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 border="0" cellpadding="0" cellspacing="0" style="width:1024px;" width="1023">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti- SSEA-1/CD15 antibody, APC conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">561716</td>
			<td style="width:371px;">use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti- TFCP2L1 antibody</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">HPA029708</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-beta-Actin antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab6276</td>
			<td>use at 1:5000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD146 antibody,&#160; PE conjugated&#160;</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">550315</td>
			<td>use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD31 antibody, APC conjugated</td>
			<td style="width:161px;">eBioscience</td>
			<td style="width:119px;">17-0319-42</td>
			<td>use 2&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD31 microbead kit</td>
			<td style="width:161px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-NANOG antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab109250</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-NR5A2 antibody</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">HPA005455</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-p44/42 MAPK (Erk1/2) antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">4695</td>
			<td>use at 1:1000 (Western blot), for detection of total protein</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204 antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">4370</td>
			<td>use at 1:1000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-phospho-STAT3 (Tyr705) antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">9145</td>
			<td>use at 1:1000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-rabbit immunoglobulin antibody, biotinylated</td>
			<td style="width:161px;">Agilent</td>
			<td style="width:119px;">E0432</td>
			<td>use at a 1:500-1:1000 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-SSEA-4 antibody, APC conjugated&#160;</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">FAB1435A</td>
			<td>use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-SSEA-4 GloLIVE antibody, NL493 conjugated</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">NLLC1435G</td>
			<td>use at 1:50 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-STAT3 antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">9139</td>
			<td>use at 1:1000 (Western blot), for detection of total protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-STELLA/DPPA3 antibody</td>
			<td style="width:161px;">Millipore</td>
			<td style="width:119px;">MAB4388</td>
			<td>use at a 1:50 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA-1-60 GloLIVE antibody, NL557 conjugated&#160;</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">NLLC4770R</td>
			<td>use at&#160; a 1:50 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-TRA-1-60 StainAlive Antibody, DyLight 488 conjugated</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">09-0068</td>
			<td>use at a 1:100 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-TRA-1-81 StainAlive Antibody, DyLight 488 conjugated</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">09-0069</td>
			<td>use at a 1:100 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA1-60 antibody, PE conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">560193</td>
			<td>use 10&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA1-81 antibody, PE conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">560161</td>
			<td>use 10&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">APEL2-Li</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">5271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Bovine Serum Albumin</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CellAdhere dilution buffer</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">7183</td>
			<td>dilutent for Vitronectin XF&#8482; matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CF1 mouse</td>
			<td style="width:161px;">Charles river</td>
			<td style="width:119px;">023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CHIR99021</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">L5283</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">confocal microscope system</td>
			<td style="width:161px;">Zeiss</td>
			<td style="width:119px;">LSM 510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">cord blood CD34+ derived iPSC line</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A18945</td>
			<td>also referred as 6.2 line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Corning Costar tissue culture-treated 6-well plates</td>
			<td style="width:161px;">Corning</td>
			<td style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Countess&#160; cell counting chamber slide</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C10228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Countess automated cell counter</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)&#160;</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11995-065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">DMEM-F12</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">DMSO (dimethyl sulfoxide)</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">DR4 mouse</td>
			<td style="width:161px;">The Jackson Laboratory</td>
			<td align="right" style="width:119px;">3208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Essential 8 (E8) medium</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">5940</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Fetal bovin serum (FBS)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Forskolin</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0025</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Gelatin (porcine)</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td>resuspend in water and sterilize with an autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">KnockOut Serum Replacement</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">L-Glutamine (100X)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">MEM Non-essential amino acid (MEM NEAA) (100X)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">mTeSR1 medium</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">85850</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Nalgene cryogenic vials</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">5000-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Nunc Lab-Tek II Chamber Slide System</td>
			<td style="width:161px;">Fisher Scientific</td>
			<td style="width:119px;">154534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Paraformaldehyde (PFA) solution , 4% in PBS</td>
			<td style="width:161px;">USB Corporation&#160;</td>
			<td style="width:119px;">19943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">PD0325901</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">PZ0162</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Penicillin/streptomycin (10,000 U/mL)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Phosphate buffered saline (PBS)</td>
			<td style="width:161px;">Biological Industries</td>
			<td style="width:119px;">02-023-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Purmorphamine</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0009</td>
			<td>reconstitute at 10mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human Activin A</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">AF-120-14E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">recombinant human Bone morphogenetic protein (BMP)-4</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">120-05ET</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human FGF-basic (bFGF)</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human LIF</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">300-05</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">SB431542&#160;</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0010-05</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Stemolecule Y27632 in Solution</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0012-02</td>
			<td>ROCK inhibitor in solution (10mM)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A11105-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Streptavidin-Cy3 conjugate</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">S6402</td>
			<td>use at 1:500-1:1000 dilution (immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Thermo Scientific Mr. Frosty Freezing Container</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vascular endothelial growth factor (VEGF)-165</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vitronectin XF matrix</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">7180</td>
			<td>dilute at 40&#181;L/mL in CellAdhere&#8482; dilution buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">XAV939</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">X3004</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">&#946;-mercaptoethanol</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">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 NaÃƒÂ¯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

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

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

人类多能干细胞的模板微图案用于探测分化命运的空间组织

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

科研

发育生物学

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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

在人多能的体外干细胞向滋养层细胞

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

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...