这项研究得到了来自 Boettcher 基金会韦伯-华林生物医学研究项目的资金支持, 美国心脏协会科学家发展补助金 (13SDG17400031), 科罗拉多大学医学系杰出的早期职业生涯学者计划, 科罗拉多大学心脏科洛 Nyle 捐赠和 NIH R01HL133230 (到 K)。犍为县 R 得到了美国国立卫生研究院/NCATS 科罗拉多 CTSA 赠款号 TL1TR001081 和科罗拉多大学纤维化研究 & 翻译协会 (CFReT) 博士研究生奖学金的支持。这项研究也得到了癌症中心支持补助金 (P30CA046934), 皮肤病研究核心赠款 (P30AR057212), 以及在科罗拉多大学安舒茨医学院的流式细胞术核心的支持。

所有需要动物的实验都是由丹佛安舒茨医学院的机构动物护理和使用委员会批准的。
1. MEFs 的分离
<ol><li>在 E13 购买 C57BL/6 怀孕小鼠。船过夜。</li>
	<li>弄死母亲根据批准的 IACUC 协议 (前: ~ 1.3 升/分 CO2 , 直到动物出现死亡后, 颈部脱位)</li>
	<li>用70% 乙醇和开腹腔喷洒母亲。移除含有胚胎的子宫角, 并在10厘米的盘子中放置无菌 PBS。</li>
	<li>在胚囊中进行切口以释放胚胎。转移胚胎, 以清洁的 PBS 10 厘米菜。彻底冲洗。</li>
	<li>将身体切开肝脏并丢弃头部和上身。取出内脏并丢弃。将剩余的组织转移到一个干净的 10 cm 菜与无菌 PBS 和转移板材到生物安全水平1或2个内阁。将所有胚胎中的组织从干净的、干燥的10厘米的盘子中组合成细小的碎肉。</li>
	<li>加入6毫升 0.25% trypsin/1 毫米 EDTA 到碎胚。Triturate 几次通过吸管来分解组织。在37˚C 上孵育40分钟, 5% CO2。</li>
	<li>生长介质中的并用重悬细胞(表 1)。根据收获的胚胎数量调整培养基的体积。一般而言, 每1.2 胚胎使用大约25毫升生长培养基的比率。</li>
	<li>25毫升的悬浮细胞在15厘米的盘子里。24小时后, 吸入介质, 并在每个盘子中加入25毫升新鲜的生长培养基。</li>
	<li>72小时后, 添加2毫升 0.25% trypsin/1 毫米 EDTA 到每 15 cm 菜。当细胞从培养皿中分离出来时, 用15毫升生长培养基钝化胰蛋白酶, 并在50毫升聚苯乙烯锥形管中收集细胞。在室温下, 离心细胞在 160 x g处为3分钟。并用重悬细胞在生长培养基中的颗粒, 并通过70µm 孔细胞滤池过滤。</li>
	<li>计数细胞使用 hemocytometer 和冻结 MEFs 在200万细胞整除数在冷冻培养基 (10% 亚砜, 25% 血清在 Dulbecco 的改良鹰培养基 (DMEM)/高葡萄糖)-80 摄氏度。在24小时后将细胞转化为液氮, 贮存至可供使用。 注: MEFs 的全球基因表达通过 rna 测序 (rna 序列) 进行分离, 以确保细胞认证。从编码的 MEF 细胞的 RNA 序列数据下载从 NIH 基因表达综合 (GEO) 与加入编号 GSE93454。我们使用的 MEFs 的 RNA 序列基因表达数据被存放在 NIH 地理与加入编号 GSE71405。这两个数据集是根据常见的基因符号合并的。然后将表达式数据进行日志转换, 并生成 MEFs 和编码 MEFs 的表达式数据的散点图, 并与线性回归线(图 1A) 配合使用。使用此协议隔离的 MEFs 与其他实验室隔离的分子相似。</li>
