这项工作得到了中国国家自然科学基金 (21435008 和 21327006) 和中国科学院战略优先研究项目 (XDB14030200) 的支持。

1. 筹备工作
<ol><li>
制备1.0 毫米 PD0325901 (酮抑制剂) 和3.0 毫米 CHIR99021 (GSK3 抑制剂) 的溶液。
	<ol>
<li>重2毫克的 PD0325901 和添加4.15 毫升亚砜在琥珀玻璃瓶。</li>
		<li>重2毫克的 CHIR99021 和添加1.43 毫升亚砜在琥珀玻璃瓶。</li>
		<li>重组后, 储存整除数 (50 µL/管在200µL PCR 管) 在-20 摄氏度和保护免受光。在使用前, 从冷藏库中取出含有冰冻库存的管子, 在室温下解冻。</li>
	</ol>
</li>
	<li>将 Vc 重0.0176 克到离心管的1毫升内, 再用1毫升的无菌水将其重新构成100毫米的最后浓度, 然后再使用。</li>
	<li>灭活血清: 在56摄氏度的水浴中孵育500毫升的血清, 30 分钟。</li>
	<li>
制备600毫升培养小鼠 ES 细胞的基本培养基。
	<ol>
<li>使用一瓶 DMEM/高葡萄糖培养基 (500 毫升), 并去除40毫升。</li>
		<li>补充460毫升的 DMEM/高葡萄糖培养基与20% 灭活血清 (120 毫升), 1000 U/毫升 LIF (60 µL 107 U/毫升库存解决方案), 0.1 毫米非必需氨基酸 (NEAA) (6 毫升10毫米库存溶液), 1 毫米丙酮酸钠 (6 毫升100毫米库存溶液), 2 毫米 l-谷氨酰胺 (6 毫升200毫米的库存溶液), 0.1 毫米β-巯基乙醇 (600 µL 100 毫米的股票溶液), 33 毫升/ml 青霉素和33µg/毫升链霉素 (2 毫升青霉素-链霉素混合溶液 (1万 ml/毫升青霉素和1万µg/毫升链霉素)).将基本培养基定义为血清培养基。</li>
		<li>吸管50µL 的 PD0325901 (1.0 毫米) 和 Vc (100 毫米) 的库存溶液, 分别为50毫升的血清培养基 (最终浓度: 1.0 µM PD0325901 和100µM Vc), 并定义培养基为 Vc/PD0325901 培养基。 注: 由于 Vc 的不稳定, 在使用前准备 Vc/PD0325901 介质。</li>
		<li>使用吸管, 转移50µL 的 PD0325901 (1.0 毫米) 和 CHIR99021 (3.0 毫米) 的股票溶液, 分别为50毫升的血清培养基 (最终浓度: PD0325901 1.0 µM 和 CHIR99021 3.0 µM), 并定义培养基为2i 培养基。</li>
	</ol>
</li>
	<li>
准备明胶涂层的菜肴。
	<ol>
<li>准备0.1% 的明胶溶液: 将0.3 克明胶溶成300毫升的超纯水, 在玻璃试剂瓶中加盖, 在蒸压釜内消毒121摄氏度为20分钟. 在室温下贮存无菌溶液。</li>
		<li>在室温下孵化细胞培养皿 (6 厘米直径), 2 毫升0.1% 明胶溶液, 在室温下吸出明胶。在空气中烘干盘子, 在12小时内使用。</li>
	</ol>
</li>
	<li>在15毫升离心管中制备10毫升的小鼠 ES 细胞冷冻培养基: 90% 只血清 (9 毫升) 和10% 亚砜 (1 毫升)。在1天内使用培养基。</li>
	<li>准备冷冻容器: 将100% 异丙醇加入冷冻容器的灌装线, 并丢弃旧异丙醇。在每第五使用后直接添加新的异丙醇。</li>
	<li>使酶消化缓冲: 将1.2114 克的三个粉末重化为100毫升的超纯水, 并将浓缩的 HCl 溶液 (约12米) 加入到三溶液中, 将 pH 值调整为 7.6 (约0.6 毫升的浓缩 HCl 溶液)。</li>
	<li>准备50毫升的无线电免疫沉淀检测缓冲器 (马国贤缓冲器):20 毫米三, pH 7.4, 1 毫米氯化镁 2, 150 毫米氯化钠, 20% 甘油, 0.5% NP40, 1 毫米 EDTA, 1 毫米 EGTA.补充1毫米, 1X 蛋白酶抑制剂鸡尾酒, 和1毫米 PMSF 之前使用。添加 PMSF 后, 在30分钟内使用缓冲区。</li>
</ol>2. 在明胶膜上种植小鼠 ES 细胞
<ol><li>预热的小鼠 ES 细胞 (野生型, 129 SvEv) 培养基, 胰蛋白酶, 磷酸盐缓冲溶液 (PBS) 在水浴在37°c。</li>
	<li>
通过细胞。从盘子中完全吸入培养基, 将2毫升的 PBS 吸入到盘子里冲洗细胞。
	<ol>
<li>用吸管取出 pbs, 再用2毫升的 pbs 冲洗细胞。</li>
		<li>取出 PBS, 加入0.3 毫升的胰蛋白酶-EDTA (0.25%) 到每道菜。</li>
		<li>用胰蛋白酶快速覆盖整个盘子, 然后立即取出。</li>
		<li>把盘子放在37摄氏度的孵化器里, 把盘子里的细胞分离出来1分钟。从孵化器中取出菜肴, 加入2毫升预热的血清培养基, 终止胰蛋白酶的作用。</li>
		<li>用吸管 (5 毫升吸管尖) 将细胞菌落分离成单个细胞悬浮液, resuspending 10 次。</li>
	</ol>
</li>
	<li>转移150µL 2 毫升细胞溶液 (大约19万细胞通过计数与 hemocytometer) 入一个新的明胶涂层的菜, 并且补充以3毫升新鲜地被做的 Vc/PD0325901 培养基每 6 cm 培养皿。在37°c 孵化器中培养出 5% CO2的细胞。</li>
	<li>每24小时孵化, 去除旧培养基, 并加入3毫升的新鲜 Vc/PD0325901-medium 进入养殖盘由于 Vc 的不稳定性. 期待70–80% 融合后2–3天。</li>
	<li>在达到70-80% 融合后, 通过细胞并继续按照步骤2.2–2.4 中的描述进行培养。</li>
</ol>3. 冷冻和解冻小鼠 ES 细胞
<ol><li>
冷冻小鼠 ES 细胞。
	<ol>
<li>按照步骤 2.2, 用胰蛋白酶将细胞从盘子中分离出来。</li>
		<li>将细胞转化为15毫升的塑料管, 离心机在 800 x g 处, 室温为3分钟。</li>
		<li>将上清液轻轻倒入烧杯中, 用1毫升新鲜的冷冻培养基并用重悬细胞颗粒。在这个步骤之前, 准备冷冻介质30分钟, 以确保在使用前是室温。</li>
