这项工作得到了 nih/内 (R01EY023962)、nih/发育研究院 (R01HD082098)、RPB 斯坦创新奖、马里兰干细胞研究基金 (2018-MSCRFV-4048、2014-MSCRFE-0742)、新德科学论坛奖和莫斯利基金会赠款的支持。

所有动物程序都是按照约翰霍普金斯医学院动物保育和使用委员会 (IACUC) 批准的动物护理准则和议定书进行的。
1. 为依赖于饲养者的常规 (人类胚胎干细胞中/mef) 或天真恢复的 (LIF-3i 中/mef) hPSC 培养小鼠胚胎成纤维细胞 (mef) 的制备
<ol><li>
购买或准备从 CF1 或 CF1 x DR4 混合 E13.5 小鼠胚胎的 MEF 馈线的内部低通道供应在发布的协议4之后。
	<ol>
<li>Cryopreserve 低通道 (p1-p2) MEF 区域性预 (长期存储) 或后 (长达6月) 辐照和储存在液态氮如前所述4。</li>
		<li>辐照 (5000 rad) 大体积扩展 MEF 下 p5 使用γ或 X 射线照射器和准备整除数在 1.5 X 106细胞每瓶为短期贮存 (少于6月) 在-80 °c 在一个超低温冷藏柜。</li>
		<li>板在 1.2 x 106 (新鲜地被辐照的不保存) 和 1.5 x 106 (被辐照的保存) 之下 MEF 每一个6井糊板材为准备饲养者文化, 如下所述。</li>
	</ol>
</li>
	<li>在生物安全柜中加入1.5 毫升的无菌0.1% 明胶溶液, 制备明胶涂层的6井无菌组织培养处理板。</li>
	<li>在实验室 CO2孵化器中, 在37摄氏度的至少1小时或一夜间孵化明胶涂层板材。</li>
	<li>在第二天, 解冻低通道 (例如, P2 到 P4) 亚砜-保存和辐照 (5000 rad) MEF 根据下面所示的步骤。 注意: 非辐照的 MEF 也应在使用前立即解冻、扩展和辐照。</li>
	<li>在37摄氏度的水浴中放置一个冷冻的 MEF 整除。解冻后, 用乙醇消毒管, 并立即稀释至少10倍的亚砜 cryoprotectant 与 MEF 介质 (表 1) 内的一个生物安全柜 (例如, 转移1毫升亚砜-mef 整除到9毫升 mef 培养基中的无菌15毫升圆锥)。</li>
	<li>离心稀释的 MEF 细胞在 200 x g 5 分钟在无菌15毫升圆锥管。</li>
	<li>在生物安全柜中, 吸入并丢弃无细胞的上清液, 并在并用重悬新鲜的 MEF 培养基中进行细胞颗粒的释放。</li>
	<li>如上文所示, 轻轻丢弃明胶溶液, 并在 mef 介质中加入2毫升的 mef, 再悬浮在糊6井板的每一个井中。将 mef 单元格和板块 1.2 x 106 (新鲜辐照的非冷冻保存) 或 1.5 x 106 (辐照保存) 的每一个6井糊板的 mef 计数。 注: 所有从 CO2孵化器转移到生物安全柜的细胞培养板可以用乙醇喷纸轻轻擦拭, 但不应直接喷洒70% 乙醇溶液, 以避免在油井中乙醇的传播。</li>
	<li>在实验室 co2孵化器 (5% co2, 湿润大气) 中孵化37摄氏度的 MEF 板, 以允许附着, 使用前。</li>
</ol>2. 传统 hPSC 文化在随后的幼稚逆转中的大体积稳定, 并简要 LIF-5i 适应步骤
<ol><li>在开始 LIF-5i/LIF-3i 逆转之前, 验证所有常规的 hPSC 线是否拥有正常的核型。 注: 应避免高通道常规 hPSC 线的 LIF-3i 逆转 (例如, P > 40–50), 因为这些文化可能已经产生了基因畸变, 可能对稳定、高效和体积 LIF-3i 逆转有负面影响,convetional hPSC 线。</li>
	<li>在基于 MEF 的文化系统 (如第1节中所述) 中, 维护和扩展传统的 hPSC 文化, 并验证正常的核型, 或无馈线养殖系统 (根据调查者的喜好)。 注意: 与馈线相关的 hPSC/MEF cocultures (例如,人类胚胎干细胞介质 (表 1) 辅以 4–10 ng/mL bFGF) 或无馈线 (e., E8 5或 mTSER 6介质 (根据制造商的说明及其与受精涂层板) 方法是兼容的批量天真的逆转使用 LIF-5i/LIF-3i/MEF 系统 (图 1)。非酶方法是首选的传代常规 hPSC 之前, 准备他们的逆转。LIF-5i 和 LIF-3i 的培养基配方不含抗生素或抗真菌剂。为了避免任何细菌或真菌污染, 应严格遵守生物安全柜的标准操作规程, 不育和维护。</li>
