Name: derivation of stem cell lines from mouse preimplantation embryos
Uploaded: 2017-08-20T13:00:00.000+00:00
Duration: 12 min 59 s
Description: Watch this Scientific Journal Video about Derivation of Stem Cell Lines from Mouse Preimplantation Embryos at JoVE.com

我们感谢 Jonatan 卢卡斯他与馈线细胞培养，Servei Estabulari de la UAB 为动物保健和 Domènec Martín 为录制视频的技术援助。这项工作得到了 Ministerio 德经济 y 与两性 AGL2014-52408-R 和自治区政府大楼和加泰罗尼亚 2014 SGR 524。MVC 是 PIF UAB 研究金的受益人。

使用以下各节所述的动物已被批准，由动物和人类研究的大学 Autònoma de 巴塞罗那伦理委员会和部 d &#39; 农业，Ramaderia，以上我Alimentació 的加泰罗尼亚 （协议 #8741）。
 1.馈线细胞失活和存储 注: 包皮成纤维细胞 (h f F-1) 以前被冰冻住的冷冻液组成的 90%胎牛血清 (FBS) 和 10%二甲基亚砜 （1 毫升的二甲基亚砜），直到使用存储在液态氮。
<ol><li>解冻 h f F-1 （1 毫升） 低温瓶浸泡在 37 ° C 水浴瓶。</li>
	<li>将低温瓶解冻的内容添加到预热杜尔贝科 &#39; s 改良鹰 &#39; s 介质 (DMEM) 辅以 10%胎牛血清，使 1:10 稀释。</li>
	<li>种子在 T75 瓶 （10 毫升） 稀释的细胞和培养他们在 37 ° C，5%CO 2。</li>
	<li>第二天，改变介质以消除二甲基亚砜遗留物。删除介质和添加 10 毫升的预热 DMEM 辅以 10 %fbs。</li>
	<li>文化 HFFs 和改变介质每 48 小时直到文化达到汇合的阶段。这可能需要大约 7 到 10 天。</li>
	<li>制备灭活溶液，稀释丝裂霉素 C 在 DMEM 辅以 10%胎牛血清在终浓度为 10 微克/毫升。孵育细胞在 37 ° C，5%CO 2 3 h 的灭活解决方案。</li>
	<li>删除灭活解决方案和冲洗三次用 2 毫升的汉克斯平衡盐溶液 (HBSS) 消除丝裂霉素 C 残余细胞。</li>
	<li>要分离的灭活的单元格，添加 1 毫升的预热胰蛋白酶-EDTA 溶液和孵育 5 分钟在 37 ° C，5%CO 2。</li>
	<li>观察下倒置显微镜观察，以确保它们是分离的细胞 （10 x 目标与 0.3 的数值孔径或 NA）。如果他们不是，则把他们孵育几分钟在 37 ° C，5%CO 2。检查再下倒置显微镜观察细胞。</li>
	<li>以游离细胞块与巴斯德吸管吸管解决方案。</li>
	<li>消除酶解液加入 4 毫升的 DMEM 辅以 10%胎牛血清和重悬。</li>
	<li>细胞悬液分装，在 HBSS 让 1: 100 稀释。要确定细胞数目，加载的稀释 10 微升到 Neubauer &#39; s 分庭。位于倒置显微镜 （20 x 目标与 0.45 NA） 下室的大广场之一的单元格进行计数。
	<ol><li>重复计数总数的四个大正方形。计算总数的细胞 (N) 为 N = 单元格的平均数目计数总体积稀释 x 10 4 x.</li>
	</ol></li> <li>离心上清液的样本 200 x g.丢弃在 5 分钟的休息和重悬与冻结溶液浓度为每毫升 10 6 细胞颗粒。</li>
	<li>悬浮在低温与 10 6 个细胞 （1 毫升） 每瓶分装。低温瓶置于冻结的容器和冻结在-80 ° C.</li>
	<li>第二天，在液氮中存储低温瓶。</li>
</ol> 2。进纸器细胞培养 <ol><li>1 至 4 天前开始的推导过程，解冻低温每小瓶含 1 x 10 6 灭活 h f F 1 细胞在水浴在 37 ° C.</li>
	<li>使在解冻的 h f F 1 1:6 稀释预热 DMEM 辅以 10%胎牛血清和保持温度在 37 ° C.</li>
	<li>填充的预热 0.2%明胶从猪皮 500 微升 4 井板每个井和保持在 37 ° C 10 分钟板</li>
	<li>从水井中删除明胶 500 微升和添加的 HFFs 稀释 500 µ L。轻轻摇动板以确保 HFFs 井底部均匀分布。避免循环动作，否则细胞将集中在井中心。</li>
	<li>孵育至少 24 h 在 37 ° C，5%CO 2 获得在每口井的灭活 HFFs 单层板。</li>
	第二天，<li>（10 X 目标与 0.3 NA） 倒置显微镜下观察细胞和检查，他们附加分布均匀。更改来消除二甲基亚砜遗留的媒介。去除培养基，并添加 500 µ L 的预热 DMEM 辅以 10 %fbs。</li>
</ol> 3。胚胎收藏与文化 <ol><li>四天前胚胎集合管理 5 IU 的怀孕母马 &#39; 到 6-12 周老雌性小鼠腹腔注射血清促性腺激素 s. 