		<li>将冷冻培养基中的细胞转移到1.8 毫升的离心管中, 用1毫升吸管, 将离心管放入冷冻容器中。在冷冻容器的灌装线上加入异丙醇, 并在使用前保持室温。</li>
		<li>把冰冻容器放在-80 摄氏度的冰箱里过夜。</li>
		<li>将离心管转移到液态氮气容器中, 2–3年。 注意: 不要在冷冻培养基中长时间保持细胞, 并且由于亚砜的毒性, 迅速将细胞转移到-80 摄氏度的冷藏库中。</li>
	</ol>
</li>
	<li>
解冻小鼠 ES 细胞。
	<ol>
<li>预热50毫升的血清培养基在37°c 的水浴。</li>
		<li>从液氮容器中取出一个含有细胞的瓶子, 并将盖瓶浸入37摄氏度的水浴中。在水浴中迅速摇动以解冻。用1毫升的吸管将细胞转化为15毫升管。在管内加入2毫升预热的血清培养基, 稀释冷冻培养基, 然后离心机在 800 x g 处, 室温为3分钟。</li>
		<li>用5毫升的吸管取出上清, 用3毫升新鲜 Vc/PD0325901 培养基轻轻并用重悬细胞颗粒。</li>
		<li>将细胞从15毫升管转移到明胶涂层皿中, 培养细胞 24 h. 按照步骤2中的说明再次培养细胞。</li>
	</ol>
</li>
</ol>4. 从细胞中提取总蛋白
<ol><li>用1毫升 PBS 冲洗收集的细胞, 然后离心机在 800 x g 处, 室温为3分钟。</li>
	<li>用马国贤缓冲液, 用并用重悬、蛋白酶抑制剂鸡尾酒和 PMSF 吹打轻轻地吸出上清, 并对细胞颗粒进行彻底的补充。马国贤缓冲器体积约为细胞颗粒的5x。 注: 在冰上进行细胞泥沙。</li>
	<li>保持细胞在马国贤缓冲30分钟冰和离心机在 1.3万 x g 和4°c 5 分钟。</li>
	<li>将上清液转移到新管, 贮存整除数-80 摄氏度。</li>
	<li>用布拉德福德蛋白试剂盒测定蛋白质浓度。</li>
</ol>5. 利用酶从细胞中提取 dna 并将 dna 消化成单个核苷类
<ol><li>用胰蛋白酶收集细胞, 如步骤3.1.1 和3.1.2 所述。</li>
	<li>按照制造商的说明, 使用基因组 dna 纯化试剂盒进行 dna 提取。</li>
	<li>将提取的 DNA 储存在1毫升离心管中。在离心管中加入100µL 的超纯水, 吹打约15次溶解 DNA。通过测量 260 nm 和 280 nm15的吸光度, 确定浓度并评估 DNA 的质量。DNA 浓度约为500µL。</li>
	<li>在50µL 溶液系统中消化5µg 的 DNA: 添加5µL 100 毫米的三盐酸溶液, pH 7.6 (最终浓度:10 毫米), 2 u 小牛肠道磷酸酶, 1 u DNase i, 0.005 u 蛇毒磷酸二酯化 i, 和5µg 的 DNA (计算增加量根据安全措施红色 DNA 浓度, ~ 10 µL) 成离心管, 并增加50µL 与超纯水溶液的体积。在一夜之间孵化37摄氏度的混合物。将所消化的 DNA 溶液在1000克和室温下离心处理1分钟, 以收集管底的溶液。</li>
	<li>将被消化的 DNA 溶液从离心管转移到超滤管 (分子重量截止: 3 kDa), 离心机在1.3万克和4摄氏度时, 去除消化酶。</li>
	<li>
准备样品5mC 和5hmC 分析。
	<ol>
<li>5hmC 分析: 吸管36µL 过滤器溶液到一个新的离心管 (1 毫升) 和添加4µL 5 '-(羟甲基 d3)-2 '-脱氧胞苷 ([d3]-5hmC), 最后浓度为 3 nM。</li>
		<li>5mC 分析: 将196µL 的超纯水添加到新的离心管中, 吸管4µL 过滤器溶液 (50 倍稀释), 因为基因组 DNA 中的5mC 密度较高。将稀释溶液的36µL 转移到新的离心管上, 并添加4µL (5 '-(甲基 d3)-2 '-脱氧胞苷 ([d3]-5mC), 最后浓度为 50 nM。使用 [d3]-5hmC 和 [d3]-5mC 作为内部标准分别校准5hmC 和5mC。</li>
	</ol>
</li>
	<li>将样品溶液转移到测量管并贮存在4摄氏度。用超高性能液相色谱-三重四极质谱-多反应监测 (UHPLC/ms (MRM)) 分析5mC 和 5hmC, 如前一报告15中所述。</li>
</ol>6. 使用 UHPLC/ms15分析5mC 和5hmC
<ol><li>
使用 UHPLC 软件来设置用于分析5mC 和5hmC 的各种参数。
	<ol>
<li>建立新的方法。打开软件并单击 "文件 |新 |方法 "。将 "方法编辑器" 的消息框与内容一起显示 "是否要保存当前方法的更改", 然后单击 "否"。</li>
		<li>建立取样器的参数。点击 "取样器 |设置 |注射 "。选择 "标准注入", 并输入 "5 µL 在注塑量"。</li>
		<li>在 UHPLC 中建立泵的参数。<ol>
<li>点击 "BinPump2 |设置 "以生成泵的参数。在 "15 分钟" 设置 "0.25 毫升/分钟" 和 "停止时间" 的 "流"。</li>
			<li>在 "最大600巴" 上设置 "溶剂 B 5%" 和 "压力限制"。</li>
			<li>点击 "BinPump2 |时间表 "建立等度洗脱参数5mC 分析。单击 "追加" 以添加行。输入 "0.00" 在 "时间" 和 "5.0" 在 "B%"。再次单击 "追加" 以添加新行。输入 "15.00" 在 "时间" 和 "5.0" 在 "B%"。</li>
			<li>点击 "BinPump2 |为5hmC 分析建立梯度洗脱参数的时间表。单击 "追加" 以添加行。输入0.00 在时间和5.0 在 "B%"。再次单击 "追加" 以添加新行。输入 "3.00" 在 "时间" 和 "5.0" 在 "B%"。再单击 "追加" 6 次以添加6行。输入 "3.01" 在 "时间" 和 "15.0" 在 "B%", "6.00" 在 "时间" 和 "15.0" 在 "B%", "6.01" 在 "时间" 和 "100" 在 "B%", "10.00" 在 "时间" 和 "100.0" 在 "B%", "10.01" 在 "时间" 和 "5.0" 在 "B%", "15.00" 在 "时间" 和 "5.0" 在 "B%" 顺序。 注意: UHPLC 不同的分离条件, 分别对5mC 和5hmC 进行分析。</li>
		</ol>
</li>
