这项工作得到了全 Medizinische Forschung 在苏黎世大学的 Forschungskredit, 苏黎世大学, absciex NMSCh在研究金10.216 和 12.176, 瑞士国立心脏病学会, 瑞士国家科学依据授予 [320030-122273] 并且 [310030-143992], 第七框架节目, 生活阀门, 欧共体根据授予 [242008], 奥尔加 Mayenfisch 基础, EMDO 基础, 启动的津贴2012大学医院苏黎世和米切尔癌症研究所的内部资金。

该议定书遵循人类研究道德委员会的机构准则。病人的书面同意获得了使用羊水进行研究。
该议定书遵循南阿拉巴马大学机构动物保育和使用委员会的政策。
1. 原代羊膜间充质干细胞的分离培养
<ol><li>
羊膜液细胞的电镀
	<ol>
<li>在医生的羊膜穿刺过程中获得至少2.5 毫升的羊水。 注: 所有活细胞和组织的处理必须在无菌组织培养柜中进行, 必须使用适当的个人防护设备。熟悉基本的细胞培养和无菌技术是必需的。</li>
		<li>制备羊水和膜细胞 (AFMC) 培养基: EBM-2 基培养基, 15% 胎牛血清 (FBS), 20 ng/毫升的 bFGF, 25 ng/ml 的 EGF, 10 ng/毫升的 igf-i。对于原代羊水和羊膜干细胞的培养, 培养基应辅以抗生素-抗液。</li>
		<li>在0天, 混合2.5 毫升的羊水与3.5 毫升的 AFMC 培养基和板块成一个 T25 烧瓶。孵育在37° c 和 5% CO2为至少 48 h 不受干扰在检查之前殖民地的存在。</li>
		<li>在5天, 黏附细胞的殖民地应该存在。轻轻摇动烧瓶, 将未完全附着在底部和碎片上的细胞取出, 用巴斯德吸管抽吸所用的中/羊水混合液。替换为5毫升的新鲜 AFMC 培养基。</li>
		<li>文化再过5天。每隔一天更换介质, 如步骤1.1.4 中所述。</li>
	</ol>
</li>
	<li>
人羊膜原发间充质干细胞的分离
	<ol>
<li>出生后尽快获得胎盘, 最迟在24小时内, 以最大限度地提高细胞的完整性。切开羊膜的9厘米2段, 取出血块并在50毫升离心管中冲洗30毫升的 PBS, 辅以抗生素抗溶液。</li>
		<li>用一双手术刀在不育的10厘米的组织培养皿中切碎。细胞膜的消化和细胞的提取将通过组织离解系统来实现。遵循制造商的协议。 注意: 切碎后的组织切片越细, 消化后细胞数越多。</li>
		<li>用手术刀刀片将切碎的膜组织体转移到一个组织离解管中, 并与4.7 毫升的 RPMI 1640 培养基混合。混合离解酶 (请参见材料表)。</li>
		<li>将管子安装到组织 dissociator 上, 并运行程序 "h_tumor_01"。在37° c 的摇摆平台上孵育30分钟的管子。</li>
		<li>进一步稀释悬浮液与35毫升的 RPMI 1640 和适用于70µm 过滤器放置在50毫升收集离心管。</li>
		<li>离心机为5分钟在室温 200 x g, 丢弃上清, 重在5毫升的 RPMI 1640, 计数的细胞使用 hemacytometer, 和板在密度1万细胞/cm2到组织培养处理过的血管与新鲜的准备AFMC 培养基辅以抗生素-抗溶液。 注意: 在不完全消化的情况下, 小块组织将会存在, 单个细胞将会稀少。再次向下旋转, 将整个颗粒放入一个 T75 烧瓶中。</li>
	</ol>
</li>
	<li>
小学 AFSC 与 AMSC 文化
	<ol>
<li>AFSC/AMSC 的通道殖民地使用巴斯德吸管吸取的真空吸气培养基, 并在烧瓶中加入2毫升的细胞剥离酶。孵育在37° c 为5-8 分钟。</li>
		<li>水龙头上的烧瓶, 以帮助赶走细胞和混合的悬浮与等量的 AFMC 培养基 (抗生素-抗补充不应该是必要的, 从这一点上)。离心机在200克为4-5 分钟. 用玻璃巴斯德吸管或简单地倒置管子并将其排空到废物容器中去除上清。</li>
		<li>轻拂离心管的底部, 将小球分解成单个的悬浮液, 并将其与 AFMC 介质混合以进行电镀。板到 T 烧瓶在密度在2500和5000细胞/cm2之间。</li>
		<li>每隔一天更换一次培养基。不要把细胞系培养到超过6通道。为重新编程目的, 尽量使用低的段落。</li>
		<li>用冷冻培养基制备 AFSC 和 AMSC 的冷冻储存备份。收获文化使用细胞剥离酶, 离心在 200 g 为4° c 为 5 min。</li>
		<li>用巴斯德吸管移除上清液, 并轻拂管子底部以 singularize 颗粒中的细胞。重在 1×106/毫升和分的密度为 cryovials 的完全冻结介质中。在-80 ° c 过夜的冷冻容器中贮存。然后移动到液氮长期储存。</li>
	</ol>
</li>
</ol>2. 重新编程成多
<ol><li>获得重编程质粒
	<ol>
<li>通过一个非营利质粒存储库购买重编程粒。需要一个物质转让协议。</li>
		<li>将质粒转化为大肠杆菌合格的细胞, 并使用商业化的粒化提取试剂盒分离出粒。按照制造商的说明进行操作。</li>
		<li>用分光光度法测定质粒 DNA 的浓度。目的是高质粒浓度, 理想情况下约1µg/µL, 以避免稀释样品在转染。</li>
		<li>使用紫外分光光度计测量单个质粒的浓度, 并分它们。</li>
		<li>分别将3µg、3µg 和2µg EN2K、ET2K 和 M2L 质粒混合在一起。这是重编程质粒溶液。质粒溶液的数量足以染 1 x 106细胞。准备几个这样的等分。</li>
		<li>储存所有等分在-80 ° c。</li>
	</ol></li>
	<li>准备目标培养皿
	<ol>
<li>将一个 6-井板涂上受精-在每个井中加入1毫升的受精稀释缓冲器, 并在40µL VTN 库存溶液中混合 (1 µg/cm2)。在室温下 (RT) 或在37° c 为1小时的恒温箱中离开。</li>
		<li>真空吸出的解决方案使用巴斯德吸管和更换2毫升的 AFMC 介质在每个井。储存在37° c, 直到细胞被镀。 注: 重要: 此步骤中使用的 AFMC 介质不应含有任何抗生素或抗溶液。</li>
	</ol></li>
	<li>收获培养的初级 AFSC/AMSC 注: 扩大 AFSC/AMSC 在文化足以使冻结股票在一个低的通道数, 并致力于一个 T-75 瓶重新编程。由于10万细胞不足以进行实验, 目的是在50万细胞周围收获, 以弥补损失, 如果转染参数或不同培养条件的优化将被测试。<ol>
<li>在最早期的方便, 在低通道, 收获 AFSC/AMSC 使用细胞剥离酶混合描述的步骤1.3.1 到1.3.2。在单元格被离心后, 继续下一步。</li>
		<li>重1毫升的 PBS 和混合好, 以洗涤血清成分的颗粒。使用 hemacytometer 计数单元格。调整细胞密度 10万/毫升的 PBS 和分成1.5 毫升离心管。这只保证了细胞和用于转染的缓冲器之间的最小接触时间。</li>
		<li>把离心管放在5毫升聚苯乙烯管 (作为适配器, 将允许离心在一个规则摆动转子) 和离心机在 200 x g 4 分钟的室温。倒置管, 把上清液丢弃在废物容器内。不要使用固定角度的转子。</li>
		<li>在室温下以 200 x g 为3分钟执行额外的离心步骤。这将允许剩余的液体从管壁的底部收集。用200µL 吸管小心地吸干它</li>
	</ol></li>
	<li>重组质粒转染
	<ol>
<li>对于重新编程的实验, 一个转染系统 (见材料表) 将被用来提供重组质粒进入细胞。将转染提示、转染管、悬浮缓冲液和电解缓冲液放入组织培养柜中。试剂盒被保存在 RT, 直到他们被打开, 然后他们被存放在4° c。 注: 我们使用10µL 版本的套件。</li>
		<li>将转染装置关闭, 使其管站可以直接放置到机柜中。用3毫升的电解缓冲液填充一个转染管, 并通过将其一路推入槽内将管子装入空间站。</li>
		<li>在-80 ° c 的等分中, 采取步骤2.1.5 制备的重编程质粒溶液, 使其在培养基中的 RT 处解冻。</li>
		<li>在转染装置上, 选择以下转染参数: 950 V、40毫秒和1脉冲。</li>
		<li>重在10µL 悬浮缓冲中含有10万细胞的颗粒。从这一点上迅速工作, 因为悬浮缓冲是轻微的毒性和增加曝光时间导致明显较低的细胞活力。</li>
		<li>混合在1/10 的重编程质粒溶液 (解决方案分为总共 1 x 106细胞准备)。</li>
		<li>将转染针尖贴在转染吸管上。</li>
		<li>仔细吸取细胞悬浮进入转染尖端, 避免气泡形成。如果观察到气泡, 则将悬浮物排出并重复这一愿望。气泡会阻碍转染。</li>
		<li>将转染吸管插入转染管, 然后在转染装置的屏幕上按 "启动" 按钮。等待屏幕信息, 告知转染的成功, 并立即从管中取出吸管。</li>