	注意: 我们使用女性从 129S2 或 B6CFAF1 株，虽然可以使用从其他小鼠品系的女性。然而，当与女性从非许可菌株 （如 CBA），工作介质中应补充 2i 4.2 步所述。</li>
	<li>后 48 h，通过腹腔注射，管理 5 IU 人绒毛膜促性腺激素和交配的女性与男性，至少 8 周龄，在 1:1 的比例。女性分开男性第二天。</li>
	<li>胚胎集合前, 一天准备 35 毫米细胞培养用几个 40 µ L 滴 4 毫克/毫升牛血清白蛋白 (BSA) KSOMaag 培养基的皮氏培养皿和填充板是用矿物油，完全涵盖滴。孵化在 37 ° C，5%CO 2 平衡介质盘。</li>
	<li>准备胚胎操作移液器，热最细部分的长尖的玻璃巴斯德吸管使用喷灯。一旦软，撤出的火焰，并拉开两个端点。打破它到所需长度。顶端内径应该是 100-150 微米。</li>
	<li>对雌性小鼠颈椎脱位 45-48 小时 后 coitum (p.c.) 通过实施安乐死。</li>
	<li>解剖小鼠暴露腹腔内通过削减皮肤与曲线光滑锋利的剪刀和腹膜与直锋利的剪刀。抓住一个子宫钳夹、 切断输卵管用直锋利的剪刀，并将其置于复缓冲 CZB 介质 (HCZB) 13 37 ° C.重复上述过程，删除其他输卵管。</li>
	钟表匠钳收集 2-细胞胚胎在地方举办的 <li>同花顺 1 毫升注射器与输卵管装有 HCZB. 
	注: 准备冲洗针取 30 G 皮下注射针头，结束时，切到钝尖上磨料的石头地面。</li>
	<li>使用嘴移液器装置，拿出胚胎，洗他们的 HCZB 并培养他们在事先准备好的胚胎培养皿在 37 ° C，5%CO 2 至囊胚阶段。50 胚胎可以有多达体外每一 40 µ L 滴。胚胎发育在体视显微镜下每隔 24 小时监视。 
	注意: 另外，胚胎可以收集在胚泡期缩短 离体 胚培养。在这种情况下，对实施安乐死的女性在一天 3.5-4.5 邮编、 解剖子宫。和用 HCZB 冲洗。</li>
</ol> 4。干细胞衍生 <ol><li>的推导板制备 <ol><li>日的胚胎播种，准备推导介质通过补充 DMEM 培养基与 100 µ M 2 β 巯基乙醇，1 x 非必需氨基酸20%的血清替换和 10 3 U/mL LIF。</li>
		<li>如果需要，还添加 1 µ M 的 MEK 抑制剂 PD0325901 和 3 µ M 的 GSK3 抑制剂 CHIR99021 以获得 2i 媒介。</li>
		<li>带的灭活 HFFs （步骤 2.6） 从单层 4 井板孵化器。删除介质并将 800 µ L 的预热的推导介质添加到每个井。孵化器返回板。</li>
	</ol></li> <li>卵透明带去除和胚胎播种 <ol><li>准备 35 毫米细胞培养用酸性浸入三 30 微升滴培养皿 &#39; s 解决方案 14 和推导介质三 30 微升滴。盖与矿物油滴和保持这道菜在 37 ° C.</li>
		<li>从孵化器采取胚胎培养皿。一个接一个地从文化滴转移囊胚到酸性浸入 &#39; s 溶液降使用胚胎操作移液管。如果可能的话，选择只扩张囊胚开始的推导过程。</li>
		在体视显微镜下的 <li>显示器卵透明带消化。尽快卵透明带消失，转，囊胚推导中滴以洗出酸性浸入 &#39; s 解决方案。</li>
		<li>以每口井 4 井推导板孵化器和种子一无卵囊胚上料机从细胞。</li>
		<li>返回的孵化器和文化在 37 ° C，5%CO 2 4 井推导板。</li>
	</ol></li> <li>监测干细胞生长和介质改变 <ol><li>监测文化 （20 X 目标与 0.45 NA） 倒置显微镜下每隔 24 小时。很重要的是要注意是否有迹象表明干细胞分化，如周边产物或甚至推开送纸器细胞的肿胀屈光细胞。</li>
		<li>每隔一天，改变文化的媒介。4 井板从孵化器，从每个井，去除培养基，并添加 800 µ L 的预热的推导 LIF 和 2i，培养基，如果使用。保持文化，直到植物突起观察 （6-8 天）。</li>
	</ol></li></ol> 5。干细胞培养和维护 <ol><li>准备 · 科列 · 像控点和干细胞操纵式吸液管。遵循步骤 3.4 准备干细胞操纵吸液管内径顶端的 250-300 µ m.使用被拉扯的巴斯德吸管的另一部分准备 · 科列 · 像句柄。热和形状直到获得一个钩子。</li>
	<li>采取 4 井文化板在孵化器中的，定位在体视显微镜下的产物。使用该句柄推开分化的细胞和环绕副产物馈线。 
	注意: 避免最终分化的产物，是重要的是从边缘的副产物中移除所有分化的细胞。</li>
	<li>产物与干细胞操纵吸管吸出并将其放在 50 µ L 胰蛋白酶-EDTA 滴一道新菜。</li>
	<li>用手术刀，分列其机械和酶切产物中几个部分。</li>
	<li>马上，将产物片段转移到新鲜滴推导介质来抵消胰蛋白酶行动。</li>
	<li>将产物片段转移到新 4 孔板与单层馈线。转让每井达 10 碎片和培养他们在 37 ° C，5%CO 2 推导介质。</li>
	<li>至少六个星期前考虑设立的卓线保持干细胞样文化。更改介质其他亚文化和每天他们一周一次。</li>
</ol>

