我们希望大学由Arieh已Shalhav博士率领芝加哥科泌尿外科，泌尿研究嘉莉博士RINKER谢弗主任的支持表示感谢。我们还想大学芝加哥综合癌症中心（UCCCC）LE BEAU米歇尔博士领导的支持表示感谢。我们还与，感谢专家技术援助的人体组织资源中心的核心设施马克林根博士的带领下，和莱斯利·马丁和玛丽·乔·菲克特援助。我们也感谢李特丽免疫组化核心设备运行。这项工作是由芝加哥外科部泌尿外科，该​​科大学，美国癌症协会的研究资助机构（ACS-IRG IRG-58-004）;癌症中心的支持格兰特（P30 CA14599）;布林森基金会;阿尔文鲍姆家庭基金;大学芝加哥癌症研究基金会的董事会; S.克雷格尔由霍华德休斯医学研究所的支持：到毕业的医学研究员髋部（56006772）和癌症生物学训练津贴（T32-CA09594）。最后，我们要感谢罗伯特·克拉克，和弥敦道Stadick博士VenkateshKrishnan，都为他们的批判性评价的稿件。

这项研究进行了严格按照美国国立卫生研究院的实验室动物护理和使用指南的建议。该协议被批准大学芝加哥机构动物护理和使用委员会（IACUC，协议号码72066和72231）。所有的手术麻醉下进行，所有的努力都是为了最大限度地减少痛苦。收购WiCell（麦迪逊，威斯康星州）和培养人类胚胎干细胞系WA01（H1; NIH注册＃0043）使用独立的馈线协议使用mTeSR1媒体（干细胞技术;温哥华，不列颠哥伦比亚省）。胚胎干细胞被用来内10通道解冻。 1。从小鼠或大鼠胚胎的泌尿生殖窦分离<ol><li>安乐死定时孕鼠（17.5天）或大鼠吸入CO 2（18.5天）5分钟。也证实了在大鼠的生命体征停止死亡。然后打破它的脖子人道。在R在安乐死在这一点上。喷70％乙醇消毒腹部皮肤和侧面。切腹部的皮肤和肌肉层，然后切子宫，分离大鼠胚胎的羊膜囊，剪断脐带。胚胎转移到一个50毫升的Falcon管中含有冰冷的Hank氏平衡盐溶液（HBSS）中，并保持在冰上。胎儿安乐死斩首用手术剪刀。 </li><li>将胚胎在100毫米的培养皿。平分胚胎用剪刀在肚脐的水平（ 图1A）。取出胃和小肠。显示女性胚胎卵巢和睾丸在男性胚胎（ 图1B和1C）。 （卵巢位于尾侧磁极的肾脏，睾丸是大致水平的膀胱）。在这个发展阶段，UGSM隔离的主机的存在能够诱导前列腺上皮血统的男性和女性胚胎然而，这个发展的时间点后的睾丸激素。4，前列腺芽开始形成UGSM，和纯UGSM隔 ​​离是非常具有挑战性的。 </li><li>解剖显微镜下，在中间线与维纳斯剪刀露出腹部膀胱后壁切开腹壁。免费上生殖道的附件（雄性胚胎，切断输尿管，沃尔夫管和苗勒氏管;女性胚胎，切断输尿管和苗勒氏管）。从脐带释放膀胱。切耻骨直言分开杜蒙精尖镊子泌尿生殖窦前壁。最后，从直肠泌尿生殖窦后壁分开。在其下端（从尿道）剪切与膀胱的泌尿生殖窦和冰冷的HBSS中存储的泌尿生殖窦整块（膀胱）。 </li></ol> 2。泌尿生殖窦间质细胞的分离<ol><li&gt;解剖显微镜下取出膀胱泌尿生殖窦（ 图1D）。 </li><li>转移泌尿生殖窦含1％胰蛋白酶，钙（Ca 2 +的）和镁（Mg 2 +）的HBSS猎鹰管。管放置在冰箱中在4℃下75分钟。 </li><li>取出胰蛋白酶和10％胎儿的鲍文血清（FBS）的DMEM/F12培养基中和胰酶消化。 </li><li>小心地挑逗粘间充质套管针或的杜蒙细尖镊子（保持完好的上皮管上皮细胞间质污染的机会减少，这可能会导致啮齿动物的腺体形成，随着干细胞衍生的人类上皮管腺上皮细胞）（ 图1D）。 </li><li>转移到一个新的猎鹰管含有DMEM/F12培养基+ UGSM 10％FBS + 1％青霉素/链霉素（PEN /链球菌，0.5毫克/毫升）+ 1％非必需氨基酸（NEAA）加1nm的R1881。排位一点管的培养箱中在37℃用5％CO 2下过夜。 </li><li>组织以200×g离心，弃去上清液。洗净用磷酸盐缓冲液（PBS）（请勿打扰组织沉淀）。 </li><li>添加0.2％胶原酶的DMEM/F12重悬组织。在37°C孵育1小时，温和的摇摆。 </li><li>大力涡消化组织三次，直到它变成均匀的单细胞混合物中，并加DMEM/F12培养基+ 10％FBS的1％P / S + 1％NEAA + 1nm的R1881中的胶原酶。 </li><li>在200 XG离心，然后洗重新悬浮细胞在HBSS UGSM，重复3次充分洗净UGSM的。最后暂停的DMEM/F12间质细胞计数使用血球细胞或细胞计数装置。 </li><li>游离培养胚胎干细胞使用馈线协议（mTeSR1媒体干细胞技术，不列颠哥伦比亚省温哥华）使用的Accutase消化（Millipore公司;比尔里卡，MA）。洗净的胚胎干细胞DMEM/F12媒体在温暖他们的成长在日志记录阶段，再加入温暖的1X的Accutase解决方案，并在37℃孵育15-20分钟，直到单细胞均匀脱离殖民地。新增等量，1X定义的胰蛋白酶抑制剂（Invitrogen公司，大岛，NY）中的Accutase轻轻吸取向上和向下混合，打破了细胞团块。移液管细胞以200×g，3分钟，重新悬浮细胞沉淀中的DMEM/F12培养基到离心管中，离心。离心管3分钟在200 XG再次和去除上清，然后重新暂停在冷的HBSS或DMEM/F12所需的音量。 </li><li>胚胎干细胞与胚胎干/间充质细胞的1:2.5的比例添加。3，这将是一个蜂窝1E 5 UGSM的4E 4每10毫升注射（单个大鼠UGS通常约2E 6间充质细胞的产率）的人类胚胎干细胞的细胞组合物。离心200×g离心5分钟，然后弃去上清液。重新暂停与75％基质胶（BD Biosciences公司，加利福尼亚州圣何塞，混有25％冷的HBSS更容易处理），然后将子肾囊注射管在冰。 </li></ol> 3。移植肾包膜底下的组织重组<ol><li>准备一个无菌手术区。 </li><li>麻醉裸鼠鼠标一个男性氯胺酮/甲苯​​噻嗪IP，使用新鲜的组合，包含1:1:8的比例氯胺酮：甲苯噻嗪：生理盐水。给药将是100毫克/千克氯胺酮和2.5毫克/公斤甲苯噻嗪。一个脚趾捏反应迟钝的动物应该是完全由20-30分钟。 </li><li>手术准备剃须手术部位（如果不使用裸鼠），鼠标，清洁三个交替湿巾70％的酒精，然后用优碘溶液和应用手术悬垂。 </li><li>将眼部水分的药膏（Altalube眼膏）鼠标的眼睛，在麻醉过程中，以防止干燥。 </li><li>在手术过程中，小鼠的呼吸速率与核心体温监控。呼吸率升ULD稳定，保持在40-90次/分钟。芯体的温度进行监控，观察粘膜和颜色的粉红色的脚底。 </li><li>一个1.0厘米长的纵向皮肤切口的背。继续在0.5厘米纵切口切开腹壁。 </li><li>轻轻挤压腹部和肾肾脏保持表面湿润，用无菌生理盐水滴。 </li><li>轻轻穿刺肾包膜使用一个空的27号注射器针头。一个非常浅刺就足够了。这将是您插入您的钝针注射的细胞混合物。 </li><li>用10微升的细胞混合物中填写的28号钝的注射器针头。慢慢插入穿刺部位和穿透肾实质面积将达肾包膜下方。注入10μl的细胞混合物以形成丸剂对肾脏肾小囊的下面。提款针。 </li><li>肾脏阿卜杜接线腔。固定用4-0尼龙缝合腹膜肌肉层。 </li><li>关闭的皮肤，无论是缝合或主食。 </li><li>清洁血液从皮肤，用无菌生理盐水。 </li></ol> 4。手术后的镇痛和监测<ol><li>丁丙诺啡（0.1毫克/千克，每12小时）或酮洛芬（3-5毫克/公斤，盐水每隔12小时）注入动物管理手术后的疼痛，1毫升37°C生理盐水IP动物保持温暖并防止脱水。 </li><li>将在一个干净的笼子里的动物，并把它放在一个温和的加热垫。 </li><li>发送的动物，动物房，一旦他们恢复知觉，并正在内笼。 </li><li>意想不到的疾病和感染迹象随后三天，每天观察动物。将小鼠观察在笼子里，包括存取的食物和水后1天可操作的活动监测。如果老鼠显示活动较少，酮洛芬是腹腔广告再次伺候，每天一次。 </li><li>去除皮肤钉或缝线手术后2周。 </li><li>异种移植组织可以尽早手术后8周收获。使用小动物超声（ 图2A），可以是固定的组织切片，染色用免疫组化实验（ 图3）的各种蛋白质，异种移植物可在体内成像。 </li></ol>