		<li>将2.2 条所制备的目标6井板的悬浮液排出1井。在培养基中混入邻井, 并将悬浮液均匀地分配到两个井中 (由此产生的细胞密度为 5万/井)。</li>
		<li>重复转染的所有离心管包含 AFSC/AMSC 单独。将板放入37° c 和 5% CO2的孵化器中。</li>
	</ol></li>
	<li>转染 AFSC/AMSC 的文化
	<ol>
<li>培养转染细胞2-5 天。然后切换到重新编程培养基组成的 E8 补充100µM 的丁酸钠在3天。 注意: 可进行二次通道, 以避免源 AFSC/AMSC 过度生长。但是, 如果此参数有兴趣, 传代将禁用选项来正确计算重新编程效率。</li>
		<li>每天将重编程介质更改为每隔一天10天。每天从10天改变媒介。</li>
	</ol></li>
	<li>人工采摘完全 reprogramed 的克隆扩张菌落
	<ol>
<li>完全地被重新编程的殖民地出现在天14附近。允许殖民地扩大规模, 并成为紧凑。他们可以手动采摘和转移到新鲜的板块早在15-16 天。</li>
		<li>1 h 在采摘做法之前, 外套 24-井板材与8µL VTN 在300µL 受精稀释缓冲每井 (1 µg/cm2) 和孵育在 RT 或37° c。用无丁酸钠的 E8 培养基代替溶液。</li>
		<li>选择一个足够的大小的殖民地 (理想地超过400µm 直径) 在一个无菌文化内阁。可以使用相衬显微镜或显微镜。</li>
		<li>对于采摘, 将使用 LCD 成像显微镜放置在机柜, 因为它的显示器消除了头盔的需要。用70% 乙醇消毒显微镜阶段。</li>
		<li>使用常规的相衬细胞培养显微镜, 选择, 标记, 并记下要采摘的菌落数。这对于确保在实际领料过程中不浪费时间是很重要的。</li>
		<li>在 PBS 中填充数等于或大于30µL 0.5 毫米乙酸酸 (EDTA) 的菌落数量的 PCR 管。在电镀之前, 菌落将被放置在这些管子中进行部分离解。</li>
		<li>计划一次从板块中选取5个菌落, 使用10µL 吸管, 设置为2µL. 在菌落边缘的角度, 并小心地把吸管尖端, 并逐步刮掉整个殖民地的表面。立即将整个菌落吸进吸管尖端, 并将其转移到制备的一根 EDTA 的 PCR 管中。</li>
		<li>重复其余4殖民地。在 RT 孵育4-6 分钟。</li>
		<li>吸管用较大的吸管尖端轻轻地向上和向下, 将蜂群分解成更小的团簇。避免创建单个单元格挂起。</li>
		<li>将悬浮板直接放入一 2.6.2 24 井板的目标井中。重复与其余的殖民地。</li>
		<li>重复步骤2.6.6 通过 2.6.10, 如果超过5个殖民地是要采摘, 但不挑选超过5殖民地一次。孵育在37° c 和 5% CO2。</li>
	</ol></li>
	<li>iPSC 的克隆扩张与成熟
	<ol>
<li>允许殖民地成长并变得紧凑。3-6 天就足够了。改变文化媒介日报。使用400µL 和1毫升的 E8 培养基根据细胞密度。</li>
		<li>具有足够菌落密度的24孔板的井将扩展成6井板。1小时前传代, 外套6井板与 VTN (如在步骤 2.2.1)。然后用2毫升的 E8 介质在井中更换溶液。</li>
		<li>用1毫升吸管从源井中吸取所用培养基, 用300µL 0.5 毫米 EDTA 进行洗涤。立即用同样的吸管尖吸吸, 再用300µL 0.5 毫米 EDTA 代替, 然后在 RT 孵育5分钟, 用1毫升吸管抽吸所有液体。</li>
		<li>将1毫升吸管设置为容量, 在其上安装1毫升宽口径的尖端, 并将 E8 介质从目标井吸入到顶端。用介质的流将源 iPSC 文化洗掉。</li>
		<li>将悬浮液转移到目标井中, 并多次吸管, 将菌落分成20-50 细胞团簇。确保他们得到均匀分布在井中的温柔摇摆和摇动板和孵化在37° c 和 5% CO2。</li>
		<li>每天3至4天更换介质。任何分化的菌落都可以在相衬显微镜下标记, 并在培养柜中使用吸管尖端去除。这就允许有选择地传播高质量的纯 iPSC 文化。</li>
		<li>在传代前1小时, 用 VTN 在台阶2.2.1 上涂上一个6孔板的井。然后用2毫升的 E8 培养基代替溶液。</li>
		<li>常规传代是类似的初始传代做使用0.5 毫米 EDTA (步骤 2.7.2 2.7.5)。用1毫升 EDTA 代替6井板的一个井中的废培养基进行洗涤和丢弃。</li>
		<li>在 RT 中加入1毫升 EDTA, 孵育5-7 分钟, 部分游离。如果需要, 优化孵化时间, 确保悬浮约20至50细胞团簇产生。避免分解成单个细胞。</li>
		<li>丢弃 EDTA 溶液, 用1毫升吸管从靶井吸入1毫升的 E8 培养基, 注入宽口径的吸管尖端。</li>
		<li>用 E8 介质的流反复冲洗源 iPSC 文化, 直到它的所需部分从表面释放并转移到目标井。此部分表示拆分比率 (例如, 可以将1/8 的区域性转移为1:8 的比率。</li>
		<li>通过每3-4 天。允许 iPSC 线成熟, 培养他们至少15通道之前使用他们在下游实验</li>
	</ol></li>
</ol>3. 多的定性和确认
注: 有关流式细胞仪和共聚焦显微镜的详细信息, 请参阅补充文件。
<ol><li>
畸胎瘤形成试验
	<ol>
<li>通过畸胎瘤形成试验, 确定 iPSC 分化为所有三种细菌层的组织的能力, 遵循动物保育和使用的体制政策, 提前计划, 以便有时间提交适当的议定书文件.小鼠畸胎瘤的形成需要6-10 周。</li>
		<li>培养4口6孔板每 iPSC 线4天。向鼠标的一个侧面注入的单元格的近似数目是0.5 到 1 x 106单元格。可选: 一个额外的井可以致力于确定一个代表性的细胞数在井中通过单细胞分离酶和计数。</li>
		<li>计算 E8/BMM 混合物的体积 iPSC 团块将被悬浮在: 一只老鼠的两翼被注射, 每一个都有150µL 的丛悬浮物。三只老鼠足以测试一个 iPSC 行畸胎瘤形成。在所产生的体积内, 每针额外加150µL, 以弥补死体积损失。1针每只老鼠使用。因此总容量是 3 * (150 µL * 2 + 150 µL) = 1350 µL</li>
		<li>部分地离解 iPSC 殖民地与 EDTA, 好象他们将是传代, 如描述在步2.7.8 对2.7.10。重要的是, 这些菌落被分离成团簇, 而不是单独的细胞。</li>
		<li>使用宽口径的尖端, 用675µL 的 E8 培养基 (从步骤3.3.3 计算出的体积的一半) 冲洗 iPSC 菌落。将该丛悬浮液转化为5毫升的聚苯乙烯管。在冰上的地方。</li>
		<li>结合675µL 的钢。将产生的悬浮液保持在冰上直到注射。</li>
		<li>麻醉老鼠用异氟醚固定它们。这应由 vivarium 的熟练人员执行或指导。</li>
		<li>漩涡5毫升聚苯乙烯管短暂和吸气的丛悬浮 (450 µL 每只老鼠) 成一个装有22克针的胰岛素注射器。注入150µL 的细胞悬浮。此卷包含大约 1 x 106单元格 (请参见步骤 3.1.2)。</li>
		<li>每 iPSC 线注射3只老鼠。遵循适当的动物护理规程。</li>
		<li>每日监测小鼠的健康状况。</li>
		<li>当畸胎瘤到达终点直径1.5 至2厘米, 安乐的小鼠, 外植体畸胎瘤, 并储存在福尔马林溶液的组织固定24小时。</li>
		<li>将固定的畸胎瘤带到一个组织学核心设施苏木素曙红 (H & E) 染色。病理学家将对所有三种细菌层的组织的存在进行评分。 型的注意事项: 活 iPSC 文化应运到专门的细胞遗传学实验室, 以测试核型的完整性。建议每隔5通道进行一次测试。</li>
	</ol>
</li>
	<li>
转录分析
	<ol>
<li>培养2口6井板, 3-4 天, 获得一个 RNA 样本。只有使用高品质的文化, 小污染与区分细胞可以通过刮他们使用吸管提示来解决。</li>
		<li>根据制造商的协议, 使用商业可用的套件将 RNA 从 iPSC 的文化中分离出来。将样品运送到专门的基因组核心设施。</li>
		<li>使用微阵列获取 iPSC 线的全球转录剖面 (参见材料表支持的选择) 或 RNA 测序。</li>