<ol>
	<li>Biswas, A., Hutchins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells.">Embryonic stem cells.</a> Stem Cell Dev. 16 (2), 213-221 (2007).</li><li>Weinberger, L., Ayyash, M., Novershtern, N., Hanna, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+stem+cell+states:+naive+to+primed+pluripotency+in+rodents+and+humans.">Dynamic stem cell states: naive to primed pluripotency in rodents and humans.</a> Nat Rev Mol Cell Biol. 17 (3), 155-169 (2016).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc Nat Acad Sci U S A. 78 (12), 7634-7638 (1981).</li><li>Martin, G. R., Evans, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+clonal+lines+of+teratocarcinoma+cells:+formation+of+embryoid+bodies+in+vitro.">Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro.</a> PNAS. 72 (4), 1441-1445 (1976).</li><li>Czechanski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+characterization+of+mouse+embryonic+stem+cells+from+permissive+and+nonpermissive+strains.">Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains.</a> Nat Prot. 9 (3), 559-574 (2014).</li><li>Smith, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+pluripotential+embryonic+stem+cell+differentiation+by+purified+polypeptides.">Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.</a> Nature. 336 (6200), 688-690 (1988).</li><li>Williams, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336 (6200), 684-687 (1988).</li><li>Brook, F. A., Gardner, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+efficient+derivation+of+embryonic+stem+cells+in+the+mouse.">The origin and efficient derivation of embryonic stem cells in the mouse.</a> Proc Nat Acad Sci U S A. 94 (11), 5709-5712 (1997).</li><li>Ward, C. M., Stern, P., Willington, M. A., Flenniken, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+germline+transmission+of+mouse+embryonic+stem+cells+grown+in+synthetic+serum+in+the+absence+of+a+fibroblast+feeder+layer.">Efficient germline transmission of mouse embryonic stem cells grown in synthetic serum in the absence of a fibroblast feeder layer.</a> Lab Invest. 82 (12), 1765-1767 (2002).</li><li>Ying, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453 (7194), 519-523 (2008).</li><li>Wakayama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+establishment+of+mouse+embryonic+stem+cell+lines+from+single+blastomeres+and+polar+bodies.">Efficient establishment of mouse embryonic stem cell lines from single blastomeres and polar bodies.</a> Stem cells (Dayton, Ohio). 25 (4), 986-993 (2007).</li><li>Chatot, C. L., Ziomek, C. A., Bavister, B. D., Lewis, J. L., Torres, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+culture+medium+support+development+of+random-bred+1-cell+mouse+embryos+in+vitro.">An improved culture medium support development of random-bred 1-cell mouse embryos in vitro.</a> J Reprod Fertil. 86, 679-688 (1989).</li><li>Nagy, A., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2003).</li><li>Lee, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+and+techniques+for+mouse+embryonic+stem+cell+derivation+and+culture.">Conditions and techniques for mouse embryonic stem cell derivation and culture.</a> Pluripotent Stem Cells. , 85-115 (2013).</li><li>Ma, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+foreskin+fibroblast+produces+interleukin-6+to+support+derivation+and+self-renewal+of+mouse+embryonic+stem+cells.">Human foreskin fibroblast produces interleukin-6 to support derivation and self-renewal of mouse embryonic stem cells.</a> Stem Cell Res Ther. 3 (29), (2012).</li><li>Cheng, J., Dutra, A., Takesono, A., Garrett-Beal, L., Schwartzberg, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+generation+of+C57BL/6J+mouse+embryonic+stem+cells+in+a+defined+serum-free+media.">Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media.</a> Genesis. 39 (2), 100-104 (2004).</li><li>Chaudhry, M. A., Vitalis, T. Z., Bowen, B. D., Piret, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+medium+composition+and+serum+or+serum+replacement+concentration+influences+on+the+maintenance+of+murine+embryonic+stem+cells.">Basal medium composition and serum or serum replacement concentration influences on the maintenance of murine embryonic stem cells.</a> Cytotechnology. 58 (3), 173-179 (2008).</li><li>Kawase, E., Suemori, H., Takahashi, N., Okazaki, K., Hashimoto, K., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+difference+in+establishment+of+mouse+embryonic+stem+(ES)+cell+lines.">Strain difference in establishment of mouse embryonic stem (ES) cell lines.</a> Int. J. Dev. Biol. 38 (2), 385-390 (1994).</li><li>Gonzalez, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+culture+conditions+support+distinct+states+associated+with+different+developmental+stages+and+potency.">Embryonic stem cell culture conditions support distinct states associated with different developmental stages and potency.</a> Stem Cell Rep. 7 (2), 177-191 (2016).</li></ol>

