这项工作是由纽约州卫生署（C026878）。 MS是在纽约干细胞基金会德鲁肯米勒研究员。支持部分的March of Dimes的基础研究批准号：5-FY11-571。

1。饲养细胞的制备请注意，下面描述的所有试剂应在无菌的方式（请参阅表1和表2）。此协议采用JK1细胞系（了解Cell Biolabs公司，目录＃CBA-315），这是成年小鼠睾丸体细胞的转化的衍生物和其它地方已经描述了3作为馈线。还要注意的是，所有的动物程序应按照机构的指导方针和法规进行。 <ol><li> JK1细胞可以维持在培养，并成功地用来作为馈线通路39。文化JK1细胞在100毫米的细胞培养皿（BD隼，目录＃353003）用10毫升过滤灭菌馈线生长培养基（DMEM培养基补充有10％牛胎儿血清[胎牛血清，2mM的L-谷氨酰胺和抗生素）。当培养达到95％汇合，分裂的细胞1:6 -1:10比。 </li><li>删除介质融合的JK1饲养细胞单层。 </li><li>一次，用PBS洗细胞层，无钙和镁。 </li><li>加入预热的胰蛋白酶/ EDTA，1×（0.05％trypsin/0.53 mM的EDTA，康宁Cellgro，产品编号25-051-CI），并于37°C为5-10分钟。 </li><li>失活胰蛋白酶与馈线等体积的生长培养基中。离心收集细胞，并在300×g离心5分钟。重新悬浮细胞在馈线生长培养基。 </li><li>预涂多孔细胞培养板中，加入足够的0.4％明胶溶液覆盖，并在室温下5分钟后，移除。 </li><li>板〜250个细胞每mm 2在明胶包被的细胞培养板（ 例如，48孔或6孔的格式）。为16至24小时，在37°C孵育文化。 </li><li>将培养基井。注意：要灭活细胞的生长，添加新鲜的溶液中于10微克/毫升丝裂霉素C的DMEM中稀释到各孔中（ 例如，200μl/孔的48孔板）。丝裂霉素-C是有毒的。小心处理和处置的。孵育37℃下4小时。 </li><li>从细胞中移除丝裂霉素-C溶液。用DMEM洗涤3次之前电镀干细胞。 </li><li>删除的DMEM从饲养细胞井，并添加足够的干细胞的培养基（100μl/孔的48孔板; 见表3），以防止干燥，将进料器中的传代。添加精原干细胞在同一天，如下面的第3.1或3.4，使用指定大小的孔中馈线。 </li></ol> 2。解离的小鼠睾丸组织获取供体精原细胞<ol><li>收获成年小鼠睾丸在无菌的时尚和暂时存放在有盖培养皿（不添加任何液体）睾丸。这道菜必须立即放置在冰上，以防止使干燥，保持细胞活力。 </li><li>使用无菌细的钳和罚款剪刀在细胞培养罩，作一横切口的白膜没有完全横断的睾丸（ 即切〜½ - ¾距离THRough），并用钳子挤出板;丢弃图尼卡和连接组织到一个角落里的曲细精管。查看板置于冰上整个保持组织的冷冻，从而保持其完整性和防止蒸发。 </li><li>快速切末管细春风似剪刀至少每3分钟睾丸，同时保持冰盘上。 </li><li>收集在50毫升锥形管和肾小管片段洗〜40毫升冰鲜PBS / 1％牛血清白蛋白。 </li><li>离心60 xg离心10分钟，分离肾小管从精子和碎片的碎片。弃去上清液。 </li><li>在一个15毫升锥形管中，将沉淀重悬于3毫升每睾丸预热的离解缓冲液（DMEM培养液，0.05％胰蛋白酶，0.03％胶原酶I型，80 U / ml的DNA酶I和0.5％牛血清白蛋白，请参阅表2的离解缓冲液中的更具体的细节）。 </li><li>水平放置的锥形管的齿条在振动机中设置到150 rpm，在37℃下进行15分钟至最大化gitation。 </li><li>降速在60×g离心10分钟，未解离的组块的独立的单细胞。 </li><li>下次含颗粒的块，直到下一个步骤。收集上清从步骤2.8，并加入3毫升DMEM/10％胎牛血清中离解缓冲液。