		<li>建立 UHPLC 中柱室温度的参数。 点击 "|设置 |温度 (左) "和输入" 30 °c "。</li>
		<li>建立 QQQ/ms 的参数。<ol>
<li>点击 "MS QQQ |停止时间 |无极限/泵浦 ", 离子源 |ESI "," 时间过滤 |峰值宽度: 0.07 分钟。设置 "时间段: 开始时间: 0";"扫描类型: MRM";"Div 值: MS";"三角洲 EMV (+): 200" 和 "三角洲 EMV (-): 0"。</li>
			<li>生成监视5mC、D3-5mC、dC、5hmC 和 D3-5hmC 的转换的参数<ol>
<li>点击 "MS QQQ |收购 |扫描段: 居住:90 ";"Fragmentor:90";"碰撞能量: 5";"电池加速器电压: 4" 和 "极性: 阳性"。</li>
				<li>输入 "5mC" 在 "复合名称", "242" 在 "前体离子", "126" 在 "产品离子" 为5mC 分析。单击右键并选择 "添加行" 以添加新行。输入 "d3-5mC" 在 "复合名称", "245" 在 "前体离子", "129" 在 "产品离子" 为 D3-5mC 分析。</li>
				<li>使用相同的模式补充另外三行。输入 "dc" 在 "复合名称", "228" 在 "前体离子", "112" 在 "产品离子" 为 dC 分析。输入 "5hmC" 在 "复合名称", "258" 在 "前体离子", "142" 在 "产品离子" 为5hmC 分析。输入 d3-5hmC 在复合名称, "261" 在 "前体离子", "145" 在 "产品离子" 为 d3-5hmC 分析。</li>
			</ol>
</li>
			<li>建立气体源的参数。点击 "MS QQQ |来源 |源参数: 气体温度: 300 °c ";"气体流量:11 升/分";"雾化器:15 psi";"正毛细管: 3500 V"。</li>
			<li>保存建议的方法。点击 "MS QQQ |仪器 |将方法另存为 "。通过 "目录" 选择所建议方法的路径, 并通过输入 "方法" 中的内容为该方法命名, 例如: 5mC 和5hmC 分析。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
mononucleosides 的 UHPLC 分离及质谱分析
	<ol>
<li>为5mC 和胞嘧啶 (C) 分析准备移动相: 形成溶液 a 和 b 溶液的流动阶段 a: 500 毫升超纯水, 500 µL 甲酸 (最终浓度: 0.1%), 溶液 B: 500 毫升100% 甲醇。混合溶液 A 和 B 在 95:5 (v: v) 作为移动的阶段到洗脱5mC 和 C (设置在步 6.1.3.3)。将流速设置为0.25 毫升/分 (在步骤6.1.3.1 中设置)。</li>
		<li>用溶剂 A 和 B 为5hmC 分析准备移动相。溶剂 A 为2.0 毫米 NH4HCO3水溶液 (pH 9.0) (500 毫升), 溶剂 B 为100% 甲醇 (500 毫升)。通过优化梯度洗脱分离 5hmC: 0-3 分钟, 5.0% B 和 95% A (v: v);3-6 分钟, 15.0% B 和 85% A;6-10 分钟, 100% B;10-15 分钟, 5.0% B 和 95% A (在步骤6.1.3.4 中设置)。将流速设置为0.25 毫升/分 (在步骤6.1.3.1 中设置)。</li>
	</ol>
</li>
	<li>
利用具有 MRM 模式 (在步骤6.1.5.1 中设置) 的电喷雾 ms/毫秒分析从柱上洗脱, 采用正离子模式 (在步骤6.1 中设置 5.2. 1)。
	<ol>
<li>监控过渡: 5mC, 242→126 (碰撞能量, 5 eV);[D3]-5mC, m/z 245→129 (5 eV);直流, 228→112/z (5 eV);5hmC、258→142 (5 eV);[D3]-5hmC, m/z 261→145 (5 eV) (在步骤 6.1 (5.2. 2) 中设置)。</li>
		<li>将毛细管和碎片电压设置为 +3,500 v (在步骤6.1.5.3 中设置) 和 90 v (在步骤 6.1 (5.2. 1) 中分别设置)。</li>
		<li>将注入量设置为5µL, 并分析每个采样3次 (在步骤6.1.2 中设置)。使用相应的标准曲线计算5mC 和5hmC 的量。<ol>
<li>建立5mC 的标准曲线: 将 1–50 nM (1、5、10、20、50 nm、最终浓度) 标准溶液5mC 转化为离心管, 并将 50 nM [D3]-5mC 转管。将总容积补充到50µL. 按照5mC 示例 (步骤 6) 的说明执行标准5mC 的分析。</li>
			<li>建立5hmC 的标准曲线: 转移 1–20 nM (1, 2, 5, 10, 20 nm, 最终浓度) 标准溶液5hmC 到离心管, 并添加 3 nM [D3]-5hmC 到管。将总容积补充到50µL. 按照5hmC 示例 (步骤 6) 的说明执行标准5hmC 的分析。</li>
		</ol>
</li>
	</ol>
</li>
</ol>7. 分析统计意义
<ol><li>
使用软件进行统计分析。
	<ol>
<li>打开软件并在 "新表 & 图形" 中选择 "列"。按如下方式设置参数: "示例数据 |从空数据表开始 ";"选择图形-第一种模式";"图形复制或错误栏 |剧情 |指的是 SEM "。</li>
		<li>单击 "创建", 分别在 "A" 和 "B" 行中输入控件和 Vc/PD0325901-treated 组的三个复制。选择六数据, 然后单击 "分析" 中的 "分析"。</li>
		<li>在 "列分析" 中选择 "t测试 (和非参数测试)", 然后单击 "确定" 进入下一个接口。</li>
		<li>按如下方式设置参数: "选择测试 |测试名称 |未配对的t测试;选项 |双尾 ";"置信区间: 95%";"有效数字 |显示4个有效数字 "。单击 "确定" 以获取控件和 Vc/PD0325901 处理之间的p值。</li>
	</ol>
</li>
	<li>评估使用双尾和未配对学生的t测试16的对照组与实验小组之间的统计学意义。 注: p < 0.05 表示具有统计学意义的差异。* p < 0.05, ** p < 0.01, *** p < 0.001。</li>
</ol>