		<li>通过 Coriell 研究所的在线界面提交 "idat" 生物评估多的文件。或者, 通过约翰·霍普金斯大学的在线界面, 提交 "*" 文件, 以生物识别细胞类型, 包括多能干细胞。有关单个生物分析接受的数据类型的详细信息, 请参见材料表。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Pluripotent+Stem+Cell+Lines+Derived+from+Human+Somatic+Cells.">Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells.</a> Science. 318 (5858), 1917-1920 (2007).</li><li>Trounson, A., DeWitt, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+progressing+to+the+clinic.">Pluripotent stem cells progressing to the clinic.</a> Nat. Rev. Mol. Cell Biol. 17 (3), 194-200 (2016).</li><li>Schmidt, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatally+fabricated+autologous+human+living+heart+valves+based+on+amniotic+fluid+derived+progenitor+cells+as+single+cell+source.">Prenatally fabricated autologous human living heart valves based on amniotic fluid derived progenitor cells as single cell source.</a> Circulation. 116 (11 Suppl), I64-I70 (2007).</li><li>Weber, B., Zeisberger, S. M., Hoerstrup, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatally+harvested+cells+for+cardiovascular+tissue+engineering:+Fabrication+of+autologous+implants+prior+to+birth.">Prenatally harvested cells for cardiovascular tissue engineering: Fabrication of autologous implants prior to birth.</a> Placenta. 32, S316-S319 (2011).</li><li>Weber, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatally+engineered+autologous+amniotic+fluid+stem+cell-based+heart+valves+in+the+fetal+circulation.">Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation.</a> Biomaterials. 33 (16), 4031-4043 (2012).</li><li>Kehl, D., Weber, B., Hoerstrup, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+living+cardiac+and+venous+valve+replacements:+current+status+and+future+prospects.">Bioengineered living cardiac and venous valve replacements: current status and future prospects.</a> Cardiovasc. Pathol. 25 (4), 300-305 (2016).</li><li>Slamecka, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-integrating+episomal+plasmid-based+reprogramming+of+human+amniotic+fluid+stem+cells+into+induced+pluripotent+stem+cells+in+chemically+defined+conditions.">Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.</a> Cell Cycle. 15 (2), 234-249 (2016).</li><li>Jiang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Transgene-Free+Amniotic-Fluid-Derived+Induced+Pluripotent+Stem+Cells+for+Autologous+Cell+Therapy.">Human Transgene-Free Amniotic-Fluid-Derived Induced Pluripotent Stem Cells for Autologous Cell Therapy.</a> Stem Cells Dev. 23 (21), 2613-2625 (2014).</li><li>Pipino, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trisomy+21+mid-trimester+amniotic+fluid+induced+pluripotent+stem+cells+maintain+genetic+signatures+during+reprogramming:+implications+for+disease+modeling+and+cryobanking.">Trisomy 21 mid-trimester amniotic fluid induced pluripotent stem cells maintain genetic signatures during reprogramming: implications for disease modeling and cryobanking.</a> Cell. Reprogram. 16 (5), 331-344 (2014).</li><li>Cai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+umbilical+cord+matrix+and+amniotic+membrane+mesenchymal+cells.">Generation of human induced pluripotent stem cells from umbilical cord matrix and amniotic membrane mesenchymal cells.</a> J Biol. Chem. 285 (15), 11227-11234 (2010).</li><li>Ge, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Amniotic+Mesenchymal+Stem+Cell-Derived+Induced+Pluripotent+Stem+Cells+May+Generate+a+Universal+Source+of+Cardiac+Cells.">Human Amniotic Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells May Generate a Universal Source of Cardiac Cells.</a> Stem Cells Dev. 21 (15), 2798-2808 (2012).</li><li>Drozd, A. M., Walczak, M. P., Piaskowski, S., Stoczynska-Fidelus, E., Rieske, P., Grzela, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+iPSCs+from+cells+of+fibroblastic+and+epithelial+origin+by+means+of+the+oriP/EBNA-1+episomal+reprogramming+system.">Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system.</a> Stem Cell Res. Ther. 6 (1), (2015).</li><li>Kang, N. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+antitumor+therapeutic+strategies+of+human+amniotic+membrane+and+amniotic+fluid-derived+stem+cells.">Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.</a> Cancer Gene Ther. 19 (8), 517-522 (2012).</li><li>Moschidou, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+Acid+Confers+Functional+Pluripotency+to+Human+Amniotic+Fluid+Stem+Cells+in+a+Transgene-free+Approach.">Valproic Acid Confers Functional Pluripotency to Human Amniotic Fluid Stem Cells in a Transgene-free Approach.</a> Mol. Ther. 20 (10), 1953-1967 (2012).</li><li>Kim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+memory+in+induced+pluripotent+stem+cells.">Epigenetic memory in induced pluripotent stem cells.</a> Nature. 467 (7313), 285-290 (2010).</li><li>Chen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nature Methods. 8 (5), 424-429 (2011).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Induced+Pluripotent+Stem+Cells+Free+of+Vector+and+Transgene+Sequences.">Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences.</a> Science. 324 (5928), 797-801 (2009).</li><li>Mart&#237;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pluripotent+stem+cells.">Characterization of pluripotent stem cells.</a> Nat. Protoc. 8 (2), 223-253 (2013).</li><li>Chan, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+distinguishes+bona+fide+human+iPS+cells+from+partially+reprogrammed+cells.">Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.</a> Nat. Biotechnol. 27 (11), 1033-1037 (2009).</li><li>Adewumi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+embryonic+stem+cell+lines+by+the+International+Stem+Cell+Initiative.">Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.</a> Nat. Biotechnol. 25 (7), 803-816 (2007).</li><li>M&#252;ller, F. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioinformatic+assay+for+pluripotency+in+human+cells.">A bioinformatic assay for pluripotency in human cells.</a> Nature Methods. 8 (4), 315-317 (2011).</li><li>Cahan, P., Li, H., Morris, S. A., Lummertz da Rocha, E., Daley, G. Q., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellNet:+Network+Biology+Applied+to+Stem+Cell+Engineering.">CellNet: Network Biology Applied to Stem Cell Engineering.</a> Cell. 158 (4), 903-915 (2014).</li><li>Schopperle, W. M., DeWolf, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TRA-1-60+and+TRA-1-81+Human+Pluripotent+Stem+Cell+Markers+Are+Expressed+on+Podocalyxin+in+Embryonal+Carcinoma.">The TRA-1-60 and TRA-1-81 Human Pluripotent Stem Cell Markers Are Expressed on Podocalyxin in Embryonal Carcinoma.</a> STEM CELLS. 25 (3), 723-730 (2007).</li><li>Ohnishi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+Termination+of+Reprogramming+In+Vivo+Leads+to+Cancer+Development+through+Altered+Epigenetic+Regulation.">Premature Termination of Reprogramming In Vivo Leads to Cancer Development through Altered Epigenetic Regulation.</a> Cell. 156 (4), 663-677 (2014).</li><li>Schlaeger, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+non-integrating+reprogramming+methods.">A comparison of non-integrating reprogramming methods.</a> Nature Biotechnology. 33 (1), 58-63 (2014).</li><li>M&#252;ller, F. -. J., Goldmann, J., L&#246;ser, P., Loring, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+to+standardize+teratoma+assays+used+to+define+human+pluripotent+cell+lines.">A call to standardize teratoma assays used to define human pluripotent cell lines.</a> Cell Stem Cell. 6 (5), 412-414 (2010).</li><li>Beers, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+and+colony+expansion+of+human+pluripotent+stem+cells+by+enzyme-free+dissociation+in+chemically+defined+culture+conditions.">Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.</a> Nat. Protoc. 7 (11), 2029-2040 (2012).</li></ol>