</ol>2. 逆转录病毒的生产和 MEFs 的转导
注意: 本节中的所有步骤都必须在安全级别2的内阁中进行。由于本议定书采用逆转录病毒转导, 确保采取安全预防措施, 包括将含有10% 漂白剂的所有含病毒介质的废物处理至少20分钟. 有关重新编程的时间线, 请参阅图 1B 。
<ol><li>
利用铂 E (PE) 细胞系生产逆转录病毒酶
	<ol>
<li>在 pe 介质 (表 1) 中维护商业上可用的 pe 单元格, 其中包含 blasticidin 和嘌呤霉素选择标记, 以确保插科打诨-和可用于有效的逆转录病毒粒子生产的基因的表达 36.通道细胞每两天1:4 或1:5。注意防止细胞过度拥挤, 因为这样可以降低逆转录病毒的产量。</li>
		<li>在1天, 种子大约 5 x 106细胞每 10 cm 菜在增长的媒介 (表 1) 16-20 h 在转染之前 (参见表 2为电镀密度为其他标准细胞养殖盘大小)。轻轻地来回摇动盘子几次, 小心地放置在37摄氏度, 5% CO2孵化器, 以确保均匀电镀分布 (见图 1C为最佳电镀密度在转染之前)。 注: 在转染前, 请注意在培养基中无选择标记的平板 PE 细胞, 以确保含有生成逆转录病毒粒子的培养基不杀死 MEFs。</li>
		<li>在0天, 准备 DNA 转染。对于 10 cm 盘转染, 添加2µg 的每个重新编程因子-GATA4, Hand2, Mef2c, Tbx5, miR-1, miR-133 (GHMT2m)-到1.5 毫升管。 注: 请参阅表 2以扩展染其他标准细胞培养皿大小所需的 DNA 量。</li>
		<li>在一个单独的1.5 毫升管, 添加360µL 减少血清培养基。然后将36µL 转染试剂直接添加到减少的血清介质中。轻轻地拍打管子, 在室温下孵育5分钟。 注意: 为避免转染效率降低, 应小心地将转染试剂直接添加到减少的血清介质中。</li>
		<li>添加减少血清培养基/转染试剂混合物 GHMT2m DNA 鸡尾酒。轻轻地拍打管子, 在室温下孵育15分钟。不要旋涡。</li>
		<li>加入反应混合物滴状到 PE 细胞, 轻轻地涡流介质 10-15 s, 并放置在37°c, 5% CO2孵化器。 注: 根据制造商的数据, PE 电池的平均滴度为 106到 107传染性单位/毫升。此协议生成大约 2 x 106感染单位/毫升由 24 h 和 6 x 106感染单位/毫升 48 h 为平均教学语言4。如果需要, 转染 GFP/DsRed 也可用作转染/转导效率的替代品;转染效率预计将超过 90% 48 h (图 1C)。</li>
	</ol>
</li>
	<li>
MEFs 的转导
	<ol>
<li>在天 0, 外套菜用胶原蛋白和地方在37°c, 5% CO2孵化器为至少 2 h。</li>
		<li>从液态氮和解冻中去除 MEFs。板材 0.2 x 106细胞每60毫米盘在成长媒介。轻轻地摇板, 以确保均匀电镀分布和放置在孵化器 (见图 1C , 以最佳电镀密度和表 2的电镀密度为其他标准细胞培养皿大小)。 注: 这些电镀密度已经过多次分离 MEFs 测试;除非 MEFs 显示明显危及生存能力, 调整细胞播种密度是没有必要的。</li>