将管上的冰是暂时的。 </li><li>重复步骤2.6〜2.7与保存的颗粒从2.9步重新消化块后剩余的步骤2.8。 </li><li>加入等体积的DMEM/10％FBS中和离解缓冲液。 </li><li>从步骤保存的细胞悬液从步骤2.9 2.11重组暂停。以300×g离心5分钟。 </li><li>在一个体积为16毫升每对睾丸干细胞培养基重悬沉淀（ 见表3）。 </li></ol> 3。电镀睾丸细胞悬液<ol><li>板2毫升每孔的细胞悬浮液中的6孔板中含有饲养细胞的有丝分裂，丝裂霉素-C和地点在细胞培养箱灭活在37＆度，C在5％CO 2。 </li><li> 48小时后，吸出培养基，添加2毫升的新鲜培养基中，并返回到培养箱。 </li><li> 48小时后，向每孔加入0.5毫升新鲜干细胞培养基，并返回到培养箱。 </li><li> 48小时后，随后喂细胞每周三次，此后如下，直到大，谨慎的殖民地（&gt; 50个细胞）出现（7至21天之内）。对于第一和第二供给每星期（ 例如星期一和星期三）删除〜50％的培养基中，并添加50％（体积）的新鲜培养基。对于第三喂养（ 例如星期五），吸出所有的中型和2毫升新鲜培养基更换。这种方法是基于上的理由，避免完全培养基变化浓度的旁分泌信号被分泌到培养基中9波动最小化。 </li></ol> 4。殖民地采摘的第一通道<ol><li>删除介质的井，传代的殖民地，并添加新鲜的干细胞培养基。 </li><li>使用10倍的目标识别殖民地。稍微聚焦显微镜，SSC菌落在井沿将显示为均匀的明亮的团块，包括许多11-12微米直径的细胞，其中它是困难或不可能辨别个别单元格边框，由于细胞出现熔合在一起（ 图1A）。可能会出现非SSC菌落较暗，粒状，或不太均匀，可辨别的边界（ 图1A，插图）。 </li><li>用200μl移液管尖，与设置到50μlpipetteman，首先占用介质从阱和驱逐润湿小费。然后，轻轻地用刀尖轻移殖民地之前迅速取出50μl的培养基撞出到的前端（ 图1B）通过吸菌落。 </li><li>驱逐到一个孔的含有灭活的饲养细胞用100μl的干细胞培养基中（ 图1C）的48孔板的培养基中的菌落。 </li><li>重复步骤4.3和8殖民地同井，每口井不超过500微升。 </li><li>重复步骤4.3至4.5的48孔板准备新井，通过一个SSC殖民地。 </li><li>井将在7-14天，分裂后的大团块高达500个细胞出现（ 图1D）。 </li></ol> 5。随后的扩大和传代SSC的研制<ol><li>到新鲜的馈线，第一代的细胞传代培养后至14天的第3代和分流比为1:2 1:4 1:6（每7天），此后如下。通过跨细胞洗涤介质与1毫升移液管尖轻轻捣碎菌落关闭半汇合井（ 如 〜300,000精原干细胞/孔的6孔板）。菌落可以看出分离。液体流用力过猛，会造成不必要的饲养层细胞来过。 </li><li>在锥形管和离心收集捣碎细胞在300×g离心5分钟。注：可以安全地在这里添加一个胰蛋白酶消化步骤，如果需要的话，研究者。 </li><li>吸液上清液，并重新悬浮于新鲜的干细胞培养基彻底上下吹打扰乱菌落。 </li><li>板精原干细胞对丝裂霉素-C的新鲜制备的饲养层细胞失活，如第1节所述。 </li><li>饲料细胞3次，每周如下。对于第一和第二馈电（ 例如，星期一和星期三）中添加50％（体积）的新鲜培养基。对于第三喂养（ 例如星期五），吸出所有的中型和2毫升新鲜培养基更换。板将成为在7-10天内汇合。文化应该由98％的生殖细胞（ 即 &lt;1％的污染体细胞）5-7通道后，根据免疫组化（ 例如 ，使用小鼠生殖细胞标记GCNA）来区分生殖细胞，体细胞10。注：这些方法是在符合规定和要求，提出由该机构。人动物管理和使用委员会在威尔康乃尔医学院。 </li></ol>

