Development of this protocol was supported by the Advancing a Healthier Wisconsin and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

注：患者和/或其监护人应知情同意参与机构审查委员会批准的研究。 1.缓冲区和媒体准备<ol><li>洗涤缓冲液：向制备100毫升的洗涤缓冲液，加入1 ml 100×青霉素/链霉素溶液（链霉素100U / ml青霉素+ 100微克/毫升）的99毫升磷酸盐缓冲盐水（PBS）。 </li><li>尿祖细胞（UPC）介质：要准备UPC中，混合的角质形成细胞无血清（KSF）中等+祖细胞培养基等体积。 <ol><li> KSF培养基：制备角质形成细胞培养基中，添加的表皮生长因子5纳克/毫升（EGF），牛垂体提取物50纳克/毫升（BPE），霍乱毒素的30纳克/毫升，和青霉素/链霉素溶液（100U / ml青霉素，100μg/ ml的链霉素）至500ml KSF介质。 </li><li>祖细胞培养基：添加四分之三DMEM中以四分之一的Ham-F12培养基，并补充有10％FBS，0.4微克/ ml氢化可的松，0.1纳米霍乱毒素，5纳克/毫升的胰岛素，1.8×10 -4 M腺嘌呤，5微克/毫升转铁蛋白，2×10 -9 M 的三碘甲腺氨酸，10纳克/毫升的EGF，和青霉素/链霉素溶液中。 </li></ol></li><li>的hES培养基：添加20％KO血清替代（K-SR），1×MEM非必需氨基酸，2mM的L-谷氨酰胺，100μM的β巯基乙醇，20纳克/ ml的bFGF和青霉素/链霉素至400毫升DMEM / F12培养基。 </li></ol> 2.尿液样品采集<ol><li>指示患者饮用液体30分钟之前，采尿，以确保尿液足够量（〜30-40毫升）可以被收集。 </li><li>得到患者和/或其监护人含有下列项目的尿收集试剂盒：（1）关于如何获得无菌或清洁渔获尿样的书面说明，（2）湿润抗菌洗手间，及（3）的100毫升的无菌标本采集杯。指导患者收集样品。 </li><li>立即将尿液样品在冰上并传送到labora保守党在凉爽。立即处理的尿细胞分离。如果延迟是不可避免的，储存在冰上的样品长达4小时与细胞存活力损失最小。 </li></ol> 3.分离和扩增尿细胞注意：请在一个BSL2生物安全柜在无菌条件下进行如下操作。 <ol><li>使用无菌移液管，移尿样到无菌的50毫升离心管中。离心机在400×g下在室温下10分钟。 </li><li>吸出上清液留下1毫升在管;要小心，不要扰乱细胞沉淀。 </li><li>重悬并结合粒料从多个管为7毫升的洗涤缓冲液，并重复离心，在400×g下在室温下10分钟。 </li><li>小心吸出上清液，留下细胞团块和〜0.2-0.5上清液毫升。重悬在2-3毫升尿祖细胞（UPC）的培养基中，该悬浮液转移到4-6孔的未中涂覆的24孔板中。 </li><li> 72小时后，补充的培养，用0.5毫升每孔新鲜培养基的UPC和之后每2-3天更换培养基。红细胞和鳞状上皮细胞不附着于板将与介质的变化被移除。 </li><li>经过4-6天监测的文化。 注：2-4小细胞集落将开始出现。细胞将是圆形的或细长代表的I型或肾上皮（RE）细胞分别为3的II型。 </li><li>改变介质每2-3天，因为一旦出现细胞，它们将经历快速膨胀阶段。 </li><li>一旦细胞达到80-90％汇合时，电镀后左右9-15天后，分离和传代的细胞（15,000-18,000细胞/ cm 2）到2-4孔中的24孔平板用于进一步扩展。这标志着作为细胞传代1（P1）。 </li><li>通过流式细胞仪分析表征尿细胞谱系规范，免疫组织的iCal染色或通过逆转录酶 - 聚合酶链反应（RT-PCR）。 </li><li>重新编程分离尿液细胞（第4节）或冷冻下来在冷冻培养基（DMEM补充有10％血清和10％DMSO），用于长期cryostorage。 </li></ol> 4.尿细胞重编程使用仙台病毒注：妥善处理和使用PPE（个人防护装备）的建议，同时操纵转染试剂。执行在一个BSL2生物安全柜在无菌条件下进行如下操作。适当处理转染试剂和/或转染细胞的建议，以避免对环境和健康的危害风险。尿细胞重编程，使用仙台重编程套件改装制造商的馈线相关的协议，详情如下。 <ol><li>为了确保使用仙台病毒高效率的重新编程，使用通道1-5尿液细胞是迅速LY分。如果细胞不复制好或成为衰老，放弃他们，因为他们将不能有效地重新编程。 </li><li>种子每孔60,000个细胞在两口井6孔板。马克这是日-2。调节细胞接种密度（5× 十月四日至9日×10 4个细胞每孔）根据需要通过第0天至达到80-90％汇合。 </li><li>第0天（电镀细胞后48小时），制备含四个OSKM因素的仙台重编程载体按季。每个向量添加到1ml预热UPC培养基。使用1-1.5的MOI（5-7.5×10 5 CIU），这足够用于重编程细胞的尿。吸出培养基，并慢慢地加入1ml的UPC + SeV载体对尿小区中的一个井。使用第二以及转导阴性对照。 </li><li>第1天，更换UPC +中的SeV新鲜UPC媒介。由于病毒的细胞毒性24小时后的一些细胞死亡被看见。 </li><li>根据重新编程酷龙效率Ë形成和紧凑的形态，保持每日更换介质，直到6天或7。 </li><li>前一天转导的细胞（第5天或6），通过电镀丝裂霉素-C（10微克/毫升3小时）处理-MEF中以5×10 4个细胞/ cm 2，密度在0.1制备MEF饲养板的离解％明胶涂层百毫米2培养皿。 </li><li>次日（第6天或7），解离的细胞用0.25％胰蛋白酶，将细胞重悬于培养基中的UPC和板5×10 4 - 2个10 5细胞/ 100mm 2 MEF饲养盘。 </li><li>翌日，切换到的hES培养基每天更换介质。观察在显微镜下的细胞来监测转化的细胞。 注意：这些细胞形成克隆聚集具有特色的鹅卵石形态，并具有较高的细胞核，细胞质比例（12-18天后转）。 </li><li>在两到三个星期转导后，挑殖民地和转移到新的板块为酷龙人扩张。 </li><li>选择的iPSC克隆与TRA-1-81抗体孵育细胞1小时37进行活细胞染色 ℃下的CO 2培养箱中，然后用特定的荧光染料缀合的二级抗体计数器染色1小时，在37℃的CO 2培养箱中培养。 </li><li>使用＆G 1.5寸毫针交叉孵化较大TRA-1-81阳性的iPSC克隆成小相等大小的块（每4-6克隆件）。 </li><li>使用无菌移液管尖端来接并转印10-20交叉影线件从多个克隆到基质胶的每个孔（10微克/厘米2）包被的24孔板用的hES培养基。 注：从单独镀成单井单克隆饮片不扩大或保持多能性。 </li><li>由hES改变的iPSC介质24-48小时后的重新编程克隆附着到基质胶的表面。 </li><li>在对基质胶涂层板，手动涉农供应链的维护PE客用吸管尖，以丰富的完全重新编程和多能性营养不良的iPSC（MD-IPSC）克隆任何分化的细胞或污染MEF饲养层细胞。 </li><li>后单个克隆已达到约100微米的大小，通过用0.48毫摩尔EDTA（乙二胺四乙酸）溶液解离3分钟和电镀的MD-iPS细胞的小细胞聚集体悬浮在介质的iPSC展开克隆（补充有5微米Y27632，Rho激酶抑制剂）在新鲜的基质胶（10微克/厘米2）涂布12孔平板。每日更换的iPSC中。 </li><li>每个克隆的完整的重新编程可通过OSKM基因和多能性标记物（的Oct3 / 4和TRA-1-81）免疫组织化学染色的两个基因表达分析来确定。多能性潜力的一个严格的评估应该被用来确认完全重新编程iPSC系能够分化成三个胚层。这可以通过胚状体（EB）形成和分化测定或之三来实现atoma形成实验。 </li></ol>