		<li>在1天, 24 小时后转染, 使用30毫升注射器收集由 PE 细胞产生的逆转录病毒培养基。将介质通过0.45 µm 孔大小的过滤器进入50毫升圆锥管。添加铵溴化物转染试剂, 最终浓度为6µg/毫升。小心地添加10毫升新鲜生长培养基到 PE 细胞, 通过吹打培养基上的培养基, 以防止细胞移位。</li>
		<li>从 MEFs 中吸取培养基, 并在每道菜中加入4毫升新鲜的逆转录病毒培养基。返回 MEFs 到孵化器。将10% 漂白剂添加到与病毒介质接触的所有材料中, 以中和逆转录病毒微粒。</li>
		<li>当天 2, 48 小时后转染, 重复步骤2.2.2 和2.2.3。PE 单元格在反转录病毒培养基的 2nd集合之后被丢弃。 注: 将10% 漂白剂添加到丢弃的 PE 细胞中, 至少20分钟, 以中和不需要的逆转录病毒。</li>
		<li>在3天, 吸入培养基从 MEFs 和补充4毫升 iCM 培养基 (表 1) 包含0.5 µM A-83-01, 一 TGF β I 型受体抑制剂。每2天补充包含 A-83-01 的 iCM 介质。 注意: 如果使用 GFP 作为转导控件, 则此时间点 (图 1C) 的表达式预计将超过90%。</li>
	</ol>
</li>
</ol>3. 心脏标记的流式细胞术
<ol><li>吸入介质从9天重新编程 MEFs 和洗涤 (1x) 与 1x PBS 去除死的细胞和残骸。</li>
	<li>添加1毫升0.25% 胰蛋白酶/EDTA 5 分钟在37˚C。用光镜和摇盘检查细胞;如果细胞保持连接, 孵化5分钟, 在37˚C, 直到细胞分离。</li>
	<li>在 1x PBS 中冲洗细胞, 5% 只血清和过滤通过70µm 孔细胞过滤器。</li>
	<li>离心机在 160 x g室温下3分钟, 从颗粒中抽出液体。 注: 为了增加细胞的产量, 留出大约50µL 液体在细胞颗粒之上。</li>
	<li>用500µL 1x PBS 冲洗细胞, 含 1% BSA。</li>
	<li>固定细胞与 0.2 mL/1 百万细胞固定溶液20分钟冰。</li>
	<li>添加1毫升的1x 冷烫发/洗涤缓冲。 注: 制造商的解决方案是10x。</li>
	<li>离心细胞在 160 x g 5 分钟在4°c 和吸入上清。</li>
	<li>重复步骤3.7 和3.8 额外的时间。在 2nd清洗之后, 直接进行下一步, 或将单元格保持在4摄氏度的一夜之间。</li>
	<li>100µL 5% 山羊血清和驴血清在室温下1x 烫发/洗涤缓冲器30分钟。</li>
	<li>添加100µL 稀释的原发抗体。 注: 对于本协议, 细胞用小鼠肌钙蛋白 T (1:200) 和小鼠α actinin (1:200) 标记, 以定量测定心脏小节关键成分阳性细胞的百分比。室温下孵育1小时的原发抗体。</li>
	<li>离心细胞在 160 x g为 5 m 在4°c, 吸入上清, 并且洗涤2x 与冰冷的烫发或洗涤缓冲。</li>
	<li>添加100µL 稀释二次抗体。 注: 对于本协议, 细胞被标记为抗鼠 647 nm 二次抗体 (1:200)。在端部结束管转子室温下孵育45分钟的二级抗体。用铝箔包裹管子来保护细胞不受光线的侵害。</li>
	<li>离心细胞在 160 x g为5分钟在4°c, 吸入上清, 并且洗涤2x 与冰冷的烫发或洗涤缓冲。</li>