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作者没有披露潜在的利益冲突。

在这项工作中, 我们展示了一种新的方法, 结合 vc 和 PD0325901 维持小鼠 ES 细胞在未分化和 hypomethylated 的状态, 这是通过协同行动促进 dna 去甲基化的 Vc 和抑制从头 dna甲基化的 PD0325901。此外, 小鼠 ES 细胞在 Vc/PD0325901 培养体系下表现出很大的形态学。
为了更好地维持 Vc/PD0325901 培养系统中小鼠 ES 细胞的状态, 有一些关键步骤。首先, 需要预先筛选出血清批次, 以发现和储存最佳批次;此外, 频繁更换血清是有害的细胞增殖和维持干细胞。其次, 避免细胞吹打细胞在细胞游离过程中, 以单细胞悬浮液为单元格通道。第三, 为了减弱胰蛋白酶在细胞培养过程中的不良影响, 在细胞传代过程中, 应立即去除胰蛋白酶中的细胞。此外, 由于 Vc 的不稳定性, Vc/PD0325901 培养基应每天更换。
与传统的血清培养相比, Vc/PD0325901 条件下的小鼠 ES 细胞显示出更均匀的细胞群 (图 3B、 C) 和 hypomethylated 状态, 类似于 ICM 和 PGC。此外, 与最近开发的2i 文化相比, 我们的方法可以实现更快的动力学低甲基化, 并利用单一抑制剂 (PD0325901) 减少亚砜的输入量, 从而减少对细胞的损害。然而, 由于在我们的文化体系中加入了血清, 在长期的培养过程中, 血清的矛盾的双重作用可能导致细胞分化。总体上, 这种新型的培养系统适用于大鼠 ES 细胞的维护和扩展。