	<li>对于基于 MEF 的区域性系统, 请在 LIF-5i-adapted 常规 hPSC 文化传代之前至少一天内, 在糊6井板块中对 mef 馈线进行准备。</li>
	<li>在传统的 hPSC 文化已经达到了50% 融合 (即在最初电镀后的3–5天), 取代标准人类胚胎干细胞培养基与 LIF-5i 培养基 (2 毫升每井;表 1)。 注: 在安全柜中执行这些步骤。</li>
	<li>文化和维护常规 hPSC/MEF 在 LIF-5i2孵化器 (5% co2, 潮湿的大气) 2 天。改变 LIF-5i 媒介每日适应他们以后的段落和稳定的回归在 LIF-3i。</li>
	<li>另外, 对于无馈线传统文化 (例如, 在 E8), 文化 hPSC 在 LIF-5i 只有一夜之间传代第二天早晨。 注: LIF-5i 和 LIF-3i 的文化可以保持与大气 (21% o2) 或生理 (5% o2) 氧气水平在 co2孵化器 (5% CO2, 潮湿的大气)。</li>
	<li>在传代之前, 将培养皿放在生物安全柜中, 用 PBS 清洗 LIF-5i-adapted 常规 hPSC, 并在每个井中添加1毫升细胞离解试剂。孵化5分钟在37°c 在一个 CO2孵化器, 并轻轻地 triturate 与吸管进入单个细胞悬浮在生物安全柜。 注: 非酶解离解缓冲器可交替用于为传代制备单细胞。</li>
	<li>收集细胞悬浮在人类胚胎干细胞培养基 (至少2倍稀释) 在一个无菌15毫升圆锥管, 并轻轻地 triturate 细胞吹打获得一个单一的细胞悬浮。</li>
	<li>离心机在 300 x g 5 分钟, 吸/丢弃上清, 并并用重悬细胞颗粒在 LIF-5i 培养基在生物安全柜。使用 hemocytometer 或自动单元格计数器计数单元格。</li>
	<li>用 pbs 清洗预镀的 mef 板两次 (每井2毫升, 6 井板), 并将其 x 106单元 (在2毫升 LIF-5i 介质中) 分布到 pbs 清洗的 mef 板的1井中。</li>
	<li>调整和优化每个单独的 hPSC 线的初始电镀密度是幼稚的恢复, 因为 replating 效率可以是高度可变的。在 co2孵化器 (5% co2, 潮湿的大气) 安置板材。 注意: 不建议使用这种方法对大多数 hPSC 行采用抗凋亡试剂。LIF-5i 系统已经显著提高了常规 hPSC 线的初始批量克隆再电镀效率。然而, 一次性使用岩石抑制剂 (5–10微米 Y-27632) 可以进一步提高传统 hPSC 文化的初始 LIF-5i 克隆再镀效率的第一段, 一些不稳定的血统引 hPSC 线的倾向自发分化。</li>
	<li>如果从无馈线的文化开始 (例如, E8), 直接转移一井分离的常规 hPSC 适应 LIF-5i 入一个被辐照的 MEF 被镀的井 (即, 1:1 段落)。</li>
	<li>第二天, 轻轻地旋转盘子, 以解除所有非附着细胞, 吸入培养基 (PBS 洗涤是可选的), 并取代2毫升的 LIF-5i 培养基。每天在生物安全柜中执行此步骤3–5天, 或直到细胞60–70% 汇合 (图 1)。在 co2孵化器 (5% co2, 潮湿的大气) 安置板材。</li>
</ol>3. LIF-3i 介质中 n-hPSC 的长期维护和扩展
<ol><li>在最初的 LIF-5i 适应之后, 在生物安全柜中的每3–4天, 或者当文化成为60–70% 汇合时, 通过随后稳定的 LIF-3i 文化 (图 1)。 注: LIF-3i 文化需要严格的维护和允许 n-hPSC 培养达到高的融合/细胞密度从长时间的培养 (例如, > 4 天) 减少后续克隆再电镀效率, 并促进自发分化。</li>
	<li>在生物安全柜中, 通过轻轻添加2毫升的 PBS, 摒弃培养基, 洗净 LIF-5i/LIF-3i 文化的每一个井。丢弃 PBS 并添加1毫升的细胞脱离溶液。孵化5分钟在37°c 在 co2孵化器 (5% CO2, 潮湿的大气)。</li>
	<li>收集细胞悬浮液, 添加人类胚胎干细胞培养基 (不带抑制剂或生长因子 (表 1); 至少2倍稀释) 恢复所有 hPSC, 并通过吹打轻轻 triturate 细胞以获得单个细胞悬浮。将悬浮液转移至无菌15毫升圆锥管。</li>
	<li>离心机在300克5分钟, 吸/丢弃上清。在 LIF-3i 培养基中重新悬浮细胞颗粒。使用 hemocytometer 或自动单元格计数器计数单元格。</li>
	<li>板 ~ 2 x 105细胞每井在被辐照的 MEF 在糊6井板材为常规传代 LIF-3i 文化。为最初的 LIF-5i-adapted 文化, 板材最初更高的密度 (4 x 105细胞/井) 在第一个段落之前入 LIF-3i/MEF。</li>