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MS接收通过授权的特许权使用费威尔康乃尔医学院细胞生物实验室，，分配JK1细胞。

这种方法获得成人精原干细胞，成人睾丸源性饲养层细胞是强大的，并已成功常见的遗传背景（ 例如，FVB，C57BL6和129SV/C57Bl/6混合）以及不同的突变株时，采用2,3,7,14。事实上，所创造的文化系统中的微环境，是足以克服一些对维持精原干细胞在成人体内 （ 例如，在的情况下，PLZF - / -动物）的基因屏障。虽然我们采用JK1细胞作为供料器，非转化成人睾?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM </td><td>康宁</td><td> 10-013 </td><td>分离缓冲稀释液</td></tr><tr><td>胰蛋白酶/ EDTA </td><td>敏达</td><td> 25-051-CI </td><td></td></tr><tr><td>干细胞基础培养基（StemPro-34） </td><td> Life Technologies公司</td><td> 10639-011 </td><td>需要补充每Shinohara 等人的 。 （2003年）* </td></tr><tr><td>干细胞培养基补充</td><td>各个</td><td>见表3 </td><td>需要补充每Shinohara 等人的 。 （2003年）* </td></tr><tr><td> JK1细胞</td><td>了解Cell Biolabs公司</td><td> CBA-315 </td><td>可以代替成人睾丸间质细胞，每Seandel 等 。 （2007） </td></tr><tr><td>丝裂霉素-C（注意） </td><td> Sigma-Aldrich公司</td><td> M4287 </td><td>有毒小心处理。 </td></tr><tr><td>明胶</td><td> Sigma-Aldrich公司</td><td> G1890 </td><td>在水中的0.4％的溶液</td></tr><tr><td> EVOS xl的数码倒置显微镜</td><td>先进的显微组</td><td> - </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1中。特定的试剂和设备。 *请参阅表3 </td></tr><tr><td> DMEM </td><td>康宁</td><td> 10-013 </td><td>分离缓冲稀释液</td></tr><tr><td>胰蛋白酶（1:250） </td><td> Life Technologies公司</td><td> 27250-018 </td><td>解离缓冲区：最后的0.05％重量/体积</td></tr><tr><td>胶原酶，I型，235 U / ml的</td><td>沃辛顿</td><td> CLS1 235 </td><td>离解缓冲液：最后的0.03％重量/体积</td></tr><tr><td> DNA酶I </td><td> SIGMA-Aldrich公司</td><td> DN25 </td><td>离解缓冲液：最后80 U / ml的</td></tr><tr><td>牛血清白蛋白</td><td> ICP生物</td><td> ABRE-100G </td><td>解离缓冲区：最后的0.5％重量/体积</td></tr><tr><td></td><td></td><td></td><td> 表2中。离解缓冲液 </td></tr><tr><td> StemPro-34 SFM </td><td> Life Technologies公司</td><td> 10639-011 </td><td></td></tr><tr><td> StemPro-34营养补充</td><td> Life Technologies公司</td><td> 10639-011 </td><td></td></tr><tr><td> 额外的补充** </td><td></td><td></td><td></td></tr><tr><td>非必需氨基酸</td><td> Sigma-Aldrich公司</td><td> M7145 </td><td> 1X </td></tr><tr><td> MEM维生素解决方案</td><td> Life Technologies公司</td><td> 11120-052 </td><td> 1X </td></tr><tr><td> L-谷氨酰胺</td><td>敏达</td><td> 25-005 </td><td> 2毫米</td></tr><tr><td>牛血清白蛋白</td><td> ICP生物</td><td> ABRE </td><td> 0.50％ </td></tr><tr><td>抗生素，抗真菌的解决方案</td><td>敏达</td><td> 30-004-CI </td><td> 1X </td></tr><tr><td> D（+）葡萄糖</td><td> Sigma-Aldrich公司</td><td> G8769 </td><td> 6毫克/毫升</td></tr><tr><td> β-雌二醇</td><td> Sigma-Aldrich公司</td><td> E2758 </td><td> 30毫微克/毫升</td></tr><tr><td>黄体激素</td><td> Calbiochem公司</td><td> 5341 </td><td> 60毫微克/毫升</td></tr><tr><td>胎牛血清</td><td>变量</td><td> N / A </td><td> 1％ </td></tr><tr><td>牛全息转</td><td> Sigma-Aldrich公司</td><td> T1283 </td><td> 100微克/毫升</td></tr><tr><td>胰岛素</td><td>双子座生物制品</td><td> 700-112P </td><td> 25微克/毫升</td></tr><tr><td>人GDNF </td><td>生命技术部历史ologies </td><td> PHC7041 </td><td> 10纳克/毫升</td></tr><tr><td>人碱性成纤维细胞生长因子</td><td> Life Technologies公司</td><td> PHG0023 </td><td> 10纳克/毫升</td></tr><tr><td>鼠标EGF </td><td> Life Technologies公司</td><td> PHG0313 </td><td> 20纳克/毫升</td></tr><tr><td>腐胺</td><td>研究有机物</td><td> 0778P </td><td> 60μM </td></tr><tr><td>亚硒酸钠</td><td> Sigma-Aldrich公司</td><td> S5261 </td><td> 30海里</td></tr><tr><td>丙酮酸</td><td>阿法埃莎</td><td> A13875 </td><td> 30微克/毫升</td></tr><tr><td> DL-乳酸</td><td> JT贝克</td><td> 0196-04 </td><td> 1微克/毫升</td></tr><tr><td> β-巯基乙醇</td><td> Life Technologies公司</td><td> 21985-023 </td><td> 50μM </td></tr><tr><td>抗坏血酸</td><td> Sigma-Aldrich公司</td><td> A4544 </td><td> 100μM </td></tr><tr> &lt;TD&gt; D-生物素</td><td> Sigma-Aldrich公司</td><td> B4639 </td><td> 10微克/毫升</td></tr><tr><td></td><td></td><td></td><td> 表3中。干细胞培养基 </td></tr><tr><td></td><td></td><td></td><td> * 注意：使用前介质中添加补充。过滤消毒，并保持在4°C。该培养基是稳定至少2周。 </td></tr><tr><td></td><td></td><td></td><td> **我们所采用的不同的制造商，制剂，和/或这些试剂的批号，没有任何明显的有害影响。在一般情况下，细胞培养级试剂。 </td></tr></tbody></table>