ES 细胞来源于胚泡1的 ICM。细胞处于干细胞状态, 可以形成所有体细胞血统和生殖细胞2。ES 细胞的建立为研究体外的发育过程提供了机会, 并可根据其干细胞3生成再生医学的医学相关性细胞。
两组 seminally 在1981年建立了小鼠 ES 细胞系, 当从早期小鼠胚胎中提取的细胞以小鼠胚胎成纤维细胞 (MEFs) 的胎牛血清 (含血清) 培养, 作为进纸层1,4.MEFs 被灭活 mitotically, 在培养 ES 细胞前先在菜肴中生长。MEFs 为小鼠 ES 细胞附着提供支持, 并产生生长因子, 促进传播和抑制分化5, 而血清为细胞增殖提供必需的营养因子和荷尔蒙。随后的研究表明, 饲养细胞产生的 lif 是小鼠 ES 细胞干细胞自我更新和维持的关键细胞因子, 在培养基中加入 lif 可以替代馈线细胞6。目前, 喂食器中小鼠 ES 细胞的维持仍然是许多研究者采用的标准方法。然而, 这一古典文化方法也出现了一些问题。首先, 饲养细胞分泌过量和不受控制的因素, 可能导致致病性污染7。为避免这种干扰, 用明胶对菜肴表面进行涂敷, 并在含血清培养基中添加 LIF, 是维持不带进料层细胞的小鼠 ES 细胞的替代方法。此外, 在血清/LIF 条件下生长的小鼠 ES 细胞在细胞群中呈现形态学异质性, 甚至在干细胞相关因子的表达水平上表现为8。最近的研究表明, 在血清/生命条件下, 与干细胞相关的核心转录因子 (包括 SOX2, 北美和 OCT4) 可以维持干细胞通过 LIF 和 WNT 信号;然而, 值得注意的是, 它们还激活了成纤维细胞生长因子信号, 触发分化8。由于核心转录因子的矛盾双重作用, 在血清中培养的小鼠 ES 细胞呈现出不均一性, 由两个可互换的种群组成, 一个类似于 ICM, 另一个相似于引啮鼠动物状态8。此外, 血清中的小鼠 ES 细胞通常表现为全球甲基9, 而 ICM 和 PGCs 处于全局 hypomethylated 状态, 与干细胞9、10紧密相连。
开发新的培养小鼠 ES 细胞的方法有相当大的需求。自 2003年11以来已建立了若干改进的协议, 但仍然存在一些限制和缺点7。自2008年以来, 两个小分子激酶抑制剂 PD0325901 (丝裂原活化蛋白激酶 (MAPK)/胞外信号调节激酶 (ERK)) 和 CHIR99021 (糖原合成酶抑制剂) 的联合利用激酶 3 (GSK3)), 在 N2B27培养基与 LIF 和没有血清已经打开新的观点培养小鼠 ES 细胞12。这种新定义的培养基的特点是使用两种抑制剂 (2i)。在2干细胞培养基中培养的小鼠 ES 细胞在细胞群中比较均匀, 表达的因子也较多。此外, 2 的 i/低甲基化培养的小鼠 ES 细胞在全球范围内呈现 DNA, 它接近于 ICM 样细胞 9, 13.即便如此, 2i 文化也有其弊端。PD0325901 和 CHIR99021 不溶于水, 一般被溶于二甲基亚砜 (双砜) 的库存溶液中, 添加到培养基中。研究表明, 长期和低剂量的细胞暴露于亚砜可能导致细胞毒性14。
在这里, 我们利用了两个小分子化合物, 并开发了一种新的培养方法的小鼠 ES 细胞。这种新的培养方法结合 Vc 和甲酮抑制剂 PD0325901, 以促进 DNA 低甲基化迅速和有效地向可比水平的 PGC, 命名为 Vc/PD0325901 培养协议。Vc/PD0325901-added 血清中的小鼠 ES 细胞在形态学上表现为同质性, 并维持在基态。与2i 培养相比, Vc/PD0325901 条件下培养的小鼠 ES 细胞具有更快的 DNA 去甲基化动力学, 可达到与 PGC 相比的低甲基化水平。另外, 使用单一抑制剂 (PD0325901) 将亚砜的输入量减少到培养基中, 与 2i (PD0325901/CHIR99021) 中使用的相比, 减少了对细胞的损伤。