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The authors declare that they have no competing financial interests.

造型采用iPSCs的心血管疾病已成为了解遗传贡献4-6的常用方法。获得细胞样品从病人的一些未预料到的困难，尤其是幼儿，能够避免通过提供一种非侵入性的方法的选择，如尿液收集。在这个年轻的患者群中，通常难以收集的尿量足够的体积，以产生足够的尿细胞进行重新编程。执教年轻患者饮用液体前尿液收集30分钟，提高了尿细胞分离成功。然而，重复尿收集可能需要在该人群中。我?...

心肌病死亡的患者Duchenne型和Becker型肌营养不良症（MD）的第二大原因。虽然在X连锁的抗肌萎缩蛋白基因突变发生在1：3,500男胎，很少有人知道分子和细胞事件，导致进行性心肌损害。从肌营养不良患者衍生的人诱导多能干细胞已成为一种新的工具来研究潜在的疾病的机制和用于药物筛选1,2。 皮肤活检或血液样本的预期可能不适劝阻青少年患者和/或其监护人放弃了参与研究的同意。尿液样本是体细胞是易于进行重新编程的方法的非侵入性的来源。我们最近已表明，从肌营养不良的患者采集的尿液细胞可以是培养的，高效地重新编程成使用逆转录病毒转导的山中因素iPSCs的（的Oct3 / 4，SOX2，Klf4和c-Myc的; OSKM）1。的逆转录病毒基因递送的缺点是随机整合的重新编程的基因到宿主染色体中。为了克服这个限制，我们已经使用了非整合型仙台病毒为尿细胞重编程。 这个协议的细节由肌营养不良患者中分离尿细胞然后可以分化成心肌细胞或其它细胞类型用于进一步研究的仙台病毒重新编程。该协议也可以适用于其他患者特定的疾病。