作者没有透露。

虽然卓推导出来的线是广为人知的程序经常使用在许多实验室，其效率并不总是可以打扰全能维护的多因素预期那么高。在本文中我们表明，一步一步走，如何与高效率使用简单和可靠的协议建立卓行。这些效率相近的文献报道。
为了确保最高的效率，是重要的是控制细胞每日或每隔一天，从时间点表示只是说明性。至关重要的是，要避免过度的殖民地，因为这会促进细胞?...

胚胎干细胞 (ESC) 是多能干细胞来自胚胎植入前胚胎，保留自我更新或特定条件1下区分的能力。基于这些属性，按 esc 键已成为再生医学、 疾病建模和组织工程研究2有用工具。
按 ESC 取自于胚胎植入前胚胎，源自鼠标 ESC （卓） 线与低成功3，4第一次。多年来，推导效率仍然很低难以维持体外多向分化潜能。此外的进纸器细胞5，提高工作效率推导，存在促进殖民地附件和提高染色体稳定性6，全能维修改进协议导致的几种修改。最重要是加法白血病抑制因子 (LIF) 到卓培养基7，8，使用 129S2 宽松背景9从胚胎和卓与培养基的文化定义血清，免费的潜在分化因素10。最近，令人信服的证据表明，另外的两个小分子调节的信号传导通路在卓培养基 （称为 2i） 鸡尾酒是最好的保持多向分化潜能。2i 鸡尾酒由 MEK 抑制剂 PD0325901，减少分化信号抑制有丝分裂原活化蛋白激酶 (MAPK) 通路与都可诱发 gsk3 抑制剂 CHIR99021，增强了细胞存活在低密度的结合通过激活 Wnt 途径11。
在本文中，我们描述了一个简单的协议，有效地将卓行从小鼠囊胚。我们按照标准程序，培养从宽容菌株对 LIF 和血清更换12推导培养基中的饲养细胞的胚胎。按照此协议，卓行可以得到效率相当于那些获得 2i 介质中。尽管这样，可以添加 2i，以避免可能出现的问题，具有多向分化潜能维修或使用非许可株时。