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作者提出的工作有没有利益冲突。

组织重组使用UGSM是一个非常有用的技术来探讨前列腺，导致前列腺癌症的发生的分子事件的发展。已被用于众多应用在前列腺研究中的感应电势UGSM，其中包括提高肿瘤的前列腺细胞系和肿瘤研究上皮间质的相互作用，并形成跨物种前列腺重组体。7,17-20适当的准备UGSM然而，实验成功，将导致污染啮齿动物上皮腺腔形成，可以混淆结果是至关重要的。因此，分离的UGSM从UGSE中（步骤2.4）是?...

为更好的人体模型系统对前列腺癌有一个巨大的需要。尤其是，相关的人体模型系统的正常，非恶性前列腺组织可以操纵基因直接辨别特定基因的作用在前列腺癌的启动将是令人难以置信的信息。基因组时代的来临已经确定了许多基因可能在癌症的形成有一定的作用。然而，缺乏人体实验模型系统严重损害我们的能力，功能测试和表征候选前列腺癌易感基因。一个理想的模型系统，将有利于迅速和更迅速的癌症易感基因组合与适当的转基因啮齿动物模型系统的功能分析。此外，这种人体模型系统将使前列腺癌变的信号机制向发现和验证新疗法的分子特征预防前列腺癌的形成。 人胚胎干细胞（人胚胎干细胞）是能够形成作为异种移植的人类前列腺组织。泰勒等人在2006年报道，人类胚胎干细胞可以诱导在体内与啮齿动物的泌尿生殖窦间充质（UGSM）在8-12周的时间段之内重新组合以形成前列腺上皮细胞。1这些研究是基于以前的工作库尼亚实验室表明，啮齿类动物胚胎UGSM可以促进前列腺分化的干细胞和胚胎在体内的上皮细胞群。2,3前列腺癌的发展从一个胚胎原基称为泌尿生殖窦（UGS），胚胎17天前（E17鼠标一天E18在大鼠）UGS可以删除，并在物理上分为上皮（UGSE）和间充质（UGSM）4本组织重组方法已明显使我们进一步了解前列腺的发育和癌变，特别是光年生长因子和激素信号转导途径和前列腺基质和上皮细胞的分子之间的关系。5-8此方法涉及体外的UGSM组合与干细胞或上皮细胞中的相同或不同的种属，这些细胞/组织的重组体注入和生长经过一段时间的在体内的生长和小鼠异种移植物于主机。4,9，所述植入物包含嵌入在基质组织的前列腺上皮腺状结构。进一步的染色，也可进行，以确定这样的结构是否是真正的前列腺和人类起源。10,11

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Hank's Balanced Salt Solution (HBSS)</td>
 <td>GIBCO</td>
 <td>14170</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>GIBCO</td>
 <td>11330</td>
 <td></td>
 </tr>
 <tr>
 <td>R1881</td>
 <td>Sigma</td>
 <td>965-93-5</td>
 <td>Mix to 1 ug/ml in Ethanol (1,000x stock)</td>
 </tr>
 <tr>
 <td>NEAA</td>
 <td>GIBCO</td>
 <td>11140</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen-Strep Solution</td>
 <td>GIBCO</td>
 <td>15070</td>
 <td>100x stock</td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>BD Biosciences</td>
 <td>354230</td>
 <td></td>
 </tr>
 <tr>
 <td>KETASET (ketamine hydrochloride) </td>
 <td>Fort Dodge Animal Health</td>
 <td>NDC 0856-2013-01</td>
 <td>100 mg/ml; dilute 1:10 in sterile saline </td>
 </tr>
 <tr>
 <td>AnaSed (xylazine) </td>
 <td>VET-A-MIX, Inc.</td>
 <td>NADA 139-236</td>
 <td>20 mg/ml; dilute 1:10 in sterile saline</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>BD Biosciences</td>
 <td>215240</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase </td>
 <td>Sigma</td>
 <td>C2014</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketoprofen </td>
 <td>Fort Dodge</td>
 <td>NDC 0856-4396-01</td>
 <td>100 mg/ml; dilute 1:1,000 in sterile saline</td>
 </tr>
 <tr>
 <td>Altalube eye ointment </td>
 <td>Altaire Pharmaceuticals, Inc.</td>
 <td>NLC 56641-19850</td>
 <td></td>
 </tr>
 <tr>
 <td>Leica MZ16 F Stereomicroscope</td>
 <td>Leica</td>
 <td></td>
 <td>Any good dissecting scope can be used.</td>
 </tr>
 <tr>
 <td>Vannas spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15001-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Hamilton</td>
 <td>84855</td>
 <td></td>
 </tr>
 <tr>
 <td>Hamilton Needle, Small RN, 28 gauge, 0.5inches, Point Style #3 (Blunt)</td>
 <td>Hamilton</td>
 <td>7803-02 </td>
 <td>Custom Needle </td>
 </tr>
 <tr>
 <td>Ethanol Prep Pads</td>
 <td>Fisher Scientific</td>
 <td>06-669-62 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile Gauze Pads</td>
 <td>Fisher Scientific</td>
 <td>22-415-469 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethicon Vicryl Suture (4-0 FS-2)</td>
 <td>MedVet International</td>
 <td>J392H </td>
 <td>Needle-in, dissolvable suture </td>
 </tr>
 <tr>
 <td>Autoclip 9 mm Wound Clips</td>
 <td>Becton Dickenson</td>
 <td>427631</td>
 <td></td>
 </tr>
 <tr>
 <td>PVP Iodine Prep Pads</td>
 <td>Fisher Scientific</td>
 <td>06-669-98 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissector scissor with blunt end</td>
 <td>Fine Science Tools</td>
 <td>14072-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont fine tip forceps</td>
 <td>Fine Science Tools</td>
 <td>11252-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder with Scissor</td>
 <td>Fine Science Tools</td>
 <td>12002-14</td>
 <td></td>
 </tr>
 </tbody>
</table>