	<li>在 PBS 中加入1毫升 1% BSA, 并立即执行资产管制。 注: 在执行资产管制计划时, 首先生成一个正向散射和侧向散射图, 使之成为栅极活细胞。或者, 使用商业上可用的活/死细胞染色之前, 修复细胞, 以确定活和死亡的细胞群体。</li>
</ol>4. 染色重新编程的 MEFs
<ol><li>吸入介质从14天重新编程 MEFs 和洗涤 (1x) 与1毫升1x 冰冷 PBS。 注: 对于染色, 在12个井板中进行重新编程, 以最小化抗体溶液体积。也可使用24井板;仅有一半以下卷。</li>
	<li>500µL 2% 多聚甲醛在室温下吸入和固定细胞, 10 分钟。</li>
	<li>吸入和洗涤细胞3倍与1毫升 1x PBS (每洗5分钟室温)。</li>
	<li>Permeabilize 细胞与500µL 0.2% 通透试剂在 PBS 为15分钟室温。</li>
	<li>在室温下, 在 PBS 中吸入和阻断500µL 10% 马血清中的30分钟。</li>
	<li>在室温下, 用稀释后的初级抗体的500µL 吸出并孵育1小时。 注: 对于本协议, 细胞用小鼠肌钙蛋白 T 或α actinin (每 1:400) 标记, 并与连接蛋白 43 (1:400) 或肌钙蛋白 i (1:400) 共同标记。</li>
	<li>吸入初级抗体和洗涤三倍1毫升 1x PBS (每洗5分钟室温)。</li>
	<li>500µL 稀释的二级抗体在室温下吸入和孵育1小时。 注: 对于本协议, 细胞被标记为抗鼠 555 nm 二次抗体 (1:800), 以识别肌钙蛋白 T 或α actinin 和抗兔 488 nm 二次抗体 (1:800), 以识别连接蛋白43或肌钙蛋白 i. 此外, 细胞核染色赫斯特 (1:10,000)。通过在黑暗中孵化来保护细胞不受光线的侵害。</li>
	<li>吸入二次抗体和洗涤三次与1毫升 1x PBS (每洗涤在室温下5分钟)。包装在箔板, 以保护细胞从光和存储在4摄氏度。</li>
	<li>图像使用三通道荧光显微术。</li>
</ol>5. 钙成像
注: 如果成像在10X 放大, 标准细胞培养皿和板材适合钙成像。成像在更高的放大倍数要求细胞被镀在玻璃盖玻片或玻璃底菜。
<ol><li>吸入介质并添加2毫升 (用于6井板) 改性 Tyrode 溶液 (140 毫米氯化钠, 5 毫米氯化钾, 1.8 毫米 CaCl2, 1 毫米氯化镁2, 10 毫米葡萄糖, 10 毫米 HEPES, 0.1% BSA, 和1% 丙酮酸, pH 7.4) 包含5µM Fura-2 AM 和 0.1% Pluronic F-127 到跳动 (D14 to D20) iCMs。孵育30分钟, 在37摄氏度。 注: 保护细胞免受光照, 防止荧光指示器淬火。</li>
	<li>洗涤细胞在2毫升 Tyrode 的解决方案30分钟室温, 以允许脱酯化的 Fura-2 AM。</li>
	<li>使用 Slidebook 5.5 Ca2 +成像软件的 340 nm 和 380 nm 的旋转磁盘共焦显微图像。在室温下进行成像。</li>
	<li>钙瞬变的计算使用荧光强度的比率在 340 nm 超过荧光强度在 380 nm。</li>
	<li>如果需要, 治疗 iCMs 与1或2µM 异丙肾上腺素或10µM 硝苯地平控制钙处理后成像基线钙瞬变。将化合物局部添加到单元格和图像中。</li>
</ol>