作者声明他们没有竞争的金融利益。

胚胎干细胞 iPSC 生成的初始阶段需要从胎儿组织中提取源细胞, 它们的培养、扩展和 episomal 重编程质粒的引入。在第一个完全重新编程的殖民地可以扩展之前, 这一阶段的文化周期大约14-18 天。最后阶段是 iPSC 无性系的成熟。羊膜干细胞的初步提取是通过羊膜的联合机械和酶消化的方法来实现的。我们发现, 潜伏期为30分钟, 产生的细胞数量最多, 提取的存活率最高。消化过程可以产生小块的组织和...

诱导多能干细胞技术 (iPSC) 带来了潜在的细胞置换疗法, 疾病和发育模型, 药物和毒理学筛选1,2,3。在概念上可以通过细胞注射、体外分化组织 (如心脏贴片) 或通过组织工程进行引导再生来实现替代疗法。羊水 (AFSC) 和膜干细胞 (AMSC) 是一种极好的细胞源, 这些干预措施要么直接4,5,6,7 ,要么作为重新编程的起始单元格填充进入多8,9,10,11,12。
早期的方法使用未定义的区域性系统或重新编程的方法, 需要构建9、10、11、12。最近的一项研究采用了无异的培养基, 即使使用了较不明确的基底膜附着基质 (钢), 从羊水上皮细胞中生成 iPSC。然而, 畸胎瘤的形成方法并没有包括在研究中, 以及大量的体外和分子数据。羊水上皮细胞与新生儿成纤维细胞的13相比, 具有大约8倍的高重编程效率。在另一项研究中, 羊水中的间充质干细胞也被发现被重新编程成 iPSC, 效率更高12。
多能干细胞可以分化为所有3种细菌层的组织代表, 因此具有最广泛的潜能。儿科患者可以受益于他们的自体羊水干细胞产前和羊膜干细胞产期的收获, 重新编程和组织工程。此外, 相对较低的胚胎干细胞分化水平 (低于成人干细胞14,15) 可以从理论上帮助解决从 iPSC16中的源细胞观察到的表观遗传偏见的保留。
在这里, 我们提出了一个协议, 以重新编程羊水和膜干细胞多在化学定义的无异 E8 培养基上的重组受精17 (VTN) 使用 episomal 质粒18。羊水和膜细胞作为细胞重新编程的主要优势在于它们的可用性和产期, 因此这种方法主要有利于儿科组织工程的研究。