	<li>将 LIF-5i-adapted hPSC 重新板和分发到新的 PBS 冲洗辐照的 MEF 进纸板上 (在上一天前准备) 在 LIF-3i 介质中。每日更换 LIF-3i 介质。</li>
	<li>使用 n-hPSC 在功能研究或超低温保存前, 在 LIF-3i 培养基中至少第4-7 连续散通道的 hPSC。记录常规或 LIF-3i 介质中 n-hPSC 的通道数。 注: 不推荐使用高通道的 LIF-3i 逆转 (例如> p40) 沿袭引道, 常规 hPSC 线。应努力使传统的 hPSC 线在尽可能低的通道中恢复可用。此外, 不建议使用 LIF-3i-reverted hPSC 超过 10 LIF-3i 通道的功能研究, 因为这种 n-hPSC 的文化可能会港口 karyotypically 异常克隆由于延长克隆细胞培养选择。如果不提供 LIF-3i < 10 通道的 N hPSC 库存, 则应进行功能性研究, 逆转低通道常规 hPSC 线的新鲜 LIF-5i/LIF-3i。</li>
</ol>4. LIF-3i-reverted hPSC 的超低温保存和解冻
<ol><li>在 LIF-3i 中, 如上文所示, 在使用功能研究或长期冷冻保存之前, 将 hPSC 至少5-7 个通道展开还原。记录在常规条件和 LIF-3i 条件下, 每一个冷冻瓶的通道数量。 注: 在功能化验中不使用过量 LIF-3i-reverted n-hPSC 可在每段中保存, 但低逆转后通道(例如 g.、< p3) 的冻结可能会导致不良或高度可变的解冻后恢复效率。</li>
	<li>在生物安全柜中, 吸入培养基, 在 PBS 中洗涤细胞 (每井2毫升), 用细胞脱离溶液 (每井1毫升) 将 pbs 和离解 hPSC 菌落分离成单个细胞。在 co2孵化器 (5% co2, 潮湿的大气) 安置板材为5分钟在37°c。稀释细胞脱离溶液与 LIF-3i 培养基 (2 倍), 收集 hPSC 在一个无菌15毫升圆锥, 离心细胞在 200 x g 5 分钟, 并用重悬细胞颗粒在 LIF-3i 培养基 (每良好当量)。使用 hemocytometer 或自动单元格计数器计算单元格数。</li>
	<li>离心 hPSC 在 LIF-3i 介质中 (200 x g为5分钟) 和并用重悬细胞在一个安全柜中的冷冻解决方案 (表 2), 密度至少 1 x 106细胞/毫升。</li>
	<li>将细胞转移到长期贮存的低温管中, 放入缓冻容器中。允许样品在-80 摄氏度冷藏库过夜。</li>
	<li>第二天, 将 cryovials 转移到液氮冷藏库中, 用于长期贮存。</li>
	<li>解冻时, 将冷冻小瓶放入37摄氏度水浴中2分钟. 消毒瓶 (即乙醇喷雾), 转移 hPSC 在一个无菌15毫升圆锥和慢慢稀释细胞10倍在人类胚胎干细胞培养基 (表 1) 补充与5微米的蛋白激酶 (岩石) 抑制剂 Y-27632 在无菌生物安全罩柜内。</li>
	<li>离心机在 200 x g 5 分钟。在生物安全柜中, 丢弃无细胞上清液和并用重悬 LIF-3i 培养基中的细胞颗粒 (), 辅以5微米岩石抑制剂 Y-27632。 注意: 排除 Y-27632 将导致解冻后恢复效率较差 (图 2)。</li>
	<li>将解冻后的细胞悬浮 LIF-3i/ROCK 抑制剂转移到 PBS 洗涤的 MEF 电镀井中。Cryopreserve LIF-3i 的文化密度为每瓶 1 x 106细胞。在糊6井板的一个井中, 将这些小瓶解冻到馈线中。</li>
	<li>第二天, 开始定期 LIF-3i 中等扩张, 没有蛋白激酶抑制剂。</li>
</ol>5. hPSC 样品收集用移料器
<ol><li>要准备来自 LIF-3i/MEF (或人类胚胎干细胞/MEF 常规) 的样本 (例如, 基因表达 (例如, 定量 rt-pcr, 微阵列) 或蛋白质 (例如,西方印迹) 分析, 消耗来自 MEF 馈线的 LIF-3i 文化, 使用磁性活化细胞分选 (mac) 与 anti-TRA-1-81 抗体涂层微珠根据制造商的协议。</li>
	<li>或者, 使用以下简单的预镀方法来耗尽馈线的文化, 如下所述。</li>
	<li>在无菌生物安全罩柜中, 丢弃无细胞上清液, 用2毫升无菌 PBS 清洗颗粒。用细胞脱离溶液分离粘附 LIF-3i/MEF 培养物 (1 毫升/井)。孵化5分钟在 co2孵化器 (5% CO2, 潮湿的大气)。收集 hPSC 在一个无菌15毫升圆锥。在人类胚胎干细胞培养基中洗涤2倍, 将细胞转化为无菌15毫升锥形, 离心机在 200 x g处进行5分钟。</li>