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作者没有什么可透露的。

本研究概述了一种高效的策略, 通过 GHMT2m 重编程因子结合抑制亲纤维化信号通路, 直接将成纤维细胞重新编程为功能 iCMs。通过流式细胞术、免疫荧光成像、钙成像和跳动细胞计数, 我们表明该协议中的大多数细胞都经过成功的重编程, 并采用 CM 血统的命运。我们以前已经表明, 添加抗纤维化化合物, 包括 TGF β I 型受体抑制剂 A-83-01 产生约6倍的数量增加殴打 iCMs 相比, 转导与 GHMT2m 单独, 表明亲纤维...

缺血性心脏病是美国的主要死因1。大约80万美国人每年经历第一次或复发性心肌梗塞 (MI)1。继 MI 后, 心肌细胞 (CMs) 的死亡和心肌纤维化, 由活化的心脏成纤维形成, 损害心脏功能2,3。由于成人 CMs4、5的再生能力较差,MI 后心力衰竭的进展很大程度上是不可逆转的。虽然目前的临床治疗缓慢疾病进展和降低未来心脏事件的风险6,7,8,9, 没有治疗逆转疾病进展由于无法重新生成 CMs 后梗塞10。新的细胞疗法正在出现, 以治疗患者继 mi. 令人失望的是, 迄今为止, 通过 mi 向心脏提供干细胞的临床试验显示了不确定的再生潜能11,12,13,14,15,16,17,18。
通过对四种转录因子的过度表达, 首次证明了由高桥 & Yamanaka 产生的人源性诱导多能干细胞 (hiPSCs), 为细胞治疗的新突破打开了大门19。这些单元格可以区分为所有三个胚芽层19, 并且以前已经显示了一些高效的用于生成大量 CMs 的方法, 其中有20、21。HiPSC 衍生的 cms (髋关节 cms) 提供了一个强大的平台, 研究 cardiomyogenesis, 可能有重要的影响, 修复心脏后损伤。然而, 髋关节 CMs 目前面临的翻译障碍, 由于关注畸胎瘤形成22, 其不成熟的性质可能是亲致心律失常23。将成纤维细胞重新编程成 hiPSCs 激发了人们对直接重新编程成纤维细胞的兴趣。大意et . 表明, GATA4、Mef2c 和 Tbx5 (GMT) 在成纤维细胞中的过度表达导致了心脏谱系的直接重新编程, 尽管效率低下的24。通过添加 Hand2 (GHMT)25, 改进了重新编程效率。自这些早期研究以来, 许多出版物表明, 改变重新编程因子鸡尾酒与其他转录因子26,27,28,29,染色质修饰符30,31, microRNAs32,33, 或小分子34导致改进的重新编程效率和/或诱导心肌样细胞的成熟 (iCMs).
在这里, 我们提供了一个详细的协议, 以产生 iCMs 从小鼠胚胎成纤维细胞 (MEFs) 高效。我们以前表明, GHMT 鸡尾酒是显着改善, 增加了 miR-1 和 miR-133 (GHMT2m), 并进一步改善, 当亲纤维化信号通路, 包括转化生长因子β (TGF β) 信号或与蛋白质激酶 (岩石) 信号通路被抑制35。使用该协议, 我们表明大约60% 的细胞表达心肌肌钙蛋白 T (cTnT), 约50% 表达α actinin, 和大量的殴打细胞可以观察到11天后, 后再编程因素和治疗转导与 TGF β型 I 受体抑制剂 A-83-01。此外, 这些 iCMs 的表达间隙连接蛋白包括 43, 并呈现自发收缩和钙瞬变。与早期研究相比, 重新编程效率的显著提高表明了在脑梗塞后的内源细胞群中再生 CMs 的潜力。