<table><tbody><tr><td>1 mL sryringe</td><td>BD&amp;nbsp;</td><td>309628</td><td></td></tr><tr><td>2-&amp;beta;-mercaptoethanol</td><td>Life Technologies</td><td>31350-010</td><td></td></tr><tr><td>4-well plate</td><td>Thermo Scientific Nunc</td><td>176740</td><td></td></tr><tr><td>Acidic Tyrode&#039;s solution&amp;nbsp;</td><td>Home-made</td><td></td><td>See recipe in Nagy et al, 2003. For short-term storage (up to 1 month) keep it at 4&amp;ordm;C. Alternatively, for long-term storage keep it at -20&amp;ordm;C. If the efficiency of the solution diminishes, it should be acidified by the adition of a small drop of HCl 1M.&amp;nbsp;</td></tr><tr><td>BSA</td><td>Sigma</td><td>A3311</td><td></td></tr><tr><td>Chicken anti-Mouse IgG, Alexa Fluor 488</td><td>Molecular Probes - Invitrogen</td><td>A-21200</td><td>1:500 dilution. Secondary antibody for Oct4, Tuj1, &amp;alpha;SMA and AFP mouse antibodies.&amp;nbsp;</td></tr><tr><td>CHIR990212</td><td>Axon Medchem&amp;nbsp;</td><td>1386</td><td>Reconstitute with 2.15 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20&amp;ordm;C. Avoid freeze and thaw cycles.&amp;nbsp;</td></tr><tr><td>CryoTube</td><td>Thermo Scientific Nunc</td><td>3754518</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>41640</td><td></td></tr><tr><td>DMEM</td><td>BioWest</td><td>L0107</td><td></td></tr><tr><td>FBS</td><td>BioWest</td><td>S1810</td><td>Inactivate it prior to use by heating for 30 mins at 56&amp;ordm;C. Aliquot and store at -20&amp;ordm;C</td></tr><tr><td>Flushing needle</td><td>BD&amp;nbsp;</td><td>304000</td><td>Cut the end of the needle and ground to a blunt tip on an abrasive stone</td></tr><tr><td>Gelatin from porcine skin</td><td>Fluka</td><td>48724</td><td>Dilute at 0,2% in destilled water and autoclave it. Each dilution can be used for a month.</td></tr><tr><td>Goat anti-Rabbit IgG, Alexa Fluor 594</td><td>Molecular Probes - Invitrogen</td><td>A-11037</td><td>1:500 dilution. Secondary antibody for Sox2 rabbit antibody.&amp;nbsp;</td></tr><tr><td>HBSS</td><td>BioWest</td><td>L0611</td><td></td></tr><tr><td>Hepes-buffered CZB&amp;nbsp;</td><td>Home-made</td><td></td><td>See composition in Chatot et al, 1989</td></tr><tr><td>Human chorionic gonadotropin</td><td>Divasa-Farmavic</td><td>Veterin-Corion 3000 UI</td><td>Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&amp;ordm;C for up to two months. Once thawed do not freeze again.&amp;nbsp;</td></tr><tr><td>Human foreskin fibroblasts</td><td>ATCC</td><td>ATCCSCRC-1041</td><td>For long term storage it is recommended to store them in liquid nitrogen.</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>&amp;nbsp;10828-028</td><td>Photosensitive. It is recommended to aliquot it in small volumes such as 10 ml and store it at -20&amp;ordm;C (check the expiration date). Avoid freeze and thaw cycles.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>KSOMaag Evolve</td><td>Zenith Biotech</td><td>ZEKS-050</td><td>Supplement with 4 mg/ml BSA. Equilibrate at 37&amp;ordm;C and 5% CO2 a day prior to use.</td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Merk Millipore</td><td>ESG1106</td><td></td></tr><tr><td>Mice&amp;nbsp;</td><td>Charles River/Harlan</td><td></td><td>It is recommended to use mice from a permissive strain in terms of mESC derivation (such as 129S2 or C57BL). However, inbreed strains are less efficient producing embryos. Therefore, it is recomended to use a hybrid strain, such as 129S2 x C57BL or B6CBAF1 to take advantage of the hybrid vigor.&amp;nbsp;</td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M8410</td><td>Embryo tested. Photosensitive.&amp;nbsp;</td></tr><tr><td>Mitomycin C</td><td>Serva</td><td>2980501</td><td>Reconstitute the powder with MilliQ water at 0.5 mg/ml. Store at 4&amp;ordm;C protected from light. Attention, it is harmful and suspected of causing cancer.&amp;nbsp;</td></tr><tr><td>Mouse anti-tubulin &amp;beta; III (Tuj1)</td><td>Biolegend</td><td>MMS-435P</td><td>1:500 dilution</td></tr><tr><td>Mouse monoclonal anti-&amp;alpha;SMA</td><td>Sigma</td><td>A5228</td><td>1:200 dilution</td></tr><tr><td>Mouse monoclonal anti-AFP</td><td>R&amp;amp;D Systems</td><td>MAB1368</td><td>1:50 dilution</td></tr><tr><td>Mouse monoclonal anti Oct3/4&amp;nbsp;</td><td>Santa cruz</td><td>Sc-5279</td><td>1:50 dilution</td></tr><tr><td>Non-Essential Amino Acids</td><td>Life Technologies</td><td>11140-035</td><td></td></tr><tr><td>PD0325901</td><td>Axon Medchem&amp;nbsp;</td><td>1408</td><td>Reconstitute with 2.08 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20&amp;ordm;C. Avoid freeze and thaw cycles.</td></tr><tr><td>Petri dish (35mm)</td><td>Thermo Scientific Nunc</td><td>153066</td><td></td></tr><tr><td>Petri dish (60mm)</td><td>Thermo Scientific Nunc</td><td>150288</td><td></td></tr><tr><td>Pregnant mare&#039;s serum gonadotropin</td><td>Foligon</td><td>Foligon-1000 UI</td><td>Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&amp;ordm;C for up to two months. Once thawed do not freeze again.&amp;nbsp;</td></tr><tr><td>Rabbit policlonal anti-Sox2</td><td>Merck Millipore</td><td>AB5603</td><td>1:200 dilution</td></tr><tr><td>T75 falcon</td><td>Thermo Scientific</td><td>130190</td><td></td></tr><tr><td>Trypsin-EDTA 10x</td><td>BioWest</td><td>X0930</td><td>Dilute 1:10 in HBSS to obtain a 1x solution</td></tr></tbody></table>