	<li>在生物安全柜中, 将 LIF-3i/MEF 离解细胞的每一个井重新悬浮成2毫升的 LIF-3i 介质, 并直接转移到一个新的井中的6井板已新鲜涂有0.1% 明胶。</li>
	<li>孵化 LIF-3i/MEF hPSC 细胞为1小时在37°c 在 co2孵化器 (5% CO2, 潮湿的大气)。在新的锥形管中用吸管收集非黏附细胞。轻轻地加入2毫升的人类胚胎干细胞培养基到每个井和漩涡收集剩余的非黏附的细胞。 注: 大部分被辐照的 MEF 将附着在1小时内的糊板上, 使得大部分 hPSC 悬浮。</li>
	<li>结合和离心细胞在 300 x g 5 分钟洗在 PBS。在离心后对液氮中的细胞颗粒进行快速冻结, 或者在与下游蛋白或核酸分析相兼容的株溶藻缓冲中重新悬浮颗粒 (图 2B)。</li>
	<li>用匹配的传统引物等基因系 hPSC 控制对 LIF-3i hPSC 线进行刻画。 注意: 由于引种文化之间和内部的内在差异, 相关的控制是在文化的匹配 timepoint 或在幼稚逆转之前准备的。详细的协议和材料为下游免疫荧光 bioimaging, 流式细胞术, 西方印迹, 基因表达 (rt-pcr 检测和微阵列), 甲基化研究 (斑点印迹, CpG 甲基化芯片), OCT4 近端和远端增强剂占主导地位的记者化验和信令抑制剂化验在别处提供3。</li>
</ol>6. 后幼稚逆转: 在功能测定之前使用 LIF-3i-reverted n-hPSC 的基因组完整性和保留父母印记的验证
<ol><li>在开始 LIF-5i/LIF-3i 逆转之前, 屏幕上的起始引物, 传统的 hPSC 文化为拥有正常核型 (例如, 以姬姆萨带染色分析使用发布的方法7)。 注: 这是为了消除常规的 hPSC 种群, 可能导致异常基因组改变, 这可能会驱动 artefactual 选择生存优势在克隆 LIF-3i 条件。</li>
	<li>为了获得最佳结果, 在使用功能研究或定向分化前几个星期, 新鲜地将传统的 hPSC 文化恢复到一种天真的状态 LIF-3i。 注意: 不建议在 LIF-3i 条件下的常规长时间 "维护" 文化, 10 段以上的短文。常规的扩展和维护人类胚胎干细胞和 hiPSC 线应使用传统的文化系统 (例如,在 E8, 或 MEF/人类胚胎干细胞培养基与 bFGF)。</li>
	<li>评估后恢复的 n-hPSC 线保留正常核型5–7通道后 LIF-3i 逆转 (e., 与姬姆萨波段染色分析7, 或任何其他选择方法)。</li>
	<li>评估所有恢复的 n-hPSC 线, 以保留正常的父母基因组印记通过 DNA 甲基化分析的选择 (例如, 中央 DNA 芯片的协议 LIF-3i-reverted n-hPSC 的父母印记的分析在别处提供3) 后5–10通道 LIF-3i 逆转。</li>
</ol>7. 后幼稚逆转: 用 LIF-3i-reverted n-hPSC 定量定向分化试验设计指南
<ol><li>直接利用 LIF-3i n-hPSC 建立定向分化协议, 无需额外的细胞培养操作。 注: 重新启动 (即,将 n-hPSC 回到传统的引物条件之前, 他们使用的定向分化化验) 是不必要的 LIF-3i 方法, 不建议。</li>
	<li>为了控制单个 hPSC 线功能测试中的检测和联运变异性的影响, 通过使用至少三 hPSC 的独立分化协议, 交叉验证谱系特异分化效用来自独立遗传背景的线 (即多捐助者衍生的 hiPSC 和人类胚胎干细胞)。</li>
	<li>对于功能比较, 建立同级文化, 在相同的段落编号, 并从相同 (等基因系) hPSC 线平行于传统的血统引种和 LIF-3i-reverted hPSC 文化。在各自的媒体中保持等基因系 hPSC 文化的引导/天真的兄弟姐妹 (e., E8 vs。LIF-3i), 并同时区分使用相同的区分协议和材料, 以消除任何实验性偏差 (图 4)。</li>
	<li>为等基因系引物对幼稚 hPSC 比较, 调整和优化初始电镀密度的每一个单独的鉴别试验。 注: LIF-3i-reverted hPSC 的神经祖、确定性内胚层和换血术内皮定向分化的详细协议在别处提供3。LIF-3i-reverted n-hPSC 在定向分化检测中比常规 hPSC 具有更强的增殖和分化能力。hPSC 通常需要较低的初始电镀浓度比传统的 hPSC, 而不像他们传统的引 hPSC 同行, 不需要使用抗凋亡试剂, 以提高其克隆生存后, 酶分化测定中的消化。</li>
</ol>