<table border="0" cellpadding="0" cellspacing="0" width="1326">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">C57BL/6 Mice</td>
			<td style="width:197px;">Charles River&#39;s Laboratory</td>
			<td style="width:111px;">027</td>
			<td style="width:667px;">For MEF isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum E (PE) Cells</td>
			<td>Cell Biolabs, INC</td>
			<td>RV-101</td>
			<td>For retrovirus production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose</td>
			<td>Gibco</td>
			<td>SH30022.FS</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium 199</td>
			<td>Life Technologies</td>
			<td>11150-059</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gemini</td>
			<td>100106</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donor Horse Serum</td>
			<td>Gemini</td>
			<td>100508 500</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Essential Amino Acids, 50X</td>
			<td>Life Technologies</td>
			<td>11130051</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Sodium Pyruvate Solution, 100X</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td>Component of iCM media and for calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Non-Essential Amino Acids, 100X</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Vitamin Solution, 100X</td>
			<td>Life Technologies</td>
			<td>11120-052</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Insulin-Transferrin-Selenium</td>
			<td>Gibco</td>
			<td>41400045</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Penicilin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">GlutaMAX (L-Glutamine Supplement)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin-HCl</td>
			<td>Life Technologies</td>
			<td>A11139-03</td>
			<td>Component of PE media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Puromycin dihydrochloride</td>
			<td>Life Technologies</td>
			<td>A11138-03</td>
			<td>Component of PE media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">0.25% Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>For detaching cells from culture dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A-83-01</td>
			<td>R&#38;D Systems - Tocris</td>
			<td>2939/10</td>
			<td>Treat cells to inhibit TGF-&#946; signaling - promotes high efficiecy reprogramming. Use at 0.5 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Thermo Scientific</td>
			<td>85190</td>
			<td>For dilution and storage of A-83-01 and component of Freeze Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SureCoat</td>
			<td>Cellutron</td>
			<td>SC-9035</td>
			<td>For coating dishes to plate MEFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FuGENE 6 Transfection Reagent</td>
			<td>Promega</td>
			<td>E2692</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Media</td>
			<td>Gibco</td>
			<td>11058-021</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-GATA4</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Hand2</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Mef2c</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Tbx5</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X miR-1</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X miR-133</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X GFP</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polybrene (Hexadimethrine bromide)</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
			<td>For viral induction. Use at a concentration of 6 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Vacuum Filter + bottles (0.22 &#181;m pores)</td>
			<td>Nalgene</td>
			<td>&#160;569-0020&#160;</td>
			<td>For filtering media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringes</td>
			<td>Bd Vacutainer Labware&#160;</td>
			<td>309654</td>
			<td>For viral filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#181;m Filters</td>
			<td>Celltreat</td>
			<td>229749</td>
			<td>For viral filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m cell strainers</td>
			<td>Falcon&#160;</td>
			<td>352350</td>
			<td>For MEF isolation and Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Cytofix/Cytoperm Solution</td>
			<td>BD</td>
			<td>554722</td>
			<td>For fixation and permeabilization of cells for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">perm/wash buffer&#160;</td>
			<td>BD</td>
			<td>554723</td>
			<td>For washing cells for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS 1X</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td>For washing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>VWR</td>
			<td>0332-100g</td>
			<td>For flow cytometry and calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum</td>
			<td>Sigma&#160;</td>
			<td>G9023</td>
			<td>For blocking cells for Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey Serum</td>
			<td>Sigma</td>
			<td>D9663-10mg</td>
			<td>For blocking cells for Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Troponin T</td>
			<td>Thermo Scientific</td>
			<td>ms-295-p</td>
			<td>1:400 IF, 1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse &#945;-actinin</td>
			<td>Sigma</td>
			<td>A7811L</td>
			<td>1:400 IF, 1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit Connexin 43</td>
			<td>Sigma&#160;</td>
			<td>C6219</td>
			<td>1:400 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit Troponin I</td>
			<td>PhosphoSolutions</td>
			<td>2010-TNI</td>
			<td>1:400 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Life Technologies</td>
			<td>62249</td>
			<td>1:10000 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488, rabbit</td>
			<td>Life Technologies</td>
			<td>A-11034</td>
			<td>1:800 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 555, mouse</td>
			<td>Life Technologies</td>
			<td>A-21422</td>
			<td>1:800 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 647, mouse</td>
			<td>Life Technologies</td>
			<td>A-31571</td>
			<td>1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">27-color ZE5 Flow Cytometer&#160;</td>
			<td>Bio-RAD</td>
			<td></td>
			<td>For FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>sigma</td>
			<td>P6148-500mg</td>
			<td>For fixing cells for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td>For permeabilization of cells for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOS&#8482; FL Color Imaging System</td>
			<td>Thermo Scientific</td>
			<td>AMEFC4300</td>
			<td>For IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>RPI</td>
			<td>S23020-5000</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR</td>
			<td>395</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2</td>
			<td>Fisher</td>
			<td>C614-500</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td>VWR</td>
			<td>97061-352</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glucose</td>
			<td>sigma</td>
			<td>G7528-250g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>sigma</td>
			<td>H4034-500g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fura-2 AM</td>
			<td>Life Technologies</td>
			<td>F1221</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nifedipine</td>
			<td>Sigma</td>
			<td>N7634-1G</td>
			<td>For disruption calcium transients in iCMs - use at 10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoproterenol</td>
			<td>sigma</td>
			<td>I6504-1g</td>
			<td>For increasing number of calcium transients in iCMs - use at 1-2 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Marianas Spinning Disk Confocal microscope</td>
			<td>3i</td>
			<td></td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol</td>
			<td>Decon Laboratories</td>
			<td>2801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bleach</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tubes</td>
			<td>GREINER BIO-ONE</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical tubes</td>
			<td>GREINER BIO-ONE</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm cell culture dishes</td>
			<td>Falcon</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm cell culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm cell culture dishes</td>
			<td>GREINER BIO-ONE</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>657160&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>665180&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>662160&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