<table><tbody><tr><td>Tumor Dissociation Kit, human</td><td>Miltenyi Biotec</td><td>130-095-929</td><td>tissue dissociation system, reagent kit, includes tissue dissociation tubes and tissue dissociation enzymes</td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>tissue dissociation system, dissociator</td></tr><tr><td>Thermo Scientific&amp;trade; Shandon&amp;trade; Disposable Scalpel No. 10, Sterile, Individually Wrapped, 5.75 (14.6cm)</td><td>Thermo-Fisher</td><td>3120032</td><td></td></tr><tr><td>70 &amp;micro;m cell strainers</td><td>Corning</td><td>10054-456</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Thermo-Fisher</td><td>32404014&amp;nbsp;</td><td></td></tr><tr><td>rocking platform</td><td>VWR</td><td>40000-300</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>Thermo-Fisher</td><td>339652</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Thermo-Fisher</td><td>339650</td><td></td></tr><tr><td>EBM-2 basal medium</td><td>Lonza</td><td>CC-3156</td><td>basal medium for AFMC medium</td></tr><tr><td>FGF 2 Human (expressed in E. coli, non-glycosylated)</td><td>Prospec Bio</td><td>CYT-218</td><td>bFGF, supplement for AFMC medium</td></tr><tr><td>EGF Human, Pichia</td><td>Prospec Bio</td><td>CYT-332&amp;nbsp;</td><td>EGF, supplement for AFMC medium</td></tr><tr><td>LR3 Insulin Like Growth Factor-1 Human Recombinant</td><td>Prospec Bio</td><td>CYT-022</td><td>IGF, supplement for AFMC medium</td></tr><tr><td>Fetal Bovine Serum, embryonic stem cell-qualified</td><td>Thermo-Fisher</td><td>10439024</td><td>FBS</td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Thermo-Fisher</td><td>15240062&amp;nbsp;</td><td>for primary AFSC/AMSC, for routine AFSC/AMSC it should not be necessary, do not use in medium for transfected cells!</td></tr><tr><td>Accutase cell detachment solution</td><td>StemCell Technologies</td><td>07920</td><td>cell detachment enzyme</td></tr><tr><td>CryoStor&amp;trade; CS10</td><td>StemCell Technologies</td><td>07930</td><td>complete freezing medium</td></tr><tr><td>PBS, pH 7.4</td><td>Thermo-Fisher Scientific</td><td>10010023&amp;nbsp;</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit (10)</td><td>Qiagen</td><td>12362</td><td>for plasmid isolation</td></tr><tr><td>pEP4 E02S EN2K</td><td>Addgene</td><td>20925</td><td>EN2K, reprogramming factors Oct4+Sox2, Nanog+Klf4</td></tr><tr><td>pEP4 E02S ET2K</td><td>Addgene</td><td>20927</td><td>ET2K, reprogramming factors Oct4+Sox2, SV40LT+Klf4</td></tr><tr><td>pCEP4-M2L</td><td>Addgene</td><td>20926</td><td>M2L, reprogramming factors c-Myc+LIN28</td></tr><tr><td>NanoDrop 2000c UV-Vis Spectrophotometer</td><td>Thermo-Fisher</td><td>ND-2000C</td><td>spectrophotometer</td></tr><tr><td>Neon&amp;reg; Transfection System</td><td>Thermo-Fisher</td><td>MPK5000</td><td>transfection system, components:&lt;br/&gt; Neon pipette - transfection pipette&lt;br/&gt; Neon device - transfection device</td></tr><tr><td>Neon&amp;reg; Transfection System 10 &amp;micro;L Kit</td><td>Thermo-Fisher</td><td>MPK1025</td><td>consumables kit for the Neon Transfection System, it contains:&lt;br/&gt; Neon tip - transfection tip&lt;br/&gt; Neon tube - transfection tube&lt;br/&gt; buffer R - resuspension buffer&lt;br/&gt; buffer E - electrolytic buffer</td></tr><tr><td>Stemolecule&amp;trade; Sodium Butyrate</td><td>StemGent</td><td>04-0005</td><td>small molecule enhancer of reprogramming</td></tr><tr><td>TeSR-E8</td><td>StemCell Technologies</td><td>05940</td><td>E8 medium</td></tr><tr><td>Vitronectin XF&amp;trade;</td><td>StemCell Technologies</td><td>07180</td><td>VTN, stock concentration 250 &amp;micro;g/ml, used for coating at 1 &amp;micro;g/cm&lt;sup&gt;2&lt;/sup&gt; in vitronectin dilution (CellAdhere) buffer</td></tr><tr><td>CellAdhere&amp;trade; Dilution Buffer</td><td>StemCell Technologies</td><td>07183</td><td>vitronectin dilution buffer</td></tr><tr><td>UltraPure&amp;trade; 0.5M EDTA, pH 8.0</td><td>Thermo-Fisher</td><td>15575020</td><td>dilute with PBS to 0.5 mM before use</td></tr><tr><td>EVOS&amp;reg; FL Imaging System</td><td>Thermo-Fisher Scientific</td><td>AMF4300</td><td>LCD imaging microscope system</td></tr><tr><td>CKX53 Inverted Microscope</td><td>Olympus</td><td></td><td>phase contrast cell culture microscope</td></tr><tr><td>Pierce&amp;trade; 16% Formaldehyde (w/v), Methanol-free</td><td>Thermo-Fisher</td><td>28908</td><td>dilute to 4% with PBS before use, diluted can be stored at 2-8 &amp;deg;C for 1 week</td></tr><tr><td>Perm Buffer III</td><td>BD Biosciences</td><td>558050</td><td>permeabilization buffer, chill to -20 &amp;deg;C before use</td></tr><tr><td>Mouse IgG1, &amp;kappa; Isotype Control, Alexa Fluor&amp;reg; 488</td><td>BD Biosciences</td><td>557782</td><td>isotype control for Oct3/4A, Nanog</td></tr><tr><td>Mouse IgG1, &amp;kappa; Isotype Control, Alexa Fluor&amp;reg; 647</td><td>BD Biosciences</td><td>557783</td><td>isotype control for Sox2</td></tr><tr><td>Mouse anti-human Oct3/4 (Human Isoform A), Alexa Fluor&amp;reg; 488</td><td>BD Biosciences</td><td>561628</td><td></td></tr><tr><td>Mouse anti-human Nanog, Alexa Fluor&amp;reg; 488</td><td>BD Biosciences</td><td>560791</td><td></td></tr><tr><td>Mouse anti-human Sox-2, Alexa Fluor&amp;reg; 647</td><td>BD Biosciences</td><td>562139</td><td></td></tr><tr><td>Mouse