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根据生命技术与约翰霍普金斯大学 (JHU) 之间的许可协议, Zambidis 博士有权获得大学颁发的关于干细胞许可的版税的份额。这一安排的条款由 JHU 按照其利益冲突政策进行管理。这并不会改变作者对共享数据和材料的期刊政策的遵守。

LIF-3i 系统将经典小鼠2i 天真逆转鸡尾酒1的改良版应用于人类多潜能干细胞。hPSC 的自我更新 (不能单独扩展 2i) 在 LIF-2i 通过补充这鸡尾酒与 tankyrase 抑制剂 XAV939 稳定。LIF-3i 文化允许大量和有效地逆转传统 hPSC 到一个多能状态类似于人类植入前啮鼠动物3。虽然 XAV939 在 hPSC 中的作用机制很可能是复杂的, 并且与2i 协同, 但它们可能至少?...

2i (甲酮/ERK 和 GSK3β抑制剂) 培养系统最初开发, 以改善异种血清基小鼠胚胎干细胞 (卓制) 的文化, 以统一的地面状态, 干细胞类似于老鼠植入前啮鼠动物1。然而, 2i 不支持人类多潜能干细胞 (hPSC)2线的稳定维护。最近据报道, 各种复杂的小分子、生长因子补充和转基因方法捕获认定类似人类天真的多能分子状态2。然而, 许多用这些方法创建的 "天真的" 状态也表现出核型不稳定, epigenomic 缺陷 (例如,全球父母基因印迹的丧失), 或分化潜能受损。
相比之下, 对 GSK3β、ERK 和 tankyrase 信号和白血病抑制因子 (LIF-3i) 的三重化学抑制的鸡尾酒, 足以满足常规 hPSC 线3的一种稳定幼稚的逆转。LIF-3i-reverted 天真的 hPSC (hPSC) 维持正常的核型, 并增加其表达的幼稚特定的人类植入前啮鼠动物基因 (如北美, KLF2, NR5A2, DNMT3L, HERVH, 斯特拉 (DPPA3), KLF17, TFCP2L1)。LIF-3i 逆转也授予 hPSC 与卓制相似的天真干细胞独特的分子和生物化学特征, 包括增加磷酸化 STAT3 信号, 减少 ERK 磷酸化, 全球 5-甲基胞嘧啶 CpG低甲基化, 全基因组的 CpG 去甲基化在胚胎干细胞 (ESC) 特定的基因启动器, 和显性远端 OCT4 增强剂的使用。此外, 与其他幼稚的逆转方法, 导致 aberrantly hypomethylated 印迹基因组的基因座, LIF-3i-reverted n-hPSC 没有系统性丧失印迹的 CpG 模式或丧失 DNA 甲基表达 (例如,DNMT1, DNMT3A, DNMT3B)3。
一种直接的 LIF-3i 培养, 广泛的常规人类胚胎干细胞 (人类胚胎干细胞) 和人类诱导的多能干细胞 (hiPSC) 生长在喂食器或 E8 无饲养条件获得迅速和大量逆转到一个天真的啮鼠动物样的状态。然而, 直接 LIF-3i 幼稚逆转可能是低效的一些不稳定的常规 hPSC 线由于固有的基因组和沿袭引多变性产生的基因多样性的捐助者的背景。
因此, 为了拓宽 LIF-3i 方法的效用, 本文提出了一种渐进优化, 它允许在喂食器上培养的几乎任何常规人类胚胎干细胞或无转基因 hiPSC 线的普遍幼稚逆转。这种普及天真的逆转方法在常规 hPSC 中采用了一个瞬态初始培养步骤, 补充了 LIF-3i 鸡尾酒与另外两个小分子 (LIF-5i), 事实蛋白激酶 a (佛司可林) 和声波刺猬 (嘘) (purmorphamine) 信号。在 LIF-5i 中, 常规 hPSC 的一个初始通道将它们适应随后的稳定 LIF-3i 批量回归。最初的 LIF-5i 适应显著增加了在 E8 或喂食器上生长的常规 hPSC 的初始单细胞克隆增殖 (在其随后稳定的、连续的 LIF-3i 单独的通道之前)。传统的 hPSC 线适应了第一个通道在 LIF-5i 容忍随后批量克隆传代的天真恢复细胞在 LIF-3i 条件, 这省却需要采摘和亚克隆的稀有稳定的殖民地, 或常规使用抗凋亡分子或相关蛋白激酶 (岩石) 抑制剂。
LIF-3i 方法已成功地用于稳定地扩展和维护 > 30 独立, 基因多样性的常规 hPSC 线 > 10–30通道使用非酶或酶解离方法, 并没有诱导染色体或 epigenomic 异常的证据, 包括印迹基因位点的异常。此外, 序列 LIF-5i/LIF-3i 文化是唯一的天真的逆转方法, 迄今已报告, 改善功能干细胞的常规 hPSC 线, 通过减少其谱系引种基因表达和大大提高了他们的多向分化效力。LIF-3i 天真回归方法消除了谱系引种、常规 hPSC 线分化的内在联运变异性, 将在再生医学和细胞治疗中有很大的应用前景。