<table><tbody><tr><td>fetal bovine serum Australia source</td><td>Corning</td><td>35-076-CV</td><td>Component of mouse ES cell medium</td></tr><tr><td>DMEM/high glucose</td><td>Hyclone</td><td>SH30022</td><td>Component of mouse ES cell medium</td></tr><tr><td>LIF</td><td>Millipore</td><td>ESG1107</td><td>Component of mouse ES cell medium</td></tr><tr><td>non-essential amino acids (NEAA)</td><td>Gibco</td><td>11140050</td><td>Component of mouse ES cell medium</td></tr><tr><td>sodium pyruvate</td><td>Gibco</td><td>11360070</td><td>Component of mouse ES cell medium</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030081</td><td>Component of mouse ES cell medium</td></tr><tr><td>penicillin streptomycin solution</td><td>Gibco</td><td>15140122</td><td>Component of mouse ES cell medium</td></tr><tr><td>PBS</td><td>Sigma</td><td>P5493</td><td>Cells rinse</td></tr><tr><td>trypsin</td><td>Hyclone</td><td>SH30042</td><td>Cell dissociation</td></tr><tr><td>gelatin</td><td>Sigma</td><td>48722</td><td>Dishes coating</td></tr><tr><td>PD0325901</td><td>Stemolecule</td><td>04-0006-10</td><td>small-molecule chemical for cell culture</td></tr><tr><td>CHIR99021</td><td>Stemolecule</td><td>04-0004</td><td>small-molecule chemical for cell culture</td></tr><tr><td>vitamin C</td><td>Sinopharm Chemical Reagent Co.,Ltd&amp;nbsp;</td><td>XW00508171</td><td>small-molecule chemical for cell culture</td></tr><tr><td>Ultra Bioscience water purification systemr</td><td>Purelab</td><td>Ultra</td><td>Ultra water</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>Cell freezing medium</td></tr><tr><td>centrifuger</td><td>Eppendorf</td><td>5427R</td><td>DNA and protein extraction</td></tr><tr><td>protease inhibitor cocktail</td><td>Sigma</td><td>P8340</td><td>Component of RIPA buffer</td></tr><tr><td>PMSF Protease Inhibitor</td><td>ThermoFisher Scientific</td><td>36978</td><td>Component of RIPA buffer</td></tr><tr><td>DTT</td><td>Sigma</td><td>43815</td><td>Component of RIPA buffer</td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td>Component of RIPA buffer</td></tr><tr><td>Genomic DNA Purification Kit&amp;nbsp;</td><td>Promega</td><td>A1125</td><td>Genomic DNA extraction</td></tr><tr><td>NanoDrop 2000</td><td>ThermoFisher Scientific</td><td>NanoDrop 2000</td><td>Determination of DNA concentration</td></tr><tr><td>calf intestinal phosphatase</td><td>New England Biolabs</td><td>M0290</td><td>DNA digestion</td></tr><tr><td>DNase I</td><td>New England Biolabs</td><td>M0303</td><td>DNA digestion</td></tr><tr><td>snake venom phosphodiesterase I</td><td>Sigma</td><td>P4506</td><td>DNA digestion</td></tr><tr><td>Nanosep 3K Omega</td><td>Pall</td><td>OD003C35</td><td>filtration of digested DNA</td></tr><tr><td>1290 UHPLC system</td><td>Agilent&amp;nbsp;</td><td>1290</td><td>UHPLC separation</td></tr><tr><td>G6410B triple quadrupole mass spectrometer</td><td>Agilent&amp;nbsp;</td><td>G6410B</td><td>MS/MS analysis</td></tr><tr><td>Zorbax Eclipse Plus C18 column</td><td>Agilent&amp;nbsp;</td><td>Zorbax Eclipse Plus</td><td>colume for UHPLC separation</td></tr><tr><td>5-methylcytosine</td><td>Solarbio Life Sciences</td><td>SM8900&amp;nbsp;</td><td>standard 5mC</td></tr><tr><td>5-hydroxymethylcytosine (standard)</td><td>Toronto Research Chemicals</td><td>M295900</td><td>standard 5hmC</td></tr><tr><td>Prdm14 antibody</td><td>bioworld</td><td>BS7634</td><td>Western blot analysis-primary antibody</td></tr><tr><td>Dnmt3a antibody</td><td>bioworld</td><td>BS6587</td><td>Western blot analysis-primary antibody</td></tr><tr><td>Dnmt3b antibody</td><td>abcam</td><td>ab13604</td><td>Western blot analysis-primary antibody</td></tr><tr><td>Dnmt3l antibody</td><td>abcam</td><td>ab3493</td><td>Western blot analysis-primary antibody</td></tr><tr><td>Dnmt1 antibody</td><td>abcam</td><td>ab13537</td><td>Western blot analysis-primary antibody</td></tr><tr><td>&amp;beta;-tubulin antibody</td><td>bioworld</td><td>BS1482MH</td><td>Western blot analysis-primary antibody</td></tr><tr><td>goat anti-rabbit IgG</td><td>abcam</td><td>ab6721</td><td>Western blot analysis-secondary antibody</td></tr><tr><td>goat anti-mouse IgG</td><td>abcam</td><td>ab6789</td><td>Western blot analysis-secondary antibody</td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-026</td><td>RNA extraction</td></tr><tr><td>reverse transcription system</td><td>Promega</td><td>A3500</td><td>RNA reverse transcription</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6001</td><td>RT-PCR</td></tr><tr><td>StemTAG alkaline phosphatase staining and activity assay kit</td><td>Cells Biolabs, Inc.</td><td>CBA-302</td><td>&amp;nbsp;alkaline phosphatase staining analysis</td></tr><tr><td>mouse ES cells: WT, 129 SvEv</td><td>Provided by Professor Guoliang Xu (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China)</td><td></td><td>cell strain of mouse ES cells</td></tr><tr><td>microscope</td><td>Zeiss</td><td>LSM510</td><td>cells observation</td></tr><tr><td>GraphPad Prism 5.0</td><td>GraphPad Prism Software Inc.</td><td>5.0</td><td>statistical analysis</td></tr></tbody></table>

an alternative culture method to maintain genomic hypomethylation of mouse embryonic stem cells using mek inhibitor pd0325901 and vitamin c

胚胎干细胞有可能分化为三种细菌层 (内胚层、胚层或外胚层) 中的任何一种, 并能为再生医学产生许多血统。ES 细胞培养体外长期以来一直是人们普遍关注的问题。经典的, 小鼠 ES 细胞维持在血清和白血病抑制因子 (LIF) 的含培养基。然而, 在血清/生长条件下, 细胞表现出异质性和干细胞相关基因的表达谱, 大多处于亚稳态状态。此外, 培养的 es 细胞呈现全球甲基, 但内细胞质量 (ICM) 和原始生殖细胞 (PGCs) 的天真 ES 细胞处于全球低甲基化状态。ICM 和 PGCs 的 hypomethylated 状态与他们的干细胞紧密相关。为了改善小鼠 ES 细胞培养方法, 我们最近开发了一种新的方法, 在选择性地联合利用两个小分子化合物, 以维持 DNA hypomethylated 和多能状态。在这里, 我们提出的合作治疗维生素 C (Vc) 和 PD0325901 可以清除约 90% 5 甲基胞嘧啶 (5mC) 5 天的小鼠 ES 细胞。生成的5mC 内容与 PGCs 中的相同。机械调查显示, 通过减少 PD0325901 5mC 合成, Prdm14 表达式抑制 Dnmt3b (DNA 甲基) 和 Dnmt3l (余子). Vc 促进5mC 到 5-hydroxymethylcytosine (5hmC) 的转换主要由 Tet1 和 Tet2, 表明参与的被动和主动 DNA demethylations。此外, 在 Vc/PD0325901 条件下, 小鼠 ES 细胞呈均匀形态学和多能态。我们共同提出了一种新的化学协同培养方法来实现小鼠 ES 细胞 DNA 低甲基化和干细胞的维持。小分子化学依赖性方法克服了血清培养的主要缺陷, 并有望生成均质 ES 细胞, 进一步临床应用和研究。