derivation of stem cell lines from mouse preimplantation embryos

小鼠胚胎干细胞 （卓） 推导是哪个多能干细胞系建立了从胚胎植入前胚胎的过程。这些行保留自我更新或区分在特定条件下的能力。由于这些特性，卓在再生医学、 疾病建模和组织工程研究中是一个有用的工具。这篇文章描述了一个简单的协议，以获得高推导效率 （60-80%) 的卓行通过培养从宽容小鼠品系对白血病抑制因子定义培养基中的饲养细胞的囊胚。议定书 》 还可有效地将卓行从非许可的小鼠品系，通过简单的加法到推导介质 （2i 中等） 的两种小分子抑制剂的鸡尾酒。提供了制备及文化的饲养层细胞，收集和小鼠胚胎和推导的文化和文化的卓行的详细的过程。该协议不需要专门的设备，可以在任何与基本的哺乳动物细胞培养的专门知识的实验室进行。

继本议定书中，超过 95%的产物形成应该达到后第一周的文化 (图 1A)。卓系后六通道的建立是变量实验复制，与平均成功的 60-80%之间。2i 的加法不能显著改善结果时使用许可小鼠品系，例如那些与 129S2 的背景，但它是必要的当工作与非许可的菌株。
鼠标按 ESC 殖民地应该与扁平的形状呈现形态规则和定义...

Watch this Scientific Journal Video about Derivation of Stem Cell Lines from Mouse Preimplantation Embryos at JoVE.com

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

从小鼠胚胎植入前胚胎干细胞系的推导

这篇文章描述了一个协议，以有效地获得和囊胚阶段文化从小鼠胚胎多能干细胞系。

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

derivation-of-stem-cell-lines-from-mouse-preimplantation-embryos

Research

JoVE Journal

Developmental Biology

8.3K 次观看.  Universitat Autònoma de Barcelona. 这篇文章描述了一个协议，以有效地获得和囊胚阶段文化从小鼠胚胎多能干细胞系。

视频: Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

发育生物学

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

鼠标滋养层干细胞从胚泡的推导

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

应用小鼠部分遗传类胚胎干细胞作为精子的替代品

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

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 ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

细胞标记和注射胚胎发展小鼠心脏

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...