serial enrichment of spermatogonial stem and progenitor cells (sscs) in culture for derivation of long-term adult mouse ssc lines

精原干细胞和祖细胞（精原干细胞）的睾丸一个典型的例子成年哺乳动物干细胞，保留生育功能接近的动物的寿命。虽然确切的管理机制，自我更新和分化的体内具有挑战性的研究，已开发各种系统以前传播小鼠精原干细胞在体外结合使用专门的培养基和饲养层细胞1-3。 大多数体外先遣部队到SSC的生物学中衍生的细胞系，从新生儿，可能是由于在获得成人细胞系4的难度。但是，睾丸的不断成熟，直到〜5周龄在大多数小鼠品系中。早在产后期间，戏剧性的变化发生在睾丸的结构和在生物的体细胞和生精细胞，包括大量的干细胞相关的表达水平的改变基因。因此，出生衍生的SSC线可能无法完全概括成人精原干细胞的生物学特性，坚持成年后的睾丸已经达到了稳定状态。 有几个因素阻碍了成人SSC线的生产历史。首先，功能干细胞的比例可能会降低成年时期，无论是由于内在或外在因素5,6。此外，与其他成体干细胞，它一直难以丰富精原干细胞，占成人睾丸细胞，而无需使用相结合的免疫选择或其他排序策略的7。通常采用的策略包括隐睾小鼠作为供体细胞的来源，由于到一个更高的干细胞比其它类型的细胞8使用。基于的假设，即从最初的文化破坏去除体细胞相互作用的干细胞小生境为SSC生存所必需的，我们以前开发的方法，以获得成人的升国际核事件分级不需要免疫选择或隐睾捐助者，但而采用串行富集的精原干细胞的培养，以下简称为SESC 2,3。 下面描述的方法需要一个简单的程序，用于导出成人SSC通过分离成年供体，接着通过电镀睾丸基质细胞系（JK1）3组成的供料器上的细胞的生精小管的线。通过串口从文化，同时富集的精原干细胞传代，牢固地粘附被耗尽，污染非生殖细胞。以这种方式生产的文化包含在不同分化阶段的精原细胞的混合物，其中包含SSC的，基于长期的自我更新能力。关键的SESC方法是，它使精原干细胞在体内长期在体外的自我更新一个完全不同的微环境，使艰难的过渡，从自我更新，产生的污染长期的SSC线，免费体细胞，从而使随后的实验操作SSC的。

通路零野生型的外观，成人SSC菌落后7天是在图1中所示。三维菌落由连接的馈线或由喂料器具有多个层的生长在上面的SSC的下属的细胞外基质沉积的层的扁平细胞。虽然健康的SSC的是明亮的折射和均匀地11-12微米直径，单元格边框是难以区分的菌落的大小可能是高度可变的，在井之间以及从不同的小鼠制备的孔。是由计算机辅助可视化的菌落相显微镜，数字显示和稍微为重点的的焦平?...

Watch this Scientific Journal Video about Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines at JoVE.com

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

这里介绍一个简单的方法来获得和保持从成年小鼠精原干细胞和祖细胞株。该方法利用从体细胞室的成年小鼠睾丸的饲养层细胞。这种技术适用于常见的小鼠品系，包括转基因，基因敲除，基因敲除的小鼠。

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

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

serial-enrichment-spermatogonial-stem-progenitor-cells-sscs-culture

Research

JoVE Journal

Biology

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

13.7K 次观看.  Weill Cornell Medical College. 这里介绍一个简单的方法来获得和保持从成年小鼠精原干细胞和祖细胞株。该方法利用从体细胞室的成年小鼠睾丸的饲养层细胞。这种技术适用于常见的小鼠品系，包括转基因，基因敲除，基因敲除的小鼠。

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

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

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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 ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...