Vc/PD0325901 协同诱导的小鼠 ES 细胞的全局擦除。血清中的小鼠 ES 细胞表现出 dna 甲基, 而多能 ICM 细胞和 PGCs 显示全球对 dna 甲基化的擦除, hypomethylated 状态与干细胞9、10密切相关。
以前, 我们和其他人发现 Vc 可以增强5mC 去甲基化15, 17.同时, 还发现2i 可抑制5mC 的从头合成。在这些发现的背景下, 我们进一步提出了用 Vc 和2i 协同操作小鼠 ES 细胞。在分析 UHPLC-ms/毫秒后, 我们发现, Vc 和2i 补充在血清中的培养基可以大大减少5mC 的内容, 从3.2 到0.33 每 100 C (90% 5mC 损失) 的天11在小鼠 ES 细胞 (图 1A)。相比之下, Vc 或 2 i 处理仅导致58% 擦除与稳定水平在 1.4% 5mC 或61% 减少在水平 1.3% 5mC。
2i 培养基由甲酮抑制剂 PD0325901 和 GSK3β抑制剂 CHIR99021 组成。接下来, 我们研究在 Vc 和2i 的共同处理过程中, 哪个组件导致了全球5mC 的损失。有趣的是, Vc/PD0325901 导致 DNA 甲基化水平的下降速度比 Vc/2i 快。Vc/PD0325901 在5天后达到稳定的5mC 水平 (约 0.33% 5mC), 而 Vc/2i 达到11天的可比水平。相比之下, CHIR99021 的补充部分抑制了 Vc 触发的 DNA 去甲基化(图 1A)。这些数据清楚地表明, 2i 的 PD0325901 对小鼠 ES 细胞基因组 DNA 的全球低甲基化有贡献, 而2i 的 CHIR99021 部分抑制了5mC 的擦除。因此, 我们推测 Vc/PD0325901 相对于 Vc/2i 诱导的 DNA 去甲基化的加速可能归因于 CHIR99021 对去甲基化的部分抑制作用。
我们假设, Vc/PD0325901 诱导的小鼠 ES 细胞中5mC 的全球擦除可能与 DNA 去甲基化的增强有一定的相关性。因此, 我们研究了5mC 的基因组氧化产物。据报道, 5mC 可转换为5hmC 的催化氧化的春节家庭 dioxygenases, 这是依赖于 Fe (II) 和 2-oxoglutarate18,19。基因组5hmC 可能被 DNA 复制20稀释。此外, 5hmC 可进一步氧化的新 dioxygenases 生产 5-formylcytosine (5fC) 和 5-carboxylcytosine (5caC), 可以有效地切除嘧啶 DNA glycosylase (TDG) 恢复非甲基化胞嘧啶, 表明有源 DNA去甲基化21, 22.
与以前的工作15一致, vc (vc、Vc/2i、Vc/PD0325901、Vc/CHIR99021) 在1天后显著增加5hmC 频率 (5 hmc/106 C), 此后5hmC 级逐渐下降 (图 1B), 原因是逐渐减少5mC 底物。值得注意的是, Vc/PD0325901 和 Vc/2i 比 Vc 和 Vc/CHIR99021 更快的降低了5hmC 的动力学, 因为5mC 的速度更快。此外, 我们还观察到, 在1天, vc 处理刺激了5hmC 与 Vc/CHIR99021 的生成, 表明 CHIR99021 部分抑制了 vc 诱导的5hmC 的生产和抑制 DNA 去甲基化。5hmC. 2i 处理中的损失应归因于5mC 基板的减少。同时, 这些结果表明, Vc 增强的 DNA 去甲基化活性在一定程度上有助于协同低甲基化 Vc/PD0325901 和 Vc/2i 的共同治疗。
两个哈比比et 。23和冯 Meyenn et 等。24还报告了2i 条件下的小鼠 ES 细胞显示全球 DNA 低甲基化和天真干细胞。5mC 水平在从血清到2i 后12–14天逐渐呈下降趋势, 达到稳定状态 (约 1% 5mC)。在 Vc/PD0325901 培养系统中, 由于 Vc/PD0325901 的协同作用, 在血清培养基中添加 Vc/PD0325901 5 天后, 共处理导致 DNA 去甲基化更快, 达到稳定水平 (0.33% 5mC)。所达到的甲基化水平 (0.33% 5mC) 发出音低于哈比比 et 报告的2i 条件. 和冯 Meyenn et 等人, 而5hmC 的水平是较高的, 因为促进生成的 Vc。
PD0325901 提升 Prdm14 表达式以抑制 Dnmt3b 和 Dnmt3l, 但不 Dnmt3a25。为了研究 PD0325901 在诱导5mC 损失中的作用, 研究了一系列与 5 mc 相关的蛋白表达, 包括维护 (Dnmt1) 和从头(Dnmt3a 和 Dnmt3b) DNA 甲基转移酶及其余子 (Uhrf1 和 Dnmt3l) 和调节蛋白 (Prdm14) 8,9,26。在西方印迹分析后, Prdm14 在 PD0325901-including 文化中发出音提升 (图2)。相比之下, Dnmt3b 及其余子 Dnmt3l 在 PD0325901-including 治疗中表现出相当大的降低蛋白质表达的调节 (图 2)。CHIR99021 治疗对 Prdm14、Dnmt3b、Dnmt3l 水平无或轻微影响。Dnmt3awas 的表达没有改变, 原因不明。此外, 维持 DNA 甲基 Dnmt1 及其靶向因子 Uhrf1 也没有改变, 也没有显示数据。
最近的研究表明, Prdm14 可以招募 polycomb 压制性复杂的 2 (PRC2) 到一些基因促进者压制其表达, 如诺新 DNA 甲基转移酶 (Dnmt3a 和 Dnmt3b) 及其余子 (Dnmt3l), 并有助于在2i 条件下维护低甲基化8, 27.我们的数据显示, PD0325901 提高了 Prdm14 的表达水平, 以抑制 Dnmt3b 和 Dnmt3l, 并减少了基因组5mC 合成的机制. 
为探讨 Vc/PD0325901 条件下小鼠 ES 细胞的干细胞维护, 研究了几种核心干细胞相关因子的 mRNA 表达水平, 并确认这些因素在干细胞28中起重要作用。实时 PCR 分析发现, Oct4、SOX2、Esrrb 和 Sall4 在5天的治疗中表现出一致的表达水平, 与血清培养相比 (图 3A)。北美和 Tcf3 分别增加了1.3 倍和1.6 倍。相比之下, Klf4 显示42% 的下降调节和 Rex1 只下降了约12%。这些结果表明, Vc/PD0325901 共同治疗引起了不同的变化, 表达干细胞因素;然而, Oct4 和 SOX2, 最核心的因素, 保持几乎恒定的表达水平。