<table><tbody><tr><td>anti- SSEA-1/CD15 antibody, APC conjugated</td><td>BD Biosciences</td><td>561716</td><td>use 5&amp;micro;L per assay (FACS)</td></tr><tr><td>anti- TFCP2L1 antibody</td><td>Sigma Aldrich</td><td>HPA029708</td><td>use at a 1:100 dilution (immunostainings)</td></tr><tr><td>anti-beta-Actin antibody</td><td>Abcam</td><td>ab6276</td><td>use at 1:5000 (Western blot)</td></tr><tr><td>anti-CD146 antibody,&amp;nbsp; PE conjugated&amp;nbsp;</td><td>BD Biosciences</td><td>550315</td><td>use 5&amp;micro;L per assay (FACS)</td></tr><tr><td>anti-CD31 antibody, APC conjugated</td><td>eBioscience</td><td>17-0319-42</td><td>use 2&amp;micro;L per assay (FACS)</td></tr><tr><td>anti-CD31 microbead kit</td><td>Miltenyi Biotec</td><td>130-091-935</td><td></td></tr><tr><td>anti-NANOG antibody</td><td>Abcam</td><td>ab109250</td><td>use at a 1:100 dilution (immunostainings)</td></tr><tr><td>anti-NR5A2 antibody</td><td>Sigma Aldrich</td><td>HPA005455</td><td>use at a 1:100 dilution (immunostainings)</td></tr><tr><td>anti-p44/42 MAPK (Erk1/2) antibody</td><td>Cell Signaling</td><td>4695</td><td>use at 1:1000 (Western blot), for detection of total protein</td></tr><tr><td>anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204 antibody</td><td>Cell Signaling</td><td>4370</td><td>use at 1:1000 (Western blot)</td></tr><tr><td>anti-phospho-STAT3 (Tyr705) antibody</td><td>Cell Signaling</td><td>9145</td><td>use at 1:1000 (Western blot)</td></tr><tr><td>anti-rabbit immunoglobulin antibody, biotinylated</td><td>Agilent</td><td>E0432</td><td>use at a 1:500-1:1000 dilution (immunostainings)</td></tr><tr><td>anti-SSEA-4 antibody, APC conjugated&amp;nbsp;</td><td>R&amp;amp;D System</td><td>FAB1435A</td><td>use 5&amp;micro;L per assay (FACS)</td></tr><tr><td>anti-SSEA-4 GloLIVE antibody, NL493 conjugated</td><td>R&amp;amp;D System</td><td>NLLC1435G</td><td>use at 1:50 dilution (live and fixed immunostainings)</td></tr><tr><td>anti-STAT3 antibody</td><td>Cell Signaling</td><td>9139</td><td>use at 1:1000 (Western blot), for detection of total protein</td></tr><tr><td>anti-STELLA/DPPA3 antibody</td><td>Millipore</td><td>MAB4388</td><td>use at a 1:50 dilution (immunostainings)</td></tr><tr><td>anti-TRA-1-60 GloLIVE antibody, NL557 conjugated&amp;nbsp;</td><td>R&amp;amp;D System</td><td>NLLC4770R</td><td>use at&amp;nbsp; a 1:50 dilution (live and fixed immunostainings)</td></tr><tr><td>anti-TRA-1-60 StainAlive Antibody, DyLight 488 conjugated</td><td>Stemgent</td><td>09-0068</td><td>use at a 1:100 dilution (live and fixed immunostainings)</td></tr><tr><td>anti-TRA-1-81 StainAlive Antibody, DyLight 488 conjugated</td><td>Stemgent</td><td>09-0069</td><td>use at a 1:100 dilution (live and fixed immunostainings)</td></tr><tr><td>anti-TRA1-60 antibody, PE conjugated</td><td>BD Biosciences</td><td>560193</td><td>use 10&amp;micro;L per assay (FACS)</td></tr><tr><td>anti-TRA1-81 antibody, PE conjugated</td><td>BD Biosciences</td><td>560161</td><td>use 10&amp;micro;L per assay (FACS)</td></tr><tr><td>APEL2-Li</td><td>StemCell Technologies</td><td>5271</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A3311</td><td></td></tr><tr><td>CellAdhere dilution buffer</td><td>StemCell Technologies</td><td>7183</td><td>dilutent for Vitronectin XF&amp;trade; matrix</td></tr><tr><td>CF1 mouse</td><td>Charles river</td><td>023</td><td></td></tr><tr><td>CHIR99021</td><td>R&amp;amp;D System</td><td>L5283</td><td>reconstitute at 100mM in DMSO</td></tr><tr><td>confocal microscope system</td><td>Zeiss</td><td>LSM 510</td><td></td></tr><tr><td>cord blood CD34+ derived iPSC line</td><td>Thermo Fisher Scientific</td><td>A18945</td><td>also referred as 6.2 line</td></tr><tr><td>Corning Costar tissue culture-treated 6-well plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Countess&amp;nbsp; cell counting chamber slide</td><td>Thermo Fisher Scientific</td><td>C10228</td><td></td></tr><tr><td>Countess automated cell counter</td><td>Thermo Fisher Scientific</td><td>AMQAX1000</td><td></td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11995-065</td><td></td></tr><tr><td>DMEM-F12</td><td>Thermo Fisher Scientific</td><td>11330-032</td><td></td></tr><tr><td>DMSO (dimethyl sulfoxide)</td><td>Sigma Aldrich</td><td>D2650</td><td></td></tr><tr><td>DR4 mouse</td><td>The Jackson Laboratory</td><td>3208</td><td></td></tr><tr><td>Essential 8 (E8) medium</td><td>StemCell Technologies</td><td>5940</td><td></td></tr><tr><td>Fetal bovin serum (FBS)</td><td>Thermo Fisher Scientific</td><td>SH30071.03</td><td></td></tr><tr><td>Forskolin</td><td>Stemgent</td><td>04-0025</td><td>reconstitute at 100mM in DMSO</td></tr><tr><td>Gelatin (porcine)</td><td>Sigma Aldrich</td><td>G1890-100G</td><td>resuspend in water and sterilize with an autoclave</td></tr><tr><td>KnockOut Serum Replacement</td><td>Thermo Fisher Scientific</td><td>10828-028</td><td></td></tr><tr><td>L-Glutamine (100X)</td><td>Thermo Fisher Scientific</td><td>25030-081</td><td></td></tr><tr><td>MEM Non-essential amino acid (MEM NEAA) (100X)</td><td>Thermo Fisher Scientific</td><td>11140-050</td><td></td></tr><tr><td>mTeSR1 medium</td><td>StemCell Technologies</td><td>85850</td><td></td></tr><tr><td>Nalgene cryogenic vials</td><td>Thermo Fisher Scientific</td><td>5000-0020</td><td></td></tr><tr><td>Nunc Lab-Tek II Chamber Slide System</td><td>Fisher Scientific</td><td>154534</td><td></td></tr><tr><td>Paraformaldehyde (PFA) solution , 4% in PBS</td><td>USB Corporation&amp;nbsp;</td><td>19943</td><td></td></tr><tr><td>PD0325901</td><td>Sigma Aldrich</td><td>PZ0162</td><td>reconstitute at 100mM in DMSO</td></tr><tr><td>Penicillin/streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Biological Industries</td><td>02-023-1A</td><td></td></tr><tr><td>Purmorphamine</td><td>Stemgent</td><td>04-0009</td><td>reconstitute at 10mM in DMSO</td></tr><tr><td>recombinant human Activin A</td><td>Peprotech</td><td>AF-120-14E</td><td></td></tr><tr><td>recombinant human Bone morphogenetic protein (BMP)-4</td><td>Peprotech</td><td>120-05ET</td><td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td></tr><tr><td>recombinant human FGF-basic (bFGF)</td><td>Peprotech</td><td>100-18B</td><td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td></tr><tr><td>recombinant human LIF</td><td>Peprotech</td><td>300-05</td><td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td></tr><tr><td>SB431542&amp;nbsp;</td><td>Stemgent</td><td>04-0010-05</td><td>reconstitute at 100mM in DMSO</td></tr><tr><td>Stemolecule Y27632 in Solution</td><td>Stemgent</td><td>04-0012-02</td><td>ROCK inhibitor in solution (10mM)</td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>Thermo Fisher Scientific</td><td>A11105-01</td><td></td></tr><tr><td>Streptavidin-Cy3 conjugate</td><td>Sigma Aldrich</td><td>S6402</td><td>use at 1:500-1:1000 dilution (immunostainings)</td></tr><tr><td>Thermo Scientific Mr. Frosty Freezing Container</td><td>Thermo Fisher Scientific</td><td>5100-0001</td><td></td></tr><tr><td>Vascular endothelial growth factor (VEGF)-165</td><td>Peprotech</td><td>100-21</td><td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td></tr><tr><td>Vitronectin XF matrix</td><td>StemCell Technologies</td><td>7180</td><td>dilute at 40&amp;micro;L/mL in CellAdhere&amp;trade; dilution buffer</td></tr><tr><td>XAV939</td><td>Sigma Aldrich</td><td>X3004</td><td>reconstitute at 100mM in DMSO</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Thermo Fisher Scientific</td><td>21985-023</td><td>light sensitive</td></tr></tbody></table>