formation of human prostate epithelium using tissue recombination of rodent urogenital sinus mesenchyme and human stem cells

人源性和激素天真的模型系统，这限制了我们的能力，了解相关前列腺疾病引发的遗传和分子事件的可用性，在前列腺癌的研究进展受到严重限制。对发展更好的模型系统研究人类前列腺癌，我们和其他人利用胚胎鼠的前列腺基质亲前列腺独特的感应电势，被称为泌尿生殖窦间充质（UGSM）。当重组的目标，如胚胎干细胞的多能性的细胞群，UGSM睾酮依赖性诱导正常人类前列腺上皮细胞的形成。这种人体模型系统可用于调查和实验测试候选前列腺癌易感基因的能力比较典型的啮齿动物转基因研究加快。由于人类胚胎干细胞（胚胎）可以在全光照培养转基因克诱导基因表达或siRNA击倒向量组织重组之前，这样的模式有利于测试基因功能的后果，或基因组合，这被认为是促进或防止癌变的。 然而，分离纯人群的UGSM细胞，该技术的挑战和学习往往需要与以前的专业知识的人亲自教导。此外，肾包膜下的细胞混合物接种的免疫功能低下的主机可以是技术上的挑战。这里，我们勾勒，说明适当的隔离的UGSM从啮齿动物胚胎组织混合物形成人类前列腺上皮和肾囊的着床。这样的方法，在目前的阶段，需要在体内异种移植的胚胎干细胞，未来的应用程序可能会包括在体外腺形成或使用诱导式多能性干细胞群（iPS细胞）。

泰勒等人的精彩的 ​​报告基础上，我们的实验室已研制出一种嫁接协议使用常用的H1（NIH-WA01指定，男性基因）人类胚胎干细胞系。1这行已经严格测试，质量控制和核型正常的13，当适当的培养，胚胎干细胞可以保持，扩大，并使用无饲养层培养方法（馈线系统通过干细胞技术市售温哥华BC），冷冻保存在一个未分化和多能状态。14我们协议采用结合与单细胞悬液E...

Watch this Scientific Journal Video about Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells at JoVE.com

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

为了解开最早的分子机制启动前列腺癌，新颖创新的人体模型系统和方法是迫切需要的。诱导多能干细胞群形成的人类前列腺上皮细胞的潜力预前列腺泌尿生殖窦间充质（UGSM）是一个强大的实验工具，在前列腺研究。

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

formation-human-prostate-epithelium-using-tissue-recombination-rodent

Research

JoVE Journal

Medicine

13.6K 次观看.  University of Chicago. 为了解开最早的分子机制启动前列腺癌，新颖创新的人体模型系统和方法是迫切需要的。诱导多能干细胞群形成的人类前列腺上皮细胞的潜力预前列腺泌尿生殖窦间充质（UGSM）是一个强大的实验工具，在前列腺研究。

视频: Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

科研

医学

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

人类前列腺上皮的激光捕获显微切割的RNA分析

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

人前列腺癌的临床前原位小鼠模型

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...