其次, 进行碱性磷酸酶 (AP) 测定, 以评估小鼠 ES 细胞是否处于未分化或分化状态。单元格的照片 (图 3B 和3C) 是从连接到倒置显微镜的摄像头获得的, 目标透镜的放大倍数为10倍。在 Vc/PD0325901-added 血清培养基连续培养26天后, 小鼠 ES 细胞呈均匀形态学。此外, 几乎所有的细胞都被染成紫黑色, 并表现出一个球状的状态没有突起 (图 3C), 显示一个未分化的状态。相比之下, 在血清条件下, 26 天鼠 ES 细胞的一部分在染色后呈紫黑色, 在形态学上呈球状状态, 而另一些则呈现浅色, 用白色虚线椭圆形线 (图 3B) 标记扩张;这表明, 血清培养的小鼠 ES 细胞在形态学和多能性状态下是异构的, 与以前的报告13一致。总的来说, 数据支持 PD0325901 结合 Vc 可以保持良好的形态学和未分化的状态, 在小鼠 ES 细胞。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56391/56391fig1.jpg"> 图 1:Vc/PD0325901 协同诱导的基因组5mCin 小鼠 ES 细胞的全局擦除.(A) 5mC 频率 (5 mc/c) 和 (B) 5hmC 频率 (5 hmc/c) 在26天的治疗过程中依赖于含血清培养基中 Vc、2i、Vc/2i、Vc/PD0325901 或 Vc/CHIR99021 的连续补充。误差条显示标准偏差 (SD) 从三重复分析的 UHPLC-ms/ms C: 胞嘧啶。此数字已由 Li et修改。25.<a href="/files/ftp_upload/56391/56391fig1large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56391/56391fig2.jpg"> 图 2:Prdm14 和 DNA 甲基转移酶 (Dnmt3a、Dnmt3b 和 Dnmt3l) 的蛋白质表达改变。通过5天的血清培养 (控制)、Vc、PD0325901、CHIR99021、2i、Vc/PD0325901、Vc/CHIR99021 和 Vc/2i 的治疗, 获得了所提出的带 (从左到右)。在 PD0325901-including 文化之后, Prdm 14 的表达上调, Dnmt3b 和 Dnmt3l 下降. Dnmt3a 没有变化。β-蛋白被设置为内部参考。管制; 控制PD: PD0325901;CHIR99021。此数字已由 Li et修改。25.<a href="/files/ftp_upload/56391/56391fig2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56391/56391fig3.jpg"> 图 3:Vc/PD0325901-induced 改变多能基因 mRNA 表达和碱性磷酸酶活性.(A) 部分多能基因的 mRNA 水平通过5天的 Vc/PD0325901 治疗与血清条件 (控制) 相比较而改变。数据以平均值表示。对统计学意义进行了评价, p < 0.05 在统计学上有显著差异。* p < 0.05, ** p < 0.01, *** p < 0.001, ns 代表非显著性。PD0325901。血清中的小鼠 ES 细胞26天 (B) 表现为白色虚线椭圆形线的部分突起, 而 Vc/PD0325901-added 血清培养基保持大形态学和未分化状态 (C)。这些图像是从一个在明亮的领域与相机连接的倒置显微镜获得的。此数字已由 Li et修改。25. 缩放条 = 100 µm.<a href="/files/ftp_upload/56391/56391fig3large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>

Watch this Scientific Journal Video about An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C at JoVE.com

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

用酮抑制剂 PD0325901 和维生素 C 维持小鼠胚胎干细胞基因组低甲基化的一种替代培养方法

我们详细描述了两种基于化学的小鼠胚胎干细胞培养协议。这种新方法采用了促进甲基胞嘧啶介导氧化 (维生素 C) 的协同机制, 并抑制了 5-PD0325901 的合成, 以维持小鼠胚胎干细胞的 DNA 低甲基化和干细胞.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

an-alternative-culture-method-to-maintain-genomic-hypomethylation

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.6K 次观看.  Chinese Academy of Sciences. 我们详细描述了两种基于化学的小鼠胚胎干细胞培养协议。这种新方法采用了促进甲基胞嘧啶介导氧化 (维生素 C) 的协同机制, 并抑制了 5-PD0325901 的合成, 以维持小鼠胚胎干细胞的 DNA 低甲基化和干细胞. 

视频: An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

科研

发育生物学

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

非随机小鼠模型在非活性 X 染色体上对 Mcp2 的药理再激活

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

遗传学

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

生物学

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

诱导多能干细胞来源于小鼠的生成

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

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

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 ...