suppression of pro-fibrotic signaling potentiates factor-mediated reprogramming of mouse embryonic fibroblasts into induced cardiomyocytes

将一种体细胞类型转化为另一种躯体细胞, 具有巨大的模型和治疗人类疾病的潜力。以前的研究表明, 小鼠胚胎, 真皮和心脏成纤维细胞可以重新编程成功能性诱发心肌样的干细胞 (iCMs) 通过过度表达的心源性转录因子, 包括 GATA4, Hand2, Mef2c, 和Tbx5体外和体内。然而, 这些先前的研究显示效率相对较低。为了恢复损伤后的心脏功能, 必须阐明调节心脏重新编程的机制, 以提高 iCMs 的效率和成熟度。
我们以前证明, 抑制亲纤维化信号显着提高重新编程效率。在这里, 我们详细的方法来实现编程效率高达60%。此外, 我们描述了几种方法, 包括流式细胞术, 免疫荧光成像, 钙成像, 以量化重新编程效率和成熟的重组成纤维细胞。通过这里详细的协议, 可以进行机械研究, 以确定积极和消极的调节心脏重新编程。这些研究可以确定信号通路, 可针对促进重新编程的效率和成熟, 这可能导致新的细胞治疗人类心脏病。

使用上面概述的重新编程策略和在图 1B中, 我们生成的 iCMs 约70% 的细胞表达心肌肌钙蛋白 T 和大约55% 细胞表达心脏α actinin, 量化的流式细胞术在9天跟随 GHMT2m 的转导 (图 2A和B)。此外, 大多数细胞表达心肌肌钙蛋白 T, 肌钙蛋白 i, 和心脏α actinin 以及缝隙连接标志联结蛋白43在14天后转导 (图 2C和...

Watch this Scientific Journal Video about Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes at JoVE.com

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

通过 GATA4、Hand2、Mef2c、Tbx5、miR-1 和 miR-133 (GHMT2m) 的过度表达和 TGF β信号的抑制, 我们提出了一种健壮的方法, 将原发成纤维细胞重新编程成功能性心肌细胞。我们的协议在7天后的转导中产生跳动的心肌细胞, 效率高达60%。

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

suppression-pro-fibrotic-signaling-potentiates-factor-mediated

Research

JoVE Journal

Developmental Biology

7.2K 次观看.  University of Colorado Anschutz Medical Campus. 通过 GATA4、Hand2、Mef2c、Tbx5、miR-1 和 miR-133 (GHMT2m) 的过度表达和 TGF β信号的抑制, 我们提出了一种健壮的方法, 将原发成纤维细胞重新编程成功能性心肌细胞。我们的协议在7天后的转导中产生跳动的心肌细胞, 效率高达60%。

视频: Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

科研

发育生物学

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

诱发心肌细胞的使用多顺反子构建表达GATA4，MEF2C和Tbx5中的最优比率改善发电

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

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

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

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

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

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

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

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 ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

小鼠胚胎肾的解剖与培养

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...