IgGM, &amp;kappa; Isotype Control, Alexa Fluor&amp;reg; 488</td><td>BD Biosciences</td><td>401617</td><td>isotype control for TRA-1-60</td></tr><tr><td>Mouse IgGM, &amp;kappa; Isotype Control, Alexa Fluor&amp;reg; 647</td><td>BD Biosciences</td><td>401618</td><td>isotype control for TRA-1-81</td></tr><tr><td>Mouse anti-human TRA-1-60, Alexa Fluor&amp;reg; 488</td><td>BD Biosciences</td><td>330613</td><td></td></tr><tr><td>Mouse anti-human TRA-1-81, Alexa Fluor&amp;reg; 647</td><td>BD Biosciences</td><td>330705</td><td></td></tr><tr><td>Mouse IgG1, &amp;kappa; Isotype Control, Alexa Fluor&amp;reg; 488</td><td>BD Biosciences</td><td>400129</td><td>isotype control for SSEA-1</td></tr><tr><td>Mouse IgG3, &amp;kappa; Isotype Control, Alexa Fluor&amp;reg; 647</td><td>BD Biosciences</td><td>401321</td><td>isotype control for SSEA-4</td></tr><tr><td>Mouse anti-human SSEA-1, Alexa Fluor&amp;reg; 488</td><td>BD Biosciences</td><td>323010</td><td></td></tr><tr><td>Mouse anti-human SSEA-4, Alexa Fluor&amp;reg; 647</td><td>BD Biosciences</td><td>330407</td><td></td></tr><tr><td>Affinipure F(ab&#039;)2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor&amp;reg; 488</td><td>Jackson Immunoresearch</td><td>115-606-068</td><td>use at a dilution of 1:600 or further optimize</td></tr><tr><td>Affinipure F(ab&#039;)2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor&amp;reg; 647</td><td>Jackson Immunoresearch</td><td>115-546-068</td><td>use at a dilution of 1:600 or further optimize</td></tr><tr><td>DAPI</td><td>Thermo-Fisher Scientific</td><td>D21490</td><td>stock solution 10 mM, further dilute to 1:12.000 for a working solution</td></tr><tr><td>Corning&amp;reg; Matrigel&amp;reg; Growth Factor Reduced, Phenol Red-Free</td><td>Corning</td><td>356231</td><td>basement membrane matrix (BMM)</td></tr><tr><td>scid-beige mice, female</td><td>Taconic</td><td>CBSCBG-F</td><td></td></tr><tr><td>RNeasy Plus Mini Kit (50)</td><td>Qiagen</td><td>74134</td><td>RNA isolation kit</td></tr><tr><td>T-25 flasks, tissue culture-treated</td><td>Thermo-Fisher</td><td>156367</td><td></td></tr><tr><td>T-75 flasks, tissue culture-treated</td><td>Thermo-Fisher</td><td>156499</td><td></td></tr><tr><td>Nunc&amp;trade; tissue-culture dish</td><td>Thermo-Fisher</td><td>12-567-650&amp;nbsp;</td><td>10 cm tissue culture dish</td></tr><tr><td>6-well plates, tissue-culture treated</td><td>Thermo-Fisher</td><td>140675</td><td></td></tr><tr><td>Neubauer counting chamber (hemacytometer)</td><td>VWR</td><td>15170-173</td><td></td></tr><tr><td>Mr. Frosty&amp;trade; Freezing Container</td><td>Thermo-Fisher</td><td>5100-0001&amp;nbsp;</td><td>freezing container</td></tr><tr><td>FACS tubes, Round Bottom Polystyrene Test Tube, 5ml</td><td>Corning</td><td>352058</td><td>5 ml polystyrene tubes</td></tr><tr><td>Eppendorf tubes, 1.5 ml</td><td>Thermo-Fisher</td><td>05-402-96</td><td>1.5 ml microcentrifuge tubes</td></tr><tr><td>PCR tubes, 200 &amp;micro;l</td><td>Thermo-Fisher</td><td>14-222-262</td><td></td></tr><tr><td>pipette tips, 100 to 1250 &amp;micro;l</td><td>Thermo-Fisher</td><td>02-707-407</td><td>narrow-bore 1 mL tips</td></tr><tr><td>pipette tips, 5 to 300 &amp;micro;l</td><td>Thermo-Fisher</td><td>02-707-410</td><td></td></tr><tr><td>pipette tips, 0.1 to 10 &amp;micro;l</td><td>Thermo-Fisher</td><td>02-707-437</td><td></td></tr><tr><td>wide-bore pipette tips, 1000 &amp;micro;l</td><td>VWR</td><td>89049-166</td><td>wide-bore 1 mL tips</td></tr><tr><td>glass Pasteur pipettes</td><td>Thermo-Fisher</td><td>13-678-20A</td><td></td></tr><tr><td>ethanol, 200 proof</td><td>Thermo-Fisher</td><td>04-355-451</td><td></td></tr><tr><td>vortex mixer</td><td>VWR</td><td>10153-842</td><td></td></tr><tr><td>chambered coverglass, 8-well, 1.5mm borosilicate glass</td><td>Thermo-Fisher</td><td>155409</td><td>glass-bottom confocal-grade cultureware</td></tr><tr><td>22G needles</td><td>VWR</td><td>82002-366</td><td></td></tr><tr><td>insulin syringes</td><td>Thermo-Fisher</td><td>22-253-260</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128-4L</td><td>fixation of explanted teratomas</td></tr><tr><td>Illumina HT-12 v4 Expression BeachChip</td><td>Illumina</td><td>BD-103-0204</td><td>expression microarray, supported by PluriTest, discontinued by manufacturer</td></tr><tr><td>PrimeView Human Genome U219 Array Plate</td><td>Thermo-Fisher</td><td>901605</td><td>expression microarray (formerly Affymetrix brand), soon to be supported by PluriTest</td></tr><tr><td>GeneChip&amp;trade; Human Genome U133 Plus 2.0 Array</td><td>Thermo-Fisher</td><td>902482</td><td>expression microarray (formerly Affymetrix brand), supported by CellNet, soon to be supported by PluriTest</td></tr><tr><td>PluriTest&amp;reg;</td><td>Coriell Institute</td><td></td><td>www.pluritest.org, free service for bioinformatic assessment of pluripotency, accepts microarray data - *.idat files from HT-12 v4 platform, soon to support U133, U219 microarray and RNA sequencing data</td></tr><tr><td>CellNet</td><td>Johns Hopkins University</td><td></td><td>cellnet.hms.harvard.edu, free service for bioinformatic identification of cell type, including plutipotent stem cells, based on U133 microarray data - *.cel files, soon to support RNA sequencing data</td></tr></tbody></table>