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1144px;">List of Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 Oz. Specimen Cup with Lid</td>
			<td>STL Medical Supply</td>
			<td>M9AMSAS340</td>
			<td>For Urine Sample Collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round Glass Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-81</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CytoTune-iPS Sendai Reprogramming Kit</td>
			<td>Life Technologies</td>
			<td>A1378-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen-Strep w/o Glutamine&#160;</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/o Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>0050129SA</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 (1:1)</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene, 1:5000&#160;</td>
			<td>Life Technologies</td>
			<td>15040066</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bFGF (10 &#181;g)</td>
			<td>Life Technologies</td>
			<td>13256-029</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counter Cartridges</td>
			<td>Life Technologies</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum (500 mL Bottle)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recovery Cell Culture Freezing Medium</td>
			<td>Life Technologies</td>
			<td>12648-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, High Glucose, Glutamax</td>
			<td>Life Technologies</td>
			<td>10566-016</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ham&#39;s F-12 Nutrient Mix&#160;</td>
			<td>Life Technologies</td>
			<td>11765-054</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGF Recombinant Human Protein, Liquid Form</td>
			<td>Life Technologies</td>
			<td>PHG0311L</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin, Human Recombinant, Zinc Soln</td>
			<td>Life Technologies</td>
			<td>12585-014</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TeSR-E8</td>
			<td>Stem Cell Technologies</td>
			<td>5940</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ROCK Inhibitor (Y-27632)</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>hESC Qualified Matrix, LVED Free</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>170-8890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DreamTaq Green PCR Master Mix (2X)</td>
			<td>Thermo-Scientific</td>
			<td>K1081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS-Qualified</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ADENINE BIOREAGENT</td>
			<td>Sigma Aldrich</td>
			<td>A2786-5G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHOLERA TOXIN FROM VIBRIO CHOLERAE</td>
			<td>Sigma Aldrich</td>
			<td>C8052-.5MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HYDROCORTISONE</td>
			<td>Sigma Aldrich</td>
			<td>H0888-1G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HOLO-TRANSFERRIN FROM HUMAN</td>
			<td>Sigma Aldrich</td>
			<td>T0665-50MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,3&#39;,5-TRIIODO-L-THYRONINE SODIUM SALT&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T6397-100MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-3598</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluoromount Aquous mounting medium</td>
			<td>Sigma Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% Paraformaldehyde</td>
			<td>Alfa-Aesar</td>
			<td>43368</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">List of Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD44 - labelled with FITC&#160;</td>
			<td>BD Biosciences</td>
			<td>560977</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD146 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD73 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-&#945;-smooth muscle actin</td>
			<td>Sigma Aldrich</td>
			<td>A2547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-Vimentin</td>
			<td>Abcam</td>
			<td>ab8978-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Anti - TRA-1-81</td>
			<td>Life Technologies</td>
			<td>411100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti Oct 3/4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-9081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:415px;">Mouse Anti Dystrophin</td>
			<td>Leica Biosystems</td>
			<td>NCL-DYSB</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells

营养不良性心肌病是肌营养不良症的了解甚少的后果。产生从患者肌营养不良诱导多能干细胞（iPS细胞）可以在体外疾病模型系统的宝贵的蜂窝源和可用于药物的筛选研究。患者来源的尿细胞已被用于在成功的重编程为诱导性多能干细胞，以模拟营养不良的心肌1。寻址整合载体系统的安全问题，我们提出了一个协议使用非整合型仙台病毒载体为山的转导因子成从患者肌营养不良采集的尿液细胞。该协议产生完全重新编程2-3周内克隆。所述多能干细胞是由通道-13载体的无。这些营养不良的iPSC可以分化成心肌细胞和使用，也可以研究疾病的机制或用于药物筛选。

最祖细胞从人尿中分离的阳性尿路上皮的祖和周细胞标记物如CD44，CD73，和CD146（97.37％，97.09％，和97.3％分别; 图1A和1B）。这些细胞还表达其他间充质标记物如α-平滑肌肌动蛋白和波形蛋白（ 图1B）。 RT-PCR分析提供了细胞中，所述培养物的混合群的证据存在是细胞角蛋白-7的弱表达（CK-7）和Uroplakin（UP）-Ia＆-IIIa，尿路上皮谱系的标记物（ 图1C）。</stro...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells at JoVE.com

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

从生成性肌营养不良患者诱导多能干细胞的：尿源性细胞的有效整合，免费重编程

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

generation-induced-pluripotent-stem-cells-from-muscular-dystrophy

Research

JoVE Journal

Medicine

11.1K 次观看.  Medical College of Wisconsin. This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction. 

视频: Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

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 ...

科研

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前列腺癌患者产生派生异种移植模型从循环肿瘤细胞

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

在C2C12肌细胞造型强直性肌营养不良1

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

通过重组酶介导的盒式交换在重组人多能干细胞的快速高效生成<em&gt; AAVS1</em&gt;轨迹

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

剪接开关反义寡核苷酸诱导的 SMA 患者成纤维细胞外显子夹杂的评价

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

丙戊酸在人脐带血源性 CD34 + 细胞造血干细胞体外扩增中的作用

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...