X with the basic medium, aliquot and store at -20 &deg;C. Do not keep complete medium at 4 &deg;C for more than 1 week</td>
		</tr>
		<tr>
			<td>Nutristem hESC XF</td>
			<td>Stemgent from Biological Industries</td>
			<td>05-100-1A</td>
			<td>Thaw at 4 &deg;C O/N, aliquot and store at -20 &deg;C until use. Aliquots may be kept at 4 &deg;C for no more than a week.</td>
		</tr>
		<tr>
			<td>mFreSR</td>
			<td>Stem Cell Technologies</td>
			<td>5853</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544-25G</td>
			<td>Prepare stocks at 10 mg/ml in water. Store aliquots at -20 &deg;C in the dark. Add to EBM at the final concentration (500X)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>35 mm culture dishes</td>
			<td>Becton Dickinson</td>
			<td>353001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Becton Dickinson</td>
			<td>353043</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permanox 2-well chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>177437</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass 2-well chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>177380</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6-well plates, ultra-low-attachment surface</td>
			<td>Corning Incorporated</td>
			<td>3471</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18G needle</td>
			<td>Becton Dickinson</td>
			<td>304622</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass pasteur pipettes, 230 mm</td>
			<td>VWR International</td>
			<td>612-1702</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2.5 ml syringe</td>
			<td>Becton Dickinson</td>
			<td>300188</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipments</td>
		</tr>
		<tr>
			<td>IVF Workstation</td>
			<td>KSystem, by Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nikon SMZ1500 Stereomicroscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

generation of human cardiomyocytes: a differentiation protocol from feeder-free human induced pluripotent stem cells

为了研究心脏发育的事件驱动和确定导致心肌人类疾病的分子机制，它是必不可少的，以产生人心肌功能的CM。使用这些细胞在药物发现和毒理学研究也将是非常有益的，允许人类来源的细胞要验证的预临床上用于治疗心脏疾病的新的药物分子。的CM的可能来源，诱导多能干细胞（iPS细胞）是最有前途的，因为它们可以容易获得的患者组织直接来源于具备的特性的主体1的所有类型的细胞产生的能力。已经提出了几种方法进入CMS分化的iPS细胞，包括从古典的胚状体（EBS）聚合方法已有化学定义的协议2,3。在这篇文章中，我们提出了一种基于EBS-协议，并展示如何METHOd可以高效地生成功能CM从馈线的iPS细胞样细胞。

在我们的研究中，我们从几个iPS细胞系产生的CMS。这些线MEF饲养层上，但最初生成，立即放在基底膜使用定义的介质（要么mTESR1或Nutristem）。各种实验，用于验证的形态特征，典型的多能干细胞的标记物表达（OCT-4，SSEA4 TRA1-60），其基因组中稳定（ 图1）的维护。 感应到心脏谱系成胚体和在一个特定类型的血清和AA（ 图2A和2B）的存在下?...

Watch this Scientific Journal Video about Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells at JoVE.com

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

多能干细胞，胚胎或诱导多能干细胞（iPS），构成人类分化的细胞，包括心肌细胞的重要来源。在这里，我们将专注于心脏iPS细胞的诱导，展示如何使用它们来获得功能的人类心肌细胞通过胚状体为基础的协议。

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 ...

generation-human-cardiomyocytes-differentiation-protocol-from-feeder

Research

JoVE Journal

Biology

21.2K 次观看.  Humanitas Clinical and Research Center, Italy. 多能干细胞，胚胎或诱导多能干细胞（iPS），构成人类分化的细胞，包括心肌细胞的重要来源。在这里，我们将专注于心脏iPS细胞的诱导，展示如何使用它们来获得功能的人类心肌细胞通过胚状体为基础的协议。

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

科研

生物学

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...