reprogramming primary amniotic fluid and membrane cells to pluripotency in xeno-free conditions

随着诱导多能干细胞的引入, 自体细胞疗法在现实中得到了更进一步的研究。胚胎干细胞, 如羊水和膜间充质干细胞, 代表了一种独特的组织工程中的未分化细胞, 并为未来的儿科干预和干细胞银行重组为 iPSC。这里介绍的协议描述了一个优化的过程, 以提取和培养初级羊水和膜间充质干细胞和产生 episomal 诱导多能干细胞从这些细胞在完全化学定义的文化利用人类重组受精和 E8 培养基的条件。本文还描述了采用严格的方法-流式细胞仪、共聚焦成像、畸胎瘤形成和转录分析-的新品系的特性。新生成的 Oct3/4A、Nanog、Sox2、TRA-1-60、TRA-1-81、SSEA-4 等胚胎干细胞的标记, 同时对 SSEA-1 标记呈阴性。干细胞系形成畸胎瘤在免疫缺陷-米色小鼠6-8 周和畸胎瘤包含组织代表的所有三种细菌层。通过将全球表达微阵列数据提交到生物多评估算法中的线转录分析认为所有的线多能性, 因此, 这种方法是一个诱人的替代动物试验。新的 iPSC 线可以很容易地用于下游实验, 涉及优化分化和组织工程。

在为遗传学测试目的采集羊水之前, 患者获得了知情的书面同意, 并为研究提供了一小分。由于胎盘代表医疗废物, 在研究中使用羊膜是不需要同意的。羊水和膜干细胞表现出典型的间质性, 其细胞形态呈纺锤形, 相亮。在重新编程时, 细胞经历 mesenchymal-to-epithelial (MET) 过渡, 并获得鹅卵石样的形态学和空间组织的殖民地, 表明上皮的性质。这一过程是启动早在 48-72 h 后引进重...

Watch this Scientific Journal Video about Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions at JoVE.com

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

该协议描述了在完全化学定义的条件下, 使用 non-integrating episomal 方法将原代羊水和膜间充质干细胞重新编程为诱导多能干细胞。详细介绍了通过严格的方法提取、培养、重新编程和表征所产生的诱导多能干细胞的过程。

无异条件下多原代羊水和膜细胞重新编程

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

reprogramming-primary-amniotic-fluid-membrane-cells-to-pluripotency

Research

JoVE Journal

Developmental Biology

9.2K 次观看.  University of South Alabama. 该协议描述了在完全化学定义的条件下, 使用 non-integrating episomal 方法将原代羊水和膜间充质干细胞重新编程为诱导多能干细胞。详细介绍了通过严格的方法提取、培养、重新编程和表征所产生的诱导多能干细胞的过程。

视频: Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

科研

发育生物学

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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 ...

Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening is purely diagnostic and does not elucidate disease mechanism. Although cells from aneuploid fetuses are valuable biological material bearing the chromosomal aneuploidy, these cells are short lived, limiting their use for downstream research experiments. Generation of induced pluripotent stem cell (iPSC) models is an effective method of cell preparation for perpetual conservation of aneuploid traits. They are self-renewing and differentiate into specialized cells reminiscent of embryonic development. Thus, iPSCs serve as excellent tools to study early developmental events. Turner syndrome (TS) is a rare condition associated with a completely or partially missing X chromosome. The syndrome is characterized by infertility, short stature, endocrine, metabolic, autoimmune and cardiovascular disorders and neurocognitive defects. The following protocol describes isolation and culturing of fibroblasts from TS (45XO) fetal tissue, generation of integration free TSiPSCs through delivery of episomal reprogramming plasmids by nucleofection followed by characterization. The reprogramming TSiPSCs were initially screened by live cell alkaline phosphatase staining followed by extensive probing for pluripotency biomarkers. Selected colonies were mechanically dissected, passaged several times and stable self-renewing cells were used for further experiments. The cells expressed pluripotency transcription factors OCT4, NANOG, SOX2, cell surface markers SSEA 4 and TRA1-81 typical of pluripotent stem cells. The original 45XO karyotype was retained post reprogramming. The TSiPSCs were able to form embryoid bodies and differentiate into cells of endoderm, mesoderm and ectoderm expressing lineage specific biomarkers ((SRY BOX17), (MYOSIN VENTRICULAR HEAVY CHAIN&#945;/&#946;), (&#946;III TUBULIN)). The exogenous episomal plasmids were lost spontaneously and not detected after passage 15 in cells. These TSiPSCs are a valuable cellular resource for modelling defective molecular and cellular neurodevelopment causing neurocognitive deficits associated with Turner syndrome.

Generation of Induced Pluripotent Stem Cells from Turner Syndrome 45XO Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

This protocol describes the generation of integration free iPSCs from fetal tissue fibroblasts through delivery of episomal plasmids by nucleofection followed by description of methods used for iPSC characterization and neuronal differentiation.

从特纳综合征（45XO）胎儿细胞生成诱导多能干细胞，用于与该综合征相关的神经功能障碍的下游建模

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工程

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

生物学

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

的iPS细胞从中间体可重编程小鼠模型的细胞表面标志物介导的净化

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

无异条件下多原代羊水和膜细胞重新编程

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的iPS细胞从中间体可重编程小鼠模型的细胞表面标志物介导的净化

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