这项工作是由SMM的补助：果酸GIA0855953G，NIH 1R01 HL091105 - 01。提供了额外的支持：德国之声：国立卫生研究院RO1DK075013，DDK和Kleberg基金会的UCCC流式细胞仪核心研究院（NIH 5 P30的CA 46934-15），UCCC芯片的核心（NCI的P3​​0的CA 46934-14）。

1。肺隔离<ol><li>牺牲成年小鼠（8-10周的年龄; C57Bl6J 18-20克）小鼠异氟醚的过量使用无菌放血。资料会略有不同菌株之间。 </li><li>手术取出膜片，打开胸腔，通过鼠标两侧的肋骨横向切割。轻轻地使用磷酸盐3 5mls灌流右心室的血液从肺部冲洗缓冲液（PBS）。 </li><li>解剖肺叶用Hanks填补了培养皿中删除气管，大支气管和地方缓冲液（HBSS）。以下收集所有肺癌肺叶钳盘的盖子放到组织。然后组织到足够的液体一次性使用手术刀，保持湿​​润，但没有这么多，他们漂浮和移动的小块肉末。 </li><li>解剖小鼠后肢和孤立的骨髓染色控制使用设置FLO瓦特仪门。染色的骨髓（BM）作为先前描述1。 </li></ol> 2。从肺组织单细胞悬液的制备<ol><li>每个肺是通过优化组织重量体积比0.2克：5mls预热0.2％沃辛顿2型胶原酶（顿生化，翠湖，新泽西州，猫＃LS004202），溶解在无菌的HBSS消化。该混合物在37℃水浴30分钟2,3中浸泡。 </li><li>磨碎样品，直到肺组织消化流动很容易通过10毫升吸管（约10重复）。孵育额外的15分钟，在37 ° C到完成组织消化，并确保更均匀的单细胞悬液。 </li><li>稀释肺细胞悬液与HBSS和磨碎与5毫升吸管驱散剩余的组织碎片。 </li><li>过滤器暂停使用70μM细胞过滤器去除未消化的组织碎片。样品可分为multipl在这一点上发送锥形管，以防止超载一个细胞过滤器和减少时间。 </li><li>沉淀10分钟在1500转和倒出上清液的细胞悬液。 </li><li>轻轻重悬细胞沉淀用室温红细胞裂解液（eBiosciences，圣迭戈，CA;猫＃00-4333-57）在室温下5分钟，并培育。加入等体积的HBSS灭活裂解液和过滤器使用40微米的细胞过滤器，清除杂物和细胞聚集的细胞悬液。 </li><li>移液器的每个样品中加入10μl染色，肺和骨髓样本使用血球计数细胞数量。注：记录细胞的总量。 </li><li>颗粒的肺细胞的单细胞在10分钟1500转离心悬浮。 </li><li>肺和骨髓细胞重悬在1 × 10 6细胞/毫升在预热（37℃）的DMEM +（贝科的修改鹰的中，高血糖（Gibco公司，卡尔斯巴德，CA;猫＃11965-092）含2％胎牛血清boviNE血清（FBS）和10mm的HEPES。 </li><li>准备一个解决方案，在水中的Hoechst 33342染料（Sigma公司，圣路易斯，密苏里州;猫＃B2261）1mg/ml的股票和过滤消毒1,4。 Hoechst染料可能会储存在-80 ° C作为等分</li><li>赫司特33342染料添加到最后一个5μg/ml的浓度（200X的一个1mg/ml的股票稀释）的单细胞悬液。 </li><li>轻轻反转，并培育混合细胞在37℃水浴整整90分钟。 </li></ol> 3。染色和龙流式细胞仪分析细胞悬液的制备<ol><li> 90分钟后用Hoechst染色肺细胞的所有步骤必须执行在4 ° C或冰以防止染料外排。颗粒细胞在1500转离心10分钟，4℃，在冰冷的染色缓冲液（PBS 2％FBS的）重悬细胞。 </li><li>在使用300 -600μl的PBS含2％的浓度不超过1 × 10 7细胞/管悬浮细胞FBS和10毫米的HEPES。 </li><li>新增充分tittered CD45抗体（通常1:200稀释（BD Pharmingen公司，加利福尼亚州圣迭戈猫＃559864），CD45 - APC的肺和骨髓样本，在冰上孵育10-15分钟包括在未染色的控制。实验过程是有用的设置分析盖茨。 </li><li>颗粒细胞在4 ° C下5分钟1500转。弃上清，悬浮细胞冷冻染色缓冲液（PBS 2％FBS的）在500μL。