chemical reversion of conventional human pluripotent stem cells to a na&#239;ve-like state with improved multilineage differentiation potency

天真的人类多潜能干细胞 (n-hPSC) 具有改进的功能, 可能对再生医学产生广泛的影响。该协议的目标是有效地恢复沿袭的, 传统的人类多潜能干细胞 (hPSC) 保持在无馈线或馈线依赖的条件, 以一个天真的干细胞, 改善功能。这种化学幼稚的逆转方法采用了经典的白血病抑制因子 (LIF), GSK3β和甲酮/ERK 抑制鸡尾酒 (LIF-2i), 补充只有 tankyrase 抑制剂 XAV939 (LIF-3i)。LIF-3i 将传统的 hPSC 还原为一种稳定的多能态, 采用人类预植入啮鼠动物的生物化学、转录和表观遗传特征。这种 LIF-3i 方法需要最小的细胞培养操作, 并在广泛的人类胚胎干细胞 (人类胚胎干细胞) 和无转基因人类诱导的多潜能干细胞 (hiPSC) 线高度重现。LIF-3i 方法在分化之前不需要重新启动步骤;hPSC 可直接与极高的效率相区别, 维持核型和 epigenomic 的稳定性 (包括印迹位点)。为提高该方法的普遍性, 传统的 hPSC 首先培养在 LIF-3i 鸡尾酒补充两个额外的小分子, 事实蛋白激酶 A (佛司可林) 和声波刺猬 (嘘) (purmorphamine) 信号 (LIF-5i)。这一简短的 LIF-5i 适应步骤显著增强了传统 hPSC 的初始克隆扩展, 允许它们随后以 LIF-3i 的数量单独恢复, 从而不需要采摘/亚克隆稀有的 N hPSC殖民地以后。LIF-5i-stabilized hPSCs 随后在 LIF-3i 单独维持, 不需要抗凋亡分子。最重要的是, LIF-3i 逆转明显改善了常规 hPSC 的功能干细胞, 通过减少其谱系引种基因表达和清除联运变异的定向分化通常观察到独立的 hPSC 线。给出了 LIF-3i-reverted n-hPSC 的代表性特征, 并给出了等基因系 hPSC 在谱系引物与的功能比较的实验策略。概述了类似幼稚的国家。

该协议优化了在馈线依赖和馈线无关的传统 hPSC 文化 (图 1) 中 LIF-3i 的高效天真的回归。本文描述的详细协议概述了从馈线依赖或无馈线常规 hPSC 条件 (如E8 介质) 开始的 LIF-3i 的顺序适应。
图 1–3了几种常规人类胚胎干细胞和无转基因 hiPSC 线 LIF-3i 逆转的代表性结果。这些典型的结果?...

Watch this Scientific Journal Video about Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃ¯ve-like State with Improved Multilineage Differentiation Potency at JoVE.com

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

我们提出一项协议, 以有效, 散装, 快速化学逆转传统的谱系诱导人类多潜能干细胞 (hPSC) 成一个 epigenomically 稳定幼稚的植入啮鼠动物样多潜能状态。该方法在常规 hPSC 线的多向分化上降低了谱系引种基因的表达和显著改善。

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

chemical-reversion-conventional-human-pluripotent-stem-cells-to-nave

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.8K 次观看.  Johns Hopkins School of Medicine. 我们提出一项协议, 以有效, 散装, 快速化学逆转传统的谱系诱导人类多潜能干细胞 (hPSC) 成一个 epigenomically 稳定幼稚的植入啮鼠动物样多潜能状态。该方法在常规 hPSC 线的多向分化上降低了谱系引种基因的表达和显著改善。

视频: Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Naïve-like State with Improved Multilineage Differentiation Potency

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. 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 tumorigenicity7-10. 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 problematic11-13. 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 molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

科研

神经科学

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

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

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 ...

发育生物学

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

用DMSO对人多能干细胞的暂时性治疗促进分化

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

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 ...

生物学

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

推导与表征的转基因无人类诱导多能干细胞系，并转换成定义的临床级条件

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

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 ...