转让prechilled管理单元第聚丙烯管（Fisherbrand，绍姆堡，白细胞介素;猫＃14 - 956 - 1D）。 </li><li>加入碘化丙啶（PI，200μg/ml股票在PBS）浓度为2μg/ml最终排除死细胞样本。 PI必须结合使用的Hoechst 33342染料来实现正确的流式细胞仪分析后的荧光轮廓。 </li><li>存放在冰管，保护，直到流式细胞仪分析。 </li></ol> 4。流式细胞仪分析，以确定和隔离一个肺间质干细胞（肺MSC）的人口使用Hoechst染料外排检测侧群（SP） <ol><li>本分析有以下仪器要求： </li><ul><li> 488 nm和紫外线（350-355纳米）激光器</li><li> 2蓝色（六十五分之四百五十〇）和红色（五十分之六百七十五）过滤器的紫外激光路径上的探测器。 </li><li>红色的紫外线探测器必须具有高灵敏度的红色信号。这可能是一个旧的细胞分拣中存在的问题。 </li><li>冷热水机组，样品保持在5-10 ° C。 </li></ul><li>对齐激光器和细胞分选，按照制造商的协议。 </li><li>收集红色（X轴）和蓝色（Y轴）赫斯特信号采用线性规模。 </li><li>光电倍增管电压调整放在情节的G0/G1期的高峰，在右上角的中心，以便尾随方人口的足够的显示。部分的细胞周期的S期和整个G2可能规模上右上角的情节。蓝色信号NAL是比较光明的，而红色信号是暗淡的。典型MoFlo XDP（Beckman Coulter公司，迈阿密，佛罗里达州）的电压是425为蓝色和红色的650。 </li><li>死细胞出使用PI门控。可以使用标准的PI路径（488nm激发，发射630nm）。此外，PI是兴奋的紫外激光和死细胞人口在于规模的右侧侧群（SP）的情节。这减轻了需要补偿从任何其他如FITC和PE荧光的PI信号。原古德尔方法遵循5-7后者程序。 </li><li>在分析过程中保持相对低的压差和排序，以确保紧的一面人口解决。 </li><li> SP的出现，除了从肺细胞的主要人口侧臂。这一人群被称为赫斯特低 。 </li><li>的Hoechst 低 / SP进行分析，以独立的CD45的正面和负面的人群使用直方图 2,3（六igure 1）。 </li><li>选择和浇注的Hoechst 低 CD45的NEG肺海安人口的细胞可能被收集整理成筒或单细胞作为一个人口混杂，隔离到一个96 孔板 3的克隆。 </li><li>排序的排序流偏转板调整至25％，创造一个更垂直的排序比通常用于管排序流。 </li><li>目的排序流发送到左上角的测试流脉冲，然后右下角的一个96孔板。 </li><li>设置分拣机软件板块地图存入每口井所需的细胞数量。 </li><li>要隔离单细胞克隆是一个单肺MSC排序进入到培养基150μl（α- MEM补充含20％FBS）以及96孔板。排序中的额外100μL。 </li></ol> 5。肺癌的MSC和克隆的分离，培养和表征<ol><li> CulturE和扩大混合肺MSC人口和单细胞克隆以下排序是相同的，使用标准的文化条件（37 ° C，5％CO 2和湿度&gt; 95％ ）。细胞是坚持16小时以下隔离。媒体是每48小时更换（α- MEM含20％FBS的补充）。当细胞开始增殖更迅速，更达到75-80％汇合，他们是代（图2）之间。 </li><li>细胞是预热的HBSS冲洗，并用0.5％胰蛋白酶/ EDTA（Cellgro，弗吉尼亚州马纳萨斯的猫＃25 - 053 - CL）在37 °不到2分钟，C。细胞是通过一个倒置的范围，以确认一个细胞悬液的外观。 </li><li>龙海安，然后再除以在1:2或1:3的比率取决于对当前的文化增殖率。 </li><li>肺癌的MSC能够分化成传统的间充质谱系的成骨细胞，脂肪细胞和软骨细胞和他们的接受间质标记的细胞表面表达我小号评估每个单元的准备。细胞遗传学显带分析也进行确认总值2,3,8-11染色体异常的情况下。 </li></ol> 6。肺海安枚举使用克隆形成实验（CFU - F） <ol><li>完成MesenCult介质（干细胞技术，温哥华，不列颠哥伦比亚省）稀释到终浓度为1 × 10 6细胞/ ml的细胞。 </li><li>执行系列稀释，以实现最终浓度在6x10 4个细胞，3x10 4细胞，1.5 × 10 4细胞，并在媒体10毫升0.75 × 10 4细胞百毫米菜。分析重复或一式三份，每个单元的浓度和可能规模较小的表面积。 </li><li> 10天标准条件下培养细胞。第10天细胞，用PBS冲洗，固定为5分钟，在室温下使用100％的甲醇。 </li><li>检测经染色的CFU - F的殖民地，与0.4％W / V姬姆萨染色解决方案（西格玛化工有限公司，圣路易斯，密苏里州），用去离子水稀释1:20。允许样品风干和量化殖民地的殖民地大于25％的细胞（图3）。 </li></ol> 7。 T细胞增殖和凋亡肺癌的MSC的免疫调节特性分析<ol><li> 6.25x10 4每口井的MSC细胞肺癌接种于96孔板，静置过夜 2 。 </li><li> CD4 + T -细胞受体（TCR）转基因小鼠，卵清蛋白323-339肽与T细胞能够识别OT -二，脾脏细胞纯化消耗+，CD19 +，MHC -Ⅱ+和CD25 +细胞与CD8一个Miltenyi AutoMACS细胞分选（Miltenyi，赤褐色，CA）。抗原呈递细胞（APC）是孤立的积极排序MHC -Ⅱ+细胞12。 </li><li> APC的4％多聚甲醛是固定的，在PBS / 5％BSA/2mM EDTA洗3次，0.5μg/ml或5μg/ M加载升OVA323肽。 </li><li>模拟，被捕APC的增长是在96孔板肺海安。 </li><li>大于95％，纯的CD4 + 1 × 10 5细胞标记5μM羧基琥珀酰亚胺酯（CFSE;西格玛，密苏里州圣路易斯）的PBS孵育5分钟，在黑暗中，洗净，用PBS - 5％BSA，然后添加到APC和肺的MSC细胞在DMEM培养液5％胎牛血清媒体。细胞，然后培养96小时。 </li><li> T细胞，然后防抗CD40分子（Cy5的）; CD4（APC - Cy7）; CD8（上ViolBlue）; Vbeta 5（PE）和染色检测使用一个Miltenyi MacsQuant 7通道流式细胞仪（Miltenyi，Aurburn，CA）和FloJo分析软件（Treestar，亚什兰，或）。扩散是减少的背景相比，平均CFSE荧光强度测量，与CFSE标记的T细胞，并没有接触到APC（图4）。 T细胞计数在这个时候，可行性评估与台盼蓝排斥。 </li></ol> 8。代表性的成果： 图1流式细胞仪分析和龙的MSC定义 。肺组织消化的单细胞悬液用Hoechst 33342染色和流式细胞仪检测出的差异。前向散射（FSC）和侧向散射（SSC） 和 B。赫斯特蓝色和红色荧光。在门口看到一个侧群（SP）的存在。然后分析的Hoechst 低 SP单独的 C。 CD45 阴性肺癌海安人口。 CD45的积极肺SP负比率通常是70:30 / 60:40。 <img src="/files/ftp_upload/3159/3159fig2.jpg" alt="图2"/> 图2：龙的MSC分离和文化。以下收集的肺癌细胞分选海安，细胞镀在使用α- MEM supplem30毫米菜ented含20％FBS。新鲜排序细胞出现小的，圆而明亮，B 。经过约2-3周与间充质表型的殖民地成为明显和扩散更为明显。 <img src="/files/ftp_upload/3159/3159fig3.jpg" alt="图3"/> 每所描述的协议扩展肺海安镀CFU - F检测。 答 ：经过10天和姬姆萨染色菌落是显而易见的。B.菌落大的一个组成图3。菌落形成成纤维细胞含量的MSC中的枚举。数百个细胞C.细胞显示间充质的表型。 A和B，比例尺= 0.5厘米，C比例尺= 0.14厘米。 <img src="/files/ftp_upload/3159/3159fig4.jpg" alt="图4"/> 图4。CFSE基于T细胞增殖实验 。在肺MSC和antige存在的情况下ñ呈递细胞（APC）+ / -卵白蛋白（OVA）CD40的积极效应T细胞表现出CFSE强度下降，这表明扩散（红色圆圈） 。 CFSE荧光强度降低的T细胞膜化学计量与每一个细胞分裂，即。各个部门。在肺癌海安，APC的存在+ / - OVA，CD40阳性效应T细胞表现出CFSE强度没有显着的变化，这表明缺乏扩散（红色圆圈）。

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没有利益冲突的声明。

我们已经适应了最初的方法，用于确定骨髓造血干细胞分离的居民肺癌的MSC的具体人口。由于隔离的重复性这些​​细胞，然后很好的特点，海安。其原籍已被定义为在成年小鼠肺癌（反对骨髓衍生）的居民和表型和分子的个人资料记录2。反复隔离这一特点的人口的能力允许的生物的重要性，并在组织的动态平衡和疾病肺MSC的作用的进一步研究。在小鼠和人类的肺组织最近的人口在体内的定义，有利于发展的内源性细胞在肺损伤和疾病的修复，以方便救援指挥了治疗策略。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>磷酸盐缓冲液（PBS） </td><td>西格玛</td><td> P - 5368 </td><td></td></tr><tr><td>汉克斯缓冲盐溶液（HBSS） </td><td> Thermo Scientific的</td><td> SH30588.01 </td><td></td
></tr><tr><td> 0.2％沃辛顿，2型胶原酶</td><td>沃辛顿生物化学</td><td> LS004202 </td><td></td
></tr><tr><td>红血细胞裂解液</td><td> eBiosciences </td><td> 00-4333-57 </td><td></td
></tr><tr><td> DMEM培养液</td><td> Invitrogen公司Gibco公司</td><td> 11965-092 </td><td></td
></tr><tr><td>赫希斯特33342染料</td><td>西格玛</td><td> B2261 </td><td></td
></tr><tr><td> CD45 - APC </td><td> BD Pharmingen公司</td><td> 559864 </td><td></td
></tr><tr><td>碘化丙啶</td><td>西格玛</td><td> 81845 </td><td></td
></tr><tr><td> A - MEM </td><td> Thermo Scientific的</td><td> SH30265.01 </td><td>; </td
></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 16000-069 </td><td></td
></tr><tr><td> 0.5％胰蛋白酶/ EDTA </td><td> Cellgro </td><td> 25 - 053 - CL </td><td></td
></tr><tr><td>完整的MesenCult中等</td><td>干细胞技术</td><td> 05511 </td><td></td
></tr><tr><td> 0.4％W / V姬姆萨染色解决方案</td><td>西格玛</td><td> GS1L </td><td></td
></tr><tr><td> 4％多聚甲醛</td><td>电子显微镜</td><td> 15710 </td><td> 16％paraformeldehyde是稀释到4％，用PBS </td
></tr><tr><td>羧基荧光素琥珀酰亚胺酯（CFSE） </td><td>西格玛</td><td> 21888 </td><td></td
></tr></tbody></table>

isolation &#38; characterization of hoechstlow cd45negative mouse lung mesenchymal stem cells

组织居民间充质干细胞（MSC）是组织修复或再生，纤维化，炎症，血管生成和肿瘤形成的重要调节因子。两者合计，这些研究表明，居民肺癌的MSC发挥的作用在肺组织的动态平衡，损伤和修复过程中，如肺间质纤维化（PF）和动脉高压（PAH）的疾病。在这里，我们描述一个技术定义的居民肺癌海安人口。这部分人口在体内的肺组织的定义，使用定义的标记，方便重复隔离的一个特点是干细胞流式细胞仪和一个特定的细胞类型和功能的研究人口。

Watch this Scientific Journal Video about Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells at JoVE.com

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

在这篇文章中，我们展示了居民小鼠肺间质干细胞（肺海安），他们的扩张，表征和分析免疫属性的隔离。

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

isolation-characterization-hoechstlow-cd45negative-mouse-lung

Research

JoVE Journal

Biology

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

20.3K 次观看.  University of Colorado Denver. 在这篇文章中，我们展示了居民小鼠肺间质干细胞（肺海安），他们的扩张，表征和分析免疫属性的隔离。

视频: Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

科研

生物学

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 ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

小鼠原发性肾管上皮细胞的分离、鉴定及高通量的细胞外流量分析

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...